S. cerevisiae

Drug Chem Toxicol, 2003 May, 26(2), 75 - 85
Toxicity of metal ions used in dental alloys: a study in the yeast Saccharomyces cerevisiae; Yang HC et al.; Metal ions are released from dental alloys into the oral environment, which can cause biological responses over short and extended periods . Since most toxic metal ions are capable of inducing oxidative stress on cells through the mitochondrial respiratory chain, mitochondria may contribute to and be a target of metal toxicity . In this study, we investigated the effect of metal ions on growth of the budding yeast, Saccharomyces cerevisiae, and on the morphology and function of yeast mitochondria . Moreover, we tested whether mitochondrial respiratory activity contributes to metal toxicity . Metal ions affected yeast cell growth . The toxicity of metal ions to yeast cells, ranked in decreasing order are as follows: Hg > Ag > Au > Cu, Ni, Co, Zn . This result mostly correlates with the degree of toxicity of those metal ions to growth of human cells . The MIC90 of Hg, Ag and Au ions in synthetic complete media are 0.325, 5 and 320 microM, respectively . None of the toxic metal ions resulted in loss of mitochondrial respiratory activity . However, respiration-deficient rho0 cells appeared to be resistant to Ag ion, but not to Hg and Au ions . Furthermore, at high concentrations, Ag ion caused morphological changes in mitochondria . These studies indicate that yeast may be used as a model system to screen for toxic effect of metals ions from dental alloys, and that oxidation activity in mitochondria may play a role in acute toxicity of silver ion.

Cell Mol Biol Lett, 2003, 8(2), 261 - 8
The genetic characteristics Saccharomyces cerevisiae aci(+) mutants; Grochowalska R et al.; A series of 30 Saccharomyces cerevisiae aci(+) mutants (characterized as acidifying Ogur's glucose medium containing bromocresol purple) were isolated after EMS mutagenesis . All the mutants excreted acid metabolites to the medium after 24 or 48 hours of incubation . The character of the aci(+) mutations was defined using classical genetic techniques . Three of the aci(+) mutants were studied by molecular genetics techniques.

Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7460 - 4 Epub 2003 Jun 16.
Determination of the membrane topology of Ost4p and its subunit interactions in the oligosaccharyltransferase complex in Saccharomyces cerevisiae; Kim H et al.; Ost4p is a minimembrane protein containing only 36 amino acids and is a subunit of oligosaccharyltransferase (OT) in Saccharomyces cerevisiae . It was found previously when amino acid residues 18-25 of Ost4p were mutated to ionizable amino acids and defects were observed in the interaction between Ost4p and either Stt3p or Ost3p, two other components of OT . The transmembrane segment of Ost4p is likely to extend from residues 10-25 . This is consistent with the finding that alpha-helicity is estimated to be 36% by CD analysis of synthetic Ost4p in liposomes . This value is in reasonable agreement with the assumption that amino acids 10-25 (16 of 36 or 44%) are transmembrane . Therefore, the mutation-sensitive region (residues 18-25) is localized to only one half of the putative transmembrane domain of Ost4p . To learn where this region of Ost4p is situated in relation to the faces of endoplasmic reticulum (ER) membrane, we determined the membrane topology of Ost4p using an in vivo method and established that it is an Nlumen-Ccyto, type I membrane protein . These results indicate that the mutation-sensitive region of Ost4p is localized in the cytoplasmic leaflet of the ER membrane . In the current study, we also observed a loss of direct interaction between Ost3p and Stt3p in the presence of ost4 temperature-sensitive mutants, which indicates Ost4p, via interactions with amino acid residues in the cytosolic leaflet of the ER membrane, functions to bind these two proteins together in a subcomplex of OT.

RNA, 2003 Jul, 9(7), 871 - 80
RNA-binding activity of translation initiation factor eIF4G1 from Saccharomyces cerevisiae; Berset C et al.; We identified and mapped RNA-binding sites of yeast Saccharomyces cerevisiae translation initiation factor eIF4G1 and examined their importance for eIF4G1 function in vitro and in vivo . Yeast eIF4G1 binds to single-stranded RNA with three different sites, the regions of amino acids 1-82 (N terminus), 492-539 (middle), and 883-952 (C terminus) . The middle and C-terminal RNA-binding sites represent RS (arginine and serine)-rich domains; the N-terminal site is asparagine-, glutamine- and glycine-rich . The three RNA-binding sites have similar affinity for single-stranded RNA, whereas the affinity for single-stranded RNA full-length eIF4G1 is about 100-fold higher (approximate K(d) of 5 x 10(-8) M) . Replacement of the arginine residues in the middle RS site by alanine residues abolishes its RNA-binding activity . Deletion of individual RNA-binding sites shows that eIF4G1 molecules lacking one binding site are still active in supporting growth of yeast cells and translation in vitro, whereas eIF4G1 molecules lacking two or all three RNA-binding sites are strongly impaired or inactive . These data suggest that RNA-binding activity is required for eIF4G1 function.

Mol Biol Cell, 2003 Jun, 14(6), 2237 - 49 Epub 2003 Feb 06.
She4p/Dim1p interacts with the motor domain of unconventional myosins in the budding yeast, Saccharomyces cerevisiae; Toi H et al.; She4p/Dim1p, a member of the UNC-45/CRO1/She4p (UCS) domain-containing protein family, is required for endocytosis, polarization of actin cytoskeleton, and polarization of ASH1 mRNA in Saccharomyces cerevisiae . We show herein that She4p/Dim1p is involved in endocytosis and actin polarization through interactions with the type I myosins Myo3p and Myo5p . Two-hybrid and biochemical experiments showed that She4p/Dim1p interacts with the motor domain of Myo3/5p through its UCS domain . She4p/Dim1p was required for Myo5p localization to cortical patch-like structures . Using random mutagenesis of the motor region of MYO5, we identified four independent dominant point mutations that suppress the temperature-sensitive growth phenotype of the she4/dim1 null mutant . All of the amino acid substitutions caused by these mutations, V164I, N168I, N209S, and K377M, could suppress the defects of endocytosis and actin polarization of the she4/dim1 mutant as well . She4p/Dim1p also showed two-hybrid interactions with the motor domain of a type II myosin Myo1p and type V myosins Myo2p and Myo4p, and was required for proper localization of Myo4p, which regulates polarization of ASH1 mRNA . Our results suggest that She4p/Dim1p is required for structural integrity or regulation of the motor domain of unconventional myosins.

Mol Biol Cell, 2003 Jun, 14(6), 2226 - 36 Epub 2003 Mar 07.
Pex15p of Saccharomyces cerevisiae provides a molecular basis for recruitment of the AAA peroxin Pex6p to peroxisomal membranes; Birschmann I et al.; The gene products (peroxins) of at least 29 PEX genes are known to be necessary for peroxisome biogenesis but for most of them their precise function remains to be established . Here we show that Pex15p, an integral peroxisomal membrane protein, in vivo and in vitro binds the AAA peroxin Pex6p . This interaction functionally interconnects these two hitherto unrelated peroxins . Pex15p provides the mechanistic basis for the reversible targeting of Pex6p to peroxisomal membranes . We could demonstrate that the N-terminal part of Pex6p contains the binding site for Pex15p and that the two AAA cassettes D1 and D2 of Pex6p have opposite effects on this interaction . A point mutation in the Walker A motif of D1 (K489A) decreased the binding of Pex6p to Pex15p indicating that the interaction of Pex6p with Pex15p required binding of ATP . Mutations in Walker A (K778A) and B (D831Q) motifs of D2 abolished growth on oleate and led to a considerable larger fraction of peroxisome bound Pex6p . The nature of these mutations suggested that ATP-hydrolysis is required to disconnect Pex6p from Pex15p . On the basis of these results, we propose that Pex6p exerts at least part of its function by an ATP-dependent cycle of recruitment and release to and from Pex15p.

Genetics, 2003 Jun, 164(2), 443 - 55
Delineating the requirements for spontaneous DNA damage resistance pathways in genome maintenance and viability in Saccharomyces cerevisiae; Morey NJ et al.; Cellular metabolic processes constantly generate reactive species that damage DNA . To counteract this relentless assault, cells have developed multiple pathways to resist damage . The base excision repair (BER) and nucleotide excision repair (NER) pathways remove damage whereas the recombination (REC) and postreplication repair (PRR) pathways bypass the damage, allowing deferred removal . Genetic studies in yeast indicate that these pathways can process a common spontaneous lesion(s), with mutational inactivation of any pathway increasing the burden on the remaining pathways . In this study, we examine the consequences of simultaneously compromising three or more of these pathways . Although the presence of a functional BER pathway alone is able to support haploid growth, retention of the NER, REC, or PRR pathway alone is not, indicating that BER is the key damage resistance pathway in yeast and may be responsible for the removal of the majority of either spontaneous DNA damage or specifically those lesions that are potentially lethal . In the diploid state, functional BER, NER, or REC alone can support growth, while PRR alone is insufficient for growth . In diploids, the presence of PRR alone may confer a lethal mutation load or, alternatively, PRR alone may be insufficient to deal with potentially lethal, replication-blocking lesions.

J Med Chem, 2003 Jun 19, 46(13), 2631 - 40
Discovery and structural modification of inhibitors of methionine aminopeptidases from Escherichia coli and Saccharomyces cerevisiae; Luo QL et al.; A series of pyridine-2-carboxylic acid derivatives were synthesized according to the leads from the screening, and potent inhibitors have been obtained by structural modification . They have shown submicromolar inhibition of the enzymes (for example, for 9n, IC(50) = 130 nM for EcMetAP1 and IC(50) = 380 nM for ScMetAP1) . They represent small-molecule MetAP inhibitors with novel structures different from alkylating fumagillin derivatives and peptidic bestatin-based MetAP inhibitor.

Folia Microbiol (Praha), 2003, 48(2), 193 - 8
Trehalose, glycogen and ethanol metabolism in the gcr1 mutant of Saccharomyces cerevisiae; Seker T et al.; Since Gcr1p is pivotal in controlling the transcription of glycolytic enzymes and trehalose metabolism seems to be one of the control points of glycolysis, we examined trehalose and glycogen synthesis in response to 2% glucose pulse during batch growth in gcr1 (glucose regulation-1) mutant lacking fully functional glycolytic pathway and in the wild-type strain . An increase in both trehalose and glycogen stores was observed 1 and 2 h after the pulse followed by a steady decrease in both the wild-type and the gcr1 mutant . The accumulation was faster while the following degradation was slower in gcr1 cells compared to wild-type ones . Although there was no distinct glucose consumption in the mutant cells it seemed that the glucose repression mechanism is similar in gcr1 mutant and in wild-type strain at least with respect to trehalose and glycogen metabolism.

Genome Res, 2003 Jun, 13(6A), 1146 - 54
Multimeric threading-based prediction of protein-protein interactions on a genomic scale: application to the Saccharomyces cerevisiae proteome; Lu L et al.; MULTIPROSPECTOR, a multimeric threading algorithm for the prediction of protein-protein interactions, is applied to the genome of Saccharomyces cerevisiae . Each possible pairwise interaction among more than 6000 encoded proteins is evaluated against a dimer database of 768 complex structures by using a confidence estimate of the fold assignment and the magnitude of the statistical interfacial potentials . In total, 7321 interactions between pairs of different proteins are predicted, based on 304 complex structures . Quality estimation based on the coincidence of subcellular localizations and biological functions of the predicted interactors shows that our approach ranks third when compared with all other large-scale methods . Unlike other in silico methods, MULTIPROSPECTOR is able to identify the residues that participate directly in the interaction . Three hundred seventy-four of our predictions can be found by at least one of the other studies, which is compatible with the overlap between two different other methods . From the analysis of the mRNA abundance data, our method does not bias towards proteins with high abundance . Finally, several relevant predictions involved in various functions are presented . In summary, we provide a novel approach to predict protein-protein interactions on a genomic scale that is a useful complement to experimental methods.

Exp Cell Res, 2003 Jul 1, 287(1), 10 - 5
Generation and maintenance of synchrony in Saccharomyces cerevisiae continuous culture; Murray DB et al.; Cultures of Saccharomyces cerevisiae grown continuously produce an autonomous oscillation in many metabolic outputs . The most conveniently measured variable, i.e., dissolved oxygen concentration, oscillates with a period of 40-55 min . Previously we have identified two compounds capable of resetting phase, acetaldehyde and hydrogen sulfide . The phase-response curves constructed for acetaldehyde show a strong (Type 0) response at 3.0 mM and a weak (Type 1) response at 1.0 mM . Ammonium sulfide phase-response curves (pulse injected at 1.0 microM and 3.0 microM) revealed that sulfide is only an effective perturbation agent when endogenous sulfide concentrations are at a maximum . Also only Type 1 phase responses were observed . When the phase-response curve for sulfite (at 3.0 M) was constructed, phase responses were at a maximum at 60 degrees, indicating the possible involvement of sulfite in cell synchronization . It is concluded that endogenously produced acetaldehyde and sulfite tune the oscillation of mitochondrial energization state whereas sulfide mediates population synchrony.

Eukaryot Cell, 2003 Jun, 2(3), 651 - 3
Evidence that synthesis of the Saccharomyces cerevisiae mitochondrially encoded ribosomal protein Var1p may be membrane localized; Fiori A et al.; The 5'-untranslated leaders of mitochondrial mRNAs appear to localize translation within the organelle . VAR1 is the only yeast mitochondrial gene encoding a major soluble protein . A chimeric mRNA bearing the VAR1 untranslated regions and the coding sequence for pre-Cox2p appears to be translated at the inner membrane surface . We propose that translation of the ribosomal protein Var1p is also likely to occur in close proximity to the inner membrane.

Eukaryot Cell, 2003 Jun, 2(3), 599 - 608
Identification of in vivo enzyme activities in the cometabolism of glucose and acetate by Saccharomyces cerevisiae by using 13C-labeled substrates; dos Santos MM et al.; A detailed characterization of the central metabolic network of Saccharomyces cerevisiae CEN.PK 113-7D was carried out during cometabolism of different mixtures of glucose and acetate, using aerobic C-limited chemostats in which one of these two substrates was labeled with (13)C . To confirm the role of malic enzyme, an isogenic strain with the corresponding gene deleted was grown under the same conditions . The labeling patterns of proteinogenic amino acids were analyzed and used to estimate metabolic fluxes and/or make inferences about the in vivo activities of enzymes of the central carbon metabolism and amino acid biosynthesis . Malic enzyme flux increased linearly with increasing acetate fraction . During growth on a very-high-acetate fraction, the activity of malic enzyme satisfied the biosynthetic needs of pyruvate in the mitochondria, while in the cytosol pyruvate was supplied via pyruvate kinase . In several cases enzyme activities were unexpectedly detected, e.g., the glyoxylate shunt for a very-low-acetate fraction, phosphoenolpyruvate carboxykinase for an acetate fraction of 0.46 C-mol of acetate/C-mol of substrate, and glucose catabolism to CO(2) via the tricarboxylic acid cycle for a very-high-acetate fraction . Cytoplasmic alanine aminotransferase activity was detected, and evidence was found that alpha-isopropylmalate synthase has two active forms in vivo, one mitochondrial and the other a short cytoplasmic form.

Eukaryot Cell, 2003 Jun, 2(3), 552 - 9
Rapamycin treatment results in GATA factor-independent hyperphosphorylation of the proline utilization pathway activator in Saccharomyces cerevisiae; Saxena D et al.; Treatment of Saccharomyces cerevisiae cells with the immunosuppressive drug rapamycin results in a variety of cellular changes in response to perceived nutrient deprivation . Among other effects, rapamycin treatment results in the nuclear localization of the global nitrogen activators Gln3p and Nil1p/Gat1p, which leads to expression of nitrogen assimilation genes . The proline utilization (Put) pathway genes were shown to be among the genes induced by rapamycin . Having previously shown that the Put pathway activator Put3p is differentially phosphorylated in response to the quality of the nitrogen source, we examined the phosphorylation status of Put3p after rapamycin treatment . Treatment with rapamycin resulted in the hyperphosphorylation of Put3p, which was independent of Gln3p, Nil1p, and Ure2p . The relative contributions of global nitrogen (Gln3p and Nil1p) and pathway-specific (Put3p) activators to rapamycin-induced expression of the target gene PUT1 were also examined . We found that Nil1p and Put3p, but not Gln3p, play major roles in rapamycin-induced PUT1 expression . Our findings show that perceived nitrogen deprivation triggered by rapamycin treatment and steady-state growth in nitrogen-derepressing conditions are associated with hyperphosphorylation of Put3p and increased PUT1 expression . Rapamycin treatment and nitrogen derepression may share some, but not all, regulatory elements, since Gln3p and Nil1p do not participate identically in both processes and are not required for hyperphosphorylation . A complex relationship exists among the global and pathway-specific regulators, depending on the nature and quality of the nitrogen source.

Eukaryot Cell, 2003 Jun, 2(3), 534 - 41
A Saccharomyces cerevisiae mutant unable to convert glucose to glucose-6-phosphate accumulates excessive glucose in the endoplasmic reticulum due to core oligosaccharide trimming; Miseta A et al.; D-Glucose is the preferred carbon and energy source for most eukaryotic cells . Immediately following its uptake, glucose is rapidly phosphorylated to glucose-6-phosphate (Glc-6-P) . The yeast Saccharomyces cerevisiae has three enzymes (Hxk1p, Hxk2p, and Glk1p) that convert glucose to Glc-6-P . In the present study, we found that yeast mutants lacking any two of these enzymes retain the ability to efficiently convert glucose to Glc-6-P and thus maintain a low level of cellular glucose . However, a mutant strain lacking all three glucose-phosphorylating enzymes contained up to 225-fold more intracellular glucose than normal . Drugs that inhibit the synthesis or the trimming of the lipid-linked core oligosaccharide Glu(3)Man(9)GlcNac(2) effectively reduced the accumulation of glucose . Similarly, mutations that block the addition of glucose residues to the core oligosaccharide moiety, such as alg5Delta or alg6Delta, also diminished glucose accumulation . These results indicate that the intracellular glucose accumulation observed in the glucose phosphorylation mutant results primarily from the trimming of glucose residues from core oligosaccharide chains within the endoplasmic reticulum (ER) . Consistent with this conclusion, both {(14)C}glucose exchange and subcellular fractionation experiments indicate that much of the accumulated glucose is retained within an intracellular compartment, suggesting that the efficient transport of glucose from the ER to the cytosol in yeast may be coupled to its rephosphorylation to Glc-6-P . The high level of cellular glucose was associated with an increased level of protein glycation and the release of glucose into the culture medium via its transit through the secretory pathway . Finally, we also found that the accumulation of glucose may lead to a subtle alteration in ion homeostasis, particularly Ca(2+) uptake . This suggests that this mutant strain may serve as a useful model to study the consequences of excessive glucose accumulation and protein glycation.

Eukaryot Cell, 2003 Jun, 2(3), 431 - 45
Ady4p and Spo74p are components of the meiotic spindle pole body that promote growth of the prospore membrane in Saccharomyces cerevisiae; Nickas ME et al.; Spore formation in Saccharomyces cerevisiae occurs via the de novo synthesis of the prospore membrane during the second meiotic division . Prospore membrane formation is triggered by assembly of a membrane-organizing center, the meiotic outer plaque (MOP), on the cytoplasmic face of the spindle pole body (SPB) during meiosis . We report here the identification of two new components of the MOP, Ady4p and Spo74p . Ady4p and Spo74p interact with known proteins of the MOP and are localized to the outer plaque of the SPB during meiosis II . MOP assembly and prospore membrane formation are abolished in spo74Delta/spo74Delta cells and occur aberrantly in ady4Delta/ady4Delta cells . Spo74p and the MOP component Mpc70p are mutually dependent for recruitment to SPBs during meiosis . In contrast, both Ady4p and Spo74p are present at SPBs, albeit at reduced levels, in cells that lack the MOP component Mpc54p . Our findings suggest a model for the assembled MOP in which Mpc54p, Mpc70p, and Spo74p make up a core structural unit of the scaffold that initiates synthesis of the prospore membrane, and Ady4p is an auxiliary component that stabilizes the plaque.

Yeast, 2003 Jun, 20(8), 739 - 46
Construction of a set of Saccharomyces cerevisiae vectors designed for recombinational cloning; Van Mullem V et al.; The Gateway technology is becoming an increasingly popular method for cloning ORFs by recombination . It allows the transfer of any ORF flanked by specific recombination sites into any vectors harbouring the corresponding sites . Here we describe the construction of a set of 20 Saccharomyces cerevisiae Gateway compatible vectors . These plasmids bear an URA3 or TRP1 selection marker . They are designed for expression without tag sequence or for C- or N-terminal protein tagging with 3HA (haemagglutinin), 13MYC, 4TAP (tandem affinity purification) or GST (glutathione S-transferase) epitopes . The centromeric vectors allow expression of DNA sequence in yeast under tetracycline-regulatable promoters, while expression from the high copy vectors is driven by PGK promoter . To test their applicability, the genes encoding the RNA polymerase I subunit Rpa12p or the TFIIS transcription factor were cloned in these vectors . Their expression was demonstrated using Western blotting or complementation assays .

J Med Virol, 2003 Aug, 70(4), 513 - 9
Saccharomyces cerevisiae-derived HBsAg preparations differ in their attachment to monocytes, immune-suppressive potential, and T-cell immunogenicity; Vanlandschoot P et al.; Expression of the hepatitis B virus S protein results in the formation of a lipoprotein particle, the hepatitis B surface antigen (HBsAg) . Such particles, produced in Saccharomyces cerevisiae, bind to the cell surface of monocytes through interaction with the lipopolysaccharide binding protein and the lipopolysaccharide receptor, CD14 . This attachment is suggested to depend on the presence of charged phospholipids in the particles . In addition, such particles interfere with the lipopolysaccharide and interleukin-2-induced activation of monocytes . In the present study, it is reported that of three Saccharomyces cerevisiae-derived HBsAg preparations, two have a reduced capacity to bind to monocytes . A correlation with a reduced potential to inhibit the lipopolysaccharide-induced activation of monocytes and an increased potential to stimulate HBsAg-specific T-cell proliferation is observed . Surprisingly, differences in phospholipid content that might explain these observations, were not detected .

Int J Oncol, 2003 Jul, 23(1), 229 - 33
WD-40 repeat containing rat lethal giant larvae recessive oncogene, but not m-tomosyn, restores the salt sensitivity in Saccharomyces cerevisiae; Kim YS et al.; Recently, we cloned rgl-1, a putative rat homologue of the Drosophila tumor suppressor oncogene lethal (2) giant larvae (l(2)gl), from the rat brain . Sequence analysis revealed that rgl-1 encodes a 1,036 amino acid polypeptide containing a domain characteristic of WD-40 repeat sequence . It has been suggested that m-tomosyn is expressed in the rat brain, encodes a 1,116 amino acid polypeptide, contains WD-40 repeat sequence, and is homologous to the Drosophila l(2)gl and mouse mgl-1 genes . The comparison of amino acid sequence for m-tomosyn with l(2)gl and mgl-1 proteins using the clustal method showed only 17.8% and 20% amino acid identity, respectively . However, amino acid identity of rgl-1 protein to l(2)gl and mgl-1 proteins is much higher (30.6% and 96.8%, respectively) . Therefore, we investigated their biological function in the absence of Saccharomyces cerevisiae SOP1 and SOP2, the yeast homologues of the Drosophila l(2)gl . Functional analysis showed that the expression of the rgl-1 cDNA, but not m-tomosyn, restored partially salt sensitivity in yeast, indicating that rgl-1 is a homologue of lgl family members.

J Biol Chem, 2003 Aug 29, 278(35), 32692 - 701 Epub 2003 Jun 04.
Functional interactions between Hsp90 and the co-chaperones Cns1 and Cpr7 in Saccharomyces cerevisiae; Tesic M et al.; Hsp90 complexes contain a class of co-chaperones characterized by a tetratricopeptide repeat (TPR) domain, which mediates binding to a carboxyl-terminal EEVD region in Hsp90 . Among Hsp90 TPR co-chaperones in Saccharomyces cerevisiae, only Cns1 is essential . The amino terminus of Cns1, which harbors the TPR domain, is sufficient for viability when overexpressed . In a screen for temperature-sensitive alleles of CNS1, we identified mutations resulting in substitutions of conserved residues in the TPR domain . Mutations in CNS1 disrupt in vitro and in vivo interaction with Hsp90 and reduce Hsp90 function, indicating that Cns1 is a bona fide co-chaperone . Genetic interactions between CNS1 and another Hsp90 co-chaperone, CPR7, suggest that the two co-chaperones share an essential role in the cell . Although both the TPR and the isomerase domains of the cyclophilin Cpr7 are required for viability of cns1 mutant cells, this requirement does not depend on the catalytic function of the isomerase domain . Instead, hydrophilic residues on the surface of this domain appear to be important for the common Cns1.Cpr7 function . Although both co-chaperones interact with Hsp90 primarily through the carboxyl terminus (EEVD), Cns1 and Cpr7 are mostly found in complexes distinct from Hsp90 . EEVD is required for normal growth in cns1 mutant cells, demonstrating for the first time in vivo requirement for this conserved region of Hsp90 . Overall, our findings reveal a considerable degree of complexity in the interactions not only between Hsp90 and its co-chaperones, but also among the co-chaperones themselves.

Biochem Biophys Res Commun, 2003 Jun 20, 306(1), 5 - 9
Genome-wide expression analysis of NAP1 in Saccharomyces cerevisiae; Ohkuni K et al.; Nap1 is a nucleosome assembly protein which is necessary to keep proper nucleosome structures in transcription and replication in vitro . In Saccharomyces cerevisiae, additional functions have been ascribed to Nap1, as it has been shown to interact with Clb2 (B type cyclin) and Gin4 (septum formation) . In this study, we investigate genome-wide expression in the Deltanap1 cells using DNA microarrays . About 10% of all yeast open reading frames changed the transcription level more than 2-fold in the Deltanap1 strain, compared with wild-type strain, in one experiment . Interestingly, these genes, whose expressions are up- or down-regulated in the Deltanap1 cells, are clustered . This result suggests that yeast NAP1 is required for the maintenance of cumulative nucleosome formation in vivo and the loss of Nap1 leads to a change in the gene expression level in a cluster.

Mutat Res, 2003 Jun 19, 527(1-2), 37 - 48
Validation of a novel assay for checkpoint responses: characterization of camptothecin derivatives in Saccharomyces cerevisiae; Zhang H et al.; The evolutionary conservation of pathways preserving genetic stability supports the use of a lower eukaryote such as the yeast Saccharomyces cerevisiae in screening for novel anti-neoplastic agents . Yeast is already established as a model system to characterize the cellular effects of the topoisomerase inhibitor and anti-cancer agent camptothecin (CPT) . Here, we demonstrate that a recently developed two-hybrid based plate assay that visualizes the DNA damage-induced homomeric complex formation of the yeast checkpoint protein Rad17 correctly predicts the biological activity of the tested camptothecin derivatives . The used criteria for biological activity include lethality, cell cycle arrest and Rad53p phosphorylation, an essential signaling event during checkpoint activation . Surprisingly, although responsive to camptothecin and not without influence on drug sensitivity, Rad17p appears to be dispensable for cell cycle arrest and for Rad53p phosphorylation following treatment with camptothecin . Such a role is only uncovered if double-strand break repair is compromised.

Biotechnol Bioeng, 2003 Aug 5, 83(3), 335 - 43
Multicriteria optimization of biochemical systems by linear programming: application to production of ethanol by Saccharomyces cerevisiae; Vera J et al.; In this study we present a method for simultaneous optimization of several metabolic responses of biochemical pathways . The method, based on the use of the power law formalism to obtain a linear system in logarithmic coordinates, is applied to ethanol production by Saccharomyces cerevisiae . Starting from an experimentally based kinetic model, we translated it to its power law equivalent . With this new model representation, we then applied the multiobjective optimization method . Our intent was to maximize ethanol production and minimize each of the internal metabolite concentrations . To ensure cell viability, all optimizations were carried out under imposed constraints . The different solutions obtained, which correspond to alternative patterns of enzyme overexpression, were implemented in the original model . We discovered few discrepancies between the S-system-optimized steady state and the corresponding optimized state in the original kinetic model, thus demonstrating the suitability of the S-system representation as the basis for the optimization procedure . In all optimized solutions, the ATP level reached its maximum and any increase in its activity positively affected the optimization process . This work illustrates that in any optimization study no single criteria is of general application being the multiobjective and constrained task the proper way to address it . It is concluded that the proposed multiobjective method can serve to carry out, in a single study, the general pattern of behavior of a given metabolic system with regard to its control and optimization .

FEBS Lett, 2003 Jun 5, 544(1-3), 160 - 4
Saccharomyces cerevisiae 14-3-3 proteins Bmh1 and Bmh2 participate in the process of catabolite inactivation of maltose permease; Mayordomo I et al.; In this study we show that Reg1, the regulatory subunit of the Reg1/Glc7 protein phosphatase (PP1) complex, interacts physically with the two yeast members of the 14-3-3 protein family, Bmh1 and Bmh2 . By using different fragments of the Reg1 protein we mapped the interaction domain at the N-terminal part of the protein . We also show that Reg1 and yeast 14-3-3 proteins participate actively in the regulation of the glucose-induced degradation of maltose permease (Mal61).

FEBS Lett, 2003 Jun 5, 544(1-3), 148 - 52
Mitochondrial and cytosolic expression of human peroxiredoxin 5 in Saccharomyces cerevisiae protect yeast cells from oxidative stress induced by paraquat; Tien Nguyen-nhu N et al.; Human peroxiredoxin 5 is a recently discovered mitochondrial, peroxisomal and cytosolic thioredoxin peroxidase able to reduce hydrogen peroxide and alkyl hydroperoxides . To gain insight into peroxiredoxin 5 antioxidant role in cell protection, we investigated the resistance of yeast cells expressing human peroxiredoxin 5 in mitochondria or in the cytosol against oxidative stress induced by paraquat . The herbicide paraquat is a redox active drug known to generate superoxide anions in mitochondria and the cytosol of yeast and mammalian cells leading to the formation of several reactive oxygen species . Here, we report that mitochondrial and cytosolic human peroxiredoxin 5 protect yeast cells from cytotoxicity and lipid peroxidation induced by paraquat.

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1093 - 5 Epub 2003 May 23.
Crystallization and X-ray diffraction data analysis of leukotriene A4 hydrolase from Saccharomyces cerevisiae; Andersson B et al.; The Saccharomyces cerevisiae leukotriene A4 (LTA4) hydrolase (scLTA4 hydrolase) has been crystallized in order to study the two activities of LTA4 hydrolase in an evolutionary perspective . Single well diffracting crystals are obtained after switching from the hanging-drop method to liquid-liquid diffusion in capillaries using PEG 8000 as precipitant . These crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.8, b = 98.1, c = 99.2 A . Intensity data to 2.3 A resolution were collected from a native scLTA4 hydrolase crystal using synchrotron radiation . A molecular-replacement solution was obtained using the human LTA4 hydrolase structure and the program BEAST.

Mol Cell Biol, 2003 Jun, 23(12), 4207 - 18
Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II; Krogan NJ et al.; Set2 methylates Lys36 of histone H3 . We show here that yeast Set2 copurifies with RNA polymerase II (RNAPII) . Chromatin immunoprecipitation analyses demonstrated that Set2 and histone H3 Lys36 methylation are associated with the coding regions of several genes that were tested and correlate with active transcription . Both depend, as well, on the Paf1 elongation factor complex . The C terminus of Set2, which contains a WW domain, is also required for effective Lys36 methylation . Deletion of CTK1, encoding an RNAPII CTD kinase, prevents Lys36 methylation and Set2 recruitment, suggesting that methylation may be triggered by contact of the WW domain or C terminus of Set2 with Ser2-phosphorylated CTD . A set2 deletion results in slight sensitivity to 6-azauracil and much less beta-galactosidase produced by a reporter plasmid, resulting from a defect in transcription . In synthetic genetic array (SGA) analysis, synthetic growth defects were obtained when a set2 deletion was combined with deletions of all five components of the Paf1 complex, the chromodomain elongation factor Chd1, the putative elongation factor Soh1, the Bre1 or Lge1 components of the histone H2B ubiquitination complex, or the histone H2A variant Htz1 . SET2 also interacts genetically with components of the Set1 and Set3 complexes, suggesting that Set1, Set2, and Set3 similarly affect transcription by RNAPII.

Biophys J, 2003 Jun, 84(6), 3917 - 23
15N-1H Residual dipolar coupling analysis of native and alkaline-K79A Saccharomyces cerevisiae cytochrome c; Assfalg M et al.; Residual dipolar couplings (RDCs) and pseudocontact shifts are experimentally accessible properties in nuclear magnetic resonance that are related to structural parameters and to the magnetic susceptibility anisotropy . We have determined RDCs due to field-induced orientation of oxidized-K79A and reduced cytochrome c at pH 7.0 and oxidized-K79A cytochrome c at pH 11.1 through measurements of amide (15)N-(1)H (1)J couplings at 800 and 500 MHz . The pH 7.0 RDCs for Fe(III)- and Fe(II)-cytochrome c together with available nuclear Overhauser effects were used to recalculate solution structures that were consistent with both sets of constraints . Molecular magnetic susceptibility anisotropy values were calculated for both redox states of the protein . By subtracting the residual dipolar couplings (RDCs) of the reduced form from those of the oxidized form measured at the same magnetic field (800 MHz), we found the RDC contribution of the paramagnetic metal ion in the oxidized protein . The magnetic susceptibility anisotropy, which was calculated from the structure, was found to be the same as that of the paramagnetic metal ion obtained independently from pseudocontact shifts, thereby indicating that the elements of secondary structure either are rigid or display the same mobility in both oxidation states . The residual dipolar coupling values of the alkaline-K79A form are small with respect to those of oxidized native cytochrome, whereas the pseudocontact shifts are essentially of the same magnitude, indicating local mobility . Importantly, this is the first time that mobility has been found through comparison of RDCs with pseudocontact shifts.

J Biol Chem, 2003 Aug 8, 278(32), 30051 - 6 Epub 2003 May 23.
Cloning and characterization of hCTF18, hCTF8, and hDCC1 . Human homologs of a Saccharomyces cerevisiae complex involved in sister chromatid cohesion establishment; Merkle CJ et al.; A growing body of evidence suggests that establishment of sister chromatid cohesion is dependent on replication fork passage over a precohesion area . In Saccharomyces cerevisiae, this process involves an alternative replication factor C (RFC) complex that contains the four small RFC subunits as well as CTF18, CTF8, and DCC1 . Here, we show that an evolutionarily conserved homologous complex exists in the nucleus of human cells . We demonstrate that hCTF18, hCTF8, and hDCC1 interact with each other as well as with the p38 subunit of RFC . This alternative RFC-containing complex interacts with proliferating cell nuclear antigen but not with the Rad9/Rad1/Hus1 complex, a proliferating cell nuclear antigen-like clamp involved in the DNA damage response . hCTF18 preferentially binds chromatin during S phase, suggesting a role during replication . Our data provide evidence for the existence of an alternative RFC complex with a probable role in mammalian sister chromatid cohesion establishment.

Biochimie, 2003 Jan-Feb, 85(1-2), 133 - 43
Study on agglutinating factors from flocculent Saccharomyces cerevisiae strains; Ngondi-Ekome J et al.; The lectin-like theory suggest that yeast flocculation is mediated by an aggregating lectinic factor . In this study we isolated an agglutinating factor, which corresponds to lectin, from whole cells by treating the flocculent wild-type Saccharomyces cerevisiae NCYC 625 strain and its weakly flocculent mutant {rho degrees } with EDTA and two non-ionic surfactants (Hecameg and HTAC) . The dialysed crude extracts obtained in this way agglutinated erythrocytes and this hemagglutination was specifically inhibited by mannose and mannose derivatives . However, SDS-PAGE profiles showed that the three reagents had different effects on the yeast cells . The non-ionic surfactants appeared to be the most efficient, as their extracts possessed the highest specific agglutinating activity . The products released by the wild-type strain presented a higher specific agglutinating activity than those released by the {rho degrees } mutant . Purification of the agglutinating factor from extracts of both strains by affinity chromatography revealed two active bands of relative mass of 26 and 47 kDa on SDS-PAGE . Mass spectrometry analysis by MALDI-TOF, identified a 26 kDa band as the triose phosphate isomerase (TPI) whereas a 47 kDa band was identical to enolase . Edman degradation showed that the N-terminal sequences of these proteins were similar to TPI and enolase, respectively . The difference in the flocculation behaviour of the two strains is due to changes in the protein composition of the cell wall and in the protein structure involved in cell-cell recognition.

Biotechniques, 2003 May, 34(5), 1024 - 8, 1033
Specific targeted integration of kanamycin resistance-associated nonselectable DNA in the genome of the yeast Saccharomyces cerevisiae; Waghmare SK et al.; Sophisticated genome manipulation requires the possibility to modify any intergenic or intragenic DNA sequence at will, without leaving large amounts of undesired vector DNA at the site of alteration . To this end, a series of vectors was developed from a previous gene knockout plasmid system to integrate nonselectable foreign DNA at any desired genomic location in yeast, with a minimum amount of residual plasmid DNA . These vectors have two mutated Flp recognition targets (FRT) sequences flanking the KanMX4 gene and multiple sites for subcloning the DNA fragment to be integrated . The selectable marker can be recycled by Flp site-specific excision between the identical FRTs, thereby allowing the integration of further DNA fragments . With this system, the NLS-tetR-GFP and DsRed genes were successfully integrated at the thr1 locus, and the RVB1 gene was tagged at the C-terminus with the V5-epitope-6-histidine tag . This plasmid system provides for a new molecular tool to integrate any DNA fragment at any genome location in {cir+} yeast strains . Moreover, the system can be extrapolated to other eukaryotic cells in which the FLP/FRT system functions efficiently.

Curr Genet, 2003 Aug, 43(5), 327 - 36 Epub 2003 May 23.
Suppressor mutations define two regions in the Cbp1 protein important for mitochondrial cytochrome b mRNA stability in Saccharomyces cerevisiae; Islas-Osuna MA et al.; Nuclear-encoded Cbp1 stabilizes and promotes translation of mitochondrial cytochrome b (COB) mRNA . A CCG triplet within the 5'UTR of COB mRNA is essential for Cbp1-dependent stability . Like cbp1 mutations, mutation of any nucleotide in CCG results in degradation of COB transcripts . In this study, CBP1-linked pseudorevertants of the temperature-sensitive CCU strain were isolated . The suppressors are missense mutations within a central cluster or a carboxyl cluster in the linear sequence of Cbp1 . Strains with mutations in the carboxyl half of the central cluster or the carboxyl cluster respire better than those with mutations in the amino half of the central cluster . COB mRNA levels in the suppressor strains were increased compared with that in the CCU strain and were positively correlated with respiratory capability . This correlation supports a model in which the primary role of Cbp1 is to protect COB mRNAs and deliver them to the mitochondrial translational apparatus.

Antimicrob Agents Chemother, 2003 Jun, 47(6), 2012 - 4
Comparative roles of the cell wall and cell membrane in limiting uptake of xenobiotic molecules by Saccharomyces cerevisiae; Aouida M et al.; Using reversible electropermeabilization of cells and spheroplasts, we show that the cell wall and plasma membrane partly account for bleomycin resistance by acting as two independent barriers . We also report on the presence of a membrane protein that may be responsible for bleomycin internalization and toxicity in Saccharomyces cerevisiae.

Curr Genet, 2003 Aug, 43(5), 337 - 50 Epub 2003 May 21.
Characterization of mutations that are synthetic lethal with pol3-13, a mutated allele of DNA polymerase delta in Saccharomyces cerevisiae; Chanet R et al.; The pol3-13 mutation is located in the C-terminal end of POL3, the gene encoding the catalytic subunit of polymerase delta, and confers thermosensitivity onto the Saccharomyces cerevisiae mutant strain . To get insight about DNA replication control, we performed a genetic screen to identify genes that are synthetic lethal with pol3-13 . Mutations in genes encoding the two other subunits of DNA polymerase delta (HYS2, POL32) were identified . Mutations in two recombination genes (RAD50, RAD51) were also identified, confirming that homologous recombination is necessary for pol3-13 mutant strain survival . Other mutations were identified in genes involved in repair and genome stability (MET18/ MMS19), in the control of origin-firing and/or transcription (ABF1, SRB7), in the S/G2 checkpoint (RAD53), in the Ras-cAMP signal transduction pathway (MKS1), in nuclear pore metabolism (SEH1), in protein degradation (DOC1) and in folding (YDJ1) . Finally, mutations in three genes of unknown function were isolated (NBP35, DRE2, TAH18) . Synthetic lethality between pol3-13 and each of the three mutants pol32, mms19 and doc1 could be suppressed by a rad18 deletion, suggesting an important role of ubiquitination in DNA replication control . We propose that the pol3-13 mutant generates replicative problems that need both homologous recombination and an intact checkpoint machinery to be overcome.

Biochem J, 2003 Sep 1, 374(Pt 2), 513 - 9
Protein S-thiolation targets glycolysis and protein synthesis in response to oxidative stress in the yeast Saccharomyces cerevisiae; Shenton D et al.; The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, a process by which protein SH groups form mixed disulphides with low-molecular-mass thiols such as glutathione . We report here the target proteins which are modified in yeast cells in response to H(2)O(2) . In particular, a range of glycolytic and related enzymes (Tdh3, Eno2, Adh1, Tpi1, Ald6 and Fba1), as well as translation factors (Tef2, Tef5, Nip1 and Rps5) are identified . The oxidative stress conditions used to induce S-thiolation are shown to inhibit GAPDH (glyceraldehyde-3-phosphate dehydrogenase), enolase and alcohol dehydrogenase activities, whereas they have no effect on aldolase, triose phosphate isomerase or aldehyde dehydrogenase activities . The inhibition of GAPDH, enolase and alcohol dehydrogenase is readily reversible once the oxidant is removed . In addition, we show that peroxide stress has little or no effect on glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, the enzymes that catalyse NADPH production via the pentose phosphate pathway . Thus the inhibition of glycolytic flux is proposed to result in glucose equivalents entering the pentose phosphate pathway for the generation of NADPH . Radiolabelling is used to confirm that peroxide stress results in a rapid and reversible inhibition of protein synthesis . Furthermore, we show that glycolytic enzyme activities and protein synthesis are irreversibly inhibited in a mutant that lacks glutathione, and hence cannot modify proteins by S-thiolation . In summary, protein S-thiolation appears to serve an adaptive function during exposure to an oxidative stress by reprogramming metabolism and protecting protein synthesis against irreversible oxidation.

Mol Microbiol, 2003 May, 48(4), 1119 - 30
TFIIB and subunits of the SAGA complex are involved in transcriptional activation of phospholipid biosynthetic genes by the regulatory protein Ino2 in the yeast Saccharomyces cerevisiae; Dietz M et al.; In the yeast Saccharomyces cerevisiae, genes involved in phospholipid biosynthesis are activated by ICRE (inositol/choline-responsive element) up-stream motifs and the corresponding heterodimeric binding factor, Ino2 + Ino4 . Both Ino2 and Ino4 contain basic helix-loop-helix (bHLH) domains required for ICRE binding, whereas transcriptional activation is mediated exclusively by Ino2 . In this work, we describe a molecular analysis of functional minimal domains responsible for specific DNA recognition and transcriptional activation (TAD1 and TAD2) . We also define the importance of individual amino acids within the more important activation domain TAD1 . Random mutagenesis at five amino acid positions showed the importance of acidic as well as hydrophobic residues within this minimal TAD . We also investigated the contribution of known general transcription factors and co-activators for Ino2-dependent gene activation . Although an ada5 single mutant and a gal11 paf1 double mutant were severely affected, a partial reduction in activation was found for gcn5 and srb2 . Ino2 interacts physically with the basal transcription factor Sua7 (TFIIB of yeast) . Interestingly, interaction is mediated by the HLH dimerization domain of Ino2 and by two non-overlapping domains within Sua7 . Thus, Sua7 may compete with Ino4 for binding to the Ino2 activator, creating the possibility of positive and negative influence of Sua7 on ICRE-dependent gene expression.

Proc Natl Acad Sci U S A, 2003 May 27, 100(11), 6640 - 5 Epub 2003 May 15.
Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability; Myung K et al.; Some spontaneous gross chromosomal rearrangements (GCRs) seem to result from DNA-replication errors . The chromatin-assembly factor I (CAF-I) and replication-coupling assembly factor (RCAF) complexes function in chromatin assembly during DNA replication and repair and could play a role in maintaining genome stability . Inactivation of CAF-I or RCAF increased the rate of accumulating different types of GCRs including translocations and deletion of chromosome arms with associated de novo telomere addition . Inactivation of CAF-I seems to cause damage that activates the DNA-damage checkpoints, whereas inactivation of RCAF seems to cause damage that activates the DNA-damage and replication checkpoints . Both defects result in increased genome instability that is normally suppressed by these checkpoints, RAD52-dependent recombination, and PIF1-dependent inhibition of de novo telomere addition . Treatment of CAF-I- or RCAF-defective cells with methyl methanesulfonate increased the induction of GCRs compared with that seen for a wild-type strain . These results indicate that coupling of chromatin assembly to DNA replication and DNA repair is critical to maintaining genome stability.

J Biol Chem, 2003 Jul 25, 278(30), 27372 - 81 Epub 2003 May 15.
Evidence for a role of MCM (mini-chromosome maintenance)5 in transcriptional repression of sub-telomeric and Ty-proximal genes in Saccharomyces cerevisiae; Dziak R et al.; The MCM (mini-chromosome maintenance) genes have a well established role in the initiation of DNA replication and in the elongation of replication forks in Saccharomyces cerevisiae . In this study we demonstrate elevated expression of sub-telomeric and Ty retrotransposon-proximal genes in two mcm5 strains . This pattern of up-regulated genes resembles the genome-wide association of MCM proteins to chromatin that was reported earlier . We link the altered gene expression in mcm5 strains to a reversal of telomere position effect (TPE) and to remodeling of sub-telomeric and Ty chromatin . We also show a suppression of the Ts phenotype of a mcm5 strain by the high copy expression of the TRA1 component of the chromatin-remodeling SAGA/ADA (SPT-ADA-GCN5 acetylase/ADAptor) . We propose that MCM proteins mediate the establishment of silent chromatin domains around telomeres and Ty retrotransposons.

Genetics, 2003 May, 164(1), 65 - 79
Characterization of the hyperrecombination phenotype of the pol3-t mutation of Saccharomyces cerevisiae; Galli A et al.; The DNA polymerase delta (Pol3p/Cdc2p) allele pol3-t of Saccharomyces cerevisiae has previously been shown to increase the frequency of deletions between short repeats (several base pairs), between homologous DNA sequences separated by long inverted repeats, and between distant short repeats, increasing the frequency of genomic deletions . We found that the pol3-t mutation increased intrachromosomal recombination events between direct DNA repeats up to 36-fold and interchromosomal recombination 14-fold . The hyperrecombination phenotype of pol3-t was partially dependent on the Rad52p function but much more so on Rad1p . However, in the double-mutant rad1 Delta rad52 Delta, the pol3-t mutation still increased spontaneous intrachromosomal recombination frequencies, suggesting that a Rad1p Rad52p-independent single-strand annealing pathway is involved . UV and gamma-rays were less potent inducers of recombination in the pol3-t mutant, indicating that Pol3p is partly involved in DNA-damage-induced recombination . In contrast, while UV- and gamma-ray-induced intrachromosomal recombination was almost completely abolished in the rad52 or the rad1 rad52 mutant, there was still good induction in those mutants in the pol3-t background, indicating channeling of lesions into the above-mentioned Rad1p Rad52p-independent pathway . Finally, a heterozygous pol3-t/POL3 mutant also showed an increased frequency of deletions and MMS sensitivity at the restrictive temperature, indicating that even a heterozygous polymerase delta mutation might increase the frequency of genetic instability.

Genetics, 2003 May, 164(1), 47 - 64
Multiple roles for Saccharomyces cerevisiae histone H2A in telomere position effect, Spt phenotypes and double-strand-break repair; Wyatt HR et al.; Telomere position effects on transcription (TPE, or telomeric silencing) are nucleated by association of nonhistone silencing factors with the telomere and propagated in subtelomeric regions through association of silencing factors with the specifically modified histones H3 and H4 . However, the function of histone H2A in TPE is unknown . We found that deletion of either the amino or the carboxyltails of H2A substantially reduces TPE . We identified four H2A modification sites necessary for wild-type efficiency of TPE . These "hta1tpe" alleles also act as suppressors of a delta insertion allele of LYS2, suggesting shared elements of chromatin structure at both loci . Interestingly, we observed combinatorial effects of allele pairs, suggesting both interdependent acetylation and deacetylation events in the amino-terminal tail and a regulatory circuit between multiple phosphorylated residues in the carboxyl-terminal tail . Decreases in silencing and viability are observed in most hta1tpe alleles after treatment with low and high concentrations, respectively, of bleomycin, which forms double-strand breaks (DSBs) . In the absence of the DSB and telomere-binding protein yKu70, the bleomycin sensitivity of hta1tpe alleles is further enhanced . We also provide data suggesting the presence of a yKu-dependent histone H2A function in TPE . These data indicate that the amino- and carboxyl-terminal tails of H2A are essential for wild-type levels of yKu-mediated TPE and DSB repair.

Proteomics, 2003 May, 3(5), 752 - 63
Fragmentation of dihydroxyacetone kinase 1 from Saccharomyces cerevisiae indicates a two-domain structure; Molin M et al.; Global protein expression in Saccharomyces cerevisiae strains either deleted for both yeast dihydroxyacetone kinases (DAK1 and DAK2) or overexpressing DAK1, was characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) . We found protein expression in the double deletion strain to be highly similar to wild-type . In the strain overexpressing Dak1p, nine spots representing fragments of the Dak1p protein in the size range 40-20 kDa and amounting to approximately 30% of total Dak1p, were discovered (native size Dak1p migrates at roughly 60 kDa) . Fragments were characterized by matrix-assisted laser desorption/ionization mass spectrometry and electrospray mass spectrometry analyses to represent either the N- or the C-terminal part of the DAK1 protein . Cleavage points, predicted from mass spectrometry and 2-D PAGE data, mapped almost exclusively in the middle region showing low sequence conservation between Dak1p and its closest homologues . We hypothesize that observed Dak1p fragments represent stable structural domains shielded from access by native endoproteases . Furthermore, overexpressing Dak1p with the non-native N-terminus (M)A-, resulted in native size Dak1p and N-terminal Dak1p fragments appearing in two major 2-D PAGE forms of approximately equal size and abundance, but with slightly different isoelectric points . However, when overexpressing Dak1p with the native N-terminus (M)S-, only the more acidic 2-D PAGE form appeared . In the N-terminal acetyltransferase mutant nat1delta, (M)A-Dak1p species were converted into the basic form, arguing twin spots to represent forms with acetylated and deacetylated N-termini . Data thus indicated that (M)A-N-termini, in the Dak1p context, were NatA substrates recognized with 50% lower efficiency than (M)S-N-termini.

Mol Cell Biol, 2003 Jun, 23(11), 3721 - 34
The Rad51 pathway of telomerase-independent maintenance of telomeres can amplify TG1-3 sequences in yku and cdc13 mutants of Saccharomyces cerevisiae; Grandin N et al.; In the yeast Saccharomyces cerevisiae, Cdc13, Yku, and telomerase define three parallel pathways for telomere end protection that prevent chromosome instability and death by senescence . We report here that cdc13-1 yku70delta mutants generated telomere deprotection-resistant cells that, in contrast with telomerase-negative senescent cells, did not display classical crisis events . cdc13-1 yku70delta cells survived telomere deprotection by exclusively amplifying TG(1-3) repeats (type II recombination) . In a background lacking telomerase (tlc1delta), this process predominated over type I recombination (amplification of subtelomeric Y' sequences) . Strikingly, inactivation of the Rad50/Rad59 pathway (which is normally required for type II recombination) in cdc13-1 yku70delta or yku70delta tlc1delta mutants, but also in cdc13-1 YKU70(+) tlc1delta mutants, still permitted type II recombination, but this process was now entirely dependent on the Rad51 pathway . In addition, delayed senescence was observed in cdc13-1 yku70delta rad51delta and cdc13-1 tlc1delta rad51delta cells . These results demonstrate that in wild-type cells, masking by Cdc13 and Yku prevents the Rad51 pathway from amplifying telomeric TG(1-3) sequences . They also suggest that Rad51 is more efficient than Rad50 in amplifying the sequences left uncovered by the absence of Cdc13 or Yku70.

J Biol Chem, 2003 Jul 25, 278(30), 27605 - 11 Epub 2003 May 14.
Saccharomyces cerevisiae PIP2 mediating oleic acid induction and peroxisome proliferation is regulated by Adr1p and Pip2p-Oaf1p; Rottensteiner H et al.; Saccharomyces cerevisiae genes involved in fatty acid degradation contain in their promoters oleate response elements (OREs) and type 1 upstream activation sequences (UAS1s) that bind Pip2p-Oaf1p and Adr1p, respectively . The promoter of the PIP2 gene was found to contain a potential UAS1 that consists of a tandem array of CYCCRR half-sites in an overlapping arrangement with a previously characterized ORE . Electrophoretic mobility shift analysis demonstrated that Adr1p bound to UAS1PIP2, and Northern analysis in combination with a lacZ reporter gene confirmed that Adr1p influenced the transcription of PIP2 . Immunoprecipitation showed that, in adr1delta mutant cells grown on oleic acid, Pip2p was less abundant compared with the corresponding wild-type . In addition, the amount of Pip2p-Oaf1p that bound to a target ORE in vitro was reduced in mutant extracts compared with the wild-type . Transcription of the oleic acid-inducible genes SPS19 and CTA1, which rely on both Pip2p-Oaf1p and Adr1p for their regulation, was reduced in adr1delta mutant cells . However, by ectopically restoring levels of Pip2p in adr1delta cells grown on oleic acid medium, transcription of both genes increased 2-fold compared with the control . This partial suppression of the adr1delta mutant phenotype was additionally manifested by moderate utilization of oleic acid . Hence, both the expression as well as the action of the two transcription factors, Adr1p and Pip2p-Oaf1p, are interconnected, which allows for an elaborate control of fatty acid-inducible genes.

J Cell Biol, 2003 May 12, 161(3), 483 - 8
Spindle orientation in Saccharomyces cerevisiae depends on the transport of microtubule ends along polarized actin cables; Hwang E et al.; Microtubules and actin filaments interact and cooperate in many processes in eukaryotic cells, but the functional implications of such interactions are not well understood . In the yeast Saccharomyces cerevisiae, both cytoplasmic microtubules and actin filaments are needed for spindle orientation . In addition, this process requires the type V myosin protein Myo2, the microtubule end-binding protein Bim1, and Kar9 . Here, we show that fusing Bim1 to the tail of the Myo2 is sufficient to orient spindles in the absence of Kar9, suggesting that the role of Kar9 is to link Myo2 to Bim1 . In addition, we show that Myo2 localizes to the plus ends of cytoplasmic microtubules, and that the rate of movement of these cytoplasmic microtubules to the bud neck depends on the intrinsic velocity of Myo2 along actin filaments . These results support a model for spindle orientation in which a Myo2-Kar9-Bim1 complex transports microtubule ends along polarized actin cables . We also present data suggesting that a similar process plays a role in orienting cytoplasmic microtubules in mating yeast cells.

Nature, 2003 May 8, 423(6936), 181 - 5
Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae; Anderson RM et al.; Calorie restriction extends lifespan in a broad range of organisms, from yeasts to mammals . Numerous hypotheses have been proposed to explain this phenomenon, including decreased oxidative damage and altered energy metabolism . In Saccharomyces cerevisiae, lifespan extension by calorie restriction requires the NAD+-dependent histone deacetylase, Sir2 (ref . 1) . We have recently shown that Sir2 and its closest human homologue SIRT1, a p53 deacetylase, are strongly inhibited by the vitamin B3 precursor nicotinamide . Here we show that increased expression of PNC1 (pyrazinamidase/nicotinamidase 1), which encodes an enzyme that deaminates nicotinamide, is both necessary and sufficient for lifespan extension by calorie restriction and low-intensity stress . We also identify PNC1 as a longevity gene that is responsive to all stimuli that extend lifespan . We provide evidence that nicotinamide depletion is sufficient to activate Sir2 and that this is the mechanism by which PNC1 regulates longevity . We conclude that yeast lifespan extension by calorie restriction is the consequence of an active cellular response to a low-intensity stress and speculate that nicotinamide might regulate critical cellular processes in higher organisms.

J Mol Microbiol Biotechnol, 2003, 5(2), 78 - 81
Maize Activator (AC) transposase (TPase) is expressed in Saccharomyces cerevisiae from a genomic clone . detection via Elisa, and proposed use in complementation studies; MacRae AF; The maize Activator (Ac) transposase (TPase) was expressed as a Histidine (His)-tagged protein in Saccharomyces cerevisiae from a full length genomic clone . Expression was demonstrated via the highly specific nickel-coated Elisa plate method, using an anti-His antibody and 2 separate anti-Ac TPase antibodies, to Ac residues 103-465 and 189-807 . AC TPase expression in Saccharomyces is important for two reasons: (a) because the expression from a genomic clone herein permits the future study of RNA splicing mechanisms in common between maize and yeast systems, and (b) because a yeast system can easily be used for demonstrating complementation of function . Thus, such transformed yeast systems could be used in future, to experimentally test whether Ac TPase could complement various yeast mutations . Specifically, Ac TPase may be able to complement (i.e . provide the same function) to yeast transcription factor mutants or to genes mutated in other essential yeast functions . If confirmed, this would lend support to Barbara McClintock's hypothesis that transposable elements can serve as 'controlling elements' within the genome, by their ability to supplement other essential genes' functions, as needed . Work herein is contrasted with existing studies on Ac in yeast .

Nucleic Acids Res, 2003 May 15, 31(10), 2576 - 85
Multiple recombination pathways for sister chromatid exchange in Saccharomyces cerevisiae: role of RAD1 and the RAD52 epistasis group genes; Dong Z et al.; Sister chromatid exchange (SCE) can occur by several recombination mechanisms, including those directly initiated by double-strand breaks (DSBs), such as gap repair and break-induced replication (BIR), and those initiated when DNA polymerases stall, such as template switching . To elucidate SCE recombination mechanisms, we determined whether spontaneous and DNA damage-associated SCE requires specific genes within the RAD52 and RAD3 epistasis groups in Saccharomyces cerevisiae strains containing two his3 fragments, his3-Delta5' and his3-Delta3'::HOcs . SCE frequencies were measured after cells were exposed to UV, X-rays, 4-nitroquinoline 1-oxide (4-NQO) and methyl methanesulfonate (MMS), or when an HO endonuclease-induced DSB was introduced at his3-Delta3'::HOcs . Our data indicate that genes involved in gap repair, such as RAD55, RAD57 and RAD54, are required for DNA damage-associated SCE but not for spontaneous SCE . RAD50 and RAD59, genes required for BIR, are required for X-ray-associated SCE but not for SCE stimulated by HO-induced DSBs . In comparison with wild type, rates of spontaneous SCE are 10-fold lower in rad51 rad1 but not in either rad51 rad50 or rad51 rad59 double mutants . We propose that gap repair mechanisms are important in DNA damage-associated recombination, whereas alternative pathways, including a template switch pathway, play a role in spontaneous SCE.

Nucleic Acids Res, 2003 May 15, 31(10), 2524 - 33
RRP20, a component of the 90S preribosome, is required for pre-18S rRNA processing in Saccharomyces cerevisiae; Senapin S et al.; A strain of Saccharomyces cerevisiae, defective in small subunit ribosomal RNA processing, has a mutation in YOR145c ORF that converts Gly235 to Asp . Yor145c is a nucleolar protein required for cell viability and has been reported recently to be present in 90S pre-ribosomal particles . The Gly235Asp mutation in YOR145c is found in a KH-type RNA-binding domain and causes a marked deficiency in 18S rRNA production . Detailed studies by northern blotting and primer extension analyses show that the mutant strain impairs the early pre-rRNA processing cleavage essentially at sites A1 and A2, leading to accumulation of a 22S dead-end processing product that is found in only a few rRNA processing mutants . Furthermore, U3, U14, snR10 and snR30 snoRNAs, involved in early pre-rRNA cleavages, are not destabilized by the YOR145c mutation . As the protein encoded by YOR145c is found in pre-ribosomal particles and the mutant strain is defective in ribosomal RNA processing, we have renamed it as RRP20.

Curr Genet, 2003 Jul, 43(4), 263 - 72 Epub 2003 May 07.
Rsf1p, a protein required for respiratory growth of Saccharomyces cerevisiae; Lu L et al.; A central problem in our understanding of mitochondrial (mt) function remains the question of how coordinate transcriptional control is accomplished between nucleus and mitochondria . Here, we report the initial characterization of a protein of previously unknown function, the product of the YMR030 W gene, that appears to mediate such coordinate gene expression . Expression of YMR030 W is glucose-repressible; a deletion mutant for this gene shows a severe growth defect on glycerol-, but not glucose- or ethanol-based medium . In that mutant, transcript levels from GUT1 and GUT2 are highly attenuated compared with those of the wild-type parent when both are grown on glycerol-based medium . Under the same growth conditions, transcripts from the mt OLI1 gene, which has one copy of a mt upstream activating sequence (UAS) in its 5'-flanking region, are attenuated in the DeltaYMR030 W mutant, but mRNA from the mt COX3 ( OXI2) gene, which lacks the mt UAS, are not . Some nuclear genes encoding mt-related proteins also show low transcript levels in the DeltaYMR030 W mutant in comparison with those of the wild-type parent strain during glycerol-based growth . Localization of the protein, via its expression fused to green fluorescent protein, indicates that it is present in both nucleus and mitochondria, supporting a respiration-related transcriptional role for this gene product in both cellular genetic compartments . Because of its role in both respiratory growth and mt function, we designate the YMR030 W coding sequence RSF1 (respiration factor 1).

Mol Genet Genomics, 2003 Jun, 269(3), 370 - 80 Epub 2003 May 06.
Merging of multiple signals regulating delta9 fatty acid desaturase gene transcription in Saccharomyces cerevisiae; Nakagawa Y et al.; Fatty acid desaturation, which requires molecular oxygen (O2) as an electron acceptor, is catalyzed by delta9 fatty acid desaturase, which is encoded by OLE1 in Saccharomyces cerevisiae . Transcription of the OLE1 gene is repressed by unsaturated fatty acids (UFAs) and activated by hypoxia and low temperatures via the endoplasmic reticulum membrane protein Mga2p . We previously reported the isolation of the nfo3-1 (negative factor for OLE1) mutant, which exhibits enhanced expression of OLE1 in the presence of UFA and under aerobic conditions . In this work, we demonstrated that the NFO3 gene is identical to OLE1 and that the nfo3-1 mutation (renamed ole1-101) alters arginine-346, in the vicinity of the conserved histidine-rich motif essential for the catalytic function of the Ole1 protein, to lysine . The ratio of UFAs to total fatty acids in the ole1-101 mutant was 60%, compared to 75% in the wild type, suggesting that the reduction in relative levels of intracellular UFAs activates OLE1 transcription . However, in ole1-101 cells grown in the presence of oleic acid, the level of OLE1 expression remained high, although the relative amount of UFAs in the ole1-101 mutant cells was almost the same as that in wild-type cells growing under the same conditions . By contrast, when cells were grown with linoleic acid, which has a lower melting point than oleic acid, the elevation of the OLE1 expression level due to the ole1-101 mutation was almost completely suppressed . These observations suggest that the ole1-101 cells activate OLE1 transcription by sensing not only the intracellular UFA level, but also membrane fluidity or the nature of the UFA species itself . Furthermore, we found that not only the fatty acid- regulated (FAR) element but also the O2- regulated (O2R) element in the OLE1 promoter was involved in the activation of OLE1 transcription by the ole1-101 mutation, and that the effects of the low-oxygen signal and the ole1-101-generated signal on OLE1 expression were not additive . Taken together, these findings suggest that signals associated with hypoxia, low temperatures and intracellular UFA depletion activate OLE1 transcription by a common pathway.

J Cell Biol, 2003 May 12, 161(3), 497 - 505 Epub 2003 May 05.
Oscillatory nucleocytoplasmic shuttling of the general stress response transcriptional activators Msn2 and Msn4 in Saccharomyces cerevisiae; Jacquet M et al.; Msn2 and Msn4 are two related transcriptional activators that mediate a general response to stress in yeast Saccharomyces cerevisiae by eliciting the expression of specific sets of genes . In response to stress or nutritional limitation, Msn2 and Msn4 migrate from the cytoplasm to the nucleus . Using GFP-tagged constructs and high-resolution time-lapse video microscopy on single cells, we show that light emitted by the microscope also triggers this migration . Unexpectedly, the population of Msn2 or Msn4 molecules shuttles repetitively into and out of the nucleus with a periodicity of a few minutes . A large heterogeneity in the oscillatory response to stress is observed between individual cells . This periodic behavior, which can be induced by various types of stress, at intermediate stress levels, is not dependent upon protein synthesis and persists when the DNA-binding domain of Msn2 is removed . The cAMP-PKA pathway controls the sensitivity of the oscillatory nucleocytoplasmic shuttling . In the absence of PKA, Msn4 continues to oscillate while Msn2 is maintained in the nucleus . We show that a computational model based on the possibility that Msn2 and Msn4 participate in autoregulatory loops controlling their subcellular localization can account for the oscillatory behavior of the two transcription factors.

Electrophoresis, 2003 May, 24(9), 1458 - 70
Proteome analysis of Saccharomyces cerevisiae under metal stress by two-dimensional differential gel electrophoresis; Hu Y et al.; The defense mechanism by which cells combat metal stress remains poorly understood . By utilizing a newly developed technique - the differential gel electrophoresis (DIGE) - we evaluated the biological alterations of metal stress on Saccharomyces cerevisiae at its translational level . By simultaneously comparing the differential expression profiles of thousands of proteins as results of 15 different metal treatments, we were able to closely examine the response of a large number of proteins within the yeast proteome towards individual metals, as well as the response of the same proteins towards different metals . This, to our knowledge, is the first case which demonstrates the potential of DIGE as a high-throughput tool for large-scale proteome analysis . From our studies, where yeast cells were exhaustively treated with exogenous metals, 20-30% of all proteins detected showed statistically significant changes . According to different effects (up-/downregulation) of protein expression levels observed, we were able to tentatively divide the 15 metals into three groups . By mass spectrometric analysis, more than 50 protein spots were positively identified, both quantitatively and qualitatively . One of the proteins was identified to be Cu/Zn superoxide dismutase (SOD1), and its expression levels as a result of 15 different metal treatments was further examined in greater details . Significant changes in SOD1 expression were observed throughout all 15 DIGE gels.

Int Microbiol, 2003 Mar, 6(1), 49 - 55 Epub 2003 Jan 25.
Intracellular transport of a heterologous membrane protein, the human transferrin receptor, in Saccharomyces cerevisiae; Prinz B et al.; We have analyzed the intracellular behavior of the human transferrin receptor (TfR) in Saccharomyces cerevisiae . The major part of the heterologously expressed TfR, which has previously been used as a model for heterologous expression of membrane proteins in yeast, is localized in the endoplasmic reticulum (ER) membranes; a minor fraction is present in the plasma membrane (PM) . The stability of the TfR depends on vacuolar proteases, implying that it is degraded in the vacuolar compartment . Degradation is further dependent on favorable transport conditions to this compartment . The main bottleneck of transport seems to be the transition from the ER to the PM . The chaperone Cne1p, which is involved in quality control in the ER, plays a role in regulating the amount of heterologous TfR, as deletion of CNE1 leads to significant accumulation of the protein . This is the first demonstration of the involvement of CNE1 in regulating the level of heterologous membrane proteins.

J Nutr, 2003 May, 133(5 Suppl 1), 1532S - 5S
Multiple regulatory mechanisms maintain zinc homeostasis in Saccharomyces cerevisiae; Eide DJ; Zinc is an essential nutrient, yet it is toxic if it accumulates in excess amounts within cells; therefore the intracellular labile zinc content of cells is tightly controlled . In Saccharomyces cerevisiae, zinc homeostasis is regulated by the controlled activity of zinc uptake transporters in the plasma membrane and transporters responsible for intracellular zinc compartmentalization . The activity of these transporters is regulated at both transcriptional and posttranscriptional levels in response to zinc . These different mechanisms work together to precisely balance zinc uptake and its storage and utilization.

J Biol Chem, 2003 Jul 4, 278(27), 24636 - 43 Epub 2003 May 02.
Nuclear thiol peroxidase as a functional alkyl-hydroperoxide reductase necessary for stationary phase growth of Saccharomyces cerevisiae; Cha MK et al.; Yeast nucleus-localized thiol peroxidase (nTPx) was characterized as a functional peroxidase . There are two cysteine residues in nTPx . Replacement of Cys-106 or Cys-111 with serine resulted in a complete loss of thioredoxin-linked peroxidase activity . However, when their activities were measured in terms of the ability to inhibit oxidation of glutamine synthetase, C111S showed the same antioxidant activity as the wild type protein . SDS-PAGE gel analysis revealed that only C111S existed as the dimer form . In addition to the identification of Cys-106 as the primary catalytic site, these data suggest the formation of the intradisulfide bond as a part of the catalytic cycle between nTPx and thioredoxin . nTPx preferentially reduced alkyl-hydroperoxides rather than H2O2 . Furthermore, a nTPx mutant strain showed higher sensitivity toward alkyl-hydroperoxide than hydrogen peroxide . Also, reduction of the viability of nTPx mutant strain against various oxidants supports an in vivo antioxidant role for nTPx . nTPx transcriptional activity was not significantly detectable in log phase yeast, but the activity was exponentially increased after the diauxic shift . The transcriptional activity was highly induced even in the log phase yeast grown in nonfermentable carbon source . Deletion of Tor1p, Ras1p, and Ras2p resulted in considerable induction when compared with their parent strains, demonstrating the activation of the transcription of nTPx gene at the diauxic shift . Transcription of nTPx gene was induced in response to oxidative stress . Viability of a stationary phase nTPx mutant was considerably reduced compared with the isogenic strain . Collectively, these data demonstrate that nTPx is a thiol peroxidase family acting as alkyl-hydroperoxide reductase in the nucleus during post-diauxic growth.

Biosci Biotechnol Biochem, 2003 Feb, 67(2), 448 - 50
Cooperation of Sly1/SM-family protein and sec18/NSF of Saccharomyces cerevisiae in disassembly of cis-SNARE membrane-protein complexes; Kosodo Y et al.; Assembly and disassembly of the SNARE membrane-protein complexes plays a key role in vesicular trafficking . The SM-family Slyl protein binds to the tSNARE Sed5 protein and stimulates its assembly into a trans-SNARE complex . Disassembly of the resulting cis-SNARE complex containing Sed5 was retarded in a temperature-sensitive yeast mutant of Slyl protein with a defect in binding to Sed5 . A temperature-sensitive mutation (sec18-1) of Sec18/NSF disassembly ATPase showed synthetic lethality with the sly1(ts) mutation . These results suggest that Slyl and Sec18 proteins work cooperatively and that the binding of Slyl to Sed5 stimulates the disassembly of the cis-SNARE complex by Sec18 ATPase.

FEMS Microbiol Lett, 2003 Apr 25, 221(2), 249 - 55
Optimisation of interdelta analysis for Saccharomyces cerevisiae strain characterisation; Legras JL et al.; A new primer pair (delta12-delta21) for polymerase chain reaction-based yeast typing was designed using the yeast genome sequence . The specificity of this primer pair was checked by the comparison of the electrophoresis pattern with a virtual profile calculated from Blast data . The analysis of 53 commercial and laboratory Saccharomyces cerevisiae yeast strains showed a clear improvement of interdelta analysis using the newly designed primers.

FEMS Microbiol Lett, 2003 Apr 25, 221(2), 151 - 4
Human TSG101 does not replace Saccharomyces cerevisiae VPS23 role in the quality control of plasma membrane proteins; Blanco S et al.; The Saccharomyces cerevisiae VPS23 (STP22) gene is implicated in the control of vesicle movement and quality of plasma membrane proteins . VPS23 mutants have defects either in removing defective membrane proteins such as alpha-mating factor receptor and arginine permease . The human ortholog TSG101 and its variants, isolated from tumor cells, do not substitute VPS23 in its ability to rescue the phenotype of defective plasma membrane proteins.

Mol Cell Biol, 2003 May, 23(10), 3681 - 91
Telomeric position effect variegation in Saccharomyces cerevisiae by Caenorhabditis elegans linker histones suggests a mechanistic connection between germ line and telomeric silencing; Jedrusik MA et al.; Linker histones are nonessential for the life of single-celled eukaryotes . Linker histones, however, can be important components of specific developmental programs in multicellular animals and plants . For Caenorhabditis elegans a single linker histone variant (H1.1) is essential in a chromatin silencing process which is crucial for the proliferation and differentiation of the hermaphrodite germ line . In this study we analyzed the whole linker histone complement of C . elegans by telomeric position effect variegation in budding yeast . In this assay an indicator gene (URA3) placed close to the repressive telomeric chromatin structure is subject to epigenetically inherited gene inactivation . Just one out of seven C . elegans linker histones (H1.1) was able to enhance the telomeric position effect in budding yeast . Since these results reflect the biological function of H1.1 in C . elegans, we suggest that chromatin silencing in C . elegans is governed by molecular mechanisms related to the telomere-dependent silencing in budding yeast . We confirmed this hypothesis by testing C . elegans homologs of three yeast genes which are established modifiers of the yeast telomeric chromatin structure (SIR2, SET1, and RAD17) for their influence on repeat-dependent transgene silencing for C . elegans.

Mol Cell Biol, 2003 May, 23(10), 3405 - 16
Rad53 checkpoint kinase phosphorylation site preference identified in the Swi6 protein of Saccharomyces cerevisiae; Sidorova JM et al.; Rad53 of Saccharomyces cerevisiae is a checkpoint kinase whose structure and function are conserved among eukaryotes . When a cell detects damaged DNA, Rad53 activity is dramatically increased, which ultimately leads to changes in DNA replication, repair, and cell division . Despite its central role in checkpoint signaling, little is known about Rad53 substrates or substrate specificity . A number of proteins are implicated as Rad53 substrates; however, the evidence remains indirect . Previously, we have provided evidence that Swi6, a subunit of the Swi4/Swi6 late-G(1)-specific transcriptional activator, is a substrate of Rad53 in the G(1)/S DNA damage checkpoint . In the present study we identify Rad53 phosphorylation sites in Swi6 in vitro and demonstrate that at least one of them is targeted by Rad53 in vivo . Mutations in these phosphorylation sites in Swi6 shorten but do not eliminate the Rad53-dependent delay of the G(1)-to-S transition after DNA damage . We derive a consensus for Rad53 site preference at positions -2 and +2 (-2/+2) and identify its potential substrates in the yeast proteome . Finally, we present evidence that one of these candidates, the cohesin complex subunit Scc1 undergoes DNA damage-dependent phosphorylation, which is in part dependent on Rad53.

Microbiology, 2003 May, 149(Pt 5), 1205 - 16
Synergistic inhibition of APC/C by glucose and activated Ras proteins can be mediated by each of the Tpk1-3 proteins in Saccharomyces cerevisiae; Bolte M et al.; Proteolysis triggered by the anaphase-promoting complex/cyclosome (APC/C) is essential for the progression through mitosis . APC/C is a highly conserved ubiquitin ligase whose activity is regulated during the cell cycle by various factors, including spindle checkpoint components and protein kinases . The cAMP-dependent protein kinase (PKA) was identified as negative regulator of APC/C in yeast and mammalian cells . In the yeast Saccharomyces cerevisiae, PKA activity is induced upon glucose addition or by activated Ras proteins . This study shows that glucose and the activated Ras2(Val19) protein synergistically inhibit APC/C function via the cAMP/PKA pathway in yeast . Remarkably, Ras2 proteins defective in the interaction with adenylate cyclase fail to influence APC/C, implying that its function is regulated exclusively by PKA, but not by alternative Ras pathways . Furthermore, it is shown that the three PKAs in yeast, Tpk1, Tpk2 and Tpk3, have redundant functions in regulating APC/C in response to glucose medium . Single or double deletions of TPK genes did not prevent inhibition of APC/C, suggesting that each of the Tpk proteins can take over this function . However, Tpk2 seems to inhibit APC/C function more efficiently than Tpk1 and Tpk3 . Finally, evidence is provided that Cdc20 is involved in APC/C regulation by the cAMP/PKA pathway.

Microbiology, 2003 May, 149(Pt 5), 1193 - 204
Response to high osmotic conditions and elevated temperature in Saccharomyces cerevisiae is controlled by intracellular glycerol and involves coordinate activity of MAP kinase pathways; Wojda I et al.; In the yeast Saccharomyces cerevisiae, response to an increase in external osmolarity is mediated by the HOG (high osmolarity glycerol) MAP kinase pathway . HOG pathway mutant strains display osmosensitive phenotypes . Recently evidence has been obtained that the osmosensitivity of HOG pathway mutants is reduced during growth at elevated temperature (37 degrees C) . A notable exception is the ste11ssk2ssk22 mutant, which displays hypersensitivity to osmotic stress at 37 degrees C . This paper reports that overexpression of FPS1 or GPD1 (encoding the glycerol transport facilitator and glycerol-3-phosphate dehydrogenase, respectively, and both affecting intracellular glycerol levels) reduces the hypersensitivity to osmotic stress of ste11ssk2ssk22 at 37 degrees C . Although in this particular HOG pathway mutant a correlation between suppression of the phenotype and glycerol content could be demonstrated, the absolute level of intracellular glycerol per se does not determine whether a strain is osmosensitive or not . Rather, evidence was obtained that the glycerol level may have an indirect effect, viz . by influencing signalling through the PKC (protein kinase C) MAP kinase pathway, which plays an important role in maintenance of cellular integrity . In order to validate the data obtained with a HOG pathway mutant strain for wild-type yeast cells, MAP kinase signalling under different growth conditions was examined in wild-type strains . PKC pathway signalling, which is manifest at elevated growth temperature by phosphorylation of MAP kinase Mpk1p, is rapidly lost when cells are shifted to high external osmolarity conditions . Expression of bck1-20 or overexpression of WSC3 in wild-type cells resulted in restoration of PKC signalling . Both PKC and HOG signalling, cell wall phenotypes and high osmotic stress responses in wild-type cells were found to be influenced by the growth temperature . The data taken together indicate the intricate interdependence of growth temperature, intracellular glycerol, cell wall structure and MAP kinase signalling in the hyperosmotic stress response of yeast.

Biosci Biotechnol Biochem, 2003 Mar, 67(3), 517 - 24
Characterization of a Saccharomyces cerevisiae mutant with pseudohyphae and cloning of a gene complementing the mutation; Maneesri J et al.; Screening for morphological mutants of a haploid strain of Saccharomyces cerevisiae was done on the basis of their cell-shape on a solid medium containing isoamyl alcohol, which causes cell elongation, to obtain information on the morphogenesis . Mutant J19, which had pseudohyphae in liquid medium even in the absence of isoamyl alcohol, had many elongated cells . Few reports exist of haploid cells growing as pseudohyphae in liquid culture . Cell-wall analysis showed that J19 had ordinary amounts of alkali-insoluble glucan and chitin, but that isoamyl alcohol in the medium caused structural changes in the cell wall . Addition of a DNA fragment that included the wild-type SCL1 gene to J19 complemented its morphological phenotype . Sequencing of J19 SCL1 showed that the glycine at position 226 in the Scl1 protein had been replaced by asparatic acid, suggesting that this mutation in the protein, a subunit of proteasomes, may be involved in the morphological change.

J Biol Chem, 2003 Jul 11, 278(28), 25308 - 16 Epub 2003 Apr 29.
The Saccharomyces cerevisiae COQ6 gene encodes a mitochondrial flavin-dependent monooxygenase required for coenzyme Q biosynthesis; Gin P et al.; Coenzyme Q (Q) is a lipid that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes . There are eight complementation groups of Q-deficient Saccharomyces cerevisiae mutants, designated coq1-coq8 . Here we have isolated the COQ6 gene by functional complementation and, in contrast to a previous report, find it is not an essential gene . coq6 mutants are unable to grow on nonfermentable carbon sources and do not synthesize Q but instead accumulate the Q biosynthetic intermediate 3-hexaprenyl-4-hydroxybenzoic acid . The Coq6 polypeptide is imported into the mitochondria in a membrane potential-dependent manner . Coq6p is a peripheral membrane protein that localizes to the matrix side of the inner mitochondrial membrane . Based on sequence homology to known proteins, we suggest that COQ6 encodes a flavin-dependent monooxygenase required for one or more steps in Q biosynthesis.

Protein Sci, 2003 May, 12(5), 1136 - 40
A novel member of the split betaalphabeta fold: Solution structure of the hypothetical protein YML108W from Saccharomyces cerevisiae; Pineda-Lucena A et al.; As part of the Northeast Structural Genomics Consortium pilot project focused on small eukaryotic proteins and protein domains, we have determined the NMR structure of the protein encoded by ORF YML108W from Saccharomyces cerevisiae . YML108W belongs to one of the numerous structural proteomics targets whose biological function is unknown . Moreover, this protein does not have sequence similarity to any other protein . The NMR structure of YML108W consists of a four-stranded beta-sheet with strand order 2143 and two alpha-helices, with an overall topology of betabetaalphabetabetaalpha . Strand beta1 runs parallel to beta4, and beta2:beta1 and beta4:beta3 pairs are arranged in an antiparallel fashion . Although this fold belongs to the split betaalphabeta family, it appears to be unique among this family; it is a novel arrangement of secondary structure, thereby expanding the universe of protein folds.

Mol Genet Genomics, 2003 Apr, 269(1), 78 - 89 Epub 2003 Feb 12.
Disruption of the Saccharomyces cerevisiae cell-wall pathway gene SLG1 causes hypersensitivity to the antitumor drug bleomycin; Leduc A et al.; Bleomycin is an antitumor drug that damages DNA via a free radical-dependent mechanism, and yeast mutants defective in DNA repair are hypersensitive to the drug . To identify possible pathways that may contribute to bleomycin resistance in yeast, we characterized a panel of bleomycin-sensitive mutants that were previously isolated by insertion mutagenesis using the transposon miniTn3::Leu2::LacZ::AMP( R) . One of these mutants harbored a single insertion in the SLG1 gene, which encodes a cell membrane protein that senses cell wall stress, and functions to maintain cell wall function by activating the protein kinase C signaling pathway . Deletion of the SLG1 gene in parental strains caused hypersensitivity to bleomycin, and this correlated with an accumulation of damaged DNA . A plasmid that expresses the native SLG1 gene or that increases PKC1 gene dosage restored bleomycin resistance to the slg1Delta mutant . Two-dimensional gel electrophoresis revealed that exposure to bleomycin triggered the expression of certain proteins, presumably to maintain cell wall function, in a Slg1-dependent manner . In addition, mutants lacking cell wall function were found to be hypersensitive to bleomycin . We conclude that mutants deficient in proteins that maintain cell wall function are severely compromised in their ability to limit bleomycin entry into the cell . Therefore, these mutants are burdened with increased genotoxicity upon exposure to bleomycin in the medium . Our results show that major mechanisms other than DNA repair are operating in yeast to mediate bleomycin resistance.

Nucleic Acids Res, 2003 May 1, 31(9), 2289 - 96
Identification of stop codon readthrough genes in Saccharomyces cerevisiae; Namy O et al.; We specifically sought genes within the yeast genome controlled by a non-conventional translation mechanism involving the stop codon . For this reason, we designed a computer program using the yeast database genomic regions, and seeking two adjacent open reading frames separated only by a unique stop codon (called SORFs) . Among the 58 SORFs identified, eight displayed a stop codon bypass level ranging from 3 to 25% . For each of the eight sequences, we demonstrated the presence of a poly(A) mRNA . Using isogenic {PSI(+)} and {psi(-)} yeast strains, we showed that for two of the sequences the mechanism used is a bona fide readthrough . However, the six remaining sequences were not sensitive to the PSI state, indicating either a translation termination process independent of eRF3 or a new stop codon bypass mechanism . Our results demonstrate that the presence of a stop codon in a large ORF may not always correspond to a sequencing error, or a pseudogene, but can be a recoding signal in a functional gene . This emphasizes that genome annotation should take into account the fact that recoding signals could be more frequently used than previously expected.

Fish Shellfish Immunol, 2003 May, 14(5), 375 - 88
A mannose-receptor is possibly involved in the phagocytosis of Saccharomyces cerevisiae by seabream (Sparus aurata L.) leucocytes; Rodriguez A et al.; In this paper the possible involvement of the mannose-receptor on the non-specific recognition and phagocytosis of heat killed yeast cells (Saccharomyces cerevisiae) by gilthead seabream (Sparus aurata L.) head-kidney leucocytes was established by studying the ability of different sugars to inhibit the uptake of the yeast cells by leucocytes . Leucocytes were preincubated for 30min with different concentrations of sugar (alpha-mannan, d-mannose, d-fucose, l-fucose, d-glucose, d-glucosamine and n-acetyl-glucosamine, all of them described as specific ligands of the vertebrate mannose-receptor) and afterwards incubated with FITC-labelled yeast cells for phagocytosis assays . The phagocytic ability (percentage of cells with one or more ingested yeast cells within the total cell population) and capacity (number of ingested yeast cells per cell) of leucocytes was analysed by flow cytometry . The results demonstrate the potential existence of a specific receptor-sugar or receptor-yeast cell binding process, which was saturable, specific and dose-dependent . More specifically, when leucocytes were preincubated with appropriate doses of d-mannose, d- or l-fucose, d-glucose or n-acetyl-glucosamine the phagocytosis of yeast cells by head-kidney leucocytes was partially blocked . Seabream leucocytes were also preincubated with chloroquine, a lysosomotropic drug which downregulates (in a nonspecific manner) the expression of mannose-receptors in mammals, before phagocytosis assays were performed . The results demonstrated that the phagocytosis of yeast was completely blocked by this substance . The overall results seem to corroborate the presence of the mannose-receptor in seabream phagocytes, which is involved in the non-specific binding and phagocytosis of yeast cells by head-kidney leucocytes.

J Biol Chem, 2003 Jun 27, 278(26), 23610 - 6 Epub 2003 Apr 22.
Phosphorylation of Saccharomyces cerevisiae CTP synthetase at Ser424 by protein kinases A and C regulates phosphatidylcholine synthesis by the CDP-choline pathway; Choi MG et al.; The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinases A and C . Previous studies have revealed that Ser424 is the target site for protein kinase A . Using a purified S424A mutant CTP synthetase enzyme, we examined the effect of Ser424 phosphorylation on protein kinase C phosphorylation . The S424A mutation in CTP synthetase caused a 50% decrease in the phosphorylation of the enzyme by protein kinase C and an 80% decrease in the stimulatory effect on CTP synthetase activity by protein kinase C . The S424A mutation caused increases in the apparent Km values of CTP synthetase and ATP of 20-and 2-fold, respectively, in the protein kinase C reaction . The effect of the S424A mutation on the phosphorylation reaction was dependent on time and protein kinase C concentration . A CTP synthetase synthetic peptide (SLGRKDSHSA) containing Ser424 was a substrate for protein kinase C . Comparison of phosphopeptide maps of the wild type and S424A mutant CTP synthetase enzymes phosphorylated by protein kinases A and C indicated that Ser424 was also a target site for protein kinase C . Phosphorylation of Ser424 accounted for 10% of the total phosphorylation of CTP synthetase by protein kinase C . The incorporation of {methyl-3H}choline into phosphocholine, CDP-choline, and phosphatidylcholine in cells carrying the S424A mutant CTP synthetase enzyme was reduced by 48, 32, and 46%, respectively, when compared with control cells . These data indicated that phosphorylation of Ser424 by protein kinase A or by protein kinase C was required for maximum phosphorylation and stimulation of CTP synthetase and that the phosphorylation of this site played a role in the regulation of phosphatidylcholine synthesis by the CDP-choline pathway.

Eur J Biochem, 2003 May, 270(9), 2013 - 22
Saccharomyces cerevisiae Pip2p-Oaf1p regulates PEX25 transcription through an adenine-less ORE; Rottensteiner H et al.; The role of the Saccharomyces cerevisiae Pip2p-Oaf1p transcription factor was examined in reference to the regulation of the peroxin gene PEX25 involved in peroxisome proliferation . The PEX25 promoter contains an oleate response element (ORE)-like sequence comprising a CGG palindrome lacking a canonical adenine, which is considered critical for element function and Pip2p-Oaf1p binding . Pex25p levels were higher in wild-type cells grown on oleic acid medium than in those grown on ethanol, but this induction was abolished in cells devoid of Pip2p-Oaf1p . Studies based on lacZ reporter genes and in vitro protein-DNA interactions revealed that the PEX25 ORE could bind Pip2p-Oaf1p and confer activation on a basal promoter . These findings reinforced the central role played by Pip2p-Oaf1p in regulating peroxisome proliferation . We also investigated whether Pip2p-Oaf1p is important for regulating genes encoding peroxins involved in protein import into the peroxisomal matrix . Pip2p-Oaf1p was able to bind efficiently to the PEX5 ORE but not to an ORE-like CGG palindrome in the PEX14 promoter . However, immunoblotting revealed that both Pex5p and Pex14p (as well as Pex7p and Pex13p) were not more abundant in cells grown on oleic acid medium compared with ethanol . These data on a functional, adenine-less, PEX25 ORE and a nonfunctional N13-spaced ORE-like sequence in the PEX14 promoter capable of binding Pip2p-Oaf1p prompts readjustment of the ORE consensus to comprise CGGN3TNA/(R)N8-12CCG.

Chromosoma, 2003 Apr, 111(7), 417 - 28 Epub 2003 Mar 18.
Analysis of the distribution of the kinetochore protein Ndc10p in Saccharomyces cerevisiae using 3-D modeling of mitotic spindles; Muller-Reichert T et al.; Ndc10p is one of the DNA-binding constituents of the kinetochore in Saccharomyces cerevisiae but light microscopy analysis suggests that Ndc10p is not limited to kinetochore regions . We examined the localization of Ndc10p using immunoelectron microscopy and showed that Ndc10p is associated with spindle microtubules from S-phase through anaphase . By serial section reconstruction of mitotic spindles combined with immunogold detection, we showed that Ndc10p interacts with microtubules laterally as well as terminally . About 50% of the gold label in serial section reconstructions of short mitotic spindles was associated with the walls of spindle microtubules . Interaction of kinetochore components with microtubule walls was also shown for kinetochore protein Ndc80p . Our data suggest that at least a subset of kinetochore-associated protein is dispersed throughout the mitotic spindle.

Genetics, 2003 Apr, 163(4), 1287 - 98
Mode of selection and experimental evolution of antifungal drug resistance in Saccharomyces cerevisiae; Anderson JB et al.; We show that mode of selection, degree of dominance of mutations, and ploidy are determining factors in the evolution of resistance to the antifungal drug fluconazole in yeast . In experiment 1, yeast populations were subjected to a stepwise increase in fluconazole concentration over 400 generations . Under this regimen, two mutations in the same two chromosomal regions rose to high frequency in parallel in three replicate populations . These mutations were semidominant and additive in their effect on resistance . The first of these mutations mapped to PDR1 and resulted in the overexpression of the ABC transporter genes PDR5 and SNQ2 . These mutations had an unexpected pleiotropic effect of reducing the residual ability of the wild type to reproduce at the highest concentrations of fluconazole . In experiment 2, yeast populations were subjected to a single high concentration of fluconazole . Under this regimen, a single recessive mutation appeared in each of three replicate populations . In a genome-wide screen of approximately 4700 viable deletion strains, 13 were classified as resistant to fluconazole (ERG3, ERG6, YMR102C, YMR099C, YPL056C, ERG28, OSH1, SCS2, CKA2, SML1, YBR147W, YGR283C, and YLR407W) . The mutations in experiment 2 all mapped to ERG3 and resulted in the overexpression of the gene encoding the drug target ERG11, but not PDR5 and SNQ2 . Diploid hybrids from experiments 1 and 2 were less fit than the parents in the presence of fluconazole . In a variation of experiment 2, haploids showed a higher frequency of resistance than diploids, suggesting that degree of dominance and ploidy are important factors in the evolution of antifungal drug resistance.

Genetics, 2003 Apr, 163(4), 1273 - 86
Crossover interference in Saccharomyces cerevisiae requires a TID1/RDH54- and DMC1-dependent pathway; Shinohara M et al.; Two RecA-like recombinases, Rad51 and Dmc1, function together during double-strand break (DSB)-mediated meiotic recombination to promote homologous strand invasion in the budding yeast Saccharomyces cerevisiae . Two partially redundant proteins, Rad54 and Tid1/Rdh54, act as recombinase accessory factors . Here, tetrad analysis shows that mutants lacking Tid1 form four-viable-spore tetrads with levels of interhomolog crossover (CO) and noncrossover recombination similar to, or slightly greater than, those in wild type . Importantly, tid1 mutants show a marked defect in crossover interference, a mechanism that distributes crossover events nonrandomly along chromosomes during meiosis . Previous work showed that dmc1Delta mutants are strongly defective in strand invasion and meiotic progression and that these defects can be partially suppressed by increasing the copy number of RAD54 . Tetrad analysis is used to show that meiotic recombination in RAD54-suppressed dmc1Delta cells is similar to that in tid1; the frequency of COs and gene conversions is near normal, but crossover interference is defective . These results support the proposal that crossover interference acts at the strand invasion stage of recombination.

FEMS Yeast Res, 2001 Apr, 1(1), 57 - 65
The essential and ancillary role of glutathione in Saccharomyces cerevisiae analysed using a grande gsh1 disruptant strain; Lee JC et al.; A grande gsh1 disruptant mutant of Saccharomyces cerevisiae was generated by crossing a petite disruptant to a wild-type grande strain . This strain was relatively stable, but generated petites at an elevated frequency, illustrating the ancillary role of glutathione (GSH) in the maintenance of the genetic integrity of the mitochondrial genome . The availability of the grande gsh1 deletant enabled an evaluation of the role of GSH in the cellular response to hydrogen peroxide independent of the effects of a petite mutation . The mutant strain was more sensitive to hydrogen peroxide than the wild-type strain but was still capable of producing an adaptive stress response to this compound . GSH was found to be essential for growth and sporulation of the yeast, but the intracellular level needed to support growth was at least two orders of magnitude less than that normally present in wild-type cells . This surprising result indicates that there is an essential role for GSH but only very low amounts are needed for growth . This result was also found in anaerobic conditions, thus this essential function does not involve protection from oxidative stress . Suppressors of the gsh1 deletion mutation were isolated by ethylmethanesulfonate mutagenesis . These were the result of a single recessive mutation (sgr1, suppressor for glutathione requirement) that relieved the requirement for GSH for growth on minimal medium but did not affect the sensitivity to H(2)O(2) stress . Interestingly, the gsh1 sgr1 mutant generated petites at a lower rate than the gsh1 mutant . Thus, it is suggested that the essential role of GSH is involved in the maintenance of the mitochondrial genome.

FEMS Yeast Res, 2001 Apr, 1(1), 47 - 55
The impact of GAL6, GAL80, and MIG1 on glucose control of the GAL system in Saccharomyces cerevisiae; Ostergaard S et al.; The role of the proteins encoded by the GAL80 gene, the MIG1 gene and the GAL6 gene in glucose control of galactose consumption by Saccharomyces cerevisiae was studied by physiological characterisation of various GAL mutant strains . Dynamic experiments with the CEN.PK 113-7D wild-type strain and a deltagal80deltamig1 double-mutant strain in aerobic nitrogen-limited continuous cultivations at a dilution rate of 0.1 h(-1), showed simultaneous glucose and galactose consumption by the deltagal80deltamig1 strain . The wild-type strain did not consume galactose in the presence of glucose . Aerobic batch cultivations on glucose-galactose mixtures with the wild-type strain and with recombinant strains with a de-regulated GAL system (the deltagal80deltamig1 strain, a deltagal6 deleted strain, a deltagal6deltagal80deltamig1 triple mutant, and a deltagal6deltagal80deltamig1 triple mutant harbouring a GAL4 high-copy vector) were carried out . Generally, a reduction of glucose control lowered the maximum specific growth rate on glucose and increased the ethanol yield obtained on galactose with more than 100% . In contrast to the wild-type strain, the deltagal6deltagal80deltamig1 triple mutant strain consumed glucose and galactose simultaneously, and this strain also showed the highest ethanol production with an overall ethanol yield of 0.35 g g-1 sugar, which is 17% higher than the yield on glucose obtained with the wild-type strain . GAL80 and MIG1 were demonstrated to be responsible for the majority of the glucose control on the GAL system, whereas GAL6 has a minor role in glucose control . Deletion of GAL6 was shown to have a major impact on biomass and ethanol formation when cells were grown on galactose, and from the data obtained we speculate that Gal6 may be involved in mRNA degradation of the GAL gene transcripts.

FEMS Yeast Res, 2001 Apr, 1(1), 33 - 45
The role of hexose transport and phosphorylation in cAMP signalling in the yeast Saccharomyces cerevisiae; Rolland F et al.; Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detection via the Gpr1-Gpa2 G-protein coupled receptor system and intracellular glucose-sensing that depends on glucose uptake and phosphorylation . The glucose uptake requirement can be fulfilled by any glucose carrier including the Gal2 permease or by intracellular hydrolysis of maltose . Hence, the glucose carriers do not seem to play a regulatory role in cAMP signalling . Also the glucose carrier homologues, Snf3 and Rgt2, are not required for glucose-induced cAMP synthesis . Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling . This indicates that a regulatory function may be associated with the hexose kinases . Consistently, intracellular acidification, another known trigger of cAMP synthesis, can bypass the glucose uptake requirement but not the absence of a functional hexose kinase . This may indicate that intracellular acidification can boost a downstream effect that amplifies the residual signal transmitted via the hexose kinases when glucose uptake is too low.

FEMS Yeast Res, 2003 Apr, 3(2), 201 - 9
Chronological and replicative lifespan of polyploid Saccharomyces cerevisiae (syn . S . pastorianus); Maskell DL et al.; Chronological lifespan may be defined as the result of accumulation of irreversible damage to intracellular components during extended stationary phase, compromising cellular integrity and leading to death and autolysis . In contrast, replicative lifespan relates to the number of divisions an individual cell has undertaken before entering a non-replicative state termed senescence, leading to cell death and autolysis . Both forms of lifespan have been considered to represent models of ageing in higher eukaryotes, yet the relation between chronologically and replicatively aged populations has not been investigated . In this study both forms of lifespan have been investigated in Saccharomyces cerevisiae (Syn . S . pastorianus) to establish the relationship between chronological and replicative ageing.

FEMS Yeast Res, 2001 Dec, 1(3), 225 - 32
Phospholipase D1 is required for efficient mating projection formation in Saccharomyces cerevisiae; Hairfield ML et al.; Phospholipase D1 (PLD1) is an important enzyme involved in lipid signal transduction in eukaryotes . A role for PLD1 in signaling in Saccharomyces cerevisiae was examined . Pheromone response in yeast is controlled by a well-characterized protein kinase cascade . Loss of PLD1 activity was found to impair pheromone-induced changes in cellular morphology that result in formation of mating projections . The rate at which projections appeared following pheromone treatment was delayed, suggesting that PLD1 facilitates the execution of a rate-limiting step in morphogenesis . Mutants were found to be less sensitive to pheromone, again arguing that PLD1 is acting at a rate-limiting step . The fact that morphogenesis is most dramatically affected indicates that PLD1 functions primarily in the morphogenic branch of the pheromone response pathway.

FEMS Yeast Res, 2001 Dec, 1(3), 169 - 75
The glutamate synthase (GOGAT) of Saccharomyces cerevisiae plays an important role in central nitrogen metabolism; Guillamon JM et al.; Central nitrogen metabolism contains two pathways for glutamate biosynthesis, glutaminases and glutamate synthase (GOGAT), using glutamine as the sole nitrogen source . GOGAT's importance for cellular metabolism is still unclear . For a further physiological characterisation of the GOGAT function in central nitrogen metabolism, a GOGAT-negative (Deltaglt1) mutant strain (VWk274 LEU(+)) was studied in glutamine-limited continuous cultures . As reference, we did the same experiments with a wild-type strain (VWk43) . Intracellular and extracellular metabolites were analysed during different steady states in both strains . The redox state of the cell was taken into account and the NAD(H) and NADP(H) concentrations were determined as well as the reduced and oxidised forms of glutathione (GSH and GSSG, respectively) . The results of this study confirm an earlier suggestion, based on a metabolic network model, that GOGAT may be a link between the carbon catabolic reactions (energy production) and nitrogen anabolic reactions (biomass production) by working as a shuttle between cytosol and mitochondria.

FEMS Yeast Res, 2002 Jan, 1(4), 307 - 14
Catabolite inactivation of the sugar transporters in Saccharomyces cerevisiae is inhibited by the presence of a nitrogen source; Lucero P et al.; Saccharomyces cerevisiae uses glucose preferentially to any other carbon source and this preferential use is ensured by control mechanisms triggered by glucose . The consensus is that inactivation of sugar transporters other than glucose transporters is one of these mechanisms . This inactivation is called catabolite inactivation because of its apparent analogy with the catabolite inactivation of gluconeogenic enzymes . Recently, doubt has been cast on the role of the inactivation of the sugar transporters in controlling the use of glucose because this inactivation neither is specifically triggered by glucose nor specifically affects non-glucose sugar transporters . Based on the fact that this inactivation has been almost exclusively investigated using nitrogen-starved cells, it has been proposed that it might be due to the stimulation of the protein turnover that follows nitrogen starvation . The results obtained in this work support this possibility since they show that the presence of a nitrogen source in the medium strongly inhibited the inactivation . It is concluded that, in growing yeast cells, the contribution of the inactivation by glucose of the non-glucose sugar transporters to the preferential use of glucose is much lower than generally believed.

FEMS Yeast Res, 2002 Mar, 2(1), 31 - 7
The isoprenoid biosynthetic pathway in Saccharomyces cerevisiae is affected in a maf1-1 mutant with altered tRNA synthesis; Kaminska J et al.; tRNA isopentenylation is a branch of an isoprenoid pathway in yeast . There is a competition for a substrate between isoprenoid biosynthetic enzyme Erg20p and tRNA isopentenyltransferase . Here we studied the direct effect of elevated tRNA biosynthesis on ERG20 expression . The maf1-1 mutant of Saccharomyces cerevisiae that has enhanced cellular tRNA levels was used . We show that both ERG20 transcript and Erg20 protein levels are increased in maf1-1 . Additionally, maf1-1 leads to decreased ergosterol content in the cells . These effects of maf1-1 are dependent on functional tRNA isopentenyltransferase . Our results indicate that a complex regulation of the isoprenoid pathway involves also an effect of changes in tRNA biosynthesis.

FEMS Yeast Res, 2002 May, 2(2), 87 - 91
Functional differences between RSC1 and RSC2, components of a for growth essential chromatin-remodeling complex of Saccharomyces cerevisiae, during the sporulation process; Yukawa M et al.; RSC, a for growth essential chromatin-remodeling complex of Saccharomyces cerevisiae, is composed of 15 subunits . Rsc1p and Rsc2p are highly homologous proteins and are contained in distinct RSC complexes . We found that both rsc1Delta and rsc2Delta homozygous diploids showed reduced sporulation with decreased expression of IME2 and that rsc1Delta, but not rsc2Delta, produced aberrant asci containing one to three spores . Overexpression of RSC2 in rsc1Delta recovered the sporulation efficiency but not the production of aberrant asci . In contrast, overexpression of RSC1 in rsc2Delta did not alleviate its sporulation defect . These results suggest that both Rsc1p and Rsc2p share overlapping functions on IME2 expression, with a prominent role for Rsc2p, whereas Rsc1p has an additional function in the late steps of the sporulation process.

FEMS Yeast Res, 2002 Aug, 2(3), 283 - 91
Determination of in vivo kinetics of the starvation-induced Hxt5 glucose transporter of Saccharomyces cerevisiae; Buziol S et al.; We have investigated the role and the kinetic properties of the Hxt5 glucose transporter of Saccharomyces cerevisiae . The HXT5 gene was not expressed during growth of the yeast cells in rich medium with glucose or raffinose . However, it became strongly induced during nitrogen or carbon starvation . We have constructed yeast strains constitutively expressing only Hxt5, Hxt1 (low affinity) or Hxt7 (high affinity), but no other glucose transporters . Aerobic fed-batch cultures at quasi steady-state conditions, and aerobic and anaerobic chemostat cultures at steady-state conditions of these strains were used for estimation of the kinetic properties of the individual transporters under in vivo conditions, by investigating the dynamic responses of the strains to changes in extracellular glucose concentration . The K(m) value and the growth properties of the HXT5 single expression strain indicate that Hxt5 is a transporter with intermediate affinity.

FEMS Yeast Res, 2002 Aug, 2(3), 267 - 76
A novel mitochondrial DEAD box protein (Mrh4) required for maintenance of mtDNA in Saccharomyces cerevisiae; Schmidt U et al.; In a screen of nuclear genes that assist splicing of mitochondrial localized group II introns in yeast we isolated low-copy number suppressors of splicing and respiratory-deficient point mutants of intron aI5gamma, the last intron of the gene encoding cytochrome c oxidase subunit I . One of the genes found contains the open reading frame (ORF) YGL064c that has previously been proposed to encode a putative RNA helicase of the DEAD box family . Deletion of the ORF gives rise to 100% cytoplasmic petites, indicating that the protein plays an essential role in the mitochondrial RNA metabolism . Overexpression of YGL064c-GFP fusions clearly revealed a mitochondrial localization of the protein . The gene encodes the fourth putative RNA helicase of Saccharomyces cerevisiae implicated in a mitochondrial function and was therefore termed MRH4 (for mitochondrial RNA helicase).

FEMS Yeast Res, 2002 Aug, 2(3), 259 - 65
Dolichol biosynthesis in the yeast Saccharomyces cerevisiae: an insight into the regulatory role of farnesyl diphosphate synthase; Grabinska K et al.; Dolichol, an isoprenoid lipid, known mainly for its function in protein glycosylation, is synthesised in the mevalonate pathway . The pathway is highly regulated, on multiple levels, by sterol and non-sterol derivatives of mevalonic acid . Farnesyl diphosphate (FPP) and/or FPP-derived molecules have been identified as the main non-sterol compounds regulating degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, one of the regulatory enzymes in the mevalonate pathway . In the present review we concentrate on the effect of overexpression of farnesyl diphosphate synthase on dolichol biosynthesis in yeast . In this context the role of the Yta7 protein, belonging to the AAA ATPase family, in the regulation of FPP flux to the dolichol branch of the mevalonate pathway is discussed, and the effect of FPP and/or derived molecules on the transcription of genes encoding the first enzyme committed to dolichol biosynthesis, i.e . cis-prenyl transferase.

FEMS Yeast Res, 2002 Dec, 2(4), 539 - 50
Characterisation of glucose transport in Saccharomyces cerevisiae with plasma membrane vesicles (countertransport) and intact cells (initial uptake) with single Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 or Gal2 transporters; Maier A et al.; The yeast glucose transporters Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 and Gal2, individually expressed in an hxt1-7 null mutant strain, demonstrate the phenomenon of countertransport . Thus, these transporters, which are the most important glucose transporters in Saccharomyces cerevisiae, are facilitated diffusion transporters . Apparent K(m)-values from high to low affinity, determined from countertransport and initial-uptake experiments, respectively, are: Hxt6 0.9+/-0.2 and 1.4+/-0.1 mM, Hxt7 1.3+/-0.3 and 1.9+/-0.1 mM, Gal2 1.5 and 1.6+/-0.1 mM, Hxt2 2.9+/-0.3 and 4.6+/-0.3 mM, Hxt4 6.2+/-0.5 and 6.2+/-0.3 mM, Hxt3 28.6+/-6.8 and 34.2+/-3.2 mM, and Hxt1 107+/-49 and 129+/-9 mM . From both independent methods, countertransport and initial uptake, the same range of apparent K(m)-values was obtained for each transporter . In contrast to that in human erythrocytes, the facilitated diffusion transport mechanism of glucose in yeast was symmetric . Besides facilitated diffusion there existed in all single glucose transport mutants, except for the HXT1 strain, significant first-order behaviour.

FEMS Yeast Res, 2003 Mar, 3(1), 119 - 26
Activation state of protein kinase A as measured in permeabilised Saccharomyces cerevisiae cells correlates with PKA-controlled phenotypes in vivo; Portela P et al.; Protein kinase A (PKA) activity was measured in situ in permeabilised Saccharomyces cerevisiae cells in the absence and the presence of cAMP . Four strains genetically predicted to have differential PKA-dependent phenotypes were used: a wild-type strain and a strain containing a bcy1-14 mutation (with almost constitutively active PKA), and the same strains with overexpression of the wild-type or mutant BCY1 gene, respectively . Cells were grown on galactose or glucose . The measured phenotypic characteristics were: trehalose and glycogen levels and the activity of a reporter gene under control of the NTH1 promoter . The 'endogenous' PKA activity (measured in situ in the absence of cAMP) showed the best correlation with the PKA-dependent phenotypes determined in vivo . We propose that this parameter offers a good estimate for the degree of activation of PKA in vivo.

FEMS Yeast Res, 2003 Mar, 3(1), 77 - 84
New mutations of Saccharomyces cerevisiae that partially relieve both glucose and galactose repression activate the protein kinase Snf1; Rodriguez C et al.; We isolated from Saccharomyces cerevisiae two mutants, esc1-1 and ESC3-1, in which genes FBP1, ICL1 or GDH2 were partially derepressed during growth in glucose or galactose . The isolation was done starting with a triple mutant pyc1 pyc2 mth1 unable to grow in glucose-ammonium medium and selecting for mutants able to grow in the non-permissive medium . HXT1 and HXT2 which encode glucose transporters were expressed at high glucose concentrations in both esc1-1 and ESC3-1 mutants, while derepression of invertase at low glucose concentrations was impaired . REG1, cloned as a suppressor of ESC3-1, was not allelic to ESC3-1 . Two-hybrid analysis showed an increased interaction of the protein kinase Snf1 with Snf4 in the ESC3-1 mutant; this was not due to mutations in SNF1 or SNF4 . ESC3-1 did not bypass the requirement of Snf1 for derepression . We hypothesize that ESC3-1 either facilitates activation of Snf1 or interferes with its glucose-dependent inactivation.

J Biol Chem, 2003 Jun 27, 278(26), 23317 - 23 Epub 2003 Apr 07.
YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae; Athenstaedt K et al.; Previous work from our laboratory (Athenstaedt, K., Zweytick, D., Jandrositz, A., Kohlwein, S . D., and Daum, G . (1999) J . Bacteriol . 181, 6441-6448) showed that the gene product of YMR313c (named Tgl3p) is a component of yeast lipid particles, and deletion of this gene led to an increase in the cellular level of triacylglycerols (TAG) . These observations suggested that TGL3 may encode a TAG lipase of Saccharomyces cerevisiae . Here we demonstrate by cell fractionation and by microscopic inspection of a strain bearing a Tgl3p-GFP hybrid that this polypeptide is highly enriched in the lipid particle fraction but virtually absent from other organelles . The entire TAG lipase activity of lipid particles is attributed to Tgl3p, because the activity in this organelle is completely absent in a Deltatgl3 deletion mutant, whereas it is significantly enhanced in a strain overexpressing Tgl3p . A His6-tagged Tgl3p hybrid purified close to homogeneity from a yeast strain overexpressing this fusion protein exhibited high TAG lipase activity . Most importantly, experiments in vivo using the fatty acid synthesis inhibitor cerulenin demonstrated that deletion of TGL3 resulted in a decreased mobilization of TAG from lipid particles . The amino acid sequence deduced from the open reading frame YMR313c contains the consensus sequence motif GXSXG typical for lipolytic enzymes . Otherwise, Tgl3p has no significant sequence homology to other lipases identified so far . In summary, our data identified Tgl3p as a novel yeast TAG lipase at the molecular level and by function in vivo and in vitro.

J Biol Chem, 2003 Jul 18, 278(29), 26450 - 7 Epub 2003 Apr 15.
Multiple positive and negative elements involved in the regulation of expression of GSY1 in Saccharomyces cerevisiae; Unnikrishnan I et al.; GSY1 is one of the two genes encoding glycogen synthase in Saccharomyces cerevisiae . Both the GSY1 message and the protein levels increased as cells approached stationary phase . A combination of deletion analysis and site-directed mutagenesis revealed a complex promoter containing multiple positive and negative regulatory elements . Expression of GSY1 was dependent upon the presence of a TATA box and two stress response elements (STREs) . Expression was repressed by Mig1, which mediates responses to glucose, and Rox1, which mediates responses to oxygen . Characterization of the GSY1 promoter also revealed a novel negative element . This element, N1, can repress expression driven by either an STRE or a heterologous element, the UAS of CYC1 . Repression by N1 is dependent on the number of these elements that are present, but is independent of their orientation . N1 repressed expression when placed either upstream or downstream of the UAS, although the latter position is more effective . Gel shift analysis detected a factor that appears to bind to the N1 element . The complexity of the GSY1 promoter, which includes two STREs and three distinct negative elements, was surprising . This complexity may allow GSY1 to respond to a wide range of environmental stresses.

BMC Cell Biol . 2003 Mar 28;4(1):3.
Characterization of Saccharomyces cerevisiae protein Ser/Thr phosphatase T1 and comparison to its mammalian homolog PP5; Jeong JY et al.; BACKGROUND: Protein Ser/Thr phosphatase 5 (PP5) and its Saccharomyces cerevisiae homolog protein phosphatase T1 (Ppt1p) each contain an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain that is related to the catalytic subunits of protein phosphatases 1 and 2A, and calcineurin . Analysis of yeast Ppt1p could provide important clues to the function of PP5 and its homologs, however it has not yet been characterized at the biochemical or cellular level . RESULTS: The specific activity of recombinant Ppt1p toward the artificial substrates 32P-myelin basic protein (MBP) and 32P-casein was similar to that of PP5 . Dephosphorylation of 32P-MBP, but not 32P-casein, was stimulated by unsaturated fatty acids and by arachidoyl coenzyme A . Limited proteolysis of Ppt1p removed the TPR domain and abrogated lipid stimulation . The remaining catalytic fragment exhibited a two-fold increase in activity toward 32P-MBP, but not 32P-casein . Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment . Ppt1p was localized throughout the cell including the nucleus . Levels of PPT1 mRNA and protein peaked in early log phase growth . CONCLUSIONS: Many characteristics of Ppt1p are similar to those of PP5, including stimulation of phosphatase activity with some substrates by lipids, and peak expression during periods of rapid cell growth . Unlike PP5, however, proteolytic removal of the TPR domain or C-terminal truncation only modestly increased its activity . In addition, C-terminal truncation did not prevent further activation by lipid . This suggests that these regions play only a minor role in controlling its activity compared to PP5 . Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments . The observation that Ppt1p is most highly expressed during early log phase growth suggests that this enzyme is involved in cell growth or its expression is controlled by metabolic or nutritional signals.

J Biol Chem, 2003 Jun 20, 278(25), 22537 - 45 Epub 2003 Apr 10.
Rio2p, an evolutionarily conserved, low abundant protein kinase essential for processing of 20 S Pre-rRNA in Saccharomyces cerevisiae; Geerlings TH et al.; Saccharomyces cerevisiae Rio2p (encoded by open reading frame Ynl207w) is an essential protein of unknown function that displays significant sequence similarity to Rio1p/Rrp10p . The latter was recently shown to be an evolutionarily conserved, predominantly cytoplasmic serine/threonine kinase whose presence is required for the final cleavage at site D that converts 20 S pre-rRNA into mature 18 S rRNA . A data base search identified homologs of Rio2p in a wide variety of eukaryotes and Archaea . Detailed sequence comparison and in vitro kinase assays using recombinant protein demonstrated that Rio2p defines a subfamily of protein kinases related to, but both structurally and functionally distinct from, the one defined by Rio1p . Failure to deplete Rio2p in cells containing a GAL-rio2 gene and direct analysis of Rio2p levels by Western blotting indicated the protein to be low abundant . Using a GAL-rio2 gene carrying a point mutation that reduces the kinase activity, we found that depletion of this mutant protein blocked production of 18 S rRNA due to inhibition of the cleavage of cytoplasmic 20 S pre-rRNA at site D . Production of the large subunit rRNAs was not affected . Thus, Rio2p is the second protein kinase that is essential for cleavage at site D and the first in which the processing defect can be linked to its enzymatic activity . Contrary to Rio1p/Rrp10p, however, Rio2p appears to be localized predominantly in the nucleus.

FEMS Yeast Res, 2003 May, 3(3), 261 - 8
Trehalose and glycogen accumulation is related to the duration of the G1 phase of Saccharomyces cerevisiae; Paalman JW et al.; Several factors may control trehalose and glycogen synthesis, like the glucose flux, the growth rate, the intracellular glucose-6-phosphate level and the glucose concentration in the medium . Here, the possible relation of these putative inducers to reserve carbohydrate accumulation was studied under well-defined growth conditions in nitrogen-limited continuous cultures . We showed that the amounts of accumulated trehalose and glycogen were regulated by the growth rate imposed on the culture, whereas other implicated inducers did not exhibit a correlation with reserve carbohydrate accumulation . Trehalose accumulation was induced at a dilution rate (D)</=0.10 h(-1), whereas glycogen accumulation gradually increased at decreasing growth rates . The growth rate dependency of trehalose accumulation was supported by studies in cells overexpressing the G(1)-cyclin CLN3 . The trehalose level appeared to be dependent on the duration of the G(1) phase, as trehalose was only accumulated at a G(1) phase duration of more than 5 h in both wild-type and CLN3-overexpressing cells . On the other hand, the glycogen level was reduced by CLN3 overexpression in a cell cycle-independent manner . A possible regulatory mechanism that links trehalose and glycogen accumulation to the growth rate is discussed.

FEMS Yeast Res, 2003 May, 3(3), 233 - 8
Exopolyphosphatases of the yeast Saccharomyces cerevisiae; Lichko LP et al.; Separate compartments of the yeast cell possess their own exopolyphosphatases differing from each other in their properties and dependence on culture conditions . The low-molecular-mass exopolyphosphatases of the cytosol, cell envelope, and mitochondrial matrix are encoded by the PPX1 gene, while the high-molecular-mass exopolyphosphatase of the cytosol and those of the vacuoles, mitochondrial membranes, and nuclei are presumably encoded by their own genes . Based on recent works, a preliminary classification of the yeast exopolyphosphatases is proposed.

Nucleic Acids Res, 2003 Apr 15, 31(8), 2056 - 65
Subunit interactions in the assembly of Saccharomyces cerevisiae DNA polymerase alpha; Biswas SB et al.; Eukaryotic DNA polymerase (pol) alpha is a complex of four subunits . The subunits in the yeast Saccharomyces cerevisiae are: 167, 79, 62 and 48 kDa polypeptides . The p79 subunit has no known enzymatic functions, but it is essential for growth and chromosomal DNA replication . We have analyzed the interaction between the subunits of yeast pol alpha, particularly p167 and p79, using a yeast two-hybrid screen and deletion analysis . We have identified the interaction sites in each of these two subunits leading to p167.p79 complex formation, and correlated our results with the available genetic data . A detailed two-hybrid analysis, using the p79 gene as the bait and a yeast genomic DNA library, identified two independent groups of positive clones . One group that displayed strong positive interaction (delta1) with p79 represented a fusion of the p167 open reading frame at 3502 bp (Ile1168), and the second group, displaying weak positive interaction (delta2) with p79, had a fusion at 3697 bp (Asn1233) with the DNA-binding domain of the yeast Gal4 transcription factor . A detailed deletion analysis of the downstream region indicated the existence of two subdomains that interact with p79 . Subdomain I encompasses a 65 amino acid segment between Ile1168 and Phe1232, and subdomain II is a 25 amino acid segment between Glu1259 and Leu1283 . Deletion and two-hybrid interaction analysis of the p79 subunit of pol alpha revealed a complementary region with two subdomains: a 67 amino acid segment between Asn189 and Gln255 (I) and a 68 amino acid segment between Glu256 and Met323 (II) . The p79 subdomains I and II appeared to interact with the p167 subdomains I and II, respectively . Analysis of interaction between p62 and various deletion clones of p167 did not result in an unambiguous and stable positive interaction in the two-hybrid screen between these two subunits . A strong interaction between p167 and p62 would probably require the presence of either p79 or p48 in the complex.

Biometals, 2003 Sep, 16(3), 369 - 78
The Saccharomyces cerevisiae Arr4p is involved in metal and heat tolerance; Shen J et al.; Homologues of the bacterial ArsA ATPase are found in nearly every organism . While the enzyme is involved in arsenic detoxification in bacteria, the roles of eukaryotic homologues have not been identified . This article reports the function of the Saccharomyces cerevisiae homologue encoded by ARR4 gene (YDL100c ORF) . Disruption of ARR4 was not lethal, but the disrupted strain displayed increased sensitivity to As3+, As5+, Co2+, Cr3+, Cu2+ or VO4(3-) salts and temperature . A plasmid-encoded copy of a wild-type ARR4 gene could complement the heat- or metal-related stress responses . Mutation of a codon within the consensus sequence for the nucleotide-binding site resulted in loss of complementation of the disrupted strain and produced a dominant negative phenotype in a wild type strain . Wild type and mutant Arr4p were purified from Escherichia coli . The wild type protein exhibited a low level of ATPase activity, and the mutant was inactive . The purified ATPase eluted as a dimer of 80-kDa species . A fusion of ARR4 and the GFP (green fluorescent protein) gene was constructed . The gene fusion was able to complement stress-related phenotype of the ARR4 disruption . Under non-stress conditions, GFP fluorescence was found diffusely in the cytosol . Under stress conditions GFP was localized in a few punctate bodies resembling late endosomes . It is proposed that under heat or metal stress, the soluble ATPase becomes membrane-associated, perhaps through interaction with a partner protein, and that this complex is involved in stress tolerance.

Br J Biomed Sci, 2003, 60(1), 22 - 5
Gene expression study of Saccharomyces cerevisiae with the Agilent 2100 bioanalyser; Liu CH et al.; This study explores the restriction display-polymerase chain reaction (RD-PCR) application of a new chip-based nucleic acid analysis system (Agilent 2100 bioanalyser) in a gene differential expression study . Total RNAs is extracted from Saccharomyces cerevisiae, double-stranded complementary DNA (cDNA) is synthesised by reverse transcription from the purified messenger RNA (mRNA), RD-PCR conducted to obtain the cDNA fragments and bioanalyser and agarose gel electrophoresis compared for the analysis of RD-PCR products . The bioanalyser proved to be faster and more sensitive in separating and detecting gene fragments, and was also able to compare different gene fragments quantitatively . Using this technology, comparison of several differential gene fragments is performed.

Mol Biol Cell, 2003 Apr, 14(4), 1652 - 63
Catabolite degradation of fructose-1,6-bisphosphatase in the yeast Saccharomyces cerevisiae: a genome-wide screen identifies eight novel GID genes and indicates the existence of two degradation pathways; Regelmann J et al.; Metabolic adaptation of Saccharomyces cerevisiae cells from a nonfermentable carbon source to glucose induces selective, rapid breakdown of the gluconeogenetic key enzyme fructose-1,6-bisphosphatase (FBPase), a process called catabolite degradation . Herein, we identify eight novel GID genes required for proteasome-dependent catabolite degradation of FBPase . Four yeast proteins contain the CTLH domain of unknown function . All of them are Gid proteins . The site of catabolite degradation has been controversial until now . Two FBPase degradation pathways have been described, one dependent on the cytosolic ubiquitin-proteasome machinery, and the other dependent on vacuolar proteolysis . Interestingly, three of the novel Gid proteins involved in ubiquitin-proteasome-dependent degradation have also been reported by others to affect the vacuolar degradation pathway . As shown herein, additional genes suggested to be essential for vacuolar degradation are unnecessary for proteasome-dependent degradation . These data raise the question as to whether two FBPase degradation pathways exist that share components . Detailed characterization of Gid2p demonstrates that it is part of a soluble, cytosolic protein complex of at least 600 kDa . Gid2p is necessary for FBPase ubiquitination . Our studies have not revealed any involvement of vesicular intermediates in proteasome-dependent FBPase degradation . The influence of Ubp14p, a deubiquitinating enzyme, on proteasome-dependent catabolite degradation was further uncovered.

J Biol Chem, 2003 Jun 20, 278(25), 22513 - 22 Epub 2003 Apr 09.
Dna2 helicase/nuclease causes replicative fork stalling and double-strand breaks in the ribosomal DNA of Saccharomyces cerevisiae; Weitao T et al.; We have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae and contributes to the shortened lifespan of dna2 mutants . Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis . We show directly that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB . Both pausing and breakage are elevated in the early aging, hypomorphic dna2-2 helicase mutant . Deletion of FOB1, encoding the fork barrier protein, suppresses the elevated pausing and DSB formation, and represses initiation at rDNA ARSs . The dna2-2 mutation is synthetically lethal with deltarrm3, encoding another DNA helicase involved in rDNA replication . It does not appear to be the case that the rDNA is the only determinant of genome stability during the yeast lifespan however since strains carrying deletion of all chromosomal rDNA but with all rDNA supplied on a plasmid, have decreased rather than increased lifespan . We conclude that the replication-associated defects that we can measure in the rDNA are symbolic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.

Mol Genet Genomics, 2003 May, 269(2), 290 - 8 Epub 2003 Mar 26.
Functional differentiation and selective inactivation of multiple Saccharomyces cerevisiae genes involved in very-long-chain fatty acid synthesis; Rossler H et al.; While de novo fatty acid synthesis uses acetyl-CoA, fatty acid elongation uses longer-chain acyl-CoAs as primers . Several mutations that interfere with fatty acid elongation in yeast have already been described, suggesting that there may be different elongases for medium- and long-chain acyl-CoA primers . In the present study, an experimental approach is described that allows differential characterization of the various yeast elongases in vitro . Based on their characteristic primer specificities and product patterns, at least three different yeast elongases are defined . Elongase I extends C12-C16 fatty acyl-CoAs to C16-C18 fatty acids . Elongase II elongates palmitoyl-CoA and stearoyl-CoA up to C22 fatty acids, and elongase III synthesizes 20-26-carbon fatty acids from C18-CoA primers . Elongases I, II and III are specifically inactivated in, respectively, elo1, elo2 and elo3 mutants . Elongases II and III share the same 3-ketoacyl reductase, which is encoded by the YBR159w gene . Inactivation of YBR159w inhibits in vitro fatty acid elongation after the first condensation reaction . Although in vitro elongase activity is absent, the mutant nevertheless contains 10-30% of normal VLCFA levels . On the basis of this finding, an additional elongating activity is inferred to be present in vivo . ybr159Delta cells show synthetic lethality in the presence of cerulenin, which inactivates fatty acid synthase . An involvement of FAS in VLCFA synthesis may account for these findings, but remains to be demonstrated directly . Alternatively, a vital role for C18 and C20 hydroxyacids, which are dramatically overproduced in ybr159Delta cells, may be postulated.

Mol Genet Genomics, 2003 Jun, 269(3), 321 - 30 Epub 2003 Mar 29.
Dissecting the pet18 mutation in Saccharomyces cerevisiae: HTL1 encodes a 7-kDa polypeptide that interacts with components of the RSC complex; Lu YM et al.; The yeast pet18 mutant exhibits three distinct phenotypes: temperature-sensitive lethality, failure to maintain a dsRNA virus, and respiration deficiency . We have isolated a yeast mutant, H53, with phenotypes identical to those of pet18 . Based on PCR and Southern hybridization analysis, H53 was found to result from a large chromosomal deletion extending from YCR019w to YCR028c on chromosome III . Genetic analysis was carried out on H53 to correlate individual loci with each of the observed phenotypes . Disruption of YCR020c-a/MAK31 brought about a loss of dsRNA without affecting the temperature sensitive phenotype . The loss of YCR020w-b/HTL1, which encodes a hypothetical protein of 78 amino acids in length, was shown to be responsible for the temperature-sensitive lethality of the H53 mutant . Using immunoblotting, we demonstrated that a 7-kDa protein was indeed expressed in wild-type yeast, but not in a HTL1 deletion mutant . Moreover, the significance of HTL1 was investigated by isolating genes that are functionally associated with HTL1 . We demonstrated that Rsc8p interacts physically with Htl1p, and that the genes RSC3, STH1 and RSC30 interact with HTL1 . Thus, HTL1 may play a role in the function of the RSC complex.

J Biol Inorg Chem, 2003 Jul, 8(6), 611 - 20 Epub 2003 Apr 09.
Targeted suppression of the ferroxidase and iron trafficking activities of the multicopper oxidase Fet3p from Saccharomyces cerevisiae; Wang TP et al.; The Fet3 protein in Saccharomyces cerevisiae is a multicopper oxidase tethered to the outer surface of the yeast plasma membrane . Fet3p catalyzes the oxidation of Fe(2+) to Fe(3+); this ferroxidation reaction is an obligatory first step in high-affinity iron uptake through the permease Ftr1p . Here, kinetic analyses of several Fet3p mutants identify residues that contribute to the specificity that Fet3p has for Fe(2+), one of which is essential also to the coupling of the ferroxidase and uptake processes . The spectral and kinetic properties of the D278A, E185D and A, Y354F and A, and E185A/Y354A mutants of a soluble form of Fet3p showed that all of the mutants exhibited the normal absorbance at 330 nm and 608 nm due to the type 3 and type 1 copper sites in Fet3p, respectively . The EPR spectra of the mutants were also equivalent to wild-type, showing that the type 1 and type 2 Cu(II) sites in the proteins were not perturbed . The only marked kinetic defects measured in vitro were increases in K(M) for Fe(2+) exhibited by the D278A, E185A, Y354A, and E185A/Y354A mutants . These results suggest that these three residues contribute to the ferroxidase specificity site in Fet3p . In vivo analysis of these mutant proteins in their membrane-bound form showed that only E185 mutants exhibited kinetic defects in (59)Fe uptake . For the Fet3p(E185D) mutant, K(M) for iron was 300-fold greater than the wild-type K(M), while Fet3p(E185A) was completely inactive in support of iron uptake . In situ fluorescence demonstrated that all of the mutant Fet3 proteins, in complex with an Ftr1p:YFP fusion protein, were trafficked normally to the plasma membrane . These results suggest that E185 contributes to Fe(2+ )binding to Fet3p and to the subsequent trafficking of the Fe(3+) produced to Ftr1p.

Curr Genet, 2003 Apr, 43(1), 62 - 9 Epub 2003 Feb 05.
Pitfalls of the synthetic lethality screen in Saccharomyces cerevisiae: an improved design; Koren A et al.; The colony color assay in yeast enables the visual identification of plasmid-loss events . In combination with a plasmid-dependence assay, it is commonly used to identify synthetic interactions between functionally related genes . Frequently, the plasmid carries the ADE3 gene and mutants are recognized as red colonies that fail to produce sectors . In these assays, a high percentage of false-positives is obtained, most of which result from synthetic lethality with the ade3 mutation . Here, we study the nature of these mutants . We report that mutations in the HIP1 and SHM1 genes exhibit synthetic lethality with ade3 deletions . A similar interaction is found between the fur1 and ura3 mutations . Lethality in the absence of the mitochondrial Shm1 and the cytoplasmic Ade3 enzymes indicates that, under certain circumstances, these cellular compartments cooperate in carrying out essential metabolic processes . In addition, we report the identification of a truncated ADE3 allele with a unique coloration phenotype and show that it can be used to improve synthetic lethal screens.

Mol Cell Proteomics, 2003 Mar, 2(3), 191 - 204 Epub 2003 Apr 07.
The Transcriptome and Its Translation during Recovery from Cell Cycle Arrest in Saccharomyces cerevisiae; Serikawa KA et al.; Complete genome sequences together with high throughput technologies have made comprehensive characterizations of gene expression patterns possible . While genome-wide measurement of mRNA levels was one of the first applications of these advances, other important aspects of gene expression are also amenable to a genomic approach, for example, the translation of message into protein . Earlier we reported a high throughput technology for simultaneously studying mRNA level and translation, which we termed translation state array analysis, or TSAA . The current studies test the proposition that TSAA can identify novel instances of translation regulation at the genome-wide level . As a biological model, cultures of Saccharomyces cerevisiae were cell cycle-arrested using either alpha-factor or the temperature-sensitive cdc15-2 allele . Forty-eight mRNAs were found to change significantly in translation state following release from alpha-factor arrest, including genes involved in pheromone response and cell cycle arrest such as BAR1, SST2, and FAR1 . After the shift of the cdc15-2 strain from 37 degrees C to 25 degrees C, 54 mRNAs were altered in translation state, including the products of the stress genes HSP82, HSC82, and SSA2 . Thus, regulation at the translational level seems to play a significant role in the response of yeast cells to external physical or biological cues . In contrast, surprisingly few genes were found to be translationally controlled as cells progressed through the cell cycle . Additional refinements of TSAA should allow characterization of both transcriptional and translational regulatory networks on a genomic scale, providing an additional layer of information that can be integrated into models of system biology and function.

Eukaryot Cell, 2003 Apr, 2(2), 306 - 17
Saccharomyces cerevisiae Dap1p, a novel DNA damage response protein related to the mammalian membrane-associated progesterone receptor; Hand RA et al.; The response to damage is crucial for cellular survival, and eukaryotic cells require a broad array of proteins for an intact damage response . We have found that the YPL170W (DAP1 {for damage response protein related to membrane-associated progesterone receptors}) gene is required for growth in the presence of the methylating agent methyl methanesulfonate (MMS) . The DAP1 open reading frame shares homology with a broadly conserved family of membrane-associated progesterone receptors (MAPRs) . Deletion of DAP1 leads to sensitivity to MMS, elongated telomeres, loss of mitochondrial function, and partial arrest in sterol synthesis . Sensitivity of dap1 strains to MMS is not due to loss of damage checkpoints . Instead, dap1 cells are arrested as unbudded cells after MMS treatment, suggesting that Dap1p is required for cell cycle progression following damage . Dap1p also directs resistance to itraconazole and fluconazole, inhibitors of sterol synthesis . We have found that dap1 cells have slightly decreased levels of ergosterol but increased levels of the ergosterol intermediates squalene and lanosterol, indicating that dap1 cells have a partial defect in sterol synthesis . This is the first evidence linking a MAPR family member to sterol regulation or the response to damage, and these functions are probably conserved in a variety of eukaryotes.

Eukaryot Cell, 2003 Apr, 2(2), 284 - 94
Ceramide/long-chain base phosphate rheostat in Saccharomyces cerevisiae: regulation of ceramide synthesis by Elo3p and Cka2p; Kobayashi SD et al.; Sphingolipid precursors, namely, ceramide and long-chain base phosphates (LCBPs), are important growth regulators with often opposite effects on mammalian cells . A set of enzymes that regulate the levels of these precursors, referred to as a ceramide/LCBP rheostat, is conserved in all eukaryotes . In order to gain further insight into the function of the rheostat in Saccharomyces cerevisiae, we searched for mutants that are synthetically lethal with a deletion of the LCB3 gene encoding LCBP phosphatase . In addition to acquiring expected mutants lacking the LCBP lyase, the screen revealed elo3 (sur4) mutants that were defective in fatty acid elongation and cka2 mutants lacking the alpha' subunit of the protein kinase CK2 (casein kinase) . Both mutations affected the in vivo activity of the acyl coenzyme A (acyl-CoA)-dependent and fumonisin B(1)-sensitive ceramide synthase (CS) . The Elo3 protein is necessary for synthesis of C(26)-CoA, which in wild-type yeast is a source of C(26) fatty acyls found in the ceramide moieties of all sphingolipids . In the in vitro assay, CS had a strong preference for acyl-CoAs containing longer acyl chains . This finding suggests that a block in the formation of C(26)-CoA in yeast may cause a reduction in the conversion of LCBs into ceramides and lead to an overaccumulation of LCBPs that is lethal in strains lacking the Lcb3 phosphatase . In fact, elo3 mutants were found to accumulate high levels of LCBs and LCBPs . The cka2 mutants, on the other hand, exhibited only 25 to 30% of the in vitro CS activity found in wild-type membranes, indicating that the alpha' subunit of CK2 kinase is necessary for full activation of CS . The cka2 mutants also accumulated high levels of LCBs and had elevated levels of LCBPs . In addition, both the elo3 and cka2 mutants showed increased sensitivity to the CS inhibitors australifungin and fumonisin B(1) . Together, our data demonstrate that the levels of LCBPs in yeast are regulated by the rate of ceramide synthesis, which depends on CK2 kinase activity and is also strongly affected by the supply of C(26)-CoA . This is the first evidence indicating the involvement of protein kinase in the regulation of de novo sphingolipid synthesis in any organism.

Proc Natl Acad Sci U S A, 2003 Apr 15, 100(8), 4457 - 62 Epub 2003 Apr 04.
Isolation and identification of short nucleotide sequences that affect translation initiation in Saccharomyces cerevisiae; Zhou W et al.; In previous studies, we demonstrated the sufficiency of short nucleotide sequences to facilitate internal initiation of translation in mammalian cells . By using a selection methodology, we have now identified comparable sequences in Saccharomyces cerevisiae . For these studies, a library of constructs expressing dicistronic mRNAs with the HIS3 gene as the second cistron and 18 random nucleotides in the intercistronic region was introduced into a yeast strain in which the endogenous HIS3 gene was deleted . Untransformed cells or those containing the parent construct failed to grow on medium lacking histidine . Intercistronic sequences recovered from cells that did grow were evaluated by using various criteria . Fifty-six of the 18-nt sequences (approximately 1/400,000) functioned as synthetic internal ribosome entry sites (IRESes) . The 14 most active sequences allowed growth in the presence of 0.1-0.6 mM 3-amino-1,2,4-triazole, a competitive inhibitor of the HIS3 gene product . In addition, eight sequences were identified that were not IRESes, but that enhanced HIS3 expression by an alternative mechanism that depended on the 5' end of the mRNA and appeared to involve either shunting or reinitiation . Comparisons among the 56 selected IRESes identified eight significant sequence matches containing up to 10 nucleotides . Many of the selected sequences also contained extensive complementary matches to yeast 18S rRNA, some at overlapping sites . The identification of cis sequences that facilitate translation initiation in yeast enables detailed biochemical and genetic analyses of underlying mechanisms and may have practical applications for bioengineering.

J Bioenerg Biomembr, 2002 Dec, 34(6), 507 - 16
The key role of the energized state of Saccharomyces cerevisiae mitochondria in modulations of the outer membrane channels by the intermembrane space proteins; Stobienia O et al.; Mitochondria of the yeast Saccharomyces cerevisiae constitute a perfect model to study the outer membrane channel modulation as besides the TOM complex channel they contain only a single isoform of the VDAC channel and it is possible to obtain viable mutants devoid of the channel . Here, we report that the fraction of the intermembrane space isolated from wild type and the VDAC channel-depleted yeast mitochondria, except of the well-known VDAC channel modulator activity, displays also the TOM complex channel modulating activity as measured in the reconstituted system and with intact mitochondria . The important factor influencing the action of both modulating activities is the energized state of mitochondria . Moreover, the presence of the VDAC channel itself seems to be crucial to properties of the intermembrane space protein(s) able to modulate the outer membrane channels because in the case of intact mitochondria quantitative differences are observed between modulating capabilities of the fractions isolated from wild type and mutant mitochondria.

J Bioenerg Biomembr, 2002 Dec, 34(6), 499 - 506
NADH is specifically channeled through the mitochondrial porin channel in Saccharomyces cerevisiae; Averet N et al.; In many kinds of permeabilized cells, the restriction of metabolite diffusion by a mitochondrial porin "closed state" has been shown to control the respiration rate . However, since in isolated mitochondria the porin appears to be always "open," the physiological relevance of a putative regulation via this channel status is now a subject of discussion . In Saccharomyces cerevisiae, in which some of the NADH dehydrogenase active sites are facing the intermembrane space, this regulatory mechanism might play an important role for the regulation of the cytosolic redox status . Using permeabilized spheroplasts from wild-type and porin-deficient mutant, we show that the NADH produced in the cytosol is channeled to the mitochondrial NADH dehydrogenases through a metabolic network involving the porin channel . Thus, the control exerted by the porin (i.e., "open" or "closed" state) seems to be determined through its participation or not in organized metabolic networks.

J Biol Chem, 2003 Jun 20, 278(25), 22828 - 37 Epub 2003 Apr 03.
A role for Saccharomyces cerevisiae Cul8 ubiquitin ligase in proper anaphase progression; Michel JJ et al.; We have undertaken a study of the yeast cullin family members Cul3 and Cul8, as little is known about their biochemical and physiological functions . We demonstrate that these cullins are associated in vivo with ubiquitin ligase activity . We show that Cul3 and Cul8 are functionally distinct from Cdc53 and do not interact with ySkp1, suggesting that they target substrates by Skp1- and possibly F-box protein-independent mechanisms . Whereas null mutants of CUL3 appear normal, yeast cells lacking CUL8 have a slower growth rate and are delayed in their progress through anaphase . The anaphase delay phenotype can be complemented by ectopic expression of Cul8 but not by any other yeast or human cullins, nor by a cul8 mutant deficient in binding to RING finger protein Roc1 . Deletion of the RAD9 gene suppressed the anaphase delay phenotype of cul8delta, suggesting that loss of Cul8 function may compromise genomic integrity . These results indicate that in addition to the anaphase promoting complex, mitotic progression may involve another E3 ubiquitin ligase mediated by Cul8 protein.

Appl Environ Microbiol, 2003 Apr, 69(4), 1999 - 2005
Expression of heteropolymeric ferritin improves iron storage in Saccharomyces cerevisiae; Kim HJ et al.; Saccharomyces cerevisiae was engineered to express different amount of heavy (H)- and light (L)-chain subunits of human ferritin by using a low-copy integrative vector (YIp) and a high-copy episomal vector (YEp) . In addition to pep4::HIS3 allele, the expression host strain was bred to have the selection markers leu2(-) and ura3(-) for YIplac128 and YEp352, respectively . The heterologous expression of phytase was used to determine the expression capability of the host strain . Expression in the new host strain (2805-a7) was as high as that in the parental strain (2805), which expresses high levels of several foreign genes . Following transformation, Northern and Western blot analyses demonstrated the expression of H- and L-chain genes . The recombinant yeast was more iron tolerant, in that transformed cells formed colonies on plates containing more than 25 mM ferric citrate, whereas none of the recipient strain cells did . Prussian blue staining indicated that the expressed isoferritins were assembled in vivo into a complex that bound iron . The expressed subunits showed a clear preference for the formation of heteropolymers over homopolymers . The molar ratio of H to L chains was estimated to be 1:6.8 . The gel-purified heteropolymer took up iron faster than the L homopolymer, and it took up more iron than the H homopolymer did . The iron concentrations in transformants expressing the heteropolymer, L homopolymer, and H homopolymer were 1,004, 760, and 500 micro g per g (dry weight) of recombinant yeast cells, respectively . The results indicate that heterologously expressed H and L subunits coassemble into a heteropolymer in vivo and that the iron-carrying capacity of yeast is further enhanced by the expression of heteropolymeric isoferritin.

Yeast, 2003 Apr 15, 20(5), 407 - 15
Screening and characterization of transposon-insertion mutants in a pseudohyphal strain of Saccharomyces cerevisiae; Suzuki C et al.; Strains of Saccharomyces cerevisiae with sigma1278b background are widely used for elucidation of pseudohyphal differentiation and signal transduction . However, information and resources on the strains are limited compared to the S288C strains . To facilitate functional analysis of strains with sigma1278b background, mutant strains were generated by using a mini-transposon3-3 x HA/LacZ (mTn3)-mutagenized library . Mutants with mTn3 insertions were screened for expression of beta-galactosidase activity under nitrogen starvation and the insertion sites were identified . One hundred and five heterozygous diploid strains were selected and subjected to tetrad analysis . Insertion of mTn3 in 11 genes was lethal in the strain, including three genes, HAC1, TPS1 and UME6, which are non-essential genes according to the Saccharomyces Genome Database . Viable haploid strains with mTn3 insertions were examined for invasive growth, which is a differentiation process in haploid strains including agar penetration on rich medium, and cell morphology during invasive growth . We also examined homozygous diploid strains with mTn3-insertions for filamentous growth and sporulation .

Yeast, 2003 Apr 15, 20(5), 397 - 406
trans-Complementation assay establishes the role of proregion hydrophobic amino acid residues in the biosynthesis of Saccharomyces cerevisiae Kex2p endoprotease; Lesage G et al.; The proregion of Saccharomyces cerevisiae endoprotease Kex2p is essential for the biosynthesis of an active enzyme . It has been suggested that the proregion acts in the endoplasmic reticulum to catalyse folding of the enzyme . To identify amino acid residues important for proregion function, we used an in vivo system in which the Kex2p proregion can act in trans to activate a Kex2p enzyme synthesized without its proregion . Activation of Kex2p by wild-type and mutated proregions revealed the essential role of hydrophobic residues F(37), V(39) and F(70) in enzyme activation . Further exploration of the role of these residues by in vitro inhibition of Kex2p activity by its proregion indicated that they are essential to form the proregion/enzyme bimolecular complex . In contrast, basic residues K(108) and R(109), located in the C-terminus of the proregion, are not involved in complex formation but are necessary for the biosynthesis of an active enzyme .

J Biol Chem, 2003 Jun 6, 278(23), 20785 - 94 Epub 2003 Apr 01.
Phosphorylation of CTP synthetase on Ser36, Ser330, Ser354, and Ser454 regulates the levels of CTP and phosphatidylcholine synthesis in Saccharomyces cerevisiae; Park TS et al.; The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinase C . We examined the hypothesis that Ser36, Ser330, Ser354, and Ser454, contained in a protein kinase C sequence motif in CTP synthetase, were target sites for the kinase . Synthetic peptides containing a phosphorylation motif at these serine residues served as substrates for protein kinase C in vitro . Ser --> Ala (S36A, S330A, S354A, and S454A) mutations in CTP synthetase were constructed by site-directed mutagenesis and expressed normally in a ura7 ura8 double mutant that lacks CTP synthetase activity . The CTP synthetase activity in extracts from cells bearing the S36A, S354A, and S454A mutant enzymes was reduced when compared with cells bearing the wild type enzyme . Kinetic analysis of purified mutant enzymes showed that the S36A and S354A mutations caused a decrease in the Vmax of the reaction . This regulation could be attributed in part by the effects phosphorylation has on the nucleotide-dependent oligomerization of CTP synthetase . In contrast, CTP synthetase activity in cells bearing the S330A mutant enzyme was elevated, and kinetic analysis of purified enzyme showed that the S330A mutation caused an elevation in the Vmax of the reaction . In vitro data indicated that phosphorylation of CTP synthetase at Ser330 affected the phosphorylation of the enzyme at another site . The phosphorylation of CTP synthetase at Ser36, Ser330, Ser354, and Ser454 residues was physiologically relevant . Cells bearing the S36A, S354A, and S454A mutations had reduced CTP levels, whereas cells with the S330A mutation had elevated CTP levels . The alterations in CTP levels correlated with the regulatory effects CTP has on the pathways responsible for the synthesis of the membrane phospholipid phosphatidylcholine.

Cell Biol Educ, 2002 Winter, 1(4), 173 - 92
Analysis of protein localization and secretory pathway function using the yeast Saccharomyces cerevisiae; Vallen E; The isolation and characterization of mutants has been crucial in understanding a number of processes in the field of cell biology . In this exercise, students examine the effects of mutations in the secretory pathway on protein localization . Yeast strains deficient for synthesis of histidinol dehydrogenase are transformed with a plasmid encoding a chimeric protein . The chimera contains a signal sequence fused to histidinol dehydrogenase . A strain with a defect in the translocation of secretory proteins into the endoplasmic reticulum (ER) accumulates sufficient histidinol dehydrogenase in the cytoplasm to grow on media lacking histidine . In contrast, yeast proficient for secretion, or yeast with secretion defects later in the pathway, are unable to grow on media lacking histidine . Student analysis of the experimental yeast transformants and appropriate controls allows investigation into the effects of conditional defects in the secretory pathway on both cell viability and protein localization . The exercise is usually performed in a manner that allows students to execute a number of techniques common in molecular biology laboratories, including plasmid minipreps, restriction digestions, and Southern blots . Student understanding and enjoyment of the exercise was assessed by laboratory reports, oral and written examinations, and questionnaires . After completion of these experiments, students can describe the utility of protein fusions, the roles of mutant analysis in cell biology, and the steps taken by proteins transiting the secretory pathway.

J Cell Sci, 2003 May 15, 116(Pt 10), 1893 - 903 Epub 2003 Mar 18.
Bro1 is an endosome-associated protein that functions in the MVB pathway in Saccharomyces cerevisiae; Odorizzi G et al.; Multivesicular bodies are late endosomal compartments containing lumenal vesicles that are formed by inward budding of the limiting endosomal membrane . In the yeast Saccharomyces cerevisiae, integral membrane proteins are sorted into the lumenal vesicles of multivesicular bodies, and this process requires the class E subset of VPS genes . We show that one of the class E VPS genes, BRO1/VPS31, encodes a cytoplasmic protein that associates with endosomal compartments . The dissociation of Bro1 from endosomes requires another class E Vps protein, Vps4, which is an ATPase that also regulates the endosomal dissociation of ESCRT-III, a complex of four class E Vps proteins (Vps2, Vps20, Vps24 and Snf7/Vps32) that oligomerize at the endosomal membrane . We also show that the endosomal association of Bro1 is specifically dependent on one of the ESCRT-III components, Snf7 . Our data suggest that the function of Bro1 in the MVB pathway takes place on endosomal membranes and occurs in concert with or downstream of the function of the ESCRT-III complex.

Biochim Biophys Acta, 2003 Apr 7, 1621(1), 22 - 30
Overexpression of GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae corrects defects in dolichol-linked saccharide formation and protein glycosylation; Janik A et al.; Thermosensitive mutants of Saccharomyces cerevisiae, affected in the endoplasmic reticulum (ER) located glycosylation, i.e . in Dol-P-Man synthase (dpm1), in beta-1,4 mannosyl transferase (alg1) and in alpha-1,3 mannosyltransferase (alg2), were used to assess the role of GDP-Man availability for the synthesis of dolichol-linked saccharides . The mutants were transformed with the yeast gene MPG1 (PSA1/VIG9) encoding GDP-Man pyrophosphorylase catalyzing the final step of GDP-Man formation . We found that overexpression of MPG1 allows growth at non-permissive temperature and leads to an increase in the cellular content of GDP-Man . In the alg1 and alg2 mutants, complemented with MPG1 gene, N-glycosylation of invertase was in part restored, to a degree comparable to that of the wild-type control . In the dpm1 mutant, the glycosylation reactions that depend on the formation of Dol-P-Man, i.e . elongation of Man(5)GlcNAc(2)-PP-Dol, O-mannosylation of chitinase and synthesis of GPI anchor were normal when MPG1 was overexpressed.Our data indicate that an increased level of GDP-Man is able to correct defects in mannosylation reactions ascribed to the ER and to the Golgi.

Biochemistry, 2003 Apr 8, 42(13), 3655 - 65
Solution structure and interdomain interactions of the Saccharomyces cerevisiae "TATA binding protein" (TBP) probed by radiolytic protein footprinting; Rashidzadeh H et al.; Although atomic-resolution crystal structures of the conserved C-terminal domain of several species of TBP and their complexes with DNA have been determined, little information is available concerning the structure in solution of full-length TBP containing both the conserved C-terminal and nonconserved N-terminal domains . Quantitation of the amino acid side chain oxidation products generated by synchrotron X-ray radiolysis by mass spectrometry has been used to determine the solvent accessibility of individual residues in monomeric Saccharomyces cerevisiae TATA binding protein (TBP) free in solution and in the TBP-DNA complex . Amino acid side chains within the C-terminal domain of unliganded full-length TBP that are predicted to be accessible from crystal structures of the isolated domain are protected from oxidation . Residues within the N-terminal domain are also protected from oxidation in both the absence and presence of DNA . Some residues within the DNA-binding "saddle" of the C-terminal domain are protected upon formation of a TBP-DNA complex as expected, while others are protected in both the absence and presence of bound DNA . In addition, residues on the upper side of the beta-sheets undergo reactivity changes as a function of DNA binding . These data suggest that the DNA-binding saddle of monomeric unliganded yeast TBP is only partially accessible to solvent, the N-terminal domain is partially structured, and the N- and C-terminal domains form a different set of contacts in the free and DNA-bound protein . The functional implications of these results are discussed.

Mol Cell Biol, 2003 Apr, 23(8), 2733 - 48
Saccharomyces cerevisiae DNA polymerase epsilon and polymerase sigma interact physically and functionally, suggesting a role for polymerase epsilon in sister chromatid cohesion; Edwards S et al.; The large subunit of Saccharomyces cerevisiae DNA polymerase epsilon, Pol2, comprises two essential functions . The N terminus has essential DNA polymerase activity . The C terminus is also essential, but its function is unknown . We report here that the C-terminal domain of Pol2 interacts with polymerase sigma (Pol sigma), a recently identified, essential nuclear nucleotidyl transferase encoded by two redundant genes, TRF4 and TRF5 . This interaction is functional, since Pol sigma stimulates the polymerase activity of the Pol epsilon holoenzyme significantly . Since Trf4 is required for sister chromatid cohesion as well as for completion of S phase and repair, the interaction suggested that Pol epsilon, like Pol sigma, might form a link between the replication apparatus and sister chromatid cohesion and/or repair machinery . We present evidence that pol2 mutants are defective in sister chromatid cohesion . In addition, Pol2 interacts with SMC1, a subunit of the cohesin complex, and with ECO1/CTF7, required for establishing sister chromatid cohesion; and pol2 mutations act synergistically with smc1 and scc1 . We also show that trf5 Delta mutants, like trf4 Delta mutants, are defective in DNA repair and sister chromatid cohesion.

J Biol Chem, 2003 Jun 6, 278(23), 20946 - 53 Epub 2003 Mar 28.
The beta subunit of the Sec61p endoplasmic reticulum translocon interacts with the exocyst complex in Saccharomyces cerevisiae; Toikkanen JH et al.; The exocyst is a conserved protein complex proposed to mediate vesicle tethering at the plasma membrane . Previously, we identified SEB1/SBH1, encoding the beta subunit of the Sec61p ER translocation complex, as a multicopy suppressor of the sec15-1 mutant, defective for one subunit of the exocyst complex . Here we show the functional and physical interaction between components of endoplasmic reticulum translocon and the exocytosis machinery . We show that overexpression of SEB1 suppresses the growth defect in all exocyst sec mutants . In addition, overexpression of SEC61 or SSS1 encoding the other two components of the Sec61p complex suppressed the growth defects of several exocyst mutants . Seb1p was coimmunoprecipitated from yeast cell lysates with Sec15p and Sec8p, components of the exocyst complex, and with Sec4p, a secretory vesicle associated Rab GTPase that binds to Sec15p and is essential for exocytosis . The interaction between Seb1p and Sec15p was abolished in sec15-1 mutant and was restored upon SEB1 overexpression . Furthermore, in wild type cells overexpression of SEB1 as well as SEC4 resulted in increased production of secreted proteins . These findings propose a novel functional and physical link between the endoplasmic reticulum translocation complex and the exocyst.

Genetics, 2003 Mar, 163(3), 875 - 94
A Saccharomyces cerevisiae genome-wide mutant screen for altered sensitivity to K1 killer toxin; Page N et al.; Using the set of Saccharomyces cerevisiae mutants individually deleted for 5718 yeast genes, we screened for altered sensitivity to the antifungal protein, K1 killer toxin, that binds to a cell wall beta-glucan receptor and subsequently forms lethal pores in the plasma membrane . Mutations in 268 genes, including 42 in genes of unknown function, had a phenotype, often mild, with 186 showing resistance and 82 hypersensitivity compared to wild type . Only 15 of these genes were previously known to cause a toxin phenotype when mutated . Mutants for 144 genes were analyzed for alkali-soluble beta-glucan levels; 63 showed alterations . Further, mutants for 118 genes with altered toxin sensitivity were screened for SDS, hygromycin B, and calcofluor white sensitivity as indicators of cell surface defects; 88 showed some additional defect . There is a markedly nonrandom functional distribution of the mutants . Many genes affect specific areas of cellular activity, including cell wall glucan and mannoprotein synthesis, secretory pathway trafficking, lipid and sterol biosynthesis, and cell surface signal transduction, and offer new insights into these processes and their integration.

Genetics, 2003 Mar, 163(3), 843 - 56
Ku-dependent and Ku-independent end-joining pathways lead to chromosomal rearrangements during double-strand break repair in Saccharomyces cerevisiae; Yu X et al.; Chromosomal double-strand breaks (DSBs) can be repaired by either homology-dependent or homology-independent pathways . Nonhomologous repair mechanisms have been relatively less well studied, despite their potential importance in generating chromosomal rearrangements . We have developed a Saccharomyces cerevisiae-based assay to identify and characterize homology-independent chromosomal rearrangements associated with repair of a unique DSB generated within an engineered URA3 gene . Approximately 1% of successfully repaired cells have accompanying chromosomal rearrangements consisting of large insertions, deletions, aberrant gene conversions, or other more complex changes . We have analyzed rearrangements in isogenic wild-type, rad52, yku80, and rad52 yku80 strains, to determine the types of events that occur in the presence or absence of these key repair proteins . Deletions were found in all strain backgrounds, but insertions were dependent upon the presence of Yku80p . A rare RAD52- and YKU80-independent form of deletion was present in all strains . These events were characterized by long one-sided deletions (up to 13 kb) and extensive imperfect overlapping sequences (7-22 bp) at the junctions . Our results demonstrate that the frequency and types of repair events depend on the specific genetic context . This approach can be applied to a number of problems associated with chromosome stability.

Proc Natl Acad Sci U S A, 2003 Apr 1, 100(7), 3889 - 94 Epub 2003 Mar 26.
Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae; Arava Y et al.; We have analyzed the translational status of each mRNA in rapidly growing Saccharomyces cerevisiae . mRNAs were separated by velocity sedimentation on a sucrose gradient, and 14 fractions across the gradient were analyzed by quantitative microarray analysis, providing a profile of ribosome association with mRNAs for thousands of genes . For most genes, the majority of mRNA molecules were associated with ribosomes and presumably engaged in translation . This systematic approach enabled us to recognize genes with unusual behavior . For 43 genes, most mRNA molecules were not associated with ribosomes, suggesting that they may be translationally controlled . For 53 genes, including GCN4, CPA1, and ICY2, three genes for which translational control is known to play a key role in regulation, most mRNA molecules were associated with a single ribosome . The number of ribosomes associated with mRNAs increased with increasing length of the putative protein-coding sequence, consistent with longer transit times for ribosomes translating longer coding sequences . The density at which ribosomes were distributed on each mRNA (i.e., the number of ribosomes per unit ORF length) was well below the maximum packing density for nearly all mRNAs, consistent with initiation as the rate-limiting step in translation . Global analysis revealed an unexpected correlation: Ribosome density decreases with increasing ORF length . Models to account for this surprising observation are discussed.

Mol Genet Genomics, 2003 Mar, 268(6), 825 - 31 Epub 2003 Feb 25.
Mutations in GCR1 affect SUC2 gene expression in Saccharomyces cerevisiae; Turkel S et al.; Transcription of SUC2, the gene that encodes the cytoplasmic and secreted forms of the enzyme invertase, is controlled by glucose repression and derepression mechanisms in Saccharomyces cerevisiae . Several regulatory factors such as the Mig1p-Tup1p-Ssn6p repressor complex and the Snf1p kinase complex have been identified previously as regulators of SUC2 expression . We show that, in addition to these factors, expression of SUC2 is affected by mutations in the gene GCR1 that encodes the glycolysis regulatory protein Gcr1p . Expression of Suc2-LacZ was not repressed by glucose in gcr1 mutant yeast cells exposed to glucose . Furthermore, secreted invertase activity was constitutively expressed under glucose-repressed and derepressed conditions in gcr1 mutants . DNA gel mobility shift assays and in-vitro DNase I protection experiments mapped a DNA binding site for Gcr1p in the transcriptional control region of the SUC2 gene, next to a previously mapped Mig1p binding site . However, the mechanism by which gcr1 mutations relieve glucose repression remains obscure.

Cell Mol Biol Lett, 2003, 8(1), 111 - 20
The dual mechanism of the antifungal effect of new lysosomotropic agents on the Saccharomyces cerevisiae RXII strain; Krasowska A et al.; Quinacrine was used to visualize the intracellular pH changes in the yeast strain Saccharomyces cerevisiae RXII occurring after exposure to four recently-synthesized lysosomotropic drugs: DM-11, PY-11, PYG-12s and DMAL-12s . The cells took up quinacrine, mostly accumulating it in their vacuoles . DM-11 and PY-11 gave rise to diffuse quinacrine fluorescence throughout the cells, with the vacuoles staining to a somewhat greater extent than the cytosol . This quinacrine-detected overall acidification of the cell interior is very probably caused by blocking of plasma membrane H(+)-ATPase . PYG-12s gave rise to a strong vacuolar accumulation of the dye . Like the vacuolar ATPase inhibitor bafilomycin A(1), DMAL-12s strongly lowered the intensity of quinacrine fluorescence . Owing to its low pK(a), it can penetrate rapidly into the cells and may inhibit vacuolar H(+)-ATPase and prevent quinacrine-detectable vacuolar acidification without causing strong cell acidification . Since these drugs were found to penetrate into the cells, their lack of effect may reflect a higher resistance of both plasma membrane H(+)-ATPase and vacuolar ATPase to the drugs . Our data indicate that the lysosomotropic drugs under study have a dual action . On entering the cell, they cause intracellular acidification, very probably by inhibiting plasma membrane H(+)-ATPase and curtailing active proton pumping from the cells . Furthermore, they interfere with the function of V-type ATPase, causing vacuolar alkalinization and eventually cell death.

Nucleic Acids Res, 2003 Apr 1, 31(7), 1969 - 73
Transcription of genes encoding trans-acting factors required for rRNA maturation/ribosomal subunit assembly is coordinately regulated with ribosomal protein genes and involves Rap1 in Saccharomyces cerevisiae; Miyoshi K et al.; We demonstrate that the genes encoding trans- acting factors essential for pre-rRNA processing/ribosomal subunit assembly are responsive to various kinds of stresses such as heat shock, nitrogen deprivation and a secretory defect, in coordination with ribosomal protein genes in Saccharomyces cerevisiae . The rap1-17 mutation, which produces the C-terminally truncated protein of a transcriptional factor Rap1p, affects transcriptional repression of the trans-acting factor genes due to a secretory defect as shown previously for both ribosomal protein and rRNA genes.

Eur J Biochem, 2003 Apr, 270(7), 1578 - 89
H2O2, but not menadione, provokes a decrease in the ATP and an increase in the inosine levels in Saccharomyces cerevisiae . An experimental and theoretical approach; Osorio H et al.; When Saccharomyces cerevisiae cells, grown in galactose, glucose or mannose, were treated with 1.5 mm hydrogen peroxide (H2O2) for 30 min, an important decrease in the ATP, and a less extensive decrease in the GTP, CTP, UTP and ADP-ribose levels was estimated . Concomitantly a net increase in the inosine levels was observed . Treatment with 83 mm menadione promoted the appearance of a compound similar to adenosine but no appreciable changes in the nucleotide content of yeast cells, grown either in glucose or galactose . Changes in the specific activities of the enzymes involved in the pathway from ATP to inosine, in yeast extracts from (un)treated cells, could not explain the effect of H2O2 on the levels of ATP and inosine . Application of a mathematical model of differential equations previously developed in this laboratory pointed to a potential inhibition of glycolysis as the main reason for that effect . This theoretical consideration was reinforced both by the lack of an appreciable effect of 1.5 mm (or even higher concentrations) H2O2 on yeast grown in the presence of ethanol or glycerol, and by the observed inhibition of the synthesis of ethanol promoted by H2O2 . Normal values for the adenylic charge, ATP and inosine levels were reached at 5, 30 and 120 min, respectively, after removal of H2O2 from the culture medium . The strong decrease in the ATP level upon H2O2 treatment is an important factor to be considered for understanding the response of yeast, and probably other cell types, to oxidative stress.

Biochemistry, 2003 Apr 1, 42(12), 3567 - 74
Regulation of c-Fes tyrosine kinase activity by coiled-coil and SH2 domains: analysis with Saccharomyces cerevisiae; Takashima Y et al.; The c-Fes protein-tyrosine kinase regulates the growth and differentiation of diverse cell types, including myeloid hematopoietic cells, vascular endothelial cells, and neurons . Structurally, Fes is composed of a unique N-terminal region with coiled-coil oligomerization motifs, followed by SH2 and kinase domains . Although Fes kinase activity is tightly regulated in cells, the structural basis for its negative regulation is not clear . In this report, c-Fes was expressed in Saccharomyces cerevisiae to determine whether regulation is kinase-intrinsic or dependent upon protein factors found in mammalian cells . Wild-type Fes kinase activity was completely repressed in yeast and did not affect cell growth . Mutation or deletion of the more N-terminal c-Fes coiled-coil domain reversed negative regulation, leading to strong kinase activation and suppression of yeast cell growth . Similarly, replacement of the wild-type SH2 domain with that of v-Src induced strong kinase activation and the growth-inhibitory phenotype . Immunoblotting with phosphospecific antibodies shows that activation of Fes by either mechanism induced autophosphorylation of the activation loop tyrosine residue (Tyr 713) . These data support the idea that Fes naturally adopts an inactive conformation in vivo, and that maintenance of the inactive structure requires the coiled-coil and SH2 domains.

Biotechnol Bioeng, 2003 Jun 5, 82(5), 506 - 16
Impact of operating variables on the expanded bed adsorption of Saccharomyces cerevisiae cells using a concanavalin A derivatized perfluorocarbon; Clemmitt RH et al.; The use of fluidizable affinity adsorbents for the adsorption of cells in expanded mode is investigated . Affinity adsorbents have been synthesized by immobilizing the lectin Concanavalin A onto the surface of triazine-activated perfluorocarbon-solids . The adsorbents were found to adsorb Saccharomyces cerevisiae cells from solution with adsorption capacities of up to 6.8 x 10(9) cells mL(-1) . Adsorption kinetics were rapid with a time constant of </=8 min . The adsorbed cells could be eluted using 500 mM methyl alpha,D-mannopyranoside, although the kinetics of release were slowed by the multipoint nature of the interaction . The dynamic capacity of the Con A PVA FEP in expanded mode was up to 4.5 x 10(9) cells mL(-1) . The operating parameters of bed height, application flow rate, and adsorbent size distribution were investigated for any potential improvements in throughput, which may improve utility for more fragile cells . A decrease in settled-bed height from 20 to 5 cm resulted in a decrease in dynamic capacity of 27% from 4.5 to 3.3 x 10(9) cells mL(-1) . An increase in application flow rate from 0.7 to 2.0 mL/min(-1) (resulting in an expansion increase from two- to fourfold) resulted in a 40% decrease in dynamic capacity from 4.0 to 2.4 x 10(9) cells mL(-1) . An increase in the mean size distribution of the perfluorocarbon from 42 to 69 microm and therefore the flow rate needed for twofold expansion of 0.7 to 1.5 mL/min(-1) resulted in a 56% decrease in dynamic capacity from 4.0 to 1.8 x 10(9) cells mL(-1) . The expanded bed, using certain combinations of the operating parameters, therefore shows significant potential for the robust, high efficiency and high capacity capture and separation of cells .

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Mar 25, 786(1-2), 187 - 95
Optimisation of expression and purification of the recombinant Yol066 (Rib2) protein from Saccharomyces cerevisiae; Urban A et al.; Yeast protein Yol066 (encoded by YOL066 ORF, also known as Rib2) possesses two distinct sequence domains: C-terminal deaminase domain and N-terminal part related to RNA:pseudouridine (psi)-synthases . The deaminase domain is implicated in the riboflavine biosynthesis, while the exact function of the RNA:Psi-synthase domain remains obscure . Here we report the optimisation of growth conditions and purification scheme for recombinant His(6)-tagged Yol066 expressed in E . coli BL21(DE3) using pET28 plasmid . Production of soluble Yol066 protein is best at low temperature (18 degrees C) and IPTG concentration (50 micro M) and Yol066 purification was achieved using metal-affinity and ion-exchange chromatography . This optimised protocol yields about 10 mg of highly purified recombinant Yol066 from 3 l of E . coli culture.

FEBS Lett, 2003 Mar 27, 539(1-3), 68 - 72
Cu,Zn-superoxide dismutase of Saccharomyces cerevisiae is required for resistance to hyperosmosis; Garay-Arroyo A et al.; Here we analyzed the role of the antioxidant response in Saccharomyces cerevisiae adaptation to hyperosmotic stress . We show that Cu,Zn-superoxide dismutase (SOD1) plays a fundamental role in this adaptation process since under hyperosmosis SOD1 mutants lead to high protein oxidation levels and show a sensitive phenotype, which is reversed by the addition of N-acetylcysteine to the medium . Pretreatment with MnCl(2), a superoxide scavenger, improves the survival of the sod1 strain upon hyperosmosis . Additionally, we show that upon hyperosmotic shock there is a small and transient increase in SOD1 transcript levels, regulated by the protein kinase A-cAMP and SKN7 pathways.

RNA, 2003 Apr, 9(4), 419 - 31
Antagonistic signals within the COX2 mRNA coding sequence control its translation in Saccharomyces cerevisiae mitochondria; Williams EH et al.; Translation of the mitochondrially coded COX2 mRNA within the organelle in yeast produces the precursor of Cox2p (pre-Cox2p), which is processed and assembled into cytochrome c oxidase . The mRNA sequence of the first 14 COX2 codons, specifying the pre-Cox2p leader peptide, was previously shown to contain a positively acting element required for translation of a mitochondrial reporter gene, ARG8(m), fused to the 91st codon of COX2 . Here we show that three relatively short sequences within the COX2 mRNA coding sequence, or structures they form in vivo, inhibit translation of the reporter in the absence of the positive element . One negative element was localized within codons 15 to 25 and shown to function at the level of the mRNA sequence, whereas two others are within predicted stem-loop structures formed by codons 22-44 and by codons 46-74 . All three of these inhibitory elements are antagonized in a sequence-specific manner by reintroduction of the upstream positive-acting sequence . These interactions appear to be independent of 5'- and 3'-untranslated leader sequences, as they are also observed when the same reporter constructs are expressed from the COX3 locus . Overexpression of MRS2, which encodes a mitochondrial magnesium carrier, partially suppresses translational inhibition by each isolated negatively acting element, but does not suppress them in combination . We hypothesize that interplay among these signals during translation in vivo may ensure proper timing of pre-Cox2p synthesis and assembly into cytochrome c oxidase.

J Biol Chem, 2003 May 30, 278(22), 19751 - 6 Epub 2003 Mar 18.
ATP22, a nuclear gene required for expression of the F0 sector of mitochondrial ATPase in Saccharomyces cerevisiae; Helfenbein KG et al.; Expression of the mitochondrial proton-translocating ATPase of Saccharomyces cerevisiae has been shown to depend on chaperones that target the F1 and F0 sectors of this inner membrane complex . Here we report a new gene, designated ATP22 (reading frame YDR350C on chromosome IV), that provides an essential function in the assembly of F0 . ATP22 was cloned by transformation of C208/L2, a strain previously assigned to complementation group G99 of a collection of respiration-defective nuclear pet mutants . C208/L2 and the other atp22 mutants have oligomycin-insensitive F1-ATPase, suggesting that the lesion is confined to F0 . This is supported by the sedimentation properties of the mutant ATPase and results of immunochemical analysis of F0 subunit polypeptides . Northern analysis of ATPase transcripts and in vivo pulse labeling of the mitochondrial translation products in the mutant indicate normal expression of subunits 6, 8, and 9, the three mitochondrial gene products of F0 . Atp22p therefore functions at a post-translational stage in assembly of F0 . Localization studies indicate Atp22p to be a component of the mitochondrial inner membrane . Protease protection experiments further indicate that Atp22p faces the matrix side of the membrane where most of the ATPase proteins are located and assembled.

J Biol Chem, 2003 May 30, 278(22), 20345 - 57 Epub 2003 Mar 18.
Genome-wide analysis of the response to cell wall mutations in the yeast Saccharomyces cerevisiae; Lagorce A et al.; Perturbations of the yeast cell wall trigger a repair mechanism that reconfigures its molecular structure to preserve cell integrity . To investigate this mechanism, we compared the global gene expression in five mutant strains, each bearing a mutation (i.e . fks1, kre6, mnn9, gas1, and knr4 mutants) that affects in a different manner the cell wall construction . Altogether, 300 responsive genes were kept based on high stringency criteria during data processing . Functional classification of these differentially expressed genes showed a substantial subset of induced genes involved in cell wall construction and an enrichment of metabolic, energy generation, and cell defense categories, whereas families of genes belonging to transcription, protein synthesis, and cellular growth were underrepresented . Clustering methods isolated a single group of approximately 80 up-regulated genes that could be considered as the stereotypical transcriptional response of the cell wall compensatory mechanism . The in silico analysis of the DNA upstream region of these co-regulated genes revealed pairwise combinations of DNA-binding sites for transcriptional factors implicated in stress and heat shock responses (Msn2/4p and Hsf1p) with Rlm1p and Swi4p, two PKC1-regulated transcription factors involved in the activation genes related to cell wall biogenesis and G1/S transition . Moreover, this computational analysis also uncovered the 6-bp 5'-AGCCTC-3' CDRE (calcineurin-dependent response element) motif in 40% of the co-regulated genes . This motif was recently shown to be the DNA binding site for Crz1p, the major effector of calcineurin-regulated gene expression in yeast . Taken altogether, the data presented here lead to the conclusion that the cell wall compensatory mechanism, as triggered by cell wall mutations, integrates three major regulatory systems: namely the PKC1-SLT2 mitogen-activated protein kinase-signaling module, the "global stress" response mediated by Msn2/4p, and the Ca2+/calcineurin-dependent pathway . The relative importance of these regulatory systems in the cell wall compensatory mechanism is discussed.

J Biol Chem, 2003 Mar 14, 278(11), 9345 - 52
Replication-independent MCB gene induction and deoxyribonucleotide accumulation at G1/S in Saccharomyces cerevisiae; Koc A et al.; In Saccharomyces cerevisiae, many genes encoding enzymes involved in deoxyribonucleotide synthesis are expressed preferentially near the G1/S boundary of the cell cycle . The relationship between the induction of deoxyribonucleotide-synthesizing genes, deoxyribonucleoside triphosphate levels, and replication initiation was investigated using factor-synchronized wild-type yeast or dbf4 yeast that are temperature-sensitive for replication initiation . Neither the timing nor extent of gene induction was inhibited when factor-arrested dbf4 cells were released into medium containing the ribonucleotide reductase inhibitor hydroxyurea, which blocks replication fork progression, or were released at 37 degrees C, which blocks replication origin firing . Thus, the induction of deoxyribonucleotide-synthesizing genes at G1/S was fully independent of DNA chain elongation or initiation . Deoxyribonucleoside triphosphate levels increased severalfold at G1/S in wild-type cells and in dbf4 mutants incubated at the non-permissive temperature . Thus, deoxyribonucleoside triphosphate accumulation, like the induction of deoxyribonucleotide-synthesizing genes, was not dependent on replication initiation . Deoxyribonucleoside triphosphate accumulation at G1/S was suppressed in cells lacking Swi6, a transcription factor required for normal cell cycle regulation of deoxyribonucleotide-synthesizing genes . The results suggest that cells use gene induction at G1/S as a mechanism to pre-emptively, rather than reflexively, increase the synthesis of DNA precursors to meet the demand of the replication forks for deoxyribonucleotides.

J Cell Biol, 2003 Mar 17, 160(6), 833 - 43
Differential kinetochore protein requirements for establishment versus propagation of centromere activity in Saccharomyces cerevisiae; Mythreye K et al.; Dicentric chromosomes undergo a breakage-fusion-bridge cycle as a consequence of having two centromeres on the same chromatid attach to opposite spindle poles in mitosis . Suppression of dicentric chromosome breakage reflects loss of kinetochore function at the kinetochore-microtubule or the kinetochore-DNA interface . Using a conditionally functional dicentric chromosome in vivo, we demonstrate that kinetochore mutants exhibit quantitative differences in their degree of chromosome breakage . Mutations in chl4/mcm17/ctf17 segregate dicentric chromosomes through successive cell divisions without breakage, indicating that only one of the two centromeres is functional . Centromere DNA introduced into the cell is unable to promote kinetochore assembly in the absence of CHL4 . In contrast, established centromeres retain their segregation capacity for greater than 25 generations after depletion of Chl4p . The persistent mitotic stability of established centromeres reveals the presence of an epigenetic component in kinetochore segregation . Furthermore, this study identifies Chl4p in the initiation and specification of a heritable chromatin state.

Mol Biol Cell, 2003 Mar, 14(3), 987 - 1001
Short telomeres induce a DNA damage response in Saccharomyces cerevisiae; IJpma AS et al.; Telomerase-deficient Saccharomyces cerevisiae cells show a progressive decrease in telomere length . When grown for several days in log phase, the tlc1Delta cells initially display wild-type growth kinetics with subsequent loss of growth potential after which survivors are generated via RAD52-dependent homologous recombination . We found that chromosome loss in these telomerase-deficient cells only increased after a significant decline in growth potential of the culture . At earlier stages of growth, as the telomerase-deficient cells began to show loss of growth potential, the cells arrested in G2/M and showed RNR3 induction and Rad53p phosphorylation . These responses were dependent on RAD24 and MEC1, suggesting that short telomeres are recognized as DNA damage and signal G2/M arrest.

Yeast, 2003 Mar, 20(4), 351 - 67
Cyclic AMP mediates the cell cycle dynamics of energy metabolism in Saccharomyces cerevisiae; Muller D et al.; We have investigated the role of 3',5'-cyclic-adenosine-monophosphate (cAMP) in mediating the coupling between energy metabolism and cell cycle progression in both synchronous cultures and oscillating continuous cultures of Saccharomyces cerevisiae . For the first time, a peak in intracellular cAMP was shown to precede the observed breakdown of trehalose and glycogen during cell cycle-related oscillations . Measurements in synchronous cultures demonstrated that this peak can be associated with the cell cycle dynamics of cAMP under conditions of glucose-limited growth, which was found to differ significantly from that observed in synchronous glucose-repressed cultures . Our results support the notion that cAMP plays a major role in mediating the integration of energy metabolism and cell cycle progression, both in the single cell and during cell cycle-related oscillations in continuous culture, respectively . Evidence is presented that the dynamic behaviour of intracellular cAMP during the cell cycle is modulated depending on nutrient supply . The implications of these findings regarding the role of cAMP in regulating cell cycle progression and energy metabolism are discussed .

Yeast, 2003 Mar, 20(4), 315 - 30
Four ARF GAPs in Saccharomyces cerevisiae have both overlapping and distinct functions; Zhang CJ et al.; Previous studies in yeast have revealed the presence of four proteins with a conserved, cysteine-rich, ARF GAP domain that share the ability to suppress the conditional growth defect of the arf1-3 mutant . Three of these proteins have been shown previously to be ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs) . We now demonstrate that the fourth also exhibits in vitro ARF GAP activity and correlates the suppressor and ARF GAP activities for all four . Because the four ARF GAP proteins are quite diverse outside the ARF GAP domain, a genetic analysis was undertaken to define the level of functional cross-talk between them . A large number of synthetic defects were observed that point to a high degree of functional overlap among the four ARF GAPs . However, several differences were also noted in the ability of each gene to suppress the synthetic defects of others and in the impact of single or combined deletions on assays of membrane traffic . We interpret these results as supportive evidence for roles of ARF GAPs in a number of distinct, essential cellular processes that include cell growth, protein secretion, endocytosis and cell cycling . The description of the specificities of the ARF GAPs for the different responses is viewed as a necessary first step in dissecting biologically relevant pathways through a functionally overlapping family of signalling proteins .

Proc Natl Acad Sci U S A, 2003 Mar 18, 100(6), 3095 - 100 Epub 2003 Mar 07.
A Saccharomyces cerevisiae mutant strain defective in acetyl-CoA carboxylase arrests at the G2/M phase of the cell cycle; Al-Feel W et al.; To elucidate the essential functions of acetyl-CoA carboxylase (ACC1FAS3) in Saccharomyces cerevisiae, a temperature-sensitive mutant (acc1(ts)) was constructed . When the acc1(ts) cells were synchronized in G(1) phase with alpha-factor at the permissive temperature of 24 degrees C and then released from the blockade and incubated at the restrictive temperature of 37 degrees C, 95% of the cell population became arrested at the G(2)M phase of the cell cycle despite the presence of fatty acids (C(14)-C(26)) in the medium . These cells developed large undivided nuclei, and the spindles of the arrested mutant cells were short . Shifting the G(2) arrested cells back to the permissive temperature resulted in a reversal of the cell-cycle arrest, with cells initiating mitosis . However, after 3 h of incubation at 37 degrees C, G(2) arrested mutant cells lost viability and displayed a uniquely altered nuclear envelope . Using {1-(14)C}acetate as a precursor for fatty acids synthesis, we identified the phospholipids and sphingolipids derived from acc1(ts) cells and wild-type cells at 24 degrees C and 37 degrees C, respectively . The levels of inositol-ceramides {IPC, MIPC, and M(IP)(2)C} and very long-chain fatty acids C(24) and C(26) declined sharply in the G(2)M arrested cells because of ACC inactivation . Shifting the acc1(ts) cells to 24 degrees C after 2 h of incubation at 37 degrees C resulted in reactivation of the ACC and elevation of the ceramides and very long-chain fatty acid syntheses with normal cell-cycle progression . In contrast, synthesis of wild-type inositol-ceramides, C(24) and C(26), fatty acids were elevated on incubation at 37 degrees C and declined when the cells shifted to the permissive temperature of 24 degrees C.

Nucleic Acids Res, 2003 Mar 15, 31(6), 1715 - 24
The ATM-related Tel1 protein of Saccharomyces cerevisiae controls a checkpoint response following phleomycin treatment; Nakada D et al.; MEC1 and TEL1 encode ATR- and ATM-related proteins in the budding yeast Saccharomyces cerevisiae, respectively . Phleomycin is an agent that catalyzes double-strand breaks in DNA . We show here that both Mec1 and Tel1 regulate the checkpoint response following phleomycin treatment . MEC1 is required for Rad53 phosphorylation and cell-cycle progression delay following phleomycin treatment in G1, S or G2/M phases . The tel1Delta mutation confers a defect in the checkpoint responses to phleomycin treatment in S phase . In addition, the tel1Delta mutation enhances the mec1 defect in activation of the phleomycin-induced checkpoint pathway in S phase . In contrast, the tel1Delta mutation confers only a minor defect in the checkpoint responses in G1 phase and no apparent defect in G2/M phase . Methyl methanesulfonate (MMS) treatment also activates checkpoints, inducing Rad53 phosphorylation in S phase . MMS-induced Rad53 phosphorylation is not detected in mec1Delta mutants during S phase, but occurs in tel1Delta mutants similar to wild-type cells . Finally, Xrs2 is phosphorylated after phleomycin treatment in a TEL1-dependent manner during S phase, whereas no significant Xrs2 phosphorylation is detected after MMS treatment . Together, our results support a model in which Tel1 contributes to checkpoint control in response to phleomycin-induced DNA damage in S phase.

Genetika, 2003 Jan, 39(1), 18 - 24
{Genetic analysis of spontaneous suppressors of the pho85 mutation in the yeast Saccharomyces cerevisiae}; Sambuk EV et al.; Chaperones are known to play an important role in complexation of cyclin-dependent kinases with cyclins . In yeast cells growing in the presence of phosphate, cyclin-dependent kinase Pho85p and cyclin Pho80p form a complex and phosphorylate activator Pho4p . As a result, Pho4p is exported from the nucleus, and the PHO5 gene is not transcribed . The mutations suppressing the pho85 mutation were analyzed in order to identify genes which code for chaperones involved in the formation of the Pho80p-Pho85p complex in the presence of environmental phosphate . Dominant mutations DSP1, DSP2, and DSP4-6 were found . It is shown that the DSP1 gene is 2.1 cM away from the PHO85 gene on chromosome XVI and probably coincides with the EGD1 gene coding for a chaperone.

Cryobiology, 2003 Feb, 46(1), 33 - 42
Influence of cooling rate on Saccharomyces cerevisiae destruction during freezing: unexpected viability at ultra-rapid cooling rates; Dumont F et al.; The purpose of this work was to study cell viability as a function of cooling rate during freezing . Cooling rate strongly influences the viability of cells during cold thermal stress . One of the particularities of this study was to investigate a large range of cooling rates and particularly very rapid cooling rates (i.e., faster than 20000 degrees C min (-1)) . Four distinct ranges of cooling rates were identified . The first range (A(')) corresponds to very slow cooling rates (less than 5 degrees C min (-1)), and results in high cell mortality . The second range (A) corresponds to low cooling rates (5-100 degrees C min (-1)), at which cell water outflow occurs slowly and does not damage the cells . The third range (B) corresponds to rapid cooling rates (100-2000 degrees C min (-1)), at which there is competition between heat flow and water flow . In this case, massive water outflow, which is related to the increase in extracellular osmotic pressure and the membrane-lipid phase transition, can cause cell death . The fourth range (C) corresponds to very high cooling rates (more than 5000 degrees C min (-1)), at which the heat flow is very rapid and partially prevents water exit, which seems to preserve cell viability.

Genes Cells, 2003 Mar, 8(3), 235 - 50
The upstream regulator, Rsr1p, and downstream effectors, Gic1p and Gic2p, of the Cdc42p small GTPase coordinately regulate initiation of budding in Saccharomyces cerevisiae; Kawasaki R et al.; BACKGROUND: Cdc42p, a Rho family small GTPase, is essential for budding initiation in the yeast Saccharomyces cerevisiae . The homologous proteins Gic1p and Gic2p (Gic1/2p) are effectors of Cdc42p, but their precise functions remain unknown . Rsr1p/Bud1p is a Ras family small GTPase that controls the selection of the budding site . Previous observations suggested that Rsr1p-GTP recruits Cdc24p, a GDP/GTP exchange factor for Cdc42p, at the incipient bud site . However, this model only addresses how Rsr1p determines the budding site, because the rsr1 mutant normally initiates budding . RESULTS: Here we show that a rsr1 gic1 gic2 mutant fails to initiate budding, resulting in unbudded, large, and multinucleated cells . Expression of a dominant active or dominant negative mutant of RSR1 also inhibited the growth of the gic1 gic2 mutant, suggesting that cycling of Rsr1p between the GTP- and GDP-bound forms is required for budding initiation in the gic1 gic2 mutant . Among the mutations in effectors of CDC42, only the gic1 gic2 mutation demonstrated a synthetic lethal interaction with rsr1 . Increased gene dosage of CDC42 suppressed defects in budding initiation of rsr1 gic1 gic2 mutants containing additional mutations in other effectors of CDC42, including BNI1, CLA4 or STE20 . The polarized localization of Bni1p-GFP (green fluorescent protein) and Cla4p-GFP was lost after depletion of Gic1p in the rsr1 gic2 mutant . CONCLUSION: We propose that Gic1/2p may stabilize or maintain a complex consisting of Cdc42p-GTP and its effectors at the budding site, which are assembled by the action of the Rsr1p-Cdc24p system.

Appl Environ Microbiol, 2003 Mar, 69(3), 1623 - 8
Improved anaerobic use of arginine by Saccharomyces cerevisiae; Martin O et al.; Anaerobic arginine catabolism in Saccharomyces cerevisiae was genetically modified to allow assimilation of all four rather than just three of the nitrogen atoms in arginine . This was accomplished by bypassing normal formation of proline, an unusable nitrogen source in the absence of oxygen, and causing formation of glutamate instead . A pro3 ure2 strain expressing a PGK1 promoter-driven PUT2 allele encoding Delta(1)-pyrroline-5-carboxylate dehydrogenase lacking a mitochondrial targeting sequence produced significant cytoplasmic activity, accumulated twice as much intracellular glutamate, and produced twice as much cell mass as the parent when grown anaerobically on limiting arginine as sole nitrogen source.

FEMS Microbiol Lett, 2003 Feb 28, 219(2), 187 - 94
Utilization of glutathione as an exogenous sulfur source is independent of gamma-glutamyl transpeptidase in the yeast Saccharomyces cerevisiae: evidence for an alternative gluathione degradation pathway; Kumar C et al.; gamma-Glutamyl transpeptidase (gamma-GT) is the only enzyme known to be responsible for glutathione degradation in living cells . In the present study we provide evidence that the utilization of glutathione can occur in the absence of gamma-GT . When disruptions in the CIS2 gene encoding gamma-GT were created in met15Delta strains, which require organic sulfur sources for growth, the cells were able to grow well with glutathione as the sole sulfur source suggesting that a gamma-GT-independent pathway for glutathione degradation exists in yeast cells . The CIS2 gene was strongly repressed by ammonium and derepressed in glutamate medium, and was found to be regulated by the nitrogen regulatory circuit . The utilization of glutathione as a sulfur source was, however, independent of the nitrogen source in the medium, further underlining that the two degradatory pathways were distinct.

Biosci Biotechnol Biochem, 2003 Jan, 67(1), 186 - 91
Putative homologs of SSK22 MAPKK kinase and PBS2 MAPK kinase of Saccharomyces cerevisiae encoded by os-4 and os-5 genes for osmotic sensitivity and fungicide resistance in Neurospora crassa; Fujimura M et al.; We cloned and characterized Neurospora NcSSK22 and NcPBS2 genes, similar to yeast SSK22 mitogen-activated protein (MAP) kinase kinase kinase and the PBS2 MAP kinase kinase genes, respectively . Disruptants of the NcSSK22 gene were sensitive to osmotic stress and resistant to iprodione and fludioxonil . Their phenotypes were similar to those of osmotic-sensitive (os) mutants os-1, os-2, os-4, and os-5 . The os-4 mutant strain transformed with the wild-type NcSSK22 gene grew on a medium containing 4% NaCl and was sensitive to iprodione and fludioxonil . In contrast, the NcPBS2 gene complemented the osmotic sensitivity and fungicide resistance of the os-5 mutant strain . We sequenced the NcPBS2 gene of the os-5 mutant strain (NM216o) and found five nucleotides deleted within the kinase domain . This result suggests that the gene products of os-4 and os-5 are components of the MAP kinase cascade, which is probably regulated upstream by two-component histidine kinase encoded by the os-1/nik1 gene.

Genetics, 2003 Feb, 163(2), 527 - 38
Targeted nucleotide repair of cyc1 mutations in Saccharomyces cerevisiae directed by modified single-stranded DNA oligonucleotides; Brachman EE et al.; Modified single-stranded DNA oligonucleotides have been used to direct base changes in the CYC1 gene of Saccharomyces cerevisiae . In this process, the oligonucleotide is believed to hybridize to the target site through the action of a DNA recombinase and, once bound, DNA repair enzymes act to excise the nucleotide, replace it, and revert the gene to wild-type status . Nucleotide exchange exhibits a strand bias as, in most cases, a higher level of base reversal appears in cells in which the oligonucleotide is designed to hybridize to the nontemplate strand . But, in one case, a higher level was observed when an oligonucleotide complementary to the transcribed strand was used . Mutant haploid and diploid strains are reverted to wild type at this locus with approximately the same frequency and all strains take up the oligonucleotide with approximately equal efficiency . Some repair preference for certain base mismatches was observed; for example, T/T and C/C mispairs exhibited the highest degree of reactivity . Finally, we demonstrate that proteins involved in DNA pairing can enhance the repair activity up to 22-fold, while others affect the reaction minimally . Taken together, these results confirm the importance and versatility of yeast as a model system to elucidate the factors regulating the frequency of nucleotide exchange directed by oligonucleotides.

Genetics, 2003 Feb, 163(2), 515 - 26
MLH1 mutations differentially affect meiotic functions in Saccharomyces cerevisiae; Hoffmann ER et al.; To test whether missense mutations in the cancer susceptibility gene MLH1 adversely affect meiosis, we examined 14 yeast MLH1 mutations for effects on meiotic DNA transactions and gamete viability in the yeast Saccharomyces cerevisiae . Mutations analogous to those associated with hereditary nonpolyposis colorectal cancer (HNPCC) or those that reduce Mlh1p interactions with ATP or DNA all impair replicative mismatch repair as measured by increased mutation rates . However, their effects on meiotic heteroduplex repair, crossing over, chromosome segregation, and gametogenesis vary from complete loss of meiotic functions to no meiotic defect, and mutants defective in one meiotic process are not necessarily defective in others . DNA binding and ATP binding but not ATP hydrolysis are required for meiotic crossing over . The results reveal clear separation of different Mlh1p functions in mitosis and meiosis, and they suggest that some, but not all, MLH1 mutations may be a source of human infertility.

Genetics, 2003 Feb, 163(2), 507 - 14
Std1p (Msn3p) positively regulates the Snf1 kinase in Saccharomyces cerevisiae; Kuchin S et al.; The Snf1 protein kinase of the glucose signaling pathway in Saccharomyces cerevisiae is regulated by an autoinhibitory interaction between the regulatory and catalytic domains of Snf1p . Transitions between the autoinhibited and active states are controlled by an upstream kinase and the Reg1p-Glc7p protein phosphatase 1 . Previous studies suggested that Snf1 kinase activity is also modulated by Std1p (Msn3p), which interacts physically with Snf1p and also interacts with glucose sensors . Here we address the relationship between Std1p and the Snf1 kinase . Two-hybrid assays showed that Std1p interacts with the catalytic domain of Snf1p, and analysis of mutant kinases suggested that this interaction is incompatible with the autoinhibitory interaction of the regulatory and catalytic domains . Overexpression of Std1p increased the two-hybrid interaction of Snf1p with its activating subunit Snf4p, which is diagnostic of an open, uninhibited conformation of the kinase complex . Overexpression of Std1p elevated Snf1 kinase activity in both in vitro and in vivo assays . These findings suggest that Std1p stimulates the Snf1 kinase by an interaction with the catalytic domain that antagonizes autoinhibition and promotes an active conformation of the kinase.

Genetics, 2003 Feb, 163(2), 495 - 506
Saccharomyces cerevisiae Hsp70 mutations affect {PSI+} prion propagation and cell growth differently and implicate Hsp40 and tetratricopeptide repeat cochaperones in impairment of {PSI+}; Jones GW et al.; We previously described an Hsp70 mutant (Ssa1-21p), altered in a conserved residue (L483W), that dominantly impairs yeast {PSI(+)} prion propagation without affecting growth . We generated new SSA1 mutations that impaired {PSI(+)} propagation and second-site mutations in SSA1-21 that restored normal propagation . Effects of mutations on growth did not correlate with {PSI(+)} phenotype, revealing differences in Hsp70 function required for growth and {PSI(+)} propagation and suggesting that Hsp70 interacts differently with {PSI(+)} prion aggregates than with other cellular substrates . Complementary suppression of altered activity between forward and suppressing mutations suggests that mutations that impair {PSI(+)} affect a similar Hsp70 function and that suppressing mutations similarly overcome this effect . All new mutations that impaired {PSI(+)} propagation were located in the ATPase domain . Locations and homology of several suppressing substitutions suggest that they weaken Hsp70's substrate-trapping conformation, implying that impairment of {PSI(+)} by forward mutations is due to altered ability of the ATPase domain to regulate substrate binding . Other suppressing mutations are in residues important for interactions with Hsp40 or TPR-containing cochaperones, suggesting that such interactions are necessary for the impairment of {PSI(+)} propagation caused by mutant Ssa1p.

Curr Genet, 2003 Mar, 42(6), 313 - 21 Epub 2003 Feb 01.
Ionizing irradiation effects on S-phase in checkpoint mutants of the yeast Saccharomyces cerevisiae; King WR et al.; In mammalian cells, gamma-irradiation activates checkpoint controls to delay entry into, or passage through S-phase, while chronic exposure to methyl methanesulfonate or hydroxyurea causes a similar delay in yeast . In yeast, at least five genes are involved: RAD9, RAD17, RAD24, RAD53 and MEC1, a homologue of ATM . Here, using flow cytometry analysis and alkaline sucrose gradient centrifugation of labeled, newly made DNA, we demonstrate, in synchronized RAD wild-type Saccharomyces cerevisiae cells, that: (1) gamma-irradiation at START delays entry into S-phase, (2) irradiation shortly before or during early S-phase delays completion of S-phase and (3) the latter response is largely a consequence of replicon initiation inhibition . The delay produced by irradiation during early S-phase depends on the function of the checkpoint genes RAD9, RAD17, RAD24, RAD53, MEC1 and MEC3 . However, at least four, RAD17, RAD53, MEC1, MEC3, are not needed to delay S-phase progression when cells are irradiated shortly before S-phase begins.

Mol Cell Biol, 2003 Mar, 23(6), 2083 - 95
Late cytoplasmic maturation of the small ribosomal subunit requires RIO proteins in Saccharomyces cerevisiae; Vanrobays E et al.; Numerous nonribosomal trans-acting factors involved in pre-rRNA processing have been characterized, but few of them are specifically required for the last cytoplasmic steps of 18S rRNA maturation . We have recently demonstrated that Rrp10p/Rio1p is such a factor . By BLAST analysis, we identified the product of a previously uncharacterized essential gene, YNL207W/RIO2, called Rio2p, that shares 43% sequence similarity with Rrp10p/Rio1p . Rio2p homologues were identified throughout the Archaea and metazoan species . We show that Rio2p is a cytoplasmic-nuclear protein and that its depletion blocks 18S rRNA production, leading to 20S pre-rRNA accumulation . In situ hybridization reveals that in Rio2p-depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect . We also show that both Rio1p and Rio2p accumulate in the nucleus of crm1-1 cells at the nonpermissive temperature . Nuclear as well as cytoplasmic Rio2p and Rio1p cosediment with pre-40S particles . These results strongly suggest that Rio2p and Rrp10p/Rio1p are shuttling proteins which associate with pre-40S particles in the nucleus and they are not necessary for export of the pre-40S complexes but are absolutely required for the cytoplasmic maturation of 20S pre-rRNA at site D, leading to mature 40S ribosomal subunits.

J Biol Chem, 2003 May 9, 278(19), 16698 - 705 Epub 2003 Feb 27.
Mnd2 and Swm1 are core subunits of the Saccharomyces cerevisiae anaphase-promoting complex; Hall MC et al.; The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that regulates the metaphase-anaphase transition and exit from mitosis in eukaryotic cells . Eleven subunits have been previously identified in APC from budding yeast . We have identified two additional subunits, Mnd2 and Swm1, by mass spectrometry . Both Mnd2 and Swm1 were found specifically associated with a highly purified preparation of APC from haploid yeast whole cell extract . Moreover, the APC co-purified with epitope-tagged Mnd2 and Swm1 . Both proteins were present in APC preparations from haploid cells arrested in G(1), S, and M phases and from meiotic diploid cells, indicating that they are constitutive components of the complex throughout the yeast cell cycle . Mnd2 interacted strongly with Cdc23, Apc5, and Apc1 when coexpressed in an in vitro transcription/translation reaction . Swm1 also interacted with Cdc23 and Apc5 in this system . Previous studies described meiotic defects for mutations in MND2 and SWM1 . Here, we show that mnd2delta and swm1delta haploid strains exhibit slow growth and accumulation of G(2)/M cells comparable with that seen in apc9delta or apc10Delta strains and consistent with an APC defect . Taken together, these results demonstrate that Swm1 and Mnd2 are functional components of the yeast APC.

DNA Repair (Amst), 2003 Apr 2, 2(4), 375 - 86
Transcription, nucleosome positioning and protein binding modulate nucleotide excision repair of the Saccharomyces cerevisiae MET17 promoter; Powell NG et al.; We have assessed how transcription, chromatin structure and protein binding modulate nucleotide excision repair in the upstream regulatory region and early coding region of the endogenous Saccharomyces cerevisiae gene MET17 . Removal of UV-induced cyclobutane pyrimidine dimers was measured from these regions, in which transcription and chromatin structure could be regulated independently of each other . Distinct repair trends were apparent depending on transcriptional state . When transcription was repressed nucleosome positioning and protein binding as determined by chromatin immunoprecipitation and quantitative real-time PCR, were significant factors . Nucleosome positioning and/or protein binding effects were most apparent on the strand that becomes transcribed, with repair occurring fastest in a nucleosome free region but being retarded where regulatory proteins bound within this region . When transcription was derepressed the rate of repair increased on both strands in a region beginning 200 bp upstream of the TATA box and extending downstream into the coding region . This effect overrode the influences of nucleosome positioning and protein binding.

Biotechnol Appl Biochem, 2003 Aug, 38(Pt 1), 43 - 51
A strategic study using mutant-strain entrapment in calcium alginate for the production of Saccharomyces cerevisiae cells with high invertase activity; Rossi-Alva JC et al.; Entrapped cells and entrapped cells grown inside of a calcium alginate matrix as well as free cells have been investigated using Saccharomyces cerevisiae mutant strains with regard to their pattern of growth and invertase activity . The repression of invertase by glucose and glucose-consumption ability were considered in the selection process of the mutants . Efficient sucrose bioconversion due to high invertase activity was obtained when entrapped mutant strain Q6R2 cells were grown within calcium alginate gel beads using sucrose plus glucose as the carbon source . Under these conditions, 1 mg (dry weight) of entrapped cells is able to produce 20 micromol of inverted sugar in 3 min (the maximum activity obtained was 20 units x mg(-1)) . The experiments were carried out for 6 months without appreciable loss of either bead integrity or invertase activity . The biocatalyst was also stored at 4 degrees C for 6 months without appreciable loss of the invertase activity . This work shows that entrapped yeast cells with a weak ability to consume sugar may be used to produce inverted sugar.

Mol Microbiol, 2003 Mar, 47(5), 1329 - 39
F1-catalysed ATP hydrolysis is required for mitochondrial biogenesis in Saccharomyces cerevisiae growing under conditions where it cannot respire; Lefebvre-Legendre L et al.; Mutant strains of yeast Saccharomyces cerevisiae lacking a functional F1-ATPase were found to grow very poorly under anaerobic conditions . A single amino acid replacement (K222 > E222) that locally disrupts the adenine nucleotide catalytic site in the beta-F1 subunit was sufficient to compromise anaerobic growth . This mutation also affected growth in aerated conditions when ethidium bromide (an intercalating agent impairing mtDNA propagation) or antimycin (an inhibitor of respiration) was included in the medium . F1-deficient cells forced to grow in oxygen-limited conditions were shown to lose their mtDNA completely and to accumulate Hsp60p mainly under its precursor form . Fluorescence microscopy analyses with a modified GFP containing a mitochondrial targeting presequence revealed that aerobically growing F1-deficient cells stopped importing the GFP when antimycin was added to the medium . Finally, after total inactivation of the catalytic alpha3beta3 subcomplex of F1, mitochondria could no longer be energized by externally added ATP because of either a block in assembly or local disruption of the adenine nucleotide processing site . Altogether these data strengthen the notion that in the absence of respiration, and whether the proton translocating domain (F0) of complex V is present or not, F1-catalysed hydrolysis of ATP is essential for the occurrence of vital cellular processes depending on the maintenance of an electrochemical potential across the mitochondrial inner membrane.

Eur J Biochem, 2003 Mar, 270(5), 1014 - 24
The N-acetylglutamate synthase/N-acetylglutamate kinase metabolon of Saccharomyces cerevisiae allows co-ordinated feedback regulation of the first two steps in arginine biosynthesis; Pauwels K et al.; In Saccharomyces cerevisiae, which uses the nonlinear pathway of arginine biosynthesis, the first two enzymes, N-acetylglutamate synthase (NAGS) and N-acetylglutamate kinase (NAGK), are controlled by feedback inhibition . We have previously shown that NAGS and NAGK associate in a complex, essential to synthase activity and protein level {Abadjieva, A., Pauwels, K., Hilven, P . & Crabeel, M . (2001) J . Biol . Chem.276, 42869-42880} . The NAGKs of ascomycetes possess, in addition to the catalytic domain that is shared by all other NAGKs and whose structure has been determined, a C-terminal domain of unknown function and structure . Exploring the role of these two domains in the synthase/kinase interaction, we demonstrate that the ascomycete-specific domain is required to maintain synthase activity and protein level . Previous results had suggested a participation of the third enzyme of the pathway, N-acetylglutamylphosphate reductase, in the metabolon . Here, genetic analyses conducted in yeast at physiological level, or in a heterologous background, clearly demonstrate that the reductase is dispensable for synthase activity and protein level . Most importantly, we show that the arginine feedback regulation of the NAGS and NAGK enzymes is mutually interdependent . First, the kinase becomes less sensitive to arginine feedback inhibition in the absence of the synthase . Second, and as in Neurospora crassa, in a yeast kinase mutant resistant to arginine feedback inhibition, the synthase becomes feedback resistant concomitantly . We conclude that the NAGS/NAGK metabolon promotes the co-ordination of the catalytic activities and feedback regulation of the first two, flux controlling, enzymes of the arginine pathway.

Biochem J, 2003 May 15, 372(Pt 1), 247 - 52
Transmembrane segments 1, 5, 7 and 8 are required for high-affinity glucose transport by Saccharomyces cerevisiae Hxt2 transporter; Kasahara T et al.; Hxt2 is a high-affinity facilitative glucose transporter of Saccharomyces cerevisiae and belongs to the major facilitator superfamily . Hxt1 shares approximately 70% amino acid identity with Hxt2 in its transmembrane segments (TMs) and inter-TM loops, but transports D-glucose with an affinity about one-tenth of that of Hxt2 . To determine which TMs of Hxt2 are important for high-affinity glucose transport, we constructed chimaeras of Hxt2 and Hxt1 by randomly replacing each of the 12 TMs of Hxt2 with the corresponding segment of Hxt1, for a total of 4096 different transporters . Among > 20000 yeast transformants screened, 39 different clones were selected by plate assays of high-affinity glucose-transport activity and sequenced . With only two exceptions, the selected chimaeras contained Hxt2 TMs 1, 5, 7 and 8 . We then constructed chimaeras corresponding to all 16 possible combinations of Hxt2 TMs 1, 5, 7 and 8 . Only one chimaera, namely that containing all four Hxt2 TMs, exhibited transport activity comparable with that of Hxt2 . The K (m) and V (max) values for D-glucose transport, and the substrate specificity of this chimaera were almost identical with those of Hxt2 . These results indicate that TMs 1, 5, 7 and 8 are necessary for exhibiting high-affinity glucose-transport activity of Hxt2.

Intervirology, 2002, 45(4-6), 308 - 17
Generation of recombinant virus-like particles of human and non-human polyomaviruses in yeast Saccharomyces cerevisiae; Sasnauskas K et al.; OBJECTIVES: Non-viral methods of gene transfer have been preferred in gene therapy approaches for several reasons, particularly for their safety, simplicity and convenience in introducing heterologous DNA into cells . Polyomavirus virus-like particles (VLPs) represent a promising carrier for encapsidation of foreign nucleic acids for gene therapy . For the development of such gene delivery systems as well as for providing reagents for improving virus diagnostics, an efficient yeast expression system for the generation of different polyomavirus VLPs was established . METHODS: A galactose-inducible Saccharomyces cerevisiae yeast expression system was used . Formation of empty VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy . Cross-reactivity of the major capsid proteins (VP1) of different polyomaviruses was analyzed by Western blot using rabbit and mice sera raised against the VP1 proteins . RESULTS: VP1 of polyomaviruses from humans (JC polyomavirus and serotypes AS and SB of BK polyomavirus), rhesus monkeys (simian virus 40), hamsters (hamster polyomavirus), mice (murine polyomavirus) and birds (budgerigar fledgling disease virus) were expressed at high levels in yeast . Empty VLPs formed by all yeast-expressed VP1 proteins were dissociated into pentamers and reassociated into VLPs by defined ion and pH conditions . Different patterns of cross-reactivity of the VP1 proteins with heterologous mice and rabbit sera were observed . CONCLUSION: The developed heterologous yeast expression system is suitable for high-level production of polyomavirus VLPs . Yeast-derived VLPs are generally free of toxins, host cell DNA and proteins . These VLPs might be useful for the generation of new diagnostical tools, gene delivery systems and antiviral vaccines.

J Biol Chem, 2003 May 2, 278(18), 16414 - 22 Epub 2003 Feb 24.
Vectorial acylation in Saccharomyces cerevisiae . Fat1p and fatty acyl-CoA synthetase are interacting components of a fatty acid import complex; Zou Z et al.; In Saccharomyces cerevisiae Fat1p and fatty acyl-CoA synthetase (FACS) are hypothesized to couple import and activation of exogenous fatty acids by a process called vectorial acylation . Molecular genetic and biochemical studies were used to define further the functional and physical interactions between these proteins . Multicopy extragenic suppressors were selected in strains carrying deletions in FAA1 and FAA4 or FAA1 and FAT1 . Each strain is unable to grow under synthetic lethal conditions when exogenous long-chain fatty acids are required, and neither strain accumulates the fluorescent long-chain fatty acid C(1)-BODIPY-C(12) indicating a fatty acid transport defect . By using these phenotypes as selective screens, plasmids were identified encoding FAA1, FAT1, and FAA4 in the faa1Delta faa4Delta strain and encoding FAA1 and FAT1 in the faa1Delta fat1Delta strain . Multicopy FAA4 could not suppress the growth defect in the faa1Delta fat1Delta strain indicating some essential functions of Fat1p cannot be performed by Faa4p . Chromosomally encoded FAA1 and FAT1 are not able to suppress the growth deficiencies of the fat1Delta faa1Delta and faa1Delta faa4Delta strains, respectively, indicating Faa1p and Fat1p play distinct roles in the fatty acid import process . When expressed from a 2-mu plasmid, Fat1p contributes significant oleoyl-CoA synthetase activity, which indicates vectorial esterification and metabolic trapping are the driving forces behind import . Evidence of a physical interaction between Fat1p and FACS was provided using three independent biochemical approaches . First, a C-terminal peptide of Fat1p deficient in fatty acid transport exerted a dominant negative effect against long-chain acyl-CoA synthetase activity . Second, protein fusions employing Faa1p as bait and portions of Fat1p as trap were active when tested using the yeast two-hybrid system . Third, co-expressed, differentially tagged Fat1p and Faa1p or Faa4p were co-immunoprecipitated . Collectively, these data support the hypothesis that fatty acid import by vectorial acylation in yeast requires a multiprotein complex, which consists of Fat1p and Faa1p or Faa4p.

Protein Expr Purif, 2003 Feb, 27(2), 357 - 64
Identification and characterization of a second NMN adenylyltransferase gene in Saccharomyces cerevisiae; Emanuelli M et al.; The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT) (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD(+) . On the basis of a remarkable structural similarity with previously described Saccharomyces cerevisiae NMNAT (yNMNAT-1), the YGR010-encoded protein was identified as a second isoform of yeast NMNAT (yNMNAT-2) . The YGR010 gene was isolated, cloned into a T7-based vector, and successfully expressed in Escherichia coli BL21 cells, yielding high level of NMN adenylyltransferase activity . The purification procedure reported in this paper, consisting of two chromatographic steps, allowed the isolation of 3mg of electrophoretically homogeneous yNMNAT-2 from 1 liter of E . coli culture . Under SDS/PAGE, the recombinant protein resulted in a single polypeptide of 46 kDa, in agreement with the molecular mass of the hypothetical protein encoded by YGR010 gene . The N-terminal sequence of the purified recombinant yNMNAT-2 exactly corresponds to the predicted sequence . Molecular and kinetic properties of recombinant yNMNAT-2 are reported and compared with those already known for yNMNAT-1 .

Biosci Biotechnol Biochem, 2002 Dec, 66(12), 2706 - 9
Substrate inhibition of L-cysteine alpha,beta-elimination reaction catalyzed by L-cystathionine gamma-lyase of Saccharomyces cerevisiae; Yamagata S et al.; The alpha,beta-elimination of L-cysteine catalyzed by Saccharomyces cerevisiae L-cystathionine gamma-lyase (EC 4.4.1.1) was inhibited by the substrate . The absorption spectrum of the holoenzyme in the presence of L-cysteine showed that the substrate inhibition observed in this reaction was due mainly to removal of the cofactor.

FEMS Microbiol Lett, 2003 Feb 14, 219(1), 137 - 42
The ldb1 mutant of Saccharomyces cerevisiae is defective in Pmr1p, the yeast secretory pathway/Golgi Ca(2+)/Mn(2+)-ATPase; Olivero I et al.; The LDB1 gene of Saccharomyces cerevisiae was identified by complementation of the ldb1 mutant phenotype with a genomic library . We found that the ldb1 defect is complemented by PMR1 which codes for the yeast secretory pathway/Golgi Ca(2+)/Mn(2+)-ATPase . Besides that, the analysis of a null mutation of the PMR1 gene revealed a phenotype identical to that of ldb1 mutant . Thus, LDB1 must be considered a synonym of PMR1.

FEMS Microbiol Lett, 2003 Feb 14, 219(1), 99 - 104
Involvement of oxidative stress in the regulation of H(2)S production during ultradian metabolic oscillation of Saccharomyces cerevisiae; Kwak WJ et al.; Periodic evolution of H(2)S during aerobic chemostat culture of Saccharomyces cerevisiae resulted in ultradian metabolic oscillation via periodic inhibition of respiratory activity . To understand the nature of periodic H(2)S evolution, we investigated whether oxidative stress is associated with H(2)S production . The cellular oxidative states represented by intracellular level of lipid peroxides oscillated out of phase with the oscillation of dissolved O(2) . Pulse addition of antioxidant, oxidative agent or inhibitor of antioxidation enzymes perturbed metabolic oscillation producing changes in H(2)S evolution . Analysis of H(2)S production profiles during perturbation of oscillation revealed that the amount of H(2)S production is closely linked with cellular oxidative states . Based on these results and our previous reports, we suggest that oxidative stresses result in periodic depletion of glutathione and cysteine, which in turn causes stimulation of the sulfate assimilation pathway and H(2)S production.

BMC Mol Biol . 2003 Feb 10;4(1):2.
Viable nonsense mutants for the essential gene SUP45 of Saccharomyces cerevisiae; Moskalenko SE et al.; BACKGROUND: Termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs) - eRF1 and eRF3 . The highly conserved translation termination factor eRF1 in Saccharomyces cerevisiae is encoded by the essential gene SUP45 . RESULTS: We have isolated five sup45-n (n from nonsense) mutations that cause nonsense substitutions in the following amino acid positions of eRF1: Y53 --> UAA, E266 --> UAA, L283 --> UAA, L317 --> UGA, E385 --> UAA . We found that full-length eRF1 protein is present in all mutants, although in decreased amounts . All mutations are situated in a weak termination context . All these sup45-n mutations are viable in different genetic backgrounds, however their viability increases after growth in the absence of wild-type allele . Any of sup45-n mutations result in temperature sensitivity (37 degrees C) . Most of the sup45-n mutations lead to decreased spore viability and spores bearing sup45-n mutations are characterized by limited budding after germination leading to formation of microcolonies of 4-20 cells . CONCLUSIONS: Nonsense mutations in the essential gene SUP45 can be isolated in the absence of tRNA nonsense suppressors.

Curr Genet, 2003 Jan, 42(4), 185 - 98 Epub 2002 Nov 29.
Mitotic recombination in Saccharomyces cerevisiae; Prado F et al.; Mitotic homologous recombination (HR) is an important mechanism for the repair of double-strand breakS and errors occurring during DNA replication . It is likely that the recombinational repair of DNA lesions occurs preferentially by sister chromatid exchanges that have no genetic consequences . However, most genetically detectable HR events occur between homologous DNA sequences located at allelic positions in homologous chromosomes, or between DNA repeats located at ectopic positions in either the same, homologous or heterologous chromosomes . Mitotic recombination may occur by multiple mechanisms, including double-strand break repair, synthesis-dependent strand annealing, break-induced replication and single-strand annealing . The occurrence of one recombination mechanism versus another depends on different elements, including the position of the homologous partner, the initiation event, the length of homology of the recombinant molecules and the genotype . The genetics and molecular biology of the yeast Saccharomyces cerevisiae have proved essential for the understanding of mitotic recombination mechanisms in eukaryotes . Here, we review recent genetic yeast data that contribute to our understanding of the different mechanisms of mitotic recombination and the in vivo role of the recombination proteins.

Mol Biol Cell, 2003 Feb, 14(2), 730 - 47
Cdc50p, a conserved endosomal membrane protein, controls polarized growth in Saccharomyces cerevisiae; Misu K et al.; During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and the growth of cell surface are polarized, mediating bud emergence, bud growth, and cytokinesis . We identified CDC50 as a multicopy suppressor of the myo3 myo5-360 temperature-sensitive mutant, which is defective in organization of cortical actin patches . The cdc50 null mutant showed cold-sensitive cell cycle arrest with a small bud as reported previously . Cortical actin patches and Myo5p, which are normally localized to polarization sites, were depolarized in the cdc50 mutant . Furthermore, actin cables disappeared, and Bni1p and Gic1p, effectors of the Cdc42p small GTPase, were mislocalized in the cdc50 mutant . As predicted by its amino acid sequence, Cdc50p appears to be a transmembrane protein because it was solubilized from the membranes by detergent treatment . Cdc50p colocalized with Vps21p in endosomal compartments and was also localized to the class E compartment in the vps27 mutant . The cdc50 mutant showed defects in a late stage of endocytosis but not in the internalization step . It showed, however, only modest defects in vacuolar protein sorting . Our results indicate that Cdc50p is a novel endosomal protein that regulates polarized cell growth.

Mol Biol Cell, 2003 Feb, 14(2), 556 - 70
Telomeric protein distributions and remodeling through the cell cycle in Saccharomyces cerevisiae; Smith CD et al.; In Saccharomyces cerevisiae, telomeric DNA is protected by a nonnucleosomal protein complex, tethered by the protein Rap1 . Rif and Sir proteins, which interact with Rap1p, are thought to have further interactions with conventional nucleosomic chromatin to create a repressive structure that protects the chromosome end . We showed by microarray analysis that Rif1p association with the chromosome ends extends to subtelomeric regions many kilobases internal to the terminal telomeric repeats and correlates strongly with the previously determined genomic footprints of Rap1p and the Sir2-4 proteins in these regions . Although the end-protection function of telomeres is essential for genomic stability, telomeric DNA must also be copied by the conventional DNA replication machinery and replenished by telomerase, suggesting that transient remodeling of the telomeric chromatin might result in distinct protein complexes at different stages of the cell cycle . Using chromatin immunoprecipitation, we monitored the association of Rap1p, Rif1p, Rif2p, and the protein component of telomerase, Est2p, with telomeric DNA through the cell cycle . We provide evidence for dynamic remodeling of these components at telomeres.

Mol Biol Cell, 2003 Feb, 14(2), 370 - 83
Synthetic lethal interaction of the mitochondrial phosphatidylethanolamine biosynthetic machinery with the prohibitin complex of Saccharomyces cerevisiae; Birner R et al.; The majority of mitochondrial phosphatidylethanolamine (PtdEtn), a phospholipid essential for aerobic growth of yeast cells, is synthesized by phosphatidylserine decarboxylase 1 (Psd1p) in the inner mitochondrial membrane (IMM) . To identify components that become essential when the level of mitochondrial PtdEtn is decreased, we screened for mutants that are synthetically lethal with a temperature-sensitive (ts) allele of PSD1 . This screen unveiled mutations in PHB1 and PHB2 encoding the two subunits of the prohibitin complex, which is located to the IMM and required for the stability of mitochondrially encoded proteins . Deletion of PHB1 and PHB2 resulted in an increase of mitochondrial PtdEtn at 30 degrees C . On glucose media, phb1Delta psd1Delta and phb2Delta psd1Delta double mutants were rescued only for a limited number of generations by exogenous ethanolamine, indicating that a decrease of the PtdEtn level is detrimental for prohibitin mutants . Similar to phb mutants, deletion of PSD1 destabilizes polypeptides encoded by the mitochondrial genome . In a phb1Delta phb2Delta psd1(ts) strain the destabilizing effect is dramatically enhanced . In addition, the mitochondrial genome is lost in this triple mutant, and nuclear-encoded proteins of the IMM are assembled at a very low rate . At the nonpermissive temperature mitochondria of phb1Delta phb2Delta psd1(ts) were fragmented and aggregated . In conclusion, destabilizing effects triggered by low levels of mitochondrial PtdEtn seem to account for synthetic lethality of psd1Delta with phb mutants.

Mol Cell Biol, 2003 Mar, 23(5), 1750 - 63
Far3 and five interacting proteins prevent premature recovery from pheromone arrest in the budding yeast Saccharomyces cerevisiae; Kemp HA et al.; In budding yeast, diffusible mating pheromones initiate a signaling pathway that culminates in several responses, including cell cycle arrest . Only a handful of genes required for the interface between pheromone response and the cell cycle have been identified, among them FAR1 and FAR3; of these, only FAR1 has been extensively characterized . In an effort to learn about the mechanism by which Far3 acts, we used the two-hybrid method to identify interacting proteins . We identified five previously uncharacterized open reading frames, dubbed FAR7, FAR8, FAR9, FAR10, and FAR11, that cause a far3-like pheromone arrest defect when disrupted . Using two-hybrid and coimmunoprecipitation analysis, we found that all six Far proteins interact with each other . Moreover, velocity sedimentation experiments suggest that Far3 and Far7 to Far11 form a complex . The phenotype of a sextuple far3far7-far11 mutant is no more severe than any single mutant . Thus, FAR3 and FAR7 to FAR11 all participate in the same pathway leading to G1 arrest . These mutants initially arrest in response to pheromone but resume budding after 10 h . Under these conditions, wild-type cells fail to resume budding even after several days whereas far1 mutant cells resume budding within 1 h . We conclude that the FAR3-dependent arrest pathway is functionally distinct from that which employs FAR1.

Mol Cell Biol, 2003 Mar, 23(5), 1558 - 68
In exponentially growing Saccharomyces cerevisiae cells, rRNA synthesis is determined by the summed RNA polymerase I loading rate rather than by the number of active genes; French SL et al.; Genes encoding rRNA are multicopy and thus could be regulated by changing the number of active genes or by changing the transcription rate per gene . We tested the hypothesis that the number of open genes is limiting rRNA synthesis by using an electron microscopy method that allows direct counting of the number of active genes per nucleolus and the number of polymerases per active gene . Two strains of Saccharomyces cerevisiae were analyzed during exponential growth: a control strain with a typical number of rRNA genes ( approximately 143 in this case) and a strain in which the rRNA gene number was reduced to approximately 42 but which grows as well as controls . In control strains, somewhat more than half of the genes were active and the mean number of polymerases/gene was approximately 50 +/- 20 . In the 42-copy strain, all rRNA genes were active with a mean number of 100 +/- 29 polymerases/gene . Thus, an equivalent number of polymerases was active per nucleolus in the two strains, though the number of active genes varied by twofold, showing that overall initiation rate, and not the number of active genes, determines rRNA transcription rate during exponential growth in yeast . Results also allow an estimate of elongation rate of approximately 60 nucleotides/s for yeast Pol I and a reinitiation rate of less than 1 s on the most heavily transcribed genes.

Genetics, 2003 Jan, 163(1), 447 - 52
Identification of a functional domain within the essential core of histone H3 that is required for telomeric and HM silencing in Saccharomyces cerevisiae; Thompson JS et al.; Fourteen novel single-amino-acid substitution mutations in histone H3 that disrupt telomeric silencing in Saccharomyces cerevisiae were identified, 10 of which are clustered within the alpha1 helix and L1 loop of the essential histone fold . Several of these mutations cause derepression of silent mating locus HML, and an additional subset cause partial loss of basal repression at the GAL1 promoter . Our results identify a new domain within the essential core of histone H3 that is required for heterochromatin-mediated silencing.

Genetics, 2003 Jan, 163(1), 69 - 77
Specialization of function among aldehyde dehydrogenases: the ALD2 and ALD3 genes are required for beta-alanine biosynthesis in Saccharomyces cerevisiae; White WH et al.; The amino acid beta-alanine is an intermediate in pantothenic acid (vitamin B(5)) and coenzyme A (CoA) biosynthesis . In contrast to bacteria, yeast derive the beta-alanine required for pantothenic acid production via polyamine metabolism, mediated by the four SPE genes and by the FAD-dependent amine oxidase encoded by FMS1 . Because amine oxidases generally produce aldehyde derivatives of amine compounds, we propose that an additional aldehyde-dehydrogenase-mediated step is required to make beta-alanine from the precursor aldehyde, 3-aminopropanal . This study presents evidence that the closely related aldehyde dehydrogenase genes ALD2 and ALD3 are required for pantothenic acid biosynthesis via conversion of 3-aminopropanal to beta-alanine in vivo . While deletion of the nuclear gene encoding the unrelated mitochondrial Ald5p resulted in an enhanced requirement for pantothenic acid pathway metabolites, we found no evidence to indicate that the Ald5p functions directly in the conversion of 3-aminopropanal to beta-alanine . Thus, in Saccharomyces cerevisiae, ALD2 and ALD3 are specialized for beta-alanine biosynthesis and are consequently involved in the cellular biosynthesis of coenzyme A.

Genetics, 2003 Jan, 163(1), 55 - 67
The Rad27 (Fen-1) nuclease inhibits Ty1 mobility in Saccharomyces cerevisiae; Sundararajan A et al.; Although most Ty1 elements in Saccharomyces cerevisiae are competent for retrotransposition, host defense genes can inhibit different steps of the Ty1 life cycle . Here, we demonstrate that Rad27, a structure-specific nuclease that plays an important role in DNA replication and genome stability, inhibits Ty1 at a post-translational level . We have examined the effects of various rad27 mutations on Ty1 element retrotransposition and cDNA recombination, termed Ty1 mobility . The point mutations rad27-G67S, rad27-G240D, and rad27-E158D that cause defects in certain enzymatic activities in vitro result in variable increases in Ty1 mobility, ranging from 4- to 22-fold . The C-terminal frameshift mutation rad27-324 confers the maximum increase in Ty1 mobility (198-fold), unincorporated cDNA, and insertion at preferred target sites . The null mutation differs from the other rad27 alleles by increasing the frequency of multimeric Ty1 insertions and cDNA recombination with a genomic element . The rad27 mutants do not markedly alter the levels of Ty1 RNA or the TyA1-gag protein . However, there is an increase in the stability of unincorporated Ty1 cDNA in rad27-324 and the null mutant . Our results suggest that Rad27 inhibits Ty1 mobility by destabilizing unincorporated Ty1 cDNA and preventing the formation of Ty1 multimers.

Genetics, 2003 Jan, 163(1), 47 - 54
Large-scale functional genomic analysis of sporulation and meiosis in Saccharomyces cerevisiae; Enyenihi AH et al.; We have used a single-gene deletion mutant bank to identify the genes required for meiosis and sporulation among 4323 nonessential Saccharomyces cerevisiae annotated open reading frames (ORFs) . Three hundred thirty-four sporulation-essential genes were identified, including 78 novel ORFs and 115 known genes without previously described sporulation defects in the comprehensive Saccharomyces Genome (SGD) or Yeast Proteome (YPD) phenotype databases . We have further divided the uncharacterized sporulation-essential genes into early, middle, and late stages of meiosis according to their requirement for IME1 induction and nuclear division . We believe this represents a nearly complete identification of the genes uniquely required for this complex cellular pathway . The set of genes identified in this phenotypic screen shows only limited overlap with those identified by expression-based studies.

Genetics, 2003 Jan, 163(1), 21 - 33
Loss of CDC5 function in Saccharomyces cerevisiae leads to defects in Swe1p regulation and Bfa1p/Bub2p-independent cytokinesis; Park CJ et al.; In many organisms, polo kinases appear to play multiple roles during M-phase progression . To provide new insights into the function of budding yeast polo kinase Cdc5p, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal domain . Here we show that, at a semipermissive temperature, the cdc5-3 mutant exhibited a synergistic bud elongation and growth defect with loss of HSL1, a component important for normal G(2)/M transition . Loss of SWE1, which phosphorylates and inactivates the budding yeast Cdk1 homolog Cdc28p, suppressed the cdc5-3 hsl1Delta defect, suggesting that Cdc5p functions at a point upstream of Swe1p . In addition, the cdc5-4 and cdc5-7 mutants exhibited chained cell morphologies with shared cytoplasms between the connected cell bodies, indicating a cytokinetic defect . Close examination of these mutants revealed delayed septin assembly at the incipient bud site and loosely organized septin rings at the mother-bud neck . Components in the mitotic exit network (MEN) play important roles in normal cytokinesis . However, loss of BFA1 or BUB2, negative regulators of the MEN, failed to remedy the cytokinetic defect of these mutants, indicating that Cdc5p promotes cytokinesis independently of Bfa1p and Bub2p . Thus, Cdc5p contributes to the activation of the Swe1p-dependent Cdc28p/Clb pathway, normal septin function, and cytokinesis.

Genetics, 2003 Jan, 163(1), 9 - 20
Genetic analysis of the interface between Cdc42p and the CRIB domain of Ste20p in Saccharomyces cerevisiae; Ash J et al.; Mutagenesis was used to probe the interface between the small GTPase Cdc42p and the CRIB domain motif of Ste20p . Members of a cluster of hydrophobic residues of Cdc42p were changed to alanine and/or arginine . The interaction of the wild-type and mutant proteins was measured using the two-hybrid assay; many, but not all, changes reduced interaction between Cdc42p and the target CRIB domain . Mutations in conserved residues in the CRIB domain were also tested for their importance in the association with Cdc42p . Two conserved CRIB domain histidines were changed to aspartic acid . These mutants reduced mating, as well as responsiveness to pheromone-induced gene expression and cell cycle arrest, but did not reduce in vitro the kinase activity of Ste20p . GFP-tagged mutant proteins were unable to localize to sites of polarized growth . In addition, these point mutants were synthetically lethal with disruption of CLA4 and blocked the Ste20p-Cdc42p two-hybrid interaction . Compensatory mutations in Cdc42p that reestablished the two-hybrid association with the mutant Ste20p CRIB domain baits were identified . These mutations improved the pheromone responsiveness of cells containing the CRIB mutations, but did not rescue the lethality associated with the CRIB mutant CLA4 deletion interaction . These results suggest that the Ste20p-Cdc42p interaction plays a direct role in Ste20p kinase function and that this interaction is required for efficient activity of the pheromone response pathway.

Chemosphere, 2003 Apr, 51(1), 47 - 54
Toxicity of chlorinated phenoxyacetic acid herbicides in the experimental eukaryotic model Saccharomyces cerevisiae: role of pH and of growth phase and size of the yeast cell population; Cabral MG et al.; The inhibitory effect of the herbicides 2-methyl-4-chlorophenoxyacetic acid (MCPA) and 2,4-dichlorophenoxyacetic acid (2,4-D) in Saccharomyces cerevisiae growth is strongly dependent on medium pH (range 2.5-6.5) . Consistent with the concept that the toxic form is the liposoluble undissociated form, at values close to their pK(a) (3.07 and 2.73, respectively) the toxicity is high, decreasing with the increase of external pH . In addition, the toxicity of identical concentrations of the undissociated acid form is pH independent, as observed with 2,4-dichlorophenol (2,4-DCP), an intermediate of 2,4-D degradation . Consequently, at pH values above 3.5 (approximately one unit higher than 2,4-D pK(a)), 2,4-DCP becomes more toxic than the original herbicide . A dose-dependent inhibition of growth kinetics and increased duration of growth latency is observed following sudden exposure of an unadapted yeast cell population to the presence of the herbicides . This contrasts with the effect of 2,4-DCP, which essentially affects growth kinetics . Experimental evidences suggest that the acid herbicides toxicity is not exclusively dependent on the liposolubility of the toxic form, as may essentially be the case of 2,4-DCP . An unadapted yeast cell population at the early stationary-phase of growth under nutrient limitation is significantly more resistant to short-term herbicide induced death than an exponential-phase population . Consequently, the duration of growth latency is reduced, as observed with the increase of the size of the herbicide stressed population . However, these physiological parameters have no significant effect either on growth kinetics, following growth resumption under herbicide stress, or on the growth curve of yeast cells previously adapted to the herbicides, indicating that their role is exerted at the level of cell adaptation.

Biotechnol Bioeng, 2003 Apr 20, 82(2), 143 - 51
Free intracellular amino acid pools during autonomous oscillations in Saccharomyces cerevisiae; Hans MA et al.; In the present work dynamic changes of free intracellular amino acid pools during autonomous oscillations of Saccharomyces cerevisiae were quantified in glucose-limited continuous cultivations . At a dilution rate of D = 0.22 h(-1) cyclic changes with a period of 120 min were found for many variables such as carbon dioxide production rate, dissolved oxygen, pH, biomass content, and various metabolite concentrations . On the basis of the observed dynamic patterns, free intracellular amino acids were classified to show oscillatory, stationary, or chaotic behavior . Amino acid pools such as serine, alanine, valine, leucine, or lysine were subjected to clear oscillations with a frequency of 120 min, identical to that of other described cultivation variables, indicating that there is a direct correlation between the periodic changes of amino acid concentrations and the metabolic oscillations on the cellular level . The oscillations of these amino acids were unequally phase-delayed and had different amplitudes of oscillation . Accordingly, they exhibited different patterns in phase plane plots vs . intracellular trehalose . Despite the complex and marked metabolic changes during oscillation, selected intracellular amino acids such as histidine, threonine, isoleucine, or arginine remained about constant . Concentrations of glutamate and glutamine showed a chaotic behavior . However, the ratio of glutamate to glutamine concentration was found to be oscillatory, with a period of 60 min and a corresponding figure eight-shaped pattern in a plot vs . trehalose concentration . Considering the described diversity, it can be concluded that the observed periodic changes are neither just the consequence of low or high rates of protein biosynthesis/degradation nor correlated to changing cell volumes during oscillation . The ratio between doubling time (189 min) and period of oscillation of intracellular amino acids (120 min) was 1:6 . The fact that there is a close relationship between doubling time and period of oscillation underlines that the described autonomous oscillations are cell-cycle-associated .

Nucleic Acids Res, 2003 Feb 15, 31(4), 1164 - 73
Gene conversion tracts in Saccharomyces cerevisiae can be extremely short and highly directional; Palmer S et al.; Gene conversion is a common outcome of double-strand break (DSB) repair in yeast . Prior studies revealed that DSB-induced gene conversion tracts are often short (<53 bp), unidirectional, and biased toward promoter-proximal (5') markers . In those studies, broken ends had short, non-homologous termini . For the present study we created plasmid x chromosome, chromosomal direct repeat and allelic recombination substrates in which donor alleles carried mutant HO sites (HOinc--not cleaved) at the same position as cleavable HO sites in recipient alleles . In these substrates, broken ends are almost completely homologous to donor alleles, differing only at the three HOinc mutations . These mutations serve as markers very close to, or within, the four-base overhang produced by HO nuclease . We identified extremely short tracts (<12 bp) and many tracts were highly directional, extending <2 bp on one side of the DSB . We thought that terminal homology would promote bidirectional tracts, but found instead that unidirectional tracts were more frequent . Interestingly, substrates with terminal homology displayed enhanced 3' conversion, and in several cases conversion bias was reversed toward 3' markers . These results are discussed in relation to factors that may influence tract length and directionality, including heteroduplex DNA formation, transcription, replication and mismatch repair.

Eukaryot Cell, 2003 Feb, 2(1), 143 - 9
Glucose regulation of Saccharomyces cerevisiae cell cycle genes; Newcomb LL et al.; Nutrient-limited Saccharomyces cerevisiae cells rapidly resume proliferative growth when transferred into glucose medium . This is preceded by a rapid increase in CLN3, BCK2, and CDC28 mRNAs encoding cell cycle regulatory proteins that promote progress through Start . We have tested the ability of mutations in known glucose signaling pathways to block glucose induction of CLN3, BCK2, and CDC28 . We find that loss of the Snf3 and Rgt2 glucose sensors does not block glucose induction, nor does deletion of HXK2, encoding the hexokinase isoenzyme involved in glucose repression signaling . Rapamycin blockade of the Tor nutrient sensing pathway does not block the glucose response . Addition of 2-deoxy glucose to the medium will not substitute for glucose . These results indicate that glucose metabolism generates the signal required for induction of CLN3, BCK2, and CDC28 . In support of this conclusion, we find that addition of iodoacetate, an inhibitor of the glyceraldehyde-3-phosphate dehydrogenase step in yeast glycolysis, strongly downregulates the levels CLN3, BCK2, and CDC28 mRNAs . Furthermore, mutations in PFK1 and PFK2, which encode phosphofructokinase isoforms, inhibit glucose induction of CLN3, BCK2, and CDC28 . These results indicate a link between the rate of glycolysis and the expression of genes that are critical for passage through G(1).

Eukaryot Cell, 2003 Feb, 2(1), 134 - 42
mRNAs encoding telomerase components and regulators are controlled by UPF genes in Saccharomyces cerevisiae; Dahlseid JN et al.; Telomeres, the chromosome ends, are maintained by a balance of activities that erode and replace the terminal DNA sequences . Furthermore, telomere-proximal genes are often silenced in an epigenetic manner . In Saccharomyces cerevisiae, average telomere length and telomeric silencing are reduced by loss of function of UPF genes required in the nonsense-mediated mRNA decay (NMD) pathway . Because NMD controls the mRNA levels of several hundred wild-type genes, we tested the hypothesis that NMD affects the expression of genes important for telomere functions . In upf mutants, high-density oligonucleotide microarrays and Northern blots revealed that the levels of mRNAs were increased for genes encoding the telomerase catalytic subunit (Est2p), in vivo regulators of telomerase (Est1p, Est3p, Stn1p, and Ten1p), and proteins that affect telomeric chromatin structure (Sas2p and Orc5p) . We investigated whether overexpressing these genes could mimic the telomere length and telomeric silencing phenotypes seen previously in upf mutant strains . Increased dosage of STN1, especially in combination with increased dosage of TEN1, resulted in reduced telomere length that was indistinguishable from that in upf mutants . Increased levels of STN1 together with EST2 resulted in reduced telomeric silencing like that of upf mutants . The half-life of STN1 mRNA was not altered in upf mutant strains, suggesting that an NMD-controlled transcription factor regulates the levels of STN1 mRNA . Together, these results suggest that NMD maintains the balance of gene products that control telomere length and telomeric silencing primarily by maintaining appropriate levels of STN1, TEN1, and EST2 mRNA.

Eukaryot Cell, 2003 Feb, 2(1), 19 - 26
Yap1 accumulates in the nucleus in response to carbon stress in Saccharomyces cerevisiae; Wiatrowski HA et al.; Yap1 is a transcription factor of the AP-1 family that is required for the adaptive response to oxidative stress in Saccharomyces cerevisiae . We recovered Yap1 in a two-hybrid screen for proteins that interact with the Sip2 subunit of the Snf1 protein kinase, which is required for the adaptation of cells to glucose limitation . Yap1 becomes enriched in the nucleus when cells are subjected to oxidative stress . We show that the localization of Yap1 is similarly sensitive to carbon stress . When glucose-grown cells were shifted to medium containing glycerol or no added carbon source, green fluorescent protein (GFP)-Yap1 accumulated in the nucleus . After adaptation to growth in glycerol, GFP-Yap1 was again primarily cytoplasmic . Nuclear accumulation was independent of respiration and of the Snf1, PKA, TOR, and Yak1 pathways, and the mechanism is distinct from that involved in the response to hydrogen peroxide . Addition of glutathione to the medium inhibited nuclear accumulation of GFP-Yap1 in response to carbon stress but did not affect the relocalization of Gal83 or Mig1 . Other stresses such as increased temperature, acidic pH, and ionic stress did not cause nuclear enrichment of GFP-Yap1 . These findings suggest a role for Yap1 in the response to carbon stress.

Mol Microbiol, 2003 Feb, 47(4), 1163 - 81
Inorganic phosphate is sensed by specific phosphate carriers and acts in concert with glucose as a nutrient signal for activation of the protein kinase A pathway in the yeast Saccharomyces cerevisiae; Giots F et al.; Yeast cells starved for inorganic phosphate on a glucose-containing medium arrest growth and enter the resting phase G0 . We show that re-addition of phosphate rapidly affects well known protein kinase A targets: trehalase activation, trehalose mobilization, loss of heat resistance, repression of STRE-controlled genes and induction of ribosomal protein genes . Phosphate-induced activation of trehalase is independent of protein synthesis and of an increase in ATP . It is dependent on the presence of glucose, which can be detected independently by the G-protein coupled receptor Gpr1 and by the glucose-phosphorylation dependent system . Addition of phosphate does not trigger a cAMP signal . Despite this, lowering of protein kinase A activity by mutations in the TPK genes strongly reduces trehalase activation . Inactivation of phosphate transport by deletion of PHO84 abolishes phosphate signalling at standard concentrations, arguing against the existence of a transport-independent receptor . The non-metabolizable phosphate analogue arsenate also triggered signalling . Constitutive expression of the Pho84, Pho87, Pho89, Pho90 and Pho91 phosphate carriers indicated pronounced differences in their transport and signalling capacities in phosphate-starved cells . Pho90 and Pho91 sustained highest phosphate transport but did not sustain trehalase activation . Pho84 sustained both transport and rapid signalling, whereas Pho87 was poor in transport but positive for signalling . Pho89 displayed very low phosphate transport and was negative for signalling . Although the results confirmed that rapid signalling is independent of growth recovery, long-term mobilization of trehalose was much better correlated with growth recovery than with trehalase activation . These results demonstrate that phosphate acts as a nutrient signal for activation of the protein kinase A pathway in yeast in a glucose-dependent way and they indicate that the Pho84 and Pho87 carriers act as specific phosphate sensors for rapid phosphate signalling.

J Biol Chem, 2003 Apr 25, 278(17), 15397 - 405 Epub 2003 Feb 10.
Identification and characterization of a novel RanGTP-binding protein in the yeast Saccharomyces cerevisiae; Braunwarth A et al.; The small Ras-like GTPase Ran plays an essential role in the transport of macromolecules in and out of the nucleus and has been implicated in spindle (1,2 ) and nuclear envelope formation (3,4 ) during mitosis in higher eukaryotes . We identified Saccharomyces cerevisiae open reading frame YGL164c encoding a novel RanGTP-binding protein, termed Yrb30p . The protein competes with yeast RanBP1 (Yrb1p) for binding to the GTP-bound form of yeast Ran (Gsp1p) and is, like Yrb1p, able to form trimeric complexes with RanGTP and some of the karyopherins . In contrast to Yrb1p, Yrb30p does not coactivate but inhibits RanGAP1(Rna1p)-mediated GTP hydrolysis on Ran, like the karyopherins . At steady state, Yrb30p localizes exclusively to the cytoplasm, but the presence of a functional nuclear export signal and the localization of truncated forms of Yrb30p suggest that the protein shuttles between nucleus and cytoplasm and is exported via two alternative pathways, dependent on the nuclear export receptor Xpo1p/Crm1p and on RanGTP binding . Whereas overproduction of the full-length protein and complete deletion of the open reading frame reveal no obvious phenotype, overproduction of C-terminally truncated forms of the protein inhibits yeast vegetative growth . Based on these results and the exclusive conservation of the protein in the fungal kingdom, we hypothesize that Yrb30p represents a novel modulator of the Ran GTPase switch related to fungal lifestyle.

Biochemistry, 2003 Feb 11, 42(5), 1266 - 73
NMR structure of the DNA-binding domain of the cell cycle protein Mbp1 from Saccharomyces cerevisiae; Nair M et al.; The three-dimensional solution structure of the DNA-binding domain of Mlu-1 box binding protein (Mbp1) has been determined by multidimensional NMR spectroscopy . Mbp1 is a cell cycle transcription factor from Saccharomyces cerevisiae and consists of an N-terminal DNA-binding domain, a series of ankyrin repeats, and a heterodimerization domain at the C-terminus . A set of conformers comprising 19 refined structures was calculated via a molecular dynamics simulated annealing protocol using distance, dihedral angle, and residual dipolar coupling restraints derived from either double or triple resonance NMR experiments . The solution structure consists of a six-stranded beta-sheet segment folded against two pairs of alpha-helices in the topology of the winged helix-turn-helix family of proteins and is in agreement with the X-ray structures . In addition, the solution structure shows that the C-terminal tail region of this domain folds back and makes specific interactions with the N-terminal beta-strand of the protein . This C-terminal region is essential for full DNA-binding activity but appears in the X-ray structure to be disordered . The fold-back structure extends the region of positive electrostatic potential, and this may enhance the nonspecific contribution to binding by favorable electrostatic interactions with the DNA backbone.

J Biol Chem, 2003 Apr 11, 278(15), 13390 - 7 Epub 2003 Jan 31.
Sip2, an N-myristoylated beta subunit of Snf1 kinase, regulates aging in Saccharomyces cerevisiae by affecting cellular histone kinase activity, recombination at rDNA loci, and silencing; Lin SS et al.; Saccharomyces cerevisiae has evolved a number of mechanisms for sensing glucose . In the present study we examine the mechanism by which one of these pathways, involving Snf1, regulates cellular aging . Snf1 is a heterotrimer composed of a catalytic alpha subunit (Snf1p) that phosphorylates target proteins at Ser/Thr residues, an activating gamma subunit (Snf4p), and a beta subunit (Sip1p, Sip2p, or Gal83) . We previously showed that forced expression of Snf1p or loss of Sip2p, but not the other beta subunits, causes accelerated aging, while removal of Snf4p extends life span (Ashrafi, K., Lin, S . S., Manchester, J . K., and Gordon, J . I . (2000) Genes Dev . 14, 1872-1885) . We now demonstrate that in wild type cells, there is an age-associated shift in Sip2p from the plasma membrane to the cytoplasm, a prominent redistribution of Snf4p from the plasma membrane to the nucleus, a modest increase in nuclear Snf1p, and a concomitant increase in cellular Snf1 histone H3 kinase activity . Covalent attachment of myristate to the N-terminal Gly of Sip2p is essential for normal cellular life span . When plasma membrane association of Sip2p is abolished by a mutation that blocks its N-myristoylation, Snf4p is shifted to the nucleus . Rapidly aging sip2 Delta cells have higher levels of histone H3 kinase activity than their generation-matched isogenic wild type counterparts . Increased Snf1 activity is associated with augmented recombination at rDNA loci, plus desilencing at sites affected by Snf1-catalyzed Ser(10) phosphorylation of histone H3 (the INO1 promoter plus targets of the transcription factor Adr1p) . The rapid-aging phenotype of sip2 Delta cells is fully rescued by blocking recombination at rDNA loci with a fob1 Delta allele; rescue is not accompanied by amelioration of an age-associated shift toward gluconeogenesis and glucose storage . Together, these findings suggest that Sip2p acts as a negative regulator of nuclear Snf1 activity in young cells by sequestering its activating gamma subunit at the plasma membrane and that loss of Sip2p from the plasma membrane to the cytoplasm in aging cells facilities Snf4p entry into the nucleus so that Snf1 can modify chromatin structure.

Nucleic Acids Res, 2003 Feb 1, 31(3), 1013 - 23
Genome instability in rad54 mutants of Saccharomyces cerevisiae; Schmuckli-Maurer J et al.; The RAD54 gene of Saccharomyces cerevisiae encodes a conserved dsDNA-dependent ATPase of the Swi2/Snf2 family with a specialized function during recombinational DNA repair . Here we analyzed the consequences of the loss of Rad54 function in vegetative (mitotic) cells . Mutants in RAD54 exhibited drastically reduced rates of spontaneous intragenic recombination but were proficient for spontaneous intergenic recombinant formation . The intergenic recombinants likely arose by a RAD54-independent pathway of break-induced replication . Significantly increased rates of spontaneous chromosome loss for diploid rad54/rad54 cells were identified in several independent assays . Inter estingly, the increase in chromosome loss appeared to depend on the presence of a homolog . In addition, the rate of complex genetic events involving chromosome loss were drastically increased in diploid rad54/rad54 cells . Together, these data suggest a role for Rad54 protein in the repair of spontaneous damage, where in the absence of Rad54 protein, homologous recombination is initiated but not properly terminated, leading to misrepair and chromosome loss.

Nucleic Acids Res, 2003 Feb 1, 31(3), 893 - 8
Sir2p suppresses recombination of replication forks stalled at the replication fork barrier of ribosomal DNA in Saccharomyces cerevisiae; Benguria A et al.; In the ribosomal DNA (rDNA) of Saccharomyces cerevisiae replication forks progressing against transcription stall at a polar replication fork barrier (RFB) located close to and downstream of the 35S transcription unit . Forks blocked at this barrier are potentially recombinogenic . Plasmids bearing the RFB sequence in its active orientation integrated into the chromosomal rDNA in sir2 mutant cells but not in wild-type cells, indicating that the histone deacetylase silencing protein Sir2 (Sir2p), which also modulates the aging process in yeast, suppresses the recombination competence of forks blocked at the rDNA RFB . Orientation of the RFB sequence in its inactive course or its abolition by FOB1 deletion avoided plasmid integration in sir2 mutant cells, indicating that stalling of the forks in the plasmid context was required for recombination to take place . Altogether these results strongly suggest that one of the functions of Sir2p is to modulate access of the recombination machinery to the forks stalled at the rDNA RFB.

Gene, 2003 Jan 16, 303, 63 - 8
Nucleo-mitochondrial interactions in Saccharomyces cerevisiae: characterization of a nuclear gene suppressing a defect in mitochondrial tRNA(Asp) processing; Rinaldi T et al.; We utilized the heat-sensitive mutant strain (Ts932), bearing a mutation at position 61 in the mitochondrial tRNA(Asp) gene, to identify nuclear genes involved in tRNA biogenesis; this mutant is defective in 3'-end processing and consequently in the production of mature mitochondrial tRNA(Asp) . We transformed this strain with a yeast nuclear library and we isolated among other suppressors, an unknown, non-essential gene (called SMM1, corresponding to open reading frame YNR015w), which restored the growth on glycerol and a normal amount of processed tRNA(Asp) in the mutant . The gene contains a domain highly conserved in evolution from bacteria to human and its product has been recently shown to have dihydrouridine synthase activity.

Mol Cell Biol, 2003 Feb, 23(4), 1403 - 17
Molecular dissection of mitotic recombination in the yeast Saccharomyces cerevisiae; Aylon Y et al.; Recombination plays a central role in the repair of broken chromosomes in all eukaryotes . We carried out a systematic study of mitotic recombination . Using several assays, we established the chronological sequence of events necessary to repair a single double-strand break . Once a chromosome is broken, yeast cells become immediately committed to recombinational repair . Recombination is completed within an hour and exhibits two kinetic gaps . By using this kinetic framework we also characterized the role played by several proteins in the recombinational process . In the absence of Rad52, the broken chromosome ends, both 5' and 3', are rapidly degraded . This is not due to the inability to recombine, since the 3' single-stranded DNA ends are stable in a strain lacking donor sequences . Rad57 is required for two consecutive strand exchange reactions . Surprisingly, we found that the Srs2 helicase also plays an early positive role in the recombination process.

Mol Cell Biol, 2003 Feb, 23(4), 1135 - 50
Effect of the pheromone-responsive G(alpha) and phosphatase proteins of Saccharomyces cerevisiae on the subcellular localization of the Fus3 mitogen-activated protein kinase; Blackwell E et al.; The mating-specific G(alpha) protein of Saccharomyces cerevisiae, Gpa1, stimulates adaptation to pheromone by a mechanism independent of G(beta gamma) sequestration . Genetic evidence suggests that Gpa1 targets the Fus3 mitogen-activated protein kinase, and it has recently been shown that the two proteins interact in cells responding to pheromone . To test the possibility that Gpa1 downregulates the mating signal by affecting the localization of Fus3, we created a Fus3-green fluorescent protein (GFP) fusion protein . In vegetative cells, Fus3-GFP was found in both the cytoplasm and the nucleus . Pheromone stimulated a measurable increase in the ratio of nuclear to cytoplasmic Fus3-GFP . In contrast, the relative level of nuclear Fus3-GFP decreased as cells recovered from pheromone arrest and did not increase when cells adapted to chronic stimulus were challenged again . Accumulation of Fus3-GFP in the nuclei of stimulated cells was also inhibited by overexpression of either wild-type Gpa1, the E364K hyperadaptive mutant form of Gpa1, or the Msg5 dually specific phosphatase . The effects of Gpa1 and Msg5 on Fus3 are partially interdependent . In a genetic screen for adaptive defective mutants, a nonsense allele of the nucleocytoplasmic transport receptor, Kap104, was identified . Truncation of the Kap104 cargo-binding domain blocked the effect of both Gpa1(E364K) and Msg5 on Fus3-GFP localization . Based on these results, we propose that Gpa1 and Msg5 work in concert to downregulate the mating signal and that they do so by inhibiting the pheromone-induced increase of Fus3 in the nucleus . Kap104 is required for the G(alpha)/phosphatase-mediated effect on Fus3 localization.

Biochem Cell Biol, 2002, 80(6), 789 - 96
Cellular resistance to bleomycin in Saccharomyces cerevisiae is not affected by changes in bleomycin hydrolase levels; Wang H et al.; Bleomycin is a glycopeptide drug that exerts potent genotoxic potential and is highly effective in the treatment of certain cancers when used in combination therapy . Unfortunately, however, tumors often develop resistance against bleomycin, and the mechanism of this resistance remains unclear . It has been postulated that bleomycin hydrolase, a protease encoded by the BLH1 gene in humans, may account for tumor resistance to bleomycin . In support of such a notion, earlier studies showed that exogenous expression of yeast Blh1 in human cells can enhance resistance to bleomycin . Here we show that (i) yeast blh1delta mutants are not sensitive to bleomycin, (ii) bleomycin-hypersensitive yeast mutants were no more sensitive to this agent upon deletion of the BLH1/LAP3/GAL6 gene, and (iii) overproduction of Blhl in either the parent or bleomycin-hypersensitive mutants did not confer additional resistance to these strains . Therefore, yeast Blh1 apparently has no direct role in protecting this organism from the lethal effects of bleomycin, even though the enzyme can degrade the drug in vitro . Clearly, additional studies are required to establish the actual biological role of Blh1 in yeast.

Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 334 - 7 Epub 2003 Jan 23.
Crystallization and preliminary X-ray analysis of NADP(H)-dependent alcohol dehydrogenases from Saccharomyces cerevisiae and Rana perezi; Valencia E et al.; Different crystal forms diffracting to high resolution have been obtained for two NADP(H)-dependent alcohol dehydrogenases, members of the medium-chain dehydrogenase/reductase superfamily: ScADHVI from Saccharomyces cerevisiae and ADH8 from Rana perezi . ScADHVI is a broad-specificity enzyme, with a sequence identity lower than 25% with respect to all other ADHs of known structure . The best crystals of ScADHVI diffracted beyond 2.8 A resolution and belonged to the trigonal space group P3(1)21 (or to its enantiomorph P3(2)21), with unit-cell parameters a = b = 102.2, c = 149.7 A, gamma = 120 degrees . These crystals were produced by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant . Packing considerations together with the self-rotation function and the native Patterson map seem to indicate the presence of only one subunit per asymmetric unit, with a Volume solvent content of about 80% . ADH8 from R . perezi is the only NADP(H)-dependent ADH from vertebrates characterized to date . Crystals of ADH8 obtained both in the absence and in the presence of NADP(+) using polyethylene glycol and lithium sulfate as precipitants diffracted to 2.2 and 1.8 A, respectively, using synchrotron radiation . These crystals were isomorphous, space group C2, with approximate unit-cell parameters a = 122, b = 79, c = 91 A, beta = 113 degrees and contain one dimer per asymmetric unit, with a Volume solvent content of about 50%.

Mol Biochem Parasitol, 2003 Jan, 126(1), 81 - 92
The PmSOD1 gene of the protistan parasite Perkinsus marinus complements the sod2Delta mutant of Saccharomyces cerevisiae, and directs an iron superoxide dismutase to mitochondria; Schott EJ et al.; The facultative intracellular oyster parasite, Perkinsus marinus, taxonomically related to both dinoflagellates and apicomplexan parasites, possesses at least two distinct genes (PmSOD1 and PmSOD2) predicted to encode iron-containing superoxide dismutases (Fe-SOD) . The present study demonstrates that PmSOD1 complements a Saccharomyces cerevisiae mutant lacking the mitochondrial manganese-containing SOD (Mn-SOD), whereas PmSOD2 complements an Escherichia coli mutant lacking genes for cytosolic SOD activities . Mitochondria isolated from complemented yeast contain an SOD activity susceptible to inhibition by hydrogen peroxide, but resistant to cyanide, both characteristics of Fe-SODs . In cultured P . marinus trophozoites, indirect immunofluorescence using anti-PmSOD1 antibodies shows colocalization of PmSOD1 product with the mitochondrial marker MitoTracker Red . Further analysis of the leader sequence of the predicted PmSOD1 product revealed similarities to a mitochondrial targeting domain, an unusual observation for Fe-SODs, which are typically localized in the cytoplasm . These results suggest that PmSOD1 encodes a mitochondrial Fe-SOD, which may contribute to P . marinus resistance to exogenous oxidative damage in host phagocytes . The present study constitutes the first report of an endogenous Fe-SOD that is directed to the mitochondria, and suggests that mitochondria targeting sequences have been conserved among diverse branches of the eukaryotes, including the early protista . It also illustrates the potential of complementation-based approaches for further gene discovery and characterization in P . marinus.

Eur J Cell Biol, 2002 Dec, 81(12), 635 - 46
A novel phospholipid-binding protein from the yeast Saccharomyces cerevisiae with dual binding specificities for the transport GTPase Ypt7p and the Sec1-related Vps33p; Lazar T et al.; The gene product of the Saccharomyces cerevisiae open reading frame YDR229w (named IVY1 for: Interacting with Vps33p and Ypt7p) was found to interact with both the GTPase Ypt7p and the Sec1-related Vps33 protein . While deletion of IVY1 does not lead to any recognized change in phenotype, overexpression of Ivy1p leads to fragmentation of the vacuole, missorting of the vacuolar enzyme carboxypeptidase Y (CPY) to the exterior of the cell, and an accumulation of multivesicular bodies inside the cell . All effects caused by the overexpression of Ivy1p can be reset by simultaneously raising the amount of Vps33p . This suppression activity of Vps33p suggests that Ivy1p and Vps33p at least partially counteract the action of each other in the cell . The intracellular level of Ivy1p increases in cells approaching stationary growth phase at which part of the protein is located at the rim of the vacuole . In addition to its specific interactions with members of two regulatory protein families, Ivy1p in vitro shows a marked propensity for binding phospholipids with high affinity.

Plant Cell Physiol, 2003 Jan, 44(1), 3 - 9
Plant RelA/SpoT homolog confers salt tolerance in Escherichia coli and Saccharomyces cerevisiae; Yamada A et al.; To analyze the mechanisms of salt tolerance in the halophyte Suaeda japonica, Escherichia coli was used as a host organism to undertake functional screening of cDNAs encoding proteins that may play an important role for the salt-tolerance mechanisms . A transformant expressing RelA/SpoT homolog (Sj-RSH) was found to have enhanced salt tolerance . In E . coli, RelA/SpoT controlled the amount of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are the effectors of the bacterial stringent response . Complementation analysis using the relA mutant of E . coli showed that Sj-RSH conferred the phenotype associated with (p)ppGpp synthesis . Furthermore, expression of Sj-RSH driven by the GAL1 promoter also gave rise to enhanced salt tolerance in yeast . Northern blot analyses of the yeast transformant revealed that the transcriptional levels of stress responsive genes including GPD1, VMA6, BMH1, HYP1 and HOG1 were clearly enhanced in the Sj-RSH transformant when compared with an empty vector transformant under stress-free and 1.5 M NaCl stress conditions . These results suggest that (p)ppGpp synthesis mediated by plant RelA/SpoT homologs plays a critical role for the transcriptional induction of several stress responsive genes, directly or indirectly in yeast, and that the conserved stress-resistance system may exist in higher plants.

Wei Sheng Wu Xue Bao, 1998 Aug, 38(4), 289 - 94
{Isolation, purification and identification of metallthionein from strain BD 101 of Saccharomyces cerevisiae}; Lin Z et al.; Selection of Saccharomyces cerevisiae BD101 having resistance to metal ions and producing metallothionein by inducer from Saccharomyces . These proteins were isolated and purified by Sephadex G-50 and subsequent DEAE Sepharose CL-4B, then Sephadex G-25 for desalination . There were two iso-MTs for Cu-induced . The, molecular weights were 7 kD and had 60 amino acids . Both Cu-MTs were rich in Cysteine (10%) . 4 atom Cu/6 mole Cys/mole Cu-MTs.

Wei Sheng Wu Xue Bao, 1998 Apr, 38(2), 92 - 7
{Comparative research on two different killer strains of Saccharomyces cerevisiae}; Qin Y et al.; Using two different killer strains SK4(K1 type) and ERR1(K2 type), their killer characters were analysed and find two strains compete with each other when they are mixed and killer characters are related with the growth of strains . Different M-dsRNA plasmids in different killer strains have different molecular lengths, being 1.7 kb and 1.5 kb in SK4 and ERR1, respectively . L-dsRNA plasmids in both strains have the same molecular lengths, 4.0 kb . Cured by high temperature (38 degrees C) and U.V . (15 W, 30 cm, 5 min), killer strains lose their killer characters with the disappearance of M-dsRNA, the curing rate of killer activity is associated with strains and curing conditions . Killer toxins produced by two strains have different killer effects . Toxins of SK4 and ERR1 have the different highest killer activity at pH 4.8, 16 degrees C and pH4.2, 22 degrees C, but both of two toxins have the greatest effect on the sensitive strain in the late-log phase.

Wei Sheng Wu Xue Bao, 1998 Jun, 38(3), 229 - 32
{Purification and properties of intercellular inorganic pyrophosphatase from Saccharomyces cerevisiae}; Gou P et al.; An inorganic pyrophosphatase (EC3.6.1.1) from Saccharomyces cerevisiae was purified to PAGE homogeneity by sonication disruption, (NH4)2SO4 fractionation and DEAE-cellulose column chromatography . The optimum pH and temperature of the enzyme were 7.4-7.8 and 60 degrees C, respectively . The Km was 19.3 mmol/L . The enzyme required Mg2+ as a cofactor for hydrolysis of pyrophosphate and was inhibited by Ca2+, Hg2+, Pb2+, Mn2+.

Wei Sheng Wu Xue Bao, 2000 Feb, 40(1), 105 - 7
{Research of toxin and plasmids of Saccharomyces cerevisiae}; Qin Y et al.; Killer toxin from Saccharomyces cerevisiae SK was isolated by ultrafiltration of culture supernatants and purified by poly(ethylene glycol) . The toxin migrates as one single protein band on SDS-PAGE and its molecular weight is 15 kD . The SK toxin has the greatest lethal effect on the sensitive yeast strain in the lat-lag phase . Extraction and purification of killer heretity factor(dsRNA) from SK found that M-dsRNA plasmid and L-dsRNA plasmid have different molecular lengths being 1.7 kb and 4.0 kb.

DNA Repair (Amst), 2003 Mar 1, 2(3), 315 - 23
hSMUG1 can functionally compensate for Ung1 in the yeast Saccharomyces cerevisiae; Elateri I et al.; There are at least four distinct families of enzymes that recognize and remove uracil from DNA . Family-3 (SMUG1) enzymes have recently been identified and have a preference for uracil in single-stranded DNA when assayed in vitro . Here we investigate the in vivo function of SMUG1 using the yeast Saccharomyces cerevisiae as a model system . These organisms lack a SMUG1 homologue and use a single enzyme, Ung1 to carry out uracil-repair . When a wild-type strain is treated with antifolate agents to induce uracil misincorporation into DNA, S-phase arrest and cellular toxicity occurs . The arrest is characteristic of checkpoint activation due to single-strand breaks caused by continuous uracil removal and self-defeating DNA repair . When uracil-DNA glycosylase is deleted (deltaung1), cells continue through S-phase and arrest at G(2)/M, presumably due to the effects of stable uracil misincorporation in DNA . Pulsed field gel electrophoresis (PFGE) demonstrates that cells are able to complete DNA replication with uracil-substituted DNA and do not experience the extensive strand breakage attributed to uracil-DNA glycosylase-mediated repair . As a result, these cells experience early protection from antifolate-induced cytotoxicity . When either UNG1 or SMUG1 functions are reintroduced back into the null strain and then subjected to antifolate treatment, the cells revert back to the wild-type phenotype as shown by a restored sensitivity to drug and S-phase arrest . The arrest is accompanied by the accumulation of replication intermediates as determined by PFGE . Collectively, these data indicate that SMUG1 can act as a functional homolog of the family-1 uracil-DNA glycosylase enzymes.

DNA Repair (Amst), 2003 Mar 1, 2(3), 243 - 58
Induction of genome instability by DNA damage in Saccharomyces cerevisiae; Myung K et al.; The accumulation of gross chromosomal rearrangements (GCRs) is a characteristic of many types of cancer cells, although it is unclear what defects cause these rearrangements and how the different types of GCRs observed are formed . In the present study, we have used a Saccharomyces cerevisiae system for measuring GCRs to analyze the ability of a variety of DNA damaging agents to induce GCRs . The two most potent inducers of GCRs observed were methyl methane sulfonate (MMS) and HO-endonuclease-induced double strand breaks (DSBs) . Bleomycin, camptothecan and gamma-irradiation induced intermediate levels of GCRs and cisplatin induced very low levels of GCRs whereas N-methyl-NPRIME;-nitro-N-nitrosoguanidine (MNNG) and ethyl methane sulfonate (EMS) primarily induced base substitution mutations . MMS treatment primarily induced rearrangements in which the end of a chromosome was deleted and a new telomere was added (telomere additions) and also induced translocations . Consistent with this GCR spectrum, the formation of MMS-induced GCRs was primarily dependent on telomere maintenance functions and were completely eliminated in mutants that were defective for both telomere maintenance functions and non-homologous end joining (NHEJ) . In contrast, HO-endonuclease DSBs induced mostly translocations and interstitial deletions whereas few telomere additions were observed . Genetic analysis indicated that HO DSB-induced GCRs were suppressed by a number of pathways including the DNA damage checkpoints, DSB repair pathways and NHEJ.

Biochem Soc Trans, 2003 Feb, 31(Pt 1), 242 - 6
Role of the Saccharomyces cerevisiae Rad9 protein in sensing and responding to DNA damage; Toh GW et al.; Eukaryotic cells have evolved surveillance mechanisms, known as DNA-damage checkpoints, that sense and respond to genome damage . DNA-damage checkpoint pathways ensure co-ordinated cellular responses to DNA damage, including cell cycle delays and activation of repair mechanisms . RAD9, from Saccharomyces cerevisiae, was the first damage checkpoint gene to be identified, although its biochemical function remained unknown until recently . This review examines briefly work that provides significant insight into how Rad9 activates the checkpoint signalling kinase Rad53.

J Antibiot (Tokyo), 2002 Nov, 55(11), 962 - 8
Isolation of temperature-sensitive Saccharomyces cerevisiae with a mutation in erg25 for C-4 sterol methyl oxidase; Nose H et al.; C-4 sterol methyl oxidase encoded by the ERG25 gene is a key enzyme in the ergosterol biosynthetic pathway in fungi . ERG25p contains three histidine clusters common to nonheme iron binding enzymes and endoplasmic reticulum retrieval signal . In order to characterize ERG25p, we generated a series of temperature-sensitive(ts) erg25 mutants by random mutagenesis . One of the resulting mutants, the mERG25 strain, accumulated 4,4-dimethlzymosterol at the nonpermissive temperature . Sequence analysis of the mERG25 mutant indicated three amino acid substitutions in ERG25p, namely N48D, V133A, and F135S . These results indicate that the ERG25 gene product is a new antifungal target.

J Ind Microbiol Biotechnol, 2003 Jan, 30(1), 57 - 69 Epub 2003 Jan 03.
Microarray analyses of the metabolic responses of Saccharomyces cerevisiae to organic solvent dimethyl sulfoxide; Zhang W et al.; The toxic effects that organic solvents have on whole cells are important drawbacks in the application of these solvents in the production of fine chemicals by whole-cell stereoselective biotransformations . Although early studies found that organic solvents mainly destroyed the integrity of cell membranes by accumulating in the lipid bilayer of plasma membranes, the cellular metabolic responses to the presence of an organic solvent remain unclear . With the rapid development of genomics, it is possible to study cellular metabolism under perturbed conditions at the genome level . In this paper, the global gene expression profiles of Saccharomyces cerevisiae BY4743 grown in media with a high concentration of the organic solvent dimethyl sulfoxide (DMSO) were determined by microarray analysis of ~6,200 yeast open reading frames (ORFs) . From cells grown in SD minimal medium containing 1.0% (v/v) DMSO, changes in transcript abundance greater than or equal to 2.5-fold were classified . Genomic analyses showed that 1,338 genes were significantly regulated by the presence of DMSO in yeast . Among them, only 400 genes were previously found to be responsive to general environmental stresses, such as temperature shock, amino acid starvation, nitrogen source depletion, and progression into stationary phase . The DMSO-responsive genes were involved in a variety of cellular functions, including carbohydrate, amino acid and lipid metabolism, cellular stress responses, and energy metabolism . Most of the genes in the lipid biosynthetic pathways were down-regulated by DMSO treatment, whereas genes involved in amino acid biosynthesis were mostly up-regulated . The results demonstrate that the application of microarray technology allows better interpretation of metabolic responses, and the information obtained will be useful for the construction of engineered yeast strains with better tolerance of organic solvents.

Free Radic Biol Med, 2003 Feb 1, 34(3), 385 - 93
Regulation of antioxidant enzymes gene expression in the yeast Saccharomyces cerevisiae during stationary phase; Cyrne L et al.; Gene expression of three antioxidant enzymes, Mn superoxide dismutase (MnSOD), Cu,Zn superoxide dismutase (Cu,ZnSOD), and glutathione reductase (GR) was investigated in stationary phase Saccharomyces cerevisiae during menadione-induced oxidative stress . Both GR and Cu,ZnSOD mRNA steady state levels increased, reaching a plateau at about 90 min exposure to menadione . GR mRNA induction was higher than that of Cu,ZnSOD (about 14-fold and 9-fold after 90 min, respectively) . A different pattern of response was obtained for MnSOD mRNA, with a peak at about 15 min (about 8-fold higher) followed by a decrease to a plateau approximately 4-fold higher than the control value . However, these increased mRNA levels did not result in increased protein levels and activities of these enzymes . Furthermore, exposure to menadione decreased MnSOD activity to half its value, indicating that the enzyme is partially inactivated due to oxidative damage . Cu,ZnSOD protein levels were increased 2-fold, but MnSOD protein levels were unchanged after exposure to menadione in the presence of the proteolysis inhibitor phenylmethylsulfonyl fluoride . These results indicate that the rates of Cu,ZnSOD synthesis and proteolysis are increased, while the rates of MnSOD synthesis and proteolysis are unchanged by exposure to menadione . Also, the translational efficiency for both enzymes is probably decreased, since increases in protein levels when proteolysis is inhibited do not reflect the increases in mRNA levels . Our results indicate that oxidative stress modifies MnSOD, Cu,ZnSOD, and GR gene expression in a complex way, not only at the transcription level but also at the post-transcriptional, translational, and post-translational levels.

In Silico Biol, 2002, 2(3), 263 - 73
Mining putative regulatory elements in promoter regions of Saccharomyces cerevisiae; Horng JT et al.; The availability of genome-wide gene expression data provides a unique set of genes from which we can decipher the mechanisms underlying the common transcriptional response . Transcription factors, which can bind to specific DNA sites, cooperatively regulate the transcription of genes . This study attempts to mine putative binding sites to investigate how combinations of the sites predicted from known sites and over-represented repetitive elements are distributed in the promoter regions of groups of functionally related genes . The over-represented repetitive elements appearing in the associations are possible transcription factor binding sites . The deduced association rules would facilitate to predict putative regulatory elements and to identify genes which are potentially co-regulated by the putative regulatory elements . Our proposed approach is applied to Saccharomyces cerevisiae and the promoter regions of yeast ORFs.

Genome Biol . 2003;4(1):R3 . Epub 2002 Dec 17.
Hap4p overexpression in glucose-grown Saccharomyces cerevisiae induces cells to enter a novel metabolic state; Lascaris R et al.; BACKGROUND: Metabolic and regulatory gene networks generally tend to be stable . However, we have recently shown that overexpression of the transcriptional activator Hap4p in yeast causes cells to move to a state characterized by increased respiratory activity . To understand why overexpression of HAP4 is able to override the signals that normally result in glucose repression of mitochondrial function, we analyzed in detail the changes that occur in these cells . RESULTS: Whole-genome expression profiling and fingerprinting of the regulatory activity network show that HAP4 overexpression provokes changes that also occur during the diauxic shift . Overexpression of HAP4, however, primarily acts on mitochondrial function and biogenesis . In fact, a number of nuclear genes encoding mitochondrial proteins are induced to a greater extent than in cells that have passed through a normal diauxic shift: in addition to genes required for mitochondrial energy conservation they include genes encoding mitochondrial ribosomal proteins . CONCLUSIONS: We show that overproduction of a single nuclear transcription factor enables cells to move to a novel state that displays features typical of, but clearly not identical to, other derepressed states.

Nat Biotechnol, 2003 Feb, 21(2), 163 - 70 Epub 2003 Jan 21.
Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library; Feldhaus MJ et al.; A nonimmune library of 10(9) human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow-cytometric sorting . The yeast library can be amplified 10(10)-fold without measurable loss of clonal diversity, allowing its effectively indefinite expansion . The expression, stability, and antigen-binding properties of >50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents . The ability to use multiplex library screening demonstrates the usefulness of this approach for high-throughput antibody isolation for proteomics applications.

Mol Microbiol, 2003 Feb, 47(3), 745 - 54
Domestication of wild Saccharomyces cerevisiae is accompanied by changes in gene expression and colony morphology; Kuthan M et al.; Although colonies from Saccharomyces cerevisiae laboratory strains are smooth, those isolated from nature exhibit a structured fluffy pattern . Environmental scanning electron microscopy shows that the cells within wild fluffy colonies are connected by extracellular matrix (ECM) material . This material contains a protein of about 200 kDa unrelated to the flocculins, proteins involved in cell-cell adhesion in liquid media . The matrix material binds to concanavalin A . Within a few passages on rich agar medium, the wild strains switch from the fluffy to the smooth colony morphology . This domestication is accompanied by loss of the ECM and by extensive changes in gene expression as detected by DNA microarrays . The expression of about 320 genes was changed in smooth colonies . The major changes comprise carbohydrate metabolism, cell wall, water channels, Ty-transposons and subtelomeric genes, iron homeostasis, vitamin metabolism and cell cycle and polarity . The growth in fluffy colonies may represent a metabolic strategy for survival of yeast under unfavourable conditions that is switched off under felicitous laboratory conditions.

Bull Exp Biol Med, 2002 Oct, 134(4), 370 - 3
Comparative study of Saccharomyces cerevisiae LPS; Gordonova IK et al.; LPS of yeast strains producing human epidermal growth factor were studied . Experiments demonstrated the absence of essential differences in the characteristics of these LPS and LPS of nonrecombinant Saccharomyces cerevisiae strains, which allowed us to develop a universal complex technology of simultaneous preparation of heterologous proteins and highly active immunomodulating LPS.

DNA Repair (Amst), 2002 Nov 3, 1(11), 895 - 912
Characterization of nuclease-dependent functions of Exo1p in Saccharomyces cerevisiae; Tran PT et al.; Exo1p is a member of the Rad2p family of structure-specific nucleases that contain conserved N and I nuclease domains . Exo1p has been implicated in numerous DNA metabolic processes, such as recombination, double-strand break repair and DNA mismatch repair (MMR) . In this report, we describe in vitro and in vivo characterization of full-length wild-type and mutant forms of Exo1p . Herein, we demonstrate that full-length yeast Exo1p possesses an intrinsic 5'-3' exonuclease activity as reported previously, but also possesses a flap-endonuclease activity . Our study indicates that Exo1p shares similar, but not identical structure-function relationships to other characterized members of the Rad2p family in the N and I nuclease domains . The two exo1p mutants we examined, showed deficiencies for both double-stranded DNA (dsDNA) 5'-3' exonuclease and flap-endonuclease activities . Examining the genetic interaction of these two exo1 mutations with rad27Delta suggest that the Exo1p flap-endonuclease activity and not the dsDNA 5'-3' exonuclease is redundant to Rad27p for viability . In addition, our in vivo results also indicate that many exo1Delta phenotypes are dependent on the complete catalytic activities of Exo1p . Finally, our findings plus those of other investigators suggest that Exo1p functions both in a catalytic and a structural capacity during DNA MMR.

Mol Biol Cell, 2003 Jan, 14(1), 324 - 33
Interactions among COX1, COX2, and COX3 mRNA-specific translational activator proteins on the inner surface of the mitochondrial inner membrane of Saccharomyces cerevisiae; Naithani S et al.; The core of the cytochrome c oxidase complex is composed of its three largest subunits, Cox1p, Cox2p, and Cox3p, which are encoded in mitochondrial DNA of Saccharomyces cerevisiae and inserted into the inner membrane from the inside . Mitochondrial translation of the COX1, COX2, and COX3 mRNAs is activated mRNA specifically by the nuclearly coded proteins Pet309p, Pet111p, and the concerted action of Pet54p, Pet122p, and Pet494p, respectively . Because the translational activators recognize sites in the 5'-untranslated leaders of these mRNAs and because untranslated mRNA sequences contain information for targeting their protein products, the activators are likely to play a role in localizing translation . Herein, we report physical associations among the mRNA-specific translational activator proteins, located on the matrix side of the inner membrane . These interactions, detected by coimmune precipitation and by two-hybrid experiments, suggest that the translational activator proteins could be organized on the surface of the inner membrane such that synthesis of Cox1p, Cox2p, and Cox3p would be colocalized in a way that facilitates assembly of the core of the cytochrome c oxidase complex . In addition, we found interactions between Nam1p/Mtf2p and the translational activators, suggesting an organized delivery of mitochondrial mRNAs to the translation system.

Mol Biol Cell, 2003 Jan, 14(1), 129 - 41
Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae; Roberts P et al.; Nucleus-vacuole (NV) junctions in Saccharomyces cerevisiae are formed through specific interactions between Vac8p on the vacuole membrane and Nvj1p in the nuclear envelope . Herein, we report that NV junctions in yeast promote piecemeal microautophagy of the nucleus (PMN) . During PMN, teardrop-like blebs are pinched from the nucleus, released into the vacuole lumen, and degraded by soluble hydrolases . PMN occurs in rapidly dividing cells but is induced to higher levels by carbon and nitrogen starvation and is under the control of the Tor kinase nutrient-sensing pathway . Confocal and biochemical assays demonstrate that Nvj1p is degraded in a PMN-dependent manner . PMN occurs normally in apg7-delta cells and is, therefore, not dependent on macroautophagy . Transmission electron microscopy reveals that portions of the granular nucleolus are often sequestered into PMN structures . These results introduce a novel mode of selective microautophagy that targets nonessential components of the yeast nucleus for degradation and recycling in the vacuole.

Mol Cell Biol, 2003 Feb, 23(3), 873 - 86
Systematic mutagenesis of the Saccharomyces cerevisiae MLH1 gene reveals distinct roles for Mlh1p in meiotic crossing over and in vegetative and meiotic mismatch repair; Argueso JL et al.; In eukaryotic cells, DNA mismatch repair is initiated by a conserved family of MutS (Msh) and MutL (Mlh) homolog proteins . Mlh1 is unique among Mlh proteins because it is required in mismatch repair and for wild-type levels of crossing over during meiosis . In this study, 60 new alleles of MLH1 were examined for defects in vegetative and meiotic mismatch repair as well as in meiotic crossing over . Four alleles predicted to disrupt the Mlh1p ATPase activity conferred defects in all functions assayed . Three mutations, mlh1-2, -29, and -31, caused defects in mismatch repair during vegetative growth but allowed nearly wild-type levels of meiotic crossing over and spore viability . Surprisingly, these mutants did not accumulate high levels of postmeiotic segregation at the ARG4 recombination hotspot . In biochemical assays, Pms1p failed to copurify with mlh1-2, and two-hybrid studies indicated that this allele did not interact with Pms1p and Mlh3p but maintained wild-type interactions with Exo1p and Sgs1p . mlh1-29 and mlh1-31 did not alter the ability of Mlh1p-Pms1p to form a ternary complex with a mismatch substrate and Msh2p-Msh6p, suggesting that the region mutated in these alleles could be responsible for signaling events that take place after ternary complex formation . These results indicate that mismatches formed during genetic recombination are processed differently than during replication and that, compared to mismatch repair functions, the meiotic crossing-over role of MLH1 appears to be more resistant to mutagenesis, perhaps indicating a structural role for Mlh1p during crossing over.

Biochim Biophys Acta, 2003 Jan 3, 1625(1), 88 - 97
Expression of human TFIIA subunits in Saccharomyces cerevisiae identifies regions with conserved and species-specific functions; Upadhyaya AB et al.; The transcription factor TFIIA stabilizes the interaction between the TATA-binding protein (TBP) and promoter DNA and facilitates activator function . In yeast, TFIIA is composed of large (TOA1) and small (TOA2) subunits that interact to form a beta-barrel domain and a helix bundle domain . Here we report plasmid shuffle experiments showing that the human subunits (TFIIAalpha/beta, ALF, and TFIIAgamma) are not able to support growth in yeast and that the failure is associated with morphological abnormalities related to cell division . To determine the regions responsible for species specificity, we examined a series of chimeric yeast-human subunits . The results showed that yeast-human hybrids that contained the N-termini of TFIIAgamma or TFIIAalpha/beta were viable, presumably because they could form a functional interspecies alpha-helical bundle . Likewise, a TOA1 hybrid that contained the nonconserved internal region from TFIIAalpha/beta also had no effect on TFIIA function . However, hybrids that contained the acidic region III or C-terminal region IV from TFIIAalpha/beta grew more slowly than the wild-type TOA1 subunit, and if both regions were exchanged, this effect was far more severe . Although these hybrids exchanged sequences which are involved in beta-barrel formation and interactions with TBP, they were all active in a TBP-dependent mobility shift assay . The results suggest that the growth phenotypes of these hybrids might be due to a failure to interact with components of the yeast transcription machinery other than TBP . Finally, we show that sequences from region III of TFIIA large subunits fall into classes that are either highly acidic or that are divergent and nonacidic, and provide the first evidence to suggest that, at least in yeast, this region is important for TFIIA function.

J Virol, 2003 Feb, 77(3), 2116 - 23
Replication of Carnation Italian ringspot virus defective interfering RNA in Saccharomyces cerevisiae; Pantaleo V et al.; Two plasmids from which the sequences coding for the 36- and 95-kDa proteins of Carnation Italian ringspot virus (CIRV) could be transcribed in vivo in the yeast Saccharomyces cerevisiae under the control of the ADH1 promoter and terminator were constructed . The two proteins, which constitute the viral replicase, were correctly translated and integrated into membranes of the yeast cells . An additional plasmid was introduced in yeasts expressing the CIRV replicase, from which a defective interfering (DI) RNA (DI-7 RNA) could be transcribed under the control of the GAL1 promoter and terminated by the Tobacco ringspot virus satellite ribozyme, which cleaved 19 nucleotides downstream of the 3' end of DI RNA . The DI-7 RNA transcripts were amplified by the viral replicase as demonstrated by the restoration of the authentic 3' end, the requirement of a specific cis-acting signal at this terminus, the preferential accumulation of molecules with the authentic 5' terminus (AGAAA), the synthesis of head-to-tail dimers, the presence of negative strands, and the incorporation of 5-bromo-UTP . Additionally, transformation with a dimeric construct of DI-7 RNA led to the synthesis of monomers, mimicking the activity of the viral replicase in plant cells.

J Biol Chem, 2003 Mar 21, 278(12), 10264 - 72 Epub 2003 Jan 13.
Ser3p (Yer081wp) and Ser33p (Yil074cp) are phosphoglycerate dehydrogenases in Saccharomyces cerevisiae; Albers E et al.; Two genes YER081W and YIL074C, renamed SER3 and SER33, respectively, which encode phosphoglycerate dehydrogenases in Saccharomyces cerevisiae were identified . These dehydrogenases catalyze the first reaction of serine and glycine biosynthesis from the glycolytic metabolite 3-phosphoglycerate . Unlike either single mutant, the ser3Delta ser33Delta double mutant lacks detectable phosphoglycerate dehydrogenase activity and is auxotrophic for serine or glycine for growth on glucose media . However, the requirement for the SER-dependent "phosphoglycerate pathway" is conditional since the "glyoxylate" route of serine/glycine biosynthesis is glucose-repressed . Thus, in cells grown on ethanol both expression and activity of all SER-encoded proteins are low, including the remaining enzymes of the phosphoglycerate pathway, Ser1p and Ser2p . Moreover the available nitrogen source regulates the expression of SER genes . However, for only SER33, and not SER3, expression was regulated in relation to the available nitrogen source in a coordinated fashion with SER1 and SER2 . Based on these mRNA data together with data on enzyme activities, Ser33p is likely to be the main isoenzyme of the phosphoglycerate pathway during growth on glucose . Moreover, since phosphoglycerate dehydrogenase activity requires NAD(+) as cofactor, deletion of SER3 and SER33 markedly affected redox metabolism as shown by substrate and product analysis.

J Biol Chem, 2003 Mar 21, 278(12), 10208 - 13 Epub 2003 Jan 10.
Topology models for 37 Saccharomyces cerevisiae membrane proteins based on C-terminal reporter fusions and predictions; Kim H et al.; We provide experimentally based topology models for 37 integral membrane proteins from Saccharomyces cerevisiae . A C-terminal fusion to a dual Suc2/His4C topology reporter has been used to determine the location of the C terminus of each protein relative to the endoplasmic reticulum membrane, and this information is used in conjunction with theoretical topology prediction methods to arrive at a final topology model . We propose that this approach may be used to produce reliable topology models on a proteome-wide scale.

Genetics, 2002 Dec, 162(4), 1583 - 94
Differential processing of leading- and lagging-strand ends at Saccharomyces cerevisiae telomeres revealed by the absence of Rad27p nuclease; Parenteau J et al.; Saccharomyces cerevisiae strains lacking the Rad27p nuclease, a homolog of the mammalian FEN-1 protein, display an accumulation of extensive single-stranded G-tails at telomeres . Furthermore, the lengths of telomeric repeats become very heterogeneous . These phenotypes could be the result of aberrant Okazaki fragment processing of the C-rich strand, elongation of the G-rich strand by telomerase, or an abnormally high activity of the nucleolytic activities required to process leading-strand ends . To distinguish among these possibilities, we analyzed strains carrying a deletion of the RAD27 gene and also lacking genes required for in vivo telomerase activity . The results show that double-mutant strains died more rapidly than strains lacking only telomerase components . Furthermore, in such strains there is a significant reduction in the signals for G-tails as compared to those detected in rad27delta cells . The results from studies of the replication intermediates of a linear plasmid in rad27delta cells are consistent with the idea that only one end of the plasmid acquires extensive G-tails, presumably the end made by lagging-strand synthesis . These data further support the notion that chromosome ends have differential requirements for end processing, depending on whether the ends were replicated by leading- or lagging-strand synthesis.

Genetics, 2002 Dec, 162(4), 1557 - 71
Defects in SPT16 or POB3 (yFACT) in Saccharomyces cerevisiae cause dependence on the Hir/Hpc pathway: polymerase passage may degrade chromatin structure; Formosa T et al.; Spt16/Cdc68, Pob3, and Nhp6 collaborate in vitro and in vivo as the yeast factor SPN, which is homologous to human FACT . SPN/FACT complexes mediate passage of polymerases through nucleosomes and are important for both transcription and replication . An spt16 mutation was found to be intolerable when combined with a mutation in any member of the set of functionally related genes HIR1, HIR2/SPT1, HIR3/HPC1, or HPC2 . Mutations in POB3, but not in NHP6A/B, also display strong synthetic defects with hir/hpc mutations . A screen for other mutations that cause dependence on HIR/HPC genes revealed genes encoding members of the Paf1 complex, which also promotes transcriptional elongation . The Hir/Hpc proteins affect the expression of histone genes and also promote normal deposition of nucleosomes; either role could explain an interaction with elongation factors . We show that both spt16 and pob3 mutants respond to changes in histone gene numbers, but in opposite ways, suggesting that Spt16 and Pob3 each interact with histones but perhaps with different subsets of these proteins . Supporting this, spt16 and pob3 mutants also display different sensitivities to mutations in the N-terminal tails of histones H3 and H4 and to mutations in enzymes that modulate acetylation of these tails . Our results support a model in which SPN/FACT has two functions: it disrupts nucleosomes to allow polymerases to access DNA, and it reassembles the nucleosomes afterward . Mutations that impair the reassembly activity cause chromatin to accumulate in an abnormally disrupted state, imposing a requirement for a nucleosome reassembly function that we propose is provided by Hir/Hpc proteins.

Genetics, 2002 Dec, 162(4), 1545 - 56
A defect of Kap104 alleviates the requirement of mitotic exit network gene functions in Saccharomyces cerevisiae; Asakawa K et al.; A subgroup of the karyopherin beta (also called importin beta) protein that includes budding yeast Kap104 and human transportin/karyopherin beta2 is reported to function as a receptor for the transport of mRNA-binding proteins into the nucleus . We identified KAP104 as a responsible gene for a suppressor mutation of cdc15-2 . We found that the kap104-E604K mutation suppressed the temperature-sensitive growth of cdc15-2 cells by promoting the exit from mitosis and suppressed the temperature sensitivity of various mitotic-exit mutations . The cytokinesis defect of these mitotic-exit mutants was not suppressed by kap104-E604K . Furthermore, the kap104-E604K mutation delays entry into DNA synthesis even at a permissive temperature . In cdc15-2 kap104-E604K cells, SWI5 and SIC1, but not CDH1, became essential at a high temperature, suggesting that the kap104-E604K mutation promotes mitotic exit via the Swi5-Sic1 pathway . Interestingly, SPO12, which is involved in the release of Cdc14 from the nucleolus during early anaphase, also became essential in cdc15-2 kap104-E604K cells at a high temperature . The kap104-E604K mutation caused a partial delocalization of Cdc14 from the nucleolus during interphase . This delocalization of Cdc14 was suppressed by the deletion of SPO12 . These results suggest that a mutation in Kap104 stimulates exit from mitosis through the activation of Cdc14 and implies a novel role for Kap104 in cell-cycle progression in budding yeast.

Biochem J, 2003 Apr 15, 371(Pt 2), 533 - 40
Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase; Shelton SN et al.; The yeast Saccharomyces cerevisiae contains two known phosphoinositide 4-kinases (PI 4-kinases), which are encoded by PIK1 and STT4; both are essential . Pik1p is important for exocytic transport from the Golgi, whereas Stt4p plays a role in cell-wall integrity and cytoskeletal rearrangements . In the present study, we report that cells have a third PI 4-kinase activity encoded by LSB6, a protein identified previously in a two-hybrid screen as interacting with LAS17p . Although Pik1p and Stt4p are closely related members of the Type III class of PI 4-kinases, Lsb6p belongs to the distinct Type II class, based on its amino acid sequence, its sensitivity to inhibition by adenosine and its insensitivity to wortmannin . Lsb6p is the first fungal Type II enzyme cloned . The protein was expressed and purified from Sf9 cells and used to define kinetic parameters . As commonly observed for surface-active enzymes, activities varied both with substrate concentration and lipid/detergent molar ratios . Maximal activities of approx . 100 min(-1) were obtained at the PI/Triton X-100 ratio of 1:5 . The K (m) value for ATP was 266 microM, intermediate between the values reported for mammalian Type II and III kinases . Epitope-tagged protein, expressed in yeast, was entirely particulate, and about half of it could be extracted with non-ionic detergent . Lsb6p-green fluorescent protein was found both on vacuolar membranes and on the plasma membrane, suggesting a role in endocytic or exocytic pathways.

J Biol Chem, 2003 Mar 14, 278(11), 9382 - 7 Epub 2003 Jan 08.
Checkpoint arrest signaling in response to UV damage is independent of nucleotide excision repair in Saccharomyces cerevisiae; Zhang H et al.; The recognition of DNA double-stranded breaks or single-stranded DNA gaps as a precondition for cell cycle checkpoint arrest has been well established . However, how bulky base damage such as UV-induced pyrimidine dimers elicits a checkpoint response has remained elusive . Nucleotide excision repair represents the main pathway for UV dimer removal that results in strand interruptions . However, we demonstrate here that Rad53p hyperphosphorylation, an early event of checkpoint signaling in Saccharomyces cerevisiae, is independent of nucleotide excision repair (NER), even if replication as a source of secondary DNA damage is excluded . Thus, our data hint at primary base damage or at UV damage (primary or secondary) that does not need to be processed by NER as the relevant substrate of damage-sensing checkpoint proteins.

Yeast, 2003 Jan 30, 20(2), 117 - 32
Metabolic flux analysis of RQ-controlled microaerobic ethanol production by Saccharomyces cerevisiae; Franzen CJ; Microaerobic ethanol production by Saccharomyces cerevisiae CBS 8066 was investigated at different growth rates in respiratory quotient (RQ)-controlled continuous culture . The RQ was controlled by changing the inlet gas composition by a feedback controller while keeping other parameters constant . The ethanol yield increased slightly from the anaerobic values with decreasing RQ, reaching a broad maximum at RQ 20 to 12 . There was little or no glycerol production at RQ values below 17 over a wide range of dilution rates . Metabolic flux analysis revealed that a decrease in the ethanol yield at RQ 6 coincided with the cyclic, oxidative operation of the TCA cycle reactions . The model indicated that respiratory dissimilation of glucose only occurs when the oxygen uptake rate is high enough to completely substitute for glycerol formation . The cytosolic and the mitochondrial NADH balances were important for determining the flux distributions . The smallest deviations between estimated and measured product yields were obtained when the unknown co-factor requirements of a limited number of biosynthetic reactions were chosen so that the amount of excess NADH formed in biosynthesis was minimized . The biomass yield was positively correlated with the net amount of NADH reoxidized in respiration and glycerol formation, indicating that the turnover of excess NADH from biosynthesis is an important factor influencing the biomass yield under oxygen-limiting conditions .

Yeast, 2003 Jan 30, 20(2), 97 - 108
Casein kinase II phosphorylates translation initiation factor 5 (eIF5) in Saccharomyces cerevisiae; Maiti T et al.; Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S initiation complex (40S-eIF3-mRNA-Met-tRNA(f)-eIF2-GTP) to promote the hydrolysis of ribosome-bound GTP . In Saccharomyces cerevisiae, eIF5 is encoded by a single-copy essential gene, TIF5, that is required for cell growth and viability . In this work, we show that eIF5 immunoprecipitated from cell-free extracts of (32)P-labelled yeast cells is phosphorylated on multiple serine residues . Phosphopeptide mapping reveals four major sites of phosphorylation that appear to be identical to recombinant yeast eIF5 sites phosphorylated in vitro by casein kinase II . Furthermore, analysis of eIF5 isolated from a yeast strain having a conditional mutant of casein kinase II indicates that phosphorylation of eIF5 is completely abolished at the non-permissive temperature . Additionally, haploid yeast strains were constructed to contain Ser-to-Ala mutations at the five casein kinase II consensus sequences in eIF5; in these cells, eIF5 phosphorylation was absent . Surprisingly, substitution of the TIF5 gene mutated at these sites for the wild-type gene had no obvious effect on cell growth under normal growth conditions . The implications of these results in eIF5 function are discussed .

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Jan, 35(1), 86 - 91
{Involvement of PHO80 and PHO85 genes in Saccharomyces cerevisiae ion tolerance}; Mao XC et al.; PHO85 is a versatile gene in Saccharomyces cerevisiae, which is involved in metabolism of inorganic phosphate and usage of carbon source, accumulation of glycogen, regulation of protein stability and cell cycle control . The viability of wild type budding yeast strain YPH499 and its derivative pho85Delta mutant, pho80 mutant, and pap1(pcl-7)Delta mutant in different cations were investigated and their tolerance to the cations(LC(50)) was measured . The results showed that the deletion of PHO85 or PHO80 gene both increased sensibility of Sacchromyces cerevisiae to ions K(+), Mg(2+), Zn(2+), Ca(2+) and Mn(2+), while the deletion of pap1(pcl-7) gene did not lead to such phenotype . The difference between the patterns of relative growth curve of the mutants and wild type strain in the above ions also implied that PHO80 was the unique PCLs in complex with PHO85 CDK, that were contributed to K(+) and Mg(2+) ion homeostasis control and there were some other PCLs besides PHO80 that were involved in Zn(2+), Ca(2+) and Mn(2+) tolerance regulation as cyclin of PHO85 CDK . Furthermore, the amount of the total cellular calcium of pho85Delta mutant, pho80Delta mutant and YPH499 indicated that the ability of calcium accumulation of pho85 mutant and pho80Delta mutant was impaired.

J Biol Chem, 2003 Mar 7, 278(10), 8219 - 23 Epub 2003 Jan 03.
Bipartite signals mediate subcellular targeting of tail-anchored membrane proteins in Saccharomyces cerevisiae; Beilharz T et al.; Tail-anchored proteins have an NH(2)-terminal cytosolic domain anchored to intracellular membranes by a single, COOH-terminal, transmembrane segment . Sequence analysis identified 55 tail-anchored proteins in Saccharomyces cerevisiae, with several novel proteins, including Prm3, which we find is required for karyogamy and is tail-anchored in the nuclear envelope . A total of six tail-anchored proteins are present in the mitochondrial outer membrane and have relatively hydrophilic transmembrane segments that serve as targeting signals . The rest, by far the majority, localize via a bipartite system of signals: uniformly hydrophobic tail anchors are first inserted into the endoplasmic reticulum, and additional segments within the cytosolic domain of each protein can dictate subsequent sorting to a precise destination within the cell.

Appl Environ Microbiol, 2003 Jan, 69(1), 212 - 9
L-proline accumulation and freeze tolerance of Saccharomyces cerevisiae are caused by a mutation in the PRO1 gene encoding gamma-glutamyl kinase; Morita Y et al.; We previously isolated a mutant which showed a high tolerance to freezing that correlated with higher levels of intracellular L-proline derived from L-proline analogue-resistant mutants . The mutation responsible for the analogue resistance and L-proline accumulation was a single nuclear dominant mutation . By introducing the mutant-derived genomic library into a non-L-proline-utilizing strain, the mutant was found to carry an allele of the wild-type PRO1 gene encoding gamma-glutamyl kinase, which resulted in a single amino acid replacement; Asp (GAC) at position 154 was replaced by Asn (AAC) . Interestingly, the allele of PRO1 was shown to enhance the activities of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase, both of which catalyze the first two steps of L-proline synthesis from L-glutamate and which together may form a complex in vivo . When cultured in liquid minimal medium, yeast cells expressing the mutated gamma-glutamyl kinase were found to accumulate intracellular L-proline and showed a prominent increase in cell viability after freezing at -20 degrees C compared to the viability of cells harboring the wild-type PRO1 gene . These results suggest that the altered gamma-glutamyl kinase results in stabilization of the complex or has an indirect effect on gamma-glutamyl phosphate reductase activity, which leads to an increase in L-proline production in Saccharomyces cerevisiae . The approach described in this paper could be a practical method for breeding novel freeze-tolerant yeast strains.

Mol Cell Biol, 2003 Jan, 23(2), 677 - 86
The transcription factor Rim101p governs ion tolerance and cell differentiation by direct repression of the regulatory genes NRG1 and SMP1 in Saccharomyces cerevisiae; Lamb TM et al.; Environmental pH changes have broad consequences for growth and differentiation . The best-understood eukaryotic pH response pathway acts through the zinc-finger transcription factor PacC of Aspergillus nidulans, which activates alkaline pH-induced genes directly . We show here that Saccharomyces cerevisiae Rim101p, the pH response regulator homologous to PacC, functions as a repressor in vivo . Chromatin immunoprecipitation assays show that Rim101p is associated in vivo with the promoters of seven Rim101p-repressed genes . A reporter gene containing deduced Rim101p binding sites is negatively regulated by Rim101p and is associated with Rim101p in vivo . Deletion mutations of the Rim101p repression targets NRG1 and SMP1 suppress rim101Delta mutant defects in ion tolerance, haploid invasive growth, and sporulation . Therefore, transcriptional repression is the main biological function of Rim101p . The Rim101p repression target Nrg1p is in turn required for repression of two alkaline pH-inducible genes, including the Na+ pump gene ENA1, which is required for ion tolerance . Thus, Nrg1p, a known transcriptional repressor, functions as an inhibitor of alkaline pH responses . Our findings stand in contrast to the well-characterized function of PacC as a direct activator of alkaline pH-induced genes yet explain many aspects of Rim101p and PacC function in other organisms.

DNA Repair (Amst), 2002 Jan 22, 1(1), 27 - 40
Complementation between N-terminal Saccharomyces cerevisiae mre11 alleles in DNA repair and telomere length maintenance; Lee SE et al.; In Saccharomyces cerevisiae, Mre11p, Rad50p, and Xrs2p function as a multiprotein complex that has a central role in several DNA repair mechanisms . Though Mre11p has both single-stranded and double-stranded 3'-5' exonuclease activity in vitro, null mutants of MRE11, RAD50, and XRS2 exhibit reduced 5'-3' resection of HO-induced double-strand breaks (DSBs) in vivo . In this study, we analyzed four mre11 mutants harboring changes in the N-terminus of Mre11p where the four phosphoesterase motifs specify the in vitro nuclease activities of Mre11p and its homologues . We find that the 5'-3' resection defects in vivo do not correlate with several mitotic phenotypes: non-homologous end-joining (NHEJ), telomere length maintenance, and adaptation to the DNA damage-inducible G2/M checkpoint . Overexpression of the 5'-3' exonuclease Exo1p in a mre11Delta strain partially increased 5'-3' resection and partially suppressed both methyl methanesulfonate (MMS) hypersensitivity and adaptation phenotypes, but did not affect telomere length or NHEJ . Surprisingly, the co-expression of two alleles, mre11-58S and mre11-N113S, each of which confers MMS hypersensitivity and short telomeres, can fully complement the MMS sensitivity and shortened telomere length of mre11Delta cells . We propose that at least two separate activities associated with the N-terminus of Mre11p are required for its mitotic function.

DNA Repair (Amst), 2002 Apr 29, 1(4), 287 - 97
HSM2 (HMO1) gene participates in mutagenesis control in yeast Saccharomyces cerevisiae; Alekseev SY et al.; We have previously reported about a new Saccharomyces cerevisiae mutation, hsm2-1, that results in increase of both spontaneous and UV-induced mutation frequencies but does not alter UV-sensitivity . Now HSM2 gene has been genetically and physically mapped and identified as a gene previously characterized as HMO1, a yeast homologue of human high mobility group genes HMG1/2 . We found that hsm2 mutant is slightly deficient in plasmid-borne mismatch repair . We tested UV-induced mutagenesis in double mutants carrying hsm2-1 mutation and a mutation in a gene of principal damaged DNA repair pathways (rad2 and rev3) or in a mismatch repair gene (pms1 and recently characterized in our laboratory hsm3) . The frequency of UV-induced mutations in hsm2 rev3 was not altered in comparison with single rev3 mutant . In contrast, the interaction of hsm2-1 with rad2 and pms1 was characterized by an increased frequency of UV-induced mutations in comparison with single rad2 and pms1 mutants . The UV-induced mutation frequency in double hsm2 hsm3 mutant was lower than in the single hsm2 and hsm3 mutants . The role of the HSM2 gene product in control of mutagenesis is discussed.

J Cell Sci, 2003 Feb 1, 116(Pt 3), 561 - 9
Nuclear oscillations and nuclear filament formation accompany single-strand annealing repair of a dicentric chromosome in Saccharomyces cerevisiae; Thrower DA et al.; Dicentric chromosomes undergo breakage during mitosis as a result of the attachment of two centromeres on one sister chromatid to opposite spindle poles . Studies utilizing a conditional dicentric chromosome III in Saccharomyces cerevisiae have shown that dicentric chromosome repair occurs primarily by deletion of one centromere via a RAD52-dependent recombination pathway . We report that dicentric chromosome resolution requires RAD1, a gene involved in the single-strand annealing DNA repair pathway . We additionally show that single-strand annealing repair of a dicentric chromosome can occur in the absence of RAD52 . RAD52-independent repair requires the adaptation-defective cdc5-ad allele of the yeast polo kinase and the DNA damage checkpoint gene RAD9 . Dicentric chromosome breakage in cdc5-ad rad52 mutant cells is associated with a prolonged mitotic arrest, during which nuclei undergo microtubule-dependent oscillations, accompanied by dynamic changes in nuclear morphology . We further demonstrate that the frequency of spontaneous direct repeat recombination is suppressed in yeast cells treated with benomyl, a drug that perturbs microtubules . Our findings indicate that microtubule-dependent processes facilitate recombination.

Folia Microbiol (Praha), 2002, 47(5), 507 - 10
Effect of Brestan on Saccharomyces cerevisiae during continuous cultivation; Razmovski R et al.; An increase in Brestan concentration in nutrient media decreased the content of protein, phosphorus, total ribonucleic acid, activity of pyruvate carboxylase and isocitrate lyase in cells of Saccharomyces cerevisiae parent strain and respiratory deficient (RD) mutant while the trehalose content increased . The respiration quotient value for the RD mutant was higher than for the parent strain . The RD mutant lacked cytochrome aa3; cytochrome c and b contents were lower than those of the parent strain.

Genes Dev, 2002 Dec 15, 16(24), 3142 - 57
Nob1p is required for biogenesis of the 26S proteasome and degraded upon its maturation in Saccharomyces cerevisiae; Tone Y et al.; Nob1p is a nuclear protein that forms a complex with the 19S regulatory particle of the 26S proteasome and with uncharacterized nuclear protein Pno1p . Overexpression of NOB1 overrode the defects in maturation of the 20S proteasome of ump1Delta cells, and temperature-sensitive nob1 and pno1 mutants exhibited defects in the processing of the beta subunits and in the assembly of the 20S and the 26S proteasomes . A defect in either NOB1 or PNO1 caused accumulation of newly formed Pre6p in the cytoplasm, whereas Pre6p of the ump1Delta strain accumulated in the nucleus irrespective of the temperature . Here we present a model proposing that (1) Nob1p serves as a chaperone to join the 20S proteasome with the 19S regulatory particle in the nucleus and facilitates the maturation of the 20S proteasome and degradation of Ump1p, and (2) Nob1p is then internalized into the 26S proteasome and degraded to complete 26S proteasome biogenesis.

J Gen Appl Microbiol, 1998 Aug, 44(4), 275 - 281
Region specificity of chromosome III on gene expression in the yeast Saccharomyces cerevisiae; Yamane S et al.; A single copy of a reporter gene cassette, such as PGKP-lacZ-LEU2 (promoter-reporter-marker gene) cassette, was inserted into one of 32 positions along chromosome III in Saccharomyces cerevisiae with an interval of approximately 10 kb . The amounts of translational gene product (beta-galactosidase) synthesized by the cassette-transformed cells were then determined . The region specificity in chromosome III could be demonstrated in gene expression: two higher-expressed regions (hot regions), 133 and 199 (MAT) regions, and seven lower-expressed regions (cold regions) . For the steady and high production of polypeptide, foreign gene products, by yeast, we would like to state that we hope for an insertion of the artificially prepared gene cassette {(promoter)-(foreign gene)-(marker gene) } into a hot region, such as 199 (MAT) region of chromosome III.

J Gen Appl Microbiol, 1997 Jun, 43(3), 157 - 162
A glutamine synthetase mutant of Saccharomyces cerevisiae shows defect in cell wall; Shimizu J et al.; To study the organization and biosynthesis of the yeast cell wall, hypo-osmolarity-sensitive mutants of Saccharomyces cerevisiae were analyzed . Cells of JS4 were irregular in shape and fragile . Calcofluor staining and quantitative analysis indicated that the chitin content was reduced . By DNA cloning and genetic analysis, the mutation hpo1-1 was found to be allelic to GLN1 which encodes glutamine synthetase . The glutamine content was significantly low in JS4, and the mutant was recovered from the cell wall defect by supplying glutamine in the medium . Partial inhibition of glutamine synthetase by phosphinothricin also induced defects in the cell wall . These results indicate that the shortage of glutamine affects cell wall integrity prior to other cellular functions.

Biochemistry, 2002 Dec 31, 41(52), 15536 - 42
The solution structure of apocalmodulin from Saccharomyces cerevisiae implies a mechanism for its unique Ca2+ binding property; Ishida H et al.; We have determined the solution structure of calmodulin (CaM) from yeast (Saccharomyces cerevisiae) (yCaM) in the apo state by using NMR spectroscopy . yCaM is 60% identical in its amino acid sequence with other CaMs, and exhibits its unique biological features . yCaM consists of two similar globular domains (N- and C-domain) containing three Ca(2+)-binding motifs, EF-hands, in accordance with the observed 3 mol of Ca(2+) binding . In the solution structure of yCaM, the conformation of the N-domain conforms well to the one of the expressed N-terminal half-domains of yCaM {Ishida, H., et al . (2000) Biochemistry 39, 13660-13668} . The conformation of the C-domain basically consists of a pair of helix-loop-helix motifs, though a segment corresponding to the forth Ca(2+)-binding site of CaM deviates in its primary structure from a typical EF-hand motif and loses the ability to bind Ca(2+) . Thus, the resulting conformation of each domain is essentially identical to the corresponding domain of CaM in the apo state . A flexible linker connects the two domains as observed for CaM . Any evidence for the previously reported interdomain interaction in yCaM was not observed in the solution structure of the apo state . Hence, the interdomain interaction possibly occurs in the course of Ca(2+) binding and generates a cooperative Ca(2+) binding among all three sites . Preliminary studies on a mutant protein of yCaM, E104Q, revealed that the Ca(2+)-bound N-domain interacts with the apo C-domain and induces a large conformational change in the C-domain.

J Biol Chem, 2003 Mar 7, 278(10), 7810 - 21 Epub 2002 Dec 22.
Intravacuolar membrane lysis in Saccharomyces cerevisiae . Does vacuolar targeting of Cvt17/Aut5p affect its function?
Epple UD, Eskelinen EL, Thumm M.
The integral membrane protein Cvt17/Aut5p is a putative lipase essential for intravacuolar lysis of autophagic bodies . It is localized at the endoplasmic reticulum, from which it is targeted via the multivesicular body (MVB) pathway to intravacuolar MVB vesicles . Proteinase protection experiments now demonstrate that the Aut5 amino terminus is located in the cytosol, and the carboxyl terminus is located inside the ER lumen . In contrast to procarboxypeptidase S, targeting of Cvt17/Aut5p to MVB vesicles is not blocked in cells lacking the ubiquitin ligase Tul1p or the deubiquitinating enzyme Doa4p . Also, truncation of the amino-terminal cytosolic Cvt17/Aut5p domain does not inhibit its targeting to MVB vesicles . These findings suggest that similar to Sna3p sorting of Cvt17/Aut5p to MVB vesicles is independent of ubiquitination . By fusing the ER retention/retrieval signal HDEL to the carboxyl terminus of Cvt17/Aut5p, we generated a construct that is held back at the ER . Detailed analysis of this construct suggests an essential role of vacuolar targeting of Cvt17/Aut5p for its function . Consistently, aut5Delta cells are found impaired in vacuolar degradation of autophagocytosed peroxisomes . Importantly, biochemical and morphological data further suggest involvement of Cvt17/Aut5p in disintegration of intravacuolar MVB vesicles . This points to a general function of Cvt17/Aut5p in intravacuolar membrane breakdown.

J Biol Chem, 2003 Mar 7, 278(10), 8028 - 34 Epub 2002 Dec 23.
The catabolism of amino acids to long chain and complex alcohols in Saccharomyces cerevisiae; Dickinson JR et al.; The catabolism of phenylalanine to 2-phenylethanol and of tryptophan to tryptophol were studied by (13)C NMR spectroscopy and gas chromatography-mass spectrometry . Phenylalanine and tryptophan are first deaminated (to 3-phenylpyruvate and 3-indolepyruvate, respectively) and then decarboxylated . This decarboxylation can be effected by any of Pdc1p, Pdc5p, Pdc6p, or Ydr380wp; Ydl080cp has no role in the catabolism of either amino acid . We also report that in leucine catabolism Ydr380wp is the minor decarboxylase . Hence, all amino acid catabolic pathways studied to date use a subtly different spectrum of decarboxylases from the five-membered family that comprises Pdc1p, Pdc5p, Pdc6p, Ydl080cp, and Ydr380wp . Using strains containing all possible combinations of mutations affecting the seven AAD genes (putative aryl alcohol dehydrogenases), five ADH genes, and SFA1, showed that the final step of amino acid catabolism (conversion of an aldehyde to a long chain or complex alcohol) can be accomplished by any one of the ethanol dehydrogenases (Adh1p, Adh2p, Adh3p, Adh4p, Adh5p) or by Sfa1p (formaldehyde dehydrogenase.)

Mol Cancer Res, 2002 Dec, 1(2), 103 - 12
Damage recovery pathways in Saccharomyces cerevisiae revealed by genomic phenotyping and interactome mapping; Begley TJ et al.; We have generated a genomic phenotyping database identifying hundreds of Saccharomyces cerevisiae genes important for viable cellular recovery after mutagen exposure . Systematic phenotyping of 1,615 gene deletion strains produced distinctive signatures for each of four mutagens . Integration of the phenotyping database with mutagen-induced transcriptional profiling data demonstrated that being transcriptionally responsive to a mutagen does not predict whether or not a gene contributes to recovery from exposure to that mutagen . Computational integration of the database with 4,025 interacting proteins, comprising the yeast interactome, identified several multiprotein networks important for damage recovery . Some networks were associated with DNA metabolism and cell cycle control functions, but most were associated with unexpected functions such as cytoskeleton remodeling, chromatin remodeling, protein, RNA, and lipid metabolism . Hence, a plethora of responses other than the DNA damage response is important for recovery . These network mapping results demonstrate how systematic phenotypic assays may be linked directly to underlying molecular mechanisms.

J Biol Chem, 2003 Feb 28, 278(9), 6731 - 40 Epub 2002 Dec 19.
Domain analysis of the Saccharomyces cerevisiae heterogeneous nuclear ribonucleoprotein, Nab2p . Dissecting the requirements for Nab2p-facilitated poly(A) RNA export; Marfatia KA et al.; Mature poly(A) RNA transcripts are exported from the nucleus in complex with heterogeneous nuclear ribonucleoproteins (hnRNPs) . Nab2p is an essential Saccharomyces cerevisiae hnRNP protein that interacts with poly(A) RNA and shuttles between the nucleus and cytoplasm . Functional Nab2p is required for export of poly(A) RNA from the nucleus . The Nab2 protein consists of the following four domains: a unique N-terminal domain, a glutamine-rich domain, an arginine-glycine (RGG) domain, and a zinc finger domain . We generated Nab2p deletion mutants to analyze the contribution of each domain to the in vivo function of Nab2p . We first tested whether the deletion mutants could replace the essential NAB2 gene . We then examined the impact of these mutations on Nab2p localization, poly(A) RNA localization, and association of Nab2p with poly(A) RNA . Our analyses revealed that the N-terminal domain is required for nuclear export of both poly(A) RNA and Nab2p . We confirm that the RGG domain is important for Nab2p import in vivo . Finally, the zinc finger domain is critical for the interaction between Nab2p and poly(A) RNA in vivo . Our data support a model where Nab2p associates with poly(A) RNA in the nucleus through the zinc finger domain and facilitates the export of the poly(A) RNA through protein interactions mediated by the N-terminal domain.

Int J Cancer, 2003 Feb 20, 103(5), 624 - 30
Analysis of the human APC mutation spectrum in a saccharomyces cerevisiae strain with a mismatch repair defect; Otsuka K et al.; Somatic APC mutations in colorectal tumors with an RER phenotype reflect excessive frameshift mutations, especially in simple repetition tracts within the coding sequence . Because this type of mutation is characteristic of cells with a deficient DNA MMR system, the APC mutation signature of RER tumors may be attributable to a defect in the MMR system . However, there is little experimental evidence to prove that the spectrum of mutations and the APC gene distribution are directly influenced by MMR system defects . We therefore examined the mutation spectrum of the MCR of the APC gene after transfection into both MMR-proficient and MMR-deficient yeast strains and compared it with a previously reported human APC mutation database . Small insertions or deletions in mono- or dinucleotide repeats were more common in the MMR-deficient than in the MMR-proficient strain (91.2% vs . 38.1%, Fisher's exact test p < 0.0001) . Furthermore, the 2 mutation hot spots, 4385-4394(AG)(5) and 4661-4666(A)(6), found in the yeast system corresponded with those in human tumors . Combining our data with those from human tumors, there appears to be hypermutable mutations in specific simple repetitive sequences within the MCR, which are more prevalent in MMR-deficient cells and RER tumors than in MMR-proficient cells and non-RER tumors . We therefore consider that the differences in the spectra of RER and non-RER tumors are attributable at least in part to the MMR system of the host cells .

J Biol Chem, 2003 Feb 28, 278(9), 7325 - 34 Epub 2002 Dec 18.
Lcb4p is a key regulator of ceramide synthesis from exogenous long chain sphingoid base in Saccharomyces cerevisiae; Funato K et al.; Long chain sphingoid bases (LCBs) and their phosphates (LCBPs) are not only important intermediates in ceramide biosynthesis but also signaling molecules in the yeast, Saccharomyces cerevisiae . Their cellular levels, which control multiple cellular events in response to external and intrinsic signals, are tightly regulated by coordinated action of metabolic enzymes such as LCB kinase and LCBP phosphatase . However, little is known about the mechanisms by which the two enzymes generate biosynthetic or signaling outputs . It has been shown that the LCBP phosphatase, Lcb3p, is required for efficient ceramide synthesis from exogenous LCB . Here we present direct evidence that the major LCB kinase, Lcb4p, but not the minor kinase, Lcb5p, regulates synthesis of ceramide from exogenously added LCB . Surprisingly, our biochemical evidence suggests that the LCBP used for ceramide synthesis must be generated on the membrane . Our data show that Lcb4p is tightly associated with membranes and is localized to the endoplasmic reticulum where it can work in concert with Lcb3p . These results raise the conceptually attractive possibility that membrane-associated and cytosolic Lcb4p play distinct roles to differentially generate biosynthetic and signaling pools of LCBP.

Mol Microbiol, 2003 Jan, 47(1), 223 - 34
Amiodarone induces a caffeine-inhibited, MID1-depedent rise in free cytoplasmic calcium in Saccharomyces cerevisiae; Courchesne WE et al.; Calcium signalling is involved in myriad cellular processes such as mating morphogenesis . Mating in yeast induces changes in cell morphology with a concomitant increase in calcium uptake that is dependent on the MID1 and CCH1 genes . Mid1p and Cch1p are believed to function in a capacitive calcium entry (CCE)-like process . Amiodarone alters mammalian calcium channel activity but, despite its clinical importance, its molecular mechanisms are not clearly defined . We have shown previously that amiodarone has fungicidal activity against a broad array of fungi . We show here that amiodarone causes a dramatic increase in cytoplasmic calcium ({Ca2+}cyt) in Saccharomyces cerevisiae . The majority of this increase is dependent on extracellular Ca2+ nonetheless, a significant increase in {Ca2+}cyt is still induced by amiodarone when no uptake of extracellular Ca2+ can occur . The influx of extracellular Ca2+ may be a direct effect of amiodarone on a membrane transporter or may be by a CCE mechanism . Uptake of the extracellular Ca2+ is inhibited by caffeine and reduced in strains deleted for the mid1 gene, but not in cells deleted for cch1 . Our data are the first demonstrating control of yeast calcium channels by amiodarone and caffeine.

Mol Microbiol, 2003 Jan, 47(1), 119 - 34
Mss11p is a transcription factor regulating pseudohyphal differentiation, invasive growth and starch metabolism in Saccharomyces cerevisiae in response to nutrient availability; Gagiano M et al.; In Saccharomyces cerevisiae, the cell surface protein, Muc1p, was shown to be critical for invasive growth and pseudohyphal differentiation . The transcription of MUC1 and of the co-regulated STA2 glucoamylase gene is controlled by the interplay of a multitude of regulators, including Ste12p, Tec1p, Flo8p, Msn1p and Mss11p . Genetic analysis suggests that Mss11p plays an essential role in this regulatory process and that it functions at the convergence of at least two signalling cascades, the filamentous growth MAPK cascade and the cAMP-PKA pathway . Despite this central role in the control of filamentous growth and starch metabolism, the exact molecular function of Mss11p is unknown . We subjected Mss11p to a detailed molecular analysis and report here on its role in transcriptional regulation, as well as on the identification of specific domains required to confer transcriptional activation in response to nutritional signals . We show that Mss11p contains two independent transactivation domains, one of which is a highly conserved sequence that is found in several proteins with unidentified function in mammalian and invertebrate organisms . We also identify conserved amino acids that are required for the activation function.

Curr Genet, 2002 Dec, 42(3), 140 - 6 Epub 2002 Nov 29.
The influence of the mismatch-repair system on stationary-phase mutagenesis in the yeast Saccharomyces cerevisiae; Halas A et al.; Stationary-phase (also called adaptive) mutation occurs in non-dividing cells during prolonged non-lethal selective pressure, e.g . starvation for an essential amino acid . Because in such conditions no DNA replication is observed, mutations probably arise as a result of inefficient DNA repair . In order to understand the role of the yeast mismatch-repair (MMR) system in the mutagenesis of stationary-phase cells, we studied the effects of deletions in genes encoding MutS- and MutL-related proteins on the reversion frequency of the lys2 Delta Bgl frameshift mutation . We found that the level of Lys(+) reversion was increased in all MMR mutants, with the strongest effect observed in a MSH2 (MUTS homologue)-deprived strain . Disruption of the MSH3 or MSH6 genes (also MUTS homologues) resulted in elevation of the mutation frequency and rate, but to a lesser degree than that caused by the inactivation of MSH2 . MutL-related proteins were also required for mutation avoidance in stationary-phase cells, but to a lesser extent than MutS homologues . Among MutL homologues, Mlh1 seems to play the major role in this process, while Pms1 and Mlh3 are partially redundant and appear to substitute for each other . These data suggest that MMR proteins, particularly MutS homologues, are involved in the control of mutability in stationary-phase yeast cells.

Nucleic Acids Res . 2002 Dec 15;30(24):e136.
Promoter-trapping in Saccharomyces cerevisiae by radiation-assisted fragment insertion; Kiechle M et al.; Non-homologous insertion (NHI) of DNA fragments into genomic DNA is a method widely used in insertional mutagenesis screens . In the yeast Saccharomyces cerevisiae, the efficiency of NHI is very low . Here we report that its efficiency can be increased by gamma-irradiation of recipient cells at the time of transformation . Radiation-assisted NHI depends on YKU70, but its efficiency is not improved by inactivation of RAD5 or RAD52 . In a pilot study, we generated 102 transformant clones expressing a lacZ reporter gene under standard conditions (30 degrees C, rich medium) . The site of insertion was determined in a subset of eight clones in which lacZ expression was altered by UV-irradiation . A comparison with published data revealed that three of the eight genes identified in our screen have not been targeted by large-scale transposon-based insertion screens . This suggests that radiation-assisted NHI offers a more homogeneous coverage of the genome than methods relying on transposons or retroviral elements.

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