Yeasts

J Biol Chem, 2001 Mar 16, 276(11), 7705 - 8 Epub 2001 Jan 19.
A novel family of phosphatidylinositol 4-kinases conserved from yeast to humans; Barylko B et al.; Phosphatidylinositolpolyphosphates (PIPs) are centrally involved in many biological processes, ranging from cell growth and organization of the actin cytoskeleton to endo- and exocytosis . Phosphorylation of phosphatidylinositol at the D-4 position, an essential step in the biosynthesis of PIPs, appears to be catalyzed by two biochemically distinct enzymes . However, only one of these two enzymes has been molecularly characterized . We now describe a novel class of phosphatidylinositol 4-kinases that probably corresponds to the missing element in phosphatidylinositol metabolism . These kinases are highly conserved evolutionarily, but unrelated to previously characterized phosphatidylinositol kinases, and thus represent the founding members of a new family . The novel phosphatidylinositol 4-kinases, which are widely expressed in cells, only phosphorylate phosphatidylinositol, are potently inhibited by adenosine, but are insensitive to wortmannin or phenylarsine oxide . Although they lack an obvious transmembrane domain, they are strongly attached to membranes by palmitoylation . Our data suggest that independent pathways for phosphatidylinositol 4-phosphate synthesis emerged during evolution, possibly to allow tight temporal and spatial control over the production of this key signaling molecule.

J Bacteriol, 2001 Apr, 183(7), 2306 - 15
Functional domains of yeast plasmid-encoded Rep proteins; Sengupta A et al.; Both of the Saccharomyces cerevisiae 2 microm circle-encoded Rep1 and Rep2 proteins are required for efficient distribution of the plasmid to daughter cells during cellular division . In this study two-hybrid and in vitro protein interaction assays demonstrate that the first 129 amino acids of Rep1 are sufficient for self-association and for interaction with Rep2 . Deletion of the first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still allowed some self-association, suggesting that different but overlapping domains specify these interactions . Amino- or carboxy-terminally truncated Rep1 fusion proteins were unable to complement defective segregation of a 2 microm-based stability vector with rep1 deleted, supporting the idea of the requirement of Rep protein interaction for plasmid segregation but indicating a separate required function for the carboxy-terminal portion of Rep1 . The results of in vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both of which are capable of mediating Rep2 self-association . The amino-terminal domain interacts with Rep1, while the carboxy-terminal domain was shown by Southwestern analysis to have DNA-binding activity . The overlapping Rep1 and Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1, Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2 microm circle plasmid stability locus, forming a structure that mediates plasmid segregation . In this model, competition between Rep1 and Rep2 for association with Rep1 determines the formation or disassembly of the segregation complex.

Biochem Biophys Res Commun, 2001 Mar, 281(5), 1170 - 5
Analysis of molecular interactions of the p53-family p51(p63) gene products in a yeast two-hybrid system: homotypic and heterotypic interactions and association with p53-regulatory factors; Kojima T et al.; p51 in the p53 tumor suppressor family, also referred to as p63, encodes multiple isoforms including p51A (TAp63gamma) and p51B (TAp63alpha) . The p53 protein forms a tetramer, and its stability and activity are regulated by molecular association with viral and cellular proteins and by biochemical modifications . Using a yeast two-hybrid system, the p51A and p51B isoforms were examined for homotypic and heterotypic interactions in the p53 family proteins and for their affinity to the p53-regulatory factors . Results indicate a homotypic interaction dependent on the presumed oligomerization domain of the p51 proteins . The possibility of a weak heterotypic interaction between p51 and p73 proteins was suggested, while association between p51 and p53 appeared improbable . Furthermore, unlike p53, the p51 proteins failed to display an affinity to SV40 large T antigen or MDM2-family proteins . Having several features in common with p53, the p51 proteins may function in biological processes apart from p53 .

J Mol Biol, 2001 Mar 16, 307(1), 247 - 57
Crystal structure of unligated guanylate kinase from yeast reveals GMP-induced conformational changes; Blaszczyk J et al.; The crystal structure of guanylate kinase (GK) from yeast (Saccharomyces cerevisiae) with a non-acetylated N terminus has been determined in its unligated form (apo-GK) as well as in complex with GMP (GK.GMP) . The structure of apo-GK was solved with multiwavelength anomalous diffraction data and refined to an R-factor of 0.164 (R(free)=0.199) at 2.3 A resolution . The structure of GK.GMP was determined using the crystal structure of GK with an acetylated N terminus as the search model and refined to an R-factor of 0.156 (R(free)=0.245) at 1.9 A . GK belongs to the family of nucleoside monophosphate (NMP) kinases and catalyzes the reversible phosphoryl transfer from ATP to GMP . Like other NMP kinases, GK consists of three dynamic domains: the CORE, LID, and NMP-binding domains . Dramatic movements of the GMP-binding domain and smaller but significant movements of the LID domain have been revealed by comparing the structures of apo-GK and GK.GMP . apo-GK has a much more open conformation than the GK.GMP complex . Systematic analysis of the domain movements using the program DynDom shows that the large movements of the GMP-binding domain involve a rotation around an effective hinge axis approximately parallel with helix 3, which connects the GMP-binding and CORE domains . The C-terminal portion of helix 3, which connects to the CORE domain, has strikingly higher temperature factors in GK.GMP than in apo-GK, indicating that these residues become more mobile upon GMP binding . The results suggest that helix 3 plays an important role in domain movement . Unlike the GMP-binding domain, which moves toward the active center of the enzyme upon GMP binding, the LID domain moves away from the active center and makes the presumed ATP-binding site more open . Therefore, the LID domain movement may facilitate the binding of MgATP . The structure of the recombinant GK.GMP complex superimposes very well with that of the native GK.GMP complex, indicating that N-terminal acetylation does not have significant impact on the three-dimensional structure of GK .

Gene Expr, 2000, 9(3), 145 - 56
An antiprion effect of the anticytoskeletal drug latrunculin A in yeast; Bailleul-Winslett PA et al.; Prions are infectious aggregation-prone isoforms of the normal proteins, supposedly able to seed aggregation of the normal cellular counterparts . In vitro, prion proteins form amyloid fibers, resembling cytoskeletal structures . Yeast prion {PSI}, which is a cytoplasmically inherited aggregated isoform of the translation termination factor Sup35p (eRF3), serves as a useful model for studying mechanisms of prion diseases and other amyloidoses . The previously described interaction between Sup35p and cytoskeletal assembly protein Sla1p points to the possible relationships between prions and cytoskeletal networks . Although the Sup35PSI+ aggregates do not colocalize with actin patches, we have shown that yeast cells are efficiently cured of the {PSI} prion by prolonged incubation with latrunculin A, a drug disrupting the actin cytoskeleton . On the other hand, treatments with sodium azide or cycloheximide, agents blocking yeast protein synthesis and cell proliferation but not disrupting the cytoskeleton, do not cause a significant loss of {PSI} . Moreover, simultaneous treatment with sodium azide or cycloheximide blocks {PSI} curing by latrunculin A, indicating that prion loss in the presence of latrunculin A requires a continuation of protein synthesis during cytoskeleton disruption . The sodium azide treatment also decreases the toxic effect of latrunculin A . Latrunculin A influences neither the levels of total cellular Sup35p nor the levels of chaperone proteins, such as Hsp104 and Hsp70, which were previously shown to affect {PSI} . This makes an indirect effect of latrunculin A on {PSI} via induction of Hsps unlikely . Fluorescence microscopy detects changes in the structure and/or localization of the Sup35PSI+ aggregates in latrunculin A-treated cells . We conclude that the stable maintenance of the {PSI} prion aggregates in the protein-synthesizing yeast cells partly depends on an intact actin cytoskeleton, suggesting that anticytoskeletal treatments could be used to counteract some aggregation-related disorders.

Biochemistry (Mosc), 2001 Jan, 66(1), 47 - 54
Effect of zinc ions on conformational stability of yeast alcohol dehydrogenase; Yang Y et al.; Yeast alcohol dehydrogenase preparations were prepared with the conformational zinc ion removed (Apo-I YADH) and with both the conformational and catalytic zinc ions removed (Apo-II YADH) . The unfolding of Apo-I YADH and Apo-II YADH during denaturation in urea solutions was then followed by fluorescence emission, circular dichroism, and second-derivative optical spectroscopies . Compared with the native enzyme, Apo-I YADH incurred some slight unfolding, and its stability against urea was markedly decreased, while Apo-II YADH incurred marked unfolding but contained residual ordered structure even at high urea concentrations . The results show that native YADH is more conformationally stable against urea denaturation than Apo-I YADH, indicating that the conformational Zn(2+) plays an important role in stabilizing the conformation of the YADH molecule . However, unfolding of the region around the conformational zinc ion is shown not to be the rate limited step in the unfolding of the molecule by the fact that the unfolding and inactivation rate constants of native and Apo-I YADH are the same . It is suggested that the catalytic zinc ion is more important in maintaining the structure of YADH . YADH lost its cooperative unfolding ability after the zinc ions were removed . The shape of the transition curves of Apo-I YADH suggests the existence of an unfolding intermediate . For both native and Apo-I YADH, inactivation occurs at much lower urea concentrations than that needed to produce significant conformational changes of the enzyme molecule . At urea concentration above 4 M, the inactivation rate constants are much higher than those of the fast phase of the reaction of unfolding . These results support the suggestion of flexibility at the active site of the enzyme (C . L . Tsou (1986) Trends Biochem . Sci., 11, 427-429; (1993) Science, 262, 308-381).

Mol Cell Biol, 2001 Mar, 21(6), 2098 - 106
Interactions of Isw2 chromatin remodeling complex with nucleosomal arrays: analyses using recombinant yeast histones and immobilized templates; Gelbart ME et al.; To facilitate the biochemical characterization of chromatin-associated proteins in the budding yeast Saccharomyces cerevisiae, we have developed a system to assemble nucleosomal arrays on immobilized templates using recombinant yeast core histones . This system enabled us to analyze the interaction of Isw2 ATP-dependent chromatin remodeling complex with nucleosomal arrays . We found that Isw2 complex interacts efficiently with both naked DNA and nucleosomal arrays in an ATP-independent manner, suggesting that ATP is required at steps subsequent to this physical interaction . We identified the second subunit of Isw2 complex, encoded by open reading frame YGL 133w (herein named ITC1), and found that both subunits of the complex, Isw2p and Itc1p, are essential for efficient interaction with DNA and nucleosomal arrays . Both subunits are also required for nucleosome-stimulated ATPase activity and chromatin remodeling activity of the complex . Finally, we found that ITC1 is essential for function of Isw2 complex in vivo, since isw2 and itc1 deletion mutants exhibit virtually identical phenotypes . These results demonstrate the utility of our in vitro system in studying interactions between chromatin-associated proteins and nucleosomal arrays.

Mol Cell Biol, 2001 Mar, 21(6), 2057 - 69
Identification of the Sin3-binding site in Ume6 defines a two-step process for conversion of Ume6 from a transcriptional repressor to an activator in yeast; Washburn BK et al.; The DNA-binding protein Ume6 is required for both repression and activation of meiosis-specific genes, through interaction with the Sin3 corepressor and Rpd3 histone deacetylase and the meiotic activator Ime1 . Here we show that fusion of a heterologous activation domain to Ume6 is unable to convert it into a constitutive activator of early meiotic gene transcription, indicating that an additional function is needed to overcome repression at these promoters . Mutations in UME6 allowing the fusion to activate lie in a predicted amphipathic alpha helix and specifically disrupt interaction with Sin3 but not with Teal, an activator of Ty transcription also found to interact with Ume6 in a two-hybrid screen . The mutations cause a loss of repression by Ume6 and precisely identify the Ume6 Sin3-binding domain, which we show interacts with the paired amphipathic helix 2 region of Sin3 . Analysis of these mutants indicates that conversion of Ume6 to an activator involves two genetically distinct steps that act to relieve Sin3-mediated repression and provide an activation domain to Ume6 . The mutants further demonstrate that premature expression and lack of subsequent rerepression of Ume6-Sin3-regulated genes are not deleterious to meiotic progression and suggest that the essential role of Sin3 in meiosis is independent of Ume6 . The model for Ume6 function arising from these studies indicates that Ume6 is similar in many respects to metazoan regulators that utilize Sin3, such as the Myc-Mad-Max system and nuclear hormone receptors, and provides new insights into the control of transcriptional repression and activation by the Ume6-URS1 regulatory complex in yeast.

Mol Cell Biol, 2001 Mar, 21(6), 2018 - 25
Acidic residues critical for the activity and biological function of yeast DNA polymerase eta; Kondratick CM et al.; Rad30 is a member of the newly discovered UmuC/DinB/Rad30 family of DNA polymerases . The N-terminal regions of these proteins are highly homologous, and they contain five conserved motifs, I to V, while their C-terminal regions are quite divergent . We examined the contributions of the C-terminal and N-terminal regions of Rad30 to its activity and biological function . Although deletion of the last 54 amino acids has no effect on DNA polymerase or thymine-thymine (T-T) dimer bypass activity, this C-terminal deletion-containing protein is unable to perform its biological function in vivo . The presence of a bipartite nuclear targeting sequence within this region suggests that at least one function of this portion of Rad30 is nuclear targeting . To identify the active-site residues of Rad30 important for catalysis, we generated mutations of nine acidic residues that are invariant or highly conserved among Rad30 proteins from different eukaryotic species . Mutations of the Asp30 and Glu39 residues present in motif I and of the Asp155 residue present in motif III to alanine completely inactivated the DNA polymerase and T-T dimer bypass activities, and these mutations did not complement the UV sensitivity of the rad30Delta mutation . Mutation of Glu156 in motif III to alanine confers a large reduction in the efficiency of nucleotide incorporation, whereas the remaining five Rad30 mutant proteins retain wild-type levels of DNA polymerase and T-T dimer bypass activities . From these observations, we suggest a role for the Asp30, Glu39, and Asp155 residues in the binding of two metal ions required for the reaction of the incoming deoxynucleoside 5'-triphosphate with the 3'-hydroxyl in the primer terminus, while Glu156 may participate in nucleotide binding.

Mol Cell Biol, 2001 Mar, 21(5), 1854 - 65
HIRA, the human homologue of yeast Hir1p and Hir2p, is a novel cyclin-cdk2 substrate whose expression blocks S-phase progression; Hall C et al.; Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP) . To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs . One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p . Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase . First, HIRA bound to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner . Second, HIRA was phosphorylated in vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin A-cdk2 in vitro . Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21(cip1) . Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active . Fifth, HIRA was localized preferentially to the nucleus, where active cyclin A- and E-cdk2 are located . Finally, ectopic expression of HIRA in cells caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control of the cell cycle.

Mol Cell Biol, 2001 Mar, 21(5), 1841 - 53
Histone folds mediate selective heterodimerization of yeast TAF(II)25 with TFIID components yTAF(II)47 and yTAF(II)65 and with SAGA component ySPT7; Gangloff YG et al.; We show that the yeast TFIID (yTFIID) component yTAF(II)47 contains a histone fold domain (HFD) with homology to that previously described for hTAF(II)135 . Complementation in vivo indicates that the yTAF(II)47 HFD is necessary and sufficient for vegetative growth . Mutation of highly conserved residues in the alpha1 helix of the yTAF(II)47 HFD results in a temperature-sensitive phenotype which can be suppressed by overexpression of yTAF(II)25, as well as by yTAF(II)40, yTAF(II)19, and yTAF(II)60 . In yeast two-hybrid and bacterial coexpression assays, the yTAF(II)47 HFD selectively heterodimerizes with yTAF(II)25, which we show contains an HFD with homology to the hTAF(II)28 family We additionally demonstrate that yTAF(II)65 contains a functional HFD which also selectively heterodimerizes with yTAF(II)25 . These results reveal the existence of two novel histone-like pairs in yTFIID . The physical and genetic interactions described here show that the histone-like yTAF(II)s are organized in at least two substructures within TFIID rather than in a single octamer-like structure as previously suggested . Furthermore, our results indicate that ySPT7 has an HFD homologous to that of yTAF(II)47 which selectively heterodimerizes with yTAF(II)25, defining a novel histone-like pair in the SAGA complex.

Mol Cell Biol, 2001 Mar, 21(5), 1710 - 8
Checkpoint adaptation precedes spontaneous and damage-induced genomic instability in yeast; Galgoczy DJ et al.; Despite the fact that eukaryotic cells enlist checkpoints to block cell cycle progression when their DNA is damaged, cells still undergo frequent genetic rearrangements, both spontaneously and in response to genotoxic agents . We and others have previously characterized a phenomenon (adaptation) in which yeast cells that are arrested at a DNA damage checkpoint eventually override this arrest and reenter the cell cycle, despite the fact that they have not repaired the DNA damage that elicited the arrest . Here, we use mutants that are defective in checkpoint adaptation to show that adaptation is important for achieving the highest possible viability after exposure to DNA-damaging agents, but it also acts as an entree into some forms of genomic instability . Specifically, the spontaneous and X-ray-induced frequencies of chromosome loss, translocations, and a repair process called break-induced replication occur at significantly reduced rates in adaptation-defective mutants . This indicates that these events occur after a cell has first arrested at the checkpoint and then adapted to that arrest . Because malignant progression frequently involves loss of genes that function in DNA repair, adaptation may promote tumorigenesis by allowing genomic instability to occur in the absence of repair.

Mol Cell Biol, 2001 Mar, 21(5), 1656 - 61
3'-phosphodiesterase and 3'-->5' exonuclease activities of yeast Apn2 protein and requirement of these activities for repair of oxidative DNA damage; Unk I et al.; In Saccharomyces cerevisiae, the AP endonucleases encoded by the APN1 and APN2 genes provide alternate pathways for the removal of abasic sites . Oxidative DNA-damaging agents, such as H(2)O(2), produce DNA strand breaks which contain 3'-phosphate or 3'-phosphoglycolate termini . Such 3' termini are inhibitory to synthesis by DNA polymerases . Here, we show that purified yeast Apn2 protein contains 3'-phosphodiesterase and 3'-->5' exonuclease activities, and mutation of the active-site residue Glu59 to Ala in Apn2 inactivates both these activities . Consistent with these biochemical observations, genetic studies indicate the involvement of APN2 in the repair of H(2)O(2)-induced DNA damage in a pathway alternate to APN1, and the Ala59 mutation inactivates this function of Apn2 . From these results, we conclude that the ability of Apn2 to remove 3'-end groups from DNA is paramount for the repair of strand breaks arising from the reaction of DNA with reactive oxygen species.

Mol Cell Biol, 2001 Mar, 21(5), 1647 - 55
Pir1p mediates translocation of the yeast Apn1p endonuclease into the mitochondria to maintain genomic stability; Vongsamphanh R et al.; The mitochondrial genome is continuously subject to attack by reactive oxygen species generated through aerobic metabolism . This leads to the formation of a variety of highly genotoxic DNA lesions, including abasic sites . Yeast Apn1p is localized to the nucleus, where it functions to cleave abasic sites, and apn1 Delta mutants are hypersensitive to agents such as methyl methanesulfonate (MMS) that induce abasic sites . Here we demonstrate for the first time that yeast Apn1p is also localized to the mitochondria . We found that Pir1p, initially isolated as a cell wall constituent of unknown function, interacts with the C-terminal end of Apn1p, which bears a bipartite nuclear localization signal . Further analysis revealed that Pir1p is required to cause Apn1p mitochondrial localization, presumably by competing with the nuclear transport machinery . pir1 Delta mutants displayed a striking (approximately 3-fold) increase of Apn1p in the nucleus, which coincided with drastically reduced levels in the mitochondria . To explore the functional consequences of the Apn1p-Pir1p interaction, we measured the rate of mitochondrial mutations in the wild type and pir1 Delta and apn1 Delta mutants . pir1 Delta and apn1 Delta mutants exposed to MMS exhibited 3.6- and 5.8-fold increases, respectively, in the rate of mitochondrial mutations, underscoring the importance of Apn1p in repair of the mitochondrial genome . We conclude that Pir1p interacts with Apn1p, at the level of either the cytoplasm or nucleus, and facilitates Apn1p transport into the mitochondria to repair damaged DNA.

Genetics, 2001 Mar, 157(3), 1159 - 68
The git5 Gbeta and git11 Ggamma form an atypical Gbetagamma dimer acting in the fission yeast glucose/cAMP pathway; Landry S et al.; Fission yeast adenylate cyclase, like mammalian adenylate cyclases, is regulated by a heterotrimeric G protein . The gpa2 Galpha and git5 Gbeta are both required for glucose-triggered cAMP signaling . The git5 Gbeta is a unique member of the Gbeta family in that it lacks an amino-terminal coiled-coil domain shown to be essential for mammalian Gbeta folding and interaction with Ggamma subunits . Using a git5 bait in a two-hybrid screen, we identified the git11 Ggamma gene . Co-immunoprecipitation studies confirm the composition of this Gbetagamma dimer . Cells deleted for git11 are defective in glucose repression of both fbp1 transcription and sexual development, resembling cells lacking either the gpa2 Galpha or the git5 Gbeta . Overexpression of the gpa2 Galpha partially suppresses loss of either the git5 Gbeta or the git11 Ggamma, while mutational activation of the Galpha fully suppresses loss of either Gbeta or Ggamma . Deletion of gpa2 (Galpha), git5 (Gbeta), or git11 (Ggamma) confer quantitatively distinct effects on fbp1 repression, indicating that the gpa2 Galpha subunit remains partially active in the absence of the Gbetagamma dimer and that the git5 Gbeta subunit remains partially active in the absence of the git11 Ggamma subunit . The addition of the CAAX box from the git11 Ggamma to the carboxy-terminus of the git5 Gbeta partially suppresses the loss of the Ggamma . Thus the Ggamma in this system is presumably required for localization of the Gbetagamma dimer but not for folding of the Gbeta subunit . In mammalian cells, the essential roles of the Gbeta amino-terminal coiled-coil domains and Ggamma partners in Gbeta folding may therefore reflect a mechanism used by cells that express multiple forms of both Gbeta and Ggamma subunits to regulate the composition and activity of its G proteins.

Genetics, 2001 Mar, 157(3), 1141 - 58
Yeast frameshift suppressor mutations in the genes coding for transcription factor Mbf1p and ribosomal protein S3: evidence for autoregulation of S3 synthesis; Hendrick JL et al.; The SUF13 and SUF14 genes were identified among extragenic suppressors of +1 frameshift mutations . SUF13 is synonymous with MBF1, a single-copy nonessential gene coding for a POLII transcription factor . The suf13-1 mutation is a two-nucleotide deletion in the SUF13/MBF1 coding region . A suf13::TRP1 null mutant suppresses +1 frameshift mutations, indicating that suppression is caused by loss of SUF13 function . The suf13-1 suppressor alters sensitivity to aminoglycoside antibiotics and reduces the accumulation of his4-713 mRNA, suggesting that suppression is mediated at the translational level . The SUF14 gene is synonymous with RPS3, a single-copy essential gene that codes for the ribosomal protein S3 . The suf14-1 mutation is a missense substitution in the coding region . Increased expression of S3 limits the accumulation of SUF14 mRNA, suggesting that expression is autoregulated . A frameshift mutation in SUF14 that prevents full-length translation eliminated regulation, indicating that S3 is required for regulation . Using CUP1-SUF14 and SUF14-lacZ fusions, run-on transcription assays, and estimates of mRNA half-life, our results show that transcription plays a minor role if any in regulation and that the 5'-UTR is necessary but not sufficient for regulation . A change in mRNA decay rate may be the primary mechanism for regulation.

Genetics, 2001 Mar, 157(3), 1117 - 40
Overlapping functions of the yeast oxysterol-binding protein homologues; Beh CT et al.; The Saccharomyces cerevisiae genome encodes seven homologues of the mammalian oxysterol-binding protein (OSBP), a protein implicated in lipid trafficking and sterol homeostasis . To determine the functions of the yeast OSBP gene family (OSH1-OSH7), we used a combination of genetics, genomics, and sterol lipid analysis to characterize OSH deletion mutants . All 127 combinations and permutations of OSH deletion alleles were constructed . Individual OSH genes were not essential for yeast viability, but the elimination of the entire gene family was lethal . Thus, the family members shared an essential function . In addition, the in vivo depletion of all Osh proteins disrupted sterol homeostasis . Like mutants that affect ergosterol production, the viable combinations of OSH deletion alleles exhibited specific sterol-related defects . Although none of the single OSH deletion mutants was defective for growth, gene expression profiles revealed that each mutant had a characteristic molecular phenotype . Therefore, each gene performed distinct nonessential functions and contributed to a common essential function . Our findings indicated that OSH genes performed a multitude of nonessential roles defined by specific subsets of the genes and that most shared at least one essential role potentially linked to changes in sterol lipid levels.

Genetics, 2001 Mar, 157(3), 1089 - 105
Mutations in the YRB1 gene encoding yeast ran-binding-protein-1 that impair nucleocytoplasmic transport and suppress yeast mating defects; Kunzler M et al.; We identified two temperature-sensitive (ts) mutations in the essential gene, YRB1, which encodes the yeast homolog of Ran-binding-protein-1 (RanBP1), a known coregulator of the Ran GTPase cycle . Both mutations result in single amino acid substitutions of evolutionarily conserved residues (A91D and R127K, respectively) in the Ran-binding domain of Yrb1 . The altered proteins have reduced affinity for Ran (Gsp1) in vivo . After shift to restrictive temperature, both mutants display impaired nuclear protein import and one also reduces poly(A)+ RNA export, suggesting a primary defect in nucleocytoplasmic trafficking . Consistent with this conclusion, both yrb1ts mutations display deleterious genetic interactions with mutations in many other genes involved in nucleocytoplasmic transport, including SRP1 (alpha-importin) and several beta-importin family members . These yrb1ts alleles were isolated by their ability to suppress two different types of mating-defective mutants (respectively, fus1Delta and ste5ts), indicating that reduction in nucleocytoplasmic transport enhances mating proficiency . Indeed, in both yrb1ts mutants, Ste5 (scaffold protein for the pheromone response MAPK cascade) is mislocalized to the cytosol, even in the absence of pheromone . Also, both yrb1ts mutations suppress the mating defect of a null mutation in MSN5, which encodes the receptor for pheromone-stimulated nuclear export of Ste5 . Our results suggest that reimport of Ste5 into the nucleus is important in downregulating mating response.

Genes Dev, 2001 Mar 1, 15(5), 619 - 26
In vivo chromatin remodeling by yeast ISWI homologs Isw1p and Isw2p; Kent NA et al.; Isw1p and Isw2p are budding yeast homologs of the Drosophila ISWI chromatin-remodeling ATPase . Using indirect-end-label and chromatin immunoprecipitation analysis, we show both independent and cooperative Isw1p- and Isw2p-mediated positioning of short nucleosome arrays in gene-regulatory elements at a variety of transcription units in vivo . We present evidence that both yeast ISWI complexes regulate developmental responses to starvation and that for Isw2p, recruitment by different DNA-binding proteins controls meiosis and haploid invasive growth.

Biochem J, 2001 Mar 15, 354(Pt 3), 655 - 61
Identification of Cdc6 protein domains involved in interaction with Mcm2 protein and Cdc4 protein in budding yeast cells; Jang SW et al.; The Cdc6 protein (Cdc6p) has essential roles in regulating initiation of DNA replication . Cdc6p is recruited to origins of replication by the origin recognition complex (ORC) late in mitosis; Cdc6p in turn recruits minichromosome maintenance (Mcm) proteins to form the pre-replicative complex . Cdc6p is thought to interact with one or more Mcm proteins but this point has not yet been demonstrated . In the present study we observed that Cdc6p interacted significantly only with Mcm2p out of six Mcm proteins in yeast two-hybrid cells . Our results indicate that the interaction of Cdc6p with Mcm2p is specific, although we cannot exclude the possibility that the interaction might not be direct . In attempts to identify domains of Cdc6p important for interaction with Mcm2p, we tested interactions of various deleted versions of Cdc6p with Mcm2p and also with Cdc4p, which was previously known to interact with Cdc6p . The portion of Cdc6p from amino acid residues 51 to 394 was able to interact with Mcm2p . During the course of the studies we also discovered a previously undetected Cdc4p interaction domain between residues 51 and 394 . Interestingly, when all six putative Cdc28 phosphorylation sites in Cdc6p were changed to alanine, a 6-7-fold increase in binding to Mcm2p was observed . This result suggests that unphosphorylated Cdc6p has higher affinity than phosphorylated Cdc6p for Mcm2p; this might partly explain the previous observation that Cdc6p failed to load Mcm proteins on replication origins during S phase when the cyclin-dependent protein kinase was active, thus helping to prevent the reinitiation of activated replicons.

Prikl Biokhim Mikrobiol, 2001 Jan-Feb, 37(1), 29 - 35
{Nucleoprotein complexes of yeast and their biological effect on T-lymphocytes}; Sinitskaia NS et al.; Complexes of nucleic acids and acid nuclear proteins that are active toward human T-lymphocytes were isolated from cells of bakers' yeast Saccharomyces cerevisiae . The conditions of isolation of nucleoprotein complexes by acid extraction followed by microfiltration for concentration of macromolecular components were optimized . Gel filtration and electrophoresis were used to study the composition and molecular weights of components of the preparations obtained . It was shown that nucleoprotein complex had a molecular weight of 1430 kDa . However, only one zone was determined by electrophoresis of the protein component with a molecular weight of 30 kDa.

Nat Biotechnol, 2001 Mar, 19(3), 242 - 7
Large-scale analysis of the yeast proteome by multidimensional protein identification technology; Washburn MP et al.; We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT) . MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date . A total of 1,484 proteins were detected and identified . Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases . Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins . MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.

Eur J Biochem, 2001 Mar, 268(5), 1155 - 62
Effects of mutations in mitochondrial cytochrome b in yeast and man . Deficiency, compensation and disease; Fisher N et al.; The mitochondrial cytochrome bc(1) complex is a key protonmotive component of eukaryotic respiratory chains . The mitochondrially encoded cytochrome b forms, with cytochrome c(1) and the iron--sulfur protein, the catalytic core of this multimeric enzyme . Mutations of cytochrome b have been reported in association with human diseases . In the highly homologous yeast cytochrome b, several mutations that impair the respiratory function, and reversions that correct the defect, have been described . In this paper, we re-examine the mutations in the light of the atomic structure of the complex, and discuss the possible effect, at enzyme level, of the human cytochrome b mutations and the correcting effect of the reversions.

Curr Biol, 2001 Jan 23, 11(2), 88 - 97
B-type cyclins CLB5 and CLB6 control the initiation of recombination and synaptonemal complex formation in yeast meiosis; Smith KN et al.; BACKGROUND: The life cycle of most eukaryotic organisms includes a meiotic phase, in which diploid parental cells produce haploid gametes . During meiosis a single round of DNA replication is followed by two rounds of chromosome segregation . In the first, or reductional, division (meiosis I), which is unique to meiotic cells, homologous chromosomes segregate from one another, whereas in the second, or equational, division (Meiosis II) sister centromeres disjoin . Meiotic DNA replication precedes the initiation of recombination by programmed Spo11-dependent DNA double-strand breaks . Recent reports that meiosis-specific cohesion is established during meiotic S phase and that the length of S phase is modified by recombination factors (Spo11 and Rec8) raise the possibility that replication plays a fundamental role in the recombination process . RESULTS: To address how replication influences the initiation of recombination, we have used mutations in the B-type cyclin genes CLB5 and CLB6, which specifically prevent premeiotic replication in the yeast Saccharomyces cerevisiae . We find that clb5 and clb5 clb6 but not clb6 mutants are defective in DSB induction and prior associated changes in chromatin accessibility, heteroallelic recombination, and SC formation . The severity of these phenotypes in each mutant reflects the extent of replication impairment . CONCLUSIONS: This assemblage of phenotypes reveals roles for CLB5 and CLB6 not only in DNA replication but also in other key events of meiotic prophase . Links between the function of CLB5 and CLB6 in activating meiotic DNA replication and their effects on subsequent events are discussed.

Plant Cell Physiol, 2001 Feb, 42(2), 245 - 9
Improved salt tolerance of transgenic tobacco expressing apoplastic yeast-derived invertase; Fukushima E et al.; We investigated the salt tolerance of transgenic tobacco, in which yeast invertase is expressed in the apoplastic (Apo-Inv) spaces . Whereas photosynthetic activities in wild-type tobacco in light were inhibited under salt stress, transgenic Apo-Inv tobacco maintained constant photosynthetic activities . The physical appearance of plants under salt stress also indicates that yeast invertase expression in the apoplastic space is beneficial for inducing salt tolerance . Apo-Inv tobacco had a much higher osmotic pressure increase in the cell sap than did wild-type tobacco under this type of stress . The physiological importance of sucrose metabolism under salt stress is discussed.

EMBO J, 2001 Mar 1, 20(5), 1074 - 85
GSK-3 kinase Mck1 and calcineurin coordinately mediate Hsl1 down-regulation by Ca2+ in budding yeast; Mizunuma M et al.; The Ca2+-activated pathways of Saccharomyces cerevisiae induce a delay in the onset of mitosis through the activation of Swe1, a negative regulatory kinase that inhibits the Cdc28-Clb complex . Calcineurin and Mpk1 activate Swe1 at the transcriptional and post-translational level, respectively, and both pathways are essential for the cell cycle delay . Our genetic screening identified the MCK1 gene, which encodes a glycogen synthetase kinase-3 family protein kinase, as a component of the Ca2+ signaling pathway . Genetic analyses indicated that Mck1 functions downstream of the Mpk1 pathway and down-regulates Hsl1, an inhibitory kinase of Swe1 . In medium with a high concentration of Ca2+, Hsl1 was delocalized from the bud neck and destabilized in a manner dependent on both calcineurin and Mck1 . Calcineurin was required for the dephosphorylation of autophosphorylated Hsl1 . The E3 ubiquitin ligase complex SCF(Cdc4), but not the anaphase-promoting complex (APC), was essential for Hsl1 destabilization . The Ca2+-activated pathway may play a role in the rapid inactivation of Hsl1 at the cell cycle stage(s) when APC activity is low.

Fresenius J Anal Chem, 2000 Nov, 368(5), 471 - 4
Stability of inorganic mercury and methylmercury on yeast-silica gel microcolumns: field sampling capabilities; Perez-Corona MT et al.; The stability of methylmercury and inorganic mercury retained on yeast-silica gel microcolumns was established and compared with the stability of these species in solution . Yeast-silica gel columns with the retained analytes were stored for two months at three different temperatures: -20 degrees C, 4 degrees C and room temperature . At regular time intervals, both mercury species were eluted and quantified by cold vapor atomic absorption spectrometry (CVAAS) . Methylmercury was found stable in the columns over the two-month period at the three different temperatures tested while the concentration of inorganic mercury decreased after one week's storage even at -20 degrees C . These results are of great interest since the use of these microcolumns allows the preconcentration and storage of mercury species until analysis, thus saving laboratory space and avoiding the problems associated with maintaining species integrity in aqueous solution.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2375 - 80 Epub 2001 Feb 20.
An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated beta-sheet structure for amyloid; Balbirnie M et al.; X-ray diffraction and other biophysical tools reveal features of the atomic structure of an amyloid-like crystal . Sup35, a prion-like protein in yeast, forms fibrillar amyloid assemblies intrinsic to its prion function . We have identified a polar peptide from the N-terminal prion-determining domain of Sup35 that exhibits the amyloid properties of full-length Sup35, including cooperative kinetics of aggregation, fibril formation, binding of the dye Congo red, and the characteristic cross-beta x-ray diffraction pattern . Microcrystals of this peptide also share the principal properties of the fibrillar amyloid, including a highly stable, beta-sheet-rich structure and the binding of Congo red . The x-ray powder pattern of the microcrystals, extending to 0.9-A resolution, yields the unit cell dimensions of the well-ordered structure . These dimensions restrict possible atomic models of this amyloid-like structure and demonstrate that it forms packed, parallel-stranded beta-sheets . The unusually high density of the crystals shows that the packed beta-sheets are dehydrated, despite the polar character of the side chains . These results suggest that amyloid is a highly intermolecularly bonded, dehydrated array of densely packed beta-sheets . This dry beta-sheet could form as Sup35 partially unfolds to expose the peptide, permitting it to hydrogen-bond to the same peptide of other Sup35 molecules . The implication is that amyloid-forming units may be short segments of proteins, exposed for interactions by partial unfolding.

EMBO J, 2001 Jan 15, 20(1-2), 262 - 71
Sec63p and Kar2p are required for the translocation of SRP-dependent precursors into the yeast endoplasmic reticulum in vivo; Young BP et al.; The translocation of secretory polypeptides into the endoplasmic reticulum (ER) occurs at the translocon, a pore-forming structure that orchestrates the transport and maturation of polypeptides at the ER membrane . In yeast, targeting of secretory precursors to the translocon can occur by two distinct pathways that are distinguished by their dependence upon the signal recognition particle (SRP) . The SRP-dependent pathway requires SRP and its membrane-bound receptor, whereas the SRP-independent pathway requires a separate receptor complex consisting of Sec62p, Sec63p, Sec71p, Sec72p plus lumenal Kar2p/BiP . Here we demonstrate that Sec63p and Kar2p are also required for the SRP-dependent targeting pathway in vivo . Furthermore, we demonstrate multiple roles for Sec63p, at least one of which is exclusive to the SRP-independent pathway.

EMBO J, 2001 Jan 15, 20(1-2), 222 - 30
Reconstitution of Epstein-Barr virus-based plasmid partitioning in budding yeast; Kapoor P et al.; The EBNA1 protein of Epstein-Barr virus (EBV) mediates the partitioning of EBV episomes and EBV-based plasmids during cell division by a mechanism that appears to involve binding to the cellular EBP2 protein on human chromosomes . We have investigated the ability of EBNA1 and the EBV segregation element (FR) to mediate plasmid partitioning in Saccharomyces cerevisiae . EBNA1 expression alone did not enable the stable segregation of FR-containing plasmids in yeast, but segregation was rescued by human EBP2 . The reconstituted segregation system required EBNA1, human EBP2 and the FR element, and functionally replaced a CEN element . An EBP2 binding mutant of EBNA1 and an EBNA1 binding mutant of EBP2 each failed to support FR-plasmid partitioning, indicating that an EBNA1-EBP2 interaction is required . The results provide direct evidence of the role of hEBP2 in EBNA1-mediated segregation and demonstrate that heterologous segregation systems can be reconstituted in yeast.

EMBO J, 2001 Jan 15, 20(1-2), 197 - 209
A cytosolic NAD-dependent deacetylase, Hst2p, can modulate nucleolar and telomeric silencing in yeast; Perrod S et al.; In budding yeast, the silent information regulator Sir2p is a nuclear NAD-dependent deacetylase that is essential for both telomeric and rDNA silencing . All eukaryotic species examined to date have multiple homologues of Sir two (HSTs), which share a highly conserved globular core domain . Here we report that yeast Hst2p and a mammalian Hst2p homologue, hSirT2p, are cytoplasmic in yeast and human cells, in contrast to yHst1p and ySir2p which are exclusively nuclear . Although yHst2p cannot restore silencing in a sir2 deletion, overexpression of yHst2p influences nuclear silencing events in a SIR2 strain, derepressing subtelomeric silencing while increasing repression in the rDNA . In contrast, a form of ySir2p carrying a point mutation in the conserved core domain disrupts both telomeric position effect (TPE) and rDNA repression at low expression levels . This argues that non-nuclear yHst2p can compete for a substrate or ligand specifically required for telomeric, and not rDNA repression.

EMBO J, 2001 Jan 15, 20(1-2), 82 - 90
Specificity of Cdk activation in vivo by the two Caks Mcs6 and Csk1 in fission yeast; Hermand D et al.; Activating phosphorylation of cyclin-dependent kinases (Cdks) is mediated by at least two structurally distinct types of Cdk-activating kinases (Caks): the trimeric Cdk7-cyclin H-Mat1 complex in metazoans and the single-subunit Cak1 in budding yeast . Fission yeast has both Cak types: Mcs6 is a Cdk7 ortholog and Csk1 a single-subunit kinase . Both phosphorylate Cdks in vitro and rescue a thermosensitive budding yeast CAK1 strain . However, this apparent redundancy is not observed in fission yeast in vivo . We have identified mutants that exhibit phenotypes attributable to defects in either Mcs6-activating phosphorylation or in Cdc2-activating phosphorylation . Mcs6, human Cdk7 and budding yeast Cak1 were all active as Caks for Cdc2 when expressed in fission yeast . Although Csk1 could activate Mcs6, it was unable to activate Cdc2 . Biochemical experiments supported these genetic results: budding yeast Cak1 could bind and phosphorylate Cdc2 from fission yeast lysates, whereas fission yeast Csk1 could not . These results indicate that Mcs6 is the direct activator of Cdc2, and Csk1 only activates Mcs6 . This demonstrates in vivo specificity in Cdk activation by Caks.

J Membr Biol, 2001 Jan 15, 179(2), 143 - 53
Functional asymmetry of the sodium-D-glucose cotransporter expressed in yeast secretory vesicles; Firnges MA et al.; The sodium-D-glucose cotransporter (SGLT1) was expressed in a yeast mutant strain NY 17 (sec6-4) that accumulates secretory vesicles at a nonpermissive temperature because of a block in the delivery of these vesicles to the plasma membrane . By differential centrifugation a microsomal fraction enriched in secretory vesicles was prepared with a high specific activity of the vanadate-sensitive H+-ATPase and invertase . In this membrane fraction one protein band of an apparent molecular weight of 55 kDa representing the nonglycosylated SGLT1 protein could be detected by immunochemical analysis . In addition, higher molecular weight protein bands probably representing dimers and aggregates were found . In transport studies with the microsomes D-glucose fluxes showed asymmetric properties: efflux experiments revealed the typical properties of the SGLT1 such as sodium dependence, inhibition by phlorizin and potential dependence . Influx of D-glucose showed no dependence on sodium and was not inhibited by phlorizin . Furthermore, the transporter exhibited a striking asymmetry with regard to the D-glucose affinity and the sugar specificity . These results suggest that the orientation of the SGLT1 expressed in yeast secretory vesicles is, indeed, inverted with regard to its configuration in the plasma membrane of epithelial cells . Moreover, there are striking functional differences between the periplasmic and cytoplasmic face of the transporter.

Acta Crystallogr D Biol Crystallogr, 2001 Mar, 57(Pt 3), 441 - 4
Structural studies of a yeast quaternary transcription-initiation complex; Wang SM et al.; A 96.7 kDa quaternary transcription-factor complex consisting of the conserved core domains of yeast TBP, TFIIA, TFIIB and TATA-box DNA has been assembled from purified components . Crystals of the complex were obtained by the hanging-drop vapor-diffusion method . Native data sets were collected at synchrotron sources . Crystal form I belongs to space group R32, with unit-cell parameters a = b = 173.2, c = 164.1 A, and diffracts to 2.5 resolution, but contains substoichiometric amounts of TFIIB . Crystal form II was assembled with a longer piece of DNA than that used for form I crystals and contains the complete quaternary complex . These crystals belong to space group P42(1)2, with unit-cell parameters a = b = 141.7, c = 112.0 A, and diffract to 3.6 A resolution.

Nucleic Acids Res . 2001 Mar 1;29(5):E28.
Identification of partial loss of function p53 gene mutations utilizing a yeast-based functional assay; Kovvali GK et al.; Missense mutations within the central DNA binding region of p53 are the most prevalent mutations found in human cancer . Numerous studies indicate that 'hot-spot' p53 mutants (which comprise approximately 30% of human p53 gene mutations) are largely devoid of transcriptional activity . However, a growing body of evidence indicates that some non-hot-spot p53 mutants retain some degree of transcriptional activity in vivo, particularly against strong p53 binding sites . We have modified a previously described yeast-based p53 functional assay to readily identify such partial loss of function p53 mutants . We demonstrate the utility of this modified p53 functional assay using a diverse panel of p53 mutants.

Nucleic Acids Res, 2001 Mar 1, 29(5), 1222 - 7
Translational control of human p53 expression in yeast mediated by 5'-UTR-ORF structural interaction; Mokdad-Gargouri R et al.; We have expressed human p53 cDNA in the yeast Saccharomyces cerevisiae and shown that the level of production and the length of the p53 protein depends on the presence of untranslated mRNA regions (UTRs) . The expression of the ORF alone leads to a p53 protein of correct size (53 kDa) that accumulates to high levels, concomitantly with the presence of a small amount of a p40 protein (40 kDa) . However, when either the entire 5'-UTR and a part of the 3'- or 5'-UTR alone is used, this leads to the production of small amounts of the 40 kDa truncated form only . The p40 protein corresponds to a truncated form of p53 at the C-terminal extremity since it reacts only with a monoclonal antibody recognising the N-terminal epitope . This effect on the amount and length of p53 protein had no correlation at the mRNA level, suggesting that translational control probably occurs through the 5'-UTR . We propose a model of structural interaction between this UTR and a part of the ORF mRNA for the regulation of p53 expression in this heterologous context.

Nucleic Acids Res, 2001 Mar 1, 29(5), 1144 - 55
Characterization and mutational analysis of yeast Dbp8p, a putative RNA helicase involved in ribosome biogenesis; Daugeron MC et al.; RNA helicases of the DEAD box family are involved in almost all cellular processes involving RNA molecules . Here we describe functional characterization of the yeast RNA helicase Dbp8p (YHR169w) . Our results show that Dbp8p is an essential nucleolar protein required for biogenesis of the small ribosomal subunit . In vivo depletion of Dbp8p resulted in a ribosomal subunit imbalance due to a deficit in 40S ribosomal subunits . Subsequent analyses of pre-rRNA processing by pulse-chase labeling, northern hybridization and primer extension revealed that the early steps of cleavage of the 35S precursor at sites A(1) and A(2) are inhibited and delayed at site A(0) . Synthesis of 18S rRNA, the RNA moiety of the 40S subunit, is thereby blocked in the absence of Dbp8p . The involvement of Dbp8p as a bona fide RNA helicase in ribosome biogenesis is strongly supported by the loss of Dbp8p in vivo function obtained by site-directed mutagenesis of some conserved motifs carrying the enzymatic properties of the protein family.

Nucleic Acids Res, 2001 Mar 1, 29(5), 1107 - 13
Excision of 8-oxoguanine within clustered damage by the yeast OGG1 protein; David-Cordonnier MH et al.; Clustered damages are formed in DNA by ionising radiation and radiomimetic anticancer agents and are thought to be biologically severe . 7,8-dihydro-8-oxoguanine (8-oxoG), a major DNA damage resulting from oxidative attack, is highly mutagenic leading to a high level of G.C-->T.A transversions if not previously excised by OGG1 DNA glycosylase/AP lyase proteins in eukaryotes . However, 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell . The ability of yeast OGG1 to excise 8-oxoG was determined when another type of damage {dihydrothymine, uracil, 8-oxoG, abasic (AP) site or various types of single-strand breaks (SSBs)} is present on the complementary strand 1, 3 or 5 bases 5' or 3' opposite to 8-oxoG . Base damages have little or no influence on the excision of 8-oxoG by yeast OGG1 (yOGG1) whereas an AP site has a strong inhibitory effect . Various types of SSBs, obtained using either oligonucleotides with 3'- and 5'-phosphate termini around a gap or through conversion of an AP site with either endonuclease III or human AP endonuclease 1, strongly inhibit excision of 8-oxoG by yOGG1 . Therefore, this large inhibitory effect of an AP site or a SSB may minimise the probability of formation of a double-strand break in the processing of 8-oxoG within clustered damages.

J Exp Biol, 2001 Mar, 204(Pt 6), 1053 - 61
Properties of the mammalian and yeast metal-ion transporters DCT1 and Smf1p expressed in Xenopus laevis oocytes; Sacher A et al.; Transition metals are essential for many metabolic processes, and their homeostasis is crucial for life . Metal-ion transporters play a major role in maintaining the correct concentrations of the various metal ions in living cells . Little is known about the transport mechanism of metal ions by eukaryotic cells . Some insight has been gained from studies of the mammalian transporter DCT1 and the yeast transporter Smf1p by following the uptake of various metal ions and from electrophysiological experiments using Xenopus laevis oocytes injected with RNA copies (c-RNA) of the genes for these transporters . Both transporters catalyze the proton-dependent uptake of divalent cations accompanied by a 'slippage' phenomenon of different monovalent cations unique to each transporter . Here, we further characterize the transport activity of DCT1 and Smf1p, their substrate specificity and their transport properties . We observed that Zn(2+) is not transported through the membrane of Xenopus laevis oocytes by either transporter, even though it inhibits the transport of the other metal ions and enables protons to 'slip' through the DCT1 transporter . A special construct (Smf1p-s) was made to enhance Smf1p activity in oocytes to enable electrophysiological studies of Smf1p-s-expressing cells . 54Mn(2+) uptake by Smf1p-s was measured at various holding potentials . In the absence of Na(+) and at pH 5.5, metal-ion uptake was not affected by changes in negative holding potentials . Elevating the pH of the medium to 6.5 caused metal-ion uptake to be influenced by the holding potential: ion uptake increased when the potential was lowered . Na(+) inhibited metal-ion uptake in accordance with the elevation of the holding potential . A novel clutch mechanism of ion slippage that operates via continuously variable stoichiometry between the driving-force pathway (H(+)) and the transport pathway (divalent metal ions) is proposed . The possible physiological advantages of proton slippage through DCT1 and of Na(+) slippage through Smf1p are discussed.

Naunyn Schmiedebergs Arch Pharmacol, 2001 Feb, 363(2), 203 - 8
Sanguinarine induces K+ outflow from yeast cells expressing mammalian sodium pumps; Scheiner-Bobis G; Sanguinarine, an alkaloid from Sanguinaria canadensis, has no effect on the yeast Saccharomyces cerevisiae at concentrations of up to 225 microM . Yeast cells become sensitive to sanguinarine and lose cytosolic K+ in a time- and concentration-dependent manner when they express the mammalian Na+,K+-ATPase (sodium pump) . Dose-response studies show that sanguinarine induces K+ outflow from cells expressing wild-type sodium pumps with an EC50 of 29.3+/-1.2 microM . A similar effect with a comparable EC50) of 26.8+/-1.3 microM is obtained with cells expressing an Asp369Ala mutant of the sodium pump alpha1 subunit . Since this sodium pump mutant does not hydrolyze ATP, it can be excluded that the observed sanguinarine-induced outflow of K+ is an active ion transport process . Ouabain inhibits the sanguinarine effect at concentrations higher than 1 mM . In contrast, proscillaridin A inhibits the sanguinarine-induced K+ outflow from cells expressing the wild-type sodium pump with an IC50 of 48.9+/-1.3 microM . A similar IC50 of 52.2+/-3.0 microM is obtained with cells expressing the Asp369Ala mutant . These data, together with the fact that sanguinarine inhibits the binding of {3H}ouabain to microsomes prepared from yeast cells expressing the sodium pump with an IC50 of 94.5+/-4.3 microM, all indicate that sanguinarine specifically targets the sodium pump, and that the observed K+ outflow is tightly associated with the presence of the enzyme.

Indian J Pathol Microbiol, 2000 Apr, 43(2), 143 - 5
Comparison of Vitek Yeast Biochemical Card with conventional methods for speciation of Candida; Sood P et al.; The ability of the Vitek Yeast Biochemical Card to identify yeast isolates was compared with conventional methods . Of the fifty yeast isolates tested same species identification was obtained in thirty-four isolates . The Vitek yeast biochemical card identified 13 isolates which could not be identified by the conventional tests . Though the Vitek Yeast biochemical card gave a good rapid identification the high cost of each test severely limits its routine use in most of the laboratories.

J Environ Pathol Toxicol Oncol, 2001, 20(1), 39 - 45
Effect of caffeine on the genotoxic effects of gamma radiation and 4-NQO in diploid yeast; Anjaria KB et al.; Caffeine is an environmental agent to which people are commonly exposed through medicines, drinks, food items, etc . It has been shown to be mutagenic in a number of test systems . In addition, it has also been shown to modify the mutagenic response of ionizing radiation, UV, and several chemical mutagens in a number of test systems . We have studied the effect of caffeine on gamma radiation and 4-Nitroquinoline 1-oxide (4-NQO)-induced gene conversion in the yeast Saccharomyces cerevisiae D7 . Stationary phase cells were either exposed to 100-600 Gy of 60Co gamma radiation or treated with 0.15-0.3 microM 4-NQO (30 degrees C, 1 hour), after which they were plated on synthetic complete or minimal media with or without caffeine . Caffeine concentrations ranged from 5 to 15 mM . The results indicated that caffeine at 5 and 10 mM decreased gamma radiation-induced gene conversion frequencies significantly at 400 and 600 Gy . At 600 Gy, the decrease was about 30% and 50% with caffeine concentrations of 5 and 10 mM, respectively . In contrast, caffeine was found to increase the induced gene conversion frequency when cells treated with 0.15, 0.225, and 0.3 microM 4-NQO were plated on media containing caffeine . The increase with 5, 10, and 15 mM caffeine was approximately 1.5, 2, and 2.5, respectively, times the value of 4-NQO alone . The results indicate that the posttreatment repair processes following gamma irradiation or 4-NQO treatment are modified via different pathways.

RNA, 2001 Jan, 7(1), 133 - 42
Yeast U1 snRNP-pre-mRNA complex formation without U1snRNA-pre-mRNA base pairing; Du H et al.; Base pairing between the 5' end of U1 snRNA and the conserved 5' splice site of pre-mRNA is important for commitment complex formation in vitro . However, the biochemical mechanisms by which pre-mRNA is initially recognized by the splicing machinery is not well understood . To evaluate the role of this base pairing interaction, we truncated U1 snRNA to eliminate the RNA-RNA interaction and surprisingly found that U1 snRNP can still form a nearly normal RNA-protein complex and maintain sequence specificity . We propose that some feature of U1 snRNP, perhaps one or more protein factors, is more important than the base pairing for initial 5' splice site recognition . In addition, at least five sets of interactions contribute to complex formation or stability . Only one of these is base pairing between the 5' splice site and the 5' end of U1 snRNA, without which the U1 snRNP-pre-mRNA complex is less stable and has a somewhat altered conformation.

Cell Mol Life Sci, 1999 Nov 30, 56(9-10), 807 - 16
Yeast aging research: recent advances and medical relevance; Sinclair DA; The molecular mechanisms of aging are most fully understood for the budding yeast Saccharomyces cerevisiae . Recent advances in our understanding of aging in this organism have enabled researchers to answer some fundamental questions about the aging process . Is aging due to a multitude of 'mechanisms' or can there be a key few? Can we design single-gene mutations that will prolong life? Can we prolong life whilst maintaining health and fecundity? The various contributing factors to yeast longevity, uncovered thus far, fall into three classes: DNA metabolism, heterochromatin, and metabolic activity . However, these separate classes may actually represent different aspects of the same aging mechanism based on genome stability . This review examines the recent advances in our understanding of yeast aging and discusses their relevance, if any, to the human condition.

Cell Mol Life Sci, 1999 Oct 30, 56(5-6), 415 - 26
Molecular mechanism of heme signaling in yeast: the transcriptional activator Hap1 serves as the key mediator; Zhang L et al.; Heme is a key molecule in mediating the effects of oxygen on various molecular and cellular processes in many living organisms . In the yeast Saccharomyces cerevisiae, heme serves as a secondary signal for oxygen; intracellular heme synthesis directly correlates with oxygen tension in the environment . In yeast, oxygen sensing and heme signaling are primarily mediated by the heme activator protein Hap1, which, in response to heme, activates the transcription of genes required for respiration and for controlling oxidative damage . Heme regulation of many genes required for anaerobic growth is mediated by the aerobic repressor Rox1, whose expression is controlled by heme . In this review, we summarize recent knowledge about (i) how heme synthesis may be controlled by oxygen tension, (ii) how heme precisely and stringently controls Hap1 activity and (iii) whether other transcriptional activators can also mediate heme action.

Int J Syst Evol Microbiol, 2001 Jan, 51(Pt 1), 231 - 5
Sporobolomyces yunnanensis sp . nov., a Q-10(H2)-containing yeast species with a close phylogenetic relationship to Erythrobasidium hasegawianum; Bai FY et al.; A ballistoconidia-forming yeast strain, CH 2.141T, isolated from a semi-dried leaf sample collected in Yunnan, China, was found to have Q-10(H2) as its major ubiquinone . Molecular phylogenetic analysis based on the nucleotide sequences of small subunit (18S) rDNA and the internal transcribed spacer region (including 5.8S rDNA) indicated that the strain was closely related to the two described Q-10(H2)-containing yeast species, Erythrobasidium hasegawianum and Sporobolomyces elongatus, with a closer relationship to the former . A DNA-DNA reassociation experiment showed that strain CH 2.141T represents a new yeast species, for which the name Sporobolomyces yunnanensis sp . nov . is proposed.

J Dairy Sci, 2001 Jan, 84(1), 204 - 12
Effects of forage neutral detergent fiber and yeast culture on performance of cows during early lactation; Wang Z et al.; Sixty Holstein cows were assigned to two treatments at 21 d before calving and were group-fed a prepartum diet with or without yeast culture . After parturition, cows were individually fed one of five treatments for 140 d: 1) 21% forage neutral detergent fiber (NDF) without yeast culture, 2) 21% forage NDF with yeast culture, 3) 17% forage NDF without yeast culture, 4) 17% forage NDF with yeast culture, and 5) 25% forage NDF with yeast culture for 30 d and then switched to diet 4 for 110 d . Cows fed yeast culture prepartum were also fed yeast culture postpartum (60 g/d) . A quadratic increase to 25, 21, and 17% forage NDF occurred during the first 30 d in milk (DIM) for dry matter intake, milk yield, and milk protein yield . No differences were observed for yeast culture or interaction of yeast culture and forage NDF for the first 30 DIM . Feeding 17 versus 21% forage NDF increased milk protein percentage and tended to increase dry matter intake as a percentage of body weight from 31 to 140 DIM . During this period, yeast culture tended to increase milk fat percentage and appeared to have positive effects on dry matter intake, milk yield, and milk fat yield when supplemented to diets with 21% forage NDF but not with 17% forage NDF . Feeding 17% forage NDF may be too low for the first 30 DIM but may improve animal performance after 30 DIM compared to 21% forage NDF.

Biol Chem, 2000 Dec, 381(12), 1175 - 83
Yeast translational activator Cbs2p: mitochondrial targeting and effect of overexpression; Tzschoppe K et al.; The yeast translational activator protein Cbs2p is imported into mitochondria without obvious proteolytic processing . To test the importance of amino-terminal amino acids for mitochondrial targeting we fused varying portions of the N-terminus with green fluorescent protein and examined the intracellular distribution of the reporter protein . We show that the 25 N-terminal amino acids are sufficient to direct the majority of the fusion protein into mitochondria . Cbs2p derivatives lacking 9 to 35 amino acids from the N-terminus fail to complement the respiratory deficiency of a deltacbs2 strain, but are still imported into mitochondria . Therefore Cbs2p contains at least one independent mitochondrial targeting information in addition to the N-terminal signal . We further analyzed the effect of over-expression of Cbs2p on mitochondrial function . Elevated concentrations of Cbs2p lead to slightly impaired mitochondrial gene expression, probably as the result of the formation of inactive Cbs2p aggregates.

Biochem Cell Biol, 2000, 78(6), 683 - 90
A comparative hybridization analysis of yeast DNA with Paramecium parafusin- and different phosphoglucomutase-specific probes; Wyroba E et al.; Molecular probes designed for the parafusin (PFUS), the Paramecium exocytic-sensitive phosphoglycoprotein, gave distinct hybridization patterns in Saccharomyces cerevisiae genomic DNA when compared with different phosphoglucomutase specific probes . These include two probes identical to segments of yeast phosphoglucomutase (PGM) genes 1 and 2 . Neither of the PGM probes revealed the 7.4 and 5.9 kb fragments in Bgl II-cut yeast DNA digest detected with the 1.6 kb cloned PFUS cDNA and oligonucleotide constructed to the PFUS region (insertion 3--I-3) not found in other species . PCR amplification with PFUS-specific primers generated yeast DNA-species of the predicted molecular size which hybridized to the I-3 probe . A search of the yeast genome database produced an unassigned nucleotide sequence that showed 55% identity to parafusin gene and 37% identity to PGM2 (the major isoform of yeast phosphoglucomutase) within the amplified region.

Nature, 2001 Jan 25, 409(6819), 533 - 8
Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF; Iyer VR et al.; Proteins interact with genomic DNA to bring the genome to life; and these interactions also define many functional features of the genome . SBF and MBF are sequence-specific transcription factors that activate gene expression during the G1/S transition of the cell cycle in yeast . SBF is a heterodimer of Swi4 and Swi6, and MBF is a heterodimer of Mbpl and Swi6 (refs 1, 3) . The related Swi4 and Mbp1 proteins are the DNA-binding components of the respective factors, and Swi6 mayhave a regulatory function . A small number of SBF and MBF target genes have been identified . Here we define the genomic binding sites of the SBF and MBF transcription factors in vivo, by using DNA microarrays . In addition to the previously characterized targets, we have identified about 200 new putative targets . Our results support the hypothesis that SBF activated genes are predominantly involved in budding, and in membrane and cell-wall biosynthesis, whereas DNA replication and repair are the dominant functions among MBF activated genes . The functional specialization of these factors may provide a mechanism for independent regulation of distinct molecular processes that normally occur in synchrony during the mitotic cell cycle.

Protein Sci, 2000 Dec, 9(12), 2354 - 65
Backbone dynamics of sequence specific recognition and binding by the yeast Pho4 bHLH domain probed by NMR; Cave JW et al.; Backbone dynamics of the basic/helix-loop-helix domain of Pho4 from Saccharomyces cerevisae have been probed by NMR techniques, in the absence of DNA, nonspecifically bound to DNA and bound to cognate DNA . Alpha proton chemical shift indices and nuclear Overhauser effect patterns were used to elucidate the secondary structure in these states . These secondary structures are compared to the co-crystal complex of Pho4 bound to a cognate DNA sequence (Shimizu T . Toumoto A, Ihara K, Shimizu M, Kyogou Y, Ogawa N, Oshima Y, Hakoshima T, 1997, EMBO J 15: 4689-4697) . The dynamic information provides insight into the nature of this DNA binding domain as it progresses from free in solution to a specifically bound DNA complex . Relative to the unbound form, we show that formation of either the nonspecific and cognate DNA bound complexes involves a large change in conformation and backbone dynamics of the basic region . The nonspecific and cognate complexes, however, have nearly identical secondary structure and backbone dynamics . We also present evidence for conformational flexibility at a highly conserved glutamate basic region residue . These results are discussed in relation to the mechanism of sequence specific recognition and binding.

Ukr Biokhim Zh, 2000 May-Jun, 72(3), 25 - 30
{Riboflavin biosynthesis in yeast as a model for estimating mechanisms of regulating synthesis of biologically active substances}; Fedorovych DV et al.; General principles of regulation of transcription in eucaryotic cells are characterized . Investigations on riboflavin biosynthesis and regulation of this process in yeasts are summarized . The data obtained permit to consider riboflavin biosynthesis control as an interesting and convenient model capable to estimate the mechanisms various transcription factors action . These investigations are required in order to reveal general features of vitamins and relative compounds biosynthesis regulation.

Bioresour Technol, 2001 Feb, 76(3), 253 - 8
Utilization of brewer's yeast cells for the production of food-grade yeast extract . Part 1: Effects of different enzymatic treatments on solid and protein recovery and flavor characteristics; Chae HJ et al.; Yeast extract was produced from brewer's yeast of a beer factory by combined enzymatic treatments using endoprotease, exoprotease, 5'-phosphodiesterase, and adenosine monophosphate (AMP)-deaminase . Effects of enzyme combination, enzyme dosages and treatment sequence on the recovery of solid and protein, flavor and compositional characteristics were investigated . Exoprotease dosage strongly affected the recovery of protein and degree of hydrolysis (DH) and sensory characteristics . When the yeast cells were treated using optimal combination of endoprotease and exoprotease (0.6% Protamex and 0.6% Flavourzyme), high solid recovery (48.3-53.1%) and the best flavor profile were obtained . Among various treatment sequences using multiple enzymes, treatment with protease followed by nuclease resulted in the highest 5'-guanosine monophosphate (5'-GMP) content . The optimal concentrations of both 5'-phosphodiesterase and AMP-deaminase were found to be 0.03% . After treatments using optimal combination of enzyme, enzyme dosages and treatment sequence for four enzymes, a high solid yield of 55.1% and 5'-nucleotides content of 3.67% were obtained.

Cancer Res, 2001 Jan 1, 61(1), 64 - 70
Human homologue of yeast Rad23 protein A interacts with p300/cyclic AMP-responsive element binding (CREB)-binding protein to down-regulate transcriptional activity of p53; Zhu Q et al.; The tumor suppressor protein p53 regulates various cellular responses to DNA damage and plays a significant role in DNA repair . The nuclear p300/cyclic AMP-responsive element binding (CREB)-binding protein (CBP) proteins act as coactivators in supporting the transcription function of p53 . We examined the role of the human homologue of yeast Rad23 protein A (hHR23A), one of the two human homologues of the Saccharomyces cerevisiae nucleotide excision repair gene product Rad23, in the p300/CBP-associated regulation of p53 activity . Overexpression of wild-type hHR23A inhibits the p53 transcriptional activity and results in a decreased steady-state protein level of cellular p53 . The inhibitory effect of hHR23A can be overcome by the concomitant expression of p300, CBP, and p300 segments harboring C/H1 domain and neutralized by the coexpression of HIV accessory protein Vpr, which binds COOH terminus of hHR23A/B . Additionally, hHR23A was shown to interact in vitro and in vivo with p300 segments harboring C/H1 domain . These studies provide evidence for the involvement of hHR23A in the regulation of p53 activity through p300/CBP . Although the precise direct role of hHR23 proteins in regulation of p53 and DNA repair remains to be elucidated, our data suggest that the interaction between hHR23A and p300/CBP has important implications in cross-talk between the p53 pathway and DNA repair.

Mikrobiologiia, 2000 Nov-Dec, 69(6), 801 - 4
{A new species of psychrophilic basidiomycetes yeast Leucosporidium fasciculatum sp . Nov.}; Bab'eva IP et al.; A psychrophilic yeast with a basidiomycetous developmental cycle and properties corresponding to the genus Leucosporidium Fell et al . was isolated from the fruiting body of the edible spring mushroom Gyromitra esculenta Pers . picked near Moscow . However, the isolate differed from all Leucosporidium species described to date in a number of characteristics . The results of the study of the developmental cycle and of the cultural, morphological, physiological, and biochemical properties of the new isolate, strain KBP Y-3696, allow it to be assigned to a new species of the genus Leucosporidium.

Mikrobiologiia, 2000 Nov-Dec, 69(6), 790 - 5
{Composition of lipids from yeast-like and mycelial cells of the fungus Mucor hiemalis, grown in the presence of 4-chloroaniline}; Mysiakina IS et al.; The fungus Mucor hiemalis, which is commonly thought to be monomorphic, produced two types of cells, yeastlike and mycelial, during growth in a medium containing 4-chloroaniline . Among the polar lipids of yeastlike cells, diphosphatidylglycerol was dominant, while phosphatidylcholine and phosphatidylethanolamine were present in minor amounts . Conversely, mycelial cells mainly contained phosphatidylcholine and phosphatidylethanolamine, whereas the content of diphosphatidylglycerol was low . The neutral lipids of yeastlike cells were dominated by diacylglycerides, sterols, and fatty acids . The content of triacylglycerides and sterol esters was low . Yeastlike cells contained higher amounts of saturated fatty acids and lower amounts of unsaturated fatty acids than the mycelium . The content of stearic acid in the fatty acids of the mycelium grown in the presence of 4-chloroaniline was as high as 25.3-29.9%.

Hokkaido Igaku Zasshi, 2000 Nov, 75(6), 385 - 97
{Development of a detection system (APC yeast color assay) of APC mutations by color change of yeast}; Furuuchi K; The author developed a sensitive yeast-based color assay which expresses APC-ADE2 (reporter) fusion protein in yeast and can screen almost the entire coding region of the APC gene . In this assay, the wild-type APC coding sequence of 8.5 kb is divided into 5 overlapping regions which are respectively ligated in-frame with an ADE2 open reading frame . The resulting five constructs containing a part of wild-type APC gene preserve the ADE2(+) phenotype (white yeast colony) when introduced into the yeast, whereas the yeast transfected with plasmids containing frameshift mutations of the APC gene shows an ADE2(-) phenotype (red yeast colony) . Six human colon cancer cell lines were analyzed by this yeast color assay . HCT116 cells with wild-type APC and normal colonic mucosa gave low percentages of red colonies (0-9.9%) in all the regions . On the other hand, more than 96% red colonies were observed in one of the five regions in SW480, Colo201 and Colo320DM cells . Sequence analysis demonstrated the clonal APC mutations at codon 1,338 in SW480, 1,554 in Colo201 and 811 in Colo320DM . Moreover, the assay detected a germline mutation of the APC gene in polyps of a familial adenomatous polyposis (FAP) patient which gave about 50% red colonies . For testing the assay for clinical utilization, 18 colon cancer tissues were subjected to the assay . Eleven cancers (61%) gave more than 10% red colonies (17-57%) and clonal mutations were detected in all these samples . The same mutations were demonstrated in both DNA and RNA samples derived from idendical tissues . These results suggest that this APC yeast color assay is powerful means for detection of APC mutations in clinical samples.

Biologist (London), 2000 Feb, 47(1), 15 - 8
Yeast as factory and factotum; Dixon B; After centuries of vigorous activity in making fine wines, beers and breads, Saccharomyces cerevisiae is now acquiring a rich new portfolio of skills, bestowed by genetic manipulation . As shown in a recent shop-window of research supported by the European Commission, yeasts will soon be benefiting industries as diverse as fish farming, pharmaceuticals and laundering.

IUBMB Life, 2000 Aug, 50(2), 105 - 13
A CD2-based model of yeast alpha-agglutinin elucidates solution properties and binding characteristics; Grigorescu A et al.; We have previously shown that the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin has sequence characteristics of immunoglobulin-like proteins and have successfully modeled residues 200-325, based on the structure of immunoglobulin variable-type domains . Alignments matching residues 20-200 of alpha-agglutinin with domains I and II of members of the CD2/CD4 subfamily of the immunoglobulin superfamily showed > 80% conservation of key residues despite low sequence similarity overall . Three-dimensional models of two alpha-agglutinin domains constructed on the basis of these alignments were shown to conform to peptide mapping data and biophysical properties of alpha-agglutinin . In addition, the residue volume and surface accessibility characteristics of these models resembled those of the well-packed structures of related proteins . Residue-by-residue analysis showed that packing and accessibility anomalies were largely confined to glycosylated and protease-susceptible loop regions of the domains . Surface accessibility of hydrophobic residues was typical of proteins with extensive domain interactions, a finding compatible with the hydrodynamic properties of alpha -agglutinin and the hydrophobic nature of binding to its peptide ligand alpha-agglutinin . The procedures used to align the alpha-agglutinin sequence and test the quality of the model may be applicable to other proteins, especially those that resist crystallization because of extensive glycosylation.

Traffic, 2001 Jan, 2(1), 37 - 50
NBD-labeled phosphatidylcholine and phosphatidylethanolamine are internalized by transbilayer transport across the yeast plasma membrane; Grant AM et al.; The internalization and distribution of fluorescent analogs of phosphatidylcholine (M-C6-NBD-PC) and phosphatidylethanolamine (M-C6-NBD-PE) were studied in Saccharomyces cerevisiae . At normal growth temperatures, M-C6-NBD-PC was internalized predominantly to the vacuole and degraded . M-C6-NBD-PE was internalized to the nuclear envelope/ER and mitochondria, was not transported to the vacuole, and was not degraded . At 2 degrees C, both were internalized to the nuclear envelope/ER and mitochondria by an energy-dependent, N-ethylmaleimide-sensitive process, and transport of M-C6-NBD-PC to and degradation in the vacuole was blocked . Internalization of neither phospholipid was reduced in the endocytosis-defective mutant, end4-1 . However, following pre-incubation at 37 degrees C, internalization of both phospholipids was inhibited at 2 degrees C and 37 degrees C in sec mutants defective in vesicular traffic . The sec18/NSF mutation was unique among the sec mutations in further blocking M-C6-NBD-PC translocation to the vacuole suggesting a dependence on membrane fusion . Based on these and previous observations, we propose that M-C6-NBD-PC and M-C6-NBD-PE are transported across the plasma membrane to the cytosolic leaflet by a protein-mediated, energy-dependent mechanism . From the cytosolic leaflet, both phospholipids are spontaneously distributed to the nuclear envelope/ER and mitochondria . Subsequently, M-C6-NBD-PC, but not M-C6-NBD-PE, is sorted by vesicular transport to the vacuole where it is degraded by lumenal hydrolases.

Traffic, 2000 May, 1(5), 425 - 34
A novel pathway for transport and metabolism of a fluorescent phosphatidic acid analog in yeast; Trotter PJ; Phosphatidic acid is a central intermediate of biosynthetic lipid metabolism as well as an important signaling molecule in the cell . These studies assess the internalization, or retrograde transport, and metabolism of phosphatidic acid in yeast using a fluorescent analog . An analog of phosphatidic acid fluorescently labeled at the sn-2 position with N-4-nitrobenz-2-oxa-1, 3-diazole-aminocaproic acid (NBD-phosphatidic acid) was introduced to yeast cells by spontaneous transfer from phospholipid vesicles . Transport and metabolism of the NBD-phosphatidic acid were then monitored by fluorescence spectrophotometry, fluorescence microscopy and routine biochemical methods . Primary metabolites of the NBD-phosphatidic acid in yeast were found to be NBD-diacylgycerol and NBD-phosphatidylinositol . Experiments in cells possessing different levels of phosphatidate phosphatase activity suggest that conversion of the NBD-phosphatidic acid to NBD-diacylglycerol is not a pre-requisite for internalization in yeast . Internalization is sensitive to decreased temperature, but neither ATP depletion nor a sec6-4 mutation, which interrupts endocytosis, has an affect . Thus, internalization of NBD-phosphatidic acid apparently occurs via a non-endocytic route . These characteristics of retrograde transport of NBD-phosphatidic acid in yeast differ significantly from transport of other NBD-phospholipids in yeast as well as NBD-phosphatidic acid transport in mammalian fibroblasts.

Traffic, 2000 Mar, 1(3), 259 - 69
Pep12p is a multifunctional yeast syntaxin that controls entry of biosynthetic, endocytic and retrograde traffic into the prevacuolar compartment; Gerrard SR et al.; Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of the endosomal syntaxin, Pep12p . Many vacuolar proteins, such as the soluble vacuolar hydrolase, carboxypeptidase Y (CPY), traverse the prevacuolar compartment (PVC) en route to the vacuole . Here we show that deletion of the carboxy-terminal transmembrane domain of Pep12p results in a temperature-conditional block in transport of CPY to the PVC . The PVC also receives traffic from the early endosome and the vacuole, and mutation in PEP12 also blocks these other trafficking pathways into the PVC . Therefore, Pep12p is a multifunctional syntaxin that is required for all known trafficking pathways into the yeast PVC . Finally, we found that the internalized pheromone receptor, Ste3p, can cycle out of the PVC in a VPS27-independent fashion.

Traffic, 2000 Mar, 1(3), 248 - 58
Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking; Babst M et al.; The mammalian tumor susceptibility gene tsg101 encodes the homologue of Vps23p, a class E Vps protein essential for normal membrane trafficking in the late endosome/multivesicular body of yeast . Both proteins assemble into large (approximately 350 kDa) cytosolic protein complexes and we show that the yeast complex contains another class E Vps protein, Vps28p . tsg101 mutant cells exhibit defects in sorting and proteolytic maturation of the lysosomal hydrolase cathepsin D, as well as in the steady-state distribution of the mannose-6-phosphate receptor . Additionally, endocytosed EGF receptors that are normally sorted to the lysosome are instead rapidly recycled back to the cell surface in tsg101 mutant cells . We propose that tsg101 mutant cells are defective in the delivery of cargo proteins to late endosomal compartments . One consequence of this endosomal trafficking defect is the delayed down-regulation/degradation of activated cell surface receptors, resulting in prolonged signaling . This may contribute to the tumorigenic phenotype exhibited by the tsg101 mutant fibroblasts.

Traffic, 2000 Feb, 1(2), 172 - 83
Sec7p directs the transitions required for yeast Golgi biogenesis; Deitz SB et al.; Endoplasmic reticulum (ER)-to-Golgi traffic in yeast proceeds by the maturation of membrane compartments from post-ER vesicles to intermediate small vesicle tubular clusters (VTCs) to Golgi nodular membrane networks (Morin-Ganet et al., Traffic 2000; 1: 56-68) . The balance between ER and Golgi compartments is maintained by COPII- and COPI-mediated anterograde and retrograde traffic, which are dependent on Sec7p and ARF function . The sec7-4 temperature-sensitive allele is a mutation in the highly conserved Sec7 domain (Sec7d) found in all ARF-guanine nucleotide exchange factor proteins . Post-ER trafficking is rapidly inactivated in sec7-4 mutant yeast at the restrictive temperature . This conditional defect prevented the normal production of VTCs and instead generated Golgi-like tubes emanating from the ER exit sites . These tubes progressively developed into stacked cisternae defining the landmark sec7 mutant phenotype . Consistent with the in vivo results, a Sec7d peptide inhibited ER-to-Golgi transport and displaced Sec7p from its membrane anchor in vitro . The similarities in the consequences of inactivating Sec7p or ARFs in vivo was revealed by genetic disruption of yeast ARFs or by addition of brefeldin A (BFA) to whole cells . These treatments, as in sec7-4 yeast, affected the morphology of membrane compartments in the ER-Golgi transition . Further evidence for Sec7p involvement in the transition for Golgi biogenesis was revealed by in vitro binding between distinct domains of Sec7p with ARFs, COPI and COPII coat proteins . These results suggest that Sec7p coordinates membrane transitions in Golgi biogenesis by directing and scaffolding the binding and disassembly of coat protein complexes to membranes, both at the VTC transition from ER exit sites to form Golgi elements and for later events in Golgi maturation.

Traffic, 2000 Oct, 1(10), 763 - 8
The use of yeast two-hybrid screens in studies of protein:protein interactions involved in trafficking; Stephens DJ et al.; The yeast two-hybrid system has provided a convenient means to both screen for proteins that interact with a protein of interest and to characterise the known interaction between two proteins . Several groups with an interest in the molecular mechanisms that underlie discrete steps along trafficking pathways have exploited the yeast two-hybrid system . Here, we provide a brief background to the technology, attempt to point out some of the pitfalls and benefits of the different systems that can be employed, and mention some of the areas (within the trafficking field) where yeast two-hybrid interaction assays have been particularly informative.

Traffic, 2000 Jan, 1(1), 56 - 68
Morphogenesis and dynamics of the yeast Golgi apparatus; Morin-Ganet MN et al.; A kinetic and morphometric study was conducted with the electron microscope to clarify the biogenesis and structural diversity of the Golgi apparatus in the yeast Saccharomyces cerevisiae . Secretion was synchronized by inhibiting protein synthesis and/or by subjecting thermosensitive secretory mutants to double temperature shifts . Five membrane-bounded structures disappeared or reappeared in an orderly manner at approximately the rate of secretory protein flow . 1) The first detectable post-ER intermediates were very short-lived clusters of small vesicles that appeared next to the endoplasmic reticulum (ER) . 2) Their constituent small vesicles were rapidly bridged by membrane tubules in a SEC18-dependent manner, giving short-lived tubular clusters of small vesicles, analogous to mammalian vesicular-tubular clusters . 3) Fine and 4) large nodular networks (coated with the Golgi protein Sec7), and 5) secretory granules . Upon relieving a secretory block, each structure successively reappeared, seemingly by transformation of the previous one . When no secretory cargo was to be transported, these structures were not renewed . They disappeared more than five times faster than some Golgi enzymes such as Och1p, implying that the latter are recycled and perhaps partially retained . Retention could arise from intra-compartmental flow of cargo/carrier, hinted at by the varying calibers within a single nodular network.

Traffic, 2000 Jan, 1(1), 45 - 55
The yeast endosomal t-SNARE, Pep12p, functions in the absence of its transmembrane domain; Gerrard SR et al.; Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of two distinct SNARE complexes . Pep12p and Vam3p are both t-SNAREs of the syntaxin family that are components of these SNARE complexes . We have used a genetic approach to address the role of Pep12p in vacuolar protein transport . Our screen for temperature-sensitive pep12 mutants yielded six alleles that were rapidly inactivated upon exposure to the non-permissive temperature . Surprisingly, the proteins encoded by these alleles were all truncated immediately prior to the transmembrane domain . Here we demonstrate that Pep12p requires its transmembrane domain for proper localization, but not for its role in vesicle fusion . In addition, we show that although Pep12p can replace Vam3p in the vacuolar SNARE complex, its transmembrane domain is required to function at this step . Therefore, the transmembrane domain of Pep12p performs different roles in the prevacuolar and vacuolar SNARE complexes.

Bull Exp Biol Med, 2000 Nov, 130(11), 1084 - 6
Allergen-containing drug from Malassezia spp . yeast; Arzumanyan VG et al.; Malassezia spp . yeast habituating the skin of healthy humans can be a source of allergens for patients with atopic dermatitis . We proposed a method for obtaining allergen-containing preparation by trimming outer cell wall proteins with 1% sodium dodecyl sulfate . The resultant preparation contained 36 and 67 kD proteins known as Malassezia allergens . IgE antibodies to these proteins were detected in the sera of young people with atopic dermatitis.

Yeast, 2001 Feb, 18(3), 239 - 49
Pho85 kinase, a yeast cyclin-dependent kinase, regulates the expression of UGP1 encoding UDP-glucose pyrophosphorylase; Nishizawa M et al.; The PHO85 gene is a negative regulator of the PHO system in the yeast Saccharomyces cerevisiae and encodes a protein kinase (Pho85) highly homologous to the Cdc28 kinase (Cdc28) . Ten cyclin-like proteins are known to interact with Pho85, and combination with different cyclins is believed to be responsible for distinct Pho85 functions, including phosphate metabolism, carbon source utilization and cell cycle regulation . However, only a limited number of substrates of Pho85 kinase, including Pho4, Gsy2 and Sicl, have so far been identified . To search for more targets of Pho85 and to clarify the genetic control mechanisms by Pho85 kinase in these cellular functions, we carried out a genome-wide analysis of the effect of a pho85Delta mutation on gene expression . We found that expression of various genes involved in carbon metabolism are affected by the mutation and that among them, UGP1 promoter activity was increased in the absence of Pho85 kinase . This increase in the promoter activity was not observed in a pho4Delta mutant or with a mutant UGP1 promoter that is devoid of putative Pho4 and Bas2 binding sites, suggesting that UGP1 expression is modulated by Pho85 through Pho4 . We also found that expression of several Pho85-cyclin genes were altered by the carbon source, the growth phase and Pho85 kinase itself.

Mol Biol Cell, 2001 Feb, 12(2), 323 - 37
Remodeling of yeast genome expression in response to environmental changes; Causton HC et al.; We used genome-wide expression analysis to explore how gene expression in Saccharomyces cerevisiae is remodeled in response to various changes in extracellular environment, including changes in temperature, oxidation, nutrients, pH, and osmolarity . The results demonstrate that more than half of the genome is involved in various responses to environmental change and identify the global set of genes induced and repressed by each condition . These data implicate a substantial number of previously uncharacterized genes in these responses and reveal a signature common to environmental responses that involves approximately 10% of yeast genes . The results of expression analysis with MSN2/MSN4 mutants support the model that the Msn2/Msn4 activators induce the common response to environmental change . These results provide a global description of the transcriptional response to environmental change and extend our understanding of the role of activators in effecting this response.

EMBO J, 2001 Feb 15, 20(4), 777 - 91
The budding yeast proteins Spc24p and Spc25p interact with Ndc80p and Nuf2p at the kinetochore and are important for kinetochore clustering and checkpoint control; Janke C et al.; Here, we show that the budding yeast proteins Ndc80p, Nuf2p, Spc24p and Spc25p interact at the kinetochore . Consistently, Ndc80p, Nuf2p, Spc24p and Spc25p associate with centromere DNA in chromatin immunoprecipitation experiments, and SPC24 interacts genetically with MCM21 encoding a kinetochore component . Moreover, although conditional lethal spc24-2 and spc25-7 cells form a mitotic spindle, the kinetochores remain in the mother cell body and fail to segregate the chromosomes . Despite this defect in chromosome segregation, spc24-2 and spc25-7 cells do not arrest in metaphase in response to checkpoint control . Furthermore, spc24-2 cells showed a mitotic checkpoint defect when microtubules were depolymerized with nocodazole, indicating that Spc24p has a function in checkpoint control . Since Ndc80p, Nuf2p and Spc24p are conserved proteins, it is likely that similar complexes are part of the kinetochore in other organisms.

J Biochem (Tokyo), 2001 Feb, 129(2), 297 - 302
Study on the biosynthesis of dolichol in yeast: recognition of the prenyl chain length in polyprenol reduction; Tateyama S et al.; We synthesized three water-soluble biotin-tagged compounds with different prenyl chain lengths, biotinylated farnesal (BF), biotinylated C(55)-polyprenal (BP55), and biotinylated C(80)-polyprenal (BP80), and examined their effects on in vitro dolichol synthesis from farnesyl diphosphate . BF and BP55 did not affect the dolichol synthesis, whereas BP80 inhibited the reduction pathway from polyprenol to dolichol, accompanied by a decrease in the entire polyprenol and dolichol synthesis . Comparison of BP80 with eighteen detergents, including Triton X-100, CHAPS, octylglucoside, deoxycholate, and Tween 80, revealed the specific effect of BP80 on the reduction pathway . On SDS-polyacrylamide gel electrophoresis, BP80 was detected in an associated form with a 50 kDa protein . These results suggest that the reduction of polyprenol to dolichol in the dolichol biosynthetic pathway proceeds with the recognition of the polyprenol chain length by a 50 kDa protein.

Biochemistry (Mosc), 2000 Dec, 65(12), 1362 - 6
Interaction of catalytic domains in cytochrome P450scc--adrenodoxin reductase--adrenodoxin fusion protein imported into yeast mitochondria; Novikova LA et al.; We have constructed plasmids for yeast expression of the fusion protein pre-cytochrome P450scc--adrenodoxin reductase-adrenodoxin (F2) and a variant of F2 with the yeast CoxIV targeting presequence . Mitochondria isolated from transformed yeast cells contained the F2 fusion protein at about 0.5% of total protein and showed cholesterol hydroxylase activity with 22(R)-hydroxycholesterol . The activity increased 17- or 25-fold when sonicated mitochondria were supplemented with an excess of purified P450scc or a mixture of adrenodoxin (Adx) and adrenodoxin reductase (AdxRed), respectively . These data suggest that, at least in yeast mitochondria, the interactions of the catalytic domains of P450scc, Adx, and AdxRed in the common polypeptide chain are restricted.

Biochemistry (Mosc), 2000 Dec, 65(12), 1352 - 6
The Ca(2+)-transport system of yeast (Endomyces magnusii) mitochondria: independent pathways for Ca(2+) uptake and release; Deryabina YI et al.; Some features of the Ca(2+)-transport system in mitochondria of the yeast Endomyces magnusii are considered . The Ca(2+) uniporter was shown to be specifically activated by low concentrations of physiological modulators such as ADP, NADH, spermine, and Ca(2+) itself . The Na(+)-independent system responsible for Ca(2+) release from Ca(2+)-preloaded yeast mitochondria was characterized . The rate of the Ca(2+) release was proportional to the Ca(2+) load, insensitive to cyclosporin A and to Na(+), inhibited by La(3+), TPP(+), P(i), and nigericin, while being activated by spermine . We conclude that Ca(2+) release from preloaded E . magnusii yeast mitochondria is mediated by a Na(+)-independent pathway, very similar to that in mitochondria from nonexcitable mammalian tissues . A scheme describing an arrangement of the Ca(2+) transport system of yeast mitochondria is proposed.

J Gen Virol, 2001 Mar, 82(Pt 3), 677 - 86
Identification of an N-terminal domain of the plum pox potyvirus CI RNA helicase involved in self-interaction in a yeast two-hybrid system; Lopez L et al.; Potyvirus CI RNA helicase is a protein involved in RNA genome replication and virus movement . The protein aggregates in the cytoplasm of infected cells to form typical cylindrical inclusions . A yeast two-hybrid system was used to analyse interactions of the CI RNA helicase from plum pox potyvirus (PPV) with itself and with other viral proteins . No interactions could be detected of full-length CI protein with itself or with PPV P3/6K1, NIa, NIb or CP proteins . However, positive self-interactions were detected for N-terminal fragments of the CI protein, allowing the mapping of a CI-CI binding domain to the N-terminal 177 aa of the protein . Further deletion analysis suggested that several regions of this domain contribute to the interaction . Moreover, pull-down experiments demonstrate that, at least in vitro, full-length PPV CI protein is able to self-interact in the absence of other virus or plant factors.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1501 - 6
Molecular cloning of a cytochrome P450 taxane 10 beta-hydroxylase cDNA from Taxus and functional expression in yeast; Schoendorf A et al.; The early steps in the biosynthesis of Taxol involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation at C5, acetylation of this intermediate, and a second cytochrome P450-dependent hydroxylation at C10 to yield taxadien-5 alpha-acetoxy-10 beta-ol . Subsequent steps of the pathway involve additional cytochrome P450 catalyzed oxygenations and CoA-dependent acylations . The limited feasibility of reverse genetic cloning of cytochrome P450 oxygenases led to the use of Taxus cell cultures induced for Taxol production and the development of an approach based on differential display of mRNA-reverse transcription-PCR, which ultimately provided full-length forms of 13 unique but closely related cytochrome P450 sequences . Functional expression of these enzymes in yeast was monitored by in situ spectrophotometry coupled to in vivo screening of oxygenase activity by feeding taxoid substrates . This strategy yielded a family of taxoid-metabolizing enzymes and revealed the taxane 10 beta-hydroxylase as a 1494-bp cDNA that encodes a 498-residue cytochrome P450 capable of transforming taxadienyl acetate to the 10 beta-hydroxy derivative; the identity of this latter pathway intermediate was confirmed by chromatographic and spectrometric means . The 10 beta-hydroxylase represents the initial cytochrome P450 gene of Taxol biosynthesis to be isolated by an approach that should provide access to the remaining oxygenases of the pathway.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1459 - 64 Epub 2001 Feb 06.
The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p; Umland TC et al.; The yeast nonchromosomal gene {URE3} is due to a prion form of the nitrogen regulatory protein Ure2p . It is a negative regulator of nitrogen catabolism and acts by inhibiting the transcription factor Gln3p . Ure2p residues 1--80 are necessary for prion generation and propagation . The C-terminal fragment retains nitrogen regulatory activity, albeit somewhat less efficiently than the full-length protein, and it also lowers the frequency of prion generation . The crystal structure of this C-terminal fragment, Ure2p(97--354), at 2.3 A resolution is described here . It adopts the same fold as the glutathione S-transferase superfamily, consistent with their sequence similarity . However, Ure2p(97--354) lacks a properly positioned catalytic residue that is required for S-transferase activity . Residues within this regulatory fragment that have been indicated by mutational studies to influence prion generation have been mapped onto the three-dimensional structure, and possible implications for prion activity are discussed.

Biochem Soc Trans, 2000 Dec, 28(6), 935 - 7
Utility of the Arabidopsis FAE1 and yeast SLC1-1 genes for improvements in erucic acid and oil content in rapeseed; Katavic V et al.; High-erucic acid (HEA) Brassica napus cultivars are regaining interest in industrial contexts . Erucic acid and its derivatives are important renewable raw materials utilized in the manufacture of plastic films, in the synthesis of Nylon 13,13, and in the lubricant and emollient industries . Theoretically, the highest level of erucic acid that can be achieved by means of classical breeding is 66 mol%; however, using new approaches on the basis of genetic engineering, it might be possible to develop a B . napus cultivar containing levels of erucic acid significantly above 66 mol% (>80 mol%) . In an attempt to increase the amounts of very-long-chain fatty acids (VLCFAs), and erucic acid in particular, in Canadian HEA B . napus cultivars, we have focused on two targets using a transgenic approach . We examined both the role/function of the Arabidopsis thaliana FAE1 (fatty acid elongase) gene by expressing it under the control of the seed-specific napin promoter in B . napus germplasm with analysis of the changes in VLCFA content in the seed oil of transgenic lines, and the performance of the yeast SLC1-1 (sphingolipid compensation mutant) in B . napus cv . Hero transgenic progeny in the field . Here, we report analyses of the contents of 22:1, total VLCFAand oil in the seed oil, as well as seed yield of the field-grown FAE1 and SLC1-1 B . napus cv . Hero progeny.

Biochem Soc Trans, 2000 Dec, 28(6), 692 - 5
Expression in yeast of an acyl-CoA:diacylglycerol acyltransferase cDNA from Caenorhabditis elegans; Bouvier-Nave P et al.; We have identified a cDNA from the nematode worm Caenorhabditis elegans that encodes an acyl-CoA:diacylglycerol acyltransferase (DGAT) . Its expression in Saccharomyces cerevisiae resulted in an increase both in triacylglycerol content and in microsomal oleyl-CoA:diacylglycerol acyltransferase activity . Such effects were similar to those of characterized plant DGAT genes . This is the first DGAT gene isolated from an invertebrate . The phylogenetic relationships between DGATs and animal and yeast acyl-CoA:sterol acyltransferases are illustrated.

Biochem J, 2001 Mar 1, 354(Pt 2), 407 - 12
Site-directed mutations in the mitochondrially encoded subunits I and III of yeast cytochrome oxidase; Meunier B; Since yeast is amenable to mitochondrial transformation, designed mutations can be introduced in the mitochondrially encoded subunits of the respiratory complexes . In the present work, six mutations have been introduced by the biolistic method into yeast (Saccharomyces cerevisiae) cytochrome oxidase subunits I and III . The effects of these mutations on respiratory growth competence, cytochrome oxidase activity and optical properties were then characterized . Firstly, the conserved glutamate Glu-243 in the D-channel of subunit I was replaced by an asparagine or an aspartate residue . The effects of the mutations showed that Glu-243, which is essential for proton movement in bacterial oxidases, is also required for the activity of the eukaryotic enzyme . Secondly, four mutations associated with human disease were introduced in yeast, allowing detailed analysis of their deleterious effects on cytochrome oxidase function: Met-273-->Thr, Ile-280-->Thr and Gly-317-->Ser, affecting residues located in or near the K-channel in subunit I, and a short in-frame deletion comprising residues Phe-102 to Phe-106 in subunit III (DeltaF102-F106) . The subunit III mutation was highly deleterious and abolished enzyme assembly . The change Gly-317-->Ser had no effect on respiratory function . However, mutations Met-273-->Thr and Ile-280-->Thr were mildly deleterious, decreased cytochrome oxidase activity and slightly perturbed the properties of the binuclear centre.

Biochem J, 2001 Feb 1, 353(Pt 3), 681 - 8
Multiple polyamine transport systems on the vacuolar membrane in yeast; Tomitori H et al.; We recently identified a gene (TPO1, YLL028w) that encodes a polyamine transport protein on the vacuolar membrane in yeast {Tomitori, Kashiwagi, Sakata, Kakinuma and Igarashi (1999) J . Biol . Chem . 274, 3265-3267} . Because the existence of one or more other genes for a polyamine transport protein on the vacuolar membrane was expected, we searched sequence databases for homologues of the protein encoded by TPO1 . Membrane proteins encoded by the open reading frames YGR138c (TPO2), YPR156c (TPO3) and YOR273c (TPO4) were postulated to be polyamine transporters and, indeed, were subsequently shown to be polyamine transport proteins on the vacuolar membrane . Cells overexpressing these genes were resistant to polyamine toxicity and showed an increase in polyamine uptake activity and polyamine content in vacuoles . Furthermore, cells in which these genes were disrupted showed an increased sensitivity to polyamine toxicity and a decrease in polyamine uptake activity and polyamine content in vacuoles . Resistance to polyamine toxicity in cells overexpressing the genes was overcome by bafilomycin A(1), an inhibitor of the vacuolar H(+)-ATPase . Among the four polyamine transporters, those encoded by TPO2 and TPO3 were specific for spermine, whereas those encoded by TPO1 and TPO4 recognized spermidine and spermine . These results suggest that polyamine content in the cytoplasm of yeast is elaborately regulated by several polyamine transport systems in vacuoles . Furthermore, it was shown that Glu-207, Glu-324 (or Glu-323) and Glu-574 of TPO1 protein were important for the transport activity.

Mol Cell Biol Res Commun, 2000 Aug, 4(2), 81 - 9
Adr1p-dependent regulation of the oleic acid-inducible yeast gene SPS19 encoding the peroxisomal beta-oxidation auxiliary enzyme 2,4-dienoyl-CoA reductase; Gurvitz A et al.; The role of Saccharomyces cerevisiae Adr1p was examined with respect to the transcriptional regulation of the SPS19 gene encoding the peroxisomal beta-oxidation auxiliary enzyme 2,4-dienoyl-CoA reductase . The SPS19 promoter contains both an oleate response element that binds the Pip2p-Oaf1p transcription factor as well as a canonical Adr1p-binding element, termed UAS1(SPS19) . Northern analysis demonstrated that transcriptional up-regulation of SPS19 was abolished in cells devoid of Adr1p . Expression of an SPS19-lacZ reporter gene was shown to be quiescent in the adr1Delta mutant and abnormally elevated in cells containing multiple ADR1 copies . UAS1(SPS19) was able to compete for formation of a specific complex between recombinant Adr1p-LacZ and UAS1(CTA1) representing the corresponding Adr1p-binding element in the promoter of the catalase A gene, and to interact directly with this fusion protein . We conclude that in the presence of fatty acids in the medium transcription of SPS19 is directly regulated by both Pip2p-Oaf1p and Adr1p .

Microsc Res Tech, 2000 Dec 15, 51(6), 601 - 12
Protein localisation approaches for understanding yeast cell wall biogenesis; Molina M et al.; Yeast cells are surrounded by the cell wall, a rigid but dynamic structure that is essential for their viability . The complexity and functionality of this structure suggest that a high number of proteins must be involved in the biogenesis of the cell wall architecture and, as a consequence, in the maintenance of cell integrity . Among them, a high percentage is assumed to be located at the cell surface, mostly as structural or enzymatic components of the cell wall . Therefore, the presence of a protein in the cell wall is suggestive of its cell wall-related function . Different techniques can be used to specifically detect the cell wall localisation of a given protein or to identify cell wall proteins in large-scale analyses . These include the detection of proteins in whole cells or specific cell wall fractions by immunological, biochemical, microscopic, or genetic approaches, as well as the emerging proteomic technology . The advantages, limitations, and usefulness of these techniques are discussed and illustrated with some examples .

Microsc Res Tech, 2000 Dec 15, 51(6), 584 - 600
Peroxisome biogenesis and degradation in yeast: a structure/function analysis; Veenhuis M et al.; In yeast, peroxisomes are the site of specific catabolic pathways that characteristically include hydrogen peroxide producing oxidases and catalase . During the last 10 years, much progress has been made in unravelling the molecular mechanisms involved in the biogenesis of this organelle . At present, 23 different genes (PEX genes) have been identified that are involved in different aspects of peroxisome biogenesis (e.g., proliferation, formation of the peroxisomal membrane, import of matrix proteins) . The principles of peroxisome degradation are still much less understood . Recently, the first yeast mutants affected in this process have become available and used to clone corresponding genes by functional complementation . In this paper, an overview is presented of the research on yeast peroxisomes, focusing on recent achievements in the molecular aspects of peroxisome development, function, and turnover .

Microsc Res Tech, 2000 Dec 15, 51(6), 573 - 83
Yeast mitochondrial dynamics: fusion, division, segregation, and shape; Jensen RE et al.; Mitochondria are essential organelles found in virtually all eukaryotic cells that play key roles in a variety of cellular processes . Mitochondria show a striking heterogeneity in their number, location, and shape in many different cell types . Although the dynamic nature of mitochondria has been known for decades, the molecules and mechanisms that mediate these processes are largely unknown . Recently, several laboratories have isolated and analyzed mutants in the yeast Saccharomyces cerevisiae defective in mitochondrial fusion and division, in the segregation of mitochondria to daughter cells, and in the establishment and maintenance of mitochondrial shape . These studies have identified several proteins that appear to mediate different aspects of mitochondrial morphogenesis . Although it is clear that many additional components have yet to be identified, some of the newly discovered proteins raise intriguing possibilities for how the processes of mitochondrial division, fusion, and segregation occur . Below we summarize our current understanding of the molecules known to be required for yeast mitochondrial dynamics .

Microsc Res Tech, 2000 Dec 15, 51(6), 511 - 29
The beauty of the yeast: live cell microscopy at the limits of optical resolution; Kohlwein SD; The yeast Saccharomyces cerevisiae is a very powerful system for cell biological research . Recent advances in electronic light microscopy together with the application of green fluorescent protein and other in vivo staining techniques have allowed novel and exciting insights into structural organization and dynamics of cells as small as yeast . Methods for staining yeast for microscopic inspection and for introducing tags for localization studies of proteins in living or fixed cells are summarized . Electronic light microscopy, video/deconvolution methods, and confocal laser scanning microscopy as novel tools for structural analyses, and their practical applications in yeast, are discussed .

Microsc Res Tech, 2000 Dec 15, 51(6), 496 - 510
Transmission electron microscopy of yeast; Wright R; The challenges of sample preparation can limit a researcher's selection of transmission electron microcopy (TEM) for analysis of yeast . However, with the exception of thin sectioning, preparation of well-fixed and infiltrated samples of yeast cells is achievable by any reasonably equipped laboratory . This review presents a general overview of TEM sample preparation methods and detailed protocols for chemical fixation of yeast for ultrastructural analysis and immunolabeling . For ultrastructural analysis, the most commonly used chemical fixation involves treatment with glutaraldehyde followed by either potassium permanganate or osmium . Prior to osmium postfixation, the cell wall must be enzymatically digested to allow optimal fixation and embedding . Freeze substitution methods continue to provide the highest quality of fixation, but equipment needed for these protocols is not generally available to many labs . The low viscosity of Spurr's resin makes it the resin of choice for ultrastructure studies . Immunoelectron microscopy has enjoyed great success in analysis of yeast molecular organization . For immunoelectron microscopy, glutaraldehyde/formaldehyde-fixed cells are embedded in LR White resin . The thin sections are then treated in much the same way as an immunoblot: following blocking, they are incubated in primary antiserum, washed, and then incubated in gold-labeled secondary antiserum .

Yeast, 2001 Jan 30, 18(2), 163 - 72
Identification of yeast deletion strains that are hypersensitive to brefeldin A or monensin, two drugs that affect intracellular transport; Muren E et al.; We have screened the Eurofan deletion strain collection for mutants that are either sensitive or resistant to three drugs known to affect intracellular transport: brefeldin A, monensin and C(2)-ceramide . Drug-sensitive mutants were analysed by complementation with cognate clones and tetrad analysis to confirm that the phenotypes are linked to the deletions . Out of 620 deletion strains, we found 18 mutants that were sensitive to either brefeldin A, monensin or both . Several of these mutants are deleted for genes that are known to be involved in intracellular transport, membrane biogenesis and/or cell wall biosynthesis . Among such previously known genes were VAM6, VAC7, SYS1, TLG2, RCY1, ERG4, ALG9 and ALG12 . Some other genes recovered in our screen were not previously implicated in intracellular transport, but are related to other yeast genes with such a function . Still other genes encode proteins with no obvious link to intracellular transport . Several of these are putative transcription factors or RNA-binding proteins, which suggests that they may affect drug sensitivity by modulating the expression of other genes or proteins .

Mol Microbiol, 2001 Feb, 39(3), 533 - 41
Role of the glutathione/glutaredoxin and thioredoxin systems in yeast growth and response to stress conditions; Grant CM; Sulphydryl groups (-SH) play a remarkably broad range of roles in the cell, and the redox status of cysteine residues can affect both the structure and the function of numerous enzymes, receptors and transcription factors . The intracellular milieu is usually a reducing environment as a result of high concentrations of the low-molecular-weight thiol glutathione (GSH) . However, reactive oxygen species (ROS), which are the products of normal aerobic metabolism, as well as naturally occurring free radical-generating compounds, can alter this redox balance . A number of cellular factors have been implicated in the regulation of redox homeostasis, including the glutathione/glutaredoxin and thioredoxin systems . Glutaredoxins and thioredoxins are ubiquitous small heat-stable oxidoreductases that have proposed functions in many cellular processes, including deoxyribonucleotide synthesis, repair of oxidatively damaged proteins, protein folding and sulphur metabolism . This review describes recent findings in the lower eukaryote Saccharomyces cerevisiae that are leading to a better understanding of their role in redox homeostasis in eukaryotic cell metabolism.

Curr Opin Biotechnol, 2001 Feb, 12(1), 28 - 34
Large-scale mutagenesis: yeast genetics in the genome era; Vidan S et al.; The completion of the DNA sequence of the budding yeast Saccharomyces cerevisiae resulted in the identification of a large number of genes . However, the function of most of these genes is not known . One of the best ways to determine gene function is to carry out mutational and phenotypic analysis . In recent years, several approaches have been developed for the mutational analysis of yeast genes on a large scale . These include transposon-based insertional mutagenesis, and systematic deletions using PCR-based approaches . These projects have produced collections of yeast strains and plasmid alleles that can be screened using novel approaches . Analysis of these collections by the scientific community promises to reveal a great deal of biological information about this organism.

Curr Biol, 2001 Jan 9, 11(1), R16 - 20
Yeast mating: getting close to membrane merger; White JM et al.; The machinery that mediates membrane fusion during yeast mating has remained elusive . But now a post-genomics approach has provided a powerful wedge into this difficult problem: a pheromone-induced multimembrane spanning protein has been identified as a key part of the mating machine.

Curr Biol, 2001 Jan 9, 11(1), 50 - 4
Nuclear exclusion of Cdc25 is not required for the DNA damage checkpoint in fission yeast; Lopez-Girona A et al.; Maintenance of genome integrity requires a checkpoint that restrains mitosis in response to DNA damage {1} . This checkpoint is enforced by Chk1, a protein kinase that targets Cdc25 {2--7} . Phosphorylated Cdc25 associates with 14-3-3 proteins, which appear to occlude a nuclear localization signal (NLS) and thereby inhibit Cdc25 nuclear import {6, 8--14} . Proficient checkpoint arrest is thought to require Cdc25 nuclear exclusion, although definitive evidence for this model is lacking . We have tested this hypothesis in fission yeast . We show that elimination of an NLS in Cdc25 causes Cdc25 nuclear exclusion and a mitotic delay, as predicted by the model . Attachment of an exogenous NLS forces nuclear inclusion of Cdc25 in damaged cells . However, forced nuclear localization of Cdc25 fails to override the damage checkpoint . Thus, nuclear exclusion of Cdc25 is unnecessary for checkpoint enforcement . We propose that direct inhibition of Cdc25 phosphatase activity by Chk1, as demonstrated in vitro with fission yeast and human Chk1 {15, 16}, is sufficient for proficient checkpoint regulation of Cdc25 and may be the primary mechanism of checkpoint enforcement in fission yeast.

FEMS Microbiol Lett, 2001 Jan 15, 194(2), 207 - 14
The basal turnover of yeast branched-chain amino acid permease Bap2p requires its C-terminal tail; Omura F et al.; The branched-chain amino acid permease Bap2p is a transport system for leucine, isoleucine, and valine in Saccharomyces cerevisiae, and its synthesis is regulated transcriptionally . However, the downregulation mechanisms of Bap2p have not been established . Here we demonstrate that the C-terminal region of Bap2p plays a pivotal role in its basal turnover . Truncation of the C-terminal 29 residues caused the stabilization and accumulation in the plasma membrane of Bap2p . Furthermore, when the Bap2p C-terminal region was fused to green fluorescent protein, the fusion protein localized to the plasma membrane, suggesting the existence of a possible degradation-related acceptor site for the C-terminal tail of Bap2p.

FEMS Microbiol Lett, 2001 Jan 15, 194(2), 171 - 4
Evidence for multiple nitrate uptake systems in the yeast Hansenula polymorpha; Machin F et al.; Hansenula polymorpha mutants disrupted in the high-affinity nitrate transporter gene (YNT1) are still able to grow in nitrate . To detect the nitrate transporter(s) responsible for this growth a strain containing disruption of the nitrate assimilation gene cluster and expressing nitrate reductase gene (YNR1) under the control of H . polymorpha MOX1 (methanol oxidase) promoter was used (FM31 strain) . In this strain nitrate taken up is transformed into nitrite by nitrate reductase and excreted to the medium where it is easily detected . Nitrate uptake which is neither induced by nitrate nor repressed by reduced nitrogen sources was detected in the FM31 strain . Likewise, nitrate uptake detected in the strain FM31 is independent of both Ynt1p and Yna1p and is not affected by ammonium, glutamine or chlorate . The inhibition of nitrite extrusion by extracellular nitrite suggests that the nitrate uptake system shown in the FM31 strain could also be involved in nitrite uptake.

Mol Cell, 2000 Dec, 6(6), 1501 - 7
Mismatch repair blocks expansions of interrupted trinucleotide repeats in yeast; Rolfsmeier ML et al.; Disease-causing expansions of trinucleotide repeats (TNRs) can occur very frequently . In contrast, expansions are rare if the TNR is interrupted (imperfect) . The molecular mechanism stabilizing interrupted alleles and thereby preventing disease has been elusive . We show that mismatch repair is the major stabilizing force for interrupted TNRs in Saccharomyces cerevisiae . Interrupted alleles expand much more often when mismatch repair is blocked by mutation or by poorly corrected mispairs . These results suggest that interruptions lead to mismatched expansion precursors . In normal cells, expansions are prevented in trans by mismatch repair, which coexcises the mismatches plus the aberrant, TNR-mediated secondary structure that otherwise resists removal . This study indicates a novel role for mismatch repair in mutation avoidance and, potentially, in disease prevention.

Mol Cell, 2000 Dec, 6(6), 1377 - 87
The spike of S phase cyclin Cig2 expression at the G1-S border in fission yeast requires both APC and SCF ubiquitin ligases; Yamano H et al.; We describe a novel set of oscillation mechanisms for the fission yeast S phase cyclin Cig2, which contains an authentic destruction box and is destroyed at anaphase via the APC/cyclosome (APC/C) . Unlike the mitotic cyclin Cdc13, however, Cig2 mRNA and protein peak at the G1/S boundary and decline to low levels in G2 and M phases . We show here that SCF(Pop1, Pop2) plays a role in transcriptional periodicity, as pop mutations result in constitutive cig2(+) transcripts . The instability of Cig2 during G2 and M is independent of either the APC/C or Pop1/Pop2, but requires Skp1, a core component of SCF . These data indicate that the APC/C and SCF control Cig2 levels differentially at different stages of the cell cycle.

Mol Cell, 2000 Dec, 6(6), 1297 - 307
Coordinate regulation of yeast ribosomal protein genes is associated with targeted recruitment of Esa1 histone acetylase; Reid JL et al.; The Esa1-containing NuA4 histone acetylase complex can interact with activation domains in vitro and stimulate transcription on reconstituted chromatin templates . In yeast cells, Esa1 is targeted to a small subset of promoters in an activator-specific manner . Esa1 is specifically recruited to ribosomal protein (RP) promoters, and this recruitment appears to require binding by Rap1 or Abf1 . Esa1 is important for RP transcription, and Esa1 recruitment to RP promoters correlates with coordinate regulation of RP genes in response to growth stimuli . However, following Esa1 depletion, H4 acetylation decreases dramatically at many loci, but transcription is not generally affected . Therefore, the transcription-associated targeted recruitment of Esa1 to RP promoters occurs in a background of more global nontargeted acetylation that is itself not required for transcription.

Curr Opin Genet Dev, 2001 Feb, 11(1), 83 - 90
Mitotic checkpoints: from yeast to cancer; Wassmann K et al.; Separation of chromosomes during mitosis is monitored by a checkpoint that leads to cell-cycle arrest if the chromosomes are not properly attached to the mitotic spindle . Molecular mechanisms controlling this checkpoint have been identified . In addition, loss of this checkpoint has been shown to result in chromosome missegregation in higher eukaryotes and may contribute to the genomic instability observed in human cancers.

Biochem Biophys Res Commun, 2001 Feb 9, 280(5), 1346 - 51
Inhibitory effects of detergents on rat CYP1A1-dependent monooxygenase: comparison of mixed and fused systems consisting of rat CYP 1A1 and yeast NADPH-P450 reductase; Inouye K et al.; Inhibitory effects of detergents Triton X-100 and Chaps on 7-ethoxycoumarin O-deethylation activity were examined in the recombinant microsomes containing both rat CYP1A1 and yeast NADPH-P450 reductase (the mixed system) and their fused enzyme (the fused system) . Triton X-100 showed competitive inhibition in both mixed and fused systems with K(i) values of 24.6 and 21.5 microM, respectively . These results strongly suggest that Triton X-100 binds to the substrate-binding pocket of CYP1A1 . These K(i) values are far below the critical micelle concentration of Triton X-100 (240 microM) . Western blot analysis revealed no disruption of the microsomal membrane by Triton X-100 in the presence of 0-77 microM Triton X-100 . On the other hand, Chaps gave distinct inhibitory effects to the mixed and fused systems . In the fused system, a mixed-type inhibition was observed with K(i) and K(i)' values of 1.2 and 5.4 mM of Chaps, respectively . However, in the mixed system, multiple inhibition modes by Chaps were observed . Western blot analysis revealed that the solubilized fused enzyme by Chaps preserved the activity whereas the solubilized CYP1A1 and NADPH-P450 reductase reductase showed no activity in the mixed system . Thus, the comparison of the mixed and fused systems appears quite useful to elucidate inhibition mechanism of detergents.

Biochem Biophys Res Commun, 2001 Jan 26, 280(3), 700 - 6
Functional analysis of mutant human carnitine acylcarnitine translocases in yeast; IJlst L et al.; Long chain fatty acids are translocated as carnitine esters across the mitochondrial inner membrane by carnitine acylcarnitine translocase (CACT) . We report functional studies on the mutant CACT proteins from a severe and a mild patient with CACT deficiency . CACT activities in fibroblasts of both patients were markedly deficient with some residual activity (<1%) in the milder patient . Palmitate oxidation activity in cells from the severe patient was less than 5% but in the milder patient approximately 27% residual activity was found . Sequencing of the CACT cDNAs revealed a c.241G>A (G81R) in the severe and a c.955insC mutation (C-terminal extension of 21 amino acids (CACT(+21aa)) in the milder patient . The effect of both mutations on the protein was studied in a sensitive expression system based on the ability of human CACT to functionally complement a CACT-deletion strain of yeast . Expression in this strain revealed significant residual activity for CACT(+21aa), while the CACT(G81R) was inactive .

Biochem Biophys Res Commun, 2001 Jan 12, 280(1), 363 - 7
Two isoforms of trimming glucosidase II exist in mammalian tissues and cell lines but not in yeast and insect cells; Ziak M et al.; We previously cloned glucosidase II and provided in vivo evidence for its involvement in protein folding quality control . DNA-sequencing of different clones demonstrated the existence of two isoforms of glucosidase II which differed by 66 nucleotides due to alternative splicing . The existence of two enzyme isoforms in various organs of pig and rat as well as human, bovine, rat, and mouse cell lines could be demonstrated by RT-PCR and Western blotting . Furthermore, the two isoforms of glucosidase II could be detected in embryonic and postnatal rat kidney and liver . In yeast, Saccharomyces cerevisiae, and in insects, Drosophila S2 cells, only one isoforms of the enzyme was detectable . The ubiquitous occurrence of the two glucosidase II isoforms in mammalian tissues and cell lines might be indicative of a special function of each isoform .

J Mol Biol, 2001 Jan 26, 305(4), 829 - 38
The bZIP-like motif of hnRNP C directs the nuclear accumulation of pre-mRNA and lethality in yeast; Tan JH et al.; The hnRNP C protein tetramer cooperatively binds 230 nt increments of pre-mRNA in vitro in a salt-resistant manner and is located along the length of vertebrate transcripts in vivo . Based on these and other findings it has been suggested that hnRNP C functions as a chaperonin to maintain long lengths of RNA topologically single-stranded and accessible to splicing factors . We report here that human C protein is lethal when expressed in the yeast Saccharomyces cerevisiae . Through a series of fluorescent immunolocalization studies, lethality was observed to be associated with the rapid nuclear accumulation of both C protein and yeast pre-mRNA . Studies using various protein constructs and the two hybrid assay reveal that these events are dependent on the basic 40 residue high-affinity RNA binding domain and its contiguous leucine zipper-like motif (the bZLM, residues 140-214) . Additionally, equilibrium binding studies have shown that the bZLM is the determinant of C protein's salt-resistant RNA binding mode . Taken together, these findings further distinguish the bZIP-like domain as the major determinant of C protein's high-affinity interaction with RNA, oligomerization, and its highly cooperative RNA binding activity . Finally, these findings indicate that yeast and vertebrates may possess a conserved mechanism for general import of RNP although a true homolog to vertebrate C protein appears not to exist in yeast . Lethality is likely due to the absence in yeast of specific mechanisms for the removal of human C protein from nascent transcripts .

Nucleic Acids Res, 2001 Feb 1, 29(3), 598 - 603
Stable expression in yeast of the mature form of human telomerase RNA depends on its association with the box H/ACA small nucleolar RNP proteins Cbf5p, Nhp2p and Nop10p; Dez C et al.; Telomerase is a ribonucleoprotein (RNP) particle required for the replication of telomeres . The RNA component, termed hTR, of human telomerase contains a domain structurally and functionally related to box H/ACA small nucleolar RNAs (snoRNAs) . Furthermore, hTR is known to be associated with two core components of H/ACA snoRNPs, hGar1p and Dyskerin (the human counterpart of yeast Cbf5p) . To assess the functional importance of the association of hTR with H/ACA snoRNP core proteins, we have attempted to express hTR in a genetically tractable system, Saccharomyces cerevisiae . Both mature non-polyadenylated and polyadenylated forms of hTR accumulate in yeast . The former is associated with all yeast H/ACA snoRNP core proteins, unlike TLC1 RNA, the endogenous RNA component of yeast telomerase . We show that the presence of the H/ACA snoRNP proteins Cbf5p, Nhp2p and Nop10p, but not Gar1p, is required for the accumulation of mature non-polyadenylated hTR in yeast, while accumulation of TLC1 RNA is not affected by the absence of any of these proteins . Our results demonstrate that yeast telomerase is unrelated to H/ACA snoRNPs . In addition, they show that the accumulation in yeast of the mature RNA component of human telomerase depends on its association with three of the four core H/ACA snoRNP proteins . It is likely that this is the case in human cells as well.

Mol Biol Cell, 2001 Jan, 12(1), 221 - 38
Multiple roles of Arf1 GTPase in the yeast exocytic and endocytic pathways; Yahara N et al.; ADP-ribosylation factors, a family of small GTPases, are believed to be key regulators of intracellular membrane traffic . However, many biochemical in vitro experiments have led to different models for their involvement in various steps of vesicular transport, and their precise role in living cells is still unclear . We have taken advantage of the powerful yeast genetic system and screened for temperature-sensitive (ts) mutants of the ARF1 gene from Saccharomyces cerevisiae . By random mutagenesis of the whole open reading frame of ARF1 by error-prone PCR, we isolated eight mutants and examined their phenotypes . arf1 ts mutants showed a variety of transport defects and morphological alterations in an allele-specific manner . Furthermore, intragenic complementation was observed between certain pairs of mutant alleles, both for cell growth and intracellular transport . These results demonstrate that the single Arf1 protein is indeed involved in many different steps of intracellular transport in vivo and that its multiple roles may be dissected by the mutant alleles we constructed.

J Pharmacol Exp Ther, 2001 Feb, 296(2), 329 - 37
Assessment of the effects of metabolism on the estrogenic activity of xenoestrogens: a two-stage approach coupling human liver microsomes and a yeast estrogenicity assay; Elsby R et al.; Concern that the reproductive health of humans is being affected by exposure to xenoestrogens has led to the development of various in vitro and in vivo screening assays for the identification of suspected xenoestrogens . However, the estrogenic activity of a chemical determined in vitro may not necessarily predict its activity in vivo if the chemical is metabolized during the assay and/or in vivo . Therefore, to investigate the role of metabolism in modulating the estrogenic activity of suspected xenoestrogens, we have devised a two-stage approach coupling incubations with either human or rat hepatic microsomes with a yeast estrogenicity (transcription) assay . We have assessed the activity of the proestrogenic pesticide 99.5% methoxychlor {1,1,1-trichloro-2,2-bis-(4-methoxyphenyl)ethane, MXC} (EC(50) = 4.45 +/- 1.9 ,icroM, n = 6) and a structural analog, methoxybisphenol A {2,2-bis-(4-methoxyphenyl) propane, MBPA}, in the yeast estrogenicity assay and also established that yeast (Saccharomyces cerevisiae), unlike human liver microsomes, are not able to demethylate MXC or MBPA to estrogenic metabolites . This indicates that the proestrogen MXC has weak intrinsic estrogenic activity . Using 99.5% MXC and 17beta-estradiol as paradigms, we have demonstrated how metabolism can enhance or suppress, respectively, estrogenic activity . The effect of metabolism on the activities of the weak xenoestrogens 3,17beta-bisdesoxyestradiol {1,3,5(10)-estratriene} and 6-hydroxytetralin (5,6,7,8-tetrahydro-2-naphthol) was also assessed . This two-stage approach can distinguish the estrogenic activity of a suspect chemical from the activity due to its more, or less, active metabolites and will aid in the evaluation of novel xenoestrogens and, more importantly, proestrogens.

Hum Mol Genet, 2001 Feb 1, 10(3), 259 - 69
Distinct roles for two N-terminal cleaved domains in mitochondrial import of the yeast frataxin homolog, Yfh1p; Gordon DM et al.; The yeast frataxin homolog (Yfh1p) participates in mitochondrial iron homeostasis . The phenotypic defects of the Delta yfh1 mutant include drastic accumulation of iron in mitochondria and slow growth . The Yfh1p precursor protein contains two N-terminal domains that are sequentially cleaved by the matrix processing peptidase on import into mitochondria, generating the mature protein . We have precisely mapped these two cleavage sites . Mutations blocking the first or the second cleavage of Yfh1p do not interfere with its in vitro import or with its ability to complement phenotypes of the Delta yfh1 mutant strain . Distinct roles have been ascertained for the two cleaved domains of Yfh1p . The first cleaved domain (domain I) is sufficient for in vitro mitochondrial import of a non-mitochondrial passenger protein . However, neither domain I nor other matrix-targeting signals alone can support efficient in vitro import of mature Yfh1p . The second cleaved domain (domain II) is required as a spacer between a targeting signal and mature Yfh1p . Likewise, when Yfh1p constructs lacking domain I or II are expressed in vivo, they fail to attain appreciable steady-state amounts in mitochondria and cannot complement phenotypes of the Delta yfh1 mutant.

Glycobiology, 2000 Dec, 10(12), 1271 - 5
Phosphatidylethanolamine is the donor of the phosphorylethanolamine linked to the alpha1,4-linked mannose of yeast GPI structures; Imhof I et al.; Glycosylphosphatidylinositol (GPI) anchors of all species contain the core structure protein-CO-NH-(CH(2))(2)-PO(4)-Manalpha1-2Manalpha1-6Manalpha1-4GlcNalpha1-6inositol-PO(4)-lipid . In recent studies in yeast it was found that gpi10-1 mutants accumulate M2, an abnormal intermediate having the structure Manalpha1-6{NH(2)-(CH(2))(2)-PO(4)-->}Manalpha1-4GlcNalpha1-6(acyl-->)inositol-PO(4)-lipid . It thus was realized that yeast GPI lipids, as their mammalian counterparts, contain an additional phosphorylethanolamine side chain on the alpha1,4-linked mannose . The biosynthetic origin of this phosphorylethanolamine group was investigated using gpi10-1 Deltaept1 Deltacpt1, a strain which is unable to synthesize phosphatidylethanolamine by transferring phosphorylethanolamine from CDP-ethanolamine onto diacylglycerol, but which still can make phosphatidylethanolamine by decarboxylation of phosphatidylserine . Gpi10-1 Deltaept1 Deltacpt1 triple mutants are unable to incorporate {(3)H}ethanolamine into M2 although metabolic labeling with {(3)H}inositol demonstrates that they make as much M2 as gpi10-1 . In contrast, when labeled with {(3)H}serine, the triple mutant incorporates more label into M2 than gpi10-1 . This result establishes that the phosphorylethanolamine group on the alpha1,4-linked mannose is derived from phosphatidylethanolamine and not from CDP-ethanolamine.

Biophys J, 2001 Jan, 80(1), 427 - 34
Tryptophan fluorescence of yeast actin resolved via conserved mutations; Doyle TC et al.; Actin contains four tryptophan residues, W79, W86, W340, and W356, all located in subdomain 1 of the protein . Replacement of each of these residues with either tyrosine (W79Y and W356Y) or phenylalanine (W86F and W340F) generated viable proteins in the yeast Saccharomyces cerevisiae, which, when purified, allowed the analysis of the contribution of these residues to the overall tryptophan fluorescence of actin . The sum of the relative contributions of these tryptophans was found to account for the intrinsic fluorescence of wild-type actin, indicating that energy transfer between the tryptophans is not the main determinant of their quantum yield, and that these mutations induce little conformational change to the protein . This was borne out by virtually identical polymerization rates and similar myosin interactions of each of the mutants and the wild-type actin . In addition, these mutants allowed the dissection of the microenvironment of each tryptophan as actin undergoes conformational changes upon metal cation exchange and polymerization . Based on the relative tryptophan contributions determined from single mutants, a triple mutant of yeast actin (W79) was generated that showed small intrinsic fluorescence and should be useful for studies of actin interactions with actin-binding proteins.

Mol Cell Biol, 2001 Feb, 21(4), 1260 - 71
Rrb1p, a yeast nuclear WD-repeat protein involved in the regulation of ribosome biosynthesis; Iouk TL et al.; Ribosome biogenesis is regulated by environmental cues that coordinately modulate the synthesis of ribosomal components and their assembly into functional subunits . We have identified an essential yeast WD-repeat-containing protein, termed Rrb1p, that has a role in both the assembly of the 60S ribosomal subunits and the transcriptional regulation of ribosomal protein (RP) genes . Rrb1p is located in the nucleus and is concentrated in the nucleolus . Its presence is required to maintain normal cellular levels of 60S subunits, 80S ribosomes, and polyribosomes . The function of Rrb1p in ribosome biogenesis appears to be linked to its association with the ribosomal protein rpL3 . Immunoprecipitation of Rrb1p from nuclear extracts revealed that it physically interacts with rpL3 . Moreover, the overproduction of Rrb1p led to increases in cellular levels of free rpL3 that accumulated in the nucleus together with Rrb1p . The concentration of these proteins within the nucleus was dependent on ongoing protein translation . We also showed that overexpression of RRB1 led to an increase in the expression of RPL3 while all other examined RP genes were unaffected . In contrast, depletion of RRB1 caused an increase in the expression of all RP genes examined except RPL3 . These results suggest that Rrb1p regulates RPL3 expression and uncouples it from the coordinated expression of other RP genes.

Mol Cell Biol, 2001 Feb, 21(4), 1145 - 54
Region of yeast TAF 130 required for TFIID to associate with promoters; Mencia M et al.; TFIID, a multiprotein complex comprising the TATA-binding protein (TBP) and TBP-associated factors (TAFs), associates specifically with core promoters and nucleates the assembly the RNA polymerase II transcription machinery . In yeast cells, TFIID is not generally required for transcription, although it plays an important role at many promoters . Understanding of the specific functions and physiological roles of individual TAFs within TFIID has been hampered by the fact that depletion or thermal inactivation of individual TAFs generally results in dissociation of the TFIID complex . We describe here C-terminally deleted derivatives of yeast TAF130 that assemble into normal TFIID complexes but are transcriptionally inactive in vivo . In vivo, these mutant TFIID complexes are dramatically reduced in their ability to associate with all promoters tested . In vitro, a TFIID complex containing a deleted form of TAF130 associates poorly with DNA, but it is unaffected for interacting with transcriptional activation domains . These results suggest that the C-terminal region of TAF130 is required for TFIID to associate with promoters.

Mol Cell Biol, 2001 Feb, 21(4), 1089 - 97
The yeast mitochondrial carrier Leu5p and its human homologue Graves' disease protein are required for accumulation of coenzyme A in the matrix; Prohl C et al.; The transport of metabolites, coenzymes, and ions across the mitochondrial inner membrane is still poorly understood . In most cases, membrane transport is facilitated by the so-called mitochondrial carrier proteins . The yeast Saccharomyces cerevisiae contains 35 members of the carrier family, but a function has been identified for only 13 proteins . Here, we investigated the yeast carrier Leu5p (encoded by the gene YHR002w) and its close human homologue Graves' disease protein . Leu5p is inserted into the mitochondrial inner membrane along the specialized import pathway used by carrier proteins . Deletion of LEU5 (strain Deltaleu5) was accompanied by a 15-fold reduction of mitochondrial coenzyme A (CoA) levels but did not affect the cytosolic CoA content . As a consequence, the activities of several mitochondrial CoA-dependent enzymes were strongly decreased in Deltaleu5 cells . Our in vitro and in vivo analyses assign a function to Leu5p in the accumulation of CoA in mitochondria, presumably by serving as a transporter of CoA or a precursor thereof . Expression of the Graves' disease protein in Deltaleu5 cells can replace the function of Leu5p, demonstrating that the human protein represents the orthologue of yeast Leu5p . The function of the human protein might not be directly linked to the disease, as antisera derived from patients with active Graves' disease do not recognize the protein after expression in yeast, suggesting that it does not represent a major autoantigen . The two carrier proteins characterized herein are the first components for which a role in the subcellular distribution of CoA has been identified.

Mol Cell Biol, 2001 Feb, 21(4), 1011 - 23
Splicing factor slt11p and its involvement in formation of U2/U6 helix II in activation of the yeast spliceosome; Xu D et al.; Slt11p is a new splicing factor identified on the basis of synthetic lethality with a mutation in the 5' end of U2 snRNA, a region that is involved in intermolecular U2/U6 helix II interaction . Slt11p is required for spliceosome assembly . Our genetic results suggest that Slt11p is involved in the base-pairing interaction of U2/U6 helix II in vivo . We showed that the recombinant protein binds to RNAs with some degree of structural specificity . Slt11p also anneals RNA and binds to the resulting duplexes, which contain two separated helical regions . These RNA structures are reminiscent of U2/U6 helix II, which is formed concomitantly with U4/U6 stem II, and suggest that Slt11p facilitates the cooperative formation of helix II in association with stem II in the spliceosome . We show that Slt11p and Slu7p, a second-step factor, interact with each other both in vivo and in vitro and that the binding of Slu7p to Slt11p impairs the RNA-binding activity of the latter . These results suggest that the function of Slt11p is regulated by Slu7p in the spliceosome.

EMBO J, 2001 Feb 1, 20(3), 552 - 61
Post-transcriptional regulation through the HO 3'-UTR by Mpt5, a yeast homolog of Pumilio and FBF; Tadauchi T et al.; Drosophila Pumilio (Pum) and Caenorhabditis elegans FBF bind to the 3'-untranslated region (3'-UTR) of their target mRNAs and repress translation . Pum and FBF are members of a large and evolutionarily conserved protein family, the Puf family, found in Drosophila, C.elegans, humans, and yeasts . Budding yeast, Saccharomyces cerevisiae, has five proteins with conserved Puf motifs: Mpt5/Uth4, Ygl014w, Yll013c, Jsn1, and Ypr042c . Here we report that Mpt5 negatively regulates expression of the HO gene . Loss of MPT5 increased expression of reporter genes integrated into the ho locus, whereas overexpression of MPT5 decreased expression . Repression required the 3'-UTR of HO, which contains a tetranucleotide, UUGU, also found in the binding sites of Pum and FBF . Mutation of UUGU to UACU in the HO 3'-UTR abolished Mpt5-mediated repression . Studies using a three-hybrid assay for RNA binding indicate that Mpt5 binds to the 3'-UTR of HO mRNA containing a UUGU sequence but not a UACU sequence . These observations suggest that the yeast Puf homolog, Mpt5, negatively regulates HO expression post-transcriptionally.

Genetics, 2001 Feb, 157(2), 579 - 89
Homologous recombinational repair of double-strand breaks in yeast is enhanced by MAT heterozygosity through yKU-dependent and -independent mechanisms; Clikeman JA et al.; DNA double-strand breaks (DSBs) are repaired by homologous recombination (HR) and nonhomologous end-joining (NHEJ) . NHEJ in yeast chromosomes has been observed only when HR is blocked, as in rad52 mutants or in the absence of a homologous repair template . We detected yKu70p-dependent imprecise NHEJ at a frequency of approximately 0.1% in HR-competent Rad+ haploid cells . Interestingly, yku70 mutation increased DSB-induced HR between direct repeats by 1.3-fold in a haploid strain and by 1.5-fold in a MAT homozygous (a/a) diploid, but yku70 had no effect on HR in a MAT heterozygous (a/alpha) diploid . yku70 might increase HR because it eliminates the competing precise NHEJ (religation) pathway and/or because yKu70p interferes directly or indirectly with HR . Despite the yku70-dependent increase in a/a cells, HR remained 2-fold lower than in a/alpha cells . Cell survival was also lower in a/a cells and correlated with the reduction in HR . These results indicate that MAT heterozygosity enhances DSB-induced HR by yKu-dependent and -independent mechanisms, with the latter mechanism promoting cell survival . Surprisingly, yku70 strains survived a DSB slightly better than wild type . We propose that this reflects enhanced HR, not by elimination of precise NHEJ since this pathway produces viable products, but by elimination of yKu-dependent interference of HR.

Genetics, 2001 Feb, 157(2), 533 - 43
Generation of an isogenic collection of yeast actin mutants and identification of three interrelated phenotypes; Whitacre J et al.; A large collection of yeast actin mutations has been previously isolated and used in numerous studies of actin cytoskeletal function . However, the various mutations have been in congenic, rather than isogenic, backgrounds, making it difficult to compare the subtle phenotypes that are characteristic of these mutants . We have therefore placed 27 mutations in an isogenic background . We used a subset of these mutants to compare the degree to which different actin alleles are defective in sporulation, endocytosis, and growth on NaCl-containing media . We found that the three phenotypes are highly correlated . The correlations are specific and not merely a reflection of general growth defects, because the phenotypes are not correlated with growth rates under normal conditions . Significantly, those actin mutants exhibiting the most severe phenotypes in all three processes have altered residues that cluster to a small region of the actin crystal structure previously defined as the fimbrin (Sac6p)-binding site . We examined the relationship between endocytosis and growth on salt and found that shifting wild-type or actin mutant cells to high salt reduces the rate of alpha-factor internalization . These results suggest that actin mutants may be unable to grow on salt because of additive endocytic defects (due to mutation and salt).

Genetics, 2001 Feb, 157(2), 503 - 18
Fine structure analysis of the yeast centrin, Cdc31p, identifies residues specific for cell morphology and spindle pole body duplication; Ivanovska I et al.; Centrin/Cdc31p is a Ca2+-binding protein related to calmodulin found in the MTOC of diverse organisms . In yeast, Cdc31p localizes to the SPB where it interacts with Kar1p and is required for SPB duplication . Recent findings suggest that centrin also functions elsewhere in the cell . To dissect the functions of Cdc31p, we generated cdc31 mutations chosen only for temperature sensitivity, but otherwise unbiased as to phenotype . Three phenotypes of the cdc31 mutants, temperature sensitivity, G2/M arrest, and cell lysis, were not well correlated, indicating that the mutations may differentially affect Cdc31p's interactions with other proteins . Alleles near the C-terminal region exhibited high G2/M arrest and genetic interactions with kar1-Delta17, suggesting that this region modulates an SPB-related function . Alleles causing high lysis and reduced Kic1p kinase activity mapped to the middle of the gene, suggesting disruption of a KIC1-like function and defects in activating Kic1p . A third region conferred temperature sensitivity without affecting cell lysis or G2/M arrest, suggesting that it defines a third function . Mutations in the C-terminal region were also defective for interaction with Kic1p . Mapping the alleles onto a predicted structure of Cdc31p, we have identified surfaces likely to be important for interacting with both Kar1p and Kic1p.

J Biol Chem, 2000 Apr 14, 275(15), 11092 - 9
Structural basis of BFL-1 for its interaction with BAX and its anti-apoptotic action in mammalian and yeast cells; Zhang H et al.; BFL-1 is the smallest member of the BCL-2 family and has been shown to retard apoptosis in various cell lines . However, the structural basis for its function remains unclear . Molecular modeling showed that BFL-1 could have a similar core structure as BCL-xL, consisting of seven alpha helices, although both proteins share only the conserved BCL-2 homology domains (BH1 and BH2 domains), but otherwise have very limited sequence homology, particularly in the N-terminal region . We demonstrated in the yeast two-hybrid system that BFL-1 interacts strongly with human BAX but is not able to form homodimers nor to interact with human BCL-2 or BCL-xL . Overexpression experiments in REF52 rat fibroblasts showed that BFL-1 conferred increased resistance to apoptosis induced by serum deprivation . BFL-1 had also the ability to neutralize BAX lethality in yeast . BAX requires the BH3 domain for interaction with BFL-1 . However, the minimal region of BFL-1 for the interaction with BAX in coimmunoprecipitation experiments was not sufficient to protect cells from apoptosis . Further examination of BFL-1 and several other anti-apoptotic proteins suggests a more general type of structure based on structural motifs, i.e . a hydrophobic pocket for the binding of proapoptotic proteins, rather than extended sequence homologies.

J Biol Chem, 2000 Apr 14, 275(15), 11071 - 4
Mutation of Arg(273) to Leu alters the specificity of the yeast N-glycan processing class I alpha1,2-mannosidase; Romero PA et al.; Class I alpha1,2-mannosidases (glycosyl hydrolase family 47) involved in the processing of N-glycans during glycoprotein maturation have different specificities . Enzymes in the endoplasmic reticulum of yeast and mammalian cells remove a single mannose from Man(9)GlcNAc(2) to form Man(8)GlcNAc(2) isomer B (lacking the alpha1, 2-mannose residue of the middle alpha1, 3-arm), whereas other alpha1,2-mannosidases, including Golgi alpha1,2-mannosidases IA and IB, can convert Man(9)GlcNAc(2) to Man(5)GlcNAc(2) . In the present work, it is demonstrated that with a single mutation in its catalytic domain (Arg(273) --> Leu) the yeast endoplasmic reticulum alpha1,2-mannosidase acquires the ability to transform Man(9)GlcNAc to Man(5)GlcNAc . High resolution proton nuclear magnetic resonance analysis of the products shows that the order of removal of mannose from Man(9)GlcNAc is different from that of other alpha1, 2-mannosidases that remove four mannose from Man(9)GlcNAc . These results demonstrate that Arg(273) is in part responsible for the specificity of the endoplasmic reticulum alpha1,2-mannosidase and that small differences in non-conserved amino acids interacting with the oligosaccharide substrate in the active site of class I alpha1, 2-mannosidases are responsible for the different specificities of these enzymes.

Curr Biol, 2000 Apr 6, 10(7), 373 - 82
MAP kinase signaling induces nuclear reorganization in budding yeast; Stone EM et al.; BACKGROUND: During the mating pheromone response in budding yeast, activation of a mitogen-activated protein kinase (MAP kinase) cascade results in well-characterized changes in cytoskeletal organization and gene expression . Spatial reorganization of genes within the nucleus has been documented during cell-type differentiation in mammalian cells, but no information was previously available on the morphology of the yeast nucleus during the major transcriptional reprogramming that accompanies zygote formation . RESULTS: We find that in response to mating pheromone, budding yeast nuclei assume an unusual dumbbell shape, reflecting a spatial separation of chromosomal and nucleolar domains . Within the chromosomal domain, telomeric foci persist and maintain their associated complement of Sir proteins . The nucleolus, on the other hand, assumes a novel cup-shaped morphology and a position distal to the mating projection tip . Although microtubules are required for this orientation with respect to the projection tip, neither microtubules nor actin polymerization are necessary for the observed changes in nuclear shape . We find that activation of the pheromone-response MAP kinase pathway by ectopic expression of STE4 or STE11 leads to identical nuclear and nucleolar reorganization in the absence of pheromone . Mutation of downstream effector MAP kinases Fus3p and Kss1p, or of the transcriptional regulator Ste12p, blocks nuclear shape changes, whereas overexpression of Ste12p promotes dumbbell-shaped nuclei in the absence of pheromone . CONCLUSIONS: Nuclear remodeling occurs when the MAP kinase cascade is activated by yeast pheromone, but it is independent of the cytoskeletal reorganization regulated by the same signaling pathway . Activation of the Ste12p transcription factor is necessary, and may be sufficient, for the changes in nuclear structure that coincide with developmentally significant changes in gene expression.

Mutat Res, 2000 Feb 14, 447(2), 209 - 20
Transcriptional slippage of p53 gene enhanced by cellular damage in rat liver: monitoring the slippage by a yeast functional assay; Ba Y et al.; The Long-Evans Cinnamon (LEC) rat is a mutant strain characterized by abnormal copper metabolism and a high incidence of hepatitis and hepatoma . Using a yeast-based assay which scores mutants in p53 gene transcripts as red colonies, we detected frequent mutations in the liver of LEC rats . The majority (50-60%) of these were frameshift mutations caused by the insertion of an extra adenine (A) in the regions containing six consecutive adenines . The rate of A insertion was calculated to be 6.9-9.0% of the total p53 cDNA . Insertions of an extra adenine were found almost exclusively in the mRNA (cDNA), especially in the (A)(6) tract located at the most 5'-side (exon 4) among the three (A)(6) tracts (exons 4, 7, and 8), but rarely in the corresponding sites of genomic DNA . Wild-type p53 cDNA was transcribed in vitro into mRNA with the use of SP6 RNA polymerase and tested by the yeast functional assay . Subsequent sequencing detected A insertions at an overall rate of 1.6% in exons 7 and 8 but none in exon 4 . This indicates that the A insertion in the exon 4 (A)(6) tract was an in vivo phenomenon rather than an artifact in reverse transcription or polymerase chain reaction . The percentage of red colonies increased sharply to about 20% of the liver samples in the acute hepatitis stage, and returned to control level of those in the chronic hepatitis stage, and increased again slightly to those in the neoplastic stage . The percentage of red colonies correlated with the serum GOT level (r=0.96, p<0.001) but not with the contents of copper and 8-hydroxydeoxyguanosine in the liver of LEC rats . Ethanol treatment of hepatic cell lines also increased the rate of transcriptional slippage at the (A)(6) tract . These findings indicate that cellular damage is responsible for the increase in the rate of mutation at the transcriptional level, and suggest that cellular damage degrades transcriptional fidelity, thereby further impairing cellular functions.

J Agric Food Chem, 1998 Jan 19, 46(1), 354 - 360
Heterologous Expression of a Candida molischiana Anthocyanin-beta-glucosidase in a Wine Yeast Strain; Sanchez-Torres P et al.; A recombinant wine yeast strain expressing the Candidamolischiana bgln gene encoding a beta-glucosidase/anthocyanase under the control of the Saccharomyces cerevisiae actin gene promoter has been constructed . The corresponding protein, BGLN, was mainly located on the cell wall . BGLN was purified in a single chromotagraphic step, and different physicochemical and kinetic properties have been determined . BGLN showed maximum activity against the artificial substrate p-nitrophenyl beta-D-glucopyranoside . It also hydrolyzed salicin, p-nitrophenyl beta-D-xyloside, cellobiose, and arbutin to a lesser extent . Fructose and SO(2) did not affect enzyme activity, which was activated by ethanol, while glucose was a strong competitive inhibitor . The purified BGLN showed a novel anthocyanase decolorizing capability on red wines . This anthocyanase activity was readily observed during microvinification experiments . However, the physicochemical characteristics of the wines obtained with the recombinant wine yeast strain were indistinguishable from those obtained with the parental strain.

J Biol Chem, 2001 Apr 6, 276(14), 10634 - 40 Epub 2001 Jan 04.
Relationship between genotype, activity, and galactose sensitivity in yeast expressing patient alleles of human galactose-1-phosphate uridylyltransferase; Riehman K et al.; Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosemia; the biochemical basis of pathophysiology in galactosemia remains unknown . We have applied a yeast expression system for human GALT to test the hypothesis that genotype will correlate with GALT activity measured in vitro and with metabolite levels and galactose sensitivity measured in vivo . In particular, we have determined the relative degree of functional impairment associated with each of 16 patient-derived hGALT alleles; activities ranged from null to essentially normal . Next, we utilized strains expressing these alleles to demonstrate a clear inverse relationship between GALT activity and galactose sensitivity . Finally, we monitored accumulation of galactose-1-P, UDP-gal, and UDP-glc in yeast expressing a subset of these alleles . As reported for humans, yeast deficient in GALT, but not their wild type counterparts, demonstrated elevated levels of galactose 1-phosphate and diminished UDP-gal upon exposure to galactose . These results present the first clear evidence in a genetically and biochemically amenable model system of a relationship between GALT genotype, enzyme activity, sensitivity to galactose, and aberrant metabolite accumulation . As such, these data lay a foundation for future studies into the underlying mechanism(s) of galactose sensitivity in yeast and perhaps other eukaryotes, including humans.

J Agric Food Chem, 2000 Dec, 48(12), 5929 - 32
Immobilization of yeast cells with ionic hydrogel carriers by adhesion-multiplication; Zhaoxin L et al.; The mixture of an ionic monomer, 2-acrylamido 2-methylpropanesulfonic acid (TBAS), and a series of poly(ethylene glycol) dimethacrylate (nG) monomers were copolymerized with 60Co gamma-rays, and the produced ionic hydrogel polymers were used for immobilization of yeast cells . The cells were adhered onto the surface of the hydrogel polymers and intruded into the interior of the polymers with growing . The immobilized yeast cells with these hydrogel polymers had higher ethanol productivity than that of free cells . The yield of ethanol with poly(TBAS-14G) carrier was the highest and increased by 3.5 times compared to the free cells . It was found that the ethanol yield increased with the increase of glycol number in poly(ethylene glycol) dimethacrylate . The state of the immobilized cells was observed with microscope, and it was also found that the difference in the ethanol productivity is mainly due to the difference in the internal structure and properties of polymer carrier, such as surface charge, hydrophilicity, and swelling ability of polymer carrier.

J Biol Chem, 2001 Mar 30, 276(13), 9640 - 8 Epub 2001 Jan 02.
Relationships of the antiproliferative proteins BTG1 and BTG2 with CAF1, the human homolog of a component of the yeast CCR4 transcriptional complex: involvement in estrogen receptor alpha signaling pathway; Prevot D et al.; We have reported previously the physical interaction of B-cell translocation gene proteins (BTG)1 and BTG2 with the mouse protein CAF1 (CCR4-associated factor 1) and suggested that these proteins may participate, through their association with CAF1, in transcription regulation . Here we describe the in vitro and in vivo association of these proteins with hPOP2, the human paralog of hCAF1 . The physical and functional relationships between the BTG proteins and their partners hCAF1 and hPOP2 were investigated to find out how these interactions affect cellular processes, and in particular transcription regulation . We defined their interaction regions and examined their expression in various human tissues . We also show functional data indicating their involvement in estrogen receptor alpha (ERalpha)-mediated transcription regulation . We found that BTG1 and BTG2, probably through their interaction with CAF1 via a CCR4-like complex, can play both positive or negative roles in regulating the ERalpha function . In addition, our results indicate that two LXXLL motifs, referred to as nuclear receptor boxes, present in both BTG1 and BTG2, are involved in the regulation of ERalpha-mediated activation.

J Biol Chem, 2001 Mar 23, 276(12), 8681 - 94 Epub 2000 Dec 27.
An oxysterol-derived positive signal for 3-hydroxy- 3-methylglutaryl-CoA reductase degradation in yeast; Gardner RG et al.; Sterol synthesis by the mevalonate pathway is modulated, in part, through feedback-regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) . In mammals, both a non-sterol isoprenoid signal derived from farnesyl diphosphate (FPP) and a sterol-derived signal appear to act together to positively regulate the rate of HMGR degradation . Although the nature and number of sterol-derived signals are not clear, there is growing evidence that oxysterols can serve in this capacity . In yeast, a similar non-sterol isoprenoid signal generated from FPP acts to positively regulate HMGR degradation, but the existence of any sterol-derived signal has thus far not been revealed . We now demonstrate, through the use of genetic and pharmacological manipulation of oxidosqualene-lanosterol cyclase, that an oxysterol-derived signal positively regulated HMGR degradation in yeast . The oxysterol-derived signal acted by specifically modulating HMGR stability, not endoplasmic reticulum-associated degradation in general . Direct biochemical labeling of mevalonate pathway products confirmed that oxysterols were produced endogenously in yeast and that their levels varied appropriately in response to genetic or pharmacological manipulations that altered HMGR stability . Genetic manipulation of oxidosqualene-lanosterol cyclase did result in the buildup of detectable levels of 24,25-oxidolanosterol by gas chromatography, gas chromatography-mass spectroscopy, and NMR analyses, whereas no detectable amounts were observed in wild-type cells or cells with squalene epoxidase down-regulated . In contrast to mammalian cells, the yeast oxysterol-derived signal was not required for HMGR degradation in yeast . Rather, the function of this second signal was to enhance the ability of the FPP-derived signal to promote HMGR degradation . Thus, although differences do exist, both yeast and mammalian cells employ a similar strategy of multi-input regulation of HMGR degradation.

Genes Dev, 2001 Jan 1, 15(1), 42 - 9
Deletion of MUD2, the yeast homolog of U2AF65, can bypass the requirement for sub2, an essential spliceosomal ATPase; Kistler AL et al.; Mammalian U2AF65 and UAP56 are required for prespliceosome (PS) formation . We tested the predictions that the yeast UAP56 homolog, SUB2, is required for the same step and functions collaboratively with MUD2, the yeast homolog of U2AF65 . Unexpectedly, sub2-1 extracts accumulate PS-like complexes . Moreover, deletion of MUD2 exacerbates the cs phenotype of sub2 alleles yet suppresses both the ts sub2-1 and the lethal Deltasub2 phenotypes . We propose that Sub2 functionally interacts with Mud2 both before and after PS formation . In the absence of Mud2, Sub2 function becomes dispensable.

Genes Dev, 2001 Jan 1, 15(1), 30 - 5
Identification and characterization of yUAP/Sub2p, a yeast homolog of the essential human pre-mRNA splicing factor hUAP56; Zhang M et al.; The human 56-kD U2AF(65)-associated protein (hUAP56), a member of the DExD/H box protein family of RNA-dependent ATPases, is required for the stable binding of U2 snRNP to the pre-mRNA branchpoint . Here we identify a highly conserved Saccharomyces cerevisiae homolog of hUAP56, yUAP/Sub2p . yUAP/Sub2p can be functionally substituted for by hUAP56 and, like its human counterpart, is an essential pre-mRNA splicing factor . yUAP/Sub2p is required for formation of the commitment complex, the precursor for U2 snRNP addition . In conjunction with previous studies, we conclude that at least two DExD/H box proteins, Prp5p and yUAP/Sub2p, mediate the U2 snRNP-branchpoint interaction.

Acta Virol, 2000 Jun-Aug, 44(3), 199 - 202
Analysis of interaction between molecules of Bombyx mori nucleopolyhedrovirus IE-2 using a yeast two-hybrid system; Imai N et al.; Baculovirus IE-2 protein is one of well-known transactivators . In this report, we demonstrate that Bombyx mori nucleopolyhedrovirus (BmNPV) IE-2 interacts with itself . Several clones were obtained from a yeast two-hybrid screening system using IE-2 as bait and were found to encode IE-2 protein . Nucleotide sequencing of these clones showed that they contained C-terminal regions in common . Further analyses suggest that BmNPV IE-2 protein interacts with itself through 80 amino acid residues of coiled-coil domain in C-terminus.

J Dermatol Sci, 2001 Jan, 25(1), 45 - 52
Doxorubicin toxicity to the skin: possibility of protection with antioxidants enriched yeast; Korac B et al.; The possibility of skin protection against doxorubicin toxicity was examined after oral antioxidative pretreatment of the rats with yeast supplemented with selenium and vitamins E, C and A for 15 days . The activity and level of antioxidative defense components were monitored in the skin and blood 48 h after i.v . applied doxorubicin . In the blood, increased glutathione peroxidase activity in the erythrocytes, and amounts of vitamin E and glutathione in the plasma were found after the antioxidative treatment . It also led to an increase of the reductive capacity in the skin (increased thioredoxin reductase activity and reduced glutathione level) . Doxorubicin alone, depleted reductive capacity, i.e . decreased the activity of thioredoxin reductase in the skin, as well as the content of reduced glutathione both in the skin and blood plasma . Depletion of reductive capacity represents one of the first harmful doxorubicin effects to the skin at the time when the changes of other antioxidative enzyme activities were not detectable . Reductive capacity in the skin of animals given antioxidative pretreatment was maintained elevated upon doxorubicin application in comparison with the corresponding control . Oral supplementation with antioxidants thus prevents toxic effects of doxorubicin in the skin and may contribute to the alleviation of its secondary cytotoxicity during the chemotherapy.

Mol Cell Biol, 2001 Feb, 21(3), 940 - 51
Inactivation of DNA mismatch repair by increased expression of yeast MLH1; Shcherbakova PV et al.; Inactivation of DNA mismatch repair by mutation or by transcriptional silencing of the MLH1 gene results in genome instability and cancer predisposition . We recently found (P . V . Shcherbakova and T . A . Kunkel, Mol . Cell . Biol . 19:3177-3183, 1999) that an elevated spontaneous mutation rate can also result from increased expression of yeast MLH1 . Here we investigate the mechanism of this mutator effect . Hybridization of poly(A)(+) mRNA to DNA microarrays containing 96.4% of yeast open reading frames revealed that MLH1 overexpression did not induce changes in expression of other genes involved in DNA replication or repair . MLH1 overexpression strongly enhanced spontaneous mutagenesis in yeast strains with defects in the 3'-->5' exonuclease activity of replicative DNA polymerases delta and epsilon but did not enhance the mutation rate in strains with deletions of MSH2, MLH1, or PMS1 . This suggests that overexpression of MLH1 inactivates mismatch repair of replication errors . Overexpression of the PMS1 gene alone caused a moderate increase in the mutation rate and strongly suppressed the mutator effect caused by MLH1 overexpression . The mutator effect was also reduced by a missense mutation in the MLH1 gene that disrupted Mlh1p-Pms1p interaction . Analytical ultracentrifugation experiments showed that purified Mlh1p forms a homodimer in solution, albeit with a K(d) of 3.14 microM, 36-fold higher than that for Mlh1p-Pms1p heterodimerization . These observations suggest that the mismatch repair defect in cells overexpressing MLH1 results from an imbalance in the levels of Mlh1p and Pms1p and that this imbalance might lead to formation of nonfunctional mismatch repair complexes containing Mlh1p homodimers.

Mol Cell Biol, 2001 Feb, 21(3), 814 - 26
Genetic and biochemical analysis of the yeast plasma membrane Ssy1p-Ptr3p-Ssy5p sensor of extracellular amino acids; Forsberg H et al.; Ssy1p and Ptr3p are known components of a yeast plasma membrane system that functions to sense the presence of amino acids in the extracellular environment . In response to amino acids, this sensing system initiates metabolic signals that ultimately regulate the functional expression of several amino acid-metabolizing enzymes and transport proteins, including multiple, genetically distinct amino acid permeases . We have found that SSY5 encodes a third component of this amino acid sensing system . Mutations in SSY5 manifest phenotypes that are indistinguishable from those resulting from either single ssy1 and ptr3 mutations or ssy5 ssy1 and ssy5 ptr3 double mutations . Although Ssy5p is predicted to be a soluble protein, it exhibits properties indicating that it is a peripherally associated plasma membrane protein . Each of the three sensor components, Ssy1p, Ptr3p, and Ssy5p, adopts conformations and modifications that are dependent upon the availability of amino acids and on the presence of the other two components . These results suggest that these components function as part of a sensor complex localized to the plasma membrane . Consistent with a sensor complex, the overexpression of SSY1 or the unique N-terminal extension of this amino acid permease homologue inactivates the amino acid sensor in a dominant-negative manner . Each of the components of the Ssy1p-Ptr3p-Ssy5p (SPS) signaling system undergoes rapid physical changes, reflected in altered electrophoretic mobility, when leucine is added to cells grown in media lacking amino acids . Furthermore, the levels of each SPS sensor component present in whole-cell extracts diminish upon leucine addition . The rapid physical alterations and reduced levels of sensor components are consistent with their being downregulated in response to amino acid availability . These results reveal the dynamic nature of the amino acid-initiated signals transduced by the SPS sensor.

J Cell Biol, 2001 Jan 8, 152(1), 27 - 40
A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1; Jensen S et al.; In Saccharomyces cerevisiae, the metaphase-anaphase transition is initiated by the anaphase-promoting complex-dependent degradation of Pds1, whereby Esp1 is activated to promote sister chromatid separation . Although this is a fundamental step in the cell cycle, little is known about the regulation of Esp1 and how loss of cohesion is coordinated with movement of the anaphase spindle . Here, we show that Esp1 has a novel role in promoting anaphase spindle elongation . The localization of Esp1 to the spindle apparatus, analyzed by live cell imaging, is regulated in a manner consistent with a function during anaphase B . The protein accumulates in the nucleus in G2 and is mobilized onto the spindle pole bodies and spindle midzone at anaphase onset, where it persists into midanaphase . Association with Pds1 occurs during S phase and is required for efficient nuclear targeting of Esp1 . Spindle association is not fully restored in pds1 mutants expressing an Esp1-nuclear localization sequence fusion protein, suggesting that Pds1 is also required to promote Esp1 spindle binding . In agreement, Pds1 interacts with the spindle at the metaphase-anaphase transition and a fraction remains at the spindle pole bodies and the spindle midzone in anaphase cells . Finally, mutational analysis reveals that the conserved COOH-terminal region of Esp1 is important for spindle interaction.

Biochemistry, 2001 Jan 16, 40(2), 422 - 8
Interactions between yeast iso-1-cytochrome c and its peroxidase; Pielak GJ et al.; Isothermal titration calorimetry was used to study the formation of 19 complexes involving yeast iso-1-ferricytochrome c (Cc) and ferricytochrome c peroxidase (CcP) . The complexes comprised combinations of the wild-type proteins, six CcP variants, and three Cc variants . Sixteen protein combinations were designed to probe the crystallographically defined interface between Cc and CcP . The data show that the high-affinity sites on Cc and CcP coincide with the crystallographically defined sites . Changing charged residues to alanine increases the enthalpy of complex formation by a constant amount, but the decrease in stability depends on the location of the amino acid substitution . Deleting methyl groups has a small effect on the binding enthalpy and a larger deleterious effect on the binding free energy, consistent with model studies of the hydrophobic effect, and showing that nonpolar interactions also stabilize the complex . Double-mutant cycles were used to determine the coupling energies for nine Cc-CcP residue pairs . Comparing these energies to the crystal structure of the complex leads to the conclusion that many of the substitutions induce a rearrangement of the complex.

J Cell Physiol, 2001 Jan, 186(1), 124 - 35
Mutated-gamma-actin restores growth to a yeast amino acid transport defective mutant; Khamessan A et al.; A mutated yeast cell 22574d lacking all three proline transporters, PUT4, UGA4, and GAP1, and incapable of growth on proline recovers its lost ability to grow on proline as sole nitrogen source when transformed with a mutagenized mouse gamma-actin cDNA (M-gamma-A) . Native mouse gamma-actin cDNA is ineffective . The 3'-region of gamma-actin cDNA was mutagenized to resemble E51 cDNA previously isolated from Ehrlich tumor cells . The E51 cDNA has an extended reading frame in the 3'-region compared to that in native gamma-actin . The extension of the open reading frame in E51 cDNA, was found to be due to an additional pair of bases (TG) at position 1104 of E51 cDNA . After site-directed mutagenesis of the 3'-region of native gamma-actin cDNA to resemble that of E51 cDNA, the construct, M-gamma-A cDNA, was expressed in the 22574d yeast . While the transformation with M-gamma-A increased the uptake of both proline and gamma-amino butyric acid, the transport of five other solutes was not changed by this transformation . Northern blotting of the nontransformed and the M-gamma-A-transformed 22574d cells with gene-specific probes for the three proline transporters showed the expression of an mRNA for UGA4 in both transformed and nontransformed cells but no evidence for the expression of GAP1 or PUT4 . The mRNA for UGA4 was expressed at a lower level in strain 22574d than in the parent yeast sigma1278b . Furthermore, the message in the mutated cells is smaller in size by about 15% . These results are consistent with the synthesis of a mutated transporter which requires the coexpression of M-gamma-A, but not native gamma-actin, to restore physiological function, i.e., proline or gamma-amino acid transport.

Nat Cell Biol, 2000 Dec, 2(12), 958 - 63
A yeast DNA J protein required for uncoating of clathrin-coated vesicles in vivo; Pishvaee B et al.; Clathrin-coated vesicles mediate diverse processes such as nutrient uptake, downregulation of hormone receptors, formation of synaptic vesicles, virus entry, and transport of biosynthetic proteins to lysosomes . Cycles of coat assembly and disassembly are integral features of clathrin-mediated vesicular transport (Fig . 1a) . Coat assembly involves recruitment of clathrin triskelia, adaptor complexes and other factors that influence coat assembly, cargo sequestration, membrane invagination and scission (Fig . 1a) . Coat disassembly is thought to be essential for fusion of vesicles with target membranes and for recycling components of clathrin coats to the cytoplasm for further rounds of vesicle formation . In vitro, cytosolic heat-shock protein 70 (Hsp70) and the J-domain co-chaperone auxilin catalyse coat disassembly . However, a specific function of these factors in uncoating in vivo has not been demonstrated, leaving the physiological mechanism and significance of uncoating unclear . Here we report the identification and characterization of a Saccharomyces cerevisiae J-domain protein, Aux1 . Inactivation of Aux1 results in accumulation of clathrin-coated vesicles, impaired cargo delivery, and an increased ratio of vesicle-associated to cytoplasmic clathrin . Our results demonstrate an in vivo uncoating function of a J domain co-chaperone and establish the physiological significance of uncoating in transport mediated by clathrin-coated vesicles.

Nat Cell Biol, 2001 Jan, 3(1), E23 - 7
The spindle cycle in budding yeast; Winey M et al.; The mitotic spindle of the budding yeast Saccharomyces cerevisiae will probably be the first such organelle to be understood in molecular detail . Here we describe the mitotic spindle cycle of budding yeast using electron-microscope-derived structures and dynamic live-cell imaging . Recent work has revealed that many general aspects of mitosis are conserved, making budding yeast an excellent model for the study of mitosis.

RNA, 2000 Dec, 6(12), 1859 - 69
Disruption of the 5' stem-loop of yeast U6 RNA induces trimethylguanosine capping of this RNA polymerase III transcript in vivo; Kwan S et al.; Primary transcripts made by RNA polymerase II (Pol II), but not Pol I or Pol III, are modified by addition of a 7-methylguanosine (m7G) residue to the triphosphate 5' end shortly after it emerges from the polymerase . The m7G "caps" of small nuclear and small nucleolar RNAs, but not messenger RNAs, are subsequently hypermethylated to a 2,2,7-trimethylguanosine (TMG) residue . U6 RNA, the only small nuclear RNA synthesized by Pol III in most eukaryotes, does not receive a methylguanosine cap . However, human U6 RNA is O-methylated on the 5'-terminal (gamma) phosphate by an enzyme that recognizes the 5' stem-loop of U6 . Here we show that variant yeast U6 RNAs truncated or substituted within the 5' stem-loop are TMG capped in vivo . Accumulation of the most efficiently TMG-capped U6 RNA variant is strongly inhibited by a conditional mutation in the largest subunit of Pol III, confirming that it is indeed synthesized by Pol III . Thus, methylguanosine capping and cap hypermethylation are not exclusive to Pol II transcripts in yeast . We propose that TMG capping of variant U6 RNAs occurs posttranscriptionally due to exposure of the 5' triphosphate by disruption of protein binding and/or gamma-methyl phosphate capping . 5' truncation and TMG capping of U6 RNA does not appear to affect its normal function in splicing, suggesting that assembly and action of the spliceosome is not very sensitive to the 5' end structure of U6 RNA.

Mol Cell Biol, 2001 Jan, 21(2), 534 - 47
Remodeling of yeast CUP1 chromatin involves activator-dependent repositioning of nucleosomes over the entire gene and flanking sequences; Shen CH et al.; The yeast CUP1 gene is activated by the copper-dependent binding of the transcriptional activator, Ace1p . An episome containing transcriptionally active or inactive CUP1 was purified in its native chromatin structure from yeast cells . The amount of RNA polymerase II on CUP1 in the purified episomes correlated with its transcriptional activity in vivo . Chromatin structures were examined by using the monomer extension technique to map translational positions of nucleosomes . The chromatin structure of an episome containing inactive CUP1 isolated from ace1Delta cells is organized into clusters of overlapping nucleosome positions separated by linkers . Novel nucleosome positions that include the linkers are occupied in the presence of Ace1p . Repositioning was observed over the entire CUP1 gene and its flanking regions, possibly over the entire episome . Mutation of the TATA boxes to prevent transcription did not prevent repositioning, implicating a chromatin remodeling activity recruited by Ace1p . These observations provide direct evidence in vivo for the nucleosome sliding mechanism proposed for remodeling complexes in vitro and indicate that remodeling is not restricted to the promoter but occurs over a chromatin domain including CUP1 and its flanking sequences.

Mol Cell Biol, 2001 Jan, 21(2), 511 - 23
Nucleocytoplasmic distribution of budding yeast protein kinase A regulatory subunit Bcy1 requires Zds1 and is regulated by Yak1-dependent phosphorylation of its targeting domain; Griffioen G et al.; In Saccharomyces cerevisiae the subcellular distribution of Bcy1 is carbon source dependent . In glucose-grown cells, Bcy1 is almost exclusively nuclear, while it appears more evenly distributed between nucleus and cytoplasm in carbon source-derepressed cells . Here we show that phosphorylation of its N-terminal domain directs Bcy1 to the cytoplasm . Biochemical fractionation revealed that the cytoplasmic fraction contains mostly phosphorylated Bcy1, whereas unmodified Bcy1 is predominantly present in the nuclear fraction . Site-directed mutagenesis of two clusters (I and II) of serines near the N terminus to alanine resulted in an enhanced nuclear accumulation of Bcy1 in ethanol-grown cells . In contrast, substitutions to Asp led to a dramatic increase of cytoplasmic localization in glucose-grown cells . Bcy1 modification was found to be dependent on Yak1 kinase and, consequently, in ethanol-grown yak1 cells the Bcy1 remained nuclear . A two-hybrid screen aimed to isolate genes encoding proteins that interact with the Bcy1 N-terminal domain identified Zds1 . In ethanol-grown zds1 cells, cytoplasmic localization of Bcy1 was largely absent, while overexpression of ZDS1 led to increased cytoplasmic Bcy1 localization . Zds1 does not regulate Bcy1 modification since this was found to be unaffected in zds1 cells . However, in zds1 cells cluster II-mediated, but not cluster I-mediated, cytoplasmic localization of Bcy1 was found to be absent . Altogether, these results suggest that Zds1-mediated cytoplasmic localization of Bcy1 is regulated by carbon source-dependent phosphorylation of cluster II serines, while cluster I acts in a Zds1-independent manner.

Proc Natl Acad Sci U S A, 2001 Jan 16, 98(2), 415 - 20 Epub 2000 Dec 26.
Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: Evidence for acetyl transfer from substrate to an NAD breakdown product; Tanny JC et al.; The Saccharomyces cerevisiae silencing protein Sir2 is the founding member of a universally conserved family of proteins that have been shown to possess NAD-dependent histone deacetylation and ADP-ribosylation activities . Here we show that histone deacetylation by Sir2 is coupled to cleavage of the high-energy bond that links the ADP-ribose moiety of NAD to nicotinamide . Analysis of the NAD cleavage products revealed the presence of nicotinamide, ADP-ribose, and a third product that appeared to be related to ADP-ribose . With the use of label transfer experiments, we show that the acetyl group in the histone substrate is transferred to this NAD breakdown product during deacetylation, forming a product that we conclude to be O-acetyl-ADP-ribose . Detection of this species strongly argues for obligate coupling of histone deacetylation to NAD breakdown by Sir2 . We propose reaction mechanisms that could account for this coupling via acetyl-ADP-ribose formation . The unprecedented coupling of amide bond cleavage to cleavage of a high-energy bond raises the possibility that NAD breakdown by Sir2 plays an important role in silencing that is independent of its requirement for deacetylation.

Lipids, 2000 Nov, 35(11), 1205 - 14
Evidence for the mitochondrial biosynthesis of 3R-hydroxy-5Z,8Z,11Z,1 4Z-eicosatetraenoic acid in the yeast Dipodascopsis uninucleata; Fox SR et al.; The biosynthesis of 3R-hydroxy-5Z, 8Z, 11Z,14Z-eicosatetraenoic acid (3R-HETE) from arachidonic acid (20:4n-6) by the hyphal-forming yeast, Dipodascopsis uninucleata, in cell-free enzyme extracts required CoASH, ATP, NAD+ and Mg2+; 3R-HETE was present as the CoA derivative in enzyme extracts and its biosynthesis was associated with mitochondria . Its synthesis was high from arachidonoyl-CoA (15% conversion of the substrate; 22 nmol mg protein(-1) x h), but significantly higher from trans-2-arachidonoyl-CoA (53 nmol mg protein(-1) x min) . Aspirin, an inhibitor of prostaglandin endoperoxide synthase synthase (cyclooxygenase), did not significantly inhibit 3R-HETE biosynthesis in enzyme extracts, as opposed to antimycin A (46% inhibition) . The chirality of 3-HETE was 95% R and 5% S . 3R-HETE has the same chirality as the products of peroxisomal enoyl-CoA hydratases of Neurospora crassa and Saccharomyces cerevisiae; the difference appears to be that in D . uninucleata the Renantiomers are synthesized in mitochondria . Exogenously supplied eicosapentaenoic acid was converted to 3-hydroxy 5Z,11Z,14Z,17Z-eicosapentaenoic acid by cell-free enzyme extracts though there was no requirement for a 5Z,8Z-diene structure for the biosynthesis of 3-hydroxylated fatty acids as 3-hydroxy-8Z,11Z,14Z, and 3-hydroxy-11Z,14Z,17Z-eicosatrienoic acids were synthesized from the corresponding fatty acids . We found no evidence for the synthesis of the prostaglandins F2alpha and E2.

Biosci Biotechnol Biochem, 2000 Oct, 64(10), 2025 - 33
Expression of human cytochromes P450 1A1 and P450 1A2 as fused enzymes with yeast NADPH-cytochrome P450 oxidoreductase in transgenic tobacco plants; Shiota N et al.; Among 11 isoforms of the human cytochrome P450 enzymes metabolizing xenobiotics, CYP 1A1 and CYP 1A2 were major P450 species in the metabolism of the herbicides chlortoluron and atrazine in a yeast expression system . CYP1A2 was more active in the metabolism of both herbicides than CYP1A1 . The fused enzymes of CYP1A1 and CYP1A2 with yeast NADPH-cytochrome P450 oxidoreductase were functionally active in the microsomal fraction of the yeast Saccharomyces cerevisiae and showed increased specific activity towards 7-ethoxyresorufin as compared to CYP1A1 and CYP1A2 alone . Then, both fused enzymes were each expressed in the microsomes of tobacco (Nicotiana tabacum cv . Samsun NN) plants . The transgenic plants expressing the CYP1A2 fusion enzyme had higher resistance to the herbicide chlortoluron than the plants expressing the CYP1A1 fusion enzyme did . The transgenic plants expressing the CYP1A2 fused enzyme metabolized chlortoluron to a larger extent to its non-phytotoxic metabolites through N-demethylation and ring-methyl hydroxylation as compared to the plants expressing the CYP1A1 fused enzyme . Thus, the possibility of increasing the herbicide resistance in the transgenic plants by the selection of P450 species and the fusion with P450 reductase is discussed.

Mol Gen Genet, 2000 Nov, 264(4), 378 - 91
Allele-specific interactions between the yeast RFC1 and RFC5 genes suggest a basis for RFC subunit-subunit interactions; Beckwith W et al.; Replication factor C (RFC) is an essential, multi-subunit ATPase that functions in DNA replication, DNA repair, and DNA metabolism-related checkpoints . In order to investigate how the individual RFC subunits contribute to these functions in vivo, we undertook a genetic analysis of RFC genes from budding yeast . We isolated and characterized mutations in the RFC5 gene that could suppress the cold-sensitive phenotype of rfc1-1 mutants . Analysis of the RFC5 suppressors revealed that they could not suppress the elongated telomere phenotype, the sensitivity to DNA damaging agents, or the mutator phenotype of rfc1-1 mutants . Unlike the checkpoint-defective rfc5-1 mutation, the RFC5 suppressor mutations did not interfere with the methylmethane sulfonate- or hydroxyurea-induced phosphorylation of Rad53p . The Rfc5p suppressor substitutions mapped to amino acid positions in the conserved RFC box motifs IV-VII . Comparisons of the structures of related RFC box-containing proteins suggest that these RFC motifs may function to coordinate interactions between neighboring subunits of multi-subunit ATPases.

Philos Trans R Soc Lond B Biol Sci, 2000 Jul 29, 355(1399), 873 - 7
New concepts in fission yeast morphogenesis; Brunner D et al.; The ability to generate spatial form is a fundamental characteristic of all living organisms, which has been much studied by successive generations of developmental biologists . In recent years increasing numbers of cell biologists have turned their attention to the mechanisms by which cells generate their spatial form . These include the mechanisms that position components in different places within the cell, that specify the position of these components, and that generate the overall shape of these components . These problems are entirely analogous to those studied by developmental biologists, although usually at the level of the whole organism, organ or tissue . Because the organization of all cells is basically similar, it is possible that the concepts and the underlying molecular mechanisms of cell morphogenesis may be highly conserved . In this article we consider the generation of spatial form within the fission yeast cell, focusing on emerging new concepts, which may be applicable to the morphogenesis of other cells.

Biotechniques, 2000 Dec, 29(6), 1226 - 31
Tightly regulated, beta-estradiol dose-dependent expression system for yeast; Gao CY et al.; We have refined the regulated expression of UASGAL1, 10-driven genes in yeast by modifying a vector encoding the beta-estradiol inducible activator, GAL4.ER.VP16 (GEV) . The expression of GEV was placed under the regulation of the low-level, constitutive MRP7 promoter, and beta-estradiol-regulated expression was monitored by the expression of an integrated UASGAL10-lacZ reporter and by immunoblot analysis of a UASGAL1-regulated gene product . Target gene expression regulated by low levels of GEV has several advantages over the standard galactose-inducible expression systems . (i) Most importantly, the target gene expression is undetectable in the absence of hormone; (ii) target gene expression is beta-estradiol dose-dependent, and variable levels of target gene expression from low to several hundred-fold induction can be achieved; and (iii) induction or depletion studies can be conducted independent of carbon source in gal4 delta strains . In addition, any UASGAL1,10 expression construct can be used without modification of the target gene or many gal4 delta host strains, and GEV vectors are compatible with other inducible yeast expression systems . This method may be useful to researchers investigating the functions of essential genes, dominant negative mutants, mitochondrial genes, and viral, plant, and mammalian genes in yeast assay systems.

Yeast, 2001 Jan 15, 18(1), 69 - 80
Characterization of 16 novel human genes showing high similarity to yeast sequences; Stanchi F et al.; The entire set of open reading frames (ORFs) of Saccharomyces cerevisiae has been used to perform systematic similarity searches against nucleic acid and protein databases: with the aim of identifying interesting homologies between yeast and mammalian genes . Many similarities were detected: mostly with known genes . However: several yeast ORFs were only found to match human partial sequence tags: indicating the presence of human transcripts still uncharacterized that have a homologous counterpart in yeast . About 30 such transcripts were further studied and named HUSSY (human sequence similar to yeast) . The 16 most interesting are presented in this paper along with their sequencing and mapping data . As expected: most of these genes seem to be involved in basic metabolic and cellular functions (lipoic acid biosynthesis: ribulose-5-phosphate-3-epimerase: glycosyl transferase: beta-transducin: serine-threonine-kinase: ABC proteins: cation transporters) . Genes related to RNA maturation were also found (homologues to DIM1: ROK1-RNA-elicase and NFS1) . Furthermore: five novel human genes were detected (HUSSY-03: HUSSY-22: HUSSY-23: HUSSY-27: HUSSY-29) that appear to be homologous to yeast genes whose function is still undetermined . More information on this work can be obtained at the website

J Mol Biol, 2000 Dec 15, 304(5), 941 - 51
Structure of the FHA1 domain of yeast Rad53 and identification of binding sites for both FHA1 and its target protein Rad9; Liao H et al.; Forkhead-associated (FHA) domains have been shown to recognize both pThr and pTyr-peptides . The solution structures of the FHA2 domain of Rad53 from Saccharomyces cerevisiae, and its complex with a pTyr peptide, have been reported recently . We now report the solution structure of the other FHA domain of Rad53, FHA1 (residues 14-164), and identification of binding sites of FHA1 and its target protein Rad9 . The FHA1 structure consists of 11 beta-strands, which form two large twisted anti-parallel beta-sheets folding into a beta-sandwich . Three short alpha-helices were also identified . The beta-strands are linked by several loops and turns . These structural features of free FHA1 are similar to those of free FHA2, but there are significant differences in the loops . Screening of a peptide library {XXX(pT)XXX} against FHA1 revealed an absolute requirement for Asp at the +3 position and a preference for Ala at the +2 position . These two criteria are met by a pThr motif (192)TEAD(195) in Rad9 . Surface plasmon resonance analysis showed that a pThr peptide containing this motif, (188)SLEV(pT)EADATFVQ(200) from Rad9, binds to FHA1 with a K(d) value of 0.36 microM . Other peptides containing pTXXD sequences also bound to FHA1, but less tightly (K(d)=4-70 microM) . These results suggest that Thr192 of Rad9 is the likely phosphorylation site recognized by the FHA1 domain of Rad53 . The tight-binding peptide was then used to identify residues of FHA1 involved in the interaction with the pThr peptide . The results are compared with the interactions between the FHA2 domain and a pTyr peptide derived from Rad9 reported previously .

Chem Res Toxicol, 2000 Dec, 13(12), 1214 - 22
Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis; Vinggaard AM et al.; The use of recycled paper for the manufacture of food contact materials is widespread, but very little is known about the presence of potential contaminants in the paper . The purpose of this study was to assess the worst-case migration of estrogenic active compounds using extracts of paper for household use . Twenty different brands of kitchen rolls, nine of which were made from recycled paper and the remainder from virgin paper, were obtained from retail shops . Paper extracts were subjected to (a) determination of the total estrogenic activity by using an in vitro estrogen screen based on yeast cells stably transfected with the human estrogen receptor alpha and (b) chemical analysis and quantification by GC/MS, GC/FTIR/MS, and GC/FID for detection of a variety of estrogenic compounds . A marked estrogenic response was observed in nine of the extracts, seven of which were made from recycled paper and two from virgin paper . The chemical analysis revealed that extracts made from recycled paper contained levels of bisphenol A ranging from 0.6 to 24 mg/kg of kitchen roll, whereas extracts from virgin paper contained no bisphenol A or only negligible amounts . In contrast, 4-tert-octylphenol, 4-nonylphenols, and di-n-butyl and diisobutyl phthalate were present to a varying degree in both recycled and virgin paper with no apparent preferable distribution between the two paper types . The estrogenic response of the two extracts made from virgin paper appeared to be due partly to the presence of the preservative propyl paraben . Diisopropylnaphthalene, which turned out to be weakly estrogenic active in vitro (EC(50) = 53 microM), was detected in minor amounts in most of the extracts with the major part, ranging from 0.3 to 4.7 mg/kg of paper, found in recycled paper . Our findings that recycled kitchen rolls contain bisphenol A and other xenoestrogens may apply to other types of recycled paper used for food packaging and emphasize the importance of identifying this and other contaminants in recycled paper in general . These data indicate that bisphenol A may be useful as a purity indicator for recycled paper.

Mol Microbiol, 2001 Jan, 39(1), 37 - 46
The relationship between visible intracellular aggregates that appear after overexpression of Sup35 and the yeast prion-like elements {PSI(+)} and {PIN(+)}; Zhou P et al.; Overproduced fusions of Sup35 or its prion domain with green fluorescent protein (GFP) have previously been shown to form frequent dots in {PSI(+)} cells . Rare foci seen in {psi(-)} cells were hypothesized to indicate the de novo induction of {PSI(+)} caused by the overproduced prion domain . Here, we describe novel ring-type aggregates that also appear in {psi(-)} cultures upon Sup35 overproduction and show directly that dot and ring aggregates only appear in cells that have become {PSI(+)} . The formation of either type of aggregate requires {PIN(+)}, an element needed for the induction of {PSI(+)} . Although aggregates are visible predominantly in stationary-phase cultures, {PSI(+)} induction starts in exponential phase, suggesting that much smaller aggregates can also propagate {PSI(+)} . Such small aggregates are probably present in {PSI(+)} cells and, upon Sup35-GFP overproduction, facilitate the frequent formation of dot aggregates, but only the occasional appearance of ring aggregates . In contrast, rings are very frequent when {PSI(+)} cultures, including those lacking {PIN(+)}, are grown in the presence of GuHCl or excess Hsp104 while overexpressing Sup35-GFP . Thus, intermediates formed during {PSI(+)} curing seem to facilitate ring formation . Surprisingly, GuHCl and excess Hsp104, which are known to promote loss of {PSI(+)}, did not prevent the de novo induction of {PSI(+)} by excess Sup35 in {psi(-)}{PIN(+)} strains.

J Appl Microbiol, 2000 Dec, 89(6), 1009 - 17
Solute stresses affect growth patterns, endogenous water potentials and accumulation of sugars and sugar alcohols in cells of the biocontrol yeast Candida sake; Abadias M et al.; AIM: To evaluate the effect of modifications of water activity (aw 0 . 996-0.92) of a molasses medium with different solutes (glycerol, glucose, NaCl, proline or sorbitol) on growth, intracellular water potentials (psi(c)) and endogenous accumulation of polyols/sugars in the biocontrol yeast Candida sake . METHODS AND RESULTS: Modification of solute stress significantly influenced growth, psi(c) and accumulation of sugars (glucose/trehalose) and polyols (glycerol, erythritol, arabitol and mannitol) in the yeast cells . Regardless of the solute used to modify aw, growth was always decreased as water stress increased . Candida sake cells grew better in glycerol- and proline-amended media, but were sensitive to NaCl . The psi(c) measured using psychrometry showed a significant effect of solutes, aw and time . Cells from the 0.96 aw NaCl treatment presented the lowest psic value (- 5.20 MPa) while cells from unmodified media (aw = 0 . 996) had the highest value (- 0.30 MPa) . In unmodified medium, glycerol was the predominant reserve accumulated . Glycerol and arabitol were the major compounds accumulated in media modified with glucose or NaCl . In proline media, the concentration of arabitol increased . In glycerol- and sorbitol-amended media, the concentration of glycerol rose . Some correlations were obtained between compatible solutes and psi(c) . CONCLUSIONS AND SIGNIFICANCE: This study demonstrates that subtle changes in physiological parameters significantly affect the endogenous contents of C . sake cells . It may be possible to utilize such physiological information to develop biocontrol inocula with improved quality.

Immunology, 2000 Dec, 101(4), 563 - 9
Virulence of Sporothrix schenckii conidia and yeast cells, and their susceptibility to nitric oxide; Fernandes KS et al.; The involvement of nitric oxide (NO) in macrophage (M phi) fungicidal activity against Sporothrix schenckii, and the relationship between NO susceptibility and the differential virulence of conidia and yeast cells, were investigated . Confirming a previously reported correlation between the length of time in culture and virulence of S . schenckii, conidia isolated from 12-day mycelial cultures (Ss-12) were less virulent to mice than conidia from 7-day cultures (Ss-7) or yeast cells . Indicative of NO production, infected animals showed a significant increase in serum levels of nitrite that was lower in mice infected with Ss-12 than in mice infected with Ss-7 or yeast . Stimulation of murine M phi with interferon-gamma (IFN-gamma) induced NO production and inhibition of fungal growth . The cytotoxic activity of M phi against Ss-12 was significantly greater than against Ss-7 or yeast cells, the highly virulent fungal forms . The addition of NO synthase inhibitors abrogated M phi cytotoxic activity against all fungal forms . The phagocytic activity of M phi against Ss-7 was significantly lower than against Ss-12 or yeast cells . Although the ingestion of fungal cells triggered the oxidative burst in M phi, the fungicidal activity was not altered in the presence of superoxide dismutase (SOD) and catalase . In addition, Ss-12 and yeast cells were more susceptible than Ss-7 to the direct fungicidal activity of the NO donors S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitrosoglutathione (GSNO) and 3-morpholinosydnonimine (SIN-1) . The results of this study indicate that NO is a key cytotoxic mediator involved in the murine M phi defence against S . schenckii, and that the virulence of Ss-7, Ss-12 and yeast cells may be related to a differential susceptibility to NO.

J Struct Biol, 2000 Oct, 132(1), 1 - 5
Yeast nuclear pore complex assembly defects determined by nuclear envelope reconstruction; Gomez-Ospina N et al.; Assembly of nuclear pore complexes (NPCs) is a critical yet poorly understood cellular function . One approach to studying NPC assembly is to identify yeast mutants defective in this process . This requires robust assays for NPC assembly that can be used for phenotypic analysis . We have previously reconstructed yeast nuclei from electron micrographs of serially sectioned cells to precisely determine the number of NPCs (Winey et al., 1997) . Here we report the analysis of strains mutant in either of two nucleoporin-encoding genes, NIC96 (Zabel et al., 1996) and NUP192 (Kosova et al., 1999) . Using conditional alleles of either gene, we have found that the NPC number falls significantly following shift to the restrictive temperature . We conclude that the drop in NPC number results from the failure to assemble new NPCs during cell divisions, leading to the dilution of NPCs that existed when the cells were shifted to the restrictive temperature . We are also able to document a subtle defect in NPC numbers in nup192-15 cells at their permissive temperature . The data presented here quantitatively demonstrate that NPC numbers fall in nic96-1 and nup192-15 strains upon shifting to the restrictive temperature, indicating that these gene products are required for NPC assembly .

Nat Toxins, 1999, 7(6), 401 - 6
A yeast bioassay for trichothecenes; Binder J; Like all eucaryotic cells, yeasts are sensitive to trichothecenes, especially T-2 toxin and verrucarin A . Based on this sensitivity, a yeast bioassay was developed to evaluate the toxicity of corn samples . The bioassay was optimized using spiked maize extracts . The toxicity of samples was defined as toxicity equivalent to a certain concentration of T-2 toxin standards . The assay can be performed on crude extracts, but the results are more precise after column clean-up . The test can also be used for the screening of trichothecene toxicity in general . The relative standard deviation (RSD) at 85 % growth inhibition (EC85) was 4.5% for the T-2 toxin standards (n = 8) . This corresponds to an initial T-2 toxin concentration of approximately 58 ppb in the corn sample . Samples containing 188 and 113 ppb T-2 toxin caused a growth inhibition higher than 85%, whereas samples with toxin concentrations of 56 and 19 ppb had a growth inhibition less than 85% . Therefore the test can be used for the qualitative evaluation of corn samples up to a level of 58 ppb +/- 2.8 ppb . The bioassay is easy to perform with minimum requirements for equipment . Results can be obtained within 24 h and a large number of samples can be analysed daily . The costs are low and the results obtained are repeatable . With some modifications this test can be used for toxicity studies on trichothecene metabolites as well as for extracts with unknown compounds with properties similar to trichothecenes.

Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14500 - 5
HO endonuclease-induced recombination in yeast meiosis resembles Spo11-induced events; Malkova A et al.; In meiosis, gene conversions are accompanied by higher levels of crossing over than in mitotic cells . To determine whether the special properties of meiotic recombination can be attributed to the way in which Spo11p creates double-strand breaks (DSBs) at special hot spots in Saccharomyces cerevisiae, we expressed the site-specific HO endonuclease in meiotic cells . We could therefore compare HO-induced recombination in a well-defined region both in mitosis and meiosis, as well as compare Spo11p- and HO-induced meiotic events . HO-induced gene conversions in meiosis were accompanied by crossovers at the same high level (52%) as Spo11p-induced events . Moreover, HO-induced crossovers were reduced 3-fold by a msh4Delta mutation that similarly affects Spo11p-promoted events . In a spo11Delta diploid, where the only DSB is made by HO, crossing over was significantly higher (27%) than in mitotic cells (</=7%) . This single meiotic DSB failed to induce the formation of a synaptonemal complex . We also show that HO-induced gene conversion tract lengths are shorter in meiotic than in mitotic cells . We conclude that a hallmark of meiotic recombination, the production of crossovers, is independent of the nature of Spo11p-generated DSBs at special hotspots, but some functions of Spo11p are required in trans to achieve maximum crossing over.

Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14370 - 5
Yin6, a fission yeast Int6 homolog, complexes with Moe1 and plays a role in chromosome segregation; Yen HC et al.; The INT6 gene has been implicated in human breast cancer formation, but its function is unknown . We isolated an Int6 homolog from fission yeast, Yin6, by its binding to a conserved protein in the Ras pathway, Moe1 . Yin6 and Moe1 converge on the same protein complex to promote microtubule instability/disassembly . Yin6 and Moe1 interact cooperatively: when either protein is absent, the other becomes mislocalized with decreased protein levels . Furthermore, whereas full-length human Int6 rescues the phenotypes of the yin6-null (yin6Delta) mutant cells and binds human Moe1, truncated Int6 proteins found in tumors do not . Importantly, yin6Delta alone impairs chromosome segregation weakly, but yin6Delta together with ras1Delta causes severe chromosome missegregation . These data support a model in which INT6 mutations in humans either alone or together with additional mutations, such as a RAS mutation, may contribute to tumorigenesis by altering genome stability.

Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14115 - 20
Stepwise assembly of initiation proteins at budding yeast replication origins in vitro; Seki T et al.; The initiation of DNA replication in the budding yeast Saccharomyces cerevisiae occurs in two sequential and mutually exclusive steps . Prereplicative complexes (pre-RCs) containing origin recognition complex (ORC), Cdc6p, and the MCM2-7 proteins assemble only under conditions of low cyclin-dependent kinase (Cdk) activity during G(1), whereas origin activation is driven by the increase in Cdk activity at the end of G(1) . As a first step toward the reconstitution of this two-step process in vitro, we describe a system in which extracts prepared from G(1)-arrested cells promote sequential assembly of ORC, Cdc6p, and MCM2-7 proteins onto exogenously added origin-containing DNA . This reaction requires an intact ARS consensus sequence and requires ATP for two distinct steps . Extracts from cells arrested in mitosis also can support the binding of ORC but are unable to load either Cdc6p or MCM2-7 proteins . This system should be useful for studying the mechanism and regulation of pre-RC assembly.

FEBS Lett, 2000 Dec 15, 486(3), 300 - 4
Inositol polyphosphate kinase activity of Arg82/ArgRIII is not required for the regulation of the arginine metabolism in yeast; Dubois E et al.; Arg82, a nuclear regulator of diverse cellular processes in yeast, is an inositol polyphosphate kinase . Some defects such as the regulation of arginine metabolism observed in an arg82Delta, result from a lack of Mcm1 and Arg80 stability . We show here that neither the kinase activity of Arg82 nor inositol phosphates are required for the control of arginine metabolism . Arg82 mutations keeping kinase active affect the expression of arginine genes, whereas mutations in the kinase domain do not impair this metabolic control.

Biochim Biophys Acta, 2000 Dec 11, 1499(1-2), 85 - 100
Mutations in SPC110, encoding the yeast spindle pole body calmodulin-binding protein, cause defects in cell integrity as well as spindle formation; Stirling DA et al.; The 110 kDa spindle pole body component, Spc110p, is an essential target of calmodulin in budding yeast . Cells with mutations which reduce calmodulin binding to Spc110p are unable to form a mitotic spindle and die . Here we show that these effects can be overcome either directly by increasing extracellular calcium or calmodulin expression, which reverse the primary spindle defect, or indirectly through increased extracellular osmolarity or high dosage of MID2 or SLG1/HCS77/WSC1 which preserve viability . We propose that overcoming a cell integrity defect associated with the mitotic arrest enables the defective spindle pole bodies to provide sufficient function for proliferation of a large proportion of mutant cells . Our findings demonstrate a role for calcium in the Spc110p-calmodulin interaction in vivo and have important general implications for the interpretation of genetic interactions involving cell integrity genes.

Biochim Biophys Acta, 2000 Dec 20, 1509(1-2), 189 - 94
The cell wall integrity/remodeling MAPK cascade is involved in glucose activation of the yeast plasma membrane H(+)-ATPase; de la Fuente N et al.; Glucose triggers transcriptional and post-transcriptional mechanisms that increase the amount and the activity of Saccharomyces cerevisiae plasma membrane H(+)-ATPase . In a previous study, we found that a mutation in the Rsp5 ubiquitin-protein ligase enzyme affected the post-transcriptional activation of the enzyme by glucose . Mutations at the RSP5 locus alter the glucose-triggered K(m) decrease . In a genetic screening for multicopy suppressors of the rsp5 mutation, we identified the WSC2/YNL283c gene . Deletion of the WSC2 gene disturbs ATPase activation by glucose, abolishing the K(m) decrease that occurs during this process . Wsc2 is a component of the PKC1-MPK1 mitogen-activated protein kinase (MAPK) signaling pathway that controls the cell wall integrity . Deletion of the MPK1/SLT2 gene disturbs the glucose-triggered K(m) decrease in ATPase.

Biochim Biophys Acta, 2000 Dec 20, 1509(1-2), 86 - 94
Involvement of the TOM complex in external NADH transport into yeast mitochondria depleted of mitochondrial porin1; Kmita H et al.; The protein(s) responsible for metabolite transport through the outer membrane of the yeast Saccharomyces cerevisiae mitochondria depleted of mitochondrial porin (also known as voltage-dependent anion selective channel), termed here porin1, is (are) still unidentified . It is postulated that the transport may be supported by the protein import machinery of the outer membrane, the TOM complex (translocase of the outer membrane) . We demonstrate here that in the absence of functional porin1, the blockage of the TOM complex by the fusion protein termed pb(2)-DHFR (consisting of the first 167 amino acids of yeast cytochrome b(2) preprotein connected to mouse dihydrofolate reductase) limits the access of external NADH to mitochondria . It was measured by the ability of the blockage to inhibit external NADH oxidation by the proper dehydrogenase located at the outer surface of the inner membrane . The inhibition depends on external NADH concentration and increases with decreasing amounts of the substrate . In the presence of 1 microg of pb(2)-DHFR per 50 microg of mitochondrial protein almost quantitative inhibition was observed when external NADH was applied at the concentration of 70 nmol per mg of mitochondrial protein . On the other hand, external NADH decreases the levels of pb(2)-DHFR binding at the trans site of the TOM complex in porin1-depleted mitochondria in a concentration-dependent fashion . Our data define an important role of the TOM complex in the transport of external NADH across the outer membrane of porin1-depleted mitochondria.

Biochem Biophys Res Commun, 2000 Dec 20, 279(2), 468 - 74
Functional complementation by wheat eIF2alpha in the yeast GCN2-mediated pathway; Chang LY et al.; Translational control by specific eIF2alpha phosphorylation on serine 51 has been characterized in all eukaryotes with the significant exception of plants . In order to evaluate the capability of plant eIF2alpha to functionally control translation, the wild type (51S) and a nonphosphorylatable mutant (51A) of wheat eIF2alpha were expressed in a yeast genetic system . Expression of either wheat protein did not handicap growth under conditions that repress the eIF2alpha phosphorylation pathway . However, under conditions that induce specific eIF2alpha phosphorylation only strains expressing wheat 51S were able to grow between 2 and 4 days . Growth was dependent upon activity of yeast eIF2alpha kinase GCN2 and resulted in the increased translation of GCN4 . The association between plant eIF2alpha and yeast eIF2B is supported by their specific coimmunoprecipitation from transgenic yeast cells . These data support the similarity among eukaryotic translational initiation processes and strengthen the concept that plants may contain an eIF2alpha phosphorylation pathway .

Biochem Biophys Res Commun, 2000 Dec 20, 279(2), 445 - 50
Dissociation of Kar2p/BiP from an ER sensory molecule, Ire1p, triggers the unfolded protein response in yeast; Okamura K et al.; The unfolded protein response (UPR) is a signal transduction pathway induced by a variety of endoplasmic reticulum (ER) stresses and functions to maintain homeostasis of the cellular membrane in eukaryotes . Various ER stresses result in the accumulation of unfolded proteins in the ER, which is sensed by the transmembrane protein kinase/ribonuclease Ire1p that transmits a signal from the ER to the nucleus in Saccharomyces cerevisiae . Here we report that the yeast ER chaperone Kar2p/BiP, a member of the HSP70 family found in the ER, directly regulates the UPR by the interaction with Ire1p . In the absence of ER stress, Kar2p binds the lumenal domain of Ire1p and keeps Ire1p in an inactive unphosphorylated state . Upon exposure of cells to ER stresses, Kar2p is released from Ire1p, resulting in activation of Ire1p and signal transduction to the nucleus . Subsequently, KAR2 mRNA is induced and Kar2p accumulates in the ER in a time-dependent manner, restoring the system to the basal state . This negative autoregulation is similar to the regulation of mammalian cytosolic chaperone Hsp70 via its interaction with heat shock factor 1 .

Enzyme Microb Technol, 2000 Dec, 27(10), 806 - 811
Preparation of high activity whole cell biocatalyst by permeabilization of recombinant flocculent yeast with alcohol; Kondo A et al.; Flocculent yeast Saccharomyces cerevisiae YF234 (MATa ura3-52 trp1Delta2 his ade 2-1 can1-100 sta1 FLO8) cells overexpressing glyoxalase I and having strong flocculation ability were permeabilized with isopropyl alcohol and ethanol under various conditions . The treatment with 40% isopropyl alcohol significantly improves the initial reaction rates of recombinant flocculent yeast cells . Moreover, the reactivity of permeabilized flocculent yeast cells was similar to that of dispersed cells with EDTA . On the other hand, the flocculation ability of yeast cells was not affected by the treatment with alcohol solutions of various concentrations and treatment time length . Therefore, the recombinant flocculent yeast cells permeabilized with alcohol are very effective whole cell biocatalysts.

J Biol Chem, 2001 Mar 16, 276(11), 8616 - 22 Epub 2000 Dec 15.
Nam1p, a protein involved in RNA processing and translation, is coupled to transcription through an interaction with yeast mitochondrial RNA polymerase; Rodeheffer MS et al.; Alignment of three fungal mtRNA polymerases revealed conserved amino acid sequences in an amino-terminal region of the Saccharomyces cerevisiae enzyme implicated previously as harboring an important functional domain . Phenotypic analysis of deletion and point mutations, in conjunction with a yeast two-hybrid assay, revealed that Nam1p, a protein involved in RNA processing and translation in mitochondria, binds specifically to this domain . The significance of this interaction in vivo was demonstrated by the fact that the temperature-sensitive phenotype of a deletion mutation (rpo41Delta2), which impinges on this amino-terminal domain, is suppressed by overproducing Nam1p . In addition, mutations in the amino-terminal domain result specifically in decreased steady-state levels of mature mitochondrial CYTB and COXI transcripts, which is a primary defect observed in NAM1 null mutant yeast strains . Finally, one point mutation (R129D) did not abolish Nam1p binding, yet displayed an obvious COX1/CYTB transcript defect . This mutation exhibited the most severe mitochondrial phenotype, suggesting that mutations in the amino-terminal domain can perturb other critical interactions, in addition to Nam1p binding, that contribute to the observed phenotypes . These results implicate the amino-terminal domain of mtRNA polymerases in coupling additional factors and activities involved in mitochondrial gene expression directly to the transcription machinery.

EMBO J, 2000 Dec 15, 19(24), 6686 - 96
Asymmetrically localized Bud8p and Bud9p proteins control yeast cell polarity and development; Taheri N et al.; Diploid strains of the budding yeast Saccharomyces cerevisiae change the pattern of cell division from bipolar to unipolar when switching growth from the unicellular yeast form (YF) to filamentous, pseudohyphal (PH) cells in response to nitrogen starvation . The functions of two transmembrane proteins, Bud8p and Bud9p, in regulating YF and PH cell polarity were investigated . Bud8p is highly concentrated at the distal pole of both YF and PH cells, where it directs initiation of cell division . Asymmetric localization of Bud8p is independent of the Rsr1p/Bud1p GTPase . rsr1/bud1 mutations are epistatic to bud8 mutations, placing Rsr1p/Bud1p downstream of Bud8p . In YF cells, Bud9p is also localized at the distal pole, yet deletion of BUD9 favours distal bud initiation . In PH cells, nutritional starvation for nitrogen efficiently prevents distal localization of Bud9p . Because Bud8p and Bud9p proteins associate in vivo, we propose Bud8p as a landmark for bud initiation at the distal cell pole, where Bud9p acts as inhibitor . In response to nitrogen starvation, asymmetric localization of Bud9p is averted, favouring Bud8p-mediated cell division at the distal pole.

J Biol Chem, 2001 Mar 9, 276(10), 7643 - 53 Epub 2000 Dec 14.
The yeast inositol polyphosphate 5-phosphatase Inp54p localizes to the endoplasmic reticulum via a C-terminal hydrophobic anchoring tail: regulation of secretion from the endoplasmic reticulum; Wiradjaja F et al.; The budding yeast Saccharomyces cerevisiae has four inositol polyphosphate 5-phosphatase (5-phosphatase) genes, INP51, INP52, INP53, and INP54, all of which hydrolyze phosphatidylinositol (4,5)-bisphosphate . INP54 encodes a protein of 44 kDa which consists of a 5-phosphatase domain and a C-terminal leucine-rich tail, but lacks the N-terminal SacI domain and proline-rich region found in the other three yeast 5-phosphatases . We report that Inp54p belongs to the family of tail-anchored proteins and is localized to the endoplasmic reticulum via a C-terminal hydrophobic tail . The hydrophobic tail comprises the last 13 amino acids of the protein and is sufficient to target green fluorescent protein to the endoplasmic reticulum . Protease protection assays demonstrated that the N terminus of Inp54p is oriented toward the cytoplasm of the cell, with the C terminus of the protein also exposed to the cytosol . Null mutation of INP54 resulted in a 2-fold increase in secretion of a reporter protein, compared with wild-type yeast or cells deleted for any of the SacI domain-containing 5-phosphatases . We propose that Inp54p plays a role in regulating secretion, possibly by modulating the levels of phosphatidylinositol (4,5)-bisphosphate on the cytoplasmic surface of the endoplasmic reticulum membrane.

Plant Physiol, 2000 Dec, 124(4), 1814 - 27
The ACA4 gene of Arabidopsis encodes a vacuolar membrane calcium pump that improves salt tolerance in yeast; Geisler M et al.; Several lines of evidence suggest that regulation of intracellular Ca(2+) levels is crucial for adaptation of plants to environmental stress . We have cloned and characterized Arabidopsis auto-inhibited Ca(2+)-ATPase, isoform 4 (ACA4), a calmodulin-regulated Ca(2+)-ATPase . Confocal laser scanning data of a green fluorescent protein-tagged version of ACA4 as well as western-blot analysis of microsomal fractions obtained from two-phase partitioning and Suc density gradient centrifugation suggest that ACA4 is localized to small vacuoles . The N terminus of ACA4 contains an auto-inhibitory domain with a binding site for calmodulin as demonstrated through calmodulin-binding studies and complementation experiments using the calcium transport yeast mutant K616 . ACA4 and PMC1, the yeast vacuolar Ca(2+)-ATPase, conferred protection against osmotic stress such as high NaCl, KCl, and mannitol when expressed in the K616 strain . An N-terminally modified form of ACA4 specifically conferred increased NaCl tolerance, whereas full-length ATPase had less effect.

Plant Physiol, 2000 Dec, 124(4), 1800 - 13
The isolation and characterization in yeast of a gene for Arabidopsis S-adenosylmethionine:phospho-ethanolamine N-methyltransferase; Bolognese CP et al.; Saccharomyces cerevisiae opi3 mutant strains do not have the phospholipid N-methyltransferase that catalyzes the two terminal methylations in the phosphatidylcholine (PC) biosynthetic pathway . This results in a build up of the intermediate phosphatidylmonomethylethanolamine, causing a temperature-sensitive growth phenotype . An Arabidopsis cDNA library was used to isolate three overlapping plasmids that complemented the temperature-sensitive phenotype . Phospholipid analysis showed that the presence of the cloned cDNA caused a 65-fold reduction in the level of phosphatidylmonomethylethanolamine and a significant, though not equivalent, increase in the production of PC . Sequence analysis established that the cDNA was not homologous to OPI3 or to CHO2, the only other yeast phospholipid N-methyltransferase, but was similar to several other classes of methyltransferases . S-adenosyl-Met:phospho-base N-methyltransferase assays revealed that the cDNA catalyzed the three sequential methylations of phospho-ethanolamine to form phospho-choline . Phospho-choline is converted to PC by the CDP-choline pathway, explaining the phenotype conferred upon the yeast mutant strain by the cDNA . In accordance with this the gene has been named AtNMT1 . The identification of this enzyme and the failure to isolate a plant phospholipid N-methyltransferase suggests that there are fundamental differences between the pathways utilized by yeast and by some plants for synthesis of PC.

Plant J, 2000 Nov, 24(4), 489 - 501
A member of a novel Arabidopsis thaliana gene family of candidate Mg2+ ion transporters complements a yeast mitochondrial group II intron-splicing mutant; Schock I et al.; Autocatalytic activity of some group II introns has been demonstrated in vitro, but helper functions such as the yeast MRS2 protein are essential for splicing in vivo . In our search for such helper factors in plants, we pursued the cloning of two Arabidopsis thaliana homologues, atmrs2-1 and atmrs2-2 . Atmrs2-1, but not atmrs2-2, complements the yeast deletion mutant of mrs2, and this is congruent with the prediction of two adjacent transmembrane stretches in AtMRS2-1 and yeast MRS2 but not in AtMRS2-2 . This complementation depends on fusion of the native yeast mitochondrial import sequence to atmrs2-1 . A differing, non-mitochondrial, cellular targeting in Arabidopsis is supported by the analysis of green fluorescent protein fusion constructs after transient transformation into plant protoplasts . Further members of what now appears to be a family of 10 mrs2 homologues are identified in the Arabidopsis genome . Similarity searches with the PSI-BLAST algorithm in the protein database fail to identify homologues of this novel gene family in any eukaryotes other than yeasts, but do identify its distant relatedness to the corA group of bacterial magnesium transporters . In line with this observation, intramitochondrial magnesium concentrations are indeed restored to wild-type levels in the yeast mutant on complementation with atmrs2-1.

Mol Microbiol, 2000 Nov, 38(4), 879 - 90
A Rox1-independent hypoxic pathway in yeast . Antagonistic action of the repressor Ord1 and activator Yap1 for hypoxic expression of the SRP1/TIR1 gene; Bourdineaud JP et al.; Hypoxic SRP1/TIR1 gene expression depends on the absence of haem but is independent of Rox1-mediated repression . We have found a new hypoxic pathway involving an antagonistic interaction between the Ixr1/Ord1 repressor and the Yap1 factor, a transcriptional activator involved in oxidative stress response . Here, we show that Ord1 repressed SRP1 gene expression under normoxia and hypoxia, whereas Yap1 activated it . Ord1 and Yap1 have been shown to bind the SRP1 promoter in a region extending from -299 to -156 bp upstream of the start codon . A typical AP-1 responsive element lying from -247 to -240 bp allows Yap1 binding . Internal deletion of sequences within the SRP1 promoter were introduced . Two regions were characterized at positions -299/-251 and -218/-156 that, once removed, resulted in a constitutive expression of SRP1 in a wild-type strain under normoxic conditions . Deletion of both these two sequences allowed the bypass of YAP1 requirement in a Deltayap1 strain, whereas these two internal deletions did not yield increased expression in a Deltaord1 strain compared with the full-length promoter . Both a single Deltaord1 mutant and a doubly disrupted Deltayap1 Deltaord1 strain yielded normoxic constitutive SRP1 expression and increased hypoxic SRP1 induction, thereby demonstrating that ord1 is epistatic to yap1 . Thus, Yap1 is not directly involved in SRP1 induction by hypoxia, but is necessary to counteract the Ord1 effect.

Genes Dev, 2000 Dec 1, 14(23), 2962 - 75
The unfolded protein response represses nitrogen-starvation induced developmental differentiation in yeast; Schroder M et al.; Diploid budding yeast exhibits two developmental programs in response to nitrogen starvation, pseudohyphal growth, and sporulation . Here we show that both programs are repressed by activation of the unfolded protein response (UPR), a stress-signal transduction pathway responsible for induction of endoplasmic reticulum (ER)-resident chaperones when protein folding in the ER is impaired . Pseudohyphal growth was derepressed in ire1Delta/ire1Delta and hac1Delta/hac1Delta strains . Activation of the UPR or overexpression of the transcription factor Hac1(i)p, the product of an unconventional splicing reaction regulated by the UPR, was sufficient for repression of pseudohyphal growth and meiosis . HAC1 splicing occurred in a nitrogen-rich environment but ceased rapidly on nitrogen starvation . Further, addition of ammonium salts to nitrogen-starved cells was sufficient to rapidly reactivate HAC1 splicing . We propose that high translation rates in a nitrogen-rich environment are coupled to limited protein unfolding in the ER, thereby activating the UPR . An activated UPR then represses pseudohyphal growth and meiosis . Nitrogen starvation slows translation rates, allowing for more efficient folding of nascent polypeptide chains, down-regulation of the UPR, and subsequent derepression of pseudohyphal growth and meiosis . These findings significantly broaden the range of physiological functions of the UPR and define a role for the UPR in nitrogen sensing.

Curr Biol, 2000 Nov 30, 10(23), 1519 - 22
Yeast Eap1p, an eIF4E-associated protein, has a separate function involving genetic stability; Chial HJ et al.; A rate-limiting step during translation initiation in eukaryotic cells involves binding of the initiation factor eIF4E to the 7-methylguanosine-containing cap of mRNAs . Overexpression of eIF4E leads to malignant transformation {1-3}, and eIF4E is elevated in many human cancers {4-7} . In mammalian cells, three eIF4E-binding proteins each interact with eIF4E and inhibit its function {8-10} . In yeast, EAP1 encodes a protein that binds eIF4E and inhibits cap-dependent translation in vitro {11} . A point mutation in the canonical eIF4E-binding motif of Eap1p blocks its interaction with eIF4E {11} . Here, we characterized the genetic interactions between EAP1 and NDC1, a gene whose function is required for duplication of the spindle pole body (SPB) {12}, the centrosome-equivalent organelle in yeast that functions as the centrosome . We found that the deletion of EAP1 is lethal when combined with the ndc1-1 mutation . Mutations in NDC1 or altered NDC1 gene dosage lead to genetic instability {13,14} . Yeast strains lacking EAP1 also exhibit genetic instability . We tested whether these phenotypes are due to loss of EAP1 function in regulating translation . We found that both the synthetic lethal phenotype and the genetic instability phenotypes are rescued by a mutant allele of EAP1 that is unable to bind eIF4E . Our findings suggest that Eap1p carries out an eIF4E-independent function to maintain genetic stability, most likely involving SPBs.

Curr Biol, 2000 Nov 30, 10(23), 1497 - 506
The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle of budding yeast; Beach DL et al.; BACKGROUND: Two genetic 'pathways' contribute to the fidelity of nuclear segregation during the process of budding in the yeast Saccharomyces cerevisiae . An early pathway, involving Kar9p and other proteins, orients the mitotic spindle along the mother-bud axis . Upon the onset of anaphase, cytoplasmic dynein provides the motive force for nuclear movement into the bud . Loss of either pathway results in nuclear-migration defects; loss of both is lethal . Here, to visualize the functional steps leading to correct spindle orientation along the mother-bud axis, we imaged live yeast cells expressing Kar9p and dynein as green fluorescent protein fusions . RESULTS: Transport of Kar9p into the bud was found to require the myosin Myo2p . Kar9p interacted with microtubules through the microtubule-binding protein Bim1p and facilitated microtubule penetration into the bud . Once microtubules entered the bud, Kar9p provided a platform for microtubule capture at the bud cortex . Kar9p was also observed at sites of microtubule shortening in the bud, suggesting that Kar9p couples microtubule shortening to nuclear migration . CONCLUSIONS: Thus, Kar9p provides a key link between the actin cytoskeleton and microtubules early in the cell cycle . A cooperative mechanism between Kar9p and Myo2p facilitates the pre-anaphase orientation of the spindle . Later, Kar9p couples microtubule disassembly with nuclear migration.

Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14307 - 10
Structural organization of yeast and mammalian mediator complexes; Dotson MR et al.; Structures of yeast Mediator complex, of a related complex from mouse cells and of thyroid hormone receptor-associated protein complex from human cells have been determined by three-dimensional reconstruction from electron micrographs of single particles . All three complexes show a division in two parts, a "head" domain and a combined "middle-tail" domain . The head domains of the three complexes appear most similar and interact most closely with RNA polymerase II . The middle-tail domains show the greatest structural divergence and, in the case of the tail domain, may not interact with polymerase at all . Consistent with this structural divergence, analysis of a yeast Mediator mutant localizes subunits that are not conserved between yeast and mammalian cells to the tail domain . Biochemically defined Rgr1 and Srb4 modules of yeast Mediator are then assigned to the middle and head domains.

Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14548 - 53
Ama1p is a meiosis-specific regulator of the anaphase promoting complex/cyclosome in yeast; Cooper KF et al.; Meiosis is the developmental program by which diploid organisms produce haploid gametes capable of sexual reproduction . Here we describe the yeast gene AMA1, a new member of the Cdc20 protein family that regulates the multisubunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C) . AMA1 is developmentally regulated in that its transcription and splicing occur only in meiotic cells . The meiosis-specific processing of AMA1 mRNA depends on the previously described MER1 splicing factor . Several results indicate that Ama1p is required for APC/C function during meiosis . First, coimmunoprecipitation assays indicate that Ama1p associates with the APC/C in vivo . Second, Ama1p is required for the degradation of the B-type cyclin Clb1p, an APC/C substrate in both meiotic and mitotic cells . Third, ectopic overexpression of AMA1 is able to stimulate ubiquitination of Clb1p in vitro and degradation of Clb1p in vivo . Mutants lacking AMA1 revealed that it is required for the first meiotic division but not the mitotic-like meiosis II . In addition, ama1 mutants are defective for both spore wall assembly and the expression of late meiotic genes . In conclusion, this study indicates that Ama1p directs a meiotic APC/C that functions solely outside mitotic cell division . The requirement of Ama1p only for meiosis I and spore morphogenesis suggests a function for APC/C(Ama1) specifically adapted to germ cell development.

J Cell Sci, 2001 Jan, 114(Pt 1), 207 - 218
Cdc28-Clb mitotic kinase negatively regulates bud site assembly in the budding yeast; Padmashree CG et al.; In the budding yeast Saccharomyces cerevisiae, a prospective mother normally commences the formation of a daughter (the bud) only in the G(1) phase of the cell division cycle . This suggests a strict temporal regulation of the processes that initiate the formation of a new bud . Using cortical localization of bud site components Spa2 and Bni1 as an indicator of bud site assembly, we show that cells assemble a bud site following inactivation of the Cdc28-Clb mitotic kinase but prior to START . Interestingly, an untimely inactivation of the mitotic kinase is sufficient to drive cells to assemble a new bud site inappropriately in G(2) or M phases . The induction of Cdc28/Clb kinase activity in G(1), on the other hand, dramatically reduces a cell's ability to construct an incipient bud site . Our findings strongly suggest that the Cdc28-Clb kinase plays a critical role in the mechanism that restricts the timing of bud formation to the G(1) phase of the cell cycle.

Biochemistry, 2000 Dec 19, 39(50), 15333 - 43
Determinants of allosteric activation of yeast pyruvate kinase and identification of novel effectors using computational screening; Bond CJ et al.; We have analyzed the structural determinants of the allosteric activation of yeast pyruvate kinase (YPK) by mutational and kinetic analysis and initiated a structure-based design project to identify novel effectors that modulate its allosteric response by binding to the allosteric site for fructose-1,6-bisphosphate (FBP) . The wild-type enzyme is strongly activated by fructose-1,6-bisphosphate and weakly activated by both fructose-1-phosphate and fructose-6-phosphate; the strength of the activation response is proportional to the affinity of the allosteric effector . A point mutation within the 6'-phosphate binding loop of the allosteric site (T403E) abolishes activation of the enzyme by fructose-1, 6-bisphosphate . The mutant enzyme is also not activated by F1P or F6P . The mutation alone (which incorporates a glutamic acid that is strictly conserved in mammalian M1 isozymes) slightly reduces cooperativity of substrate binding . Three novel compounds were identified that effect the allosteric regulation of YPK by FBP and/or act as novel allosteric activators of the enzyme . One is a physiologically important diphospho sugar, while the other two are hydrophobic compounds that are dissimilar to the natural effector . These results demonstrate that novel allosteric effectors may be identified using structure-based screening and are indicative of the potential of this strategy for drug discovery . Regulatory sites are generally more divergent than catalytic sites and therefore offer excellent opportunities for discrimination and specificity between different organisms or between different tissue types.

J Biol Chem, 2001 Mar 16, 276(11), 7762 - 8 Epub 2000 Dec 08.
Mitochondrial control of iron homeostasis . A genome wide analysis of gene expression in a yeast frataxin-deficient strain; Foury F et al.; Deletion of YFH1, the yeast frataxin homologue gene, elicits mitochondrial iron accumulation and alters cellular iron homeostasis . Here, we report a genome wide analysis of gene expression in a yfh1(DeltaYFH1) deleted strain . Frataxin deficiency results in enhanced expression of some 70 genes including a set of genes, called the iron regulon, that are under the control of the iron-sensing transcription factor AFT1 . Five new AFT1-dependent genes, YOR382w, YOR383c, YDR534c, YLR136c, and YLR205c were found . The first three genes presumably encode cell-wall glycosylphosphatidylinositol anchor proteins and exhibit a 30-100-fold increased expression . The triple deletion of these genes decreases efficiency in utilization of the iron of ferrioxamine B by the yeast cell . YLR136c bears homology to tristetraproline proteins, which are post-transcriptional regulators in mammalian cells . Deletion of YLR136c increases the mRNA levels of iron regulon members . YLR205c bears homology to heme oxygenases . Our data show that frataxin deficiency elicits iron mobilization from all iron sources in an AFT1-dependent manner . Wild-type and DeltaYFH1 glycerol-grown cells exhibit similar high respiration rates, no mitochondrial iron accumulation, and high expression of the iron regulon, suggesting that under these conditions little iron is extruded from mitochondria . These data suggest that the activity of Yfh1p is not essential in cells grown on glycerol . This study has also revealed unexpected links between mitochondria and remote metabolic pathways since frataxin deficiency also enhances the expression of genes such as HSP30, that escape to AFT1 control . Finally, no oxidative stress gene is induced.

Biochim Biophys Acta, 2000 Dec 15, 1529(1-3), 155 - 63
Conservation of eukaryotic sterol homeostasis: new insights from studies in budding yeast; Sturley SL; The model eukaryote Saccharomyces cerevisiae (budding yeast) has provided significant insight into sterol homeostasis . The study of sterol metabolism in a genetically amenable model organism such as yeast is likely to have an even greater impact and relevance to human disease with the advent of the complete human genome sequence . In addition to definition of the sterol biosynthetic pathway, almost to completion, the remarkable conservation of other components of sterol homeostasis are described in this review.

Science, 2000 Dec 8, 290(5498), 1975 - 8
Multigenerational cortical inheritance of the Rax2 protein in orienting polarity and division in yeast; Chen T et al.; Diploid yeast cells repeatedly polarize and bud from their poles, probably because of highly stable marks of unknown composition . Here, Rax2, a membrane protein, was shown to behave as such a mark . The Rax2 protein itself was inherited immutably at the cell cortex for multiple generations, and Rax2 was shown to have a half-life exceeding several generations . The persistent inheritance of cortical protein markers would provide a means to couple a cell's history to the future development of a precise morphogenetic form.

J Biol Chem, 2001 Mar 16, 276(11), 8314 - 20 Epub 2000 Dec 07.
Nuclear localization of yeast Nfs1p is required for cell survival; Nakai Y et al.; Saccharomyces cerevisiae Nfs1p is mainly found in the mitochondrial matrix and has been shown to participate in iron-sulfur cluster assembly . We show here that Nfs1p contains a potential nuclear localization signal, RRRPR, in its mature part . When this sequence was mutated to RRGSR, the mutant protein could not restore cell growth under chromosomal NFS1-depleted conditions . However, this mutation did not affect the function of Nfs1p in biogenesis of mitochondrial iron-sulfur proteins . The growth defect of the mutant was complemented by simultaneous expression of the mature Nfs1p, which contains the intact nuclear localization signal but lacks its mitochondrial-targeting presequence . These results suggest that a fraction of Nfs1p is localized in the nucleus and is essential for cell viability.

Mol Cell, 2000 Nov, 6(5), 1195 - 205
Crystal structure of yeast Esa1 suggests a unified mechanism for catalysis and substrate binding by histone acetyltransferases; Yan Y et al.; Esa1 is the catalytic subunit of the NuA4 histone acetylase (HAT) complex that acetylates histone H4, and it is a member of the MYST family of HAT proteins that includes the MOZ oncoprotein and the HIV-1 Tat interacting protein Tip60 . Here we report the X-ray crystal structure of the HAT domain of Esa1 bound to coenzyme A and investigate the protein's catalytic mechanism . Our data reveal that Esa1 contains a central core domain harboring a putative catalytic base, and flanking domains that are implicated in histone binding . Comparisons with the Gcn5/PCAF and Hat1 proteins suggest a unified mechanism of catalysis and histone binding by HAT proteins, whereby a structurally conserved core domain mediates catalysis, and sequence variability within a structurally related N- and C-terminal scaffold determines substrate specificity.

RNA, 2000 Nov, 6(11), 1660 - 71
Yeast Rrp9p is an evolutionarily conserved U3 snoRNP protein essential for early pre-rRNA processing cleavages and requires box C for its association; Venema J et al.; Pre-rRNA processing in eukaryotic cells requires participation of several snoRNPs . These include the highly conserved and abundant U3 snoRNP, which is essential for synthesis of 18S rRNA . Here we report the characterization of Rrp9p, a novel yeast U3 protein, identified via its homology to the human U3-55k protein . Epitope-tagged Rrp9p specifically precipitates U3 snoRNA, but Rrp9p is not required for the stable accumulation of this snoRNA . Genetic depletion of Rrp9p inhibits the early cleavages of the primary pre-rRNA transcript at A0, A1, and A2 and, consequently, production of 18S, but not 25S and 5.8S, rRNA . The hU3-55k protein can partially complement a yeast rrp9 null mutant, indicating that the function of this protein has been conserved . Immunoprecipitation of extracts from cells that coexpress epitope-tagged Rrp9p and various mutant forms of U3 snoRNA limits the region required for association of Rrp9p to the U3-specific box B/C motif . Box C is essential, whereas box B plays a supportive role.

Genome Biol . 2000;1(1):REVIEWS103 . Epub 2000 Apr 27.
A cultivated taste for yeast; Brenner C; The availability of complete genomic sequences of Saccharomyces cerevisiae has catalyzed a cultural change in the practice of yeast biology, providing opportunities to develop high throughput techniques to define protein function, to define drug targets, and to discover and characterize drugs.

J Biol Chem, 2001 Mar 9, 276(10), 7114 - 21 Epub 2000 Dec 04.
Characterization of phosphatidylserine transport to the locus of phosphatidylserine decarboxylase 2 in permeabilized yeast; Wu WI et al.; In yeast, nascent phosphatidylserine (PtdSer) can be transported to the mitochondria and Golgi/vacuole for decarboxylation to synthesize phosphatidylethanolamine (PtdEtn) . In strains with a psd1Delta allele for the mitochondrial PtdSer decarboxylase, the conversion of nascent PtdSer to PtdEtn can serve as an indicator of lipid transport to the locus of PtdSer decarboxylase 2 (Psd2p) in the Golgi/vacuole . We have followed the metabolism of {(3)H}serine into PtdSer and PtdEtn to study lipid transport in permeabilized psd1Delta yeast . The permeabilized cells synthesize (3)H-PtdSer and, after a 20-min lag, decarboxylate it to form {(3)H}PtdEtn . Formation of {(3)H}PtdEtn is linear between 20 and 100 min of incubation and does not require ongoing PtdSer synthesis . PtdSer transport can be resolved into a two-component system using washed, permeabilized psd1Delta cells as donors and membranes isolated by ultracentrifugation as acceptors . With this system, the transport-dependent decarboxylation of nascent PtdSer is dependent upon the concentration of acceptor membranes, requires Mn(2+) but not nucleotides, and is inhibited by EDTA . High speed membranes isolated from a previously identified PtdSer transport mutant, pstB2, contain normal Psd2p activity but fail to reconstitute PtdSer transport and decarboxylation . Reconstitution with permutations of wild type and pstB2Delta donors and acceptors identifies the site of the mutant defect as the acceptor side of the transport reaction.

Mol Biol Cell, 2000 Dec, 11(12), 4241 - 57
Genomic expression programs in the response of yeast cells to environmental changes; Gasch AP et al.; We explored genomic expression patterns in the yeast Saccharomyces cerevisiae responding to diverse environmental transitions . DNA microarrays were used to measure changes in transcript levels over time for almost every yeast gene, as cells responded to temperature shocks, hydrogen peroxide, the superoxide-generating drug menadione, the sulfhydryl-oxidizing agent diamide, the disulfide-reducing agent dithiothreitol, hyper- and hypo-osmotic shock, amino acid starvation, nitrogen source depletion, and progression into stationary phase . A large set of genes (approximately 900) showed a similar drastic response to almost all of these environmental changes . Additional features of the genomic responses were specialized for specific conditions . Promoter analysis and subsequent characterization of the responses of mutant strains implicated the transcription factors Yap1p, as well as Msn2p and Msn4p, in mediating specific features of the transcriptional response, while the identification of novel sequence elements provided clues to novel regulators . Physiological themes in the genomic responses to specific environmental stresses provided insights into the effects of those stresses on the cell.

Mol Biol Cell, 2000 Dec, 11(12), 4105 - 16
Human orthologs of yeast vacuolar protein sorting proteins Vps26, 29, and 35: assembly into multimeric complexes; Haft CR et al.; Sorting nexin (SNX) 1 and SNX2 are mammalian orthologs of Vps5p, a yeast protein that is a subunit of a large multimeric complex, termed the retromer complex, involved in retrograde transport of proteins from endosomes to the trans-Golgi network . We report the cloning and characterization of human orthologs of three additional components of the complex: Vps26p, Vps29p, and Vps35p . The close structural similarity between the yeast and human proteins suggests a similarity in function . We used both yeast two-hybrid assays and expression in mammalian cells to define the binding interactions among these proteins . The data suggest a model in which hVps35 serves as the core of a multimeric complex by binding directly to hVps26, hVps29, and SNX1 . Deletional analyses of hVps35 demonstrate that amino acid residues 1-53 and 307-796 of hVps35 bind to the coiled coil-containing domain of SNX1 . In contrast, hVps26 binds to amino acid residues 1-172 of hVps35, whereas hVps29 binds to amino acid residues 307-796 of hVps35 . Furthermore, hVps35, hVps29, and hVps26 have been found in membrane-associated and cytosolic compartments . Gel filtration chromatography of COS7 cell cytosol showed that both recombinant and endogenous hVps35, hVps29, and hVps26 coelute as a large complex ( approximately 220-440 kDa) . In the absence of hVps35, neither hVps26 nor hVps29 is found in the large complex . These data provide the first insights into the binding interactions among subunits of a putative mammalian retromer complex.

Genetics, 2000 Dec, 156(4), 1727 - 36
The oxen gene of Drosophila encodes a homolog of subunit 9 of yeast ubiquinol-cytochrome c oxidoreductase complex: evidence for modulation of gene expression in response to mitochondrial activity; Frolov MV et al.; A P-element insertion in the oxen gene, ox(1), has been isolated in a search for modifiers of white gene expression . The mutation preferentially exerts a negative dosage effect upon the expression of three genes encoding ABC transporters involved in pigment precursor transport, white, brown, and scarlet . A precise excision of the P element reverts the mutant phenotype . Five different transcription units were identified around the insertion site . To distinguish a transcript responsible for the mutant phenotype, a set of deletions within the oxen region was generated . Analysis of gene expression within the oxen region in the case of deletions as well as generation of transgenic flies allowed us to identify the transcript responsible for oxen function . It encodes a 6.6-kD homolog of mitochondrial ubiquinol cytochrome c oxidoreductase (QCR9), subunit 9 of the bc(1) complex in yeast . In addition to white, brown, and scarlet, oxen regulates the expression of three of seven tested genes . Thus, our data provide additional evidence for a cellular response to changes in mitochondrial function . The oxen mutation provides a model for the genetic analysis in multicellular organisms of the effect of mitochondrial activity on nuclear gene expression.

Nat Struct Biol, 2000 Dec, 7(12), 1165 - 71
The structure and oligomerization of the yeast arginine methyltransferase, Hmt1; Weiss VH et al.; Protein methylation at arginines is ubiquitous in eukaryotes and affects signal transduction, gene expression and protein sorting . Hmt1/Rmt1, the major arginine methyltransferase in yeast, catalyzes methylation of arginine residues in several mRNA-binding proteins and facilitates their export from the nucleus . We now report the crystal structure of Hmt1 at 2.9 A resolution . Hmt1 forms a hexamer with approximate 32 symmetry . The surface of the oligomer is dominated by large acidic cavities at the dimer interfaces . Mutation of dimer contact sites eliminates activity of Hmt1 both in vivo and in vitro . Mutating residues in the acidic cavity significantly reduces binding and methylation of the substrate Npl3.

Nat Biotechnol, 2000 Dec, 18(12), 1257 - 61
A network of protein-protein interactions in yeast; Schwikowski B et al.; A global analysis of 2,709 published interactions between proteins of the yeast Saccharomyces cerevisiae has been performed, enabling the establishment of a single large network of 2,358 interactions among 1,548 proteins . Proteins of known function and cellular location tend to cluster together, with 63% of the interactions occurring between proteins with a common functional assignment and 76% occurring between proteins found in the same subcellular compartment . Possible functions can be assigned to a protein based on the known functions of its interacting partners . This approach correctly predicts a functional category for 72% of the 1,393 characterized proteins with at least one partner of known function, and has been applied to predict functions for 364 previously uncharacterized proteins.

EMBO J, 2000 Dec 1, 19(23), 6602 - 11
The Puf3 protein is a transcript-specific regulator of mRNA degradation in yeast; Olivas W et al.; Eukaryotic post-transcriptional regulation is often specified by control elements within mRNA 3'- untranslated regions (3'-UTRs) . In order to identify proteins that regulate specific mRNA decay rates in Saccharomyces cerevisae, we analyzed the role of five members of the Puf family present in the yeast genome (referred to as JSN1/PUF1, PUF2, PUF3, PUF4 and MPT5/PUF5) . Yeast strains lacking all five Puf proteins showed differential expression of numerous yeast mRNAs . Examination of COX17 mRNA indicates that Puf3p specifically promotes decay of this mRNA by enhancing the rate of deadenylation and subsequent turnover . Puf3p also binds to the COX17 mRNA 3'-UTR in vitro . This indicates that the function of Puf proteins as specific regulators of mRNA deadenylation has been conserved throughout eukaryotes . In contrast to the case in Caenorhabditis elegans and Drosophila, yeast Puf3p does not affect translation of COX17 mRNA . These observations indicate that Puf proteins are likely to play a role in the control of transcript-specific rates of degradation in yeast by interacting directly with the mRNA turnover machinery.

Nature, 2000 Nov 23, 408(6811), 495 - 8
Global histone acetylation and deacetylation in yeast; Vogelauer M et al.; Histone acetyltransferases and deacetylases can be targeted to promoters to activate or repress genes . For example, the histone acetyltransferase GCN5 is part of a yeast multiprotein complex that is recruited by the DNA-binding activator protein GCN4 (refs 1-3) . The histone deacetylase RPD3 complex is recruited to DNA by the repressor UME6 (refs 4, 5); similar mechanisms exist in other eukaryotes . However, deletion of RPD3 also increases expression of the PHO5 gene that is repressed by nucleosomes, and regulated by GCN5 (ref . 10) but not by UME6 . We have determined whether acetylation and deacetylation are promoter specific at PHO5, by using antibodies against acetylated lysine residues and chromatin immunoprecipitation to examine the acetylation state of a 4.25-kilobase region surrounding the PHO5 gene . Here we show that this region is acetylated extensively by ESA1 and GCN5 and deacetylated by HDA1 and RPD3, and that widespread histone modification affects three separate chromosomal regions examined, which total 22kb . Our data indicate that targeted modification occurs in a background of global acetylation and deacetylation that not only reduces basal transcription, but also allows a rapid return to the initial state of acetylation when targeting is removed.

J Biol Chem, 2001 Mar 9, 276(10), 7593 - 601 Epub 2000 Nov 30.
Effects of anoxia and the mitochondrion on expression of aerobic nuclear COX genes in yeast: evidence for a signaling pathway from the mitochondrial genome to the nucleus; Dagsgaard C et al.; Eucaryotic cells contain at least two general classes of oxygen-regulated nuclear genes: aerobic genes and hypoxic genes . Hypoxic genes are induced upon exposure to anoxia while aerobic genes are down-regulated . Recently, it has been reported that induction of some hypoxic nuclear genes in mammals and yeast requires mitochondrial respiration and that cytochrome-c oxidase functions as an oxygen sensor during this process . In this study, we have examined the role of the mitochondrion and cytochrome-c oxidase in the expression of yeast aerobic nuclear COX genes . We have found that the down-regulation of these genes in anoxic cells is reflected in reduced levels of their subunit polypeptides and that cytochrome-c oxidase subunits I, II, III, Vb, VI, VII, and VIIa are present in promitochondria from anoxic cells . By using nuclear cox mutants and mitochondrial rho(0) and mit(-) mutants, we have found that neither respiration nor cytochrome-c oxidase is required for the down-regulation of these genes in cells exposed to anoxia but that a mitochondrial genome is required for their full expression under both normoxic and anoxic conditions . This requirement for a mitochondrial genome is unrelated to the presence or absence of a functional holocytochrome-c oxidase . We have also found that the down-regulation of these genes in cells exposed to anoxia and the down-regulation that results from the absence of a mitochondrial genome are independent of one another . These findings indicate that the mitochondrial genome, acting independently of respiration and oxidative phosphorylation, affects the expression of the aerobic nuclear COX genes and suggest the existence of a signaling pathway from the mitochondrial genome to the nucleus.

Proc Natl Acad Sci U S A, 2000 Dec 5, 97(25), 13708 - 13
Genomewide studies of histone deacetylase function in yeast; Bernstein BE et al.; The trichostatin A (TSA)-sensitive histone deacetylase (HDAC) Rpd3p exists in a complex with Sin3p and Sap30p in yeast that is recruited to target promoters by transcription factors including Ume6p . Sir2p is a TSA-resistant HDAC that mediates yeast silencing . The transcription profile of rpd3 is similar to the profiles of sin3, sap30, ume6, and TSA-treated wild-type yeast . A Ume6p-binding site was identified in the promoters of genes up-regulated in the sin3 strain . Two genes appear to participate in feedback loops that modulate HDAC activity: ZRT1 encodes a zinc transporter and is repressed by RPD3 (Rpd3p is zinc-dependent); BNA1 encodes a nicotinamide adenine dinucleotide (NAD)-biosynthesis enzyme and is repressed by SIR2 (Sir2p is NAD-dependent) . Although HDACs are transcriptional repressors, deletion of RPD3 down-regulates certain genes . Many of these are down-regulated rapidly by TSA, indicating that Rpd3p may also activate transcription . Deletion of RPD3 previously has been shown to repress ("silence") reporter genes inserted near telomeres . The profiles demonstrate that 40% of endogenous genes located within 20 kb of telomeres are down-regulated by RPD3 deletion . Rpd3p appears to activate telomeric genes sensitive to histone depletion indirectly by repressing transcription of histone genes . Rpd3p also appears to activate telomeric genes repressed by the silent information regulator (SIR) proteins directly, possibly by deacetylating lysine 12 of histone H4 . Finally, bioinformatic analyses indicate that the yeast HDACs RPD3, SIR2, and HDA1 play distinct roles in regulating genes involved in cell cycle progression, amino acid biosynthesis, and carbohydrate transport and utilization, respectively.

Proc Natl Acad Sci U S A, 2000 Dec 5, 97(25), 13643 - 8
Active recycling of yeast Golgi mannosyltransferase complexes through the endoplasmic reticulum; Todorow Z et al.; Mnn9p is a component of two distinct multiprotein complexes in the Saccharomyces cerevisiae cis-Golgi that have both been shown to have alpha-1,6-mannosyltransferase activity in vitro . In one of these complexes, Mnn9p associates with four other membrane proteins, Anp1p, Mnn10p, Mnn11p, and Hoc1p, whereas the other complex consists of Mnn9p and Van1p . Members of the Mnn9p-containing complexes were incorporated into COPII vesicles made in vitro from endoplasmic reticulum (ER) membranes isolated from cycloheximide-treated cells . This behavior is consistent with an active Golgi to ER recycling process . To examine this path in vivo, we monitored retrograde transport of subunits of the complex in cells blocked in anterograde transport from the ER . In this situation, specific relocation of the proteins from the Golgi to the ER was observed in the absence of new protein synthesis . Conversely, when retrograde transport was blocked in vivo, subunits of the mannosyltransferase complex accumulated in the vacuole . Packaging of Mnn9p in COPI-coated vesicles from purified Golgi membranes was also investigated using a coatomer-dependent vesicle budding assay . Gradient fractionation experiments showed that Mnn9p and the retrograde v-SNARE, Sec22p, were incorporated into COPI-coated vesicles . These observations indicate that the Mnn9p-containing mannosyltransferase complexes cycle back and forth between the ER and Golgi.

FEMS Microbiol Lett, 2000 Dec 1, 193(1), 171 - 7
DNA probes specific for the yeast species Debaryomyces hansenii: useful tools for rapid identification; Corredor M et al.; We developed a rapid and sensitive identification method for the halotolerant yeast Debaryomyces hansenii, based on the hybridization of species-specific sequences . These sequences were first identified in a survey of D . hansenii strains by random amplification of polymorphic DNA (RAPD) as giving conserved bands in all isolates tested . Two such conserved RAPD products, termed F01pro and M18pro, were cloned from the type strain CBS 767 . The specificity of these probes was assessed by hybridizing them to DNA from various species of yeasts commonly found in cheese . F01pro and M18pro hybridized to the DNA of all D . hansenii var . hansenii tested, but not to DNA of other yeast species including the closely related strain of D . hansenii var . fabryii CBS 789 . Hybridization patterns of F01pro and M18pro on digested genomic DNA of D . hansenii indicated that the sequences were repeated in the genome of all D . hansenii var . hansenii tested, and gave distinct polymorphic patterns . The single F01pro probe generated 11 different profiles for 24 strains by restriction fragment length polymorphism, using one restriction enzyme . F01pro represents a new type of repeated element found in fungi, useful for both identification and typing of D . hansenii and, together with M18pro, simplifies the study of this species in complex flora.

Mol Cell Biol, 2000 Dec, 20(24), 9376 - 90
The yeast inositol polyphosphate 5-phosphatases inp52p and inp53p translocate to actin patches following hyperosmotic stress: mechanism for regulating phosphatidylinositol 4,5-bisphosphate at plasma membrane invaginations; Ooms LM et al.; The Saccharomyces cerevisiae inositol polyphosphate 5-phosphatases (Inp51p, Inp52p, and Inp53p) each contain an N-terminal Sac1 domain, followed by a 5-phosphatase domain and a C-terminal proline-rich domain . Disruption of any two of these 5-phosphatases results in abnormal vacuolar and plasma membrane morphology . We have cloned and characterized the Sac1-containing 5-phosphatases Inp52p and Inp53p . Purified recombinant Inp52p lacking the Sac1 domain hydrolyzed phosphatidylinositol 4,5-bisphosphate {PtdIns(4,5)P(2)} and PtdIns(3, 5)P(2) . Inp52p and Inp53p were expressed in yeast as N-terminal fusion proteins with green fluorescent protein (GFP) . In resting cells recombinant GFP-tagged 5-phosphatases were expressed diffusely throughout the cell but were excluded from the nucleus . Following hyperosmotic stress the GFP-tagged 5-phosphatases rapidly and transiently associated with actin patches, independent of actin, in both the mother and daughter cells of budding yeast as demonstrated by colocalization with rhodamine phalloidin . Both the Sac1 domain and proline-rich domains were able to independently mediate translocation of Inp52p to actin patches, following hyperosmotic stress, while the Inp53p proline-rich domain alone was sufficient for stress-mediated localization . Overexpression of Inp52p or Inp53p, but not catalytically inactive Inp52p, which lacked PtdIns(4,5)P(2) 5-phosphatase activity, resulted in a dramatic reduction in the repolarization time of actin patches following hyperosmotic stress . We propose that the osmotic-stress-induced translocation of Inp52p and Inp53p results in the localized regulation of PtdIns(3,5)P(2) and PtdIns(4,5)P(2) at actin patches and associated plasma membrane invaginations . This may provide a mechanism for regulating actin polymerization and cell growth as an acute adaptive response to hyperosmotic stress.

Mol Cell Biol, 2000 Dec, 20(24), 9307 - 16
The essential interaction between yeast mRNA capping enzyme subunits is not required for triphosphatase function in vivo; Takase Y et al.; The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5'-triphosphatase (Cet1) and an mRNA guanylyltransferase (Ceg1) . In yeast, the capping enzyme is recruited to the RNA polymerase II (Pol II) transcription complex via an interaction between Ceg1 and the phosphorylated carboxy-terminal domain of the Pol II largest subunit . Previous in vitro experiments showed that the Cet1 carboxy-terminal region (amino acids 265 to 549) carries RNA triphosphatase activity, while the region containing amino acids 205 to 265 of Cet1 has two functions: it mediates dimerization with Ceg1, but it also allosterically activates Ceg1 guanylyltransferase activity in the context of Pol II binding . Here we characterize several Cet1 mutants in vivo . Mutations or deletions of Cet1 that disrupt interaction with Ceg1 are lethal, showing that this interaction is essential for proper capping enzyme function in vivo . Remarkably, the interaction region of Ceg1 becomes completely dispensable when Ceg1 is substituted by the mouse guanylyltransferase, which does not require allosteric activation by Cet1 . Although no interaction between Cet1 and mouse guanylyltransferase is detectable, both proteins are present at yeast promoters in vivo . These results strongly suggest that the primary physiological role of the Ceg1-Cet1 interaction is to allosterically activate Ceg1, rather than to recruit Cet1 to the Pol II complex.

J Biol Chem, 2001 Mar 2, 276(9), 6789 - 96 Epub 2000 Nov 28.
Identification of a nuclear gene (FMC1) required for the assembly/stability of yeast mitochondrial F(1)-ATPase in heat stress conditions; Lefebvre-Legendre L et al.; We have identified a yeast nuclear gene (FMC1) that is required at elevated temperatures (37 degrees C) for the formation/stability of the F(1) sector of the mitochondrial ATP synthase . Western blot analysis showed that Fmc1p is a soluble protein located in the mitochondrial matrix . At elevated temperatures in yeast cells lacking Fmc1p, the alpha-F(1) and beta-F(1) proteins are synthesized, transported, and processed to their mature size . However, instead of being incorporated into a functional F(1) oligomer, they form large aggregates in the mitochondrial matrix . Identical perturbations were reported previously for yeast cells lacking either Atp12p or Atp11p, two specific assembly factors of the F(1) sector (Ackerman, S . H., and Tzagoloff, A . (1990) Proc . Natl . Acad . Sci . U . S . A . 87, 4986--4990), and we show that the absence of Fmc1p can be efficiently compensated for by increasing the expression of Atp12p . However, unlike Atp12p and Atp11p, Fmc1p is not required in normal growth conditions (28--30 degrees C) . We propose that Fmc1p is required for the proper folding/stability or functioning of Atp12p in heat stress conditions.

J Biol Chem, 2001 Feb 23, 276(8), 6016 - 22 Epub 2000 Nov 28.
Rvs161p and Rvs167p, the two yeast amphiphysin homologs, function together in vivo; Lombardi R et al.; Mutations in RVS161 and RVS167, the two yeast amphiphysin homologs, cause very similar growth phenotypes, a depolarized actin cytoskeleton, and a defect in the internalization step of endocytosis . Rvs161p and Rvs167p have been shown to interact in the two-hybrid system, but their localization in the cell may be different thus raising the question whether the interaction is physiologically relevant . Here we demonstrate that the two proteins function together in vivo . We find that the steady state level of Rvs167p is strongly reduced in an rvs161Delta strain . Similarly, the level of Rvs161p is strongly reduced in an rvs167Delta strain . We demonstrate that these reduced protein levels at steady state are due to a decreased stability of either Rvs protein in the absence of the other protein . Furthermore, we find that the amount and ratio of Rvs161p and Rvs167p are critical parameters for receptor-mediated endocytosis . In addition, by using the two-hybrid system we show that the interaction of Rvs167p with actin is not abolished in an abp1Delta strain suggesting that Abp1p is not essential for this interaction.

Biochemistry (Mosc), 2000 Oct, 65(10), 1206 - 11
On the effect of cholesterol on the fate of CYP11A1 imported into yeast mitochondria in vivo; Kovaleva IE et al.; It has earlier been shown that CYP11A1 (cytochrome P450scc precursor), synthesized in yeast cells, is imported into yeast mitochondria . However, in large part the foreign protein undergoes degradation or aggregates . In this work, we tried to prevent aggregation of CYP11A1 and stimulate its insertion into the mitochondrial inner membrane by substituting cholesterol (a substrate for cytochrome P450scc) for ergosterol in yeast cells . To this end, an ergosterol-deficient Saccharomyces cerevisiae mutant, growing in the presence of cholesterol and expressing a modified bovine CYP11A1 gene, was used . Under defined conditions, the mitochondrial respiratory system developed in this yeast and CYP11A1 with the CoxIV targeting presequence was imported into the mitochondria, being then proteolytically processed . However, substitution of cholesterol for ergosterol did not result in lowered aggregation of the imported CYP11A1 and its increased content in the SMP fraction . Hence, the presence of cholesterol is not instrumental in proper intramitochondrial compartmentalization and folding of CYP11A1.

Biochemistry (Mosc), 2000 Oct, 65(10), 1167 - 74
Ca2+-release pathways from mitochondria of the yeast Endomyces magnusii; Deryabina YI et al.; Ca2+-release pathways from Ca2+-preloaded mitochondria of the yeast Endomyces magnusii were studied . In the presence of phosphate as a permeant anion, Ca2+ was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis . Intensive aeration of the mitochondrial suspension rapidly inhibited the efflux of Ca2+ and induced its reuptake . The Ca2+ release was not affected by cyclosporin A, an inhibitor of the nonselective permeability transition of mammalian mitochondria . With acetate as the permeant anion, a spontaneous net Ca2+ efflux began after uptake of about 75% of the added cation . The rate of this efflux was insensitive to cyclosporin A, aeration, and Na+ and was proportional to the Ca2+ load . The Ca2+ release was inhibited by La3+, Mn2+, Mg2+, TPP+, and nigericin (in the presence of KCl) and activated by spermine and hypotonicity . We conclude that Ca2+ efflux from preloaded E . magnusii mitochondria is very similar to the Na+-independent specific pathway for Ca2+ release operative in mitochondria from nonexcitable mammalian tissues.

Biochem Genet, 2000 Jun, 38(5-6), 181 - 200
Functional correlation in amino acid residue mutations of yeast iso-2-cytochrome c that is consistent with the prediction of the concomitantly variable codon theory in cytochrome c evolution; Fisher A et al.; Fitch and Markowitz' theory of concomitantly variable codons (covarions) in evolution predicted the existence of functional correlation in amino acid residue mutations among present-day cytochromes c . Mutational analysis was carried out on yeast iso-2-cytochrome c, where hydrophobic core residues I20, M64, L85, and M98 and surface residue L9 were mutated, in selected combinations, to those found in mammalian and bird cytochromes c . The functionality assay is based upon the ability of yeast cells to grow in YPGE medium . Furthermore, experiments on the single M64L and M98L mutations as well as the double M64L/M98L mutation using NMR showed that the effects of these mutations are to perturb the structural integrity of the protein . We identified functional correlation in two cases of a pair of residue mutations, the I20-->V and M98-->L pair and the L9-->I and L85-->I pair . In both cases, only one of the two alternative, putative evolutionary pathways leads to a functional protein and the corresponding pairs of residue mutations are among those found in present-day cytochromes c . Since valine is predicted to be at position 20 in the ancestral form of cytochrome c, the present data provide an explanation for the ancient requirement of leucine rather than methionine in position 98 . The present data provide further evidence for the role of those specific atom-atom interactions in directing a pathway in the evolutionary changes of the amino acid sequence that have taken place in cytochrome c, in accordance with Fitch and Markowitz.

Mol Cell, 2000 Oct, 6(4), 947 - 52
Telomerase-independent lengthening of yeast telomeres occurs by an abrupt Rad50p-dependent, Rif-inhibited recombinational process; Teng SC et al.; Type II survivors arise in Saccharomyces cells lacking telomerase by a recombinational pathway that results in very long and heterogeneous length telomeres . Here we show that type II telomeres appeared abruptly in a population of cells with very short telomeres . Once established, these long telomeres progressively shortened . Short telomeres were substrates for rare, one-step lengthening events . The generation of type II survivors was absolutely Rad50p dependent . In a telomerase-proficient cell, the telomere-binding Rif proteins inhibited telomerase lengthening of telomeres . In a telomerase-deficient strain, Rif proteins, especially Rif2p, inhibited type II recombination . These data argue that only short telomeres are substrates for type II recombination and suggest that the donor for this recombination is not a chromosomal telomere.

Nature, 2000 Nov 9, 408(6809), 221 - 5
PCNA connects DNA replication to epigenetic inheritance in yeast; Zhang Z et al.; Formation of a heterochromatin-like structure results in transcriptional silencing at the HM mating-type loci and telomeres in Saccharomyces cerevisiae . Once formed, such epigenetically determined structures are inherited for many mitotic divisions . Here we show that mutations in the proliferating cell nuclear antigen (PCNA), an essential component at the DNA replication fork, reduced repression of genes near a telomere and at the silent mating-typelocus, HMR . The pol30-8 mutant displayed coexistence of both repressed (pink) and de-repressed (white) cells within a single colony when assayed with the ADE2 gene inserted at HMR . Unlike pol30-8, the pol30-6 and pol30-79 mutants partially reduced gene silencing at telomeres and the HMR and synergistically decreased silencing in cells lacking chromatin assembly factor 1 (CAF-1) . All silencing defective mutants showed reduced binding to CAF-1 in vitro and altered chromatin association of the CAF-1 large subunit in vivo . Thus, PCNA participates in inheritance of both DNA and epigenetic chromatin structures during the S phase of the cell cycle, the latter by at least two mechanisms.

Curr Opin Genet Dev, 2000 Dec, 10(6), 617 - 23
The yeast genome: on the road to the Golden Age; Johnston M; Having the complete genome sequence of Saccharomyces cerevisiae makes us aware of the ultimate goal of yeast molecular biology: the 'solution' of the cell, that is, an understanding of the function of all approximately 6000 proteins (and a few RNAs) and how they interact with each other and the environment . The recent development of 'genomic' approaches for studying gene function makes this goal seem reachable in the foreseeable future . When this is accomplished, we will have entered a Golden Age, when we will have the information necessary for designing truly incisive experiments to reveal biological function.

Biochim Biophys Acta, 2000 Nov 30, 1543(1), 174 - 88
Isolation and physico-chemical characterization of a cytochrome c from the methylotrophic yeast Hansenula polymorpha; Borsari M et al.; Cytochrome c from the methylotrophic yeast Hansenula polymorpha was isolated and purified to homogeneity for the first time . The final yield of the highly purified protein from 1.4 kg (wet weight) cells was about 20 mg . The hemoprotein has an apparent molecular mass of 12 kDa and isoelectric point (pI) of 9.3 . The purified protein was characterized by electronic, EPR and NMR spectroscopies . The redox potential of the cytochrome, E degrees, measured by cyclic voltammetry measurements at neutral pH, is 0.302 V . Both NMR spectroscopy and electrochemical measurements confirm the presence in the solution of several acid-base equilibria, the most pronounced being characterized by a pK(a) of 8.3 . The latter pK(a) was attributed to the detachment of the iron(III) ion-coordinated methionine and its replacement by a lysine residue . The electrochemically derived thermodynamic parameters for neutral and alkaline protein species (DeltaS degrees (rc) and DeltaH degrees (rc)) were obtained from the temperature dependence of the redox potential.

Proc Natl Acad Sci U S A, 2000 Nov 21, 97(24), 13080 - 5
Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase; Caruthers JM et al.; The eukaryotic translation initiation factor 4A (eIF4A) is a member of the DEA(D/H)-box RNA helicase family, a diverse group of proteins that couples an ATPase activity to RNA binding and unwinding . Previous work has provided the structure of the amino-terminal, ATP-binding domain of eIF4A . Extending those results, we have solved the structure of the carboxyl-terminal domain of eIF4A with data to 1.75 A resolution; it has a parallel alpha-beta topology that superimposes, with minor variations, on the structures and conserved motifs of the equivalent domain in other, distantly related helicases . Using data to 2.8 A resolution and molecular replacement with the refined model of the carboxyl-terminal domain, we have completed the structure of full-length eIF4A; it is a "dumbbell" structure consisting of two compact domains connected by an extended linker . By using the structures of other helicases as a template, compact structures can be modeled for eIF4A that suggest (i) helicase motif IV binds RNA; (ii) Arg-298, which is conserved in the DEA(D/H)-box RNA helicase family but is absent from many other helicases, also binds RNA; and (iii) motifs V and VI "link" the carboxyl-terminal domain to the amino-terminal domain through interactions with ATP and the DEA(D/H) motif, providing a mechanism for coupling ATP binding and hydrolysis with conformational changes that modulate RNA binding.

Protein Expr Purif, 2000 Dec, 20(3), 485 - 91
Production of full-length human pre-elafin, an elastase specific inhibitor, from yeast requires the absence of a functional yapsin 1 (Yps1p) endoprotease; Bourbonnais Y et al.; Pre-elafin, also known as trappin-2, is an elastase-specific inhibitor that belongs to the trappin gene family . A chimeric gene encoding polyhistidine-tagged human pre-elafin fused to the yeast alpha-factor precursor was expressed in Saccharomyces cerevisiae . The chimera was engineered to keep a single copy of the mature alpha-factor peptide . This enabled the use of a simple bioassay (mating assay) to assess the relative efficiency of both the expression and the secretion of the recombinant molecule . We found that pre-elafin is processed both in vivo and in vitro by yapsin 1, the yeast aspartyl endoprotease encoded by YPS1 . Cleavage by yapsin 1 occurred C-terminal to a subset of single lysine residues . Expression in a yapsin 1-deficient yeast strain was an indispensable condition to allow the efficient production of full-length human pre-elafin . The recombinant inhibitor was purified from concentrated culture medium by ammonium sulfate precipitation, affinity purification on a Ni(2+) resin, and cation exchange chromatography . Recombinant human pre-elafin was fully active and showed the same inhibitory profile toward different serine proteases to that reported for mature elafin .

Pharm Res, 2000 Sep, 17(9), 1049 - 55
The yeast two-hybrid system and its pharmaceutical significance; Topcu Z et al.; The detected phenotypes in many diseases are caused from dysfunction in protein-protein, protein-DNA and receptor-ligand interactions . Therefore, determination of these molecular interactions followed by designing or screening the compounds to target these interactions provides a significant challenge in drug development . This review aims to highlight the yeast two-hybrid system in determination of protein-protein interactions and its possible outcomes in pharmaceutical research . The variations of the basic methodology as one- and three-hybrid systems are also discussed in relation to their potential pharmaceutical applications.

FEBS Lett, 2000 Nov 17, 485(1), 19 - 24
Mitochondrial copper metabolism in yeast: interaction between Sco1p and Cox2p; Lode A et al.; Yeast mitochondrial Sco1p is required for the formation of a functional cytochrome c oxidase (COX) . It was suggested that Sco1p aids copper delivery to the catalytic center of COX . Here we show by affinity chromatography and coimmunoprecipitation that Sco1p interacts with subunit Cox2p . In addition we provide evidence that Sco1p can form homomeric complexes . Both homomer formation and binding of Cox2p are neither dependent on the presence of copper nor affected by mutations of His-239, Cys-148 or Cys-152 . These amino acids, which are conserved among the members of the Sco1p family, have been suggested to act in the reduction of the cysteines in the copper binding center of Cox2p and are discussed as ligands for copper.

Biochem J, 2000 Dec 1, 352 Pt 2, 593 - 9
Glucose and the ATP paradox in yeast; Somsen OJ et al.; A sustained decrease in the intracellular ATP concentration has been observed when extra glucose was added to yeast cells growing aerobically under glucose limitation . Because glucose degradation is the main source of ATP-derived free energy, this is a counter-intuitive phenomenon, which cannot be attributed to transient ATP consumption in the initial steps of glycolysis . We present a core model for aerobic growth in which glucose supplies carbon, as well as free energy, for biosynthesis . With Metabolic Control Analysis and numerical simulations, we demonstrate that the decrease in the ATP concentration can be reproduced if the biosynthetic route is more strongly activated by carbon substrates than is the catabolic (ATP-producing) route.

J Biol Chem, 2001 Jan 19, 276(3), 2083 - 7 Epub 2000 Nov 20.
Degradation of lipid vesicles in the yeast vacuole requires function of Cvt17, a putative lipase; Teter SA et al.; The vacuole/lysosome serves an essential role in allowing cellular components to be degraded and recycled under starvation conditions . Vacuolar hydrolases are key proteins in this process . In Saccharyomces cerevisiae, some resident vacuolar hydrolases are delivered by the cytoplasm to vacuole targeting (Cvt) pathway, which shares mechanistic features with autophagy . Autophagy is a degradative pathway that is used to degrade and recycle cellular components under starvation conditions . Both the Cvt pathway and autophagy employ double-membrane cytosolic vesicles to deliver cargo to the vacuole . As a result, these pathways share a common terminal step, the degradation of subvacuolar vesicles . We have identified a protein, Cvt17, which is essential for this membrane lytic event . Cvt17 is a membrane glycoprotein that contains a motif conserved in esterases and lipases . The active-site serine of this motif is required for subvacuolar vesicle lysis . This is the first characterization of a putative lipase implicated in vacuolar function in yeast.

Mol Gen Genet, 2000 Oct, 264(3), 306 - 16
spo12 is a multicopy suppressor of mcs3 that is periodically expressed in fission yeast mitosis; Samuel JM et al.; Hyperactivation of Cdc2 in fission yeast causes cells to undergo a lethal premature mitosis, a phenomenon called mitotic catastrophe . This phenotype is observed in cdc2-3w wee1-50 cells at high temperature and is suppressed by a single recessive mutant, mcs3-12 . Mcs3 acts independently of the Wee1 kinase and Cdc25 phosphatase, two major regulators of Cdc2 . We have isolated multicopy suppressors of the cell cycle arrest phenotype of mcs3-12 wee1-50 cdc25-22 cells, but did not identify the mcs3 gene itself . Instead several known mitotic regulators were isolated, including the Cdc25 phosphatase, Wis2 cyclophilin, Cek1 kinase, and an Hsp90 homologue, Swo1 . We also isolated clones encoding non-functional, truncated forms of the Wee1 kinase and Dis2 type 1 phosphatase . In addition we identified a multicopy suppressor that encodes a structural homologue of the budding yeast SPO12 gene . We find that overexpression of fission yeast spo12 not only suppresses the phenotype of the mcs3-12 wee1-50 cdc25-22 strain, but also that of a win1-1 wee1-50 cdc25-22 strain at high temperature, indicating that the function of spo12 is not directly related to mcs3 . We show that spo12 mRNA is periodically expressed during the fission yeast cell cycle, peaking at the G2/M transition coincidently with cdc15 . Deletion of spo12, however, has no overt effect on either the mitotic or meiotic cell cycles, except when the function of the major B type cyclin, Cdc13, is compromised.

Mol Gen Genet, 2000 Oct, 264(3), 300 - 5
Simultaneous expression of both MAT loci in haploid cells suppresses mutations in yeast microtubule motor genes; Steinberg-Neifach O et al.; The kinesin-related Cin8p and cytoplasmic dynein are microtubule-associated motor proteins required for anaphase spindle elongation in the yeast Saccharomyces cerevisiae . Cells deleted for DYN1 (the gene encoding the dynein heavy chain) and carrying the temperature-sensitive allele cin8-3 cannot grow at temperatures above 35 degrees C . Here, we report that the temperature sensitivity of haploid cin8-3 dyn1delta cells is suppressed by the simultaneous presence of the loci MATa and MATalpha, which contain the regulatory genes that determine mating-type and ploidy-dependent phenotypes . The presence of the two MAT loci also rendered haploid cells more resistant to the antimicrotubule drug benomyl . Our results suggest that, in preparation for handling double the amount of DNA in mitosis, properties of microtubules in diploid cells are modified in a pathway controlled by the mating-type regulatory genes.

Curr Biol, 2000 Nov 2, 10(21), 1349 - 58
The auxilin-like phosphoprotein Swa2p is required for clathrin function in yeast; Gall WE et al.; BACKGROUND: In eukaryotic cells, clathrin-coated vesicles transport specific cargo from the plasma membrane and trans-Golgi network to the endosomal system . Removal of the clathrin coat in vitro requires the uncoating ATPase Hsc70 and its DnaJ cofactor auxilin . To date, a requirement for auxilin and Hsc70 in clathrin function in vivo has not been demonstrated . RESULTS: The Saccharomyces cerevisiae SWA2 gene, previously identified in a synthetic lethal screen with arf1, was cloned and found to encode a protein with a carboxy-terminal DnaJ domain which is homologous to that of auxilin . Like auxilin, Swa2p has a clathrin-binding domain and is able to stimulate the ATPase activity of Hsc70 . The swa2-1 allele recovered from the original screen carries a point mutation in its tetratricopeptide repeat (TPR) domain, a motif not found in auxilin but known in other proteins to mediate interaction with heat-shock proteins . Swa2p fractionates in the cytosol and appears to be heavily phosphorylated . Disruption of SWA2 causes slow growth and several phenotypes that are very similar to those exhibited by clathrin mutants . Furthermore, the swa2Delta mutant exhibits a significant increase in membrane- associated or -assembled clathrin relative to a wild-type strain . CONCLUSIONS: These results indicate that Swa2p is a clathrin-binding protein required for normal clathrin function in vivo . They suggest that Swa2p is the yeast ortholog of auxilin and has a role in disassembling clathrin, not only in uncoating clathrin-coated vesicles but perhaps in preventing unproductive clathrin assembly in vivo.

Int J Pharm, 2000 Nov 19, 209(1-2), 69 - 77
Application of acid-treated yeast cell wall (AYC) as a pharmaceutical additive . II: effects of curing on the medicine release from AYC-coated tablets; Yuasa H et al.; Acid-treated yeast cell wall (AYC) was newly prepared by acidifying brewers' yeast cell wall . Core tablets containing 3% of acetaminophen (AAP) were coated with the AYC aqueous dispersion containing 5% (w/v) of AYC and 0.35% (w/v) of glycerol . The curing of AYC-coated tablets was performed at various curing periods of time and temperatures . The effects of curing on AAP release from AYC-coated tablets, the weight and thickness of the coated layer of AYC and the water sorption into the AYC-coated tablets were studied . The tensile strength and pore size distribution of the AYC cast film were measured . In the case of 60, 80, or 100 degrees C curing, AAP release from AYC-coated tablets showed a sigmoidal release profile with an initial lag time . The duration of the lag time increased with the increasing curing time and temperature, though the release rate after the lag time hardly changed . At 120 degrees C curing, the release rate after the lag time decreased with the increasing curing time and a sustained release was observed . The weight and thickness of the AYC-coated layer and the water sorption rate into AYC-coated tablets decreased with the increasing curing time and temperature . The tensile strength of the AYC cast film increased with increasing the curing temperature, particularly at 120 degrees C curing . It is considered that the water was evaporated from the AYC-coated layer and the adhesion force between AYC particles increased during curing, making the structure of the AYC-coated layer densely firm . The changes in the duration of lag time and the release rate may be due to changes in the structure of the AYC-coated layer caused by curing . These results show that it is feasible to control the lag time and the release rate of AAP from AYC-coated tablets by varying the curing time and temperature.

Biosystems, 2000 Aug-Sep, 57(3), 139 - 45
On the doublet formation in the flocculation process of the yeast cells; Stan S et al.; The combination of single cells to form doublets is regarded as the rate-limiting step of flocculation and requires the presence of surface proteins in active form . The process of activation of the flocculation proteins of yeast cells is described in the frame of the autocrine interaction regime (Science 224 (1984) 1312) . The influence of several effectors (the cell efficiency to use sugars, the calcium content in the external medium and the probability that free cells collide each other under thermal motion conditions) on the initial rate of flocculation and on the fraction of remaining free cells in the steady state is briefly discussed in the paper . The present model offers a useful tool for further quantitative investigations in this topic . Also, it indicates qualitatively a way in which the regulation of flocculation might be controlled at the level of the expression of cell-surface activation abilities.

J Biol Chem, 2001 Mar 9, 276(10), 7011 - 6 Epub 2000 Nov 17.
A genetic investigation of the essential role of glutathione: mutations in the proline biosynthesis pathway are the only suppressors of glutathione auxotrophy in yeast; Spector D et al.; In an attempt to elucidate the essential function of glutathione in Saccharomyces cerevisiae, we searched for suppressors of the GSH auxotrophy of Deltagsh1, a strain lacking the rate-limiting enzyme of glutathione biosynthesis . We found that specific mutations of PRO2, the second enzyme in proline biosynthesis, permitted the growth of Deltagsh1 in the absence of exogenous GSH . The suppression mechanism by alleles of PRO2 involved the biosynthesis of a trace amount of glutathione . Deletion of PRO1, the first enzyme of the proline biosynthesis pathway, or PRO2 eliminated the suppression, suggesting that gamma-glutamyl phosphate, the product of Pro1 and the physiological substrate of Pro2, is required as an obligate substrate of suppressor alleles of PRO2 for glutathione synthesis . A mutagenesis of a Deltagsh1 strain also lacking the proline pathway failed to generate any suppressor mutants under either aerobic or anaerobic conditions, confirming that glutathione is essential in yeast . This essential function is not related to DNA synthesis based on the terminal phenotype of glutathione-depleted cells or to toxic accumulation of non-native protein disulfides . Analysis of the suppressor strain demonstrates that normal glutathione levels are required for the tolerance to oxidants under acute, but not chronic stress conditions.

J Cell Sci, 2000 Dec, 113 Pt 24, 4545 - 55
Identification of Rgp1p, a novel Golgi recycling factor, as a protein required for efficient localization of yeast casein kinase 1 to the plasma membrane; Panek HR et al.; The Yck1p and Yck2p casein kinase 1 isoforms in yeast are essential peripheral plasma membrane-associated protein kinases with roles in endocytosis, cellular morphogenesis and cytokinesis . The membrane targeting of these cytoplasmically oriented protein kinases requires normal secretory pathway function, but specific targeting factors have not been identified . To learn more about Yckp targeting, we characterized mutations that cause synthetic lethality with impairment of Yck function . We report here that these include mutations in two gene products that function in protein trafficking . One of these is the previously described t-SNARE Tlg2p, which participates in recycling of proteins to the Golgi . The other is a previously uncharacterized protein, Rgp1p, which appears to have a similar function . Loss of either Tlg2p or Rgp1p causes inefficient localization of Yck2p, suggesting that its transport may be directed, in part, by a targeting factor that must be recycled back to the Golgi.

Genome, 2000 Oct, 43(5), 910 - 7
Improving synthetic lethal screens by regulating the yeast centromere sequence; Barbour L et al.; The synthetic lethal screen is a useful method in identifying novel genes functioning in an alternative pathway to the gene of interest . The current synthetic lethal screen protocol in yeast is based on a colony-sectoring assay that allows direct visualization of mutant colonies among a large population by their inability to afford plasmid loss . This method demands an appropriate level of stability of the plasmid carrying the gene of interest . YRp-based plasmids are extremely unstable and complete plasmid loss occurs within a few generations . Consequently, YCp plasmids are the vector of choice for synthetic lethal screens . However, we found that the high-level stability of YCp plasmids resulted in a large number of false positives that must be further characterized . In this study, we attempt to improve the existing synthetic lethal screen protocol by regulating the plasmid stability and copy number . It was found that by placing a yeast centromere sequence under the control of either inducible or constitutive promoters, plasmid stability can be significantly decreased . Hence, altering the conditions under which yeast cells carrying the plasmid PGAL1-CEN4 were cultivated allowed us to develop a method that eliminated virtually 100% of false positives and drastically reduced the time required to carry out a synthetic lethal screen.

Cell, 2000 Oct 27, 103(3), 375 - 86
Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast; Uhlmann F et al.; In eukaryotic cells, replicated DNA strands remain physically connected until their segregation to opposite poles of the cell during anaphase . This "sister chromatid cohesion" is essential for the alignment of chromosomes on the mitotic spindle during metaphase . Cohesion depends on the multisubunit cohesin complex, which possibly forms the physical bridges connecting sisters . Proteolytic cleavage of cohesin's Sccl subunit at the metaphase to anaphase transition is essential for sister chromatid separation and depends on a conserved protein called separin . We show here that separin is a cysteine protease related to caspases that alone can cleave Sccl in vitro . Cleavage of Sccl in metaphase arrested cells is sufficient to trigger the separation of sister chromatids and their segregation to opposite cell poles.

EMBO J, 2000 Nov 15, 19(22), 6218 - 29
Yeast snoRNA accumulation relies on a cleavage-dependent/polyadenylation-independent 3'-processing apparatus; Fatica A et al.; In Saccharomyces cerevisiae, snoRNAs are encoded by independent genes and within introns . Despite this heterogenous organization, snoRNA biosynthesis relies on a common theme: entry sites for 5'-3' and 3'-5' exonucleases are created on precursor molecules allowing the release of mature snoRNAs . In independently transcribed snoRNAs, such entry sites are often produced by the Rnt1p endonuclease . In many cases, cleavage sites are absent in the 3' portion of the pre-snoRNAs, suggesting that processing starts from the 3' end of the primary transcript . Here we show that cleavage/polyadenylation sites driving efficient polyadenylation, such as CYC1, prevent production of mature and functional snoRNPs . With these sites, snoRNA accumulation is restored only if polyadenylation activity is inhibited . Analysis of sequences downstream of snoRNA-coding units and the use of strains carrying mutations in RNA polymerase II (polII) cleavage/polyadenylation activities allowed us to establish that formation of snoRNA mature 3' ends requires only the cleavage activity of the polII 3'-processing machinery . These data indicate that, in vivo, uncoupling of cleavage and polyadenylation is necessary for an essential cellular biosynthesis.

EMBO J, 2000 Nov 15, 19(22), 6098 - 111
The fission yeast gamma-tubulin complex is required in G(1) phase and is a component of the spindle assembly checkpoint; Vardy L et al.; Microtubule polymerization is initiated from the microtubule organizing centre (MTOC), which contains the gamma-tubulin complex . We have identified fission yeast Alp4 and Alp6, which are homologues of the gamma-tubulin-interacting proteins Sc.Spc97/Hs.Gcp2 and Sc . Spc98/Hs.Gcp3, respectively . The size of the fission yeast gamma-tubulin complex is large (>2000 kDa), comparable to that in metazoans . Both Alp4 and Alp6 localize to the spindle pole body (SPB) and also to the equatorial MTOC . Temperature-sensitive (ts) alp4 and alp6 mutants show two types of microtubular defects . First, monopolar mitotic spindles form . Secondly, abnormally long cytoplasmic microtubules appear that do not stop at the cell tips and are still associated with the SPB . Alp4 function is required in G(1) phase and ts mutants become lethal before S-phase . alp4 and alp6 mutants are hypersensitive to the microtubule- destabilizing drug thiabendazole (TBZ) and show a lethal 'cut' phenotype in its presence . Furthermore, alp4mad2 double mutants show an exaggerated multiple septation phenotype in TBZ . These results indicate that Alp4 and Alp6 may play a crucial role in the spindle pole-mediated checkpoint pathway.

EMBO J, 2000 Nov 15, 19(22), 6075 - 84
Phosphorylation and N-terminal region of yeast ribosomal protein P1 mediate its degradation, which is prevented by protein P2; Nusspaumer G et al.; The stalk proteins P1 and P2, which are fundamental for ribosome activity, are the only ribosomal components for which there is a cytoplasmic pool . Accumulation of these two proteins is differentially regulated in Saccharomyces cerevisiae by degradation . In the absence of P2, the amount of P1 is drastically reduced; in contrast, P2 proteins are not affected by a deficiency in P1 . However, association with P2 protects P1 proteins . The half-life of P1 is a few minutes, while that of P2 is several hours . The proteasome is not involved in the degradation of P1 proteins . The different sensitivity to degradation of these two proteins is associated with two structural features: phosphorylation and N-terminus structure . A phosphorylation site at the C-terminus is required for P1 proteolysis . P2 proteins, despite being phosphorylated, are protected by their N-terminal peptide . An exchange of the first five amino acids between the two types of protein makes P1 resistant and P2 sensitive to degradation.

J Biol Chem, 2001 Mar 16, 276(11), 8415 - 26 Epub 2000 Nov 16.
Solution structure of the yeast copper transporter domain Ccc2a in the apo and Cu(I)-loaded states; Banci L et al.; Ccc2 is an intracellular copper transporter in Saccharomyces cerevisiae and is a physiological target of the copper chaperone Atx1 . Here we describe the solution structure of the first N-terminal MTCXXC metal-binding domain, Ccc2a, both in the presence and absence of Cu(I) . For Cu(I)-Ccc2a, 1944 meaningful nuclear Overhauser effects were used to obtain a family of 35 structures with root mean square deviation to the average structure of 0.36 +/- 0.06 A for the backbone and 0.79 +/- 0.05 A for the heavy atoms . For apo-Ccc2a, 1970 meaningful nuclear Overhauser effects have been used with 35 (3)J(HNHalpha) to obtain a family of 35 structures with root mean square deviation to the average structure of 0.38 +/- 0.06 A for the backbone and 0.82 +/- 0.07 A for the heavy atoms . The protein exhibits a betaalphabetabetaalphabeta, ferrodoxin-like fold similar to that of its target Atx1 and that of a human counterpart, the fourth metal-binding domain of the Menkes protein . The overall fold remains unchanged upon copper loading, but the copper-binding site itself becomes less disordered . The helical context of the copper-binding site, and the copper-induced conformational changes in Ccc2a differ from those in Atx1 . Ccc2a presents a conserved acidic surface which complements the basic surface of Atx1 and a hydrophobic surface . These results open new mechanistic aspects of copper transporter domains with physiological copper donor and acceptor proteins.

J Biol Chem, 2001 Mar 16, 276(11), 8602 - 7 Epub 2000 Nov 16.
Bovine coupling factor 6, with just 14.5% shared identity, replaces subunit h in the yeast ATP synthase; Velours J et al.; The mammalian mitochondrial ATP synthase is composed of at least 16 polypeptides . With the exception of coupling factor F(6), there are likely yeast homologs for each of these polypeptides . There are no obvious yeast homologs of F(6), as predicted from primary sequence comparison of the putative peptides encoded by the open reading frames in the yeast genome . In this manuscript, we demonstrate that expression of bovine F(6) complements a null mutant in ATP14 gene in yeast Saccharomyces cerevisiae . Subunit h of the yeast ATP synthase is encoded by ATP14 and is just 14.5% identical to bovine F(6) . Expression of bovine F(6) in an atp14 null mutant strain recovers oxidative phosphorylation, and the ATP synthase is active, although functioning with a lower efficiency than the wild type enzyme . Like subunit h, bovine F(6) is shown to interact mainly with subunit 4 (subunit b), a component of the second stalk of the enzyme . These data indicated the subunit h is the yeast homolog of mammalian coupling factor F(6).

Proc Natl Acad Sci U S A, 2000 Nov 21, 97(24), 13015 - 20
The human SWI/SNF-B chromatin-remodeling complex is related to yeast rsc and localizes at kinetochores of mitotic chromosomes; Xue Y et al.; The SWI/SNF family of chromatin-remodeling complexes facilitates gene expression by helping transcription factors gain access to their targets in chromatin . SWI/SNF and Rsc are distinctive members of this family from yeast . They have similar protein components and catalytic activities but differ in biological function . Rsc is required for cell cycle progression through mitosis, whereas SWI/SNF is not . Human complexes of this family have also been identified, which have often been considered related to yeast SWI/SNF . However, all human subunits identified to date are equally similar to components of both SWI/SNF and Rsc, leaving open the possibility that some or all of the human complexes are rather related to Rsc . Here, we present evidence that the previously identified human SWI/SNF-B complex is indeed of the Rsc type . It contains six components conserved in both Rsc and SWI/SNF . Importantly, it has a unique subunit, BAF180, that harbors a distinctive set of structural motifs characteristic of three components of Rsc . Of the two mammalian ATPases known to be related to those in the yeast complexes, human SWI/SNF-B contains only the homolog that functions like Rsc during cell growth . Immunofluorescence studies with a BAF180 antibody revealed that SWI/SNF-B localizes at the kinetochores of chromosomes during mitosis . Our data suggest that SWI/SNF-B and Rsc represent a novel subfamily of chromatin-remodeling complexes conserved from yeast to human, and could participate in cell division at kinetochores of mitotic chromosomes.

J Cell Biol, 2000 Nov 13, 151(4), 789 - 800
Fission yeast myosin-I, Myo1p, stimulates actin assembly by Arp2/3 complex and shares functions with WASp; Lee WL et al.; Fission yeast myo1(+) encodes a myosin-I with all three tail homology domains (TH1, 2, 3) found in typical long-tailed myosin-Is . Myo1p tail also contains a COOH-terminal acidic region similar to the A-domain of WASp/Scar proteins and other fungal myosin-Is . Our analysis shows that Myo1p and Wsp1p, the fission yeast WASp-like protein, share functions and cooperate in controlling actin assembly . First, Myo1p localizes to cortical patches enriched at tips of growing cells and at sites of cell division . Myo1p patches partially colocalize with actin patches and are dependent on an intact actin cytoskeleton . Second, although deletion of myo1(+) is not lethal, Deltamyo1 cells have actin cytoskeletal defects, including loss of polarized cell growth, delocalized actin patches, and mating defects . Third, additional disruption of wsp1(+) is synthetically lethal, suggesting that these genes may share functions . In mapping the domains of Myo1p tail that share function with Wsp1p, we discovered that a Myo1p construct with just the head and TH1 domains is sufficient for cortical localization and to rescue all Deltamyo1 defects . However, it fails to rescue the Deltamyo1 Deltawsp1 lethality . Additional tail domains, TH2 and TH3, are required to complement the double mutant . Fourth, we show that a recombinant Myo1p tail binds to Arp2/3 complex and activates its actin nucleation activity.

Biochemistry, 2000 Nov 14, 39(45), 13931 - 8
Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase; Belogurov GA et al.; Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4 . 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4 . 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3) . These data confirmed that enzyme-PP(i) intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP(i) release . The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PP(i) intermediate, presumably nucleophilic water, from >7 to 5.85 . Protonation of the isomerized enzyme-PP(i) intermediate decelerates PP(i) hydrolysis but accelerates PP(i) release by affecting the back isomerization . The binding of two Mg(2+) ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3 . An acidic group with a pK(a) approximately 9 is modulatory in PP(i) hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.

Biochemistry, 2000 Nov 14, 39(45), 13660 - 8
Solution structures of the N-terminal domain of yeast calmodulin: Ca2+-dependent conformational change and its functional implication; Ishida H et al.; We have determined solution structures of the N-terminal half domain (N-domain) of yeast calmodulin (YCM0-N, residues 1-77) in the apo and Ca(2+)-saturated forms by NMR spectroscopy . The Ca(2+)-binding sites of YCM0-N consist of a pair of helix-loop-helix motifs (EF-hands), in which the loops are linked by a short beta-sheet . The binding of two Ca(2+) causes large rearrangement of the four alpha-helices and exposes the hydrophobic surface as observed for vertebrate calmodulin (CaM) . Within the observed overall conformational similarity in the peptide backbone, several significant conformational differences were observed between the two proteins, which originated from the 38% disagreement in amino acid sequences . The beta-sheet in apo YCM0-N is strongly twisted compared with that in the N-domain of CaM, while it turns to the normal more stable conformation on Ca(2+) binding . YCM0-N shows higher cooperativity in Ca(2+) binding than the N-domain of CaM, and the observed conformational change of the beta-sheet is a possible cause of the highly cooperative Ca(2+) binding . The hydrophobic surface on Ca(2+)-saturated YCM0-N appears less flexible due to the replacements of Met51, Met71, and Val55 in the hydrophobic surface of CaM with Leu51, Leu71, and Ile55, which is thought to be one of reasons for the poor activation of target enzymes by yeast CaM.

Methods Enzymol, 2000, 328, 430 - 44
Yeast surface display for directed evolution of protein expression, affinity, and stability; Boder ET et al.; The described protocols enable thorough screening of polypeptide libraries with high confidence in the isolation of improved clones . It should be emphasized that the protocols have been fashioned for thoroughness, rather than speed . With library plasmid DNA in hand, the time to plated candidate yeast display mutants is typically 2-3 weeks . Each of the experimental approaches required for this method is fairly standard: yeast culture, immunofluorescent labeling, flow cytometry . Protocols that are more rapid could conceivably be developed by using solid substrate separations with magnetic beads, for instance . However, loss of the two-color normalization possible with flow cytometry would remove the quantitative advantage of the method . Yeast display complements existing polypeptide library methods and opens the possibility of examining extracellular eukaryotic proteins, an important class of proteins not generally amenable to yeast two-hybrid or phage display methodologies.

Nucleic Acids Res, 2000 Nov 15, 28(22), 4460 - 6
A network of yeast basic helix-loop-helix interactions; Robinson KA et al.; The Ino4 protein belongs to the basic helix-loop-helix (bHLH) family of proteins . It is known to form a dimer with Ino2p, which regulates phospholipid biosynthetic genes . Mammalian bHLH proteins have been shown to form multiple dimer combinations . However, this flexibility in dimerization had not been documented for yeast bHLH proteins . Using the yeast two-hybrid assay and a biochemical assay we show that Ino4p dimerizes with the Pho4p, Rtg1p, Rtg3p and Sgc1p bHLH proteins . Screening a yeast cDNA library identified three additional proteins that interact with Ino4p: Bck2p, YLR422W and YNR064C . The interaction with Bck2p prompted us to examine if any of the Bck2p-associated functions affect expression of phospholipid biosynthetic genes . We found that hyperosmotic growth conditions altered the growth phase regulation of a phospholipid biosynthetic gene, CHO1 . There are two recent reports of initial whole genome yeast two-hybrid interactions . Interestingly, one of these reports identified five proteins that interact with Ino4p: Ino2p, Hcs1p, Apl2p, YMR317W and YNL279W . Ino2p is the only protein in common with the data presented here . Our finding that Ino4p interacts with five bHLH proteins suggests that Ino4p is likely to be a central player in the coordination of multiple biological processes.

Mol Biol Cell, 2000 Nov, 11(11), 4033 - 49
Mutational analysis suggests that activation of the yeast pheromone response mitogen-activated protein kinase pathway involves conformational changes in the Ste5 scaffold protein; Sette C et al.; Ste5 is essential for pheromone response and binds components of a mitogen-activated protein kinase (MAPK) cascade: Ste11 (MEKK), Ste7 (MEK), and Fus3 (MAPK) . Pheromone stimulation releases Gbetagamma (Ste4-Ste18), which recruits Ste5 and Ste20 (p21-activated kinase) to the plasma membrane, activating the MAPK cascade . A RING-H2 domain in Ste5 (residues 177-229) negatively regulates Ste5 function and mediates its interaction with Gbetagamma . Ste5(C177A C180A), carrying a mutated RING-H2 domain, cannot complement a ste5Delta mutation, yet supports mating even in ste4Delta ste5Delta cells when artificially dimerized by fusion to glutathione S-transferase (GST) . In contrast, wild-type Ste5 fused to GST permits mating of ste5Delta cells, but does not allow mating of ste4Delta ste5Delta cells . This differential behavior provided the basis of a genetic selection for STE5 gain-of-function mutations . MATa ste4Delta ste5Delta cells expressing Ste5-GST were mutagenized chemically and plasmids conferring the capacity to mate were selected . Three independent single-substitution mutations were isolated . These constitutive STE5 alleles induce cell cycle arrest, transcriptional activation, and morphological changes normally triggered by pheromone, even when Gbetagamma is absent . The first, Ste5(C226Y), alters the seventh conserved position in the RING-H2 motif, confirming that perturbation of this domain constitutively activates Ste5 function . The second, Ste5(P44L), lies upstream of a basic segment, whereas the third, Ste5(S770K), is situated within an acidic segment in a region that contacts Ste7 . None of the mutations increased the affinity of Ste5 for Ste11, Ste7, or Fus3 . However, the positions of these novel-activating mutations suggested that, in normal Ste5, the N terminus may interact with the C terminus . Indeed, in vitro, GST-Ste5(1-518) was able to associate specifically with radiolabeled Ste5(520-917) . Furthermore, both the P44L and S770K mutations enhanced binding of full-length Ste5 to GST-Ste5(1-518), whereas they did not affect Ste5 dimerization . Thus, binding of Gbetagamma to the RING-H2 domain may induce a conformational change that promotes association of the N- and C-terminal ends of Ste5, stimulating activation of the MAPK cascade by optimizing orientation of the bound kinases and/or by increasing their accessibility to Ste20-dependent phosphorylation (or both) . In accord with this model, the novel Ste5 mutants copurified with Ste7 and Fus3 in their activated state and their activation required Ste20.

Mol Biol Cell, 2000 Nov, 11(11), 3993 - 4003
A fission yeast homolog of Int-6, the mammalian oncoprotein and eIF3 subunit, induces drug resistance when overexpressed; Crane R et al.; Through a screen to identify genes that induce multi-drug resistance when overexpressed, we have identified a fission yeast homolog of Int-6, a component of the human translation initiation factor eIF3 . Disruption of the murine Int-6 gene by mouse mammary tumor virus (MMTV) has been implicated previously in tumorigenesis, although the underlying mechanism is not yet understood . Fission yeast Int6 was shown to interact with other presumptive components of eIF3 in vivo, and was present in size fractions consistent with its incorporation into a 43S translation preinitiation complex . Drug resistance induced by Int6 overexpression was dependent on the AP-1 transcription factor Pap1, and was associated with increased abundance of Pap1-responsive mRNAs, but not with Pap1 relocalization . Fission yeast cells lacking the int6 gene grew slowly . This growth retardation could be corrected by the expression of full length Int6 of fission yeast or human origin, or by a C-terminal fragment of the fission yeast protein that also conferred drug resistance, but not by truncated human Int-6 proteins corresponding to the predicted products of MMTV-disrupted murine alleles . Studies in fission yeast may therefore help to explain the ways in which Int-6 function can be perturbed during MMTV-induced mammary tumorigenesis.

Mol Biol Cell, 2000 Nov, 11(11), 3949 - 61
Dynamic positioning of mitotic spindles in yeast: role of microtubule motors and cortical determinants; Yeh E et al.; In the budding yeast Saccharomyces cerevisiae, movement of the mitotic spindle to a predetermined cleavage plane at the bud neck is essential for partitioning chromosomes into the mother and daughter cells . Astral microtubule dynamics are critical to the mechanism that ensures nuclear migration to the bud neck . The nucleus moves in the opposite direction of astral microtubule growth in the mother cell, apparently being "pushed" by microtubule contacts at the cortex . In contrast, microtubules growing toward the neck and within the bud promote nuclear movement in the same direction of microtubule growth, thus "pulling" the nucleus toward the bud neck . Failure of "pulling" is evident in cells lacking Bud6p, Bni1p, Kar9p, or the kinesin homolog, Kip3p . As a consequence, there is a loss of asymmetry in spindle pole body segregation into the bud . The cytoplasmic motor protein, dynein, is not required for nuclear movement to the neck; rather, it has been postulated to contribute to spindle elongation through the neck . In the absence of KAR9, dynein-dependent spindle oscillations are evident before anaphase onset, as are postanaphase dynein-dependent pulling forces that exceed the velocity of wild-type spindle elongation threefold . In addition, dynein-mediated forces on astral microtubules are sufficient to segregate a 2N chromosome set through the neck in the absence of spindle elongation, but cytoplasmic kinesins are not . These observations support a model in which spindle polarity determinants (BUD6, BNI1, KAR9) and cytoplasmic kinesin (KIP3) provide directional cues for spindle orientation to the bud while restraining the spindle to the neck . Cytoplasmic dynein is attenuated by these spindle polarity determinants and kinesin until anaphase onset, when dynein directs spindle elongation to distal points in the mother and bud.

Mol Biol Cell, 2000 Nov, 11(11), 3885 - 96
The yeast nucleoporin Nup53p specifically interacts with Nic96p and is directly involved in nuclear protein import; Fahrenkrog B et al.; The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore complex (NPC) which, in yeast, is composed of approximately 30 different proteins (nucleoporins) . Pre-embedding immunogold-electron microscopy revealed that Nic96p, an essential yeast nucleoporin, is located about the cytoplasmic and the nuclear periphery of the central channel, and near or at the distal ring of the yeast NPC . Genetic approaches further implicated Nic96p in nuclear protein import . To more specifically explore the potential role of Nic96p in nuclear protein import, we performed a two-hybrid screen with NIC96 as the bait against a yeast genomic library to identify transport factors and/or nucleoporins involved in nuclear protein import interacting with Nic96p . By doing so, we identified the yeast nucleoporin Nup53p, which also exhibits multiple locations within the yeast NPC and colocalizes with Nic96p in all its locations . Whereas Nup53p is directly involved in NLS-mediated protein import by its interaction with the yeast nuclear import receptor Kap95p, it appears not to participate in NES-dependent nuclear export.

Mol Biol Cell, 2000 Nov, 11(11), 3849 - 58
Testing the 3Q:1R "rule": mutational analysis of the ionic "zero" layer in the yeast exocytic SNARE complex reveals no requirement for arginine; Katz L et al.; The crystal structure of the synaptic SNARE complex reveals a parallel four-helix coiled-coil arrangement; buried in the hydrophobic core of the complex is an unusual ionic layer composed of three glutamines and one arginine, each provided by a separate alpha-helix . The presence of glutamine or arginine residues in this position is highly conserved across the t- and v-SNARE families, and it was recently suggested that a 3Q:1R ratio is likely to be a general feature common to all SNARE complexes . In this study, we have used genetic and biochemical assays to test this prediction with the yeast exocytic SNARE complex . We have determined that the relative position of Qs and Rs within the layer is not critical for biological activity and that Q-to-R substitutions in the layer reduce complex stability and result in lethal or conditional lethal growth defects . Surprisingly, SNARE complexes composed of four glutamines are fully functional for assembly in vitro and exocytic function in vivo . We conclude that the 3Q:1R layer composition is not required within the yeast exocytic SNARE complex because complexes containing four Q residues in the ionic layer appear by all criteria to be functionally equivalent . The unexpected flexibility of this layer suggests that there is no strict requirement for the 3Q:1R combination and that the SNARE complexes at other stages of transport may be composed entirely of Q-SNAREs or other noncanonical combinations.

Mol Biol Cell, 2000 Nov, 11(11), 3737 - 49
Polar transmembrane domains target proteins to the interior of the yeast vacuole; Reggiori F et al.; Membrane proteins transported to the yeast vacuole can have two fates . Some reach the outer vacuolar membrane, whereas others enter internal vesicles, which form in late endosomes, and are ultimately degraded . The vacuolar SNAREs Nyv1p and Vam3p avoid this fate by using the AP-3-dependent pathway, which bypasses late endosomes, but the endosomal SNARE Pep12p must avoid it more directly . Deletion analysis revealed no cytoplasmic sequences necessary to prevent the internalization of Pep12p in endosomes . However, introduction of acidic residues into the cytoplasmic half of the transmembrane domain created a dominant internalization signal . In other contexts, this same feature diverted proteins from the Golgi to endosomes and slowed their exit from the endoplasmic reticulum . The more modestly polar transmembrane domains of Sec12p and Ufe1p, which normally serve to hold these proteins in the endoplasmic reticulum, also cause Pep12p to be internalized, as does that of the vacuolar protein Cps1p . It seems that quality control mechanisms recognize polar transmembrane domains at multiple points in the secretory and endocytic pathways and in endosomes sort proteins for subsequent destruction in the vacuole . These mechanisms may minimize the damaging effects of abnormally exposed polar residues while being exploited for the localization of some normal proteins.

Proc Natl Acad Sci U S A, 2000 Nov 7, 97(23), 12672 - 7
TEP1, the yeast homolog of the human tumor suppressor gene PTEN/MMAC1/TEP1, is linked to the phosphatidylinositol pathway and plays a role in the developmental process of sporulation; Heymont J et al.; PTEN/MMAC1/TEP1 (PTEN, phosphatase deleted on chromosome ten; MMAC1, mutated in multiple advanced cancers; TEP1, tensin-like phosphatase) is a major human tumor suppressor gene whose suppressive activity operates on the phosphatidylinositol pathway . A single homologue of this gene, TEP1 (YNL128w), exists in the budding yeast Saccharomyces cerevisiae . Yeast strains deleted for TEP1 exhibit essentially no phenotype in haploids; however, diploids exhibit resistance to the phosphatidylinositol-3-phosphate kinase inhibitor wortmannin and to lithium ions . Although rates of cancer increase with age, neither tep1 haploids nor diploids have altered life spans . TEP1 RNA is present throughout the cell cycle, and levels are dramatically up-regulated during meiotic development . Although homozygous tep1 mutants initiate the meiotic program and form spores with wild-type kinetics, analysis of the spores produced in tep1 mutants indicates a specific defect in the trafficking or deposition of dityrosine, a major component of yeast spore walls, to the surface . Introduction of a common PTEN mutation found in human tumors into the analogous position in Tep1p produces a nonfunctional protein based on in vivo activity . These studies implicate Tep1p in a specific developmental trafficking or deposition event and suggest that Tep1p, like its mammalian counterpart, impinges on the phosphatidylinositol pathway.

Proc Natl Acad Sci U S A, 2000 Nov 7, 97(23), 12583 - 8
Interaction of yeast kinetochore proteins with centromere-protein/transcription factor Cbf1; Hemmerich P et al.; The centromere-kinetochore complex of Saccharomyces cerevisiae is a specialized chromosomal substructure that mediates attachment of duplicated chromosomes to the mitotic spindle by a regulated network of protein-DNA and protein-protein interactions . We have used in vitro assays to analyze putative molecular interactions between components of the yeast centromerekinetochore complex . Glutathione S-transferase pull-down experiments showed the direct interaction of in vitro translated p110, p64, and p58 of the essential CBF3 kinetochore protein complex with Cbf1p, a basic region helix-loop-helix zipper protein (bHLHzip) that specifically binds to the CDEI region on the centromere DNA . Furthermore, recombinant p64 and p23 each stimulated the in vitro DNA binding activity of Cbf1p . The N-terminal 70 amino acids of p23 were sufficient to mediate this effect . P64 could also promote the multimerization activity of Cbf1p in the presence of centromere DNA in vitro . These results show the direct physical interaction of Cbf1p and CBF3 subunits and provide evidence that CBF3 components can promote the binding of Cbf1p to its binding site in the yeast kinetochore . A functional comparison of the centromere binding proteins with transcription factors binding at MET16 promoters reveals the strong analogy between centromeres and the MET16 promoter.

Genes Dev, 2000 Nov 1, 14(21), 2757 - 70
Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase; Tomonaga T et al.; Cohesin complex acts in the formation and maintenance of sister chromatid cohesion during and after S phase . Budding yeast Scc1p/Mcd1p, an essential subunit, is cleaved and dissociates from chromosomes in anaphase, leading to sister chromatid separation . Most cohesin in higher eukaryotes, in contrast, is dissociated from chromosomes well before anaphase . The universal role of cohesin during anaphase thus remains to be determined . We report here initial characterization of four putative cohesin subunits, Psm1, Psm3, Rad21, and Psc3, in fission yeast . They are essential for sister chromatid cohesion . Immunoprecipitation demonstrates stable complex formation of Rad21 with Psm1 and Psm3 but not with Psc3 . Chromatin immunoprecipitation shows that cohesin subunits are enriched in broad centromere regions and that the level of centromere-associated Rad21 did not change from metaphase to anaphase, very different from budding yeast . In contrast, Rad21 containing similar cleavage sites to those of Scc1p/Mcd1p is cleaved specifically in anaphase . This cleavage is essential, although the amount of cleaved product is very small (<5%) . Mis4, another sister chromatid cohesion protein, plays an essential role for loading Rad21 on chromatin . A simple model is presented to explain the specific behavior of fission yeast cohesin and why only a tiny fraction of Rad21 is sufficient to be cleaved for normal anaphase.

Curr Biol, 2000 Oct 19, 10(20), 1256 - 64
Nuclear export of yeast signal recognition particle lacking Srp54p by the Xpo1p/Crm1p NES-dependent pathway; Ciufo LF et al.; BACKGROUND: The movement of macromolecules through the nuclear pores requires energy and transport receptors that bind both cargo and nuclear pores . Different molecules/complexes often require different transport receptors . The signal recognition particle (SRP) is a conserved cytosolic ribonucleoprotein that targets proteins to the endoplasmic reticulum . Previous studies have shown that the export of SRP RNA from the nucleus requires trans-acting factors and that SRP may be at least partly assembled in the nucleus, but little else is known about how it is assembled and exported into the cytoplasm . RESULTS: Of the six proteins that constitute the yeast SRP, we found that all except Srp54p were imported into the nucleus . Four of these had nucleolar pools . The same four proteins are required for stability of the yeast SRP RNA scR1, suggesting that they assemble with the RNA in the nucleus to form a central core SRP . This core SRP was a competent export substrate . Of the remaining components, Sec65p entered the nucleus and was assembled onto the core particle there, whereas Srp54p was solely cytoplasmic . The export of SRP from the nucleus required the transport receptor Xpo1p/Crm1p and Yrb2p, both components of the pathway that exports leucine-rich nuclear export signal (NES)-containing proteins from the nucleus . CONCLUSIONS: The SRP is assembled in the nucleus into a complex lacking only Srp54p . It is then exported through the NES pathway into the cytoplasm where Srp54p binds to it . This transport route for a ribonucleoprotein complex is so far unique in yeast.

J Biol Chem, 2001 Feb 9, 276(6), 4424 - 32 Epub 2000 Nov 06.
Identification of a conserved motif in the yeast golgi GDP-mannose transporter required for binding to nucleotide sugar; Gao XD et al.; Glycoproteins and lipids in the Golgi complex are modified by the addition of sugars . In the yeast Saccharomyces cerevisiae, these terminal Golgi carbohydrate modifications primarily involve mannose additions that utilize GDP-mannose as the substrate . The transport of GDP-mannose from its site of synthesis in the cytosol into the lumen of the Golgi is mediated by the VRG4 gene product, a nucleotide sugar transporter that is a member of a large family of related membrane proteins . Loss of VRG4 function leads to lethality, but several viable vrg4 mutants were isolated whose GDP-mannose transport activity was reduced but not obliterated . Mutations in these alleles mapped to a region of the Vrg4 protein that is highly conserved among other GDP-mannose transporters but not other types of nucleotide sugar transporters . Here, we present evidence that suggest an involvement of this region of the protein in binding GDP-mannose . Most of the mutations that were introduced within this conserved domain, spanning amino acids 280-291 of Vrg4p, lead to lethality, and none interfere with Vrg4 protein stability, localization, or dimer formation . The null phenotype of these mutant vrg4 alleles can be complemented by their overexpression . Vesicles prepared from vrg4 mutant strains were reduced in luminal GDP-mannose transport activity, but this effect could be suppressed by increasing the concentration of GDP-mannose in vitro . Thus, either an increased substrate concentration, in vitro, or an increased Vrg4 protein concentration, in vivo, can suppress these vrg4 mutant phenotypes . Vrg4 proteins with alterations in this region were reduced in binding to guanosine 5'-{gamma-(32)P}triphosphate gamma-azidoanilide, a photoaffinity substrate analogue whose binding to Vrg4-HAp was specifically inhibited by GDP-mannose . Taken together, these data are consistent with the model that amino acids in this region of the yeast GDP-mannose transporter mediate the recognition of or binding to nucleotide sugar prior to its transport into the Golgi.

Microbiology, 2000 Nov, 146 ( Pt 11), 2833 - 43
Mutational and hyperexpression-induced disruption of bipolar budding in yeast; Freedman T et al.; Analysis of bud-site selection in the yeast Saccharomyces cerevisiae has helped to identify many genes that are generally important for eukaryotic cell polarization . Colony morphology screens were used to identify factors relevant to the process of bipolar budding in yeast . Mutants defective in bipolar budding were identified by virtue of their inability to grow as pseudohyphae in a haploid bud3 background . A mutant allele of the MYO2 gene, encoding a class-V unconventional myosin was identified that perturbs bipolar budding without affecting axial budding and without grossly affecting the role of Myo2p in secretion and maintenance of the actin cytoskeleton . Several genes were also identified whose products, when overexpressed, are capable of disrupting bipolar budding . Among these are the actin-monomer-binding protein profilin and the Aip3p/Bud6p-interacting protein Atc1p . The results strongly support involvement of the actin cytoskeleton in the establishment of bipolar budding and in the maintenance of pseudohyphal growth.

FEMS Microbiol Lett, 2000 Nov 15, 192(2), 243 - 8
Cytochemical evaluation of localization and secretion of a heterologous enzyme displayed on yeast cell surface; Shibasaki Y et al.; A starch-utilizing Saccharomyces cerevisiae strain was constructed by cell surface engineering . Distribution of the heterologous glucoamylase-alpha-agglutinin fusion protein on the yeast cell was analyzed by indirect fluorescence microscopy using an anti-glucoamylase antibody . Most of the intense fluorescence was first localized in the small bud, then observed on the entire cell wall of the daughter and mother cells . Fluorescence also accumulated at the neck region . These observations suggest that the display of the heterologous protein on the cell surface is carried with other cell wall components to the areas in which the cell wall is newly synthesized; the distribution is controlled by the cell cycle . Then, the heterologous protein-encoding gene was expressed in a sec1 mutant, in which secretory vesicles accumulate under restrictive temperature, and the produced protein was detected by immunoelectron microscopy . Most of the gold particles that reacted with the fusion protein were not localized in vesicles but in expanding endoplasmic reticulum . This phenomenon may be due to overproduction of the heterologous protein which was designed to be displayed on the cell wall . Artificial production of heterologous protein may have caused a relative shortage of glycosyl phosphatidylinositol anchors.

Curr Opin Cell Biol, 2000 Dec, 12(6), 710 - 5
Microarrays and cell cycle transcription in yeast; Futcher B; Microarrays have been used to characterize gene expression through the yeast cell cycle . Computational methods have been applied to the microarray data to identify coregulated clusters of genes, and motif-finding algorithms have found promoter elements characteristic of each cluster . The functional relevance of these promoter elements can be tested using chromatin immunoprecipitation, additional microarrays and other molecular techniques . The yeast forkhead proteins have been successfully identified as cell cycle transcription factors for an important cluster of genes by this and other approaches.

J Biol Chem, 2000 Dec 29, 275(52), 40667 - 70
Mutations in yeast ARV1 alter intracellular sterol distribution and are complemented by human ARV1; Tinkelenberg AH et al.; Intracellular cholesterol redistribution between membranes and its subsequent esterification are critical aspects of lipid homeostasis that prevent free sterol toxicity . To identify genes that mediate sterol trafficking, we screened for yeast mutants that were inviable in the absence of sterol esterification . Mutations in the novel gene, ARV1, render cells dependent on sterol esterification for growth, nystatin-sensitive, temperature-sensitive, and anaerobically inviable . Cells lacking Arv1p display altered intracellular sterol distribution and are defective in sterol uptake, consistent with a role for Arv1p in trafficking sterol into the plasma membrane . Human ARV1, a predicted sequence ortholog of yeast ARV1, complements the defects associated with deletion of the yeast gene . The genes are predicted to encode transmembrane proteins with potential zinc-binding motifs . We propose that ARV1 is a novel mediator of eukaryotic sterol homeostasis.

Genetics, 2000 Nov, 156(3), 953 - 61
Yeast GMP kinase mutants constitutively express AMP biosynthesis genes by phenocopying a hypoxanthine-guanine phosphoribosyltransferase defect; Lecoq K et al.; We have characterized a new locus, BRA3, leading to deregulation of the yeast purine synthesis genes (ADE genes) . We show that bra3 mutations are alleles of the GUK1 gene, which encodes GMP kinase . The bra3 mutants have a low GMP kinase activity, excrete purines in the medium, and show vegetative growth defects and resistance to purine base analogs . The bra3 locus also corresponds to the previously described pur5 locus . Several lines of evidence indicate that the decrease in GMP kinase activity in the bra3 mutants results in GMP accumulation and feedback inhibition of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), encoded by the HPT1 gene . First, guk1 and hpt1 mutants share several phenotypes, such as adenine derepression, purine excretion, and 8-azaguanine resistance . Second, overexpression of HPT1 allows suppression of the deregulated phenotype of the guk1 mutants . Third, we show that purified yeast HGPRT is inhibited by GMP in vitro . Finally, incorporation of hypoxanthine into nucleotides is similarly diminished in hpt1 and guk1 mutants in vivo . We conclude that the decrease in GMP kinase activity in the guk1 mutants results in deregulation of the ADE gene expression by phenocopying a defect in HGPRT . The possible occurrence of a similar phenomenon in humans is discussed.

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