Yeasts

Nature, 2002 Feb 7, 415(6872), 651 - 5 Epub 2002 Jan 23.
S-Cdk-dependent phosphorylation of Sld2 essential for chromosomal DNA replication in budding yeast; Masumoto H et al.; Cyclin-dependent protein kinases (Cdks) in eukaryotic cells work as a key enzyme at various points in the cell cycle . At the onset of S phase, active S-phase Cdks (S-Cdks) are essential for chromosomal DNA replication . Although several replication proteins are phosphorylated in a Cdk-dependent manner, the biological effects of phosphorylation of these proteins on the activation of DNA replication have not been elucidated . Here we show that Sld2 (ref . 4) (also known as Drc1; ref . 5), one of the replication proteins of budding yeast (Saccharomyces cerevisiae), is phosphorylated in S phase in an S-Cdk-dependent manner, and mutant Sld2 lacking all the preferred Cdk phosphorylation sites (All-A) is defective in chromosomal DNA replication . Moreover, the complex that contains, at least, Sld2 and Dpb11 (ref . 6) (the Sld2-Dpb11 complex) is formed predominantly in S phase; the All-A protein is defective in this complex formation . Because this complex is suggested to be essential for chromosomal DNA replication, it seems likely that S-Cdk positively regulates formation of the Sld2-Dpb11 complex and, consequently, chromosomal DNA replication.

J Cell Biol, 2002 Jan 21, 156(2), 271 - 85 Epub 2002 Jan 21.
A subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway; Harsay E et al.; Exocytic vesicles that accumulate in a temperature-sensitive sec6 mutant at a restrictive temperature can be separated into at least two populations with different buoyant densities and unique cargo molecules . Using a sec6 mutant background to isolate vesicles, we have found that vacuolar protein sorting mutants that block an endosome-mediated route to the vacuole, including vps1, pep12, vps4, and a temperature-sensitive clathrin mutant, missort cargo normally transported by dense exocytic vesicles, such as invertase, into light exocytic vesicles, whereas transport of cargo specific to the light exocytic vesicles appears unaffected . Immunoisolation experiments confirm that missorting, rather than a changed property of the normally dense vesicles, is responsible for the altered density gradient fractionation profile . The vps41Delta and apl6Delta mutants, which block transport of only the subset of vacuolar proteins that bypasses endosomes, sort exocytic cargo normally . Furthermore, a vps10Delta sec6 mutant, which lacks the sorting receptor for carboxypeptidase Y (CPY), accumulates both invertase and CPY in dense vesicles . These results suggest that at least one branch of the yeast exocytic pathway transits through endosomes before reaching the cell surface . Consistent with this possibility, we show that immunoisolated clathrin-coated vesicles contain invertase.

J Cell Biol, 2002 Jan 21, 156(2), 241 - 8 Epub 2002 Jan 21.
The conserved Pkh-Ypk kinase cascade is required for endocytosis in yeast; deHart AK et al.; Internalization of activated signaling receptors by endocytosis is one way cells downregulate extracellular signals . Like many signaling receptors, the yeast alpha-factor pheromone receptor is downregulated by hyperphosphorylation, ubiquitination, and subsequent internalization and degradation in the lysosome-like vacuole . In a screen to detect proteins involved in ubiquitin-dependent receptor internalization, we identified the sphingoid base-regulated serine-threonine kinase Ypk1 . Ypk1 is a homologue of the mammalian serum- and glucocorticoid-induced kinase, SGK, which can substitute for Ypk1 function in yeast . The kinase activity of Ypk1 is required for receptor endocytosis because mutations in two residues important for its catalytic activity cause a severe defect in alpha-factor internalization . Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination . Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1 . The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in alpha-factor internalization and fluid-phase endocytosis . These observations demonstrate that Ypk1 acts downstream of the Pkh kinases to control endocytosis by phosphorylating components of the endocytic machinery.

Biophys J, 2002 Feb, 82(2), 781 - 92
Carboxyl tail prevents yeast K(+) channel closure: proposal of an integrated model of TOK1 gating; Loukin SH et al.; TOK1 encodes the channel responsible for the prominent outward K(+) current of the yeast plasma membrane . It can dwell in several impermeable states, including a rapidly transiting, K(+)-electromotive-force-dependent "R" (rectifying) state, a voltage-independent "IB" (interburst) state, and a set of {K(+)}(ext) and voltage-dependent "C" (closed) states . Whereas evidence suggests that the C states result from the constriction of an inner gate at the cytosolic end of the pore, R is most likely an intrinsic gating property of the K(+) filter . Here, we present evidence that Tok1's carboxyl-tail domain also plays an intimate role in channel gating by dynamically preventing inner-gate closures . We present an integrated model of TOK1 gating in which the filter gate, inner gate, and carboxyl tail interact to produce the various phenomenological states . Both wild-type and tailless behaviors can be replicated using Monte Carlo computer simulations based on this model.

Curr Genet, 2001 Dec, 40(4), 251 - 9
The role of DSC1 components cdc10+, rep1+ and rep2+ in MCB gene transcription at the mitotic G1-S boundary in fission yeast; White S et al.; In this paper, we describe the transcription profile of a group of genes at the G1-S boundary of fission yeast in synchronously dividing mitotic cells, under a variety of different conditions . This transcription profile is unaffected in cells where either cdc10+ or cdc10-C4 are constitutively overexpressed . In contrast, overexpression of either rep1+ or rep2+ results in constitutive expression of MCB-regulated genes, suggesting that these polypeptides have important regulatory properties in controlling MCB transcription . Finally, we examine the pattern of MCB-regulated transcription in cells where the G1 period is extended . Surprisingly, we find that the wee1-50 mutation causes MCB transcription throughout the cell cycle, whereas cells re-fed after nitrogen starvation have normal expression patterns . The implications of these observations for understanding MCB-regulated transcription are discussed.

Nature, 2002 Jan 10, 415(6868), 141 - 7
Functional organization of the yeast proteome by systematic analysis of protein complexes; Gavin AC et al.; Most cellular processes are carried out by multiprotein complexes . The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units . We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae . We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies . Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation . Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment . Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions . This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.

Proc Natl Acad Sci U S A, 2002 Jan 22, 99(2), 751 - 6
Actin cable dynamics in budding yeast; Yang HC et al.; Actin cables, bundles of actin filaments that align along the long axis of budding yeast, are crucial for establishment of cell polarity . We fused green fluorescent protein (GFP) to actin binding protein 140 (Abp140p) and visualized actin cable dynamics in living yeast . We detected two populations of actin cables: (i) bud-associated cables, which extend from the bud along the mother-bud axis, and (ii) randomly oriented cables, which are relatively short . Time-lapse imaging of Abp140p-GFP revealed an apparent increase in the length of bud-associated actin cables . Analysis of movement of Abp140p-GFP fiduciary marks on bud-associated cables and fluorescence loss in photobleaching experiments revealed that this apparent elongation occurs by assembly of new material at the end of the cable within the bud and movement of the opposite end of the cable toward the tip of the mother cell distal to the bud . The rate of extension of the tip of an elongating actin cable is 0.29 +/- 0.08 microm/s . Latrunculin A (Lat-A) treatment completely blocked this process . We also observed movement of randomly oriented cables around the cortex of cells at a rate of 0.59 +/- 0.14 microm/s . Mild treatment with Lat-A did not affect the velocity of movement of randomly oriented cables . However, Lat-A treatment did increase the number of randomly oriented, motile cables per cell . Our observations suggest that establishment of bud-associated actin cables during the cell cycle is accomplished not by realignment of existing cables but by assembly of new cables within the bud or bud neck, followed by elongation.

Proc Natl Acad Sci U S A, 2002 Jan 22, 99(2), 745 - 50
Budding yeast Dsk2p is a polyubiquitin-binding protein that can interact with the proteasome; Funakoshi M et al.; Dsk2p from Saccharomyces cerevisiae belongs to the class of proteins that contain a ubiquitin-like (UbL) domain at the N terminus together with a ubiquitin-associated (UBA) domain at the C terminus . We show here that the C-terminal UBA domain of Dsk2p binds to K48-linked polyubiquitin chains, and the N-terminal UbL domain of Dsk2p interacts with the proteasome . Overexpression of Dsk2p caused the accumulation of large amounts of polyubiquitin, and extragenic suppressors of the Dsk2p-mediated lethality proved to be temperature-sensitive mutations in two proteasome subunits, rpn1 and pre2 . K48-linked ubiquitin-dependent degradation was impaired by disruption of the DSK2 gene . These results indicate that Dsk2p is K48-linked polyubiquitin-binding protein and also interacts with the proteasome . We discuss a possible role of adaptor function of Dsk2p via its UbL and UBA domains in the ubiquitin-proteasome pathway.

J Biol Chem, 2002 Apr 5, 277(14), 11845 - 52 Epub 2002 Jan 22.
Repair of active and silenced rDNA in yeast: the contributions of photolyase and transcription-couples nucleotide excision repair; Meier A et al.; DNA repair by photolyase (photoreactivation) and nucleotide excision repair (NER) are the major pathways to remove UV-induced cyclobutane-pyrimidine dimers (CPDs) . The nucleolus is a nuclear subcompartment containing the ribosomal RNA genes (rDNA) of which a fraction is transcribed by RNA polymerase I (RNAP-I), and the rest is silenced . Here yeast was used to investigate how photoreactivation and NER contribute to repair of active and inactive rDNA . Cells were irradiated with UV light and exposed to different repair conditions . Nuclei were isolated, and the active genes were separated from the inactive genes by restriction endonuclease digestion . CPDs were measured in total rDNA, in both fractions, and in the GAL10 gene . Repair in rDNA was as efficient as in GAL10 indicating that both pathways have unrestricted access to the nucleolus . Photoreactivation was much faster than NER and therefore was the predominant repair pathway . Active genes were faster repaired by photolyase than were silenced genes providing evidence for an open chromatin structure during repair . The transcribed strands of active genes, but not of inactive genes, were slightly faster repaired by NER providing evidence for transcription-coupled repair by RNAP-I . There was no pronounced inhibition of photoreactivation by RNAP-I in the transcribed strand, which is in contrast to genes transcribed by RNAP-II and suggests different stabilities of RNAP-I and RNAP-II stalled at CPDs.

Genetics, 2002 Jan, 160(1), 123 - 36
Precocious S-phase entry in budding yeast prolongs replicative state and increases dependence upon Rad53 for viability; Sidorova JM et al.; Precocious entry into S phase due to overproduction of G1 regulators can cause genomic instability . The mechanisms of this phenomenon are largely unknown . We explored the consequences of precocious S phase in yeast by overproducing a deregulated form of Swi4 (Swi4-t) . Swi4 is a late G1-specific transcriptional activator that, in complex with Swi6, binds to SCB elements and activates late G1-specific genes, including G1 cyclins . We find that wild-type cells tolerate Swi4-t, whereas checkpoint-deficient rad53-11 cells lose viability within several divisions when Swi4-t is overproduced . Rad53 kinase activity is increased in cells overproducing Swi4-t, indicating activation of the checkpoint . We monitored the transition from G1 to S in cells with Swi4-t and found that there is precocious S-phase entry and that the length of S phase is extended . Moreover, there were more replication intermediates, and firing of at least a subset of origins may have been more extensive in the cells expressing Swi4-t . Our working hypothesis is that Rad53 modulates origin firing based upon growth conditions to optimize the rate of S-phase progression without adversely affecting fidelity . This regulation becomes essential when S phase is influenced by Swi4-t.

Genetics, 2002 Jan, 160(1), 49 - 62
Differential suppression of DNA repair deficiencies of Yeast rad50, mre11 and xrs2 mutants by EXO1 and TLC1 (the RNA component of telomerase); Lewis LK et al.; Rad50, Mre11, and Xrs2 form a nuclease complex that functions in both nonhomologous end-joining (NHEJ) and recombinational repair of DNA double-strand breaks (DSBs) . A search for highly expressed cDNAs that suppress the DNA repair deficiency of rad50 mutants yielded multiple isolates of two genes: EXO1 and TLC1 . Overexpression of EXO1 or TLC1 increased the resistance of rad50, mre11, and xrs2 mutants to ionizing radiation and MMS, but did not increase resistance in strains defective in recombination (rad51, rad52, rad54, rad59) or NHEJ only (yku70, sir4) . Increased Exo1 or TLC1 RNA did not alter checkpoint responses or restore NHEJ proficiency, but DNA repair defects of yku70 and rad27 (fen) mutants were differentially suppressed by the two genes . Overexpression of Exo1, but not mutant proteins containing substitutions in the conserved nuclease domain, increased recombination and suppressed HO and EcoRI endonuclease-induced killing of rad50 strains . exo1 rad50 mutants lacking both nuclease activities exhibited a high proportion of enlarged, G2-arrested cells and displayed a synergistic decrease in DSB-induced plasmid:chromosome recombination . These results support a model in which the nuclease activity of the Rad50/Mre11/Xrs2 complex is required for recombinational repair, but not NHEJ . We suggest that the 5'-3' exo activity of Exo1 is able to substitute for Rad50/Mre11/Xrs2 in rescission of specific classes of DSB end structures . Gene-specific suppression by TLC1, which encodes the RNA subunit of the yeast telomerase complex, demonstrates that components of telomerase can also impact on DSB repair pathways.

Mol Cell, 2002 Jan, 9(1), 31 - 44
Composition and functional characterization of the yeast spliceosomal penta-snRNP; Stevens SW et al.; Pre-mRNA introns are spliced in a macromolecular machine, the spliceosome . For each round of splicing, the spliceosome assembles de novo in a series of ATP-dependent steps involving numerous changes in RNA-RNA and RNA-protein interactions . As currently understood, spliceosome assembly proceeds by addition of discrete U1, U2, and U4/U6*U5 snRNPs to a pre-mRNA substrate to form functional splicing complexes . We characterized a 45S yeast penta-snRNP which contains all five spliceosomal snRNAs and over 60 pre-mRNA splicing factors . The particle is functional in extracts and, when supplied with soluble factors, is capable of splicing pre-mRNA . We propose that the spliceosomal snRNPs associate prior to binding of a pre-mRNA substrate rather than with pre-mRNA via stepwise addition of discrete snRNPs.

Int Rev Cytol, 2002, 212, 133 - 207
Genes involved in the initiation of DNA replication in yeast; Calzada A et al.; Replication and segregation of the information contained in genomic DNA are strictly regulated processes that eukaryotic cells alternate to divide successfully . Experimental work on yeast has suggested that this alternation is achieved through oscillations in the activity of a serine/threonine kinase complex, CDK, which ensures the timely activation of DNA synthesis . At the same time, this CDK-mediated activation sets up the basis of the mechanism that ensures ploidy maintenance in eukaryotes . DNA synthesis is initiated at discrete sites of the genome called origins of replication on which a prereplicative complex (pre-RC) of different protein subunits is formed during the G1 phase of the cell division cycle . Only after pre-RCs are formed is the genome competent to be replicated . Several lines of evidence suggest that CDK activity prevents the assembly of pre-RCs ensuring single rounds of genome replication during each cell division cycle . This review offers a descriptive discussion of the main molecular events that a unicellular eukaryote such as the budding yeast Saccharomyces cerevisiae undergoes to initiate DNA replication.

Biochem J, 2002 Feb 1, 361(Pt 3), 587 - 95
The human homologue of the yeast polyubiquitination factor Ufd2p is cleaved by caspase 6 and granzyme B during apoptosis; Mahoney JA et al.; In the present study, we demonstrate that a human homologue of Ufd2p (a yeast protein that catalyses the formation of long polyubiquitin chains, and is implicated in responses to environmental stress), UFD2 (ubiquitin fusion degradation protein-2), is cleaved during apoptosis induced by multiple stimuli, including UVB irradiation, Fas ligation, staurosporine treatment and cytotoxic lymphocyte granule-induced death . Caspase 6 and granzyme B efficiently cleave UFD2 {k(cat)/K(m)=(4-5) x 10(4) M(-1) x s(-1)} at Asp(123), whereas caspases 3 and 7 cleave UFD2 approx . 10-fold less efficiently immediately upstream at Asp(109) . Thus UFD2 is added to the growing list of proteins with closely spaced caspase and granzyme B cleavage sites, suggesting the presence of a previously unrecognized, conserved motif . Both cleavage sites are contained and conserved within a novel 300-amino-acid N-terminal domain present in apparent UFD2 orthologues in mice and zebrafish, but absent in all UFD2 family members in lower eukaryotes . Full-length recombinant UFD2 exhibited ubiquitin-protein ligase ('E3')-like ubiquitination activity in vitro, but this activity was abolished in recombinant UFD2 truncated at the granzyme B/caspase 6 cleavage site . Cleavage of UFD2 by caspases or granzyme B within this putative regulatory N-terminal domain might have important functional consequences within the apoptotic cascade.

J Cell Sci, 2002 Jan 1, 115(Pt 1), 217 - 26
Casein kinase I controls a late step in the endocytic trafficking of yeast uracil permease; Marchal C et al.; The modification of yeast uracil permease by phosphorylation at the plasma membrane is a key mechanism for regulating transporter endocytosis . Uracil permease is phosphorylated at several serine residues within a well characterized PEST sequence . The phosphorylation of these residues facilitates the ubiquitination and internalization of the permease . Following endocytosis, the permease is targeted to the lysosome/vacuole for proteolysis . We have shown that in casein kinase 1 (CK1)-deficient cells, the permease is poorly phosphorylated, poorly ubiquitinated and that Yck activity may play a direct role in phosphorylating the permease . We show here that CK1-deficient cells accumulated permease that was subjected to endocytosis in an internal compartment on its way to the vacuole . Uracil permease, produced as a fusion protein with green fluorescent protein in CK1-deficient cells, was detected in dots adjacent to the vacuole . These dots probably correspond to the late endosome/prevacuolar compartment because they were partially colocalized with the Pep12p marker . This accumulation was abolished by mutations affecting the adaptor-related complex, AP-3 . The CPY and ALP pathways to the vacuole were both unaffected in CK1-deficient cells . Our analysis provides the first evidence that CK1 is important for the delivery of proteins to the vacuole after endocytosis.

J Biol Chem, 2002 Apr 5, 277(14), 12288 - 93 Epub 2002 Jan 18.
The yeast Vps10p cytoplasmic tail mediates lysosomal sorting in mammalian cells and interacts with human GGAs; Dennes A et al.; Yeast Vps10p is a receptor for transport of the soluble vacuolar hydrolase carboxypeptidase Y to the lysosome-like vacuole . Its functional equivalents in mammalian cells are the mannose 6-phosphate receptors that mediate sorting to lysosomes of mannose 6-phosphate-containing lysosomal proteins . A chimeric receptor was constructed by substituting the cytoplasmic domain of M(r) 300,000 mannose 6-phosphate receptor with the Vps10p cytoplasmic tail . Expression of the chimera in cells lacking endogenous mannose 6-phosphate receptors resulted in a subcellular receptor distribution and an efficiency in sorting of lysosomal enzymes similar to that of the wild type M(r) 300,000 mannose 6-phosphate receptor . Moreover, the cytoplasmic tail of the Vps10p was found to interact with GGA1 and GGA2, two mammalian members of a recently discovered family of clathrin-binding cytosolic proteins that participate in trans-Golgi network-endosome trafficking in both mammals and yeast . Our findings suggest a conserved machinery for Golgi-endosome/vacuole sorting and may serve as a model for future studies of yeast proteins.

Med Mycol, 2001, 39 Suppl 1, 111 - 21
The yeast cell-wall salvage pathway; Popolo L et al.; The integrity of the cell wall depends on the synthesis and correct assembly of its individual components . Several environmental factors, such as temperature up-shift, treatments with mating factors or with specific cell wall-perturbing drugs, or genetic factors, such as inactivation of cell wall-related genes (for example FKS1 or GAS1) can impair construction of the cell wall . As the cell wall is essential for preserving the osmotic integrity of the cell, several responses are triggered in response to cell-wall damage . This review focuses on the activation of salvage pathways that guarantee cell survival through remodeling of the extracellular matrix . These researches have useful implication for the study of similar pathways in human fungal pathogens, and for the evaluation of the efficacy of new antifungal drugs.

J Biol Chem, 2002 Apr 5, 277(14), 11645 - 52 Epub 2002 Jan 16.
Essential hydrophilic carboxyl-terminal regions including cysteine residues of the yeast stretch-activated calcium-permeable channel Mid1; Maruoka T et al.; The yeast Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca(2+)-permeable nonselective cation channel composed of 548 amino acid residues . A physiological role of the Mid1 channel is known to maintain the viability of yeast cells exposed to mating pheromone, but its structural basis remains to be clarified . To solve this problem, we identified the mutation sites of mid1 mutant alleles generated by in vivo ethyl methanesulfonate mutagenesis and found that two mid1 alleles have nonsense mutations at the codon for Trp(441), generating a truncated Mid1 protein lacking two-thirds of the intracellular carboxyl-terminal region from Asn(389) to Thr(548) . In vitro random mutagenesis with hydroxylamine also showed that the carboxyl-terminal region is essential . To identify the functional portion of the carboxyl-terminal region in detail, we performed a progressive carboxyl-terminal truncation followed by functional analyses and found that the truncated protein produced from the mid1 allele bearing the amber mutation at the codon for Phe(522) (F522Am) complemented the mating pheromone-induced death phenotype of the mid1 mutant and increased its Ca(2+) uptake activity to a wild-type level, whereas N521Am did not . This result indicates that the carboxyl-terminal domain spanning from Asn(389) to Asn(521) is required for Mid1 function . Interestingly, this domain is cysteine-rich, and alanine-scanning mutagenesis revealed that seven out of 10 cysteine residues are unexchangeable . These results clearly indicate that the carboxyl-terminal domain including the cysteine residues is important for Mid1 function.

Funct Integr Genomics, 2000 Nov, 1(3), 186 - 92
Dynamic architecture of the yeast cell cycle uncovered by wavelet decomposition of expression microarray data; Klevecz RR; Wavelet analysis has been applied to yeast cell cycle expression microchip data to reveal large-scale temporal structures and ubiquitous oscillations in mRNA levels . Discrete intervals in time within the cell cycle when expression levels changed were visualized as contour maps in which points of transition in gene expression among all 6178 genes were plotted as a function of cell cycle time . Time-frequency analysis using wavelet transforms supported the direct visualization and led to the conclusion that the predominant period is not the cell cycle but a higher frequency, 40 min, submultiple of the cycle . Each of the 6178 gene expression profiles was dissected by wavelet decomposition into all permitted frequencies from the Nyquist limit to roughly twice the cell cycle length . Transitions associated with maximum up- or down-regulation of mRNA levels appear as bands at circa 40-min intervals, half the length of the cycle, through two cell cycles . More than two thirds of the genes, including many of the cyclins, showed this half-cycle periodicity . Gene expression and events within the yeast cell cycle may be regulated by an attractor whose fundamental period is an emergent property of dynamic interactions within the yeast transcriptome.

Funct Integr Genomics, 2000 Sep, 1(2), 99 - 113
Functional analysis of the yeast genome; Ross-Macdonald P; Just as Saccharomyces cerevisiae itself provides a model for so many processes essential to eukaryotic life, we anticipate that the methods and the mindset that have moved yeast biological research "beyond the genome" provide a prototype for making similar progress in other organisms . In this review I describe the experimental processes, results and utility of the current large-scale experimental approaches that use genomic data to provide a functional analysis of the yeast genome.

J Biol Chem, 2002 Mar 22, 277(12), 10547 - 54 Epub 2002 Jan 15.
Retention of the yeast Sac1p phosphatase in the endoplasmic reticulum causes distinct changes in cellular phosphoinositide levels and stimulates microsomal ATP transport; Konrad G et al.; The yeast phosphoinositide phosphatase Sac1p localizes to endoplasmic reticulum (ER) and Golgi membranes and has compartment-specific functions in these organelles . In this study we analyzed in detail the topology of Sac1p . Our data show that Sac1p is a type II transmembrane protein with a large N-terminal cytosolic domain, which is anchored in the membrane by the two potential transmembrane helices near the C terminus . Based on this topology, we created a mutation that caused retention of Sac1p in the ER and as a consequence showed specific alterations in cellular phosphoinositide levels . Our results suggest that Sac1p controls a pool of phosphatidylinositol 3-phosphate and phosphatidylinositol 4-phosphate in the ER . Retention of Sac1p in the ER also stimulates ATP transport into the ER lumen but causes the same Golgi-specific defects that are seen in a sac1 null mutant . Taken together this study provides evidence that Sac1p is an important 4-phosphatase in the ER controlling different aspects of ER-based protein processing and secretion.

Genome Biol . 2001;2(12):RESEARCH0052 . Epub 2001 Nov 15.
Development of an optimized interaction-mating protocol for large-scale yeast two-hybrid analyses; Soellick TR et al.; BACKGROUND: Protein-protein interactions have decisive roles in almost all aspects of the structural and functional organization of cells . But in spite of the increasing amount of complete genome sequence data, the ability to predict protein function from sequences alone is limited . Therefore comprehensive analysis of protein-protein interactions, as derived from the yeast two-hybrid mating system, will yield valuable information for functional biology on a proteomic scale . RESULTS: We have developed an optimized interaction mating protocol for the yeast two-hybrid system, which gives increased mating efficiencies . This significantly reduces the effort and cost of cDNA library screening and allows multiple parallel approaches . Improved preincubation conditions before mating, and optimal cell densities and cell ratios enable almost quantitative mating of the yeast cells carrying the cDNA library . We have proved the applicability of this technology using 20 bait proteins to screen an Arabidopsis thaliana cDNA library, in spite of bait-dependent variations in mating efficiency . CONCLUSIONS: The improved yeast two-hybrid interaction-mating protocol presented here allows the multiple parallel screening of cDNA libraries . It can be carried out without specialized equipment and has the potential to be standardized and automated.

Yeast, 2002 Jan 30, 19(2), 141 - 9
Marker-fusion PCR for one-step mutagenesis of essential genes in yeast; Kitazono AA et al.; We describe a one-step gene replacement method based on fusion PCR that can be used to mutagenize essential genes at their endogenous locus . Marker-fusion PCR can facilitate transfer of alleles between strains as well as PCR-based techniques, such as site-directed and error-prone PCR mutagenesis, all without cloning or strain constructions . With this method, PCR is used to fuse a mutagenized fragment to an overlapping fragment containing a selectable marker flanked by regions of homology to the target . By transforming yeast with these PCR products, specific mutations are introduced at the endogenous locus through homologous recombination . We tested the 'marker-fusion PCR' method using the budding yeast CDC28 gene and were able to efficiently introduce site-directed mutations and integrate genomic or plasmid-borne mutant alleles . As a further application for this method, we used a spiked oligonucleotide to randomize the coding sequence for a single domain of CDC28 and were able to construct highly mutagenized libraries for this region .

Nucleic Acids Res . 2002 Jan 15;30(2):E8.
A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast; Noskov V et al.; Transformation-associated recombination (TAR) is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library . This technique involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5' and 3' gene targeting sequences (hooks) . Typically, TAR cloning produces positive YAC recombinants at a frequency of approximately 0.5%; the positive clones are identified by PCR or colony hybridization . This paper describes a novel TAR cloning procedure that selects positive clones by positive and negative genetic selection . This system utilizes a TAR vector with two targeting hooks, HIS3 as a positive selectable marker, URA3 as a negative selectable marker and a gene-specific sequence called a loop sequence . The loop sequence lies distal to a targeting hook sequence in the chromosomal target, but proximal to the targeting hook and URA3 in the TAR vector . When this vector recombines with chromosomal DNA at the gene-specific targeting hook, the recombinant YAC product carries two copies of the loop sequence, therefore, the URA3 negative selectable marker becomes mitotically unstable and is lost at high frequency by direct repeat recombination involving the loop sequence . Positive clones are identified by selecting against URA3 . This method produces positive YAC recombinants at a frequency of approximately 40% . This novel TAR cloning method provides a powerful tool for structural and functional analysis of complex genomes.

Infection, 2001 Dec, 29(6), 342 - 4
Safety and immunogenicity of a yeast-derived recombinant hepatitis B vaccine in Bulgarian newborns; Kojouharova M et al.; BACKGROUND: In 1998, Bulgaria adopted a recombinant DNA yeast-derived hepatitis B (HB) vaccine (Euvax B) for universal vaccination of all Bulgarian newborns on a 0-1-6 month schedule, the first dose to be given within 24 h of birth . MATERIALS AND METHODS: We evaluated the safety, immunogenicity and effectiveness of this vaccine in over 40,000 healthy infants from July 1998 to December 1999 . Standard safety information was collected for all infants vaccinated, subsets being followed for solicited local and systemic adverse events (n = 200) and antibodies to HB surface antigen (anti-HBsAg) 1-3 months after the third dose (n = 140) . RESULTS: No serous adverse events were registered for any vaccinee, solicited local reactions were rare (< 1.5%), mild and transient . The overall geometric mean titer (GMT) was 1,012 mIU/ml (95% CI: 786; 1,302), the seroprotection rate being 98.6% . CONCLUSION: These surveillance data, obtained under the conditions of universal infant immunization show the novel recombinant HB vaccine, Euvax B, is safe and well-tolerated with an immunogenicity similar to other recombinant HB vaccines.

Biochim Biophys Acta, 2001 Dec 30, 1534(2-3), 65 - 77
Regulation of vesicle trafficking, transcription, and meiosis: lessons learned from yeast regarding the disparate biologies of phosphatidylcholine; Howe AG et al.; Phosphatidylcholine (PtdCho) is the major phospholipid present in eukaryotic cell membranes generally comprising 50% of the phospholipid mass of most cells and their requisite organelles . PtdCho has a major structural role in maintaining cell and organelle integrity, and thus its synthesis must be tightly monitored to ensure appropriate PtdCho levels are present to allow for its coordination with cell growth regulatory mechanisms . One would also expect that there needs to be coordinated regulation of PtdCho synthesis with its transport from its site of synthesis to cellular organelles to ensure organellar structures and functions are maintained . Each of these processes need to be intimately coordinated with cellular growth decision making processes . To this end, it has recently been revealed that ongoing PtdCho synthesis is required for global transcriptional regulation of phospholipid synthesis . PtdCho is also a major component of intracellular transport vesicles and the synthesis of PtdCho is intimately involved in the regulation of vesicle transport from the Golgi apparatus to the cell surface and the vacuole (yeast equivalent of the mammalian lysosome) . This review details some of the more recent advances in our knowledge concerning the role of PtdCho in the regulation of global lipid homeostasis through (i) its restriction of the trafficking of intracellular vesicles that distribute lipids and proteins from their sites of synthesis to their ultimate cellular destinations, (ii) its regulation of specific transcriptional processes that coordinate lipid biosynthetic pathways, and (iii) the role of PtdCho catabolism in the regulation of meiosis . Combined, these regulatory roles for PtdCho ensure vesicular, organellar, and cellular membrane biogenesis occur in a coordinated manner.

FEMS Microbiol Lett, 2002 Jan 2, 206(1), 121 - 5
Heme-regulated expression of two yeast acyl-CoA:sterol acyltransferases is involved in the specific response of sterol esterification to anaerobiosis; Valachovic M et al.; Sterol esterification in Saccharomyces cerevisiae is catalyzed by two acyl-CoA:sterol acyltransferases encoded by the genes ARE1 and ARE2 . Using double mutants in the HEM1 gene and individual ARE genes we demonstrated that the relative contribution of these two enzymes to sterol esterification was dependent on cellular heme status . Observed changes in sterol esterification could be explained by a different effect of heme on the transcription of both genes: while the ARE1 transcript level was elevated in heme-deficient and anaerobic cells, the ARE2 gene transcript was more abundant in aerobic cells competent for heme synthesis . Our results indicate that transcriptional regulation of ARE genes by heme and specific substrate preferences of Are1p and Are2p may be involved in the adaptation of yeast sterol metabolism to hypoxia.

Mikrobiol Z, 2001 May-Jun, 63(3), 57 - 64
{Use of associative cultures of cyanobacteria for tertiary treatment of sewage from yeast and alcohol plants}; Koshel' MI et al.; The associative cultures of cyanobacteria were selected which may be used for development of technology of tertiary treatment of sewage for yeast and alcohol production plants . Growth parameters of associations were studied during periodic and continuous cultivation . When studying the influence of flowing speed for some exponents of treatment it was shown that cultivation of cyanobacteria should be carried out with the change of volume not more than 0.01 h . Final treatment 0.010 h-1 in order to return sewage to natural water reservoirs may be carried out by means of Oscillatoria sp . in bioponds.

Proc Natl Acad Sci U S A, 2002 Jan 22, 99(2), 649 - 54 Epub 2002 Jan 08.
Transcription-coupled repair in RNA polymerase I-transcribed genes of yeast; Conconi A et al.; Nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in the individual strands of transcriptionally active and inactive ribosomal genes of yeast . Ribosomal genes (rDNA) are present in multiple copies, but only a fraction of them is actively transcribed . Restriction enzyme digestion was used to specifically release the transcriptionally active fraction from yeast nuclei, and selective psoralen crosslinking was used to distinguish between active and inactive rDNA chromatin . Removal of CPDs was followed in both rDNA populations, and the data clearly show that strand-specific repair occurs in transcriptionally active rDNA while being absent in the inactive rDNA fraction . Thus, transcription-coupled repair occurs in RNA polymerase I-transcribed genes in yeast . Moreover, the nontranscribed strand of active rDNA is repaired faster than either strand of inactive rDNA, implying that NER has preferred access to the active, non-nucleosomal rDNA chromatin . Finally, restriction enzyme accessibility to active rDNA varies during NER, suggesting that there is a change in ribosomal gene chromatin structure during or soon after CPD removal.

Genes Dev, 2002 Jan 1, 16(1), 101 - 13
Ctf3p, the Mis6 budding yeast homolog, interacts with Mcm22p and Mcm16p at the yeast outer kinetochore; Measday V et al.; The budding yeast kinetochore is composed of an inner and outer protein complex, which binds to centromere (CEN) DNA and attaches to microtubules . We performed a genetic synthetic dosage lethality screen to identify novel kinetochore proteins in a collection of chromosome transmission fidelity mutants . Our screen identified several new kinetochore-related proteins including YLR381Wp/Ctf3p, which is a member of a conserved family of centromere-binding proteins . Ctf3p interacts with Mcm22p, Mcm16p, and the outer kinetochore protein Ctf19p . We used chromatin immunoprecipitation to demonstrate that Ctf3p, Mcm22p, and Mcm16p bind to CEN DNA in a Ctf19p-dependent manner . In addition, Ctf3p, Mcm22p, and Mcm16p have a localization pattern similar to other kinetochore proteins . The fission yeast Ctf3p homolog, Mis6, is required for loading of a CENP-A centromere specific histone, Cnp1, onto centromere DNA . We find however that Ctf3p is not required for loading of the budding yeast CENP-A homolog, Cse4p, onto CEN DNA . In contrast, Ctf3p and Ctf19p fail to bind properly to the centromere in a cse4-1 mutant strain . We conclude that the requirements for CENP-A loading onto centromere DNA differ in fission versus budding yeast.

Nat Cell Biol, 2001 Dec, 3(12), E277 - 86
The evolution of diverse biological responses to DNA damage: insights from yeast and p53; Wahl GM et al.; The cellular response to ionizing radiation provides a conceptual framework for understanding how a yeast checkpoint system, designed to make binary decisions between arrest and cycling, evolved in a way as to allow reversible arrest, senescence or apoptosis in mammals . We propose that the diversity of responses to ionizing radiation in mammalian cells is possible because of the addition of a new regulatory control module involving the tumour-suppressor gene p53 . We review the complex mechanisms controlling p53 activity and discuss how the p53 regulatory module enables cells to grow, arrest or die by integrating DNA damage checkpoint signals with the response to normal mitogenic signalling and the aberrant signalling engendered by oncogene activation.

Nat Cell Biol, 2001 Dec, 3(12), 1051 - 9
MAP kinase dynamics in response to pheromones in budding yeast; van Drogen F et al.; Although scaffolding is a major regulator of mitogen-activated protein kinase (MAPK) pathways, scaffolding proteins are poorly understood . During yeast mating, MAPK Fus3p is phosphorylated by MAPKK Ste7p, which is activated by MAPKKK Ste11p . This MAPK module interacts with the scaffold molecule Ste5p . Here we show that Ste11p and Ste7p were predominantly cytoplasmic proteins, while Ste5p and Fus3p were found in the nucleus and the cytoplasm . Ste5p, Ste7p and Fus3p also localized to tips of mating projections in pheromone-treated cells . Using fluorescence recovery after photobleaching (FRAP), we demonstrate that Fus3p rapidly shuttles between the nucleus and the cytoplasm independently of pheromones, Fus3p phosphorylation and Ste5p . Membrane-bound Ste5p can specifically recruit Fus3p and Ste7p to the cell cortex . Ste5p remains stably bound at the plasma membrane, unlike activated Fus3p, which dissociates from Ste5p and translocates to the nucleus.

J Cell Biol, 2002 Jan 7, 156(1), 29 - 34 Epub 2002 Jan 07.
Internal Ca(2+) release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue; Denis V et al.; Calcium ions, present inside all eukaryotic cells, are important second messengers in the transduction of biological signals . In mammalian cells, the release of Ca(2+) from intracellular compartments is required for signaling and involves the regulated opening of ryanodine and inositol-1,4,5-trisphosphate (IP3) receptors . However, in budding yeast, no signaling pathway has been shown to involve Ca(2+) release from internal stores, and no homologues of ryanodine or IP3 receptors exist in the genome . Here we show that hyperosmotic shock provokes a transient increase in cytosolic Ca(2+) in vivo . Vacuolar Ca(2+), which is the major intracellular Ca(2+) store in yeast, is required for this response, whereas extracellular Ca(2+) is not . We aimed to identify the channel responsible for this regulated vacuolar Ca(2+) release . Here we report that Yvc1p, a vacuolar membrane protein with homology to transient receptor potential (TRP) channels, mediates the hyperosmolarity induced Ca(2+) release . After this release, low cytosolic Ca(2+) is restored and vacuolar Ca(2+) is replenished through the activity of Vcx1p, a Ca(2+)/H(+) exchanger . These studies reveal a novel mechanism of internal Ca(2+) release and establish a new function for TRP channels.

J Biol Chem, 2002 Mar 22, 277(12), 10194 - 200 Epub 2002 Jan 07.
Mutations in the yeast LCB1 and LCB2 genes, including those corresponding to the hereditary sensory neuropathy type I mutations, dominantly inactivate serine palmitoyltransferase; Gable K et al.; It was recently demonstrated that mutations in the human SPTLC1 gene, encoding the Lcb1p subunit of serine palmitoyltransferase (SPT), cause hereditary sensory neuropathy type I . As a member of the subfamily of pyridoxal 5'-phosphate enzymes known as the alpha-oxoamine synthases, serine palmitoyltransferase catalyzes the committed step of sphingolipid synthesis . The residues that are mutated to cause hereditary sensory neuropathy type I reside in a highly conserved region of Lcb1p that is predicted to be a catalytic domain of Lcb1p on the basis of alignments with other members of the alpha-oxoamine synthase family . We found that the corresponding mutations in the LCB1 gene of Saccharomyces cerevisiae reduce serine palmitoyltransferase activity . These mutations are dominant and decrease serine palmitoyltransferase activity by 50% when the wild-type and mutant LCB1 alleles are coexpressed . We also show that serine palmitoyltransferase is an Lcb1p small middle dotLcb2p heterodimer and that the mutated Lcb1p proteins retain their ability to interact with Lcb2p . Modeling studies suggest that serine palmitoyltransferase is likely to have a single active site that lies at the Lcb1p small middle dotLcb2p interface and that the mutations in Lcb1p reside near the lysine in Lcb2p that is expected to form the Schiff's base with the pyridoxal 5'-phosphate cofactor . Furthermore, mutations in this lysine and in a histidine residue that is also predicted to be important for pyridoxal 5'-phosphate binding to Lcb2p also dominantly inactivate SPT similar to the hereditary sensory neuropathy type 1-like mutations in Lcb1p.

Biochemistry, 2002 Jan 15, 41(2), 451 - 61
Solvent kinetic isotope effects monitor changes in hydrogen bonding at the active center of yeast pyruvate decarboxylase concomitant with substrate activation: the substituent at position 221 can control the state of activation; Wei W et al.; Substrate activation of yeast pyruvate decarboxylase has been studied extensively in the authors' laboratories providing strong evidence that interaction of substrate with residue C221 provides the trigger, and the information is then transmitted along the C221 to H92 to E91 to W412 to G413 pathway to the 4'-amino nitrogen of the thiamin diphosphate cofactor . Earlier, it was found that the C221S substitution reduced the Hill coefficient from 2.0 to 0.8-0.9, the C221A substitution to 1.0, even though C221 is located on the beta domain some 20 A from the active center thiamin diphosphate cofactor, which is at the interface of the alpha and gamma domains . Here are reported experiments on the C221D/C222A and C221E/C222A variants, in which a negative charge is built onto the C221 side chain, to better mimic the effect of a pyruvate molecule covalently bonded to C221 as a thiohemiketal . Both variants were purified to an optimal activity of 70% of the wild-type enzyme, higher activity than that with the earlier uncharged substitutions at this position . The Hill coefficient for both variants is exactly 1.0 . The deuterium solvent kinetic isotope effects (SKIE) on k(cat) for these variants were similar to that for the wild-type enzyme and the C221A/C222A variant, suggesting that starting with the first irreversible step (decarboxylation) the rate-limiting transition states are very similar for all of these enzyme forms . In contrast, such SKIE on k(cat)/K(m) are quite different for the C221A/C222A variant (0.62) than for the C221E/C222A or C221D/C222A variants (0.80-0.82), clearly indicating the effect of the C221 substitutions on transition states starting with the binding of the first substrate to the enzyme and terminating with the decarboxylation step . The results provide strong additional evidence for the involvement of residue C221 in the substrate activation process and suggest that the C221D (C221E) substitution shifts the enzyme into a conformation that resembles the activated conformation . A comparison with SKIE for the wild-type enzyme provides insight to changes in hydrogen bonding at the active center as a result of substrate activation.

Nat Cell Biol, 2002 Jan, 4(1), 89 - 93
Recruitment of cohesin to heterochromatic regions by Swi6/HP1 in fission yeast; Nonaka N et al.; Fission yeast centromeres, like those of higher eukaryotes, are composed of repeated DNA structures and associated heterochromatin protein complexes, that have a critical function in the faithful segregation of chromosomes during cell division . Cohesin protein complexes, which are essential for sister-chromatid cohesion and proper chromosome segregation, are enriched at centromeric repeats . We have identified a functional and physical link between heterochromatin and cohesin . We find that the preferential localization of cohesins at the centromeric repeats is dependent on Swi6, a conserved heterochromatin protein that is required for proper kinetochore function . Cohesin is also enriched at the mating-type heterochromatic region in a manner that depends on Swi6 and is required to preserve the genomic integrity of this locus . We provide evidence that a cohesin subunit Psc3 interacts with Swi6 and its mouse homologue HP1 . These data define a conserved function of Swi6/HP1 in recruitment of cohesin to heterochromatic regions, promoting the proper segregation of chromosomes.

Genome Res, 2002 Jan, 12(1), 190 - 7
Linker-mediated recombinational subcloning of large DNA fragments using yeast; Raymond CK et al.; The homologous recombination pathway in yeast is an ideal tool for the sequence-specific assembly of plasmids . Complementary 80-nucleotide oligonucleotides that overlap a vector and a target fragment were found to serve as efficient recombination linkers for fragment subcloning . Using electroporation, single-stranded 80-mers were adequate for routine plasmid construction . A cycloheximide-based counterselection was introduced to increase the specificity of cloning by homologous recombination relative to nonspecific vector background . Reconstruction experiments suggest this counterselection increased cloning specificity by 100-fold . Cycloheximide counterselection was used in conjunction with 80-bp linkers to subclone targeted regions from bacterial artificial chromosomes . This technology may find broad application in the final stages of completing the Human Genome Sequencing Project and in applications of BAC clones to the functional analysis of complex genomes.

Genetics, 2001 Dec, 159(4), 1467 - 78
Counteracting regulation of chromatin remodeling at a fission yeast cAMP response element-related recombination hotspot by stress-activated protein kinase, cAMP-dependent kinase and meiosis regulators; Mizuno K et al.; In fission yeast, an ATF/CREB-family transcription factor Atf1-Pcr1 plays important roles in the activation of early meiotic processes via the stress-activated protein kinase (SAPK) and the cAMP-dependent protein kinase (PKA) pathways . In addition, Atf1-Pcr1 binds to a cAMP responsive element (CRE)-like sequence at the site of the ade6-M26 mutation, which results in local enhancement of meiotic recombination and chromatin remodeling . Here we studied the roles of meiosis-inducing signal transduction pathways in M26 chromatin remodeling . Chromatin analysis revealed that persistent activation of PKA in meiosis inhibited M26 chromatin remodeling, suggesting that the PKA pathway represses M26 chromatin remodeling . The SAPK pathway activated M26 chromatin remodeling, since mutants lacking a component of this pathway, the Wis1 or Spc1/Sty1 kinases, had no M26 chromatin remodeling . M26 chromatin remodeling also required the meiosis regulators Mei2 and Mei3 but not the subsequently acting regulators Sme2 and Mei4, suggesting that induction of M26 chromatin remodeling needs meiosis-inducing signals before premeiotic DNA replication . Similar meiotic chromatin remodeling occurred meiotically around natural M26 heptamer sequences . These results demonstrate the coordinated action of genetic and physiological factors required to remodel chromatin in preparation for high levels of meiotic recombination and eukaryotic cellular differentiation.

Cell, 2001 Dec 28, 107(7), 917 - 27
Yeast DNA polymerase eta utilizes an induced-fit mechanism of nucleotide incorporation; Washington MT et al.; DNA polymerase eta (Poleta) is unique among eukaryotic DNA polymerases in its proficient ability to replicate through distorting DNA lesions, and Poleta synthesizes DNA with a low fidelity . Here, we use pre-steady-state kinetics to investigate the mechanism of nucleotide incorporation by Poleta and show that it utilizes an induced-fit mechanism to selectively incorporate the correct nucleotide . Poleta discriminates poorly between the correct and incorrect nucleotide at both the initial nucleotide binding step and at the subsequent induced-fit conformational change step, which precedes the chemical step of phosphodiester bond formation . This property enables Poleta to bypass lesions with distorted DNA geometries, and it bestows upon the enzyme a low fidelity.

J Mol Biol, 2002 Jan 11, 315(2), 213 - 27
Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates; Galani D et al.; The Saccharomyces cerevisiae non-Mendelian factor {URE3} propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2 . The N-terminal prion domain (PrD) of Ure2 is required for prion activity in vivo and amyloid formation in vitro . However, the molecular mechanism of the prion-like activity remains obscure . Here we measure the kinetics of folding of Ure2 and two N-terminal variants that lack all or part of the PrD . The kinetic folding behaviour of the three proteins is identical, indicating that the PrD does not change the stability, rates of folding or folding pathway of Ure2 . Both unfolding and refolding kinetics are multiphasic . An intermediate is populated during unfolding at high denaturant concentrations resulting in the appearance of an unfolding burst phase and "roll-over" in the denaturant dependence of the unfolding rate constants . During refolding the appearance of a burst phase indicates formation of an intermediate during the dead-time of stopped-flow mixing . A further fast phase shows second-order kinetics, indicating formation of a dimeric intermediate . Regain of native-like fluorescence displays a distinct lag due to population of this on-pathway dimeric intermediate . Double-jump experiments indicate that isomerisation of Pro166, which is cis in the native state, occurs late in refolding after regain of native-like fluorescence . During protein refolding there is kinetic partitioning between productive folding via the dimeric intermediate and a non-productive side reaction via an aggregation prone monomeric intermediate . In the light of this and other studies, schemes for folding, aggregation and prion formation are proposed .

Biochem Biophys Res Commun, 2002 Jan 11, 290(1), 457 - 62
Soybean ascorbate peroxidase suppresses Bax-induced apoptosis in yeast by inhibiting oxygen radical generation; Moon H et al.; Bax, a mammalian proapoptotic member of the Bcl-2 family, can induce cell death when expressed in yeast or plant cells . To identify plant Bax inhibitors, we cotransformed a soybean cDNA library and the Bax gene into yeast cells and screened for expressed genes that prevented Bax-induced apoptosis . From the Bax-inhibiting genes isolated, ascorbate peroxidase (sAPX) was selected for characterization . The transcription of sAPX in plants was specifically induced by oxidative stress . Moreover, overexpression of sAPX partially suppressed the H(2)O(2)-sensitive phenotype of yeast cytosolic catalase T (Deltactt)- and thermosensitive phenotype of cytochrome c peroxidase (Deltaccp)-deleted mutant cells . Examination of reactive oxygen species (ROS) production using the fluorescence method of dihydrorhodamine 123 oxidation revealed that expression of Bax in yeast cells generated ROS, which was greatly reduced by coexpression with sAPX . Our results collectively suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Bax-induced cell death in yeast . (c)2002 Elsevier Science.

Appl Microbiol Biotechnol, 2001 Dec, 57(5-6), 697 - 701
Cell surface-engineered yeast displaying a histidine oligopeptide (hexa-His) has enhanced adsorption of and tolerance to heavy metal ions; Kuroda K et al.; A histidine oligopeptide (hexa-His) with the ability to chelate divalent heavy metal ions was displayed on the yeast cell surface for the purpose of enhanced adsorption of heavy metal ions . We genetically fused a hexa-His-encoding gene with the gene encoding the C-terminal half of alpha-agglutinin that includes a glycosylphosphatidylinositol anchor attachment signal sequence and attached the hexa-His peptide on the cell wall of Saccharomyces cerevisiae . This surface-engineered yeast adsorbed three to eight times more copper ions than the parent strain and was more resistant to copper (4 mM) than the parent (below 1 mM at pH 7.8) . It was possible to recover about a half of the copper ions adsorbed by whole cells with EDTA treatment without disintegrating the cells . Thus, we succeeded in constructing a novel yeast cell with both tolerance to toxic contaminants and enhanced adsorption of metal ions onto the cell surface.

J Cell Biol, 2002 Jan 7, 156(1), 23 - 8 Epub 2002 Jan 03.
Polo boxes and Cut23 (Apc8) mediate an interaction between polo kinase and the anaphase-promoting complex for fission yeast mitosis; May KM et al.; The fission yeast plo1(+) gene encodes a polo-like kinase, a member of a conserved family of kinases which play multiple roles during the cell cycle . We show that Plo1 kinase physically interacts with the anaphase-promoting complex (APC)/cyclosome through the noncatalytic domain of Plo1 and the tetratricopeptide repeat domain of the subunit, Cut23 . A new cut23 mutation, which specifically disrupts the interaction with Plo1, results in a metaphase arrest . This arrest can be rescued by high expression of Plo1 kinase . We suggest that this physical interaction is crucial for mitotic progression by targeting polo kinase activity toward the APC.

J Biol Chem, 2002 Mar 15, 277(11), 8979 - 88 Epub 2002 Jan 03.
Mutational analysis of the subunit C (Vma5p) of the yeast vacuolar H+-ATPase; Curtis KK et al.; Subunit C is a V(1) sector subunit found in all vacuolar H(+)-ATPases (V-ATPases) that may be part of the peripheral stalk connecting the peripheral V(1) sector with the membrane-bound V(0) sector of the enzyme (Wilkens, S., Vasilyeva, E., and Forgac, M . (1999) J . Biol . Chem . 274, 31804--31810) . To elucidate subunit C function, we performed random and site-directed mutagenesis of the yeast VMA5 gene . Site-directed mutations in the most highly conserved region of Vma5p, residues 305--325, decreased catalytic activity of the V-ATPase by up to 48% without affecting assembly . A truncation mutant (K360stop) identified by random mutagenesis suggested a small region near the C terminus of the protein (amino acids 382--388) might be important for subunit stability . Site-directed mutagenesis revealed that three aromatic amino acids in this region (Tyr-382, Phe-385, and Tyr-388) in addition to four other conserved aromatic amino acids (Phe-260, Tyr-262, Phe-296, Phe-300) are essential for stable assembly of V(1) with V(0), although alanine substitutions at these positions support some activity in vivo . Surprisingly, three mutations (F260A, Y262A, and F385A) greatly decrease the stability of the V-ATPase in vitro but increase its k(cat) for ATP hydrolysis and proton transport by at least 3-fold . The peripheral stalk of V-ATPases must balance the stability essential for productive catalysis with the dynamic instability involved in regulation; these three mutations may perturb that balance.

J Biol Chem, 2002 Mar 8, 277(10), 8178 - 86 Epub 2002 Jan 02.
The SANT domain of Ada2 is required for normal acetylation of histones by the yeast SAGA complex; Sterner DE et al.; Transcription is regulated through chromatin remodeling and histone modification, mediated by large protein complexes . Histone and nucleosome interaction has been shown to be mediated by specific chromatin domains called bromodomains and chromodomains . Here we provide evidence for a similar function of two additional domains within the yeast SAGA complex, containing the histone acetyltransferase Gcn5 . We have analyzed deletion and substitution mutations within Gcn5 and Ada2, an interacting protein within SAGA, and have identified substrate recognition functions within the SANT domain of Ada2 and regions of the histone acetyltransferase domain of Gcn5 that are distinct from catalytic function itself . These results suggest that histone and nucleosomal substrate recognition by SAGA involves multiple conserved domains and proteins, beyond those that have been previously identified.

Fresenius J Anal Chem, 2001 Nov, 371(6), 775 - 81
Two-dimensional gel electrophoresis of selenized yeast and autoradiography of 75Se-containing proteins; Chery CC et al.; Two-dimensional high-resolution gel electrophoresis (2DE) has been applied to the fractionation of 75Se-containing proteins in yeast, grown in 75Se-containing medium, and autoradiography was used for detection of the 75Se-containing proteins . Gel filtration and ultrafiltration were used to check whether the selenium side-chains were stable in the presence of the chemicals used for lysis and 2DE . The mass distribution of the selenium-containing proteins was estimated by use of gel filtration and the results were compared with the distribution obtained by 2DE . A 2DE map of selenium-containing proteins in yeast is presented, and compared with a total protein map of yeast.

Int J Food Microbiol, 2001 Dec 4, 71(1), 45 - 51
Structural and ultrastructural changes in yeast cells during autolysis in a model wine system and in sparkling wines; Martinez-Rodriguez AJ et al.; This study shows the changes that occur during the autolysis of yeast in a model wine medium and in a sparkling wine after 12 months of aging, using Nomarsky Light Microscopy and Low Temperature Scanning Electron Microscopy (LTSEM) . The size of the yeasts after 24 h of autolysis in a model medium is much smaller than when they are in the growth stage . With LTSEM . a large number of folds can be observed on the surface of the yeast and practically empty cells . Greater morphological changes, both structural and ultrastructural, can be observed in the yeast after 12 months of aging in wine than in the yeast after 24 h of induced autolysis . However, less of the cytoplasmic content of the yeast that has undergone autolysis in the wine was solubilized than that of the yeast after 24 h of autolysis in the model wine system . These findings indicate that autolysis of yeast in wine is a long-lasting process, which continues for at least 12 months.

DNA Seq, 2001, 12(2), 91 - 6
Cloning, mapping and expression analysis of C15orf4, a novel human gene with homology to the yeast mitochondrial ribosomal protein Ym130 gene; Carim-Todd L et al.; We have identified C15orf4, a novel human gene showing homology to the yeast mitochondrial ribosomal protein YmL30 . C15orf4 encodes a transcript of 1,006 nt with an ORF of 279 amino acids and a predicted protein size of 31.7 kDa . Expression of C15orf4 is enriched in testis . C15orf4 was positioned to chromosome 15q24 by radiation hybrid mapping . We have identified the C15orf4 mouse orthologue as well as homologues in other species.

J Chemother, 2001 Oct, 13(5), 555 - 62
Sertaconazole: in-vitro antifungal activity against vaginal and other superficial yeast isolates; Carrillo-Munoz AJ et al.; The in vitro susceptibilities of 183 clinical yeast isolates to sertaconazole (STZ) were compared to their susceptibilities to clotrimazole (CTZ), econazole (ECZ), ketoconazole (KTZ), miconazole (MNZ), fluconazole (FLZ), itraconazole (ITZ), tioconazole (TCZ), amphotericin B (AMB) and flucytosine (5FC) by using a commercial agar diffusion method . Strains were isolated from vaginal and other superficial clinical samples (18 species of Candida and five strains belonging to other yeast genera) . Only one strain (0.5%) was resistant to STZ out of 87.4% of susceptible strains (n=160) . The percentage of susceptible strains was higher than those obtained with the other agents evaluated and the percentage of resistant strains was lower than for most of the other antifungals . The pattern of susceptibility of C . albicans to STZ, TCZ, ITZ and CLZ was similar and superior to the pattern of susceptibility of this species to MNZ, ECZ, FLZ, 5FC and KTZ . C . dubliniensis was more susceptible to STZ, MNZ, MNZ, FLZ, ITZ, CLZ than to TCZ, ECZ, 5FC, AMB or KTZ . Ten susceptible strains to STZ were resistant to FLZ and one strain was resistant to ITZ . The overall antifungal activity of STZ in vitro against a wide range of clinically important yeasts from vaginal and cutaneous samples indicates the therapeutic potential of this agent for the treatment of infections caused by these fungi . However, the activity of STZ and the clinical value of in vitro data need to be verified in human clinical trials.

J Biol Rhythms, 2001 Dec, 16(6), 516 - 22
Clockless yeast and the gears of the clock: how do they mesh?
Baler R.
In spite of its apparent weakness as a clock model, the budding yeast has spawned a technique that has revolutionized our ability to study specific protein-protein interactions like those at the core of the molecular timekeeping mechanisms . Here, the author will summarize the evolution, power, and limitations of this technique and highlight its potential and actual contributions to the field of chronobiology.

Int J Food Microbiol, 2001 Oct 22, 70(1-2), 1 - 10
Study of the authenticity of commercial wine yeast strains by molecular techniques; Fernandez-Espinar MT et al.; mtDNA restriction analysis has been carried out with 45 different commercial Saccharomyces wine yeast strains . The analysis with Hinf I provided unique profiles for 17 of the 45 strains and can therefore be considered as individual strains . Nevertheless, among the remaining 28 strains, only eight mtDNA restriction patterns appeared . These strains were subjected to electrophoretic karyotyping and PCR amplification of delta sequences . We concluded that the maximum discriminatory power was obtained when the results of the three techniques were combined, giving 13 different composite patterns for the 28 strains under study . The results showed evidence of mistakes during production or fraudulent practices by yeast producers, since only 30 individual strains have been identified among the 45 Saccharomyces wine yeast strains commercialised by different companies . Additionally, commercial starters of Saccharomyces uvarum and Saccharomyces bayanus have been re-identified as Saccharomyces cerevisiae.

Biosci Biotechnol Biochem, 2001 Oct, 65(10), 2353 - 7
Purification and some properties of an inhibitor for a yeast pleiotropic drug resistant pump from Kitasatospora sp . E-420; Wanigasekera A et al.; A strain producing an inhibitor for pleiotropic drug resistant 5 (Pdr5) was isolated using our original screening system in yeast . The strain was classified and named as Kitasatospora sp . E-420 . The purified inhibitor (molecular weight = 1,193 by FAB-MS) inhibited a Pdr5-mediated efflux of cycloheximide and cerulenin . The intracellular accumulation of a fluorescent dye, rhodamine 123, by the inhibitor was also confirmed . Some physicochemical data suggested that the Pdr5-specific inhibitor was different from an immunosuppressant, FK506, reported as the only inhibitor of Pdr5 in vivo.

J Cell Biol, 2001 Dec 24, 155(7), 1239 - 50 Epub 2001 Dec 17.
The Gcs1 and Age2 ArfGAP proteins provide overlapping essential function for transport from the yeast trans-Golgi network; Poon PP et al.; Many intracellular vesicle transport pathways involve GTP hydrolysis by the ADP-ribosylation factor (ARF) type of monomeric G proteins, under the control of ArfGAP proteins . Here we show that the structurally related yeast proteins Gcs1 and Age2 form an essential ArfGAP pair that provides overlapping function for TGN transport . Mutant cells lacking the Age2 and Gcs1 proteins cease proliferation, accumulate membranous structures resembling Berkeley bodies, and are unable to properly process and localize the vacuolar hydrolase carboxypeptidase (CPY) and the vacuolar membrane protein alkaline phosphatase (ALP), which are transported from the TGN to the vacuole by distinct transport routes . Immunofluorescence studies localizing the proteins ALP, Kex2 (a TGN resident protein), and Vps10 (the CPY receptor for transport from the TGN to the vacuole) suggest that inadequate function of this ArfGAP pair leads to a fragmentation of TGN, with effects on secretion and endosomal transport . Our results demonstrate that the Gcs1 + Age2 ArfGAP pair provides overlapping function for transport from the TGN, and also indicate that multiple activities at the TGN can be maintained with the aid of a single ArfGAP.

Yeast, 2002 Jan 15, 19(1), 43 - 54
SRC1: an intron-containing yeast gene involved in sister chromatid segregation; Rodriguez-Navarro S et al.; Analysis of a three-member gene family in the yeast Saccharomyces cerevisiae has allowed the discovery of a new gene that comprises two contiguous open reading frames previously annotated as YML034w and YML033w . The gene contains a small intron with two alternative 5' splicing sites . It is specifically transcribed during G(2)/M in the cell cycle and after several hours of meiosis induction . Splicing of the mRNA is partially dependent on NAM8 but does not vary during meiosis or the cell cycle . Deletion of the gene induces a shortening of the anaphase and aggravates the phenotype of scc1 and esp1 conditional mutants, which suggests a direct role of the protein in sister chromatid separation .

Nat Biotechnol, 2002 Jan, 20(1), 58 - 63
An integrated approach for finding overlooked genes in yeast; Kumar A et al.; We report here the discovery of 137 previously unappreciated genes in yeast through a widely applicable and highly scalable approach integrating methods of gene-trapping, microarray-based expression analysis, and genome-wide homology searching . Our approach is a multistep process in which expressed sequences are first trapped using a modified transposon that produces protein fusions to beta-galactosidase (beta-gal); non-annotated open reading frames (ORFs) translated as beta-gal chimeras are selected as a candidate pool of potential genes . To verify expression of these sequences, labeled RNA is hybridized against a microarray of oligonucleotides designed to detect gene transcripts in a strand-specific manner . In complement to this experimental method, novel genes are also identified in silico by homology to previously annotated proteins . As these methods are capable of identifying both short ORFs and antisense ORFs, our approach provides an effective supplement to current gene-finding schemes . In total, the genes discovered using this approach constitute 2% of the yeast genome and represent a wealth of overlooked biology.

Nucleic Acids Res, 2002 Jan 1, 30(1), 80 - 3
YPL.db: the Yeast Protein Localization database; Habeler G et al.; The Yeast Protein Localization database (YPL.db) contains information about the localization patterns of yeast proteins resulting from microscopic analyses . The data and parameters of the experiments to obtain the localization information, together with images from confocal or video microscopy, are stored in a relational database, building an archive of, and the documentation for, all experiments . The database can be queried based on gene name, protein localization, growth conditions and a number of additional parameters . All experiment parameters are selectable from predefined lists to ensure database integrity and conformity across different investigators . The database provides a structure reference resource to allow for better characterization of unknown or ambiguous localization patterns . Links to MIPS, YPD and SGD databases are provided to allow fast access to further information not contained in the localization database itself . YPL.db is available at http://ypl.tugraz.at.

Nucleic Acids Res, 2002 Jan 1, 30(1), 76 - 9
yMGV: helping biologists with yeast microarray data mining; Le Crom S et al.; yMGV (yeast Microarray Global Viewer) was designed to provide biologists with meaningful information from genome-wide yeast expression data . The database includes most of the available expression data published on yeast microarrays over the last 4 years . It provides customizable tools for the rapid visualization of expression profiles associated with a set of genes from all published experiments . It also allows users to compare the results from different publications so that they can identify genes with common expression profiles . We used yMGV to perform global analyses to find a gene expression profile specific for given biological conditions and to locate functional gene clusters on chromosomes . Other organisms will be added to this database . yMGV is accessible on the web at http://transcriptome.ens.fr/ymgv.

Nucleic Acids Res, 2002 Jan 1, 30(1), 73 - 5
The TRIPLES database: a community resource for yeast molecular biology; Kumar A et al.; TRIPLES is a web-accessible database of TRansposon-Insertion Phenotypes, Localization and Expression in Saccharomyces cerevisiae-a relational database housing nearly half a million data points generated from an ongoing study using large-scale transposon mutagenesis to characterize gene function in yeast . At present, TRIPLES contains three principal data sets (i.e . phenotypic data, protein localization data and expression data) for over 3500 annotated yeast genes as well as several hundred non-annotated open reading frames . In addition, the TRIPLES web site provides online order forms linked to each data set so that users may request any strain or reagent generated from this project free of charge . In response to user requests, the TRIPLES web site has undergone several recent modifications . Our localization data have been supplemented with approximately 500 fluorescent micrographs depicting actual staining patterns observed upon indirect immunofluorescence analysis of indicated epitope-tagged proteins . These localization data, as well as all other data sets within TRIPLES, are now available in full as tab-delimited text . To accommodate increased reagent requests, all orders are now cataloged in a separate database, and users are notified immediately of order receipt and shipment . Also, TRIPLES is one of five sites incorporated into the new functional analysis tool Function Junction provided by the Saccharomyces Genome Database . TRIPLES may be accessed from the Yale Genome Analysis Center (YGAC) homepage at http://ygac.med.yale.edu.

J Biol Chem, 2002 Mar 15, 277(11), 8877 - 81 Epub 2001 Dec 18.
The DGA1 gene determines a second triglyceride synthetic pathway in yeast; Oelkers P et al.; Diacylglycerol esterification provides an excellent target for the pharmacological reduction of triglyceride accumulation in several human disease states . We have used Saccharomyces cerevisiae as a model system to study this critical component of triglyceride synthesis . Recent studies of an oleaginous fungus, Mortierella ramanniana, identified a new family of enzymes with in vitro acyl-CoA:diacylglycerol acyltransferase activity . We show here that DGA1, the sole member of this gene family in yeast, has a physiological role in triglyceride synthesis . Metabolic labeling of DGA1 deletion strains with triglyceride precursors detected significant reductions in triglyceride synthesis . Triglyceride synthesis was virtually abolished in four different growth conditions when DGA1 was deleted in concert with LRO1, an enzyme that esterifies diacylglycerol from a phospholipid acyl donor . The relative contributions of the two enzymes depended on growth conditions . The residual synthesis was lost when ARE2, encoding an acyl-CoA:sterol acyltransferase, was deleted . In vitro microsomal assays verified that DGA1 and ARE2 mediate acyl-CoA:diacylglycerol acyltransferase reactions . Three enzymes can thus account for diacylglycerol esterification in yeast . Yeast strains deficient in both diacylglycerol and sterol esterification showed only a slight growth defect indicating that neutral lipid synthesis is dispensable under common laboratory conditions.

J Biol Chem, 2002 Mar 1, 277(9), 7430 - 7 Epub 2002 Jan 07.
The molecular chaperone Hsp90 mediates heme activation of the yeast transcriptional activator Hap1; Lee HC et al.; Hsp90 plays critical roles in the proper functioning of a wide array of eukaryotic signal transducers such as steroid receptors and tyrosine kinases . Hap1 is a naturally occurring substrate of Hsp90 in Saccharomyces cerevisiae . Hap1 transcriptional activity is precisely and stringently controlled by heme . Previous biochemical studies suggest that in the absence of heme, Hap1 is bound to Hsp90 and other proteins, forming a higher order complex termed HMC (high molecular weight complex), and is repressed . Heme promotes the disruption of the HMC and activates Hap1, permitting Hap1 to bind to DNA with high affinity and to stimulate transcription . By lowering the expression levels of wild-type Hsp90, using a highly specific Hsp90 inhibitor, and by examining the effects of various Hsp90 mutants on Hap1, we show that Hsp90 is critical for Hap1 activation by heme . Furthermore, we show that many Hsp90 mutants exert differential effects on Hap1 and steroid receptors . Notably, mutant G313N weakens Hsp90 steroid receptor interaction but strongly enhances Hsp90-Hap1 interaction and increases Hap1 resistance to protease digestion . Additionally, we found that a heme-independent Hap1 mutant still depends on Hsp90 for high activity . These experiments together suggest that Hsp90 promotes Hap1 activation by inducing or maintaining Hap1 in a transcriptionally active conformation.

EMBO Rep, 2002 Jan, 3(1), 76 - 81 Epub 2001 Dec 19.
The {URE3} phenotype: evidence for a soluble prion in yeast; Fernandez-Bellot E et al.; The aggregation of the two yeast proteins Sup35p and Ure2p is widely accepted as a model for explaining the prion propagation of the phenotypes {PSI+} and {URE3}, respectively . Here, we demonstrate that the propagation of {URE3} cannot simply be the consequence of generating large aggregates of Ure2p, because such aggregation can be found in some conditions that are not related to the prion state of Ure2p . A comparison of {PSI+} and {URE3} aggregation demonstrates differences between these two prion mechanisms . Our findings lead us to propose a new unifying model for yeast prion propagation.

FEBS Lett, 2001 Dec 14, 509(3), 430 - 4
Cytosolic thioredoxin peroxidase I is essential for the antioxidant defense of yeast with dysfunctional mitochondria; Demasi AP et al.; The specific role of cytosolic thioredoxin peroxidase I (cTPx I), encoded by TSA1 (thiol-specific antioxidant), was investigated in the oxidative stress response of Saccharomyces cerevisiae . In most cases, deletion of TSA1 has showed only a slight effect on hydrogen peroxide sensitivity . However, when the functional state of the mitochondria was compromised, the necessity of TSA1 in cell protection against this oxidant was much more evident . All the procedures used to disrupt the mitochondrial respiratory chain promoted increases in the generation of H(2)O(2) in cells, which could be related to their elevated sensitivity to oxidative stress . In fact, TSA1 is highly expressed when cells with respiratory deficiency are exposed to H(2)O(2) . In conclusion, our results indicate that cTPx I is a key component of the antioxidant defense in respiratory-deficient cells.

Proc R Soc Lond B Biol Sci, 2001 Dec 22, 268(1485), 2537 - 42
Outcrossed sex allows a selfish gene to invade yeast populations; Goddard MR et al.; Homing endonuclease genes (HEGs) in eukaryotes are optional genes that have no obvious effect on host phenotype except for causing chromosomes not containing a copy of the gene to be cut, thus causing them to be inherited at a greater than Mendelian rate via gene conversion . These genes are therefore expected to increase in frequency in outcrossed populations, but not in obligately selfed populations . In order to test this idea, we compared the dynamics of the VDE HEG in six replicate outcrossed and inbred populations of yeast (Saccharomyces cerevisiae) . VDE increased in frequency from 0.21 to 0.55 in four outcrossed generations, but showed no change in frequency in the inbred populations . The absence of change in the inbred populations indicates that any effect of VDE on mitotic replication rates is less than 1% . The data from the outcrossed populations best fit a model in which 82% of individuals are derived from outcrossing and VDE is inherited by 74% of the meiotic products from heterozygotes (as compared with 50% for Mendelian genes) . These results empirically demonstrate how a host mating system plays a key role in determining the population dynamics of a selfish gene.

J Biol Chem, 2002 Mar 1, 277(9), 6881 - 7 Epub 2001 Dec 03.
Functional expression of heteromeric calcitonin gene-related peptide and adrenomedullin receptors in yeast; Miret JJ et al.; The ability of G protein-coupled receptors (GPCRs) to form homo- and heteromeric complexes has important implications for the regulation of cellular events . A notable example of heteromer formation is the interaction of the calcitonin receptor-like receptor (CRLR) with different members of the receptor activity modifying protein (RAMP) family, which results in the formation of two different receptors, a calcitonin gene-related peptide (CGRP) receptor and an adrenomedullin receptor . To analyze the role of RAMPs in determining ligand specificity, we have co-expressed CRLR and RAMP proteins in the yeast Saccharomyces cerevisiae, which provides a null system to study the function of mammalian receptors . Co-expression of RAMP1 and CRLR reconstituted a CGRP receptor that was able to activate the pheromone-signaling pathway with pharmacological properties similar to those observed previously in mammalian cells . Co-expression of CRLR with RAMP2 or RAMP3 resulted in a response with the pharmacological properties of an adrenomedullin receptor . These data indicate that RAMPs are necessary and sufficient to determine ligand specificity of CRLR . Contrary to observations in mammalian cells, the glycosylation of CRLR was not affected by the presence of RAMPs in yeast, indicating that glycosylation of CRLR is not the prime determinant of ligand specificity . The first functional reconstitution of a heteromeric seven transmembrane receptor in yeast suggests this organism as a useful research tool to study the molecular nature of other heteromeric receptors.

Yeast, 2001 Dec, 18(16), 1505 - 14
Expression of the yeast glycogen phosphorylase gene is regulated by stress-response elements and by the HOG MAP kinase pathway; Sunnarborg SW et al.; Yeast glycogen metabolism responds to environmental stressors such as nutrient limitation and heat shock . This response is mediated, in part, by the regulation of the glycogen metabolic genes . Environmental stressors induce a number of glycogen metabolic genes, including GPH1, which encodes glycogen phosphorylase . Primer extension analysis detected two start sites for GPH1, one of which predominated . Sequences upstream of these sites included a possible TATA element . Mutation of this sequence reduced GPH1 expression by a factor of 10 but did not affect start site selection . This mutation also did not affect the relative induction of GPH1 upon entry into stationary phase . Three candidates for stress response elements (STREs) were found upstream of the TATA sequence . Mutation of the STREs showed that they were required for regulation of GPH1 expression in early stationary phase, and in response to osmotic shock and heat shock . These elements appeared to act synergistically, since the intact promoter exhibited 30-fold more expression in stationary phase than the sum of that observed for each element acting independently . HOG1, which encodes a MAP kinase, has been implicated in control mediated by STREs . For GPH1, induction by osmotic shock depended on a functional HOG1 allele . In contrast, induction upon entry into stationary phase was only partially dependent on HOG1 . Furthermore, the heat shock response, which can also be mediated by STREs, was independent of HOG1 . These observations suggest that the GPH1 STREs respond to more than one pathway, only one of which requires HOG1 .

Yeast, 2001 Dec, 18(16), 1457 - 70
Genetic analysis of the karyotype instability in natural wine yeast strains; Carro D et al.; Yeast strains isolated from the wild may show high rates of changes in their karyotypes during vegetative growth . We analysed over 500 karyotypes from mitotic and meiotic derivatives of strain DC5, which has a chromosome rearrangement rate of 8.2 x 10(-3) changes/generation . About 70% of the meiotic derivatives of DC5 had low rearrangement rates, with an average of 5.8 x 10(-4) changes/generation, suggesting that karyotype instability behaved as a dominant phenotype . Diploid derivatives with low karyotype variability in mitosis also had low rates of chromosomal rearrangement during meiosis, suggesting that the two phenotypes may be linked . DC5 and some of its meiotic derivatives (both with high and low karyotype variability) had chromosome XII hypervariable bands . Their distribution among the meiotic products indicates that they are not indicators for genetic instability . To our knowledge, data in this paper are the first to indicate that karyotypically unstable yeast strains may give stable progeny at high rates . Understanding of the relevant mechanism(s) may allow the design of genetic strategies to stabilize karyotypes from natural and/or industrial wine yeasts with unacceptable karyotype rearrangement rates .

Cell, 2001 Dec 14, 107(6), 739 - 50
Yeast Cbk1 and Mob2 activate daughter-specific genetic programs to induce asymmetric cell fates; Colman-Lerner A et al.; In Saccharomyces cerevisiae, mothers and daughters have distinct fates . We show that Cbk1 kinase and its interacting protein Mob2 regulate this asymmetry by inducing daughter-specific genetic programs . Daughter-specific expression is due to Cbk1/Mob2-dependent activation and localization of the Ace2 transcription factor to the daughter nucleus . Ectopic localization of active Ace2 to mother nuclei is sufficient to activate daughter-specific genes in mothers . Eight genes are daughter-specific under the tested conditions, while two are daughter-specific only in saturated cultures . Some daughter-specific gene products contribute to cell separation by degrading the cell wall . These experiments define programs of gene expression specific to daughters and describe how those programs are controlled.

Biochemistry, 2001 Dec 25, 40(51), 15562 - 9
Identification of yeast cofilin residues specific for actin monomer and PIP2 binding; Ojala PJ et al.; Cofilin/ADF is a ubiquitous actin-binding protein that is important for rapid actin dynamics in vivo . The long alpha-helix (helix 3 in yeast cofilin) forms the most highly conserved region in cofilin/ADF proteins, and residues in the NH2-terminal half of this alpha-helix have been shown to be essential for actin binding in cofilin/ADF . Recent studies also suggested that the basic residues in the COOH-terminal half of this alpha-helix would play an important role in F-actin binding . In contrast to these studies, we show here that the charged residues in the COOH-terminal half of helix 3 are not important for actin filament binding in yeast cofilin . Mutations in these residues, however, result in a small defect in actin monomer interactions . We also show that yeast cofilin can differentiate between various phosphatidylinositides, and mapped the PI(4,5)P2 binding site by using a collection of cofilin mutants . The PI(4,5)P2 binding site of yeast cofilin is a large positively charged surface that consists of residues in helix 3 as well as residues in other parts of the cofilin molecule . This suggests that cofilin/ADF proteins probably interact simultaneously with more than one PI(4,5)P2 molecule . The PI(4,5)P2-binding site overlaps with areas that are important for F-actin binding, explaining why the actin-related activities of cofilin/ADF are inhibited by PI(4,5)P2 . The biological roles of actin and PI(4,5)P2 interactions of cofilin are discussed in light of phenotypes of specific yeast strains carrying mutations in residues that are important for actin and PI(4,5)P2 binding.

Arch Biochem Biophys, 2002 Jan 1, 397(1), 28 - 39
Kinetic and allosteric consequences of mutations in the subunit and domain interfaces and the allosteric site of yeast pyruvate kinase; Fenton AW et al.; The mechanism by which pyruvate kinase (PK) is allosterically activated by fructose-1,6-bisphosphate (FBP) is poorly understood . To identify residues key to allostery of yeast PK, a point mutation strategy was used . T403E and R459Q mutations in the FBP binding site caused reduced FBP affinity . Introducing positive charges at the 403, 458, and 406 positions in the FBP binding site had little consequence . The mutation Q299N in the A {bond} A subunit interface caused the enzyme response to ADP to be sensitive to FBP . The T311M A {bond} A interface mutant has a decreased affinity for PEP and FBP, and is dependent on FBP for activity . The R369A mutation in the C {bond} C interface only moderately influenced allostery . Creating an E392A mutation in the C {bond} C subunit interface eliminated all cooperativity and allosteric regulation . None of the seven A {bond} C domain interface mutations altered allostery . A model that includes a central role for E392 in allosteric regulation of yeast PK is proposed.

Yeast, 2001 Nov, 18(15), 1397 - 412
Large-scale phenotypic analysis reveals identical contributions to cell functions of known and unknown yeast genes; Bianchi MM et al.; Sequencing of the yeast genome has shown that about one-third of the yeast ORFs code for unknown proteins . Many other have similarity to known genes, but still the cellular functions of the gene products are unknown . The aim of the B1 Consortium of the EUROFAN project was to perform a qualitative phenotypic analysis on yeast strains deleted for functionally orphan genes . To this end we set up a simple approach to detect growth defects of a relatively large number of strains in the presence of osmolytes, ethanol, high temperature, inhibitory compounds or drugs affecting protein biosynthesis, phosphorylation level or nucleic acids biosynthesis . We have now developed this procedure to a semi-quantitative level, we have included new inhibitors, such as hygromycin B, benomyl, metals and additional drugs interfering with synthesis of nucleic acids, and we have performed phenotypic analysis on the deleted strains of 564 genes poorly characterized in respect to their cellular functions . About 30% of the deleted strains showed at least one phenotype: many of them were pleiotropic . For many gene deletions, the linkage between the deletion marker and the observed phenotype(s) was studied by tetrad analysis and their co-segregation was demonstrated . Co-segregation was found in about two-thirds of the analysed strains showing phenotype(s) .

J Biol Chem, 2002 Mar 1, 277(9), 7246 - 54 Epub 2001 Dec 13.
Detection of a {3Fe-4S} cluster intermediate of cytosolic aconitase in yeast expressing iron regulatory protein 1 . Insights into the mechanism of Fe-S cluster cycling; Brown NM et al.; Interconversion of iron regulatory protein 1 (IRP1) with cytosolic aconitase (c-aconitase) occurs via assembly/disassembly of a {4Fe-4S} cluster . Recent evidence implicates oxidants in cluster disassembly . We investigated H(2)O(2)-initiated Fe-S cluster disassembly in c-aconitase expressed in Saccharomyces cerevisiae . A signal for {3Fe-4S} c-aconitase was detected by whole-cell EPR of aerobically grown, aco1 yeast expressing wild-type IRP1 or a S138A-IRP1 mutant (IRP1(S138A)), providing the first direct evidence of a 3Fe intermediate in vivo . Exposure of yeast to H(2)O(2) increased this 3Fe c-aconitase signal up to 5-fold, coincident with inhibition of c-aconitase activity . Untreated yeast expressing IRP1(S138D) or IRP1(S138E), which mimic phosphorylated IRP1, failed to give a 3Fe signal . H(2)O(2) produced a weak 3Fe signal in yeast expressing IRP1(S138D) . Yeast expressing IRP1(S138D) or IRP1(S138E) were the most sensitive to inhibition of aconitase-dependent growth by H(2)O(2) and were more responsive to changes in media iron status . Ferricyanide oxidation of anaerobically reconstituted c-aconitase yielded a strong 3Fe EPR signal with wild-type and S138A c-aconitases . Only a weak 3Fe signal was obtained with S138D c-aconitase, and no signal was obtained with S138E c-aconitase . This, paired with loss of c-aconitase activity, strongly argues that the Fe-S clusters of these phosphomimetic c-aconitase mutants undergo more complete disassembly upon oxidation . Our results demonstrate that 3Fe c-aconitase is an intermediate in H(2)O(2)-initiated Fe-S cluster disassembly in vivo and suggest that cluster assembly/disassembly in IRP1 is a dynamic process in aerobically growing yeast . Further, our results support the view that phosphorylation of IRP1 can modulate its response to iron through effects on Fe-S cluster stability and turnover.

J Biol Chem, 2002 Feb 22, 277(8), 6353 - 8 Epub 2001 Dec 13.
Post-translational modification of plant plasma membrane H(+)-ATPase as a requirement for functional complementation of a yeast transport mutant; Jahn TP et al.; Many heterologous membrane proteins expressed in the yeast Saccharomyces cerevisiae fail to reach their normal cellular location and instead accumulate in stacked internal membranes . Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 (AHA2) is expressed predominantly in yeast internal membranes and fails to complement a yeast strain devoid of its endogenous H(+)-ATPase Pma1 . We observed that phosphorylation of AHA2 in the heterologous host and subsequent binding of 14-3-3 protein is crucial for the ability of AHA2 to substitute for Pma1 . Thus, mutants of AHA2, complementing pma1, showed increased phosphorylation at the penultimate residue (Thr(947)), which creates a binding site for endogenous 14-3-3 protein . Only a pool of ATPase in the plasma membrane is phosphorylated . Double mutants carrying in addition a T947A substitution lost their ability to complement pma1 . However, mutants affected in both autoinhibitory regions of the C-terminal regulatory domain complemented pma1 irrespective of their ability to become phosphorylated at Thr(947) . This demonstrates that it is the activity status of the mutant enzyme and neither redirection of trafficking nor 14-3-3 binding per se that determines the ability of H(+)-pumps to rescue pma1.

Mech Ageing Dev, 2002 Feb, 123(4), 287 - 95
Loss of prohibitins, though it shortens the replicative life span of yeast cells undergoing division, does not shorten the chronological life span of G0-arrested cells; Piper PW et al.; Prohibitin proteins have been implicated in cell proliferation, ageing and the maintenance of mitochondrial integrity . The yeast prohibitins, Phb1p and Phb2p, are close in sequence to their two human counterparts, prohibitin and BAP37 . Mutants of Saccharomyces cerevisiae that lack these prohibitins have a shortened replicative (budding) life span . Nevertheless, their chronological life span, measured as the survival of stationary phase (G0) cells over time, is essentially normal . Loss of prohibitins does not hypersensitise cells to their endogenous free radical production, though it does slightly increase their sensitivity to ethanol . It is unlikely, therefore, that the influences of prohibitins over replicative senescence involve free radicals, despite the evidence from many systems linking ageing to the long-term effects of oxidative stress . Yeast phb1 and phb2 mutants and also the phb1, phb2 double mutant, tend to lose respiration competence when in G0-arrest, indicating that nondividing cells lacking prohibitins have problems maintaining a functional mitochondrial electron transport chain . This may reflect an imbalance in the turnover of components of the respiratory chain in G0 cells, since the Phb1/2p complex is known to help stabilise these components . Such losses of respiratory function in G0-arrested cells are greater with the loss of Phb1p than with the loss of Phb2p, revealing the Phb1p null and Phb2p null phenotypes to be nonidentical.

Biochem J, 2002 Jan 1, 361(Pt 1), 27 - 34
Direct interactions between molecular chaperones heat-shock protein (Hsp) 70 and Hsp40: yeast Hsp70 Ssa1 binds the extreme C-terminal region of yeast Hsp40 Sis1; Qian X et al.; Heat-shock protein 40 (Hsp40) enables Hsp70 to play critical roles in a number of cellular processes, such as protein folding, assembly, degradation and translocation in vivo . Hsp40 recognizes and binds non-native polypeptides and delivers them to Hsp70 . Then Hsp40 stimulates the ATPase activity of Hsp70 to fold the polypeptides . By using yeast Hsp40 Sis1 and yeast Hsp70 Ssa1 as our model proteins, we found that the Sis1 peptide-binding fragment interacts directly with the full-length Ssa1 in vitro . Further studies showed that the C-terminal lid domain of Ssa1 could interact with Sis1 peptide-binding domain physically in vitro . The Sis1 peptide-binding fragment forms a stable complex with the Ssa1 C-terminal lid domain in solution . The interactions between these two proteins appear to be charge-charge interactions because high-ionic-strength buffer can dissociate the complex . Further mapping studies showed that the Sis1 peptide-binding fragment binds the extreme C-terminal 15 amino acid residues of Ssa1 . A flexible glycine-rich region is followed by these 15 residues in the Ssa1 primary sequence . Atomic force microscopy of the Sis1-Ssa1 complex showed that only one end of the Ssa1 lid domain binds the Sis1 peptide-binding-fragment dimer at the upper level of the huge groove within the Sis1 dimer . Based on the data, we propose an "anchoring and docking" model to illustrate the mechanisms by which Hsp40 interacts with Hsp70 and delivers the non-native polypeptide to Hsp70.

Chem Res Toxicol, 2001 Dec, 14(12), 1584 - 9
Mechanisms of nitrogen oxide-mediated disruption of metalloprotein function: an examination of the copper-responsive yeast transcription factor Ace1; Shinyashiki M et al.; Nitric oxide (NO) has been found to inhibit the copper-responsive yeast transcription factor Ace1 in an oxygen-dependent manner . However, the mechanism responsible for NO-dependent inhibition of Ace1 remains unestablished . In the present study, the chemical interaction of nitrogen oxide species with Ace1 was examined using a yeast reporter system . Exposure of yeast to various nitrogen oxides, under a variety of conditions, revealed that the oxygen-dependent inhibition of Ace1 is due to the reaction of NO with O(2) . The nitrosating nitrogen oxide species N(2)O(3) is likely to be the disrupter of Ace1 activity . Considering the similarity of metal-thiolate ligation in Ace1 with other mammalian metalloproteins such as metallothionein, metal chaperones, and zinc-finger proteins, these results help to understand the biochemical interactions of NO with those mammalian metalloproteins.

Anal Biochem, 2002 Jan 1, 300(1), 27 - 33
Phosphate is required to maintain the outer membrane integrity and membrane potential of respiring yeast mitochondria; Janssen MJ et al.; The buffer requirements to maintain mitochondrial intactness and membrane potential in in vitro studies were investigated, using gradient purified yeast mitochondria . It was found that the presence of phosphate is crucial for generation of a stable membrane potential and for preserving the intactness of the outer membrane, as assessed by probing the accessibility of Tom40p to trypsin and the leakage of cytochrome b2 from the intermembrane space . Upon addition of respiratory substrate in the absence of phosphate, mitochondria generate a membrane potential that collapses within 1 min . Under the same conditions, the mitochondrial outer membrane is disrupted . The presence of phosphate prevents both phenomena . The DeltapH component of the proton motive force appears to be responsible for the compromised outer membrane integrity . The collapse of the membrane potential is reversible to a limited extent . Only when phosphate is added soon enough after the addition of exogenous respiratory substrate can a stable membrane potential be obtained again . Within a few minutes, this capacity is lost . The presence of Mg(2+) prevents rupture of the outer membrane, but does not prevent rapid dissipation of the membrane potential . Similar results were obtained for mitochondria isolated and stored in the presence of dextran or bovine serum albumin . (c)2001 Elsevier Science.

Science, 2001 Dec 14, 294(5550), 2364 - 8
Systematic genetic analysis with ordered arrays of yeast deletion mutants; Tong AH et al.; In Saccharomyces cerevisiae, more than 80% of the approximately 6200 predicted genes are nonessential, implying that the genome is buffered from the phenotypic consequences of genetic perturbation . To evaluate function, we developed a method for systematic construction of double mutants, termed synthetic genetic array (SGA) analysis, in which a query mutation is crossed to an array of approximately 4700 deletion mutants . Inviable double-mutant meiotic progeny identify functional relationships between genes . SGA analysis of genes with roles in cytoskeletal organization (BNI1, ARP2, ARC40, BIM1), DNA synthesis and repair (SGS1, RAD27), or uncharacterized functions (BBC1, NBP2) generated a network of 291 interactions among 204 genes . Systematic application of this approach should produce a global map of gene function.

J Biol Chem, 2002 Feb 22, 277(8), 6478 - 82 Epub 2001 Dec 10.
Storage lipid synthesis is non-essential in yeast; Sandager L et al.; Steryl esters and triacylglycerol (TAG) are the main storage lipids in eukaryotic cells . In the yeast Saccharomyces cerevisiae, these storage lipids accumulate during stationary growth phase within organelles known as lipid bodies . We have used single and multiple gene disruptions to study storage lipid synthesis in yeast . Four genes, ARE1, ARE2, DGA1, and LRO1, were found to contribute to TAG synthesis . The most significant contribution is made by DGA1, which encodes a novel acyl-CoA:diacylglycerol acyltransferase . Two of the genes, ARE1 and ARE2, are also involved in steryl ester synthesis . A yeast strain that lacks all four genes is viable and has no apparent growth defects under standard conditions . The strain is devoid of both TAG and steryl esters, and fluorescence microscopy revealed that it also lacks lipid bodies . We conclude that neither storage lipids nor lipid bodies are essential for growth in yeast.

J Biol Chem, 2002 Feb 15, 277(7), 5120 - 5 Epub 2001 Dec 07.
Cadmium resistance conferred to yeast by a non-metallothionein-encoding gene of the earthworm Enchytraeus; Tschuschke S et al.; The earthworm Enchytraeus is able to survive in cadmium (Cd)-polluted environments . Upon Cd exposure, the worms express a gene encoding the putative non-metallothionein 25-kDa cysteine-rich protein (CRP), which contains eight repeats with highly conserved cysteines in Cys-X-Cys and Cys-Cys arrangements exhibiting 36-53% identities to the 6-7-kDa metallothioneins of different organisms . Here, we demonstrate that the CRP protein confers a highly Cd-resistant phenotype to a Cd-hypersensitive yeast strain . Cd resistance increases with increasing numbers of expressed CRP repeats, but even one 3-kDa CRP repeat still mediates Cd resistance . Site-directed mutagenesis reveals that each single cysteine within a given repeat is important for Cd resistance, though to a different extent . However, replacement of other conserved amino acids such as Pro(136) and Asp(196) at the CRP repeat junctions does not affect Cd resistance . Our data indicate (i) that the non-metallothionein CRP protein is able to detoxify Cd and (ii) that this is dependent on the availability of sulfhydryl groups of the conserved cysteines.

J Biol Chem, 2002 Feb 22, 277(8), 6422 - 7 Epub 2001 Dec 06.
Functional expression in yeast of the human secretory pathway Ca(2+), Mn(2+)-ATPase defective in Hailey-Hailey disease; Ton VK et al.; The discovery and biochemical characterization of the secretory pathway Ca(2+)-ATPase, PMR1, in Saccharomyces cerevisiae, has paved the way for identification of PMR1 homologues in many species including rat, Caenorhabditis elegans, and Homo sapiens . In yeast, PMR1 has been shown to function as a high affinity Ca(2+)/Mn(2+) pump and has been localized to the Golgi compartment where it is important for protein sorting, processing, and glycosylation . However, little is known about PMR1 homologues in higher organisms . Loss of one functional allele of the human gene, hSPCA1, has been linked to Hailey-Hailey disease, characterized by skin ulceration and improper keratinocyte adhesion . We demonstrate that expression of hSPCA1 in yeast fully complements pmr1 phenotypes of hypersensitivity to Ca(2+) chelators and Mn(2+) toxicity . Similar to PMR1, epitope-tagged hSPCA1 also resides in the Golgi when expressed in yeast or in chinese hamster ovary cells . (45)Ca(2+) transport by hSPCA1 into isolated yeast Golgi vesicles shows an apparent Ca(2+) affinity of 0.26 microm, is inhibitable by Mn(2+), but is thapsigargin-insensitive . In contrast, heterologous expression of vertebrate sarcoplasmic reticulum and plasma membrane Ca(2+)-ATPases in yeast complement the Ca(2+)- but not Mn(2+)-related phenotypes of the pmr1-null strain, suggesting that high affinity Mn(2+) transport is a unique feature of the secretory pathway Ca(2+)-ATPases.

Mol Cell, 2001 Nov, 8(5), 1075 - 83
Targeting an mRNA for decapping: displacement of translation factors and association of the Lsm1p-7p complex on deadenylated yeast mRNAs; Tharun S et al.; The major pathway of eukaryotic mRNA decay involves deadenylation-dependent decapping followed by 5' to 3' exonucleolytic degradation . By examining interactions among mRNA decay factors, the mRNA, and key translation factors, we have identified a critical transition in mRNP organization that leads to decapping and degradation of yeast mRNAs . This transition occurs after deadenylation and includes loss of Pab1p, eIF4E, and eIF4G from the mRNA and association of the decapping activator complex, Lsm1p-7p, which enhances the coimmunoprecipitation of a decapping enzyme complex (Dcp1p and Dcp2p) with the mRNA . These results define an important rearrangement in mRNP organization and suggest that deadenylation promotes mRNA decapping by both the loss of Pab1p and the recruitment of the Lsm1p-7p complex.

Neurobiol Dis, 2001 Dec, 8(6), 982 - 92
Neuropathological characterization of mutant amyloid precursor protein yeast artificial chromosome transgenic mice; Kulnane LS et al.; Mutations in the amyloid precursor protein (APP) gene result in elevated production and deposition of the 42 amino acid beta-amyloid (Abeta1-42) peptide and early-onset Alzheimer's disease (AD) . To accurately examine the effect of the APP FAD mutations in vivo, we introduced yeast artificial chromosomes (YACs) containing the entire genomic copy of human APP harboring FAD mutations into transgenic mice . Our current results demonstrate that mutant APP YAC transgenic mice exhibit many features characteristic of human AD, including regional deposition of Abeta with preferential deposition of Abeta1-42, extensive neuritic abnormalities as evidenced by staining with APP, ubiquitin, neurofilament, and hyperphosphorylated tau antibodies, increased markers of inflammation, and the overlapping deposition of Abeta with apolipoproteins E and J . Our results also suggest that APP YAC transgenic mice possess unique pathological attributes when compared to other transgenic mouse models of AD that may reflect the experimental design of each model.

Nat Cell Biol, 2002 Jan, 4(1), 42 - 50
Yeast formins regulate cell polarity by controlling the assembly of actin cables; Sagot I et al.; Formins are conserved Rho-GTPase effectors that communicate Rho-GTPase signals to the cytoskeleton . We found that formins were required for the assembly of one of the three budding yeast actin structures: polarized arrays of actin cables . A dominant-active formin induced the assembly of actin cables . The activation and localization of the formin Bni1p required components of the polarisome complex . These findings potentially define the cellular function of formins in budding yeast and explain their involvement in the generation of cell polarity . A requirement for formins in constructing specific actin structures might be the basis for the diverse activities of formins in development.

Nat Cell Biol, 2002 Jan, 4(1), 32 - 41
Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast; Evangelista M et al.; Formins have been implicated in the regulation of cytoskeletal structure in animals and fungi . Here we show that the formins Bni1 and Bnr1 of budding yeast stimulate the assembly of actin filaments that function as precursors to tropomyosin-stabilized cables that direct polarized cell growth . With loss of formin function, cables disassemble, whereas increased formin activity causes the hyperaccumulation of cable-like filaments . Unlike the assembly of cortical actin patches, cable assembly requires profilin but not the Arp2/3 complex . Thus formins control a distinct pathway for assembling actin filaments that organize the overall polarity of the cell.

Science, 2001 Dec 7, 294(5549), 2181 - 6
Chromosome dynamics in the yeast interphase nucleus; Heun P et al.; Little is known about the dynamics of chromosomes in interphase nuclei . By tagging four chromosomal regions with a green fluorescent protein fusion to lac repressor, we monitored the movement and subnuclear position of specific sites in the yeast genome, sampling at short time intervals . We found that early and late origins of replication are highly mobile in G1 phase, frequently moving at or faster than 0.5 micrometers/10 seconds, in an energy-dependent fashion . The rapid diffusive movement of chromatin detected in G1 becomes constrained in S phase through a mechanism dependent on active DNA replication . In contrast, telomeres and centromeres provide replication-independent constraint on chromatin movement in both G1 and S phases.

Mol Biol Cell, 2001 Dec, 12(12), 4129 - 38
Plasma membrane proton ATPase Pma1p requires raft association for surface delivery in yeast; Bagnat M et al.; Correct sorting of proteins is essential to generate and maintain the identity and function of the different cellular compartments . In this study we demonstrate the role of lipid rafts in biosynthetic delivery of Pma1p, the major plasma membrane proton ATPase, to the cell surface . Disruption of rafts led to mistargeting of Pma1p to the vacuole . Conversely, Pma1-7, an ATPase mutant that is mistargeted to the vacuole, was shown to exhibit impaired raft association . One of the previously identified suppressors, multicopy AST1, not only restored surface delivery but also raft association of Pma1-7 . Ast1p, which is a peripheral membrane protein, was found to directly interact with Pma1p inducing its clustering into a SDS/Triton X100-resistant oligomer . We suggest that clustering facilitates partition of Pma1p into rafts and transport to the cell surface.

Mol Biol Cell, 2001 Dec, 12(12), 4044 - 53
Localization of fission yeast type II myosin, Myo2, to the cytokinetic actin ring is regulated by phosphorylation of a C-terminal coiled-coil domain and requires a functional septation initiation network; Mulvihill DP et al.; Myo2 truncations fused to green fluorescent protein (GFP) defined a C-terminal domain essential for the localization of Myo2 to the cytokinetic actin ring (CAR) . The localization domain contained two predicted phosphorylation sites . Mutation of serine 1518 to alanine (S(1518)A) abolished Myo2 localization, whereas Myo2 with a glutamic acid at this position (S(1518)E) localized to the CAR . GFP-Myo2 formed rings in the septation initiation kinase (SIN) mutant cdc7-24 at 25 degrees C but not at 36 degrees C . GFP-Myo2S(1518)E rings persisted at 36 degrees C in cdc7-24 but not in another SIN kinase mutant, sid2-250 . To further examine the relationship between Myo2 and the SIN pathway, the chromosomal copy of myo2(+) was fused to GFP (strain myo2-gc) . Myo2 ring formation was abolished in the double mutants myo2-gc cdc7.24 and myo2-gc sid2-250 at the restrictive temperature . In contrast, activation of the SIN pathway in the double mutant myo2-gc cdc16-116 resulted in the formation of Myo2 rings which subsequently collapsed at 36 degrees C . We conclude that the SIN pathway that controls septation in fission yeast also regulates Myo2 ring formation and contraction . Cdc7 and Sid2 are involved in ring formation, in the case of Cdc7 by phosphorylation of a single serine residue in the Myo2 tail . Other kinases and/or phosphatases may control ring contraction.

Mol Biol Cell, 2001 Dec, 12(12), 3973 - 86
Dominant-lethal alpha-tubulin mutants defective in microtubule depolymerization in yeast; Anders KR et al.; The dynamic instability of microtubules has long been understood to depend on the hydrolysis of GTP bound to beta-tubulin, an event stimulated by polymerization and necessary for depolymerization . Crystallographic studies of tubulin show that GTP is bound by beta-tubulin at the longitudinal dimer-dimer interface and contacts particular alpha-tubulin residues in the next dimer along the protofilament . This structural arrangement suggests that these contacts could account for assembly-stimulated GTP hydrolysis . As a test of this hypothesis, we examined, in yeast cells, the effect of mutating the alpha-tubulin residues predicted, on structural grounds, to be involved in GTPase activation . Mutation of these residues to alanine (i.e., D252A and E255A) created poisonous alpha-tubulins that caused lethality even as minor components of the alpha-tubulin pool . When the mutant alpha-tubulins were expressed from the galactose-inducible promoter of GAL1, cells rapidly acquired aberrant microtubule structures . Cytoplasmic microtubules were largely bundled, spindle assembly was inhibited, preexisting spindles failed to completely elongate, and occasional, stable microtubules were observed unattached to spindle pole bodies . Time-lapse microscopy showed that microtubule dynamics had ceased . Microtubules containing the mutant proteins did not depolymerize, even in the presence of nocodazole . These data support the view that alpha-tubulin is a GTPase-activating protein that acts, during microtubule polymerization, to stimulate GTP hydrolysis in beta-tubulin and thereby account for the dynamic instability of microtubules.

Mol Biol Cell, 2001 Dec, 12(12), 3933 - 46
Dynamic behavior of microtubules during dynein-dependent nuclear migrations of meiotic prophase in fission yeast; Yamamoto A et al.; During meiotic prophase in fission yeast, the nucleus migrates back and forth between the two ends of the cell, led by the spindle pole body (SPB) . This nuclear oscillation is dependent on astral microtubules radiating from the SPB and a microtubule motor, cytoplasmic dynein . Here we have examined the dynamic behavior of astral microtubules labeled with the green fluorescent protein during meiotic prophase with the use of optical sectioning microscopy . During nuclear migrations, the SPB mostly follows the microtubules that extend toward the cell cortex . SPB migrations start when these microtubules interact with the cortex and stop when they disappear, suggesting that these microtubules drive nuclear migrations . The microtubules that are followed by the SPB often slide along the cortex and are shortened by disassembly at their ends proximal to the cortex . In dynein-mutant cells, where nuclear oscillations are absent, the SPB never migrates by following microtubules, and microtubule assembly/disassembly dynamics is significantly altered . Based on these observations, together with the frequent accumulation of dynein at a cortical site where the directing microtubules interact, we propose a model in which dynein drives nuclear oscillation by mediating cortical microtubule interactions and regulating the dynamics of microtubule disassembly at the cortex.

Mol Biol Cell, 2001 Dec, 12(12), 3919 - 32
Two related kinesins, klp5+ and klp6+, foster microtubule disassembly and are required for meiosis in fission yeast; West RR et al.; The kinesin superfamily of microtubule motor proteins is important in many cellular processes, including mitosis and meiosis, vesicle transport, and the establishment and maintenance of cell polarity . We have characterized two related kinesins in fission yeast, klp5+ and klp6+,, that are amino-terminal motors of the KIP3 subfamily . Analysis of null mutants demonstrates that neither klp5+ nor klp6+, individually or together, is essential for vegetative growth, although these mutants have altered microtubule behavior . klp5Delta and klp6Delta are resistant to high concentrations of the microtubule poison thiabendazole and have abnormally long cytoplasmic microtubules that can curl around the ends of the cell . This phenotype is greatly enhanced in the cell cycle mutant cdc25-22, leading to a bent, asymmetric cell morphology as cells elongate during cell cycle arrest . Klp5p-GFP and Klp6p-GFP both localize to cytoplasmic microtubules throughout the cell cycle and to spindles in mitosis, but their localizations are not interdependent . During the meiotic phase of the life cycle, both of these kinesins are essential . Spore viability is low in homozygous crosses of either null mutant . Heterozygous crosses of klp5Delta with klp6Delta have an intermediate viability, suggesting cooperation between these proteins in meiosis.

Mol Biol Cell, 2001 Dec, 12(12), 3783 - 96
Golgi-to-endoplasmic reticulum (ER) retrograde traffic in yeast requires Dsl1p, a component of the ER target site that interacts with a COPI coat subunit; Reilly BA et al.; DSL1 was identified through its genetic interaction with SLY1, which encodes a t-SNARE-interacting protein that functions in endoplasmic reticulum (ER)-to-Golgi traffic . Conditional dsl1 mutants exhibit a block in ER-to-Golgi traffic at the restrictive temperature . Here, we show that dsl1 mutants are defective for retrograde Golgi-to-ER traffic, even under conditions where no anterograde transport block is evident . These results suggest that the primary function of Dsl1p may be in retrograde traffic, and that retrograde defects can lead to secondary defects in anterograde traffic . Dsl1p is an ER-localized peripheral membrane protein that can be extracted from the membrane in a multiprotein complex . Immunoisolation of the complex yielded Dsl1p and proteins of approximately 80 and approximately 55 kDa . The approximately 80-kDa protein has been identified as Tip20p, a protein that others have shown to exist in a tight complex with Sec20p, which is approximately 50 kDa . Both Sec20p and Tip20p function in retrograde Golgi-to-ER traffic, are ER-localized, and bind to the ER t-SNARE Ufe1p . These findings suggest that an ER-localized complex of Dsl1p, Sec20p, and Tip20p functions in retrograde traffic, perhaps upstream of a Sly1p/Ufe1p complex . Last, we show that Dsl1p interacts with the delta-subunit of the retrograde COPI coat, Ret2p, and discuss possible roles for this interaction.

Mol Biol Cell, 2001 Dec, 12(12), 3759 - 72
Regulation of yeast actin cytoskeleton-regulatory complex Pan1p/Sla1p/End3p by serine/threonine kinase Prk1p; Zeng G et al.; The serine/threonine kinase Prk1p is known to be involved in the regulation of the actin cytoskeleton organization in budding yeast . One possible function of Prk1p is the negative regulation of Pan1p, an actin patch regulatory protein that forms a complex in vivo with at least two other proteins, Sla1p and End3p . In this report, we identified Sla1p as another substrate for Prk1p . The phosphorylation of Sla1p by Prk1p was established in vitro with the use of immunoprecipitated Prk1p and in vivo with the use of PRK1 overexpression, and was further supported by the finding that immunoprecipitated Sla1p contained PRK1- and ARK1-dependent kinase activities . Stable complex formation between Prk1p and Sla1p/Pan1p in vivo could be observed once the phosphorylation reaction was blocked by mutation in the catalytic site of Prk1p . Elevation of Prk1p activities in wild-type cells resulted in a number of deficiencies, including those in colocalization of Pan1p and Sla1p, endocytosis, and cell wall morphogenesis, likely attributable to a disintegration of the Pan1p/Sla1p/End3p complex . These results lend a strong support to the model that the phosphorylation of the Pan1p/Sla1p/End3p complex by Prk1p is one of the important mechanisms by which the organization and functions of the actin cytoskeleton are regulated.

Mol Biol Cell, 2001 Dec, 12(12), 3744 - 58
Structure-function analysis of fission yeast Hus1-Rad1-Rad9 checkpoint complex; Kaur R et al.; Hus1, Rad1, and Rad9 are three evolutionarily conserved proteins required for checkpoint control in fission yeast . These proteins are known to form a stable complex in vivo . Recently, computational studies have predicted structural similarity between the individual proteins of Hus1-Rad1-Rad9 complex and the replication processivity factor proliferating cell nuclear antigen (PCNA) . This has led to the proposal that the Hus1-Rad1-Rad9 complex may form a PCNA-like ring structure, and could function as a sliding clamp during checkpoint control . In the present study, we have attempted to test the predictions of this model by asking whether the PCNA alignment identifies functionally important residues or explains mutant phenotypes of hus1, rad1, or rad9 alleles . Although some of our results are consistent with the PCNA alignment, others indicate that the Hus1-Rad1-Rad9 complex possesses unique structural and functional features.

Mol Cell Biol, 2002 Jan, 22(1), 298 - 308
Multiple roles of the tau131 subunit of yeast transcription factor IIIC (TFIIIC) in TFIIIB assembly; Dumay-Odelot H et al.; Yeast transcription factor IIIC (TFIIIC) plays a key role in assembling the transcription initiation factor TFIIIB on class III genes after TFIIIC-DNA binding . The second largest subunit of TFIIIC, tau131, is thought to initiate TFIIIB assembly by interacting with Brf1/TFIIIB70 . In this work, we have analyzed a TFIIIC mutant (tau131-DeltaTPR2) harboring a deletion in tau131 removing the second of its 11 tetratricopeptide repeats . Remarkably, this thermosensitive mutation was selectively suppressed in vivo by overexpression of B"/TFIIIB90, but not Brf1 or TATA-binding protein . In vitro, the mutant factor preincubated at restrictive temperature bound DNA efficiently but lost transcription factor activity . The in vitro transcription defect was abolished at high concentrations of B" but not Brf1 . Copurification experiments of baculovirus-expressed proteins confirmed a direct physical interaction between tau131 and B" . tau131, therefore, appears to be involved in the recruitment of both Brf1 and B".

Mol Cell Biol, 2002 Jan, 22(1), 286 - 97
Yeast hnRNP K-like genes are involved in regulation of the telomeric position effect and telomere length; Denisenko O et al.; Mammalian heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA- and DNA-binding protein implicated in the regulation of gene expression processes . To better understand its function, we studied two Saccharomyces cerevisiae homologues of the human hnRNP K, PBP2 and HEK2 (heterogeneous nuclear RNP K-like gene) . pbp2Delta and hek2Delta mutations inhibited expression of a marker gene that was inserted near telomere but not at internal chromosomal locations . The telomere proximal to the ectopic marker gene became longer, while most of the other telomeres were not altered in the double mutant cells . We provide evidence that telomere elongation might be the primary event that causes enhanced silencing of an adjacent reporter gene . The telomere lengthening could, in part, be explained by the inhibitory effect of hek2Delta mutation on the telomeric rapid deletion pathway . Hek2p was detected in a complex with chromosome regions proximal to the affected telomere, suggesting a direct involvement of this protein in telomere maintenance . These results identify a role for hnRNP K-like genes in the structural and functional organization of telomeric chromatin in yeast.

Mol Cell Biol, 2002 Jan, 22(1), 1 - 11
Protein kinase A regulates sexual development and gluconeogenesis through phosphorylation of the Zn finger transcriptional activator Rst2p in fission yeast; Higuchi T et al.; Protein kinase A (PKAi a cyclic AMP-dependent protein kinase) negatively regulates sexual development and gluconeogenesis in fission yeast by suppressing the transcription of ste11 required for the former and the transcription of fbp1 required for the latter . Here we show that Rst2p, a zinc finger protein that can bind to the upstream region of ste11 and fbp1 via the STREP motif, mediates the activity of PKA to transcription of these genes . A simple reporter system confirmed that PKA could cause its negative effect on transcription through the combination of Rst2p and STREP . Rst2p was phosphorylated by PKA in vitro at two consensus sequences on it . Substitution of the target threonine residues by alanine made the protein active even in the presence of high PKA activity . Rst2p underwent hyperphosphorylation in the medium lacking glucose, and PKA inhibited this hyperphosphorylation . Rst2p was mainly cytoplasmic under high PKA activity but was concentrated in the nucleus when this activity was lowered, suggesting that PKA might regulate ste11 and fbp1 negatively by excluding Rst2p from the nucleus . However, the shift of Rst2p localization was not perfect under physiological conditions, leaving the possibility that PKA inhibits Rst2p function in another way as well . Although the PKA-Rst2p-STREP pathway is apparently central to the regulation of ste11 and fbp1 transcription in accordance with nutritional conditions, some additional paths are likely to connect nitrogen to repression of ste11 and glucose to repression of fbp1 . These paths may ensure the specificity between the type of nutrients in shortage and the type of genes to be expressed.

Mutat Res, 2001 Dec 19, 487(3-4), 137 - 47
Deletion of the MAG1 DNA glycosylase gene suppresses alkylation-induced killing and mutagenesis in yeast cells lacking AP endonucleases; Xiao W et al.; DNA base excision repair (BER) is initiated by DNA glycosylases that recognize and remove damaged bases . The phosphate backbone adjacent to the resulting apurinic/apyrimidinic (AP) site is then cleaved by an AP endonuclease or glycosylase-associated AP lyase to invoke subsequent BER steps . We have used a genetic approach in Saccharomyces cerevisiae to address whether AP sites are blocks to DNA replication and the biological consequences if AP sites persist in the genome . We found that yeast cells deficient in the two AP endonucleases (apn1 apn2 double mutant) are extremely sensitive to killing by methyl methanesulfonate (MMS), a model DNA alkylating agent . Interestingly, this sensitivity can be reduced up to 2500-fold by deleting the MAG1 3-methyladenine DNA glycosylase gene, suggesting that Mag1 not only removes lethal base lesions, but also benign lesions and possibly normal bases, and that the resulting AP sites are highly toxic to the cells . This rescuing effect appears to be specific for DNA alkylation damage, since the mag1 mutation reduces killing effects of two other DNA alkylating agents, but does not alter the sensitivity of apn cells to killing by UV, gamma-ray or H(2)O(2) . Our mutagenesis assays indicate that nearly half of spontaneous and almost all MMS-induced mutations in the AP endonuclease-deficient cells are due to Mag1 DNA glycosylase activity . Although the DNA replication apparatus appears to be incapable of replicating past AP sites, Polzeta-mediated translesion synthesis is able to bypass AP sites, and accounts for all spontaneous and MMS-induced mutagenesis in the AP endonuclease-deficient cells . These results allow us to delineate base lesion flow within the BER pathway and link AP sites to other DNA damage repair and tolerance pathways.

Exp Gerontol, 2001 Dec, 37(1), 27 - 31
Apoptosis in yeast: a new model for aging research; Frohlich KU et al.; Apoptosis is a form of programmed cell death with a central role in development and homeostasis of metazoan organisms . Recent research indicates the presence of an apoptotic cell death program in unicellular eukaryotes . Yeast can be killed by expression of mammalian proapoptotic genes or in response to oxygen stress, which is an inducer of mammalian apoptosis . The dying yeast cells show morphological alterations typical for apoptosis . Yeast provides a simple model for cellular aging . The observation that old yeast cells produce oxygen radicals and die apoptotically may provide clues to a similar sequence of events in mammalian aging.

Traffic, 2001 Dec, 2(12), 896 - 907
Proteolytic function of GPI-anchored plasma membrane protease Yps1p in the yeast vacuole and Golgi; Sievi E et al.; Yps1p is a member of the GPI-anchored aspartic proteases which reside at the plasma membrane of Saccharomyces cerevisiae . Here we show that in Delta erg6 cells, where a late biosynthetic step of the membrane lipid ergosterol is blocked, part of Yps1p was targeted to the vacuole . There it overtook proteolytic functions of the Pep4p protease, resulting in processing of pro-CPY to CPY in cells lacking the PEP4 gene . Yps1p was enriched in membrane microdomains, as it could be isolated in detergent-insoluble complexes from both normal and Delta erg6 cells . Vacuolar Yps1 caused degradation of a mammalian sialyltransferase ectodomain fusion protein (ST6Ne), which was directed from the Golgi to the vacuole in both normal and Delta erg6 cells . Unexpectedly, ST6Ne was degraded also when arrested in the Golgi in a temperature-sensitive sec7-1 mutant . Newly synthesized Yps1p, in transit to the plasma membrane, was also involved in the Golgi-associated degradation . These data show that GPI-anchored proteases, whose biological roles are unknown, may reside and function in different subcellular locations.

J Cell Biol, 2001 Dec 10, 155(6), 949 - 59 Epub 2001 Dec 03.
Vesicular and nonvesicular transport of ceramide from ER to the Golgi apparatus in yeast; Funato K et al.; Transport and sorting of lipids must occur with specific mechanisms because the membranes of intracellular organelles differ in lipid composition even though most lipid biosynthesis begins in the ER . In yeast, ceramide is synthesized in the ER and transferred to the Golgi apparatus where inositolphosphorylceramide (IPC) is formed . These two facts imply that ceramide can be transported to the Golgi independent of vesicular traffic because IPC synthesis still continues when vesicular transport is blocked in sec mutants . Nonvesicular IPC synthesis in intact cells is not affected by ATP depletion . Using an in vitro assay that reconstitutes the nonvesicular pathway for transport of ceramide, we found that transport is temperature and cytosol dependent but energy independent . Preincubation of ER and Golgi fractions together at 4 degrees C, where ceramide transport does not occur, rendered the transport reaction membrane concentration independent, providing biochemical evidence that ER-Golgi membrane contacts stimulate ceramide transport . A cytosolic protease-sensitive factor is required after establishment of ER-Golgi contacts.

Proc Natl Acad Sci U S A, 2001 Dec 4, 98(25), 14322 - 7
A second iron-regulatory system in yeast independent of Aft1p; Rutherford JC et al.; Iron homeostasis in the yeast Saccharomyces cerevisiae is regulated at the transcriptional level by Aft1p, which activates the expression of its target genes in response to low-iron conditions . The yeast genome contains a paralog of AFT1, which has been designated AFT2 . To establish whether AFT1 and AFT2 have overlapping functions, a mutant containing a double aft1Deltaaft2Delta deletion was generated . Growth assays established that the single aft2Delta strain exhibited no iron-dependent phenotype . However, the double-mutant aft1Deltaaft2Delta strain was more sensitive to low-iron growth conditions than the single-mutant aft1Delta strain . A mutant allele of AFT2 (AFT2-1(up)), or overexpression of the wild-type AFT2 gene, led to partial complementation of the respiratory-deficient phenotype of the aft1Delta strain . The AFT2-1(up) allele also increased the uptake of (59)Fe in an aft1Delta strain . DNA microarrays were used to identify genes regulated by AFT2 . Some of the AFT2-regulated genes are known to be regulated by Aft1p; however, AFT2-1(up)-dependent activation was independent of Aft1p . The kinetics of induction of two genes activated by the AFT2-1(up) allele are consistent with Aft2p acting as a direct transcriptional factor . Truncated forms of Aft1p and Aft2p bound to a DNA duplex containing the Aft1p binding site in vitro . The wild-type allele of AFT2 activated transcription in response to growth under low-iron conditions . Together, these data suggest that yeast has a second regulatory pathway for the iron regulon, with AFT1 and AFT2 playing partially redundant roles.

Biochemistry, 2001 Dec 11, 40(49), 14942 - 51
The role of redox-active amino acids on compound I stability, substrate oxidation, and protein cross-linking in yeast cytochrome C peroxidase; Pfister TD et al.; The role of two tryptophans (Trp51 and Trp191) and six tyrosines (Tyr36, Tyr39, Tyr42, Tyr187, Tyr229, and Tyr236) in yeast cytochrome c peroxidase (CcP) has been probed by site-directed mutagenesis . A series of sequential mutations of these redox-active amino acid residues to the corresponding, less oxidizable residues in lignin peroxidase (LiP) resulted in an increasingly more stable compound I, with rate constants for compound I decay decreasing from 57 s(-1) for CcP(MI, W191F) to 7 s(-1) for CcP(MI, W191F,W51F,Y187F,Y229F,Y236F,Y36F,Y39E,Y42F) . These results provide experimental support for the proposal that the stability of compound I depends on the number of endogenous oxidizable amino acids in proteins . The higher stability of compound I in the variant proteins also makes it possible to observe its visible absorption spectroscopic features more clearly . The effects of the mutations on oxidation of ferrocytochrome c and 2,6-dimethoxyphenol were also examined . Since the first mutation in the series involved the change of Trp191, a residue that plays a critical role in the electron transfer pathway between CcP and cyt c, the ability to oxidize cyt c was negligible for all mutant proteins . On the other hand, the W191F mutation had little effect on the proteins' ability to oxidize 2,6-dimethoxyphenol . Instead, the W51F mutation resulted in the largest increase in the k(cat)/K(M), from 2.1 x 10(2) to 5.0 x 10(3) M(-1) s(-1), yielding an efficiency that is comparable to that of manganese peroxidase (MnP) . The effect in W51F mutation can be attributed to the residue's influence on the stability and thus reactivity of the ferryl oxygen of compound II, whose substrate oxidation is the rate-determining step in the reaction mechanism . Finally, out of all mutant proteins in this study, only the variant containing the Y36F, Y39E, and Y42F mutations was found to prevent covalent protein cross-links in the presence of excess hydrogen peroxide and in the absence of exogenous reductants . This finding marks the first time a CcP variant is incapable of forming protein cross-links and confirms that one of the three tyrosines must be involved in the protein cross-linking.

Genes Genet Syst, 2001 Aug, 76(4), 257 - 69
Characterization of a fission yeast mutant which displays defects in cell wall integrity and cytokinesis; Ishiguro J et al.; The fission yeast cps6-153 mutant was originally isolated based on its hypersensitivity to the spindle poison isopropyl N-3-chlorophenyl carbamate (CIPC) . The mutant also shows defects in both cell wall integrity and cytokinesis, resulting in the accumulation of unseparated cells with weakened cell walls . The arrested cells display a disoriented alignment of cytoplasmic microtubules . When the mutant cells are cultivated at high temperature (35 degrees C), both cell walls and septa become very thick . Electron microscopy revealed the disorganized structure of the thickened cell walls and septa, in which fibrillar components were not completely masked with an amorphous matrix . rad25+ was cloned from a genomic library by complementation of the mutant phenotypes, suggesting the involvement of Rad25p, one of two 14-3-3 proteins in S . pombe, in the pathway of cell wall integrity and cytokinesis.

Genome Res, 2001 Dec, 11(12), 2009 - 19
Evolution of gene order in the genomes of two related yeast species; Fischer G et al.; Changes in gene order between the genomes of two related yeast species, Saccharomyces cerevisiae and Saccharomyces bayanus var . uvarum were studied . From the dataset of a previous low coverage sequencing of the S . bayanus var . uvarum genome, 35 different synteny breakpoints between neighboring genes and two cases of local gene inversion were characterized in detail . The number and the type of the chromosomal rearrangements that have led to these differences were identified . We show that evolution of gene order in the genomes of these two yeast species is driven mainly by gene duplication onto different chromosomes followed by differential loss of the repeated copies . In addition, local gene inversions also would result from a mechanism of gene duplication, but in an inverted orientation, followed by loss of the original copy . The identification of traces of anciently duplicated genes, called relics, show that the loss of duplicates is more frequently caused by the accumulation of numerous mutations in one of the two copies than by DNA deletion . Surprisingly, gross chromosomal rearrangements such as translocations have only a minor effect on gene order reshuffling as they account for <10% of the synteny breakpoints.

Genes Dev, 2001 Dec 1, 15(23), 3155 - 68
The yeast SAS (something about silencing) protein complex contains a MYST-type putative acetyltransferase and functions with chromatin assembly factor ASF1; Osada S et al.; It is well established that acetylation of histone and nonhistone proteins is intimately linked to transcriptional activation . However, loss of acetyltransferase activity has also been shown to cause silencing defects, implicating acetylation in gene silencing . The something about silencing (Sas) 2 protein of Saccharomyces cerevisiae, a member of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, promotes silencing at HML and telomeres . Here we identify a ~450-kD SAS complex containing Sas2p, Sas4p, and the tf2f-related Sas5 protein . Mutations in the conserved acetyl-CoA binding motif of Sas2p are shown to disrupt the ability of Sas2p to mediate the silencing at HML and telomeres, providing evidence for an important role for the acetyltransferase activity of the SAS complex in silencing . Furthermore, the SAS complex is found to interact with chromatin assembly factor Asf1p, and asf1 mutants show silencing defects similar to mutants in the SAS complex . Thus, ASF1-dependent chromatin assembly may mediate the role of the SAS complex in silencing.

Genes Dev, 2001 Dec 1, 15(23), 3118 - 29
The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint; Biggins S et al.; The spindle checkpoint prevents cell cycle progression in cells that have mitotic spindle defects . Although several spindle defects activate the spindle checkpoint, the exact nature of the primary signal is unknown . We have found that the budding yeast member of the Aurora protein kinase family, Ipl1p, is required to maintain a subset of spindle checkpoint arrests . Ipl1p is required to maintain the spindle checkpoint that is induced by overexpression of the protein kinase Mps1 . Inactivating Ipl1p allows cells overexpressing Mps1p to escape from mitosis and segregate their chromosomes normally . Therefore, the requirement for Ipl1p in the spindle checkpoint is not a consequence of kinetochore and/or spindle defects . The requirement for Ipl1p distinguishes two different activators of the spindle checkpoint: Ipl1p function is required for the delay triggered by chromosomes whose kinetochores are not under tension, but is not required for arrest induced by spindle depolymerization . Ipl1p localizes at or near kinetochores during mitosis, and we propose that Ipl1p is required to monitor tension at the kinetochore.

Curr Opin Microbiol, 2001 Dec, 4(6), 720 - 7
Cell cycle control of yeast filamentous growth; Rua D et al.; Great progress has been made toward dissecting the signal transduction pathways and transcriptional outputs regulating yeast pseudohyphal growth . However, the mechanism underlying polarized morphogenesis in filamentous growth remains unclear . A synthesis of the data suggests that the ultimate target of these pathways is to repress the activity of the mitotic cyclin Clb2 as an antagonist of polarized growth . Here, we discuss how this cell cycle regulation, in concert with control of transcription, ubiquitin-dependent proteolysis and cytoskeletal polarity, may mediate the switch to filamentous growth.

Curr Opin Microbiol, 2001 Dec, 4(6), 703 - 12
Modular complexes that regulate actin assembly in budding yeast; Goode BL et al.; The actin cytoskeleton of budding yeast contains an extensive set of actin-associated proteins with conserved mammalian counterparts . For more than 20 years, yeast has been used as a model organism to dissect the in vivo functions of these factors, revealing an intricate web of genetic interactions in the cell . Now, a surge of biochemical reports is defining the physical interactions and activities of these proteins and providing mechanistic insights into their cellular roles . The emerging view is that most actin-associated proteins do not act alone but, rather, associate to form modular protein complexes that regulate actin assembly and organization.

Curr Opin Microbiol, 2001 Dec, 4(6), 690 - 5
Assembly and regulation of the cytokinetic apparatus in budding yeast; Tolliday N et al.; The identification of an actomyosin-based contractile ring in budding yeast has recently established this organism as a general model for studying cytokinesis . Work over the past three years has provided important new insights into the conserved mechanisms underlying the assembly and regulation of the cytokinetic structures . This review covers the recent progress in studying cytokinesis in budding yeast.

Curr Opin Microbiol, 2001 Dec, 4(6), 681 - 9
The septin cortex at the yeast mother-bud neck; Gladfelter AS et al.; A specialized cortical domain is organized by the septins at the necks of budding yeast cells . Recent findings suggest that this domain serves as a diffusion barrier and also as a local cell-shape sensor . We review these findings along with what is known about the organization of the septin cortex and its regulation during the cell cycle.

Biol Cell, 2001 Sep, 93(1-2), 63 - 70
MAP kinase dynamics in yeast; van Drogen F et al.; MAP kinase pathways play key roles in cellular responses towards extracellular signals . In several cases, the three core kinases interact with a scaffold molecule, but the function of these scaffolds is poorly understood . They have been proposed to contribute to signal specificity, signal amplification, or subcellular localization of MAP kinases . Several MAP kinases translocate to the nucleus in response to their activation, suggesting that nuclear transport may provide a regulatory mechanism . Here we describe new applications for Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP), to study dynamic translocations of MAPKs between different subcellular compartments . We have used these methods to measure the nuclear/cytoplasmic dynamics of several yeast MAP kinases, and in particular to address the role of scaffold proteins for MAP-kinase signaling.

J Biol Chem, 2002 Feb 8, 277(6), 4056 - 61 Epub 2001 Nov 29.
Metabolic engineering in yeast demonstrates that S-adenosylmethionine controls flux through the methylenetetrahydrofolate reductase reaction in vivo; Roje S et al.; One-carbon flux into methionine and S-adenosylmethionine (AdoMet) is thought to be controlled at the methylenetetrahydrofolate reductase (MTHFR) step . Mammalian MTHFRs are inhibited by AdoMet in vitro, and it has been proposed that methyl group biogenesis is regulated in vivo by this feedback loop . In this work, we used metabolic engineering in the yeast Saccharomyces cerevisiae to test this hypothesis . Like mammalian MTHFRs, the yeast MTHFR encoded by the MET13 gene is NADPH-dependent and is inhibited by AdoMet in vitro . This contrasts with plant MTHFRs, which are NADH-dependent and AdoMet-insensitive . To manipulate flux through the MTHFR reaction in yeast, the chromosomal copy of MET13 was replaced by an Arabidopsis MTHFR cDNA (AtMTHFR-1) or by a chimeric sequence (Chimera-1) comprising the yeast N-terminal domain and the AtMTHFR-1 C-terminal domain . Chimera-1 used both NADH and NADPH and was insensitive to AdoMet, supporting the view that the C-terminal domain is responsible for AdoMet inhibition . Engineered yeast expressing Chimera-1 accumulated 140-fold more AdoMet and 7-fold more methionine than did the wild-type and grew normally . Yeast expressing AtMTHFR-1 accumulated 8-fold more AdoMet . This is the first in vivo evidence that the AdoMet sensitivity and pyridine nucleotide preference of MTHFR control methylneogenesis . (13)C labeling data indicated that glycine cleavage becomes a more prominent source of one-carbon units when Chimera-1 is expressed . Possibly related to this shift in one-carbon fluxes, total folate levels are doubled in yeast cells expressing Chimera-1.

Genetics, 2001 Nov, 159(3), 919 - 28
A filamentous growth response mediated by the yeast mating pathway; Erdman S et al.; Haploid cells of the budding yeast Saccharomyces cerevisiae respond to mating pheromones by arresting their cell-division cycle in G1 and differentiating into a cell type capable of locating and fusing with mating partners . Yeast cells undergo chemotactic cell surface growth when pheromones are present above a threshold level for morphogenesis; however, the morphogenetic responses of cells to levels of pheromone below this threshold have not been systematically explored . Here we show that MATa haploid cells exposed to low levels of the alpha-factor mating pheromone undergo a novel cellular response: cells modulate their division patterns and cell shape, forming colonies composed of filamentous chains of cells . Time-lapse analysis of filament formation shows that its dynamics are distinct from that of pseudohyphal growth; during pheromone-induced filament formation, daughter cells are delayed relative to mother cells with respect to the timing of bud emergence . Filament formation requires the RSR1(BUD1), BUD8, SLK1/BCK1, and SPA2 genes and many elements of the STE11/STE7 MAP kinase pathway; this response is also independent of FAR1, a gene involved in orienting cell polarization during the mating response . We suggest that mating yeast cells undergo a complex response to low levels of pheromone that may enhance the ability of cells to search for mating partners through the modification of cell shape and alteration of cell-division patterns.

FEBS Lett, 2001 Nov 23, 508(3), 375 - 8
The Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP) can function as a dominant suppressor of Bax-induced cell death of yeast; Pan L et al.; We identified genes based on screening of an Arabidopsis cDNA library for functional suppressors of mouse Bax-induced cell death of yeast cells . Interestingly, the cDNA encoding AtEBP, known as Arabidopsis thaliana ethylene-responsive element binding protein, was isolated numerous times in the functional screen (82% of all suppressors) . Full-length AtEBP and its localization to the nucleus were essential for the suppression of Bax-induced cell death . Morphological abnormality of intracellular network that is a hallmark of Bax-induced cell death was attenuated by expression of AtEBP.

Curr Biol, 2001 Nov 27, 11(23), 1885 - 90
A yeast homolog of chromatin assembly factor 1 is involved in early ribosome assembly; Schaper S et al.; Cells have a recurrent need for the correct assembly of protein-nucleic acid complexes . We have studied a yeast homolog of the smallest subunit of chromatin assembly factor 1 (CAF1), encoded by YMR131c and termed "RRB1" . Unlike other yeast homologs, Msi1p, and Hat2p, Rrb1p is essential for cell viability . Impairment of Rrb1p function results in decreased levels of free 60S ribosomal subunits and the appearance of half-mer polysomes, suggesting its involvement in ribosome biogenesis . Using tandem affinity purification (TAP ) combined with mass spectrometry, we show that Rrb1p is associated with ribosomal protein L3 . A fraction of Rrb1p is also found in a protein-precursor rRNA complex containing at least ten other early-assembling ribosomal proteins . We propose that Rrb1p is required for proper assembly of preribosomal particles during early ribosome biogenesis, presumably by targeting L3 onto the 35S precursor rRNA . This action may resemble the mechanism by which CAF1 assembles histones H3/H4 onto newly replicated DNA.

Prog Drug Res, 2001, 57, 117 - 62
Applications of yeast in drug discovery; Ma D; The yeast Saccharomyces cerevisiae is perhaps the best-studied eukaryotic organism . Its experimental tractability, combined with the remarkable conservation of gene function throughout evolution, makes yeast the ideal model genetic organism . Yeast is a non-pathogenic model of fungal pathogens used to identify antifungal targets suitable for drug development and to elucidate mechanisms of action of antifungal agents . As a model of fundamental cellular processes and metabolic pathways of the human, yeast has improved our understanding and facilitated the molecular analysis of many disease genes . The completion of the Saccharomyces genome sequence helped launch the post-genomic era, focusing on functional analyses of whole genomes . Yeast paved the way for the systematic analysis of large and complex genomes by serving as a test bed for novel experimental approaches and technologies, tools that are fast becoming the standard in drug discovery research

Nat Genet, 2001 Dec, 29(4), 426 - 34
Genes required for ionizing radiation resistance in yeast; Bennett CB et al.; The ability of Saccharomyces cerevisiae to tolerate ionizing radiation damage requires many DNA-repair and checkpoint genes, most having human orthologs . A genome-wide screen of diploid mutants homozygous with respect to deletions of 3,670 nonessential genes revealed 107 new loci that influence gamma-ray sensitivity . Many affect replication, recombination and checkpoint functions . Nearly 90% were sensitive to other agents, and most new genes could be assigned to the following functional groups: chromatin remodeling, chromosome segregation, nuclear pore formation, transcription, Golgi/vacuolar activities, ubiquitin-mediated protein degradation, cytokinesis, mitochondrial activity and cell wall maintenance . Over 50% share homology with human genes, including 17 implicated in cancer, indicating that a large set of newly identified human genes may have related roles in the toleration of radiation damage.

EMBO J, 2001 Dec 3, 20(23), 6783 - 92
Sphingoid base signaling via Pkh kinases is required for endocytosis in yeast; Friant S et al.; In yeast, sphingoid base synthesis is required for the internalization step of endocytosis and organization of the actin cytoskeleton . We show that overexpression of either one of the two kinases Pkh1p or Pkh2p, that are homologous to mammalian 3-phosphoinositide-dependent kinase-1 (PDK1), can specifically suppress the sphingoid base synthesis requirement for endocytosis . Pkh1p and Pkh2p have an overlapping function because only a mutant with impaired function of both kinases is defective for endocytosis . Pkh1/2p kinases are activated in vitro by nanomolar concentrations of sphingoid base . These results suggest that Pkh1/2p kinases are part of a sphingoid base-mediated signaling pathway that is required for the internalization step of endocytosis . The Pkc1p kinase that is phosphorylated by Pkh1/2p kinases and plays a role in endocytosis was identified as one of the downstream effectors of this signaling cascade.

EMBO J, 2001 Dec 3, 20(23), 6683 - 91
Yeast prion protein derivative defective in aggregate shearing and production of new 'seeds'; Borchsenius AS et al.; According to the nucleated polymerization model, in vivo prion proliferation occurs via dissociation (shearing) of the huge prion polymers into smaller oligomeric 'seeds', initiating new rounds of prion replication . Here, we identify the deletion derivative of yeast prion protein Sup35 (Sup35-Delta22/69) that is specifically defective in aggregate shearing and 'seed' production . This derivative, {PSI+}, previously thought to be unable to turn into a prion state, in fact retains the ability to form a prion ({PSI+}(Delta22/69)) that can be maintained in selective conditions and transmitted by cytoplasmic infection (cytoduction), but which is mitotically unstable in non-selective conditions . MorePSI+}(Delta22/69) retains its mitotic stability defect . The {PSI+}(Delta22/69) cells contain more Sup35 protein in the insoluble fraction and form larger Sup35 aggregates compared with the conventional {PSI+} cells . Moderate excess of Hsp104 disaggregase increases transmission of the {PSI+}(Delta22/69) prion, while excess Hsp70-Ssa chaperone antagonizes it, opposite to their effects on conventional {PSI+} . Our results shed light on the mechanisms determining the differences between transmissible prions and non-transmissible protein aggregates.

EMBO J, 2001 Dec 3, 20(23), 6591 - 600
Specific roles of protein-phospholipid interactions in the yeast cytochrome bc1 complex structure; Lange C et al.; Biochemical data have shown that specific, tightly bound phospholipids are essential for activity of the cytochrome bc1 complex (QCR), an integral membrane protein of the respiratory chain . However, the structure and function of such phospholipids are not yet known . Here we describe five phospholipid molecules and one detergent molecule in the X-ray structure of yeast QCR at 2.3 A resolution . Their individual binding sites suggest specific roles in facilitating structural and functional integrity of the enzyme . Interestingly, a phosphatidylinositol molecule is bound in an unusual interhelical position near the flexible linker region of the Rieske iron-sulfur protein . Two possible proton uptake pathways at the ubiquinone reduction site have been identified: the E/R and the CL/K pathway . Remarkably, cardiolipin is positioned at the entrance to the latter . We propose that cardiolipin ensures structural integrity of the proton-conducting protein environment and takes part directly in proton uptake . Site-directed mutagenesis of ligating residues confirmed the importance of the phosphatidylinositol- and cardiolipin-binding sites.

Am J Respir Cell Mol Biol, 2001 Dec, 25(6), 692 - 8
Expression of yeast apurinic/apyrimidinic endonuclease (APN1) protects lung epithelial cells from bleomycin toxicity; He YH 2nd et al.; Bleomycin is a well-established anti-tumor drug . Its major untoward effect, pulmonary toxicity, has limited its usage . In this study, we used a DNA repair protein, yeast apurinic/apyrimidinic endonuclease (APN1) to reduce the toxicity of bleomycin on lung cells . A549 cells, an alveolar epithelial cell line, were transduced by MIEG3 retroviral vector encoding both enhanced green fluorescent protein (EGFP) and APN1 . Transduced cells were sorted by fluorescent-activated cell sorter (FACS) analysis and were cloned . The APN1 expression of transduced A549 cell population and four selected clones expressing different levels of EGFP was confirmed by Northern, Western, and apurinic/apyrimidinic (AP) endonuclease activity analyses . The expression of APN1 was positively correlated with the expression of EGFP . The protective effect of APN1 against bleomycin was determined by single cell gel electrophoresis/Comet assay and by clonogenic survival assay following bleomycin treatment . The A549 population expressing APN1 showed a significant reduction of DNA damage in the presence of 20, 50, and 100 microg/ml bleomycin; similarly, the APN1-expressing A549 population also demonstrated increased survival in the presence of bleomycin compared with the vector-transduced A549 population . In selected clones, three of four APN1-expressing clones resulted in significantly improved cell survival . The current study suggests that the yeast DNA repair protein, APN1, can reduce bleomycin toxicity to target lung cells.

J Cell Biol, 2001 Nov 26, 155(5), 725 - 31 Epub 2001 Nov 26.
A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast; Pavlov EV et al.; During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms . Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis . The pore diameter inferred from the largest conductance state of this channel is approximately 4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins . The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties . Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2 . Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax . These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

J Cell Biol, 2001 Nov 26, 155(5), 711 - 8 Epub 2001 Nov 26.
Correct spindle elongation at the metaphase/anaphase transition is an APC-dependent event in budding yeast; Severin F et al.; At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids . Subsequently, spindles elongate, separating the sister chromosomes into two sets . Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication . We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation . Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation . Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1 . Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1.

Biochemistry, 2001 Dec 4, 40(48), 14669 - 78
Phosphorylation and inactivation of yeast 6-phosphofructo-2-kinase contribute to the regulation of glycolysis under hypotonic stress; Dihazi H et al.; Phosphorylation of yeast 6-phosphofructo-2-kinase and its role for the regulation of glycolysis under hypoosmotic conditions were investigated . 6-Phosphofructo-2-kinase was found to be phosphorylated in vitro by protein kinase C at serine 652 and thereby inactivated . Protein phosphatase 2A reversed the phosphorylative inhibition of the enzyme . When yeast cells were shifted to hypotonic media, 6-phosphofructo-2-kinase was found to be phosphorylated and inactivated . Under in vivo conditions, two phosphate residues were incorporated into the enzyme . One of them is bound to serine 652, indicating that this modification was probably caused by yeast protein kinase C1 . The second phosphate is bound to Ser8 within the N-terminal peptide T(1-41) which contains several serine residues but no protein kinase C recognition sequence . Site-directed mutagenesis confirmed that the phosphorylation of serine 652 but not the N-terminal modification is responsible for the in vivo inactivation of 6-phosphofructo-2-kinase . The obtained results suggest that the phosphorylation of 6-phosphofructo-2-kinase mediates a response of the cells to an activation of the hypoosmolarity MAP kinase pathway . Via a suppression of glycolysis, the inactivation of 6-phosphofructo-2-kinase is expected to be responsible for the observed accumulation of glucose 6-phosphate, an essential precursor of the cell wall glucans, and the decrease of glycerol, an important osmolyte.

Arch Virol, 2001 Oct, 146(10), 1919 - 34
Thermal blockage of viruslike particle formation for the yeast retrotransposon Ty3 reveals differences in the cellular stress response; Sadeghi N et al.; The long terminal repeat (LTR) retrotransposons of the yeast Saccharomyces cerevisiae are similar in their structures and life cycles to animal retroviruses . The yeast LTR retrotransposon Ty3 does not transpose under conditions where the cellular stress response is activated . During stress, mature Ty3 proteins, indicative of the formation of intracellular Ty3 viruslike particles (VLPs), do not accumulate . In order to examine the role of stress proteins in Ty3 transposition, a sensitive genetic assay was developed to measure VLP formation . The assay employs a Ty3 element marked with a mutant allele of the yeast HIS3 gene (his3AI) . To create a stable His+ phenotype, Ty3 must form VLPs, reverse transcribe Ty3 RNA into cDNA, and then insert the cDNA into either chromosomal or plasmid DNA . Using this assay, thermal inhibition of Ty3 transposition was evident at temperatures as low as 30 degrees C . The level of production of mature Ty3 proteins parallels the transposition frequency . Although overexpression of the yeast UBP3 gene allows VLPs to form and transposition to occur in the constitutively stressed ssa1 ssa2 strain, it does not alleviate the inhibition of these processes during stress induced by heat or ethanol . This suggests that the genetic and physical modes of stress response induction are not equivalent.

RNA, 2001 Nov, 7(11), 1566 - 77
A cis-acting element known to block 3' mRNA degradation enhances expression of polyA-minus mRNA in wild-type yeast cells and phenocopies a ski mutant; Brown JT et al.; mRNA lacking a 3' polyA tail is not translated efficiently in wild-type eukaryotic cells, but is translated efficiently in yeast ski mutants . This enhanced expression could be due to altered translational specificity . However, as the SKI genes are required for 3' mRNA degradation, it could be a consequence of inhibition of 3' mRNA decay . Therefore, we asked if inhibition of 3' decay of a polyA-minus mRNA in cis would allow its efficient expression in wild-type cells . Capped in vitro reporter transcripts were prepared with or without a 3' cis-acting element known to inhibit 3' degradation (oligoG) and electroporated into yeast cells . The addition of oligoG to a polyA-minus mRNA enhanced expression 30-fold in wild-type cells . This level of expression was the same as that for an oligoG-minus, polyA-minus transcript in a ski mutant . The addition of oligoG did not significantly enhance the expression of polyA-minus mRNA in a ski mutant . The oligoG-dependent increase in expression was due to an increase in initial rate of translation and an increase in the functional half-life of the mRNA, similar to the effects observed in a ski mutant . The enhanced expression of the oligoG-containing RNA did not require Pab1p . We conclude that the enhanced translation of polyA-minus RNA in a ski mutant is due to inhibition of 3' mRNA degradation . Furthermore, a polyA-minus mRNA is expressed in wild-type cells when terminated in an element known to inhibit 3' decay in cis.

RNA, 2001 Nov, 7(11), 1554 - 65
The yeast U5 snRNP coisolated with the U1 snRNP has an unexpected protein composition and includes the splicing factor Aar2p; Gottschalk A et al.; We describe the purification and characterization of a 16S U5 snRNP from the yeast Saccharomyces cerevisiae and the identification of its proteins . In contrast to the human 20S U5 snRNP, it has a comparatively simple protein composition . In addition to the Sm core proteins, it contains only two of the U5 snRNP specific proteins, Prp8p and Snu114p . Interestingly, the 16S U5 snRNP contains also Aar2p, a protein that was previously implicated in splicing of the two introns of the MATa1 pre-mRNA . Here, we demonstrate that Aar2p is essential and required for in vivo splicing of U3 precursors . However, it is not required for splicing in vitro . Aar2p is associated exclusively with this simple form of the U5 snRNP (Aar2-U5), but not with the {U4/U6.U5} tri-snRNP or spliceosomal complexes . Consistent with this, we show that depletion of Aar2p interferes with later rounds of splicing, suggesting that it has an effect when splicing depends on snRNP recycling . Remarkably, the Aar2-U5 snRNP is invariably coisolated with the U1 snRNP regardless of the purification protocol used . This is consistent with the previously suggested cooperation between the U1 and U5 snRNPs prior to the catalytic steps of splicing . Electron microscopy of the Aar2-U5 snRNP revealed that, despite the comparatively simple protein composition, the yeast Aar2-U5 snRNP appears structurally similar to the human 20S U5 snRNP . Thus, the basic structural scaffold of the Aar2-U5 snRNP seems to be essentially determined by Prp8p, Snu114p, and the Sm proteins.

Curr Biol, 2001 Nov 13, 11(22), 1794 - 8
Promoter-specific activation defects by a novel yeast TBP mutant compromised for TFIIB interaction; Virbasius CM et al.; TFIIB is an RNA polymerase II general transcription factor (GTF) that has also been implicated in the mechanism of action of certain promoter-specific activators (see, for examples, {1-11}) . TFIIB enters the preinitiation complex (PIC) primarily through contact with the TATA box binding protein (TBP), an interaction mediated by three TBP residues {12-14} . To study the role of TFIIB in transcription activation in vivo, we randomly mutagenized these three residues in yeast TBP and screened for promoter-specific activation mutants . One mutant bearing a single conservative substitution, TBP-E186D, is the focus of this study . As expected, TBP-E186D binds normally to the TATA box but fails to support the entry of TFIIB into the PIC . Cells expressing TBP-E186D are viable but have a severe slow-growth phenotype . Whole-genome expression analysis indicates that transcription of 17% of yeast genes are compromised by this mutation . Chimeric promoter analysis indicates that the region of the gene that confers sensitivity to the TBP-E186D mutation is the UAS (upstream activating sequence), which contains the activator binding sites . Most interestingly, other TBP mutants that interfere with different interactions (TFIIB, TFIIA, or the TATA box) and a TFIIB mutant defective for interaction with TBP all manifest distinct and selective promoter-specific activation defects . Our results implicate the entry of TFIIB into the PIC as a critical step in the activation of certain promoters and reveal diverse mechanisms of transcription activation.

J Bacteriol, 2001 Dec, 183(24), 7253 - 9
Clock control of ultradian respiratory oscillation found during yeast continuous culture; Murray DB et al.; A short-period autonomous respiratory ultradian oscillation (period approximately 40 min) occurs during aerobic Saccharomyces cerevisiae continuous culture and is most conveniently studied by monitoring dissolved O(2) concentrations . The resulting data are high quality and reveal fundamental information regarding cellular dynamics . The phase diagram and discrete fast Fourier transformation of the dissolved O(2) values revealed a square waveform with at least eight harmonic peaks . Stepwise changes in temperature revealed that the oscillation was temperature compensated at temperatures ranging from 27 to 34 degrees C when either glucose (temperature quotient {Q(10)} = 1.02) or ethanol (Q(10) = 0.82) was used as a carbon source . After alteration of the temperature beyond the temperature compensation region, phase coherence events for individual cells were quickly lost . As the cell doubling rate decreased from 15.5 to 9.2 h (a factor of 1.68), the periodicity decreased by a factor of 1.26 . This indicated that there was a degree of nutrient compensation . Outside the range of dilution rates at which stable oscillation occurred, the mode of oscillation changed . The oscillation in respiratory output is therefore under clock control.

J Biol Chem, 2002 Feb 15, 277(7), 4778 - 81 Epub 2001 Nov 19.
Acetylation of the yeast histone H4 N terminus regulates its binding to heterochromatin protein SIR3; Carmen AA et al.; Heterochromatin at yeast telomeres and silent mating (HM) loci represses adjacent genes and is formed by the binding and spreading of silencing information regulators (SIR proteins) along histones . This involves the interaction between the C terminus of SIR3 and the N terminus of histone H4 . Since H4 is hypoacetylated in heterochromatin we wished to determine whether acetylation is involved in regulating the contacts between SIR3 and H4 . Binding of H4 peptide (residues 1-34) acetylated at lysines Lys-5, Lys-8, Lys-12, and Lys-16 to an immobilized SIR3 protein fragment (residues 510-970) was investigated using surface plasmon resonance . We find that acetylation of H4 lysines reduces binding (K(a)) of H4 to SIR3 in a cumulative manner so that the fully acetylated peptide binding is decreased approximately 50-fold relative to unacetylated peptide . Thus, by affecting SIR3-H4 binding, acetylation may regulate the formation of heterochromatin . These data help explain the hypoacetylated state of histone H4 in heterochromatin of eukaryotes.

J Agric Food Chem, 2001 Nov, 49(11), 5397 - 408
Definitive evidence for the actual contribution of yeast in the transformation of neutral precursors of grape aromas; Delfini C et al.; Experiments were designed to demonstrate the actual contribution of yeast in the formation of the primary aroma during the vinification of neutral grapes . Ruche was chosen as the model wine to study because of its unique fragrance . A yeast strain specific for Ruche was selected using a new and rapid isolation method for red wines . The results of this study can be summarized as follows: Skins from nonaromatic white or red grapes apparently contain most of the primary aroma compounds that are revealed in the must only after contact with yeast cells under defined conditions . Similar results were obtained with the pulp and seeds fractions; however, the olfactory notes, although well characterized, differed from those obtained with skins alone . Clarification, filtration, and centrifugation of the pulp and seed fractions or sonification of the skins produce different and well-characterized olfaction notes during the contact with yeast . The primary aroma of nonaromatic white and red grapes contained in the skins can be revealed within 24-48 h of yeast contact in a synthetic nutrient medium (SNM) . The primary aroma precursors extracted from the skins with methanol, water-saturated butanol, or aqueous buffer at pH 3.2, concentrated and eluted from a C18 resin column, can be transformed to the free form wine aroma markers within 6 h of contact with yeast cells in SNM . By contrast, prolonged maceration of the skins in aqueous alcoholic buffer at pH 3.2 or 1.1, at 50 or 70 degrees C did not release primary odors typical of wine . The individual primary aroma compounds, identified by GC-MS analysis in Ruche wine samples or in Ruche skin-yeast-SNM samples, could not explain the complexity of the typical Ruche wine odor . Only odors common to many wine varieties were identified by GC-olfactometry analysis.

Mol Genet Genomics, 2001 Nov, 266(3), 505 - 13
The PH domain of the yeast GEF Rom2p serves an essential function in vivo; Lorberg A et al.; In a screen designed to identify new upstream components of the Pkc1p-MAP kinase signal transduction pathway that responds to cell wall damage in yeast, we identified a new mutant allele of the ROM2 gene, which encodes a GDP/GTP exchange factor that acts on Rho1p . This allele, originally termed ubk1 (upstream of Bck1p) encodes a truncated protein that lacks the putative PH domain . Complementation experiments showed that genes coding for several known components of the pathway are able to suppress the ubk1 mutation to various degrees when introduced on low- or high-copy-number vectors . Analysis of several rom2 mutants showed that mutants in which the PH domain is deleted result in a phenotype indistinguishable from that of a strain deleted for the entire gene, indicating that this domain fulfills an essential function in vivo . Furthermore, we found that the growth phenotype of rom2 mutants is highly dependent on the strain background . Surprisingly, analysis of the phosphorylation status of Mpk1p in these mutants showed an elevated level of doubly phosphorylated Mpk1 protein, indicating that the growth defect of rom2 mutants is not due to an inability to activate the MAP kinase module, but rather to lack of a function of the Rom2 protein that has yet to be identified precisely.

Mol Genet Genomics, 2001 Nov, 266(3), 481 - 96
Yeast cys3 and gsh1 mutant cells display overlapping but non-identical symptoms of oxidative stress with regard to subcellular protein localization and CDP-DAG metabolism; Matiach A et al.; In a screen for temperature-sensitive (37 degrees C) mutants of Saccharomyces cerevisiae that are defective in the proper localization of the Golgi transmembrane protein Emp47p, we uncovered a constitutive loss-of-function mutation in CYS3/STR1, the gene coding for cystathionine-gamma-lyase . We showed by immunofluorescence, sucrose-gradient analysis and quantitative Western analysis that the mutant mislocalized Emp47p to the vacuole at high temperature, while Golgi structures were apparently normal and biosynthetic routing of the vacuolar carboxypeptidase Y (CPY) and the plasma membrane GPI-anchored protein Gas1p were unaffected . The effect of high temperature on Emp47p localization, as well as the temperature sensitivity of the mutant strain on rich medium, appear to be caused by oxidative stress and are correlated with severe reductions in the intracellular levels of low-molecular-weight thiols . In accordance with this conclusion, cys3-2 mutant cells were more sensitive to the oxidizing agent 1-chloro-2,4-dinitrobenzene, which also aggravated the mislocalization of Emp47p observed at high temperature . Furthermore, all the phenotypes of the mutant were completely complemented by exogenous supply of the main low-molecular-weight thiol, glutathione (GSH) and, importantly, the thiol beta-mercaptoethanol reversed the temperature sensitivity of the mutant . A comparison of our mutant with a mutant defective in GSH synthesis showed that gsh1Delta cells were similar to wild-type cells under the stress conditions tested, with the exception of one novel oxidative stress-related phenotype that is observed in both cys3-2 and gsh1Delta mutant cells - a defect in CDP-DAG metabolism upon shift to the non-permissive temperature . As most of the stress-related phenotypes of cys3-2 mutant cells are more severe than those seen in gsh1Delta cells, we conclude that cysteine as such is required and sufficient to confer some degree of protection from oxidative stress in yeast cells.

Mol Genet Genomics, 2001 Nov, 266(3), 436 - 44
Two alternative cell cycle checkpoint pathways differentially control DNA damage-dependent induction of MAG1 and DDI1 expression in yeast; Zhu Y et al.; Eukaryotic cells respond to DNA damage by activating damage checkpoint pathways, which arrest cell cycle progression and induce gene expression . In order to understand how damage checkpoints control the expression of DNA damage-inducible genes, the transcript level of two closely clustered genes, MAG1 and DDI1, was examined in a number of checkpoint mutants . We previously reported that MAG1 induction was abolished in pol2 and rad53 mutants, but not in the mec1-1 mutant . In this study, we found that mec1Delta and dun1Delta null mutants were defective in MAG1 induction, suggesting that MAG1 shares a common regulatory pathway with the RNR1,2,3,4 genes, which are also regulated by the POL2-MEC1-RAD53-DUN1 checkpoint pathway, and that the mec1-1 mutation probably represents a separation-of-function mutation . However, MAG1 is not activated in precisely the same way as the RNR genes, since mutations in CRT1, TUP1 and SSN6, which encode repressors of RNR genes, did not affect basal or induced expression of MAG1 . In contrast, the DDI1 transcript level was not affected by any of the above checkpoint mutations . Interestingly, simultaneous inactivation of RAD53 or DUN1 with PDS1, a newly identified checkpoint gene, resulted in severe down-regulation of DDI1 expression, suggesting that DDI1 is controlled by two damage checkpoint pathways, one mediated by POL2-MEC1-RAD53-DUN1 and the other by CHK1-PDS1 . On the other hand, deletion of TEL1, a structural homologue of MEC1, did not affect expression of MAG1, DDI1 or RNR3, suggesting that TEL1 plays no role in induction by DNA damage . Based on these and previous studies, we present a model for the role of checkpoint genes in transcriptional regulation in response to DNA damage.

Nucleic Acids Res, 2001 Nov 15, 29(22), 4625 - 32
A downstream regulatory element located within the coding sequence mediates autoregulated expression of the yeast fatty acid synthase gene FAS2 by the FAS1 gene product; Wenz P et al.; The fatty acid synthase genes FAS1 and FAS2 of the yeast Saccharomyces cerevisiae are transcriptionally co-regulated by general transcription factors (such as Reb1, Rap1 and Abf1) and by the phospholipid-specific heterodimeric activator Ino2/Ino4, acting via their corresponding upstream binding sites . Here we provide evidence for a positive autoregulatory influence of FAS1 on FAS2 expression . Even with a constant FAS2 copy number, a 10-fold increase of FAS2 transcript amount was observed in the presence of FAS1 in multi-copy, compared to a fas1 null mutant . Surprisingly, the first 66 nt of the FAS2 coding region turned out as necessary and sufficient for FAS1-dependent gene expression . FAS2-lacZ fusion constructs deleted for this region showed high reporter gene expression even in the absence of FAS1, arguing for a negatively-acting downstream repression site (DRS) responsible for FAS1-dependent expression of FAS2 . Our data suggest that the FAS1 gene product, in addition to its catalytic function, is also required for the coordinate biosynthetic control of the yeast FAS complex . An excess of uncomplexed Fas1 may be responsible for the deactivation of an FAS2-specific repressor, acting via the DRS.

Mol Cell Biol, 2001 Dec, 21(24), 8651 - 6
Requirement for yeast RAD26, a homolog of the human CSB gene, in elongation by RNA polymerase II; Lee SK et al.; Mutations in the human CSB gene cause Cockayne syndrome (CS) . In addition to increased photosensitivity, CS patients suffer from severe developmental abnormalities, including growth retardation and mental retardation . Whereas a deficiency in the preferential repair of UV lesions from the transcribed strand accounts for the increased photosensitivity of CS patients, the reason for developmental defects in these individuals has remained unclear . Here we provide in vivo evidence for a role of RAD26, the counterpart of the CSB gene in Saccharomyces cerevisiae, in transcription elongation by RNA polymerase II, and in addition we show that under conditions requiring rapid synthesis of new mRNAs, growth is considerably reduced in cells lacking RAD26 . These findings implicate a role for CSB in transcription elongation, and they strongly suggest that impaired transcription elongation is the underlying cause of the developmental problems in CS patients.

Mol Cell Biol, 2001 Dec, 21(24), 8638 - 50
EBP2 is a member of the yeast RRB regulon, a transcriptionally coregulated set of genes that are required for ribosome and rRNA biosynthesis; Wade C et al.; In an effort to identify sets of yeast genes that are coregulated across various cellular transitions, gene expression data sets derived from yeast cells progressing through the cell cycle, sporulation, and diauxic shift were analyzed . A partitioning algorithm was used to divide each data set into 24 clusters of similar expression profiles, and the membership of the clusters was compared across the three experiments . A single cluster of 189 genes from the cell cycle experiment was found to share 65 genes with a cluster of 159 genes from the sporulation data set . Many of these genes were found to be clustered in the diauxic-shift experiment as well . The overlapping set was enriched for genes required for rRNA biosynthesis and included genes encoding RNA helicases, subunits of RNA polymerases I and III, and rRNA processing factors . A subset of the 65 genes was tested for expression by a quantitative-relative reverse transcriptase PCR technique, and they were found to be coregulated after release from alpha factor arrest, heat shock, and tunicamycin treatment . Promoter scanning analysis revealed that the 65 genes within this ribosome and rRNA biosynthesis (RRB) regulon were enriched for two motifs: the 13-base GCGATGAGATGAG and the 11-base TGAAAAATTTT consensus sequences . Both motifs were found to be important for promoting gene expression after release from alpha factor arrest in a test rRNA processing gene (EBP2), which suggests that these consensus sequences may function broadly in the regulation of a set of genes required for ribosome and rRNA biosynthesis.

Mol Cell Biol, 2001 Dec, 21(24), 8346 - 56
Mpe1, a zinc knuckle protein, is an essential component of yeast cleavage and polyadenylation factor required for the cleavage and polyadenylation of mRNA; Vo LT et al.; In Saccharomyces cerevisiae, in vitro mRNA cleavage and polyadenylation require the poly(A) binding protein, Pab1p, and two multiprotein complexes: CFI (cleavage factor I) and CPF (cleavage and polyadenylation factor) . We characterized a novel essential gene, MPE1 (YKL059c), which interacts genetically with the PCF11 gene encoding a subunit of CFI . Mpe1p is an evolutionarily conserved protein, a homolog of which is encoded by the human genome . The protein sequence contains a putative RNA-binding zinc knuckle motif . MPE1 is implicated in the choice of ACT1 mRNA polyadenylation site in vivo . Extracts from a conditional mutant, mpe1-1, or from a wild-type extract immunoneutralized for Mpe1p are defective in 3'-end processing . We used the tandem affinity purification (TAP) method on strains TAP-tagged for Mpe1p or Pfs2p to show that Mpe1p, like Pfs2p, is an integral subunit of CPF . Nevertheless a stable CPF, devoid of Mpe1p, was purified from the mpe1-1 mutant strain, showing that Mpe1p is not directly involved in the stability of this complex . Consistently, Mpe1p is also not necessary for the processive polyadenylation, nonspecific for the genuine pre-mRNA 3' end, displayed by the CPF alone . However, a reconstituted assay with purified CFI, CPF, and the recombinant Pab1p showed that Mpe1p is strictly required for the specific cleavage and polyadenylation of pre-mRNA . These results show that Mpe1p plays a crucial role in 3' end formation probably by promoting the specific link between the CFI/CPF complex and pre-mRNA.

Mol Cells, 2001 Oct 31, 12(2), 244 - 9
Tyrosine phosphorylation-dependent yeast two-hybrid system for the identification of the SH2 domain-binding proteins; Park D et al.; In this paper, we established a modified yeast two-hybrid system, which is specialized for the detection of SH2 domain-binding proteins . The employment of the SH2 domain-tyrosine kinase fusion protein as bait allowed the efficient identification of SH2 domain-binding proteins . The general applicability of the system was tested using various combinations of SH2-kinase fusion bait and prey . The results indicate that the system specifically detected the previously reported in vivo interactions between the SH2 domains and their binding partners . In addition, using this system, we found the interaction between the adaptor protein, Lad, and the SH2 domain of Grb2 or PLC-gamma1 . The binding of Lad to Grb2 was further confirmed in mammalian cells by a co-immunoprecipitation study . The conclusion is that the established tyrosine phosphorylation-dependent yeast two-hybrid system provides a novel and efficient way to define the SH2 domain-binding molecules.

Dev Cell, 2001 Nov, 1(5), 621 - 31
Phosphorylation of gamma-tubulin regulates microtubule organization in budding yeast; Vogel J et al.; gamma-Tubulin is essential for microtubule nucleation in yeast and other organisms; whether this protein is regulated in vivo has not been explored . We show that the budding yeast gamma-tubulin (Tub4p) is phosphorylated in vivo . Hyperphosphorylated Tub4p isoforms are restricted to G1 . A conserved tyrosine near the carboxy terminus (Tyr445) is required for phosphorylation in vivo . A point mutation, Tyr445 to Asp, causes cells to arrest prior to anaphase . The frequency of new microtubules appearing in the SPB region and the number of microtubules are increased in tub4-Y445D cells, suggesting this mutation promotes microtubule assembly . These data suggest that modification of gamma-tubulin is important for controlling microtubule number, thereby influencing microtubule organization and function during the yeast cell cycle.

Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 103 - 10
RACK1 protein interacts with Helicobacter pylori VacA cytotoxin: the yeast two-hybrid approach; Hennig EE et al.; The VacA toxin is the major virulence factor of Helicobacter pylori . The studies on VacA intracellular expression suggest that it interacts with cytosolic proteins and that this interaction contributes significantly to vacuolization . The aim of this study was to identify the host protein(s) that interacts with the VacA protein . We used the fragments of VacA protein fused with GAL4-BD as the baits in the yeast two-hybrid approach . The yeast transformed with plasmids encoding bait proteins were screened with human gastric mucosa cDNA library, encoded C-terminal fusion proteins with GAL4-AD . Three independent His-beta-Gal-positive clones were identified in VacA-b1 screen; they matched two different lengths of cDNA encoding RACK1 protein . The specific activity of beta-galactosidase found in the yeast expressing both VacA-b1 and RACK1 fusion proteins was 12-19 times higher compared to all negative controls used . VacA is capable of binding the RACK1 in vitro as was confirmed by the pull-down assay with GST fusion VacA protein and {(35)S}Met-labeled RACK1 protein fragments .

EMBO J, 2001 Nov 15, 20(22), 6485 - 98
Hyperactivation of the yeast DNA damage checkpoint by TEL1 and DDC2 overexpression; Clerici M et al.; The evolutionarily conserved yeast Mec1 and Tel1 protein kinases, as well as the Mec1-interacting protein Ddc2, are involved in the DNA damage checkpoint response . We show that regulation of Tel1 and Ddc2-Mec1 activities is important to modulate both activation and termination of checkpoint-mediated cell cycle arrest . In fact, overproduction of either Tel1 or Ddc2 causes a prolonged cell cycle arrest and cell death in response to DNA damage, impairing the ability of cells to recover from checkpoint activation . This cell cycle arrest is independent of Mec1 in UV-irradiated Tel1-overproducing cells, while it is strictly Mec1 dependent in similarly treated DDC2-overexpressing cells . The Rad53 checkpoint kinase is instead required in both cases for cell cycle arrest, which correlates with its enhanced and persistent phosphorylation, suggesting that unscheduled Rad53 phosphorylation might prevent cells from re-entering the cell cycle after checkpoint activation . In addition, Tel1 overproduction results in transient nuclear division arrest and concomitant Rad53 phosphorylation in the absence of exogenous DNA damage independently of Mec1 and Ddc1.

EMBO J, 2001 Nov 15, 20(22), 6464 - 74
A novel eIF2B-dependent mechanism of translational control in yeast as a response to fusel alcohols; Ashe MP et al.; Fusel alcohols are natural products of amino acid catabolism in the yeast Saccharomyces cerevisiae that cause morphological changes similar to those seen during pseudohyphal growth . We have discovered that certain of these alcohols, including butanol and isoamyl alcohol, bring about a rapid inhibition of translation at the initiation step . This inhibition is strain specific and is not explained by previously described translational control pathways . Using genetic mapping, we have identified a proline to serine allelic variation at amino acid 180 of the GCD1 gene product as the genetic locus that allows translational regulation upon butanol addition . Gcd1p forms part of the eIF2B guanine nucleotide complex that is responsible for recycling eIF2-GDP to eIF2-GTP . This represents one of the key limiting steps of translation initiation and we provide evidence that fusel alcohols target eIF2B in order to bring about translational regulation.

FEBS Lett, 2001 Nov 9, 508(1), 117 - 20
The C-terminal domain of yeast Ero1p mediates membrane localization and is essential for function; Pagani M et al.; In eukaryotes, members of the Ero1 family control oxidative protein folding in the endoplasmic reticulum (ER) . Yeast Ero1p is tightly associated with the ER membrane, despite cleavage of the leader peptide, the only hydrophobic sequence that could mediate lipid insertion . In contrast, human Ero1-Lalpha and a yeast mutant (Ero1pDeltaC) lacking the 127 C-terminal amino acids are soluble when expressed in yeast . Neither Ero1-Lalpha nor Ero1pDeltaC complements an ERO1 disrupted strain . Appending the yeast C-terminal tail to human Ero1-Lalpha restores membrane association and allows growth of ERO1 disrupted cells . Therefore, the tail of Ero1p mediates membrane association and is crucial for function.

FEBS Lett, 2001 Nov 9, 508(1), 39 - 43
Functional identification of sterol-4alpha-methyl oxidase cDNAs from Arabidopsis thaliana by complementation of a yeast erg25 mutant lacking sterol-4alpha-methyl oxidation; Darnet S et al.; Specific primers derived from both genomic sequence data and EST cDNA sequences were used to polymerase chain reaction amplify two full-length cDNA sequences (AtSMO1 and AtSMO2), 801 and 783 bp, respectively, from an Arabidopsis thaliana cDNA library . The predicted proteins show 32 and 29% identity to the ERG25 gene from Saccharomyces cerevisiae which encodes the sterol-4alpha-methyl oxidase (SMO), a membrane-bound non-heme di-iron oxygenase involved in lipid metabolism . Heterologous expression of AtSMO1 and AtSMO2 in a yeast erg25 ergosterol auxotroph, lacking SMO activity, restored growth and endogenous ergosterol synthesis . These results represent the first functional identification of SMO genes from plants.

J Cell Biol, 2001 Nov 12, 155(4), 581 - 92 Epub 2001 Nov 12.
Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud; Adamo JE et al.; The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells . In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane . We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization . cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane . This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput . Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis . Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus . Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

J Biol Chem, 2002 Jan 25, 277(4), 2406 - 12 Epub 2001 Nov 12.
Yeast oxidosqualene cyclase (Erg7p) is a major component of lipid particles; Milla P et al.; Oxidosqualene cyclase of the yeast encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis . In a previous study (Athenstaedt, K., Zweytick, D., Jandrositz, A, Kohlwein, S . D., and Daum, G . (1999) J . Bacteriol . 181, 6441-6448), Erg7p was identified as a component of yeast lipid particles . Here, we present evidence that Erg7p is almost exclusively associated with this compartment as shown by analysis of enzymatic activity, Western blot analysis, and in vivo localization of Erg7p-GFP . Occurrence of oxidosqualene cyclase in other organelles including the endoplasmic reticulum is negligible . In an erg7 deletion strain or in wild-type cells treated with an inhibitor of oxidosqualene cyclase, the substrate of Erg7p, oxidosqualene, accumulated mostly in lipid particles . Storage in lipid particles of this intermediate produced in excess may provide a possibility to exclude this membrane-perturbing component from other organelles . Thus, our data provide evidence that lipid particles are not only a depot for neutral lipids, but also participate in coordinate sterol metabolism and trafficking and serve as a storage site for compounds that may negatively affect membrane integrity.

J Biol Chem, 2002 Jan 18, 277(3), 2360 - 7 Epub 2001 Nov 08.
Moe1 and spInt6, the fission yeast homologues of mammalian translation initiation factor 3 subunits p66 (eIF3d) and p48 (eIF3e), respectively, are required for stable association of eIF3 subunits; Bandyopadhyay A et al.; The protein encoded by the fission yeast gene, moe1(+) is the homologue of the p66/eIF3d subunit of mammalian translation initiation factor eIF3 . In this study, we show that in fission yeast, Moe1 physically associates with eIF3 core subunits as well as with 40 S ribosomal particles as a constituent of the eIF3 protein complex that is similar in size to multisubunit mammalian eIF3 . However, strains lacking moe1(+) (Deltamoe1) are viable and show no gross defects in translation initiation, although the rate of translation in the Deltamoe1 cells is about 30-40% slower than wild-type cells . Mutant Deltamoe1 cells are hypersensitive to caffeine and defective in spore formation . These phenotypes of Deltamoe1 cells are similar to those reported previously for deletion of the fission yeast int6(+) gene that encodes the fission yeast homologue of the p48/Int6/eIF3e subunit of mammalian eIF3 . Further analysis of eIF3 subunits in Deltamoe1 or Deltaint6 cells shows that in these deletion strains, while all the eIF3 subunits are bound to 40 S particles, dissociation of ribosome-bound eIF3 results in the loss of stable association between the eIF3 subunits . In contrast, eIF3 isolated from ribosomes of wild-type cells are associated with one another in a protein complex . These observations suggest that Moe1 and spInt6 are each required for stable association of eIF3 subunits in fission yeast.

Nippon Ishinkin Gakkai Zasshi, 2001, 42(4), 218 - 20
{Relationship between Malassezia Yeast and Infantile Seborrhoeic Dermatitis}; Nakabayashi A et al.; We examined 52 patients with infantile seborrhoeic dermatitis (ISD) and 47 healthy 1-month-old infants . Yeast cells on the right side of the face were counted by direct microscopic examination, and isolates from the left side of the face were identified by Tween test . Yeast cells were more numerous patients with ISD than in the healthy infants . M . furfur and M . globosa were isolated from ISD patients at significantly higher rates than from healthy infants.

Dev Cell, 2001 Sep, 1(3), 401 - 9
Ssh1p determines the translocation and dislocation capacities of the yeast endoplasmic reticulum; Wilkinson BM et al.; Sec61p is required both for protein translocation and dislocation across the membrane of the endoplasmic reticulum (ER) . However, the cellular role of the Sec61p homolog Ssh1p has not been clearly defined . We show that deltassh1 mutant cells have strong defects in both SRP-dependent and -independent translocation . Moreover, these cells were also found to be induced for the unfolded protein response and to be defective in dislocation of a misfolded ER protein . In addition, deltassh1 mutant cells rapidly became respiratory deficient . The other defects discussed above were suppressed in the respiratory-deficient state or under conditions where the rate of polypeptide translation was artificially reduced . These data identify Ssh1p as a component of a second, functionally distinct translocon in the yeast ER membrane.

Dev Cell, 2001 Sep, 1(3), 389 - 99
Phosphorylation of Mei2 and Ste11 by Pat1 kinase inhibits sexual differentiation via ubiquitin proteolysis and 14-3-3 protein in fission yeast; Kitamura K et al.; Fission yeast Pat1 kinase inhibits sexual differentiation by phosphorylating the meiotic inducer Mei2 and the transcription factor Ste11 . Here, we show how Pat1 downregulates these proteins . Mei2 is degraded via a ubiquitin-proteasome pathway in a phosphorylation-dependent fashion . The E2 Ubc2 and the E3 Ubr1 are required for this proteolysis . In addition, Pat1 negatively regulates Ste11 via Rad24/14-3-3, thereby repressing mei2+ transcription . The Pat1 phosphorylation sites of Ste11 match the consensus recognition sequence for 14-3-3 . Rad24 binds preferentially to phosphorylated Ste11, and this binding results in inhibition of the transcriptional activation capacity of Ste11 . Overall, therefore, these results show that Pat1 coordinates concerted molecular mechanisms that govern the sexual differentiation developmental decision.

J Biol Chem, 2002 Jan 11, 277(2), 1195 - 202 Epub 2001 Nov 07.
Inhibitory analogs of ubiquinol act anti-cooperatively on the Yeast cytochrome bc1 complex . Evidence for an alternating, half-of-the-sites mechanism of ubiquinol oxidation; Gutierrez-Cirlos EB et al.; The cytochrome bc(1) complex is a dimeric enzyme that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is re-reduced at a second center, referred to as center N . To understand better the mechanism of ubiquinol oxidation, we have examined the interaction of several inhibitory analogs of ubiquinol with the yeast cytochrome bc(1) complex . Stigmatellin and methoxyacrylate stilbene, two inhibitors that block ubiquinol oxidation at center P, inhibit the yeast enzyme with a stoichiometry of 0.5 per bc(1) complex, indicating that one molecule of inhibitor is sufficient to fully inhibit the dimeric enzyme . This stoichiometry was obtained when the inhibitors were titrated in cytochrome c reductase assays and in reactions of quinol with enzyme in which the inhibitors block pre-steady state reduction of cytochrome b . As an independent measure of inhibitor binding, we titrated the red shift in the optical spectrum of ferrocytochrome b with methoxyacrylate stilbene and thus confirmed the results of the inhibition of activity titrations . The titration curves also indicate that the binding is anti-cooperative, in that a second molecule of inhibitor binds with much lower affinity to a dimer in which an inhibitor molecule is already bound . Because these inhibitors bind to the ubiquinol oxidation site in the bc(1) complex, we propose that the yeast cytochrome bc(1) complex oxidizes ubiquinol by an alternating, half-of-the-sites mechanism.

Biochem Biophys Res Commun, 2001 Nov 16, 288(5), 1112 - 8
Differential effect of 1,10-phenanthroline on mammalian, yeast, and parasite glycosylphosphatidylinositol anchor synthesis; Sevlever D et al.; Glycosylphosphatidylinositol (GPI) anchoring of proteins to the plasma membrane is a common mechanism utilized by all eukaryotes including mammals, yeast, and the Trypanosoma brucei parasite . We have previously shown that in mammals phenanthroline (PNT) blocks the attachment of phosphoethanolamine (P-EthN) groups to mannose residues in GPI anchor intermediates, thus preventing the synthesis of mammalian GPI anchors . Therefore, PNT is likely to inhibit GPI-phosphoethanolamine transferases (GPI-PETs) . Here we report that in yeast, PNT also inhibits the synthesis of the GPI anchor as well as GPI-anchored proteins . Interestingly, the mechanism of PNT inhibition of GPI synthesis is different from that of YW3548, another putative GPI-PET inhibitor . In contrast to mammals and yeast, the synthesis of GPIs in T . brucei is not affected by PNT . Our results indicate that the T . brucei GPI-PET could be a potential target for antiparasitic drugs .

Proc Natl Acad Sci U S A, 2001 Nov 20, 98(24), 13675 - 80 Epub 2001 Nov 06.
Yeast Dam1p has a role at the kinetochore in assembly of the mitotic spindle; Jones MH et al.; During mitosis, replicated chromosomes are separated to daughter cells by the microtubule-based mitotic spindle . Chromosomes attach to the mitotic spindle through specialized DNA/protein structures called kinetochores, but the mechanism of attachment is not well understood . We show here that the yeast microtubule-binding protein, Dam1p, associates physically and functionally with kinetochores, suggesting a role in kinetochore attachment to the spindle . An epitope-tagged version of Dam1p colocalizes with the integral kinetochore component Ndc10p/Cbf2p in immunofluorescence analysis of chromosome spreads . In addition, Dam1p is associated preferentially with centromeric DNA as shown by chromatin immunoprecipitation experiments, and this association depends on Ndc10p/Cbf2p . We also demonstrate genetic interactions between DAM1 and CTF19 or SLK19 genes encoding kinetochore proteins . Although the defect caused by the dam1-1 mutation leads to activation of the spindle checkpoint surveillance system and consequent persistence of sister chromatid cohesion, the metaphase arrest spindle abnormally elongates, resulting in virtually complete chromosome missegregation . Execution point experiments indicate that Dam1p has a role in formation of a metaphase spindle and in anaphase spindle elongation . Finally, we have observed that the protein encoded by the dam1-1 allele becomes delocalized at the nonpermissive temperature, correlating with the subsequent onset of the mutant phenotype . Our studies are consistent with a role for Dam1p in attachment of sister chromatids through the kinetochore to the mitotic spindle before chromosome segregation.

Curr Opin Cell Biol, 2001 Dec, 13(6), 762 - 9
The role of the yeast spindle pole body and the mammalian centrosome in regulating late mitotic events; Pereira G et al.; Centrosomes of vertebrate cells and spindle pole bodies (SPBs) of fungi were first recognized through their ability to organize microtubules . Recent studies suggest that centrosomes and SPBs also have a function in the regulation of cell cycle progression, in particular in controlling late mitotic events . Regulators of mitotic exit and cytokinesis are associated with the SPB of budding and fission yeast . Elucidation of the molecular roles played by these regulators is helping to clarify the function of the SPB in controlling progression though mitosis.

Exp Cell Res, 2001 Nov 15, 271(1), 1 - 9
Yeast as a model system for studying endocytosis; Shaw JD et al.; Endocytosis is the membrane trafficking process by which plasma membrane components and extracellular material are internalized into cytoplasmic vesicles and delivered to early and late endosomes, eventually either recycling back to the plasma membrane or arriving at the lysosome/vacuole . The budding yeast Saccharomyces cerevisiae has proven to be an invaluable system for identifying proteins involved in endocytosis and elucidating the mechanisms underlying internalization and postinternalization events . Through genetic studies in yeast and biochemical studies in mammalian cells, it has become apparent that multiple cellular processes are linked to endocytosis, including actin cytoskeletal dynamics, ubiquitylation, lipid modification, and signal transduction . In this review, we will highlight the most exciting recent findings in the field of yeast endocytosis . Specifically, we will address the involvement of the actin cytoskeleton in internalization, the role of ubiquitylation as a regulator of multiple steps of endocytosis in yeast, and the sorting of endocytosed proteins into the recycling and vacuolar pathways .

Arch Biochem Biophys, 2001 Nov 15, 395(2), 239 - 45
Protein phosphatase methyltransferase 1 (Ppm1p) is the sole activity responsible for modification of the major forms of protein phosphatase 2A in yeast; Kalhor HR et al.; Protein phosphatase 2A (PP2A) is a major threonine/serine phosphatase that is involved in regulating a variety of cellular processes . It has been shown in both yeast and mammals that the PP2A catalytic subunit (PP2Ac) is methyl-esterified at the conserved C-terminal Leu residue . The recent characterization of a mammalian PP2A carboxyl methyltransferase has led to the identification of two ORFs in Saccharomyces cerevisiae as potential orthologues of the mammalian PP2A methyltransferase: protein phosphatase methyltransferase 1 (PPM1) and protein phosphatase methyltransferase 2 (PPM2) . To experimentally identify the PP2A methyltransferase in yeast, we obtained deletion mutants of PPM1 and PPM2 and then constructed double mutants . Using in vivo-labeling techniques, we demonstrate that only the PPM1 gene is required for PP2Ac methylation at the C-terminus . Because yeast has at least three homologues of PP2Ac (PPH21, PPH22, and PPH3), we then asked whether all of these catalytic subunits are methylated by the PPM1 and/or PPM2 putative methyltransferases . We modified the segment corresponding to the N-terminal coding region of all three PP2Ac genomic genes with a hemagglutinin (HA) tag in the parent, ppm1, ppm2, and ppm1ppm2 mutant genetic backgrounds . Using immuoprecipitation with anti-HA antibodies followed by methyl ester analysis, we showed that only in the ppm1 mutant were both Pph21p and Pph22p not methylated . We did not detect any methylesterification of Pph3p under our conditions . Our results indicate that PPM1 is the sole methyltransferase responsible for methylating the two major homologues of PP2Ac in yeast . The function of the PPM2 gene product remains unclear .

Curr Biol, 2001 Oct 30, 11(21), 1706 - 10
C . elegans clk-2, a gene that limits life span, encodes a telomere length regulator similar to yeast telomere binding protein Tel2p; Lim CS et al.; An important quest in modern biology is to identify genes involved in aging . Model organisms such as the nematode Caenorhabditis elegans are particularly useful in this regard . The C . elegans genome has been sequenced {1}, and single gene mutations that extend adult life span have been identified {2} . Among these longevity-controlling loci are four apparently unrelated genes that belong to the clk family . In mammals, telomere length and structure can influence cellular, and possibly organismal, aging . Here, we show that clk-2 encodes a regulator of telomere length in C . elegans.

Biochemistry, 2001 Nov 13, 40(45), 13564 - 73
Crystal structures of the yeast prion Ure2p functional region in complex with glutathione and related compounds; Bousset L et al.; The {URE3} phenotype in yeast Saccharomyces cerevisiae is due to an altered prion form of Ure2p, a protein involved in nitrogen catabolism . To understand possible conformational changes at the origin of prion propagation, we previously solved the crystal structure of the Ure2p functional region {Bousset et al . (2001) Structure 9, 39-46} . We showed the protein to have a fold similar to that of the beta class of glutathione S-transferases (GSTs) . Here we report crystal structures of the Ure2p functional region (extending from residues 95-354) in complex with glutathione (GSH), the substrate of all GSTs, and two widely used GST inhibitors, namely, S-hexylglutathione and S-p-nitrobenzylglutathione . In a manner similar to what is observed in many GSTs, ligand binding is not accompanied by a significant change in the conformation of the protein . We identify one GSH and one hydrophobic electrophile binding site per monomer as observed in all other GSTs . The sulfur group of GSH, that conjugates electrophiles, is located near the amide group of Asn124, allowing a hydrogen bond to be formed . Biochemical data indicate that GSH binds to Ure2p with high affinity . Its binding affects Ure2p oligomerization but has no effect on the assembly of the protein into amyloid fibrils . Despite results indicating that Ure2p lacks GST activity, we propose that Ure2p is a member of the GST superfamily that may describe a novel GST class . Our data bring new insights into the function of the Ure2p active region.

J Bioenerg Biomembr, 2001 Jun, 33(3), 187 - 96
Exploring the catalytic core of complex I by Yarrowia lipolytica yeast genetics; Kerscher S et al.; We have developed Yarrowia lipolytica as a model system to study mitochondrial complex I that combines the application of fast and convenient yeast genetics with efficient structural and functional analysis of its very stable complex I isolated by his-tag affinity purification with high yield . Guided by a structural model based on homologies between complex I and {NiFe} hydrogenases mutational analysis revealed that the 49 kDa subunit plays a central functional role in complex I . We propose that critical parts of the catalytic core of complex I have evolved from the hydrogen reactive site of {NiFe} hydrogenases and that iron-sulfur cluster N2 resides at the interface between the 49 kDa and PSST subunits . These findings are in full agreement with the "semiquinone switch" mechanism according to which coupling of electron and proton transfer in complex I is achieved by a single integrated pump comprising cluster N2, the binding site for substrate ubiquinone, and a tightly bound quinone or quinoid group.

Mol Biol Cell, 2001 Nov, 12(11), 3668 - 79
In vivo role for actin-regulating kinases in endocytosis and yeast epsin phosphorylation; Watson HA et al.; The yeast actin-regulating kinases Ark1p and Prk1p are signaling proteins localized to cortical actin patches, which may be sites of endocytosis . Interactions between the endocytic proteins Pan1p and End3p may be regulated by Prk1p-dependent threonine phosphorylation of Pan1p within the consensus sequence {L/I}xxQxTG . We identified two Prk1p phosphorylation sites within the Pan1p-binding protein Ent1p, a yeast epsin homologue, and demonstrate Prk1p-dependent phosphorylation of both threonines . Converting both threonines to either glutamate or alanine mimics constitutively phosphorylated or dephosphorylated Ent1p, respectively . Synthetic growth defects were observed in a pan1-20 ENT1(EE) double mutant, suggesting that Ent1p phosphorylation negatively regulates the formation/activity of a Pan1p-Ent1p complex . Interestingly, pan1-20 ent2 Delta but not pan1-20 ent1 Delta double mutants had improved growth and endocytosis over the pan1-20 mutant . We found that actin-regulating Ser/Thr kinase (ARK) mutants exhibit endocytic defects and that overexpressing either wild-type or alanine-substituted Ent1p partially suppressed phenotypes associated with loss of ARK kinases, including growth, endocytosis, and actin localization defects . Consistent with synthetic growth defects of pan1-20 ENT1(EE) cells, overexpressing glutamate-substituted Ent1p was deleterious to ARK mutants . Surprisingly, overexpressing the related Ent2p protein could not suppress ARK kinase mutant phenotypes . These results suggest that Ent1p and Ent2p are not completely redundant and may perform opposing functions in endocytosis . These data support the model that, as for clathrin-dependent recycling of synaptic vesicles, yeast endocytic protein phosphorylation inhibits endocytic functions.

Mol Biol Cell, 2001 Nov, 12(11), 3589 - 600
Enhanced cell polarity in mutants of the budding yeast cyclin-dependent kinase Cdc28p; Ahn SH et al.; The yeast cyclin-dependent kinase Cdc28p regulates bud morphogenesis and cell cycle progression via the antagonistic activities of Cln and Clb cyclins . Cln G1 cyclins direct polarized growth and bud emergence, whereas Clb G2 cyclins promote isotropic growth of the bud and chromosome segregation . Using colony morphology as a screen to dissect regulation of polarity by Cdc28p, we identified nine point mutations that block the apical-isotropic switch while maintaining other functions . Like a clb2 Delta mutation, each confers tubular bud shape, apically polarized actin distribution, unipolar budding, and delayed anaphase . The mutations are all suppressed by CLB2 overexpression and are synthetically lethal with a CLB2 deletion . However, defects in multiple independent pathways may underlie their common phenotype, because the mutations are scattered throughout the CDC28 sequence, complement each other, and confer diverse biochemical properties . Glu12Gly, a mutation that alters a residue involved in Swe1p inhibition of Cdc28p, was unique in being suppressed by deficiency of SWE1 or CLN1 . With wild-type CDC28, filament formation induced by CLN1 overexpression was markedly decreased in a SWE1 deletion . These results suggest that Swe1p, via inhibition of Clb2p/Cdc28p, may mediate much of the effect of Cln1p on filamentous morphogenesis.

Mol Biol Cell, 2001 Nov, 12(11), 3515 - 26
Interactions among a fimbrin, a capping protein, and an actin-depolymerizing factor in organization of the fission yeast actin cytoskeleton; Nakano K et al.; We report studies of the fission yeast fimbrin-like protein Fim1, which contains two EF-hand domains and two actin-binding domains (ABD1 and ABD2) . Fim1 is a component of both F-actin patches and the F-actin ring, but not of F-actin cables . Fim1 cross-links F-actin in vitro, but a Fim1 protein lacking either EF-hand domains (Fim1A12) or both the EF-hand domains and ABD1 (Fim1A2) has no actin cross-linking activity . Overexpression of Fim1 induced the formation of F-actin patches throughout the cell cortex, whereas the F-actin patches disappear in cells overexpressing Fim1A12 or Fim1A2 . Thus, the actin cross-linking activity of Fim1 is probably important for the formation of F-actin patches . The overexpression of Fim1 also excluded the actin-depolymerizing factor Adf1 from the F-actin patches and inhibited the turnover of actin in these structures . Thus, Fim1 may function in stabilizing the F-actin patches . We also isolated the gene encoding Acp1, a subunit of the heterodimeric F-actin capping protein . fim1 acp1 double null cells showed more severe defects in the organization of the actin cytoskeleton than those seen in each single mutant . Thus, Fim1 and Acp1 may function in a similar manner in the organization of the actin cytoskeleton . Finally, genetic studies suggested that Fim1 may function in cytokinesis in cooperation with Cdc15 (PSTPIP) and Rng2 (IQGAP), respectively.

Mol Biol Cell, 2001 Nov, 12(11), 3476 - 88
pkl1(+)and klp2(+): Two kinesins of the Kar3 subfamily in fission yeast perform different functions in both mitosis and meiosis; Troxell CL et al.; We have identified Klp2p, a new kinesin-like protein (KLP) of the KAR3 subfamily in fission yeast . The motor domain of this protein is 61% identical and 71% similar to Pkl1p, another fission yeast KAR3 protein, yet the two enzymes are different in behavior and function . Pkl1p is nuclear throughout the cell cycle, whereas Klp2p is cytoplasmic during interphase . During mitosis Klp2p enters the nucleus where it forms about six chromatin-associated dots . In metaphase-arrested cells these migrate back and forth across the nucleus . During early anaphase they segregate with the chromosomes into two sets of about three, fade, and are replaced by other dots that form on the spindle interzone . Neither klp2(+) nor pkl1(+) is essential, and the double deletion is also wild type for both vegetative and sexual reproduction . Each deletion rescues different alleles of cut7(ts), a KLP that contributes to spindle formation and elongation . When either or both deletions are combined with a dynein deletion, vegetative growth is normal, but sexual reproduction fails: klp2 Delta,dhc1-d1 in karyogamy, pkl1 Delta,dhc1-d1 in multiple phases of meiosis, and the triple deletion in both . Deletion of Klp2p elongates a metaphase-arrested spindle, but pkl1 Delta shortens it . The anaphase spindle of klp2 Delta becomes longer than the cell, leading it to curl around the cell's ends . Apparently, Klp2p promotes spindle disassembly and contributes to the behavior of mitotic chromosomes.

Mol Biol Cell, 2001 Nov, 12(11), 3375 - 85
The N-terminal domain of the t-SNARE Vam3p coordinates priming and docking in yeast vacuole fusion; Laage R et al.; Homotypic fusion of yeast vacuoles requires a regulated sequence of events . During priming, Sec18p disassembles cis-SNARE complexes . The HOPS complex, which is initially associated with the cis-SNARE complex, then mediates tethering . Finally, SNAREs assemble into trans-complexes before the membranes fuse . The t-SNARE of the vacuole, Vam3p, plays a central role in the coordination of these processes . We deleted the N-terminal region of Vam3p to analyze the role of this domain in membrane fusion . The truncated protein (Vam3 Delta N) is sorted normally to the vacuole and is functional, because the vacuolar morphology is unaltered in this strain . However, in vitro vacuole fusion is strongly reduced due to the following reasons: Assembly, as well as disassembly of the cis-SNARE complex is more efficient on Vam3 Delta N vacuoles; however, the HOPS complex is not associated well with the Vam3 Delta N cis-complex . Thus, primed SNAREs from Vam3 Delta N vacuoles cannot participate efficiently in the reaction because trans-SNARE pairing is substantially reduced . We conclude that the N-terminus of Vam3p is required for coordination of priming and docking during homotypic vacuole fusion.

J Biol Chem, 2002 Jan 4, 277(1), 862 - 8 Epub 2001 Nov 01.
Pathways mediating the nuclear import of histones H3 and H4 in yeast; Mosammaparast N et al.; The correct assembly of chromatin is necessary for the maintenance of genomic stability in eukaryotic cells . A critical step in the assembly of new chromatin is the cell cycle-regulated synthesis and nuclear import of core histones . Here we demonstrate that the nuclear import pathway of histones H3 and H4 is mediated by at least two karyopherins/importins, Kap123p and Kap121p . Cytosolic H4 is found associated with Kap123p and H3 . Kap121p is also present in the H4-PrA-associated fractions, albeit in lesser amounts than Kap123p, suggesting that this Kap serves as an additional import receptor . We further demonstrate that cytosolic Kap123p is associated with acetylated H3 and H4 . H3 and H4 each contain a nuclear localization signal (NLS) in their amino-terminal domains . These amino-terminal domains were found to be essential for the nuclear accumulation of H3 and H4-green fluorescent protein reporters . Each NLS mediated direct binding to Kap123p and Kap121p, and decreased nuclear accumulation of H3 and H4 NLS-green fluorescent protein reporters was observed in specific kap mutant strains . H3 and H4 are the first histones to be assembled onto DNA, and these results show that their import is mediated by at least two import pathways.

Genome Res, 2001 Nov, 11(11), 1899 - 912
A streamlined process to phenotypically profile heterologous cDNAs in parallel using yeast cell-based assays; Tugendreich S et al.; To meet the demands of developing lead drugs for the profusion of human genes being sequenced as part of the human genome project, we developed a high-throughput assay construction method in yeast . A set of optimized techniques allows us to rapidly transfer large numbers of heterologous cDNAs from nonyeast plasmids into yeast expression vectors . These high- or low-copy yeast expression plasmids are then converted quickly into integration-competent vectors for phenotypic profiling of the heterologous gene products . The process was validated first by testing proteins of diverse function, such as p38, poly(ADP-ribose) polymerase-1, and PI 3-kinase, by making active-site mutations and using existing small molecule inhibitors of these proteins . For less well-characterized genes, a novel random mutagenesis scheme was developed that allows a combination selection/screen for mutations that retain full-length expression and yet reverse a growth phenotype in yeast . A broad range of proteins in different functional classes has been profiled, with an average yield for growth interference phenotypes of approximately 30% . The ease of manipulation of the yeast genome affords us the opportunity to approach drug discovery and exploratory biology on a genomic scale and shortens assay development time significantly.

Genes Dev, 2001 Nov 1, 15(21), 2803 - 8
Whi3 binds the mRNA of the G1 cyclin CLN3 to modulate cell fate in budding yeast; Gari E et al.; Eukaryotic cells commit in G1 to a new mitotic cycle or to diverse differentiation processes . Here we show that Whi3 is a negative regulator of Cln3, a G1 cyclin that promotes transcription of many genes to trigger the G1/S transition in budding yeast . Whi3 contains an RNA-recognition motif that specifically binds the CLN3 mRNA, with no obvious effects on Cln3 levels, and localizes the CLN3 mRNA into discrete cytoplasmic foci . This is the first indication that G1 events may be regulated by locally restricting the synthesis of a cyclin . Moreover, Whi3 is also required for restraining Cln3 function in meiosis, filamentation, and mating, thus playing a key role in cell fate determination in budding yeast.

Nat Biotechnol, 2001 Nov, 19(11), 1042 - 6
A yeast sensor of ligand binding; Tucker CL et al.; We describe a biosensor that reports the binding of small-molecule ligands to proteins as changes in growth of temperature-sensitive yeast . The yeast strains lack dihydrofolate reductase (DHFR) and are complemented by mouse DHFR containing a ligand-binding domain inserted in a flexible loop . Yeast strains expressing two ligand-binding domain fusions, FKBP12-DHFR and estrogen receptor-alpha (ERalpha)-DHFR, show increased growth in the presence of their corresponding ligands . We used this sensor to identify mutations in residues of ERalpha important for ligand binding, as well as mutations generally affecting protein activity or expression . We also tested the sensor against a chemical array to identify ligands that bind to FKBP12 or ERalpha . The ERalpha sensor was able to discriminate among estrogen analogs, showing different degrees of growth for the analogs that correlated with their relative binding affinities (RBAs) . This growth assay provides a simple and inexpensive method to select novel ligands and ligand-binding domains.

Mol Cell Biol, 2001 Dec, 21(23), 8157 - 67
Isolation and characterization of point mutations in mismatch repair genes that destabilize microsatellites in yeast; Sia EA et al.; The stability of simple repetitive DNA sequences (microsatellites) is a sensitive indicator of the ability of a cell to repair DNA mismatches . In a genetic screen for yeast mutants with elevated microsatellite instability, we identified strains containing point mutations in the yeast mismatch repair genes, MSH2, MSH3, MLH1, and PMS1 . Some of these mutations conferred phenotypes significantly different from those of null mutations in these genes . One semidominant MSH2 mutation was identified . Finally we showed that strains heterozygous for null mutations of mismatch repair genes in diploid strains in yeast confer subtle defects in the repair of small DNA loops.

Mol Cell Biol, 2001 Dec, 21(23), 8082 - 94
Multiple interactions in Sir protein recruitment by Rap1p at silencers and telomeres in yeast; Moretti P et al.; Initiation of transcriptional silencing at mating type loci and telomeres in Saccharomyces cerevisiae requires the recruitment of a Sir2/3/4 (silent information regulator) protein complex to the chromosome, which occurs at least in part through its association with the silencer- and telomere-binding protein Rap1p . Sir3p and Sir4p are structural components of silent chromatin that can self-associate, interact with each other, and bind to the amino-terminal tails of histones H3 and H4 . We have identified a small region of Sir3p between amino acids 455 and 481 that is necessary and sufficient for association with the carboxyl terminus of Rap1p but not required for Sir complex formation or histone binding . SIR3 mutations that delete this region cause a silencing defect at HMR and telomeres . However, this impairment of repression is considerably less than that displayed by Rap1p carboxy-terminal truncations that are defective in Sir3p binding . This difference may be explained by the ability of the Rap1p carboxyl terminus to interact independently with Sir4p, which we demonstrate by in vitro binding and two-hybrid assays . Significantly, the Rap1p-Sir4p two-hybrid interaction does not require Sir3p and is abolished by mutation of the carboxyl terminus of Rap1p . We propose that both Sir3p and Sir4p can directly and independently bind to Rap1p at mating type silencers and telomeres and suggest that Rap1p-mediated recruitment of Sir proteins operates through multiple cooperative interactions, at least some of which are redundant . The physical separation of the Rap1p interaction region of Sir3p from parts of the protein required for Sir complex formation and histone binding raises the possibility that Rap1p can participate directly in the maintenance of silent chromatin through the stabilization of Sir complex-nucleosome interactions.

Mol Cell Biol, 2001 Dec, 21(23), 8045 - 55
Rna15 interaction with the A-rich yeast polyadenylation signal is an essential step in mRNA 3'-end formation; Gross S et al.; In Saccharomyces cerevisiae, four factors {cleavage factor I (CF I), CF II, polyadenylation factor I (PF I), and poly(A) polymerase (PAP)} are required for maturation of the 3' end of the mRNA . CF I and CF II are required for cleavage; a complex of PAP and PF I, which includes CF II subunits, participates in polyadenylation, along with CF I . These factors are directed to the appropriate site on the mRNA by two sequences: one A-rich and one UA-rich . CF I contains five proteins, two of which, Rna15 and Hrp1, interact with the mRNA through RNA recognition motif-type RNA binding motifs . Previous work demonstrated that the UV cross-linking of purified Hrp1 to RNA required the UA-rich element, but the contact point of Rna15 was not known . We show here that Rna15 does not recognize a particular sequence in the absence of other proteins . However, in complex with Hrp1 and Rna14, Rna15 specifically interacts with the A-rich element . The Pcf11 and Clp1 subunits of CF I are not needed to position Rna15 at this site . This interaction is essential to the function of CF I . A mutant Rna15 with decreased affinity for RNA is defective for in vitro RNA processing and lethal in vivo, while an RNA with a mutation in the A-rich element is not processed in vitro and can no longer be UV cross-linked to the Rna15 subunit assembled into CF I . Thus, the recognition of the A-rich element depends on the tethering of Rna15 through an Rna14 bridge to Hrp1 bound to the UA-rich motif . These results illustrate that the yeast 3' end is defined and processed by a mechanism surprisingly different from that used by the mammalian system.

Mol Cell Biol, 2001 Dec, 21(23), 7981 - 94
Structural requirements for function of yeast GGAs in vacuolar protein sorting, alpha-factor maturation, and interactions with clathrin; Mullins C et al.; The GGAs (Golgi-localized, gamma-ear-containing, ARF-binding proteins) are a family of multidomain adaptor proteins involved in protein sorting at the trans-Golgi network of eukaryotic cells . Here we present results from a functional characterization of the two Saccharomyces cerevisiae GGAs, Gga1p and Gga2p . We show that deletion of both GGA genes causes defects in sorting of carboxypeptidase Y (CPY) and proteinase A to the vacuole, vacuolar morphology, and maturation of alpha-factor . A structure-function analysis reveals a requirement of the VHS, GAT, and hinge for function, while the GAE domain is less important . We identify putative clathrin-binding motifs in the hinge domain of both yeast GGAs . These motifs are shown to mediate clathrin binding in vitro . While mutation of these motifs alone does not block function of the GGAs in vivo, combining these mutations with truncations of the hinge and GAE domains diminishes function, suggesting functional cooperation between different clathrin-binding elements . Thus, these observations demonstrate that the yeast GGAs play important roles in the CPY pathway, vacuole biogenesis, and alpha-factor maturation and identify structural determinants that are critical for these functions.

Mol Cell Biol, 2001 Dec, 21(23), 7901 - 12
Yeast AMP pathway genes respond to adenine through regulated synthesis of a metabolic intermediate; Rebora K et al.; In Saccharomyces cerevisiae, AMP biosynthesis genes (ADE genes) are transcriptionally activated in the absence of extracellular purines by the Bas1p and Bas2p (Pho2p) transcription factors . We now show that expression of the ADE genes is low in mutant strains affected in the first seven steps of the pathway, while it is constitutively derepressed in mutant strains affected in later steps . Combined with epistasy studies, these results show that 5'-phosphoribosyl-4-succinocarboxamide-5-aminoimidazole (SAICAR), an intermediate metabolite of the pathway, is needed for optimal activation of the ADE genes . Two-hybrid studies establish that SAICAR is required to promote interaction between Bas1p and Bas2p in vivo, while in vitro experiments suggest that the effect of SAICAR on Bas1p-Bas2p interaction could be indirect . Importantly, feedback inhibition by ATP of Ade4p, catalyzing the first step of the pathway, appears to regulate SAICAR synthesis in response to adenine availability . Consistently, both ADE4 dominant mutations and overexpression of wild-type ADE4 lead to deregulation of ADE gene expression . We conclude that efficient transcription of yeast AMP biosynthesis genes requires interaction between Bas1p and Bas2p which is promoted in the presence of a metabolic intermediate whose synthesis is controlled by feedback inhibition of Ade4p acting as the purine nucleotide sensor within the cell.

J Virol, 2001 Dec, 75(23), 11344 - 53
Functional interactions of human immunodeficiency virus type 1 integrase with human and yeast HSP60; Parissi V et al.; Integration of human immunodeficiency virus type 1 (HIV-1) proviral DNA in the nuclear genome is catalyzed by the retroviral integrase (IN) . In addition to IN, viral and cellular proteins associated in the high-molecular-weight preintegration complex have been suggested to be involved in this process . In an attempt to define host factors interacting with IN, we used an in vitro system to identify cellular proteins in interaction with HIV-1 IN . The yeast Saccharomyces cerevisiae was chosen since (i) its complete sequence has been established and the primary structure of all the putative proteins from this eucaryote has been deduced, (ii) there is a significant degree of homology between human and yeast proteins, and (iii) we have previously shown that the expression of HIV-1 IN in yeast induces a lethal phenotype . Strong evidences suggest that this lethality is linked to IN activity in infected human cells where integration requires the cleavage of genomic DNA . Using IN-affinity chromatography we identified four yeast proteins interacting with HIV-1 IN, including the yeast chaperonin yHSP60, which is the counterpart of human hHSP60 . Yeast lethality induced by HIV-1 IN was abolished when a mutated HSP60 was coexpressed, therefore suggesting that both proteins interact in vivo . Besides interacting with HIV-1 IN, the hHSP60 was able to stimulate the in vitro processing and joining activities of IN and protected this enzyme from thermal denaturation . In addition, the functional human HSP60-HSP10 complex in the presence of ATP was able to recognize the HIV-1 IN as a substrate.

J Basic Microbiol, 2001, 41(5), 289 - 303
Isolation and characterization of a novel leaf-inhabiting osmo-, salt-, and alkali-tolerant Yarrowia lipolytica yeast strain; Zvyagilskaya R et al.; Salt-excreting leaves of Atriplex halimus plants harvested in the central Negev Highlands of Israel were screened for yeasts inhabiting their surfaces . Several aerobic, moderately salt- and alkali-tolerant yeasts were isolated . One of the isolates (tentatively designated S-8) was identified as Yarrowia lipolytica (Wick.) van der Walt and Arx, on the basis of its morphological, biochemical/physiological characteristics, and of quantitative chemotaxonomic and molecular marker analyses . However, the strain is distinguished from the known members of the type Y . lipolytica strain by its pronounced osmo-, salt-, and pH tolerance . Cells displayed a unique capacity to grow over a wide pH range (from 3.5 to 11.5) with a pH optimum at 4.5 to 7.5 . It is proposed that the S-8 strain be assigned to a single Y . lipolytica species as its anamorpha, or as a new variety, Y . lipolytica var . alkalitolerance . The ecophysiological properties and biotechnological potentials of the new strain are discussed.

J Ind Microbiol Biotechnol, 2001 Oct, 27(4), 195 - 8
Selection of a high-biomass, chromium-rich yeast strain and optimization of cultivation conditions; Liu J et al.; Saccharomyces cerevisiae LZ-53 was selected from 240 primary yeast strains from different genera and species, whose chromium (Cr) resistance and biomass were higher than others were . The highest biomass and Cr content of the strain was obtained in 30 h at 28 degrees C and 200 rpm, when 20 ml of the culture in 250-ml shake flasks was grown in wort containing 1200 microg/ml Cr . The initial pH was adjusted to 6.0 . The optimal inoculum volume was 10% (v/v) . The Cr content of the cells was determined by neutron activation analysis . Under the optimized cultivation conditions, the Cr content reached 3248 microg/g.

Proc Natl Acad Sci U S A, 2001 Nov 6, 98(23), 13201 - 6 Epub 2001 Oct 30.
Altered transcription in yeast expressing expanded polyglutamine; Hughes RE et al.; Expanded polyglutamine tracts are responsible for at least eight fatal neurodegenerative diseases . In mouse models, proteins with expanded polyglutamine cause transcriptional dysregulation before onset of symptoms, suggesting that this dysregulation may be an early event in polyglutamine pathogenesis . Transcriptional dysregulation and cellular toxicity may be due to interaction between expanded polyglutamine and the histone acetyltransferase CREB-binding protein . To determine whether polyglutamine-mediated transcriptional dysregulation occurs in yeast, we expressed polyglutamine tracts in Saccharomyces cerevisiae . Gene expression profiles were determined for strains expressing either a cytoplasmic or nuclear protein with 23 or 75 glutamines, and these profiles were compared to existing profiles of mutant yeast strains . Transcriptional induction of genes encoding chaperones and heat-shock factors was caused by expression of expanded polyglutamine in either the nucleus or cytoplasm . Transcriptional repression was most prominent in yeast expressing nuclear expanded polyglutamine and was similar to profiles of yeast strains deleted for components of the histone acetyltransferase complex Spt/Ada/Gcn5 acetyltransferase (SAGA) . The promoter from one affected gene (PHO84) was repressed by expanded polyglutamine in a reporter gene assay, and this effect was mitigated by the histone deacetylase inhibitor, Trichostatin A . Consistent with an effect on SAGA, nuclear expanded polyglutamine enhanced the toxicity of a deletion in the SAGA component SPT3 . Thus, an early component of polyglutamine toxicity, transcriptional dysregulation, is conserved in yeast and is pharmacologically antagonized by a histone deacetylase inhibitor . These results suggest a therapeutic approach for treatment of polyglutamine diseases and provide the potential for yeast-based screens for agents that reverse polyglutamine toxicity.

Proc Natl Acad Sci U S A, 2001 Nov 6, 98(23), 13213 - 8 Epub 2001 Oct 30.
A novel Rtg2p activity regulates nitrogen catabolism in yeast; Pierce MM et al.; The inactivity of Ure2p, caused by either a ure2 mutation or the presence of the {URE3} prion, increases DAL5 transcription and thus enables Saccharomyces cerevisiae to take up ureidosuccinate (USA+) . Rtg2p regulates transcription of glutamate-repressible genes by facilitation of the nuclear entry of the Rtg1 and Rtg3 proteins . We find that rtg2 Delta cells take up USA even without the presence of {URE3} . Thus, the USA+ phenotype of rtg2 Delta strains is not the result generation of the {URE3} prion but is a regulatory effect . Because rtg1 Delta or rtg3 Delta mutations or the presence of glutamate do not produce the USA+ phenotype, this is a novel function of Rtg2p . The USA+ phenotype of rtg2 Delta strains depends on GLN3, is caused by overexpression of DAL5, and is blocked by mks1 Delta, but not by overexpression of Ure2p . These characteristics suggest that Rtg2p acts in the upstream part of the nitrogen catabolism regulation pathway.

Annu Rev Cell Dev Biol, 2001, 17, 297 - 310
RNP localization and transport in yeast; Chartrand P et al.; The localization of mRNAs is used by various types of polarized cells to locally translate specific proteins, which restricts their distribution to a particular sub-region of the cytoplasm . This mechanism of protein sorting is involved in major biological processes such as asymmetric cell division, oogenesis, cellular motility, and synapse formation . With the finding of localized mRNAs in the yeast Saccharomyces cerevisiae, it is now possible to benefit from the powerful yeast laboratory tools to explore the molecular basis of RNA localization . Because mRNA transport and localization in yeast share many features with RNA localization in higher eukaryotes, including the formation of a large ribonucleoprotein (RNP) localization complex, the requirement of a polarized cytoskeleton and molecular motors, and the role of nuclear RNA-binding proteins in cytoplasmic localization, the yeast can be used as a paradigm for unraveling the molecular aspects of this process . This review summarizes the current knowledge on RNP transport and localization in yeast.

J Biochem (Tokyo), 2001 Nov, 130(5), 687 - 93
The region from phenylalanine-17 to phenylalanine-28 of a yeast mitochondrial ATPase inhibitor is essential for its ATPase inhibitory activity; Ichikawa N et al.; Mitochondrial ATP synthase (F(1)F(o)-ATPase) is regulated by an intrinsic ATPase inhibitor protein . In the present study, we investigated the structure-function relationship of the yeast ATPase inhibitor by amino acid replacement . A total of 22 mutants were isolated and characterized . Five mutants (F17S, R20G, R22G, E25A, and F28S) were entirely inactive, indicating that the residues, Phe17, Arg20, Arg22, Glu25, and Phe28, are essential for the ATPase inhibitory activity of the protein . The activity of 7 mutants (A23G, R30G, R32G, Q36G, L37G, L40S, and L44G) decreased, indicating that the residues, Ala23, Arg30, Arg32, Gln36, Leu37, Leu40, and Leu44, are also involved in the activity . Three mutants, V29G, K34Q, and K41Q, retained normal activity at pH 6.5, but were less active at pH 7.2, indicating that the residues, Val29, Lys34, and Lys41, are required for the protein's action at higher pH . The effects of 6 mutants (D26A, E35V, H39N, H39R, K46Q, and K49Q) were slight or undetectable, and the residues Asp26, Glu35, His39, Lys46, and Lys49 thus appear to be dispensable . The mutant E21A retained normal ATPase inhibitory activity but lacked pH-sensitivity . Competition experiments suggested that the 5 inactivated mutants (F17S, R20G, R22G, E25A, and F28S) could still bind to the inhibitory site on F(1)F(o)-ATPase . These results show that the region from the position 17 to 28 of the yeast inhibitor is the most important for its activity and is required for the inhibition of F(1), rather than binding to the enzyme.

Fungal Genet Biol, 2001 Nov, 34(2), 63 - 82
Comparison of morphogenetic networks of filamentous fungi and yeast; Wendland J; Fungi generally display either of two growth modes, yeast-like or filamentous, whereas dimorphic fungi, upon environmental stimuli, are able to switch between the yeast-like and the filamentous growth mode . Signal transduction pathways have been elucidated in the budding yeast Saccharomyces cerevisiae, establishing a morphogenetic network that links cell-cycle events with cellular morphogenesis . Recent molecular genetic studies in several filamentous fungal model systems revealed key components required for distinct steps from fungal spore germination to the maintenance of polar hyphal growth, mycelium formation, and nuclear division . This allows a mechanistic comparison of yeast-like and hyphal growth and the establishment of a core model morphogenetic network for filamentous growth including signaling via the cAMP pathway, Rho modules, and cell cycle kinases . Appreciating similarities between morphogenetic networks of the unicellular yeasts and the multicellular filamentous fungi will open new research directions, help in isolating the central network components, and ultimately pave the way to elucidate the central differences (of many) that distinguish, e.g., the growth mode of filamentous fungi from that of their yeast-like relatives, the role of cAMP signaling, and nuclear division .

J Cell Sci, 2001 Aug, 114(Pt 16), 2911 - 20
Conserved Wat1/Pop3 WD-repeat protein of fission yeast secures genome stability through microtubule integrity and may be involved in mRNA maturation; Ochotorena IL et al.; Accurate chromosome segregation is dependent upon the integrity of mitotic spindles, which pull each pair of sister chromatids towards opposite poles . In this study, we have characterised fission yeast pop3-5235, a diploidising mutant that is impaired in genome stability . Pop3 is the same as Wat1, a conserved protein containing 7 WD repeats . Pop3/Wat1 has also been isolated from a two-hybrid screen as a binding partner to Prp2, the large subunit of the essential splicing factor U2AF . In wat1 mutants, the cellular amount of alpha-tubulin is decreased to very low levels, which results in compromised microtubules and spindles, consequently leading to unequal chromosome separation . Further analysis shows that, in spite of the binding between Wat1 and Prp2, Wat1 may not be involved directly in splicing reactions per se . Instead, we find that Wat1 is required for the maintenance of alpha-tubulin mRNA levels; moreover, transcript levels of genes other than the alpha-tubulin gene are also equally decreased in this mutant . Wild-type Wat1, but not the mutant protein, forms a large complex in the cell with several other proteins, suggesting that Wat1 functions as a structural linker in the complex . The results suggest that Wat1 plays a role in mRNA maturation as a coupling protein between splicing and synthesis and/or stabilisation.

Phytochemistry, 2001 Nov, 58(6), 841 - 7
Characterization of native and yeast-expressed tomato fruit fructokinase enzymes; Petreikov M et al.; Three fructokinase isozymes (FKI, FKII, FKIII) were separated from both immature and ripe tomato fruit pericarp . All three isozymes were specific for fructose with undetectable activity towards glucose or mannose . The three isozymes could be distinguished from one another with respect to response to fructose, Mg and nucleotide donor concentrations and this allowed the comparison of the fruit enzymes with the gene products of the two known cloned tomato fructokinase genes, LeFRK1 and LeFRK2 . FKI was characterized by both substrate (fructose), as well as Mg, inhibition; FKII was inhibited by neither fructose nor Mg; and FKIII was inhibited by fructose but not by Mg . ATP was the preferred nucleotide donor for all three FKs and FKI showed inhibition by CTP and GTP above 1 mM . All three FKs showed competitive inhibition by ADP . During the maturation of the tomato fruit total FK activity decreased dramatically . There were decreases in activity of all three FKs, nevertheless, all were still observed in the ripe fruit . The two tomato LeFRK genes were expressed in yeast and the gene products were characterized with respect to the distinguishing characteristics of fructose, Mg and nucleotide inhibition . Our results indicate that FKI is the gene product of LeFRK2 and FKII is probably the gene product of LeFRK1.

Genes Cells, 2001 Oct, 6(10), 837 - 49
Interaction of fission yeast ORC with essential adenine/thymine stretches in replication origins; Takahashi T et al.; BACKGROUND: Eukaryotic DNA replication is initiated from distinct regions on the chromosome . However, the mechanism for recognition of replication origins is not known for most eukaryotes . In fission yeast, replication origins are isolated as autonomously replicating sequences (ARSs) . Multiple adenine/thymine clusters are essential for replication, but no short consensus sequences are found . In this paper, we examined the interaction of adenine/thymine clusters with the replication initiation factor ORC . RESULTS: The SpOrc1 or SpOrc2 immunoprecipitates (IPs) containing at least four subunits of SpORC, interacted with the ars2004 fragment, which is derived from a predominant replication origin on the chromosome . SpORC-IPs preferentially interacted with two regions of the ars2004, which consist of consecutive adenines and AAAAT repeats and are essential for ARS activity . The nucleotide sequences required for the interaction with SpORC-IPs correspond closely to those necessary for in vivo ARS activity . CONCLUSION: Our results suggest that the SpORC interacts with adenine/thymine stretches, which have been shown to be the most important component in the fission yeast replication origin . The presence of multiple SpORC-binding sites, with certain sequence variations, is characteristic for the fission yeast replication origins.

J Cell Sci, 2001 Aug, 114(Pt 15), 2843 - 53
Live observation of fission yeast meiosis in recombination-deficient mutants: a study on achiasmate chromosome segregation; Molnar M et al.; Regular segregation of homologous chromosomes during meiotic divisions is essential for the generation of viable progeny . In recombination-proficient organisms, chromosome disjunction at meiosis I generally occurs by chiasma formation between the homologs (chiasmate meiosis) . We have studied meiotic stages in living rec8 and rec7 mutant cells of fission yeast, with special attention to prophase and the first meiotic division . Both rec8 and rec7 are early recombination mutants, and in rec7 mutants, chromosome segregation at meiosis I occurs without any recombination (achiasmate meiosis) . Both mutants showed distinct irregularities in nuclear prophase movements . Additionally, rec7 showed an extended first division of variable length and with single chromosomes changing back and forth between the cell poles . Two other early recombination deficient mutants (rec14 and rec15) showed very similar phenotypes to rec7 during the first meiotic division, and the fidelity of achiasmate chromosome segregation slightly exceeded the expected random level . We discuss possible regulatory mechanisms of fission yeast to deal with achiasmate chromosome segregation.

J Cell Sci, 2001 Jul, 114(Pt 14), 2641 - 8
Evidence for a nuclear passage of nascent polypeptide-associated complex subunits in yeast; Franke J et al.; The nascent polypeptide-associated complex (NAC) has been found quantitatively associated with ribosomes in the cytosol by means of cell fractionation or fluorescence microscopy . There have been reports, however, that single NAC subunits may be involved in transcriptional regulation . We reasoned that the cytosolic location might only reflect a steady state equilibrium and therefore investigated the yeast NAC proteins for their ability to enter the nucleus . We found that single subunits of yeast NAC can indeed be transported into the nucleus and that this transport is an active process depending on different nuclear import factors . Translocation into the nucleus was only observed when binding to ribosomes was inhibited . We identified a domain of the ribosome-binding NAC subunit essential for nuclear import via the importin Kapl23p/Pselp-dependent import route . We hypothesize that newly translated NAC proteins travel into the nucleus to bind stoichiometrically to ribosomal subunits and then leave the nucleus together with these subunits to concentrate in the cytosol.

J Cell Sci, 2001 Oct, 114(Pt 19), 3565 - 74
Loss of the mitochondrial Hsp70 functions causes aggregation of mitochondria in yeast cells; Kawai A et al.; Ssc1p, a member of the Hsp70 family in the mitochondrial matrix of budding yeast, mediates protein import into mitochondria and prevents irreversible aggregation of proteins in the mitochondrial matrix during folding/assembly or at elevated temperature . Here, we show that functional inactivation of the mitochondrial Hsp70 system causes aggregation of mitochondria . When temperature-sensitive mitochondrial Hsp70 mutant cells were incubated at restrictive temperature, a tubular network of mitochondria was collapsed to form aggregates . Inhibition of protein synthesis in the cytosol did not suppress the mitochondrial aggregation and functional impairment of Tim23, a subunit of mitochondrial protein translocator in the inner membrane, did not cause mitochondrial aggregation . Therefore defects of the Hsp70 function in protein import into mitochondria or resulting accumulation of precursor forms of mitochondrial proteins outside the mitochondria are not the causal reason for the aberrant mitochondrial morphology . By contrast, deletion of Mdj1p, a functional partner for mitochondrial Hsp70 in prevention of irreversible protein aggregation in the matrix, but not in protein import into mitochondria, caused aggregation of mitochondria, which was enhanced at elevated temperature (37 degrees C) . The aggregation of mitochondria at 37 degrees C was reversed when the temperature was lowered to 23 degrees C unless protein synthesis was blocked . On the basis of these results, we propose that the mitochondrial matrix contains a protein that is responsible for the maintenance of mitochondrial morphology and requires mitochondrial Hsp70 for its function.

RNA, 2001 Oct, 7(10), 1416 - 24
Poly(A)-binding proteins regulate both mRNA deadenylation and decapping in yeast cytoplasmic extracts; Wilusz CJ et al.; The pathway of mRNA degradation has been extensively studied in the yeast, Saccharomyces cerevisiae, and it is now clear that many mRNAs decay by a deadenylation-dependent mechanism . Although several of the factors required for mRNA decay have been identified, the regulation and precise roles of many of the proteins involved remains unclear . We have developed an in vitro system that recapitulates both the deadenylation and the decapping steps of mRNA decay . Furthermore, both deadenylation and decapping are inhibited by poly(A) binding proteins in our assay . Our system has allowed us to separate the decay process from translation and we have shown that the poly(A) tail is capable of inhibiting decapping in an eIF4E-independent manner . Our in vitro system should prove invaluable in dissecting the mechanisms of mRNA turnover.

Mol Microbiol, 2001 Oct, 42(1), 215 - 28
The role of the yeast plasma membrane SPS nutrient sensor in the metabolic response to extracellular amino acids; Forsberg H et al.; In response to discrete environmental cues, Saccharomyces cerevisiae cells adjust patterns of gene expression and protein activity to optimize metabolism . Nutrient-sensing systems situated in the plasma membrane (PM) of yeast have only recently been discovered . Ssy1p is one of three identified components of the Ssy1p-Ptr3p-Ssy5 (SPS) sensor of extracellular amino acids . SPS sensor-initiated signals are known to modulate the expression of a number of amino acid and peptide transporter genes (i.e . AGP1, BAP2, BAP3, DIP5, GAP1, GNP1, TAT1, TAT2 and PTR2) and arginase (CAR1) . To obtain a better understanding of how cells adjust metabolism in response to extracellular amino acids in the environment and to assess the consequences of loss of amino acid sensor function, we investigated the effects of leucine addition to wild-type and ssy1 null mutant cells using genome-wide transcription profile analysis . Our results indicate that the previously identified genes represent only a subset of the full spectrum of Ssy1p-dependent genes . The expression of several genes encoding enzymes in amino acid biosynthetic pathways, including the branched-chain, lysine and arginine, and the sulphur amino acid biosynthetic pathways, are modulated by Ssy1p . Additionally, the proper transcription of several nitrogen-regulated genes, including NIL1 and DAL80, encoding well-studied GATA transcription factors, is dependent upon Ssy1p . Finally, several genes were identified that require Ssy1p for wild-type expression independently of amino acid addition . These findings demonstrate that yeast cells require the SPS amino acid sensor component, Ssy1p, to adjust diverse cellular metabolic processes properly.

Curr Biol, 2001 Oct 16, 11(20), 1611 - 7
Nej1p, a cell type-specific regulator of nonhomologous end joining in yeast; Kegel A et al.; Mutant yeast strains lacking the silencing proteins Sir2p, Sir3p, or Sir4p have a defect in a DNA double-strand break (DSB) repair pathway, called nonhomologous end joining (NHEJ) . Mutations in sir genes also lead to the simultaneous expression of a and alpha mating type information, thus generating a nonmating haploid cell type with many properties shared with a/alpha diploids . We addressed whether cell type or Sir proteins per se regulate NHEJ by investigating the role of a novel haploid-specific gene in NHEJ . This gene, NEJ1, was required for efficient NHEJ, and transcription of NEJ1 was completely repressed in a/alpha diploid and sir haploid strains . The NEJ1 promoter contained a consensus binding site for the a1/alpha2 repressor, explaining the cell type-specific expression . Expression of Nej1p from a constitutive promoter in a/alpha diploid and sir mutant strains completely rescued the defect in NHEJ, thus showing that Sir proteins per se were dispensable for NHEJ . Nej1p and Lif1(P), the yeast XRCC4 homolog, interacted in two independent assays, and Nej1p localized to the nucleus, suggesting that Nej1p may have a direct role in NHEJ.

Biochem Biophys Res Commun, 2001 Nov 2, 288(3), 603 - 9
Functional conservation between the human, nematode, and yeast CK2 cell cycle genes; Dotan I et al.; Protein kinase CK2 (formerly casein kinase II) is a highly conserved serine/threonine protein kinase ubiquitous in eukaryotic organisms . Previously, we have shown that CK2 is required for cell cycle progression and essential for the viability of the yeast Saccharomyces cerevisiae . We now report that either the human or the nematode Caenorhabditis elegans CK2alpha catalytic subunit can substitute for the yeast catalytic subunits . Additionally, expression of the human CK2 regulatory subunit (CK2beta) can suppress the temperature sensitivity of either of the two yeast CK2 mutant catalytic subunits . Taken together, these observations reinforce the view that the CK2 cell cycle progression genes have been highly conserved during evolution from yeast to humans, not only in structure but also in function .

J Biol Chem, 2001 Dec 21, 276(51), 47801 - 6 Epub 2001 Oct 22.
Ca(2+) efflux in mitochondria from the yeast Endomyces magnusii; Deryabina YI et al.; Calcium release pathways in Ca(2+)-preloaded mitochondria from the yeast Endomyces magnusii were studied . In the presence of phosphate as a permeant anion, Ca(2+) was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis . Ca(2+) release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition . Aeration of the mitochondrial suspension inhibited the efflux of Ca(2+) and induced its re-uptake . With acetate as the permeant anion, a spontaneous net Ca(2+) efflux set in after uptake of approximately 150 nmol of Ca(2+)/mg of protein . The rate of this efflux was proportional to the Ca(2+) load and insensitive to aeration, protonophorous uncouplers, and Na(+) ions . Ca(2+) efflux was inhibited by La(3+), Mn(2+), Mg(2+), tetraphenylphosphonium, inorganic phosphate, and nigericin and stimulated by hypotonicity, spermine, and valinomycin in the presence of 4 mm KCl . Atractyloside and t-butyl hydroperoxide were without effect . Ca(2+) efflux was associated with contraction, but not with mitochondrial swelling . We conclude that the permeability transition pore is not involved in Ca(2+) efflux in preloaded E . magnusii mitochondria . The efflux occurs via an Na(+)-independent pathway, in many ways similar to the one in mammalian mitochondria.

J Org Chem, 1997 Jan 24, 62(2), 331 - 336
Synthetic Utility of Yeast Hexokinase . Substrate Specificity, Cofactor Regeneration, and Product Isolation; Chenault HK et al.; Yeast hexokinase (EC 2.7.1.1) catalyzes the phosphorylation of pyranose and furanose analogs of glucose at 0.01-125% of the rate of glucose . The enzyme is highly tolerant of structural changes at C-2 and C-3 of glucopyranose and less tolerant of changes at C-1 and C-4 . Preparative phosphorylations were performed on compounds having 0.01-100% of the activity of glucose, using phosphoenolpyruvate and pyruvate kinase to regenerate ATP . The effects of inhibition of hexokinase by phosphoenolpyruvate and acetyl phosphate on cofactor regeneration are discussed.

Biochemistry, 2001 Oct 30, 40(43), 13097 - 106
Kinetic linked-function analysis of the multiligand interactions on Mg(2+)-activated yeast pyruvate kinase; Bollenbach TJ et al.; The multiligand interactions governing the allosteric response of Mg(2+)-activated yeast pyruvate kinase (YPK) during steady-state turnover were quantitated by kinetic linked-function analysis . The substrate, PEP, the enzyme-bound divalent metal, Mg(2+), and the allosteric effector, FBP, positively influence each other's interaction with the enzyme in the presence of saturating concentrations of the second substrate, MgADP . The presence of Mg(2+) enhances the interaction of PEP and of FBP with YPK by -2.0 and -1.0 kcal/mol, respectively . The simultaneous interaction of PEP, Mg(2+), and FBP with YPK is favored by -4.1 kcal/mol over the sum of their independent binding free energies . The coupling free energies measured for Mg(2+)-activated YPK are weaker than the corresponding coupling free energies measured for Mn(2+)-activated YPK {Mesecar, A., and Nowak, T . (1997) Biochemistry 36, 6792, 6803}, but are consistent with results of thermodynamic measurements with the Mg(2+)-YPK complex {Bollenbach, T . J., and Nowak, T . (2001) Biochemistry 36, 13088-13096} . A comparison of ligand binding data measured by kinetic and thermodynamic linked-function analyses reveals that the MgADP complex modulates both the binding of the other three ligands and the two- and three-ligand coupling interactions between the other three ligands . Enzyme-bound Mg(2+) does not influence the homotropic cooperativity in PEP binding to YPK . It is the MgADP complex that induces homotropic cooperativity in PEP binding . It is the enzyme-bound Mn(2+) that induces homotropic binding of PEP with Mn(2+)-activated YPK . These results lend support to the hypothesis that divalent metals modulate the interactions of ligands on YPK and that divalent metals play a role in regulation of the glycolytic pathway.

Biochemistry, 2001 Oct 30, 40(43), 13088 - 96
Thermodynamic linked-function analysis of Mg(2+)-activated yeast pyruvate kinase; Bollenbach TJ et al.; Yeast pyruvate kinase (YPK) is regulated by intermediates of the glycolytic pathway {e.g., phosphoenolpyruvate (PEP), fructose 1,6-bisphosphate (FBP), and citrate} and by the ATP charge of the cell . Recent kinetic and thermodynamic data with Mn(2+)-activated YPK show that Mn(2+) mediates the allosteric communication between the substrate, PEP, and the allosteric effector, FBP {Mesecar, A., and Nowak, T . (1997) Biochemistry 36, 6792, 6803} . These results indicate that divalent cations modulate multiligand interactions, and hence cooperativity with YPK . The nature of multiligand interactions on YPK was investigated in the presence of the physiological divalent activator Mg(2+) . The binding interactions of PEP, Mg(2+), and FBP were monitored by fluorescence spectroscopy . The binding data were subject to thermodynamic linked-function analysis to determine the magnitudes of the multiligand interactions governing the allosteric activation of YPK . The two ligand coupling free energies between PEP and Mg(2+), PEP and FBP, and FBP and Mg(2+) are 0.88, -0.38, and -0.75 kcal/mol, respectively . The two-ligand coupling free energies between PEP and Mn(2+) and FBP and Mn(2+) are more negative than those with Mg(2+) as the cation . This indicates that the interactions between the divalent cation and PEP with YPK are different for Mg(2+) and Mn(2+) and that the interaction is not simply electrostatic in nature, as originally hypothesized . The magnitude of the heterotropic interaction between the metal and FBP is similar with Mg(2+) and Mn(2+) . The simultaneous binding of Mg(2+), PEP, and FBP to YPK is favored by 3.21 kcal/mol compared to independent binding . This complex is destabilized by 3.30 kcal/mol relative to the analogous YPK-Mn(2+)-PEP-FDP complex . Interpretation of K(d) values when cooperative binding occurs must be done with care as these are not simple thermodynamic constants . These data demonstrate that the divalent metal, which activates phosphoryl transfer in YPK, plays a key role in modulating the various multiligand interactions that define the overall allosteric properties of the enzyme.

Biotechnol Bioeng, 2001 Nov, 76(3), 241 - 6
Effects of charged water-soluble polymers on the stability and activity of yeast alcohol dehydrogenase and subtilisin Carlsberg; Foreman TM et al.; Remarkable increases in enzyme catalytic stability resulting from addition of charged water-soluble polymers have recently been reported, suggesting that use of these polymers may be an attractive general strategy for enzyme stabilization . To test the proposed hypothesis that coulombic forces between water-soluble polymers and enzymes are primarily responsible for enzyme stabilization, we examined the catalytic stability and activity of two enzymes in the presence of polymers differing in net charge . All polymers tested increased enzyme lifetimes, regardless of their net charge, suggesting that stabilization of these enzymes by water-soluble polymers is not solely dependent on simple electrostatic interactions between the polymers and enzymes .

J Biol Chem, 2002 Feb 15, 277(7), 5290 - 8 Epub 2001 Oct 19.
Unusual binding properties of the SH3 domain of the yeast actin-binding protein Abp1: structural and functional analysis; Fazi B et al.; Abp1p is an actin-binding protein that plays a central role in the organization of Saccharomyces cerevisiae actin cytoskeleton . By a combination of two-hybrid and phage-display approaches, we have identified six new ligands of the Abp1-SH3 domain . None of these SH3-mediated novel interactions was detected in recent all genome high throughput protein interaction projects . Here we show that the SH3-mediated association of Abp1p with the Ser/Thr kinases Prk1p and Ark1p is essential for their localization to actin cortical patches . The Abp1-SH3 domain has a rather unusual binding specificity, because its target peptides contain the tetrapentapeptide +XXXPXXPX+PXXL with positive charges flanking the polyproline core on both sides . Here we present the structure of the Abp1-SH3 domain solved at 1.3-A resolution . The peptide-binding pockets in the SH3 domain are flanked by two acidic residues that are uncommon at those positions in the SH3 domain family . We have shown by site-directed mutagenesis that one of these negatively charged side chains may be the key determinant for the preference for non-classical ligands.

J Org Chem, 1996 Nov 1, 61(22), 7738 - 7745
Photoactive Analogs of Farnesyl Pyrophosphate Containing Benzoylbenzoate Esters: Synthesis and Application to Photoaffinity Labeling of Yeast Protein Farnesyltransferase; Gaon I et al.; Farnesyl pyrophosphate (FPP) is involved in a large number of cellular processes including the prenylation of transforming mutants of Ras proteins implicated in cancer . Photoactive analogs could provide useful information about enzyme active sites that bind farnesyl pyrophosphate; however, the availability of such compounds is extremely limited . Molecules that incorporate benzophenone moieties are attractive photoaffinity labeling reagents because of their useful photochemical properties . Here, the syntheses of two compounds, 3a and 3b, containing para- and meta-substituted benzoylbenzoates are described . Compounds 3a and 3b are competitive inhibitors (with respect to FPP) of yeast protein farnesyltransferase (PFTase) with K(i) values of 910 and 380 nM, respectively . Both compounds inactivate PFTase upon photolysis, resulting in as much as 44% inactivation of enzyme activity . Photolysis of PFTase in the presence of {(32)P}3a or of {(32)P}3b results in preferential labeling of the beta subunit, suggesting that this subunit is involved in prenyl group recognition . These compounds should be valuable tools for studying enzymes that utilize FPP as a substrate.

Inorg Chem, 1996 Mar 13, 35(6), 1692 - 1700
New Type 2 Copper-Cysteinate Proteins . Copper Site Histidine-to-Cysteine Mutants of Yeast Copper-Zinc Superoxide Dismutase; Lu Y et al.; Preparation and characterization of two new site-directed mutant copper-zinc superoxide dismutase proteins from Saccharomyces cerevisiae, i.e., His46Cys (H46C) and His120Cys (H120C), in which individual histidyl ligands in the copper-binding site were replaced by cysteine, are reported here . These two mutant CuZnSOD proteins may be described as type 2 (or normal) rather than type 1 (or blue) copper-cysteinate proteins and are characterized by their yellow rather than blue color, resulting from intense copper-to-sulfur charge transfer bands around 400 nm, their type 2 EPR spectra, with large rather than small nuclear hyperfine interactions, and their characteristic type 2 d-d electronic absorption spectra . An interesting difference between these two copper site His-to-Cys mutations is that the imidazolate bridge between the two metal sites that is characteristic of the wild-type protein remains intact in the case of the H46C mutant but is not present in the case of the H120C mutant.

Ukr Biokhim Zh, 2001 Mar-Apr, 73(2), 33 - 8
{Effect of interferonogenic molecular complex of yeast RNA--tilorone on DNA, RNA and protein synthesis in vitro}; Karpov OV et al.; In the experiments in vitro using the primary mononuclear cells (MNC) culture of the human peripheral blood the influence of interferonogenic yeast RNA-tilorone molecular complex on the DNA, RNA and protein synthesis was studied . The complex was shown to inhibit the insertion of 3H-thymidine, 3H-uridine and 3H-leucine into DNA, RNA and protein of MNC total pool (by 13, 1 and 40% respectively); that was practically conformed with this synthesis inhibition upon to a natural origin polynucleotide interferon inducers--lariphan (9, 0 and 57% respectively) and ridostin (9, 0 and 56% respectively) action, and at the same time rather less than poly(I)-poly(C) (14, 5 and 62% respectively) . In the case of preliminary cell stimulation by the mitogen PHA the complex revealed comitogenic action at a concentration 25 micrograms/ml, that corresponded to optimal for interferonogenesis; the increase of the doses till 100-1000 micrograms/ml lead to in the reversal effect . To proceed from mutual relation between interferonogen preparations influence on the mentioned synthesis and their cytotoxicity the conclusion was about made the complex promising usage as an interferon inducer both in vitro and in vivo conditions.

Proc Natl Acad Sci U S A, 2001 Nov 6, 98(23), 13114 - 9 Epub 2001 Oct 23.
Redundant control of rereplication in fission yeast; Gopalakrishnan V et al.; The initiation of DNA replication at replication origins in eukaryotic cells is tightly controlled to ensure that the genome is duplicated only once each cell cycle . We present evidence that in fission yeast, independent regulation of two essential components of the initiation complex, Cdc18 and Cdt1, contributes to the prevention of reinitiation of DNA replication . Cdc18 is negatively controlled by cyclin-dependent kinase (CDK) phosphorylation, but low level expression of a mutant form of Cdc18 lacking CDK phosphorylation sites (Cdc18(CDK)) is not sufficient to induce rereplication . Similar to Cdc18, Cdt1 is expressed periodically in the cell cycle, accumulating in the nucleus in G(1) and declining in G(2) . When Cdt1 is expressed constitutively from an ectopic promoter, it accumulates in the nucleus throughout the cell cycle but does not promote reinitiation . However, constitutive expression of Cdt1, together with Cdc18(CDK), is sufficient to induce extra rounds of DNA replication in the absence of mitosis . Significantly greater levels of rereplication can be induced by coexpression of Cdc18(CDK) and a Cdt1 mutant lacking a conserved C-terminal motif . In contrast, uncontrolled DNA replication does not occur when either mutant protein is expressed in the absence of the other . Constitutive expression of wild-type or mutant Cdt1 also leads to an increase in the levels of Cdc18(CDK), possibly as a result of increased protein stability . Our data are consistent with the hypothesis that control of rereplication depends on a redundant mechanism in which negative regulation of Cdt1 functions in parallel with the negative regulation of Cdc18.

Genetics, 2001 Oct, 159(2), 515 - 25
The yeast recombinational repair protein Rad59 interacts with Rad52 and stimulates single-strand annealing; Davis AP et al.; The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks . In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA . Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions . Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts . Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA) . Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52 . The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59 . These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing . Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.

Radiats Biol Radioecol, 2001 Jul-Aug, 41(4), 361 - 5
{Comparative study of RBE of densely ionizing radiation for various types of cell death in yeast}; Shvedenko VI et al.; A comparative study of the relative biological effectiveness (RBE) of alpha-particles 249Pu for reproductive and interphase forms of killing of haploid and diploid yeast cells of wild-type and their radiosensitive mutants has been carried out . The correlation between the RBE of alpha-particles and cell repair capacity was confirmed for reproductive death: it was the highest for diploid cells, smaller for haploid cells and the smallest for their radiosensitive mutants . To achieve the interphase cell killing much higher irradiation doses were used after which cells were incapable of liquid-holding recovery during the storing of exposed cells in non-nutrient media at 30 degrees C . The RBE values for this form of killing were significantly lower in comparison with reproductive death . These data are an additional argument supporting the point of view that the RBE of densely ionizing radiation is determined not merely by physical processes of energy absorption as it is traditionally believed but also by ability of cells to recover from DNA damages inflicted by ionizing radiation.

Mol Cell Biol, 2001 Nov, 21(22), 7787 - 95
Srg3, a mouse homolog of yeast SWI3, is essential for early embryogenesis and involved in brain development; Kim JK et al.; Srg3 (SWI3-related gene product) is a mouse homolog of yeast SWI3, Drosophila melanogaster MOIRA (also named MOR/BAP155), and human BAF155 and is known as a core subunit of SWI/SNF complex . This complex is involved in the chromatin remodeling required for the regulation of transcriptional processes associated with development, cellular differentiation, and proliferation . We generated mice with a null mutation in the Srg3 locus to examine its function in vivo . Homozygous mutants develop in the early implantation stage but undergo rapid degeneration thereafter . An in vitro outgrowth study revealed that mutant blastocysts hatch, adhere, and form a layer of trophoblast giant cells, but the inner cell mass degenerates after prolonged culture . Interestingly, about 20% of heterozygous mutant embryos display defects in brain development with abnormal organization of the brain, a condition known as exencephaly . Histological examination suggests that exencephaly is caused by the failure in neural fold elevation, resulting in severe brain malformation . Our findings demonstrate that Srg3 is essential for early embryogenesis and plays an important role in the brain development of mice.

Mol Cell Biol, 2001 Nov, 21(22), 7696 - 706
Identification of a role for the sialomucin CD164 in myogenic differentiation by signal sequence trapping in yeast; Lee YN et al.; Determination and differentiation of skeletal muscle precursors requires cell-cell contact, but the full range of cell surface proteins that mediate this requirement and the mechanisms by which they work are not known . To identify participants in cell contact-mediated regulation of myogenesis, genes that encode secreted proteins specifically upregulated during differentiation of C2C12 myoblasts were identified by the yeast signal sequence trap method (K . A . Jacobs, L . A . Collins-Racie, M . Colbert, M . Duckett, M . Golden-Fleet, K . Kelleher, R . Kriz, E . R . La Vallie, D . Merberg, V . Spaulding, J . Stover, M . J . Williamson, and J . M . McCoy, Gene 198:289-296, 1997), followed by RNA expression analysis . We report here the identification of CD164 as a gene expressed in proliferating C2C12 cells that is upregulated during differentiation . CD164 encodes a widely expressed cell surface sialomucin that has been implicated in regulation of cell proliferation and adhesion during hematopoiesis . Stable overexpression of CD164 in C2C12 and F3 myoblasts enhanced their differentiation, as assessed by both morphological and biochemical criteria . Furthermore, expression of antisense CD164 or soluble extracellular regions of CD164 inhibited myogenic differentiation . Treatment of C2C12 cells with sialidase or O-sialoglycoprotease, two enzymes previously reported to destroy functional epitopes on CD164, also inhibited differentiation . These data indicate that (i) CD164 may play a rate-limiting role in differentiation of cultured myoblasts, (ii) sialomucins represent a class of potential effectors of cell contact-mediated regulation of myogenesis, and (iii) carbohydrate-based cell recognition may play a role in mediating this phenomenon.

J Cell Biol, 2001 Oct 15, 155(2), 261 - 70 Epub 2001 Oct 15.
A two-tiered mechanism by which Cdc42 controls the localization and activation of an Arp2/3-activating motor complex in yeast; Lechler T et al.; The establishment of cell polarity in budding yeast involves assembly of actin filaments at specified cortical domains . Elucidation of the underlying mechanism requires an understanding of the machinery that controls actin polymerization and how this machinery is in turn controlled by signaling proteins that respond to polarity cues . We showed previously that the yeast orthologue of the Wiskott-Aldrich Syndrome protein, Bee1/Las17p, and the type I myosins are key regulators of cortical actin polymerization . Here, we demonstrate further that these proteins together with Vrp1p form a multivalent Arp2/3-activating complex . During cell polarization, a bifurcated signaling pathway downstream of the Rho-type GTPase Cdc42p recruits and activates this complex, leading to local assembly of actin filaments . One branch, which requires formin homologues, mediates the recruitment of the Bee1p complex to the cortical site where the activated Cdc42p resides . The other is mediated by the p21-activated kinases, which activate the motor activity of myosin-I through phosphorylation . Together, these findings provide insights into the essential processes leading to polarization of the actin cytoskeleton.

J Cell Biol, 2001 Oct 15, 155(2), 251 - 60 Epub 2001 Oct 15.
Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin; Palmgren S et al.; Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells . To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells . Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers . A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin . The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the alpha and beta subunits of capping protein, is deleted . Purified twinfilin and capping protein form a complex on native gels . Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI{4,5}P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2 . Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.

BMC Biotechnol . 2001;1(1):5 . Epub 2001 Sep 11.
Mannosylerythritol lipid, a yeast extracellular glycolipid, shows high binding affinity towards human immunoglobulin G; Im JH et al.; BACKGROUND: There have been many attempts to develop new materials with stability and high affinity towards immunoglobulins . Some of glycolipids such as gangliosides exhibit a high affinity toward immunoglobulins . However, it is considerably difficult to develop these glycolipids into the practical separation ligand due to their limited amounts . We thus focused our attention on the feasible use of "mannosylerythritol lipid A", a yeast glycolipid biosurfactant, as an alternative ligand for immunoglobulins, and undertook the investigation on the binding between mannosylerythritol lipid A (MEL-A) and human immunoglobulin G (HIgG) . RESULTS: In ELISA assay, MEL-A showed nearly the same binding affinity towards HIgG as that of bovine ganglioside GM1 . Fab of human IgG was considered to play a more important role than Fc in the binding of HIgG by MEL-A . The bound amount of HIgG increased depending on the attached amount of MEL-A onto poly (2-hydroxyethyl methacrylate) (polyHEMA) beads, whereas the amount of human serum albumin slightly decreased . Binding-amount and -selectivity of HIgG towards MEL-A were influenced by salt species, salt concentration and pH in the buffer solution . The composite of MEL-A and polyHEMA, exhibited a significant binding constant of 1.43 x 10(6) (M(-1)) for HIgG, which is approximately 4-fold greater than that of protein A reported . CONCLUSIONS: MEL-A shows high binding-affinity towards HIgG, and this is considered to be due to "multivalent effect" based on the binding molar ratio . This is the first report on the binding of a natural human antibody towards a yeast glycolipid.

FEBS Lett, 2001 Oct 12, 506(3), 277 - 80
The yeast cyclins Pc16p and Pc17p are involved in the control of glycogen storage by the cyclin-dependent protein kinase Pho85p; Wang Z et al.; Pho85p is a yeast cyclin-dependent protein kinase (Cdk) that can interact with 10 cyclins (Pcls) to form multiple protein kinases . The functions of most of the Pcls, including Pc16p and Pc17p, are poorly defined . We report here that Pc16p and Pc17p are involved in the metabolism of the branched storage polysaccharide glycogen under certain conditions and deletion of PCL6 and PCL7 restores glycogen accumulation to a snf1 pcl8 pcl10 triple mutant, paradoxically activating both glycogen synthase and phosphorylase . Pho85p thus affects glycogen accumulation through multiple Cdks composed of different cyclin partners.

Appl Microbiol Biotechnol, 2001 Sep, 56(5-6), 681 - 6
Spacer-mediated display of active lipase on the yeast cell surface; Washida M et al.; We have constructed a Saccharomyces cerevisiae strain displaying an active lipase on the cell surface by cell surface engineering . The gene encoding Rhizopus oryzae lipase (ROL) was fused with the genes encoding the pre-alpha-factor leader sequence and the C-terminal half of alpha-agglutinin including the glycosylphosphatidylinositol-anchor attachment signal . The constructed gene was overexpressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter . Linker peptides (spacers) consisting of the Gly/Ser repeat sequence were inserted at the C-terminal portion of ROL to enhance lipase activity by preserving the conformation of the active site near the C-terminal portion . Localization of the expressed ROL on the cell surface was confirmed by immunofluorescence microscopy . The ROL displayed on the yeast cell wall exhibited activity toward soluble 2,3-dimercaptopropan-1-ol tributyl ester (BALB) and insoluble triolein . The insertion of linker peptides effected the activity towards BALB, thereby demonstrating that the optimal length of linker peptides was present . The activity towards triolein was higher in lipases with longer linker peptides . ROL displayed on the cell wall exhibited a comparable and/or higher activity towards triolein than the secreted form of the enzyme . This is the first report of an active lipase displayed on the cell surface . Furthermore, insertion of a linker peptide of the appropriate length as a spacer may be an improved method to effectively display enzymes, especially those having the active region at the C-terminal portion, on the cell surface.

Nucleic Acids Res . 2001 Oct 15;29(20):E99.
A novel yeast system for in vivo selection of recognition sequences: defining an optimal c-Myb-responsive element; Berge T et al.; Yeast (Saccharomyces cerevisiae) has proved to be a highly valuable tool in a range of screening methods . We present in this work the design and use of a novel yeast effector-reporter system for selection of sequences recognised by DNA-binding proteins in vivo . A dual HIS3-lacZ reporter under the control of a single randomised response element facilitates both positive growth selection of binding sequences and subsequent quantification of the strength of the selected sequence . A galactose-inducible effector allows discrimination between reporter activation caused by the protein under study and activation due to endogenous factors . The system mimics the physiological gene dosage relationship between transcription factor and target genes in vivo by using a low copy effector plasmid and a high copy reporter plasmid, favouring sequence selectivity . The utility of the novel yeast screening system was demonstrated by using it to refine the definition of an optimal recognition element for the c-Myb transcription factor (MRE) . We present screening data supporting an extended MRE consensus closely mimicking known strong response elements and where a sequence of 11 nt influences activity . Novel features include a more strict sequence requirement in the second half-site of the MRE where a T-rich sequence is preferred in vivo.

J Infect Dis, 2001 Nov 1, 184(9), 1183 - 6 Epub 2001 Sep 27.
Neutralization of human papillomavirus type 11 (HPV-11) by serum from women vaccinated with yeast-derived HPV-11 L1 virus-like particles: correlation with competitive radioimmunoassay titer; Brown DR et al.; Neutralization of human papillomavirus type 11 (HPV-11) has been demonstrated using serum and cervical secretions from primates vaccinated with virus-like particles (VLPs) . Theoretically, neutralizing antibodies could protect women from HPV infection . The immunogenicity of a yeast-derived HPV-11 L1 VLP vaccine was tested in women . Serum specimens were evaluated for HPV-11 titer by competitive radioimmunoassay (cRIA) and for neutralization by use of the athymic mouse xenograft system . Analysis of serum from 104 subjects showed a dose response in HPV-11 cRIA titers and neutralization . Overall, 68 (82.9%) of 82 postimmunization serum specimens from VLP recipients were 100% neutralizing when used in the assay at a 1:50 dilution . Of 69 serum specimens, 63 (91.3%) with cRIA titers >200 milliMerck units per milliliter were neutralizing . Immunization with HPV VLPs elicits a vigorous serum immune response in a high percentage of women . The HPV-11 cRIA titer appears to be a surrogate marker for neutralization.

Mol Biol Cell, 2001 Oct, 12(10), 3175 - 90
A novel mechanism for localizing membrane proteins to yeast trans-Golgi network requires function of synaptojanin-like protein; Ha SA et al.; Localization of resident membrane proteins to the yeast trans-Golgi network (TGN) involves both their retrieval from a prevacuolar/endosomal compartment (PVC) and a "slow delivery" mechanism that inhibits their TGN-to-PVC transport . A screen for genes required for the slow delivery mechanism uncovered INP53, a gene encoding a phosphoinositide phosphatase . A retrieval-defective model TGN protein, A(F-->A)-ALP, was transported to the vacuole in inp53 mutants approximately threefold faster than in wild type . Inp53p appears to function in a process distinct from PVC retrieval because combining inp53 with mutations that block retrieval resulted in a much stronger phenotype than either mutation alone . In vps27 strains defective for both anterograde and retrograde transport out of the PVC, a loss of Inp53p function markedly accelerated the rate of transport of TGN residents A-ALP and Kex2p into the PVC . Inp53p function is cargo specific because a loss of Inp53p function had no effect on the rate of Vps10p transport to the PVC in vps27 cells . The rate of early secretory pathway transport appeared to be unaffected in inp53 mutants . Cell fractionation experiments suggested that Inp53p associates with Golgi or endosomal membranes . Taken together, these results suggest that a phosphoinositide signaling event regulates TGN-to-PVC transport of select cargo proteins.

Mol Biol Cell, 2001 Oct, 12(10), 2987 - 3003
Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p; Gasch AP et al.; Eukaryotic cells respond to DNA damage by arresting the cell cycle and modulating gene expression to ensure efficient DNA repair . The human ATR kinase and its homolog in yeast, MEC1, play central roles in transducing the damage signal . To characterize the role of the Mec1 pathway in modulating the cellular response to DNA damage, we used DNA microarrays to observe genomic expression in Saccharomyces cerevisiae responding to two different DNA-damaging agents . We compared the genome-wide expression patterns of wild-type cells and mutants defective in Mec1 signaling, including mec1, dun1, and crt1 mutants, under normal growth conditions and in response to the methylating-agent methylmethane sulfonate (MMS) and ionizing radiation . Here, we present a comparative analysis of wild-type and mutant cells responding to these DNA-damaging agents, and identify specific features of the gene expression responses that are dependent on the Mec1 pathway . Among the hundreds of genes whose expression was affected by Mec1p, one set of genes appears to represent an MEC1-dependent expression signature of DNA damage . Other aspects of the genomic responses were independent of Mec1p, and likely independent of DNA damage, suggesting the pleiotropic effects of MMS and ionizing radiation . The complete data set as well as supplemental materials is available at http://www-genome.stanford.edu/mec1.

EMBO J, 2001 Oct 15, 20(20), 5779 - 90
Establishment and maintenance of sister chromatid cohesion in fission yeast by a unique mechanism; Tanaka K et al.; During S phase, chromatid cohesion is established only between nascent sisters and with faithful pairing along their entire region, but how this is ensured is unknown . Here we report that sister chromatid cohesion is formed and maintained by a unique mechanism . In fission yeast, Eso1p, functioning in close coupling to DNA replication, establishes sister chromatid cohesion whereas the newly identified Cohesin-associated protein Pds5p hinders the establishment of cohesion until counteracted by Eso1p, yet stabilizes cohesion once it is established . Eso1p interacts physically with Pds5p via its Ctf7p/Eco1p-homologous domain.

EMBO J, 2001 Oct 15, 20(20), 5657 - 65
Cdc42p functions at the docking stage of yeast vacuole membrane fusion; Muller O et al.; Membrane fusion reactions have been considered to be primarily regulated by Rab GTPases . In the model system of homotypic vacuole fusion in the yeast Saccharomyces cerevisiae, we show that Cdc42p, a member of the Rho family of GTPases, has a direct role in membrane fusion . Genetic evidence suggested a relationship between Cdc42p and Vtc1p/Nrf1p, a central part of the vacuolar membrane fusion machinery . Vacuoles from cdc42 temperature-sensitive mutants are deficient for fusion at the restrictive temperature . Specific amino acid changes on the Cdc42p protein surface in these mutants define the putative interaction domain that is crucial for its function in membrane fusion . Affinity-purified antibodies to this domain inhibited the in vitro fusion reaction . Using these antibodies in kinetic analyses and assays for subreactions of the priming, docking and post-docking phase of the reaction, we show that Cdc42p action follows Ypt7p-dependent tethering, but precedes the formation of trans-SNARE complexes . Thus, our data define an effector binding domain of Cdc42p by which it regulates the docking reaction of vacuole fusion.

Zhongguo Zhong Yao Za Zhi, 1998 Mar, 23(3), 171 - 3, inside backcover
{Antivirus effect of polysaccharides of brewer yeast in vitro}; Li F et al.; The antivirus effect of polysaccharides of brewer yeast from yeast mud on 13 kinds of viruses including DNA and RNA virus along with their mechanisms were studied . The result showed that this effect was remarkable on the infections with poliovirus III, adenovirus III, ECHO6 virus, enterovirus 71, vesicular stomatitis virus, herpesvirus I, II, coxsackie A16 virus and coxsackie B3 virus . The polysaccharides of brewer yeast could also inhibit the development of cytopathic effect(CPE) and protect cultural cells from being infected with the above viruses.

Curr Opin Neurobiol, 2001 Oct, 11(5), 615 - 20
Profiling brain transcription: neurons learn a lesson from yeast; Cao Y et al.; The application of microarray technologies to the brain poses unique challenges, because of the complexity of the central nervous system and the availability of resources . Nevertheless, recent studies using DNA chips have made inroads into the molecular characterization of regional and functional brain units, the identification of developmental gene expression patterns, and the discovery of transcriptional differences associated with behavioral and neuropathological traits.

Biochem Biophys Res Commun, 2001 Oct 19, 288(1), 16 - 21
U86, a novel snoRNA with an unprecedented gene organization in yeast; Filippini D et al.; The Xenopus laevis Nop56 gene (XNOP56), coding for a snoRNP-specific factor, belongs to the 5'-TOP gene family . XNOP56, as many 5'-TOP genes, contains an intron-encoded snoRNA . This previously unidentified RNA, named U86, was found as a highly conserved species in yeast and human . While in human it is also encoded in an intron of the hNop56 gene, in yeast it has an unprecedented gene organization: it is encoded inside an open-reading frame . Both in X . laevis and yeast, the synthesis of U86 snoRNA appears to be alternative to that of the cotranscribed mRNA . Despite the overall homology, the three U86 snoRNAs do not show strong conservation of the sequence upstream from the box D and none of them displays significant sequence complementarity to rRNA or snRNA sequences, suggesting a role different from that of methylation .

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1927 - 31
Metschnikowia koreensis sp . nov., a novel yeast species isolated from flowers in Korea; Hong SG et al.; A novel ascomycetous yeast was isolated from flowers of Lilium sp . and Ipomoea sp . in Korea . The name Metschnikowia koreensis sp . nov . (type strain SG99-34T = CBS 8854T = KCTC 7998T) is proposed for this novel species based on comparative sequence analyses of the D1/D2 domain of 26S rDNA and phenotypic characteristics.

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1917 - 25
Phylogenetic analysis of the psychrophobic yeast Arxiozyma telluris and the reinstatement of Candida pintolopesii (van Uden) Meyer et Yarrow and Candida slooffii van Uden et do Carmo Sousa; James SA et al.; A phylogenetic analysis was conducted upon ten strains of the psychrophobic yeast species Arxiozyma telluris using nuclear rDNA (18S and 26S) and mitochondrial cytochrome-c oxidase subunit II (COX2) gene sequences . Strains examined included those described originally as Candida slooffii, Torulopsis bovina (= Candida bovina) and Torulopsis pintolopesii (= Candida pintolopesii), which are all currently accepted as synonyms of Arxiozyma telluris . Comparative 18S rDNA sequence analysis showed that these strains formed a genealogically highly related group, which was phylogenetically distinct from any other ascomycetous species studied . The results showed that A . telluris, as currently described, appears to be composed of a complex of closely related but nevertheless separate taxa . rDNA and COX2 gene sequence data revealed that CBS 1787T, the type strain of C . pintolopesii, the currently recognized asexual form (anamorph) of A . telluris, along with strains CBS 2676 and CBS 2985 formed a distinct taxon that is phylogenetically separate from A . telluris . Similarly, the sequence data also showed that C . slooffii is a distinct taxon and support the reinstatement of this species . However, with regard to the relationship between the type strains of A . telluris (CBS 2685T) and C bovina (CBS 2760T), discrepancies were observed between the rDNA and COX2 sequence datasets, and these results are discussed in more detail.

Proc Natl Acad Sci U S A, 2001 Oct 23, 98(22), 12397 - 402 Epub 2001 Oct 09.
Arg-735 of the 100-kDa subunit a of the yeast V-ATPase is essential for proton translocation; Kawasaki-Nishi S et al.; The vacuolar (H(+))-ATPases (V-ATPases) are ATP-dependent proton pumps that acidify intracellular compartments and pump protons across specialized plasma membranes . Proton translocation occurs through the integral V(0) domain, which contains five different subunits (a, d, c, c', and c") . Proton transport is critically dependent on buried acidic residues present in three different proteolipid subunits (c, c', and c") . Mutations in the 100-kDa subunit a have also influenced activity, but none of these residues has proven to be required absolutely for proton transport . On the basis of previous observations on the F-ATPases, we have investigated the role of two highly conserved arginine residues present in the last two putative transmembrane segments of the yeast V-ATPase a subunit (Vph1p) . Substitution of Asn, Glu, or Gln for Arg-735 in TM8 gives a V-ATPase that is fully assembled but is totally devoid of proton transport and ATPase activity . Replacement of Arg-735 by Lys gives a V-ATPase that, although completely inactive for proton transport, retains 24% of wild-type ATPase activity, suggesting a partial uncoupling of proton transport and ATP hydrolysis in this mutant . By contrast, nonconservative mutations of Arg-799 in TM9 lead to both defective assembly of the V-ATPase complex and decreases in activity of the assembled V-ATPase . These results suggest that Arg-735 is absolutely required for proton transport by the V-ATPases and is discussed in the context of a revised model of the topology of the 100-kDa subunit a.

J Biol Chem, 2001 Dec 14, 276(50), 47150 - 3 Epub 2001 Oct 08.
Quantitation of the RNA polymerase II transcription machinery in yeast; Borggrefe T et al.; TAP tags and dot blot analysis have been used to measure the amounts of RNA polymerase II transcription proteins in crude yeast extracts . The measurements showed comparable amounts of RNA polymerase II, TFIIE, and TFIIF, lower levels of TBP and TFIIB, and still lower levels of Mediator and TFIIH . These findings are consistent with the presumed roles of the transcription proteins, but do not support the idea of their recruitment in a single large complex to RNA polymerase II promoters . The approach employed here can be readily extended to quantitative analysis of the entire yeast proteome.

J Bacteriol, 2001 Nov, 183(21), 6372 - 83
Peroxisomal catalase in the methylotrophic yeast Candida boidinii: transport efficiency and metabolic significance; Horiguchi H et al.; In this study we cloned CTA1, the gene encoding peroxisomal catalase, from the methylotrophic yeast Candida boidinii and studied targeting of the gene product, Cta1p, into peroxisomes by using green fluorescent protein (GFP) fusion proteins . A strain from which CTA1 was deleted (cta1Delta strain) showed marked growth inhibition when it was grown on the peroxisome-inducing carbon sources methanol, oleate, and D-alanine, indicating that peroxisomal catalase plays an important nonspecific role in peroxisomal metabolism . Cta1p carries a peroxisomal targeting signal type 1 (PTS1) motif, -NKF, in its carboxyl terminus . Using GFP fusion proteins, we found that (i) Cta1p is transported to peroxisomes via its PTS1 motif, -NKF; (ii) peroxisomal localization is necessary for Cta1p to function physiologically; and (iii) Cta1p is bimodally distributed between the cytosol and peroxisomes in methanol-grown cells but is localized exclusively in peroxisomes in oleate- and D-alanine-grown cells . In contrast, the fusion protein GFP-AKL (GFP fused to another typical PTS1 sequence, -AKL), in the context of CbPmp20 and D-amino acid oxidase, was found to localize exclusively in peroxisomes . A yeast two-hybrid system analysis suggested that the low transport efficiency of the -NKF sequence is due to a level of interaction between the -NKF sequence and the PTS1 receptor that is lower than the level of interaction with the AKL sequence . Furthermore, GFP-Cta1pDeltankf coexpressed with Cta1p was successfully localized in peroxisomes, suggesting that the oligomer was formed prior to peroxisome import and that it is not necessary for all four subunits to possess a PTS motif . Since the main physiological function of catalase is degradation of H2O2, suboptimal efficiency of catalase import may confer an evolutionary advantage . We suggest that the PTS1 sequence, which is found in peroxisomal catalases, has evolved in such a way as to give a higher priority for peroxisomal transport to peroxisomal enzymes other than to catalases (e.g., oxidases), which require a higher level of peroxisomal transport efficiency.

Genome Res, 2001 Oct, 11(10), 1730 - 5
A human cDNA expression library in yeast enriched for open reading frames; Holz C et al.; We developed a high-throughput technique for the generation of cDNA libraries in the yeast Saccharomyces cerevisiae which enables the selection of cloned cDNA inserts containing open reading frames (ORFs) . For direct screening of random-primed cDNA libraries, we have constructed a yeast shuttle/expression vector, the so-called ORF vector pYEXTSH3, which allows the enriched growth of protein expression clones . The selection system is based on the HIS3 marker gene fused to the C terminus of the cDNA insert . The cDNAs cloned in-frame result in histidine prototrophic yeast cells growing on minimal medium, whereas clones bearing the vector without insert or out-of-frame inserts should not grow on this medium . A randomly primed cDNA library from human fetal brain tissue was cloned in this novel vector, and using robot technology the selected clones were arrayed in microtiter plates and were analyzed by sequencing and for protein expression . In the constructed cDNA expression library, about 60% of clones bear an insert in the correct reading frame . In comparison to unselected libraries it was possible to increase the clones with inserts in the correct reading frame more than fourfold, from 14% to 60% . With the expression system described here, we could avoid time-consuming and costly techniques for identification of clones expressing protein by using antibody screening on high-density filters and subsequently rearraying the selected clones in a new "daughter" library . The advantage of this ORF vector is that, in a one-step screening procedure, it allows the generation of expression libraries enriched for clones with correct reading frames as sources of recombinant proteins.

Biotechnol Bioeng, 2001 Nov 5, 75(3), 305 - 12
Multiple stable states and hysteresis in continuous, oscillating cultures of budding yeast; Zamamiri AQ et al.; The conditions that precede the onset of autonomous oscillations in continuous yeast cultures were studied in three different types of experiments . It was found that the final state of the culture depended on the protocol used to start up the reactor . Batch cultures, switched to continuous operation at different stages of the batch growth curve, all exhibited similar dynamics-ethanol depletion followed by autonomous oscillations . Small perturbations of the distribution of states in the reactor, achieved by addition of externally grown cells, were able to quench the oscillatory dynamics . Reaching the desired operating point by slow dilution rate changes gave rise to different final states, two oscillatory states and one steady state, depending on the rate of change in dilution rate . The multiplicity of stable states at a single operating point is not explained by any current distributed model and points toward a segregated mechanism of these oscillations .

Eur J Biochem, 2001 Oct, 268(19), 5107 - 18
Role of loops in the folding and stability of yeast phosphoglycerate kinase; Collinet B et al.; Yeast phosphoglycerate kinase (yPGK) is a monomeric two domain protein used as folding model representative of large proteins . We inserted short unstructured sequences (four Gly or four Thr) into the connections between secondary structure elements and studied the consequences of these insertions on the folding process and stability of yPGK . All the mutated proteins can refold efficiently . The effect per residue on stability is larger for the first inserted residue . Insertion in two long betaalpha loops (at residue positions 71 and 129) is more destabilizing than an insertion in a short alphabeta loop (at residue position 89) located on the opposite side of the N-terminal domain . The effect on stability is mainly due to a large increase of the unfolding rate rather than a decrease of the folding rate . This suggests that these connections between secondary structure elements do not play an active role in directing the folding process . Insertion into the short alphabeta loop (position 89) has limited effects on stability and results in the detection of a kinetic phase not previously seen with the wild-type protein, suggesting that insertions in this particular loop do qualitatively affect the folding process without a large effect on folding efficiency . For the two long betaalpha loops (positions 71 and 129) located in the inner surface of the N-terminal domain, the effects on stability are possibly associated with decoupling of the two domains as observed by differential scanning calorimetry during thermal unfolding.

Eur J Paediatr Neurol, 2001, 5 Suppl A, 89 - 93
Analysis of CLN3-protein interactions using the yeast two-hybrid system; Leung KY et al.; Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a childhood neurodegenerative disease that is caused by mutations in the CLN3 gene . The protein encoded by CLN3 has no homology with any proteins of known function and its cellular role remains elusive . In order to investigate the role played by the CLN3 protein we aimed to identify interacting proteins . Here, we describe the yeast two-hybrid system as the approach taken to investigate such protein-protein interactions . CLN3 was expressed as a fusion protein with a DNA-binding domain and used to screen a library of human fetal brain cDNAs fused to a transcriptional activation domain . Owing to low level expression of the full length CLN3 fusion protein, truncated regions corresponding to the predicted hydrophilic regions were also tested . No proteins that interact with CLN3 were detected, nor was there any evidence for CLN3-CLN3 interactions . Potential interaction of CLN3 with subunit c of mitochondrial ATP synthase, the major component of the storage material that accumulates in Batten disease patients, was also tested . No interaction was detected suggesting that the accumulation of subunit c does not result from loss of a process that requires a direct interaction with CLN3 . We conclude that either CLN3 does not interact with other proteins or such interactions cannot be detected using the two-hybrid system.

Science, 2001 Oct 5, 294(5540), 115 - 21
Replication dynamics of the yeast genome; Raghuraman MK et al.; Oligonucleotide microarrays were used to map the detailed topography of chromosome replication in the budding yeast Saccharomyces cerevisiae . The times of replication of thousands of sites across the genome were determined by hybridizing replicated and unreplicated DNAs, isolated at different times in S phase, to the microarrays . Origin activations take place continuously throughout S phase but with most firings near mid-S phase . Rates of replication fork movement vary greatly from region to region in the genome . The two ends of each of the 16 chromosomes are highly correlated in their times of replication . This microarray approach is readily applicable to other organisms, including humans.

Gene, 2001 Sep 19, 275(2), 223 - 31
A novel factor required for the SUMO1/Smt3 conjugation of yeast septins; Takahashi Y et al.; SUMO1/Smt3, a ubiquitin-like protein modifier, is known to be conjugated to other proteins and modulate their functions in various important processes . Similar to the ubiquitin system, SUMO1/Smt3 is activated in an ATP-dependent reaction by thioester bond formation with E1 (activating enzyme), transferred to E2 (conjugating enzyme), and passed to a substrate lysine . It remained unknown, however, whether any SUMO1/Smt3 ligases (E3s) are involved in the final transfer of this modifier . Here we report a novel factor Siz1 (YDR409w) required for septin-sumoylation of budding yeast, possibly acting as E3 . Siz1 is a member of a new family (Miz1, PIAS3, etc.) containing a conserved domain with a similarity to a zinc-binding RING-domain, often found in ubiquitin ligases . In the siz1 mutant septin-sumoylation was completely abolished . A conserved cysteine residue in the domain was essential for this conjugation . Furthermore, Siz1 was localized at the mother-bud neck in the M-phase and physically bound to both E2 and the target proteins.

Biochem Biophys Res Commun, 2001 Oct 12, 287(5), 1045 - 50
The N-terminal domain of the yeast permease Bap2p plays a role in its degradation; Omura F et al.; The amino acid permease Bap2p in Saccharomyces cerevisiae mediates a major part of the uptake of leucine, isoleucine, and valine from media containing a preferred nitrogen source . Although the transcriptional controls of BAP2 have been well studied, the posttranslational down-regulation mechanisms for Bap2p have not been established . Here we show that Bap2p is subject to a starvation-induced degradation upon rapamycin treatment or cultivation with proline as the sole nitrogen source . The starvation-induced degradation of Bap2p was dependent on the cellular functions of ubiquitination and endocytosis . Down-regulation of the permease required the most probable ubiquitination sites, the lysine residues situated in the N-terminal 49 residues, as well as the C-terminal domain . Furthermore, when the N-terminal domain of Bap2p was fused to the general amino acid permease Gap1p, the resultant chimeric permease became susceptible to the starvation-induced degradation, indicating that the Bap2p N-terminus contains a determinant responsive to the starvation signals .

Mol Cell Biol, 2001 Nov, 21(21), 7277 - 86
Molecular basis for telomere repeat divergence in budding yeast; Forstemann K et al.; Telomerase is a ribonucleoprotein enzyme that adds repetitive sequences to the ends of linear chromosomes, thereby counteracting nucleotide loss due to incomplete replication . A short region of the telomerase RNA subunit serves as template for nucleotide addition onto the telomere 3' end . Although Saccharomyces cerevisiae contains only one telomerase RNA gene, telomere repeat sequences are degenerate in this organism . Based on a detailed analysis of the telomere sequences specified by wild-type and mutant RNA templates in vivo, we show that the divergence of telomere repeats is due to abortive reverse transcription in the 3' and 5' regions of the template and due to the alignment of telomeres in multiple registers within the RNA template . Through the interpretation of wild-type telomere sequences, we identify nucleotides in the template that are not accessible for base pairing during substrate annealing . Rather, these positions become available as templates for reverse transcription only after alignment with adjacent nucleotides has occurred, indicating that a conformational change takes place upon substrate binding . We also infer that the central part of the template region is reverse transcribed processively . The inaccessibility of certain template positions for alignment and the processive polymerization of the central template portion may serve to reduce the possible repeat diversification and enhance the incorporation of binding sites for Rap1p, the telomere binding protein of budding yeast.

J Biol Chem, 2001 Dec 7, 276(49), 46111 - 7 Epub 2001 Oct 03.
Mitochondrial and cytosolic isoforms of yeast fumarase are derivatives of a single translation product and have identical amino termini; Sass E et al.; We have previously proposed that a single translation product of the FUM1 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae and that all fumarase translation products are targeted and processed in mitochondria before distribution . Alternative models for fumarase distribution have been proposed that require more than one translation product . In the current work (i) we show by using sequential Edman degradation and mass spectrometry that fumarase cytosolic and mitochondrial isoenzymes have an identical amino terminus that is formed by cleavage by the mitochondrial processing peptidase, (ii) we have generated fumarase mutants in which the second potential translation initiation codon (Met-24) has been substituted, yet the protein is processed efficiently and retains its ability to be distributed between the cytosol and mitochondria, and (iii) we show that although a signal peptide is required for fumarase targeting to mitochondria the specific fumarase signal peptide and the sequence immediately downstream to the cleavage site are not required for the dual distribution phenomenon . Our results are discussed in light of our model of fumarase targeting and distribution that suggests rapid folding into an import-incompetent state and retrograde movement of the processed protein back to the cytosol through the translocation pore.

Am J Pathol, 2001 Oct, 159(4), 1239 - 45
Development of a yeast stop codon assay readily and generally applicable to human genes; Kataoka A et al.; We established a yeast-based method to screen chain-terminating mutations that is readily applicable to any gene of interest . Based on the finding that 18- to 24-base-long homologous sequences are sufficient for gap repair in vivo in yeast, we used a strategy to amplify a test-gene fragment with addition of 24-bp sequences homologous to both cut-ends of a yeast expression vector, pMT18 . After co-transformation with the amplified fragment and the linearized pMT18, each yeast (Saccharomyces cerevisiae) cell automatically forms a single-copy circular plasmid (because of CEN/ARS), which expresses a test-gene::ADE2 chimera protein . When the reading frame of the test-gene contains a nonsense or frameshift mutation, truncation of the chimera protein results in lack of ADE2 activity, leading to formation of a red colony . By using a nested polymerase chain reaction using proofreading Pfu polymerase to ensure specificity of the product, the assay achieved a low background (false positivity) . We applied the assay to BRCA1, APC, hMSH6, and E-cadherin genes, and successfully detected mutations in mRNA and genomic DNA . Because this method--universal stop codon assay--requires only 4 to 5 days to screen a number of samples for any target gene, it may serve as a high-throughput screening system of general utility for chain-terminating mutations that are most prevalent in human genetic diseases.

Mol Cell, 2001 Sep, 8(3), 683 - 91
Specificity of MAP kinase signaling in yeast differentiation involves transient versus sustained MAPK activation; Sabbagh W Jr et al.; Signals transmitted by common components often elicit distinct (yet appropriate) outcomes . In yeast, two developmental options-mating and invasive growth-are both regulated by the same MAP kinase cascade . Specificity has been thought to result from specialized roles for the two MAP kinases, Kss1 and Fus3, and because Fus3 prevents Kss1 from gaining access to the mating pathway . Kss1 has been thought to participate in mating only when Fus3 is absent . Instead, we show that Kss1 is rapidly phosphorylated and potently activated by mating pheromone in wild-type cells, and that this is required for normal pheromone-induced gene expression . Signal identity is apparently maintained because active Fus3 limits the extent of Kss1 activation, thereby preventing inappropriate signal crossover.

Mol Cell, 2001 Sep, 8(3), 505 - 15
Composition and functional characterization of yeast 66S ribosome assembly intermediates; Harnpicharnchai P et al.; The pathway and complete collection of factors that orchestrate ribosome assembly are not clear . To address these problems, we affinity purified yeast preribosomal particles containing the nucleolar protein Nop7p and developed means to separate their components . Nop7p is associated primarily with 66S preribosomes containing either 27SB or 25.5S plus 7S pre-rRNAs . Copurifying proteins identified by mass spectrometry include ribosomal proteins, nonribosomal proteins previously implicated in 60S ribosome biogenesis, and proteins not known to be involved in ribosome production . Analysis of strains mutant for eight of these proteins not previously implicated in ribosome biogenesis showed that they do participate in this pathway . These results demonstrate that proteomic approaches in concert with genetic tools provide powerful means to purify and characterize ribosome assembly intermediates.

J Biol Chem, 2001 Dec 14, 276(50), 46798 - 806 Epub 2001 Oct 01.
The multispanning membrane protein Ste24p catalyzes CAAX proteolysis and NH2-terminal processing of the yeast a-factor precursor; Tam A et al.; Saccharomyces cerevisiae Ste24p is a multispanning membrane protein implicated in the CAAX proteolysis step that occurs during biogenesis of the prenylated a-factor mating pheromone . Whether Ste24p acts directly as a CAAX protease or indirectly to activate a downstream protease has not yet been established . In this study, we demonstrate that purified, detergent-solubilized Ste24p directly mediates CAAX proteolysis in a zinc-dependent manner . We also show that Ste24p mediates a separate proteolytic step, the first NH(2)-terminal cleavage in a-factor maturation . These results establish that Ste24p functions both as a bona fide COOH-terminal CAAX protease and as an a-factor NH(2)-terminal protease . Importantly, this study is the first to directly demonstrate that a eukaryotic multispanning membrane protein can possess intrinsic proteolytic activity.

Biochemistry, 2001 Oct 9, 40(40), 12193 - 206
Allosteric regulation provides a molecular mechanism for preferential utilization of the fully assembled dolichol-linked oligosaccharide by the yeast oligosaccharyltransferase; Karaoglu D et al.; The oligosaccharyltransferase (OST) preferentially utilizes the fully assembled dolichol-linked oligosaccharide Glc(3)Man(9)GlcNAc(2)-PP-Dol as the donor for N-linked glycosylation of asparagine residues in N-X-T/S consensus sites in newly synthesized proteins . A wide variety of assembly intermediates (Glc(0-2)Man(0-9)GlcNAc(2)-PP-Dol) can serve as the donor substrate for N-linked glycosylation of peptide acceptor substrates in vitro or of nascent glycoproteins in mutant cells that are defective in donor substrate assembly . A kinetic mechanism that can account for the selection of the fully assembled donor substrate from a complex mixture of dolichol-linked oligosaccharides (OS-PP-Dol) has not been elucidated . Here, the steady-state kinetic properties of the OST were reinvestigated using a proteoliposome assay system consisting of the purified yeast enzyme, near-homogeneous preparations of a dolichol-linked oligosaccharide (Glc(3)Man(9)GlcNAc(2)-PP-Dol or Man(9)GlcNAc(2)-PP-Dol) and an (125)I-labeled tripeptide as the acceptor substrate . The K(m) of the OST for the acceptor tripeptide was only slightly enhanced when Glc(3)Man(9)GlcNAc(2)-PP-Dol was the donor substrate relative to when Man(9)GlcNAc(2)-PP-Dol was the donor substrate . Evaluation of the kinetic data for both donor substrates showed deviations from typical Michaelis-Menten kinetics . Sigmoidal saturation curves, Lineweaver-Burk plots with upward curvature, and apparent Hill coefficients of about 1.4 suggested a substrate activation mechanism involving distinct regulatory (activator) and catalytic binding sites for OS-PP-Dol . Results of competition experiments using either oligosaccharide donor as an alternative substrate were also consistent with this hypothesis . We propose that binding of either donor substrate to the activator site substantially enhances Glc(3)Man(9)GlcNAc(2)-PP-Dol occupancy of the enzyme catalytic site via allosteric activation.

Nippon Yakurigaku Zasshi, 2001 Sep, 118(3), 203 - 10
{Bioassay for endocrine disruptors by using yeast two-hybrid system}; Nishihara T et al.; One of the urgent tasks in understanding endocrine disruptors (EDs) is to compile a list of suspected substances among the huge number of chemicals by using the screening test method . An in vitro screening test is a more useful tool for primary selection of suspected EDs . We have developed an assay for EDs using the yeast two-hybrid system . The assay is based on the ligand-dependent interaction of two proteins, a hormone receptor and a coactivator, and the hormonal activity is detected by beta-galactosidase activity . This assay is a very simple and inexpensive test method with high repeatability to detect the agonist, and it is applicable for the detection of antagonist and active compounds after metabolism . Accordingly, it has been used in more than 40 laboratories in Japan . To date, we have tested the estrogenic activity of more than 500 chemicals including natural substances, medicines, pesticides and industrial chemicals . Sixty-four compounds were evaluated as positive and most of these possessed a common structure: phenol with a hydrophobic moiety at the para-position without bulky groups at the ortho-position . These results are expected to facilitate further risk assessment of chemicals, especially EDs.

J Biol Chem, 2001 Dec 28, 276(52), 48973 - 7 Epub 2001 Sep 27.
Yeast Ull1/Siz1 is a novel SUMO1/Smt3 ligase for septin components and functions as an adaptor between conjugating enzyme and substrates; Takahashi Y et al.; SUMO1/Smt3, a ubiquitin-like protein modifier, is known to conjugate to other proteins and modulate their functions in various important processes . Similar to the ubiquitin conjugation system, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme) . In our previous report (Takahashi, Y., Toh-e, A., and Kikuchi, Y . (2001) Gene 275, 223-231), we showed that Siz1/Ull1 (YDR409w) of budding yeast, a member of the human PIAS family containing a RING-like domain, is a strong candidate for SUMO1/Smt3 ligase because the SUMO1/Smt3 modification of septin components was abolished in the ull1 mutant and Ull1 associated with E2 (Ubc9) and the substrates (septin components) in immunoprecipitation experiments . Here we have developed an in vitro Smt3 conjugation system for a septin component (Cdc3) using purified recombinant proteins . In this system, Ull1 is additionally required as well as E1 (Sua1.Uba2 complex), E2 (Ubc9), and ATP . A cysteine residue of the RING-like domain was essential for the conjugation both in vivo and in vitro . Furthermore, a region containing the RING-like domain directly interacted with Ubc9 and Cdc3 . Thus, this SUMO/Smt3 ligase functions as an adaptor between E2 and the target proteins.

FEBS Lett, 2001 Sep 21, 505(3), 389 - 92
Glucose-dependent and -independent signalling functions of the yeast glucose sensor Snf3; Dlugai S et al.; The yeast Snf3 protein has been described to function as a sensor for low concentrations of extracellular glucose . We have found that Snf3 is able to transduce a signal in the complete absence of extracellular glucose . High basal activity of the HXT7 promoter during growth on ethanol required Snf3 as well as other components of the signalling pathway activated by Snf3 . Moreover, the C-terminal domain of Snf3 was sufficient to complement the role of Snf3 in this regulation . As the C-terminal tail of Snf3 interacted with other components at the plasma membrane independent of the carbon source, our data suggest that Snf3 is involved in signalling complexes which can be activated by other signals than extracellular glucose.

Traffic, 2001 Oct, 2(10), 705 - 16
Yeast ribosomes bind to highly purified reconstituted Sec61p complex and to mammalian p180; Morrow MW et al.; To determine whether the yeast Sec61p translocation pore is a high-affinity ribosome receptor in the endoplasmic reticulum, we isolated the Sec61p complex using an improved protocol in which contaminants found previously to be associated with the complex are absent . The purified complex, which contains Sec61p with an amino terminal hexahistidine tag, was active since it rescued a sec61-3 post-translational translocation defect in a reconstituted system . Co-reconstitution of the Sec61p and Sec63p complexes into liposomes failed to support post-translational translocation, suggesting that Sec62p is required for this process . By Scatchard analysis, the purified Sec61p complex bound to yeast ribosomes when reconstituted into liposomes with a KD of 5.6 nM, a value similar to the KD obtained when ribosome binding to total microsomal protein was measured (2.7 nM) . In addition, a mammalian protein, p180, which has been proposed to be a ribosome receptor, was expressed in yeast, and endoplasmic reticulum-derived microsomes isolated from this strain exhibited approximately 2.3-fold greater binding to yeast ribosomes . Despite this increase in ribosome binding, neither co- nor post-translational translocation was compromised in vivo . In sum, our data suggest that the Sec61p complex is a ribosome receptor in the yeast endoplasmic reticulum membrane.

J Mol Biol, 2001 Sep 28, 312(4), 637 - 47
High affinity cooperative DNA binding by the yeast Mlh1-Pms1 heterodimer; Hall MC et al.; We demonstrate here that the Saccharomyces cerevisiae Mlh1-Pms1 heterodimer required for DNA mismatch repair and other cellular processes is a DNA binding protein . Binding was evaluated using a variety of single and double-stranded DNA molecules . Mlh1-Pms1 bound short substrates with low affinity and showed a slight preference for single-stranded DNA . In contrast, Mlh1-Pms1 exhibited a much higher affinity for long DNA molecules, suggesting that binding is cooperative . High affinity binding required a duplex DNA length greater than 241 base-pairs . The rate of association with DNA was rapid and dissociation of protein-DNA complexes following extensive dilution was very slow . However, in competition experiments, we observed a rapid active transfer of Mlh1-Pms1 from labeled to unlabeled DNA . Binding was non-sequence specific and highly sensitive to salt type and concentration, suggesting that Mlh1-Pms1 primarily interacts with the DNA backbone via ionic contacts . Cooperative binding was observed visually by atomic force microscopy as long, continuous tracts of Mlh1-Pms1 protein bound to duplex DNA . These images also showed that Mlh1-Pms1 simultaneously interacts with two different regions of duplex DNA . Taken together, the atomic force microscope images and DNA binding assays provide strong evidence that Mlh1-Pms1 binds duplex DNA with positive cooperativity and that there is more than one DNA binding site on the heterodimer . These DNA binding properties of Mlh1-Pms1 may be relevant to its participation in DNA mismatch repair, recombination and cellular responses to DNA damage.

J Clin Microbiol, 2001 Oct, 39(10), 3617 - 22
Multiplex PCR using internal transcribed spacer 1 and 2 regions for rapid detection and identification of yeast strains; Fujita SI et al.; Multiplex PCR amplification followed by either agarose gel electrophoresis (PCR-AGE) or microchip electrophoresis (PCR-ME) was used to test a total of 120 fungal strains . The internal transcribed spacer 1 (ITS1) and ITS2 regions and the 5.8S ribosomal DNA (rDNA) region of the fungi were amplified by using universal primers ITS1 and ITS4 . The ITS2 region was simultaneously amplified by using universal primers ITS3 and ITS4 . Since Trichosporon asahi and T . asteroides showed similar lengths for two amplicons, 29 different gel patterns were demonstrated for 30 yeast species tested on the basis of differences in the lengths of one or two amplicons . Of 75 yeast isolates from clinical materials, 5 isolates (6.8%) which were incompletely identified or not identified by the phenotypic method were identified with our PCR-based method (2 isolates as Candida guilliermondii, 2 as C . krusei, and 1 as C . zeylanoides) . No differences in discriminating power or sensitivity were observed between the PCR-AGE method and the PCR-ME method . These methods, prospectively applied to 24 yeast-positive blood culture bottles (16 patients), resulted in the correct detection of 24 yeast strains . In conclusion, multiplex PCR followed by electrophoresis seems to be a promising tool for the rapid identification of common and uncommon yeast strains from culture colonies and from yeast-positive blood culture bottles (5.5 h for the PCR-AGE method and 3 h for the PCR-ME method).

J Biol Chem, 2001 Dec 7, 276(49), 46165 - 71 Epub 2001 Sep 26.
Complementing yeast rho1 mutation groups with distinct functional defects; Saka A et al.; Saccharomyces cerevisiae is a multifunctional molecular switch involved in establishment of cell morphogenesis . We systematically characterized isolated temperature-sensitive mutations in the RHO1 gene and identified two groups of rho1 mutations (rho1A and rho1B) possessing distinct functional defects . Biochemical and cytological analyses demonstrated that mutant cells of the rho1A and rho1B groups have defects in activation of the Rho1p effectors Pkc1p kinase and 1,3-beta-glucan synthase, respectively . Heteroallelic diploid strains with rho1A and rho1B mutations were able to grow even at the restrictive temperature of the corresponding homoallelic diploid strains, showing intragenic complementation . The ability to activate both of the essential Rho1p effector proteins was restored in the heteroallelic diploid . Thus, each of the complementing rho1 mutation groups abolishes a distinct function of Rho1p, activation of Pkc1p kinase or 1,3-beta-glucan synthase activity.

Biochem Soc Symp, 2001, (68), 35 - 43
Self-perpetuating changes in Sup35 protein conformation as a mechanism of heredity in yeast; Serio TR et al.; Recently, a novel mode of inheritance has been described in the yeast Saccharomyces cerevisiae . The mechanism is based on the prion hypothesis, which posits that self-perpetuating changes in the conformation of single protein, PrP, underlie the severe neurodegeneration associated with the transmissible spongiform enchephalopathies in mammals . In yeast, two prions, {URE3} and {PSI+}, have been identified, but these factors confer unique phenotypes rather than disease to the organism . In each case, the prion-associated phenotype has been linked to alternative conformations of the Ure2 and Sup35 proteins . Remarkably, Ure2 and Sup35 proteins existing in the alternative conformations have the unique capacity to transmit this physical state to the newly synthesized protein in vivo . Thus, a mechanism exists to ensure replication of the conformational information that underlies protein-only inheritance . We have characterized the mechanism by which Sup35 conformational information is replicated in vitro . The assembly of amyloid fibres by a region of Sup35 encompassing the N-terminal 254 amino acids faithfully recapitulates the in vivo propagation of {PSI+} . Mutations that alter {PSI+} inheritance in vivo change the kinetics of amyloid assembly in vitro in a complementary fashion, and lysates from {PSI+} cells, but not {psi-} cells, accelerate assembly in vitro . Using this system we propose a mechanism by which the alternative conformation of Sup35 is adopted by an unstructured oilgomeric intermediate at the time of assembly.

J Cell Biochem, 2001 Aug 1-9, 83(2), 249 - 58
Molecular cloning and characterization of a RING-H2 finger protein, ANAPC11, the human homolog of yeast Apc11p; Chan AH et al.; Yeast Apc11p together with Rbx1 and Roc2/SAG define a new class of RING-H2 fingers in a superfamily of E3 ubiquitin ligases . The human homolog of Apc11p, ANAPC11 was identified during a large-scale partial sequencing of a human liver cancer cDNA library and partial characterization was performed . This 514 bp full-length cDNA has a predicted open reading frame (ORF) encoding 84 amino acids . The ORF codes for ANAPC11, the human anaphase promoting complex subunit 11 (yeast APC11 homolog), which possesses a RING-H2 finger motif and exhibits sequence similarity to subunits of E3 ubiquitin ligase complexes . In Northern blot hybridization with poly(A) RNA of various human tissues using radio-labelled ANAPC11 cDNA probe, we found strong signals detected in skeletal muscle and heart; moderate signals detected in brain, kidney, and liver; and detectable but low signals in colon, thymus, spleen, small intestine, placenta, lung, and peripheral blood leukocyte . The ANAPC11 gene is located at the human chromosome 17q25 . ANAPC11 is distributed diffusely in the cytoplasm and nucleus with discrete accumulation in granular structures in all the cell lines (AML 12, HepG2, and C2C12) transfected . Expression level of ANAPC11 is found higher in certain types of cancer determined in the RNA dot blot experiment .

Cell, 2001 Sep 21, 106(6), 697 - 708
Serial regulation of transcriptional regulators in the yeast cell cycle; Simon I et al.; Genome-wide location analysis was used to determine how the yeast cell cycle gene expression program is regulated by each of the nine known cell cycle transcriptional activators . We found that cell cycle transcriptional activators that function during one stage of the cell cycle regulate transcriptional activators that function during the next stage . This serial regulation of transcriptional activators forms a connected regulatory network that is itself a cycle . Our results also reveal how the nine transcriptional regulators coordinately regulate global gene expression and diverse stage-specific functions to produce a continuous cycle of cellular events . This information forms the foundation for a complete map of the transcriptional regulatory network that controls the cell cycle.

Yeast, 2001 Oct, 18(14), 1331 - 8
Graded mode of transcriptional induction in yeast pheromone signalling revealed by single-cell analysis; Poritz MA et al.; Signalling pathways typically convert a graded, analogue signal into a binary cellular output . In the several eukaryotic systems that have been investigated to date, including MAP kinase cascade activation in Xenopus oocytes, analogue-to-digital conversion occurs at points in the pathway between receptor activation and the effector mechanism . We used flow cytometry combined with an intracellular fluorescent reporter to examine the characteristics of the yeast pheromone-response pathway . Surprisingly, pheromone response in yeast, which relies on the MAP kinase cascade, behaved in a fundamentally graded manner . Expression of certain exogenous dominant inhibitors of the pathway converted the response to graded-or-none behaviour . These results have implications for the dissection of biological response mechanisms in cells and illustrate how signalling pathways, even homologous ones, may have strikingly different signal propagation/amplification characteristics .

Yeast, 2001 Oct, 18(14), 1301 - 7
A minisatellite sequence in the upstream region of the DURA3 gene from the halotolerant yeast Debaryomyces hansenii; Bansal PK et al.; The URA3 gene of Debaryomyces hansenii, encoding orotidine 5'-phosphate decarboxylase enzyme, was isolated by complementation in the yeast Saccharomyces cerevisiae . The deduced amino acid sequence is highly similar to Ura3 proteins from other yeast and fungal species . Analysis of the region upstream of the coding sequence revealed the presence of AG-rich minisatellite DNA sequences . In addition, upstream of the DURA3 sequence, we have found the 3'-terminal of a gene encoding a GEA2-like protein .

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