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