Yeasts

J Biol Chem, 2001 Mar 9, 276(10), 7169 - 75
Yeast nuclear extract contains two major forms of RNA polymerase II mediator complexes; Liu Y et al.; The yeast Mediator complex is required for transcription by RNA polymerase II (pol II) in vivo and in vitro . This complex of over 20 polypeptides associates with pol II and is recruited to transcription complexes at promoters . Previous isolations of yeast Mediator-containing complexes in different laboratories have identified several distinct complexes . To identify the major forms of Mediator in yeast, Mediator was isolated from nuclear extracts using a two-step chromatographic procedure, avoiding ion exchange chromatography and high salt conditions to prevent dissociation of subunits during purification . Components of the Mediator complexes were identified by mass spectrometry and Western analysis . The major form of Mediator, termed pol II x Med, contained pol II and Mediator, including the Srb8-11 module . A second lower molecular size complex was also identified, termed Mediator core (Medc), which lacked pol II, Srb8-11, Rox3, Nut1, and the Rgrl module . Both of these complexes were active in transcription in vitro, although the Medc complex had significantly lower activity and could compete with the activity of the pol II x Med complex in vitro.

J Biol Chem, 2001 Aug 31, 276(35), 32437 - 45 Epub 2001 May 29.
TcSCA complements yeast mutants defective in Ca2+ pumps and encodes a Ca2+-ATPase that localizes to the endoplasmic reticulum of Trypanosoma cruzi; Furuya T et al.; Intracellular Ca(2+) in Trypanosoma cruzi is mainly located in an acidic compartment named the acidocalcisome, which among other pumps and exchangers possesses a plasma membrane-type Ca(2+)-ATPase . Evidence for an endoplasmic reticulum-located Ca(2+) uptake has been more elusive and based on indirect results . Here we report the cloning and sequencing of a gene encoding a sarcoplasmic-endoplasmic reticulum-type Ca(2+)-ATPase from T . cruzi . The protein (TcSCA) predicted from the nucleotide sequence of the gene has 1006 amino acids and a molecular mass of 109.7 kDa . Several sequence motifs found in sarcoplasmic-endoplasmic reticulum-type Ca(2+)-ATPases were present in TcSCA . Expression of TcSCA in yeast mutants deficient in the Golgi and vacuolar Ca(2+) pumps (pmr1 pmc1 cnb 1) restored growth on EGTA . Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with TcSCA, and it was found that the TcSCA polypeptide formed a Ca(2+)-dependent and hydroxylamine-sensitive (32)P-labeled phosphoprotein of 110 kDa in the presence of {gamma-(32)P}ATP . Cyclopiazonic acid, but not thapsigargin, blocked this phosphoprotein formation . Transgenic parasites expressing constructs of TcSCA with green fluorescent protein exhibited co-localization of TcSCA with the endoplasmic reticulum proteins BiP and calreticulin . An endoplasmic reticulum location was also found in amastigotes and trypomastigotes using a polyclonal antibody against a COOH-terminal region of the protein . The ability of TcSCA to restore growth of mutant pmr1 pmc1 cnb 1 on medium containing Mn(2+) suggests that TcSCA may also regulate Mn(2+) homeostasis by pumping Mn(2+) into the endoplasmic reticulum of T . cruzi.

J Biol Chem, 2001 Aug 3, 276(31), 29210 - 7 Epub 2001 May 29.
Yol082p, a novel CVT protein involved in the selective targeting of aminopeptidase I to the yeast vacuole; Leber R et al.; The yeast vacuolar enzyme aminopeptidase I (API) is synthesized in the cytoplasm as a precursor (pAPI) . Upon its assembly into dodecamers, pAPI is wrapped by double-membrane saccular structures for its further transport within vesicles that fuse with the vacuolar membrane and release their content in the vacuolar lumen . Targeting of API to the vacuole occurs by two alternative transport routes, the cvt and the autophagy pathways, which although mechanistically similar specifically operate under vegetative growth or nitrogen starvation conditions, respectively . We have studied the role of Yol082p, a protein identified by its ability to interact with API, in the transport of its precursor to the vacuole . We show that Yol082p interacts with mature API, an interaction that is strengthened by the amino extension of the API protein . Yol082p is required for targeting of pAPI to the vacuole, both under growing and short term nitrogen starvation conditions . Absence of Yol082p does not impede the assembly of pAPI into dodecamers, but precludes the enclosure of pAPI within transport vesicles . Microscopy studies show that during vegetative growth Yol082p is distributed between a cytoplasmic pool and a variable number of 0.13--0.27-microm round, mobile structures, which are no longer observed under conditions of nitrogen starvation, and become larger in cells expressing the inactive Yol082 Delta C32p, or lacking Apg12p . In contrast to the autophagy mutants involved in API transport, a Delta yol082 strain does not lose viability under nitrogen starvation conditions, indicating normal function of the autophagy pathway . The data are consistent with a role of Yol082p in an early step of the API transport, after its assembly into dodecamers . Because Yol082p fulfills the functional requisites that define the CVT proteins, we propose to name it Cvt19.

J Cell Biol, 2001 May 28, 153(5), 1121 - 6
The yeast class V myosins, Myo2p and Myo4p, are nonprocessive actin-based motors; Reck-Peterson SL et al.; The motor properties of the two yeast class V myosins, Myo2p and Myo4p, were examined using in vitro motility assays . Both myosins are active motors with maximum velocities of 4.5 microm/s for Myo2p and 1.1 microm/s for Myo4p . Myo2p motility is Ca(2+) insensitive . Both myosins have properties of a nonprocessive motor, unlike chick myosin-Va (M5a), which behaves as a processive motor when assayed under identical conditions . Additional support for the idea that Myo2p is a nonprocessive motor comes from actin cosedimentation assays, which show that Myo2p has a low affinity for F-actin in the presence of ATP and Ca(2+), unlike chick brain M5a . These studies suggest that if Myo2p functions in organelle transport, at least five molecules of Myo2p must be present per organelle to promote directed movement.

J Cell Biol, 2001 May 28, 153(5), 1061 - 70
Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation; Nishikawa SI et al.; Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome . Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined . To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro-alpha-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro . We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain-containing proteins Jem1p and Scj1p are deleted . In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein . These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.

Yeast, 2001 Jun, 18(8), 697 - 709
Ypt protein prenylation depends on the interplay among levels of Rab escort protein and geranylgeranyl diphosphate in yeast cells; Miaczynska M et al.; Farnesyl diphosphate (FPP), an intermediate of the sterol biosynthetic pathway, is used by farnesyl transferase to farnesylate, among others, the Ras proteins, and by geranylgeranyl diphosphate synthase to produce geranylgeranyl diphosphate (GGPP) . GGPP is then transferred by geranylgeranyl transferase II (GGTase II) to Rab/Ypt members of the Ras superfamily known to be required at all stages of vesicle transport in both mammals and yeast . Formation of a complex between a Rab/Ypt protein and an accessory protein named the Rab escort protein (REP) is a prerequisite for GGTase II substrate recognition . Little is known about the factors that regulate GGTase II activity in living cells but, based on available data, it seems possible that vesicle transport in higher eukaryotes is regulated by the levels of prenylated Rab/Ypt proteins in the cells . Here we show that the levels of REP play an important role in regulating GGTase II activity in yeast cells if sufficient substrates are present . Moreover, overexpression of REP causes, directly or indirectly, an increased level of Ypt substrates available for prenylation, which in turn leads to the depletion of the GGPP pool in the cell . Overall our data suggest that the levels of REP and the availability of GGPP play a role in regulating Ypt protein prenylation .

Curr Biol, 2001 May 15, 11(10), 803 - 7
A localized GTPase exchange factor, Bud5, determines the orientation of division axes in yeast; Marston AL et al.; GTPases are widespread in directing cytoskeletal rearrangements and affecting cellular organization . How they do so is not well understood . Yeast cells divide by budding, which occurs in two spatially programmed patterns, axial or bipolar {1-3} . Cytoskeletal polarization to form a bud is governed by the Ras-like GTPase, Bud1/Rsr1, in response to cortical landmarks . Bud1 is uniformly distributed on the plasma membrane, so presumably its regulators, Bud5 GTPase exchange factor and Bud2 GTPase activating protein, impart spatial specificity to Bud1 action {4} . We examined the localizations of Bud5 and Bud2 . Both Bud1 regulators associate with cortical landmarks designating former division sites . In haploids, Bud5 forms double rings that encircle the mother-bud neck and split upon cytokinesis so that each progeny cell inherits Bud5 at the axial division remnant . Recruitment of Bud5 into these structures depends on known axial landmark components . In cells undergoing bipolar budding, Bud5 associates with multiple sites, in response to the bipolar landmarks . Like Bud5, Bud2 associates with the axial division remnant, but rather than being inherited, Bud2 transiently associates with the remnant in late G1, before condensing into a patch at the incipient bud site . The relative timing of Bud5 and Bud2 localizations suggests that both regulators contribute to the spatially specific control of Bud1 GTPase.

Curr Biol, 2001 May 15, 11(10), 784 - 8
Order of function of the budding-yeast mitotic exit-network proteins Tem1, Cdc15, Mob1, Dbf2, and Cdc5; Lee SE et al.; The Dbf2 protein kinase functions as part of the mitotic-exit network (MEN), which controls the inactivation of the Cdc28-Clb2 kinase in late mitosis {1} . The MEN includes the Tem1 GTP binding protein; the kinases Cdc15 and Cdc5; Mob1, a protein of unknown function; and the phosphatase Cdc14 {2} . Here we have used Dbf2 kinase activity to investigate the regulation and order of function of the MEN . We find that Tem1 acts at the top of the pathway, upstream of Cdc15, which in turn functions upstream of Mob1 and Dbf2 . The Cdc5 Polo-like kinase impinges at least twice on the MEN since it negatively regulates the network, probably upstream of Tem1, and is also required again for Dbf2 kinase activation . Furthermore, we find that regulation of Dbf2 kinase activity and actin ring formation at the bud neck are causally linked . In metaphase-arrested cells, the MEN inhibitor Bub2 restrains both Dbf2 kinase activity {3} and actin ring formation {4} . We find that the MEN proteins that are required for Dbf2 kinase activity are also required for actin ring formation . Thus, the MEN is crucial for the regulation of cytokinesis, as well as mitotic exit.

Trends Genet, 2001 Jun, 17(6), 302 - 3
Origin of the duplicated regions in the yeast genomes; Piskur J; The genome of Saccharomyces cerevisiae contains several duplicated regions . The recent sequencing results of several yeast species suggest that the duplicated regions found in the modern Saccharomyces species are probably the result of a single gross duplication, as well as a series of sporadic independent short-segment duplications . The gross duplication might coincide with the origin of the ability to grow under anaerobic conditions.

Nucleic Acids Res, 2001 Jun 1, 29(11), 2382 - 94
Yeast telomerase appears to frequently copy the entire template in vivo; Ray A et al.; Telomeres derived from the same formation event in wild type strains of Saccharomyces cerevisiae possess the same, precise TG(1-3) sequence for the most internal approximately 100 bp of the 250-350 bp TG(1-3) repeats . The conservation of this internal domain is thought to reflect the fact that telomere lengthening and shortening, and thus alteration of the precise TG(1-3) sequence, is confined to the terminal region of the telomere . The internal domains of telomeres from yku70 and tel1 mutants, whose entire telomeres are only approximately 100 bp, were examined by analyzing 5.1 kb of cloned TG(1-3) sequences from telomeres formed during transformation of wild type, yku70 and tel1 cells . The internal domains were 97-137 bp in wild type cells, 27-36 bp in yku70 cells and 7-9 bp in tel1 cells . These data suggest that the majority of the tel1 cell TG(1-3) repeats may be resynthesized during shortening and lengthening reactions while a portion of the yku70 cell telomeres are protected . TG(1-3) sequences are synthesized by telomerase repeatedly copying an internal RNA template, which introduces a sequence bias into TG(1-3) repeats . Analysis of in vivo-derived telomeres revealed that of the many possible high affinity binding sites for the telomere protein Rap1p in TG(1-3) repeats, only those consistent with telomere hybridization to the ACACAC in the 3'-region of the telomerase RNA template followed by copying of most of the template were present . Copies of the telomerase RNA template made up 40-60% of the TG(1-3) sequences from each strain and could be found in long, tandem repeats . The data suggest that in vivo yeast telomerase frequently allows telomeres to hybridize to the 3'-region of RNA template and copy most of it prior to dissociation, or that in vivo telomere processing events result in the production of TG(1-3) sequences that mimic this process.

Nucleic Acids Res, 2001 Jun 1, 29(11), 2217 - 22
DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1; Boutabout M et al.; The fidelity of the yeast retrotransposon Ty1 reverse transcriptase (RT) was determined by an assay based on gel electrophoresis . Steady-state kinetics analyses of deoxyribonucleotide (dNTP) incorporation at a defined primer-template site indicate that Ty1 RT misincorporates dNTP at a frequency of 0.45 x 10(-5) for the A(t):A mispair in which dATP is misincorporated opposite a template A to 6.27 x 10(-5) for the C(t):A mispair . The G(t):G and T(t):T mispairs are formed with very low efficiency . The fidelity parameters of Ty1 RT do not depend on whether RNA or DNA are copied . Relative to lentiviral RTs (HIV-1, HIV-2 or EIAV) Ty1 RT is approximately 10-fold less error prone . Our data also show that the Ty1 RT is able to recapitulate two error-generating mechanisms: extension of mismatches and non-templated addition of nucleotides at the end of a blunt-end primer-template.

J Biol Chem, 2001 Aug 3, 276(31), 29382 - 92 Epub 2001 May 25.
Single amino acid substitutions and deletions that alter the G protein coupling properties of the V2 vasopressin receptor identified in yeast by receptor random mutagenesis; Erlenbach I et al.; To facilitate structure-function relationship studies of the V2 vasopressin receptor, a prototypical G(s)-coupled receptor, we generated V2 receptor-expressing yeast strains (Saccharomyces cerevisiae) that required arginine vasopressin-dependent receptor/G protein coupling for cell growth . V2 receptors heterologously expressed in yeast were unable to productively interact with the endogenous yeast G protein alpha subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal G alpha(q) sequence (Gq5) . In contrast, the V2 receptor efficiently coupled to a Gpa1p/G alpha(s) hybrid subunit containing the C-terminal G alpha(s) sequence (Gs5), indicating that the V2 receptor retained proper G protein coupling selectivity in yeast . To gain insight into the molecular basis underlying the selectivity of V2 receptor/G protein interactions, we used receptor saturation random mutagenesis to generate a yeast library expressing mutant V2 receptors containing mutations within the second intracellular loop . A subsequent yeast genetic screen of about 30,000 mutant receptors yielded four mutant receptors that, in contrast to the wild-type receptor, showed substantial coupling to Gq5 . Functional analysis of these mutant receptors, followed by more detailed site-directed mutagenesis studies, indicated that single amino acid substitutions at position Met(145) in the central portion of the second intracellular loop of the V2 receptor had pronounced effects on receptor/G protein coupling selectivity . We also observed that deletion of single amino acids N-terminal of Met(145) led to misfolded receptor proteins, whereas single amino acid deletions C-terminal of Met(145) had no effect on V2 receptor function . These findings highlight the usefulness of combining receptor random mutagenesis and yeast expression technology to study mechanisms governing receptor/G protein coupling selectivity and receptor folding.

EMBO Rep, 2001 May, 2(5), 423 - 30
Htm1p, a mannosidase-like protein, is involved in glycoprotein degradation in yeast; Jakob CA et al.; Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytoplasm and degraded by the proteasome . Processing intermediates of N-linked oligosaccharides on incompletely folded glycoproteins have an important role in their folding/refolding, and also in their targeting to proteolytic degradation . In Saccharomyces cerevisiae, we have identified a gene coding for a non-essential protein that is homologous to mannosidase I (HTM1) and that is required for degradation of glycoproteins . Deletion of the HTM1 gene does not affect oligosaccharide trimming . However, deletion of HTM1 does reduce the rate of degradation of the mutant glycoproteins such as carboxypeptidase Y, ABC-transporter Pdr5-26p and oligosaccharyltransferase subunit Stt3-7p, but not of mutant Sec61-2p, a non-glycoprotein . Our results indicate that although Htm1p is not involved in processing of N-linked oligosaccharides, it is required for their proteolytic degradation . We propose that this mannosidase homolog is a lectin that recognizes Man8GlcNAc2 oligosaccharides that serve as signals in the degradation pathway.

Folia Histochem Cytobiol, 2001, 39(2), 147 - 8
Detection of P53 abnormalities in non-small cell lung cancer by yeast functional assay; Niklinska W et al.; We assessed the status of P53 in 32 surgically treated non-small cell lung cancers (NSCLC) by using yeast functional assay . For functional assay, total RNA extracted from fresh-frozen specimens was reverse transcribed and P53 cDNAs were PCR-amplified using Pfu DNA polymerase (Stratagene) . The transcriptional competence of the P53 cDNA was then tested in a yeast reporter strain . 20 of the 32 (69%) NSCLC patients contained mutant P53 in the yeast functional assay with the higher frequency in squamous cell carcinoma (14/17, 82%) than in adenocarcinoma (5/10, 50%) and large cell carcinoma (3/5, 60%) (p<0.01, chi2 test) . No significant difference was observed with respect to the TNM . Preliminary survival analysis showed that patients scoring positive for the yeast test had shorter disease-free survival (median = 10 months) than those that scored negative (median > 21 months) . Our results suggest that yeast functional assay is not only an improved method to examine the status of P53, but might hopefully improve understanding of the role of mutant P53 in the clinical evaluation of NSCLC.

Mol Biol Evol, 2001 Jun, 18(6), 995 - 1000
Insect symbiosis: derivation of yeast-like endosymbionts within an entomopathogenic filamentous lineage; Suh SO et al.; Yeast-like endosymbionts (YLSs) of insects often are restricted to specific hosts and are essential to the host's survival . For example, in planthoppers (Homoptera: Delphacidae), endosymbionts function in sterol utilization and nitrogen recycling for the hosts . Our study, designed to investigate evolutionary changes in the YLS lineage involved in the planthopper association, strongly suggests an origin of the YLSs from within the filamentous ascomycetes (Euascomycetes), not the true yeasts (Saccharomycetes), as their morphology might indicate . During divergence of the planthopper YLSs, dramatic changes would have occurred in the insect-fungus interaction and the fungal morphology that have previously been undescribed in filamentous ascomycetes . Phylogenetic trees were based on individual and combined data sets of 2.6 kb of the nuclear small- and large-subunit ribosomal RNA genes for YLSs from three rice planthoppers (Laodelphax striatellus, Nilaparvata lugens, and Sogatella furcifera) compared with 56 other fungi . Parsimony analysis placed the planthopper YLSs within Cordyceps (Euascomycetes: Hypocreales: Clavicipitaceae), a genus of filamentous insects and a few fungal pathogenic ascomycetes . Another YLS species restricted to the aphid Hamiltonaphis styraci (Homoptera: Aphididae) was a sister taxon to the planthopper YLSS: Filamentous insect pathogens (Metarhizium and Beauveria) specific to the same species of insect hosts as the YLSs also formed lineages within the Clavicipitaceae, but these were distinct from the clade comprising YLS species . Trees constrained to include the YLSs in families of the Hypocreales other than the Clavicipitaceae were rejected by the Kishino-Hasegawa test . In addition, the results of this study support a hypothesis of two independent origins of insect-associated YLSs from among filamentous ascomycetes: the planthopper YLSs in the Clavicipitaceae and the YLSs associated with anobiid beetles (Symbiotaphrina species) . Several lineages of true yeasts (Saccharomycetes) also formed endosymbiotic associations with beetles, but they were not closely related to either group derived from the filamentous ascomycetes.

Cell, 2001 May 18, 105(4), 459 - 72
Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast; Alexandru G et al.; At the onset of anaphase, a caspase-related protease (separase) destroys the link between sister chromatids by cleaving the cohesin subunit Scc1 . During most of the cell cycle, separase is kept inactive by binding to an inhibitory protein called securin . Separase activation requires proteolysis of securin, which is mediated by an ubiquitin protein ligase called the anaphase-promoting complex . Cells regulate anaphase entry by delaying securin ubiquitination until all chromosomes have attached to the mitotic spindle . Though no longer regulated by this mitotic surveillance mechanism, sister separation remains tightly cell cycle regulated in yeast mutants lacking securin . We show here that the Polo/Cdc5 kinase phosphorylates serine residues adjacent to Scc1 cleavage sites and strongly enhances their cleavage . Phosphorylation of separase recognition sites may be highly conserved and regulates sister chromatid separation independently of securin.

Mol Genet Genomics, 2001 Mar, 265(1), 126 - 34
Hsp90 levels affect telomere length in yeast; Grandin N et al.; Cdc13 is a Saccharomyces cerevisiae protein that binds to telomeric single-stranded DNA and regulates telomerase activity . Stnl has been shown by two-hybrid analysis to form a physical complex with Cdc13 . Temperature-sensitive mutations in CDC13 and STN1, which are both essential genes, activate a DNA damage-dependent checkpoint which is the cause of the arrest seen in the mutant strains . The stn1-13 mutation induces dramatic telomere elongation which is telomerase dependent, as shown here . Additional mutants for STN1, which show a tighter arrest phenotype than stn1-13, were generated in order to perform genetic screens aiming at uncovering new regulators of telomerase . HSC82, which encodes a conserved molecular chaperone of the Hsp90 family, was thus isolated as a high-dosage suppressor of a temperature-sensitive mutation in STN1 . Overexpression of HSC82 also partially suppressed the growth defect of cdc13-1 cells . Overexpression of HSC82 was found to correct the telomeric defect associated with stn1 mutations . Shortening of telomeres was also observed in wild-type cells upon overexpression of HSC82, or of its temperature-inducible homologue, HSP82 . These results identify Hsc82/Hsp82 as potential regulators of telomerase in yeast cells.

Biotechnol Bioeng, 2001 Jul 20, 74(2), 96 - 107
Prediction of the pilot-scale recovery of a recombinant yeast enzyme using integrated models; Varga EG et al.; This article describes the rapid prediction of recovery process performance for a new recombinant enzyme product on the basis of a broad portfolio of computer models and highly targeted experimentation . A process model for the recombinant system was generated by linking unit operation models in an integrated fashion, with required parameter estimation and physical property determination accomplished using data from scale-down studies . This enabled the generic modeling framework established for processing of a natural enzyme from bakers' yeast to be applied . An experimental study of the same operations at the pilot scale showed that the process model gave a conservative prediction of recombinant enzyme recovery . The model successfully captured interactions leading to a low overall product yield and indicated the need for further study of precipitate breakage in the feed zone of a disc stack centrifuge in order to improve performance . The utility of scale-down units as an aid to fast model generation and the advantage of integrating computer modeling and scale-down studies to accelerate bioprocess development are highlighted .

Protein Sci, 2001 Jun, 10(6), 1113 - 23
Environmentally induced reversible conformational switching in the yeast cell adhesion protein alpha-agglutinin; Zhao H et al.; The yeast cell adhesion protein alpha-agglutinin is expressed on the surface of a free-living organism and is subjected to a variety of environmental conditions . Circular dichroism (CD) spectroscopy shows that the binding region of alpha-agglutinin has a beta-sheet-rich structure, with only approximately 2% alpha-helix under native conditions (15-40 degrees C at pH 5.5) . This region is predicted to fold into three immunoglobulin-like domains, and models are consistent with the CD spectra as well as with peptide mapping and site-specific mutagenesis . However, secondary structure prediction algorithms show that segments comprising approximately 17% of the residues have high alpha-helical and low beta-sheet potential . Two model peptides of such segments had helical tendencies, and one of these peptides showed pH-dependent conformational switching . Similarly, CD spectroscopy of the binding region of alpha-agglutinin showed reversible conversion from beta-rich to mixed alpha/beta structure at elevated temperatures or when the pH was changed . The reversibility of these changes implied that there is a small energy difference between the all-beta and the alpha/beta states . Similar changes followed cleavage of peptide or disulfide bonds . Together, these observations imply that short sequences of high helical propensity are constrained to a beta-rich state by covalent and local charge interactions under native conditions, but form helices under non-native conditions.

Curr Biol, 2001 Apr 17, 11(8), 537 - 49
M phase-specific kinetochore proteins in fission yeast: microtubule-associating Dis1 and Mtc1 display rapid separation and segregation during anaphase; Nakaseko Y et al.; BACKGROUND: Kinetochore microtubules are made early in mitosis and link chromosomal kinetochores to the spindle poles . They are required later to move the separated sister chromatids toward the opposite poles upon the onset of anaphase . Very little is known about proteins that are responsible for the connection between kinetochores and mitotic microtubules . RESULTS: We here show that fission yeast Dis1 and the related protein Mtc1/Alp14 are both able to bind microtubules in vitro and share an essential function for viability in vivo . The deletion of mtc1+ results in an instability of cytoplasmic microtubules that can be suppressed by the ectopic expression of dis1+ . Dis1 and Mtc1 are localized along interphase cytoplasmic microtubules and are mobilized onto the spindle upon mitotic commitment . In chromatin immunoprecipitation (CHIP) experiments Dis1 coprecipitated with the central centromeric DNA in an M phase-specific manner . Consistently, observations of both living cells in which the native, genomic copy of dis1+ tagged with GFP and cells fixed by immunostaining established that Dis1 behaves as a kinetochore protein during the progression from metaphase to anaphase . The central and C-terminal regions of Dis1 are sufficient for interactions with microtubules and the kinetochore, respectively . In anaphase, the GFP signals of both Dis1 and Mtc1 suddenly separate and move quickly toward opposite spindle poles . CONCLUSIONS: Fission yeast Dis1 and Mtc1 are members of an evolutionarily conserved microtubule binding protein family that includes frog XMAP215 . Dis1 and Mtc1 are implicated in stabilizing kinetochore microtubules in metaphase and so counteract the action of microtubule destabilizing factors that dominate in anaphase . Dis1 may play a dual role by becoming a part of the kinetochores in an M phase-specific manner, and it may possibly generate connections between kinetochores and microtubules.

Arch Biochem Biophys, 2000 Dec 15, 384(2), 379 - 82
Identification of the self-interaction of rat TCTP/IgE-dependent histamine-releasing factor using yeast two-hybrid system; Yoon T et al.; To further understand the biological function of translationally controlled tumor protein (TCTP), also known as IgE-dependent histamine-releasing factor (HRF), the yeast two-hybrid system was used to screen interacting molecules . We isolated cDNA clones coding for TCTP/HRF, suggesting that it may have a self-interacting property . Domain mapping of the interaction revealed that the C-terminal region of residue 126-172 is involved in self-interaction . The self-interacting property of TCTP/HRF was further supported by FPLC gel-filtration chromatography and coimmunoprecipitation analysis from transfected COS-7 cells . Our data suggests that TCTP/HRF may have a potential to self-interact through the C-terminal region, and the self-interaction property may be related to its biological function.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1998 Jun, 20(3), 173 - 8
{Screening of proteins interact with FMR1 by yeast two-hybrid system}; Chen Y et al.; OBJECTIVE: To establish yeast two-hybrid system for screening of protein(s) interacted with the fragile X metal retardation protein (FMRP) . METHODS: Fragment of exon 11 to 15 of the FMR1 cDNA was recombined with DNA-binding domain of the pBTM116 vector as bait to screen a mouse embryo cDNA library . RESULTS: Thirteen clones were confirmed to be able to specifically interact with FMRP bait . Sequence analysis showed that 12 clones are overlapping ones containing cDNA fragments of the mouse ubiquitin-conjugating enzyme gene (mUBC9) . CONCLUSIONS: The interaction between UBC9 and FMRP is supported both by similarity search of the amino acid sequences of the mouse and human UBC9 and by expression characteristics of the two proteins . The biological significance of the interaction is to be further studied.

Nucleic Acids Res, 2001 May 15, 29(10), 2106 - 16
Role of the ITS2-proximal stem and evidence for indirect recognition of processing sites in pre-rRNA processing in yeast; Cote CA et al.; Eucaryotic ribosome biogenesis involves many cis-acting sequences and trans-acting factors, including snoRNAS: We have used directed mutagenesis of rDNA plasmids in yeast to identify critical sequence and structural elements within and flanking the ITS2-proximal stem . This base paired structure, present in the mature ribosome, is formed between the 5'-end of 25S and the 3'-end of 5.8S rRNAS: Previously we demonstrated that formation of this structure was critical for pre-rRNA processing in yeast . Here we show that there are no sequence-specific recognition elements within the ITS2-proximal stem, rather the structure of this stem is critical for processing . This stem cannot exceed a specific length, but there are different length restrictions for different regions within this tripartite stem . Neither the conserved unpaired nucleotides within the stem nor the sequence of the mature rRNA at the processing sites are required for processing . Collectively, these results suggest a measuring model whereby initial cleavage within ITS2 at the C2 processing site and termination of subsequent exonuclease activity yielding the mature termini are affected by the relative position of sequence and structural elements within the ITS2-proximal stem.

Treatmentupdate, 1998 May, 10(3), 2 - 3
Itraconazole solution for oral yeast infections; The tetratricopeptide repeat domains of human et al.; Vienna Biocenter, Institut fur Biochemie und Molekulare Zellbiologie der Universitat Wien and Ludwig Boltzmann-Forschungsstelle fur Biochemie, AustriaIn the yeast Saccharomyces cerevisiae, beta-oxidation of fatty acids is compartmentalised in peroxisomes . Most yeast peroxisomal matrix proteins contain a type 1C-terminal peroxisomal targeting signal (PTS1) consisting of the tripeptide SKL or a conservative variant thereof . PTS1-terminated proteins are imported by Pex5p, which interacts with the targeting signal via a tetratricopeptide repeat (TPR) domain . Yeast cells devoid of Pex5p are unable to import PTS1-containing proteins and cannot degrade fatty acids . Here, the PEX5-TPR domains from human, tobacco, and nematode were inserted into a TPR-less yeast Pex5p construct to generate Pex5p chimaeras . These hybrid proteins were examined for functional complementation of the pex5delta mutant phenotype . Expression of the Pex5p chimaeras in pex5delta mutant cells restored peroxisomal import of PTS1-terminated proteins . Chimaera expression also re-established degradation of oleic acid, allowing growth on this fatty acid as a sole carbon source . We conclude that, in the context of Pex5p chimaeras, the human, tobacco, and nematode Pex5p-TPR domains are functionally interchangeable with the native domain for the peroxisomal import of yeast proteins terminating with canonical PTS1s . Non-conserved yeast PTS1s, such as HRL and HKL, did not interact with the tobacco PEX5-TPR domain in the two-hybrid system . HRL occurs at the C-terminus of the peroxisomal protein Eci1p, which is required for growth on unsaturated fatty acids . Although mutant pex5delta cells expressing a yeast/tobacco Pex5p chimaera failed to import a GFP-Eci1p reporter protein, they were able to grow on oleic acid . We reason that this is due to a cryptic PTS in native Eci1p that can function in a redundant system with the C-terminal HRL.

Mol Biol Cell, 2001 May, 12(5), 1303 - 14
Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast; Zhang Y et al.; Membrane and secretory proteins fold in the endoplasmic reticulum (ER), and misfolded proteins may be retained and targeted for ER-associated protein degradation (ERAD) . To elucidate the mechanism by which an integral membrane protein in the ER is degraded, we studied the fate of the cystic fibrosis transmembrane conductance regulator (CFTR) in the yeast Saccharomyces cerevisiae . Our data indicate that CFTR resides in the ER and is stabilized in strains defective for proteasome activity or deleted for the ubiquitin-conjugating enzymes Ubc6p and Ubc7p, thus demonstrating that CFTR is a bona fide ERAD substrate in yeast . We also found that heat shock protein 70 (Hsp70), although not required for the degradation of soluble lumenal ERAD substrates, is required to facilitate CFTR turnover . Conversely, calnexin and binding protein (BiP), which are required for the proteolysis of ER lumenal proteins in both yeast and mammals, are dispensable for the degradation of CFTR, suggesting unique mechanisms for the disposal of at least some soluble and integral membrane ERAD substrates in yeast.

Mol Biol Cell, 2001 May, 12(5), 1257 - 74
Regulation of initiation of S phase, replication checkpoint signaling, and maintenance of mitotic chromosome structures during S phase by Hsk1 kinase in the fission yeast; Takeda T et al.; Hsk1, Saccharomyces cerevisiae Cdc7-related kinase in Shizosaccharomyces pombe, is required for G1/S transition and its kinase activity is controlled by the regulatory subunit Dfp1/Him1 . Analyses of a newly isolated temperature-sensitive mutant, hsk1-89, reveal that Hsk1 plays crucial roles in DNA replication checkpoint signaling and maintenance of proper chromatin structures during mitotic S phase through regulating the functions of Rad3 (ATM)-Cds1 and Rad21 (cohesin), respectively, in addition to expected essential roles for initiation of mitotic DNA replication through phosphorylating Cdc19 (Mcm2) . Checkpoint defect in hsk1-89 is indicated by accumulation of cut cells at 30 degrees C . hsk1-89 displays synthetic lethality in combination with rad3 deletion, indicating that survival of hsk1-89 depends on Rad3-dependent checkpoint pathway . Cds1 kinase activation, which normally occurs in response to early S phase arrest by nucleotide deprivation, is largely impaired in hsk1-89 . Furthermore, Cds1-dependent hyperphosphorylation of Dfp1 in response to hydroxyurea arrest is eliminated in hsk1-89, suggesting that sufficient activation of Hsk1-Dfp1 kinase is required for S phase entry and replication checkpoint signaling . hsk1-89 displays apparent defect in mitosis at 37 degrees C leading to accumulation of cells with near 2C DNA content and with aberrant nuclear structures . These phenotypes are similar to those of rad21-K1 and are significantly enhanced in a hsk1-89 rad21-K1 double mutant . Consistent with essential roles of Rad21 as a component for the cohesin complex, sister chromatid cohesion is partially impaired in hsk1-89, suggesting a possibility that infrequent origin firing of the mutant may affect the cohesin functions during S phase.

Mol Biol Cell, 2001 May, 12(5), 1239 - 55
Isolation and characterization of effector-loop mutants of CDC42 in yeast; Gladfelter AS et al.; The highly conserved small GTPase Cdc42p is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells . Multiple effectors of Cdc42p have been identified, although it is unclear how their activities are coordinated to produce particular cell behaviors . One strategy used to address the contributions made by different effector pathways downstream of small GTPases has been the use of "effector-loop" mutants of the GTPase that selectively impair only a subset of effector pathways . We now report the generation and preliminary characterization of a set of effector-loop mutants of Saccharomyces cerevisiae CDC42 . These mutants define genetically separable pathways influencing actin or septin organization . We have characterized the phenotypic defects of these mutants and the binding defects of the encoded proteins to known yeast Cdc42p effectors in vitro . The results suggest that these effectors cannot account for the observed phenotypes, and therefore that unknown effectors exist that affect both actin and septin organization . The availability of partial function alleles of CDC42 in a genetically tractable system serves as a useful starting point for genetic approaches to identify such novel effectors.

Mol Biol Cell, 2001 May, 12(5), 1215 - 26
Yeast rab GTPase-activating protein Gyp1p localizes to the Golgi apparatus and is a negative regulator of Ypt1p; Du LL et al.; A family of related proteins in yeast Saccharomyces cerevisiae is known to have in vitro GTPase-activating protein activity on the Rab GTPases . However, their in vivo function remains obscure . One of them, Gyp1p, acts on Sec4p, Ypt1p, Ypt7p, and Ypt51p in vitro . Here, we present data to reveal its in vivo substrate and the role that it plays in the function of the Rab GTPase . Red fluorescent protein-tagged Gyp1p is concentrated on cytoplasmic punctate structures that largely colocalize with a cis-Golgi marker . Subcellular fractionation of a yeast lysate confirmed that Gyp1p is peripherally associated with membranes and that it cofractionates with Golgi markers . This localization suggests that Gyp1p may only act on Rab GTPases on the Golgi . A gyp1Delta strain displays a growth defect on synthetic medium at 37 degrees C . Overexpression of Ypt1p, but not other Rab GTPases, strongly inhibits the growth of gyp1Delta cells . Conversely, a partial loss-of-function allele of YPT1, ypt1-2, can suppress the growth defect of gyp1Delta cells . Furthermore, deletion of GYP1 can partially suppress growth defects associated with mutants in subunits of transport protein particle complex, a complex that catalyzes nucleotide exchange on Ypt1p . These results establish that Gyp1p functions on the Golgi as a negative regulator of Ypt1p.

Genes Dev, 2001 May 15, 15(10), 1217 - 28
Yak1p, a DYRK family kinase, translocates to the nucleus and phosphorylates yeast Pop2p in response to a glucose signal; Moriya H et al.; POP2 protein of Saccharomyces cerevisiae is a component of a protein complex that regulates the transcription of many genes . We found that the 97th threonine residue (Thr 97) of Pop2p was phosphorylated upon glucose limitation . The Thr 97 phosphorylation occurred within 2 min after removing glucose and was reversed within 1 min after the readdition of glucose . The effects of hexokinase mutations and glucose analogs indicate that this phosphorylation is dependent on glucose phosphorylating activity . We purified a protein kinase that phosphorylates a peptide containing Thr 97 of Pop2p and identified it as Yak1p, a DYRK family kinase . Phosphorylation of Pop2p was barely detectable in a yak1Delta strain . We found that Yak1p interacted with Bmh1p and Bmh2p only in the presence of glucose . A GFP-Yak1p fusion protein shuttled rapidly between the nucleus and the cytoplasm in response to glucose . A strain with alanine substituted for Thr 97 in Pop2p showed overgrowth in the postdiauxic transition and failed to stop the cell cycle at G(1) phase in response to glucose deprivation . Thus, Yak1p and Pop2p are part of a novel glucose-sensing system in yeast that is involved in growth control in response to glucose availability.

Cancer Res, 2001 May 15, 61(10), 4175 - 83
Novel inhibitors of poly(ADP-ribose) polymerase/PARP1 and PARP2 identified using a cell-based screen in yeast; Perkins E et al.; Multicellular organisms must have means of preserving their genomic integrity or face catastrophic consequences such as uncontrolled cell proliferation or massive cell death . One response is a modification of nuclear proteins by the addition and removal of polymers of ADP-ribose that modulate the properties of DNA-binding proteins involved in DNA repair and metabolism . These ADP-ribose units are added by poly(ADP-ribose) polymerase (PARP) and removed by poly(ADP-ribose) glycohydrolase . Although budding yeast Saccharomyces cerevisiae does not possess proteins with significant sequence similarity to the human PARP family of proteins, we identified novel small molecule inhibitors against two family members, PARP1 and PARP2, using a cell-based assay in yeast . The assay was based on the reversal of growth inhibition caused by the heterologous expression of either PARP1 or PARP2 . Validation of the assay was achieved by showing that the growth inhibition was relieved by a mutation in a single residue in the catalytic site of PARP1 or PARP2 or exposure of yeast to a known PARP1 inhibitor, 6(5H)-phenanthridinone . In separate experiments, when a putative protein regulator of PARP activity, human poly(ADP-ribose) glycohydrolase, was coexpressed with PARP1 or PARP2, yeast growth was restored . Finally, the inhibitors identified by screening the yeast assay are active in a mammalian PARP biochemical assay and inhibit PARP1 and PARP2 activity in yeast cell extracts . Thus, our data reflect the strength of using yeast to identify small molecule inhibitors of therapeutically relevant gene families, including those that are not found in yeast, such as PARP . The resultant inhibitors have two critical uses (a) as leads for drug development and (b) as tools to dissect cellular function.

J Mol Med, 2001 Apr, 79(2-3), 121 - 7
Identification and characterization of trypanocides by functional expression of an adenosine transporter from Trypanosoma brucei in yeast; Maser P et al.; The causative agents of sleeping sickness, Trypanosoma brucei rhodesiense and T . brucei gambiense, do not synthesize purines de novo but salvage purine bases and nucleosides from their hosts . We used yeast as an expression system for functional characterization of the trypanosomal adenosine transporter TbAT1 . A selection of purine analogs and flavonoids were tested for their ability to interfere with adenosine transport, with the aims of identifying (a) trypanocidal TbAT1 substrates, and (b) inhibitors of trypanosomal purine transport . Cordycepin (3'-deoxyadenosine) was a TbAT1 substrate of high activity against T . brucei rhodesiense (IC50 0.2 nM) . Inhibitors of mammalian nucleoside transport were not active, while the flavonol silibinin was a potent, noncompetitive inhibitor of TbAT1-mediated adenosine transport in yeast . Silibinin also inhibited melarsen-induced lysis of bloodstream form trypanosomes . IC50 values to T . brucei rhodesiense and to human carcinoma cells were 0.6 and 140 microM, respectively, indicating a good selectivity towards the parasites . Further studies are necessary to elucidate the effects of flavonoids on trypanosomal purine transport and their potential as trypanocides.

Folia Microbiol (Praha), 2000, 45(5), 429 - 33
The respiratory complex I in yeast: isolation of a gene NUO51 coding for the nucleotide-binding subunit of NADH:ubiquinone oxidoreductase from the obligately aerobic yeast Yarrowia lipolytica; Rycovska A et al.; We have isolated a gene NUO51 coding for a homologue of the nucleotide-binding subunit of mitochondrial respiratory chain linked NADH:ubiquinone oxidoreductase from the obligately aerobic yeast Yarrowia lipolytica . DNA sequencing revealed a 1464 bp open reading frame encoding a protein with predicted molar mass of about 53.7 kDa . The sequence is highly conserved with its counterparts from filamentous fungi and represents the first yeast homologue of the NADH-binding subunit (51 kDa) of the respiratory complex 1 . In addition, PFGE and Southern hybridization analysis indicate that NUO51 is a single copy gene in the genome of Y . lipolytica . The expression of NUO51 by Northern blot analysis was also examined.

FEMS Microbiol Lett, 2001 May 15, 199(1), 97 - 102
Cloning of the Aspergillus niger pmrA gene, a homologue of yeast PMR1, and characterization of a pmrA null mutant; Yang J et al.; The pmrA gene, a yeast PMR1 homologue, was isolated from Aspergillus niger . Sequence analysis of the pmrA cDNA and the genomic DNA revealed that two introns exist in the coding region, and that an open reading frame in the cDNA encodes a polypeptide of 1056 amino acids containing all the conserved regions present in P-type Ca2+-ATPases . The predicted pmrA protein exhibited a high degree of sequence similarity to the Pmr1 proteins from yeasts and mammalians (50-59% identity) . The expression of the pmrA cDNA partially restored the growth defect of Yarrowia lipolytica pmr1 null mutant on EGTA-containing medium . This indicates that the A . niger pmrA gene encodes a functional homologue of the yeast P-type Ca2+-ATPase involved in the secretory pathway . An A . niger pmrA null mutant exhibited growth retardation on EGTA-containing medium and the growth defect was overcome by adding Ca2+ or Mn2+ into the medium . This suggests an involvement of the pmrA protein in Ca2+ and Mn2+ homeostasis in A . niger.

Mol Cells, 2001 Apr 30, 11(2), 129 - 36
The yeast mediator; Bjorklund S et al.; The large number of signaling pathways and regulatory proteins that affect transcription highlights a need for funneling of information since transcription of all protein encoding genes is executed by the same set of general transcription factors and RNA polymerase II . This demand is met by large protein complexes such as Mediator that interact with the basic RNA polymerase II machinery and thus adds diversity simply by increasing the surface that is exposed to the incoming signals . The recent description of Mediator-like complexes in metazoans identifies it as a key player in transcriptional regulation.

Biotechniques, 2001 May, 30(5), 984 - 8
Rapid selection against truncation mutants in yeast reverse two-hybrid screens; Puthalakath H et al.; The yeast reverse two-hybrid system is a powerful technique for isolating mutations in a protein that abolish its interaction with a known partner . Selection is based on abrogation of growth suppression imposed when wild-type interactions confer 5-fluoroorotic acid (5-FOA) sensitivity to yeast cells . A laborious component of this system is to eliminate those mutations that cause protein truncation . By fusing the green fluorescent protein (GFP) to the C-terminus of a protein of interest, dynein light chain (LC8), we were able to rapidly isolate mutations that did not result in protein truncation.

Biotechniques, 2001 May, 30(5), 1000 - 4
Streamlined beta-galactosidase assay for analysis of recombinant yeast response to estrogens; Le Guevel R et al.; Here, we describe a rapid, convenient, and quantitative beta-galactosidase assay in liquid culture of recombinant yeast that expresses the estrogen receptor . This assay allows large-scale screening of chemicals (more than 600 samples/day) for the evaluation of their direct estrogenic potency and accurate determination of their EC50 with minimal manipulations . The assay, which is based on digestion of the yeast cell wall by lyticase (zymolase), a beta-glucanase isolated from Arthrobacter luteus, followed by a hypoosmotic shock lysis, is performed completely in 96-well plates . This protocol for using recombinant yeast with the two-hybrid technology significantly advances recombinant yeast manipulation.

Bioorg Med Chem, 2001 Apr, 9(4), 955 - 60
Malassezin--A novel agonist of the arylhydrocarbon receptor from the yeast Malassezia furfur; Wille G et al.; The yeast Malassezia furfur converts tryptophan into several indole compounds . One of these, malassezin, was identified as 2-(1H-indol-3-ylmethyl)-1H-indole-3-carbaldehyde (1) . It was synthesized from N-Boc-indole-3-carbaldehyde in five steps with 12% overall yield . The compound easily cyclizes to indolo{3,2-b}carbazole (7) which is known to interact with the arylhydrocarbon receptor (AHR) . Similarly, malassezin was found to induce cytochrome P450 as an agonist of AHR (EC50 = 1.57 microM) in rat hepatocytes.

J Biol Chem, 2001 Jul 13, 276(28), 26708 - 14 Epub 2001 May 15.
Overexpression of type I topoisomerases sensitizes yeast cells to DNA damage; Nitiss JL et al.; DNA topoisomerases play essential roles in many DNA metabolic processes . It has been suggested that topoisomerases play an essential role in DNA repair . Topoisomerases can introduce DNA damage upon exposure to drugs that stabilize the covalent protein-DNA intermediate of the topoisomerase reaction . Lesions in DNA are also able to trap topoisomerase-DNA intermediates, suggesting that topoisomerases have the potential to either assist in DNA repair by locating sites of damage or exacerbating DNA damage by generation of additional damage at the site of a lesion . We have shown that overexpression of yeast topoisomerase I (TOP1) conferred hypersensitivity to methyl methanesulfonate and other DNA-damaging agents, whereas expression of a catalytically inactive enzyme did not . Overexpression of topoisomerase II did not change the sensitivity of cells to these DNA-damaging agents . Yeast cells lacking TOP1 were not more resistant to DNA damage than cells expressing wild type levels of the enzyme . Yeast topoisomerase I covalent complexes can be trapped efficiently on UV-damaged DNA . We suggest that TOP1 does not participate in the repair of DNA damage in yeast cells . However, the enzyme has the potential of exacerbating DNA damage by forming covalent DNA-protein complexes at sites of DNA damage.

Plant Mol Biol, 2001 Mar, 45(4), 489 - 500
Tobacco and Arabidiopsis SLT1 mediate salt tolerance of yeast; Matsumoto TK et al.; A tobacco cDNA (NtSLT1, for Nicotiana tabacum sodium- and lithium-tolerant) was isolated by functional complementation of the salt-sensitive phenotype of a calcineurin (CaN)-deficient yeast mutant (cnb delta, regulatory subunit null) . CaN is a Ca2+/calmodulin-dependent type 2B protein phosphatase that regulates Na+ homeostasis in yeast . This phosphatase modulates plasma membrane K+/Na+ selectivity through the activation of high-affinity K+ transport, and increaseses extracellular Na+ efflux by activation and transcriptional induction of the Na+/Li+ translocating P-type ATPase encoded by ENA1 . Expression of N-terminally truncated NtSLT1 (Met-304), but not full-length protein, suppressed salt sensitivity of cnb1 . Truncated NtSLT1 also increased salt tolerance of wild-type yeast, indicating functional sufficiency . NtSLT1 encodes a protein of yet unknown function but experimentation in yeast confirms it as a salt tolerance determinant . The Arabidopsis thaliana orthologue, AtSLT1, also suppressed salt sensitivity of cnb delta but only when expressed without the N-terminus (Met-301), suggesting that this region of the proteins from these evolutionarily diverse plant species contains an autoinhibitory domain . NtSLT1 enhanced transcription of the CaN-dependent ENA1 gene promoter and compensated the salt sensitivity of a mutant deficient in TCN1--a transcription factor that is activated by CaN and then induces ENA1 expression . NtSLT1 partially suppressed the salt sensitivity of ena1-4 indicating that NtSLT1 has both ENA-dependent and independent functions . NtSLT1 suppressed spk1 hal4 (SPK1/HAL4 which encodes a serine-threonine kinase that regulates TRK1-2 transporters to have high K+/Na+ selectivity) but not ena1-4 trk1-2 implicating the ENA-independent function to be through TRK1-2 . Together, these results implicate SLT1 as a signal regulatory molecule that mediates salt tolerance by modulating Na+ homeostasis.

Plant Mol Biol, 2001 Mar, 45(4), 469 - 76
Identification and functional expression in yeast of a prenylcysteine alpha-carboxyl methyltransferase gene from Arabidopsis thaliana; Crowell DN et al.; Most isoprenylated proteins are alpha-carboxyl-methylated . However, despite numerous studies linking protein isoprenylation in plants to cell cycle control, meristem development, and phytohormone signaling, alpha-carboxyl methylation of isoprenylated plant proteins has not been characterized in detail . Here, we report the cloning of a prenylcysteine alpha-carboxyl methyltransferase gene (AtSTE14) from Arabidopsis thaliana . AtSTE14 restores fertility and enzymatic activity to a ste14 mutant of Saccharomyces cerevisiae, confirming its identity as a bona fide prenylcysteine alpha-carboxyl methyltransferase gene . Furthermore, the presence of AtSTE14 transcripts in various Arabidopsis organs suggests a ubiquitous role for the AtSTE14 protein in plant growth and development . These results demonstrate that Arabidopsis thaliana possesses a functional prenylcysteine alpha-carboxyl methyltransferase involved in post-isoprenylation protein processing.

Plant Mol Biol, 2001 Mar, 45(4), 437 - 48
Two iron-regulated cation transporters from tomato complement metal uptake-deficient yeast mutants; Eckhardt U et al.; Although iron deficiency poses severe nutritional problems to crop plants, to date iron transporters have only been characterized from the model plant Arabidopsis thaliana . To extend our molecular knowledge of Fe transport in crop plants, we have isolated two cDNAs (LeIRT1 and LeIRT2) from a library constructed from roots of iron-deficient tomato (Lycopersicon esculentum) plants, using the Arabidopsis iron transporter cDNA, IRTI, as a probe . Their deduced polypeptides display 64% and 62% identical amino acid residues to the IRT1 protein, respectively . Transcript level analyses revealed that both genes were predominantly expressed in roots . Transcription of LeIRT2 was unaffected by the iron status of the plant, while expression of LeIRT1 was strongly enhanced by iron limitation . The growth defect of an iron uptake-deficient yeast (Saccharomyces cerevisiae) mutant was complemented by LeIRT1 and LeIRT2 when ligated to a yeast expression plasmid . Transport assays revealed that iron uptake was restored in the transformed yeast cells . This uptake was temperature-dependent and saturable, and Fe2+ rather than Fe3+ was the preferred substrate . A number of divalent metal ions inhibited Fe2+ uptake when supplied at 100-fold or 10-fold excess . Manganese, zinc and copper uptake-deficient yeast mutants were also rescued by the two tomato cDNAs, suggesting that their gene products have a broad substrate range . The gene structure was determined by polymerase chain reaction experiments and, surprisingly, both genes are arranged in tandem with a tail-to-tail orientation.

J Biol Chem, 2001 May 18, 276(20), 17591 - 6 Epub 2001 Feb 28.
Budding yeast GCN1 binds the GI domain to activate the eIF2alpha kinase GCN2; Kubota H et al.; When starved for a single amino acid, the budding yeast Saccharomyces cerevisiae activates the eukaryotic initiation factor 2alpha (eIF2alpha) kinase GCN2 in a GCN1-dependent manner . Phosphorylated eIF2alpha inhibits general translation but selectively derepresses the synthesis of the transcription factor GCN4, which leads to coordinated induction of genes involved in biosynthesis of various amino acids, a phenomenon called general control response . We recently demonstrated that this response requires binding of GCN1 to the GI domain occurring at the N terminus of GCN2 (Kubota, H., Sakaki, Y., and Ito, T . (2000) J . Biol . Chem . 275, 20243-20246) . Here we provide the first evidence for the involvement of GCN1-GCN2 interaction in activation of GCN2 per se . We identified a C-terminal segment of GCN1 sufficient to bind the GI domain and used a novel dual bait two-hybrid method to identify mutations rendering GCN1 incapable of interacting with GCN2 . The yeast bearing such an allele, gcn1-F2291L, fails to display derepression of GCN4 translation and hence general control response, as does a GI domain mutant, gcn2-Y74A, defective in association with GCN1 . Furthermore, we demonstrated that phosphorylation of eIF2alpha is impaired in both mutants . Since GCN2 is the sole eIF2alpha kinase in yeast, these findings indicate a critical role of GCN1-GCN2 interaction in activation of the kinase in vivo.

Traffic, 2001 May, 2(5), 297 - 302
Clathrin function in yeast endocytosis; Baggett JJ et al.; The process of endocytosis is a complex series of events involving the coordinated activity of many proteins . In animal cells, clathrin plays a vital role in the invagination of the plasma membrane leading to formation of vesicles during endocytosis . The study of endocytosis in yeast cells has been hindered by a debate about the role of clathrin in early internalization steps . This review summarizes the evidence for and against clathrin's involvement in internalization from the yeast plasma membrane.

Biochem Biophys Res Commun, 2001 May 18, 283(4), 876 - 82
A distinct subnuclear localization of mammalian DNA topoisomerase IIbeta in yeast; Sakaguchi A et al.; Mammalian topoisomerase II isoforms alpha and beta are diverged in their C-terminal domain (CTD), but both isoforms complement the yeast top2 mutation . In this study, mammalian topoisomerase IIalpha-CTD and IIbeta-CTD were tagged with yellow fluorescent protein (YFP), expressed in yeast cells, and their localization was examined . YFP tagged-topoisomerase IIalpha-CTD was distributed evenly throughout the nucleus, while YFP tagged-topoisomerase IIbeta-CTD was sequestered into a subnuclear compartment . Deletion analysis revealed that two regions (amino acids 1207-1234 and 1513-1573) of the topoisomerase IIbeta-CTD are essential for specific localization of the beta isoform: if either of the two regions is removed, the mutant topoisomerase IIbeta-CTD distributes evenly throughout the nucleus . The data suggest that yeast cells distinguish the nuclear and subnuclear localization signals associated with these two mammalian topoisomerase II isoforms .

Science, 2001 May 11, 292(5519), 1171 - 5
Pot1, the putative telomere end-binding protein in fission yeast and humans; Baumann P et al.; Telomere proteins from ciliated protozoa bind to the single-stranded G-rich DNA extensions at the ends of macronuclear chromosomes . We have now identified homologous proteins in fission yeast and in humans . These Pot1 (protection of telomeres) proteins each bind the G-rich strand of their own telomeric repeat sequence, consistent with a direct role in protecting chromosome ends . Deletion of the fission yeast pot1+ gene has an immediate effect on chromosome stability, causing rapid loss of telomeric DNA and chromosome circularization . It now appears that the protein that caps the ends of chromosomes is widely dispersed throughout the eukaryotic kingdom.

J Biol Chem, 2001 Jul 27, 276(30), 28598 - 605 Epub 2001 May 10.
Functional analysis of conserved structural elements in yeast syntaxin Vam3p; Wang Y et al.; Vam3p, a syntaxin-like SNARE protein involved in yeast vacuole fusion, is composed of a three-helical N-terminal domain, a canonical SNARE motif, and a C-terminal transmembrane region (TMR) . Surprisingly, we find that the N-terminal domain of Vam3p is not essential for fusion, although analogous domains in other syntaxins are indispensible for fusion and/or protein-protein interactions . In contrast to the N-terminal domain, mutations in the SNARE motif of Vam3p or replacement of the SNARE motif of Vam3p with the SNARE motif from other syntaxins inhibited fusion . Furthermore, the precise distance between the SNARE motif and the TMR was critical for fusion . Insertion of only three residues after the SNARE motif significantly impaired fusion and insertion of 12 residues abolished fusion . As judged by co-immunoprecipitation experiments, the SNARE motif mutations and the insertions did not alter the association of Vam3p with Vam7p, Vti1p, Nyv1p, and Ykt6p, other vacuolar SNARE proteins implicated in fusion . In contrast, the SNARE motif substitutions interfered with the stable formation of Vam3p complexes with Nyv1p and Vti1p, although Vam3p complexes with Vam7p and Ykt6p were still present . Our data suggest that in contrast to previously characterized syntaxins, Vam3p contains only two domains essential for fusion, the SNARE motif and the TMR, and these domains have to be closely coupled to function in fusion.

Mech Ageing Dev, 2001 Jul 15, 122(9), 865 - 82
New clues to old yeast; Jazwinski SM; The yeast Saccharomyces cerevisiae has been used as an experimental model for the genetic and molecular dissecton of the aging process for the past decade . This period has seen the implication of some 30 genes in yeast aging . These genes encode a wide array of biochemical functions, suggesting the participation of multiple molecular mechanisms of aging . However, four principles appear to be at play: metabolism, stress resistance, gene dysregulation, and genetic instability . They unite the broad physiological aspects of yeast aging with those in other species . Genes and environment are not the only players; stochastic change also appears important in determining life span . This element of chance provides opportunities for an integrative approach, which is beginning to appear in yeast aging research.

Folia Microbiol (Praha), 2000, 45(4), 339 - 42
Anticancer drugs as inducers of thermotolerance in yeast; Miligkos V et al.; Yeast cell viability was evaluated microscopically following exposure to heat shock for 30 min at 53 degrees C . The cells were previously grown in the presence of potential stressors (anticancer drugs; e.g., 5-fluorouracil, methotrexate, cisplatin, bleomycin, mitomycin-C and camptothecin-11) . The induction of thermotolerance was documented by significantly increased viability after heat shock . This effect, which was reversed by cycloheximide, was comparable to that observed following exposure to a mild heat stress . These data demonstrate that pretreatment with sub-toxic concentrations of some of the clinically used antineoplastic agents conferres thermotolerance to yeast, possibly through the synthesis of protein components.

Proc Natl Acad Sci U S A, 2001 May 8, 98(10), 5625 - 30
Rck2, a member of the calmodulin-protein kinase family, links protein synthesis to high osmolarity MAP kinase signaling in budding yeast; Teige M et al.; Rck2, a yeast Ser/Thr protein kinase homologous to mammalian calmodulin kinases, requires phosphorylation for activation . We provide evidence that in budding yeast, this step can be executed by the osmostress-activated mitogen-activated protein kinase Hog1 . Rck2 phosphorylation was transiently increased during osmostress or in mutants with a hyperactive high osmolarity glycerol (HOG) pathway . This modification depended on catalytically active Hog1 kinase and two putative mitogen-activated protein kinase phosphorylation sites in Rck2 . Immunokinase assays showed that Hog1 can directly phosphorylate Rck2 to stimulate its enzymatic activity toward translation elongation factor 2 . We demonstrate that Hog1 and Rck2 are necessary for attenuation of protein synthesis in response to osmotic challenge and show that modification of elongation factor 2 induced by osmostress depends on Rck2 and Hog1 in vivo . Therefore, we propose that the transient down-regulation of protein synthesis after osmotic shock is a response not to damage but to an extracellular signal mediated by Hog1 and Rck2.

J Biol Chem, 2001 Jul 13, 276(28), 26189 - 96 Epub 2001 May 08.
Essential role of Sna41/Cdc45 in loading of DNA polymerase alpha onto minichromosome maintenance proteins in fission yeast; Uchiyama M et al.; Assembly of replication complexes at the replication origins is strictly regulated . Cdc45p is known to be a part of the active replication complexes . In Xenopus egg extracts, Cdc45p was shown to be required for loading of DNA polymerase alpha onto chromatin . The fission yeast cdc45 homologue was identified as a suppressor for nda4 and named sna41 . Nevertheless, it is not known how Cdc45p facilitates loading of DNA polymerase alpha onto chromatin, particularly to prereplicative complexes . To gain novel insight into the function of this protein in fission yeast, we characterized the fission yeast Cdc45 homologue, Sna41p . We have constructed C-terminally epitope-tagged Sna41p and Pol alpha p and replaced the endogenous genes with the corresponding tagged genes . Analyses of protein-protein interactions in vivo by the use of these tagged strains revealed the following: Sna41p interacts with Pol alpha p throughout the cell cycle, whereas it interacts with Mis5p/Mcm6p in the chromatin fractions at the G(1)-S boundary through S phase . In an initiation-defective sna41 mutant, sna41(goa1), interaction of Pol alpha p with Mis5p is not observed, although Pol alpha p loading onto the chromatin that occurs before G(1) START is not affected . These results show that fission yeast Sna41p facilitates the loading of Pol alpha p onto minichromosome maintenance proteins . Our results are consistent with a model in which loading of Pol alpha p onto replication origins occurs through two steps, namely, loading onto chromatin at preSTART and association with prereplicative complexes at G(1)-S through Sna41p, which interacts with minichromosome maintenance proteins in a cell cycle-dependent manner.

Curr Opin Cell Biol, 2001 Jun, 13(3), 349 - 55
Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals; Patil C et al.; Cellular survival of endoplasmic reticulum stress requires the unfolded protein response (UPR), a stress response first elucidated genetically in yeast . While we continue to refine our knowledge of the yeast system, especially the breadth and significance of the transcriptional response, conservation of the system's elements has allowed identification of corresponding and additional components of the mammalian UPR . Recent results reveal that the output of the mammalian UPR reaches beyond transcriptional regulation of secretory pathway components to control of general translation, the cell cycle and programmed cell death.

Nature, 2001 Jan 4, 409(6816), 109 - 13
Telomere looping permits gene activation by a downstream UAS in yeast; de Bruin D et al.; In yeast (Saccharomyces cerevisiae), transcriptional activators, such as Gal4 and Gal4-VP16, work ordinarily from sites located in the upstream activating sequence (UAS) positioned about 250 base pairs upstream of the transcription start site . In contrast to their behaviour in mammalian cells, however, such activators fail to work when positioned at distances greater than approximately 600-700 base pairs upstream, or anywhere downstream of the gene . Here we show that, in yeast, a gene bearing an enhancer positioned 1-2 kilobases downstream of the gene is activated if the reporter is linked to a telomere, but not if it is positioned at an internal chromosomal locus . These observations are explained by the finding that yeast telomeres form back-folding, or looped, structures . Because yeast telomeric regions resemble the heterochromatin found in higher eukaryotes, these findings might also explain why transcription of some higher eukaryotic genes depends on their location in heterochromatin.

Nucleic Acids Res, 2001 May 1, 29(9), 1884 - 91
Cloning the human and mouse MMS19 genes and functional complementation of a yeast mms19 deletion mutant; Queimado L et al.; The MMS19 gene of the yeast Saccharomyces cerevisiae encodes a polypeptide of unknown function which is required for both nucleotide excision repair (NER) and RNA polymerase II (RNAP II) transcription . Here we report the molecular cloning of human and mouse orthologs of the yeast MMS19 gene . Both human and Drosophila MMS19 cDNAs correct thermosensitive growth and sensitivity to killing by UV radiation in a yeast mutant deleted for the MMS19 gene, indicating functional conservation between the yeast and mammalian gene products . Alignment of the translated sequences of MMS19 from multiple eukaryotes, including mouse and human, revealed the presence of several conserved regions, including a HEAT repeat domain near the C-terminus . The presence of HEAT repeats, coupled with functional complementation of yeast mutant phenotypes by the orthologous protein from higher eukaryotes, suggests a role of Mms19 protein in the assembly of a multiprotein complex(es) required for NER and RNAP II transcription . Both the mouse and human genes are ubiquitously expressed as multiple transcripts, some of which appear to derive from alternative splicing . The ratio of different transcripts varies in several different tissue types.

Mol Cell Biol, 2001 Jun, 21(11), 3714 - 24
Xbp1-mediated repression of CLB gene expression contributes to the modifications of yeast cell morphology and cell cycle seen during nitrogen-limited growth; Miled C et al.; Yeast cells undergo morphological transformations in response to diverse environmental signals . One such event, called pseudohyphal differentiation, occurs when diploid yeast cells are partially starved for nitrogen on a solid agar medium . The nitrogen-starved cells elongate, and a small fraction form filaments that penetrate the agar surface . The molecular basis for the changes in cell morphology and cell cycle in response to nitrogen limitation are poorly defined, in part because the heterogeneous growth states of partially starved cells on agar media are not amenable to biochemical analysis . In this work, we used chemostat cultures to study the role of cell cycle regulators with respect to yeast differentiation in response to nitrogen limitation under controlled, homogeneous culture conditions . We found that Clb1, Clb2, and Clb5 cyclin levels are reduced in nitrogen-limited chemostat cultures compared to levels in rich-medium cultures, whereas the Xbp1 transcriptional repressor is highly induced under these conditions . Furthermore, the deletion of XBP1 prevents the drop in Clb2 levels and inhibits cellular elongation in nitrogen-limited chemostat cultures as well as inhibiting pseudohyphal growth on nitrogen-limited agar media . Deletion of the CLB2 gene restores an elongated morphology and filamentation to the xbp1Delta mutant in response to nitrogen limitation . Transcriptional activation of the XBP1 gene and the subsequent repression of CLB gene expression are thus key responses of yeast cells to nitrogen limitation.

Mikrobiologiia, 2001 Jan-Feb, 70(1), 29 - 33
{Level of cyclic AMP during induction of alternative oxidase in Yarrowia lipolytica yeast cells}; Medentsev AG et al.; The effect of cyanide, antimycin A, ethanol, and acetate on the induction of alternative oxidase in the yeast Yarrowia lipolytica VKM Y-155 sensitive to cyanide, in the presence of the aforementioned compounds led to the development of cyanide-resistant respiration, which could be suppressed by benzohydroxamic acid, an inhibitor of alternative oxidases . The incubation of cells with cyanide, ethanol, or acetate raised the intracellular pool of cAMP, which attained maximal values after a 2- to 3-min incubation, then rapidly decreased to the initial value and did not change over the next three hours of incubation . The possible role of cAMP in the induction of alternative oxidase in yeast cells is discussed.

Vestn Ross Akad Med Nauk, 2001, (2), 29 - 31
{The yeast malassezia on the skin of healthy individuals and patients with atopic dermatitis}; Arzumanian VG; The lipophilic yeast Malassezia spp . from the skin of 32 healthy individuals and 21 patients with atopic dermatitis was isolated and identified . Malassezia sympodialis was shown to be most common in both groups, M . Globosa was less frequently encountered . Moreover, no Malassezia was found in 34 and 53% of cases, respectively . There was a relationship of the skin colonization of Malassezia to the patients' age . Malassezia spp . cultures whose appurtenance was impossible to identified by the Guillot-Gueho method were detected.

J Biol Chem, 2001 Jul 13, 276(28), 26472 - 8 Epub 2001 May 03.
Identification of novel pheromone-response regulators through systematic overexpression of 120 protein kinases in yeast; Burchett SA et al.; Protein kinases are well known to transmit and regulate signaling pathways . To identify additional regulators of the pheromone signaling apparatus in yeast, we evaluated an array of 120 likely protein kinases encoded by the yeast genome . Each kinase was fused to glutathione S-transferase, overexpressed, and tested for changes in pheromone responsiveness in vivo . As expected, several known components of the pathway (YCK1, STE7, STE11, FUS3, and KSS1) impaired the growth arrest response . Seven other kinases also interfered with pheromone-induced growth arrest; in rank order they are as follows: YKL116c (renamed PRR1) = YDL214c (renamed PRR2) > YJL141c (YAK1, SRA1) > YNR047w = YCR091w (KIN82) = YIL095w (PRK1) > YCL024w (KCC4) . Inhibition of pheromone signaling by PRR1, but not PRR2, required the glutathione S-transferase moiety . Both kinases inhibited gene transcription after stimulation with pheromone, a constitutively active kinase mutant STE11-4, or overexpression of the transcription factor STE12 . Neither protein altered the ability of the mitogen-activated protein kinase (MAPK) Fus3 to feedback phosphorylate a known substrate, the MAPK kinase Ste7 . These results reveal two new components of the pheromone-signaling cascade in yeast, each acting at a point downstream of the MAPK.

J Biol Chem, 2001 Jul 13, 276(28), 25715 - 26 Epub 2001 May 03.
Requirement for yeast TAF145 function in transcriptional activation of the RPS5 promoter that depends on both core promoter structure and upstream activating sequences; Tsukihashi Y et al.; The general transcription factor TFIID has been shown to be involved in both core promoter recognition and the transcriptional activation of eukaryotic genes . We recently isolated TAF145 (one of TFIID subunits) temperature-sensitive mutants in yeast, in which transcription of the TUB2 gene is impaired at restrictive temperatures due to a defect in core promoter recognition . Here, we show in these mutants that the transcription of the RPS5 gene is impaired, mostly due to a defect in transcriptional activation rather than to a defect in core promoter recognition, although the latter is slightly affected as well . Surprisingly, the RPS5 core promoter can be activated by various activation domains fused to a GAL4 DNA binding domain, but not by the original upstream activating sequence (UAS) of the RPS5 gene . In addition, a heterologous CYC1 core promoter can be activated by RPS5-UAS at normal levels even in these mutants . These observations indicate that a distinct combination of core promoters and activators may exploit alternative activation pathways that vary in their requirement for TAF145 function . In addition, a particular function of TAF145 that is deleted in our mutants appears to be involved in both core promoter recognition and transcriptional activation.

J Biol Chem, 2001 Jul 20, 276(29), 26923 - 30 Epub 2001 May 03.
Yeast 1,3-beta-glucan synthase activity is inhibited by phytosphingosine localized to the endoplasmic reticulum; Abe M et al.; 1,3-beta-D-Glucan, a major filamentous component of the cell wall in the budding yeast Saccharomyces cerevisiae, is synthesized by 1,3-beta-glucan synthase (GS) . Although a yeast gene whose product is required for GS activity in vitro, GNS1, was isolated and characterized, its role in GS function has remained unknown . In the current study we show that Deltagns1 cells accumulate a non-competitive and non-proteinous inhibitor(s) in the membrane fraction . Investigations of inhibitory activity on GS revealed that the inhibitor(s) is mainly present in the sphingolipid fraction . It is shown that Deltagns1 cells contain phytosphingosine (PHS), an intermediate in the sphingolipid biosynthesis, 30-fold more than wild-type cells do . The membrane fraction isolated from Deltasur2 cells contains an increased amount of dihydrosphingosine (DHS) and also exhibits reduced GS activity . Among constituents of the sphingolipid fraction, PHS and DHS show striking inhibition in a non-competitive manner . The intracellular level of DHS is much lower than that of PHS in wild-type cells, suggesting that PHS is the primary inhibitor of GS in vivo . The localization of PHS to the endoplasmic reticulum in wild-type cells coincides with that of the inhibitor(s) in Deltagns1 cells . Taken together, our results indicate that PHS is a potent inhibitor of yeast GS in vivo.

Mol Cell, 2001 Apr, 7(4), 705 - 13
An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint; van Brabant AJ et al.; Checkpoint controls coordinate entry into mitosis with the completion of DNA replication . Depletion of nucleotide precursors by treatment with the drug hydroxyurea triggers such a checkpoint response . However, it is not clear whether the signal for this hydroxyurea-induced checkpoint pathway is the presence of unreplicated DNA, or rather the persistence of single-stranded or damaged DNA . In a yeast artificial chromosome (YAC) we have engineered an approximately 170 kb region lacking efficient replication origins that allows us to explore the specific effects of unreplicated DNA on cell cycle progression . Replication of this YAC extends the length of S phase and causes cells to engage an S/M checkpoint . In the absence of Rad9 the YAC becomes unstable, undergoing deletions within the origin-free region.

Mol Cell, 2001 Apr, 7(4), 695 - 704
Short telomeres in yeast are highly recombinogenic; McEachern MJ et al.; We report that recombination rates specifically increase by up to 10(3) near shortened telomeres in K . lactis cells . This occurs in cells lacking telomerase that undergo growth senescence as well as in cells with stably shortened telomeres that cause little effect on cell growth . The high rates of gene conversion allowed a subtelomeric marker, initially present at a single telomere, to efficiently spread to most or all other telomeres in the cell . We propose that short telomeres in K . lactis are not fully competent at capping chromosome ends and hence are occasionally processed by proteins that normally act to repair broken DNA ends through recombination . This helps explain how recombination can be frequent enough to permit maintenance of telomeres in yeast cells lacking telomerase.

FEBS Lett, 2001 Apr 27, 495(3), 148 - 53
A yeast two-hybrid study of human p97/Gab2 interactions with its SH2 domain-containing binding partners; Crouin C et al.; p97/Gab2 is a recently characterized member of a large family of scaffold proteins that play essential roles in signal transduction . Gab2 becomes tyrosine-phosphorylated in response to a variety of growth factors and forms multimolecular complexes with SH2 domain-containing signaling molecules such as the p85-regulatory subunit of the phosphoinositide-3-kinase (p85-PI3K), the tyrosine phosphatase SHP-2 and the adapter protein CrkL . To characterize the interactions between Gab2 and its SH2-containing binding partners, we designed a modified yeast two-hybrid system in which the Lyn tyrosine kinase is expressed in a regulated manner in yeast . Using this assay, we demonstrated that p97/Gab2 specifically interacts with the SH2 domains of PI3K, SHP-2 and CrkL . Interaction with p85-PI3K is mediated by tyrosine residues Y452, Y476 and Y584 of Gab2, while interaction with SHP-2 depends exclusively on tyrosine Y614 . CrkL interaction is mediated by its SH2 domain recognizing Y266 and Y293, despite the latter being in a non-consensus (YTFK) environment.

Genetics, 2001 May, 158(1), 187 - 96
Multiple functional interactions between components of the Lsm2-Lsm8 complex, U6 snRNA, and the yeast La protein; Pannone BK et al.; The U6 small nuclear ribonucleoprotein is a critical component of the eukaryotic spliceosome . The first protein that binds the U6 snRNA is the La protein, an abundant phosphoprotein that binds the 3' end of many nascent small RNAs . A complex of seven Sm-like proteins, Lsm2-Lsm8, also binds the 3' end of U6 snRNA . A mutation within the Sm motif of Lsm8p causes Saccharomyces cerevisiae cells to require the La protein Lhp1p to stabilize nascent U6 snRNA . Here we describe functional interactions between Lhp1p, the Lsm proteins, and U6 snRNA . LSM2 and LSM4, but not other LSM genes, act as allele-specific, low-copy suppressors of mutations in Lsm8p . Overexpression of LSM2 in the lsm8 mutant strain increases the levels of both Lsm8p and U6 snRNPs . In the presence of extra U6 snRNA genes, LSM8 becomes dispensable for growth, suggesting that the only essential function of LSM8 is in U6 RNA biogenesis or function . Furthermore, deletions of LSM5, LSM6, or LSM7 cause LHP1 to become required for growth . Our experiments are consistent with a model in which Lsm2p and Lsm4p contact Lsm8p in the Lsm2-Lsm8 ring and suggest that Lhp1p acts redundantly with the entire Lsm2-Lsm8 complex to stabilize nascent U6 snRNA.

Genetics, 2001 May, 158(1), 155 - 66
Expansions and contractions in 36-bp minisatellites by gene conversion in yeast; Paques F et al.; The instability of simple tandem repeats, such as human minisatellite loci, has been suggested to arise by gene conversions . In Saccharomyces cerevisiae, a double-strand break (DSB) was created by the HO endonuclease so that DNA polymerases associated with gap repair must traverse an artificial minisatellite of perfect 36-bp repeats or a yeast Y' minisatellite containing diverged 36-bp repeats . Gene conversions are frequently accompanied by changes in repeat number when the template contains perfect repeats . When the ends of the DSB have nonhomologous tails of 47 and 70 nucleotides that must be removed before repair DNA synthesis can begin, 16% of gene conversions had rearrangements, most of which were contractions, almost always in the recipient locus . When efficient removal of nonhomologous tails was prevented in rad1 and msh2 strains, repair was reduced 10-fold, but among survivors there was a 10-fold reduction in contractions . Half the remaining events were expansions . A similar decrease in the contraction rate was observed when the template was modified so that DSB ends were homologous to the template; and here, too, half of the remaining rearrangements were expansions . In this case, efficient repair does not require RAD1 and MSH2, consistent with our previous observations . In addition, without nonhomologous DSB ends, msh2 and rad1 mutations did not affect the frequency or the distribution of rearrangements . We conclude that the presence of nonhomologous ends alters the mechanism of DSB repair, likely through early recruitment of repair proteins including Msh2p and Rad1p, resulting in more frequent contractions of repeated sequences.

Genetics, 2001 May, 158(1), 87 - 93
Multiple functions of the nonconserved N-terminal domain of yeast TATA-binding protein; Lee M et al.; The TATA-binding protein (TBP) is composed of a highly conserved core domain sufficient for TATA-element binding and preinitiation complex formation as well as a highly divergent N-terminal region that is dispensable for yeast cell viability . In vitro, removal of the N-terminal region domain enhances TBP-TATA association and TBP dimerization . Here, we examine the effects of truncation of the N-terminal region in the context of yeast TBP mutants with specific defects in DNA binding and in interactions with various proteins . For a subset of mutations that disrupt DNA binding and the response to transcriptional activators, removal of the N-terminal domain rescues their transcriptional defects . By contrast, deletion of the N-terminal region is lethal in combination with mutations on a limited surface of TBP . Although this surface is important for interactions with TFIIA and Brf1, TBP interactions with these two factors do not appear to be responsible for this dependence on the N-terminal region . Our results suggest that the N-terminal region of TBP has at least two distinct functions in vivo . It inhibits the interaction of TBP with TATA elements, and it acts positively in combination with a specific region of the TBP core domain that presumably interacts with another protein(s).

RNA, 2001 Mar, 7(3), 374 - 81
A mammalian sequence-dependent upstream open reading frame mediates polyamine-regulated translation in yeast; Mize GJ et al.; In mammals, control of S-adenosylmethionine decarboxylase (AdoMetDC) translation is one component of a feedback network that regulates intracellular levels of the polyamines, spermidine, and spermine . AdoMetDC mRNA from mammals contains a highly conserved upstream open reading frame (uORF) within its leader sequence that confers polyamine-regulated suppression of translation on the associated downstream cistron . This regulation is mediated through an interaction that depends on the amino acid sequence of the uORF-encoded hexapeptide . It remains to be shown whether polyamines participate directly in this interaction or indirectly through a specialized signal transduction pathway . We show that Saccharomyces cerevisiae does not have a uORF associated with its AdoMetDC gene (SPE2) and that ribosome loading on the SPE2 mRNA is not positively influenced by polyamine depletion, as it is in mammalian cells . Nevertheless, the mammalian AdoMetDC uORF, when introduced into a polyamine auxotroph of yeast, conferred polyamine regulation of both translational efficiency and ribosome loading on the associated mRNA . This regulatory activity depended on the amino acid sequence encoded by the fourth and fifth codons of the uORF, as in mammalian cells . The fact that the regulatory properties of this mammalian translational control element are quite similar in both mammalian and yeast cells suggests that a specialized signal transduction pathway is not required . Rather, it seems likely that polyamines may be directly participating in an interaction between the uORF-encoded peptide and a constitutive component of the translation machinery, which leads to inhibition of ribosome activity.

Nat Cell Biol, 2001 May, 3(5), 522 - 6
Fission yeast Bub1 is essential in setting up the meiotic pattern of chromosome segregation; Bernard P et al.; In meiosis, sister-chromatids move to the same spindle pole during the first division (MI) and to opposite poles during the second division (MII) . This requires that MI sister kinetochores are co-orientated and form an apparent single functional unit that only interacts with microtubules from one pole, and that sister-chromatids remain associated through their centromeres until anaphase II . Here we investigate the function of Bub1 and Mad2, which are components of the mitotic-spindle checkpoint, on chromosome segregation during meiosis . Both proteins are required to prevent the occurrence of non-disjunction events in MI, which is consistent with recent findings that components of the mitotic-spindle checkpoint also operate during meiosis . However, Bub1 has several functions that are not shared with Mad2 . When the bub1 gene is deleted, sister chromatids often move to opposite spindle poles during MI, indicating that sister kinetochores are disunited . Furthermore, the cohesin Rec8 is never retained at centromeres at anaphase I and sister-chromatid cohesion is lost . Our results show that Bub1, besides its functions in monitoring chromosome attachment, is essential for two other significant aspects of MI - unification of sister kinetochores and retention of centromeric cohesion.

EMBO J, 2001 May 1, 20(9), 2111 - 9
Oligopeptide repeats in the yeast protein Sup35p stabilize intermolecular prion interactions; Parham SN et al.; The nuclear-encoded Sup35p protein is responsible for the prion-like {PSI(+)} determinant of yeast, with Sup35p existing largely as a high molecular weight aggregate in {PSI(+)} strains . Here we show that the five oligopeptide repeats present at the N-terminus of Sup35p are responsible for stabilizing aggregation of Sup35p in vivo . Sequential deletion of the oligopeptide repeats prevented the maintenance of {PSI(+)} by the truncated Sup35p, although deletants containing only two repeats could be incorporated into pre-existing aggregates of wild-type Sup35p . The mammalian prion protein PrP also contains similar oligopeptide repeats and we show here that a human PrP repeat (PHGGGWGQ) is able functionally to replace a Sup35p oligopeptide repeat to allow stable {PSI(+)} propagation in vivo . Our data suggest a model in which the oligopeptide repeats in Sup35p stabilize intermolecular interactions between Sup35p proteins that initiate establishment of the aggregated state . Modulating repeat number therefore alters the rate of yeast prion conversion in vivo . Furthermore, there appears to be evolutionary conservation of function of the N-terminally located oligopeptide repeats in prion propagation.

J Biol Chem, 2001 Jul 6, 276(27), 24855 - 61 Epub 2001 Apr 30.
Microtubules are involved in glucose-dependent dissociation of the yeast vacuolar {H+}-ATPase in vivo; Xu T et al.; The vacuolar {H(+)}-ATPases (V-ATPases) are composed of a peripheral V(1) domain and a membrane-embedded V(0) domain . Reversible dissociation of the V(1) and V(0) domains has been observed in both yeast and insects and has been suggested to represent a general regulatory mechanism for controlling V-ATPase activity in vivo . In yeast, dissociation of the V-ATPase is triggered by glucose depletion, but the signaling pathways that connect V-ATPase dissociation and glucose metabolism have not been identified . We have found that nocodazole, an agent that disrupts microtubules, partially blocked dissociation of the V-ATPase in response to glucose depletion in yeast . By contrast, latrunculin, an agent that disrupts actin filaments, had no effect on glucose-dependent dissociation of the V-ATPase complex . Neither nocodazole nor latrunculin blocked reassembly of the V-ATPase upon re-addition of glucose to the medium . The effect of nocodazole appears to be specifically through disruption of microtubules since glucose-dependent dissociation of the V-ATPase was not blocked by nocodazole in yeast strains bearing a mutation in tubulin that renders it resistant to nocodazole . Because nocodazole has been shown to arrest cells in the G(2) phase of the cell cycle, it was of interest to determine whether nocodazole exerted its effect on dissociation of the V-ATPase through cell cycle arrest . Glucose-dependent dissociation of the V-ATPase was examined in four yeast strains bearing temperature-sensitive mutations that arrest cells in different stages of the cell cycle . Because dissociation of the V-ATPase occurred normally at both the permissive and restrictive temperatures in these mutants, the results suggest that in vivo dissociation is not dependent upon cell cycle phase.

Biosci Biotechnol Biochem, 2001 Mar, 65(3), 522 - 6
Improving the freeze tolerance of bakers' yeast by loading with trehalose; Hirasawa R et al.; We examined the freeze tolerance of bakers' yeast loaded with exogenous trehalose . Freeze-tolerant and freeze-sensitive compressed bakers' yeast samples were soaked at several temperatures in 0.5 M and 1 M trehalose and analyzed . The intracellular trehalose contents in both types of bakers' yeast increased with increasing soaking period . The initial trehalose-accumulation rate increased with increasing exogenous trehalose concentration and soaking temperature . The maximum trehalose content was almost identical (200-250 mg/g of dry cells) irrespective of the soaking temperature and the type of bakers' yeast, but depended on the exogenous trehalose concentration . The leavening ability of both types of bakers' yeast loaded with trehalose was almost identical to that of the respective original cells, irrespective of the soaking conditions . The freeze-tolerant ratio (FTR) of both types of bakers' yeast increased with increasing intracellular trehalose content . However, FTR decreased during over-soaking after the maximum amount of trehalose had accumulated . FTR of the freeze-sensitive bakers' yeast was more efficiently improved than that of the freeze-tolerant type.

Cell Biochem Biophys, 2000, 32 Spring, 333 - 7
Disruption of a yeast very-long-chain acyl-CoA synthetase gene simulates the cellular phenotype of X-linked adrenoleukodystrophy; Watkins PA et al.; X-linked adrenoleukodystrophy (X-ALD) is characterized biochemically by elevated levels of saturated very long-chain fatty acids (VLCFAs) in plasma and tissues . In X-ALD, peroxisomal very-long-chain acyl-CoA synthetase (VLCS) fails to activate VLCFAs, preventing their degradation via beta-oxidation . However, the product of the defective XALD gene (ALDP) is not a VLCS, but rather a peroxisomal membrane protein (PMP) . Disruption of either or both of two yeast PMP genes related to the XALD gene did not produce a biochemical phenotype resembling that found in X-ALD fibroblasts . The authors identified a candidate yeast VLCS gene (the FAT1 locus) by its homology to rat liver VLCS . Disruption of this gene decreased VLCS activity, but had no effect on long-chain acyl-CoA synthetase activity . In FAT1-disruption strains, VLCS activity was reduced to 30-40% of wild-type in both a microsome-rich 27,000 g supernatant fraction and a peroxisome- and mitochondria-rich pellet fraction of yeast spheroplast homogenates . Separation of the latter organelles by density gradient centrifugation revealed that VLCS activity was peroxisomal and not mitochondrial . VLCS gene-disruption strains had increased cellular VLCFA levels, compared to wild-type yeast . The extent of both the decrease in peroxisomal VLCS activity and the VLCFA accumulation in this yeast model resembles that observed in cells from X-ALD patients . Characterization of the gene(s) responsible for the residual peroxisomal VLCS activity may suggest new therapeutic approaches in X-ALD.

Cell Biochem Biophys, 2000, 32 Spring, 21 - 6
Peroxisome biogenesis in the yeast Yarrowia lipolytica; Titorenko VI et al.; Extensive peroxisome proliferation during growth on oleic acid, combined with the availability of excellent genetic tools, makes the dimorphic yeast, Yarrowia lipolytica, a powerful model system to study the molecular mechanisms involved in peroxisome biogenesis . A combined genetic, biochemical, and morphological approach has revealed that the endoplasmic reticulum (ER) plays an essential role in the assembly of functional peroxisomes in this yeast . The trafficking of some membrane proteins to the peroxisomes occurs via the ER, results in their glycosylation in the ER lumen, does not involve transit through the Golgi, and requires the products of the SEC238, SRP54, PEX1, and PEX6 genes . The authors' data suggest a model for protein import into peroxisomes via two subpopulations of ER-derived vesicles that are distinct from secretory vesicles . A kinetic analysis of the trafficking of peroxisomal proteins in vivo has demonstrated that membrane and matrix proteins are initially targeted to multiple vesicular precursors that represent intermediates in the assembly pathway of peroxisomes . The authors have also recently identified a novel cytosolic chaperone, Pex20p, that assists in the oligomerization of thiolase in the cytosol and promotes its targeting to the peroxisome . These data provide the first evidence that a chaperone-assisted folding and oligomerization of thiolase in the cytosol is required for the import of this protein into the peroxisomal matrix.

Nat Med, 2001 May, 7(5), 625 - 9
Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity; Stubbs AC et al.; There is currently a need for vaccines that stimulate cell-mediated immunity-particularly that mediated by CD8+ cytotoxic T lymphocytes (CTLs)-against viral and tumor antigens . The optimal induction of cell-mediated immunity requires the presentation of antigens by specialized cells of the immune system called dendritic cells (DCs) . DCs are unique in their ability to process exogenous antigens via the major histocompatibility complex (MHC) class I pathway as well as in their ability to activate naive, antigen-specific CD8+ and CD4+ T cells . Vaccine strategies that target or activate DCs in order to elicit potent CTL-mediated immunity are the subject of intense research . We report here that whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens potently induced antigen-specific, CTL responses, including those mediating tumor protection, in vaccinated animals . Interactions between yeast and DCs led to DC maturation, IL-12 production and the efficient priming of MHC class I- and class II-restricted, antigen-specific T-cell responses . Yeast exerted a strong adjuvant effect, augmenting DC presentation of exogenous whole-protein antigen to MHC class I- and class II-restricted T cells . Recombinant yeast represent a novel vaccine strategy for the induction of broad-based cellular immune responses.

J Biol Chem, 2001 Jul 6, 276(27), 25598 - 604 Epub 2001 Apr 27.
F-actin-like ATPase activity in a polymerization-defective mutant yeast actin (V266G/L267G); Yao X et al.; Polymerization increases a low level G-actin ATPase activity yielding ADP-P(i) F-actin and then ADP F-actin following release of P(i) . By monitoring P(i) release, we explored the relationship between the ATPase activity and polymerization characteristics of a mutant yeast actin, GG . In this mutant, two hydrophobic residues at the tip of a proposed hydrophobic plug between actin subdomains 3 and 4, Val(266) and Leu(267), were mutated to Gly . Although GG-actin does not polymerize by itself in vitro, GG cells are viable . We show that GG-actin ATPase activity increases under normal polymerization conditions, although stable filaments do not form . A plot of P(i) release rate versus actin concentration yields an apparent critical concentration, like that seen for actin polymerization, of approximately 8 microm for Mg(2+) GG-actin and 11 microm for Ca(2+) GG-actin . In contrast to WT-actin, P(i) release from GG-actin is cold-sensitive, reflecting the temperature sensitivity associated with mutations that decrease hydrophobicity in this region . Thus, under polymerization conditions, GG-actin exhibits a continuous F-actin-like ATPase activity resulting from the temperature-sensitive formation of unstable cycling F-actin oligomers . Tropomyosin limits the extent and rate of this activity and restores polymerization by capturing and stabilizing these oligomers rather than enhancing filament nucleation.

J Biochem (Tokyo), 2001 May, 129(5), 699 - 708
The human homologue of fission Yeast cdc27, p66, is a component of active human DNA polymerase delta; Shikata K et al.; An essential eukaryotic DNA polymerase, DNA polymerase delta (pol delta), synthesizes DNA processively in the presence of proliferating cell nuclear antigen (PCNA) . Recently, a 66 kDa polypeptide (p66) that displays significant homology within its PCNA binding domain to that of fission yeast cdc27 was identified as a component of mouse and calf thymus pol delta . Our studies show that p66 interacts tightly with other subunits of pol delta during size fractionation of human cell extracts, and co-immunoprecipitates with these subunits along with PCNA-dependent polymerase activity . Active human pol delta could be reconstituted by co-expressing p125, p50, and p66 recombinant baculoviruses, but not by co-expressing p125 and p50 alone . Interaction studies demonstrated that p66 stabilizes the association between p125 and p50 . Pull-down assays with PCNA-linked beads demonstrated that p66 increases the overall affinity of pol delta for PCNA . These results indicate that p66 is a functionally important subunit of human pol delta that stabilizes the pol delta complex and increases the affinity of pol delta for PCNA.

Biochemistry, 2001 Feb 13, 40(6), 1528 - 39
Solution structure of the Cu(I) and apo forms of the yeast metallochaperone, Atx1; Arnesano F et al.; The (1)H NMR solution structure of the Cu(I)-bound form of Atx1, a 73-amino acid metallochaperone protein from the yeast Saccharomyces cerevisiae, has been determined . Ninety percent of the (1)H and 95% of the (15)N resonances were assigned, and 1184 meaningful NOEs and 42 (3)J(HNH)(alpha) and 60 (1)J(HN) residual dipolar couplings provided a family of structures with rmsd values to the mean structure of 0.37 +/- 0.07 A for the backbone and 0.83 +/- 0.08 A for all heavy atoms . The structure is constituted by four antiparallel beta strands and two alpha helices in a betaalphabetabetaalphabeta fold . Following EXAFS data {Pufahl, R., Singer, C . P., Peariso, K . L., Lin, S.-J., Schmidt, P . J., Fahrni, C . J., Cizewski Culotta, V., Penner-Hahn, J . E., and O'Halloran, T . V . (1997) Science 278, 853-856}, a copper ion can be placed between two sulfur atoms of Cys15 and Cys18 . The structure of the reduced apo form has also been determined with similar resolution using 1252 meaningful NOEs (rmsd values for the family to the mean structure are 0.67 +/- 0.12 A for the backbone and 1.00 +/- 0.12 A for all heavy atoms) . Comparison of the Cu(I) and apo conformations of the protein reveals that the Cu(I) binding cysteines move from a buried site in the bound metal form to a solvent-exposed conformation on the surface of the protein after copper release . Furthermore, copper release leads to a less helical character in the metal binding site . Comparison with the Hg(II)-Atx1 solid-state structure {Rosenzweig, A . C., Huffman, D . L., Hou, M . Y., Wernimont, A . K., Pufahl, R . A., and O'Halloran, T . V . (1999) Structure 7, 605-617} provides insights into the copper transfer mechanism, and a pivotal role for Lys65 in the metal capture and release process is proposed.

Histochem Cell Biol, 2001 Mar, 115(3), 189 - 94
Localization of mouse CLC-6 and CLC-7 mRNA and their functional complementation of yeast CLC gene mutant; Kida Y et al.; CLC-6 and CLC-7 belong to the family of voltage-dependent chloride channels . To learn more about the in vivo roles of CLC-6 and CLC-7, we performed in situ hybridization of these CLC channels in various mouse organs . Mouse CLC-6 (mCLC-6) was expressed in the peripheral region of seminiferous tubules in the testis, tracheal epithelium, epithelium of bronchioles, alveolar cells in the lung, acinar cells in the pancreas, and intestinal epithelium, but we could not detect signals from pancreatic islets . Mouse CLC-7 (mCLC-7) was expressed in neurons in the medulla oblongata, Purkinje cells in the cerebellum, proximal tubules in the kidney, and hepatocytes in the liver . The distribution of mCLC-6 and mCLC-7 were similar in the lung, pancreas, and testis . mCLC-6 functionally complemented the gef1 phenotype of a yeast strain in which a single CLC channel (GEF1) had been disrupted by homologous recombination . In contrast, mCLC-7 did not complement this gef1 phenotype . This study identified the cell types that express mCLC-6 and mCLC-7 in the mouse tissues, and the complementation assay suggested that mCLC-6 functions as an intracellular chloride channel.

J Bacteriol, 2001 May, 183(10), 3251 - 5
Cloning and expression of two genes coding for sodium pumps in the salt-tolerant yeast Debaryomyces hansenii; Almagro A et al.; Two genes encoding Na(+)-ATPases from Debaryomyces hansenii were cloned and sequenced . The genes, designated ENA1 from D . hansenii (DhENA1) and DhENA2, exhibited high homology with the corresponding genes from Schwanniomyces occidentalis . DhENA1 was expressed in the presence of high Na(+) concentrations, while the expression of DhENA2 also required high pH . A mutant of Saccharomyces cerevisiae lacking the Na(+) efflux systems and sensitive to Na(+), when transformed with DhENA1 or DhENA2, recovered Na(+) tolerance and also the ability to extrude Na(+).

Cancer Res, 2001 May 1, 61(9), 3787 - 94
Antitumor drug adozelesin differentially affects active and silent origins of DNA replication in yeast checkpoint kinase mutants; Wang Y et al.; The antitumor drug adozelesin is a potent cytotoxic DNA-damaging agent . Here we determined how adozelesin affects chromosomal DNA replication at a molecular level in a yeast model system and examined the influence of checkpoint kinase genes, the human homologues of which are mutated in cancer . Analysis of replication intermediates using two-dimensional gel electrophoresis showed that adozelesin inhibited the activity of a replication origin and stalled replication fork progression through chromosomal DNA at the origin . RAD53 and MEC1 protein kinase genes, homologues of human CHK2 and ATM, respectively, regulate an intra-S-phase DNA damage checkpoint and, when mutated, permit unchecked replication of damaged DNA in S-phase . Mutations in these genes did not abrogate adozelesin-induced inhibition of origin activity and fork progression at the replication origin . However, novel replication intermediates indicative of DNA breaks were detected only in the rad53 mutant, suggesting a role for the wild-type gene in maintaining chromosome integrity in the presence of the drug . In contrast to the inhibition of the active replication origin by adozelesin, normally silent origins present in the same chromosome were activated by adozelesin in rad53 and mec1 mutant cells . Thus, an antitumor drug that damages DNA can induce an abnormal replication pattern in a chromosome by activating silent origins, depending upon defects in yeast checkpoint kinase genes, the homologues of which are mutated in cancer . Implications of an abnormal replication pattern for the epigenetic regulation of gene expression are discussed.

Biochim Biophys Acta, 2001 May 3, 1526(2), 191 - 8
Protective role of superoxide dismutases against ionizing radiation in yeast; Lee JH et al.; The protective role of superoxide dismutases (SODs) against ionizing radiation, which generates reactive oxygen species (ROS) harmful to cellular function, was investigated in the wild-type and in mutant yeast strains lacking cytosolic CuZnSOD (sod1Delta), mitochondrial MnSOD (sod2Delta), or both SODs (sod1Deltasod2Delta) . Upon exposure to ionizing radiation, there was a distinct difference between these strains in regard to viability and the level of protein carbonyl content, which is the indicative marker of oxidative damage to protein, intracellular H2O2 level, as well as lipid peroxidation . When the oxidation of 2',7'-dichlorofluorescin was used to examine the hydroperoxide production in yeast cells, the SOD mutants showed a higher degree of increase in fluorescence upon exposure to ionizing radiation as compared to wild-type cells . These results indicated that mutants deleted for SOD genes were more sensitive to ionizing radiation than isogenic wild-type cells . Induction and inactivation of other antioxidant enzymes, such as catalase, glucose 6-phosphate dehydrogenase, and glutathione reductase, were observed after their exposure to ionizing radiation both in wild-type and in mutant cells . However, wild-type cells maintained significantly higher activities of antioxidant enzymes than did mutant cells . These results suggest that both CuZnSOD and MnSOD may play a central role in protecting cells against ionizing radiation through the removal of ROS, as well as in the protection of antioxidant enzymes.

Int J Syst Evol Microbiol, 2001 Mar, 51(Pt 2), 699 - 705
Candida orba sp . nov., a new cactus-specific yeast species from Queensland, Australia; Starmer WT et al.; A new species of yeast from decaying cladodes of Opuntia cactus, Candida orba, is described . This species is a member of a four-species clade of cactophilic yeasts . The new species has only been found in one region of Queensland, Australia, where it was presumably introduced during attempts to eradicate prickly pear cactus . DNA-DNA relatedness, phylogenetic analysis, physiological differences, killer-sensitivity profiles and mating reactions establish the distinctness of the taxon as a new species . C . orba is most closely related to Phaffomyces thermotolerans, a species found associated with columnar cacti in the North American Sonoran Desert . The type strain of C . orba, isolated from rotting cladodes of Opuntia stricta in the State of Queensland, Australia, is strain UCD-FST 84-833.1T (= CBS 8782T = NRRL Y-27336T = ATCC MYA-341) . Only the h- mating type of the species has been recovered . The lack of the opposite mating type could be the result of a bottleneck during its introduction to Australia . The original geographic/host distribution of this species in the Americas is unknown.

Ann Hematol, 2001 Mar, 80(3), 178 - 9
Candida ciferrii, a new fluconazole-resistant yeast causing systemic mycosis in immunocompromised patients; Gunsilius E et al.; Systemic infections related to fluconazole-resistant yeasts are increasingly observed in immunocompromised patients receiving fluconazole as a prophylactic antifungal treatment . Here, we report a case of invasive candidiasis caused by Candida ciferrii in a patient with acute myeloid leukemia and who suffered a relapse after autologous peripheral blood progenitor cell transplantation . Erythematous skin papulae and spotted pulmonary infiltrations were present . A skin biopsy led to the diagnosis of invasive candidiasis, emphasizing the diagnostic usefulness of this procedure . The yeast was identified as Candida ciferrii and in vitro susceptibility testing revealed its resistance to fluconazole . Until now, Candida ciferrii has not been known to cause invasive fungal infections in humans . Thus, we add another fungus to the list of flucanozole-resistant yeasts and suggest that in vitro susceptibility testing of isolated fungi should be performed for the selection of appropriate antimycotic drugs.

Acta Crystallogr D Biol Crystallogr, 2001 May, 57(Pt 5), 748 - 50 Epub 2001 Apr 24.
Cloning, expression, purification and preliminary X-ray crystallographic studies of yeast Hsp40 Sis1 complexed with Hsp70 Ssa1 C-terminal lid domain; Qian X et al.; Heat-shock protein 70 (Hsp70) plays essential roles in a number of cellular processes such as protein folding, assembly and translocation . Heat-shock protein 40 (Hsp40) transiently interacts with Hsp70 and facilitates Hsp70 functions in these processes within cells . Hsp40 recognizes and binds non-native polypeptide and delivers it to Hsp70 . Hsp40 can then stimulate the ATPase activity of Hsp70 to refold the polypeptide . To investigate the molecular mechanism by which Hsp40 interacts with Hsp70 to transport the non-native polypeptide, Saccharomyces cerevisiae Hsp40 Sis1 C-terminal peptide-binding fragment complexed with Hsp70 Ssa1 C-terminal lid domain has been produced and crystallized . The complex crystals diffract to 3.3 A and belong to the space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 112.17, c = 171.31 A . Structure determination by the MAD method is under way.

Anal Biochem, 2001 May 1, 292(1), 8 - 16
Yeast cell permeabilization by osmotic shock allows determination of enzymatic activities in situ; Crotti LB et al.; Yeast cells were permeabilized by incubation in 0.8 M sorbitol followed by suspension in dilute buffer . A preincubation with 2-mercaptoethanol was also included for optimal permeabilization . More than 90% of the treated cells were stainable with methylene blue . Determinations of cell wall-synthesizing enzymes (beta(1 --> 3)glucan and chitin synthases) and cytosolic enzymes in permeabilized cells yielded similar or higher activities than those in cell extracts . With chitin synthase III, the activity obtained with cells was 4- to 6-fold higher than in membrane preparations . Little protein leaks from the cells during permeabilization; yet the cells appear to be readily permeable to substrates and even proteins . Thus, these preparations may be of wide use for the study of enzymes and of biological processes in situ.

J Biol Chem, 2001 Jun 29, 276(26), 24253 - 60 Epub 2001 Apr 23.
A mammalian homolog of yeast MOB1 is both a member and a putative substrate of striatin family-protein phosphatase 2A complexes; Moreno CS et al.; Striatin and S/G(2) nuclear autoantigen (SG2NA) are related proteins that contain membrane binding domains and associate with protein phosphatase 2A (PP2A) and many additional proteins that may be PP2A regulatory targets . Here we identify a major member of these complexes as class II mMOB1, a mammalian homolog of the yeast protein MOB1, and show that its phosphorylation appears to be regulated by PP2A . Yeast MOB1 is critical for cytoskeletal reorganization during cytokinesis and exit from mitosis . We show that mMOB1 associated with PP2A is not detectably phosphorylated in asynchronous murine fibroblasts . However, treatment with the PP2A inhibitor okadaic acid induces phosphorylation of PP2A-associated mMOB1 on serine . Moreover, specific inhibition of PP2A also results in hyperphosphorylation of striatin, SG2NA, and three unidentified proteins, suggesting that these proteins may also be regulated by PP2A . Indirect immunofluorescence produced highly similar staining patterns for striatin, SG2NA, and mMOB1, with the highest concentrations for each protein adjacent to the nuclear membrane . We also present evidence that these complexes may interact with each other . These data are consistent with a model in which PP2A may regulate mMOB1, striatin, and SG2NA to modulate changes in the cytoskeleton or interactions between the cytoskeleton and membrane structures.

Genes Dev, 2001 Apr 15, 15(8), 945 - 54
Roles of yeast DNA polymerases delta and zeta and of Rev1 in the bypass of abasic sites; Haracska L et al.; Abasic (AP) sites are one of the most frequently formed lesions in DNA, and they present a strong block to continued synthesis by the replicative DNA machinery . Here we show efficient bypass of an AP site by the combined action of yeast DNA polymerases delta and zeta . In this reaction, Poldelta inserts an A nucleotide opposite the AP site, and Polzeta subsequently extends from the inserted nucleotide . Consistent with these observations, sequence analyses of mutations in the yeast CAN1s gene indicate that A is the nucleotide inserted most often opposite AP sites . The nucleotides C, G, and T are also incorporated, but much less frequently . Enzymes such as Rev1 and Poleta may contribute to the insertion of these other nucleotides; the predominant role of Rev1 in AP bypass, however, is likely to be structural . Steady-state kinetic analyses show that Polzeta is highly inefficient in incorporating nucleotides opposite the AP site, but it efficiently extends from nucleotides, particularly an A, inserted opposite this lesion . Thus, in eukaryotes, bypass of an AP site requires the sequential action of two DNA polymerases, wherein the extension step depends solely upon Polzeta, but the insertion step can be quite varied, involving not only the predominant action of the replicative DNA polymerase, Poldelta, but also the less prominent role of various translesion synthesis polymerases.

J Basic Microbiol, 2001, 41(1), 7 - 16
Natural zeolite clinoptilolite increases the concentrations of sphingoid bases in the yeast Yarrowia lipolytica; Bauman M et al.; In the present paper, we studied the effect of natural zeolite clinoptilolite on sphingolipid metabolism in the yeast Yarrowia lipolytica . We also investigated if zeolite addition had any impact on cell shape and size, as well as on the pH alterations during the culture growth . High performance liquid chromatography analysis of sphingoid bases obtained by acid hydrolysis of complex sphingolipids from Y . lipolytica showed that their concentrations markedly rose upon the zeolite addition . The largest increase among the identified molecular species of sphingoid bases was seen in C18 phytosphingosine, whose levels rose 6.2-fold and 22.3-fold after culturing cells for 24 and 36 hours respectively in the presence of finely ground zeolite . pH measurements of the culture medium showed a similarity between pH profiles of control and zeolite-supplemented cells, suggesting that ion-exchange capacity was not probably responsible for the observed change in sphingolipid metabolism . Scanning electron microscopy revealed that zeolite affected cell size and shape . Y . lipolytica cells grown in the absence of zeolite were oval-shaped with an average cell size of 0.7-2.7 microns, whereas when cultured with zeolite, they were round-shaped and larger, having an average cell size of 1.3-2.9 microns.

Oncogene, 2001 Jan 25, 20(4), 501 - 13
p53 mutants exhibiting enhanced transcriptional activation and altered promoter selectivity are revealed using a sensitive, yeast-based functional assay; Inga A et al.; Changes in promoter specificity and binding affinity that may be associated with p53 mutations or post-translational modifications are useful in understanding p53 structure/function relationships and categorizing tumor mutations . We have exploited variable expression of human p53 in yeast to identify mutants with novel phenotypes that would correspond to altered promoter selectivity and affinity . The p53 cDNA regions coding for the DNA binding and tetramerization domains were subjected to random PCR mutagenesis and were cloned directly by recombination in yeast into a vector with a GAL1 promoter whose level of expression could be easily varied . p53 variants exhibiting higher than wild type levels of transactivation (supertrans) for the RGC responsive element were identified at low level of p53 protein expression . All the p53 mutants obtained with this screen were located in the DNA binding domain . Two out of 17 supertrans mutants have been found in tumors . Six mutations were in the L1 loop region between amino acids 115 and 124 . The transactivation potential of a panel of supertrans p53 mutants on different promoters was evaluated using the p53 responsive elements, RGC, PIG3, p21 and bax . Although all mutants retained some activity with all promoters, we found different patterns of induction based on strength and promoter specificity . In particular none of the mutants was supertrans for the p21 responsive element . Interestingly, further analysis in yeast showed that the transactivation function could be retained even in the presence of dominant-negative p53 tumor mutations that could inhibit wild type p53 . Five mutants were also characterized in human cells in terms of growth suppression and transactivation of various promoters . These novel supertrans p53 mutants may be useful in studies aimed at dissecting p53 downstream pathways, understanding specific interactions between p53 and the DNA, and could replace wild type p53 in cancer gene therapy protocols . The approach may also prove useful in identifying p53 tumor mutations.

Mol Cell Biol, 2001 May, 21(10), 3491 - 502
A bipartite yeast SSRP1 analog comprised of Pob3 and Nhp6 proteins modulates transcription; Brewster NK et al.; The FACT complex of vertebrate cells, comprising the Cdc68 (Spt16) and SSRP1 proteins, facilitates transcription elongation on a nucleosomal template and modulates the elongation-inhibitory effects of the DSIF complex in vitro . Genetic findings show that the related yeast (Saccharomyces cerevisiae) complex, termed CP, also mediates transcription . The CP components Cdc68 and Pob3 closely resemble the FACT components, except that the C-terminal high-mobility group (HMG) box domain of SSRP1 is not found in the yeast homolog Pob3 . We show here that Nhp6a and Nhp6b, small HMG box proteins with overlapping functions in yeast, associate with the CP complex and mediate CP-related genetic effects on transcription . Absence of the Nhp6 proteins causes severe impairment in combination with mutations impairing the Swi-Snf chromatin-remodeling complex and the DSIF (Spt4 plus Spt5) elongation regulator, and sensitizes cells to 6-azauracil, characteristic of elongation effects . An artificial SSRP1-like protein, created by fusing the Pob3 and Nhp6a proteins, provides both Pob3 and Nhp6a functions for transcription, and competition experiments indicate that these functions are exerted in association with Cdc68 . This particular Pob3-Nhp6a fusion protein was limited for certain Nhp6 activities, indicating that its Nhp6a function is compromised . These findings suggest that in yeast cells the Cdc68 partners may be both Pob3 and Nhp6, functioning as a bipartite analog of the vertebrate SSRP1 protein.

Mol Cell Biol, 2001 May, 21(10), 3398 - 404
Threonine-11, phosphorylated by Rad3 and atm in vitro, is required for activation of fission yeast checkpoint kinase Cds1; Tanaka K et al.; Fission yeast Cds1 is phosphorylated and activated when DNA replication is interrupted by nucleotide starvation or DNA damage . Cds1 enforces the S-M checkpoint that couples mitosis (M) to the completion of DNA synthesis (S) . Cds1 also controls replicational stress tolerance mechanisms . Cds1 is regulated by a group of proteins that includes Rad3, a kinase related to human checkpoint kinase ATM (ataxia telangiectasia mutated) . ATM phosphorylates serine or threonine followed by glutamine (SQ or TQ) . Here we show that in vitro, Rad3 and ATM phosphorylate the N-terminal domain of Cds1 at the motif T(11)Q(12) . Substitution of threonine-11 with alanine (T11A) abolished Cds1 activation that occurs when DNA replication is inhibited by hydroxyurea (HU) treatment . The cds1-T11A mutant was profoundly sensitive to HU, although not quite as sensitive as a cds1(-) null mutant . Cds1(T11A) was unable to enforce the S-M checkpoint . These results strongly suggest that Rad3-dependent phosphorylation of Cds1 at threonine-11 is required for Cds1 activation and function.

Mol Cell Biol, 2001 May, 21(10), 3289 - 301
Fission yeast Rad17 associates with chromatin in response to aberrant genomic structures; Kai M et al.; Fission yeast checkpoint protein Rad17 is required for the DNA integrity checkpoint responses . A fraction of Rad17 is chromatin bound independent of the other checkpoint proteins throughout the cell cycle . Here we show that in response to DNA damage induced by either methyl methanesulfonate treatment or ionizing radiation, increased levels of Rad17 bind to chromatin . Following S-phase stall induced by hydroxyurea or a cdc22 mutation, the chromatin-bound Rad17 progressively dissociates from the chromatin . After S-phase arrest by hydroxyurea in cds1Delta or rad3Delta cells or by replication mutants, Rad17 remains chromatin bound . Rad17 is able to complex in vivo with an Rfc small subunit, Rfc2, but not with Rfc1 . Furthermore, cells with rfc1Delta are checkpoint proficient, suggesting that Rfc1 does not have a role in checkpoint function . A checkpoint-defective mutant protein, Rad17(K118E), which has similar nuclear localization to that of the wild type, is unable to bind ATP and has reduced ability in chromatin binding . Mutant Rad17(K118E) protein also has reduced ability to complex with Rfc2, suggesting that Lys(118) of Rad17 plays a role in Rad17-Rfc small-subunit complex formation and chromatin association . However, in the rad17.K118E mutant cells, Cds1 can be activated by hydroxyurea . Together, these results suggest that Rad17 binds to chromatin in response to an aberrant genomic structure generated from DNA damage, replication mutant arrest, or hydroxyurea arrest in the absence of Cds1 . Rad17 is not required to bind chromatin when genomic structures are protected by hydroxyurea-activated Cds1 . The possible checkpoint events induced by chromatin-bound Rad17 are discussed.

J Biol Chem, 2001 Jun 29, 276(26), 23486 - 91 Epub 2001 Apr 19.
Yeast ERV2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family; Gerber J et al.; Saccharomyces cerevisiae Erv2p was identified previously as a distant homologue of Erv1p, an essential mitochondrial protein exhibiting sulfhydryl oxidase activity . Expression of the ERV2 (essential for respiration and vegetative growth 2) gene from a high-copy plasmid cannot substitute for the lack of ERV1, suggesting that the two proteins perform nonredundant functions . Here, we show that the deletion of the ERV2 gene or the depletion of Erv2p by regulated gene expression is not associated with any detectable growth defects . Erv2p is located in the microsomal fraction, distinguishing it from the mitochondrial Erv1p . Despite their distinct subcellular localization, the two proteins exhibit functional similarities . Both form dimers in vivo and in vitro, contain a conserved YPCXXC motif in their carboxyl-terminal part, bind flavin adenine dinucleotide (FAD) as a cofactor, and catalyze the formation of disulfide bonds in protein substrates . The catalytic activity, the ability to form dimers, and the binding of FAD are associated with the carboxyl-terminal domain of the protein . Our findings identify Erv2p as the first microsomal member of the Erv1p/Alrp protein family of FAD-linked sulfhydryl oxidases . We propose that Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential, FAD-dependent oxidase of protein disulfide isomerase.

Biotechnol Prog, 2001 Mar-Apr, 17(2), 362 - 5
Remarkable antiagglomeration effect of a yeast biosurfactant, diacylmannosylerythritol, on ice-water slurry for cold thermal storage; Kitamoto D et al.; Antiagglomeration effects of different surfactants on ice slurry formation were examined to improve the efficiency of an ice-water slurry system to be used for cold thermal storage . Among the chemical surfactants tested, a nonionic surfactant, poly(oxyethylene) sorbitan dioleate, was found to show a greater antiagglomeration effect on the slurry than anionic, cationic, or amphoteric surfactants . More interestingly, diacylmannosylerythritol, a glycolipid biosurfactant produced by a yeast strain of Candida antarctica, exhibited a remarkable effect on the slurry, attaining a high ice packing factor (35%) for 8 h at a biosurfactant concentration of 10 mg/L . These nonionic glycolipid surfactants are likely to effectively adsorb on the ice surface in a highly regulated manner to suppress the agglomeration or growth of the ice particles . This is the first report on the utilization of biosurfactant for thermal energy storage, which may significantly expand the commercial applications of the highly environmentally friendly slurry system.

Gene, 2001 Apr 4, 267(1), 31 - 6
Involvement of yeast carboxy-terminal domain kinase I (CTDK-I) in transcription elongation in vivo; Jona G et al.; Yeast cells lacking transcription elongation factor genes such as PPR2 (TFIIS) and ELP (Elongator) are viable and show deleterious phenotypes only when transcription is rendered less effective by RNA polymerase mutations or by decreasing nucleotide pools . Here we demonstrate that deletion of the CTK1 gene, encoding the kinase subunit of RNA polymerase II carboxy-terminal domain kinase I (CTDK-I), is synthetically lethal when combined with deletion of PPR2 or ELP genes . The inviability of ctk1 elp3 double mutants can be rescued by expression of an Elp3 mutant that has retained its ability to form the Elongator complex but has severely diminished histone acetyltransferase activity, suggesting that the functional overlap between CTDK-I and Elongator is in assembly of RNA polymerase II elongation complexes . Our results suggest that CTDK-I plays an important role in transcriptional elongation in vivo, possibly by creating a form of RNA polymerase that is less prone to transcriptional arrest.

Biochem J, 2001 May 1, 355(Pt 3), 805 - 17
Mammalian inositol polyphosphate 5-phosphatase II can compensate for the absence of all three yeast Sac1-like-domain-containing 5-phosphatases; O'Malley CJ et al.; Phosphatidylinositol 4,5-bisphosphate {PtdIns(4,5)P(2)} plays a complex role in generating intracellular signalling molecules, and also in regulating actin-binding proteins, vesicular trafficking and vacuolar fusion . Four inositol polyphosphate 5-phosphatases (hereafter called 5-phosphatases) have been identified in Saccharomyces cerevisiae: Inp51p, Inp52p, Inp53p and Inp54p . Each enzyme contains a 5-phosphatase domain which hydrolyses PtdIns(4,5)P(2), forming PtdIns4P, while Inp52p and Inp53p also express a polyphosphoinositide phosphatase domain within the Sac1-like domain . Disruption of any two yeast 5-phosphatases containing a Sac1-like domain results in abnormalities in actin polymerization, plasma membrane, vacuolar morphology and bud-site selection . Triple null mutant 5-phosphatase strains are non-viable . To investigate the role of PtdIns(4,5)P(2) in mediating the phenotype of double and triple 5-phosphatase null mutant yeast, we determined whether a mammalian PtdIns(4,5)P(2) 5-phosphatase, 5-phosphatase II, which lacks polyphosphoinositide phosphatase activity, could correct the phenotype of triple 5-phosphatase null mutant yeast and restore cellular PtdIns(4,5)P(2) levels to near basal values . Mammalian 5-phosphatase II expressed under an inducible promoter corrected the growth, cell wall, vacuolar and actin polymerization defects of the triple 5-phosphatase null mutant yeast strains . Cellular PtdIns(4,5)P(2) levels in various 5-phosphatase double null mutant strains demonstrated significant accumulation (4.5-, 3- and 2-fold for Deltainp51Deltainp53, Deltainp51Deltainp52 and Deltainp52Deltainp53 double null mutants respectively), which was corrected significantly following 5-phosphatase II expression . Collectively, these studies demonstrate the functional and cellular consequences of PtdIns(4,5)P(2) accumulation and the evolutionary conservation of function between mammalian and yeast PtdIns(4,5)P(2) 5-phosphatases.

J Cell Biol, 2001 Apr 16, 153(2), 307 - 18
An exclusively nuclear RNA-binding protein affects asymmetric localization of ASH1 mRNA and Ash1p in yeast; Long RM et al.; The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae . A localization element that is predominantly in the 3'-untranslated region (UTR) can direct this mRNA to the bud . Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3'-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs . LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA . Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain . Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm . We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.

Mol Microbiol, 2001 Apr, 40(2), 422 - 32
Overlapping and distinct roles of the duplicated yeast transcription factors Ace2p and Swi5p; Doolin MT et al.; The yeast transcription factors Ace2p and Swi5p regulate the expression of several target genes involved in mating type switching, exit from mitosis and cell wall function . We describe the analysis of 12 novel targets, some regulated by Ace2p or Swi5p alone and some by both . We show that Ace2p is the major regulator of four genes (CTS1, YHR143W, SCW11 and YER124C) . Expression of all four is inhibited by Swi5p . Like Cts1p and Scw11p, the two new Ace2p targets are associated with cell wall metabolism . Yhr143p is localized to the cell wall, and deletion affects cell separation and enhances pseudohyphal growth . Deleting YER124C also affects cell separation and sensitivity to drugs targeted against the cell wall . Expression of PIR1, YPL158C and YNL046W is dependent on Swi5p alone . In contrast, expression of YBR158W, YNL078W and YOR264W is minimized when both ace2 and swi5 are disrupted . We propose that, although Ace2p and Swi5p co-operate to induce the expression of a subset of genes, some functional divergence has occurred . This results in a delay in the expression of those genes predominantly regulated by Ace2p, compared with those predominantly regulated by Swi5p.

Biol Chem, 2001 Feb, 382(2), 321 - 8
Biosynthesis of lipid-linked oligosaccharides in yeast: the ALG3 gene encodes the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase; Sharma CB et al.; The formation of N-glycosidic linkages of glycoproteins involves the ordered assembly of the common Glc3Man9GlcNAc2 core-oligosaccharide on the lipid carrier dolichyl pyrophosphate . Whereas early mannosylation steps occur on the cytoplasmic side of the endoplasmic reticulum with GDP-Man as donor, the final reactions from Man5GlcNAc2-PP-Dol to Man9GlcNAc2-PP-Dol on the lumenal side use Dol-P-Man . We have investigated these later stages in vitro using a detergent-solubilized enzyme extract from yeast membranes . Mannosyltransfer from Dol-P-Man to {3H}Man5GlcNAc2-PP-Dol with formation of all intermediates up to Man9GlcNAc2-PP-Dol occured in a rapid, time- and protein-dependent fashion . We find that the initial reaction from Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol is independent of metal ions, but further elongations need Mn2+ that can be partly replaced by Mg2+ or Ca2+ . Zn2+ or Cd2+ ions were found to inhibit formation of Man(7-9)GlcNAc2-PP-Dol, but do not affect synthesis of Man6GlcNAc2-PP-Dol . Extension did not occur when the acceptor was added as a free Man5GlcNAc2 oligosaccharide or when GDP-Man was used as mannosyl donor . The alg3 mutant was described to accumulate Man5GlcNAc2-PP-Dol . We expressed a functional active HA-epitope tagged ALG3 fusion and succeeded to selectively immunoprecipitate the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase activity from the other enzymes of the detergent extract involved in the subsequent mannosylation reactions . This demonstrates that Alg3p represents the mannosyltransferase itself and not an accessory protein involved in the reaction.

Cytogenet Cell Genet, 2001, 92(1-2), 85 - 8
The functional myosin light chain kinase (MYLK) gene localizes with marker D3S3552 on human chromosome 3q21 in a >5-Mb yeast artificial chromosome region and is not linked to olfactory receptor genes; Giorgi D et al.; The myosin light chain kinase (MYLK) gene is duplicated on human chromosome 3 (3q13-->q21; 3p13), two sites known to contain olfactory receptor (OR) genes . The 3p13 site contains a MYLK pseudogene (MYLKP) associated with a cluster of OR pseudogenes and therefore could have arisen from the duplication of a large region in 3q13-->q21 . Here, we present the localization of the MYLK gene in a >5-Mb region of the chromosome 3q21 integrated map . MYLK colocalizes with marker D3S3552 . OR genes are absent from this region, suggesting that the 3p13 duplicated region incurred further rearrangements during evolution.

J Biol Chem, 2001 Jun 22, 276(25), 22485 - 90 Epub 2001 Apr 16.
Stalk segment 5 of the yeast plasma membrane H+-ATPase: mutational evidence for a role in glucose regulation; Miranda M et al.; In P(2)-type ATPases, a stalk region connects the cytoplasmic part of the molecule, which binds and hydrolyzes ATP, to the membrane-embedded part through which cations are pumped . The present study has used cysteine scanning mutagenesis to examine structure-function relationships within stalk segment 5 (S5) of the yeast plasma-membrane H(+)-ATPase . Of 29 Cys mutants that were made and examined, two (G670C and R682C) were blocked in biogenesis, presumably due to protein misfolding . In addition, one mutant (S681C) had very low ATPase activity, and another (F685C) displayed a 40-fold decrease in sensitivity to orthovanadate, reflecting a shift in equilibrium from the E(2) conformational state toward E(1) . By far the most striking group of mutants (F666C, L671C, I674C, A677C, I684C, R687C, and Y689C) were constitutively activated even in the absence of glucose, with rates of ATP hydrolysis and kinetic properties normally seen only in glucose-metabolizing cells . Previous work has suggested that activation of the wild-type H(+)-ATPase results from kinase-mediated phosphorylation in the auto-inhibitory C-terminal region of the 100-kDa polypeptide . The seven residues identified in the present study are located on one face of the S5 alpha-helix, consistent with the idea that mutations along this face serve to release the auto-inhibition.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 583 - 95
Structural and functional properties of aldose xylose reductase from the D-xylose-metabolizing yeast Candida tenuis; Nidetzky B et al.; The primary structure of the aldose xylose reductase from Candida tenuis (CtAR) is shown to be 39% identical to that of human aldose reductase (hAR) . The catalytic tetrad of hAR is completely conserved in CtAR (Tyr51, Lys80, Asp46, His113) . The amino acid residues involved in binding of NADPH by hAR (D.K . Wilson, et al., Science 257 (1992) 81-84) are 64% identical in CtAR . Like hAR the yeast enzyme is specific for transferring the 4-pro-R hydrogen of the coenzyme . These properties suggest that CtAR is a member of the aldo/keto reductase superfamily . Unlike hAR the enzyme from C . tenuis has a dual coenzyme specificity and shows similar specificity constants for NADPH and NADH . It binds NADP(+) approximately 250 times less tightly than hAR . Typical turnover numbers for aldehyde reduction by CtAR (15-20 s(-1)) are up to 100-fold higher than corresponding values for hAR, probably reflecting an overall faster dissociation of NAD(P)(+) in the reaction catalyzed by the yeast enzyme.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 417 - 23
The activity of yeast ADH I and ADH II with long-chain alcohols and diols; Dickinson FM et al.; The activities of yeast ADH I and ADH II towards long chain alcohols and diols were studied using rather unusual conditions (1.0 M Tris pH 8.75, approximately 0.3 mg/ml enzyme and {S}<<<K(m) ) where the alcohols are oxidised quantitatively in a first-order manner . Plots of the apparent first-order rate constant versus primary alcohol chain length show double peaks with similar values for ethanol and 1-decanol and relatively low values for 1-butanol through to 1-octanol . With the alpha,omega diols only one peak of activity was observed with 1,14-tetradecanediol, the preferred substrate, being oxidised about the same rate as ethanol . Both enzymes were essentially inactive with short-chain diols (C(2)-C(8)) . For all of these assays normalised rates with ADH II were about threefold faster than with ADH I.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 173 - 9
Making an Oriental equivalent of the yeast cytosolic aldehyde dehydrogenase as well as making one with positive cooperativity in coenzyme binding by mutations of glutamate 492 and arginine 480; Wei B et al.; Yeast has at least three partially characterized aldehyde dehydrogenases . Previous studies by gene disrupted in our laboratory revealed that the Saccharomyces cerevisiae cytosol ALDH1 played an important role in ethanol metabolism as did the class 2 mitochondrial enzyme . To date, few mutagenesis studies have been performed with the yeast enzymes . An important human variant of ALDH is one found in Asian People . In it, the glutamate at position 487 is replaced by a lysine . This glutamate interacts with an arginine (475) that is located in the subunit that makes up the dimer pair in the tetrameric enzyme . Sequence alignment shows that these two residues are located at positions 492 and 480, respectively, in the yeast class 1 enzyme which shares just 45% sequence identity with the human enzymes . Mutating glutamate 492 to lysine produced an enzyme with altered kinetic properties when compared to the wild-type glutamate-enzyme . The K(m) for NADP of E492K increased to nearly 3600 microM compare to 40 microM for wild-type enzyme . The specific activity decreased more than 10-fold with respect to the recombinant wild-type yeast enzyme . Moreover, substituting a glutamine for a glutamate was not detrimental in that the E492Q had wild-type-like K(m) for NADP and V(max) . These properties were similar to the changes found with the human class 2 E487K mutant form . Further, mutating arginine 480 to glutamine produced an enzyme that exhibited positive cooperativity in NADP binding . The K(m) for NADP increased 11-fold with a Hill coefficient of 1.6 . The NADP-dependent activity of R480Q mutant was 60% of wild-type enzyme . Again, these results are very similar to what we recently showed to occur with the human enzyme {Biochemistry 39 (2000) 5295-5302} . These findings show that the even though the glutamate and arginine residues are not conserved, similar changes occur in both the human and the yeast enzyme when either is mutated.

Methods Enzymol, 2001, 332, 270 - 7
Isolation of effector-selective Ras mutants by yeast two-hybrid screening; Kaur KJ et al.; Ras mutants displaying selective target interactions will often display partial loss of function phenotypes when expressed in cells . Thus the isolation of mutations in Ras and Ras family members has proved to be a productive approach for testing the requirement of specific target interactions to mediate downstream responses . The procedures outlined here greatly simplify the isolation of such mutants, and it is hoped will contribute to a better understanding of Ras effector function.

J Biol Chem, 2001 Apr 20, 276(16), 12636 - 44
The interactions of yeast SWI/SNF and RSC with the nucleosome before and after chromatin remodeling; Sengupta SM et al.; Interactions of the yeast chromatin-remodeling complexes SWI/SNF and RSC with nucleosomes were probed using site-specific DNA photoaffinity labeling . 5 S rDNA was engineered with photoreactive nucleotides incorporated at different sites in DNA to scan for the subunits of SWI/SNF in close proximity to DNA when SWI/SNF is bound to the 5 S nucleosome or to the free 5 S rDNA . The Swi2/Snf2 and Snf6 subunits of SWI/SNF were efficiently cross-linked at several positions in the nucleosome, whereas only Snf6 was efficiently cross-linked when SWI/SNF was bound to free DNA . DNA photoaffinity labeling of RSC showed that the Rsc4 subunit is in close proximity to nucleosomal DNA and not when RSC is bound to free DNA . After remodeling, the Swi2/Snf2 and Rsc4 subunits are no longer detected near the nucleosomal DNA and are evidently displaced from the surface of the nucleosome, indicating significant changes in SWI/SNF and RSC contacts with DNA after remodeling.

J Mol Biol, 2001 Apr 20, 308(1), 49 - 58
Stoichiometry of the Sm proteins in yeast spliceosomal snRNPs supports the heptamer ring model of the core domain; Walke S et al.; Seven Sm proteins (B/B', D1, D2, D3, E, F and G proteins) containing a common sequence motif form a globular core domain within the U1, U2, U5 and U4/U6 spliceosomal snRNPs . Based on the crystal structure of two Sm protein dimers we have previously proposed a model of the snRNP core domain consisting of a ring of seven Sm proteins . This model postulates that there is only a single copy of each Sm protein in the core domain . In order to test this model we have determined the stoichiometry of the Sm proteins in yeast spliceosomal snRNPs . We have constructed seven different yeast strains each of which produces one of the Sm proteins tagged with a calmodulin-binding peptide (CBP) . Further, each of these strains was transformed with one of seven different plasmids coding for one of the seven Sm proteins tagged with protein A . When one Sm protein is expressed as a CBP-tagged protein from the chromosome and a second protein was produced with a protein A-tag from the plasmid, the protein A-tag was detected strongly in the fraction bound to calmodulin beads, demonstrating that two different tagged Sm proteins can be assembled into functional snRNPs . In contrast when the CBP and protein A-tagged forms of the same Sm protein were co-expressed, no protein A-tag was detectable in the fraction bound to calmodulin . These results indicate that there is only a single copy of each Sm protein in the spliceosomal snRNP core domain and therefore strongly support the heptamer ring model of the spliceosomal snRNP core domain .

Eur J Cell Biol, 2001 Feb, 80(2), 151 - 5
ERGIC-53 KKAA signal mediates endoplasmic reticulum retrieval in yeast; Dogic D et al.; Studies on the ERGIC-53 KKAA signal have revealed a new mechanism for static retention of mammalian proteins in the endoplasmic reticulum (Andersson, H., Kappeler, F., Hauri, H . P . (1999): Protein targeting to endoplasmic reticulum by dilysine signals involves direct retention in addition to retrieval . J . Biol . Chem . 274,15080 - 15084) . To test if this mechanism was conserved in yeast, the ERGIC-53 KKAA signal was transferred on two different yeast reporter proteins . Making use of a genetic assay, we demonstrate that this signal induces COPI-dependent ER retrieval . ER retention of KKAA-tagged proteins was impaired in yeast mutants affected in COPI subunits . Furthermore, biochemical analysis of post-ER carbohydrate modifications detected on reporter proteins indicated that KKAA-tagged proteins recycle continuously within early compartments of the secretory pathway . Therefore in yeast, the KKAA signal might only function as a classical dilysine ER retrieval signal.

Reprod Fertil Dev, 2000, 12(3-4), 191 - 9
Screening the foods of an endangered parrot, the kakapo (Strigops habroptilus), for oestrogenic activity using a recombinant yeast bioassay; Fidler AE et al.; In recent years the possibility of environmental oestrogens affecting the reproduction of vertebrates has become an issue of both public and scientific interest . Although the significance of such chemicals remains controversial there is clear evidence that, in some contexts, environmental oestrogens can influence the fertility of vertebrates . Highly endangered species represent a situation in which even modest reductions in the fertility of key individuals may have implications for the survival of the entire species . This paper reports the screening of both natural and supplementary foods of the kakapo (Strigops habroptilus), a critically endangered New Zealand nocturnal parrot, for oestrogenic activity using a recombinant yeast based bioassay . Low levels of oestrogenic activity were detected in one of the 'chick-raising' foods, but no oestrogenic activity was detected in the adult supplementary foods . The oestrogenicity of a range of phytochemicals possibly associated with the kakapo natural diet was also examined . Two such phytochemicals, podocarpic acid and its reduced derivative podocarpinol, showed weak oestrogenic activity (approximately 10(-6) and 10(-4) of the activity of 17-beta-oestradiol, respectively).

Neuron, 2001 Mar, 29(3), 657 - 67
Yeast screen for constitutively active mutant G protein-activated potassium channels; Yi BA et al.; GIRK2 is a major contributor to G protein-activated inward rectifier potassium channels in the mammalian brain . How GIRK channels open upon contact with Gbetagamma remains unknown . Using a yeast genetic screen to select constitutively active mutants from a randomly mutagenized GIRK2 library, we identified five gating mutations at four residues in the transmembrane domain . Further mutagenesis indicates that GIRK channel opening involves a rotation of the transmembrane segments, bringing one of these residues (V188) to a pore-lining position in the open conformation . Combined with double-mutant studies, these findings suggest that GIRK channels gate by moving from the open conformation inferred from our yeast study of Kir2.1 to a closed conformation perhaps resembling the known KcsA structure.

Arch Pathol Lab Med, 2001 May, 125(5), 663 - 4
Blastomyces dermatitidis with large yeast forms; Hussain Z et al.; Yeast forms of Blastomyces dermatitidis typically range from 8 to 20 microm in largest diameter . We report a rare case of primary pulmonary blastomycosis with an unusual morphology, in which we found significant numbers of large yeast forms ranging from 30 to 35 microm in diameter . To our knowledge, this is only the second reported case of giant forms of B dermatitidis . We also review the literature and discuss the possible association of this unusual morphology with immunosuppression in general and glucocorticoid use in particular.

Bioorg Chem, 2001 Apr, 29(2), 96 - 106
A reexamination of the substrate utilization of 2-thioorotidine-5'-monophosphate by yeast orotidine-5'-monophosphate decarboxylase; Smiley JA et al.; A potential alternate substrate for orotidine-5'-monophosphate decarboxylase, 2- thio-orotidine-5'-monophosphate, was synthesized enzymatically and purified by a modification of a previous account (K . Shostak, and M . E . Jones 1992, Biochemistry 31, 12155-12161) . Characterization of the product was confirmed by mass spectrometry, (31)P NMR, and utilization by orotate phosphoribosyltransferase in the direction of pyrophosphorolysis . The previous work probably did not result in the purification of the desired compound, as evidenced by our observation of 2-thioOMP's sensitivity to high temperature, as used previously . Using a very sensitive HPLC assay for the potential decarboxylated product 2-thioUMP, no measurable activity of ODCase toward the alternate substrate was observed, representing a decarboxylation rate decreased by 10(-7) from the k(cat) for ODCase toward OMP . Additionally, 2-thioOMP effects no inhibition of ODCase decarboxylation of OMP at a concentration of 50 microM, indicating a poor ability to bind to the ODCase active site . The results bear implications for proposed mechanisms for catalysis by ODCase .

Eur J Biochem, 2001 Apr, 268(8), 2351 - 61
A subfraction of the yeast endoplasmic reticulum associates with the plasma membrane and has a high capacity to synthesize lipids; Pichler H et al.; Large parts of the endoplasmic reticulum of the yeast, Saccharomyces cerevisiae, are located close to intracellular organelles, i.e . mitochondria and the plasma membrane, as shown by fluorescence and electron microscopy . Here we report the isolation and characterization of the subfraction of the endoplasmic reticulum that is closely associated with the plasma membrane . This plasma membrane associated membrane (PAM) is characterized by its high capacity to synthesize phosphatidylserine and phosphatidylinositol . As such, PAM is reminiscent of MAM, a mitochondria associated membrane fraction of the yeast {Gaigg, B., Simbeni, R., Hrastnik, C., Paltauf, F . & Daum, G . (1995) Biochim . Biophys . Acta 1234, 214-220}, although the specific activity of phosphatidylserine synthase and phosphatidylinositol synthase in PAM exceeds several-fold the activity in MAM and also in the bulk endoplasmic reticulum . In addition, several enzymes involved in ergosterol biosynthesis, namely squalene synthase (Erg9p), squalene epoxidase (Erg1p) and steroldelta24-methyltransferase (Erg6p), are highly enriched in PAM . A possible role of PAM in the supply of lipids to the plasma membrane is discussed.

J Appl Microbiol, 2001 Mar, 90(3), 372 - 9
Production of brown tyrosine pigments by the yeast Yarrowia lipolytica; Carreira A et al.; AIMS: To study the mechanism of production of brown pigments from tyrosine in the yeast Yarrowia lipolytica . METHODS AND RESULTS: Pigment formation was followed during growth in tyrosine medium, and the presence of the pigment precursor in the medium was assessed by evaluating pigment formation after removing the cells at different times of incubation . It was observed that the pigment precursor accumulated outside the cells during the exponential phase of growth, but pigment formation only occurred during the stationary phase of growth and resulted from the oxidation of the precursor . Pigment formation was repressed by glucose and L-glutamine, and promoted by lactic acid, L-asparagine and glycine . Spectra of 1H and 13C-NMR revealed that the brown pigment was derived from tyrosine and was a polymer composed of a core of aromatic residues . CONCLUSION: The results indicate that pigments result from the extracellular accumulation and auto-oxidation of an intermediate of tyrosine catabolism . SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the mechanism of pigment production from tyrosine in a yeast species.

FEBS Lett, 2001 Apr 6, 494(1-2), 79 - 84
Characterization of a novel human putative mitochondrial transporter homologous to the yeast mitochondrial RNA splicing proteins 3 and 4; Li FY et al.; We report here a novel human gene, hMRS3/4, encoding a putative mitochondrial transporter structurally and functionally homologous to the yeast mitochondrial RNA splicing proteins 3 and 4 . These proteins belong to the family of mitochondrial carrier proteins (MCF) and are likely to function as solute carriers . hMRS3/4 spans approximately 10 kb of genomic DNA on chromosome 10q24 and consists of four exons that encode a 364-aa protein with six transmembrane domains . A putative splice variant, encoding a 177-aa protein with three transmembrane domains, was also identified . hMRS3/4 has a well-conserved signature sequence of MCF and is targeted into the mitochondria . When expressed in yeast, hMRS3/4 efficiently restores the mitochondrial functions in mrs3(o)mrs4(o) knock-out mutants . Ubiquitous expression in human tissues and a well-conserved structure and function suggest an important role for hMRS3/4 in human cells.

J Gen Virol, 2001 May, 82(Pt 5), 1245 - 51
An Arabidopsis thaliana protein interacts with a movement protein of Turnip crinkle virus in yeast cells and in vitro; Lin B et al.; Plant virus movement proteins bind host components to promote virus movement from initially infected cells to neighbouring cells . In this study, cDNA clones encoding p8 and p9, two small proteins required for the movement of Turnip crinkle virus, were used as 'bait' in a yeast two-hybrid system to screen an Arabidopsis thaliana cDNA library for interactive proteins . One A . thaliana clone was identified that encodes a protein, designated Atp8, which interacted with p8 in yeast cells and in vitro . The apparent full-length of Atp8 mRNA was sequenced and shown to encode a protein with two possible transmembrane helices, several potential phosphorylation sites and two 'RGD' sequences.

J Biol Chem, 2001 Apr 27, 276(17), 13579 - 86 Epub 2001 Jan 31.
Its8, a fission yeast homolog of Mcd4 and Pig-n, is involved in GPI anchor synthesis and shares an essential function with calcineurin in cytokinesis; Yada T et al.; In fission yeast, calcineurin is required for cytokinesis and ion homeostasis; however, most of its physiological roles remain obscure . To identify genes that share an essential function with calcineurin, we screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and high temperature and isolated the mutant its8-1 . its8(+) encodes a homolog of the budding yeast MCD4 and human Pig-n that are involved in glycosylphosphatidylinositol (GPI) anchor synthesis . Consistently, reduced inositol labeling of proteins suggested impaired GPI anchor synthesis in its8-1 mutants . The temperature upshift induced a further decrease in inositol labeling and caused dramatic increases in the frequency of septation in its8-1 mutants . BE49385A, an inhibitor of MCD4 and Pig-n, also increased the septation index of the wild-type cell . Osmotic stabilization suppressed these morphological defects, indicating that cell wall weakness caused by impaired GPI anchor synthesis resulted in abnormal cytokinesis . Furthermore, calcineurin-deleted cells exhibited hypersensitivity to BE49385A, and FK506 exacerbated the cytokinesis defects of the its8-1 mutant . Thus, calcineurin and Its8 may share an essential function in cytokinesis and cell viability through the regulation of cell wall integrity.

Biochemistry, 2001 Mar 27, 40(12), 3629 - 38
Altering the intermediate in the equilibrium folding of unmodified yeast tRNAPhe with monovalent and divalent cations; Shelton VM et al.; The isothermal equilibrium folding of the unmodified yeast tRNA(Phe) is studied as a function of Na(+), Mg(2+), and urea concentration with hydroxyl radical protection, circular dichroism, and diethyl pyrocarbonate (DEPC) modification . These assays indicate that this tRNA folds in Na(+) alone . Similar to folding in Mg(2+), folding in Na(+) can be described by two transitions, unfolded-to-intermediate-to-native . The I-to-N transition has a Na(+) midpoint of approximately 0.5 M and a Hill constant of approximately 4 . Unexpectedly, the urea m-value, the dependence of free energy on urea concentration, for the I-to-N transition is significantly smaller in Na(+) than in Mg(2+), 0.4 versus 1.7 kcal mol(-1) M(-1), indicating that more structure is formed in the Mg(2+)-induced transition . DEPC modification indicates that the I state in Na(+)-induced folding contains all four helices of tRNA and the I-to-N transition primarily corresponds to the formation of the tertiary structure . In contrast, the intermediate in Mg(2+)-induced folding contains only three helices, and the I-to-N transition corresponds to the formation of the acceptor stem plus tertiary structure . The cation dependence of the intermediates arises from the differences in the stability of the acceptor stem and the tertiary structure . The acceptor stem is stable at a lower Na(+) concentration than required for the tertiary structure formation . The relative stability is reversed in Mg(2+) so that the acceptor stem and the tertiary structure form simultaneously in the I-to-N transition . These results demonstrate that formation of the RNA secondary structure can be independent or coupled to the formation of the tertiary structure depending on their relative stability in monovalent and divalent ions.

Clin Cancer Res, 2001 Mar, 7(3), 600 - 6
Detection of p53 gene mutations in human esophageal squamous cell carcinomas using a p53 yeast functional assay: possible difference in esophageal carcinogenesis between the young and the elderly group; Okuda E et al.; A p53 yeast functional assay, which cannot only detect p53 gene mutations but also can assess p53 gene function, was used to screen for p53 gene dysfunction in human esophageal squamous cell carcinomas . Surgically resected frozen tissues of esophageal squamous cell carcinomas from 57 patients were examined for p53 gene mutation . Because the mean age of the patients diagnosed with esophageal squamous cell carcinoma was 64 years, we classified those who were <65 years of age as the Young Group and classified the others as the Elderly Group . The incidence of p53 gene mutations was 43 of 57 (75%) . The incidence of p53 gene mutations observed in the Young Group was significantly higher than in the Elderly Group (P = 0.0007) . Alcohol and smoking status did not relate to p53 gene mutation expression . Survival rate after surgery was not significantly associated with the presence of p53 gene mutation . However, in the Young Group with p53 gene mutation, those who had null mutations had a significantly shorter survival than those without null mutations (P = 0.0455) . No other clinicopathological factors were associated with p53 gene mutations . Possibly, there may be a difference in esophageal carcinogenesis between the Young and the Elderly groups, because the incidence of p53 gene mutations is different between the two groups . In the Young Group, p53 gene mutation may cause esophageal carcinogenesis, and null mutation for p53 gene is a significant prognostic factor.

Microbios, 2001, 104(408), 99 - 104
Induction for the expression of yeast metallothionein gene, CUP1, by cobalt; Tohoyama H et al.; Induction for the expression of the metallothionein gene, CUP1, in the yeast Saccharomyces cerevisiae by cobalt was examined using a reporter gene with the promoter of this gene fused to the coding region of lacZ . The expression of the gene was induced by cobalt as well as by copper and silver ions . The activity of beta-galactosidase showed high levels after treatment with 1.0 mM cobalt chloride . It has been reported that the induction for the transcription of CUP1 by copper and silver is mediated by the Ace1 transcription factor . However, the expression of the gene by cobalt occurred in yeast cells lacking the Ace1 factor . These results suggest the presence of a novel cobalt-specific transcription factor for the CUP1 gene.

Biochimie, 2001 Mar-Apr, 83(3-4), 333 - 40
Genetic dissection of the yeast 26S proteasome: cell cycle defects caused by the Deltarpn9 mutation; Takeuchi J et al.; Rpn9 is one of the subunits of the regulatory particle of the yeast 26S proteasome and is needed for stability or efficient assembly of the 26S proteasome . As anticipated from the fact that the rpn9 disruptant grew at 25 degrees C but arrested in G2/M phase at 37 degrees C, the CDK inhibitor Sic1p was found to be degraded at the G1/S boundary in the Deltarpn9 cells . The degradation of the anaphase inhibitor Pds1p was delayed in the Deltarpn9 cells . Clb2p in M phase, as well as that ectopically expressed in G1 and S phases, was degraded more slowly in the Deltarpn9 cells than in the wild type cells, indicating that the 26S proteasome lacking Rpn9 uses Sic1p as a better substrate than Pds1p and Clb2p . These results, in addition to the fact that multiubiquitinated proteins were accumulated in the Deltarpn9 cells incubated at 37 degrees C, strongly suggest that Rpn9 is involved in the proteolysis of a subset of the substrates degraded by the 26S proteasome . The Deltarpn9 Deltapds1 double mutant was unable to elongate spindle at a restrictive temperature, suggesting that some protein(s) other than Scc1 (cohesin) should be degraded during progression of anaphase.

Mol Biol Cell, 2001 Apr, 12(4), 1177 - 88
Cadherin sequences that inhibit beta-catenin signaling: a study in yeast and mammalian cells; Simcha I et al.; Drosophila Armadillo and its mammalian homologue beta-catenin are scaffolding proteins involved in the assembly of multiprotein complexes with diverse biological roles . They mediate adherens junction assembly, thus determining tissue architecture, and also transduce Wnt/Wingless intercellular signals, which regulate embryonic cell fates and, if inappropriately activated, contribute to tumorigenesis . To learn more about Armadillo/beta-catenin's scaffolding function, we examined in detail its interaction with one of its protein targets, cadherin . We utilized two assay systems: the yeast two-hybrid system to study cadherin binding in the absence of Armadillo/beta-catenin's other protein partners, and mammalian cells where interactions were assessed in their presence . We found that segments of the cadherin cytoplasmic tail as small as 23 amino acids bind Armadillo or beta-catenin in yeast, whereas a slightly longer region is required for binding in mammalian cells . We used mutagenesis to identify critical amino acids required for cadherin interaction with Armadillo/beta-catenin . Expression of such short cadherin sequences in mammalian cells did not affect adherens junctions but effectively inhibited beta-catenin-mediated signaling . This suggests that the interaction between beta-catenin and T cell factor family transcription factors is a sensitive target for disruption, making the use of analogues of these cadherin derivatives a potentially useful means to suppress tumor progression.

Mol Biol Cell, 2001 Apr, 12(4), 1117 - 29
Evidence for an intrinsic toxicity of phosphatidylcholine to Sec14p-dependent protein transport from the yeast Golgi complex; Xie Z et al.; Yeast phosphatidylinositol-transfer protein (Sec14p) is essential for Golgi secretory function and cell viability . This requirement of Sec14p is relieved by genetic inactivation of the cytidine diphosphate-choline pathway for phosphatidycholine (PtdCho) biosynthesis . Standard phenotypic analyses indicate that inactivation of the phosphatidylethanolamine (PtdEtn) pathway for PtdCho biosynthesis, however, does not rescue the growth and secretory defects associated with Sec14p deficiency . We now report inhibition of choline uptake from the media reveals an efficient "bypass Sec14p" phenotype associated with PtdEtn-methylation pathway defects . We further show that the bypass Sec14p phenotype associated with PtdEtn-methylation pathway defects resembles other bypass Sec14p mutations in its dependence on phospholipase D activity . Finally, we find that increased dosage of enzymes that catalyze phospholipase D-independent turnover of PtdCho, via mechanisms that do not result in a direct production of phosphatidic acid or diacylglycerol, effect a partial rescue of sec14-1(ts)-associated growth defects . Taken together, these data support the idea that PtdCho is intrinsically toxic to yeast Golgi secretory function.

Mol Biol Cell, 2001 Apr, 12(4), 1047 - 59
Uptake of the ATP-binding cassette (ABC) transporter Ste6 into the yeast vacuole is blocked in the doa4 Mutant; Losko S et al.; Previous experiments suggested that trafficking of the a-factor transporter Ste6 of Saccharomyces cerevisiae to the yeast vacuole is regulated by ubiquitination . To define the ubiquitination-dependent step in the trafficking pathway, we examined the intracellular localization of Ste6 in the ubiquitination-deficient doa4 mutant by immunofluorescence experiments, with a Ste6-green fluorescent protein fusion protein and by sucrose density gradient fractionation . We found that Ste6 accumulated at the vacuolar membrane in the doa4 mutant and not at the cell surface . Experiments with a doa4 pep4 double mutant showed that Ste6 uptake into the lumen of the vacuole is inhibited in the doa4 mutant . The uptake defect could be suppressed by expression of additional ubiquitin, indicating that it is primarily the result of a lowered ubiquitin level (and thus of reduced ubiquitination) and not the result of a deubiquitination defect . Based on our findings, we propose that ubiquitination of Ste6 or of a trafficking factor is required for Ste6 sorting into the multivesicular bodies pathway . In addition, we obtained evidence suggesting that Ste6 recycles between an internal compartment and the plasma membrane.

Mol Biol Cell, 2001 Apr, 12(4), 957 - 69
Deletion of yeast p24 genes activates the unfolded protein response; Belden WJ et al.; Yeast cells lacking a functional p24 complex accumulate a subset of secretory proteins in the endoplasmic reticulum (ER) and increase the extracellular secretion of HDEL-containing ER residents such as Kar2p/BiP . We report that a loss of p24 function causes activation of the unfolded protein response (UPR) and leads to increased KAR2 expression . The HDEL receptor (Erd2p) is functional and traffics in p24 deletion strains as in wild-type strains, however the capacity of the retrieval pathway is exceeded . Other conditions that activate the UPR and elevate KAR2 expression also lead to extracellular secretion of Kar2p . Using an in vitro assay that reconstitutes budding from the ER, we detect elevated levels of Kar2p in ER-derived vesicles from p24 deletion strains and from wild-type strains with an activated UPR . Silencing the UPR by IRE1 deletion diminished Kar2p secretion under these conditions . We suggest that activation of the UPR plays a major role in extracellular secretion of Kar2p.

Nucleic Acids Res, 2001 Apr 15, 29(8), 1772 - 80
Identification of the yeast cytidine deaminase CDD1 as an orphan C-->U RNA editase; Dance GS et al.; Yeast co-expressing rat APOBEC-1 and a fragment of human apolipoprotein B (apoB) mRNA assembled functional editosomes and deaminated C6666 to U in a mooring sequence-dependent fashion . The occurrence of APOBEC-1-complementing proteins suggested a naturally occurring mRNA editing mechanism in yeast . Previously, a hidden Markov model identified seven yeast genes encoding proteins possessing putative zinc-dependent deaminase motifs . Here, only CDD1, a cytidine deaminase, is shown to have the capacity to carry out C-->U editing on a reporter mRNA . This is only the second report of a cytidine deaminase that can use mRNA as a substrate . CDD1-dependent editing was growth phase regulated and demonstrated mooring sequence-dependent editing activity . Candidate yeast mRNA substrates were identified based on their homology with the mooring sequence-containing tripartite motif at the editing site of apoB mRNA and their ability to be edited by ectopically expressed APOBEC-1 . Naturally occurring yeast mRNAs edited to a significant extent by CDD1 were, however, not detected . We propose that CDD1 be designated an orphan C-->U editase until its native RNA substrate, if any, can be identified and that it be added to the CDAR (cytidine deaminase acting on RNA) family of editing enzymes.

Nucleic Acids Res, 2001 Apr 15, 29(8), 1715 - 23
The wing in yeast heat shock transcription factor (HSF) DNA-binding domain is required for full activity; Cicero MP et al.; The yeast heat shock transcription factor (HSF) belongs to the winged helix family of proteins . HSF binds DNA as a trimer, and additional trimers can bind DNA co-operatively . Unlike other winged helix-turn-helix proteins, HSF's wing does not appear to contact DNA, as based on a previously solved crystal structure . Instead, the structure implies that the wing is involved in protein-protein interactions, possibly within a trimer or between adjacent trimers . To understand the function of the wing in the HSF DNA-binding domain, a Saccharomyces cerevisiae strain was created that expresses a wingless HSF protein . This strain grows normally at 30 degrees C, but shows a decrease in reporter gene expression during constitutive and heat-shocked conditions . Removal of the wing does not affect the stability or trimeric nature of a protein fragment containing the DNA-binding and trimerization domains . Removal of the wing does result in a decrease in DNA-binding affinity . This defect was mainly observed in the ability to form the first trimer-bound complex, as the formation of larger complexes is unaffected by the deletion . Our results suggest that the wing is not involved in the highly co-operative nature of HSF binding, but may be important in stabilizing the first trimer bound to DNA.

Plant Mol Biol, 2001 Feb, 45(3), 365 - 76
Further analysis of the interactions between the Brassica S receptor kinase and three interacting proteins (ARC1, THL1 and THL2) in the yeast two-hybrid system; Mazzurco M et al.; The yeast two-hybrid system was used to further characterize the interactions between the Brassica S receptor kinase (SRK) and three putative substrates, ARC1 and the two thioredoxin h proteins, THL1 and THL2 . Interactions were generally detectable with kinase domains of both Class I and Class II SRKs . Chimeric constructs were made between the SRK910 kinase domain and the non-interacting Arabidopsis RLK5 kinase domain . Only one chimeric construct, SRR2, interacted with THL1 and THL2, while none of the chimeras were able to interact with ARC1 . SRR2 is largely made up of RLK5 kinase domain with the N-terminal end being derived from the SRK910 kinase domain and was the only chimeric construct that retained kinase activity . Deletion or substitution of a conserved cysteine at the N-terminal end of the SRK910 kinase domain resulted in loss of interaction with THL1 and THL2, while the addition of this cysteine to a related receptor kinase, SFR1, conferred the ability to interact with the thioredoxin h proteins . In addition, substitution of the cysteines in the THL1 active site abolished the interaction . Lastly, the two Arabidopsis thioredoxin h clones most closely related to THL1 and THL2 were found to interact with the SRK kinase domains . Thus, the nature of the interaction of the thioredoxin h clones with SRK involves the reducing activity of these proteins and is restricted to the class of thioredoxin h proteins which have the variant CPPC active site.

Genetics, 2001 Apr, 157(4), 1425 - 36
Overexpression of translation elongation factor 1A affects the organization and function of the actin cytoskeleton in yeast; Munshi R et al.; The translation elongation factor 1 complex (eEF1) plays a central role in protein synthesis, delivering aminoacyl-tRNAs to the elongating ribosome . The eEF1A subunit, a classic G-protein, also performs roles aside from protein synthesis . The overexpression of either eEF1A or eEF1B alpha, the catalytic subunit of the guanine nucleotide exchange factor, in Saccharomyces cerevisiae results in effects on cell growth . Here we demonstrate that overexpression of either factor does not affect the levels of the other subunit or the rate or accuracy of protein synthesis . Instead, the major effects in vivo appear to be at the level of cell morphology and budding . eEF1A overexpression results in dosage-dependent reduced budding and altered actin distribution and cellular morphology . In addition, the effects of excess eEF1A in actin mutant strains show synthetic growth defects, establishing a genetic connection between the two proteins . As the ability of eEF1A to bind and bundle actin is conserved in yeast, these results link the established ability of eEF1A to bind and bundle actin in vitro with nontranslational roles for the protein in vivo.

Proc Natl Acad Sci U S A, 2001 Apr 10, 98(8), 4391 - 6 Epub 2001 Apr 03.
HDA2 and HDA3 are related proteins that interact with and are essential for the activity of the yeast histone deacetylase HDA1; Wu J et al.; Histone deacetylase HDA1, the prototype for the class II mammalian deacetylases, is likely the catalytic subunit of the HDA1-containing complex that is involved in TUP1-specific repression and global deacetylation in yeast . Although the class I RPD3-like enzymatic complexes have been well characterized, little is known about the identity and interactions of the factors that associate to form the HDA1 complex . In this paper, we identify related HDA2 and HDA3 proteins that are found in the HDA1 complex and show that HDA1 interacts with itself and with the HDA2-HDA3 subcomplex to form a likely tetramer . These interactions are necessary for catalytic activity because mutations in any of the three components disrupt activity both in vitro and in vivo . In this respect the HDA1 complex differs from yeast RPD3, which has components such as SIN3 that are not essential for activity in vitro, and yeast HOS3, which has intrinsic in vitro activity as a homodimer in the absence of other subunits.

Mol Cell Biol, 2001 May, 21(9), 3105 - 17
Skp1p and the F-box protein Rcy1p form a non-SCF complex involved in recycling of the SNARE Snc1p in yeast; Galan JM et al.; Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin-ligases composed of a core complex including Skp1p, Cdc53p, Hrt1p, the E2 enzyme Cdc34p, and one of multiple F-box proteins which are thought to provide substrate specificity to the complex . Here we show that the F-box protein Rcy1p is required for recycling of the v-SNARE Snc1p in Saccharomyces cerevisiae . Rcy1p localized to areas of polarized growth, and this polarized localization required its CAAX box and an intact actin cytoskeleton . Rcy1p interacted with Skp1p in vivo in an F-box-dependent manner, and both deletion of its F box and loss of Skp1p function impaired recycling . In contrast, cells deficient in Cdc53p, Hrt1p, or Cdc34p did not exhibit recycling defects . Unlike the case for F-box proteins that are known to participate in SCF complexes, degradation of Rcy1p required neither its F box nor functional 26S proteasomes or other SCF core subunits . Importantly, Skp1p was the only major partner that copurified with Rcy1p . Our results thus suggest that a complex composed of Rcy1p and Skp1p but not other SCF components may play a direct role in recycling of internalized proteins.

Mol Cell Biol, 2001 May, 21(9), 3037 - 46
A novel yeast U2 snRNP protein, Snu17p, is required for the first catalytic step of splicing and for progression of spliceosome assembly; Gottschalk A et al.; We have isolated and microsequenced Snu17p, a novel yeast protein with a predicted molecular mass of 17 kDa that contains an RNA recognition motif . We demonstrate that Snu17p binds specifically to the U2 small nuclear ribonucleoprotein (snRNP) and that it is part of the spliceosome, since the pre-mRNA and the lariat-exon 2 are specifically coprecipitated with Snu17p . Although the SNU17 gene is not essential, its knockout leads to a slow-growth phenotype and to a pre-mRNA splicing defect in vivo . In addition, the first step of splicing is dramatically decreased in extracts prepared from the snu17 deletion (snu17Delta) mutant . This defect is efficiently reversed by the addition of recombinant Snu17p . To investigate the step of spliceosome assembly at which Snu17p acts, we have used nondenaturing gel electrophoresis . In Snu17p-deficient extracts, the spliceosome runs as a single slowly migrating complex . In wild-type extracts, usually at least two distinct complexes are observed: the prespliceosome, or B complex, containing the U2 but not the U1 snRNP, and the catalytically active spliceosome, or A complex, containing the U2, U6, and U5 snRNPs . Northern blot analysis and affinity purification of the snu17Delta spliceosome showed that it contains the U1, U2, U6, U5, and U4 snRNPs . The unexpected stabilization of the U1 snRNP and the lack of dissociation of the U4 snRNP suggest that loss of Snu17p inhibits the progression of spliceosome assembly prior to U1 snRNP release and after {U4/U6.U5} tri-snRNP addition.

Glycobiology, 2001 Feb, 11(2), 113 - 24
Characterization of cerebrosides from the thermally dimorphic mycopathogen Histoplasma capsulatum: expression of 2-hydroxy fatty N-acyl (E)-Delta(3)-unsaturation correlates with the yeast-mycelium phase transition; Toledo MS et al.; Cerebroside (monohexosylceramide) components were identified in neutral lipids extracted from both the yeast and mycelial forms of the thermally dimorphic mycopathogen Histoplasma capsulatum . The components were purified from both forms and their structures elucidated by 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and low energy tandem collision-induced dissociation mass spectrometry (ESI-MS/CID-MS) . Both components were characterized as beta-glucopyranosylceramides (GlcCers) containing (4E,8E)-9-methyl-4,8-sphingadienine as the long-chain base, attached to 18-carbon 2-hydroxy fatty N-acyl components . However, while the fatty acid of the yeast form GlcCer was virtually all N-2'-hydroxyoctadecanoate, the mycelium form GlcCer was characterized by almost exclusive expression of N-2'-hydroxy-(E)-delta(3)-octadecenoate . These results suggest that the yeast-mycelium transition is accompanied by up-regulation of an as yet uncharacterized ceramide or cerebroside 2-hydroxy fatty N-acyl (E)-delta(3)-desaturase activity . They also constitute further evidence for the existence of two distinct pathways for ceramide biosynthesis in fungi, since glycosylinositol phosphorylceramides (GIPCs), the other major class of fungal glycosphingolipids, are found with ceramides consisting of 4-hydroxysphinganine (phytosphingosine) and longer chain 2-hydroxy fatty acids . In addition to identification of the major glucocerebroside components, minor components (< 5%) detectable by molecular weight differences in the ESI-MS profiles were also characterized by tandem ESI-MS/CID-MS analysis . These minor components were identified as variants differing in fatty acyl chain length, or the absence of the sphingoid 9-methyl group or (E)-delta(8)-unsaturation, and are hypothesized to be either biosynthetic intermediates or the result of imperfect chemical transformation by the enzymes responsible for these features . Possible implications of these findings with respect to chemotaxonomy, compartmentalization of fungal glycosphingolipid biosynthetic pathways, and regulation of morphological transitions in H.capsulatum and other dimorphic fungi are discussed.

J Cell Biol, 2001 Apr 2, 153(1), 159 - 68
The surveillance mechanism of the spindle position checkpoint in yeast; Adames NR et al.; The spindle position checkpoint in Saccharomyces cerevisiae delays mitotic exit until the spindle has moved into the mother-bud neck, ensuring that each daughter cell inherits a nucleus . The small G protein Tem1p is critical in promoting mitotic exit and is concentrated at the spindle pole destined for the bud . The presumed nucleotide exchange factor for Tem1p, Lte1p, is concentrated in the bud . These findings suggested the hypothesis that movement of the spindle pole through the neck allows Tem1p to interact with Lte1p, promoting GTP loading of Tem1p and mitotic exit . However, we report that deletion of LTE1 had little effect on the timing of mitotic exit . We also examined several mutants in which some cells inappropriately exit mitosis even though the spindle is within the mother . In some of these cells, the spindle pole body did not interact with the bud or the neck before mitotic exit . Thus, some alternative mechanism must exist to coordinate mitotic exit with spindle position . In both wild-type and mutant cells, mitotic exit was preceded by loss of cytoplasmic microtubules from the neck . Thus, the spindle position checkpoint may monitor such interactions.

Genes Chromosomes Cancer, 2001 May, 31(1), 42 - 7
Refinement within single yeast artificial chromosome clones of a minimal region commonly deleted on the short arm of chromosome 7 in Wilms tumours; Perotti D et al.; Cytogenetic and molecular data indicate an involvement of genes mapped to the proximal portion of the short arm of chromosome 7 (7p) in Wilms tumours (WTs) . We have analysed 38 WTs using a panel of eight microsatellite markers mapped to proximal 7p . Loss of heterozygosity (LOH) in tumour, compared with matched constitutional DNA, was identified in eight cases . To define better the minimal region commonly deleted in these tumours, they were analysed with nine additional markers, mapped within the region of interest . One tumour (case 30) showed LOH for only one marker (D7S510), while maintaining heterozygosity for the two immediately flanking loci (D7S555 and D7S668) . This result was confirmed by fluorescence in situ hybridisation analysis, which showed that in the majority (65%) of nuclei from tumour 30 hybridising with a bacterial artificial chromosome clone containing the D7S510 locus, only one signal was visible . Noticeably, both markers defining the limits of the observed deleted region are simultaneously present within two distinct overlapping yeast artificial chromosome (YAC) clones mapped to chromosome bands 7p13-p14 . This suggests that the maximum length of the missing DNA fragment was approximately 1.3 Mb, corresponding to the length of the smaller of the two YAC clones . In all other cases that showed LOH, the deletion encompassed the 7p13-p14 region . For this reason, we speculate that the identified interval contains a gene whose inactivation is important for the development of at least a fraction of WTs .

Yeast, 2001 Apr, 18(6), 563 - 75
A new family of yeast vectors and S288C-derived strains for the systematic analysis of gene function; Tomlin GC et al.; The yeast genome has been shown to contain a significant number of gene families with more than three members . In order to study these families it is often necessary to generate strains carrying deletions of all members of the family, which can require a wide range of auxotrophic markers . To facilitate such studies, we have generated yeast strains containing deletions of a selection of nutritional marker genes (ade2, ade4, ade8, met3 and met14) . We have also cloned the corresponding cognate genes, allowing their use in PCR-based gene disruptions . Two new pRS family Saccharomyces cerevisiae-Escherichia coli shuttle vectors containing ADE8 (one low-copy, pRS4110, and one high-copy, pRS4210) have been produced for use in conjunction with the new strains . A system for easier synthetic lethal screening using one of these new markers is also presented . The ADE8 and HIS3 genes have been cloned together on a high-copy vector (pRS4213), providing a plasmid for red-white colour screening in the ade2 Delta 0 ade8 Delta 0 strains we have generated . In contrast to some conventional systems, this plasmid allows for screening using gene libraries constructed in URA3 plasmids .

Yeast, 2001 Apr, 18(6), 499 - 509
Chronological lifespan of stationary phase yeast cells; a model for investigating the factors that might influence the ageing of postmitotic tissues in higher organisms; MacLean M et al.; Budding yeast can be considered to have two distinct lifespans: (a) a replicative (budding, non-chronological) lifespan, measured as the number of daughters produced by each actively dividing mother cell; and (ii) a chronological lifespan, measured as the ability of stationary cultures to maintain viability over time . In non-dividing cells, essential components that become damaged cannot be diluted out through cell division but must, of necessity, be turned over and renewed . By elevating stress resistances, many of the activities needed for such renewal should be elevated with commensurate reduction in the steady-state levels of damaged cell components . Therefore, chronological lifespan in particular might be expected to relate to stress resistance . For yeast to attain a full chronological lifespan requires the expression of the general stress response . It is more important, though, that the cells should be efficiently adapted to respiratory maintenance, since it is cultures grown to stationary phase on respiratory media that usually display the longest chronological lifespans . For this reason, respiration-adapted cells potentially provide a better model of chronological ageing than cultures pre-grown on glucose .

Yeast, 2001 Apr, 18(6), 489 - 97
{Psi(+)} prion generation in yeast: characterization of the 'strain' difference; Kochneva-Pervukhova NV et al.; The yeast cytoplasmically-inherited nonsense suppressor {PSI(+)} determinant is presumed to be a manifestation of the aggregated prion-like state of the Sup35 protein . Overexpression of the Sup35 protein induces generation of {PSI(+)} determinants with various suppressor efficiency and mitotic stabilities . Here, we demonstrate that the relative frequency of appearance of {PSI(+)} with different properties depends on the SUP35 allele used to induce their generation . The difference in properties of {PSI(+)} determinants was preserved after their transmission from one yeast strain to another . This difference correlated with variation in properties of the Sup35 protein . A novel type of prion instability was observed: some {PSI(+)} with weak suppressor efficiency could convert spontaneously into strong suppressor determinants .

Nat Rev Genet, 2001 Apr, 2(4), 302 - 12
Emerging technologies in yeast genomics; Kumar A et al.; The genomic revolution is undeniable: in the past year alone, the term 'genomics' was found in nearly 500 research articles, and at least 6 journals are devoted solely to genomic biology . More than just a buzzword, molecular biology has genuinely embraced genomics (the systematic, large-scale study of genomes and their functions) . With its facile genetics, the budding yeast Saccharomyces cerevisiae has emerged as an important model organism in the development of many current genomic methodologies . These techniques have greatly influenced the manner in which biology is studied in yeast and in other organisms . In this review, we summarize the most promising technologies in yeast genomics.

Proc Natl Acad Sci U S A, 2001 Apr 10, 98(8), 4569 - 74 Epub 2001 Mar 13.
A comprehensive two-hybrid analysis to explore the yeast protein interactome; Ito T et al.; Protein-protein interactions play crucial roles in the execution of various biological functions . Accordingly, their comprehensive description would contribute considerably to the functional interpretation of fully sequenced genomes, which are flooded with novel genes of unpredictable functions . We previously developed a system to examine two-hybrid interactions in all possible combinations between the approximately 6,000 proteins of the budding yeast Saccharomyces cerevisiae . Here we have completed the comprehensive analysis using this system to identify 4,549 two-hybrid interactions among 3,278 proteins . Unexpectedly, these data do not largely overlap with those obtained by the other project {Uetz, P., et al . (2000) Nature (London) 403, 623-627} and hence have substantially expanded our knowledge on the protein interaction space or interactome of the yeast . Cumulative connection of these binary interactions generates a single huge network linking the vast majority of the proteins . Bioinformatics-aided selection of biologically relevant interactions highlights various intriguing subnetworks . They include, for instance, the one that had successfully foreseen the involvement of a novel protein in spindle pole body function as well as the one that may uncover a hitherto unidentified multiprotein complex potentially participating in the process of vesicular transport . Our data would thus significantly expand and improve the protein interaction map for the exploration of genome functions that eventually leads to thorough understanding of the cell as a molecular system.

Plant Cell, 2001 Apr, 13(4), 781 - 92
Demonstration in yeast of the function of BP-80, a putative plant vacuolar sorting receptor; Humair D et al.; BP-80, later renamed VSR(PS-1), is a putative receptor involved in sorting proteins such as proaleurain to the lytic vacuole, with its N-terminal domain recognizing the vacuolar sorting determinant . Although all VSR(PS-1) characteristics and in vitro binding properties described so far favored its receptor function, this function remained to be demonstrated . Here, we used green fluorescent protein (GFP) as a reporter in a yeast mutant strain defective for its own vacuolar receptor, Vps10p . By expressing VSR(PS-1) together with GFP fused to the vacuolar sorting determinant of petunia proaleurain, we were able to efficiently redirect the reporter to the yeast vacuole . VSR(PS-1) is ineffective on GFP either alone or when fused with another type of plant vacuolar sorting determinant from a chitinase . The plant VSR(PS-1) therefore interacts specifically with the proaleurain vacuolar sorting determinant in vivo, and this interaction leads to the transport of the reporter protein through the yeast secretory pathway to the vacuole . This finding demonstrates VSR(PS-1) receptor function but also emphasizes the differences in the spectrum of ligands between Vps10p and its plant equivalent.

Mol Cell Biol, 2001 Apr, 21(8), 2736 - 42
Yeast NC2 associates with the RNA polymerase II preinitiation complex and selectively affects transcription in vivo; Geisberg JV et al.; NC2 (Dr1-Drap1 or Bur6-Ydr1) has been characterized in vitro as a general negative regulator of RNA polymerase II (Pol II) transcription that interacts with TATA-binding protein (TBP) and inhibits its function . Here, we show that NC2 associates with promoters in vivo in a manner that correlates with transcriptional activity and with occupancy by basal transcription factors . NC2 rapidly associates with promoters in response to transcriptional activation, and it remains associated under conditions in which transcription is blocked after assembly of the Pol II preinitiation complex . NC2 positively and negatively affects approximately 17% of Saccharomyces cerevisiae genes in a pattern that resembles the response to general environmental stress . Relative to TBP, NC2 occupancy is high at promoters where NC2 is positively required for normal levels of transcription . Thus, NC2 is associated with the Pol II preinitiation complex, and it can play a direct and positive role at certain promoters in vivo.

J Biol Chem, 2001 Jun 15, 276(24), 21184 - 91 Epub 2001 Mar 29.
Functional domains of the yeast splicing factor Prp22p; Schneider S et al.; The essential Saccharomyces cerevisiae PRP22 gene encodes a 1145-amino acid DEXH box RNA helicase . Prp22p plays two roles during pre-mRNA splicing as follows: it is required for the second transesterification step and for the release of mature mRNA from the spliceosome . Whereas the step 2 function of Prp22p does not require ATP hydrolysis, spliceosome disassembly is dependent on the ATPase and helicase activities . Here we delineate a minimal functional domain, Prp22(262-1145), that suffices for the activity of Prp22p in vivo when expressed under the natural PRP22 promoter and for pre-mRNA splicing activity in vitro . The biologically active domain lacks an S1 motif (residues 177-256) that had been proposed to play a role in RNA binding by Prp22p . The deletion mutant Prp22(351-1145) can function in vivo when provided at a high gene dosage . We suggest that the segment from residues 262 to 350 enhances Prp22p function in vivo, presumably by targeting Prp22p to the spliceosome . We characterize an even smaller catalytic domain, Prp22(466-1145) that suffices for ATP hydrolysis, RNA binding, and RNA unwinding in vitro and for nuclear localization in vivo but cannot by itself support cell growth . However, the ATPase/helicase domain can function in vivo if the N-terminal region Prp22(1-480) is co-expressed in trans.

J Cell Sci, 2001 Apr, 114(Pt 8), 1505 - 13
The life cycle of actin patches in mating yeast; Smith MG et al.; Actin patches are core components of the yeast actin cytoskeleton that undergo redistribution during establishment of cell polarity . Using 4D imaging, we observe the life cycle of actin patches in living yeast for the first time . We observe assembly of actin patches at sites of polarized growth, and disassembly of actin patches concomitant with movement away from those sites . The total lifetime of an actin patch is 10.9+/-4.2 seconds . These findings indicate that actin patches are labile structures, and that the localization of actin patches during establishment of cell polarity occurs by assembly of these structures at sites of polarized cell surface growth . These findings were confirmed and extended by analysis of myosin I proteins and their receptor, verprolin, proteins implicated in actin assembly in yeast . Deletion of type I myosins or their receptor has no effect on the velocity of actin patch movement . However, these mutants show a 65% reduction in number of patch movements and a three-fold increase in patch lifetime . Finally, the actin patch resident proteins Abp1p, fimbrin, and Arp2p show normal association with actin patches in myosin I and verprolin mutants . However, cofilin accumulates in abnormal 'bars' of G-actin in myo3Delta,myo5Delta and vrp1Delta strains, and Las17p/Bee1p is not associated with actin patches in vrp1Delta strains . These findings imply a multi-step process for actin patch assembly . Early events in this process, including assembly of Abp1p, fimbrin and Arp2p with F-actin, can occur throughout the cell and do not require myosin I proteins or their receptor . Later events in this process are myosin I-dependent, and are required for assembly of actin patches at sites of polarized cell surface growth.

Exp Parasitol, 2001 Feb, 97(2), 61 - 9
Plasmodium falciparum: immunogenicity of alum-adsorbed clinical-grade TBV25-28, a yeast-secreted malaria transmission-blocking vaccine candidate; Gozar MM et al.; Gozar, M . M . G., Muratova, O., Keister, D . B., Kensil, C . R., Price, V . L., and Kaslow, D . C . 2001 . Plasmodium falciparum: Immunogenicity of alum-adsorbed clinical-grade TBV25-28, a yeast-secreted malaria transmission-blocking vaccine candidate . Experimental Parasitology 97, 61-69 . The fusion of Pfs25 and Pfs28, two major surface antigens on zygotes and ookinetes of Plasmodium falciparum, as a single recombinant protein (TBV25-28) was previously shown to elicit potent transmission-blocking antibodies in mice . Clinical-grade TBV25-28 was subsequently manufactured and its potency was evaluated in rabbits . Rabbits received three doses of either clinical-grade TBV25H or clinical-grade TBV25-28 adsorbed to alum with or without QS-21 . As measured in a standard membrane-feeding assay, addition of QS-21 to the formulations appeared to enhance transmission-blocking potency of rabbit sera after two vaccinations but not after three vaccinations . Surprisingly, TBV25H elicited more potent transmission-blocking antibodies than did TBV25-28, a result strikingly different from those of previous mouse experiments using research-grade TBV25-28 . The apparent decrease in potency of clinical-grade TBV25-28 in rabbits appears to reflect an enhancement in potency of clinical-grade TBV25H in a new formulation rather than simply a species difference in immunogenicity of TBV25-28 .

Phytochemistry, 2001 Mar, 56(6), 535 - 41
Patterns of phenylpropanoids in non-inoculated and potato virus Y-inoculated leaves of transgenic tobacco plants expressing yeast-derived invertase; Baumert A et al.; The patterns of secondary metabolites in leaves of yeast invertase-transgenic tobacco plants (Nicotiana tabacum L . cv . Samsun NN) were analyzed . Plants expressing cytosolic yeast-derived invertase (cytInv) or apoplastic (cell wall associated) yeast invertase (cwInv) showed a characteristic phytochemical phenotype compared to untransformed controls (wild-type plants) . The level of phenylpropanoids decreased in the cytInv plants but increased in the cwInv plants, which showed an induced de novo synthesis of a caffeic acid amide, i.e . N-caffeoylputrescine . In addition, the level of the coumarin glucoside scopolin was markedly enhanced . Increased accumulation of scopolin in the cwInv plants is possibly correlated with the induction of defense reactions and the appearance of necrotic lesions similar to the hypersensitive response caused by avirulent pathogens . This is consistent with results from potato virus Y-infected plants . Whereas there was no additional increase in the coumarins in leaves following infection in cwInv plants, wild-type plants showed a slight increase and cytInc a marked increase.

J Biol Chem, 2001 Jun 29, 276(26), 23962 - 8 Epub 2001 Mar 28.
The human homologue of the yeast DNA repair and TFIIH regulator MMS19 is an AF-1-specific coactivator of estrogen receptor; Wu X et al.; Steroid/nuclear hormone receptors are ligand-dependent transcriptional regulators that control gene expression in a wide array of biological processes . The transcriptional activity of the receptors is mediated by an N-terminal ligand-independent transcriptional activation function AF-1 and a C-terminal ligand-dependent transcriptional activation function AF-2 . The nuclear receptor coactivator RAC3 (also known as AIB1/ACTR/pCIP/TRAM-1/SRC-3) is amplified in breast cancer cells, where it forms a complex with estrogen receptor (ER) and enhances AF-2 activity of the receptor . Here, we identify a putative human homologue of the yeast DNA repair and transcriptional regulator MMS19 as a RAC3-interacting protein . The human MMS19 interacts with the N-terminal PAS-A/B domain of RAC3 in vivo and in vitro through a conserved C-terminal domain . Interestingly, the human MMS19 also interacts with estrogen receptors in a ligand-independent manner but not with retinoic acid receptor or thyroid hormone receptor . Overexpression of the interacting domain of hMMS19 strongly inhibits ER-mediated transcriptional activation, indicating a dominant negative activity . In contrast, over expression of the full-length hMMS19 enhances ER-mediated transcriptional activation . We find that hMMS19 stimulates the AF-1 activity of ERalpha, but not the AF-2 activity, suggesting that hMMS19 may be an AF-1-specific transcriptional coactivator of estrogen receptor.

J Biol Chem, 2001 Jun 29, 276(26), 24038 - 43 Epub 2001 Mar 14.
Production of human type I collagen in yeast reveals unexpected new insights into the molecular assembly of collagen trimers; Olsen DR et al.; Substantial evidence supports the role of the procollagen C-propeptide in the initial association of procollagen polypeptides and for triple helix formation . To evaluate the role of the propeptide domains on triple helix formation, human recombinant type I procollagen, pN-collagen (procollagen without the C-propeptides), pC-collagen (procollagen without the N-propeptides), and collagen (minus both propeptide domains) heterotrimers were expressed in Saccharomyces cerevisiae . Deletion of the N- or C-propeptide, or both propeptide domains, from both proalpha-chains resulted in correctly aligned triple helical type I collagen . Protease digestion assays demonstrated folding of the triple helix in the absence of the N- and C-propeptides from both proalpha-chains . This result suggests that sequences required for folding of the triple helix are located in the helical/telopeptide domains of the collagen molecule . Using a strain that does not contain prolyl hydroxylase, the same folding mechanism was shown to be operative in the absence of prolyl hydroxylase . Normal collagen fibrils were generated showing the characteristic banding pattern using this recombinant collagen . This system offers new opportunities for the study of collagen expression and maturation.

J Biol Chem, 2001 May 11, 276(19), 16216 - 22 Epub 2001 Feb 20.
The yeast plasma membrane protein Alr1 controls Mg2+ homeostasis and is subject to Mg2+-dependent control of its synthesis and degradation; Graschopf A et al.; The Saccharomyces cerevisiae ALR1 (YOL130w) gene product Alr1p is the first known candidate for a Mg(2+) transport system in eukaryotic cells and is distantly related to the bacterial CorA Mg(2+) transporter family . Here we provide the first experimental evidence for the location of Alr1p in the yeast plasma membrane and for the tight control of its expression and turnover by Mg(2+) . Using well characterized npi1 and end3 mutants deficient in the endocytic pathway, we demonstrate that Alr1 protein turnover is dependent on ubiquitination and endocytosis . Furthermore, cells lacking the vacuolar protease Pep4p accumulated Alr1p in the vacuole . Mutants lacking Alr1p (Deltaalr1) showed a 60% reduction of total intracellular Mg(2+) compared with the wild type and failed to grow in standard media . When starved of Mg(2+), mutant and wild-type cells had similar low levels of intracellular Mg(2+); but upon addition of Mg(2+), wild-type cells replenished the intracellular Mg(2+) pool within a few hours, whereas Deltaalr1 mutant cells did not . Expression of the bacterial Mg(2+) transporter CorA in the yeast Deltaalr1 mutant partially restored growth in standard media . The results are discussed in terms of Alr1p being a plasma membrane transporter with high selectivity for Mg(2+).

J Biol Chem, 2001 May 18, 276(20), 17261 - 6 Epub 2001 Feb 13.
Structure-function analysis of the active site tunnel of yeast RNA triphosphatase; Bisaillon M et al.; Cet1, the RNA triphosphatase component of the yeast mRNA capping apparatus, catalyzes metal-dependent gamma phosphate hydrolysis within the hydrophilic interior of a topologically closed 8-strand beta barrel (the "triphosphate tunnel") . We used structure-guided alanine scanning to identify 6 side chains within the triphosphate tunnel that are essential for phosphohydrolase activity in vitro and in vivo: Arg393, Glu433, Arg458, Arg469, Asp471 and Thr473 . Alanine substitutions at two positions, Asp377 and Lys409, resulted in partial catalytic defects and a thermosensitive growth phenotype . Structure-function relationships were clarified by introducing conservative substitutions . Five residues were found to be nonessential: Lys309, Ser395, Asp397, Lys427 Asn431, and Lys474 . The present findings, together with earlier mutational analyses, reveal an unusually complex active site in which 15 individual side chains in the tunnel cavity are important for catalysis, and each of the 8 strands of the beta barrel contributes at least one functional constituent . The active site residues fall into three classes: (i) those that participate directly in catalysis via coordination of the gamma phosphate or the metal; (ii) those that make critical water-mediated contacts with the gamma phosphate or the metal; and (iii) those that function indirectly via interactions with other essential side chains or by stabilization of the tunnel structure.

J Biol Chem, 2001 May 4, 276(18), 14996 - 5002 Epub 2001 Feb 01.
Importance of homodimerization for the in vivo function of yeast RNA triphosphatase; Lehman K et al.; Saccharomyces cerevisiae RNA triphosphatase Cet1 is an essential component of the yeast mRNA capping apparatus . The active site of Cet1 resides within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel) that is supported by a globular hydrophobic core . The homodimeric quaternary structure of Cet1 is formed by a network of contacts between the partner protomers . By studying the effects of alanine-cluster mutations, we highlight the contributions of two separate facets of the crystallographic dimer interface to Cet1 function in vivo . One essential facet of the interface entails hydrophobic cross-dimer interactions of Cys(330) and Val(331) and a cross-dimer hydrogen bond of Asp(280) with the backbone amide of Gln(329) . The second functionally relevant dimer interface involves hydrophobic side-chain interactions of Phe(272) and Leu(273) . Ala-cluster mutations involving these residues elicited lethal or severe temperature-sensitive phenotypes that were suppressed completely by fusion of the mutated triphosphatases to the guanylyltransferase domain of mammalian capping enzyme . The recombinant D279A-D280A and F272A-L273A proteins retained phosphohydrolase activity but sedimented as monomers . These results indicate that a disruption of the dimer interface is uniquely deleterious when the yeast RNA triphosphatase must function in concert with the endogenous yeast guanylyltransferase . We also identify key residue pairs in the hydrophobic core of the Cet1 protomer that support the active site tunnel and stabilize the triphosphatase in vivo.

J Biol Chem, 2001 May 25, 276(21), 18450 - 6 Epub 2001 Feb 26.
Role of Arg-166 in yeast cytochrome C1; Ahmad Z et al.; A systematic screen for dominant-negative mutations of the CYT1 gene, which encodes cytochrome c(1), revealed seven mutants after testing approximately 10(4) Saccharomyces cerevisiae strains transformed with a library of mutagenized multicopy plasmids . DNA sequence analysis revealed multiple nucleotide substitutions with six of the seven altered Cyt1p having a common R166G replacement, either by itself or accompanied with other amino acid replacements . A single R166G replacement produced by site-directed mutagenesis demonstrated that this change produced a nearly nonfunctional cytochrome c(1), with diminished growth on glycerol medium and diminished respiration but with the normal or near normal level of cytochrome c(1) having an attached heme group . In contrast, R166K, R166M, or R166L replacements resulted in normal or near normal function . Arg-166 is conserved in all cytochromes c(1) and lies on the surface of Cyt1p in close proximity to the heme group but does not seem to interact directly with any of the physiological partners of the cytochrome bc(1) complex . Thus, the large size of the side chain at position 166 is critical for the function of cytochrome c(1) but not for its assembly in the cytochrome bc(1) complex.

J Biol Chem, 2001 Jun 8, 276(23), 20261 - 6 Epub 2001 Mar 07.
Starvation promotes nuclear accumulation of the hsp70 Ssa4p in yeast cells; Chughtai ZS et al.; Nuclear import of proteins that are too large to passively enter the nucleus requires soluble factors, energy, and a nuclear localization signal (NLS) . Nuclear protein transport can be regulated, and different forms of stress affect nucleocytoplasmic trafficking . As such, import of proteins containing a classical NLS is inhibited in starving yeast cells . In contrast, the hsp70 Ssa4p concentrates in nuclei upon starvation . Nuclear concentration of Ssa4p in starving cells is reversible, and transfer of stationary phase cells to fresh medium induces Ssa4p nuclear export . This export reaction represents an active process that is sensitive to oxidative stress . In starving cells, the N-terminal domain of Ssa4p mediates Ssa4p nuclear accumulation, and a short hydrophobic sequence, termed Star (for starvation), is sufficient to localize the reporter proteins green fluorescent protein or beta-galactosidase to nuclei . To determine whether nuclear accumulation of Star-beta-galactosidase depends on a specific nuclear carrier, we have analyzed its distribution in mutant yeast strains that carry a deletion of a single beta-importin gene . With this assay we have identified Nmd5p as a beta-importin required to concentrate Star-beta-galactosidase in nuclei when cells enter stationary phase.

J Biol Chem, 2001 May 25, 276(21), 17629 - 34 Epub 2001 Feb 20.
The electrophilic and leaving group phosphates in the catalytic mechanism of yeast pyrophosphatase; Zyryanov AB et al.; Binding of pyrophosphate or two phosphate molecules to the pyrophosphatase (PPase) active site occurs at two subsites, P1 and P2 . Mutations at P2 subsite residues (Y93F and K56R) caused a much greater decrease in phosphate binding affinity of yeast PPase in the presence of Mn(2+) or Co(2+) than mutations at P1 subsite residues (R78K and K193R) . Phosphate binding was estimated in these experiments from the inhibition of ATP hydrolysis at a sub-K(m) concentration of ATP . Tight phosphate binding required four Mn(2+) ions/active site . These data identify P2 as the high affinity subsite and P1 as the low affinity subsite, the difference in the affinities being at least 250-fold . The time course of five "isotopomers" of phosphate that have from zero to four (18)O during {(18)O}P(i)-{(16)O}H(2)O oxygen exchange indicated that the phosphate containing added water is released after the leaving group phosphate during pyrophosphate hydrolysis . These findings provide support for the structure-based mechanism in which pyrophosphate hydrolysis involves water attack on the phosphorus atom located at the P2 subsite of PPase.

J Biol Chem, 2001 Apr 27, 276(17), 14426 - 33 Epub 2001 Jan 22.
The yeast hsp70 homologue Ssa is required for translation and interacts with Sis1 and Pab1 on translating ribosomes; Horton LE et al.; The 70-kDa heat shock proteins are molecular chaperones that participate in a variety of cellular functions . This chaperone function is stimulated by interaction with hsp40 proteins . The Saccharomyces cerevisiae gene encoding the essential hsp40 homologue, SIS1, appears to function in translation initiation . Mutations in ribosomal protein L39 (rpl39) complement loss-of-function mutations in SIS1 as well as PAB1 (poly(A)-binding protein), suggesting a functional interaction between these proteins . However, while a direct interaction between Sis1 and Pab1 is not detectable, both of these proteins physically interact with the essential Ssa (and not Ssb) family of hsp70 proteins . This interaction is mediated by the variable C-terminal domain of Ssa . Subcellular fractionations demonstrate that the binding of Ssa to ribosomes is dependent upon its C terminus and that its interaction with Sis1 and Pab1 occurs preferentially on translating ribosomes . Consistent with a function in translation, depletion of Ssa protein produces a general translational defect that appears similar to loss of Sis1 and Pab1 function . This translational effect of Ssa appears mediated, at least in part, by its affect on the interaction of Pab1 with the translation initiation factor, eIF4G, which is dramatically reduced in the absence of functional Ssa protein.

J Biol Chem, 2001 Jun 1, 276(22), 19350 - 5 Epub 2001 Feb 26.
Pyridoxal phosphate binding sites are similar in human heme-dependent and yeast heme-independent cystathionine beta-synthases . Evidence from 31P NMR and pulsed EPR spectroscopy that heme and PLP cofactors are not proximal in the human enzyme; Kabil O et al.; Two classes of cystathionine beta-synthases have been identified in eukaryotes, the heme-independent enzyme found in yeast and the heme-dependent form found in mammals . Both classes of enzymes catalyze a pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine to produce cystathionine . The role of the heme in the human enzyme and its location relative to the PLP in the active site are unknown . (31)P NMR spectroscopy revealed that spin-lattice relaxation rates of the phosphorus nucleus in PLP are similar in both the paramagnetic ferric (T(1) = 6.34 +/- 0.01 s) and the diamagnetic ferrous (T(1) = 5.04 +/- 0.06 s) enzyme, suggesting that the two cofactors are not proximal to each other . This is also supported by pulsed EPR studies that do not provide any evidence for strong or weak coupling between the phosphorus nucleus and the ferric iron . However, the (31)P signal in the reduced enzyme moved from 5.4 to 2.2 ppm, and the line width decreased from 73 to 16 Hz, providing the first structural evidence for transmission to the active site of an oxidation state change in the heme pocket . These results are consistent with a regulatory role for the heme as suggested by previous biochemical studies from our laboratory . The (31)P chemical shifts of the resting forms of the yeast and human enzymes are similar, suggesting that despite the difference in their heme content, the microenvironment of the PLP is similar in the two enzymes . The addition of the substrate, serine, resulted in an upfield shift of the phosphorus resonance in both enzymes, signaling formation of reaction intermediates . The resting enzyme spectra were recovered following addition of excess homocysteine, indicating that both enzymes retained catalytic activity during the course of the NMR experiment.

J Biol Chem, 2001 Apr 27, 276(17), 14279 - 88 Epub 2001 Jan 30.
Antioxidant system within yeast peroxisome . Biochemical and physiological characterization of CbPmp20 in the methylotrophic yeast Candida boidinii; Horiguchi H et al.; Candida boidinii Pmp20 (CbPmp20), a protein associated with the inner side of peroxisomal membrane, belongs to a recently identified protein family of antioxidant enzymes, the peroxiredoxins, which contain one cysteine residue . Pmp20 homologs containing the putative peroxisome targeting signal type 1 have also been identified in mammals and lower eukaryotes . However, the physiological function of these Pmp20 family proteins has been unclear . In this study, we investigated the biochemical and physiological functions of recombinant CbPmp20 protein in methanol-induced peroxisomes of C . boidinii using the PMP20-deleted strain of C . boidinii (pmp20Delta strain) . The His(6)-tagged CbPmp20 fusion protein was found to have glutathione peroxidase activity in vitro toward alkyl hydroperoxides and H(2)O(2) . Catalytic activity and dimerization of His(6)-CbPmp20 depended on the only cysteine residue corresponding to Cys(53) . The pmp20Delta strain was found to have lost growth ability on methanol as a carbon and energy source . The pmp20Delta growth defect was rescued by CbPmp20, but neither CbPmp20 lacking the peroxisome targeting signal type 1 sequence nor CbPmp20 haboring the C53S mutation retrieved the growth defect . Interestingly, the pmp20Delta strain had a more severe growth defect than the cta1Delta strain, which lacks catalase, another antioxidant enzyme within the peroxisome . During incubation of these strains in methanol medium, the cta1Delta strain accumulated H(2)O(2), whereas the pmp20Delta strain did not . Therefore, it is speculated to be the main function of CbPmp20 is to decompose reactive oxygen species generated at peroxisomal membrane surface, e.g . lipid hydroperoxides, rather than to decompose H(2)O(2) . In addition, we detected a physiological level of reduced glutathione in peroxisomal fraction of C . boidinii . These results may indicate a physiological role for CbPmp20 as an antioxidant enzyme within peroxisomes rich in reactive oxygen species.

J Biol Chem, 2001 May 25, 276(21), 18633 - 9 Epub 2001 Feb 22.
Physiological levels of mammalian uncoupling protein 2 do not uncouple yeast mitochondria; Stuart JA et al.; We assessed the ability of human uncoupling protein 2 (UCP2) to uncouple mitochondrial oxidative phosphorylation when expressed in yeast at physiological and supraphysiological levels . We used three different inducible UCP2 expression constructs to achieve mitochondrial UCP2 expression levels in yeast of 33, 283, and 4100 ng of UCP2/mg of mitochondrial protein . Yeast mitochondria expressing UCP2 at 33 or 283 ng/mg showed no increase in proton conductance, even in the presence of various putative effectors, including palmitate and all-trans-retinoic acid . Only when UCP2 expression in yeast mitochondria was increased to 4 microg/mg, more than an order of magnitude greater than the highest known physiological concentration, was proton conductance increased . This increased proton conductance was not abolished by GDP . At this high level of UCP2 expression, an inhibition of substrate oxidation was observed, which cannot be readily explained by an uncoupling activity of UCP2 . Quantitatively, even the uncoupling seen at 4 microgram/mg was insufficient to account for the basal proton conductance of mammalian mitochondria . These observations suggest that uncoupling of yeast mitochondria by UCP2 is an overexpression artifact leading to compromised mitochondrial integrity.

J Biol Chem, 2001 May 11, 276(19), 16279 - 88 Epub 2001 Feb 05.
Rvb1p and Rvb2p are essential components of a chromatin remodeling complex that regulates transcription of over 5% of yeast genes; Jonsson ZO et al.; Eukaryotic Rvb1p and Rvb2p are two highly conserved proteins related to the helicase subset of the AAA+ family of ATPases . Conditional mutants in both genes show rapid changes in the transcription of over 5% of yeast genes, with a similar number of genes being repressed and activated . Both Rvb1p and Rvb2p are required for maintaining the induced state of many inducible promoters . ATP binding and hydrolysis by Rvb1p and Rvb2p is individually essential in vivo, and the two proteins are associated with each other in a high molecular weight complex that shows ATP-dependent chromatin remodeling activity in vitro . Our findings show that Rvb1p and Rvb2p are essential components of a chromatin remodeling complex and determine genes regulated by the complex.

J Biol Chem, 2001 Apr 13, 276(15), 12135 - 9 Epub 2001 Jan 12.
Biochemical characterization of Gyp6p, a Ypt/Rab-specific GTPase-activating protein from yeast; Will E et al.; Gyp6p from yeast belongs to the GYP family of Ypt/Rab-specific GTPase-activating proteins, and Ypt6p is its preferred substrate (Strom, M., Vollmer, P., Tan, T . J., and Gallwitz, D . (1993) Nature 361, 736-739) . We have investigated the kinetic parameters of Gyp6p/Ypt6p interactions and find that Gyp6p accelerates the intrinsic GTPase activity of Ypt6p (0.0002 min(-1)) by a factor of 5 x 10(6) and that they have a very low affinity for its preferred substrate (K(m) = 592 micrometer) . Substitution with alanine of several arginines, which Gyp6p shares with other GYP family members, resulted in significant inhibition of GAP activity . Replacement of arginine-155 with either alanine or lysine abolished its GAP activity, indicating a direct involvement of this strictly conserved arginine in catalysis . Physical interaction of the catalytically inactive Gyp6(R155A) mutant GAP with Ypt6 wild-type and Ypt6 mutant proteins could be demonstrated with the two-hybrid system . Short N-terminal and C-terminal truncations of Gyp6p resulted in a complete loss of GAP activity and Ypt6p binding, showing that in contrast to two other Gyp proteins studied previously, most of the 458 amino acid-long Gyp6p sequence is required to form a three-dimensional structure that allows substrate binding and catalysis.

J Biol Chem, 2001 Apr 13, 276(15), 11531 - 8 Epub 2001 Jan 19.
Analysis of gene induction and arrest site transcription in yeast with mutations in the transcription elongation machinery; Wind-Rotolo M et al.; In vitro, transcript elongation by RNA polymerase II is impeded by DNA sequences, DNA-bound proteins, and small ligands . Transcription elongation factor SII (TFIIS) assists RNA polymerase II to transcribe through these obstacles . There is however, little direct evidence that SII-responsive arrest sites function in living cells nor that SII facilitates readthrough in vivo . Saccharomyces cerevisiae strains lacking elongation factor SII and/or containing a point mutation in the second largest subunit of RNA polymerase II, which slows the enzyme's RNA elongation rate, grow slowly and have defects in mRNA metabolism, particularly in the presence of nucleotide-depleting drugs . Here we have examined transcriptional induction in strains lacking SII or containing the slow polymerase mutation . Both mutants and a combined double mutant were defective in induction of GAL1 and ENA1 . This was not due to an increase in mRNA degradation and was independent of any drug treatment, although treatment with the nucleotide-depleting drug 6-azauracil exacerbated the effect preferentially in the mutants . These data are consistent with mutants in the Elongator complex, which show slow inductive responses . When a potent in vitro arrest site was transcribed in these strains, there was no perceptible effect upon mRNA accumulation . These data suggest that an alternative elongation surveillance mechanism exists in vivo to overcome arrest.

J Biol Chem, 2001 Apr 27, 276(17), 13587 - 92 Epub 2001 Jan 30.
Decreased expression of specific genes in yeast cells lacking histone H1; Hellauer K et al.; Chromatin plays an important role in regulating eukaryotic gene expression . Chromatin is composed of DNA wrapped around a nucleosome core (consisting of two copies of the well conserved histones H2A, H2B, H3, and H4) and a more variable linker histone H1 . Various in vitro and in vivo studies have implicated histone H1 as a repressor of gene expression or as an activator, but its exact role is still unclear . Sequencing of the yeast genome has led to the identification of a putative histone H1 gene . Biochemical studies demonstrated that yeast does indeed possess a bona fide histone H1 . However, deletion of the unique yeast H1 gene is not associated with any phenotypes, and it was questioned whether it plays any role . To address this issue, we performed whole-genome microarray analysis to identify genes that are affected by H1 removal . Surprisingly, deletion of the gene encoding histone H1 does not result in increased gene expression but rather in a modest reduction . Northern blot analysis of selected genes confirmed the results obtained with the microarray analysis . A similar effect was observed with an integrated lacZ reporter . Thus, our data demonstrate that removal of yeast histone H1 only results in decreased gene expression.

J Biol Chem, 2001 May 25, 276(21), 17941 - 8 Epub 2001 Mar 02.
Yeast V-ATPase complexes containing different isoforms of the 100-kDa a-subunit differ in coupling efficiency and in vivo dissociation; Kawasaki-Nishi S et al.; The 100 kDa a-subunit of the yeast vacuolar (H(+))-ATPase (V-ATPase) is encoded by two genes, VPH1 and STV1 . These genes encode unique isoforms of the a-subunit that have previously been shown to reside in different intracellular compartments in yeast . Vph1p localizes to the central vacuole, whereas Stv1p is present in some other compartment, possibly the Golgi or endosomes . To compare the properties of V-ATPases containing Vph1p or Stv1p, Stv1p was expressed at higher than normal levels in a strain disrupted in both genes, under which conditions V-ATPase complexes containing Stv1p appear in the vacuole . Complexes containing Stv1p showed lower assembly with the peripheral V(1) domain than did complexes containing Vph1p . When corrected for this lower degree of assembly, however, V-ATPase complexes containing Vph1p and Stv1p had similar kinetic properties . Both exhibited a K(m) for ATP of about 250 microm, and both showed resistance to sodium azide and vanadate and sensitivity to nanomolar concentrations of concanamycin A . Stv1p-containing complexes, however, showed a 4-5-fold lower ratio of proton transport to ATP hydrolysis than Vph1p-containing complexes . We also compared the ability of V-ATPase complexes containing Vph1p or Stv1p to undergo in vivo dissociation in response to glucose depletion . Vph1p-containing complexes present in the vacuole showed dissociation in response to glucose depletion, whereas Stv1p-containing complexes present in their normal intracellular location (Golgi/endosomes) did not . Upon overexpression of Stv1p, Stv1p-containing complexes present in the vacuole showed glucose-dependent dissociation . Blocking delivery of Vph1p-containing complexes to the vacuole in vps21Delta and vps27Delta strains caused partial inhibition of glucose-dependent dissociation . These results suggest that dissociation of the V-ATPase complex in vivo is controlled both by the cellular environment and by the 100-kDa a-subunit isoform present in the complex.

J Biol Chem, 2001 May 18, 276(20), 17524 - 32 Epub 2001 Feb 27.
J-domain protein, Jac1p, of yeast mitochondria required for iron homeostasis and activity of Fe-S cluster proteins; Kim R et al.; J-proteins are molecular chaperones with a characteristic domain predicted to mediate interaction with Hsp70 proteins . We have previously isolated yeast mutants of the mitochondrial Hsp70, Ssq1p, in a genetic screen for mutants with altered iron homeostasis . Here we describe the isolation of mutants of the J-domain protein, Jac1p, using the same screen . Mutant jac1 alleles predicted to encode severely truncated proteins (lacking 70 or 152 amino acids) were associated with phenotypes strikingly similar to the phenotypes of ssq1 mutants . These phenotypes include activation of the high affinity cellular iron uptake system and iron accumulation in mitochondria . In contrast to iron accumulation, Fe-S proteins of mitochondria were specifically deficient . In jac1 mutants, like in ssq1 mutants, processing of the Yfh1p precursor protein from intermediate to mature forms was delayed . In the genetic backgrounds used in this study, jac1 null mutants were found to be viable, permitting analysis of genetic interactions . The Deltajac1 Deltassq1 double mutant was more severely compromised for growth than either single mutant, suggesting a synthetic or additive effect of these mutations . Overexpression of Jac1p partially suppressed ssq1 slow growth and vice versa . Similar mitochondrial localization and similar mutant phenotypes suggest that Ssq1p and Jac1p are functional partners in iron homeostasis.

J Biol Chem, 2001 Apr 13, 276(15), 11883 - 94 Epub 2001 Jan 19.
High affinity interaction of yeast transcriptional regulator, Mot1, with TATA box-binding protein (TBP); Adamkewicz JI et al.; Yeast Mot1, an essential ATP-dependent regulator of basal transcription, removes TATA box-binding protein (TBP) from TATA sites in vitro . Complexes of Mot1 and Spt15 (yeast TBP), radiolabeled in vitro, were immunoprecipitated with anti-TBP (or anti-Mot1) antibodies in the absence of DNA, showing Mot1 binds TBP in solution . Mot1 N-terminal deletions (residues 25-801) abolished TBP binding, whereas C-terminal ATPase domain deletions (residues 802-1867) did not . Complex formation was prevented above 200 mm salt, consistent with electrostatic interaction . Correspondingly, TBP variants lacking solvent-exposed positive charge did not bind Mot1, whereas a mutant lacking positive charge within the DNA-binding groove bound Mot1 . ATPase-defective mutant, Mot1(D1408N), which inhibits growth when overexpressed (but is suppressed by co-overexpression of TBP), bound TBP normally in vitro, suggesting it forms nonrecyclable complexes . N-terminal deletions of Mot1(D1408N) were not growth-inhibitory . C-terminal deletions were toxic when overexpressed, and toxicity was ameliorated by TBP co-overproduction . Residues 1-800 of Mot1 are therefore necessary and sufficient for TBP binding . The N terminus of 89B, a tissue-specific Drosophila Mot1 homolog, bound the TBP-like factor, dTRF1 . Native Mot1 and derivatives deleterious to growth localized in the nucleus, whereas nontoxic derivatives localized to the cytosol, suggesting TBP binding and nuclear transport of Mot1 are coupled.

J Biol Chem, 2001 May 25, 276(21), 17958 - 67 Epub 2001 Mar 06.
Yeast Mps1p phosphorylates the spindle pole component Spc110p in the N-terminal domain; Friedman DB et al.; The yeast spindle pole body (SPB) component Spc110p (Nuf1p) undergoes specific serine/threonine phosphorylation as the mitotic spindle apparatus forms, and this phosphorylation persists until cells enter anaphase . We demonstrate that the dual-specificity kinase Mps1p is essential for the mitosis-specific phosphorylation of Spc110p in vivo and that Mps1p phosphorylates Spc110p in vitro . Phosphopeptides generated by proteolytic cleavage were identified and sequenced by mass spectrometry . Ser(60), Thr(64), and Thr(68) are the major sites in Spc110p phosphorylated by Mps1p in vitro, and alanine substitution at these sites abolishes the mitosis-specific isoform in vivo . This is the first time that phosphorylation sites of an SPB component have been determined, and these are the first sites of Mps1p phosphorylation identified . Alanine substitution for any one of these phosphorylated residues, in conjunction with an alanine substitution at residue Ser(36), is lethal in combination with alleles of SPC97, which encodes a component of the Tub4p complex . Consistent with a specific dysfunction for the alanine substitution mutations, simultaneous mutation of all four serine/threonine residues to aspartate does not confer any defect . Sites of Mps1p phosphorylation and Ser(36) are located within the N-terminal globular domain of Spc110p, which resides at the inner plaque of the SPB and binds the Tub4p complex.

J Biol Chem, 2001 Apr 13, 276(15), 11712 - 8 Epub 2001 Jan 19.
Calcium influx and signaling in yeast stimulated by intracellular sphingosine 1-phosphate accumulation; Birchwood CJ et al.; In mammalian cells, intracellular sphingosine 1-phosphate (S1P) can stimulate calcium release from intracellular organelles, resulting in the activation of downstream signaling pathways . The budding yeast Saccharomyces cerevisiae expresses enzymes that can synthesize and degrade S1P and related molecules, but their possible role in calcium signaling has not yet been tested . Here we examine the effects of S1P accumulation on calcium signaling using a variety of yeast mutants . Treatment of yeast cells with exogenous sphingosine stimulated Ca(2+) accumulation through two distinct pathways . The first pathway required the Cch1p and Mid1p subunits of a Ca(2+) influx channel, depended upon the function of sphingosine kinases (Lcb4p and Lcb5p), and was inhibited by the functions of S1P lyase (Dpl1p) and the S1P phosphatase (Lcb3p) . The biologically inactive stereoisomer of sphingosine did not activate this Ca(2+) influx pathway, suggesting that the active S1P isomer specifically stimulates a calcium-signaling mechanism in yeast . The second Ca(2+) influx pathway stimulated by the addition of sphingosine was not stereospecific, was not dependent on the sphingosine kinases, occurred only at higher doses of added sphingosine, and therefore was likely to be nonspecific . Mutants lacking both S1P lyase and phosphatase (dpl1 lcb3 double mutants) exhibited constitutively high Ca(2+) accumulation and signaling in the absence of added sphingosine, and these effects were dependent on the sphingosine kinases . These results show that endogenous S1P-related molecules can also trigger Ca(2+) accumulation and signaling . Several stimuli previously shown to evoke calcium signaling in wild-type cells were examined in lcb4 lcb5 double mutants . All of the stimuli produced calcium signals independent of sphingosine kinase activity, suggesting that phosphorylated sphingoid bases might serve as messengers of calcium signaling in yeast during an unknown cellular response.

J Biol Chem, 2001 Apr 13, 276(15), 12100 - 12 Epub 2001 Jan 22.
Yop1p, the yeast homolog of the polyposis locus protein 1, interacts with Yip1p and negatively regulates cell growth; Calero M et al.; Rab proteins are small GTPases that are essential elements of the protein transport machinery of eukaryotic cells . Each round of membrane transport requires a cycle of Rab protein nucleotide binding and hydrolysis . We have recently characterized a protein, Yip1p, which appears to play a role in Rab-mediated membrane transport in Saccharomyces cerevisiae . In this study, we report the identification of a Yip1p-associated protein, Yop1p . Yop1p is a membrane protein with a hydrophilic region at its N terminus through which it interacts specifically with the cytosolic domain of Yip1p . Yop1p could also be coprecipitated with Rab proteins from total cellular lysates . The TB2 gene is the human homolog of Yop1p (Kinzler, K . W., Nilbert, M . C., Su, L.-K., Vogelstein, B., Bryan, T . M., Levey, D . B., Smith, K . J., Preisinger, A . C., Hedge, P., McKechnie, D., Finniear, R., Markham, A., Groffen, J., Boguski, M . S., Altschul, S . F., Horii, A., Ando, H . M., Y., Miki, Y., Nishisho, I., and Nakamura, Y . (1991) Science 253, 661-665) . Our data demonstrate that Yop1p negatively regulates cell growth . Disruption of YOP1 has no apparent effect on cell viability, while overexpression results in cell death, accumulation of internal cell membranes, and a block in membrane traffic . These results suggest that Yop1p acts in conjunction with Yip1p to mediate a common step in membrane traffic.

J Biol Chem, 2001 May 11, 276(19), 16051 - 8 Epub 2001 Feb 02.
Examination of donor substrate conversion in yeast transketolase; Fiedler E et al.; The cleavage of the donor substrate d-xylulose 5-phosphate by wild-type and H263A mutant yeast transketolase was studied using enzyme kinetics and circular dichroism spectroscopy . The enzymes are able to catalyze the cleavage of donor substrates, the first half-reaction, even in the absence of any acceptor substrate yielding d-glyceraldehyde 3-phosphate as measured in the coupled optical test according to Kochetov (Kochetov, G . A . (1982) Methods Enzymol . 90, 209-223) and compared with the H263A variant . Overall, the H263A mutant enzyme is less active than the wild-type . However, an increase in the rate constant of the release of the enzyme-bound glycolyl moiety was observed and related to a stabilization of the "active glycolaldehyde" (alpha-carbanion) by histidine 263 . Chemically synthesized dl-(alpha,beta-dihydroxyethyl)thiamin diphosphate is bound to wild-type transketolase with an apparent K(D) of 4.3 +/- 0.8 microm (racemate) calculated from titration experiments using circular dichroism spectroscopy . Both enantiomers are cleaved by the enzyme at different rates . In contrast to the enzyme-generated alpha-carbanion of (alpha,beta-dihydroxyethyl)thiamin diphosphate formed by decarboxylation of hydroxylactylthiamin diphosphate after incubation of transketolase with beta-hydroxypyruvate, the synthesized dl-(alpha,beta-dihydroxyethyl)thiamin diphosphate did not work as donor substrate when erythrose 4-phosphate is used as acceptor substrate in the coupled enzymatic test according to Sprenger (Sprenger, G . A., Schorken, U., Sprenger, G., and Sahm, H . (1995) Eur . J . Biochem . 230, 525-532).

J Biol Chem, 2001 May 4, 276(18), 15466 - 71 Epub 2001 Jan 22.
Lithium and valproate decrease inositol mass and increase expression of the yeast INO1 and INO2 genes for inositol biosynthesis; Vaden DL et al.; Bipolar affective disorder (manic-depressive illness) is a chronic, severe, debilitating illness affecting 1-2% of the population . The Food and Drug Administration-approved drugs lithium and valproate are not completely effective in the treatment of this disorder, and the mechanisms underlying their therapeutic effects have not been established . We are employing genetic and molecular approaches to identify common targets of lithium and valproate in the yeast Saccharomyces cerevisiae . We show that both drugs affect molecular targets in the inositol metabolic pathway . Lithium and valproate cause a decrease in intracellular myo-inositol mass and an increase in expression of both a structural (INO1) and a regulatory (INO2) gene required for inositol biosynthesis . The opi1 mutant, which exhibits constitutive expression of INO1, is more resistant to inhibition of growth by lithium but not by valproate, suggesting that valproate may inhibit the Ino1p-catalyzed synthesis of inositol 1-phosphate . Consistent with this possibility, growth in valproate leads to decreased synthesis of inositol monophosphate . Thus, both lithium and valproate perturb regulation of the inositol biosynthetic pathway, albeit via different mechanisms . This is the first demonstration of increased expression of genes in the inositol biosynthetic pathway by both lithium and valproate . Because inositol is a key regulator of many cellular processes, the effects of lithium and valproate on inositol synthesis have far-reaching implications for predicting genetic determinants of responsiveness and resistance to these agents.

Cell Physiol Biochem, 2001, 11(1), 55 - 60
Determination of protein-protein interactions of ICIn by the yeast two-hybrid system; Schmarda A et al.; ICln is a ubiquitously expressed eukaryotic protein . Expression of the protein in Xenopus laevis oocytes, the knocking-down of the protein in fibroblasts, or the reconstitution of the protein in lipid bilayer led to the assumption that this protein is an ionic channel or a significant part thereof . However, other possible roles for ICln in potential regulatory mechanisms have been postulated, as diverse as regulator of cell morphology by interacting with the Skb1 protein and/or interaction with core spliceosomal proteins . Here we show that ICln is able to interact with SnRNP core proteins SmD1, SmD2, SmD3, SmX5 and SmB/B'.

J Biol Chem, 2001 Jun 15, 276(24), 21924 - 31 Epub 2001 Mar 27.
Characterization of the Net1 cell cycle-dependent regulator of the Cdc14 phosphatase from budding yeast; Traverso EE et al.; In the budding yeast Saccharomyces cerevisiae, the multifunctional protein Net1 is implicated in regulating the cell cycle function of the Cdc14 protein phosphatase . Genetic and cell biological data suggest that during interphase and early mitosis Net1 holds Cdc14 within the nucleolus where its activity is suppressed . Upon its transient release from Net1 at late anaphase, active Cdc14 promotes exit from mitosis by dephosphorylating targets in the nucleus and cytoplasm . In this paper we present evidence supporting the proposed role of Net1 in regulating Cdc14 and exit from mitosis . We show that the NH(2)-terminal fragment Net1(1-600) directly binds Cdc14 in vitro and is a highly specific competitive inhibitor of its activity (K(i) = 3 nm) with five different substrates including the physiologic targets Swi5 and Sic1 . An analysis of truncation mutants indicates that the Cdc14 binding site is located within a segment of Net1 containing residues 1-341 . We propose that Net1 inhibits by occluding the active site of Cdc14 because it acts as a competitive inhibitor, binds to a site located within the catalytic domain (residues 1-374), binds with reduced affinity to a Cdc14 C283S mutant in which an active site Cys is replaced, and is displaced by tungstate, a transition state analog known to bind in the catalytic site of protein-tyrosine phosphatases.

J Biol Chem, 2001 Jun 8, 276(23), 20154 - 9 Epub 2001 Mar 27.
NatC Nalpha-terminal acetyltransferase of yeast contains three subunits, Mak3p, Mak10p, and Mak31p; Polevoda B et al.; The yeast Saccharomyces cerevisiae contains three types of N(alpha)-terminal acetyltransferases, NatA, NatB, and NatC, with each having a different catalytic subunit, Ard1p, Nat3p, and Mak3p, respectively, and each acetylating different sets of proteins with different N(alpha)-terminal regions . We show that the NatC N(alpha)-terminal acetyltransferases contains Mak10p and Mak31p subunits, in addition to Mak3p, and that all three subunits are associated with each other to form the active complex . Genetic deletion of any one of the three subunits results in identical abnormal phenotypes, including the lack of acetylation of a NatC substrate in vivo, diminished growth at 37 degrees C on media containing nonfermentable carbon sources, and the lack of maintenance or assembly of the L-A dsRNA viral particle.

J Biol Chem, 2001 Jun 29, 276(26), 23849 - 57 Epub 2001 Mar 26.
A family of yeast proteins mediating bidirectional vacuolar amino acid transport; Russnak R et al.; Seven genes in Saccharomyces cerevisiae are predicted to code for membrane-spanning proteins (designated AVT1-7) that are related to the neuronal gamma-aminobutyric acid-glycine vesicular transporters . We have now demonstrated that four of these proteins mediate amino acid transport in vacuoles . One protein, AVT1, is required for the vacuolar uptake of large neutral amino acids including tyrosine, glutamine, asparagine, isoleucine, and leucine . Three proteins, AVT3, AVT4, and AVT6, are involved in amino acid efflux from the vacuole and, as such, are the first to be shown directly to transport compounds from the lumen of an acidic intracellular organelle . This function is consistent with the role of the vacuole in protein degradation, whereby accumulated amino acids are exported to the cytosol . Protein AVT6 is responsible for the efflux of aspartate and glutamate, an activity that would account for their exclusion from vacuoles in vivo . Transport by AVT1 and AVT6 requires ATP for function and is abolished in the presence of nigericin, indicating that the same pH gradient can drive amino acid transport in opposing directions . Efflux of tyrosine and other large neutral amino acids by the two closely related proteins, AVT3 and AVT4, is similar in terms of substrate specificity to transport system h described in mammalian lysosomes and melanosomes . These findings suggest that yeast AVT transporter function has been conserved to control amino acid flux in vacuolar-like organelles.

J Biol Chem, 2001 Jun 15, 276(24), 21863 - 9 Epub 2001 Mar 23.
Nuclear import of the yeast AP-1-like transcription factor Yap1p is mediated by transport receptor Pse1p, and this import step is not affected by oxidative stress; Isoyama T et al.; The yeast AP-1-like transcription factor, Yap1p, is essential for the oxidative stress response in budding yeast . Yap1p is located predominantly in the cytoplasm; however, upon imposition of oxidative stress, Yap1p concentrates in the nucleus and activates target genes . Yap1p is constitutively transported in and out of the nucleus . Oxidative stress inhibits the Crm1p/Xpo1p-dependent nuclear export step, resulting in nuclear accumulation of Yap1p . In this study, we examined the mechanism for Yap1p nuclear import, and determined whether the import step is affected by oxidative stress . The nuclear accumulation of Yap1p required the activity of the small GTPase, Ran/Gsp1p . Under conditions in pse1-1 cells carrying a temperature-sensitive mutation of the importin beta family member PSE1/KAP121, nuclear translocation of Yap1p was inhibited dramatically . In an in vitro assay, we showed that Yap1p could directly bind to Pse1p and that this interaction was dissociated by Ran-GTP . These results indicate that Pse1p is the nuclear import receptor for Yap1p . In addition to Pse1p, we suggest that Kap123p, which is homologous to Pse1p, has a minor effect on the nuclear import of Yap1p . Furthermore, we identified the nuclear localization signal of Yap1p and demonstrated that the nuclear import of Yap1p was not affected by oxidative stress.

J Mol Biol, 2001 Mar 30, 307(3), 929 - 38
Mapping protein family interactions: intramolecular and intermolecular protein family interaction repertoires in the PDB and yeast; Park J et al.; In the postgenomic era, one of the most interesting and important challenges is to understand protein interactions on a large scale . The physical interactions between protein domains are fundamental to the workings of a cell: in multi-domain polypeptide chains, in multi-subunit proteins and in transient complexes between proteins that also exist independently . To study the large-scale patterns and evolution of interactions between protein domains, we view interactions between protein domains in terms of the interactions between structural families of evolutionarily related domains . This allows us to classify 8151 interactions between individual domains in the Protein Data Bank and the yeast Saccharomyces cerevisiae in terms of 664 types of interactions, between protein families . At least 51 interactions do not occur in the Protein Data Bank and can only be derived from the yeast data . The map of interactions between protein families has the form of a scale-free network, meaning that most protein families only interact with one or two other families, while a few families are extremely versatile in their interactions and are connected to many families . We observe that almost half of all known families engage in interactions with domains from their own family . We also see that the repertoires of interactions of domains within and between polypeptide chains overlap mostly for two specific types of protein families: enzymes and same-family interactions . This suggests that different types of protein interaction repertoires exist for structural, functional and regulatory reasons . Copyright 12001 Academic Press.

Folia Microbiol (Praha), 2000, 45(3), 231 - 8
Dimorphism in Benjaminiella poitrasii: involvement of intracellular endochitinase and N-acetylglucosaminidase activities in the yeast-mycelium transition; Ghormade VS et al.; The chitinase and N-acetylglucosaminidase activities in cell-wall-bound and free fractions in the dimorphic fungus Benjaminiella poitrasii were studied as a function of morphological (unicellular yeast-mycelium) transition . The specific activities of chitinases of cell-wall-free, particularly in the membrane fraction, were significantly different in the yeast and mycelial forms . During the yeast-mycelium transition, the N-acetylglucosaminidase activity isolated in a membrane preparation increased steadily . The activity of the yeast cells (0.83 +/- 0.17 nkat/mg protein) increased 17-fold to 14.2 +/- 1.7 nkat/mg protein in 1-d-old mycelial cells . The endochitinase activity increased 12-fold between 6 and 12 h and thereafter practically remained unchanged up to 24 h . A reverse trend in the chitinolytic activities was observed during the mycelium-yeast transition . Isoelectrofocussing (pH range 3.5-10) of mixed membrane fraction free of particulate fraction of parent and morphological (Y-5, yeast-form) mutant cells separated endochitinase and N-acetylglucosaminidase activity into two pH ranges, viz . 4.3-5.7 and 6.1-7.7, respectively . The predominant N-acetylglucosaminidase activity observed at pH 6.9 and 7.1 for the parent strain membrane fraction was undetected in the mutant preparation . The results suggested that the membrane-bound (either tightly or loosely) chitinolytic enzymes, particularly, N-acetylglucosaminidase, significantly contributed to the morphological changes in B . poitrasii.

Folia Microbiol (Praha), 2000, 45(3), 221 - 3
Two forms of yeast plasma membrane H(+)-ATPase: comparison of yield and effects of inhibitors; Lapathitis G et al.; Classical isolation procedure for plasma membrane H(+)-ATPase of Saccharomyces cerevisiae based on fractional centrifugation yielded always a roughly two-fold greater amount of membranes when starting from glucitol-preincubated than from glucose-preincubated yeast . This difference persisted all the way to the purified plasma membranes and to the purified H(+)-ATPase . The ATP-hydrolyzing activity by plasma membranes was roughly twice greater in glucose-preincubated cells than in the D-glucitol-preincubated ones while the purified enzyme was 7 times more active after glucose than after glucitol . Effects of diethylstilbestrol, suloctidil, erythrosin B, vanadate and dicarbanonaboranuide were very similar on plasma membrane-localized and purified ATPases of both forms, suggesting that both preparations contain the two ATPase forms, the glucose-preincubated one being richer in the activated form while the glucitol-preincubated one contains less of it.

EMBO Rep, 2000 Oct, 1(4), 334 - 9
Uncoupling yeast intron recognition from transcription with recursive splicing; Lopez PJ et al.; Pre-mRNA splicing has to be coordinated with other processes occurring in the nucleus including transcription, mRNA 3' end formation and mRNA export . To analyze the relationship between transcription and splicing, we constructed a network of nested introns . Introns were inserted in the 5' splice site and/or branchpoint of a synthetic yeast intron interrupting a reporter gene . The inserted introns mask the recipient intron from the cellular machinery until they are removed by splicing . Production of functional mRNA from these constructs therefore requires recognition of a spliced RNA as a splicing substrate . We show that recurrent splicing occurs in a sequential and ordered fashion in vivo . Thus, in Saccharomyces cerevisiae, intron recognition and pre-spliceosome assembly is not tightly coupled to transcription.

Genome, 2001 Feb, 44(1), 32 - 7
A physical map with yeast artificial chromosome (YAC) clones covering 63% of the 12 rice chromosomes; Saji S et al.; A new YAC (yeast artificial chromosome) physical map of the 12 rice chromosomes was constructed utilizing the latest molecular linkage map . The 1439 DNA markers on the rice genetic map selected a total of 1892 YACs from a YAC library . A total of 675 distinct YACs were assigned to specific chromosomal locations . In all chromosomes, 297 YAC contigs and 142 YAC islands were formed . The total physical length of these contigs and islands was estimated to 270 Mb which corresponds to approximately 63% of the entire rice genome (430 Mb) . Because the physical length of each YAC contig has been measured, we could then estimate the physical distance between genetic markers more precisely than previously . In the course of constructing the new physical map, the DNA markers mapped at 0.0-cM intervals were ordered accurately and the presence of potentially duplicated regions among the chromosomes was detected . The physical map combined with the genetic map will form the basis for elucidation of the rice genome structure, map-based cloning of agronomically important genes, and genome sequencing.

Curr Biol, 2001 Mar 6, 11(5), 366 - 9
Bidirectional amyloid fiber growth for a yeast prion determinant; Scheibel T et al.; The polymerization of many amyloids is a two-stage process initiated by the formation of a seeding nucleus or protofibril . Soluble protein then assembles with these nuclei to form amyloid fibers . Whether fiber growth is bidirectional or unidirectional has been determined for two amyloids . In these cases, bidirectional growth was established by time lapse atomic-force microscopy . Here, we investigated the growth of amyloid fibers formed by NM, the prion-determining region of the yeast protein Sup35p . The conformational changes in NM that lead to amyloid formation in vitro serve as a model for the self-perpetuating conformational changes in Sup35p that allow this protein to serve as an epigenetic element of inheritance in vivo . To assess the directionality of fiber growth, we genetically engineered a mutant of NM so that it contained an accessible cysteine residue that was easily labeled after fiber formation . The mutant protein assembled in vitro with kinetics indistinguishable from those of the wild-type protein and propagated the heritable genetic trait {PSI(+)} with the same fidelity . In reactions nucleated with prelabeled fibers, unlabeled protein assembled at both ends . Thus, NM fiber growth is bidirectional.

Curr Biol, 2001 Mar 6, 11(5), 345 - 50
Asymmetric spindle pole localization of yeast Cdc15 kinase links mitotic exit and cytokinesis; Menssen R et al.; The inactivation of mitotic cyclin-dependent kinases (CDKs) during anaphase is a prerequisite for the completion of nuclear division and the onset of cytokinesis {1, 2} . In the budding yeast Saccharomyces cerevisiae, the essential protein kinase Cdc15 {3} together with other proteins of the mitotic exit network (Tem1, Lte1, Cdc5, and Dbf2/Dbf20 {4-7}) activates Cdc14 phosphatase, which triggers cyclin degradation and the accumulation of the CDK inhibitor Sic1 {8} . However, it is still unclear how CDK inactivation promotes cytokinesis . Here, we analyze the properties of Cdc15 kinase during mitotic exit . We found that Cdc15 localized to the spindle pole body (SPB) in a unique pattern . Cdc15 was present at the SPB of the mother cell until late mitosis, when it also associated with the daughter pole . High CDK activity inhibited this association, while dephosphorylation of Cdc15 by Cdc14 phosphatase enabled it . The analysis of Cdc15 derivatives indicated that SPB localization was specifically required for cytokinesis but not for mitotic exit . These results show that Cdc15 has two separate functions during the cell cycle . First, it is required for the activation of Cdc14 . CD14, in turn, promotes CDK inactivation and also dephosphorylates of Cdc15 . As a consequence, Cdc15 binds to the daughter pole and triggers cytokinesis . Thus, Cdc15 helps to coordinate mitotic exit and cytokinesis.

Biochim Biophys Acta, 2001 Mar 19, 1518(1-2), 210 - 4
Identification and characterisation of the Drosophila homologue of the yeast Uba2 gene; Donaghue C et al.; We have identified the Drosophila uba2 protein (dUba2) . Analysis of the amino acid composition reveals similarity with both the mammalian (47% identity) and yeast (31% identity) homologues . dUba2 is present throughout the Drosophila life cycle but is most abundant during stages of proliferation . The protein is nucleoplasmic throughout much of the cell cycle, however it is lost from the nucleus during mitosis . The DUba2 localisation in the nucleoplasm is not uniform but is observed as concentrated patches reminiscent of the staining patterns seen for other proteins from this group . The nature of these sites is not clear, however the failure of dUba2 to localise to the sites of chorion amplification in ovaries suggests that they are not sites of ongoing DNA replication.

Nucleic Acids Res, 2001 Apr 1, 29(7), 1433 - 42
Monitoring S phase progression globally and locally using BrdU incorporation in TK(+) yeast strains; Lengronne A et al.; Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms . Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism . Here we describe a TK(+) yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules . S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes . Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci . Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8-9 kb from origins that are placed 46 kb apart on average . Quantification of total BrdU incorporation suggests that 190 'early' origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome . More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.

J Cell Biol, 2001 Jan 22, 152(2), 385 - 400
The positioning and dynamics of origins of replication in the budding yeast nucleus; Heun P et al.; We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)-tagged chromosomal domains . In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein . We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase . In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle . If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic . This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo . We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established . The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.

J Cell Biol, 2001 Jan 22, 152(2), 251 - 62
Cortical Num1p interacts with the dynein intermediate chain Pac11p and cytoplasmic microtubules in budding yeast; Farkasovsky M et al.; Num1p, a cortical 313-kD protein, controls cytoplasmic microtubule (cMT) functions and nuclear migration through the bud neck in anaphase cells . A green fluorescent protein (GFP)-Num1p fusion protein localizes at the bud tip and the distal mother pole of living cells, apparently forming cMT capture sites at late anaphase . In addition, galactose-induced GFP-Num1p is seen at the bud neck and in lateral regions of the mother cortex . The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p . Num1p associates with the dynein intermediate chain Pac11p in the presence of Dyn1p, and with the alpha-tubulin Tub3p, as shown by coimmune precipitation of tagged proteins . Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells.Our data suggest that Num1p controls nuclear migration during late anaphase by forming dynein-interacting cortical cMT capture sites at both cellular poles . In addition, Num1p may transiently cooperate with an associated Bni1p-Kar9p complex at the bud tip of early anaphase cells.

EMBO Rep, 2001 Mar, 2(3), 203 - 10
Ku-deficient yeast strains exhibit alternative states of silencing competence; Maillet L et al.; In Saccharomyces cerevisiae, efficient silencer function requires telomere proximity, i.e . compartments of the nucleoplasm enriched in silencing factors . Accordingly, silencers located far from telomeres function inefficiently . We show here that cells lacking yKu balance between two mitotically stable states of silencing competence . In one, a partial delocalization of telomeres and silencing factors throughout the nucleoplasm correlates with enhanced silencing at a non-telomeric locus, while in the other, telomeres retain their focal pattern of distribution and there is no repression at the non-telomeric locus, as observed in wild-type cells . The two states also differ in their level of residual telomeric silencing . These findings indicate the existence of a yKu-independent pathway of telomere clustering and Sir localization . Interestingly, this pathway appears to be under epigenetic control.

EMBO Rep, 2001 Mar, 2(3), 197 - 202
Telomerase subunit overexpression suppresses telomere-specific checkpoint activation in the yeast yku80 mutant; Teo SH et al.; Ku is a conserved heterodimeric DNA-binding protein that plays critical roles in DNA repair and telomere homeostasis . In Saccharomyces cerevisiae, deletion of YKU70 or YKU80 results in an inability to grow at 37 degrees C . This is suppressed by overexpression of several components of telomerase (EST1, EST2 and TLC1) . We show that overexpression of EST2 or TLC1 in yku80 mutants does not restore efficient DNA repair, or restore normal telomere function, as measured by telomere length, single-stranded G-rich strand or transcriptional silencing . Instead, yku80 mutants activate a Rad53p-dependent DNA-damage checkpoint at 37 degrees C and this is suppressed by overexpression of EST2 or TLC1 . Indeed, deletion of genes required for Rad53p activation also suppresses the yku80 temperature sensitivity . These results suggest that activation of the DNA-damage checkpoint in yku mutants at 37 degrees C does not result from reduced telomere length per se, but reflects an alteration of the telomere structure that is recognized as damaged DNA.

Environ Health Perspect, 2001 Feb, 109(2), 133 - 8
Estrogenic activity of phenolic additives determined by an in vitro yeast bioassay; Miller D et al.; We used a recombinant yeast estrogen assay to assess the activity of 73 phenolic additives that are used as sunscreens, preservatives, disinfectants, antioxidants, flavorings, or for perfumery . Thirty-two of these compounds displayed activity: 22 with potencies relative to 17beta-estradiol, ranging from 1/3,000 to < 1/3,000,000, and 10 compounds with an impaired response that could not be directly compared with 17beta-estradiol . Forty-one compounds were inactive . The major criteria for activity appear to be the presence of an unhindered phenolic OH group in a para position and a molecular weight of 140-250 Da.

EMBO Rep, 2000 Aug, 1(2), 171 - 5
TIF-IA, the factor mediating growth-dependent control of ribosomal RNA synthesis, is the mammalian homolog of yeast Rrn3p; Bodem J et al.; Cells carefully modulate the rate of rRNA transcription in order to prevent an overinvestment in ribosome synthesis under less favorable nutritional conditions . In mammals, growth-dependent regulation of RNA polymerase I (Pol I) transcription is mediated by TIF-IA, an essential initiation factor that is active in extracts from growing but not starved or cycloheximide-treated mammalian cells . Here we report the molecular cloning and functional characterization of recombinant TIF-IA, which turns out to be the mammalian homolog of the yeast factor Rrn3p . We demonstrate that TIF-IA interacts with Pol I in the absence of template DNA, augments Pol I transcription in vivo and rescues transcription in extracts from growth-arrested cells in vitro.

Biochem Biophys Res Commun, 2001 Mar 23, 282(1), 334 - 40
UCP3 expressed in yeast is primarily localized in extramitochondrial particles; Winkler E et al.; Previously it was concluded (1) that, differently from UCP1, on expression in Saccharomyces cerevisiae, UCP3, and UCP3 short (UCP3s) are in a deranged state, allowing for unregulated uncoupling . Here we show that the bulk of UCP3 and UCP3s is in extramitochondrial aggregates whether expressed with high or medium expression vectors . The evidence is based on the insolubility of most UCP3 and UCP3s in nonionic detergents such as Triton X100, in contrast to UCP1 . Using very high expression vector, macroscopic evidence for extramitochondrial UCP3 containing particles is a viscous white sediment surrounding the mitochondrial fraction which contains UCP3 as inclusion body type aggregate . Together with the previous data it is concluded that uncoupling due to small amounts of incorporated, deranged, and nucleotide insensitive UCP3 prevents incorporation of the bulk of UCP3 into mitochondria . This finding also provides a simple and stringent assay for the state of heterologously expressed in mitochondrial membrane proteins .

Biochem Biophys Res Commun, 2001 Mar 23, 282(1), 60 - 6
Abc protein transport of MRI contrast agents in canalicular rat liver plasma vesicles and yeast vacuoles; Pascolo L et al.; The mechanism of excretion into bile of hepatospecific magnetic resonance imaging (MRI) contrast media employed labeled Gd-reagents EOB.DTPA, BOPTA, B 20790 (iopanoate-linked), and B 21690 (glycocholate-linked) for measurement in rat liver canalicular plasma membrane vesicles and yeast vacuoles . The presence of ATP gave threefold greater transport of B 20790 and B 21690 than of EOB.DTPA and BOPTA . In yeast vacuoles the ATP stimulatory effect was eightfold with B 20790 and fivefold greater for B 21690, whereas in YCF1- or YLLO115w-deleted yeast cells the transport was significantly reduced and absent from double mutants, YCF1 and YLLO15w . The transport was similar in wild-type and deletant cells for B 21690; taurocholate gave 85% inhibition . These data suggest that bilary secretion of structurally related MRI agents depend on molecular structure . The findings are suggestive as of possible value for clinical diagnosis of inherited hyperbilirubinemias and other liver disorders .

EMBO Rep, 2000 Dec, 1(6), 507 - 12
The yeast mitotic cyclin Clb2 cannot substitute for S phase cyclins in replication origin firing; Donaldson AD; Cyclin-dependent kinases (CDKs) drive the cell cycle, central to which is the accurate control of chromosome replication . In Saccharomyces cerevisiae, six closely related B-type cyclins (Clb1-6) drive the events of S phase and mitosis . Either Clb5 or Clb6 can activate early-firing replication origins, whereas only Clb5 can activate late origins . Clb1-4 are expressed later in the cell cycle . Whether Clb cyclins differ only in timing of expression, or else impart different kinase specificities is under ongoing investigation . This study shows that the expression of Clb2 during S phase in cells lacking Clb5 failed to rescue late origin activation . Early expression of Clb2 in cells lacking both Clb5 and Clb6 did not activate early origins on schedule to restore the correct S phase entry time . Therefore, Clb2 cannot drive timely activation of either early or late replication origins, demonstrating that Clb2-directed CDK has a specificity distinct from that driven by Clb5 and Clb6.

Can J Microbiol, 2001 Feb, 47(2), 123 - 9
Improving water stress tolerance of the biocontrol yeast Candida sake grown in molasses-based media by physiological manipulation; Abadias M et al.; The biocontrol agent Candida sake was cultured on either an unmodified molasses-based medium (water activity, a(w) 0.996) or on water stressed media produced by the addition of glycerol, glucose, NaCl, sorbitol, or proline to 0.98, and 0.96 a(w) for 24, 48, and 72 h, to study their impact on subsequent cell viability, and on concentrations of endogenous sugars (trehalose and glucose) and polyols (glycerol, erythritol, arabitol, and mannitol) . The viability of cells of different ages cultured on these media was evaluated on NYDA medium with freely available water (a(w) 0.995), and on medium modified with polyethylene glycol to a(w) 0.95 . Regardless of solute used, viable counts of cells grown on molasses-based medium (a(w) 0.98) were equal to or higher than those obtained from the medium with water freely available . The amino acid proline stimulated growth at 10% concentration . In contrast, water stress induced by addition of NaCl, glucose, or sorbitol at a(w) 0.96 caused a significant reduction in viable counts . Older cultures were more resistant to water stress . Glycerol and arabitol were the main solutes accumulated by C . sake cells in response to lowered a(w) . Intracellular concentration of these polyols depended more on the solute used to adjust the a(w) than on the a(w) itself . Candida sake was more resistant to water stress with higher intracellular concentration of glycerol and erythritol.

Can J Microbiol, 2001 Feb, 47(2), 103 - 9
Metschnikowia lochheadii and Metschnikowia drosophilae, two new yeast species isolated from insects associated with flowers; Lachance MA et al.; Two new haplontic heterothallic species of Metschnikowia were isolated from floricolous insects and flowers . Metschnikowia lochheadii was recovered from insects found in various flowers on the Hawaiian Islands of Kauai and Maui, and from Conotelus sp . (Coleoptera: Nitidulidae) in northwestern Guanacaste Province, Costa Rica . The morphology, physiology, and sexual cycle are typical of the large-spored Metschnikowia species, and the partial ribosomal DNA large subunit (D1D2) sequences suggest that the new species is most closely related to Candida ipomoeae . Metschnikowia lochheadii is nearly indistinguishable from its ascogenous relatives and conjugates freely with Metschnikowia continentalis, forming sterile asci . It also exhibits asymmetric mating with Metschnikowia hawaiiensis . Metschnikowia drosophilae was found in morning glory (Ipomoea sp.) flowers and associated Drosophila bromeliae on Grand Cayman Island . Its nutritional profile is atypical of the genus, being the only species that does not utilize sucrose or maltose as carbon sources, and one of the few that does not utilize melezitose . D1D2 sequences show that Metschnikowia drosophilae is a sister species to Candida torresii, to which it bears considerable similarity in nutritional profile . The type cultures are: Metschnikowia lochheadii, strains UWO(PS)00-133.2 = CBS 8807 (h+, holotype) UWO(PS)99-661.1 = CBS 8808 (h-, isotype); and Metschnikowia drosophilae, strains UWO(PS)83-1135.3 = CBS 8809 (h+, holotype) and UWO(PS)83-1143.1 = CBS 8810 (h-, isotype).

J Asian Nat Prod Res, 1999, 2(1), 39 - 44
Biotransformation of 24 alpha-methylcholesterol and 24 beta-methylcholesterol by yeast mutant GL7; Cui YJ et al.; Incubation of 24 alpha- and 24 beta-methylcholesterols with yeast mutant GL7 afforded their corresponding C-22-desaturated products under the catalysis of sterol delta 22(23)-desaturase . The metabolites were identified to be 22-dehydro-24 alpha-methylcholesterol (2% yield from 24 alpha-methylcholesterol) and 22-dehydro-24 beta-methylcholesterol (51% yield from 24 beta-methylcholesterol) respectively on the basis of their chromatographic and spectral properties . It was concluded that the sterol delta 22(23)-desaturase prefers the 24 beta-methyl sterols and is highly stereospecific.

Philos Trans R Soc Lond B Biol Sci, 2001 Feb 28, 356(1406), 169 - 76
Investigating protein conformation-based inheritance and disease in yeast; Lindquist S et al.; Our work supports the hypothesis that a protein can serve as an element of genetic inheritance . This protein-only mechanism of inheritance is propagated in much the same way as hypothesized for the transmission of the protein-only infectious agent in the spongiform encephalopathies; hence these protein factors have been called yeast prions . Our work has focused on {PSI(+)}, a dominant cytoplasmically inherited factor that alters translational fidelity.This change in translation is produced by a self-perpetuating change in the conformation of the translation-termination factor, Sup35 . Most recently, we have determined that new elements of genetic inheritance can be created by deliberate genetic engineering, opening prospects for new methods of manipulating heredity . We have also uncovered evidence that other previously unknown elements of protein-based inheritance are encoded in the yeast genome . Finally, we have begun to use yeast as a model system for studying human protein folding diseases, such as Huntington's disease . Proteins responsible for some of these diseases have properties uncannily similar to those that produce protein-based mechanisms of inheritance.

Traffic, 2001 Mar, 2(3), 212 - 31
Dsl1p, an essential protein required for membrane traffic at the endoplasmic reticulum/Golgi interface in yeast; Vanrheenen SM et al.; To identify novel factors required for ER to Golgi transport in yeast we performed a screen for genes that, when mutated, confer a dependence on a dominant mutant form of the ER to Golgi vesicle docking factor Sly1p, termed Sly1-20p . DSL1, a novel gene isolated in the screen, encodes an essential protein with a predicted molecular mass of 88 kDa . DSL1 is required for transport between the ER and the Golgi because strains bearing mutant alleles of this gene accumulate the pre-Golgi form of transported proteins at the restrictive temperature . Two strains bearing temperature-sensitive alleles of DSL1 display distinct phenotypes as observed by electron microscopy at the restrictive temperature; although both strains accumulate ER membrane, one also accumulates vesicles . Interestingly, the inviability of strains bearing several mutant alleles of DSL1 can be suppressed by expression of either Erv14p (a protein required for the movement of a specific protein from the ER to the Golgi), Sec21p (the gamma-subunit of the COPI coat protein complex coatomer), or Sly1-20p . Because the strongest suppressor is SEC21, we proposed that Dsl1p functions primarily in retrograde Golgi to ER traffic, although it is possible that Dsl1p functions in anterograde traffic as well, perhaps at the docking stage, as suggested by the suppression by SLY1-20.

Genes Cells, 2001 Mar, 6(3), 201 - 14
Functional analysis of the C-terminal cytoplasmic region of the M-factor receptor in fission yeast; Hirota K et al.; BACKGROUND: Yeast mating-pheromone receptors facilitate the study of G protein-coupled signal transduction . To date, molecular dissection of the budding yeast alpha-factor receptor has been done extensively, but little analysis has been performed with pheromone receptors of fission yeast, another genetically tractable yeast species . RESULTS: We analysed the fission yeast M-factor receptor Map3p . Truncation of the C-terminal 54 amino acids made Map3p dominant-negative over the wild-type . This form, called Map3-dn9p, was competent in the induction of pheromone-dependent gene expression, although it could not direct proper conjugation . Map3-dn9p failed both to provoke the orientated projection of conjugation tubes and to induce adaptation to the pheromone signal associated with endocytosis of the receptor . Deletion and substitution analyses suggested that the integrity of the C-terminal region, rather than a specific subgroup of amino acid residues therein, was vital for the respective Map3p activities . Ubiquitination of the C-terminus was not absolutely essential for Map3p function . CONCLUSIONS: The C-terminal region of Map3p is dispensable for the pheromone signalling per se, but is pivotal for adaptation and pheromone-induced conjugation tube formation, as is true with the budding yeast alpha-factor receptor . However, the mechanisms which induce adaptation appear to differ between fission and budding yeast concerning the necessity of ubiquitination.

Genes Cells, 2001 Mar, 6(3), 187 - 99
Identification and functional analysis of the gene for type I myosin in fission yeast; Toya M et al.; BACKGROUND: Type I myosin is highly conserved among eukaryotes, and apparently plays important roles in a number of cellular processes . In the budding yeast, two myosin I species have been identified and their role in F-actin assembly has been inferred . RESULTS: We cloned the fission yeast myo1 gene, which apparently encoded a myosin I protein . Disruption of myo1 was not lethal, but it caused growth retardation at high and low temperatures, sensitivity to a high concentration of KCl, and aberrance in cell morphology associated with an abnormal distribution of F-actin patches . An abnormal deposition of cell wall materials was also seen . Homothallic myo1Delta cells could mate, but heterothallic myo1Delta cells were poor in conjugation . Myo1p was necessary for the encapsulation of spores . The tail domain of Myo1p was pivotal for its function . Calmodulin could bind to Myo1p through the IQ domain at the neck . CONCLUSIONS: Myo1p appears to control the redistribution of F-actin patches during the cell cycle . Loss of Myo1p function is likely to slow down the actin assembly/disassembly process, which results in a failure of the actin cycle to catch up with other events in both the mitotic and meiotic cell cycles, including extension of the conjugation tubes.

Mol Cell Biol, 2001 Apr, 21(7), 2292 - 7
Role of TATA binding protein (TBP) in yeast ribosomal dna transcription by RNA polymerase I: defects in the dual functions of transcription factor UAF cannot be suppressed by TBP; Siddiqi I et al.; Initiation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae involves upstream activation factor (UAF), core factor, the TATA binding protein (TBP), and Rrn3p in addition to Pol I . We found previously that yeast strains carrying deletions in the UAF component RRN9 switch completely to the use of Pol II for rRNA transcription, with no residual Pol I transcription . These polymerase-switched strains initially grow very slowly, but subsequent expansion in the number of rDNA repeats on chromosome XII leads to better growth . Recently, it was reported that TBP overexpression could bypass the requirement of UAF for Pol I transcription in vivo, producing nearly wild-type levels of growth in UAF mutant strains (P . Aprikian, B . Moorefield, and R . H . Reeder, Mol . Cell . Biol . 20:5269-5275, 2000) . Here, we demonstrate that deletions in the UAF component RRN5, RRN9, or RRN10 lead to Pol II transcription of rDNA . TBP overexpression does not suppress UAF mutation, and these strains continue to use Pol II for rRNA transcription . We do not find evidence for even low levels of Pol I transcription in UAF mutant strains carrying overexpressed TBP . In diploid strains lacking both copies of the UAF component RRN9, Pol II transcription of rDNA is more strongly repressed than in haploid strains but TBP overexpression still fails to activate Pol I . These results emphasize that UAF plays an essential role in activation of Pol I transcription and silencing of Pol II transcription of rDNA and that TBP functions to recruit the Pol I machinery in a manner completely dependent on UAF.

Biochemistry, 2001 Mar 6, 40(9), 2954 - 63
The role of copper in topa quinone biogenesis and catalysis, as probed by azide inhibition of a copper amine oxidase from yeast; Schwartz B et al.; All known copper amine oxidases (CAOs) contain 2,4,5-trihydroxyphenylalanine quinone (TPQ) as a redox cofactor . TPQ is derived posttranslationally from a specific tyrosine residue within the protein itself, and is utilized by the enzyme to oxidize amines to aldehydes . Several oxidative mechanisms for both turnover and the biogenesis of the cofactor have been proposed in recent years, which differ mainly in the nature of the interaction of oxygen with the enzyme . In this study, azide is used to probe the role of copper in catalysis and biogenesis, especially with respect to potential interactions between the metal and oxygen . During turnover, it is found that azide is a noncompetitive inhibitor with respect to O(2), most consistent mechanistically with oxygen binding off the metal prior to reaction . During biogenesis, it is found that azide likely prohibits ligation of the precursor tyrosine to the copper, thus preventing the formation of this key intermediate . This result is consistent with previous proposals, where the copper-tyrosine unit is the species that undergoes reaction with O(2) . In addition, it is found that oxygen consumption is kinetically uncoupled from TPQ formation; this leads to an expanded kinetic model for biogenesis, with important implications for previous results.

FEBS Lett, 2001 Mar 16, 492(3), 238 - 41
Nucleotides U28-A42 and A37 in unmodified yeast tRNA(Trp) as negative identity elements for bovine tryptophanyl-tRNA synthetase; Carnicelli D et al.; Wild-type bovine and yeast tRNA(Trp) are efficiently aminoacylated by tryptophanyl-tRNA synthetase both from beef and from yeast . Upon loss of modified bases in the synthetic transcripts, mammalian tRNA(Trp) retains the double recognition by the two synthetases, while yeast tRNA(Trp) loses its substrate properties for the bovine enzyme and is recognised only by the cognate synthetase . By testing chimeric bovine-yeast transcripts with tryptophanyl-tRNA synthetase purified from beef pancreas, the nucleotides responsible for the loss of charging of the synthetic yeast transcript have been localised in the anticodon arm . A complete loss of charging akin to that observed with the yeast transcript requires substitution in the bovine backbone of G37 in the anticodon loop with yeast A37 and of C28-G42 in the anticodon stem with yeast U28-A42 . Since A37 does not prevent aminoacylation of the wild-type yeast tRNA(Trp) by the beef enzyme, a negative combination apparently emerges in the synthetic transcript after unmasking of U28 by loss of pseudourydilation.

J Gen Virol, 2001 Apr, 82(Pt 4), 935 - 9
Towards a protein interaction map of potyviruses: protein interaction matrixes of two potyviruses based on the yeast two-hybrid system; Guo D et al.; A map for the interactions of the major proteins from Potato virus A (PVA) and Pea seed-borne mosaic virus (PSbMV) (members of the genus POTYVIRUS:, family POTYVIRIDAE:) was generated using the yeast two-hybrid system (YTHS) . Interactions were readily detected with five PVA protein combinations (HC-HC, HC-CI, VPg-VPg, NIa-NIb and CP-CP) and weak but reproducible interactions were detected for seven additional combinations (P1-CI, P3-NIb, NIaPro-NIb, VPg-NIa, VPg-NIaPro, NIaPro-NIa and NIa-NIa) . In PSbMV, readily detectable interactions were found in five protein combinations (HC-HC, VPg-VPg, VPg-NIa, NIa-NIa and NIa-NIb) and weaker but reproducible interactions were detected for three additional combinations (P3-NIa, NIa-NIaPro and CP-CP) . The self-interactions of HC, VPg, NIa and CP and the interactions of VPg-NIa, NIa-NIaPro and NIa-NIb were, therefore, common for the two potyviruses . The multiple protein interactions revealed in this study shed light on the co-ordinated functions of potyviral proteins involved in virus movement and replication.

J Cell Biol, 2001 Mar 19, 152(6), 1255 - 66
Budding yeast chromosome structure and dynamics during mitosis; Pearson CG et al.; Using green fluorescent protein probes and rapid acquisition of high-resolution fluorescence images, sister centromeres in budding yeast are found to be separated and oscillate between spindle poles before anaphase B spindle elongation . The rates of movement during these oscillations are similar to those of microtubule plus end dynamics . The degree of preanaphase separation varies widely, with infrequent centromere reassociations observed before anaphase . Centromeres are in a metaphase-like conformation, whereas chromosome arms are neither aligned nor separated before anaphase . Upon spindle elongation, centromere to pole movement (anaphase A) was synchronous for all centromeres and occurred coincident with or immediately after spindle pole separation (anaphase B) . Chromatin proximal to the centromere is stretched poleward before and during anaphase onset . The stretched chromatin was observed to segregate to the spindle pole bodies at rates greater than centromere to pole movement, indicative of rapid elastic recoil between the chromosome arm and the centromere . These results indicate that the elastic properties of DNA play an as of yet undiscovered role in the poleward movement of chromosome arms.

Biochem J, 2001 Apr 1, 355(Pt 1), 223 - 30
Structural and functional similarities between the central eukaryotic initiation factor (eIF)4A-binding domain of mammalian eIF4G and the eIF4A-binding domain of yeast eIF4G; Dominguez D et al.; The translation eukaryotic initiation factor (eIF)4G of the yeast Saccharomyces cerevisiae interacts with the RNA helicase eIF4A (a member of the DEAD-box protein family; where DEAD corresponds to Asp-Glu-Ala-Asp) through a C-terminal domain in eIF4G (amino acids 542-883) . Mammalian eIF4G has two interaction domains for eIF4A, a central domain and a domain close to the C-terminus . This raises the question of whether eIF4A binding to eIF4G is conserved between yeast and mammalian cells or whether it is different . We isolated eIF4G1 mutants defective in eIF4A binding and showed that these mutants are strongly impaired in translation and growth . Extracts from mutants displaying a temperature-sensitive phenotype for growth have low in vitro translation activity, which can be restored by addition of the purified eIF4G1-eIF4E complex, but not by eIF4E alone . Analysis of mutant eIF4G(542-883) proteins defective in eIF4A binding shows that the interaction of yeast eIF4A with eIF4G1 depends on amino acid motifs that are conserved between the yeast eIF4A-binding site and the central eIF4A-binding domain of mammalian eIF4G . We show that mammalian eIF4A binds tightly to yeast eIF4G1 and, furthermore, that mutant yeast eIF4G(542-883) proteins, which do not bind yeast eIF4A, do not interact with mammalian eIF4A . Despite the conservation of the eIF4A-binding site in eIF4G and the strong sequence conservation between yeast and mammalian eIF4A (66% identity; 82% similarity at the amino acid level) mammalian eIF4A does not substitute for the yeast factor in vivo and is not functional in a yeast in vitro translation system.

Yeast, 2001 Mar 30, 18(5), 481 - 8
Current awareness on yeast; Watson MD; In order to keep subscribers up-to-date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly-published material on yeasts . Each bibliography is divided into 10 sections . 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology . Within each section, articles are listed in alphabetical order with respect to author . If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted . (4 weeks journals - search completed 3rd Jan . 2001)

Yeast, 2001 Mar 30, 18(5), 403 - 21
Yeast 2 microm plasmid copy number is elevated by a mutation in the nuclear gene UBC4; Sleep D et al.; The copy number of the Saccharomyces cerevisiae endogenous 2 microm plasmid is under strict control to ensure efficient propagation to the daughter cell without significantly reducing the growth rate of the mother or the daughter cell . A recessive mutation has been identified that resulted in an elevated but stable 2 microm plasmid copy number, which could be complemented by a genomic DNA clone containing the UBC4 gene, encoding an E2 ubiquitin-conjugating enzyme . A ubc4::URA3 deletion resulted in the same elevated 2 microm plasmid copy number . An analysis of the endogenous 2 microm transcripts revealed that the steady-state abundance of REP1, REP2, FLP and RAF were all increased 4-5-fold in the mutant . Analysis of the mutant ubc4 allele identified a single base pair mutation within the UBC4 coding region, which would generate a glutamic acid to lysine amino acid substitution within a region of conserved tertiary structure located within the first alpha-helix of Ubc4p . These investigations represent the first molecular characterization of a mutation within a Saccharomyces cerevisiae nuclear gene shown to affect 2 microm steady-state plasmid copy number and implicate the ubiquitin-dependent proteolytic pathway in host control of 2 microm plasmid copy number .

Mol Gen Genet, 2001 Feb, 264(6), 842 - 51
A compromised yeast RNA polymerase II enhances UV sensitivity in the absence of global genome nucleotide excision repair; Wong JM et al.; Nucleotide excision repair is the major pathway responsible for removing UV-induced DNA damage, and is therefore essential for cell survival following exposure to UV radiation . In this report, we have assessed the contributions of some components of the RNA polymerase II (Pol II) transcription machinery to UV resistance in Saccharomyces cerevisiae . Deletion of the gene encoding the Pol II elongation factor TFIIS (SII) resulted in enhanced UV sensitivity, but only in the absence of global genome repair dependent on the RAD7 and RAD16 genes, a result seen previously with deletions of RAD26 and RAD28, yeast homologs of the human Cockayne syndrome genes CSB and CSA, respectively . A RAD7/16-dependent reduction in survival after UV irradiation was also seen in the presence of mutations in RNA Pol II that confer a defect in its response to SII, as well as with other mutations which reside in regions of the largest subunit of Pol II not involved in SII interactions . Indeed, an increase in UV sensitivity was achieved by simply decreasing the steadystate level of RNA Pol II . Truncation of the C-terminal domain and other RNA Pol II mutations conferred sensitivity to the ribonucleotide reductase inhibitor hydroxyurea and induction of RNR1 and RNR2 mRNAs after UV irradiation was attenuated in these mutant cells . That UV sensitivity can be a consequence of mutations in the RNA Pol II machinery in yeast cells suggests that alterations in transcriptional programs could underlie some of the pathophysiological defects seen in the human disease Cockayne syndrome.

Biotechniques, 2001 Mar, 30(3), 634 - 6, 638, 640 passim
Redefinition of the yeast two-hybrid system in dialogue with changing priorities in biological research; Serebriiskii IG et al.; Examination of the pattern of reagent creation and application in the two-hybrid system since 1989 reveals the expansion of a simple core technology to address increasingly sophisticated problems in protein interaction . As the technology has matured, its clear suitability for large-scale proteomic projects has made a major focus of its application the generation of global organismal protein interaction networks . In an inversion of emphasis, the increasing availability of such information now provides a master plan with the potential to specify the most promising directions for biological investigations (i.e., by directing the physiological validation of predicted critical protein-protein interactions) . Recent derivatives of the two-hybrid system enable the targeting of such key interactions by facilitating the identification of essential amino acids conferring protein interaction specificity and of small molecules that selectively disrupt defined interaction pairs . Finally, the creation of mammalian expression systems based on two-hybrid principles became a new tool to create and probe novel biological systems . Taken in sum, this trajectory emphasizes the point that the creation of tools and the evolution of the idea of what is an interesting biological problem are in intimate dialogue.

EMBO Rep, 2001 Jan, 2(1), 35 - 41
Fission yeast Prp4p kinase regulates pre-mRNA splicing by phosphorylating a non-SR-splicing factor; Schwelnus W et al.; We provide evidence that Prp4p kinase activity is required for pre-mRNA splicing in vivo and show that loss of activity impairs G1-S and G2-M progression in the cell cycle . Prp4p interacts genetically with the non-SR (serine/arginine) splicing factors Prp1p and Prp5p . Bacterially produced Prp1p is phosphorylated by Prp4p in vitro . Prp4p and Prp1p also interact in the yeast two-hybrid system . In vivo labelling studies using a strain with a mutant allele of the prp4 gene in the genetic background indicate a change in phosphorylation of the Prp1p protein . These results are consistent with the notion that Prp4p kinase is involved in the control of the formation of active spliceosomes, targeting non-SR splicing factors.

Mol Microbiol, 2001 Feb, 39(4), 914 - 23
Activation of heat shock transcription factor in yeast is not influenced by the levels of expression of heat shock proteins; Hjorth-Sorensen B et al.; Heat shock transcription factor (HSF) transiently induces the expression of a universally conserved set of proteins, the heat shock proteins (Hsps), when cells are exposed to elevated temperatures as well as to a wide range of other environmental stresses . The tight control of heat shock gene expression has prompted a model, according to which HSF activity and 'free' heat shock protein levels are tied up in a regulatory loop . Other data have indicated that HSF senses stress directly . Here, we report that yeast cells in which the basal expression levels of Hsps have been significantly increased exhibit improved thermotolerance but display no detectable difference in the temperature required for transient activation of HSF . In a separate experiment, overexpression of SSA2, a member of the Hsp70 family and a prominent candidate for the feedback regulation of HSF, did not inhibit the heat shock response . Our findings challenge the dogma that relief of the suppression of HSF activity by Hsps can account for the acute heat shock response.

Mol Microbiol, 2001 Feb, 39(4), 875 - 89
Selective expression of the virulence factor BAD1 upon morphogenesis to the pathogenic yeast form of Blastomyces dermatitidis: evidence for transcriptional regulation by a conserved mechanism; Rooney PJ et al.; Most dimorphic fungal pathogens grow as non-pathogenic moulds in soil and convert to pathogenic yeast in the host, suggesting that virulence factors are upregulated during phase transition . Such factors have been difficult to identify . We analysed BAD1 (formerly WI-1), a virulence factor in the dimorphic fungus Blastomyces dermatitidis, for expression in yeast and mycelial morphotypes . BAD1 was expressed in yeast but not in mycelia of North American strains of B . dermatitidis, and this expression pattern was confirmed for BAD1 transcript . BAD1 under the control of its promoter was transferred into African B . dermatitidis lacking a native BAD1 locus, and phase-specific expression was conserved . Sequence similarity was identified between the BAD1 promoter and the promoters of two yeast phase-specific genes in Histoplasma capsulatum . In H . capsulatum BAD1 transformants, yeast phase-specific expression of BAD1 was conserved, and no transcript was detected in mycelia . BAD1 beta-galactosidase reporter fusions analysed in B . dermatitidis and H . capsulatum confirmed that BAD1 is transcriptionally regulated in both fungi . BAD1 promoter activity and surface BAD1 expression were detected 6 h after shifting mycelia to 37 degrees C . Thus, BAD1 is expressed after transition to the pathogenic yeast morphotype and is regulated by a mechanism for phase-specific gene expression that appears to be conserved.

Int J Biol Macromol, 2001 Mar 14, 28(3), 213 - 8
A differential scanning calorimetric study of the effects of metal ions, substrate/product, substrate analogues and chaotropic anions on the thermal denaturation of yeast enolase 1; Brewer JM et al.; The thermal denaturation of yeast enolase 1 was studied by differential scanning calorimetry (DSC) under conditions of subunit association/dissociation, enzymatic activity or substrate binding without turnover and substrate analogue binding . Subunit association stabilizes the enzyme, that is, the enzyme dissociates before denaturing . The conformational change produced by conformational metal ion binding increases thermal stability by reducing subunit dissociation . 'Substrate' or analogue binding additionally stabilizes the enzyme, irrespective of whether turnover is occurring, perhaps in part by the same mechanism . More strongly bound metal ions also stabilize the enzyme more, which we interpret as consistent with metal ion loss before denaturation, though possibly the denaturation pathway is different in the absence of metal ion . We suggest that some of the stabilization by 'substrate' and analogue binding is owing to the closure of moveable polypeptide loops about the active site, producing a more 'closed' and hence thermostable conformation.

Mol Biol Cell, 2001 Mar, 12(3), 753 - 60
In vivo binding and hierarchy of assembly of the yeast RNA polymerase I transcription factors; Bordi L et al.; Transcription by RNA polymerase I in Saccharomyces cerevisiae requires a series of transcription factors that have been genetically and biochemically identified . In particular, the core factor (CF) and the upstream activation factor (UAF) have been shown in vitro to bind the core element and the upstream promoter element, respectively . We have analyzed in vivo the DNAse I footprinting of the 35S promoter in wild-type and mutant strains lacking one specific transcription factor at the time . In this way we were able to unambiguously attribute the protections by the CF and the UAF to their respective putative binding sites . In addition, we have found that in vivo a binding hierarchy exists, the UAF being necessary for CF binding . Because the CF footprinting is lost in mutants lacking a functional RNA polymerase I, we also conclude that the final step of preinitiation-complex assembly affects binding of the CF, stabilizing its contact with DNA . Thus, in vivo, the CF is recruited to the core element by the UAF and stabilized on DNA by the presence of a functional RNA polymerase I.

Mol Biol Cell, 2001 Mar, 12(3), 711 - 23
A family of small coiled-coil-forming proteins functioning at the late endosome in yeast; Kranz A et al.; The multispanning membrane protein Ste6, a member of the ABC-transporter family, is transported to the yeast vacuole for degradation . To identify functions involved in the intracellular trafficking of polytopic membrane proteins, we looked for functions that block Ste6 transport to the vacuole upon overproduction . In our screen, we identified several known vacuolar protein sorting (VPS) genes (SNF7/VPS32, VPS4, and VPS35) and a previously uncharacterized open reading frame, which we named MOS10 (more of Ste6) . Sequence analysis showed that Mos10 is a member of a small family of coiled-coil-forming proteins, which includes Snf7 and Vps20 . Deletion mutants of all three genes stabilize Ste6 and show a "class E vps phenotype." Maturation of the vacuolar hydrolase carboxypeptidase Y was affected in the mutants and the endocytic tracer FM4-64 and Ste6 accumulated in a dot or ring-like structure next to the vacuole . Differential centrifugation experiments demonstrated that about half of the hydrophilic proteins Mos10 and Vps20 was membrane associated . The intracellular distribution was further analyzed for Mos10 . On sucrose gradients, membrane-associated Mos10 cofractionated with the endosomal t-SNARE Pep12, pointing to an endosomal localization of Mos10 . The growth phenotypes of the mutants suggest that the "Snf7-family" members are involved in a cargo-specific event.

Mol Biol Cell, 2001 Mar, 12(3), 699 - 710
Interaction of the E1A oncoprotein with Yak1p, a novel regulator of yeast pseudohyphal differentiation, and related mammalian kinases; Zhang Z et al.; The C-terminal portion of adenovirus E1A suppresses ras-induced metastasis and tumorigenicity in mammalian cells; however, little is known about the mechanisms by which this occurs . In the simple eukaryote Saccharomyces cerevisiae, Ras2p, the homolog of mammalian h-ras, regulates mitogen-activated protein kinase (MAPK) and cyclic AMP-dependent protein kinase A (cAMP/PKA) signaling pathways to control differentiation from the yeast form to the pseudohyphal form . When expressed in yeast, the C-terminal region of E1A induced pseudohyphal differentiation, and this was independent of both the MAPK and cAMP/PKA signaling pathways . Using the yeast two-hybrid system, we identified an interaction between the C-terminal region of E1A and Yak1p, a yeast dual-specificity serine/threonine protein kinase that functions as a negative regulator of growth . E1A also physically interacts with Dyrk1A and Dyrk1B, two mammalian homologs of Yak1p, and stimulates their kinase activity in vitro . We further demonstrate that Yak1p is required in yeast to mediate pseudohyphal differentiation induced by Ras2p-regulated signaling pathways . However, pseudohyphal differentiation induced by the C-terminal region of E1A is largely independent of Yak1p . These data suggest that mammalian Yak1p-related kinases may be targeted by the E1A oncogene to modulate cell growth.

Mol Biol Cell, 2001 Mar, 12(3), 577 - 88
Multifaceted physiological response allows yeast to adapt to the loss of the signal recognition particle-dependent protein-targeting pathway; Mutka SC et al.; Translational control has recently been recognized as an important facet of adaptive responses to various stress conditions . We describe the adaptation response of the yeast Saccharomyces cerevisiae to the loss of one of two mechanisms to target proteins to the secretory pathway . Using inducible mutants that block the signal recognition particle (SRP) pathway, we find that cells demonstrate a physiological response to the loss of the SRP pathway that includes specific changes in global gene expression . Upon inducing the loss of the SRP pathway, SRP-dependent protein translocation is initially blocked, and cell growth is considerably slowed . Concomitantly, gene expression changes include the induction of heat shock genes and the repression of protein synthesis genes . Remarkably, within hours, the efficiency of protein sorting improves while cell growth remains slow in agreement with the persistent repression of protein synthesis genes . Our results suggest that heat shock gene induction serves to protect cells from mislocalized precursor proteins in the cytosol, whereas reduced protein synthesis helps to regain efficiency in protein sorting by reducing the load on the protein translocation apparatus . Thus, we suggest that cells trade speed in cell growth for fidelity in protein sorting to adjust to life without SRP.

Mol Biol Cell, 2001 Mar, 12(3), 539 - 49
The nuclear export receptor Xpo1p forms distinct complexes with NES transport substrates and the yeast Ran binding protein 1 (Yrb1p); Maurer P et al.; Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES) . Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore . We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran) . Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus . Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals . Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES . Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP . This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin beta-like transport receptors . A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus . Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p . This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.

Curr Opin Genet Dev, 2001 Apr, 11(2), 142 - 7
Transcription and chromatin converge: lessons from yeast genetics; Gregory PD; The control of transcription through the modification of chromatin has been a subject of intense study over the past year . The increasing use of genome-wide approaches to examine the role of chromatin and the complexes able to modify it is providing a global perspective that is profoundly altering our view of the transcription process.

Gene, 2001 Feb 21, 264(2), 249 - 56
Cloning and characterization of Chinese hamster homologue of yeast DBF4 (ChDBF4); Guo B et al.; The Dbf4 protein is the regulatory subunit of Cdc7 serine/threonine kinase, which is essential for entry into S phase . We report here the cloning and initial characterization of the Chinese hamster homologue of yeast DBF4 . The deduced ChDbf4 protein contains 676 amino acids with a predicted molecular mass of 75.8 kDa, and shares extensive identity overall with those of human (68%) and mouse (73%) . The ChDBF4 mRNA level was barely detectable in the cells arrested in the quiescent stage (G(0)) by isoleucine starvation . When cells in G(0) were released into the cell cycle, the ChDBF4 mRNA level did not significantly change until the cells reached the G(1)/S boundary, when the level rapidly increased and reached approximately 70% of the maximum level that was observed in mid to late S phase . Interestingly, gamma-irradiation rapidly and transiently downregulated the level of ChDBF4 mRNA in asynchronous cell populations . Since Dbf4-Cdc7 kinase is involved in the regulation of replication initiation, which can be transiently downregulated by irradiation (Larner et al., 1994 . Mol . Cell . Biol . 14, 1901, our data raise the possibility that the downregulation of DBF4 (and, thus, the Cdc7 kinase activity) by irradiation may play a role in the cell-cycle checkpoint that functions at the G(1)/S transition and in S phase (Lee et al., 1997 . Proc . Natl . Acad . Sci . USA 94, 526).

Biochim Biophys Acta, 2001 Mar 9, 1511(1), 74 - 9
Factors underlying membrane potential-dependent and -independent fluorescence responses of potentiometric dyes in stressed cells: diS-C(3)(3) in yeast; Gaskova D et al.; The redistribution fluorescent dye diS-C(3)(3) responds to yeast plasma membrane depolarisation or hyperpolarisation by Delta psi-dependent outflow from or uptake into the cells, reflected in changes in the fluorescence maximum lambda(max) and fluorescence intensity . Upon membrane permeabilisation the dye redistributes between the cell and the medium in a purely concentration-dependent manner, which gives rise to Delta psi-independent fluorescence responses that may mimic Delta psi-dependent blue or red shift in lambda(max) . These lambda(max) shifts after cell permeabilisation depend on probe and ion concentrations inside and outside the cells at the moment of permeabilisation and reflect (a) permeabilisation-induced Delta psi collapse, (b) changing probe binding capacity of cell constituents (inverse to the ambient ionic strength) and (c) hampering of probe equilibration by the poorly permeable cell wall . At low external ion concentrations, cell permeabilisation causes ion outflow and probe influx (hyperpolarisation-like red shift in lambda(max)) caused by an increase in the probe-binding capacity of the cell interior and, in the case of heat shock, protein denaturation unmasking additional probe-binding sites . At high external ion levels minimising net ion efflux and at high intracellular probe concentrations at the moment of permeabilisation, the Delta psi collapse causes a blue lambda(max) shift mimicking an apparent depolarisation.

Proc Natl Acad Sci U S A, 2001 Mar 13, 98(6), 3050 - 5 Epub 2001 Mar 06.
A yeast-like mRNA capping apparatus in Plasmodium falciparum; Ho CK et al.; Analysis of the mRNA capping apparatus of the malaria parasite Plasmodium falciparum illuminates an evolutionary connection to fungi rather than metazoans . We show that P . falciparum encodes separate RNA guanylyltransferase (Pgt1) and RNA triphosphatase (Prt1) enzymes and that the triphosphatase component is a member of the fungal/viral family of metal-dependent phosphohydrolases, which are structurally and mechanistically unrelated to the cysteine-phosphatase-type RNA triphosphatases found in metazoans and plants . These results highlight the potential for discovery of mechanism-based antimalarial drugs designed to specifically block the capping of Plasmodium mRNAs . A simple heuristic scheme of eukaryotic phylogeny is suggested based on the structure and physical linkage of the triphosphatase and guanylyltransferase enzymes that catalyze cap formation.

Plant Mol Biol, 2001 Jan, 45(1), 75 - 92
Molecular cloning and expression in yeast of 2,3-oxidosqualene-triterpenoid cyclases from Arabidopsis thaliana; Husselstein-Muller T et al.; A vast array of triterpenes are found in living organisms in addition to lanosterol and cycloartenol, which are involved in sterol biosynthesis in non-photosynthetic and photosynthetic eukaryotes respectively . The chemical structure of these triterpenes is determined by a single step catalysed by 2,3-oxidosqualene-triterpene cyclases . The present study describes cloning and functional expression in yeast of several OS-triterpene cyclases . Three Arabidopsis thaliana cDNAs encoding proteins (ATLUP1, ATLUP2, ATPEN1) 57%, 58% and 49% identical to cycloartenol synthase from the same plant were isolated . Expression of these cDNAs in yeast showed that the recombinant proteins catalyse the synthesis of various pentacyclic triterpenes . Whereas ATLUP1 is essentially involved in the synthesis of lupeol, ATLUP2 catalyses the production of lupeol, beta- and alpha-amyrin (in a 15:55:30 ratio) . ATLUP2 is therefore a typical multifunctional enzyme . Under the same conditions, ATPEN1 did not lead to any product . Systematic sequencing of the Arabidopsis genome has led to genomic sequences encoding proteins identical to the above triterpene synthases . ATLUPI and ATLUP2 are representative of a small subfamily (A) of at least five genes, whereas ATPEN1 is representative of a subfamily (B) of at least seven genes . The number of introns is characteristic of each subfamily . Whereas genes of family A possess 17 exons and 16 introns, genes of the subfamily B contain 14 exons and 13 introns . The size of each exon is remarkably conserved within each subfamily whereas that of each intron appears to be highly variable . Organization of the genes, sequences and functions of the deduced proteins are discussed in evolutionary terms.

Biochim Biophys Acta, 2001 Apr 2, 1504(2-3), 363 - 70
Efficient large scale purification of his-tagged proton translocating NADH:ubiquinone oxidoreductase (complex I) from the strictly aerobic yeast Yarrowia lipolytica; Kashani-Poor N et al.; Proton translocating NADH:ubiquinone oxidoreductase (complex I) is the largest membrane bound multiprotein complex of the respiratory chain and the only one for which no molecular structure is available so far . Thus, information on the mechanism of this central enzyme of aerobic energy metabolism is still very limited . As a new approach to analyze complex I, we have recently established the strictly aerobic yeast Yarrowia lipolytica as a model system that offers a complete set of convenient genetic tools and contains a complex I that is stable after isolation . For crystallization of complex I and to obtain its molecular structure it is a prerequisite to prepare large amounts of highly pure enzyme . Here we present the construction of his-tagged complex I that for the first time allows efficient affinity purification . Our protocol recovers almost 40% of complex I present in Yarrowia mitochondrial membranes . Overall, 40-80 mg highly pure and homogeneous complex I can be obtained from 10 l of an overnight Y . lipolytica culture . After reconstitution into asolectin proteoliposomes, the purified enzyme exhibits full NADH:ubiquinone oxidoreductase activity, is fully sensitive to inhibition by quinone analogue inhibitors and capable of generating a proton-motive force.

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