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