J Biol Chem, 2001 Dec 7, 276(49), 46333 - 9 Epub 2001 Sep 24. Pus1p-dependent tRNA pseudouridinylation becomes essential when tRNA biogenesis is compromised in yeast; Grosshans H et al.; Yeast Pus1p catalyzes the formation of pseudouridine (psi) at specific sites of several tRNAs, but its function is not essential for cell viability . We show here that Pus1p becomes essential when another tRNA:pseudouridine synthase, Pus4p, or the essential minor tRNA for glutamine are mutated . Strikingly, this mutant tRNA, which carries a mismatch in the T psi C arm, displays a nuclear export defect . Furthermore, nuclear export of at least one wild-type tRNA species becomes defective in the absence of Pus1p . Our data, thus, show that the modifications formed by Pus1p are essential when other aspects of tRNA biogenesis or function are compromised and suggest that impairment of nuclear tRNA export in the absence of Pus1p might contribute to this phenotype. EMBO J, 2001 Sep 17, 20(18), 5207 - 18 Structure of the yeast nucleosome core particle reveals fundamental changes in internucleosome interactions; White CL et al.; Chromatin is composed of nucleosomes, the universally repeating protein-DNA complex in eukaryotic cells . The crystal structure of the nucleosome core particle from Saccharomyces cerevisiae reveals that the structure and function of this fundamental complex is conserved between single-cell organisms and metazoans . Our results show that yeast nucleosomes are likely to be subtly destabilized as compared with nucleosomes from higher eukaryotes, consistent with the idea that much of the yeast genome remains constitutively open during much of its life cycle . Importantly, minor sequence variations lead to dramatic changes in the way in which nucleosomes pack against each other within the crystal lattice . This has important implications for our understanding of the formation of higher order chromatin structure and its modulation by post-translational modifications . Finally, the yeast nucleosome core particle provides a structural context by which to interpret genetic data obtained from yeast . Coordinates have been deposited with the Protein Data Bank under accession number 1ID3. EMBO J, 2001 Sep 17, 20(18), 5165 - 75 Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC; Schwab M et al.; Ubiquitin-mediated proteolysis has emerged as a key mechanism of regulation in eukaryotic cells . During cell division, a multi-subunit ubiquitin ligase termed the anaphase promoting complex (APC) targets critical regulatory proteins such as securin and mitotic cyclins, and thereby triggers chromosome separation and exit from mitosis . Previous studies in the yeast Saccharomyces cerevisiae identified the conserved WD40 proteins Cdc20 and Hct1 (Cdh1) as substrate-specific activators of the APC, but their precise mechanism of action has remained unclear . This study provides evidence that Hct1 functions as a substrate receptor that recognizes target proteins and recruits them to the APC for ubiquitylation and subsequent proteolysis . By co-immunoprecipitation, we found that Hct1 interacted with the mitotic cyclins Clb2 and Clb3 and the polo-related kinase Cdc5, whereas Cdc20 interacted with the securin Pds1 . Failure to interact with Hct1 resulted in stabilization of Clb2 . Analysis of Hct1 derivatives identified the C-box, a motif required for APC association of Hct1 and conserved among Cdc20-related proteins . We propose that proteins of the Cdc20 family are substrate recognition subunits of the ubiquitin ligase APC. FEBS Lett, 2001 Sep 14, 505(2), 321 - 4 Ca(2+) and H+ homeostasis in fission yeast: a role of Ca(2+)/H+ exchange and distinct V-H+-ATPases of the secretory pathway organelles; Okorokov LA et al.; We determined the H+ and Ca(2+) uptake by fission yeast membranes separated on sucrose gradient and found that (i) Ca(2+) sequestering is due to Ca(2+)/H+ antiporter(s) localized to secretory pathway organelles while Ca(2+)-ATPase activity is not detectable in their membranes; (ii) immunochemically distinct V-H+-ATPases acidify the lumen of the secretory pathway organelles . The data indicate that the endoplasmic reticulum, Golgi and vacuole form a network of Ca(2+) and H+ stores in the single cell, providing favorable conditions for such key processes as protein folding/sorting, membrane fusion, ion homeostasis and Ca(2+) signaling in a differential and local manner. Curr Biol, 2001 Sep 18, 11(18), 1462 - 7 Lack of tension at kinetochores activates the spindle checkpoint in budding yeast; Stern BM et al.; The spindle checkpoint delays the onset of anaphase until all pairs of sister chromatids are attached to the mitotic spindle . The checkpoint could monitor the attachment of microtubules to kinetochores, the tension that results from the two sister chromatids attaching to opposite spindle poles, or both . We tested the role of tension by allowing cells to enter mitosis without a prior round of DNA replication . The unreplicated chromatids are attached to spindle microtubules but are not under tension since they lack a sister chromatid that could attach to the opposite pole . Because the spindle checkpoint is activated in these cells, we conclude that the absence of tension at the yeast kinetochore is sufficient to activate the spindle checkpoint in mitosis. Curr Biol, 2001 Sep 18, 11(18), 1421 - 6 The phosphoinositide phosphatase Sac1p controls trafficking of the yeast Chs3p chitin synthase; Schorr M et al.; Phosphoinositide phosphatases play an essential but as yet not well-understood role in lipid-based signal transduction . Members of a subfamily of these enzymes share a specific domain that was first identified in the yeast Sac1 protein {1} . Sac1 homology domains were shown to exhibit 3- and 4-phosphatase activity in vitro {2, 3} and were also found, in addition to rat and yeast Sac1p, in yeast Inp/Sjl proteins {4, 5} and mammalian synaptojanins {6} . Despite the detailed in vitro characterization of the enzymatic properties of yeast Sac1p, the exact cellular function of this protein has remained obscure . We report here that Sac1p has a specific role in secretion and acts as an antagonist of the phosphatidylinositol 4-kinase Pik1p in Golgi trafficking . Elimination of Sac1p leads to excessive forward transport of chitin synthases and thus causes specific cell wall defects . Similar defects in membrane trafficking are caused by the overexpression of PIK1 . Taken together, these findings provide strong evidence that the generation of PtdIns(4)P is sufficient to trigger forward transport from the Golgi to the plasma membrane and that Sac1p is critically required for the termination of this signal. J Protein Chem, 2001 Apr, 20(3), 203 - 15 A study of the influence of the hydrophobic core residues of yeast iso-2-cytochrome c on phosphate binding: a probe of the hydrophobic core-surface charge interactions; Taniuchi H et al.; To gain insight into the role of hydrophobic core-surface charge interactions in stabilizing cytochrome c, we investigated the influence of hydrophobic core residues on phosphate binding by mutating residues in yeast iso-2-cytochrome c to those corresponding to iso-l-cytochrome c in various combinations . Heat transition of ultraviolet CD was followed as a function of pH in the presence and absence of phosphate . Thermodynamic parameters were deduced . It was found that the I20V/V43A/M98L mutation in the hydrophobic core, whose locations are remote from the putative phosphate sites, modulates phosphate interactions . The modulation is pH dependent . The I20V/ M98L and V43A mutation effects are nonadditive . The results lead to a model analogous to that of Tsao, Evans, and Wennerstrom, where a domain associated with the ordered hydrophobic core is sensitive to the fields generated by the surface charges . Such an explanation would be in accord with the observed difference in thermal stability between iso-2 and horse cytochromes c. RNA, 2001 Sep, 7(9), 1268 - 83 Bms1p, a novel GTP-binding protein, and the related Tsr1p are required for distinct steps of 40S ribosome biogenesis in yeast; Gelperin D et al.; Bms1p and Tsr1p define a novel family of proteins required for synthesis of 40S ribosomal subunits in Saccharomyces cerevisiae . Both are essential and localize to the nucleolus . Tsr1p shares two extended regions of similarity with Bms1p, but the two proteins function at different steps in 40S ribosome maturation . Inactivation of Bms1p blocks at an early step, leading to disappearance of 20S and 18S rRNA precursors . Also, slight accumulation of an aberrant 23S product and significant 35S accumulation are observed, indicating that pre-rRNA processing at sites A0, A1, and A2 is inhibited . In contrast, depletion of Tsr1p results in accumulation of 20S rRNA . Because processing of 20S to 18S rRNA occurs in the cytoplasm, this suggests that Tsr1p is required for assembly of a transport- or maturation-competent particle or is specifically required for transport of 43S pre-ribosomal particles, but not 60S ribosome precursors, from the nucleus to the cytosol . Finally, Bms1p is a GTP-binding protein, the first found to function in ribosome assembly or rRNA processing. RNA, 2001 Sep, 7(9), 1254 - 67 Bms1p, a G-domain-containing protein, associates with Rcl1p and is required for 18S rRNA biogenesis in yeast; Wegierski T et al.; Maturation of 18S rRNA and biogenesis of the 40S ribosomes in yeast requires a large number of trans-acting factors, including the U3 small nucleolar ribonucleoprotein (U3 snoRNP), and the recently characterized cyclase-like protein Rcl1p . U3 snoRNP is a key particle orchestrating early 35S rRNA cleavage events . A unique property of Rcl1p is that it specifically associates with U3 snoRNP, but this association appears to occur only at the level of nascent ribosomes and not with the U3 monoparticle . Here we report the characterization of Bms1p, a protein that associates with Rcl1p in multiple structures, including a specific complex sedimenting at around 10S . Like Rcl1p, Bms1p is an essential, evolutionarily conserved, nucleolar protein, and its depletion interferes with processing of the 35S pre-rRNA at sites A0, A1, and A2, and the formation of 40S subunits . The N-terminal domain of Bms1p has structural features found in regulatory GTPases and we demonstrate that mutations of amino acids implicated in GTP/GDP binding affect Bms1p activity in vivo . The results indicate that Bms1p may act as a molecular switch during maturation of the 40S ribosomal subunit in the nucleolus. Mol Cell Biol, 2001 Oct, 21(20), 7035 - 46 Induction of distinct {URE3} yeast prion strains; Schlumpberger M et al.; {URE3} is a non-Mendelian genetic element in Saccharomyces cerevisiae, which is caused by a prion-like, autocatalytic conversion of the Ure2 protein (Ure2p) into an inactive form . The presence of {URE3} allows yeast cells to take up ureidosuccinic acid in the presence of ammonia . This phenotype can be used to select for the prion state . We have developed a novel reporter, in which the ADE2 gene is controlled by the DAL5 regulatory region, which allows monitoring of Ure2p function by a colony color phenotype . Using this reporter, we observed induction of different {URE3} prion variants ("strains") following overexpression of the N-terminal Ure2p prion domain (UPD) or full-length Ure2p . Full-length Ure2p induced two types of {URE3}: type A corresponds to conventional {URE3}, whereas the novel type B variant is characterized by relatively high residual Ure2p activity and efficient curing by coexpression of low amounts of a UPD-green fluorescent protein fusion protein . Overexpression of UPD induced type B {URE3} but not type A . Both type A and B {URE3} strains, as well as weak and strong isolates of type A, were shown to stably maintain different prion strain characteristics . We suggest that these strain variants result from different modes of aggregation of similar Ure2p monomers . We also demonstrate a procedure to counterselect against the {URE3} state. J Mol Endocrinol, 2001 Oct, 27(2), 191 - 209 Requirement of co-factors for the ligand-mediated activity of the insect ecdysteroid receptor in yeast; Tran HT et al.; In insects, a steroid hormone 20-hydroxyecdysone has an important role in regulating critical events such as development and reproduction . The action of 20-hydroxyecdysone is mediated by its binding to the ecdysteroid receptor (EcR), which requires a heterodimeric partner, ultraspiracle protein (USP), a homologue of the retinoid X receptor (RXR) . The EcR-USP heterodimer represents a functional receptor complex capable of initiating transcription of early genes . Our goal was to establish a ligand-dependent transactivation system in yeast utilizing an insect EcR-USP heterodimer . This has been achieved using mosquito Aedes aegypti AaEcR-USP . Expression of AaEcR alone, but not USP, resulted in constitutive transcription of the ecdysone reporter gene coupled with the Drosophila heat shock protein-27 ecdysone response elements . Removal of the N-terminal A/B domain of AaEcR abolished its constitutive transcription . Constitutive transcription was also eliminated in the presence of its heterodimeric partner, AaUSPa, AaUSPb or mammalian RXR . This suggests that the A/B domain is essential for the EcR ligand-independent transactivation and its interaction with the yeast transcription complex . A ligand-mediated transactivation of Aa(Delta A/B)EcR-USP or Aa(Delta A/B)EcR-RXR heterodimers in response to an ecdysteroid agonist RH-5992 was observed only in the presence of GRIP1, a mouse co-activator . In the presence of a co-repressor, SMRT, Aa(Delta A/B)EcR-USP heterodimer exhibited a ligand-dependent repression activity . In addition, ligand-dependent transactivation systems for spruce budworm and fruit fly ecdysone receptors were also reported . This is the first report establishing the requirements of co-factors for a highly efficient ligand-dependent function of the insect EcR-USP in yeast . These findings open a way to study insect EcR-USP structure and function and to identify ligands that are specific for a certain group of insects, such as mosquitoes. Genes Dev, 2001 Sep 15, 15(18), 2445 - 56 Mechanisms controlling differential promoter-occupancy by the yeast forkhead proteins Fkh1p and Fkh2p: implications for regulating the cell cycle and differentiation; Hollenhorst PC et al.; The roles of DNA and Mcm1p interactions in determining the overlapping and distinct functions of the yeast cell cycle regulatory transcription factors Fkh1p and Fkh2p were examined . Full-length recombinant Fkh1p and Fkh2p were purified and their binding to bona fide promoters examined in vitro . Each protein bound a variety of target promoters with similar specificity in vitro, consistent with the observation that these proteins bind common promoters in vivo . However, in vivo, the Fkh1p and Fkh2p occupied different target promoters to different extents, suggesting that each was primarily responsible for controlling a different set of genes . Additional in vitro studies provided a mechanistic explanation for this differential promoter-occupancy . Specifically, the Fkh2p, but not the Fkh1p, was capable of binding cooperatively with Mcm1p . The Mcm1p-Fkh2p cooperative binding was enhanced by, but did not require, the presence of a Mcm1p-binding site within a target promoter . Consistent with these data, Mcm1p was present at Fkh-controlled promoters in vivo regardless of whether they contained Mcm1p-binding sites, suggesting a role for Mcm1p at promoters not thought previously to be under Mcm1p control . Analysis of Fkh1p and Fkh2p binding to promoter targets in vivo by use of mutant strains indicated that the two proteins compete for promoter-occupancy at a number of target promoters . We postulate that Fkh1p and a stable Fkh2p/Mcm1p complex compete for binding to target promoters and that the levels and/or binding activity of Fkh1p, but not Fkh2p, are most limiting for promoter-occupancy in vivo . Interestingly, the in vitro DNA-binding assays, using a variety of promoter targets, revealed that bona fide Fkh target promoters contained two or more Fkh-binding sites that allowed the Fkh1p and Fkh2p proteins to form multiple protein-DNA complexes in vitro . Multiple Fkh-binding sites may be a distinguishing feature of bona fide Fkh promoters in yeast and other organisms. Genes Dev, 2001 Sep 15, 15(18), 2381 - 95 D box and KEN box motifs in budding yeast Hsl1p are required for APC-mediated degradation and direct binding to Cdc20p and Cdh1p; Burton JL et al.; The precise order of molecular events during cell cycle progression depends upon ubiquitin-mediated proteolysis of cell cycle regulators . We demonstrated previously that Hsl1p, a protein kinase that inhibits the Swe1p protein kinase in a bud morphogenesis checkpoint, is targeted for ubiquitin-mediated turnover by the anaphase-promoting complex (APC) . Here, we investigate regions of Hsl1p that are critical both for binding to the APC machinery and for APC-mediated degradation . We demonstrate that Hsl1p contains both a destruction box (D box) and a KEN box motif that are necessary for Hsl1p turnover with either APC(Cdc20) or APC(Cdh1) . In coimmunoprecipitation studies, the D box of full-length Hsl1p was critical for association with Cdc20p, whereas the KEN box was important for association with Cdh1p . Fusion of a 206-amino-acid fragment of Hsl1p containing these motifs to a heterologous protein resulted in APC-dependent degradation of the fusion protein that required intact D box and KEN box motifs . Finally, this bacterially expressed Hsl1p fusion protein interacted with Cdc20p and Cdh1p either translated in vitro or expressed in and purified from insect cells . Binding to Cdc20p and Cdh1p was disrupted completely by a D box/KEN box double mutant . These results indicate that D box and KEN box motifs are important for direct binding to the APC machinery, leading to ubiquitination and subsequent protein degradation. J Am Chem Soc, 2001 Sep 26, 123(38), 9260 - 3 A distal histidine mutant (H52Q) of yeast cytochrome c peroxidase catalyzes the oxidation of H(2)O(2) instead of its reduction; Bateman L et al.; A H52Q variant of yeast cytochrome c peroxidase (CcP), in which the distal histidine is replaced by glutamine, catalyzes oxidation of H(2)O(2) instead of reduction . This redirection of catalytic action is detected by protein film voltammetry . In the presence of H(2)O(2), wild-type CcP, adsorbed on a graphite electrode, shows a strong catalytic reduction wave commencing at about 0.8V (pH 5.4); by contrast, H52Q does not exhibit this activity but instead shows a catalytic oxidation current at potentials in the region of 0.9 V . The oxidation current is partly suppressed in the presence of tetranitromethane (a superoxide scavenger) and is not observed for other mutants studied, including H52A . The only significant structural change in the H52Q variant is that the Q-52 side chain occupies the space vacated by the H-52 imidazole; specifically, the N-epsilon atom that is believed to transfer a proton and induce O--O cleavage is replaced, to within 0.75 A, by the carbamide-O . Thus, while the weakly basic amide functionality is unable to serve in the reorganization of bound H(2)O(2), it is able to facilitate its oxidation, most obviously by serving as a H-bond acceptor to assist formation of a labile superoxide intermediate. J Struct Biol, 2001 Jul, 135(1), 1 - 7 Purification, crystallization, and preliminary X-ray analysis of L-A: a dsRNA yeast virus; Naitow H et al.; TheL-A virus (LAV) particle is a specialized compartment for the transcription and replication of double-stranded RNA . It is 390 A in diameter and infects yeast . The particle is formed by a capsid containing 120 copies of a 680-residue gene product arranged with T = 1 icosahedral symmetry, approximately two copies of an RNA-directed RNA polymerase, and a 4.6-kb linear, duplex RNA . LAV crystals diffracting to at least 4.5-A resolution were grown in a combination of polyethylene glycol 8000, ethylene glycol, and lithium chloride . Following crystallization the reservoir solution was replaced by a 2x concentrated reservoir solution in order for ethylene glycol to function as a cryoprotectant even though initial crystals would not grow at sufficiently high concentrations of ethylene glycol for cryoprotection . A complete data set was collected to 6-A resolution from a frozen crystal obtained with this procedure . The crystals belong to space group P2(1) . The unit cell dimensions are a = 406.7 A, b = 403.3 A, c = 572.5 A, beta = 90.3 degrees with two virus particles in the unit cell . The particle orientation was determined with the rotation function and the particle center was estimated on the basis of packing considerations . Genetics, 2001 Sep, 159(1), 47 - 64 In vivo consequences of putative active site mutations in yeast DNA polymerases alpha, epsilon, delta, and zeta; Pavlov YI et al.; Several amino acids in the active site of family A DNA polymerases contribute to accurate DNA synthesis . For two of these residues, family B DNA polymerases have conserved tyrosine residues in regions II and III that are suggested to have similar functions . Here we replaced each tyrosine with alanine in the catalytic subunits of yeast DNA polymerases alpha, delta, epsilon, and zeta and examined the consequences in vivo . Strains with the tyrosine substitution in the conserved SL/MYPS/N motif in region II in Pol delta or Pol epsilon are inviable . Strains with same substitution in Rev3, the catalytic subunit of Pol zeta, are nearly UV immutable, suggesting severe loss of function . A strain with this substitution in Pol alpha (pol1-Y869A) is viable, but it exhibits slow growth, sensitivity to hydroxyurea, and a spontaneous mutator phenotype for frameshifts and base substitutions . The pol1-Y869A/pol1-Y869A diploid exhibits aberrant growth . Thus, this tyrosine is critical for the function of all four eukaryotic family B DNA polymerases . Strains with a tyrosine substitution in the conserved NS/VxYG motif in region III in Pol alpha, -delta, or -epsilon are viable and a strain with the homologous substitution in Rev3 is UV mutable . The Pol alpha mutant has no obvious phenotype . The Pol epsilon (pol2-Y831A) mutant is slightly sensitive to hydroxyurea and is a semidominant mutator for spontaneous base substitutions and frameshifts . The Pol delta mutant (pol3-Y708A) grows slowly, is sensitive to hydroxyurea and methyl methanesulfonate, and is a strong base substitution and frameshift mutator . The pol3-Y708A/pol3-Y708A diploid grows slowly and aberrantly . Mutation rates in the Pol alpha, -delta, and -epsilon mutant strains are increased in a locus-specific manner by inactivation of PMS1-dependent DNA mismatch repair, suggesting that the mutator effects are due to reduced fidelity of chromosomal DNA replication . This could result directly from relaxed base selectivity of the mutant polymerases due to the amino acid changes in the polymerase active site . In addition, the alanine substitutions may impair catalytic function to allow a different polymerase to compete at the replication fork . This is supported by the observation that the pol3-Y708A mutation is recessive and its mutator effect is partially suppressed by disruption of the REV3 gene. J Cell Sci, 2001 Jul, 114(Pt 13), 2427 - 35 Cytoplasmic microtubular system implicated in de novo formation of a Rabl-like orientation of chromosomes in fission yeast; Goto B et al.; Chromosomes are not packed randomly in the nucleus . The Rabl orientation is an example of the non-random arrangement of chromosomes, centromeres are grouped in a limited area near the nuclear periphery and telomeres are located apart from centromeres . This orientation is established during mitosis and maintained through subsequent interphase in a range of species . We report that a Rabl-like configuration can be formed de novo without a preceding mitosis during the transition from the sexual phase to the vegetative phase of the life cycle in fission yeast . In this process, each of the dispersed centromeres is often associated with a novel Sad1-containing body that is contacting a cytoplasmic microtubule laterally (Sad1 is a component of the spindle pole body (SPB)) . The Sad1-containing body was colocalized with other known SPB components, Kms1 and Spo15 but not with Cut12, indicating that it represents a novel SPB-related complex . The existence of the triplex structure (centromere-microtubule-Sad1 body) suggests that the clustering of centromeres is controlled by a cytoplasmic microtubular system . Accordingly, when microtubules are destabilized, clustering is markedly reduced. Free Radic Biol Med, 2001 Sep 15, 31(6), 832 - 43 Antioxidant function of cytosolic sources of NADPH in yeast; Minard KI et al.; The relative antioxidant functions of thiol-dependent mechanisms and of direct catalytic inactivation of H2O2 were examined using a collection of yeast mutants containing disruptions in single or multiple genes encoding two major enzymatic sources of NADPH {glucose-6-phosphate dehydrogenase (ZWF1) and cytosolic NADP+-specific isocitrate dehydrogenase (IDP2)} and in genes encoding two major cellular peroxidases {mitochondrial cytochrome c peroxidase (CCP1) and cytosolic catalase (CTT1)} . Both types of mechanisms were found to be important for growth in the presence of exogenous H2O2 . In the absence of exogenous oxidants, however, loss of ZWF1 and IDP2, but not loss of CTT1 and CCP1, was found to be detrimental not only to growth but also to viability of cells shifted to rich medium containing oleate or acetate . The loss in viability correlates with increased levels of intracellular oxidants apparently produced during normal metabolism of these carbon sources . Acute effects in DeltaZWF1DeltaIDP2 mutants following shifts to these nonpermissive media include an increase in the number of cells demonstrating a transient decrease in growth rate and in cells containing apparent nuclear DNA strand breaks . Cumulative effects are reflected in phenotypes, including sensitivity to acetate medium and a reduction in mating efficiency, that become more pronounced with time following disruption of the ZWF1 and IDP2 genes . These results suggest that cellular mechanisms dependent on NADPH are crucial metabolic antioxidants. Mech Ageing Dev, 2001 Oct, 122(15), 1651 - 62 The impact energy metabolism and genome maintenance have on longevity and senescence: lessons from yeast to mammals; Hasty P; The phenomenon that caloric restriction increases life span in a variety of species from yeast to mice has been the focus of much interest . Recent observations suggest that a protein important for heterochromatin formation, Sir2, is central for caloric restriction-induced longevity in lower organisms . Interestingly, Sir2 is also capable of repairing DNA double-strand breaks by nonhomologous end joining which may be important, along with proteins that repair breaks by recombinational repair, for minimizing the age-related deleterious effects of DNA damage induced by oxygen by-products of metabolism . I propose that competition between these two distinct functions could influence longevity and the onset of senescence . In addition, sequence and functional similarities between Sir2 and other chromatin metabolism proteins present the possibility that genetic components for longevity and senescence are conserved from yeast to mammals. FEBS Lett, 2001 Sep 7, 505(1), 155 - 8 Two-dimensional gel electrophoresis and FTIR spectroscopy reveal both forms of yeast plasma membrane H(+)-ATPase in activated and basal-level enzyme preparations; Lapathitis G et al.; Plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae was isolated and purified in its two forms, the activated A-ATPase from glucose-metabolizing cells, and the basal-level B-ATPase from cells with endogenous metabolism only . Using two-dimensional gel electrophoretic analysis, we showed that both enzyme preparations are actually mixtures of the non-active, i.e . non-phosphorylated, and the active, i.e . phosphorylated, forms of the enzyme . Previous deliberations suggesting that the B-ATPase displays some activity which is lower than that of A-ATPase were apparently wrong . It seems that, molecularly speaking, the B-form is actually not active at all, and what activity we measure in our preparation is due to an admixture of the true active form (A-form) . Fourier transform infrared spectroscopic study of the secondary structure and particularly thermal denaturation data suggest the possibility that the two enzyme forms interact to form complexes less stable than the single forms . On the whole then, there apparently is a different ratio of the active and inactive forms and/or complexes between the two forms present in all enzyme preparations. J Biol Chem, 2001 Nov 9, 276(45), 42003 - 10 Epub 2001 Sep 12. The structural and functional organization of the yeast mediator complex; Kang JS et al.; The Mediator complex of Saccharomyces cerevisiae is required for diverse aspects of transcription by RNA polymerase II (pol II) . Mediator is composed of two functionally distinct subcomplexes, Rgr1 and Srb4 . To identify the structures and functions of each subcomplex, we expressed recombinant proteins for each subunit and assayed their interactions with each other and with basal transcription proteins . The Rgr1 subcomplex is composed of the Gal11 module, which binds activators, and the Med9/10 module . The Med9/10 module is required for both transcriptional activation and repression, and these activities appear to be carried out by two submodules . Proteins in the Med9 submodule interact physically and genetically with Srb10/11, suggesting that the Med9 submodule mediates the repression of pol II . Purified recombinant Srb4 subcomplex stimulated basal transcription of pol II but had little effect on activated transcription and phosphorylation of the C-terminal domain of the Rpb1 subunit of pol II . Both subcomplexes of Mediator interacted with a distinct set of basal transcription factors and pol II . The modular organization of Mediator and the associated functions suggest that the Mediator complex may recruit and/or stabilize the preinitiation complex through several points of contact with transcriptional regulators and basal transcription factors. Genes Cells, 2001 Sep, 6(9), 765 - 73 Time course analysis of precocious separation of sister centromeres in budding yeast: continuously separated or frequently reassociated? Goshima G, Yanagida M. BACKGROUND: Sister kinetochores are bioriented toward the spindle poles in eukaryotic metaphase before chromosome segregation . In the budding yeast Saccharomyces cerevisiae, sister centromeres/kinetochores are separated in the early spindle, while the sister arms remain associated . Biorientation is thought to be established in this organism with precocious separation of sister centromeres in early stages of the cell cycle . It is not, however, settled whether this pre-anaphase separation is continuous or only transient and whether the transient separation has any physiological significance . RESULTS: Time-lapse observation of the behaviour of budding yeast centromeres in living cells was performed using GFP alone or in combination with CFP marking . Sixty-three per cent of the cell population showed permanent separation of centromeres for a long period of time from the small-budded stage to the onset of anaphase in the single-colour GFP-CEN construct . The remaining cell population (6 of 16) showed brief apparent reassociation of centromere signals before anaphase, but the frequency of the association was very low . In a time-lapse observation of the double-colour marked cells by GFP-CEN and CFP-SPB (the spindle pole body), the continuous separation of sister centromeres in the short medial spindle was firmly established . CONCLUSIONS: In the budding yeast, once sister centromeres separate, they rarely reassociate in pre-anaphase . Sister centromere cohesion at this stage appears to be irrelevant for normal chromosome segregation . Whether abundant cohesin in the centromere regions has any role in anaphase remains to be determined. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11108 - 13 Epub 2001 Sep 11. Association of yeast DNA topoisomerase III and Sgs1 DNA helicase: studies of fusion proteins; Bennett RJ et al.; The Sgs1 protein of the budding yeast Saccharomyces cerevisiae is a member of the RecQ DNA helicase family that includes the human Bloom, Werner, and Rothmund-Thompson syndrome proteins . The N-terminal region outside the central DNA helicase core of Sgs1, particularly the part containing the first 100 amino acid residues of the 1,447-residue protein, is known to be functionally important and has been implicated in Sgs1-DNA topoisomerase III (Top3) interaction . We show in this work that the functionality of a truncated Sgs1 lacking its N-terminal 106 residues can be restored by replacing the truncated region with Top3 . Fusion of Top3 to a mutant Sgs1 with a Val-29 to Glu substitution, which interferes with Sgs1-Top3 interaction, similarly restores the functionality of the mutant Sgs1(V29E) protein . The Top3-Sgs1(Delta1-106) and Top3-Sgs1(V29E) fusion proteins behave like wild-type Sgs1 in complementing several aspects of the sgs1 phenotype, including the hypersensitivity of sgs1 cells to methyl methanesulfonate and hydroxyurea . Complementation by the fusion proteins required both the topoisomerase activity of Top3 and the helicase activity of the Sgs1 polypeptide . These results suggest that the sole function of the N-terminal 106 amino acid residues of Sgs1 is for Top3 binding, and that the coordinated actions of Sgs1 and Top3 are important in cellular processes such as the processing of DNA after exposure of cells to DNA-damaging agents. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11289 - 94 Epub 2001 Sep 11. Serine-345 is required for Rad3-dependent phosphorylation and function of checkpoint kinase Chk1 in fission yeast; Lopez-Girona A et al.; Genome integrity is monitored by a checkpoint that delays mitosis in response to DNA damage . This checkpoint is enforced by Chk1, a protein kinase that inhibits the mitotic inducer Cdc25 . In fission yeast, Chk1 is regulated by a group of proteins that includes Rad3, a protein kinase related to human ATM and ATR . These kinases phosphorylate serine or threonine followed by glutamine (SQ/TQ) . Fission yeast and human Chk1 proteins share two conserved SQ motifs at serine-345 and serine-367 . Serine-345 of human Chk1 is phosphorylated in response to DNA damage . Here we report that Rad3 and ATM phosphorylate serine-345 of fission yeast Chk1 . Mutation of serine-345 (chk1-S345A) abrogates Rad3-dependent phosphorylation of Chk1 in vivo . The chk1-S345A cells are sensitive to DNA damage and are checkpoint defective . In contrast, mutations of serine-367 and other SQ/TQ sites do not substantially impair the checkpoint or cause damage sensitivity . These findings attest to the importance of serine-345 phosphorylation for Chk1 function and strengthen evidence that transduction of the DNA damage checkpoint signal requires direct phosphorylation of Chk1 by Rad3. Mol Biol Cell, 2001 Sep, 12(9), 2870 - 80 Control of microtubule dynamics by Stu2p is essential for spindle orientation and metaphase chromosome alignment in yeast; Kosco KA et al.; Stu2p is a member of a conserved family of microtubule-binding proteins and an essential protein in yeast . Here, we report the first in vivo analysis of microtubule dynamics in cells lacking a member of this protein family . For these studies, we have used a conditional Stu2p depletion strain expressing alpha-tubulin fused to green fluorescent protein . Depletion of Stu2p leads to fewer and less dynamic cytoplasmic microtubules in both G1 and preanaphase cells . The reduction in cytoplasmic microtubule dynamics is due primarily to decreases in both the catastrophe and rescue frequencies and an increase in the fraction of time microtubules spend pausing . These changes have significant consequences for the cell because they impede the ability of cytoplasmic microtubules to orient the spindle . In addition, recovery of fluorescence after photobleaching indicates that kinetochore microtubules are no longer dynamic in the absence of Stu2p . This deficiency is correlated with a failure to properly align chromosomes at metaphase . Overall, we provide evidence that Stu2p promotes the dynamics of microtubule plus-ends in vivo and that these dynamics are critical for microtubule interactions with kinetochores and cortical sites in the cytoplasm. Mol Biol Cell, 2001 Sep, 12(9), 2767 - 75 The domain structure of centromeres is conserved from fission yeast to humans; Kniola B et al.; The centromeric DNA of fission yeast is arranged with a central core flanked by repeated sequences . The centromere-associated proteins, Mis6p and Cnp1p (SpCENP-A), associate exclusively with central core DNA, whereas the Swi6 protein binds the surrounding repeats . Here, electron microscopy and immunofluorescence light microscopy reveal that the central core and flanking regions occupy distinct positions within a heterochromatic domain . An "anchor" structure containing the Ndc80 protein resides between this heterochromatic domain and the spindle pole body . The organization of centromere-associated proteins in fission yeast is reminiscent of the multilayered structures of human kinetochores, indicating that such domain structure is conserved in eukaryotes. J Biol Chem, 2001 Dec 7, 276(49), 46284 - 9 Epub 2001 Sep 10. Vesicle-associated membrane protein of Arabidopsis suppresses Bax-induced apoptosis in yeast downstream of oxidative burst; Levine A et al.; Programmed cell death (PCD) in many systems is controlled by relative amounts of the apoptosis-regulating proteins Bax and Bcl-2 through homo- or heterodimerization . Here we show that Bax-induced PCD of yeast was suppressed by transformation with a vesicle-associated membrane protein from Arabidopsis (AtVAMP), which was isolated by screening a cDNA expression library against sugar-induced cell death in yeast . AtVAMP expression blocked Bax-induced PCD downstream of oxidative burst . AtVAMP also prevented H(2)O(2)-induced apoptosis in yeast and in Arabidopsis cells . Reduced oxidation of lipids and plasma membrane proteins was detected in the AtVAMP-transformed yeast, suggesting improved membrane repair . Inhibition of intracellular vesicle trafficking by brefeldin A induced apoptosis from a sublethal concentration of H(2)O(2) . No protection occurred by overexpression of the yeast homolog SCN2 . However, efficient suppression of yeast PCD occurred by expression of a chimeric gene, composed of the conserved domains from yeast, fused to the variable N-terminal domain from Arabidopsis, resulting in exchange of the proline-rich N-terminal domain of SCN2 with a proline-poor Arabidopsis sequence . Our results suggest that intracellular vesicle traffic can regulate execution of apoptosis by affecting the rate of membrane recycling and that the proline-rich N-terminal domain of VAMP inhibited this process. J Biol Chem, 2001 Nov 23, 276(47), 43970 - 9 Epub 2001 Sep 10. A DnaJ-like protein homologous to the yeast co-chaperone Sis1 (TcJ6p) is involved in initiation of translation in Trypanosoma cruzi; Salmon D et al.; In eukaryotes, proteins homologous to the bacterial DnaJ protein are involved in regulation of the Hsp70 molecular chaperones, which are implicated in a variety of protein biogenesis pathways . We report herewith the molecular characterization of a T . cruzi DnaJ gene, termed TcJ6, encoding a protein that displays high sequence homology with the Saccharomyces cerevisiae Sis1 co-chaperone required for the initiation of translation . TcJ6 protein was expressed as a polypeptide of 36.5 kDa at a constant level during parasite differentiation and was associated to the cytoplasmic fraction . We showed that overexpression of TcJ6 complemented a temperature-sensitive yeast sis1 mutant . In addition, sucrose gradient sedimentation analysis of polysomes from T . cruzi and a yeast mutant overexpressing TcJ6p showed that the trypanosomal co-chaperone was closely associated with ribosomal subunits, 80 S monosomes and the smaller polysomes, as observed for Sis1p . Furthermore, in T . cruzi TcJ6p was also found to be preferentially concentrated around the nucleus, giving a speckled staining pattern . This suggests that TcJ6p is associated with the endoplasmic reticulum . Taken together, these data suggest that the trypanosomal DnaJ is involved in initiation of translation. Mol Biochem Parasitol, 2001 Sep 28, 117(1), 27 - 35 Interaction between two domains of the P . yoelii MSP-1 protein detected using the yeast two-hybrid system; Daly TM et al.; Several model systems of plasmodia have demonstrated the potential of the merozoite surface protein, MSP-1, to induce protective immunity . However, little is known about the function of this protein or its interaction with other surface molecules that may also serve as immunological targets . To identify potentially significant inter- and intra-molecular interactions involving MSP-1, we have utilized the yeast two-hybrid system . A cDNA activation domain library was constructed from the erythrocytic stages of the murine malarial parasite Plasmodium yoelii yoelii 17XL . A 795 bp region of Py17XL MSP-1 (bait), homologous to the Plasmodium falciparum MSP1(33) fragment, was inserted into a Gal4p DNA binding domain vector and used to screen the activation domain library (target) . Several randomly selected clones that demonstrated bait-target interaction were found to express overlapping regions of Py17XL MSP-1 . Deletion constructs further localized the peptide fragments retaining interaction indicating that a region within the MSP-1(38) fragment interacts with the MSP-1 bait domain . Subsequent studies confirmed this interaction, as both peptides were co-precipitated from cell lysate by a peptide tag-specific antibody . It was observed that the interaction of these two fragments significantly increased the half-life of the MSP-1(38) within yeast cells . The specific interaction described here demonstrates the potential of this approach to elucidate additional inter- or intra-molecular interactions of Py17XL MSP1 and other malarial proteins. Mutat Res, 2001 Sep 20, 496(1-2), 105 - 15 Protective effects of vitamins and selenium compounds in yeast; Bronzetti G et al.; Antimutagens and anticarcinogens are known to play an important role in decreasing damages induced by oxidants . In this study, we investigated the genotoxic and antimutagenic potential of two selenium compounds (sodium selenite: Na(2)SeO(3); seleno-DL-methionine: C(5)H(11)NO(2)Se) and Vitamins A and E in yeast cells of Saccharomyces cerevisiae . An oxidative mutagen (hydrogen peroxide (H(2)O(2)), HP) was chosen as positive control . We determined the enzymatic activities involved in the protection against oxidative damages (catalase: CAT; superoxide dismutase: SOD; glutathione peroxidase: GPx) in the cytosolic extract of yeast cells . The results demonstrated that selenium compounds exerted both mutagenic and antimutagenic effect at different concentrations . Antimutagenesis was evident both in stationary and in logarithmic phase cells . Catalase, SOD, and GPx were significantly increased in the presence of all the compounds assayed . Vitamins A (retinol) and E (alpha-tocopherol) did not have toxic or mutagenic action. Bull Exp Biol Med, 2001 Apr, 131(4), 346 - 9 Drug sensitivity of Candida yeast isolated from patients with allergic diseases; Arzumanyan VG et al.; Viability of 40 Candida spp . cultures was studied after long-term exposure to antifungal drugs in minimum inhibitory concentrations . The fungicidal effect decreased in the series: pimafucin-nitrofungin-diflucan-orungal-levorine-clotrimazole-exoderil . Nizoral in a concentration of 4 microg/ml was ineffective; in the rest cultures the effect was either fungistatic (of different degree) or null . Pimafucin, diflucan, nitrofungin, orungal, levorine, and exoderil possessed individual fungicidal effects. J Biol Chem, 2001 Sep 14, 276(37), 34792 - 800 ATP utilization by yeast replication factor C . IV . RFC ATP-binding mutants show defects in DNA replication, DNA repair, and checkpoint regulation; Schmidt SL et al.; Replication factor C is required to load proliferating cell nuclear antigen onto primer-template junctions, using the energy of ATP hydrolysis . Four of the five RFC genes have consensus ATP-binding motifs . To determine the relative importance of these sites for proper DNA metabolism in the cell, the conserved lysine in the Walker A motif of RFC1, RFC2, RFC3, or RFC4 was mutated to either arginine or glutamic acid . Arginine mutations in all RFC genes tested permitted cell growth, although poor growth was observed for rfc2-K71R . A glutamic acid substitution resulted in lethality in RFC2 and RFC3 but not in RFC1 or RFC4 . Most double mutants combining mutations in two RFC genes were inviable . Except for the rfc1-K359R and rfc4-K55E mutants, which were phenotypically similar to wild type in every assay, the mutants were sensitive to DNA-damaging agents . The rfc2-K71R and rfc4-K55R mutants show checkpoint defects, most likely in the intra-S phase checkpoint . Regulation of the damage-inducible RNR3 promoter was impaired in these mutants, and phosphorylation of Rad53p in response to DNA damage was specifically defective when cells were in S phase . No dramatic defects in telomere length regulation were detected in the mutants . These data demonstrate that the ATP binding function of RFC2 is important for both DNA replication and checkpoint function and, for the first time, that RFC4 also plays a role in checkpoint regulation. Genomics, 2001 Aug, 76(1-3), 81 - 8 Identification of MAL2, a novel member of the mal proteolipid family, though interactions with TPD52-like proteins in the yeast two-hybrid system; Wilson SH et al.; The TPD52 (tumor protein D52)-like proteins are small coiled-coil motif-bearing proteins which were first identified though their expression in human breast carcinoma . TPD52-like proteins are known to interact in hetero-and homomeric fashions, but there are no known heterologous binding partners for these proteins . We now report the cloning of a novel member of the MAL proteolipid family, named MAL2, though its interaction with a TPD52L2 bait in a yeast two-hybrid screen . MAL2 is predicted to be 176 residues (19 kDa) with four transmembrane domains and is 35.8% identical to MAL, a proteolipid required in apical vesicle transport . The MAL2 prey bound all TPD52-like baits tested in the yeast two-hybrid system and in vitro translation of MAL2 produced a single 19-kDa (35)S-labeled protein which specifically bound full-length GST-Tpd52 in GST pull-down assays . The gene MAL2, which was localized to human chromosomal band 8q23 and shown to consist of four exons, is predominantly expressed in human kidney, lung, and liver . Our study has therefore identified a novel member of the MAL proteolipid family and potentially implicates TPD52-like proteins in vesicle transport. Biochem Biophys Res Commun, 2001 Sep 14, 287(1), 147 - 52 Pressure denaturation of the yeast prion protein Ure2; Zhou JM et al.; Denaturation of the Saccharomyces cerevisiae prion protein Ure2 was investigated using hydrostatic pressure . Pressures of up to 600 MPa caused only limited perturbation of the structure of the 40-kDa dimeric protein . However, nondenaturing concentrations of GdmCl in combination with high pressure resulted in complete unfolding of Ure2 as judged by intrinsic fluorescence . The free energy of unfolding measured by pressure denaturation or by GdmCl denaturation is the same, indicating that pressure does not induce dimer dissociation or population of intermediates in 2 M GdmCl . Pressure-induced changes in 5 M GdmCl suggest residual structure in the denatured state . Cold denaturation under pressure at 200 MPa showed that unfolding begins below -5 degrees C and Ure2 is more susceptible to cold denaturation at low ionic strength . Results obtained using two related protein constructs, which lack all or part of the N-terminal prion domain, were very similar . J Biol Chem, 2001 Nov 9, 276(45), 42520 - 6 Epub 2001 Sep 06. Yeast Sco1, a protein essential for cytochrome c oxidase function is a Cu(I)-binding protein; Nittis T et al.; Sco1 is a conserved essential protein, which has been implicated in the delivery of copper to cytochrome c oxidase, the last enzyme of the electron transport chain . In this study, we show for the first time that the purified C-terminal domain of yeast Sco1 binds one Cu(I)/monomer . X-ray absorption spectroscopy suggests that the Cu(I) is ligated via three ligands, and we show that two cysteines, present in a conserved motif CXXXC, and a conserved histidine are involved in Cu(I) ligation . The mutation of any one of the conserved residues in Sco1 expressed in yeast abrogates the function of Sco1 resulting in a non-functional cytochrome c oxidase complex . Thus, the function of Sco1 correlates with Cu(I) binding . Data obtained from size-exclusion chromatography experiments with mitochondrial lysates suggest that full-length Sco1 may be oligomeric in vivo. Am J Physiol Cell Physiol, 2001 Oct, 281(4), C1355 - 64 Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells; Muller-Ehmsen J et al.; Human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) heterodimers were expressed individually in yeast, and ouabain binding and ATP hydrolysis were measured in membrane fractions . The ouabain equilibrium dissociation constant was 13-17 nM for alpha(1)beta(1) and alpha(3)beta(1) at 37 degrees C and 32 nM for alpha(2)beta(1), indicating that the human alpha-subunit isoforms have a similar high affinity for cardiac glycosides . K(0.5) values for antagonism of ouabain binding by K(+) were ranked in order as follows: alpha(2) (6.3 +/- 2.4 mM) > alpha(3) (1.6 +/- 0.5 mM) approximately alpha(1) (0.9 +/- 0.6 mM), and K(0.5) values for Na(+) antagonism of ouabain binding to all heterodimers were 9.5-13.8 mM . The molecular turnover for ATP hydrolysis by alpha(1)beta(1) (6,652 min(-1)) was about twice as high as that by alpha(3)beta(1) (3,145 min(-1)) . These properties of the human heterodimers expressed in yeast are in good agreement with properties of the human Na(+)-K(+)-ATPase expressed in Xenopus oocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-D Horisberger, L Lelievie, and K Geering . J Biol Chem 275: 1976-1986, 2000) . In contrast to Na(+) pumps expressed in Xenopus oocytes, the alpha(2)beta(1) complex in yeast membranes was significantly less stable than alpha(1)beta(1) or alpha(3)beta(1), resulting in a lower functional expression level . The alpha(2)beta(1) complex was also more easily denatured by SDS than was the alpha(1)beta(1) or the alpha(3)beta(1) complex. Mol Cell, 2001 Aug, 8(2), 473 - 9 Highly specific antibodies determine histone acetylation site usage in yeast heterochromatin and euchromatin; Suka N et al.; We have developed a highly specific antibody set for acetylation sites in yeast histones H4 (K5, K8, K12, and K16); H3 (K9, K14, K18, K23, and K27); H2A (K7); and H2B (K11 and K16) . Since ELISA does not assure antibody specificity in chromatin immunoprecipitation, we have employed additional screens against the respective histone mutations . We now show that telomeric and silent mating locus heterochromatin is hypoacetylated at all histone sites . At the INO1 promoter, RPD3 is required for strongly deacetylating all sites except H4 K16, ESA1 for acetylating H2A, H2B, and H4 sites except H4 K16, and GCN5 for acetylating H2B and H3 sites except H3 K14 . These data uncover the in vivo usage of acetylation sites in heterochromatin and euchromatin. Mol Cell, 2001 Aug, 8(2), 407 - 15 Interaction with PCNA is essential for yeast DNA polymerase eta function; Haracska L et al.; In both yeast and humans, DNA polymerase (Pol) eta functions in error-free replication of ultraviolet-damaged DNA, and Poleta promotes replication through many other DNA lesions as well . Here, we present evidence for the physical and functional interaction of yeast Poleta with proliferating cell nuclear antigen (PCNA) and show that the interaction with PCNA is essential for the in vivo function of Poleta . Poleta is highly inefficient at inserting a nucleotide opposite an abasic site, but interaction with PCNA greatly stimulates its ability for nucleotide incorporation opposite this lesion . Thus, in addition to having a pivotal role in the targeting of Poleta to the replication machinery stalled at DNA lesions, interaction with PCNA would promote the bypass of certain DNA lesions. Mol Cell, 2001 Aug, 8(2), 375 - 81 A human homolog of yeast pre-mRNA splicing gene, PRP31, underlies autosomal dominant retinitis pigmentosa on chromosome 19q13.4 (RP11); Vithana EN et al.; We report mutations in a gene (PRPF31) homologous to Saccharomyces cerevisiae pre-mRNA splicing gene PRP31 in families with autosomal dominant retinitis pigmentosa linked to chromosome 19q13.4 (RP11; MIM 600138) . A positional cloning approach supported by bioinformatics identified PRPF31 comprising 14 exons and encoding a protein of 499 amino acids . The level of sequence identity to the yeast PRP31 gene indicates that PRPF31 is also likely to be involved in pre-mRNA splicing . Mutations that include missense substitutions, deletions, and insertions have been identified in four RP11-linked families and three sporadic RP cases . The identification of mutations in a pre-mRNA splicing gene implicates defects in the splicing process as a novel mechanism of photoreceptor degeneration. Mol Cell, 2001 Aug, 8(2), 281 - 9 The arrest of secretion response in yeast: signaling from the secretory path to the nucleus via Wsc proteins and Pkc1p; Nanduri J et al.; The arrest of secretion response (ASR) in sec mutants reversibly inhibits nuclear import and relocates nuclear proteins to the cytoplasm . sec mutants also relocate nucleoporins; however, endocytic and Golgi-to-vacuole transport mutants do not cause relocation . The ASR requires Wsc membrane proteins that are trapped along the secretory path, rather than those which are at the plasma membrane . The activity of the downstream kinase, Pkc1p, is also required; however, the Pkc1p MAP kinase cascade is not . sec mutants initiate compensatory transcriptional changes distinct from those of the unfolded protein response. J Mol Biol, 2001 Sep 7, 312(1), 177 - 86 Solution structure and dynamics of yeast elongin C in complex with a von Hippel-Lindau peptide; Botuyan MV et al.; Elongin is a transcription elongation factor that stimulates the rate of elongation by suppressing transient pausing by RNA polymerase II at many sites along the DNA . It is heterotrimeric in mammals, consisting of elongins A, B and C subunits, and bears overall similarity to a class of E3 ubiquitin ligases known as SCF (Skp1-Cdc53 (cullin)-F-box) complexes . A subcomplex of elongins B and C is a target for negative regulation by the von Hippel-Lindau (VHL) tumor-suppressor protein . Elongin C from Saccharomyces cerevisiae, Elc1, exhibits high sequence similarity to mammalian elongin C . Using NMR spectroscopy we have determined the three-dimensional structure of Elc1 in complex with a human VHL peptide, VHL(157-171), representing the major Elc1 binding site . The bound VHL peptide is entirely helical . Elc1 utilizes two C-terminal helices and an intervening loop to form a binding groove that fits VHL(157-171) . Chemical shift perturbation and dynamics analyses reveal that a global conformational change accompanies Elc1/VHL(157-171) complex formation . Moreover, the disappearance of conformational exchange phenomena on the microsecond to millisecond time scale within Elc1 upon VHL peptide binding suggests a role for slow internal motions in ligand recognition . J Mol Biol, 2001 Sep 7, 312(1), 133 - 41 The x-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with substrate and three inhibitors; Erskine PT et al.; The structures of 5-aminolaevulinic acid dehydratase (ALAD) complexed with substrate (5-aminolaevulinic acid) and three inhibitors: laevulinic acid, succinylacetone and 4-keto-5-aminolaevulinic acid, have been solved at high resolution . The ligands all bind by forming a covalent link with Lys263 at the active site . The structures define the interactions made by one of the two substrate moieties that bind to the enzyme during catalysis . All of the inhibitors induce a significant ordering of the flap covering the active site . Succinylacetone appears to be unique by inducing a number of conformational changes in loops covering the active site, which may be important for understanding the co-operative properties of ALAD enzymes . Succinylacetone is produced in large amounts by patients suffering from the hereditary disease type I tyrosinaemia and its potent inhibition of ALAD also has implications for the pathology of this disease . The most intriguing result is that obtained with 4-keto-5-amino-hexanoic acid, which seems to form a stable carbinolamine intermediate with Lys263 . It appears that we have defined the structure of an intermediate of Schiff base formation that the substrate forms upon binding to the P-site of the enzyme . Genes Dev, 2001 Sep 1, 15(17), 2238 - 49 The yeast Xrs2 complex functions in S phase checkpoint regulation; D'Amours D et al.; The Nbs1 complex is an evolutionarily conserved multisubunit nuclease composed of the Mre11, Rad50, and Nbs1 proteins . Hypomorphic mutations in the NBS1 or MRE11 genes in humans result in conditions characterized by DNA damage sensitivity, cell cycle checkpoint deficiency, and high cancer incidence . The equivalent complex in the yeast Saccharomyces cerevisiae (Xrs2p complex) has been implicated in DNA double-strand break repair and in telomere length regulation . Here, we find that xrs2Delta, mre11Delta, and rad50Delta mutants are markedly defective in the initiation of the intra-S phase checkpoint in response to DNA damage . Furthermore, the absence of a functional Xrs2p complex leads to sensitivity to deoxynucleotide depletion and to an inability to efficiently slow down cell cycle progression in response to hydroxyurea . The checkpoint appears to require the nuclease activity of Mre11p and its defect is associated with the abrogation of the Tel1p/Mec1p signaling pathway . Notably, DNA damage induces phosphorylation of both Xrs2p and Mre11p in a Tel1p-dependent manner . These results indicate that the Tel1p/ATM signaling pathway is conserved from yeast to humans and suggest that the Xrs2p/Nbs1 complexes act as signal modifiers. Biochem Pharmacol, 2001 Oct 1, 62(7), 953 - 61 Transcriptional activities of estrogen receptor alpha and beta in yeast properties of raloxifene; Jisa E et al.; Raloxifene represents a potent compound for the prevention and treatment of osteoporosis and cardiovascular disease in postmenopausal women . Raloxifene exhibits targeted antiestrogenicity in breast and uterus, but acts as an agonist in bone and liver . This synthetic selective estrogen receptor modulator binds both estrogen receptors alpha and beta . The molecular mechanisms by which raloxifene exerts agonistic or antagonistic activity are still not resolved . Therefore, the binding behavior of raloxifene to estrogen receptors and its effects on DNA binding and transactivation were studied . The equilibrium binding affinity of raloxifene by displacing radiolabeled 17beta-estradiol exhibited a similar affinity behavior to that of its natural ligand . Using BIACORE technology with an immobilized estrogen response element, we showed that 17beta-estradiol and raloxifene increased the binding of estrogen receptor alpha to the DNA, suggesting a ligand-dependent dimerization . The influence of the ligands to the binding of estrogen receptor beta was lower . We may conclude that unliganded estrogen receptor alpha binds as a monomer whereas in the presence of 10(-8) M 17beta-estradiol or higher, homodimers are formed that interact with the estrogen response element . Transactivation studies in a yeast reporter system in a ligand-dependent manner resulted in a similar potency of raloxifene to estrogen receptor beta compared to the control testosterone . Subeffective doses of raloxifene combined with 17beta-estradiol did not shift the efficiency, whereas saturating concentrations of 17beta-estradiol combined with increasing concentrations of raloxifene altered the response induced by 17beta-estradiol . In this pure system, the antagonistic activity of raloxifene could not be detected as was expected by the results from ligand competition analysis. Genomics, 2001 Sep, 77(1-2), 58 - 64 Cloning, mapping, and characterization of a human homologue of the yeast longevity assurance gene LAG1; Pan H et al.; We have identified LASS2, a previously unknown human homologue of the yeast longevity assurance gene LAG1 . The LASS2 transcript is highly expressed in liver and kidney, which is very different from the expression of the previously identified human LAG1 homologue LAG1Hs-1 . Radiation hybrid mapping studies indicated that LASS2 is located on chromosome 1q11 . Yeast two-hybrid screening and glutathione S-transferase pull-down assays showed that the LASS2 protein interacts with several membrane-associated receptors or transporters . Furthermore, LASS2 protein was able to inhibit the colony formation of human hepatoma cells in vitro, which suggests that this gene may be involved in the regulation of cell growth. Annu Rev Plant Physiol Plant Mol Biol, 2000, 51, 433 - 62 Diversity and regulation of plant Ca2+ pumps: insights from expression in yeast; Sze H et al.; The spatial and temporal regulation of calcium concentration in plant cells depends on the coordinate activities of channels and active transporters located on different organelles and membranes . Several Ca2+ pumps have been identified and characterized by functional expression of plant genes in a yeast mutant (K616) . This expression system has opened the way to a genetic and biochemical characterization of the regulatory and catalytic features of diverse Ca2+ pumps . Plant Ca(2+)-ATPases fall into two major types: AtECA1 represents one of four or more members of the type IIA (ER-type) Ca(2+)-ATPases in Arabidopsis, and AtACA2 is one of seven or more members of the type IIB (PM-type) Ca(2+)-ATPases that are regulated by a novel amino terminal domain . Type IIB pumps are widely distributed on membranes, including the PM (plasma membrane), vacuole, and ER (endoplasmic reticulum) . The regulatory domain serves multiple functions, including autoinhibition, calmodulin binding, and sites for modification by phosphorylation . This domain, however, is considerably diverse among several type IIB ATPases, suggesting that the pumps are differentially regulated . Understanding of Ca2+ transporters at the molecular level is providing insights into their roles in signaling networks and in regulating fundamental processes of cell biology. Biol Sci Space, 2000 Mar, 14(1), 3 - 8 Stress-compensation by a food supplement based on yeast plasmolysate in mitogen-activated T lymphocytes under simulated low-gravity; Schwarzenberg M et al.; T lymphocyte function is strongly depressed in vitro and in vivo under low-g conditions in space as well as simulated in clinostat . Here we describe the effect of a food supplement based on yeast plasmolysate on T cells activated in vitro with Concanavalin A and cultured in a random positioning machine . The mitotic index was measured by 3H-thymidine incorporation into DNA, the expression of activation markers CD25, CD69 and HLA-DR on the cell surface by cytofluorimetry and the secretion of the IL-2R by an enzyme immunoassay . Our data indicate that the food supplement used is capable to modulate T lymphocyte function . The addition of the food supplement increased the expression of activation markers in activated and non-activated cells . Cultivation under low-gravity conditions reduced the expression of the activation markers, but this expression was partly restored or even increased upon addition of yeast plasmolysate . On the other hand, cell proliferation and secretion of soluble IL-2 receptor was reduced after addition of the food supplement in all samples. Adv Space Res, 2000, 25(10), 2085 - 94 Induction of DNA double-strand breaks in mammalian cells and yeast; Frankenberg D et al.; Induction of DNA double-strand breaks (dsb) and their distribution are dependent on the energy deposition pattern within the cell nucleus (physical structure) and the ultrastructure of the chromosomes and its variation by the cell cycle and gene activities (biological structure) . For electron radiation very similar RBE-values are observed for mammalian and yeast cells (AlK, 1.5 keV, 15 keV/micrometer: 2.6 in mammalian cells and 2.2 in yeast; CK 0.278 keV, 23 keV/micrometer: approx . 2.5 in mammalian cells and 3.8 in yeast) . In contrast, the RBE-values for the induction of dsb of 4He2+ and light ions in the LET range from about 100 keV/micrometer up to 1000 keV/micrometer are significantly higher for yeast cells compared to mammalian cells . For example, the RBE-value of alpha-particles (120 keV/micrometer) is about 1.2 for mammalian cells whereas for yeast the RBE-value is about 2.5 . The yeast chromatin has less condensed fibres compared with mammalian cells . Since a single CK photoelectron can induce only one dsb, the different condensation of the mammalian and yeast chromatin has no influence . However, particles may induce more than one dsb when traversing a chromatin fibre . The probability for the induction of closely neighboured dsb is higher the more condensed the chromatin fibres are . Since small DNA fragments (50 bp up to several kbp) are lost by standard methods of lysis, the underestimation of dsb yields increases with fibre condensation, which is in accordance with the observes dsb yields in mammalian cells and yeast . In order to obtain relevant yields of dsb (and corresponding RBE-values) the measurement of all DNA fragments down to about 50 bp are needed. Adv Space Res, 1983, 3(8), 115 - 25 Heavy ion action on yeast cells: inhibition of ribosomal-RNA synthesis, loss of colony forming ability and induction of mutants; Kiefer J et al.; The action of heavy ions (Ar to U) accelerated to specific energies up to about 10 MeV/u (u=atomic mass unit) on different functions of yeast cells was studied . Ribosomal-RNA synthesis is inhibited according to a single-hit mechanism . Inactivation cross-sections were linearly related to the ratio of the squares of the effective charge Z* and the velocity of the ions . It is concluded from the analysis that the range of the most energetic delta-electrons is larger than previously assumed . There is no such dependence for survival and induction of mutants . In both cases cross-sections increase with the ion's specific-energy indicating an important contribution of long-range delta-electrons . The analysis shows that diploid yeast is not killed by a single-hit mechanism even by very heavy ions if the track width is too small . The relative importance of the penumbral region is even more pronounced with the more sensitive strains. Adv Space Res, 1994 Oct, 14(10), 331 - 8 Mutation induction in yeast by very heavy ions; Kiefer J; Resistance to canavanine was studied in haploid yeast after exposure to heavy ions (argon to uranium) of energies between 1 and 10 MeV/u covering a LET-range up to about 10000 keV/micrometer . Mutations were found in all instances but the induction cross sections increased with ion energy . This is taken to mean that the contribution of penumbra electrons plays an important role . The probability to recover surviving mutants is highest if the cell is not directly hit by the particle . The experiments demonstrate that the geometrical dimensions of the target cell nucleus as well as its sensitivity in terms of survival have a critical influence on mutation induction with very heavy ions. Yeast, 2001 Sep 15, 18(12), 1099 - 110 Negative regulation of transcription by the yeast global transcription factors, Gal11 and Sin4; Nishizawa M; Gal11 and Sin4 proteins are yeast global transcription factors that regulate transcription of a variety of genes, both positively and negatively . Gal11, in a major part, functions in the activation of transcription, whereas Sin4 has an opposite role, yet they are reported to be present as a complex in the so-called RNA polymerase II holoenzyme . To reveal howthese auxiliary factors participate in switching transcription on and off, a complex formation between Gal11 and Sin4 and its effect on the negative regulation of transcription were studied . Using an artificial promoter that is negatively regulated by Gal11, it was shown that the presence of Sin4 or Pgd1/Hrs1/Med3 was required for Gal11 to repress both basal and activated transcription . Genetic and biochemical studies using a temperature-sensitive Gal11 mutant showed that the amino acid region 866-910 essential for Gal11 function was also important for repression of transcription and a complex formation with Sin4 . Analysis with dam methylase accessibility to the promoter region suggested that nucleosome structure may be involved in negative regulation . Based on these results, possible mechanisms by which a mediator subcomplex regulates transcription is discussed . Microbiology, 2001 Sep, 147(Pt 9), 2389 - 98 Thiamin auxotrophy in yeast through altered cofactor dependence of the enzyme acetohydroxyacid synthase; Byrne KL et al.; The THI1 gene of Saccharomyces cerevisiae has been identified and found to be allelic with the previously characterized gene ILV2 that encodes acetohydroxyacid synthase (AHAS) . This enzyme catalyses the first step in the parallel biosyntheses of the branched-chain amino acids isoleucine and valine, using thiamin pyrophosphate (TPP) as a cofactor . The ilv2-thi1 allele encodes a functional AHAS enzyme with an altered dependence for the cofactor TPP resulting in the thiamin auxotrophic phenotype . Nucleotide sequence analysis and site-directed mutagenesis revealed that the thi1 mutation is a single base substitution which causes the conserved amino acid substitution D176E in the AHAS protein . This study therefore implicates aspartate 176 as another amino acid residue important either for the efficient binding of TPP by AHAS or for the functional stability of the holoenzyme. J Biol Chem, 2001 Nov 9, 276(45), 42477 - 84 Epub 2001 Sep 04. Structures of yeast ARF2 and ARL1: distinct roles for the N terminus in the structure and function of ARF family GTPases; Amor JC et al.; Structures were determined by x-ray crystallography for two members of the ADP-ribosylation factor (ARF) family of regulatory GTPases, yeast ARF1 and ARL1, and were compared with previously determined structures of human ARF1 and ARF6 . These analyses revealed an overall conserved fold but differences in primary sequence and length, particularly in an N-terminal loop, lead to differences in nucleotide and divalent metal binding . Packing of hydrophobic residues is central to the interplay between the N-terminal alpha-helix, switch I, and the interswitch region, which along with differences in surface electrostatics provide explanations for the different biophysical and biochemical properties of ARF and ARF-like proteins. Biochemistry, 2001 Sep 11, 40(36), 10873 - 80 The reaction of yeast cystathionine beta-synthase is rate-limited by the conversion of aminoacrylate to cystathionine; Jhee KH et al.; Our studies of the reaction mechanism of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme that forms a series of intermediates in the reaction of L-serine and L-homocysteine to form L-cystathionine . To characterize these reaction intermediates, we have carried out rapid-scanning stopped-flow and single-wavelength stopped-flow kinetic measurements under pre-steady-state conditions, as well as circular dichroism and fluorescence spectroscopy under steady-state conditions . We find that the gem-diamine and external aldimine of aminoacrylate are the primary intermediates in the forward half-reaction with L-serine and that the external aldimine of aminoacrylate or its complex with L-homocysteine is the primary intermediate in the reverse half-reaction with L-cystathionine . The second forward half-reaction of aminoacrylate with L-homocysteine is rapid . No primary kinetic isotope effect was obtained in the forward half-reaction with L-serine . The results provide evidence (1) that the formation of the external aldimine of L-serine is faster than the formation of the aminoacrylate intermediate, (2) that aminoacrylate is formed by the concerted removal of the alpha-proton and the hydroxyl group of L-serine, and (3) that the rate of the overall reaction is rate-limited by the conversion of aminoacrylate to L-cystathionine . We compare our results with cystathionine beta-synthase with those of related investigations of tryptophan synthase and O-acetylserine sulfhydrylase. Nat Rev Mol Cell Biol, 2001 Sep, 2(9), 678 - 87 Evolutionary conservation between budding yeast and human kinetochores; Kitagawa K et al.; Accurate chromosome segregation during mitosis requires the correct assembly of kinetochores--complexes of centromeric DNA and proteins that link chromosomes to spindle microtubules . Studies on the kinetochore of the budding yeast Saccharomyces cerevisiae have revealed functionally novel components of the kinetochore and its regulatory complexes, some of which are highly conserved in humans. Nat Rev Genet, 2001 Sep, 2(9), 659 - 68 The art and design of genetic screens: yeast; Forsburg SL; Understanding the biology of complex systems is facilitated by comparing them with simpler organisms . Budding and fission yeasts provide ideal model systems for eukaryotic cell biology . Although they differ from one another in terms of a range of features, these yeasts share powerful genetic and genomic tools . Classical yeast genetics remains an essential element in discovering and characterizing the genes that make up a eukaryotic cell. Mol Cell Biol, 2001 Oct, 21(19), 6668 - 80 Molecular genetic dissection of TAF25, an essential yeast gene encoding a subunit shared by TFIID and SAGA multiprotein transcription factors; Kirchner J et al.; We have performed a systematic structure-function analysis of Saccharomyces cerevisiae TAF25, an evolutionarily conserved, single-copy essential gene which encodes the 206-amino-acid TAF25p protein . TAF25p is an integral subunit of both the 15-subunit general transcription factor TFIID and the multisubunit, chromatin-acetylating transcriptional coactivator SAGA . We used hydroxylamine mutagenesis, targeted deletion, alanine-scanning mutagenesis, high-copy suppression methods, and two-hybrid screening to dissect TAF25 . Temperature-sensitive mutant strains generated were used for coimmunoprecipitation and transcription analyses to define the in vivo functions of TAF25p . The results of these analyses show that TAF25p is comprised of multiple mutable elements which contribute importantly to RNA polymerase II-mediated mRNA gene transcription. Mol Cell Biol, 2001 Oct, 21(19), 6606 - 14 Targeting of the yeast Ty5 retrotransposon to silent chromatin is mediated by interactions between integrase and Sir4p; Xie W et al.; The Ty5 retrotransposons of Saccharomyces cerevisiae integrate preferentially into regions of silent chromatin at the telomeres and silent mating loci (HMR and HML) . We define a Ty5-encoded targeting domain that spans 6 amino acid residues near the C terminus of integrase (LXSSXP) . The targeting domain establishes silent chromatin when it is tethered to a weakened HMR-E silencer, and it disrupts telomeric silencing when it is overexpressed . As determined by both yeast two-hybrid and in vitro binding assays, the targeting domain interacts with the C terminus of Sir4p, a structural component of silent chromatin . This interaction is abrogated by mutations in the targeting domain that disrupt integration into silent chromatin, suggesting that recognition of Sir4p by the targeting domain is the primary determinant in Ty5 target specificity. Mol Cell Biol, 2001 Oct, 21(19), 6429 - 39 A novel upstream RNA polymerase III promoter element becomes essential when the chromatin structure of the yeast U6 RNA gene is altered; Martin MP et al.; The Saccharomyces cerevisiae U6 RNA gene, SNR6, possesses upstream sequences that allow productive binding in vitro of the RNA polymerase III (Pol III) transcription initiation factor IIIB (TFIIIB) in the absence of TFIIIC or other assembly factors . TFIIIC-independent transcription of SNR6 in vitro is highly sensitive to point mutations in a consensus TATA box at position -30 . In contrast, the TATA box is dispensable for SNR6 transcription in vivo, apparently because TFIIIC bound to the intragenic A block and downstream B block can recruit TFIIIB via protein-protein interactions . A mutant allele of SNR6 with decreased spacing between the A and B blocks, snr6-Delta42, exhibits increased dependence on the upstream sequences in vivo . Unexpectedly, we find that in vivo expression of snr6-Delta42 is much more sensitive to mutations in a (dT-dA)(7) tract between the TATA box and transcription start site than to mutations in the TATA box itself . Inversion of single base pairs in the center of the dT-dA tract nearly abolishes transcription of snr6-Delta42, yet inversion of all 7 base pairs has little effect on expression, indicating that the dA-dT tract is relatively orientation independent . Although it is within the TFIIIB footprint, point mutations in the dT-dA tract do not inhibit TFIIIB binding or TFIIIC-independent transcription of SNR6 in vitro . In the absence of the chromatin architectural protein Nhp6, dT-dA tract mutations are lethal even when A-to-B block spacing is wild type . We conclude that the (dT-dA)(7) tract and Nhp6 cooperate to direct productive transcription complex assembly on SNR6 in vivo. EMBO J, 2001 Sep 3, 20(17), 4684 - 93 Ski7p G protein interacts with the exosome and the Ski complex for 3'-to-5' mRNA decay in yeast; Araki Y et al.; Two cytoplasmic mRNA-decay pathways have been characterized in yeast, and both are initiated by shortening of the 3'-poly(A) tail . In the major 5'-to-3' decay pathway, the deadenylation triggers removal of the 5'-cap, exposing the transcript body for 5'-to-3' degradation . An alternative 3'-to-5' decay pathway also follows the deadenylation and requires two multi-complexes: the exosome containing various 3'-exonucleases and the Ski complex consisting of the RNA helicase Ski2p, Ski3p and Ski8p . In addition, Ski7p, which has an N-terminal domain and a C-terminal elongation factor 1alpha-like GTP-binding domain, is involved in the 3'-to-5' decay . However, physical interaction between the exosome and the Ski complex, together with the function of Ski7p, has remained unknown . Here we report that the N domain of Ski7p is required and sufficient for the 3'-to-5' decay . Furthermore, the exosome and the Ski complex interact with the different regions of Ski7p N domain, and both interactions are required for the 3'-to-5' decay . Thus, Ski7p G protein appears to function as a signal-coupling factor between the two multi-complexes operating in the 3'-to-5' mRNA-decay pathway. Mol Cell Biol Res Commun, 2001 Sep, 4(5), 299 - 306 A mutation in the yeast mitochondrial ribosomal protein Rml2p is associated with a defect in catalase gene expression; Hagerman RA et al.; Yeast strains containing a new temperature-sensitive allele of the RML2 gene, encoding a component of the large subunit of the mitochondrial ribosome, display normal growth on acetate, slowed growth on glycerol and an inability to grow on oleic acid . These cells, denoted rml2(fat21), have an apparent inability to induce peroxisomal function, as evidenced by a deficiency in oleic acid induction of beta-oxidation . However, the oleic acid regulation of genes encoding core enzymes of peroxisomal beta-oxidation is normal . In contrast, up-regulation of CTA1 (catalase) mRNA expression and enzyme activity is interrupted . Upon comparison of the induction requirements of catalase and the genes of beta-oxidation, we hypothesized that the rml2(fat21) mutation alters the activity of the transcription factor Adr1p . In support of this hypothesis, over-expression of ADR1 in rml2(fat21) cells restores CTA1 induction . Several assays of mitochondria from rml2(fat21) strains suggest normal mitochondrial function . Thus, the modulation of Adr1p-associated gene regulation is not due to overt mitochondrial dysfunction . Biochem Biophys Res Commun, 2001 Sep 7, 286(5), 936 - 42 Characterization of loops of the yeast mitochondrial ADP/ATP carrier facing the cytosol by site-directed mutagenesis; Hatanaka T et al.; To characterize structural features of the regions of the yeast type 2 ADP/ATP carrier (yAAC2) facing the cytosol, we prepared its Cys-less mutant, in which all four cysteine residues were replaced by alanine residues . The Cys-less mutant functioned like native yAAC2, showing that the cysteine residues are not essential . We then prepared cysteine mutants by substituting Ser(21) in the putative N-terminal region, Ala(124) and Ser(222) in the first and second loops facing cytosol, respectively, and Leu(312) in the C-terminal region of the Cys-less mutant for cysteine and examined the labeling of the substituted cysteine residues of the mutants with the membrane-impermeable SH reagent eosin-5-maleimide (EMA) from the cytosol . EMA labeled all the mutants, showing that all regions containing mutated residues faced the cytosolic side . The effects of transport inhibitors on EMA labeling were also examined . From the results, the location and conformation of the region around mutated residues were discussed . Proc Natl Acad Sci U S A, 2001 Aug 28, 98(18), 10067 - 72 Why multiple small subunits (Y2 and Y4) for yeast ribonucleotide reductase? Toward understanding the role of Y4; Ge J et al.; Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides . Class I RNRs are composed of two homodimeric subunits: R1 and R2 . R1 is directly involved in the reduction, and R2 contains the diferric-tyrosyl radical (Y*) cofactor essential for the initiation of reduction . Saccharomyces cerevisiae has two RNRs; Y1 and Y3 correspond to R1, whereas Y2 and Y4 correspond to R2 . Y4 is essential for diferric-Y* formation in Y2 from apoY2, Fe(2+), and O(2) . The actual function of Y4 is controversial . Y2 and Y4 have been further characterized in an effort to understand their respective roles in nucleotide reduction . (His)(6)-Y2, Y4, and (His)(6)-Y4 are homodimers, isolated largely in apo form . Their CD spectra reveal that they are predominantly helical . The concentrations of Y2 and Y4 in vivo are 0.5-2.3 microM, as determined by Western analysis . Incubation of Y2 and Y4 under physiological conditions generates apo Y2Y4 heterodimer, which can form a diferric-Y small middle dot when incubated with Fe(2+) and O(2) . Holo Y2Y4 heterodimer contains 0.6-0.8 Y* and has a specific activity of 0.8-1.3 micromol.min.mg . Titration of Y2 with Y4 in the presence of Fe(2+) and O(2) gives maximal activity with one equivalent of Y4 per Y2 . Models for the function of Y4 based on these data and the accompanying structure will be discussed. Appl Environ Microbiol, 2001 Sep, 67(9), 4158 - 65 Novel ring cleavage products in the biotransformation of biphenyl by the yeast Trichosporon mucoides; Sietmann R et al.; The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings . While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission . These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones . Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl . Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified . Ring cleavage of 3,4,4'-trihydroxybiphenyl resulted in the formation of {5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl}acetic acid, 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid . 2,3,4-trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl . All these ring fission products were considerably less toxic than the hydroxylated derivatives. Curr Biol, 2001 Aug 21, 11(16), 1266 - 71 MAPK specificity in the yeast pheromone response independent of transcriptional activation; Breitkreutz A et al.; The mechanisms whereby different external cues stimulate the same mitogen-activated protein kinase (MAPK) cascade, yet trigger an appropriately distinct biological response, epitomize the conundrum of specificity in cell signaling . In yeast, shared upstream components of the mating pheromone and filamentous growth pathways activate two related MAPKs, Fus3 and Kss1, which in turn regulate programs of gene expression via the transcription factor Ste12 . As fus3, but not kss1, strains are impaired for mating, Fus3 exhibits specificity for the pheromone response . To account for this specificity, it has been suggested that Fus3 physically occludes Kss1 from pheromone-activated signaling complexes, which are formed on the scaffold protein Ste5 . However, we find that genome-wide expression profiles of pheromone-treated wild-type, fus3, and kss1 deletion strains are highly correlated for all induced genes and, further, that two catalytically inactive versions of Fus3 fail to abrogate the pheromone-induced transcriptional response . Consistently, Fus3 and Kss1 kinase activity is induced to an equivalent extent in pheromone-treated cells . In contrast, both in vivo and in an in vitro-reconstituted MAPK system, Fus3, but not Kss1, exhibits strong substrate selectivity toward Far1, a bifunctional protein required for polarization and G(1) arrest . This effect accounts for the failure to repress G(1)-S specific transcription in fus3 strains and, in part, explains the mating defect of such strains . MAPK specificity in the pheromone response evidently occurs primarily at the substrate level, as opposed to specific kinase activation by dedicated signaling complexes. Curr Biol, 2001 Aug 21, 11(16), 1240 - 50 Altering telomere structure allows telomerase to act in yeast lacking ATM kinases; Chan SW et al.; BACKGROUND: Telomerase is a ribonucleoprotein that copies a short RNA template into telomeric DNA, maintaining eukaryotic chromosome ends and preventing replicative senescence . Telomeres differentiate chromosome ends from DNA double-stranded breaks . Nevertheless, the DNA damage-responsive ATM kinases Tel1p and Mec1p are required for normal telomere maintenance in Saccharomyces cerevisiae . We tested whether the ATM kinases are required for telomerase enzyme activity or whether it is their action on the telomere that allows telomeric DNA synthesis . RESULTS: Cells lacking Tel1p and Mec1p had wild-type levels of telomerase activity in vitro . Furthermore, altering telomere structure in three different ways showed that telomerase can function in ATM kinase-deleted cells: tel1 mec1 cells senesced more slowly than tel1 mec1 cells that also lacked TLC1, which encodes telomerase RNA, suggesting that tel1 mec1 cells have residual telomerase function; deleting the telomere-associated proteins Rif1p and Rif2p in tel1 mec1 cells prevented senescence; we isolated a point mutation in the telomerase RNA template domain (tlc1-476A) that altered telomeric DNA sequences, causing uncontrolled telomeric DNA elongation and increasing single strandedness . In tel1 mec1 cells, tlc1-476A telomerase was also capable of uncontrolled synthesis, but only after telomeres had shortened for >30 generations . CONCLUSION: Our results show that, without Tel1p and Mec1p, telomerase is still active and can act in vivo when the telomere structure is disrupted by various means . Hence, a primary function of the ATM-family kinases in telomere maintenance is to act on the substrate of telomerase, the telomere, rather than to activate the enzymatic activity of telomerase. Curr Genet, 2001 Jul, 39(5-6), 297 - 304 Second-site, intragenic alterations in the gene encoding subunit II of cytochrome c oxidase from yeast can suppress two different missense mutations; Machingo Q et al.; Cytochrome c oxidase, a multi-subunit enzyme complex, accepts electrons from cytochrome c and transfers them to molecular oxygen to form water . Subunit II (Cox2p) of the enzyme complex provides the initial entry site for the electrons from cytochrome c . We report here the characterization of a yeast strain bearing a mutation in the gene encoding Cox2p which abolishes the activity of the enzyme complex . The alteration, at residue 163 in the yeast polypeptide, substitutes isoleucine for threonine and leads to loss of Cox2p and loss of the ability to carry out cellular respiration . We have also characterized 55 independent revertants of the mutant which have recovered the ability to respire . Of these revertants, 37 recover the ability to respire due to a compensatory alteration at residue 163, which produces either a wild-type threonine codon or one for valine or serine . The other 18 revertants recover function due to secondary changes at four different codons within the gene encoding Cox2p . Some of these second-site, intragenic revertants occur at sites significantly distant from the position of the original mutation . In addition, alterations at two of these sites have previously been shown to suppress a completely different missense mutation in the gene. Curr Genet, 2001 Jul, 39(5-6), 291 - 6 Strains isogenic to S288C used in the yeast genome sequencing programme carry a functional KSS1 gene; Morillon A et al.; In Saccharomyces cerevisiae, the KSS1 gene encodes the MAP kinase of the invasive/filamentous growth pathway . In addition to its role in this signal transduction pathway, Kssl can replace the Fus3 MAP kinase in the pheromone-response pathway, in the absence of FUS3 . Previous work indicated that derivatives of the S288C strain carry a mutant kss1 allele . Here, we report evidence that S288C derivatives used in the Yeast Genome Sequencing Programme carry a functional KSS1 gene and can thus be used to study the regulation of gene expression by KSS1. Mol Genet Genomics, 2001 Jul, 265(5), 851 - 64 Functional analysis of the yeast Ran exchange factor Prp20p: in vivo evidence for the RanGTP gradient model; Akhtar N et al.; Numerous cellular processes rely on the movement of macromolecules into and out of the nucleus . The primary regulator of this movement is the small GTPase Ran . Like other small GTPases, the nucleotide-bound state of Ran is regulated by effectors that enhance the rate of nucleotide exchange or hydrolysis . Current models for vectorial nuclear transport suggest that it is the strict compartmentalization of these Ran effector molecules that generates a gradient of RanGTP between the nucleus and the cytoplasm to impart directionality to the transport process . Here we investigate the mechanism by which the Ran exchange factor is targeted to the nucleus, and test the impact of disrupting this nuclear compartmentalization on nucleocytoplasmic transport in vivo . Our results indicate that in Saccharomycces cerevisiae the nucleotide exchange factor Prp20p can be targeted to the nucleus via a classical nuclear localization sequence . This transport mechanism is dependent both on Ran and the receptor that recognizes the nuclear localization sequence, importin alpha . Mutations in the evolutionarily conserved nuclear localization sequence only partially inhibit nuclear import of Prp20p, suggesting the existence of a secondary mechanism for this critical nuclear targeting . In an in vivo test of the RanGTP gradient model, we demonstrate that overexpression of a functional cytoplasmic exchange factor inhibits cell growth and blocks both protein import and RNA export in wild-type cells that contain the endogenous nuclear Prp20 protein . Taken together, our results provide in vivo evidence for the idea that the compartmentalization of the exchange factor serves as a mechanism for establishing directional nuclear transport. Mol Genet Genomics, 2001 Jul, 265(5), 801 - 11 Promoter sequences regulated by the calcineurin-activated transcription factor Crz1 in the yeast ENA1 gene; Mendizabal I et al.; In Saccharomyces cerevisiae the transcription of the ENA1 gene is modulated by multiple transduction pathways that respond to osmotic, ionic and nutrient stresses . We have investigated the molecular mechanisms involved in ENA1 induction by the calcium-calcineurin-activated transcription factor Crzl/Tcn1 . We found in the ENA1 promoter a calcium-responsive, Crzl-dependent upstream activating region (UASENA1) located between -713 bp and 826 bp relative to the translation start . This region contains two separate control elements: the upstream element (5'-GAATGGCTG-3') between -813 and -821 binds Crzlp with lower affinity and mostly contributes to basal ENA1 expression, whereas the downstream element (5'-GGGTGGCTG-3') between 727 and 719 binds Crz1p with higher affinity and is a major determinant of the induction response to calcium. Mol Genet Genomics, 2001 Aug, 265(6), 1039 - 49 Sna41goa1, a novel mutation causing G1/S arrest in fission yeast, is defective in a CDC45 homolog and interacts genetically with polalpha; Uchiyama M et al.; Proteins involved in the initiation of DNA replication play critical roles in the assembly and loading of replication complexes at replication origins . To gain further insight into the regulation of initiation, we screened in fission yeast for temperature-sensitive mutants which arrested at the G1/S boundary, and isolated nine mutants which arrested with a 1C DNA content at 36 degrees C . By linkage analysis, two complementation groups were identified which were not allelic to known G1 arrest mutations . One of the mutants isolated, sna41goul, arrested with a G1 DNA content and expressed a pleiomorphic phenotype, i.e., a mixture of cut and cdc phenotypes, at 36 degrees C . The point of arrest was identified as after START but before the hydroxyurea-induced block, by taking advantage of the mutant rad26.a14, which has a defect in an early S phase-specific checkpoint, and by performing reciprocal shift experiments . sna41 goal is allelic to sna41+, which is homologous to the CDC45 gene of budding yeast, and the mutation lies in a motif that is highly conserved in Cdc45-related proteins . The temperature sensitivity of the sna41goal mutant can be suppressed to some extent by ts mutations in polalpha . Our genetic results are consistent with a model in which Cdc45 plays crucial roles in the assembly of the replication apparatus at replication origins. Plant Cell Physiol, 2001 Aug, 42(8), 813 - 22 The transcriptional activation domain of the plant-specific Dof1 factor functions in plant, animal, and yeast cells; Yanagisawa S; Maize Dof1, one of the plant-specific Dof transcription factors, is involved in light-regulated gene expression . To elucidate the molecular mechanism underlying the activity of Dof1, in vivo functional analyses were carried out using transient expression assays with maize mesophyll protoplasts . The results suggest that the Dof domain alone, the region conserved among Dof factors, can mediate interaction with DNA in vivo and distinct Dof1 activities in greening and etiolated protoplasts . A region rich in basic amino acids was identified as a nuclear localization signal . Deletion analysis defined the transcriptional activation domain of 48 amino acids located in the C-terminus of Dof1 . This activation domain was also found to be functional in both human cells and yeast, implying that Dof1 may stimulate transcription through a mechanism evolutionarily conserved among eukaryotes . A computer homology search with known transcription factors revealed that the activation domain of Dof1 displayed only a limited similarity to Activation domain II of animal transcription factor GATA-4 . Mutational analysis revealed the critical role of a tryptophan residue within the activation domain of Dof1, as had been shown in Activation domain II of GATA-4. Nucleic Acids Res, 2001 Sep 1, 29(17), 3621 - 30 ERB1, the yeast homolog of mammalian Bop1, is an essential gene required for maturation of the 25S and 5.8S ribosomal RNAs; Pestov DG et al.; We have recently shown that the mammalian nucleolar protein Bop1 is involved in synthesis of the 28S and 5.8S ribosomal RNAs (rRNAs) and large ribosome subunits in mouse cells . Here we have investigated the functions of the Saccharomyces cerevisiae homolog of Bop1, Erb1p, encoded by the previously uncharacterized open reading frame YMR049C . Gene disruption showed that ERB1 is essential for viability . Depletion of Erb1p resulted in a loss of 25S and 5.8S rRNAs synthesis, while causing only a moderate reduction and not a complete block in 18S rRNA formation . Processing analysis showed that Erb1p is required for synthesis of 7S pre-rRNA and mature 25S rRNA from 27SB pre-rRNA . In Erb1p-depleted cells these products of 27SB processing are largely absent and 27SB pre-rRNA is under-accumulated, apparently due to degradation . In addition, depletion of Erb1p caused delayed processing of the 35S pre-rRNA . These findings demonstrate that Erb1p, like its mammalian counterpart Bop1, is required for formation of rRNA components of the large ribosome particles . The similarities in processing defects caused by functional disruption of Erb1p and Bop1 suggest that late steps in maturation of the large ribosome subunit rRNAs employ mechanisms that are evolutionarily conserved throughout eukaryotes. Biochim Biophys Acta, 2001 Aug 17, 1506(2), 103 - 16 Identification of functional regions of Cbp3p, an enzyme-specific chaperone required for the assembly of ubiquinol-cytochrome c reductase in yeast mitochondria; Shi G et al.; The Cbp3 protein of Saccharomyces cerevisiae is an enzyme-specific chaperone required for the assembly of ubiquinol-cytochrome c reductase of the mitochondrial respiratory chain . To gain preliminary insight into the role of Cbp3p during assembly, 29 independently isolated mutants were examined to define functional regions of the protein . Mutants were analyzed with respect to respiratory growth, ubiquinol-cytochrome c reductase assembly, and steady state amounts of enzyme subunits and Cbp3p . Three regions essential for Cbp3p activity were identified: regions 1 and 3 were required for Cbp3p function, while region 2 was necessary for protein stability . Mutation of Glu134 in region 1 (Cys124 through Ala140) impaired the ability of the Rieske FeS protein to assemble with the enzyme complex . Mutations targeted to region 3 (Gly223 through Asp229) primarily affected the 14 kDa subunit and cytochrome c(1) assembly . Gly223 was found especially sensitive to mutation and the introduction of charged residues at this site compromised Cbp3p functional activity . Region 2 (Leu167 through Pro175) overlapped the single hydrophobic domain of Cbp3p . Mutations within this area altered the association of Cbp3p with the mitochondrial membrane resulting in enhanced protein turnover . The role of the amino-terminus in Cbp3p activity was investigated using cbp3 deletion strains Delta12-23, Delta24-54, Delta56-96 and Delta12-96 . All mutants were respiratory competent, indicating that residues 12-96 were not essential for Cbp3p function, stability or mitochondrial import . Analysis of carboxy-terminal deletion mutants demonstrated that the final 44 residues were not necessary for Cbp3p function; however, alterations in the secondary structure of the extreme carboxy-terminal 17 residues affected assembly protein activity. Biochem Biophys Res Commun, 2001 Aug 31, 286(4), 714 - 20 A comprehensive analysis of gene expression profiles in a yeast N-glycosylation mutant; Klebl B et al.; Although protein N-glycosylation is critical to many cell functions, its downstream targets remain largely unknown . In all eukaryotes, N-glycosylation utilizes the lipid-linked oligosaccharide (LLO) precursor, whose synthesis is initiated by the ALG7 gene . To elucidate the key signaling and metabolic events affected by N-glycosylation, we performed genomewide expression profiling of yeast cells carrying a hypomorphic allele of ALG7 . DNA microarrays showed that of more than 97% of known or predicted yeast genes, 29 displayed increased expression while 23 were repressed in alg7 mutants . Changes in transcript abundance were observed for a and alpha mating-type genes, for genes functioning in several mitogen-activated protein kinase (MAPK) cascades, as well as in phosphate, amino acid, carbohydrate, mitochondrial and ATP metabolism . Therefore, DNA microarrays have revealed direct and indirect targets, including internal feedback loops, through which N-glycosylation affects signaling and metabolic activities and is functionally linked with cellular regulatory circuitry . Bioseparation, 2000, 9(6), 369 - 77 Breakdown of immobilisation/separation and morphology changes of yeast suspended in water-rich ethanol mixtures exposed to ultrasonic plane standing waves; Radel S et al.; Some physiological/morphological changes have been reported before, when suspended yeasts have been irradiated with well-defined ultrasonic standing, as well as propagating, plane waves around 2.2 MHz, as used in ultrasonic coagulation, e.g., for cell filtering . Thus we used yeast as a biological model to explore the reasons for both those morphology changes and some unusual macroscopic behaviour in the case of water-rich ethanol mixtures when used as carrier liquid . When the cells were suspended in 12% (v/v) ethanol-water mixture separation was greatly reduced; the yeast cells were not retained in the pressure nodal planes of the standing wave, but mixed turbulently through the separation system . How this behaviour alters the efficiency of retention/immobilisation was measured . As the viability of the yeast was decreased as well the morphology of the cells was examined using transmission electron microscopy . Two effects, according to the type of assessment, were evident; a disruption of the cells vacuole and also damage to the cell wall/membrane complex . The extent of the alterations in vacuole structure with sonication time, utilising a fluorescent vacuole membrane dye, was measured . Transient cavitation was not detected and thus could be excluded as being responsible for the observed effects . Other possible reasons for the disruption of the intracellular compartments may be acoustic pressure, displacement or other, secondary effects like (sub) harmonic cavitation . The investigations contribute to a better understanding of the physical conditions experienced when a cell is stressed in a high-frequency ultrasonic wave in the MHz range. Bioseparation, 2000, 9(6), 329 - 41 Observation of yeast cell movement and aggregation in a small-scale MHz-ultrasonic standing wave field; Spengler JF et al.; Aggregation of suspended yeast cells in a small-scale ultrasonic standing wave field has been monitored and quantified . The aggregation effect is based on the acoustic radiation force, which concentrates the cells in clumps . The ultrasonic chamber employed (1.9 MHz, one wavelength pathlength) had a sonication volume of 60 microl . The aggregation process was observed from above the transducer through a transparent glass reflector . A distinct, reproducible, pattern of clumps formed rapidly in the sound field . The sound pressure was estimated experimentally to be of the order of 1 MPa . Microscopic observations of the formation of a single clump were recorded onto a PC . The time dependent movement patterns and travelling velocities of the cells during the aggregation process were extracted by particle image velocimetry analysis . A time dependent change was seen in the particle motion pattern during approach to its completion of clump formation after 45 s . Streaming eddies were set-up during the first couple of seconds . The scale of the eddies was consistent with Rayleigh micro-streaming theory . An increase in the travelling velocity of the cells was observed after 30 s from initially about 400 microm s(-1) to about 1 mm s(-1) . The influence of a number of mechanisms on particle behaviour (e.g . micro-streaming, particle interactions and convective flow) is considered . The experimental set-up introduced here is a powerful tool for aggregation studies in ultrasonic standing waves and lays the foundation for future quantitative experiments on the individual contributions of the different mechanisms. Genetika, 2001 Jun, 37(6), 725 - 36 {The sbr gene product in Drosophila melanogaster and its orthologs in yeast (Mex67p) and human (TAP)}; Tret'iakova IV et al.; A DNA sequence from the 9F region of Drosophila melanogaster polytene chromosomes was cloned . Sequencing the cloned region and its comparison with the known sequences of the D . melanogaster genome showed that the cloned DNA part contains gene sbr and adjacent sequences . The literature data on the structure and functions of genes TAP in humans and Mex67 in yeast are discussed . These genes are orthologous to the sbr gene of Drosophila and control mRNA export from the nucleus to the cytoplasm . The literature evidence is consistent with the recessive expression of mutation l(1)ts403 (sbr10) upon heat treatment that is manifested as impaired HSP synthesis at the posttranscriptional level . However, it fails to explain the semidominant effect of the mutation manifested in high frequency of meiotic sex-chromosome nondisjunction in heat-treated females . A comparison of amino-acid sequences corresponding to the products of the three orthologous genes, TAP, Mex67, and sbr, showed that the sbr gene product of Drosophila is more similar to the human TAP factor than to the Mex67 factor in yeast. Mol Biol Cell, 2001 Aug, 12(8), 2497 - 518 Bud8p and Bud9p, proteins that may mark the sites for bipolar budding in yeast; Harkins HA et al.; The bipolar budding pattern of a/alpha Saccharomyces cerevisiae cells appears to depend on persistent spatial markers in the cell cortex at the two poles of the cell . Previous analysis of mutants with specific defects in bipolar budding identified BUD8 and BUD9 as potentially encoding components of the markers at the poles distal and proximal to the birth scar, respectively . Further genetic analysis reported here supports this hypothesis . Mutants deleted for BUD8 or BUD9 grow normally but bud exclusively from the proximal and distal poles, respectively, and the double-mutant phenotype suggests that the bipolar budding pathway has been totally disabled . Moreover, overexpression of these genes can cause either an increased bias for budding at the distal (BUD8) or proximal (BUD9) pole or a randomization of bud position, depending on the level of expression . The structures and localizations of Bud8p and Bud9p are also consistent with their postulated roles as cortical markers . Both proteins appear to be integral membrane proteins of the plasma membrane, and they have very similar overall structures, with long N-terminal domains that are both N- and O-glycosylated followed by a pair of putative transmembrane domains surrounding a short hydrophilic domain that is presumably cytoplasmic . The putative transmembrane and cytoplasmic domains of the two proteins are very similar in sequence . When Bud8p and Bud9p were localized by immunofluorescence and tagging with GFP, each protein was found predominantly in the expected location, with Bud8p at presumptive bud sites, bud tips, and the distal poles of daughter cells and Bud9p at the necks of large-budded cells and the proximal poles of daughter cells . Bud8p localized approximately normally in several mutants in which daughter cells are competent to form their first buds at the distal pole, but it was not detected in a bni1 mutant, in which such distal-pole budding is lost . Surprisingly, Bud8p localization to the presumptive bud site and bud tip also depends on actin but is independent of the septins. Mol Biol Cell, 2001 Aug, 12(8), 2422 - 32 The proteasomal substrate Stm1 participates in apoptosis-like cell death in yeast; Ligr M et al.; We have identified the yeast gene STM1 in an overexpression screen for new proteasomal substrates . Stm1 is unstable in wild-type cells and stabilized in cells with defective proteasomal activity and thus a bona