Yeasts

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 fide substrate of the proteasome . It is localized in the perinuclear region and is required for growth in the presence of mutagens . Overexpression in cells with impaired proteasomal degradation leads to cell death accompanied with cytological markers of apoptosis: loss of plasma membrane asymmetry, chromatin condensation, and DNA cleavage . Cells lacking Stm1 display deficiency in the apoptosis-like cell death process induced by treatment with low concentrations of H(2)O(2) . We suggest that Stm1 is involved in the control of the apoptosis-like cell death in yeast . Survival is increased when Stm1 is completely missing from the cells or when inhibition of Stm1 synthesis permits proteasomal degradation to decrease its amount in the cell . Conversely, Stm1 accumulation induces cell death . In addition we identified five other genes whose overexpression in proteasomal mutants caused similar apoptotic phenotypes.

Genetics, 2001 Aug, 158(4), 1445 - 55
The yeast cytoplasmic LsmI/Pat1p complex protects mRNA 3' termini from partial degradation; He W et al.; A key aspect of understanding eukaryotic gene regulation will be the identification and analysis of proteins that bind mRNAs and control their function . Recently, a complex of seven Lsm proteins and the Pat1p have been shown to interact with yeast mRNAs and promote mRNA decapping . In this study we present several observations to indicate that the LsmI/Pat1 complex has a second distinct function in protecting the 3'-UTR of mRNAs from trimming . First, mutations in the LSM1 to LSM7, as well as PAT1, genes led to the accumulation of MFA2pG and PGK1pG transcripts that had been shortened by 10-20 nucleotides at their 3' ends (referred to as trimming) . Second, the trimming of these mRNAs was more severe at the high temperature, correlating with the inability of these mutant strains to grow at high temperature . In contrast, trimming did not occur in a dcp1 Delta strain, wherein the decapping enzyme is lacking . This indicates that trimming is not simply a consequence of the inhibition of mRNA decapping . Third, the temperature-sensitive growth of lsm and pat1 mutants was suppressed by mutations in the exosome or the functionally related Ski proteins, which are required for efficient 3' to 5' mRNA degradation of mRNA . Moreover, in lsm ski double mutants, higher levels of the trimmed mRNAs accumulated, indicating that exosome function is not required for mRNA trimming but that the exosome does degrade the trimmed mRNAs . These results raise the possibility that the temperature-sensitive growth of the lsm1-7 and pat1 mutants is at least partially due to mRNA trimming, which either inactivates the function of the mRNAs or makes them available for premature 3' to 5' degradation by the exosome.

FEBS Lett, 2001 Aug 17, 503(2-3), 196 - 200
The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors; Erskine PT et al.; The structures of 5-aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4-oxosebacic acid and 4,7-dioxosebacic acid) have been solved at high resolution . Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site . Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P-side substrate) which bind to the enzyme during catalysis . The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A- and P-side substrates) . The most intriguing result is the novel finding that 4,7-dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210 . It has been known for many years that P-side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A-side substrate involves formation of a Schiff base with the enzyme . A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A- and P-sites is proposed.

FEBS Lett, 2001 Aug 10, 503(1), 13 - 8
Reconstitution of hepatitis C virus protease activities in yeast; Mak P et al.; The hepatitis C virus (HCV) protease genes (NS2/3 and NS3) were expressed in yeast with their natural substrates fused to a ligand-dependent transcriptional activator, the retinoic acid receptor (RARbeta) . RARbeta can activate transcription in yeast cells in response to retinoic acids . We hypothesized that cis-cleavage at the NS2-3 or NS3-4A junctions by the appropriate HCV proteases would release RARbeta, thereby activating transcription of a reporter gene . Our results from Western blot analyses and reporter gene activation indicate that the wild-type NS2/3 and NS3 enzymes are catalytically active in yeast cells, whereas mutations in the catalytic domain of NS2(C993V) and NS3(S1165A) lead to inactive enzymes . We conclude that HCV NS2/3 and NS3 protease activities can be reconstituted in yeast.

Mol Cell, 2001 Jul, 8(1), 225 - 31
Intermediates of yeast meiotic recombination contain heteroduplex DNA; Allers T et al.; The formation of heteroduplex DNA features prominently in all models for homologous recombination . A central intermediate in the current double-strand break repair model contains two Holliday junctions flanking a region of heteroduplex DNA . Studies of yeast meiosis have identified such intermediates but failed to detect associated heteroduplex DNA . We show here that these intermediates contain heteroduplex DNA, providing an important validation of the double-strand break repair model . However, we also detect intermediates where both Holliday junctions are to one side of the initiating DSB site, while the intervening region shows no evidence of heteroduplex DNA . Such structures are not easily accommodated by the canonical version of the double-strand break repair model.

Mol Cell, 2001 Jul, 8(1), 189 - 99
Localization of yeast telomeres to the nuclear periphery is separable from transcriptional repression and telomere stability functions; Tham WH et al.; The left telomere of Saccharomyces chromosome VII was often localized near the nuclear periphery, even in cells lacking the silencing proteins Sir3 or Hdf1 . This association was lost in late mitotic cells and when transcription was induced through the telomeric tract . Although in silencing competent cells there was no correlation between the fraction of cells in which a telomeric gene was repressed and the fraction of cells in which it was localized to the periphery, no condition was found where the telomere was both silenced and away from the periphery . We conclude that localization of a telomere to the nuclear periphery is not sufficient for transcriptional repression nor does it affect the stability function of yeast telomeres.

Mol Cell, 2001 Jul, 8(1), 129 - 36
Budding yeast Rad9 is an ATP-dependent Rad53 activating machine; Gilbert CS et al.; We find budding yeast Rad9 in two distinct, large, and soluble complexes in cell extracts . The larger (> or =850 kDa) complex, found in nondamaged cells, contains hypophosphorylated Rad9, whereas the smaller (560 kDa) complex, which forms after DNA damage, contains hyperphosphorylated Rad9 and Rad53 . This smaller Rad9 complex is capable of catalyzing phosphorylation and release of active Rad53 kinase, a process requiring the kinase activity of Rad53 . However, Mec1 and Tel1 are no longer required once the 560 kDa complex has been formed . We propose a model whereby Mec1/Tel1-dependent hyperphosphorylation of Rad9 results in formation of the smaller Rad9 complex and recruitment of Rad53 . This complex then catalyzes activation of Rad53 by acting as a scaffold that brings Rad53 molecules into close proximity, facilitating Rad53 in trans autophosphorylation and subsequent release of activated Rad53.

Cell, 2001 Jul 27, 106(2), 183 - 94
Multiple Gln/Asn-rich prion domains confer susceptibility to induction of the yeast {PSI(+)} prion; Osherovich LZ et al.; The yeast prion {PSI(+)} results from self-propagating aggregates of Sup35p . De novo formation of {PSI(+)} requires an additional non-Mendelian trait, thought to result from a prion form of one or more unknown proteins . We find that the Gln/Asn-rich prion domains of two proteins, New1p and Rnq1p, can control susceptibility to {PSI(+)} induction as well as enhance aggregation of a human glutamine expansion disease protein . {PSI(+)} inducibility results from gain-of-function properties of New1p and Rnq1p aggregates rather than from inactivation of the normal proteins . These studies suggest a molecular basis for the epigenetic control of {PSI(+)} inducibility and may reveal a broader role for this phenomenon in the physiology of protein aggregation.

Planta Med, 2001 Aug, 67(6), 553 - 5
Estrogenic activity of phenolic compounds from Nigella damascena evaluated using a recombinant yeast screen; Agradi E et al.; We used a yeast estrogen screen (YES) containing human estrogen receptor to evaluate the estrogenic activity of both crude extracts and simple pure phenolic compounds from Nigella damascena seeds . Estrogenic activity was established in the methanolic and aqueous extracts of the seeds as well as in two simple phenolic compounds isolated from the methanolic extract, 2,4-dihydroxyphenylacetic acid, 3,4-dihydroxy-beta-phenethyl alcohol.

Mol Cell Biol, 2001 Sep, 21(18), 6292 - 311
Mechanisms controlling subcellular localization of the G(1) cyclins Cln2p and Cln3p in budding yeast; Miller ME et al.; Different G(1) cyclins confer functional specificity to the cyclin-dependent kinase (Cdk) Cdc28p in budding yeast . The Cln3p G(1) cyclin is localized primarily to the nucleus, while Cln2p is localized primarily to the cytoplasm . Both binding to Cdc28p and Cdc28p-dependent phosphorylation in the C-terminal region of Cln2p are independently required for efficient nuclear depletion of Cln2p, suggesting that this process may be physiologically regulated . The accumulation of hypophosphorylated Cln2 in the nucleus is an energy-dependent process, but may not involve the RAN GTPase . Phosphorylation of Cln2p is inefficient in small newborn cells obtained by elutriation, and this lowered phosphorylation correlates with reduced Cln2p nuclear depletion in newborn cells . Thus, Cln2p may have a brief period of nuclear residence early in the cell cycle . In contrast, the nuclear localization pattern of Cln3p is not influenced by Cdk activity . Cln3p localization requires a bipartite nuclear localization signal (NLS) located at the C terminus of the protein . This sequence is required for nuclear localization of Cln3p and is sufficient to confer nuclear localization to green fluorescent protein in a RAN-dependent manner . Mislocalized Cln3p, lacking the NLS, is much less active in genetic assays specific for Cln3p, but more active in assays normally specific for Cln2p, consistent with the idea that Cln3p localization explains a significant part of Clnp functional specificity.

Mol Cell Biol, 2001 Sep, 21(18), 6139 - 50
Regulation of the yeast Yap1p nuclear export signal is mediated by redox signal-induced reversible disulfide bond formation; Kuge S et al.; Yap1p, a crucial transcription factor in the oxidative stress response of Saccharomyces cerevisiae, is transported in and out of the nucleus under nonstress conditions . The nuclear export step is specifically inhibited by H(2)O(2) or the thiol oxidant diamide, resulting in Yap1p nuclear accumulation and induction of transcription of its target genes . Here we provide evidence for sensing of H(2)O(2) and diamide mediated by disulfide bond formation in the C-terminal cysteine-rich region (c-CRD), which contains 3 conserved cysteines and the nuclear export signal (NES) . The H(2)O(2) or diamide-induced oxidation of the c-CRD in vivo correlates with induced Yap1p nuclear localization . Both were initiated within 1 min of application of oxidative stress, before the intracellular redox status of thioredoxin and glutathione was affected . The cysteine residues in the middle region of Yap1p (n-CRD) are required for prolonged nuclear localization of Yap1p in response to H(2)O(2) and are thus also required for maximum transcriptional activity . Using mass spectrometry analysis, the H(2)O(2)-induced oxidation of the c-CRD in vitro was detected as an intramolecular disulfide linkage between the first (Cys(598)) and second (Cys(620)) cysteine residues; this linkage could be reduced by thioredoxin . In contrast, diamide induced each pair of disulfide linkage in the c-CRD, but in this case the cysteine residues in the n-CRD appeared to be dispensable for the response . Our data provide evidence for molecular mechanisms of redox signal sensing through the thiol-disulfide redox cycle coupled with the thioredoxin system in the Yap1p NES.

J Biol Chem, 2001 Oct 26, 276(43), 40254 - 62 Epub 2001 Aug 16.
The DNA-binding domain of yeast heat shock transcription factor independently regulates both the N- and C-terminal activation domains; Bulman AL et al.; The expression of heat shock proteins in response to cellular stresses is dependent on the activity of the heat shock transcription factor (HSF) . In yeast, HSF is constitutively bound to DNA; however, the mitigation of negative regulation in response to stress dramatically increases transcriptional activity . Through alanine-scanning mutagenesis of the surface residues of the DNA-binding domain, we have identified a large number of mutants with increased transcriptional activity . Six of the strongest mutations were selected for detailed study . Our studies suggest that the DNA-binding domain is involved in the negative regulation of both the N-terminal and C-terminal activation domains of HSF . These mutations do not significantly affect DNA binding . Circular dichroism analysis suggests that a subset of the mutants may have altered secondary structure, whereas a different subset has decreased thermal stability . Our findings suggest that the regulation of HSF transcriptional activity (under both constitutive and stressed conditions) may be partially dependent on the local topology of the DNA-binding domain . In addition, the DNA-binding domain may mediate key interactions with ancillary factors and/or other intramolecular regulatory regions in order to modulate the complex regulation of HSF's transcriptional activity.

Curr Biol, 2001 Jul 24, 11(14), 1124 - 7
Myosin V-mediated vacuole distribution and fusion in fission yeast; Mulvihill DP et al.; The class V myosins are actin-based motors that move a variety of cellular cargoes {1} . In budding yeast, their activity includes the relocation of a portion of the vacuole from the mother cell to the bud {2, 3} . Fission yeast cells contain numerous (approximately 80) small vacuoles . When S . pombe cells are placed in water, vacuoles fuse in response to osmotic stress {4} . Fission yeast possess two type V myosin genes, myo51(+) and myo52(+) {5} . In a myo51Delta strain, vacuoles were distributed throughout the cell, and mean vacuole diameter was identical to that seen in wild-type cells . When myo51Delta and wild-type cells were placed in water, vacuoles enlarged by fusion . In myo52Delta cells, by contrast, vacuoles were smaller and mostly clustered around the nucleus, and fusion in water was largely inhibited . When cells containing GFP-Myo52 were placed in water, Myo52 was seen to redistribute from the cell poles to the surface of the fusing vacuoles . Vacuole fusion in fission yeast was inhibited by the microtubule drug thiabendazole (TBZ) but not by the actin inhibitor latrunculin B . This is the first demonstration of the involvement of a type V myosin, possibly via an interaction with microtubules, in homotypic membrane fusion.

BMC Biochem . 2001;2(1):7 . Epub 2001 Jul 18.
The fission yeast COP9/signalosome is involved in cullin modification by ubiquitin-related Ned8p; Zhou C et al.; BACKGROUND: The function of the fission yeast cullins Pcu1p and Pcu4p requires modification by the ubiquitin-related peptide Ned8p . A recent report by Lyapina et al . shows that the COP9/signalosome (CSN), a multifunctional eight subunit complex, regulates Ned8p modification of Pcu1p . Disruption of caa1/csn1, which encodes subunit 1 of the putative S . pombe CSN, results in accumulation of Pcu1p exclusively in the modified form . However, it remained unclear whether this reflects global control of all cullins by the entire CSN complex . RESULTS: We demonstrate that multiple CSN subunits control Ned8p modification of Pcu3p, another fission yeast cullin, which, in complex with the RING domain protein Pip1p, forms a ubiquitin ligase that functions in cellular stress response . Pcu3p is modified by Ned8p on Lys 729 and accumulates exclusively in the neddylated form in cells lacking the CSN subunits 1, 3, 4, and 5 . These CSN subunits co-elute with Pcu3p in gel filtration fractions corresponding to approximately 550 kDa and specifically bind both native and Ned8p-modified Pcu3p in vivo . While CSN does not influence the subcellular localization of Pcu3p, Pcu3p-associated in vitro ubiquitin ligase activity is stimulated in the absence of CSN . CONCLUSIONS: Taken together, our data suggest that CSN is a global regulator of Ned8p modification of multiple cullins and potentially other proteins involved in cellular regulation.

Biochemistry, 2001 Aug 21, 40(33), 9758 - 69
Yeast mitochondrial dehydrogenases are associated in a supramolecular complex; Grandier-Vazeille X et al.; Separation of yeast mitochondrial complexes by colorless native polyacrylamide gel electrophoresis led to the identification of a supramolecular structure exhibiting NADH-dehydrogenase activity . Components of this complex were identified by N-terminal Edman degradation and matrix-assisted laser desorption ionization mass spectrometry . The complex was found to contain the five known intermembrane space-facing dehydrogenases, namely two external NADH-dehydrogenases Nde1p and Nde2p, glycerol-3-phosphate dehydrogenase Gut2p, D- and L-lactate-dehydrogenases Dld1p and Cyb2p, the matrix-facing NADH-dehydrogenase Ndi1p, two probable flavoproteins YOR356Wp and YPR004Cp, four tricarboxylic acids cycle enzymes (malate dehydrogenase Mdh1p, citrate synthase Cit1p, succinate dehydrogenase Sdh1p, and fumarate hydratase Fum1p), and the acetaldehyde dehydrogenase Ald4p . The association of these proteins is discussed in terms of NADH-channeling.

Res Microbiol, 2001 Jul-Aug, 152(6), 551 - 62
Genetic characterization of the nonconventional yeast Hansenula anomala; Naumov GI et al.; We describe genetic, molecular and taxonomic characteristics of the yeast Hansenula anomala . Pulsed-field gel electrophoresis of chromosomal DNAs from 19 H . anomala strains and related species indicated that H . anomala had a clearly different karyotype . Chromosome length polymorphism of the H . anomala strains was independent of their geographic origin and source of isolation . The strains were classified into four groups of similar karyotypes and one strain showed a unique profile . The sizes of chromosomes ranged from 850 to 3500 kb in different strains . The haploid chromosome number of H . anomala is at least nine . We have found RAPD primers discriminating at both the species and strain levels . All the primers tested except the M13 core sequence generated unique patterns with most strains . The results indicate the usefulness of PCR analysis with primer M13 for identification of the H . anomala species . Screening of the CBS (Utrecht) collection strains of H . anomala showed that they are rather difficult objects for genetic hybridization analysis . The strains have low fertility, viz . very poor sporulation, low mating type activities and, as a rule, nonviable ascospores . The majority of the hybrids obtained are polyploid, probably tetraploid, as judged by the segregation of control auxotrophic markers . Nevertheless, some monosporic cultures of the strains studied, including the biocontrol yeast J121, formed diploid hybrids with regular meiotic segregation of control auxotrophic markers . As a rule, H . anomala isolates are homothallic, showing delayed self-diploidization . Rare stable heterothallic strains of H . anomala also occur.

Res Microbiol, 2001 Jul-Aug, 152(6), 531 - 8
Prion model in yeast; Sapriel G; Two unusual phenotypes in Saccharomyces cerevisiae, {PSI+} and {URE3}, have been suggested to be due to prion proteins . Various research groups have shown that this is indeed the case and have characterized these yeast prions in more detail . The factors involved in prion formation, such as chaperone protein, and the intramolecular determinants of prion formation have been investigated . The ability of these yeast proteins to form prion is due to modular domains conserved throughout evolution.

Folia Microbiol (Praha), 2000, 45(6), 521 - 9
Sugar repression in the methylotrophic yeast Hansenula polymorpha studied by using hexokinase-negative, glucokinase-negative and double kinase-negative mutants; Kramarenko T et al.; Two glucose-phosphorylating enzymes, a hexokinase phosphorylating both glucose and fructose, and a glucose-specific glucokinase were electrophoretically separated in the methylotrophic yeast Hansenula polymorpha . Hexokinase-negative, glucokinase-negative and double kinase-negative mutants were isolated in H . polymorpha by using mutagenesis, selection and genetic crosses . Regulation of synthesis of the sugar-repressed alcohol oxidase, catalase and maltase was studied in different hexose kinase mutants . In the wild type and in mutants possessing either hexokinase or glucokinase, glucose repressed the synthesis of maltase, alcohol oxidase and catalase . Glucose repression of alcohol oxidase and catalase was abolished in mutants lacking both glucose-phosphorylating enzymes (i.e . in double kinase-negative mutants) . Thus, glucose repression in H . polymorpha cells requires a glucose-phosphorylating enzyme, either hexokinase or glucokinase . The presence of fructose-phosphorylating hexokinase in the cell was specifically needed for fructose repression of alcohol oxidase, catalase and maltase . Hence, glucose or fructose has to be phosphorylated in order to cause repression of the synthesis of these enzymes in H . polymorpha suggesting that sugar repression in this yeast therefore relies on the catalytic activity of hexose kinases.

J Biol Chem, 2001 Oct 5, 276(40), 37373 - 8 Epub 2001 Aug 10.
Multiple levels of control regulate the yeast cAMP-response element-binding protein repressor Sko1p in response to stress; Pascual-Ahuir A et al.; The Sko1p transcriptional repressor regulates a subset of osmoinducible stress defense genes in Saccharomyces cerevisiae by binding to cAMP-responsive elements . We have reported previously that in response to stress Sko1p is phosphorylated by the stress-activated Hog1p mitogen-activated protein kinase, which disrupts its interaction with the Ssn6p x Tup1p corepressor . Here we report that other mechanisms are essential for the regulation of the Sko1p repressor activity upon stress . The nuclear localization of Sko1p depends on the stress-inhibited protein kinase A (PKA) . Sko1p is localized in the nucleus of unstressed cells, and it redistributes to the cytosol upon severe salt stress (1 m NaCl) . Yeast mutants with low PKA activity localize Sko1p to the cytoplasm in the absence of stress and exhibit deregulated expression of cAMP-responsive element-regulated genes . The central part (315) of Sko1p, containing the PKA phosphorylation sites and the basic domain-leucine zipper domain, is essential for its nuclear localization . Salt-induced export of Sko1p from the nucleus is independent of Hog1p and of the Bcy1p regulatory subunit of PKA . Furthermore, phosphorylation by PKA slightly enhanced DNA binding affinity of Sko1p in vitro, whereas Sko1p dimerization in vivo is not regulated by stress . Sko1p repressor activity is associated to its binding to the Ssn6p x Tup1p complex . Interestingly, the Sko1p NH(2) terminus (1), containing the Hog1p phosphorylation sites, associates in vivo with Tup1p in the absence of Ssn6p, suggesting that Sko1p represses gene transcription by interacting directly with the Tup1p subunit of the Ssn6p x Tup1p complex.

J Biol Chem, 2001 Oct 12, 276(41), 38084 - 9 Epub 2001 Aug 10.
A fraction of yeast Cu,Zn-superoxide dismutase and its metallochaperone, CCS, localize to the intermembrane space of mitochondria . A physiological role for SOD1 in guarding against mitochondrial oxidative damage; Sturtz LA et al.; Cu,Zn-superoxide dismutase (SOD1) is an abundant, largely cytosolic enzyme that scavenges superoxide anions . The biological role of SOD1 is somewhat controversial because superoxide is thought to arise largely from the mitochondria where a second SOD (manganese SOD) already resides . Using bakers' yeast as a model, we demonstrate that Cu,Zn-SOD1 helps protect mitochondria from oxidative damage, as sod1Delta mutants show elevated protein carbonyls in this organelle . In accordance with this connection to mitochondria, a fraction of active SOD1 localizes within the intermembrane space (IMS) of mitochondria together with its copper chaperone, CCS . Neither CCS nor SOD1 contains typical N-terminal presequences for mitochondrial uptake; however, the mitochondrial accumulation of SOD1 is strongly influenced by CCS . When CCS synthesis is repressed, mitochondrial SOD1 is of low abundance, and conversely IMS SOD1 is very high when CCS is largely mitochondrial . The mitochondrial form of SOD1 is indeed protective against oxidative damage because yeast cells enriched for IMS SOD1 exhibit prolonged survival in the stationary phase, an established marker of mitochondrial oxidative stress . Cu,Zn-SOD1 in the mitochondria appears important for reactive oxygen physiology and may have critical implications for SOD1 mutations linked to the fatal neurodegenerative disorder, amyotrophic lateral sclerosis.

J Biol Chem, 2001 Nov 23, 276(47), 43949 - 57 Epub 2001 Aug 10.
Ubiquitin is required for sorting to the vacuole of the yeast general amino acid permease, Gap1; Soetens O et al.; In yeast, ubiquitin plays a central role in proteolysis of a multitude of proteins and serves also as a signal for endocytosis of many plasma membrane proteins . We showed previously that ubiquitination of the general amino acid permease (Gap1) is essential to its endocytosis followed by vacuolar degradation . These processes occur when NH(4)(+), a preferential source of nitrogen, is added to cells growing on proline or urea, i.e . less favored nitrogen sources . In this study, we show that Gap1 is ubiquitinated on two lysine residues in the cytosolic N terminus (positions 9 and 16) . A mutant Gap1 in which both lysines are mutated (Gap1(K9K16)) remains fully stable at the plasma membrane after NH(4)(+) addition . Furthermore, each of the two lysines harbors a poly-ubiquitin chain in which ubiquitin is linked to the lysine 63 of the preceding ubiquitin . The Gap1(K9) and Gap1(K16) mutants, in which a single lysine is mutated, are down-regulated in response to NH(4)(+) although more slowly . In proline-grown cells lacking Npr1, a protein kinase involved in the control of Gap1 trafficking, newly synthesized Gap1 is sorted from the Golgi to the vacuole without passing through the plasma membrane (accompanying article, De Craene, J.-O., Soetens, O., and Andre, B . (2001) J . Biol . Chem . 276, 43939-43948) . We show here that ubiquitination of Gap1 is also required for this direct sorting to the vacuole . In an npr1Delta mutant, neosynthesized Gap1(K9K16) is rerouted to and accumulates at the plasma membrane . Finally, Bul1 and Bul2, two proteins interacting with Npi1/Rsp5, are essential to ubiquitination and down-regulation of cell-surface Gap1, as well as to sorting of neosynthesized Gap1 to the vacuole, as occurs in an npr1Delta mutant . Our results reveal a novel role of ubiquitin in the control of Gap1 trafficking, i.e . direct sorting from the late secretory pathway to the vacuole . This result reinforces the growing evidence that ubiquitin plays an important role not only in internalization of plasma membrane proteins but also in their sorting in the endosomes and/or trans-Golgi.

J Biol Chem, 2001 Nov 23, 276(47), 43939 - 48 Epub 2001 Aug 10.
The Npr1 kinase controls biosynthetic and endocytic sorting of the yeast Gap1 permease; De Craene JO et al.; Membrane trafficking of the general amino acid permease (Gap1) of Saccharomyces cerevisiae is under nitrogen regulation . In cells growing on proline or urea as the sole nitrogen source, newly synthesized Gap1 is delivered to the plasma membrane, where it accumulates . Upon addition of NH(4)(+), a preferential nitrogen source, Gap1 is endocytosed and targeted to the vacuole, where it is degraded . This down-regulation requires ubiquitination of the permease, and this ubiquitination is dependent on the essential Npi1/Rsp5 ubiquitin ligase . In this study, we investigated the role of the Npr1 kinase in the regulation of Gap1 trafficking . We show that Npr1 is required for stabilization of Gap1 at the plasma membrane: when an npr1(ts) mutant growing on proline is shifted to the restrictive temperature, Gap1 down-regulation is triggered, as it is when NH(4)(+) is added to wild-type cells . The fate of newly synthesized Gap1 en route to the plasma membrane is also under Npr1 control: in an npr1Delta mutant, neosynthesized Gap1 is sorted from the Golgi to the vacuole without passing via the plasma membrane . Similar direct sorting of neosynthesized Gap1 to the vacuole was observed in wild-type cells grown on NH(4)(+) . Finally, Gap1 is phosphorylated in NPR1 cells, but this phosphorylation is not strictly dependent on Npr1 . Our results show that Npr1 kinase plays a central role in the physiological control of Gap1 trafficking and that this control is exerted not only on Gap1 present at the plasma membrane but also on Gap1 late in the secretory pathway . Npr1 belongs to a subgroup of protein kinases, some of which are reported to exert a positive control on the activity of other permeases . We propose that these kinases also function as regulators of permease trafficking.

EMBO J, 2001 Aug 15, 20(16), 4512 - 21
Transcription of chromosomal rRNA genes by both RNA polymerase I and II in yeast uaf30 mutants lacking the 30 kDa subunit of transcription factor UAF; Siddiqi IN et al.; UAF, a yeast RNA polymerase I transcription factor, contains Rrn5p, Rrn9p, Rrn10p, histones H3 and H4, and uncharacterized protein p30 . Mutants defective in RRN5, RRN9 or RRN10 are unable to transcribe rDNA by polymerase I and grow extremely slowly, but give rise to variants able to grow by transcribing chromosomal rDNA by polymerase II . Thus, UAF functions as both an activator of polymerase I and a silencer of polymerase II for rDNA transcription . We have now identified the gene for subunit p30 . This gene, UAF30, is not essential for growth, but its deletion decreases the cellular growth rate . Remarkably, the deletion mutants use both polymerase I and II for rDNA transcription, indicating that the silencer function of UAF is impaired, even though rDNA transcription by polymerase I is still occurring . A UAF complex isolated from the uaf30 deletion mutant was found to retain the in vitro polymerase I activator function to a large extent . Thus, Uaf30p plays only a minor role in its activator function . Possible reasons for slow growth caused by uaf30 mutations are discussed.

Appl Microbiol Biotechnol, 2001 Jul, 56(1-2), 157 - 64
Expression of mouse anticreatine kinase (MAK33) monoclonal antibody in the yeast Hansenula polymorpha; Abdel-Salam HA et al.; The methylotrophic yeast Hansenula polymorpha HM1-39 (ura 3 and leu 2) was used as a host strain for the expression of the Fab fragment of the MAK33 monoclonal antibody . The MAK33 antibody reacts specifically with creatine kinase-M . The cDNA of kappa and gamma chains were inserted between the FMD or MOX promoter and the MOX terminator within the expression plasmids . In addition, the secretion signal sequence of the mating factor-alpha (prepro segment) and a fragment from glucoamylase with its secretion signal peptide, were also inserted in the expression plasmids for efficient secretion and production of the MAK33 monoclonal antibody . The co-expression of kappa and gamma chains was achieved by double transformation with kappa and then with gamma chain-expressing plasmids . The cells of H . polymorpha HM1-39 showed high mitotic stability and both uracil+ and leucine+ phenotypic stability after double transformation . Northern analysis showed a high rate of transcription of either kappa or gamma chain mRNA but not both, when the cells were grown in an induction medium . Protein analysis of double-transformed cells showed the monomers of the MAK33 antibody (kappa and gamma chains) were not assembled into a heterodimeric functional form . The expressed proteins of light and heavy chains represent about 11-12% of total cell protein and are found more inside than outside the cell . The expressed monomers show antigen-binding affinity in the Ouchterlony diffusion test; and the binding activity exhibited by cell-free extract was more than that of the cell culture supernatant.

Anticancer Res, 2001 May-Jun, 21(3B), 1899 - 903
Alterations in DNA repair and telomere maintenance mechanism affect response to porphyrins in yeast; Izbicka E et al.; BACKGROUND: DNA quadruplex-interactive porphyrin TMPyP4, but not its isomer TMPyP2, inhibits telomerase activity and causes chromosome fusion in vivo, suggesting interference with telomere maintenance . MATERIALS AND METHODS: We examined effects of these porphyrins and hydroxyurea on growth rates of yeast Saccharomyces cerevisiae wild type and strains with defects in telomere maintenance and/or DNA repair pathways (mec1, tel1, rad9), telomere binding protein (cdc13), and anaphase control (pds1) . RESULTS: Hydroxyurea (20 mM) decreased proliferation rates only in mec1 mutant and deletion strains . TMPyP4 (200 microM) decreased growth in all strains, especially in rad9delta and mec1delta . The growth inhibition by TMPyP4 showed low growth inhibition in strains defective in cdc13 and pds1 . TMPyP2 sterically prevented from forming a planar species did not significantly inhibit growth of any strain . Overexpression of telomere binding protein Rap1 hypersensitized the mec1delta and tel1delta to TMPyP4 . CONCLUSIONS: Telomere maintenance represents a viable target for anticancer agents.

Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9660 - 5 Epub 2001 Aug 07.
The yeast mutant vps5Delta affected in the recycling of Golgi membrane proteins displays an enhanced vacuolar Mg2+/H+ exchange activity; Borrelly G et al.; Growth of the yeast vacuolar protein-sorting mutant vps5Delta affected in the endosome-to-Golgi retromer complex was more sensitive to Mg2+-limiting conditions than was the growth of the wild-type (WT) strain . This sensitivity was enhanced at acidic pH . The vps5Delta strain was also sensitive to Al3+, known to inhibit Mg2+ uptake in yeast cells . In contrast, it was found to be resistant to Ni2+ and Co2+, two cytotoxic analogs of Mg2+ . Resistance to Ni2+ did not seem to result from the alteration of plasma-membrane transport properties because mutant and WT cells displayed similar Ni2+ uptake . After plasma-membrane permeabilization, intracellular Ni2+ uptake in vps5Delta cells was 3-fold higher than in WT cells, which is consistent with the implication of the vacuole in the observed phenotypes . In reconstituted vacuolar vesicles prepared from vps5Delta, the rates of H+ exchange with Ni2+, Co2+, and Mg2+ were increased (relative to WT) by 170%, 130%, and 50%, respectively . The rates of H+ exchange with Ca2+, Cd2+, and K+ were similar in both strains, as were alpha-mannosidase and H+-ATPase activities, and SDS/PAGE patterns of vacuolar proteins . Among 14 other vacuolar protein-sorting mutants tested, only the 8 mutants affected in the recycling of trans-Golgi network membrane proteins shared the same Ni2+ resistance phenotype as vps5Delta . It is proposed that a trans-Golgi network Mg2+/H+ exchanger, mislocalized to vps5Delta vacuole, could be responsible for the phenotypes observed in vivo and in vitro.

J Cell Sci, 2001 Jun, 114(Pt 12), 2345 - 54
The Bub2-dependent mitotic pathway in yeast acts every cell cycle and regulates cytokinesis; Lee SE et al.; In eukaryotes an abnormal spindle activates a conserved checkpoint consisting of the MAD and BUB genes that results in mitotic arrest at metaphase . Recently, we and others identified a novel Bub2-dependent branch to this checkpoint that blocks mitotic exit . This cell-cycle arrest depends upon inhibition of the G-protein Tem1 that appears to be regulated by Bfa1/Bub2, a two-component GTPase-activating protein, and the exchange factor Lte1 . Here, we find that Bub2 and Bfa1 physically associate across the entire cell cycle and bind to Tem1 during mitosis and early G1 . Bfa1 is multiply phosphorylated in a cell-cycle-dependent manner with the major phosphorylation occurring in mitosis . This Bfa1 phosphorylation is Bub2-dependent . Cdc5, but not Cdc15 or Dbf2, partly controls the phosphorylation of Bfa1 and also Lte1 . Following spindle checkpoint activation, the cell cycle phosphorylation of Bfa1 and Lte1 is protracted and some species are accentuated . Thus, the Bub2-dependent pathway is active every cell cycle and the effect of spindle damage is simply to protract its normal function . Indeed, function of the Bub2 pathway is also prolonged during metaphase arrests imposed by means other than checkpoint activation . In metaphase cells Bub2 is crucial to restrain downstream events such as actin ring formation, emphasising the importance of the Bub2 pathway in the regulation of cytokinesis . Our data is consistent with Bub2/Bfa1 being a rate-limiting negative regulator of downstream events during metaphase.

J Cell Sci, 2001 Jun, 114(Pt 12), 2241 - 53
The ARF exchange factors Gea1p and Gea2p regulate Golgi structure and function in yeast; Peyroche A et al.; The Sec7 domain guanine nucleotide exchange factors (GEFs) for the GTPase ARF are highly conserved regulators of membrane dynamics . Their precise molecular roles in different trafficking steps within the cell have not been elucidated . We present a functional analysis of two members of this family, Gea1p and Gea2p, in the yeast Saccharomyces cerevisiae . Gea1p and Gea2p can functionally replace each other, but at least one is necessary for viability . Temperature sensitive gea mutants were generated and found to have defects in ER-Golgi and intra-Golgi transport . Similar to mutants in COPI subunits in yeast, gea mutants had a cargo-selective secretion defect, in that some proteins continued to be secreted whereas others were blocked in the ER or early Golgi . Like yeast arf mutants, the rate of transport of those proteins that continued to be secreted was slowed . In addition, the structure of Golgi elements was severely perturbed in gea mutants . We conclude that Gea1p and Gea2p play an important role in the structure and functioning of the Golgi apparatus in yeast.

J Cell Sci, 2001 Jun, 114(Pt 11), 2135 - 43
Fission yeast mfr1 activates APC and coordinates meiotic nuclear division with sporulation; Blanco MA et al.; Meiosis is the developmental program by which sexually reproducing diploid organisms generate haploid gametes . In yeast, meiosis is followed by spore morphogenesis . These two events are normally coordinated in such a way that spore formation is dependent upon completion of the meiotic nuclear divisions . Here we describe a meiosis-specific protein, mfr1, that is involved in this coordination . mfr1 is an activator of the anaphase-promoting complex (APC), which is necessary for the rapid degradation of the cdc13 cyclin at the end of meiosis II, prior to the formation of spores . An mfr1 null mutant completes meiosis II but remains with high levels of cdc13 and cdc2 kinase activity and has considerably delayed spore formation . By analogy with the mitotic cell cycle, where proteolysis and inactivation of cdc2 kinase are necessary to trigger mitotic exit and cytokinesis, we propose that at the end of meiosis rapid and timely proteolysis of cyclins is required to switch on the differentiation program that eventually leads to the formation of haploid gametes.

J Biol Chem, 2001 Oct 19, 276(42), 39150 - 60 Epub 2001 Aug 07.
The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmic reticulum retrieval in yeast; Andag U et al.; Sec22p is an endoplasmic reticulum (ER)-Golgi v-SNARE protein whose retrieval from the Golgi compartment to the endoplasmic reticulum (ER) is mediated by COPI vesicles . Whether Sec22p exhibits its primary role at the ER or the Golgi apparatus is still a matter of debate . To determine the role of Sec22p in intracellular transport more precisely, we performed a synthetic lethality screen . We isolated mutant yeast strains in which SEC22 gene function, which in a wild type strain background is non-essential for cell viability, has become essential . In this way a novel temperature-sensitive mutant allele, dsl1-22, of the essential gene DSL1 was obtained . The dsl1-22 mutation causes severe defects in Golgi-to-ER retrieval of ER-resident SNARE proteins and integral membrane proteins harboring a C-terminal KKXX retrieval motif, as well as of the soluble ER protein BiP/Kar2p, which utilizes the HDEL receptor, Erd2p, for its recycling to the ER . DSL1 interacts genetically with mutations that affect components of the Golgi-to-ER recycling machinery, namely sec20-1, tip20-5, and COPI-encoding genes . Furthermore, we demonstrate that Dsl1p is a peripheral membrane protein, which in vitro specifically binds to coatomer, the major component of the protein coat of COPI vesicles.

J Bacteriol, 2001 Sep, 183(17), 5102 - 9
Insertional mutagenesis in the n-alkane-assimilating yeast Yarrowia lipolytica: generation of tagged mutations in genes involved in hydrophobic substrate utilization; Mauersberger S et al.; Tagged mutants affected in the degradation of hydrophobic compounds (HC) were generated by insertion of a zeta-URA3 mutagenesis cassette (MTC) into the genome of a zeta-free and ura3 deletion-containing strain of Yarrowia lipolytica . MTC integration occurred predominantly at random by nonhomologous recombination . A total of 8,600 Ura(+) transformants were tested by replica plating for (i) growth on minimal media with alkanes of different chain lengths (decane, dodecane, and hexadecane), oleic acid, tributyrin, or ethanol as the C source and (ii) colonial defects on different glucose-containing media (YPD, YNBD, and YNBcas) . A total of 257 mutants were obtained, of which about 70 were affected in HC degradation, representing different types of non-alkane-utilizing (Alk(-)) mutants (phenotypic classes alkA to alkE) and tributyrin degradation mutants . Among Alk(-) mutants, growth defects depending on the alkane chain length were observed (alkAa to alkAc) . Furthermore, mutants defective in yeast-hypha transition and ethanol utilization and selected auxotrophic mutants were isolated . Flanking borders of the integrated MTC were sequenced to identify the disrupted genes . Sequence analysis indicated that the MTC was integrated in the LEU1 locus in N083, a leucine-auxotrophic mutant, in the isocitrate dehydrogenase gene of N156 (alkE leaky), in the thioredoxin reductase gene in N040 (alkAc), and in a peroxine gene (PEX14) in N078 (alkD) . This indicates that MTC integration is a powerful tool for generating and analyzing tagged mutants in Y . lipolytica.

Traffic, 2001 Aug, 2(8), 565 - 76
Clathrin interactions with C-terminal regions of the yeast AP-1 beta and gamma subunits are important for AP-1 association with clathrin coats; Yeung BG et al.; Heterotetrameric adaptor (AP) complexes are thought to coordinate cargo recruitment and clathrin assembly during clathrin-coated vesicle biogenesis . We have identified, and characterized the physiological significance of clathrin-binding activities in the two large subunits of the AP-1 complex in Saccharomyces cerevisiae . Using GST-fusion chromatography, two clathrin-binding sites were defined in the beta1 subunit that match consensus clathrin-binding sequences in other mammalian and yeast clathrin-binding proteins . Clathrin interactions were also identified with the C-terminal region of the gamma subunit . When introduced into chromosomal genes, point mutations in the beta1 clathrin-binding motifs, or deletion of the gamma C-terminal region, reduced association of AP-1 with clathrin in coimmunoprecipitation assays . The beta1 mutations or the gamma truncation individually produced minor effects on AP-1 distribution by subcellular fractionation . However, when beta1 and gamma mutations were combined, severe defects were observed in AP-1 association with membranes and incorporation into clathrin-coated vesicles . The combination of subunit mutations accentuated growth and alpha-factor pheromone maturation defects in chc1-ts cells, though not to the extent caused by complete loss of AP-1 activity . Our results suggest that both the beta1 and gamma subunits contribute interactions with clathrin that are important for stable assembly of AP-1 complexes into clathrin coats in vivo.

Eur J Biochem, 2001 Aug, 268(15), 4243 - 50
Characterization of novel structures of mannosylinositolphosphorylceramides from the yeast forms of Sporothrix schenckii; Loureiro y Penha CV et al.; Novel structures of glycoinositolphosphorylceramide (GIPC) from the infective yeast form of Sporothrix schenckii were determined by methylation analysis, mass spectrometry and NMR spectroscopy . The lipid portion was characterized as a ceramide composed of C-18 phytosphingosine N-acylated by either 2-hydroxylignoceric acid (80%), lignoceric (15%) or 2,3-dihydroxylignoceric acids (5%) . The ceramide was linked through a phosphodiester to myo-inositol (Ins) which is substituted on position O-6 by an oligomannose chain . GIPC-derived Ins oligomannosides were liberated by ammonolysis and characterized as: Manpalpha1-->6Ins; Manpalpha1-->3Manpalpha1-->6Ins; Manpalpha1-->6Manpalpha1-->3Manpalpha1-->3Manpalpha1-->6Ins; Manpalpha1-->2Manpalpha1-->6Manpalpha1-->3Manpalpha1-->3Manpalpha1-->6Ins . These structures comprise a novel family of fungal GIPC, as they contain the Manpalpha1-->6Ins substructure, which has not previously been characterized unambigously, and may be acylated with a 2,3 dihydroxylignoceric fatty acid, a feature hitherto undescribed in fungal lipids.

J Autoimmun, 2001 Aug, 17(1), 51 - 61
Expression of protein tyrosine phosphatase-like molecule ICA512/IA-2 induces growth arrest in yeast cells and transfected mammalian cell lines; Papakonstantinou T et al.; The ICA512/IA-2 molecule, a protein with similarity to receptor-type protein tyrosine phosphatases, was discovered during studies to identify autoantigens in Type 1 diabetes . The biological function of ICA512/IA-2 is unknown . We describe striking effects of ICA512/IA-2 on viability and growth of both yeast cells and cultured mammalian cells . In transformed yeast Saccharomyces cerevisiae cells, expression of ICA512/IA-2 induced growth retardation as judged by measurements of optical density and counts of colony-forming units . In contrast, expression of the intracellular domain (amino acids 600-979) of ICA512/IA-2 in yeast or mammalian cells had no such effects . In investigations on apoptosis, expression of ICA512/IA-2 in yeast cells caused loss of plasma membrane asymmetry, but not release of cytochrome c from mitochondria which did occur in a control system after expression of the pro-apoptotic molecule Bax . Possible interactions between ICA512/IA-2 and components of the cytoskeleton were not supported by studies on staining of fixed yeast cells with phalloidin-Texas Red . With transfected mammalian cell lines COS-7 and NIH3T3, expression of ICA512/IA-2 likewise induced growth arrest, with some of the morphological features of apoptosis . Thus obligatory expression of ICA512/IA-2 in eukaryotic cells causes disruption of cellular activities, with growth arrest in yeast and nuclear pycnosis/fragmentation in mammalian cells . A possible explanation is that growth inhibition reflects a part of the presently unknown function of ICA512/IA-2 .

Mol Cell Biol, 2001 Sep, 21(17), 6056 - 65
Partners of Rpb8p, a small subunit shared by yeast RNA polymerases I, II and III; Briand JF et al.; Rpb8p, a subunit common to the three yeast RNA polymerases, is conserved among eukaryotes and absent from noneukaryotes . Defective mutants were found at an invariant GGLLM motif and at two other highly conserved amino acids . With one exception, they are clustered on the Rpb8p structure . They all impair a two-hybrid interaction with a fragment conserved in the largest subunits of RNA polymerases I (Rpa190p), II (Rpb1p), and III (Rpc160p) . This fragment corresponds to the pore 1 module of the RNA polymerase II crystal structure and bears a highly conserved motif (P.I.KP.LW.GKQ) facing the GGLLM motif of Rpb8p . An RNA polymerase I mutant (rpa190-G728D) at the invariant glycyl of P.I.KP.LW.GKQ provokes a temperature-sensitive defect . Increasing the gene dosage of another common subunit, Rpb6p, suppresses this phenotype . It also suppresses a conditional growth defect observed when replacing Rpb8p by its human counterpart . Hence, Rpb6p and Rpb8p functionally interact in vivo . These two subunits are spatially separated by the pore 1 module and may also be possibly connected by the disorganized N half of Rpb6p, not included in the present structure data . Human Rpb6p is phosphorylated at its N-terminal Ser2, but an alanyl replacement at this position still complements an rpb6-Delta null allele . A two-hybrid interaction also occurs between Rpb8p and the product of orphan gene YGR089w . A ygr089-Delta null mutant has no detectable growth defect but aggravates the conditional growth defect of rpb8 mutants, suggesting that the interaction with Rpb8p may be physiologically relevant.

Mol Cell Biol, 2001 Sep, 21(17), 5742 - 52
Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase, and the cyclin-dependent kinase Pho85p; Wang Z et al.; In the yeast Saccharomyces cerevisiae, glycogen is accumulated as a carbohydrate reserve when cells are deprived of nutrients . Yeast mutated in SNF1, a gene encoding a protein kinase required for glucose derepression, has diminished glycogen accumulation and concomitant inactivation of glycogen synthase . Restoration of synthesis in an snf1 strain results only in transient glycogen accumulation, implying the existence of other SNF1-dependent controls of glycogen storage . A genetic screen revealed that two genes involved in autophagy, APG1 and APG13, may be regulated by SNF1 . Increased autophagic activity was observed in wild-type cells entering the stationary phase, but this induction was impaired in an snf1 strain . Mutants defective for autophagy were able to synthesize glycogen upon approaching the stationary phase, but were unable to maintain their glycogen stores, because subsequent synthesis was impaired and degradation by phosphorylase, Gph1p, was enhanced . Thus, deletion of GPH1 partially reversed the loss of glycogen accumulation in autophagy mutants . Loss of the vacuolar glucosidase, SGA1, also protected glycogen stores, but only very late in the stationary phase . Gph1p and Sga1p may therefore degrade physically distinct pools of glycogen . Pho85p is a cyclin-dependent protein kinase that antagonizes SNF1 control of glycogen synthesis . Induction of autophagy in pho85 mutants entering the stationary phase was exaggerated compared to the level in wild-type cells, but was blocked in apg1 pho85 mutants . We propose that Snf1p and Pho85p are, respectively, positive and negative regulators of autophagy, probably via Apg1 and/or Apg13 . Defective glycogen storage in snf1 cells can be attributed to both defective synthesis upon entry into stationary phase and impaired maintenance of glycogen levels caused by the lack of autophagy.

Genes Dev, 2001 Aug 1, 15(15), 1935 - 45
SAGA is an essential in vivo target of the yeast acidic activator Gal4p; Bhaumik SR et al.; Despite major advances in characterizing the eukaryotic transcriptional machinery, the function of promoter-specific transcriptional activators (activators) is still not understood . For example, in no case have the direct in vivo targets of a transcriptional activator been unambiguously identified, nor has it been resolved whether activators have a single essential target or multiple redundant targets . Here we address these issues for the prototype acidic activator yeast Gal4p . Gal4p binds to the upstream activating sequence (UAS) of GAL1 and several other GAL genes and stimulates transcription in the presence of galactose . Previous studies have shown that GAL1 transcription is dependent on the yeast SAGA (Spt/Ada/GCN5/acetyltransferase) complex . Using formaldehyde-based in vivo cross-linking, we show that the Gal4p activation domain recruits SAGA to the GAL1 UAS . If SAGA is not recruited to the UAS, the preinitiation complex (PIC) fails to assemble at the GAL1 core promoter, and transcription does not occur . SAGA, but not other transcription components, is also recruited by the Gal4p activation domain to a plasmid containing minimal Gal4p-binding sites . Recruitment of SAGA by Gal4p and stimulation of PIC assembly is dependent on several SAGA subunits but not the SAGA histone acetyl-transferase (HAT) GCN5 . Based on these and other results, we conclude that SAGA is an essential target of Gal4p that, following recruitment to the UAS, facilitates PIC assembly and transcription.

Environ Health Perspect, 2001 Jul, 109(7), 691 - 7
Optimization of a yeast estrogen screen and its applicability to study the release of estrogenic isoflavones from a soygerm powder; De Boever P et al.; Here we describe a redesigned protocol of the yeast estrogen screen developed by Routledge and Sumpter . The redesigned test comprises two steps . First, a large amount of yeast with estrogenic compounds is incubated for 24 hr . Subsequently, a mixture of cycloheximide and the chromogenic substrate chlorophenol red-beta-d-galactopyranoside (CPRG) is added . The cycloheximide stops protein synthesis and allows for an end-point measurement of beta-galactosidase activity generated during the first 24 hr . CPRG is converted to chlorophenol red and reflects beta-galactosidase activity, which is indicative of the estrogenic activity . The modifications shorten the duration of the assay at least 1 day and avoid interference of the estrogenic CPRG or chlorophenol red . The redesigned and the original protocol were used to study the estrogenic activity of bisphenol A, methoxychlor, p,p'-DDT, and isoflavones (genistein, daidzein, and glycitein) . Bisphenol A, methoxychlor, and genistein triggered higher levels of beta-galactosidase activity in the redesigned protocol . Estrogenic activity of p,p'-DDT could only be demonstrated with the redesigned protocol . Glycitein and daidzein failed to give a response with both protocols . We also studied deconjugation of beta-glycosidic isoflavones present in soygerm powder . Treatment of the soygerm powder with beta-glycosidase released isoflavones . The estrogenic response of the samples was confirmed with the redesigned protocol and correlated with the amount of genistein present . The release of isoflavones under conditions prevailing in the intestines was studied . Bacterial beta-glycosidase present in the large intestine released isoflavones, and moderate estrogenic activity could be demonstrated.

Methods Find Exp Clin Pharmacol, 2001 Mar, 23(2), 61 - 4
In vitro activity of sertaconazole, fluconazole, ketoconazole, fenticonazole, clotrimazole and itraconazole against pathogenic vaginal yeast isolates; Palacin C et al.; The in vitro activity of sertaconazole was compared with those of the most commonly used vaginal antimycotic agents--fluconazole, ketoconazole, fenticonazole, clotrimazole and itraconazole--against 94 strains of clinical isolates of Candida spp . using a macrodilution method in Casitone agar medium . The sertaconazole concentration (microgram/ml), at which 90% of the strains were inhibited, was 0.06 for C . albicans, 0.25 for C . glabrata and C . parapsilosis, 1 for C . krusei and 2 for C . tropicalis . These values show that sertaconazole is one of the most active products against yeasts causing vulvovaginal candidiasis, its activity against C . glabrata being particularly relevant.

Apoptosis, 2001 Oct, 6(5), 331 - 8
Analysis of candidate antagonists of IAP-mediated caspase inhibition using yeast reconstituted with the mammalian Apaf-1-activated apoptosis mechanism; Hawkins CJ et al.; We have reconstituted the Apaf-1-activated apoptosis mechanism in Sacchromyces cerevisiae such that the presence of a constitutively active form of Apaf-1 together with both Caspase-9 and Caspase-3 results in yeast death . This system is a good model of the Apaf-1-activated pathway in mammalian cells: MIHA (XIAP/hILP), and to a lesser degree MIHB (c-IAP1/HIAP2) and MIHC (c-IAP-2/HIAP1) can inhibit caspases in this system, and protection by IAPs (inhibitor of apoptosis) can be abrogated by coexpression of the Drosophila pro-apoptotic proteins HID and GRIM or the mammalian protein DIABLO/Smac . Using this system we demonstrate that unlike DIABLO/Smac, other proteins which interact with mammalian IAPs (TAB-1, Zap-1, Traf-1 and Traf-2) do not act to antagonise IAP- mediated caspase inhibition.

J Lipid Res, 2001 Aug, 42(8), 1282 - 91
Identification of potential substrate-binding sites in yeast and human acyl-CoA sterol acyltransferases by mutagenesis of conserved sequences; Guo Z et al.; In mammals, the esterification of sterols by ACAT plays a critical role in eukaryotic lipid homeostasis . Using the predominant isoform of the yeast ACAT-related enzyme family, Are2p, as a model, we targeted phylogenetically conserved sequences for mutagenesis in order to identify functionally important motifs . Deletion, truncation, and missense mutations implicate a regulatory role for the amino-terminal domain of Are2p and identified two carboxyl-terminal motifs as required for catalytic activity . A serine-to-leucine mutation in the (H/Y)SF motif (residues 338-340), unique to sterol esterification enzymes, nullified the activity and stability of yeast Are2p . Similarly, a tyrosine-to-alanine change in the FYxDWWN motif of Are2p (residues 523-529) produced an enzyme with decreased activity and apparent affinity for oleoyl-CoA . Mutagenesis of the tryptophan residues in this motif completely abolished activity . In human ACAT1, mutagenesis of the corresponding motifs (residues 268-270, and 403-409, respectively) also nullified enzymatic activity . On the basis of their critical roles in enzymatic activity and their sequence conservation, we propose that these motifs mediate sterol and acyl-CoA binding by this class of enzymes.

EMBO J, 2001 Aug 1, 20(15), 4204 - 13
Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein; Vanrobays E et al.; Numerous non-ribosomal trans-acting factors involved in pre-ribosomal RNA processing have been characterized, but none of them is specifically required for the last cytoplasmic steps of 18S rRNA maturation . Here we demonstrate that Rio1p/Rrp10p is such a factor . Previous studies showed that the RIO1 gene is essential for cell viability and conserved from archaebacteria to man . We isolated a RIO1 mutant in a screen for mutations synthetically lethal with a mutant allele of GAR1, an essential gene required for 18S rRNA production and rRNA pseudouridylation . We show that RIO1 encodes a cytoplasmic non-ribosomal protein, and that depletion of Rio1p blocks 18S rRNA production leading to 20S pre-rRNA accumulation . In situ hybridization reveals that, in Rio1p depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect . This strongly suggests that Rio1p is involved in the cytoplasmic cleavage of 20S pre-rRNA at site D, producing mature 18S rRNA . Thus, Rio1p has been renamed Rrp10p (ribosomal RNA processing #10) . Rio1p/Rrp10p is the first non-ribosomal factor characterized specifically required for 20S pre-rRNA processing.

Protein Expr Purif, 2001 Aug, 22(3), 369 - 80
Glycosylated and phosphorylated proteins--expression in yeast and oocytes of Xenopus: prospects and challenges--relevance to expression of thermostable proteins; Li P et al.; Phosphorylation and glycosylation are important posttranslational events in the biosynthesis of proteins . The different degrees of phosphorylation and glycosylation of proteins have been an intriguing phenomenon . Advances in genetic engineering have made it possible to control the degree of glycosylation and phosphorylation of proteins . Structural biology of phosphorylated and glycosylated proteins has been advancing at a much slower pace due to difficulties in using high-resolution NMR studies in solution phase . Major difficulties have arisen from the inherent mobilities of phosphorylated and glycosylated side chains . This paper reviews molecular and structural biology of phosphorylated and glycosylated proteins expressed in eukaryotic expression systems which are especially suited for large-scale production of these proteins . In our laboratory, we have observed that eukaryotic expression systems are particularly suited for the expression of thermostable light-activated proteins, e.g., bacteriorhodopsins and plastocyanins .

Rozhl Chir, 2001 Jun, 80(6), 320 - 3
{Occurrence of yeast infections at a surgical intensive care unit}; Motycka V et al.; The authors evaluate the occurrence of candida infections in the surgical intensive care unit (ICU) in a one-year period . From the group of 1798 patients hospitalized in a surgical ICU there are 50 patients (2.8%) with candida colonisation and even 56 patients (3.1%) with candida infection . The authors evaluate risk factors for development of candida infection, its etiologic agents and the most frequent site of infection . In addition to usual treatment the authors suggest pre-emptive therapy in patients with risk factors . In the one-year period, two patients died of multiorgan failure (MOF) in close connection with candida infection (sepsis) . The mortality of the group with proved candida infection was 3.57%.

Yeast, 2001 Aug, 18(11), 1053 - 68
The yeast Rvs161 and Rvs167 proteins are involved in secretory vesicles targeting the plasma membrane and in cell integrity; Breton AM et al.; The Rvs161 and Rvs167 proteins are known to play a role in actin cytokeleton organization and endocytosis . Moreover, Rvs167p functionally interacts with the myosin Myo2p . Therefore, we explored the involvement of the Rvs proteins in vesicle traffic and in cell integrity . The rvs mutants accumulate late secretory vesicles at sites of membrane and cell wall construction . They are synthetic-lethal with the slt2/mpk1 mutation, which affects the MAP kinase cascade controlled by Pkc1p and is required for cell integrity . The phenotype of the double mutants is close to that described for the pkc1 mutant . Synthetic defects for growth are also observed with mutation in KRE6, a gene coding for a glucan synthase, required for cell wall construction . These data support the idea that the Rvs proteins are involved in the late targeting of vesicles whose cargoes are required for cell wall construction .

Proc Natl Acad Sci U S A, 2001 Jul 31, 98(16), 9104 - 9
A mutant plasma membrane ATPase, Pma1-10, is defective in stability at the yeast cell surface; Gong X et al.; Pma1 is a plasma membrane H(+)-ATPase whose activity at the cell surface is essential for cell viability . In this paper we describe a temperature-sensitive pma1 allele, pma1-10 (with two point mutations in the first cytoplasmic loop of Pma1), in which the newly synthesized mutant protein fails to remain stable at the cell surface at 37 degrees C . Instead, Pma1-10 appears to undergo internalization for vacuolar degradation in a manner dependent on End4, Vps27, Doa4, and Pep4 . By contrast with wild-type Pma1, mutant Pma1-10 is hypophosphorylated and fails to associate with a Triton-insoluble fraction at 37 degrees C, suggesting failure to enter lipid rafts . Kinetic analysis reveals that, at the permissive temperature, newly synthesized Pma1-10 acquires Triton-insolubility before becoming stabilized . We suggest that phosphorylation and lipid raft association may play important roles in maintaining protein stability at the plasma membrane.

Clin Infect Dis, 2001 Sep 1, 33(5), E28 - 30 Epub 2001 Jul 26.
Trichosporon asahii, a non-Candida yeast that caused fatal septic shock in a patient without cancer or neutropenia; Ebright JR et al.; Trichosporon asahii (formerly Trichosporon beigelii) is an emerging fungal pathogen seen particularly in immunologically compromised patients . There are now approximately 100 reported cases of hematogenously disseminated infections with this life-threatening yeast, and no effective antifungal therapy is available . The present case is unusual because the patient did not have neutropenia or evidence of a malignancy.

J Biol Chem, 2001 Aug 3, 276(31), 28751 - 8 Epub 2001 May 24.
The yeast SEC20 gene is required for N- and O-glycosylation in the Golgi . Evidence that impaired glycosylation does not correlate with the secretory defect; Schleip I et al.; The Golgi plays a fundamental role in posttranslational glycosylation, transport, and sorting of proteins . The mechanism of protein transport through the Golgi has been seen as controversial in recent years . During the characterization of N-glycosylation-defective mutants (ngd) previously isolated by this laboratory, it was found that ngd20 is allelic to sec20 . SEC20 was reported to be required for transport from endoplasmic reticulum to Golgi, but its precise function remains to be determined . We show now that SEC20 is also required for N- and O-glycosylation in the Golgi but not in the ER, in a cargo-specific manner, and that the glycosylation defect does not correlate with the secretory defect . By pulse-chase labeling experiments in combination with mannose linkage-specific antibodies, invertase and carboxypeptidase were found to be efficiently secreted to their final compartment, even upon shift to the nonpermissive temperature, while glycosylation in the Golgi was severely impaired . Using microsomal membranes isolated from ngd20, we found that mannosyl transfer from GDP-Man to various mannose-oligosaccharides, indicative for Golgi mannosylation, was strongly diminished . Analysis of the carbohydrate component of chitinase, an exclusively O-mannosylated protein, or of the bulk mannoprotein indicates that O-mannosylation is also reduced . The results demonstrate that in addition to secretion SEC20 also affects glycosylation in the Golgi, presumably because it exerts a more general role in maintenance and function of the Golgi compartments.

Curr Issues Mol Biol, 1999, 1(1-2), 31 - 45
The yeast two-hybrid system: criteria for detecting physiologically significant protein-protein interactions; Golemis EA et al.; In vivo transcription-based assays for protein-protein interactions such as the two-hybrid system are powerful methods for identifying novel proteins based on their physical association with known proteins of biological interest, or for characterizing the degree and nature of interactions between sets of proteins . Because of the complexity inherent in assays taking place within a living organism, a key issue for the effective use of two-hybrid approaches is the ability to determine whether apparent interactions are likely to be physiologically relevant . In this article, a number of the different two-hybrid systems currently available for use will be reviewed . Then, taking as a model one such system, the Interaction Trap, examples of different reagents for use in varying the affinity range of detectable interactions will be outlined . Also set forth are a number of protocols to establish an appropriate set of conditions for either screening a library or analysing the interaction phenotype between protein sets . Finally, a number of general guidelines are suggested for trouble-shooting two-hybrid results, and for eliminating falsely positive interactions.

Nat Struct Biol, 2001 Aug, 8(8), 695 - 700
A histone fold TAF octamer within the yeast TFIID transcriptional coactivator; Selleck W et al.; Gene activity in a eukaryotic cell is regulated by accessory factors to RNA polymerase II, which include the general transcription factor complex TFIID, composed of TBP and TBP-associated factors (TAFs) . Three TAFs that contain histone fold motifs (yTAF17, yTAF60 and yTAF61) are critical for transcriptional regulation in the yeast Saccharomyces cerevisiae and are found in both TFIID and SAGA, a multicomponent histone acetyltransferase transcriptional coactivator . Although these three TAFs were proposed to assemble into a pseudooctamer complex, we find instead that yTAF17, yTAF60 and yTAF61 form a specific TAF octamer complex with a fourth TAF found in TFIID, yTAF48 . We have reconstituted this complex in vitro and established that it is an octamer containing two copies each of the four components . Point mutations within the histone folds disrupt the octamer in vitro, and temperature-sensitive mutations in the histone folds can be specifically suppressed by overexpressing the other TAF octamer components in vivo . Our results indicate that the TAF octamer is similar both in stoichiometry and histone fold interactions to the histone octamer component of chromatin.

J Biol Chem, 2001 Sep 21, 276(38), 35227 - 30 Epub 2001 Jul 25.
Strong growth polarity of yeast prion fiber revealed by single fiber imaging; Inoue Y et al.; Using the yeast prion as a model, we have developed a novel system to observe the growth of individual prion fibers directly . NM fragments, the prion-determining region of the yeast protein Sup35p, were labeled by either red or green fluorescent dyes, and the fiber growth was observed under a fluorescence microscope . When green-Sup35NM was added to the preformed fibers made of red-Sup35NM, 70-97% of green fibers grew unidirectionally, from only one end of individual red fibers, whereas the remainder grew from both ends . Similarly, the majority of red fibers grew from only one end of green fibers when the order of addition was reversed . Sonication of preformed fibers to expose fresh ends did not change the results, excluding a possibility of occasional deformation of one end as the reason of the apparent unidirectional growth . These results indicate the polarity of Sup35 prion fibers and impose constraints on the models of fiber growth.

Mech Dev, 2001 Aug, 106(1-2), 137 - 41
The Drosophila gene twister, an orthologue of the yeast helicase SKI2, is differentially expressed during development; Seago JE et al.; We have identified and characterized a Drosophila orthologue of SKI2, which, in Saccharomyces cerevisiae, is one of the key components in the cytoplasmic 3'-5' decay of mRNA . The Drosophila orthologue (twister, tst), is expressed as two transcripts which differ in the lengths of their 3'-UTRs, with the smaller transcript being particularly abundant in 0-2 h embryos and the larger transcript reaching its highest levels in 6-8 h embryos . TST protein is expressed in two forms which are differentially expressed in adult tissues and throughout development . Differential expression of TST may modulate activity of the mRNA turnover pathway and could have a major impact on the expression of target RNAs.

Curr Issues Mol Biol, 2000 Apr, 2(2), 51 - 9
{URE3} and {PSI} are prions of yeast and evidence for new fungal prions; Masison DC et al.; {URE3} and {PSI} are two non-Mendelian genetic elements discovered over 25 years ago and never assigned to a nucleic acid replicon . Their genetic properties led us to propose that they are prions, altered self-propagating forms of Ure2p and Sup35p, respectively, that cannot properly carry out the normal functions of these proteins . Ure2p is partially protease-resistant in {URE3} strains and Sup35p is aggregated specifically in {PSI} strains supporting this idea . Overexpression of Hsp104 cures {PSI}, as does the absence of this protein, suggesting that the prion change of Sup35p in {PSI} strains is aggregation . Strains of {PSI}, analogous to those described for scrapie, have now been described as well as an in vitro system for {PSI} propagation . Recently, two new potential prions have been described, one in yeast and the other in the filamentous fungus, Podospora.

In Silico Biol, 1998, 1(1), 5 - 11
Homology model building of Hho1p supports its role as a yeast histone H1 protein; Baxevanis AD et al.; Biochemical studies to date have not been able to identify the linker histone H1 protein in the budding yeast Saccharomyces cerevisiae . Database homology searching against the complete yeast genome has identified a gene, HHO1, (or YPL127C, formerly LPI17) which encodes a protein that has two regions that show similarity to the pea histone H1 globular domain . To determine whether Hho1p can assume the shape of an H1 protein, homology model building experiments were performed using the structure of chicken histone H5 globular domain as the basis for comparison . A statistically significant match between each of the two globular domains of Hho1p and the chicken histone H5 structure was obtained, and probability values indicate that there is a less than 1 in 100 chance that such a match would be the result of a random event . These findings support the proposal that Hho1p acts as an "H1 dimer" and could be responsible for the decreased linker DNA length observed between nucleosomal core particles.

J Biol Chem, 2001 Sep 28, 276(39), 36320 - 6 Epub 2001 Jul 24.
Complete reconstitution of human IkappaB kinase (IKK) complex in yeast . Assessment of its stoichiometry and the role of IKKgamma on the complex activity in the absence of stimulation; Miller BS et al.; The IkappaB kinase (IKK) complex, composed of two catalytic subunits (IKKalpha and IKKbeta) and a regulatory subunit (IKKgamma), is the key enzyme in activation of nuclear factor kappaB (NF-kappaB) . To study the mechanism and structure of the complex, we wanted to recombinantly express IKK in a model organism that lacks IKK . For this purpose, we have recombinantly reconstituted all three subunits together in yeast and have found that it is biochemically similar to IKK isolated from human cells . We show that there is one regulatory subunit per kinase subunit . Thus, the core subunit composition of IKKalpha.beta.gamma complex is alpha(1)beta(1)gamma(2), and the core subunit composition of IKKbeta.gamma is beta(2)gamma(2) . The activity of the IKK complex (alpha+beta+gamma or beta+gamma) expressed in yeast (which lack NF-kappaB and IKK) is 4-5-fold higher than an equivalent amount of IKK from nonstimulated HeLa cells . In the absence of IKKgamma, IKKbeta shows a level of activity similar to that of IKK from nonstimulated HeLa cells . Thus, IKKgamma activates IKK complex in the absence of upstream stimuli . Deleting the gamma binding domain of IKKbeta or IKKalpha prevented IKKgamma induced activation of IKK complex in yeast, but it did not prevent the incorporation of IKKgamma into IKK and large complex formation . The possibility of IKK complex being under negative control in mammalian cells is discussed.

Gene, 2001 Jul 11, 272(1-2), 275 - 81
Phylogenetic relationship and mode of evolution of yeast DNA topoisomerase II gene in the pathogenic Candida species; Kato M et al.; We have determined the nucleotide sequences of about 55% of the region of the DNA topoisomerase II gene (approximately 2.3 kb) isolated from the pathogenic Candida species, C . dubliniensis, C . parapsilosis, C . tropicalis, C . krusei, C . kefyr, C . guilliermondii and C . lusitaniae . Evolutionary relationships among nine Candida species including those of C . albicans and C . glabrata were studied based on the DNA topoisomerase II gene . The nucleotide sequences of 2192 bp, which covered two catalytic domains, ATPase and cutting/resealing, were subjected to phylogenetic analysis . Sequence comparison and evolutionary analysis have revealed that the Candida species tested here are not monophyletic, and the two strains within the species C . tropicalis and C . parapsilosis are too diverse to be in a single species . A wide variety of divergence was observed among the functional domains of DNA topoisomerase II, suggesting that Candida species were in different evolutionary paths at least as regarding the DNA topoisomerase II gene . Sequence information and the observation on the species-specific manner of molecular evolution of DNA topoisomerase II in Candida will be applied to develop a method of identification and characterization of the Candida species in both natural and clinical isolates.

Gene, 2001 Jul 11, 272(1-2), 111 - 9
Novel target genes of the yeast regulator Pdr1p: a contribution of the TPO1 gene in resistance to quinidine and other drugs; do Valle Matta MA et al.; The yeast transcription factor Pdr1p regulates the expression of a number of genes, several of which encode ATP-driven transport proteins involved in multiple drug resistance . Among 20 genes containing binding consensus sequences for the transcription factor Pdr1p in their promoter, we studied more particularly the regulation and function of PDR16 (involved in phospholipid synthesis), TPO1 (involved in vacuolar transport of polyamines), YAL061W (homologous to polyol dehydrogenases) and YLR346C (unknown function) . We found that the regulation of these four genes depends on Pdr1p, since promoter activities studied by lacZ fusion analysis and mRNA levels studied by Northern blotting analysis changed upon deletion or hyperactivation by the pdr1-3 mutant of this transcription factor . The drug sensitivity of the strains deleted for these genes revealed that TPO1, a gene previously found to be involved in spermidine resistance and vacuolar polyamine transport, is a determinant of multidrug transporter since it also mediates growth resistance to cycloheximide and quinidine . This resistance pattern overlapped with that of YOR273C, a homolog of TPO1 . These two homologous transporters are thus bona fide members of the phylogenetic subfamily DHA1 (drug/proton antiport TC 2.A.1 . 2) of the major facilitator superfamily . Both YOR273C and TPO1 as well as at least one other determinant involved in the yeast pleiotropic drug resistance network contribute to resistance to a quinoline-containing antimalarial drug.

Mutat Res, 2001 Aug 8, 479(1-2), 225 - 33
The histone deacetylase inhibitor trichostatin A reduces nickel-induced gene silencing in yeast and mammalian cells; Sutherland JE et al.; We have previously reported that nickel (Ni)-silenced expression of the URA3 gene in yeast (Saccharomyces cerevisiae) and gpt transgene in G12 Chinese hamster cells . In both cases, close proximity to a heterochromatic region was required for gene silencing . Yeast exposed to Ni exhibited reduced acetylation of the lysine residues in the N-terminal tail of histone H4 . Ni-induced silencing of the gpt gene in mammalian cells involved hypermethylation of promoter region DNA . Yeast do not employ DNA methylation to silence gene expression . To determine if histone deacetylation participates in Ni-induced silencing of the URA3 and gpt genes, we exposed yeast and G12 hamster cells to the histone deacetylase inhibitor trichostatin A (TSA) prior to and concurrently with Ni . Treatment of yeast cells with 0.2-0.6mM NiCl(2) resulted in reduced expression of the URA3 gene as assessed by increased resistance to 1g/l 5-fluorotic acid (5-FOA) . This effect was lessened when yeast were pre-treated with 50 microg TSA/ml . Similarly, treatment of G12 cells with 5 ng/ml TSA during and after exposure to 0.3 microg Ni(3)S(2)/cm(2) reduced silencing of the gpt gene as gauged by resistance to 10 microg/ml 6-thioguanine (6-TG) . The ability of TSA alone and in combination with the DNA-demethylating agent (5-AzaC) to reactivate the gpt gene in Ni-silenced variants was also assessed . Although treatment with 100 ng/ml TSA for 48 h was partially effective in reactivating the gpt gene, treatment with 5 microM 5-AzaC was more efficacious . The greatest gpt gene reversion frequencies were observed following a sequential 5-AzaC/TSA treatment . Taken all together, our data from mammalian cells suggests that both DNA methylation and histone deacetylation participate in Ni-induced silencing of the gpt gene with DNA hypermethylation playing the more dominant role in maintaining the silenced state.

Curr Biol, 2001 Jul 10, 11(13), 1074 - 8
A yeast homolog of the mammalian mannose 6-phosphate receptors contributes to the sorting of vacuolar hydrolases; Whyte JR et al.; The soluble hydrolases of the mammalian lysosome are marked for delivery to this organelle by the addition of mannose 6-phosphate to their N-glycans . Two related mannose 6-phosphate receptors (MPRs) recognize this feature in the trans Golgi network (TGN) and deliver the hydrolases to the late endosome . In contrast, the vacuolar hydrolases of the yeast Saccharomyces cerevisiae do not contain 6-phosphate monoesters on their N-glycans, and the only sorting receptor so far identified in this organism is the product of the VPS10 gene . This protein also cycles between the Golgi and the late endosome, but is unrelated to the vertebrate MPRs, and recognizes a specific amino acid sequence of carboxypeptidase Y (CPY) . This has led to the notion that although yeast and mammals share many components in Golgi to endosome traffic, they use unrelated receptor systems to sort their abundant soluble hydrolases . In this paper, we report that the yeast genome does in fact contain an uncharacterized ORF (YPR079w) that encodes a membrane protein that is distantly related to mammalian MPRs . The protein encoded by this gene (which we term MRL1) cycles through the late endosome . Moreover, there is a strong synergistic effect on the maturation of proteinases A and B when both MRL1 and VPS10 are deleted, which suggests that Mrl1p may serve as a sorting receptor in the delivery of vacuolar hydrolases.

J Mol Biol, 2001 Aug 3, 311(1), 205 - 16
Yeast cytoplasmic and mitochondrial methionyl-tRNA synthetases: two structural frameworks for identical functions; Senger B et al.; The yeast Saccharomyces cerevisiae possesses two methionyl-tRNA synthetases (MetRS), one in the cytoplasm and the other in mitochondria . The cytoplasmic MetRS has a zinc-finger motif of the type Cys-X(2)-Cys-X(9)-Cys-X(2)-Cys in an insertion domain that divides the nucleotide-binding fold into two halves, whereas no such motif is present in the mitochondrial MetRS . Here, we show that tightly bound zinc atom is present in the cytoplasmic MetRS but not in the mitochondrial MetRS . To test whether the presence of a zinc-binding site is required for cytoplasmic functions of MetRS, we constructed a yeast strain in which cytoplasmic MetRS gene was inactivated and the mitochondrial MetRS gene was expressed in the cytoplasm . Provided that methionine-accepting tRNA is overexpressed, this strain was viable, indicating that mitochondrial MetRS was able to aminoacylate tRNA(Met) in the cytoplasm . Site-directed mutagenesis demonstrated that the zinc domain was required for the stability and consequently for the activity of cytoplasmic MetRS . Mitochondrial MetRS, like cytoplasmic MetRS, supported homocysteine editing in vivo in the yeast cytoplasm . Both MetRSs catalyzed homocysteine editing and aminoacylation of coenzyme A in vitro . Thus, identical synthetic and editing functions can be carried out in different structural frameworks of cytoplasmic and mitochondrial MetRSs .

J Mol Biol, 2001 Aug 3, 311(1), 1 - 7
Domain shuffling as a tool for investigation of protein function: substitution of the cysteine-rich region of Raf kinase and PKC eta for that of yeast Pkc1p; Schmitz HP et al.; With the completion of the sequences of entire genomes, the need for functional characterisation of proteins and their domains is becoming acute . Conserved regions within proteins often share overlapping functions but despite this conservation may fulfil quite different tasks in different species . In this work, we investigated the cysteine-rich motif (C1 domain) of yeast protein kinase C (Pkc1p) as a model to establish a test system for domain function . C1 domains activate kinases through binding of either diacylglycerol and/or phosphatidylserine, as in many members of the protein kinase C (PKC) family, or by binding small GTPases, as in Raf kinase . In contrast to other members of the protein kinase C superfamily, Pkc1p of Saccharomyces cerevisiae is activated via binding of the small G-protein Rho1p to its C1 domain . We developed a system for domain shuffling to establish the function of C1 domains from human Raf kinase and rat PKC eta in yeast . Only the C1 domain from Raf kinase enabled the chimeric enzyme to bind Rho1p when substituted for the native yeast domain . Accordingly, a chimeric Pkc1p carrying the C1 from Raf kinase, but not that from PKC eta, was able to partially complement the phenotypes of a yeast pkc1 deletion mutant . We interpret these data as further evidence that interaction with a small GTPase is the main regulatory function of the C1 domain in yeast .

Arch Biochem Biophys, 2001 Aug 1, 392(1), 48 - 58
Yeast COQ4 encodes a mitochondrial protein required for coenzyme Q synthesis; Belogrudov GI et al.; The COQ4 gene coding for a component of the coenzyme Q biosynthetic pathway in the yeast Saccharomyces cerevisiae was cloned by a functional complementation of a Q-deficient mutant strain . Yeast coq4 mutant strains harboring the COQ4 gene on either single- or multicopy plasmids acquired the ability to grow on media containing a nonfermentable carbon source, synthesize Q(6), and respire . COQ4 encodes a polypeptide containing 335 amino acids with a calculated molecular mass of 38.6 kDa . By Western blot analysis with a specific antiserum, Coq4p was shown to peripherally associate with the matrix face of the mitochondrial inner membrane . The putative mitochondrial-targeting sequence present at the amino-terminus of the polypeptide efficiently imported it to mitochondria in a membrane-potential-dependent manner . Steady-state levels of COQ4 mRNA were increased during growth on glycerol-containing medium, in accordance with a function in Q biosynthesis . The function of Coq4p is unknown, although its presence is required to maintain a steady-state level of Coq7p, another component of the Q biosynthetic pathway . The results presented here, along with those available from literature, are discussed in light of the recently proposed existence of a multisubunit complex functioning in Q biosynthesis (A . Y . Hsu, T . Q . Do, P . T . Lee, and C . F . Clarke, 2000, Biochim . Biophys . Acta 1484, 287-297) .

Planta, 2001 Jun, 213(2), 223 - 30
A putative plant homolog of the yeast beta-1,3-glucan synthase subunit FKS1 from cotton (Gossypium hirsutum L.) fibers; Cui X et al.; A novel plant gene CFL1 was cloned from cotton (Gossypium hirsutum L.) fibers by expressed sequence tag (EST) database searching and 5'-RACE (rapid amplification of cDNA ends) . This gene shows sequence homology with FKS1 which has been identified as the putative catalytic subunit of the yeast beta-1,3-glucan synthase . It encodes a protein (CFL1p) of 219 kDa with 13 deduced transmembrane helices and 2 large hydrophilic domains, one of which is at the N-terminus and the other in the internal region of the polypeptide . CFL1 displays 21% identity and 41% similarity to FKS1 at the amino acid level over its entire length, with 31% identity and 52% similarity for the hydrophilic central domain . Using RNA and protein blot analysis, CFL1 was found to be expressed at higher levels in cotton fibers during primary wall development . CFL1 also had a strong expression in young roots . Using a calmodulin (CaM)-gel overlay assay, the hydrophilic N-terminal domain of CFL1p was shown to bind to CaM, while the hydrophilic central domain did not . A putative CaM-binding domain, 16 amino acids long, was predicted in the hydrophilic N-terminal domain . Moreover, a product-entrapment assay demonstrated that a protein associated with an in vitro-synthesized callose pellet could be labeled by anti-CFL1 antibodies . Our finding suggests that CFL1 is a putative plant homolog of the yeast beta-1,3-glucan synthase subunit FKS1 and could be involved in callose synthesis.

Tissue Cell, 2001 Jun, 33(3), 273 - 9
Histological observations on transovarial transmission of a yeast-like symbiote in Nilaparvata lugens Stal (Homoptera, Delphacidae); Cheng DJ et al.; Transovarial transmission of a yeast-like symbiote (YLS) in the brown planthopper, Nilaparvata lugens Stal, was observed with light and electron microscopy . Light micrographs showed that there was no YLS in testes and spermathecae of the mated females, indicating that sperm is not involved in the transovarial transmission of the symbiote . Both light and electron micrographs showed the processes of YLS transmission from fat body to the oocyte . In females, the symbiotes in mycetocytes moved out of the syncytium, which is formed from a layer of fat body cells, by exocytosis, and released into hemocoel . Then, the free YLS in hemolymph approached to the ovarioles near pedicel and were enclosed by follicle cells . They entered the follicle cells around the primary oocyte by endocytosis at epithelial plug of the ovariole . The YLS aggregated at the posterior end of the mature egg after entering, and finally formed a symbiote ball.

Biochemistry, 2001 Jul 31, 40(30), 8821 - 33
The secondary structure of the inhibited mitochondrial ADP/ATP transporter from yeast analyzed by FTIR spectroscopy; Lorenz VA et al.; Fourier transform infrared spectroscopy has been applied to the study of the carboxyatractyloside-inhibited mitochondrial ADP/ATP transporter from the yeast Saccharomyces cerevisiae, either solubilized in dodecyl maltoside or reconstituted in phosphatidylcholine liposomes . Its secondary structure has been estimated by means of Fourier self-deconvolution followed by curve fit . A Voigt function was used to fit the components of the deconvoluted spectrum, aiming to account for any distortions introduced by deconvolution . For any of the states analyzed, reconstituted or solubilized, in solution or in dry films, 60-70% of the amino acids are found to adopt alpha-helix plus unordered structures, coherent with the six transmembrane spanning helix model . Moreover, the problem of structure preservation on drying was addressed, and several observations pointed to a maintenance of the protein structure in dry films . Comparison of reconstituted and solubilized samples indicated the presence of both lipid-induced changes in the protein (decrease of the beta-sheets and increase of unordered structures) and protein-induced changes in the lipids (strong hydrogen bonding of lipid C=O groups) . To obtain a better discrimination of alpha-helix and unordered structure contributions for the reconstituted form, H/D exchange experiments were performed . Between 35% and 45% of the amino acids were finally assigned to alpha-helix structures, compatible with the existence of five or six transmembrane spanning helices in the transporter . The level of H/D exchange was determined after 15 h of exposure to D(2)O vapor to be 85%, reflecting a high accessibility of the amide hydrogens even for the carboxyatractyloside-inhibited state.

J Bacteriol, 2001 Aug, 183(16), 4761 - 70
Domain interactions in the yeast ATP binding cassette transporter Ycf1p: intragenic suppressor analysis of mutations in the nucleotide binding domains; Falcon-Perez JM et al.; The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification . Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function . Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport . To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis . Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1) . Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains . The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and V(max) of transport . The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity . We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.

Arch Latinoam Nutr, 2000 Dec, 50(4), 361 - 5
{Technology for the whole utilization of brewer's yeast in food industry}; Otero MA et al.; A flexible scheme for the fractionation of brewer's yeast was developed . The procedure allows the production of different products such as: dry yeast flakes, dry yeast pills, yeast-extract based table sauce, yeast protein concentrates and soy-like sauce . The investment required for the processing of one ton per day is below 2 million dollars with an overall profitability higher than 53% . Investment is recovered in 0.75 years . The production of food ingredients from yeast upgrades its biomass about 25 fold . Present procedure is compared with other biomass fractionation processes taking into account the utilization of all technological streams where the process becomes environmentally friendly since effluent production significantly lower than similar technologies.

Mol Cell Biol, 2001 Aug, 21(16), 5541 - 53
Yeast RNA polymerase I enhancer is dispensable for transcription of the chromosomal rRNA gene and cell growth, and its apparent transcription enhancement from ectopic promoters requires Fob1 protein; Wai H et al.; At the end of the 35S rRNA gene within ribosomal DNA (rDNA) repeats in Saccharomyces cerevisiae lies an enhancer that has been shown to greatly stimulate rDNA transcription in ectopic reporter systems . We found, however, that the enhancer is not necessary for normal levels of rRNA synthesis from chromosomal rDNA or for cell growth . Yeast strains which have the entire enhancer from rDNA deleted did not show any defects in growth or rRNA synthesis . We found that the stimulatory activity of the enhancer for ectopic reporters is not observed in cells with disrupted nucleolar structures, suggesting that reporter genes are in general poorly accessible to RNA polymerase I (Pol I) machinery in the nucleolus and that the enhancer improves accessibility . We also found that a fob1 mutation abolishes transcription from the enhancer-dependent rDNA promoter integrated at the HIS4 locus without any effect on transcription from chromosomal rDNA . FOB1 is required for recombination hot spot (HOT1) activity, which also requires the enhancer region, and for recombination within rDNA repeats . We suggest that Fob1 protein stimulates interactions between rDNA repeats through the enhancer region, thus helping ectopic rDNA promoters to recruit the Pol I machinery normally present in the nucleolus.

Mol Cell Biol, 2001 Aug, 21(16), 5359 - 73
SIR functions are required for the toleration of an unrepaired double-strand break in a dispensable yeast chromosome; Bennett CB et al.; Unrepaired DNA double-strand breaks (DSBs) typically result in G(2) arrest . Cell cycle progression can resume following repair of the DSBs or through adaptation to the checkpoint, even if the damage remains unrepaired . We developed a screen for factors in the yeast Saccharomyces cerevisiae that affect checkpoint control and/or viability in response to a single, unrepairable DSB that is induced by HO endonuclease in a dispensable yeast artificial chromosome containing human DNA . SIR2, -3, or -4 mutants exhibit a prolonged, RAD9-dependent G(2) arrest in response to the unrepairable DSB followed by a slow adaptation to the persistent break, leading to division and rearrest in the next G(2) . There are a small number of additional cycles before permanent arrest as microcolonies . Thus, SIR genes, which repress silent mating type gene expression, are required for the adaptation and the prevention of indirect lethality resulting from an unrepairable DSB in nonessential DNA . Rapid adaptation to the G(2) checkpoint and high viability were restored in sir(-) strains containing additional deletions of the silent mating type loci HML and HMR, suggesting that genes under mating type control can reduce the toleration of a single DSB . However, coexpression of MATa1 and MATalpha2 in Sir(+) haploid cells did not lead to lethality from the HO-induced DSB, suggesting that toleration of an unrepaired DSB requires more than one Sir(+) function.

J Biol Chem, 2001 Sep 14, 276(37), 35209 - 16 Epub 2001 Jul 18.
DNA sequence plays a major role in determining nucleosome positions in yeast CUP1 chromatin; Shen CH et al.; The role of DNA sequence in determining nucleosome positions in vivo was investigated by comparing the positions adopted by nucleosomes reconstituted on a yeast plasmid in vitro using purified core histones with those in native chromatin containing the same DNA, described previously . Nucleosomes were reconstituted on a 2.5 kilobase pair DNA sequence containing the yeast TRP1ARS1 plasmid with CUP1 as an insert (TAC-DNA) . Multiple, alternative, overlapping nucleosome positions were mapped on TAC-DNA . For the 58 positioned nucleosomes identified, the relative positioning strengths and the stabilities to salt and temperature were determined . These positions were, with a few exceptions, identical to those observed in native, remodeled TAC chromatin containing an activated CUP1 gene . Only some of these positions are utilized in native, unremodeled chromatin . These observations suggest that DNA sequence is likely to play a very important role in positioning nucleosomes in vivo . We suggest that events occurring in yeast CUP1 chromatin determine which positions are occupied in vivo and when they are occupied.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8355 - 60
Accuracy of lesion bypass by yeast and human DNA polymerase eta; Washington MT et al.; DNA polymerase eta (Pol eta) functions in the error-free bypass of UV-induced DNA lesions, and a defect in Pol eta in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum . Both yeast and human Pol eta replicate through a cis-syn thymine-thymine dimer (TT dimer) by inserting two As opposite the two Ts of the dimer . Pol eta, however, is a low-fidelity enzyme, and it misinserts nucleotides with a frequency of approximately 10(-2) to 10(-3) opposite the two Ts of the TT dimer as well as opposite the undamaged template bases . This low fidelity of nucleotide insertion seems to conflict with the role of Pol eta in the error-free bypass of UV lesions . To resolve this issue, we have examined the ability of human and yeast Pol eta to extend from paired and mispaired primer termini opposite a TT dimer by using steady-state kinetic assays . We find that Pol eta extends from mispaired primer termini on damaged and undamaged DNAs with a frequency of approximately 10(-2) to 10(-3) relative to paired primer termini . Thus, after the incorporation of an incorrect nucleotide, Pol eta would dissociate from the DNA rather than extend from the mispair . The resulting primer-terminal mispair then could be subject to proofreading by a 3'-->5' exonuclease . Replication through a TT dimer by Pol eta then would be more accurate than that predicted from the fidelity of nucleotide incorporation alone.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8255 - 62
Break-induced replication: a review and an example in budding yeast; Kraus E et al.; Break-induced replication (BIR) is a nonreciprocal recombination-dependent replication process that is an effective mechanism to repair a broken chromosome . We review key roles played by BIR in maintaining genome integrity, including restarting DNA replication at broken replication forks and maintaining telomeres in the absence of telomerase . Previous studies suggested that gene targeting does not occur by simple crossings-over between ends of the linearized transforming fragment and the target chromosome, but involves extensive new DNA synthesis resembling BIR . We examined gene targeting in Saccharomyces cerevisiae where only one end of the transformed DNA has homology to chromosomal sequences . Linearized, centromere-containing plasmid DNA with the 5' end of the LEU2 gene at one end was transformed into a strain in which the 5' end of LEU2 was replaced by ADE1, preventing simple homologous gene replacement to become Leu2(+) . Ade1(+) Leu2(+) transformants were recovered in which the entire LEU2 gene and as much as 7 kb of additional sequences were found on the plasmid, joined by microhomologies characteristic of nonhomologous end-joining (NHEJ) . In other experiments, cells were transformed with DNA fragments lacking an ARS and homologous to only 50 bp of ADE2 added to the ends of a URA3 gene . Autonomously replicating circles were recovered, containing URA3 and as much as 8 kb of ADE2-adjacent sequences, including a nearby ARS, copied from chromosomal DNA . Thus, the end of a linearized DNA fragment can initiate new DNA synthesis by BIR in which the newly synthesized DNA is displaced and subsequently forms circles by NHEJ.

Mutat Res, 2001 Aug 9, 486(3), 195 - 206
Interaction of the yeast Pso5/Rad16 and Sgs1 proteins: influences on DNA repair and aging; Saffi J et al.; The interaction trap method was used to isolate putative binding partners of Rad16/Pso5, a protein responsible for repair of silent DNA . One of the interactors found was Sgs1, a DNA helicase influencing the life span of Saccharomyces cerevisiae, with homology to the human BLM, WRN and RECQL4 proteins . Using the same fusion proteins from the two-hybrid screening, we show evidence that both proteins also interact in vitro . We tested isogenic strains, containing mutant alleles of the two genes in single and double mutant combination, for phenotypic similarity . Life span in sgs1Delta single and sgs1Delta rad16Delta double mutants is about 40% of that of WT, and the rad16/pso5Delta single mutant also had its life span reduced to 75% . Sensitivity to different mutagens, whose lesions are poorly repaired in rad16/pso5Delta mutants, was tested in sgs1Delta mutants . The sgs1Delta conferred sensitivity to MMS, H2O2 and was moderately sensitive to UV(254nm) (UVC) and 4-NQO . An epistatic interaction between rad16 and sgs1 mutations after UVC, 4-NQO and H2O2 was observed . Moreover, we found that in a top3 background, functional Sgs1p and Rad16p apparently channel MMS, 4-NQO and H2O2 induced lesions into aberrant DNA repair . Our results demonstrate that Sgs1 is not only involved in genome stability, somatic recombination and aging, but is also implicated, together with Rad16/Pso5, in the repair of specific DNA damage.

Plant Physiol, 2001 Jul, 126(3), 1061 - 71
The wheat cDNA LCT1 generates hypersensitivity to sodium in a salt-sensitive yeast strain; Amtmann A et al.; Salinity affects large areas of agricultural land, and all major crop species are intolerant to high levels of sodium ions . The principal route for Na(+) uptake into plant cells remains to be identified . Non-selective ion channels and high-affinity potassium transporters have emerged as potential pathways for Na(+) entry . A third candidate for Na(+) transport into plant cells is a low-affinity cation transporter represented by the wheat protein LCT1, which is known to be permeable for a wide range of cations when expressed in yeast (Saccharomyces cerevisiae) . To investigate the role of LCT1 in salt tolerance we have used the yeast strain G19, which is disrupted in the genes encoding Na(+) export pumps and as a result displays salt sensitivity comparable with wheat . After transformation with LCT1, G19 cells became hypersensitive to NaCl . We show that LCT1 expression results in a strong decrease of intracellular K(+)/Na(+) ratio in G19 cells due to the combined effect of enhanced Na(+) accumulation and loss of intracellular K(+) . Na(+) uptake through LCT1 was inhibited by K(+) and Ca(2+) at high concentrations and the addition of these ions rescued growth of LCT1-transformed G19 on saline medium . LCT1 was also shown to mediate the uptake of Li(+) and Cs(+) . Expression of two mutant LCT1 cDNAs with N-terminal truncations resulted in decreased Ca(2+) uptake and increased Na(+) tolerance compared with expression of the full-length LCT1 . Our findings strongly suggest that LCT1 represents a molecular link between Ca(2+) and Na(+) uptake into plant cells.

J Exp Bot, 2001 Jul, 52(360), 1581 - 5
Mutagenesis and heterologous expression in yeast of a plant Delta6-fatty acid desaturase; Sayanova O et al.; Membrane-bound microsomal fatty acid desaturases are known to have three conserved histidine boxes, comprising a total of up to eight histidine residues . Recently, a number of deviations from this consensus have been reported, with the substitution of a glutamine for the first histidine residue of the third histidine box being present in the so called 'front end' desaturases . These enzymes are also characterized by the presence of a cytochrome b5 domain at the protein N-terminus . Site-directed mutagenesis has been used to probe the functional importance of a number of amino acid residues which comprise the third histidine box of a 'front end' desaturase, the borage Delta6-fatty acid desaturase . This showed that the variant glutamine in the third histidine box is essential for enzyme activity and that histidine is not able to substitute for this residue.

J Biol Chem, 2001 Nov 2, 276(44), 40502 - 9 Epub 2001 Jul 16.
Free fatty acids activate a vigorous Ca(2+):2H(+) antiport activity in yeast mitochondria; Bradshaw PC et al.; The accumulation and retention of Ca(2+) by yeast mitochondria (Saccharomyces cerevisiae) mediated by ionophore ETH 129 occurs with a variable efficiency in different preparations . Ineffective Ca(2+) transport and a depressed membrane potential occur in parallel, are exacerbated in parallel by exogenous free fatty acids, and are corrected in parallel by the addition of bovine serum albumin . Bovine serum albumin is not required to develop a high membrane potential when either Ca(2+) or ETH 129 are absent, and when both are present membrane potential is restored by the addition of EGTA in a concentration-dependent manner . Respiration and swelling data indicate that the permeability transition pore does not open in yeast mitochondria that are treated with Ca(2+) and ETH 129, whereas fatty acid concentration studies and the inaction of carboxyatractyloside indicate that fatty acid-derived uncoupling does not underlie the other observations . It is concluded that yeast mitochondria contain a previously unrecognized Ca(2+):2H(+) antiporter that is highly active in the presence of free fatty acids and leads to a futile cycle of Ca(2+) accumulation and release when exogenous Ca(2+) and ETH 129 are available . It is also shown that isolated yeast mitochondria degrade their phospholipids at a relatively rapid rate . The activity responsible is also previously unrecognized . It is Ca(2+)-independent, little affected by the presence or absence of a respiratory substrate, and leads to the hydrolysis of ester linkages at both the sn-1 and sn-2 positions of the glycerophospholipids . The products of this activity, through their actions on the antiporter, explain the variable behavior of yeast mitochondria treated with Ca(2+) plus ETH 129.

Biochemistry, 2001 Jul 24, 40(29), 8479 - 86
Phosphorylation of RNA polymerase II CTD fragments results in tight binding to the WW domain from the yeast prolyl isomerase Ess1; Myers JK et al.; The yeast prolyl isomerase, Ess1, has recently been shown to interact via its WW domain with the hyperphosphorylated form of the RNA polymerase II C-terminal domain (CTD) . We have investigated folding of the Ess1 WW domain and its binding to peptides representing the CTD by circular dichroism and fluorescence . Ess1 WW folds and unfolds reversibly, but in the absence of ligand is only marginally stable with a melting temperature of 19 degrees C . The WW domain is stabilized by the addition of anionic ligands, namely, chloride, inorganic phosphate, phosphoserine, and phosphorylated CTD peptides . Dissociation constants were measured to be 70--100 microM for CTD peptides phosphorylated at one serine, and 16--21 microM for peptides with two or more phosphorylated serines . Weaker or no affinity was observed for nonphosphorylated CTD peptides . There is surprisingly little difference in the affinity for peptides phosphorylated at Ser 2 or Ser 5 of the consensus repeat, or for peptides with different patterns of multiple phosphorylation . The binding of Ess1 to phosphorylated CTD peptides is consistent with a model wherein the WW domain positions Ess1 to catalyze isomerization of the many pSer--Pro peptide bonds in the phosphorylated CTD . We suggest that cis/trans isomerization of prolyl peptide bonds plays a crucial role in CTD function during eukaryotic transcription.

Genetics, 2001 Jul, 158(3), 1013 - 25
The budding yeast Msh4 protein functions in chromosome synapsis and the regulation of crossover distribution; Novak JE et al.; The budding yeast MSH4 gene encodes a MutS homolog produced specifically in meiotic cells . Msh4 is not required for meiotic mismatch repair or gene conversion, but it is required for wild-type levels of crossing over . Here, we show that a msh4 null mutation substantially decreases crossover interference . With respect to the defect in interference and the level of crossing over, msh4 is similar to the zip1 mutant, which lacks a structural component of the synaptonemal complex (SC) . Furthermore, epistasis tests indicate that msh4 and zip1 affect the same subset of meiotic crossovers . In the msh4 mutant, SC formation is delayed compared to wild type, and full synapsis is achieved in only about half of all nuclei . The simultaneous defects in synapsis and interference observed in msh4 (and also zip1 and ndj1/tam1) suggest a role for the SC in mediating interference . The Msh4 protein localizes to discrete foci on meiotic chromosomes and colocalizes with Zip2, a protein involved in the initiation of chromosome synapsis . Both Zip2 and Zip1 are required for the normal localization of Msh4 to chromosomes, raising the possibility that the zip1 and zip2 defects in crossing over are indirect, resulting from the failure to localize Msh4 properly.

Cancer Res, 2001 Jul 15, 61(14), 5447 - 52
Multimodality therapy with a replication-conditional herpes simplex virus 1 mutant that expresses yeast cytosine deaminase for intratumoral conversion of 5-fluorocytosine to 5-fluorouracil; Nakamura H et al.; Infection of tumor cells by herpes simplex virus 1 (HSV-1) results in cell destruction and production of progeny virion in a process referred to as viral oncolysis . In this study, an HSV-1 mutant (HSV1yCD) was engineered such that the viral ribonucleotide reductase gene is disrupted by sequences encoding yeast cytosine deaminase, which efficiently metabolizes the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) . HSV1yCD-infected cells convert 5-FC to 5-FU, which enhances cytotoxicity without significantly reducing viral replication and oncolysis . Oncolysis by a replicating HSV-1 mutant combined with therapeutic transgene delivery represents a new paradigm; HSV1yCD-infected cells are destroyed by viral replication, and uninfected cells are subjected to bystander killing from both progeny virion and extracellular diffusion of 5-FU . In contrast, HSV1yCD-mediated bioactivation of another prodrug, ganciclovir, impairs viral replication . HSV1yCD administered into the portal venous system replicates preferentially in liver metastases rather than normal liver . The anti-neoplastic activity of HSV1yCD combined with systemic 5-FC administration is greater than that achieved with HSV-1 replication alone . Combination oncolysis and prodrug bioactivation leads to significant prolongation of survival in mice with diffuse liver metastases.

Curr Opin Cell Biol, 2001 Aug, 13(4), 389 - 98
A genomic view of yeast membrane transporters; Van Belle D et al.; The yeast membrane transporters play crucial roles in functions as diverse as nutrient uptake, drug resistance, salt tolerance, control of cell volume, efflux of undesirable metabolites and sensing of extracellular nutrients . A significant fraction of the many transporters inventoried after sequencing of the yeast genome has been characterised by classical experimental approaches . Post-genomic analysis has allowed a more extensive characterisation of transporter categories less tractable by genetics, for instance of transporters of intracellular membranes or transporters encoded by multigene families and displaying overlapping substrate specificities . A complete view of the role of membrane transporters in the metabolism of yeast may not be far off.

Mol Microbiol, 2001 Jul, 41(1), 155 - 66
The negative regulator Opi1 of phospholipid biosynthesis in yeast contacts the pleiotropic repressor Sin3 and the transcriptional activator Ino2; Wagner C et al.; Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are transcriptionally co-regulated by ICRE (inositol/choline-responsive element) promoter motifs . Gene activation by an ICRE is mediated by binding of the Ino2/Ino4 transcription factor, whereas repression in the presence of high concentrations of inositol and choline (IC) requires an intact Opi1 repressor . However, the mechanism of specific repression and the functional interplay among these regulators remained unclear from previous work . Using in vivo as well as in vitro interaction assays, we show binding of the pleiotropic repressor Sin3 to the pathway-specific regulator Opi1 . The paired amphipathic helix 1 (PAH1) within Sin3 and OSID (Opi1-Sin3 interaction domain) in the N-terminus of Opi1 were mapped as contact sites . The regulatory significance of the Opi1-Sin3 interaction was shown by the obvious deregulation of an ICRE-dependent reporter gene in a sin3 mutant . Opi1 also interacts with a newly identified functional domain of the transcriptional activator Ino2 (RID, repressor interaction domain) . These results define the molecular composition of the transcription complex mediating control of ICRE-dependent genes and allow a hypothesis on the flow of regulatory information in response to phospholipid precursors.

Eur J Biochem, 2001 Jul, 268(14), 4054 - 62
CYP11A1 stimulates the hydroxylase activity of CYP11B1 in mitochondria of recombinant yeast in vivo and in vitro; Cauet G et al.; In mammals, hydrocortisone synthesis from cholesterol is catalyzed by a set of five specialized enzymes, four of them belonging to the superfamily of cytochrome P-450 monooxygenases . A recombinant yeast expression system was recently developed for the CYP11B1 (P45011beta) enzyme, which performs the 11beta hydroxylation of steroids such as 11-deoxycortisol into hydrocortisone, one of the three mitochondrial cytochrome P-450 proteins involved in steroidogenesis in mammals . This heterologous system was used to test the potential interaction between CYP11B1 and CYP11A1 (P450scc), the mitochondrial cytochrome P-450 enzyme responsible for the side chain cleaving of cholesterol . Recombinant CYP11B1 and CYP11A1 were targeted to Saccharomyces cerevisiae mitochondria using the yeast cytochrome oxidase subunit 6 mitochondrial presequence fused to the mature form of the two proteins . In yeast, the presence of CYP11A1 appears to improve 11beta hydroxylase activity of CYP11B1 in vivo and in vitro . Fractionation experiments indicate the presence of the two proteins in the same membrane fractions, i.e . inner membrane and contact sites of mitochondria . Thus, yeast mitochondria provide interesting insights to study some molecular and cellular aspects of mammalian steroid synthesis . In particular, recombinant yeast should permit a better understanding of the mechanism permitting the synthesis of steroids (sex steroids, mineralocorticoids and glucocorticoids) with a minimal set of enzymes at physiological level, thus avoiding disease states.

Biochem Biophys Res Commun, 2001 Jul 20, 285(3), 696 - 701
A novel domain of the yeast heat shock factor that regulates its activation function; Sakurai H et al.; Heat shock factor Hsf1 of the yeast Saccharomyces cerevisiae binds to the heat shock element (HSE) of a subset of genes and activates their transcription in response to various environmental stresses . Hsf1 protein contains discrete domains respectively involved in DNA-binding, trimerization, transcription activation, and transcription repression . Here we have identified a novel domain rich in basic amino acids at the extreme C-terminus of Hsf1 . Deletion or point mutations of the C-terminal basic region caused an inefficient heat shock response of genes containing noncanonical HSEs such as CUP1 and HSP26 . The basic region is also essential for oxidative stress-inducible transcription of CUP1 by Hsf1 . By contrast, it was dispensable for heat induction through the canonical HSE . We suggest that the basic region is a modulator involved in regulation of the Hsf1-mediated activation depending on the architecture of its binding site .

RNA, 2001 Jul, 7(7), 1013 - 23
ATP-dependent interaction of yeast U5 snRNA loop 1 with the 5' splice site; Alvi RK et al.; Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together . In yeast, the U5 snRNA loop 1 interacts with the 5' exon before the first step of splicing and with the 5' and 3' exons before the second step . In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 5' and 3' exons for ligation during the second step . It is critical, therefore, that loop 1 contacts the 5' exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA . At present it is not known how U5 loop 1 is positioned on the 5' exon prior to the first step of splicing . To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing . We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 5' splice site that includes the invariant GU . These interactions are dependent on functional U2 and U6 snRNAs . Our results support a model where U5 snRNA loop 1 interacts with the 5' exon in two steps during its targeting to the 5' splice site.

Fukuoka Igaku Zasshi, 2001 May, 92(5), 158 - 66
{Assessment of hydroxylated metabolites of polychlorinated biphenyls and polychlorinated dibenzofurans as potential estrogens by yeast two-hybrid system}; Kuroki H et al.; The estrogenic activities of several hydroxylated metabolites of PCBs and PCDFs were investigated by yeast two-hybrid assay based on the ligand-dependent interaction of estrogen receptor with coactivator . For the hydroxylated PCBs, the order of estrogenic potency was 4-OH-2',4',6'-triCB > 4-OH-4'-monoCB, 4-OH-biphenyl . These compounds were evaluated as 10(3) to 10(4) less potent than 17 beta-estradiol based on the concentrations of test compounds showing 10% activity of 10(-7) M 17 beta-estradiol . 2-OH-3',4,4'-triCB, 4-OH-2',3,4'-triCB and 3-OH-/4-OH-2,2',5,5'-tetraCB, the metabolites of 2,2',5,5'-tetraCB were inactive as estrogens at the highest concentrations used in this study (10(-5) M) . Also 4-OH-3,3',4',5-tetraCB, the metabolite of 3,3',4,4'-tetraCB was inactive as estrogen, indicating that this hydroxylated metabolite did not take part in the estrogenic activity of 3,3',4,4'-tetraCB . OH group at 4-position of biphenyl was necessary for the expression of estrogenicity, but one or two chloro-substitution adjacent to OH group inhibited the activity . For the hydroxylated PCDFs, 8-OH-2-monoCDF, 7-OH-3,4-diCDF, 8-OH-3,4-diCDF, 8-OH-3,4,6-triCDF and 3,8-(OH)2-2-monoCDF exhibited estrogenic activity . The estrogenic activity of 3,8-(OH)2-2-monoCDF was comparable to those of 4-OH-2',4',6'-triCB and 4-nonylphenol (mixture of compounds with branched sidechain) . The order of activity was 3,8-(OH)2-monoCDF > 8-OH-3,4-diCDF, 7-OH-3,4-diCDF > 8-OH-2-monoCDF, 8-OH-3,4,6-triCDF . These compounds were evaluated as 2.5 x 10(3) to 3 x 10(4) less potent than 17 beta-estradiol . On the other hand, no estrogenic activity was observed for 2-OH-dibenzofuran, 3-OH-2,8-diCDF, 6-OH-3,4-diCDF and 9-OH-3,4-diCDF at concentrations as high as 10(-4) M . Substitution of OH group at 2(8)- or 3(7)-position of dibenzofuran and no chloro-substitution adjacent to OH group was required for the estrogenic activity.

Nucleic Acids Res, 2001 Jul 15, 29(14), 3123 - 30
Accessibility of DNA polymerases to repair synthesis during nucleotide excision repair in yeast cell-free extracts; Wu X et al.; Nucleotide excision repair (NER) removes a variety of DNA lesions . Using a yeast cell-free repair system, we have analyzed the repair synthesis step of NER . NER was proficient in yeast mutant cell-free extracts lacking DNA polymerases (Pol) beta, zeta or eta . Base excision repair was also proficient without Polbeta . Repair synthesis of NER was not affected by thermal inactivation of the temperature-sensitive mutant Polalpha (pol1-17), but was reduced after thermal inactivation of the temperature-sensitive mutant Poldelta (pol3-1) or Polvarepsilon (pol2-18) . Residual repair synthesis was observed in pol3-1 and pol2-18 mutant extracts, suggesting a repair deficiency rather than a complete repair defect . Deficient NER in pol3-1 and pol2-18 mutant extracts was specifically complemented by purified yeast Poldelta and Polvarepsilon, respectively . Deleting the polymerase catalytic domain of Polvarepsilon (pol2-16) also led to a deficient repair synthesis during NER, which was complemented by purified yeast Polvarepsilon, but not by purified yeast Poleta . These results suggest that efficient repair synthesis of yeast NER requires both Poldelta and Polvarepsilon in vitro, and that the low fidelity Poleta is not accessible to repair synthesis during NER.

Biochim Biophys Acta, 2001 Jul 9, 1548(1), 47 - 56
Studies on the function of yeast protein disulfide isomerase in renaturation of proteins; Katiyar S et al.; Renaturation of two enzymes lacking disulfide bonds, citrate synthase (CS), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and another protein containing disulfide bonds, lysozyme (LZM), were studied in order to dissect the possible chaperone function from the isomerase function of yeast protein disulfide isomerase (PDI) . Our findings suggest no independent chaperone activity of yeast PDI with respect to the two enzymes lacking disulfide bonds, GAPDH and CS, since neither of these enzymes required PDI for renaturation . In contrast, a high level of renaturation of LZM was observed in the presence of PDI . Renaturation of LZM involved formation and rearrangement of disulfide bonds . Additional studies using LZM as a substrate were done to examine the role of cysteine residues in the two active sites of PDI . Studies with a series of cysteine to serine mutants and truncation mutants of yeast PDI revealed that the two active sites of PDI were not equal in activity . An intramolecular disulfide bond in at least one active site of PDI was required for the oxidation of reduced LZM . The first cysteine in each active site was necessary for disulfide bond rearrangement, i.e., isomerization, in LZM, while the second cysteine was not.

Plant Cell, 2001 Jul, 13(7), 1609 - 24
A gene related to yeast HOS2 histone deacetylase affects extracellular depolymerase expression and virulence in a plant pathogenic fungus; Baidyaroy D et al.; A gene, HDC1, related to the Saccharomyces cerevisiae histone deacetylase (HDAC) gene HOS2, was isolated from the filamentous fungus Cochliobolus carbonum, a pathogen of maize that makes the HDAC inhibitor HC-toxin . Engineered mutants of HDC1 had smaller and less septate conidia and exhibited an approximately 50% reduction in total HDAC activity . Mutants were strongly reduced in virulence as a result of reduced penetration efficiency . Growth of hdc1 mutants in vitro was normal on glucose, slightly decreased on sucrose, and reduced by 30 to 73% on other simple and complex carbohydrates . Extracellular depolymerase activities and expression of the corresponding genes were downregulated in hdc1 mutant strains . Except for altered conidial morphology, the phenotypes of hdc1 mutants were similar to those of C . carbonum strains mutated in ccSNF1 encoding a protein kinase necessary for expression of glucose-repressed genes . These results show that HDC1 has multiple functions in a filamentous fungus and is required for full virulence of C . carbonum on maize.

J Biol Chem, 2001 Sep 7, 276(36), 34221 - 6 Epub 2001 Jul 11.
Aft2p, a novel iron-regulated transcription activator that modulates, with Aft1p, intracellular iron use and resistance to oxidative stress in yeast; Blaiseau PL et al.; The yeast, Saccharomyces cerevisiae, contains a transcription activator, Aft1p, that regulates the transcription of the high affinity iron transport system genes . This report describes the properties of Aft2p, a protein 39% homologous to Aft1p . Aft2p was found to activate transcription . Overproduction of Aft2p activates the transcription of the AFT1 target gene FET3 . The double aft1aft2 mutant was unable to grow in iron-deprived conditions . Because a fet3 mutant does not show this deficiency, the defect is not solely caused by mis-regulation of iron transport but also involves defective iron use by the cells . The aft1 cells were unable to grow in aerobic conditions on plates containing raffinose as the sole carbon source . The inability to grow on raffinose is not caused by the cell iron content being too low to sustain respiratory metabolism, because the oxygen consumption of aft1 mutants showed that their respiratory activity is 2-fold higher than that of controls . The double aft1aft2 mutant also has many phenotypes related to oxidative stress such as H(2)O(2) hypersensitivity, oxygen-dependent copper toxicity, and oxygen-dependent methionine auxotrophy, which are suppressed in anaerobiosis . These results suggest that Aft2p and Aft1p have overlapping roles in the control of iron-regulated pathway(s) connected to oxidative stress resistance in yeast.

Yeast, 2001 Jul, 18(10), 943 - 51
Yeast Lrg1p acts as a specialized RhoGAP regulating 1,3-beta-glucan synthesis; Watanabe D et al.; Selection of an extragenic suppressor of fks1-1154 Deltafks2, mutations in the catalytic subunits of yeast 1,3-beta-glucan synthase (GS) conferring temperature-sensitivity, led to the LRG1 gene, which was originally identified as a LIM-RhoGAP homologous gene . Mutations in the LRG1 gene restore impaired 1,3-beta-glucan synthesis in the fks1-1154 Deltafks2 mutant as well as that in rho1-2, a temperature-sensitive mutant of Rho-type GTPase that functions as a regulatory subunit of GS . Two-hybrid analyses of Lrg1p, which contains a sequence conserved among Rho GTPase-activating proteins (GAPs), revealed its specific interactions with the active form of Rho1p . Among eight potential yeast RhoGAPs, Lrg1p is the only member that negatively regulates GS activity: mutations in the rest of GAPs, including bem2, Deltabem3, Deltasac7, Deltabag7, Deltarga1, Deltarga2 and Deltargd1, do not suppress impairment of 1,3-beta-glucan synthesis . Analyses of Mpk1p phosphorylation revealed the inability of Lrg1p to regulate the Pkc1p-MAP kinase cascade, a distinct Rho1p-regulating signalling pathway known to be affected by the GAPs, Bem2p and Sac7p . Thus, different groups of Rho1p GAPs control the activity of different Rho1p-effector proteins .

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8720 - 5 Epub 2001 Jul 10.
Histone deacetylase-dependent transcriptional repression by pRB in yeast occurs independently of interaction through the LXCXE binding cleft; Kennedy BK et al.; We have developed a yeast model system to address transcriptional repression by the retinoblastoma protein (pRB) . When fused to the DNA-binding domain of Gal4p (DB-pRB), pRB can repress transcription of reporter genes containing Gal4p binding sites; the histone deacetylase activity encoded by yeast RPD3 is required for DB-pRB repression . Mutation of the LXCXE binding cleft in pRB, a region reported to be required for histone deacetylase recruitment, does not interfere with pRB-mediated repression . From these findings based on yeast experiments, we surmise that the small pocket region of pRB must contain an additional domain that confers histone deacetylase-dependent transcriptional repression . This hypothesis was verified by experiments examining pRB-dependent histone deacetylase association in mammalian cells . In addition to RPD3, repression by pRB in yeast requires MSI1, an ortholog of RbAp48, but not SIN3 or SAP30 . By comparing the genetic requirements of DB-pRB repression in yeast to those of other DB-repressor fusions, we can suggest a mechanism by which pRB recruits histone deacetylase activity.

J Biol Chem, 2001 Sep 21, 276(38), 35328 - 33 Epub 2001 Jul 09.
A yeast four-hybrid system identifies Cdk-activating kinase as a regulator of the XPD helicase, a subunit of transcription factor IIH; Sandrock B et al.; To understand the role of the various components of TFIIH, a DNA repair/transcription factor, a yeast four-hybrid system was designed . When the ternary Cdk-activating kinase (CAK) complex composed of Cdk7, cyclin H, and MAT1 was used as bait, the xeroderma pigmentosum (XP) D helicase of transcription factor IIH (TFIIH), among other proteins, was identified as an interacting partner . Deletion mutant analyses demonstrated that the coiled-coil and the hydrophobic domains of MAT1 interlink the CAK complex directly with the N-terminal domain of XPD . Using immunoprecipitates from cells coinfected with baculoviruses, we further validated the bridging function of XPD, which anchors CAK to the core TFIIH . In addition we show that upon interaction with MAT1, CAK inhibits the helicase activity of XPD . This inhibition is overcome upon binding to p44, a subunit of the core TFIIH . It is not surprising that under these conditions some XPD mutations affect interactions not only with p44, but also with MAT1, thus preventing either the CAK inhibitory function within CAK.XPD and/or the role of CAK within TFIIH and, consequently, explaining the variety of the XP phenotypes.

J Biol Chem, 2001 Sep 14, 276(37), 34537 - 44 Epub 2001 Jul 09.
Genetic interactions with the yeast Q-SNARE VTI1 reveal novel functions for the R-SNARE YKT6; Dilcher M et al.; SNARE proteins are required for fusion of transport vesicles with target membranes . Previously, we found that the yeast Q-SNARE Vti1p is involved in transport to the cis-Golgi, to the prevacuole/late endosome, and to the vacuole . Here we identified a previously uncharacterized gene, VTS1, and the R-SNARE YKT6 both as multicopy and as low copy suppressors of the growth and vacuolar transport defect in vti1-2 cells . Ykt6p was known to function in retrograde traffic to the cis-Golgi and homotypic vacuolar fusion . We found that VTI1 and YKT6 also interacted in traffic to the prevacuole and vacuole, indicating that these SNARE complexes contain Ykt6p, Vti1p, plus Pep12p and Ykt6p, Vti1p, Vam3p, plus Vam7p, respectively . As Ykt6p was required for several transport steps, R-SNAREs cannot be the sole determinants of specificity . To study the role of the 0 layer in the SNARE motif, we introduced the mutations vti1-Q158R and ykt6-R165Q . SNARE complexes to which Ykt6p contributed a fourth glutamine residue in the 0 layer were nonfunctional, suggesting an essential function for arginine in the 0 layer of these complexes . vti1-Q158R cells had severe defects in several transport steps, indicating that the second arginine in the 0 layer interfered with function.

FEBS Lett, 2001 Jul 6, 500(3), 177 - 82
AtBS14a and AtBS14b, two Bet1/Sft1-like SNAREs from Arabidopsis thaliana that complement mutations in the yeast SFT1 gene; Tai WC et al.; SNAREs are membrane-associated proteins that play a central role in vesicle targeting and intra-cellular membrane fusion reactions in eukaryotic cells . Here we describe the identification of AtBS14a and AtBS14b, putative SNAREs from Arabidopsis thaliana that share 60% amino acid sequence identity . Both AtBS14a and BS14b are dosage suppressors of the temperature-sensitive growth defect in sft1-1 cells and over-expression of either AtBS14a or AtBS14b can support the growth of sft1Delta cells but not bet1Delta cells . These data together with structure-function and biochemical studies presented herein suggest that AtBS14a and AtBS14b share properties that are consistent with them being members of the Bet1/Sft1 SNARE protein family.

FEBS Lett, 2001 Jul 6, 500(3), 149 - 52
Exploring the active site of yeast xylose reductase by site-directed mutagenesis of sequence motifs characteristic of two dehydrogenase/reductase family types; Klimacek M et al.; Starting from a common tyrosine, yeast xylose reductases (XRs) contain two conserved sequence motifs corresponding to the catalytic signatures of single-domain reductases/epimerases/dehydrogenases (Tyr(n)-(X)3-Lys(n+4)) and aldo/keto reductases (AKRs) (Tyr(n)-(X)28-Lys(n+29)) . Tyr(51), Lys(55) and Lys(80) of XR from Candida tenuis were replaced by site-directed mutagenesis . The purified Tyr(51)--> Phe and Lys(80)-->Ala mutants showed turnover numbers and catalytic efficiencies for NADH-dependent reduction of D-xylose between 2500- and 5000-fold below wild-type levels, suggesting a catalytic role of both residues . Replacing Lys(55) by Asn, a substitution found in other AKRs, did not detectably affect binding of coenzymes, and enzymatic catalysis to carbonyl/alcohol interconversion . The contribution of Tyr(51) to rate enhancement of aldehyde reduction conforms with expectations for the general acid catalyst of the enzymatic reaction.

J Bacteriol, 2001 Aug, 183(15), 4477 - 83
Eukaryotic translation initiation factor 4E-dependent translation is not essential for survival of starved yeast cells; Paz I et al.; The eukaryotic translation initiation factor 4E (eIF4E) interacts with the mRNA 5' cap structure (m(7)GpppX) and is essential for the appropriate translation of the vast majority of eukaryotic mRNAs . Most studies of the yeast Saccharomyces cerevisiae CDC33 gene product, eIF4E, have been carried out with logarithmically growing cells, and little is known about its role in starved, nonproliferating cells that enter the stationary phase (SP) . It has previously been found that the rate of translation in SP cells is more than 2 orders of magnitude lower than it is in dividing yeast cells . Here we show that this low rate of translation is essential for maintaining the viability of starved yeast cells that enter SP . Specifically, starved cells whose eIF4A is inactive or treated with cycloheximide rapidly lose viability . Moreover, after heat inactivation of the cdc33 temperature-sensitive product, the synthesis of most proteins is abolished and only a small group of proteins is still produced . Unexpectedly, starved cdc33 mutant cells whose eIF4E is inactive and which therefore fail to synthesize the bulk of their proteins remain viable for long periods of time, indistinguishable from their isogenic wild-type counterparts . Taken together, our results indicate that eIF4E-independent translation is necessary and sufficient for survival of yeast cells during long periods of starvation.

Mol Microbiol, 2001 Jun, 40(6), 1357 - 69
The elimination of the yeast {PSI+} prion by guanidine hydrochloride is the result of Hsp104 inactivation; Ferreira PC et al.; In the yeast Saccharomyces cerevisiae, Sup35p (eRF3), a subunit of the translation termination complex, can take up a prion-like, self-propagating conformation giving rise to the non-Mendelian {PSI+} determinant . The replication of {PSI+} prion seeds can be readily blocked by growth in the presence of low concentrations of guanidine hydrochloride (GdnHCl), leading to the generation of prion-free {psi-} cells . Here, we provide evidence that GdnHCl blocks seed replication in vivo by inactivation of the molecular chaperone Hsp104 . Although growth in the presence of GdnHCl causes a modest increase in HSP104 expression (20-90%), this is not sufficient to explain prion curing . Rather, we show that GdnHCl inhibits two different Hsp104-dependent cellular processes, namely the acquisition of thermotolerance and the refolding of thermally denatured luciferase . The inhibitory effects of GdnHCl protein refolding are partially suppressed by elevating the endogenous cellular levels of Hsp104 using a constitutive promoter . The kinetics of GdnHCl-induced {PSI+} curing could be mimicked by co-expression of an ATPase-negative dominant HSP104 mutant in an otherwise wild-type {PSI+} strain . We suggest that GdnHCl inactivates the ATPase activity of Hsp104, leading to a block in the replication of {PSI+} seeds.

Mol Microbiol, 2001 Jun, 40(6), 1345 - 56
Roles of trehalose phosphate synthase in yeast glycogen metabolism and sporulation; De Silva-Udawatta MN et al.; Trehalose is a major storage carbohydrate in budding yeast, Saccharomyces cerevisiae . Alterations in trehalose synthesis affect carbon source-dependent growth, accumulation of glycogen and sporulation . Trehalose is synthesized by trehalose phosphate synthase (TPS), which is a complex of at least four proteins . In this work, we show that the Tps1p subunit protein catalyses trehalose phosphate synthesis in the absence of other TPS components . The tps1-H223Y allele (glc6-1) that causes a semidominant decrease in glycogen accumulation exhibits greater enzyme activity than wild-type TPS1 because, unlike the wild-type enzyme, TPS activity in tps1-H223Y cells is not inhibited by phosphate . Poor sporulation in tps1 null diploids is caused by reduced expression of meiotic inducers encoded by IME1, IME2 and MCK1 . Furthermore, high-copy MCK1 or heterozygous hxk2 mutations can suppress the tps1 sporulation trait . These results suggest that the trehalose-6-phosphate inhibition of hexokinase activity is required for full induction of MCK1 in sporulating yeast cells.

J Chromatogr A, 2001 Jun 15, 919(2), 339 - 47
Gravitational field-flow fractionation for the characterisation of active dry wine yeast; Sanz R et al.; Gravitational field-flow fractionation (GrFFF) is applied to the fractionation of active dry wine yeast . An experimental approach to the analysis of the effects that field variation by changing mobile phase composition and flow-rate have on the fractionation process of standard particles (polystyrene) was first developed to further obtain effective fractionation of wine yeast by GrFFF . Scanning electron microscopy and Coulter counter particle size measurements were used to monitor the fractionation extent and capabilities of GrFFF to describe the distribution of yeast cells populations.

J Biol Chem, 2001 Aug 31, 276(35), 32905 - 16 Epub 2001 Jul 05.
Regulation of an IMP dehydrogenase gene and its overexpression in drug-sensitive transcription elongation mutants of yeast; Shaw RJ et al.; IMP dehydrogenase is a rate-limiting enzyme involved in the synthesis of GTP . In mammalian cells it is regulated with respect to growth rate and is the target of numerous therapeutic agents . Mutations in the RNA polymerase II elongation machinery render yeast sensitive to inhibitors of IMP dehydrogenase and defective in inducing transcription of one of the IMP dehydrogenase-encoding genes, IMD2 . Here we show that loss of IMD2, but not IMD1, IMD3, or IMD4, conferred upon yeast the same drug sensitivity found in elongation mutants . We tested whether the drug sensitivity of elongation mutants is due to their inability to induce IMD2 by providing them with exogenous copies of the gene . In some elongation mutants, overexpression reversed drug sensitivity and a transcriptional defect . Overexpression in mutants with a more severe phenotype partially suppressed drug sensitivity but was inconsequential in reversing a defect in transcription . These findings suggest that the drug sensitivity of elongation mutants is largely but not solely attributable to defects in the ability to induce IMD2, because transcription is compromised even when IMD2 mRNA levels are adequate . We describe two DNA sequence elements in the promoter of the gene that regulate it . We also found that IMD2 mRNA abundance is coupled to cell growth rate . These findings show that yeast possess a conserved system that gauges nucleotide pools and cell growth rate and responds through a uniquely regulated member of the IMD gene family.

Mol Cell Biol, 2001 Aug, 21(15), 5109 - 21
The TFIID components human TAF(II)140 and Drosophila BIP2 (TAF(II)155) are novel metazoan homologues of yeast TAF(II)47 containing a histone fold and a PHD finger; Gangloff YG et al.; The RNA polymerase II transcription factor TFIID comprises the TATA binding protein (TBP) and a set of TBP-associated factors (TAF(II)s) . TFIID has been extensively characterized for yeast, Drosophila, and humans, demonstrating a high degree of conservation of both the amino acid sequences of the constituent TAF(II)s and overall molecular organization . In recent years, it has been assumed that all the metazoan TAF(II)s have been identified, yet no metazoan homologues of yeast TAF(II)47 (yTAF(II)47) and yTAF(II)65 are known . Both of these yTAF(II)s contain a histone fold domain (HFD) which selectively heterodimerizes with that of yTAF(II)25 . We have cloned a novel mouse protein, TAF(II)140, containing an HFD and a plant homeodomain (PHD) finger, which we demonstrated by immunoprecipitation to be a mammalian TFIID component . TAF(II)140 shows extensive sequence similarity to Drosophila BIP2 (dBIP2) (dTAF(II)155), which we also show to be a component of Drosophila TFIID . These proteins are metazoan homologues of yTAF(II)47 as their HFDs selectively heterodimerize with dTAF(II)24 and human TAF(II)30, metazoan homologues of yTAF(II)25 . We further show that yTAF(II)65 shares two domains with the Drosophila Prodos protein, a recently described potential dTAF(II) . These conserved domains are critical for yTAF(II)65 function in vivo . Our results therefore identify metazoan homologues of yTAF(II)47 and yTAF(II)65.

Mol Cell Biol, 2001 Aug, 21(15), 4949 - 59
Cdc5 interacts with the Wee1 kinase in budding yeast; Bartholomew CR et al.; Development of a multicellular organism requires that mitosis and morphogenesis be coordinated . These processes must also be synchronized during the growth of unicellular organisms . In the yeast Saccharomyces cerevisiae, mitosis is dependent on the prior growth of a daughter cell in the form of a bud . Overexpression of wild-type Polo-like kinase Cdc5 or a catalytically inactive form resulted in the formation of multinucleate cells in budding yeast . Immunofluorescence analysis of these multinulceate cells showed that mitosis and bud formation were no longer linked . Others have shown that Swe1 is required for coupling mitosis to bud formation during a perturbed cell cycle . When the normal pathway of bud formation is perturbed, Swe1 functions to delay mitosis through negative regulation of Clb/Cdk . In cells lacking Swe1, multinucleate cells are formed in response to delays in bud formation . Affinity purification, two-hybrid analysis, and mutant characterization results suggested that Cdc5 and Swe1 interact . From these results, we conclude that multinucleate formation in response to Cdc5 overexpression is linked to titration of Swe1 function . These results also suggest that Cdc5 may be a negative regulator of Swe1.

Mol Cell Biol, 2001 Aug, 21(15), 4847 - 55
New model for the yeast RNA polymerase I transcription cycle; Aprikian P et al.; Using an immobilized template assay, we observed two steps in assembly of the yeast RNA polymerase I (Pol I) preinitiation complex: stable binding of upstream activating factor (UAF) followed by recruitment of Pol I-Rrn3p and core factor (CF) . Pol I is required for stable association of CF with the promoter and can be recruited in the absence of Rrn3p . Upon transcription initiation, Pol I-Rrn3p and CF dissociate from the promoter while UAF remains behind . These findings support a novel model in which the Pol I basal machinery cycles on and off the promoter with each round of transcription . This model accounts for previous observations that rRNA synthesis may be controlled by regulating both promoter accessibility and polymerase activity.

J Biol Chem, 2001 Aug 31, 276(35), 32696 - 703 Epub 2001 Jul 03.
Altering the DNA-binding specificity of the yeast Matalpha 2 homeodomain protein; Mathias JR et al.; Homeodomain proteins are a highly conserved class of DNA-binding proteins that are found in virtually every eukaryotic organism . The conserved mechanism that these proteins use to bind DNA suggests that there may be at least a partial DNA recognition code for this class of proteins . To test this idea, we have investigated the sequence-specific requirements for DNA binding and repression by the yeast alpha2 homeodomain protein in association with its cofactors, Mcm1 and Mata1 . We have determined the contribution for each residue in the alpha2 homeodomain that contacts the DNA in the co-crystal structures of the protein . We have also engineered mutants in the alpha2 homeodomain to alter the DNA-binding specificity of the protein . Although we were unable to change the specificity of alpha2 by making substitutions at residues 47, 54, and 55, we were able to alter the DNA-binding specificity by making substitutions at residue 50 in the homeodomain . Since other homeodomain proteins show similar changes in specificity with substitutions at residue 50, this suggests that there is at least a partial DNA recognition code at this position.

Plant Mol Biol, 2001 May, 46(1), 57 - 65
Phenotypic changes in Arabidopsis caused by expression of a yeast vacuolar Ca2+/H+ antiporter; Hirschi KD et al.; In plants, cytosolic Ca2+ levels are tightly regulated, and changes in cytosolic Ca2+ have been implicated in converting numerous signals into adapted responses . Vacuolar ion transporters are thought to be key mediators of cytosolic Ca2+ concentrations . In an attempt to interpret the role of vacuolar Ca2+ transport in plant processes, we have expressed the yeast vacuolar Ca2+/H+ antiporter, VCX1, in Arabidopsis and tobacco . This transporter localizes to the plant vacuolar membrane . VCX1-expressing Arabidopsis plants displayed increased sensitivity to sodium and other ions . These ion sensitivities could be suppressed by addition of calcium to the media . VCX1-expressing plants demonstrated increased tonoplast-enriched Ca2+/H+ antiport activity as well as increased Ca2+ accumulation . These results suggest that VCX1 expression in Arabidopsis could be a valuable tool with which to experimentally dissect the role of Ca2+ transport around the plant vacuole.

Folia Parasitol (Praha), 2001, 48(2), 149 - 53
Mortality caused by experimental infection with the yeast Candida haemulonii in the adults of Ornithodoros moubata (Acarina: Argasidae); Loosova G et al.; A relatively high rate of mortality among engorged females of Ornithodoros moubata (Murray, 1877) was observed in our laboratory colony . The general aim of the study was to identify the causative agent responsible for this mortality . The diagnostic tests were performed by Yeast Identification Service (CBS-Delft, Netherlands) and the pathogen was identified as the yeast Candida haemulonii (van Uden et Kolipinski, 1962) Meyer et Yarrovi, 1978 . The artificial infection study was performed by intrahaemocoelic inoculation of yeast suspension, resulting in a mortality of 37% . The maximum mortality of ticks infected per os by contaminated blood meal was 13% . Re-isolated yeast cells from haemolymph of dead and paralysed ticks were apparently identical with primary yeast cells, without loosing reproductive abilities . An occasional formation of elongated chains of yeast cells (pseudomycelium) was recorded . The majority of ticks infected in both experiments mentioned above survived and displayed no evident symptoms of the infection . The presence of yeast cells in the haemolymph of surviving ticks was not detected . The in vitro phagocytosis assay performed with FITC-labelled yeast cells showed that about 4% of tick haemocytes were phagocytically active against the pathogenic yeast cells . Thus phagocytosis seems to be a potent defence reaction against spreading and multiplying of the yeast C . haemulonii within the tick haemocoel.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2001 Jun, 15(2), 125 - 7
{Study on interactions between TPO molecules tested with yeast two-hybrid system}; Wang Z et al.; OBJECTIVE: To explore the interactions between TPO molecules and their locations . METHODS: Full-length TPO, TPO(N) and TPO(C) were inserted respectively into the plasmids of the yeast two-hybrid system . Six different recombinant plasmids were generated . Every 2 recombinant plasmids were transformed into the yeast cell SFY526 . The possible interactions between TPOs were then tested by the bioactivity of beta-galactosidase . RESULTS: There are interactions between full-length TPO themselves TPO(N) themselves and TPO with TPO(N), but not between TPO(C)themselves, TPO with TPO(C) and TPO(N) with TPO(C) . CONCLUSION: Naturally, the molecular interaction domain of TPO may exist in the N terminus but not in the C terminus.

J Virol, 2001 Aug, 75(15), 6769 - 75
Frameshift signal transplantation and the unambiguous analysis of mutations in the yeast retrotransposon Ty1 Gag-Pol overlap region; Lawler JF Jr et al.; The yeast retrotransposon Ty1 encodes a 7-nucleotide RNA sequence that directs a programmed, +1 ribosomal frameshifting event required for Gag-Pol translation and retrotransposition . We report mutations that block frameshifting, which can be suppressed in cis by "transplanting" the frameshift signal to a position upstream of its native location . These "frameshift transplant" mutants transpose with only a modest decrease in efficiency, suggesting that the location of the frameshift signal in a functional Ty1 element may vary . The genomic architecture of Ty1 is such that Gag, Ty1 PR (PR), and the Gag-derived p4 peptide share a common sequence . The functional independence of the movement of the frameshift signal to a new location within the Ty1 element is used to unambiguously attribute the effect of mutations deleterious to transposition in this region of overlapping coding sequences to effects on the Ty1 (PR) . This work defines the amino terminus of the Ty1 PR and introduces a new technique for studying viral genome organization.

Structure (Camb), 2001 Jun, 9(6), 539 - 46
The crystal structure of yeast thiamin pyrophosphokinase; Baker LJ et al.; BACKGROUND: Thiamin pyrophosphokinase (TPK) catalyzes the transfer of a pyrophosphate group from ATP to vitamin B1 (thiamin) to form the coenzyme thiamin pyrophosphate (TPP) . Thus, TPK is important for the formation of a coenzyme required for central metabolic functions . TPK has no sequence homologs in the PDB and functions by an unknown mechanism . The TPK structure has been determined as a significant step toward elucidating its catalytic action . RESULTS: The crystal structure of Saccharomyces cerevisiae TPK complexed with thiamin has been determined at 1.8 A resolution . TPK is a homodimer, and each subunit consists of two domains . One domain resembles a Rossman fold with four alpha helices on each side of a 6 strand parallel beta sheet . The other domain has one 4 strand and one 6 strand antiparallel beta sheet, which form a flattened sandwich structure containing a jelly-roll topology . The active site is located in a cleft at the dimer interface and is formed from residues from domains of both subunits . The TPK dimer contains two compound active sites at the subunit interface . CONCLUSIONS: The structure of TPK with one substrate bound identifies the location of the thiamin binding site and probable catalytic residues . The structure also suggests a likely binding site for ATP . These findings are further supported by TPK sequence homologies . Although possessing no significant sequence homology with other pyrophospokinases, thiamin pyrophosphokinase may operate by a mechanism of pyrophosphoryl transfer similar to those described for pyrophosphokinases functioning in nucleotide biosynthesis.

J Biotechnol, 2001 Jul 12, 88(3), 223 - 37
Estimation of kinetic parameters in a structured yeast model using regularisation; Lei F et al.; In this work, a procedure for estimating kinetic parameters in biochemically structured models was developed . The approach is applicable when the structure of a kinetic model has been set up and the kinetic parameters should be estimated . The procedure consists of five steps . First, initial values were found in or calculated from literature . Hereafter using sensitivity analysis the most sensitive parameters were identified . In the third step physiological knowledge was combined with the parameter sensitivities to manually tune the most sensitive parameters . In step four, a global optimisation routine was applied for simultaneous estimation of the most sensitive parameters identified during the sensitivity analysis . Regularisation was included in the simultaneous estimation to reduce the effect of insensitive parameters . Finally, confidence intervals for the estimated parameters were calculated . This parameter estimation approach was demonstrated on a biochemically structured yeast model containing 11 reactions and 37 kinetic constants as a case study.

Biochemistry, 2001 Jul 10, 40(27), 8101 - 8
Yeast ribosomal protein deletion mutants possess altered peptidyltransferase activity and different sensitivity to cycloheximide; Dresios J et al.; The major function of the ribosome is its ability to catalyze formation of peptide bonds, and it is carried out by the ribosomal peptidyltransferase . Recent evidence suggests that the catalyst of peptide bond formation is the 23S rRNA of the large ribosomal subunit . We have developed an in vitro system for the determination of peptidyltransferase activity in yeast ribosomes . Using this system, a kinetic analysis of a model reaction for peptidyltransferase is described with Ac-Phe-tRNA as the peptidyl donor and puromycin as the acceptor . The Ac-Phe-tRNA-poly(U)-80S ribosome complex (complex C) was isolated and then reacted with excess puromycin to give Ac-Phe-puromycin . This reaction (puromycin reaction) followed first-order kinetics . At saturating concentrations of puromycin, the first-order rate constant (k(3)) is identical to the catalytic rate constant (k(cat)) of peptidyltransferase . This k(cat) from wild-type yeast strains was equal to 2.18 min(-1) at 30 degrees C . We now present for the first time kinetic evidence that yeast ribosomes lacking a particular protein of the 60S subunit may possess significantly altered peptide bond-forming ability . The k(cat) of peptidyltransferase from mutants lacking ribosomal protein L24 was decreased 3-fold to 0.69 min(-1), whereas the k(cat) from mutants lacking L39 was slightly increased to 3.05 min(-1) and that from mutants lacking both proteins was 1.07 min(-1) . These results suggest that the presence of ribosomal proteins L24 and, to a lesser extent, L39 is required for exhibition of the normal catalytic activity of the ribosome . Finally, the L24 or L39 mutants did not affect the rate or the extent of the translocation phase of protein synthesis . However, the absence of L24 caused increased resistance to cycloheximide, a translocation inhibitor . Translocation of Ac-Phe-tRNA from the A- to P-site was inhibited by 50% at 1.4 microM cycloheximide for the L24 mutant compared to 0.7 microM for the wild type.

Nat Cell Biol, 2001 Jul, 3(7), 687 - 90
Yeast Eps15-like endocytic protein, Pan1p, activates the Arp2/3 complex; Duncan MC et al.; Longstanding evidence supports a role for actin in endocytosis; an intact actin cytoskeleton is required for endocytosis in yeast, and drugs that inhibit actin polymerization inhibit endocytosis in both yeast and mammalian cells . The yeast Arp2/3 complex is required for the internalization step of endocytosis . In addition, some early endocytic events in mammalian cells are associated with the formation of actin tails similar to those generated by activated Arp2/3 complex . However, until now no Arp2/3 complex activator has been identified among proteins known to mediate early steps in endocytosis . Here we show that the yeast endocytic protein Pan1p binds to and activates the Arp2/3 complex . Genetic interactions between PAN1 and mutants of Arp2/3 subunits, or of the Arp2/3 activator LAS17, provide evidence for this activity in vivo . We suggest that Pan1p forms the core of an endocytic complex and physically couples actin polymerization nucleated by the Arp2/3 complex to the endocytic machinery, thus providing the forces necessary for endocytosis.

Nucleic Acids Res, 2001 Jul 1, 29(13), E63 - 3
yMGV: a database for visualization and data mining of published genome-wide yeast expression data; Marc P et al.; The yeast Microarray Global Viewer (yMGV) is an on-line database providing a synthetic view of the transcriptional expression profiles of Saccharomyces cerevisiae genes in most of the published expression datasets . yMGV displays a one-screen graphical representation of gene expression variations for each published genome-wide experiment, allowing quick retrieval of experimental conditions affecting expression of this gene . yMGV also provides tools to isolate groups of genes sharing similar transcription profiles in a defined subset of experiments . Additionally, yMGV furnishes a set of statistical tools for critical assessment of published data . We therefore believe that yMGV is an efficient tool that affords a quick and comprehensive overview of microarray data and generates new gene classifications . As of 20 March 2001 the yMGV database contains 6 000 000 measurements, representing genome-wide expression comparisons of 932 experiments from 39 microarray publications . The yMGV interface is available at http://transcriptome.ens.fr/ymgv/.

Nucleic Acids Res, 2001 Jul 1, 29(13), 2875 - 83
Translesion synthesis by yeast DNA polymerase zeta from templates containing lesions of ultraviolet radiation and acetylaminofluorene; Guo D et al.; In the yeast Saccharomyces cerevisiae, DNA polymerase zeta (Polzeta) is required in a major lesion bypass pathway . To help understand the role of Polzeta in lesion bypass, we have performed in vitro biochemical analyses of this polymerase in response to several DNA lesions . Purified yeast Polzeta performed limited translesion synthesis opposite a template TT (6-4) photoproduct, incorporating A or T with similar efficiencies (and less frequently G) opposite the 3' T, and predominantly A opposite the 5' T . Purified yeast Polzeta predominantly incorporated a G opposite an acetylaminofluorene (AAF)-adducted guanine . The lesion, however, significantly inhibited subsequent extension . Furthermore, yeast Polzeta catalyzed extension DNA synthesis from primers annealed opposite the AAF-guanine and the 3' T of the TT (6-4) photoproduct with varying efficiencies . Extension synthesis was more efficient when A or C was opposite the AAF-guanine, and when G was opposite the 3' T of the TT (6-4) photoproduct . In contrast, the 3' T of a cis-syn TT dimer completely blocked purified yeast Polzeta, whereas the 5' T was readily bypassed . These results support the following dual-function model of Polzeta . First, Polzeta catalyzes nucleotide incorporation opposite AAF-guanine and TT (6-4) photoproduct with a limited efficiency . Secondly, more efficient bypass of these lesions may require nucleotide incorporation by other DNA polymerases followed by extension DNA synthesis by Polzeta.

J Biol Chem, 2001 Sep 14, 276(37), 34776 - 83 Epub 2001 Jun 29.
ATP utilization by yeast replication factor C . II . Multiple stepwise ATP binding events are required to load proliferating cell nuclear antigen onto primed DNA; Gomes XV et al.; Binding of adenosine (3-thiotriphosphate) (ATPgammaS), a nonhydrolyzable analog of ATP, to replication factor C with a N-terminal truncation (Delta2-273) of the Rfc1 subunit (RFC) was studied by filter binding . RFC alone bound 1.8 ATPgammaS molecules . However, when either PCNA or primer-template DNA were also present 2.6 or 2.7 ATPgammaS molecules, respectively, were bound . When both PCNA and DNA were present 3.6 ATPgammaS molecules were bound per RFC . Order of addition experiments using surface plasmon resonance indicate that RFC forms an ATP-mediated binary complex with PCNA prior to formation of a ternary DNA.PCNA.RFC complex . An ATP-mediated complex between RFC and DNA was not competent for binding PCNA, and the RFC.DNA complex dissociated with hydrolysis of ATP . Based on these experiments a model is proposed in which: (i) RFC binds two ATPs (RFC.ATP(2)); (ii) this complex binds PCNA (PCNA.RFC.ATP(2)), which goes through a conformational change to reveal a binding site for one additional ATP (PCNA.RFC.ATP(3)); (iii) this complex can bind DNA to yield DNA.PCNA.RFC.ATP(3); (iv) a conformational change in the latter complex reveals a fourth binding site for ATP; and (v) the DNA.PCNA.RFC.ATP(4) complex is finally competent for completion of PCNA loading and release of RFC upon hydrolysis of ATP.

J Biol Chem, 2001 Sep 14, 276(37), 34768 - 75 Epub 2001 Jun 29.
ATP utilization by yeast replication factor C . I . ATP-mediated interaction with DNA and with proliferating cell nuclear antigen; Gomes XV et al.; Eukaryotic replication factor C is the heteropentameric complex that loads the replication clamp proliferating cell nuclear antigen (PCNA) onto primed DNA . In this study we used a derivative, designated RFC, with a N-terminal truncation of the Rfc1 subunit removing a DNA-binding domain not required for clamp loading . Interactions of yeast RFC with PCNA and DNA were studied by surface plasmon resonance . Binding of RFC to PCNA was stimulated by either adenosine (3-thiotriphosphate) (ATPgammaS) or ATP . RFC bound only to primer-template DNA coated with the single-stranded DNA-binding protein RPA if ATPgammaS was also present . Binding occurred without dissociation of RPA . ATP did not stimulate binding of RFC to DNA, suggesting that hydrolysis of ATP dissociated DNA-bound RFC . However, when RFC and PCNA together were flowed across the DNA chip in the presence of ATP, a signal was observed suggesting loading of PCNA by RFC . With ATPgammaS present instead of ATP, long-lived response signals were observed indicative of loading complexes arrested on the DNA . A primer with a 3' single-stranded extension also allowed loading of PCNA; yet turnover of the reaction intermediates was dramatically slowed down . Filter binding experiments and analysis of proteins bound to DNA-magnetic beads confirmed the conclusions drawn from the surface plasmon resonance studies.

EMBO J, 2001 Jul 2, 20(13), 3389 - 401
Fission yeast ch-TOG/XMAP215 homologue Alp14 connects mitotic spindles with the kinetochore and is a component of the Mad2-dependent spindle checkpoint; Garcia MA et al.; The TOG/XMAP215-related proteins play a role in microtubule dynamics at its plus end . Fission yeast Alp14, a newly identified TOG/XMAP215 family protein, is essential for proper chromosome segregation in concert with a second homologue Dis1 . We show that the alp14 mutant fails to progress towards normal bipolar spindle formation . Intriguingly, Alp14 itself is a component of the Mad2-dependent spindle checkpoint cascade, as upon addition of microtubule-destabilizing drugs the alp14 mutant is incapable of maintaining high H1 kinase activity, which results in securin destruction and premature chromosome separation . Live imaging of Alp14-green fluorescent protein shows that during mitosis, Alp14 is associated with the peripheral region of the kinetochores as well as with the spindle poles . This is supported by ChIP (chromatin immunoprecipitation) and overlapping localization with the kinetochore marker Mis6 . An intact spindle is required for Alp14 localization to the kinetochore periphery, but not to the poles . These results indicate that the TOG/XMAP215 family may play a central role as a bridge between the kinetochores and the plus end of pole to chromosome microtubules.

J Biochem (Tokyo), 2001 Jul, 130(1), 157 - 65
Analysis of tyrosine phosphorylation-dependent protein-protein interactions in TrkB-mediated intracellular signaling using modified yeast two-hybrid system; Yamada M et al.; Activated receptor tyrosine kinases induce a large number of tyrosine phosphorylation-dependent protein-protein interactions through which they mediate their various ligand-exerted functions including regulation of proliferation, differentiation and survival . TrkB receptor tyrosine kinase activated by binding of brain-derived neurotrophic factor (BDNF) also stimulates various protein interactions in a tyrosine phosphorylation-dependent manner in neuronal cells . To examine tyrosine phosphorylation-dependent interactions stimulated by active TrkB, we developed a modified yeast two-hybrid system, which we call the yeast two-and-a-half-hybrid system . In this system, yeast was engineered to express a tyrosine kinase domain of TrkB as an effector, in addition to two fusion proteins with GAL4 DNA-binding and GAL4 activation domains as bait and prey proteins, respectively . Using this system with Shp2 as the bait, we demonstrated that Shp2 interacts directly with BIT/SHPS-1 (also called SIRP) and Grb2 depending on tyrosine phosphorylation mediated by TrkB . Furthermore, we screened an adult human brain cDNA library with the yeast two-and-a-half-hybrid system in order to identify other Shp2-binding proteins in TrkB-stimulated tyrosine phosphorylation signaling . We found that fibroblast growth factor receptor substrate 2beta (FRS2beta), also called SNT2, interacts with Shp2 dependently on TrkB-mediated tyrosine phosphorylation of FRS2beta/SNT2 . Therefore, we show that the two-and-a-half-hybrid system is a powerful tool for studying tyrosine phosphorylation-dependent protein-protein interactions in intracellular signaling pathways stimulated by TrkB receptor tyrosine kinase.

Evolution Int J Org Evolution, 2001 May, 55(5), 909 - 17
Mutational meltdown in laboratory yeast populations; Zeyl C et al.; In small or repeatedly bottlenecked populations, mutations are expected to accumulate by genetic drift, causing fitness declines . In mutational meltdown models, such fitness declines further reduce population size, thus accelerating additional mutation accumulation and leading to extinction . Because the rate of mutation accumulation is determined partly by the mutation rate, the risk and rate of meltdown are predicted to increase with increasing mutation rate . We established 12 replicate populations of Saccharomyces cerevisiae from each of two isogenic strains whose genomewide mutation rates differ by approximately two orders of magnitude . Each population was transferred daily by a fixed dilution that resulted in an effective population size near 250 . Fitness declines that reduce growth rates were expected to reduce the numbers of cells transferred after dilution, thus reducing population size and leading to mutational meltdown . Through 175 daily transfers and approximately 2900 generations, two extinctions occurred, both in populations with elevated mutation rates . For one of these populations there is direct evidence that extinction resulted from mutational meltdown: Extinction immediately followed a major fitness decline, and it recurred consistently in replicate populations reestablished from a sample frozen after this fitness decline, but not in populations founded from a predecline sample . Wild-type populations showed no trend to decrease in size and, on average, they increased in fitness.

Genetica, 2000, 109(3), 275 - 82
Dnop56, a Drosophila gene homologous to the yeast nucleolar NOP56 gene; Garcia-Planells J et al.; Small nucleolar RNAs (snoRNAs) are trans-acting factors involved in maturation of rRNA and have been classified into Box C/D and Box H/ACA families . Most of the snoRNAs occur as ribonucleoprotein complexes with snoRNA-associated proteins (snoRNPs) . All Box C/D snoRNAs in yeast form complexes with Nop1p, Nop56p and Nop58p . Similarly, it has been reported that Box H/ACA-containing snoRNAs form complexes with yeast Gar1p . Nop56p and Nop58p homologs have been described in several species . Here we report the isolation and molecular characterization of the Dnop56 genes from D . melanogaster and D . subobscura which show a very similar structure . Drosophila Nop56p proteins contain lysine-rich regions at their carboxy-terminus, and show a high degree of similarity to other Nop56p proteins from different organisms . Phylogenetic relationships among these proteins and other snoRNPs have been established.

Oncogene, 2001 Jun 14, 20(27), 3533 - 40
Amifostine (WR2721) restores transcriptional activity of specific p53 mutant proteins in a yeast functional assay; Maurici D et al.; Many p53 mutants found in human cancer have an altered ability to bind DNA and transactivate gene expression . Re-expression of functional p53 in cells in which the endogenous TP53 gene is inactivated has been demonstrated to restore a non-tumorigenic phenotype . Pharmacological modulation of p53 mutant conformation may therefore represent a mechanism to reactivate p53 function and consequently improve response to radio- and chemotherapy . We have recently reported that the radio- and chemoprotector Amifostine (WR2721, Ethyol) activates wild-type p53 in cultured mammalian cells . In the present study, we have used a yeast functional assay to investigate the effect of WR2721 on the transcriptional activity of p53 . WR2721 restored this activity in a temperature-sensitive mutant V272M (valine to methionine at codon 272) expressed at the non-permissive temperature and it also partially restored the transcriptional activity of several other conformationally flexible p53 mutants . The results indicate that the yeast functional assay may be used to identify compounds that modulate p53 activity, with potential therapeutic implications.

Biophys Chem, 2001 Jul 2, 91(2), 183 - 90
Influence of the oligomeric state of yeast hexokinase isozymes on inactivation and unfolding by urea; Morales FC et al.; The effect of the association-dissociation equilibrium on the urea-induced inactivation and unfolding of the yeast hexokinase isoforms, PI and PII, showed that these enzymes are more stable as dimers . For the monomeric PII, the inactivation and unfolding processes occurred in parallel . However, inactivation precedes the unfolding of monomeric PI or dimeric PI and PII . The unfolding transitions are biphasic for PI indicating stable intermediates, whereas for the PII isoform the unfolding occurs in a single step . Our data suggests that although PI and PII present a 78% identity in their amino acid sequences, they probably have distinct inactivation and unfolding by urea behavior.

Yeast, 2001 Jun 30, 18(9), 881 - 8
Current awareness on yeast; Goodwin TJ et al.; In order to keep subscribers up-to-date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly-published material on yeasts . Each bibliography is divided into 10 sections . 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology . Within each section, articles are listed in alphabetical order with respect to author . If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted . (4 weeks journals - search completed 4th Apr . 2001)

Yeast, 2001 Jun 30, 18(9), 841 - 8
At acidic pH, the GPA2-cAMP pathway is necessary to counteract the ORD1-mediated repression of the hypoxic SRP1/TIR1 yeast gene; Bourdineaud JP; The hypoxic SRP1/TIR1 gene encodes a stress-response cell wall mannoprotein and this gene is downregulated at acidic pH . The stress-responsive HOG pathway is necessary to maintain hypoxic TIR1 expression, but only at acidic pH . However, unlike known HOG pathway-dependent genes, TIR1 is under positive cAMP control and this effect is mediated by GPA2 but not by RAS2 . Genetic analysis showed that ord1 mutation was epistatic to the gpa2 mutation, thereby indicating that Gpa2p is needed to counteract the Ord1 factor, which is involved in the repression of hypoxic TIR1 expression, while the HOG pathway appears to be independent from Ord1 repression . In addition, an increased ORD1 gene expression was observed in the Deltagpa2 mutant cells, meaning that GPA2 maintains a low basal level of ORD1 transcripts . Thus, cAMP allows partial relief of the TIR1 repression exerted by Ord1p . However, this is contradicted at acidic pH by the HOG pathway requirement because Hog1p is activated under stress conditions when the cAMP cellular content is low . The opposite effects of the GPA2-cAMP and HOG pathways are likely to explain the diminished hypoxic expression of TIR1 at acidic pH .

J Cell Biol, 2001 Jun 25, 153(7), 1465 - 78
Nup2p dynamically associates with the distal regions of the yeast nuclear pore complex; Dilworth DJ et al.; Nucleocytoplasmic transport is mediated by the interplay between soluble transport factors and nucleoporins resident within the nuclear pore complex (NPC) . Understanding this process demands knowledge of components of both the soluble and stationary phases and the interface between them . Here, we provide evidence that Nup2p, previously considered to be a typical yeast nucleoporin that binds import- and export-bound karyopherins, dynamically associates with the NPC in a Ran-facilitated manner . When bound to the NPC, Nup2p associates with regions corresponding to the nuclear basket and cytoplasmic fibrils . On the nucleoplasmic face, where the Ran--GTP levels are predicted to be high, Nup2p binds to Nup60p . Deletion of NUP60 renders Nup2p nucleoplasmic and compromises Nup2p-mediated recycling of Kap60p/Srp1p . Depletion of Ran--GTP by metabolic poisoning, disruption of the Ran cycle, or in vitro by cell lysis, results in a shift of Nup2p from the nucleoplasm to the cytoplasmic face of the NPC . This mobility of Nup2p was also detected using heterokaryons where, unlike nucleoporins, Nup2p was observed to move from one nucleus to the other . Together, our data support a model in which Nup2p movement facilitates the transition between the import and export phases of nucleocytoplasmic transport.

J Biol Chem, 2001 Aug 17, 276(33), 31004 - 15 Epub 2001 Jun 25.
New roles for the Snp1 and Exo84 proteins in yeast pre-mRNA splicing; Awasthi S et al.; The mammalian 70K protein, a component of the U1 small nuclear ribonucleoprotein involved in pre-mRNA splicing, interacts with a number of proteins important for regulating constitutive and alternative splicing . Similar proteins that interact with the yeast homolog of the 70K protein, Snp1p, have yet to be identified . We used the two-hybrid system to find four U1-Snp1 associating (Usa) proteins . Two of these proteins physically associate with Snp1p as assayed by coimmunoprecipitation . One is Prp8p, a known, essential spliceosomal component . This interaction suggests some novel functions for Snp1p and the U1 small nuclear ribonucleoprotein late in spliceosome development . The other, Exo84p, is a conserved subunit of the exocyst, an eight-protein complex functioning in secretion . We show here that Exo84p is also involved in pre-mRNA splicing . A temperature-sensitive exo84 mutation caused increased ratios of pre-mRNA to mRNA for the Rpl30 and actin transcripts in cells incubated at the non-permissive temperature . The mutation also led to a defect in splicing and prespliceosome formation in vitro; an indication that Exo84p has a direct role in splicing . The results elucidate a surprising link between splicing and secretion.

FEMS Microbiol Lett, 2001 Jun 25, 200(2), 185 - 9
Intracellular pH-dependent efflux of the fluorescent probe pyranine in the yeast Yarrowia lipolytica; Aguedo M et al.; 8-Hydroxypyrene-1,3,6-trisulfonic acid (pyranine) can be used as a vital intracellular pH (pH(i)) indicator . In the yeast Yarrowia lipolytica, a partial efflux of the probe was detected by using the pH-independent wavelength of 415 nm . A simplified correction of the fluorescent signals was applied, enabling to show for this species a good near-neutral pH(i) maintenance capacity in a pH 3.9 medium . Octanoic acid, which is known to have toxic effects on yeast, decreased the pH(i) and increased the 260-nm-absorbing compounds leakage . However, this acid inhibited the fluorescent probe efflux linearly with its concentration suggesting a pH(i)-dependent efflux of pyranine from cells.

Bioelectromagnetics, 2001 Jul, 22(5), 345 - 50
Effects of 50 Hz magnetic field on cell cycle kinetics and the colony forming ability of budding yeast exposed to ultraviolet radiation; Markkanen A et al.; To investigate the effects of extremely low frequency magnetic fields on ultraviolet radiation (UV) exposed budding yeast, haploid yeast (Saccharomyces cerevisiae) cells of the strain SEy2101a were exposed to 50 Hz sine wave magnetic field (MF) of 120 microT with simultaneous exposure to UV radiation . Most of the UV energy was in the UVB range (280-320 nm) . The biologically weighted (CIE action spectrum) dose level for the UV radiation was 175 J/m2 . We examined whether 50 Hz MF affected the ability of UV irradiated yeast cells to form colonies (Colony Forming Units, CFUs) . In addition, the effect of coexposure on cell cycle kinetics was investigated . Although the significant effect of MF on the cell cycle phases of UV exposed yeast cells was seen only at one time point, the overall results showed that MF exposure may influence the cell cycle kinetics at the first cycle after UV irradiation . The effect of our particular MF exposure on the colony forming ability of the UV irradiated yeast cells was statistically significant 420 min after UV irradiation . Moreover, at 240, 360, and 420 min after UV irradiation, there were fewer CFUs in every experiment in (UV+MF) exposed populations than in only UV exposed yeast populations . These results could indicate that MF exposure in conjunction with UV may have some effects on yeast cell survival or growth .

Oncogene, 2001 Jun 7, 20(26), 3409 - 19
Novel human p53 mutations that are toxic to yeast can enhance transactivation of specific promoters and reactivate tumor p53 mutants; Inga A et al.; Since highly expressed human p53 can inhibit human and yeast cell growth, we predicted that p53 mutants could be generated with increased growth inhibition of the yeast Saccharomyces cerevisiae and that these would be useful for characterizing p53 functions and tumor p53 mutants . A random mutagenesis screen led to the isolation of mutations in the DNA binding domain that result in p53 being lethal even at moderate expression levels in yeast . Three independent mutants had an alanine change at the evolutionary invariant V122 in the L1 loop . The other toxic mutations affected codons 277 (C277R, C277W) and 279 (G279R) . This latter amino acid change was also reported in tumors, while all the other mutations are novel . A recently developed rheostatable GALI promoter system that provides graded increases in expression of p53 was used to examine the transactivation function of the toxic mutations when expression was greatly reduced and cells were viable . At low expression levels the toxic mutants lacked transactivation from a 3xRGC responsive element (RE) . Surprisingly some exhibited enhanced transactivation with p21 and bax REs . The V122A mutant was able to re-activate transactivation of various p53 tumor mutants and retained growth inhibition when co-expressed with dominant-negative tumor mutations . Upon expression in human Saos-2 cells the V122A p53 mutant caused growth suppression, was capable of transactivation and exhibited higher than wild type activity with the bax promoter in luciferase assays . A non-functional p53 tumor mutant was partially reactivated by V122A for both transactivation and growth suppression . Thus, the screen for toxic p53 mutants in yeast can identify novel p53 variants that may be useful in dissecting p53 regulated cellular responses and in developing p53-based cancer therapies.

Biophys J, 2001 Jul, 81(1), 446 - 54
The assembly of amyloidogenic yeast sup35 as assessed by scanning (atomic) force microscopy: an analogy to linear colloidal aggregation?
Xu S, Bevis B, Arnsdorf MF.
Amyloidosis is a class of diseases caused by protein aggregation and deposition in various tissues and organs . In this paper, a yeast amyloid-forming protein Sup35 was used as a model for understanding amyloid fiber formation . The dynamics of amyloid formation by Sup35 were studied with scanning force microscopy . We found that: 1) the assembly of Sup35 fibers begins with individual NM peptides that aggregate to form large beads or nucleation units which, in turn, form dimers, trimers, tetramers and longer linear assemblies appearing as a string of beads; 2) the morphology of the linear assemblies differ; and 3) fiber assembly suggests an analogy to the aggregation of colloidal particles . A dipole assembly model is proposed based on this analogy that will allow further experimental testing.

Photochem Photobiol, 2001 Jun, 73(6), 692 - 6
Recombinant phytochrome A in yeast differs by its spectroscopic and photochemical properties from the major phyA' and is close to the minor phyA": evidence for posttranslational modification of the pigment in plants; Sineshchekov V et al.; Previously, two pools of phytochrome A (phyA' and phyA") have been detected by in situ low-temperature fluorescence spectroscopy and photochemistry; it was suggested that they might differ in the nature of their posttranslational modification . In order to verify this possibility Arabidopsis and rice (Oryza) phyA were expressed in yeast and the pigments were assembled in vivo with phycocyanobilin (PCB) and phytochromobilin (P phi B) . The resulting recombinant phytochromes in the red-light-absorbing form (Pr) were characterized in the yeast cell by (1) the fluorescence emission spectra; (2) the temperature dependence of Pr fluorescence intensity and activation energy of fluorescence decay; and (3) the extent of photoconversion of Pr into photoproduct lumi-R (gamma 1) or far-red-light absorbing form (Pfr) (gamma 2) . Both Arabidopsis phyA/PCB and Oryza phyA/P phi B had low gamma 1 of ca 0.05, allowing their attribution to the Pr" phenomenological type of phytochrome comprising phyA", phyB and cryptogam phytochromes . The spectroscopic properties of Oryza phyA/P phi B were also very close to phyA" . However, both investigated holoproteins differed from phyA", both with respect to the character of temperature dependence of the fluorescence yield and activation energy . Thus, recombinant Oryza phyA/P phi B is similar but not identical to phyA" . The data demonstrate that the low-abundance-fraction plant phyA (phyA") comes from the same gene as the major (phyA') fraction . Because both endogenous phyA fractions differ from the phytochrome expressed in yeast, they appear to be posttranslationally modified and/or bound to partner proteins or cellular substructures . However, the character of the presumed chemical modification is different in phyA' and phyA" and its extent is more profound in the case of the former.

Genes Cells, 2001 May, 6(5), 455 - 73
Fission yeast homologues of the B' subunit of protein phosphatase 2A: multiple roles in mitotic cell division and functional interaction with calcineurin; Tanabe O et al.; BACKGROUND: Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase distributed in eukaryotes from yeast to human, and plays pivotal roles in diverse cellular functions such as metabolism, cell cycle progression, gene expression and development . PP2A holoenzyme is a heterodimer of a catalytic subunit C and a regulatory subunit A, or a heterotrimer of C, A and a variable regulatory subunit consisting of three families; B, B', and PR72 . Specific functions for each variable subunit are not well understood . RESULTS: Two fission yeast genes pbp1+ and pbp2+ homologous to the regulatory subunit B' were isolated . Physical in vivo interaction of the gene products with the catalytic subunit was demonstrated . A double disruption haploid mutant (Deltapbp1Deltapbp2) showed growth defect, cell shape and size abnormality, multiseptation and anucleated cell formation due to abnormality in septum positioning . These phenotypes were suppressed by human B' cDNA, indicating the striking conservation of the B' function from yeast to human . Over-expression of fission yeast B' led to growth defects, a loss of cell shape polarity, septal abnormality and anucleated cell formation . Deltapbp1Deltapbp2 and pbp1 null haploids were hypersensitive to calcineurin inhibitors, cyclosporin A and FK506, with which the mutants underwent arrest at post-anaphase and cell lysis . Double disruption of calcineurin and pbp1+, but not pbp2+, genes led to synthetic lethality . CONCLUSION: The fission yeast B' subunit of PP2A plays critical roles in cell shape control and septum formation, and shares essential functions with calcineurin for viability, possibly through their roles in cytokinesis and cell wall integrity.

Mol Biol Evol, 2001 Jul, 18(7), 1283 - 92
The yeast protein interaction network evolves rapidly and contains few redundant duplicate genes; Wagner A; In this paper, the structure and evolution of the protein interaction network of the yeast Saccharomyces cerevisiae is analyzed . The network is viewed as a graph whose nodes correspond to proteins . Two proteins are connected by an edge if they interact . The network resembles a random graph in that it consists of many small subnets (groups of proteins that interact with each other but do not interact with any other protein) and one large connected subnet comprising more than half of all interacting proteins . The number of interactions per protein appears to follow a power law distribution . Within approximately 200 Myr after a duplication, the products of duplicate genes become almost equally likely to (1) have common protein interaction partners and (2) be part of the same subnetwork as two proteins chosen at random from within the network . This indicates that the persistence of redundant interaction partners is the exception rather than the rule . After gene duplication, the likelihood that an interaction gets lost exceeds 2.2 x 10(-3)/Myr . New interactions are estimated to evolve at a rate that is approximately three orders of magnitude smaller . Every 300 Myr, as many as half of all interactions may be replaced by new interactions.

Mol Cell Biol, 2001 Jul, 21(14), 4656 - 69
Mechanism of prion loss after Hsp104 inactivation in yeast; Wegrzyn RD et al.; In vivo propagation of {PSI(+)}, an aggregation-prone prion isoform of the yeast release factor Sup35 (eRF3), has previously been shown to require intermediate levels of the chaperone protein Hsp104 . Here we perform a detailed study on the mechanism of prion loss after Hsp104 inactivation . Complete or partial inactivation of Hsp104 was achieved by the following approaches: deleting the HSP104 gene; modifying the HSP104 promoter that results in low level of its expression; and overexpressing the dominant-negative ATPase-inactive mutant HSP104 allele . In contrast to guanidine-HCl, an agent blocking prion proliferation, Hsp104 inactivation induced relatively rapid loss of {PSI(+)} and another candidate yeast prion, {PIN(+)} . Thus, the previously hypothesized mechanism of prion dilution in cell divisions due to the blocking of prion proliferation is not sufficient to explain the effect of Hsp104 inactivation . The {PSI(+)} response to increased levels of another chaperone, Hsp70-Ssa, depends on whether the Hsp104 activity is increased or decreased . A decrease of Hsp104 levels or activity is accompanied by a decrease in the number of Sup35(PSI+) aggregates and an increase in their size . This eventually leads to accumulation of huge agglomerates, apparently possessing reduced prion forming capability and representing dead ends of the prion replication cycle . Thus, our data confirm that the primary function of Hsp104 in prion propagation is to disassemble prion aggregates and generate the small prion seeds that initiate new rounds of prion propagation (possibly assisted by Hsp70-Ssa).

Mol Cell Biol, 2001 Jul, 21(14), 4568 - 78
GCN5 dependence of chromatin remodeling and transcriptional activation by the GAL4 and VP16 activation domains in budding yeast; Stafford GA et al.; Chromatin-modifying enzymes such as the histone acetyltransferase GCN5 can contribute to transcriptional activation at steps subsequent to the initial binding of transcriptional activators . However, few studies have directly examined dependence of chromatin remodeling in vivo on GCN5 or other acetyltransferases, and none have examined remodeling via nucleosomal activator binding sites . In this study, we have monitored chromatin perturbation via nucleosomal binding sites in the yeast episome TALS by GAL4 derivatives in GCN5(+) and gcn5Delta yeast cells . The strong activator GAL4 shows no dependence on GCN5 for remodeling TALS chromatin, whereas GAL4-estrogen receptor-VP16 shows substantial, albeit not complete, GCN5 dependence . Mini-GAL4 derivatives having weakened interactions with TATA-binding protein and TFIIB exhibit a strong dependence on GCN5 for both transcriptional activation and TALS remodeling not seen for native GAL4 . These results indicate that GCN5 can contribute to chromatin remodeling at activator binding sites and that dependence on coactivator function for a given activator can vary according to the type and strength of contacts that it makes with other factors . We also found a weaker dependence for chromatin remodeling on SPT7 than on GCN5, indicating that GCN5 can function via pathways independent of the SAGA complex . Finally, we examine dependence on GCN5 and SWI-SNF at two model promoters and find that although these two chromatin-remodeling and/or modification activities may sometimes work together, in other instances they act in complementary fashion.

Mol Cell Biol, 2001 Jul, 21(14), 4482 - 94
Deubiquitination step in the endocytic pathway of yeast plasma membrane proteins: crucial role of Doa4p ubiquitin isopeptidase; Dupre S et al.; The Fur4p uracil permease, like most yeast plasma membrane proteins, undergoes ubiquitin-dependent endocytosis and is then targeted to the vacuole (equivalent to the mammalian lysosome) for degradation . The cell surface ubiquitination of Fur4p is mediated by the essential Rsp5p ubiquitin ligase . Ubiquitination of Fur4p occurs on two target lysines, which receive two ubiquitin moieties linked through ubiquitin Lys63, a type of linkage (termed UbK63) different from that involved in proteasome recognition . We report that pep4 cells deficient for vacuolar protease activities accumulate vacuolar unubiquitinated Fur4p . In contrast, pep4 cells lacking the Doa4p ubiquitin isopeptidase accumulate ubiquitin-conjugated Fur4p . These data suggest that Fur4p undergoes Doa4p-dependent deubiquitination prior to vacuolar degradation . Compared to pep4 cells, pep4 doa4 cells have huge amounts of membrane-bound ubiquitin conjugates . This indicates that Doa4p plays a general role in the deubiquitination of membrane-bound proteins, as suggested by reports describing the suppression of some doa4 phenotypes in endocytosis and vacuolar protein sorting mutants . Some of the small ubiquitin-linked peptides that are a hallmark of Doa4 deficiency are not present in rsp5 mutant cells or after overproduction of a variant ubiquitin modified at Lys 63 (UbK63R) . These data suggest that the corresponding peptides are degradation products of Rsp5p substrates and probably of ubiquitin conjugates carrying UbK63 linkages . Doa4p thus appears to be involved in the deubiquitination of endocytosed plasma membrane proteins, some of them carrying UbK63 linkages.

EMBO Rep, 2001 Jun, 2(6), 493 - 8
An artificial transcription activator mimics the genome-wide properties of the yeast Pdr1 transcription factor; Devaux F et al.; We analysed the genome-wide regulatory properties of an artificial transcription activator in which the DNA-binding domain of the yeast transcription factor, Pdr1, was fused to the activation domain of Gal4 (Pdr1*GAD) . This Pdr1*GAD chimera was put under the control of the inducible GAL1 promoter . DNA microarray analyses showed that all the target genes upregulated by the well-studied native gain-of-function Pdr1-3 mutant were similarly activated by the chimerical factor Pdr1*GAD upon galactose induction . Additionally, this kinetic approach led us not only to confirm previously published targets, but also to define a hierarchy among members of the Pdr1 regulon . Our observations prove, for the first time at the complete genome level, that the DNA-binding domain of Pdr1 is sufficient to guide its specificity . We propose that this approach could be useful for the study of new transcription factors identified in silico from sequenced organisms . Complete data are available at www.biologie.ens.fr/yeast-publi.html.

Biochem Biophys Res Commun, 2001 Jun 29, 284(5), 1148 - 54
The fission yeast ortholog of the coregulator SKIP interacts with the small subunit of U2AF; Ambrozkova M et al.; The mode of action of transcriptional coregulators may involve the recruitment of spliceosome components . Using the two-hybrid screen, we examined the interaction partners of spSNW1, the S . pombe ortholog of the human coregulator SNW1/SKIP/NCoA-62, and found it to interact with the small subunit of the splicing factor U2AF (spU2AF23) . The interaction involves the C-terminal parts of spU2AF23 and spSNW1 . Tagged variants of both proteins were expressed in S . pombe and the interaction was proved by coprecipitation in nuclear extracts . This interaction would explain the finding of SKIP in nuclear speckles (Mintz, P . J., et al., EMBO J . 18, 4308-4320, 1999) and in reconstituted spliceosomes (Neubauer, G., et al., Nat . Genet . 20, 46-50, 1998) . We deleted the spSNW1 gene in the diploid strain and demonstrated that spSNW1 is an essential gene in S . pombe .

Brain Pathol, 2001 Jul, 11(3), 296 - 305
p73 is not mutated in meningiomas as determined with a functional yeast assay but p73 expression increases with tumor grade; Nozaki M et al.; The p53 gene is normally wild type in meningiomas . Since all three members of the p53 gene family recognize the same DNA sequence, tumors containing wild type p53 could decrease transactivation of p53 target genes by mutating either p63 or p73 . In meningiomas the most likely target is p73, because loss of heterozygosity of the chromosomal band containing p73 is the commonest genetic lesion in these tumors . To screen p73 for mutations we have developed a functional assay which tests the ability of p73 to activate transcription from a p53-responsive promoter in yeast . The assay correctly identified p73 mutants with mutations equivalent to hotspot mutations in p53, demonstrating that the assay can detect transcriptionally inactive p73 . No mutations in p73 were identified in meningiomas . p73 RNA level was higher in more advanced tumors, but there was no correlation between the expression level of p73 and p21, a known p53 target gene . The yeast assay was also used to measure the intrinsic sensitivity of the p73 protein to mutagenesis . Like p53, p73 is exceptionally easy to inactivate as a transcription factor by point mutation . Taken together, these results indicate that p53 and p73 serve very different functions in tumors.

Southeast Asian J Trop Med Public Health, 2000, 31 Suppl 1, 79 - 84
Application of yeast enolase as antigen for immunodiagnosis of malaria; Sato K et al.; In 1998, we reported that Plasmodium falciparum (Pf) enolase was useful as the capture antigen for the immunodiagnosis of malaria . In the present study, we modified a fluorescence-ELISA for the diagnosis of malaria by applying yeast enolase or rabbit muscle enolase as antigen . Sera from 67 falciparum malaria patients and 15 vivax malaria patients were tested by the method . Positivity rates of the former was 82.1% against yeast enolase antigen and 90.5% against rabbit muscle enolase antigen, and those of latter was 93.3% against both enolase antigens . Mean antibody level (RFU values) of sera from falciparum and vivax malaria patients were significantly higher than those from healthy individuals . There was a significant correlation between anti-yeast and anti-rabbit muscle enolase antibody level (RFU values) in the group of falciparum subjects (r = 0.401, p<0.001) . A significant correlation between RFU values against yeast enolase antigen and indirect fluorescent antibody titers against crude Pf antigen in the same subjects was recognized (r = 0.518, p<0.001) . Longitudinal changes of RFU values against yeast enolase for the following 4 weeks after admission were also examined for sera from falciparum malaria patients . Patients with more severe malaria showed increasing RFU values as the clinical courses progressed . However, in the mild cases, each RFU value stayed unchanged during the course . We concluded that yeast and rabbit muscle enolase could be appropriately used as antigen for the immunodiagnosis of malaria.

Altern Med Rev, 2001 Jun, 6(3), 248 - 71
Cardiovascular disease: C-reactive protein and the inflammatory disease paradigm: HMG-CoA reductase inhibitors, alpha-tocopherol, red yeast rice, and olive oil polyphenols . A review of the literature; Patrick L et al.; The current understanding of the origin of atherosclerosis is that of an inflammatory process that involves the acute phase response -an innate biological response to a disturbance in homeostasis -infection, inflammation, tissue injury, neoplasm, or immune disturbance . The activation of the acute phase response, signaled by interleukin-6, produces proteins (fibrinogen, C-reactive protein (CRP), serum amyloid A) that lead to inflammatory reactions . The tissues themselves contain elevated levels of acute phase proteins and cytokines resulting in a localized inflammatory effect . Localized inflammatory responses in the intimal layer of the arterial wall have been shown to be responsible for many of the aspects of intimal thickening and plaque disruption, leading to acute cardiovascular events . The predictive value of plasma C-reactive protein as a risk factor for cardiovascular events has led some researchers to support the use of CRP as a main cardiovascular risk assessment tool, along with total cholesterol:HDL ratios and homocysteine levels . The ability of HMG-CoA reductase inhibitors to lower C-reactive protein levels has recently brought into question the mechanisms of action of the statin drugs . Because these medications lower incidences of acute cardiovascular events as well as decreasing morbidity and mortality well before the effects of lowered LDL cholesterol can be expected to occur, questions have been asked about whether they may work independently of LDL-lowering mechanisms . Red yeast rice contains a naturally-occurring statin (lovastatin) as well as other cholesterol-lowering compounds, some with antioxidant effects . Alpha-tocopherol also significantly lowers CRP levels in diabetics and nondiabetics, and minimizes other aspects of the acute phase response and inflammatory damage involved in atherosclerosis . This may account for alpha-tocopherol's positive effect on cardiovascular morbidity and mortality . Finally, polyphenolic compounds present in virgin olive oil also have anti-inflammatory and antioxidative effects in cardiovascular disease . The phenolic compounds in virgin olive oil may explain some of the protective effects found in epidemiological studies.

Biochem Biophys Res Commun, 2001 Jun 22, 284(4), 1083 - 9
Similar subunit interactions contribute to assembly of clathrin adaptor complexes and COPI complex: analysis using yeast three-hybrid system; Takatsu H et al.; Clathrin adaptor protein (AP) complexes are heterotetramers composed of two large, one medium, and one small subunits . By exploiting the yeast three-hybrid system, we have found that an interaction between the two large subunits of the AP-1 complex, gamma-adaptin and beta1-adaptin, is markedly enhanced in the presence of the small subunit, sigma1 . Similarly, two large subunits of the AP-4 complex, epsilon-adaptin and beta4-adaptin, are found to interact with each other only in the presence of the small subunit, sigma4 . Furthermore, we have found that an interaction between two large subunits of the COPI F subcomplex, gamma-COP and beta-COP, is detectable only in the presence of zeta-COP . Because these COPI subunits have common ancestral origins to the corresponding AP subunits, these three-hybrid data, taken together with the previous two-hybrid data, suggest that the AP complexes and the COPI F subcomplex assemble by virtue of similar subunit interactions .

J Biol Chem, 2001 Aug 31, 276(35), 33257 - 64 Epub 2001 Jun 18.
The N-terminal and C-terminal domains of RAP1 are dispensable for chromatin opening and GCN4-mediated HIS4 activation in budding yeast; Yu L et al.; Repressor activator protein 1 (RAP1) assists GCN4-mediated HIS4 activation by overcoming some repressive aspect of chromatin structure to facilitate GCN4 binding . RAP1 also participates in other nuclear processes, and discrete domains of RAP1 have been shown to have specific properties including DNA binding, DNA bending, transcriptional activation, and silencing and telomere functions . To investigate whether specific domains of RAP1 are required to "open" chromatin and help GCN4 to activate the HIS4 gene, we examined the abilities of different truncated RAP1 proteins to perturb positioned nucleosomes via a nucleosomal RAP1 site in a yeast episome in vivo, and we tested HIS4 activation in yeast strains harboring truncated RAP1 mutants . We found that neither the DNA bending domain nor the putative activation domain of RAP1 is required for its ability to perturb the chromatin structure of a plasmid containing a RAP1 site . Similarly, neither the putative activation domain nor the N-terminal DNA-bending domain was required for GCN4-mediated activation of HIS4 . We also used a rap1(ts) mutant to show that continuous occupancy of the HIS4 promoter by RAP1 is required for GCN4-mediated gene activation.

FEBS Lett, 2001 Jun 8, 498(2-3), 223 - 7
What can yeast tell us about N-linked glycosylation in the Golgi apparatus?
Munro S.
The N-glycans found on eukaryotic glycoproteins occur in a vast range of different structures . A universal N-glycan core is attached to proteins during synthesis in the endoplasmic reticulum, and then diversity is generated as the proteins pass through the Golgi apparatus . Many of the Golgi-localised glycosyltransferases have now been identified in both yeast and mammalian cells, but it is still unclear how these enzymes are integrated into the Golgi and the rest of the cell so as to ensure efficient and specific processing of passing substrates . This review discusses the potential of the yeast system to address these issues.

Biochemistry, 2001 Jun 26, 40(25), 7474 - 9
The arginine finger loop of yeast and human GAP is a determinant for the specificity toward Ras GTPase; te Biesebeke R et al.; In this work, we have studied the role of the arginine finger region in determining the specificity of the GTPase activating proteins (GAPs) Saccharomyces cerevisiae Ira2p and human p120-GAP toward yeast Ras2p and human Ha-Ras p21 . It is known that p120-GAP can enhance both Ras2p and Ha-Ras GTPase activities, whereas Ira2p is strictly specific for Ras2p and fails to activate Ha-Ras GTPase . Substitution in Ira2p of the arginine following the arginine finger with alanine, the residue found in the corresponding position of p120-GAP, or by glycine as found in neurofibromin, evokes a low but significant stimulation of Ha-Ras GTPase . The stimulatory activity of Ira2p on Ha-Ras increased by substituting segments of the finger loop region with p120-GAP residues, especially with the six residues forming the tip of the arginine loop . In p120-GAP, substitution of the entire finger loop with the corresponding region of Ira2p led to a construct completely inactive on Ha-Ras GTPase but active on yeast Ras2p GTPase . Analysis of these results and modeling of Ira2p.Ras complexes emphasize the importance of the finger loop region not only for the catalytic activity but also as a structural determinant involved in the specificity of GAPs toward Ras proteins from different organisms.

Biochemistry, 2001 Jun 26, 40(25), 7410 - 20
Mutations in yeast protein phosphatase type 1 that affect targeting subunit binding; Wu X et al.; Protein phosphatase type 1 (PP1) is a major Ser/Thr protein phosphatase that is involved in many cellular processes . The activity of PP1 is controlled by regulatory subunits, many of which are thought to bind to a hydrophobic groove in PP1 via a short consensus sequence termed the V/IXF motif . To test this hypothesis, 11 variants of yeast PP1 (Glc7) were constructed in which one or more of the residues comprising the groove were changed to alanine . These variants were tested for their biological activity in vivo, for their biochemical activity in vitro, and for their ability to associate with three PP1 binding proteins . Five variants are unable to complement the essential function of PP1 in vivo although they are catalytically active in vitro . Many of the mutants are deficient in binding two V/IXF-containing subunits, Gac1 and Reg1, which regulate glycogen accumulation and glucose repression, respectively, but all retain the ability to associate with Sds22, a regulatory subunit that lacks this motif . The subcellular locations at which PP1 normally accumulates (bud neck, nucleolus, spindle pole body) were not occupied by one PP1 variant . Additionally, we provide evidence that mutations in the hydrophobic groove of PP1 affect substrate specificity . Together, these results demonstrate the importance of the hydrophobic groove for the interaction with regulatory subunits, for the proper subcellular localization of PP1 and for the substrate specificity of PP1.

Biochemistry, 2001 Jun 26, 40(25), 7382 - 403
Catalytic acid-base groups in yeast pyruvate decarboxylase . 3 . A steady-state kinetic model consistent with the behavior of both wild-type and variant enzymes at all relevant pH values; Sergienko EA et al.; The widely quoted kinetic model for the mechanism of yeast pyruvate decarboxylase (YPDC, EC 4.1.1.1), an enzyme subject to substrate activation, is based on data for the wild-type enzyme under optimal experimental conditions . The major feature of the model is the obligatory binding of substrate in the regulatory site prior to substrate binding at the catalytic site . The activated monomer would complete the cycle by irreversible decarboxylation of the substrate and product (acetaldehyde) release . Our recent kinetic studies of YPDC variants substituted at positions D28 and E477 at the active center necessitate some modification of the mechanism . It was found that enzyme without substrate activation apparently is still catalytically competent . Further, substrate-dependent inhibition of D28-substituted variants leads to an enzyme form with nonzero activity at full saturation, requiring a second major branch point in the mechanism . Kinetic data for the E477Q variant suggest that three consecutive substrate binding steps may be needed to release product acetaldehyde, unlikely if YPDC monomer is the minimal catalytic unit with only two binding sites for substrate . A model to account for all kinetic observations involves a functional dimer operating through alternation of active sites . In the context of this mechanism, roles are suggested for the active center acid-base groups D28, E477, H114, and H115 . The results underline once more the enormous importance that both aromatic rings of the thiamin diphosphate, rather than only the thiazolium ring, have in catalysis, a fact little appreciated prior to the availability of the 3-dimensional structure of these enzymes.

Biochemistry, 2001 Jun 26, 40(25), 7369 - 81
Catalytic acid-base groups in yeast pyruvate decarboxylase . 2 . Insights into the specific roles of D28 and E477 from the rates and stereospecificity of formation of carboligase side products; Sergienko EA et al.; Yeast pyruvate decarboxylase (YPDC), in addition to forming its metabolic product acetaldehyde, can also carry out carboligase reactions in which the central enamine intermediate reacts with acetaldehyde or pyruvate (instead of the usual proton electrophile), resulting in the formation of acetoin and acetolactate, respectively (typically, 1% of the total reaction) . Due to the common mechanism shared by the acetaldehyde-forming and carboligase reactions through decarboxylation, a detailed analysis of the rates and stereochemistry of the carboligase products formed by the E477Q, D28A, and D28N active center YPDC variants was undertaken . While substitution at either position led to an approximately 2-3 orders of magnitude lower catalytic efficiency in acetaldehyde formation, the rate of acetoin formation by the E477Q and D28N variants was higher than that by wild-type enzyme . Comparison of the steady-state data for acetaldehyde and acetoin formation revealed that the rate-limiting step for acetaldehyde formation by the D28A, H114F, H115F, and E477Q variants is a step post-decarboxylation . In contrast to the wild-type YPDC and the E477Q variant, the D28A and D28N variants could synthesize acetolactate as a major product . The lower overall rate of side-product formation by the D28A variant than wild-type enzyme attests to participation of D28 in steps leading up to and including decarboxylation . The results also provide insight into the state of ionization of the side chains examined . (R)-Acetoin is produced by the variants with greater enantiomeric excess than by wild-type YPDC . (S)-Acetolactate is the predominant enantiomer produced by the D28-substituted variants, the same configuration as produced by the related plant acetolactate synthase.

Biochemistry, 2001 Jun 26, 40(25), 7355 - 68
Catalytic acid-base groups in yeast pyruvate decarboxylase . 1 . Site-directed mutagenesis and steady-state kinetic studies on the enzyme with the D28A, H114F, H115F, and E477Q substitutions; Liu M et al.; The roles of four of the active center groups with potential acid-base properties in the region of pH optimum of pyruvate decarboxylase from Saccharomyces cerevisiae have been studied with the substitutions Asp28Ala, His114Phe, His115Phe, and Glu477Gln, introduced by site-directed mutagenesis methods . The steady-state kinetic constants were determined in the pH range of activity for the enzyme . The substitutions result in large changes in k(cat) and k(cat)/S(0.5) (and related terms), indicating that all four groups have a role in transition state stabilization . Furthermore, these results also imply that all four are involved in some manner in stabilizing the rate-limiting transition state(s) both at low substrate (steps starting with substrate binding and culminating in decarboxylation) and at high substrate concentration (steps beginning with decarboxylation and culminating in product release) . With the exception of some modest effects, the shapes of neither the bell-shaped k(cat)/S(0.5)-pH (and related functions) plots nor the k(cat)-pH plots are changed by the substitutions . Yet, the fractional activity still remaining after substitutions virtually rules out any of the four residues as being directly responsible for initiating the catalytic process by ionizing the C2H . There is no effect on the C2 H/D exchange rate exhibited by the D28A and E477Q substitutions . These results strongly imply that the base-induced deprotonation at C2 is carried out by the only remaining base, the iminopyrimidine tautomer of the coenzyme, via intramolecular proton abstraction . The first product is released as CO(2) rather than HCO(3)(-) by both wild-type and E477Q and D28A variants, ruling out several mechanistic alternatives.

Biol Pharm Bull, 2001 Jun, 24(6), 595 - 9
Significant expression of functional human type 1 mitochondrial ADP/ATP carrier in yeast mitochondria; Hatanaka T et al.; As a first step to characterize the unknown functional properties of the human mitochondrial ADP/ATP carrier (AAC), we tried to express human type 1 AAC (hAAC1) in Saccharomyces cerevisiae . Expression of hAAC1 in yeast mitochondrial membrane was very low, although its transcript level was high . Its expression was improved greatly by replacement of its N-terminal region with the corresponding region of yeast type 2 AAC (yAAC2), as observed with the bovine type 1 AAC (bAAC1) . This chimeric hAAC1 showed similar ADP transport activity to that of chimeric bAAC1, corresponding to the transport activity of bAAC1 in bovine heart mitochondria . These results suggested that the N-terminal region of yAAC2 is important for expression of the mammalian carriers in yeast mitochondria . Using the present expression system, studies on the functional properties of the human AAC isoforms in relation to their structures are now possible.

Nucleic Acids Res, 2001 Jun 15, 29(12), 2567 - 74
The brefeldin A resistance protein Bfr1p is a component of polyribosome-associated mRNP complexes in yeast; Lang BD et al.; The yeast gene BFR1 was originally isolated from a genetic screen for high-copy suppressors of brefeldin A-induced lethality in Saccharomyces cerevisiae . While this result suggested a possible role for the encoded protein, Bfr1p, in the secretory pathway, subsequent data have not fully supported this conclusion . Alternatively, Bfr1p has also been found by yeast two-hybrid analysis to interact with Bbp1p, a component of the spindle pole body . Finally, we have reported that Bfr1p associates with cytoplasmic mRNP complexes containing Scp160p, raising the possibility that Bfr1p may function in mRNA metabolism . Here, we have explored this possibility further . We report that Bfr1p associates with yeast polyribosomes and mRNP complexes even in the absence of Scp160p, and that its interaction with Scp160p-containing mRNP complexes is RNA-dependent . Furthermore, we have determined by fluorescence microscopy and subcellular fractionation that Bfr1p and Scp160p demonstrate similar cytoplasmic localization with enrichment around the nuclear envelope/endoplasmic reticulum . Finally, we report that loss of Bfr1p disrupts the interaction of Scp160p with polyribosomes, thereby demonstrating that the relationship between these two proteins is functional as well as physical . Considered together, these data raise the intriguing possibility that Bfr1p may provide a link between mRNA metabolism, the chromosomal segregation machinery and perhaps secretion in yeast.

Nucleic Acids Res, 2001 Jun 15, 29(12), 2448 - 55
The yeast POP2 gene encodes a nuclease involved in mRNA deadenylation; Daugeron MC et al.; The major mRNA degradation pathway involves deadenylation of the target molecule followed by decapping and, finally, 5'-->3' exonuclease digestion of the mRNA body . While yeast factors involved in the decapping and exonuclease degradation steps have been identified, the nature of the factor(s) involved in the deadenylation step remained elusive . Database searches for yeast proteins related to the mammalian deadenylase PARN identified the Pop2 protein (Pop2p) as a potential deadenylase . While Pop2p was previously identified as a factor affecting transcription, we identified a non-canonical RNase D sequence signature in its sequence . Analysis of the fate of a reporter mRNA in a pop2 mutant demonstrates that Pop2p is required for efficient mRNA degradation in vivo . Characterisation of mRNA degradation intermediates accumulating in this mutant supports the involvement of Pop2p in mRNA deadenylation in vivo . Similar phenotypes are observed in yeast strains lacking the Ccr4 protein, which is known to be associated with Pop2p . A recombinant Pop2p fragment encompassing the putative catalytic domain degrades poly(A) in vitro demonstrating that Pop2p is a nuclease . We also demonstrate that poly(A) is a better competitor than poly(G) or poly(C) of the Pop2p nuclease activity . Altogether, our study indicates that Pop2p is a nuclease subunit of the yeast deadenylase and suggests that Pop2p homologues in other species may have similar functions.

Genes Dev, 2001 Jun 15, 15(12), 1528 - 39
The 19S complex of the proteasome regulates nucleotide excision repair in yeast; Gillette TG et al.; Previous studies suggest that the amino-terminal ubiquitin-like (ubl) domain of Rad23 protein can recruit the proteasome for a stimulatory role during nucleotide excision repair in the yeast Saccharomyces cerevisiae . In this report, we show that the 19S regulatory complex of the yeast proteasome can affect nucleotide excision repair independently of Rad23 protein . Strains with mutations in 19S regulatory subunits (but not 20S subunits) of the proteasome promote partial recovery of nucleotide excision repair in vivo in rad23 deletion mutants, but not in other nucleotide excision repair-defective strains tested . In addition, a strain that expresses a temperature-degradable ATPase subunit of the 19S regulatory complex manifests a dramatically increased rate of nucleotide excision repair in vivo . These data indicate that the 19S regulatory complex of the 26S proteasome can negatively regulate the rate of nucleotide excision repair in yeast and suggest that Rad23 protein not only recruits the 19S regulatory complex, but also can mediate functional interactions between the 19S regulatory complex and the nucleotide excision repair machinery . The 19S regulatory complex of the yeast proteasome functions in nucleotide excision repair independent of proteolysis.

EMBO J, 2001 Jun 15, 20(12), 3197 - 209
Recognition of polyadenylation sites in yeast pre-mRNAs by cleavage and polyadenylation factor; Dichtl B et al.; Recognition of poly(A) sites in yeast pre-mRNAs is poorly understood . Employing an in vitro cleavage system with cleavage and polyadenylation factor (CPF) and cleavage factor IA we show that the efficiency and positioning elements are dispensable for poly(A)-site recognition within a short CYC1 substrate in vitro . Instead, U-rich elements immediately upstream and downstream of the poly(A) site mediate cleavage-site recognition within CYC1 and ADH1 pre-mRNAs . These elements act in concert with the poly(A) site to produce multiple recognition sites for the processing machinery, since combinations of mutations within these elements were most effective in cleavage inhibition . Intriguingly, introduction of a U-rich element downstream of the GAL7 poly(A) site strongly enhanced cleavage, underscoring the importance of downstream sequences in general . RNA- binding analyses demonstrate that cleavage depends on the recognition of the poly(A)-site region by CPF . Consistent with in vitro results, mutation of sequences upstream and downstream of the poly(A) site affected 3'-end formation in vivo . A model for yeast pre-mRNA cleavage-site recognition outlines an unanticipated high conservation of yeast and mammalian 3'-end processing mechanisms.

Mutat Res, 2001 Jul 1, 478(1-2), 129 - 39
Mutator effects of overproducing DNA polymerase eta (Rad30) and its catalytically inactive variant in yeast; Pavlov YI et al.; DNA polymerase eta synthesizes DNA in vitro with low fidelity . Based on this, here we report the effects of deletion or increased expression of yeast RAD30 gene, encoding for polymerase eta (Pol eta), on spontaneous mutagenesis in vivo . Deletion of RAD30 did not affect spontaneous mutagenesis . Overproduction of Rad30p was slightly mutagenic in a wild-type yeast strain and moderately mutagenic in strains with inactive 3'-->5'-exonuclease of DNA polymerase epsilon or DNA mismatch repair . These data suggest that excess Rad30p reduces replication fidelity in vivo and that the induced errors may be corrected by exonucleolytic proofreading and DNA mismatch repair . However, the magnitude of mutator effect (only up to 10-fold) suggests that the replication fork is protected from inaccurate synthesis by Pol eta in the absence of DNA damage . Overproduction of catalytically inactive Rad30p was also mutagenic, suggesting that much of the mutator effect results from indirect perturbation of replication rather than from direct misincorporation by Pol eta . Moreover, while excess wild-type Pol eta primarily induced base substitutions in the msh6 and pms1 strains, excess inactive Rad30p induced both base substitutions and frameshifts . This suggests that more than one mutagenic mechanism is operating when RAD30 is overexpressed.

Curr Genet, 2001 Apr, 39(2), 68 - 76
Contribution of Cat8 and Sip4 to the transcriptional activation of yeast gluconeogenic genes by carbon source-responsive elements; Hiesinger M et al.; The carbon source-responsive element (CSRE) functions as an activating promoter motif of gluconeogenic genes in Saccharomyces cerevisiae . The positively acting regulatory genes CAT8 and SIP4 encode CSRE-binding proteins which contribute unequally to the regulated expression of a CSRE-dependent reporter gene (85% and 15%, respectively, under conditions of glucose derepression) . Deregulated variants of Cat8 and Sip4 are able to bind to the CSRE and allow glucose-insensitive gene activation, even in the absence of the other protein, arguing against the physiological significance of heterodimer formation . Gel retardation assays provide evidence for a different binding affinity of Cat8 and Sip4 to at least some CSRE sequence variants . Both efficient biosynthesis of and transcriptional activation by Sip4 require a functional CAT8 gene, while Cat8 was not dependent on SIP4 . Thus, our data suggest that the apparent minor importance of Sip4 may be the result of autoregulatory cross-talk among the isofunctional activators Cat8 and Sip4 . The derepression deficiency of a CSRE-dependent reporter gene in a strain lacking the Cat1 (Snf1) protein kinase can be suppressed by Sip4 fused to a strong heterologous activation domain . This finding agrees with the idea that phosphorylation by Cat1 may convert Sip4 into a functional activator.

Genetics, 2001 Jun, 158(2), 627 - 34
Genetic evidence supports a role for the yeast CCR4-NOT complex in transcriptional elongation; Denis CL et al.; The CCR4-NOT complex is involved in the regulation of gene expression both positively and negatively . The repressive effects of the complex appear to result in part from restricting TBP access to noncanonical TATAA binding sites presumably through interaction with multiple TAF proteins . We provide here genetic evidence that the CCR4-NOT complex also plays a role in transcriptional elongation . First, defects in CCR4-NOT components as well as overexpression of the NOT4 gene elicited 6-azauracil (6AU) and mycophenolic acid sensitivities, hallmarks of transcriptional elongation defects . A number of other transcription initiation factors known to interact with the CCR4-NOT complex did not elicit these phenotypes nor did defects in factors that reduced mRNA degradation and hence the recycling of NTPs . Second, deletion of ccr4 resulted in severe synthetic effects with mutations or deletions in the known elongation factors RPB2, TFIIS, and SPT16 . Third, the ccr4 deletion displayed allele-specific interactions with rpb1 alleles that are thought to be important in the control of elongation . Finally, we found that a ccr4 deletion as well as overexpression of the NOT1 gene specifically suppressed the cold-sensitive phenotype associated with the spt5-242 allele . The only other known suppressors of this spt5-242 allele are factors involved in slowing transcriptional elongation . These genetic results are consistent with the model that the CCR4-NOT complex, in addition to its known effects on initiation, plays a role in aiding the elongation process.

Genetics, 2001 Jun, 158(2), 587 - 96
Yeast ASF1 protein is required for cell cycle regulation of histone gene transcription; Sutton A et al.; Transcription of the four yeast histone gene pairs (HTA1-HTB1, HTA2-HTB2, HHT1-HHF1, and HHT2-HHF2) is repressed during G1, G2, and M . For all except HTA2-HTB2, this repression requires several trans-acting factors, including the products of the HIR genes, HIR1, HIR2, and HIR3 . ASF1 is a highly conserved protein that has been implicated in transcriptional silencing and chromatin assembly . In this analysis, we show that HIR1 interacts with ASF1 in a two-hybrid analysis . Further, asf1 mutants, like hir mutants, are defective in repression of histone gene transcription during the cell cycle and in cells arrested in early S phase in response to hydroxyurea . asf1 and hir1 mutations also show very similar synergistic interactions with mutations in cac2, a subunit of the yeast chromatin assembly factor CAF-I . The results suggest that ASF1 and HIR1 function in the same pathway to create a repressive chromatin structure in the histone genes during the cell cycle.

Mol Biol Cell, 2001 Jun, 12(6), 1885 - 96
Yeast Gga coat proteins function with clathrin in Golgi to endosome transport; Costaguta G et al.; Gga proteins represent a newly recognized, evolutionarily conserved protein family with homology to the "ear" domain of the clathrin adaptor AP-1 gamma subunit . Yeast cells contain two Gga proteins, Gga1p and Gga2p, that have been proposed to act in transport between the trans-Golgi network and endosomes . Here we provide genetic and physical evidence that yeast Gga proteins function in trans-Golgi network clathrin coats . Deletion of Gga2p (gga2Delta), the major Gga protein, accentuates growth and alpha-factor maturation defects in cells carrying a temperature-sensitive allele of the clathrin heavy chain gene . Cells carrying either gga2Delta or a deletion of the AP-1 beta subunit gene (apl2Delta) alone are phenotypically normal, but cells carrying both gga2Delta and apl2Delta are defective in growth, alpha-factor maturation, and transport of carboxypeptidase S to the vacuole . Disruption of both GGA genes and APL2 results in cells so severely compromised in growth that they form only microcolonies . Gga proteins can bind clathrin in vitro and cofractionate with clathrin-coated vesicles . Our results indicate that yeast Gga proteins play an important role in cargo-selective clathrin-mediated protein traffic from the trans-Golgi network to endosomes.

Mol Biol Cell, 2001 Jun, 12(6), 1835 - 41
Perturbation of the nucleus: a novel Hog1p-independent, Pkc1p-dependent consequence of hypertonic shock in yeast; Nanduri J et al.; Hypertonic shock of Saccharomyces cerevisiae activates the Hog1p MAP kinase cascade . In contrast, protein kinase C (Pkc1p) and the "cell integrity" MAP kinase cascade are critical for the response to hypotonic shock . We observed that hypertonic shock transiently relocated many, but not all, nuclear and nucleolar proteins to the cytoplasm . We hypothesized that the relocation of nuclear proteins was due to activation of the Hog1p kinase cascade, yet, surprisingly, Hog1p was not required for these effects . In contrast, Pkc1p kinase activity was required, although the Pkc1p MAP kinase cascade and several factors known to lie upstream and downstream of Pkc1p were not . Moreover, sudden induction of a hyperactive form of Pkc1p was sufficient to relocate nuclear proteins . Taken together, these observations show that the scope of involvement of Pkc1p in the organization of the nucleus considerably exceeds what has been characterized previously . The relocation of nuclear proteins is likely to account for the profound inhibition of RNA synthesis that was observed during hypertonic shock.

Mol Biol Cell, 2001 Jun, 12(6), 1633 - 44
Dual targeting of Osh1p, a yeast homologue of oxysterol-binding protein, to both the Golgi and the nucleus-vacuole junction; Levine TP et al.; Oxysterol binding protein (OSBP) is the only protein known to bind specifically to the group of oxysterols with potent effects on cholesterol homeostasis . Although the function of OSBP is currently unknown, an important role is implicated by the existence of multiple homologues in all eukaryotes so far examined . OSBP and a subset of homologues contain pleckstrin homology (PH) domains . Such domains are responsible for the targeting of a wide range of proteins to the plasma membrane . In contrast, OSBP is a peripheral protein of Golgi membranes, and its PH domain targets to the trans-Golgi network of mammalian cells . In this article, we have characterized Osh1p, Osh2p, and Osh3p, the three homologues of OSBP in Saccharomyces cerevisiae that contain PH domains . Examination of a green fluorescent protein (GFP) fusion to Osh1p revealed a striking dual localization with the protein present on both the late Golgi, and in the recently described nucleus-vacuole (NV) junction . Deletion mapping revealed that the PH domain of Osh1p specified targeting to the late Golgi, and an ankyrin repeat domain targeting to the NV junction, the first such targeting domain identified for this structure . GFP fusions to Osh2p and Osh3p showed intracellular distributions distinct from that of Osh1p, and their PH domains appear to contribute to their differing localizations.

Mol Biol Cell, 2001 Jun, 12(6), 1611 - 21
SPO21 is required for meiosis-specific modification of the spindle pole body in yeast; Bajgier BK et al.; During meiosis II in the yeast Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body changes from a site of microtubule initiation to a site of de novo membrane formation . These membranes are required to package the haploid meiotic products into spores . This functional change in the spindle pole body involves the expansion and modification of its cytoplasmic face, termed the outer plaque . We report here that SPO21 is required for this modification . The Spo21 protein localizes to the spindle pole in meiotic cells . In the absence of SPO21 the structure of the outer plaque is abnormal, and prospore membranes do not form . Further, decreased dosage of SPO21 leaves only two of the four spindle pole bodies competent to generate membranes . Mutation of CNM67, encoding a known component of the mitotic outer plaque, also results in a meiotic outer plaque defect but does not block membrane formation, suggesting that Spo21p may play a direct role in initiating membrane formation.

Mol Biol Cell, 2001 Jun, 12(6), 1541 - 55
Ras regulates the polarity of the yeast actin cytoskeleton through the stress response pathway; Ho J et al.; Polarized growth in yeast requires cooperation between the polarized actin cytoskeleton and delivery of post-Golgi secretory vesicles . We have previously reported that loss of the major tropomyosin isoform, Tpm1p, results in cells sensitive to perturbations in cell polarity . To identify components that bridge these processes, we sought mutations with both a conditional defect in secretion and a partial defect in polarity . Thus, we set up a genetic screen for mutations that conferred a conditional growth defect, showed synthetic lethality with tpm1Delta, and simultaneously became denser at the restrictive temperature, a hallmark of secretion-defective cells . Of the 10 complementation groups recovered, the group with the largest number of independent isolates was functionally null alleles of RAS2 . Consistent with this, ras2Delta and tpm1Delta are synthetically lethal at 35 degrees C . We show that ras2Delta confers temperature-sensitive growth and temperature-dependent depolarization of the actin cytoskeleton . Furthermore, we show that at elevated temperatures ras2Delta cells are partially defective in endocytosis and show a delocalization of two key polarity markers, Myo2p and Cdc42p . However, the conditional enhanced density phenotype of ras2Delta cells is not a defect in secretion . All the phenotypes of ras2Delta cells can be fully suppressed by expression of yeast RAS1 or RAS2 genes, human Ha-ras, or the double disruption of the stress response genes msn2Deltamsn4Delta . Although the best characterized pathway of Ras function in yeast involves activation of the cAMP-dependent protein kinase A pathway, activation of the protein kinase A pathway does not fully suppress the actin polarity defects, suggesting that there is an additional pathway from Ras2p to Msn2/4p . Thus, Ras2p regulates cytoskeletal polarity in yeast under conditions of mild temperature stress through the stress response pathway.

J Biol Chem, 2001 Aug 17, 276(33), 30753 - 60 Epub 2001 Jun 14.
The protein kinase Gcn2p mediates sodium toxicity in yeast; Goossens A et al.; Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) is a conserved mechanism regulating protein synthesis in response to various stresses . A screening for negative factors in yeast salt stress tolerance has led to the identification of Gcn2p, the single yeast eIF2alpha kinase that is activated by amino acid starvation in the general amino acid control response . Mutation of other components of this regulatory circuit such as GCN1 and GCN3 also resulted in improved NaCl tolerance . The gcn2 phenotype was not accompanied by changes in sodium or potassium homeostasis . NaCl induced a Gcn2p-dependent phosphorylation of eIF2alpha and translational activation of Gcn4p, the transcription factor that mediates the general amino acid control response . Mutations that activate Gcn4p function, such as gcd7-201, cpc2, and deletion of the translational regulatory region of the GCN4 gene, also cause salt sensitivity . It can be postulated that sodium activation of the Gcn2p pathway has toxic effects on growth under NaCl stress and that this novel mechanism of sodium toxicity may be of general significance in eukaryotes.

Methods, 2001 Jul, 24(3), 201 - 17
Using the yeast interaction trap and other two-hybrid-based approaches to study protein-protein interactions; Toby GG et al.; The detection of physical interaction between two or more molecules of interest can be facilitated if the act of association between the interactive partners leads to the production of a readily observed biological or physical readout . Many interacting molecule pairs (X, Y) can be made to induce such a readout if X and Y are each fused to defined protein elements with desired properties . For example, in the yeast forward two-hybrid system, X is synthesized as a translational fusion to a DNA-binding domain (DBD), Y is synthesized as a fusion to a transcriptional activation domain (AD), and coexpression of DBD-X and AD-Y induces transcription of easily scored responsive reporters . Other approaches use paradigms based on the artificial production of two, hybrid, molecules, but substitute a variety of readouts including the repression of transcription, activation of signal transduction pathways, or reconstitution of a disrupted enzymatic activity . In this article, we summarize a number of two-hybrid-based approaches, and detail the use of the forward yeast two-hybrid system in a screen to identify novel interacting partners for a protein of interest .

Oncogene, 2001 Apr 30, 20(19), 2336 - 46
Redox control of AP-1-like factors in yeast and beyond; Toone WM et al.; Cells have evolved complex and efficient strategies for dealing with variable and often-harsh environments . A key aspect of these stress responses is the transcriptional activation of genes encoding defense and repair proteins . In yeast members of the AP-1 family of proteins are required for the transcriptional response to oxidative stress . This sub-family of AP-1 (called yAP-1) proteins are sensors of the redox-state of the cell and are activated directly by oxidative stress conditions . yAP-1 proteins are bZIP-containing factors that share homology to the mammalian AP-1 factor complex and bind to very similar DNA sequence sites . The generation of reactive oxygen species and the resulting potential for oxidative stress is common to all aerobically growing organisms . Furthermore, many of the features of this response appear to be evolutionarily conserved and consequently the study of model organisms, such as yeast, will have widespread utility . The important structural features of these factors, signaling pathways controlling their activity and the nature of the target genes they control will be discussed.

J Cell Biol, 2001 Jun 11, 153(6), 1187 - 98
Intracellular retention of newly synthesized insulin in yeast is caused by endoproteolytic processing in the Golgi complex; Zhang B et al.; An insulin-containing fusion protein (ICFP, encoding the yeast prepro-alpha factor leader peptide fused via a lysine-arginine cleavage site to a single chain insulin) has been expressed in Saccharomyces cerevisiae where it is inefficiently secreted . Single gene disruptions have been identified that cause enhanced immunoreactive insulin secretion (eis) . Five out of six eis mutants prove to be vacuolar protein sorting (vps)8, vps35, vps13, vps4, and vps36, which affect Golgi<-->endosome trafficking . Indeed, in wild-type yeast insulin is ultimately delivered to the vacuole, whereas vps mutants secrete primarily unprocessed ICFP . Disruption of KEX2, which blocks intracellular processing to insulin, quantitatively reroutes ICFP to the cell surface, whereas loss of the Vps10p sorting receptor is without effect . Secretion of unprocessed ICFP is not based on a dominant secretion signal in the alpha-leader peptide . Although insulin sorting mediated by Kex2p is saturable, Kex2p functions not as a sorting receptor but as a protease: replacement of Kex2p by truncated secretory Kex2p (which travels from Golgi to cell surface) still causes endoproteolytic processing and intracellular insulin retention . Endoproteolysis promotes a change in insulin's biophysical properties . B5His residues normally participate in multimeric insulin packing; a point mutation at this position permits ICFP processing but causes the majority of processed insulin to be secreted . The data argue that multimeric assembly consequent to endoproteolytic maturation regulates insulin sorting in the secretory pathway.

Biochemistry, 2001 Jun 19, 40(24), 7069 - 76
Interaction of yeast iso-1-cytochrome c with cytochrome c peroxidase investigated by {15N, 1H} heteronuclear NMR spectroscopy; Worrall JA et al.; The interaction of yeast iso-1-cytochrome c with its physiological redox partner cytochrome c peroxidase has been investigated using heteronuclear NMR techniques . Chemical shift perturbations for both 15N and 1H nuclei arising from the interaction of isotopically enriched 15N cytochrome c with cytochrome c peroxidase have been observed . For the diamagnetic, ferrous cytochrome c, 34 amides are affected by binding, corresponding to residues at the front face of the protein and in agreement with the interface observed in the 1:1 crystal structure of the complex . In contrast, for the paramagnetic, ferric protein, 56 amides are affected, corresponding to residues both at the front and toward the rear of the protein . In addition, the chemical shift perturbations were larger for the ferric protein . Using experimentally observed pseudocontact shifts the magnetic susceptibility tensor of yeast iso-1-cytochrome c in both the free and bound forms has been calculated with HN nuclei as inputs . In contrast to an earlier study, the results indicate that there is no change in the geometry of the magnetic axes for cytochrome c upon binding to cytochrome c peroxidase . This leads us to conclude that the additional effects observed for the ferric protein arise either from a difference in binding mode or from the more flexible overall structure causing a transmittance effect upon binding.

Biochem Biophys Res Commun, 2001 Apr 6, 282(3), 832 - 8
Isolation and characterization of acetoacetyl-CoA thiolase gene essential for n-decane assimilation in yeast Yarrowia lipolytica; Yamagami S et al.; Yarrowia lipolytica is a yeast which can utilize n-alkane as a sole carbon source . We isolated a Y . lipolytica peroxisomal acetoacetyl-CoA thiolase gene, PAT1, by complementation of a mutant that cannot utilize n-decane as a sole carbon source . We found that the putative PAT1 product had conserved features of peroxisomal acetoacetyl-CoA thiolase . We showed that the PAT1 disruptant was not able to grow on n-decane, and that n-decane-inducible acetoacetyl-CoA thiolase activity largely depended on PAT1 . The original mutant carried a mutation involving the replacement of Gly382 with Glu . This mutation inactivated the ability of PAT1 to complement the defective n-decane utilization of the disruptant . These results indicate that PAT1 encodes peroxisomal acetoacetyl-CoA thiolase and is essential for n-decane utilization in Y . lipolytica .

Genomics, 2001 Mar 1, 72(2), 158 - 68
The human mitochondrial Mrs2 protein functionally substitutes for its yeast homologue, a candidate magnesium transporter; Zsurka G et al.; We report here on the human MRS2 gene that encodes a protein, hsaMrs2p, the first molecularly characterized candidate for a magnesium transporter in metazoa . The protein, like the yeast mitochondrial Mrs2 and Lpe10 proteins, contains two predicted transmembrane domains in its carboxyl-terminus, the first of which terminates with the conserved motif F/Y-G-M-N . These are typical features of the CorA family of magnesium transporters . Expression of hsaMrs2p in mrs2-1 knock-out mutant yeast partly restores mitochondrial magnesium concentrations that are significantly reduced in this mutant . It also alleviates other defects of this mutant, which may be secondary to the reduction in magnesium concentrations . These findings suggest that hsaMrs2p and yMrs2p are functional homologues . Like its yeast homologues, hsaMrs2p has been localized in mitochondria . The hsaMRS2 gene is located on chromosome 6 (6p22.1-p22.3) and is composed of 11 exons . A low level of the transcript is detected in various mouse tissues . Copyright 2001 Academic Press.

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