Genetics, 2000 Nov, 156(3), 933 - 41 MGA2 and SPT23 are modifiers of transcriptional silencing in yeast; Dula ML et al.; Transcriptional silencing at the HM loci and telomeres in yeast depends on several trans-acting factors, including Rap1p and the Sir proteins . The SUM1-1 mutation was identified by its ability to restore silencing to strains deficient in one or more of these trans-acting factors . The mechanism by which SUM1-1 bypasses the requirement for silencing proteins is not known . We identified four loci that when reduced in dosage in diploid strains increase the ability of SUM1-1 strains to suppress silencing defects . Two of the genes responsible for this effect were found to be MGA2 and SPT23 . Mga2p and Spt23p were previously identified as functionally related transcription factors that influence chromatin structure . We find that deletion of MGA2 or SPT23 also increases the efficiency of silencing in haploid SUM1-1 strains . These results suggest that Mga2p and Spt23p are antagonists of silencing . Consistent with this proposal we find that deletion of MGA2 or SPT23 also suppresses the silencing defects caused by deletion of the SIR1 gene or by mutations in the HMR silencer sequences . However, we find that Mga2p and Spt23p can positively affect silencing in other contexts; deletion of either MGA2 or SPT23 decreases mating in strains bearing mutations in the HML-E silencer . Mga2p and Spt23p appear to be a novel class of factors that influence disparate pathways of transcriptional control by chromatin. Biochemistry, 2000 Nov 7, 39(44), 13405 - 21 Topology of yeast RNA polymerase II subunits in transcription elongation complexes studied by photoaffinity cross-linking; Wooddell CI et al.; The subunits of Saccharomyces cerevisiae RNA polymerase II (RNAP II) in proximity to the DNA during transcription elongation have been identified by photoaffinity cross-linking . In the absence of transcription factors, RNAP II will transcribe a double-stranded DNA fragment containing a 3'-extension of deoxycytidines, a "tailed template" . We designed a DNA template allowing the RNAP to transcribe 76 bases before it was stalled by omission of CTP in the transcription reaction . This stall site oriented the RNAP on the DNA template and allowed us to map the RNAP subunits along the DNA . The DNA analogue 5-{N-(p-azidobenzoyl)-3-aminoallyl}-dUTP (N(3)RdUTP) {Bartholomew, B., Kassavetis, G . A., Braun, B . R., and Geiduschek, E . P . (1990) EMBO J . 9, 2197-205} was synthesized and enzymatically incorporated into the DNA at specified positions upstream or downstream of the stall site, in either the template or nontemplate strand of the DNA . Radioactive nucleotides were positioned beside the photoactivatable nucleotides, and cross-linking by brief ultraviolet irradiation transferred the radioactive tag from the DNA onto the RNAP subunits . In addition to N(3)RdUTP, which has a photoreactive azido group 9 A from the uridine base, we used the photoaffinity cross-linker 5N(3)dUTP with an azido group directly on the uridine ring to identify the RNAP II subunits closest to the DNA at positions where multiple subunits cross-linked . In cross-linking reactions dependent on transcription, RPB1, RPB2, and RPB5 were cross-linked with N(3)RdUTP . With 5N(3)dUTP, only RPB1 and RPB2 were cross-linked . Under certain circumstances, RPB3, RPB4, and RPB7 were cross-linked . From the information obtained in this topological study, we developed a model of yeast RNAP II in a transcription elongation complex. Connect Tissue Res, 1998, 38(1-4), 257 - 67;discussion 295-303 Identification of tuftelin- and amelogenin-interacting proteins using the yeast two-hybrid system; Paine CT et al.; Biomineralization of enamel is a complex process that involves the eventual replacement of an extracellular protein matrix by hydroxyapatite crystallites . To date four different enamel matrix proteins have been identified; the amelogenins, tuftelin, enamelin and ameloblastin . Assembly of the enamel extracellular matrix from these component proteins is believed to be critical in producing a matrix competent to undergo mineral replacement . Enamel formation is a complex process and additional proteins are likely to have a role in the assembly of the extracellular matrix . In order to identify additional proteins involved in the assembly process, the yeast two-hybrid system developed by Fields and Song (1989) has been implemented . This system allows for the identification of unknown proteins that interact with proteins of interest . Typically a known protein is used as "bait" to screen a cDNA expression library of interest . In our studies, tuftelin or amelogenin have been used to screen a mouse tooth library produced from one day old pups . A library screening of six million clones with amelogenin as bait resulted in eleven positive clones all of which show high homology to the human leukocyte antigen-B (HLA-B) associated transcript (BAT) family of genes . A library screening of one million clones using tuftelin as the bait identified twenty-one tuftelin-interacting proteins . Ten of these proteins are either keratin K5 or keratin K6, four are constitutively expressed and the remaining seven are novel . Further characterization of the proteins shown to interact with amelogenin or tuftelin may shed additional light on this complex process of enamel matrix assembly. Nat Genet, 2000 Nov, 26(3), 341 - 4 The human Rhesus-associated RhAG protein and a kidney homologue promote ammonium transport in yeast; Marini AM et al.; The Rhesus blood-group antigens are defined by a complex association of membrane polypeptides that includes the non-glycosylated Rh proteins (RhD and RhCE) and the RHag glycoprotein, which is strictly required for cell surface expression of these antigens . RhAG and the Rh polypeptides are erythroid-specific transmembrane proteins belonging to the same family (36% identity) . Despite their importance in transfusion medicine, the function of RhAG and Rh proteins remains unknown, except that their absence in Rh(null) individuals leads to morphological and functional abnormalities of erythrocytes, known as the Rh-deficiency syndrome . We recently found significant sequence similarity between the Rh family proteins, especially RhAG, and Mep/Amt ammonium transporters . We show here that RhAG and also RhGK, a new human homologue expressed in kidney cells only, function as ammonium transport proteins when expressed in yeast . Both specifically complement the growth defect of a yeast mutant deficient in ammonium uptake . Moreover, ammonium efflux assays and growth tests in the presence of toxic concentrations of the analogue methylammonium indicate that RhAG and RhGK also promote ammonium export . Our results provide the first experimental evidence for a direct role of RhAG and RhGK in ammonium transport . These findings are of high interest, because no specific ammonium transport system has been characterized so far in human. Nat Genet, 2000 Nov, 26(3), 283 - 9 Analysis of yeast protein kinases using protein chips; Zhu H et al.; We have developed a novel protein chip technology that allows the high-throughput analysis of biochemical activities, and used this approach to analyse nearly all of the protein kinases from Saccharomyces cerevisiae . Protein chips are disposable arrays of microwells in silicone elastomer sheets placed on top of microscope slides . The high density and small size of the wells allows for high-throughput batch processing and simultaneous analysis of many individual samples . Only small amounts of protein are required . Of 122 known and predicted yeast protein kinases, 119 were overexpressed and analysed using 17 different substrates and protein chips . We found many novel activities and that a large number of protein kinases are capable of phosphorylating tyrosine . The tyrosine phosphorylating enzymes often share common amino acid residues that lie near the catalytic region . Thus, our study identified a number of novel features of protein kinases and demonstrates that protein chip technology is useful for high-throughput screening of protein biochemical activity. J Cell Biol, 2000 Oct 30, 151(3), 731 - 8 Recycling of the yeast a-factor receptor; Chen L et al.; The yeast a-factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand-independent pathway and a ligand-dependent uptake pathway . Whereas the constitutive pathway leads to degradation of the receptor in the vacuole, the present work finds that receptor internalized via the ligand-dependent pathway recycles . With the a-factor ligand continuously present in the culture medium, trafficking of the receptor achieves an equilibrium in which continuing uptake to endosomal compartments is balanced by its recycling return to the plasma membrane . Withdrawal of ligand from the medium leads to a net return of the internalized receptor back to the plasma membrane . Although recycling is demonstrated for receptors that lack the signal for constitutive endocytosis, evidence is provided indicating a participation of recycling in wild-type Ste3p trafficking as well: a-factor treatment both slows wild-type receptor turnover and results in receptor redistribution to intracellular endosomal compartments . Apparently, a-factor acts as a switch, diverting receptor from vacuole-directed endocytosis and degradation, to recycling . A model is presented for how the two Ste3p endocytic modes may collaborate to generate the polarized receptor distribution characteristic of mating cells. J Cell Biol, 2000 Oct 30, 151(3), 719 - 30 Prm1p, a pheromone-regulated multispanning membrane protein, facilitates plasma membrane fusion during yeast mating; Heiman MG et al.; Cell fusion occurs throughout development, from fertilization to organogenesis . The molecular mechanisms driving plasma membrane fusion in these processes remain unknown . While yeast mating offers an excellent model system in which to study cell fusion, all genes previously shown to regulate the process act at or before cell wall breakdown; i.e., well before the two plasma membranes have come in contact . Using a new strategy in which genomic data is used to predict which genes may possess a given function, we identified PRM1, a gene that is selectively expressed during mating and that encodes a multispanning transmembrane protein . Prm1p localizes to sites of cell-cell contact where fusion occurs . In matings between Deltaprm1 mutants, a large fraction of cells initiate zygote formation and degrade the cell wall separating mating partners but then fail to fuse . Electron microscopic analysis reveals that the two plasma membranes in these mating pairs are tightly apposed, remaining separated only by a uniform gap of approximately 8 nm . Thus, the phenotype of Deltaprm1 mutants defines a new step in the mating reaction in which membranes are juxtaposed, possibly through a defined adherence junction, yet remain unfused . This phenotype suggests a role for Prm1p in plasma membrane fusion. J Cell Biol, 2000 Oct 30, 151(3), 587 - 600 A selective transport route from Golgi to late endosomes that requires the yeast GGA proteins; Black MW et al.; Pep12p is a yeast syntaxin located primarily in late endosomes . Using mutagenesis of a green fluorescent protein chimera we have identified a sorting signal FSDSPEF, which is required for transport of Pep12p from the exocytic pathway to late endosomes, from which it can, when overexpressed, reach the vacuole . When this signal is mutated, Pep12p instead passes to early endosomes, a step that is determined by its transmembrane domain . Surprisingly, Pep12p is then specifically retained in early endosomes and does not go on to late endosomes.By testing appropriate chimeras in mutant strains, we found that FSDSPEF-dependent sorting was abolished in strains lacking Gga1p and Gga2p, Golgi-associated coat proteins with homology to gamma adaptin . In the gga1 gga2 double mutant endogenous Pep12p cofractionated with the early endosome marker Tlg1p, and recycling of Snc1p through early endosomes was defective . Pep12p sorting was also defective in cells lacking the clathrin heavy or light chain . We suggest that specific and direct delivery of proteins to early and late endosomes is required to maintain the functional heterogeneity of the endocytic pathway and that the GGA proteins, probably in association with clathrin, help create vesicles destined for late endosomes. Biochem Biophys Res Commun, 2000 Nov 2, 277(3), 622 - 30 Human homologues of yeast vacuolar protein sorting 29 and 35; Edgar AJ et al.; In the yeast Saccharomyces cerevisiae, a membrane coat complex is required for endosome to Golgi retrograde transport . The vacuolar protein sorting proteins Vps29p, Vps35p, and Vps26p are required for pre-vacuolar/late endosome to Golgi retrieval of the vacuolar hydrolase receptor Vps10p . They form a cargo recognition and concentration subcomplex, termed the inner shell of the retromer coat, prior to vesicle formation by the addition of the membrane-deforming outer shell . We have cloned the human and murine homologues of yeast Vps29p and the human homologue of Vps35p . They encode 182 and 796 residue proteins, with 43 and 29% identity to their respective yeast . The 10.5 kb, 5 exon, VPS29 gene is located on chromosome 12q24 and the 29.6 kb, 17 exon, VPS35 gene is on chromosome 16 . In humans, Vps29p, Vps35p, and Hbeta58, the homologue of Vps26p, may form an inner shell of the retromer coat similar to that found in yeast . Biochem Biophys Res Commun, 2000 Nov 2, 277(3), 589 - 93 Combined transformation and genetic technique verification of protein-protein interactions in the yeast two-hybrid system; Tyagi S et al.; The yeast two-hybrid system is frequently used to identify protein-protein interactions . The assay is based on the functional reconstitution of a transcriptional activator . Since an indirect phenotype of the positive clones is the basis for selection of positive interacting clones, the two-hybrid screens are vulnerable to false positives . Here we report a screening protocol based on the sequential use of the cotransformation approach followed by the genetic method for verifying true two-hybrid interactions . Using this procedure, we have screened a cDNA library and have been able to isolate true positives from the yeast two-hybrid screen . J Mol Biol, 2000 Nov 10, 303(5), 693 - 707 How the multifunctional yeast Rap1p discriminates between DNA target sites: a crystallographic analysis; Taylor HO et al.; Rap1p from Saccharomyces cerevisiae is a multifunctional, sequence-specific, DNA-binding protein involved in diverse cellular processes such as transcriptional activation and silencing, and is an essential factor for telomere length regulation and maintenance . In order to understand how Rap1p discriminates between its different DNA-binding sites, we have determined the crystal structure of the DNA-binding domain of the Rap1p (Rap1pDBD) in complex with two different DNA-binding sites . The first DNA sequence is the HMRE binding site found at silencers, which contains four base-pair substitutions in comparison to the telomeric binding site present in our earlier crystal structure of the Rap1pDBD-TeloA complex . The second complex contains an alternative telomeric binding site, TeloS, in which two half-sites are spaced closer together than in the TeloA complex . The determination of these structures was complicated by the presence of merohedral twinning in the crystals . Through identification of the twinning operator and determination of the twin fraction of the crystals, we were able to deconvolute the twinned intensities into their untwinned components, and to calculate electron density maps for both complexes . The structural information shows that the two domains present in the Rap1pDBD bind to these two biologically relevant binding sites through subtle side-chain movements at the protein-DNA interface, rather than through global domain rearrangements . EMBO J, 2000 Nov 1, 19(21), 5895 - 904 Human pre-mRNA cleavage factor II(m) contains homologs of yeast proteins and bridges two other cleavage factors; de Vries H et al.; Six different protein factors are required in vitro for 3' end formation of mammalian pre-mRNAs by endonucleolytic cleavage and polyadenylation . Five of the factors have been purified and most of their components cloned, but cleavage factor II(m) (CF II(m)) remained uncharacterized . We have purified CF II(m) from HeLa cell nuclear extract by several chromatographic steps . During purification, CF II(m) activity separated into two components, one essential (CF IIA(m)) and one stimulatory (CF IIB(m)) for the cleavage reaction . CF IIA(m) fractions contain the human homologs of two yeast 3' end processing factors, Pcf11p and Clp1p, as well as cleavage factor I(m) (CF I(m)) and several splicing and transcription factors . We report the cloning of hClp1 and show that it is a genuine subunit of CF IIA(m) . Antibodies directed against hClp1 deplete cleavage activity, but not polyadenylation activity from HeLa cell nuclear extract . hClp1 interacts with CF I(m) and the cleavage and polyadenylation specificity factor CPSF, suggesting that it bridges these two 3' end processing factors within the cleavage complex. EMBO J, 2000 Nov 1, 19(21), 5720 - 8 LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing; Kabeya Y et al.; Little is known about the protein constituents of autophagosome membranes in mammalian cells . Here we demonstrate that the rat microtubule-associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing . Two forms of LC3, called LC3-I and -II, were produced post-translationally in various cells . LC3-I is cytosolic, whereas LC3-II is membrane bound . The autophagic vacuole fraction prepared from starved rat liver was enriched with LC3-II . Immunoelectron microscopy on LC3 revealed specific labelling of autophagosome membranes in addition to the cytoplasmic labelling . LC3-II was present both inside and outside of autophagosomes . Mutational analyses suggest that LC3-I is formed by the removal of the C-terminal 22 amino acids from newly synthesized LC3, followed by the conversion of a fraction of LC3-I into LC3-II . The amount of LC3-II is correlated with the extent of autophagosome formation . LC3-II is the first mammalian protein identified that specifically associates with autophagosome membranes. J Biol Chem, 2001 Feb 16, 276(7), 5036 - 43 Epub 2000 Oct 31. The yeast gene MSC2, a member of the cation diffusion facilitator family, affects the cellular distribution of zinc; Li L et al.; The sequence of the yeast gene YDR205W places it within the family of cation diffusion facilitators: membrane proteins that transport transition metals . Deletion of YDR205W was reported to result in an increase in unequal sister chromatid recombination and was named meiotic sister chromatid recombination 2 (MSC2; Thompson, D . A., and Stahl, F . W . (1999) Genetics 153, 621-641) . We report here that a msc2 strain shows a phenotype of decreased viability in glycerol-ethanol media at 37 degrees C . Associated with decreased growth is an abnormal morphology typified by an increase in size of both cells and vacuoles . Addition of extracellular Zn2+ completely suppresses the morphological changes and partially suppresses the growth defect . Regardless of the concentration of Zn2+ in the media, the msc2 strain had a higher Zn2+ content than wild type cells . Zinquin staining also revealed that msc2 had a marked increase in fluorescence compared with the wild type, again reflecting an increase in intracellular Zn2+ . The deletion strain accumulated excess Zn2+ in nuclei-enriched membrane fractions, and when grown at 37 degrees C in glycerol-ethanol media, it showed a decreased expression of Zn2+-regulated genes . The expression of genes regulated by either Fe2+ or Cu2+ was not affected . An epitope-tagged Msc2p was localized to the endoplasmic reticulum/nucleus . These results suggest that Msc2p affects the cellular distribution of zinc and, in particular, the zinc content of nuclei. J Biol Chem, 2001 Feb 2, 276(5), 3555 - 63 Epub 2000 Oct 31. The yeast glycerol 3-phosphatases Gpp1p and Gpp2p are required for glycerol biosynthesis and differentially involved in the cellular responses to osmotic, anaerobic, and oxidative stress; Pahlman AK et al.; We have characterized the strongly homologous GPP1/RHR2 and GPP2/HOR2 genes, encoding isoforms of glycerol 3-phosphatase . Mutants lacking both GPP1 and GPP2 are devoid of glycerol 3-phosphatase activity and produce only a small amount of glycerol, confirming the essential role for this enzyme in glycerol biosynthesis . Overproduction of Gpp1p and Gpp2p did not significantly enhance glycerol production, indicating that glycerol phosphatase is not rate-limiting for glycerol production . Previous studies have shown that expression of both GPP1 and GPP2 is induced under hyperosmotic stress and that induction partially depends on the HOG (high osmolarity glycerol) pathway . We here show that expression of GPP1 is strongly decreased in strains having low protein kinase A activity, although it is still responsive to osmotic stress . The gpp1Delta/gpp2Delta double mutant is hypersensitive to high osmolarity, whereas the single mutants remain unaffected, indicating GPP1 and GPP2 substitute well for each other . Transfer to anaerobic conditions does not affect expression of GPP2, whereas GPP1 is transiently induced, and mutants lacking GPP1 show poor anaerobic growth . All gpp mutants show increased levels of glycerol 3-phosphate, which is especially pronounced when gpp1Delta and gpp1Delta/gpp2Delta mutants are transferred to anaerobic conditions . The addition of acetaldehyde, a strong oxidizer of NADH, leads to decreased glycerol 3-phosphate levels and restored anaerobic growth of the gpp1Delta/gpp2Delta mutant, indicating that the anaerobic accumulation of NADH causes glycerol 3-phosphate to reach growth-inhibiting levels . We also found the gpp1Delta/gpp2Delta mutant is hypersensitive to the superoxide anion generator, paraquat . Consistent with a role for glycerol 3-phosphatase in protection against oxidative stress, expression of GPP2 is induced in the presence of paraquat . This induction was only marginally affected by the general stress-response transcriptional factors Msn2p/4p or protein kinase A activity . We conclude that glycerol metabolism plays multiple roles in yeast adaptation to altered growth conditions, explaining the complex regulation of glycerol biosynthesis genes. Nucleic Acids Res, 2000 Nov 1, 28(21), 4090 - 6 The yeast CDP1 gene encodes a triple-helical DNA-binding protein; Musso M et al.; The formation of triple-helical DNA has been implicated in several cellular processes, including transcription, replication and recombination . While there is no direct evidence for triplexes in vivo, cellular proteins that specifically recognize triplex DNA have been described . Using a purine-motif triplex probe and southwestern library screening, we isolated five independent clones expressing the same C-terminal 210 amino acids of the Saccharomyces cerevisiae protein Cdp1p fused with beta-galactosidase . In electrophoretic mobility shift assays, recombinant Cdp1pDelta1-867 bound Pu-motif triplex DNAs with high affinity (K:(d) approximately 5 nM) and bound Py-motif triplex, duplex and single-stranded DNAs with far lower affinity (0.5-5.0 microM) . Genetic analyses revealed that the CDP1 gene product was required for proper chromosome segregation . The possible involvement of triplex DNA in this process is discussed. Nucleic Acids Res, 2000 Nov 1, 28(21), 4083 - 9 Poly(dA.dT) sequences exist as rigid DNA structures in nucleosome-free yeast promoters in vivo; Suter B et al.; Poly(dA.dT) sequences (T-tracts) are abundant genomic DNA elements with unusual properties in vitro and an established role in transcriptional regulation of yeast genes . In vitro T-tracts are rigid, contribute to DNA bending, affect assembly in nucleosomes and generate a characteristic pattern of CPDs (cyclobutane pyrimidine dimers) upon irradiation with UV light (UV photofootprint) . In eukaryotic cells, where DNA is packaged in chromatin, the DNA structure of T-tracts is unknown . Here we have used in vivo UV photofootprinting and DNA repair by photolyase to investigate the structure and accessibility of T-tracts in yeast promoters (HIS3, URA3 and ILV1) . The same characteristic photofootprints were obtained in yeast and in naked DNA, demonstrating that the unusual T-tract structure exists in living cells . Rapid repair of CPDs in the T-tracts demonstrates that these T-tracts were not folded in nucleosomes . Moreover, neither datin, a T-tract binding protein, nor Gcn5p, a histone acetyltransferase involved in nucleosome remodelling, showed an influence on the structure and accessibility of T-tracts . The data support a contribution of this unusual DNA structure to transcriptional regulation. J Cell Sci, 2000 Nov, 113 ( Pt 22), 3989 - 99 High dosage expression of a zinc finger protein, Grt1, suppresses a mutant of fission yeast slp1(+), a homolog of CDC20/p55CDC/Fizzy; Yamada HY et al.; Selective proteolysis at and after the onset of anaphase is a key cell cycle event required for sister chromatid separation as well as for exit from mitosis . It requires ubiquitination of substrates by Anaphase Promoting Complex(APC)/Cyclosome . Slp1, a WD-repeat protein, is a putative activator for APC in fission yeast . With another WD- repeat protein, Ste9/Srw1, it is thought to promote the proteolysis in a substrate-specific manner . We report here characterization of a temperature-sensitive (ts) slp1 mutant and its high-dosage suppressor, grt1(+) . In cells arrested in metaphase, wild-type Slp1 was preferentially found in a complex with hyperphosphorylated Cut9 (subunit of APC), whereas the ts Slp1 protein, lacking the last 113 amino acids, failed to interact with Cut9 . The temperature sensitivity was suppressed by high dosage expression of a zinc finger protein, Grt1 . The ts slp1 mutant was unable to maintain the normal level of Grt1 protein . The reduction in the Grt1 level may be a primary defect since high dosage expression of grt1(+) rescues the slp1 mutant . The grt1-suppression had an additive effect to ste9 and wee1-50, both of which partially suppress the ts slp1 mutant . Therefore, grt1(+) would define an independent pathway that facilitates the function of Slp1. Curr Genet, 2000 Oct, 38(3), 105 - 12 Functional analysis of the leader peptide of the yeast gene CPA1 and heterologous regulation by other fungal peptides; Delbecq P et al.; The 25-amino-acid leader peptide present at the 5' end of yeast CPA1 mRNA is responsible for the translational repression of that gene by arginine . We show here that the active domain of the yeast peptide is highly specific and extends over amino acids 6-23 . The region between amino acids 6-21 is well conserved between similar peptides present upstream of CPA1-homologous genes in other fungi . The Neurospora crassa arg-2 peptide represses the expression of CPA1, whereas the peptide from Aspergillus nidulans has only a weak regulatory effect . Such results suggest that the N- and C-terminal amino acids of the peptide may influence its regulatory activity . We also show that the transcription start sites of CPA1 are not modified when the cells are grown in the presence of arginine, nor in a strain carrying an inactive peptide. J Biochem (Tokyo), 2000 Nov, 128(5), 723 - 5 Yeast Ulp1, an Smt3-specific protease, associates with nucleoporins; Takahashi Y et al.; Yeast Smt3 is a ubiquitin-like protein similar to the mammalian SUMO-1 . Cdc3, a septin component, is known to be modified by Smt3 . The level of this modification was affected by Smt3-specific protease mutation ulp1-ts or overexpression of ULP1 . By two-hybrid screening, we isolated 5 UIP (Ulp1 interacting protein) genes . UIP1 was identical to NUP42 encoding a component of the nuclear pore complex (NPC) . Gle1, another NPC-associating component, also interacted with Ulp1 in the two-hybrid system and co-immunoprecipitation experiment . Thus Ulp1 associates with nucleoporins and may interact with septin rings in the telophase. J Biol Chem, 2001 Jan 19, 276(3), 1808 - 13 Epub 2000 Oct 30. Recruitment of the yeast Tup1p-Ssn6p repressor is associated with localized decreases in histone acetylation; Bone JR et al.; Posttranslational acetylation of histones is an important element of transcriptional regulation . The yeast Tup1p repressor is one of only a few non-enzyme proteins known to interact directly with the amino-terminal tail domains of histones H3 and H4 that are subject to acetylation . We demonstrated previously that Tup1p interacts poorly with more highly acetylated isoforms of these histones in vitro . Here we show that two separate classes of promoters repressed by Tup1p are associated with underacetylated histones in vivo . This decreased histone acetylation is dependent upon Tup1p and its partner Ssn6p and is localized to sequences near the point of Tup1p-Ssn6p recruitment . Increased acetylation of histones H3 and H4 is observed upon activation of these genes, but this increase is not dependent on transcription per se . Direct recruitment of Tup1p-Ssn6p complexes via fusion of Tup1p to the lexA DNA binding domain is sufficient to confer repression and induce decreased acetylation of H3 and H4 at a target promoter . Taken together, our results suggest that stable decreases in histone acetylation levels are directed and/or maintained by the Tup1p-Ssn6p repressor complex. Biosci Biotechnol Biochem, 2000 Sep, 64(9), 1942 - 6 A positive screening for drugs that specifically inhibit the Ca2+-signaling activity on the basis of the growth promoting effect on a yeast mutant with a peculiar phenotype; Shitamukai A et al.; An inappropriate activation of a signaling pathway in yeast often has a deleterious physiological effect and causes various defects, including growth defects . In a certain genetic background (deltazds1) of Saccharomyces cerevisiae, the cell-cycle progression in G2 is specifically blocked in the medium with CaCl2 by the hyperactivation of the Ca2+-signaling pathways . Here, we developed a novel drug screening procedure designed to detect the active compounds that specifically attenuate the Ca2+-signaling activity on the basis of the ability to abrogate the growth defect of the cells suffering from the hyperactivated Ca2+ signal . Using known calcineurin inhibitors as model compounds, we have established the screening conditions for the drugs that suppress the Ca2+-induced growth inhibition . An indicator strain with an increased drug sensitivity was constructed with a syr1/erg3 null mutation. Yeast, 2000 Nov, 16(15), 1449 - 56 Current awareness on yeast; Blasco A 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 23rd Aug . 2000) Gene, 2000 Oct 17, 257(1), 131 - 7 Yeast Pdr13p and Zuo1p molecular chaperones are new functional Hsp70 and Hsp40 partners; Michimoto T et al.; The deletion of the TOM1 gene encoding a putative ubiquitin ligase causes a temperature sensitive cellular growth in Saccharomyces cerevisiae . The arrested cells exhibit pleiotropic defects in nuclear division, maintenance of nuclear structure and heat stress responses . We previously identified a zuo1 mutation as an extragenic suppressor of the tom1 mutant . ZUO1 encodes a DnaJ-related Hsp40 . Here we show that a recessive cold sensitive mutation in PDR13 coding for an Hsp70 suppressed the tom1 mutation . The pdr13 deletion mutant was sensitive to high osmolarity, just like the zuo1 deletion mutant . A zuo1 pdr13 double deletion mutant did not show additive phenotypes . Furthermore, a tagged-Zuo1p was co-immunoprecipitated with a tagged-Pdr13p . Taken together, we propose that Pdr13p and Zuo1p are a new pair of Hsp70:Hsp40 molecular chaperones . In addition, Pdr13p co-sedimented with translating ribosomes and this association was independent of the presence of Zuo1p. J Biol Chem, 2001 Jan 19, 276(3), 2263 - 6 Epub 2000 Oct 27. Mismatch extension ability of yeast and human DNA polymerase eta; Washington MT et al.; DNA polymerase eta (Poleta) functions in error-free replication of UV-damaged DNA, and in vitro it efficiently bypasses a cis-syn T-T dimer by incorporating two adenines opposite the lesion . Steady state kinetic studies have shown that both yeast and human Poleta are low-fidelity enzymes, and they misincorporate nucleotides with a frequency of 10(-2)-10(-3) on both undamaged and T-T dimer-containing DNA templates . To better understand the role of Poleta in error-free translesion DNA synthesis, here we examine the ability of Poleta to extend from base mismatches . We find that both yeast and human Poleta extend from mismatched base pairs with a frequency of approximately 10(-3) relative to matched base pairs . In the absence of efficient extension of mismatched primer termini, the ensuing dissociation of Poleta from DNA may favor the excision of mismatched nucleotides by a proofreading exonuclease . Thus, we expect DNA synthesis by Poleta to be more accurate than that predicted from the fidelity of nucleotide incorporation alone. J Biol Chem, 2001 Jan 19, 276(3), 2243 - 8 Epub 2000 Oct 27. Circular minichromosomes become highly recombinogenic in topoisomerase-deficient yeast cells; Trigueros S et al.; In topoisomerase-deficient yeast cells, we have found that circular minichromosomes are present as broad distributions of multimeric forms, which consist of tandemly repeated copies of their monomeric sequences . This phenomenon selectively occurs in Deltatop1 cells, and is highly magnified in double mutant Deltatop1 top2-4 cells . No multimers are observed in single mutant top2-4 or Deltatop3 cells, or in Deltatop1 cells that express a plasmid-borne TOP1 gene . Interconversion among multimeric forms takes place rapidly in double mutant Deltatop1 top2-4 cells, and the multimeric distributions are readily reverted to the monomeric form when a plasmid-borne TOP1 gene is expressed from an inducible promoter . These observations are a new example of the interplay between DNA topology and genome stability, and suggest that the cell capacity to modulate DNA supercoiling is limited when DNA is organized in small topological domains . Yeast minichromosome multimerization provides an appropriate system in which to study mechanistic aspects of DNA recombination. J Biol Chem, 2001 Feb 9, 276(6), 4020 - 7 Epub 2000 Oct 27. Interorganellar communication . Altered nuclear gene expression profiles in a yeast mitochondrial dna mutant; Traven A et al.; Communication between mitochondria and the nucleus is important for a variety of cellular processes such as carbohydrate and nitrogen metabolism, mating and sporulation, and cell growth and morphogenesis . It has long been known that the functional state of mitochondria can influence nuclear gene expression . For example, in yeast cells lacking the mitochondrial genome, the expression of several nuclear genes, such as CIT2 (citrate synthase), MRP13 (mitochondrial ribosomal protein), and DLD3 (d-lactate dehydrogenase) has been reported to be altered . Here we show by microarray analysis of the genome-wide transcription profile of Saccharomyces cerevisiae that yeast petite mutants lacking mitochondrial DNA induce genes coding for mitochondrial proteins, enzymes of the glycolytic pathway and of the citric acid cycle, cell wall components, membrane transporters, and genes normally induced by nutrient deprivation and a variety of stresses . Consistent with the observed induction of genes related to cell stress and those encoding membrane transporters, yeast petite cells showed increased resistance to severe heat shock and exhibited a pleiotropic drug resistance phenotype . The observed changes in nuclear gene expression in cells lacking mitochondrial DNA may have implications for the role of mitochondria in processes such as carcinogenesis and aging. Exp Gerontol, 2000 Sep, 35(6-7), 671 - 6 Metabolic mechanisms of yeast ageing; Jazwinski SM; The genetic analysis of ageing of the yeast Saccharomyces cerevisiae points to several processes important in determining life span . Among these, metabolic control plays a leading role . An examination of the molecular mechanisms underlying metabolic control of longevity has revealed two separate pathways . The retrograde response signals mitochondrial dysfunction to the nucleus resulting in gene regulatory changes that compensate . Nutritional status also modulates life span, adjusting metabolism to efficiently utilize energy resources, in a response that closely resembles the caloric restriction paradigm described in rodents . Although the retrograde response and caloric restriction are distinct pathways of life span extension, there appears to be some overlap of the longevity effectors under their control. J Exp Bot, 2000 Oct, 51(351), 1761 - 2 CEF, a sec24 homologue of Arabidopsis thaliana, enhances the survival of yeast under oxidative stress conditions; Belles-Boix E et al.; Budding yeast strains that produced the Arabidopsis thaliana protein CEF or its amino-terminal proline-rich domain were more tolerant to hydroperoxides . CEF is homologous to animal and yeast Sec24 proteins . These data suggest that CEF plays a protective role through protein transport during growth under pro-oxidant conditions. J Bacteriol, 2000 Nov, 182(22), 6418 - 23 The yeast model for batten disease: mutations in BTN1, BTN2, and HSP30 alter pH homeostasis; Chattopadhyay S et al.; The BTN1 gene product of the yeast Saccharomyces cerevisiae is 39% identical and 59% similar to human CLN3, which is associated with the neurodegenerative disorder Batten disease . Furthermore, btn1-Delta strains have an elevated activity of the plasma membrane H(+)-ATPase due to an abnormally high vacuolar acidity during the early phase of growth . Previously, DNA microarray analysis revealed that btn1-Delta strains compensate for the altered plasma membrane H(+)-ATPase activity and vacuolar pH by elevating the expression of the two genes HSP30 and BTN2 . We now show that deletion of either HSP30 or BTN2 in either BTN1(+) or btn1-Delta strains does not alter vacuolar pH but does lead to an increased activity of the vacuolar H(+)-ATPase . Deletion of BTN1, BTN2, or HSP30 does not alter cytosolic pH but diminishes pH buffering capacity and causes poor growth at low pH in a medium containing sorbic acid, a condition known to result in disturbed intracellular pH homeostasis . Btn2p was localized to the cytosol, suggesting a role in mediating pH homeostasis between the vacuole and plasma membrane H(+)-ATPase . Increased expression of HSP30 and BTN2 in btn1-Delta strains and diminished growth of btn1-Delta, hsp30-Delta, and btn2-Delta strains at low pH reinforce our view that altered pH homeostasis is the underlying cause of Batten disease. Biophys J, 2000 Nov, 79(5), 2624 - 31 PMP1 18-38, a yeast plasma membrane protein fragment, binds phosphatidylserine from bilayer mixtures with phosphatidylcholine: a (2)H-NMR study; Roux M et al.; PMP1 is a 38-residue plasma membrane protein of the yeast Saccharomyces cerevisiae that regulates the activity of the H(+)-ATPase . The cytoplasmic domain conformation results in a specific interfacial distribution of five basic side chains, thought to strongly interact with anionic phospholipids . We have used the PMP1 18-38 fragment to carry out a deuterium nuclear magnetic resonance ((2)H-NMR) study for investigating the interactions between the PMP1 cytoplasmic domain and phosphatidylserines . For this purpose, mixed bilayers of 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphoserine (POPS) were used as model membranes (POPC/POPS 5:1, m/m) . Spectra of headgroup- and chain-deuterated POPC and POPS phospholipids, POPC-d4, POPC-d31, POPS-d3, and POPS-d31, were recorded at different temperatures and for various concentrations of the PMP1 fragment . Data obtained from POPS deuterons revealed the formation of specific peptide-POPS complexes giving rise to a slow exchange between free and bound PS lipids, scarcely observed in solid-state NMR studies of lipid-peptide/protein interactions . The stoichiometry of the complex (8 POPS per peptide) was determined and its significance is discussed . The data obtained with headgroup-deuterated POPC were rationalized with a model that integrates the electrostatic perturbation induced by the cationic peptide on the negatively charged membrane interface, and a "spacer" effect due to the intercalation of POPS/PMP1f complexes between choline headgroups. Biophys J, 2000 Nov, 79(5), 2276 - 89 Molecular dynamics of the anticodon domain of yeast tRNA(Phe): codon-anticodon interaction; Lahiri A et al.; We have studied the effect of codon-anticodon interaction on the structure and dynamics of transfer RNAs using molecular dynamics simulations over a nanosecond time scale . From our molecular dynamical investigations of the solvated anticodon domain of yeast tRNA(Phe) in the presence and absence of the codon trinucleotides UUC and UUU, we find that, although at a gross level the structures are quite similar for the free and the bound domains, there are small but distinct differences in certain parts of the molecule, notably near the Y37 base . Comparison of the dynamics in terms of interatomic or inter-residual distance fluctuation for the free and the bound domains showed regions of enhanced rigidity in the loop region in the presence of codons . Because fluorescence experiments suggested the existence of multiple conformers of the anticodon domain, which interconvert on a much larger time scale than our simulations, we probed the conformational space using five independent trajectories of 500 ps duration . A generalized ergodic measure analysis of the trajectories revealed that at least for this time scale, all the trajectories populated separate parts of the conformational space, indicating a need for even longer simulations or enhanced sampling of the conformational space to give an unequivocal answer to this question. Mol Cell Biol, 2000 Nov, 20(22), 8468 - 79 Nup2p, a yeast nucleoporin, functions in bidirectional transport of importin alpha; Solsbacher J et al.; Import of proteins containing a classical nuclear localization signal (NLS) into the nucleus is mediated by importin alpha and importin beta . Srp1p, the Saccharomyces cerevisiae homologue of importin alpha, returns from the nucleus in a complex with its export factor Cse1p and with Gsp1p (yeast Ran) in its GTP-bound state . We studied the role of the nucleoporin Nup2p in the transport cycle of Srp1p . Cells lacking NUP2 show a specific defect in both NLS import and Srp1p export, indicating that Nup2p is required for efficient bidirectional transport of Srp1p across the nuclear pore complex (NPC) . Nup2p is located at the nuclear side of the central gated channel of the NPC and provides a binding site for Srp1p via its amino-terminal domain . We show that Nup2p effectively releases the NLS protein from importin alpha-importin and beta and strongly binds to the importin heterodimer via Srp1p . Kap95p (importin beta) is released from this complex by a direct interaction with Gsp1p-GTP . These data suggest that besides Gsp1p, which disassembles the NLS-importin alpha-importin beta complex upon binding to Kap95p in the nucleus, Nup2p can also dissociate the import complex by binding to Srp1p . We also show data indicating that Nup1p, a relative of Nup2p, plays a similar role in termination of NLS import . Cse1p and Gsp1p-GTP release Srp1p from Nup2p, which suggests that the Srp1p export complex can be formed directly at the NPC . The changed distribution of Cse1p at the NPC in nup2 mutants also supports a role for Nup2p in Srp1p export from the nucleus. Mol Cell Biol, 2000 Nov, 20(22), 8397 - 408 Cdc13 cooperates with the yeast Ku proteins and Stn1 to regulate telomerase recruitment; Grandin N et al.; The Saccharomyces cerevisiae CDC13 protein binds single-strand telomeric DNA . Here we report the isolation of new mutant alleles of CDC13 that confer either abnormal telomere lengthening or telomere shortening . This deregulation not only depended on telomerase (Est2/TLC1) and Est1, a direct regulator of telomerase, but also on the yeast Ku proteins, yKu70/Hdf1 and yKu80/Hdf2, that have been previously implicated in DNA repair and telomere maintenance . Expression of a Cdc13-yKu70 fusion protein resulted in telomere elongation, similar to that produced by a Cdc13-Est1 fusion, thus suggesting that yKu70 might promote Cdc13-mediated telomerase recruitment . We also demonstrate that Stn1 is an inhibitor of telomerase recruitment by Cdc13, based both on STN1 overexpression and Cdc13-Stn1 fusion experiments . We propose that accurate regulation of telomerase recruitment by Cdc13 results from a coordinated balance between positive control by yKu70 and negative control by Stn1 . Our results represent the first evidence of a direct control of the telomerase-loading function of Cdc13 by a double-strand telomeric DNA-binding complex. Mol Cell Biol, 2000 Nov, 20(22), 8364 - 72 Sok2 regulates yeast pseudohyphal differentiation via a transcription factor cascade that regulates cell-cell adhesion; Pan X et al.; In response to nitrogen limitation, Saccharomyces cerevisiae undergoes a dimorphic transition to filamentous pseudohyphal growth . In previous studies, the transcription factor Sok2 was found to negatively regulate pseudohyphal differentiation . By genome array and Northern analysis, we found that genes encoding the transcription factors Phd1, Ash1, and Swi5 were all induced in sok2/sok2 hyperfilamentous mutants . In accord with previous studies of others, Swi5 was required for ASH1 expression . Phd1 and Ash1 regulated expression of the cell surface protein Flo11, which is required for filamentous growth, and were largely required for filamentation of sok2/sok2 mutant strains . These findings reveal that a complex transcription factor cascade regulates filamentation . These findings also reveal a novel dual role for the transcription factor Swi5 in regulating filamentous growth . Finally, these studies illustrate how mother-daughter cell adhesion can be accomplished by two distinct mechanisms: one involving Flo11 and the other involving regulation of the endochitinase Cts1 and the endoglucanase Egt2 by Swi5. J Biol Chem, 2001 Jan 12, 276(2), 1618 - 25 A role for the peroxin Pex8p in Pex20p-dependent thiolase import into peroxisomes of the yeast Yarrowia lipolytica; Smith JJ et al.; Peroxins are proteins required for peroxisome assembly . The cytosolic peroxin Pex20p binds directly to the beta-oxidation enzyme thiolase and is necessary for its dimerization and peroxisomal targeting . The intraperoxisomal peroxin Pex8p has a role in the import of peroxisomal matrix proteins, including thiolase . We report the results of yeast two-hybrid analyses with various peroxins of the yeast Yarrowia lipolytica and characterize more fully the interaction between Pex8p and Pex20p . Coimmunoprecipitation showed that Pex8p and Pex20p form a complex, while in vitro binding studies demonstrated that the interaction between Pex8p and Pex20p is specific, direct, and autonomous . Pex8p fractionates with peroxisomes in cells of a PEX20 disruption strain, indicating that Pex20p is not necessary for the targeting of Pex8p to peroxisomes . In cells of a PEX8 disruption strain, thiolase is mostly cytosolic, while Pex20p and a small amount of thiolase associate with peroxisomes, suggesting the involvement of Pex8p in the import of thiolase after docking of the Pex20p-thiolase complex to the membrane . In the absence of Pex8p, peroxisomal thiolase and Pex20p are protected from the action of externally added protease . This finding, together with the fact that Pex8p is intraperoxisomal, suggests that Pex20p may accompany thiolase into peroxisomes during import. Cell Prolif, 2000 Aug, 33(4), 209 - 18 Tuning in the transcriptome: basins of attraction in the yeast cell cycle; Klevecz RR et al.; Image processing techniques and wavelet analyses have been applied to the yeast cell cycle expression microchip data to reveal large-scale temporally coherent structures and high frequency oscillations in mRNA levels through the cycle . Because transitions in expression frequently occur in phase, they appear as peaks or troughs in colour maps and contour plots of expression levels . Although apparent in the untreated data, these transitions were identified and enhanced by convolution of a Laplacian kernel with the expression arrays of the first 4096 genes . Transitions associated with maximum up- or down-regulation of mRNA levels appear as bands at 30-40 min intervals through two cell cycles . Time-frequency analyses using wavelet transforms support these visualization techniques and lead to the conclusion that, with respect to gene expression, the dominant period is not the cell cycle (90-120 min) but, more commonly, the higher frequency 30-40 minute submultiple of the cycle period. Proc Natl Acad Sci U S A, 1997 Sep 2, 94(18), 9990 - 9995 Wheat cytosolic acetyl-CoA carboxylase complements an ACC1 null mutation in yeast; Joachimiak M et al.; Spores harboring an ACC1 deletion derived from a diploid Saccharomyces cerevisiae strain, in which one copy of the entire ACC1 gene is replaced with a LEU2 cassette, fail to grow . A chimeric gene consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat cytosolic acetyl-CoA carboxylase (ACCase) cDNA, and yeast ACC1 3' tail was used to complement a yeast ACC1 mutation . The complementation demonstrates that active wheat ACCase can be produced in yeast . At low concentrations of galactose, the activity of the "wheat gene" driven by the GAL10 promoter is low and ACCase becomes limiting for growth, a condition expected to enhance transgenic yeast sensitivity to wheat ACCase-specific inhibitors . An aryloxyphenoxypropionate and two cyclohexanediones do not inhibit growth of haploid yeast strains containing the yeast ACC1 gene, but one cyclohexanedione inhibits growth of the gene-replacement strains at concentrations below 0.2 mM . In vitro, the activity of wheat cytosolic ACCase produced by the gene-replacement yeast strain is inhibited by haloxyfop and cethoxydim at concentrations above 0.02 mM . The activity of yeast ACCase is less affected . The wheat plastid ACCase in wheat germ extract is inhibited by all three herbicides at concentrations below 0.02 mM . Yeast gene-replacement strains will provide a convenient system for the study of plant ACCases. Proc Natl Acad Sci U S A, 1996 Dec 24, 93(26), 15503 - 15507 High-resolution mapping and isolation of a yeast artificial chromosome contig containing fw2.2: A major fruit weight quantitative trait locus in tomato; Alpert KB et al.; A high-resolution physical and genetic map of a major fruit weight quantitative trait locus (QTL), fw2.2, has been constructed for a region of tomato chromosome 2 . Using an F(2) nearly isogenic line mapping population (3472 individuals) derived from Lycopersicon esculentum (domesticated tomato) x Lycopersicon pennellii (wild tomato), fw2.2 has been placed near TG91 and TG167, which have an interval distance of 0.13 +/- 0.03 centimorgan . The physical distance between TG91 and TG167 was estimated to be </= 150 kb by pulsed-field gel electrophoresis of tomato DNA . A physical contig composed of six yeast artificial chromosomes (YACs) and encompassing fw2.2 was isolated . No rearrangements or chimerisms were detected within the YAC contig based on restriction fragment length polymorphism analysis using YAC-end sequences and anchored molecular markers from the high-resolution map . Based on genetic recombination events, fw2.2 could be narrowed down to a region less than 150 kb between molecular markers TG91 and HSF24 and included within two YACs: YAC264 (210 kb) and YAC355 (300 kb) . This marks the first time, to our knowledge, that a QTL has been mapped with such precision and delimited to a segment of cloned DNA . The fact that the phenotypic effect of the fw2.2 QTL can be mapped to a small interval suggests that the action of this QTL is likely due to a single gene . The development of the high-resolution genetic map, in combination with the physical YAC contig, suggests that the gene responsible for this QTL and other QTLs in plants can be isolated using a positional cloning strategy . The cloning of fw2.2 will likely lead to a better understanding of the molecular biology of fruit development and to the genetic engineering of fruit size characteristics. J Cell Biol, 2000 Oct 16, 151(2), 439 - 52 Ordering the final events in yeast exocytosis; Grote E et al.; In yeast, assembly of exocytic soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes between the secretory vesicle SNARE Sncp and the plasma membrane SNAREs Ssop and Sec9p occurs at a late stage of the exocytic reaction . Mutations that block either secretory vesicle delivery or tethering prevent SNARE complex assembly and the localization of Sec1p, a SNARE complex binding protein, to sites of secretion . By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly . In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane . Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane. J Cell Biol, 2000 Oct 16, 151(2), 297 - 310 Sorting of yeast membrane proteins into an endosome-to-Golgi pathway involves direct interaction of their cytosolic domains with Vps35p; Nothwehr SF et al.; Resident late-Golgi membrane proteins in Saccharomyces cerevisiae are selectively retrieved from a prevacuolar-endosomal compartment, a process dependent on aromatic amino acid-based sorting determinants on their cytosolic domains . The formation of retrograde vesicles from the prevacuolar compartment and the selective recruitment of vesicular cargo are thought to be mediated by a peripheral membrane retromer protein complex . We previously described mutations in one of the retromer subunit proteins, Vps35p, which caused cargo-specific defects in retrieval . By genetic and biochemical means we now show that Vps35p directly associates with the cytosolic domains of cargo proteins . Chemical cross-linking, followed by coimmunoprecipitation, demonstrated that Vps35p interacts with the cytosolic domain of A-ALP, a model late-Golgi membrane protein, in a retrieval signal-dependent manner . Furthermore, mutations in the cytosolic domains of A-ALP and another cargo protein, Vps10p, were identified that suppressed cargo-specific mutations in Vps35p but did not suppress the retrieval defects of a vps35 null mutation . Suppression was shown to be due to an improvement in protein sorting at the prevacuolar compartment . These data strongly support a model in which Vps35p acts as a "receptor" protein for recognition of the retrieval signal domains of cargo proteins during their recruitment into retrograde vesicles. Mycoses, 2000 Sep, 43(7-8), 293 - 7 Comparison of Etest with the broth microdilution method in susceptibility testing of yeast isolates against four antifungals; Koc AN et al.; A comparative evaluation of the Etest and the broth microdilution methods for antifungal susceptibility testing of 102 clinical yeast isolates against amphotericin B, fluconazole, itraconazole, and ketoconazole was conducted . The agreements between the Etest and the broth microdilution methods were 93.1% for amphotericin B 85.2% for ketoconazole, 82.3% for itraconazole and 79.4% for fluconazole . These results suggest that the Etest approach to antifungal susceptibility testing may be a viable alternative to the NCCLS reference methods for testing yeasts, but that further evaluations are needed. Exp Cell Res, 2000 Nov 1, 260(2), 277 - 83 Nuclear envelope fission is linked to cytokinesis in budding yeast; Lippincott J et al.; We have investigated the relationship between nuclear envelope fission and cytokinesis during mitotic cell division in budding yeast . By carrying out time-lapse and optical sectioning video microscopy analysis of cells that express green fluorescent protein (GFP)-tagged nuclear envelope and actomyosin ring components, we found that nuclear division is temporally coupled to cytokinesis . Light and electron microscopy analysis also showed that nuclear envelope fission and the division of the nucleoplasm are severely delayed in cytokinesis mutants, resulting in discoupling between the nuclear division cycle and the budding cycle . These results suggest that homotypic membrane fusion may be activated by components or the mechanical action of cytokinetic structures and presents a mechanism for the equal partitioning of the nucleus and the temporal coordination of this event with chromosome segregation during mitosis . J Biol Chem, 2001 Feb 2, 276(5), 3484 - 91 Epub 2000 Oct 17. The yeast NuA4 and Drosophila MSL complexes contain homologous subunits important for transcription regulation; Eisen A et al.; In Drosophila, the MSL complex is required for the dosage compensation of X-linked genes in males and contains a histone acetyltransferase, MOF . A point mutation in the MOF acetyl-CoA-binding site results in male-specific lethality . Yeast Esa1p, a MOF homolog, is essential for cell cycle progression and is the catalytic subunit of the NuA4 acetyltransferase complex . Here we report that NuA4 purified from yeast with a point mutation in the acetyl-CoA-binding domain of Esa1p exhibits a strong decrease in histone acetyltransferase activity, yet has no effect on growth . We demonstrate that Eaf3p (Esa1p-associated factor-3 protein), a yeast protein homologous to the Drosophila dosage compensation protein MSL3, is also a stable component of the NuA4 complex . Unlike other subunits of the complex, it is not essential, and the deletion mutant has no growth phenotype . NuA4 purified from the mutant strain has a decreased apparent molecular mass, but retains wild-type levels of histone H4 acetyltransferase activity . The EAF3 deletion and the ESA1 mutation lead to a decrease in PHO5 gene expression; the EAF3 deletion also significantly reduces HIS4 and TRP4 expressions . These results, together with those previously obtained with both the MSL and NuA4 complexes, underscore the importance of targeted histone H4 acetylation for the gene-specific activation of transcription. J Biol Chem, 2001 Feb 2, 276(5), 3401 - 7 Epub 2000 Oct 17. The yeast Na+/H+ exchanger Nhx1 is an N-linked glycoprotein . Topological implications; Wells KM et al.; Nhx1, the endosomal Na(+)/H(+) exchanger of Saccharomyces cerevisiae represents the founding member of a newly emerging subfamily of intracellular Na(+)/H(+) exchangers . These proteins share significantly greater sequence homology to one another than to members of the mammalian Na(+)/H(+) exchanger (NHE) family encoding plasma membrane Na(+)/H(+) exchangers . Members of both subtypes are predicted to share a common organization, with an N-terminal transporter domain of transmembrane helices followed by a C-terminal hydrophilic tail . In the present study, we show that Nhx1 is an asparagine-linked glycoprotein and that the sites of glycosylation map to two residues within the C-terminal stretch of the polypeptide . This is the first evidence, to date, for glycosylation of the C-terminal region of any known NHE isoform . Importantly, the mapping of N-linked glycosylation to the C-terminal domain of Nhx1 is indicative of an unexpected membrane topology, particularly with regard to the orientation of the tail region . Although one recent study demonstrated that certain epitopes in the C-terminal domain of NHE3 were accessible from the exoplasmic side of the plasma membrane (Biemesderfer, D., DeGray, B., and Aronson, P . S . (1998) J . Biol . Chem . 273, 12391-12396), numerous other studies implicate a cytosolic disposition for the hydrophilic C-terminal tail of plasma membrane NHE isoforms . Our analysis of the glycosylation of Nhx1 is strongly indicative of residence of at least some portion of the hydrophilic tail domain within the endosomal lumen . These findings imply that the organization of the tail domain may be more complex than previously assumed. Mutat Res, 2000 Nov 6, 454(1-2), 53 - 62 Simultaneous measurement of the frequencies of intrachromosomal recombination and chromosome gain using the yeast DEL assay; Howlett NG et al.; The yeast DEL assay measures the frequency of intrachromosomal recombination between two partially-deleted his3 alleles on chromosome XV . The his3Delta alleles share approximately 400bp of overlapping homology, and are separated by an intervening LEU2 sequence . Homologous recombination between the his3Delta alleles results in deletion of the intervening LEU2 sequence (DEL), and reversion to histidine prototrophy . In this study we have attempted to further extend the use of the yeast DEL assay to measure the frequency of chromosome XV gain events . Reversion to His(+)Leu(+) in the haploid yeast DEL tester strain RSY6 occurs upon non-disjunction of chromosome XV sister chromatids, coupled with a subsequent DEL event . Here we have tested the ability of the yeast DEL assay to accurately predict the aneugenic potential of the diversely-acting, known or suspected aneugens actinomycin D, benomyl, chloral hydrate, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and methotrexate . Actinomycin D and benomyl strongly induced aneuploidy . EMS and methotrexate modestly induced aneuploidy, while chloral hydrate and MMS failed to illicit any significant induction . In addition, by FACS-analysis of DNA content it was shown that the majority of both spontaneous- and chemically-induced His(+)Leu(+) revertants were heterodiploid . Thus, our results indicate endoreduplication of almost entire chromosome sets as a major mechanism of aneuploidy induction in haploid Saccharomyces cerevisiae. J Biol Chem, 2001 Jan 12, 276(2), 1523 - 30 Transcriptional regulation of the yeast gmp synthesis pathway by its end products; Escobar-Henriques M et al.; AMP and GMP are synthesized from IMP by specific conserved pathways . In yeast, whereas IMP and AMP synthesis are coregulated, we found that the GMP synthesis pathway is specifically regulated . Transcription of the IMD genes, encoding the yeast homologs of IMP dehydrogenase, was repressed by extracellular guanine . Only this first step of GDP synthesis pathway is regulated, since the latter steps, encoded by the GUA1 and GUK1 genes, are guanine-insensitive . Use of mutants affecting GDP metabolism revealed that guanine had to be transformed into GDP to allow repression of the IMD genes . IMD gene transcription was also strongly activated by mycophenolic acid (MPA), a specific inhibitor of IMP dehydrogenase activity . Serial deletions of the IMD2 gene promoter revealed the presence of a negative cis-element, required for guanine regulation . Point mutations in this guanine response element strongly enhanced IMD2 expression, also making it insensitive to guanine and MPA . From these data, we propose that the guanine response element sequence mediates a repression process, which is enhanced by guanine addition, through GDP or a GDP derivative, and abolished in the presence of MPA. J Biol Chem, 2001 Jan 12, 276(2), 1503 - 9 Adrenodoxin reductase homolog (Arh1p) of yeast mitochondria required for iron homeostasis; Li J et al.; Arh1p is an essential mitochondrial protein of yeast with reductase activity . Here we show that this protein is involved in iron metabolism . A yeast strain was constructed in which the open reading frame was placed under the control of a galactose-regulated promoter . Protein expression was induced by galactose and repressed to undetectable levels in the absence of galactose, although cells grew quite well in the absence of inducer . Under noninducing conditions, cellular iron uptake was dysregulated, exhibiting a failure to repress in response to medium iron . Iron trafficking within the cell was also disturbed . Exposure of Arh1p-depleted cells to increasing iron concentrations during growth led to drastic increases in mitochondrial iron, indicating a loss of homeostatic control . Activity of aconitase, a prototype Fe-S protein, was deficient at all concentrations of mitochondrial iron, although the protein level was unaltered . Heme protein deficiencies were exacerbated in the iron-loaded mitochondria, suggesting a toxic side effect of accumulated iron . Finally, a time course correlated the cellular depletion of Arh1p with the coordinated appearance of various mutant phenotypes including dysregulated cellular iron uptake, deficiency of Fe-S protein activities in mitochondria and cytoplasm, and deficiency of hemoproteins . Thus, Arh1p is required for control of cellular and mitochondrial iron levels and for the activities of Fe-S cluster proteins. J Cell Sci, 2000 Nov, 113 Pt 21, 3871 - 82 The carboxy terminus of Tub4p is required for gamma-tubulin function in budding yeast; Vogel J et al.; The role of gamma-tubulin in microtubule nucleation is well established, however, its function in other aspects of microtubule organization is unknown . The carboxy termini of alpha/beta-tubulins influence the assembly and stability of microtubules . We investigated the role of the carboxy terminus of yeast gamma-tubulin (Tub4p) in microtubule organization . This region consists of a conserved domain (DSYLD), and acidic tail . Cells expressing truncations lacking the DSYLD domain, tail or both regions are temperature sensitive for growth . Growth defects of tub4 mutants lacking either or both carboxy-terminal domains are suppressed by the microtubule destabilizing drug benomyl . tub4 carboxy-terminal mutants arrest as large budded cells with short bipolar spindles positioned at the bud neck . Electron microscopic analysis of wild-type and CTR mutant cells reveals that SPBs are tightly associated with the bud neck/cortex by cytoplasmic microtubules in mutants lacking the tail region (tub4-delta 444, tub4-delta 448) . Mutants lacking the DSYLD residues (tub4-delta 444, tub4-delta DSYLD) form many cytoplasmic microtubules . We propose that the carboxy terminus of Tub4p is required for re-organization of the microtubules upon completion of nuclear migration, and facilitates spindle elongation into the bud. FEBS Lett, 2000 Sep 1, 480(2-3), 287 - 92 The yeast exchange assay, a new complementary method to screen for Dbl-like protein specificity: identification of a novel RhoA exchange factor; De Toledo M et al.; The target Rho GTPases of many guanine nucleotide exchange factors (GEFs) of the Dbl family remain to be identified . Here we report a new method: the yeast exchange assay (YEA), a rapid qualitative test to perform a wide range screen for GEF specificity . In this assay based on the two-hybrid system, a wild type GTPase binds to its effector only after activation by a specific GEF . We validated the YEA by activating GTPases by previously reported GEFs . We further established that a novel GEF, GEF337, activates RhoA in the YEA . GEF337 promoted nucleotide exchange on RhoA in vitro and promoted F-actin stress fiber assembly in fibroblasts, characteristic of RhoA activation. FEBS Lett, 2000 Sep 1, 480(2-3), 265 - 70 The bulk of UCP3 expressed in yeast cells is incompetent for a nucleotide regulated H+ transport; Heidkaemper D et al.; The impact of uncoupling protein (UCP) 1, UCP3 and UCP3s expressed in yeast on oxidative phosphorylation, membrane potential and H+ transport is determined . Intracellular ATP synthesis is inhibited by UCP3, much more than by UCP1, while similar levels of UCP3 and UCP1 exist in the mitochondrial fractions . Measurements of membrane potential and H+ efflux in isolated mitochondria show that, different from UCP1, with UCP3 and UCP3s there is a priori a preponderant uncoupling not inhibited by GDP . The results are interpreted to show that UCP3 and UCP3s in yeast mitochondria are in a deranged state causing uncontrolled uncoupling, which does not represent their physiological function. FEBS Lett, 2000 Oct 13, 483(1), 62 - 6 Increased synthesis and decreased stability of mitochondrial translation products in yeast as a result of loss of mitochondrial (NAD(+))-dependent isocitrate dehydrogenase; de Jong L et al.; We have previously demonstrated that the yeast Krebs cycle enzyme NAD(+)-dependent isocitrate dehydrogenase (Idh) binds specifically and with high affinity to the 5'-untranslated leader sequences of mitochondrial mRNAs in vitro and have proposed a role for the enzyme in the regulation of mitochondrial translation {Elzinga, S.D.J . et al . (2000) Curr . Genet., in press} . Although our studies initially failed to reveal any consistent correlation between idh disruption and mitochondrial translational activity, it is now apparent that compensatory extragenic suppressor mutations readily accumulate in idh disruption strains thereby masking mutant behaviour . Now, pulse-chase protein labelling of isolated mitochondria from an Idh disruption mutant lacking suppressor mutations reveals a strong (2-3-fold) increase in the synthesis of mitochondrial translation products . Strikingly, the newly synthesised proteins are more short-lived than in mitochondria from wild-type cells, their degradation occurring with a 2-3-fold reduced half-life . Enhanced degradation of translation products is also a feature of yeast mutants in which tethering/docking of mitochondrial mRNAs is disturbed . We therefore suggest that binding of Idh to mitochondrial mRNAs may suppress inappropriate translation of mitochondrial mRNAs. Biochem Biophys Res Commun, 2000 Oct 22, 277(2), 518 - 23 The CUG-binding protein binds specifically to UG dinucleotide repeats in a yeast three-hybrid system; Takahashi N et al.; The CUG-binding protein (CUG-BP) has been reported to be involved in the pathogenesis of myotonic dystrophy (DM) through binding to a CUG trinucleotide repeat located in the 3' untranslated region (3'UTR) of the DM protein kinase (DMPK) gene . We found that CUG-BP associates with long CUG trinucleotide repeats ((CUG)(11)(CUG)(12)), but not with short repeats ((CUG)(12)) in a yeast three-hybrid system . On the other hand, CUG-BP+LYLQ, an alternatively spliced isoform of CUG-BP, does not associate with CUG trinucleotide repeats regardless of the repeat length . In addition to these findings, we found that CUG-BP and CUG-BP+LYLQ strongly and specifically associate with UG dinucleotide repeats . Deletion analyse of CUG-BP revealed that the absence of the first or third RNA-binding domain (RBD I and RBD III, respectively) does not affect the interaction between CUG-BP and UG dinucleotide repeats . Loss of the second RNA-binding domain (RBD II) decreases the affinity of CUG-BP for UG dinucleotide repeats by about 40% . Unexpectedly, deletion of the linker domain most severely reduces the interaction, although this region does not contain a known RNA-binding motif . Our results suggest the possibility that both CUG-BP and CUG-BP+LYLQ associate with UG repeat-containing mRNAs and regulate such metabolic properties as mRNA localization, stability, and translation, and provide new insights into the pathogenesis of DM . J Biol Chem, 2001 Jan 5, 276(1), 434 - 41 Probing essential water in yeast pyrophosphatase by directed mutagenesis and fluoride inhibition measurements; Pohjanjoki P et al.; The pattern of yeast pyrophosphatase (Y-PPase) inhibition by fluoride suggests that it replaces active site Mg(2+)-bound nucleophilic water, for which two different locations were proposed previously . To localize the bound fluoride, we investigate here the effects of mutating Tyr(93) and five dicarboxylic amino acid residues forming two metal binding sites in Y-PPase on its inhibition by fluoride and its five catalytic functions (steady-state PP(i) hydrolysis and synthesis, formation of enzyme-bound PP(i) at equilibrium, phosphate-water oxygen exchange, and Mg(2+) binding) . D117E substitution had the largest effect on fluoride binding and made the P-O bond cleavage step rate-limiting in the catalytic cycle, consistent with the mechanism in which the nucleophile is coordinated by two metal ions and Asp(117) . The effects of the mutations on PP(i) hydrolysis (as characterized by the catalytic constant and the net rate constant for P-O bond cleavage) were in general larger than on PP(i) synthesis (as characterized by the net rate constant for PP(i) release from active site) . The effects of fluoride on the Y-PPase variants confirmed that PPase catalysis involves two enzyme.PP(i) intermediates, which bind fluoride with greatly different rates (Baykov, A . A., Fabrichniy, I . P., Pohjanjoki, P., Zyryanov, A . B., and Lahti, R . (2000) Biochemistry 39, 11939-11947) . A mechanism for the structural changes underlying the interconversion of the enzyme.PP(i) intermediates is proposed. Science, 2000 Oct 13, 290(5490), 341 - 4 Plasma membrane compartmentalization in yeast by messenger RNA transport and a septin diffusion barrier; Takizawa PA et al.; Asymmetric localization of proteins plays a key role in many cellular processes, including cell polarity and cell fate determination . Using DNA microarray analysis, we identified a plasma membrane protein-encoding mRNA (IST2) that is transported to the bud tip by an actomyosin-based process . mRNA localization created a higher concentration of IST2 protein in the bud compared with that of the mother cell, and this asymmetry was maintained by a septin-mediated membrane diffusion barrier at the mother-bud neck . These results indicate that yeast creates distinct plasma membrane compartments, as has been described in neurons and epithelial cells. Mol Cell, 2000 Sep, 6(3), 563 - 72 Superhelicity-driven homologous DNA pairing by yeast recombination factors Rad51 and Rad54; Van Komen S et al.; Yeast Rad51 recombinase has only minimal ability to form D loop . Addition of Rad54 renders D loop formation by Rad51 efficient, even when topologically relaxed DNA is used as substrate . Treatment of the nucleoprotein complex of Rad54 and relaxed DNA with topoisomerases reveals dynamic DNA remodeling to generate unconstrained negative and positive supercoils . DNA remodeling requires ATP hydrolysis by Rad54 and is stimulated by Rad51-DNA nucleoprotein complex . A marked sensitivity of DNA undergoing remodeling to P1 nuclease indicates that the negative supercoils produced lead to transient DNA strand separation . Thus, a specific interaction of Rad54 with the Rad51-ssDNA complex enhances the ability of the former to remodel DNA and allows the latter to harvest the negative supercoils generated for DNA joint formation. J Enzyme Inhib, 2000, 15(5), 487 - 96 Dye affinity labelling of yeast alcohol dehydrogenase; Labrou NE; The interaction of yeast alcohol dehydrogenase (ADH) with the reactive chlorotriazine dye Vilmafix Blue A-R (VBAR) was studied . VBAR was purified to homogeneity on lipophilic Sephadex LH-20 and characterised by reverse phase HPLC and analytical TLC . Incubation of ADH with purified VBAR at pH 8.0 and 37 degrees C resulted in a time-dependent inactivation of the enzyme . The observed rate of enzyme inactivation (kobs) exhibited a non-linear dependence on VBAR concentration from 22 to 106 nmol, with a maximum rate of inactivation (k3) of 0.134 min-1 and kD of 141.7 microM . The inhibition was irreversible and activity could not be recovered by gel-filtration chromatography . The inactivation of ADH by VBAR was competitively inhibited by the nucleotides NADH and NAD+ . These results suggest that VBAR acts as an affinity label at the nucleotide binding site of yeast ADH. J Enzyme Inhib, 2000, 15(5), 461 - 9 Expression in yeast and purification of functional recombinant human poly(ADP-ribose)polymerase (PARP) . Comparative pharmacological profile with that of the rat enzyme; Perrin D et al.; Human poly(ADP-ribose)polymerase (PARP) was expressed in the yeast line JEL1 under the control of a GAL promoter . Proteins were extracted and human recombinant PARP purified to apparent homogeneity . The pharmacological profile of this human enzyme was characterised in terms of the effects of known inhibitors of PARP belonging to various chemical families and this was compared with that of the rat enzyme purified from rat testes, using the same purification protocol . The rat and the human enzymes appeared very similar in terms of their sensitivities to those selected inhibitors. Eur J Biochem, 2000 Nov, 267(21), 6443 - 51 Topology and proximity relationships of yeast mitochondrial ATP synthase subunit 8 determined by unique introduced cysteine residues; Stephens AN et al.; We have used site-directed chemical labelling to demonstrate the membrane topology and to identify neighbouring subunits of subunit 8 (Y8) in yeast mitochondrial ATP synthase (mtATPase) . Unique cysteine residues were introduced at the N or C-terminus of Y8 by site-directed mutagenesis . Expression and targeting to mitochondria in vivo of each of these variants in a yeast Y8 null mutant was able to restore activity to an otherwise nonfunctional ATP synthase complex . The position of each introduced cysteine relative to the inner mitochondrial membrane was probed with thiol-specific nonpermeant and permeant reagents in both intact and lysed mitochondria . The data indicate that the N-terminus of Y8 is located in the intermembrane space of mitochondria whereas the C-terminus is located within the mitochondrial matrix . The proximity of Y8 to other proteins of mtATPase was tested using heterobifunctional cross-linking reagents, each with one thiol-specific reactive group and one nonspecific, photoactivatible reactive group . These experiments revealed the proximity of the C-terminal domain of Y8 to subunits d and f, and that of the N-terminal domain to subunit f . It is concluded that Y8 possesses a single transmembrane domain which extends across the inner membrane of intact mitochondria . As subunit d is a likely component of the stator stalk of mitochondrial ATP synthase, we propose, on the basis of the observed cross-links, that Y8 may also be part of the stator stalk. Mol Biol Cell, 2000 Oct, 11(10), 3629 - 43 Yeast exocytic v-SNAREs confer endocytosis; Gurunathan S et al.; In yeast, homologues of the synaptobrevin/VAMP family of v-SNAREs (Snc1 and Snc2) confer the docking and fusion of secretory vesicles at the cell surface . As no v-SNARE has been shown to confer endocytosis, we examined whether yeast lacking the SNC genes, or possessing a temperature-sensitive allele of SNC1 (SNC1(ala43)), are deficient in the endocytic uptake of components from the cell surface . We found that both SNC and temperature-shifted SNC1(ala43) yeast are deficient in their ability to deliver the soluble dye FM4-64 to the vacuole . Under conditions in which vesicles accumulate, FM4-64 stained primarily the cytoplasm as well as fragmented vacuoles . In addition, alpha-factor-stimulated endocytosis of the alpha-factor receptor, Ste2, was fully blocked, as evidenced using a Ste2-green fluorescent protein fusion protein as well as metabolic labeling studies . This suggests a direct role for Snc v-SNAREs in the retrieval of membrane proteins from the cell surface . Moreover, this idea is supported by genetic and physical data that demonstrate functional interactions with t-SNAREs that confer endosomal transport (e.g., Tlg1,2) . Notably, Snc1(ala43) was found to be nonfunctional in cells lacking Tlg1 or Tlg2 . Thus, we propose that synaptobrevin/VAMP family members are engaged in anterograde and retrograde protein sorting steps between the Golgi and the plasma membrane. Nature, 2000 Sep 28, 407(6803), 477 - 83 A yeast prion provides a mechanism for genetic variation and phenotypic diversity; True HL et al.; A major enigma in evolutionary biology is that new forms or functions often require the concerted effects of several independent genetic changes . It is unclear how such changes might accumulate when they are likely to be deleterious individually and be lost by selective pressure . The Saccharomyces cerevisiae prion {PSI+} is an epigenetic modifier of the fidelity of translation termination, but its impact on yeast biology has been unclear . Here we show that {PSI+} provides the means to uncover hidden genetic variation and produce new heritable phenotypes . Moreover, in each of the seven genetic backgrounds tested, the constellation of phenotypes produced was unique . We propose that the epigenetic and metastable nature of {PSI+} inheritance allows yeast cells to exploit pre-existing genetic variation to thrive in fluctuating environments . Further, the capacity of {PSI+} to convert previously neutral genetic variation to a non-neutral state may facilitate the evolution of new traits. Plant Physiol, 2000 Oct, 124(2), 579 - 86 The Arabidopsis UVH1 gene is a homolog of the yeast repair endonuclease RAD1; Fidantsef AL et al.; Ultraviolet radiation induces DNA damage products, largely in the form of pyrimidine dimers, that are both toxic and mutagenic . In most organisms, including Arabidopsis, these lesions are repaired both through a dimer-specific photoreactivation mechanism and through a less efficient light-independent mechanism . Several mutants defective in this "dark repair" pathway have been previously described . The mechanism of this repair has not been elucidated, but is thought to be homologous to the nucleotide excision repair mechanisms found in other eukaryotes . Here we report the complementation of the Arabidopsis uvh1 dark repair mutant with the Arabidopsis homolog of the yeast nucleotide excision repair gene RAD1, which encodes one of the subunits of the 5'-repair endonuclease . The uvh1-2 mutant allele carries a glycine-->aspartate amino acid change that has been previously identified to produce a null allele of RAD1 in yeast . Although Arabidopsis homologs of genes involved in nucleotide excision repair are readily identified by searching the genomic database, it has not been established that these homologs are actually required for dark repair in plants . The complementation of the Arabidopsis uvh1 mutation with the Arabidopsis RAD1 homolog clearly demonstrates that the mechanism of nucleotide excision repair is conserved among the plant, animal, and fungal kingdoms. Biochem Biophys Res Commun, 2000 Oct 5, 276(3), 940 - 4 Role of voltage-dependent anion channels in glutathione transport into yeast mitochondria; Cummings BS et al.; Glutathione (GSH) is imported into mitochondria from the extra-mitochondrial cytoplasm . Translocation across the inner membrane of mitochondria is thought to occur via the dicarboxylate and 2-oxoglutarate carriers; however, the means by which GSH passes through the outer membrane is unknown . Disruption of the outer membrane of yeast mitochondria using either digitonin or osmotic shock did not alter GSH accumulation as compared with accumulation in intact mitochondria . These results suggested that passage across the outer membrane was not the rate-limiting step in GSH accumulation . Mitochondria isolated from yeast strains with a disruption in the major pore-forming protein of the outer membrane, VDAC1, accumulated GSH to a greater extent than mitochondria isolated from a wild-type strain . Disruption of the gene for VDAC2 did not affect GSH import . Thus, neither VDAC form is essential for GSH translocation into mitochondria, and the participation of another outer membrane channel in GSH import is possible . Mol Cell Biol, 2000 Nov, 20(21), 8001 - 7 Replication past O(6)-methylguanine by yeast and human DNA polymerase eta; Haracska L et al.; O(6)-Methylguanine (m6G) is formed by the action of alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on DNA . m6G is a highly mutagenic and carcinogenic lesion, and it presents a block to synthesis by DNA polymerases . Here, we provide genetic and biochemical evidence for the involvement of yeast and human DNA polymerase eta (Poleta) in the replicative bypass of m6G lesions in DNA . The formation of MNNG-induced mutations is almost abolished in the rad30Delta pol32Delta double mutant of yeast, which lacks the RAD30 gene that encodes Poleta and the Pol32 subunit of DNA polymerase delta (Poldelta) . Although Poldelta can function in the mutagenic bypass of m6G lesions, our biochemical studies indicate that Poleta is much more efficient in replicating through m6G than Poldelta . Both Poleta and Poldelta insert a C or a T residue opposite from m6G; Poleta, however, is more accurate, as it inserts a C about twice as frequently as Poldelta . Alkylating agents are used in the treatment of malignant tumors, including lymphomas, brain tumors, melanomas, and gastrointestinal carcinomas, and the clinical effectiveness of these agents derives at least in part from their ability to form m6G in DNA . Inactivation of Poleta could afford a useful strategy for enhancing the effectiveness of these agents in cancer chemotherapy. Mol Cell Biol, 2000 Nov, 20(21), 7991 - 8000 Telomere folding is required for the stable maintenance of telomere position effects in yeast; de Bruin D et al.; Yeast telomeres reversibly repress the transcription of adjacent genes, a phenomenon called telomere position effect (TPE) . TPE is thought to result from Rap1 and Sir protein-mediated spreading of heterochromatin-like structures from the telomeric DNA inwards . Because Rap1p is associated with subtelomeric chromatin as well as with telomeric DNA, yeast telomeres are proposed to form fold-back or looped structures . TPE can be eliminated in trans by deleting SIR genes or in cis by transcribing through the C(1-3)A/TG(1-3) tract of a telomere . We show that the promoter of a telomere-linked URA3 gene was inaccessible to restriction enzymes and that accessibility increased both in a sir3 strain and upon telomere transcription . We also show that subtelomeric chromatin was hypoacetylated at histone H3 and at each of the four acetylatable lysines in histone H4 and that histone acetylation increased both in a sir3 strain and when the telomere was transcribed . When transcription through the telomeric tract occurred in G(1)-arrested cells, TPE was lost, demonstrating that activation of a silenced telomeric gene can occur in the absence of DNA replication . The loss of TPE that accompanied telomere transcription resulted in the rapid and efficient loss of subtelomeric Rap1p . We propose that telomere transcription disrupts core heterochromatin by eliminating Rap1p-mediated telomere looping . This interpretation suggests that telomere looping is critical for maintaining TPE. Mol Cell Biol, 2000 Nov, 20(21), 7971 - 9 Yeast Krr1p physically and functionally interacts with a novel essential Kri1p, and both proteins are required for 40S ribosome biogenesis in the nucleolus; Sasaki T et al.; Using a two-hybrid screening with TOM1, a putative ubiquitin-ligase gene of Saccharomyces cerevisiae, we isolated KRR1, a homologue of human HRB2 (for human immunodeficiency virus type 1 Rev-binding protein 2) . To characterize the gene function, we constructed temperature-sensitive krr1 mutants and isolated two multicopy suppressors . One suppressor is RPS14A, encoding a 40S ribosomal protein . The C-terminal-truncated rpS14p, which was reported to have diminished binding activity to 18S rRNA, failed to suppress the krr1 mutant . The other suppressor is a novel gene, KRI1 (for KRR1 interacting protein; YNL308c) . KRI1 is essential for viability, and Kri1p is localized to the nucleolus . We constructed a galactose-dependent kri1 strain by placing KRI1 under control of the GAL1 promoter, so that expression of KRI1 was shut off when transferring the culture to glucose medium . Polysome and 40S ribosome fractions were severely decreased in the krr1 mutant and Kri1p-depleted cells . Pulse-chase analysis of newly synthesized rRNAs demonstrated that 18S rRNA is not produced in either mutant . However, wild-type levels of 25S rRNA are made in either mutant . Northern analysis revealed that the steady-state levels of 18S rRNA and 20S pre-rRNAs were reduced in both mutants . Precursors for 18S rRNA were detected but probably very unstable in both mutants . A myc-tagged Kri1p coimmunoprecipitated with a hemagglutinin-tagged Krr1p . Furthermore, the krr1 mutant protein was defective in its interaction with Kri1p . These data lead us to conclude that Krr1p physically and functionally interacts with Kri1p to form a complex which is required for 40S ribosome biogenesis in the nucleolus. Mol Cell Biol, 2000 Nov, 20(21), 7943 - 54 Functional characterization of nuclear localization signals in yeast Sm proteins; Bordonne R; In mammals, nuclear localization of U-snRNP particles requires the snRNA hypermethylated cap structure and the Sm core complex . The nature of the signal located within the Sm core proteins is still unknown, both in humans and yeast . Close examination of the sequences of the yeast SmB, SmD1, and SmD3 carboxyl-terminal domains reveals the presence of basic regions that are reminiscent of nuclear localization signals (NLSs) . Fluorescence microscopy studies using green fluorescent protein (GFP)-fusion proteins indicate that both yeast SmB and SmD1 basic amino acid stretches exhibit nuclear localization properties . Accordingly, deletions or mutations in the NLS-like motifs of SmB and SmD1 dramatically reduce nuclear fluorescence of the GFP-Sm mutant fusion alleles . Phenotypic analyses indicate that the NLS-like motifs of SmB and SmD1 are functionally redundant: each NLS-like motif can be deleted without affecting yeast viability whereas a simultaneous deletion of both NLS-like motifs is lethal . Taken together, these findings suggest that, in the doughnut-like structure formed by the Sm core complex, the carboxyl-terminal extensions of Sm proteins may form an evolutionarily conserved basic amino acid-rich protuberance that functions as a nuclear localization determinant. Mol Cell Biol, 2000 Nov, 20(21), 7933 - 42 mRNA decapping in yeast requires dissociation of the cap binding protein, eukaryotic translation initiation factor 4E; Schwartz DC et al.; A major pathway of eukaryotic mRNA turnover occurs by deadenylation-dependent decapping that exposes the transcript to 5'-->3' exonucleolytic degradation . A critical step in this pathway is decapping, since removal of the cap structure permits 5'-->3' exonucleolytic digestion . Based on alterations in mRNA decay rate from strains deficient in translation initiation, it has been proposed that the decapping rate is modulated by a competition between the cytoplasmic cap binding complex, which promotes translation initiation, and the decapping enzyme, Dcp1p . In order to test this model directly, we examined the functional interaction of Dcp1p and the cap binding protein, eukaryotic translation initiation factor 4E (eIF4E), in vitro . These experiments indicated that eIF4E is an inhibitor of Dcp1p in vitro due to its ability to bind the 5' cap structure . In addition, we demonstrate that in vivo a temperature-sensitive allele of eIF4E (cdc33-42) suppressed the decapping defect of a partial loss-of-function allele of DCP1 . These results argue that dissociation of eIF4E from the cap structure is required before decapping . Interestingly, the temperature-sensitive allele of eIF4E does not suppress the decapping defect seen in strains lacking the decapping activators, Lsm1p and Pat1p . This indicates that these activators of decapping affect a step in mRNA turnover distinct from the competition between Dcp1 and eIF4E. Mol Cell Biol, 2000 Nov, 20(21), 7881 - 92 In yeast, the 3' untranslated region or the presequence of ATM1 is required for the exclusive localization of its mRNA to the vicinity of mitochondria; Corral-Debrinski M et al.; We isolated mitochondria from Saccharomyces cerevisiae to selectively study polysomes bound to the mitochondrial surface . The distribution of several mRNAs coding for mitochondrial proteins was examined in free and mitochondrion-bound polysomes . Some mRNAs exclusively localize to mitochondrion-bound polysomes, such as the ones coding for Atm1p, Cox10p, Tim44p, Atp2p, and Cot1p . In contrast, mRNAs encoding Cox6p, Cox5a, Aac1p, and Mir1p are found enriched in free cytoplasmic polysome fractions . Aac1p and Mir1p are transporters that lack cleavable presequences . Sequences required for mRNA asymmetric subcellular distribution were determined by analyzing the localization of reporter mRNAs containing the presequence coding region and/or the 3'-untranslated region (3'UTR) of ATM1, a gene encoding an ABC transporter of the mitochondrial inner membrane . Biochemical analyses of mitochondrion-bound polysomes and direct visualization of RNA localization in living yeast cells allowed us to demonstrate that either the presequence coding region or the 3'UTR of ATM1 is sufficient to allow the reporter mRNA to localize to the vicinity of the mitochondrion, independently of its translation . These data demonstrate that mRNA localization is one of the mechanisms used, in yeast, for segregating mitochondrial proteins. Nat Cell Biol, 2000 Oct, 2(10), 677 - 85 Chemical genetic analysis of the budding-yeast p21-activated kinase Cla4p; Weiss EL et al.; The p21-activated kinases (PAKs) are effectors for the Rho-family GTPase Cdc42p . Here we define the in vivo function of the kinase activity of the budding yeast PAK Cla4p, using cla4 alleles that are specifically inhibited by a cell-permeable compound that does not inhibit the wild-type kinase . CLA4 kinase inhibition in cells lacking the partially redundant PAK Ste20p causes reversible SWE1-dependent cell-cycle arrest and gives rise to narrow, highly elongated buds in which both actin and septin are tightly polarized to bud tips . Inhibition of Cla4p does not prevent polarization of F-actin, and cytokinesis is blocked only in cells that have not formed a bud before inhibitor treatment; cell polarization and bud emergence are not affected by Cla4p inhibition . Although localization of septin to bud necks is restored in swe1Delta cells, cytokinesis remains defective . Inhibition of Cla4p activity in swe1Delta cells causes a delay of bud emergence after cell polarization, indicating that this checkpoint may mediate an adaptive response that is capable of promoting budding when Cla4p function is reduced . Our data indicate that CLA4 PAK activity is required at an early stage of budding, after actin polarization and coincident with formation of the septin ring, for early bud morphogenesis and assembly of a cytokinesis site. Yeast, 2000 Sep 30, 17(3), 170 - 87 The dual origin of the yeast mitochondrial proteome; Karlberg O et al.; We propose a scheme for the origin of mitochondria based on phylogenetic reconstructions with more than 400 yeast nuclear genes that encode mitochondrial proteins . Half of the yeast mitochondrial proteins have no discernable bacterial homologues, while one-tenth are unequivocally of alpha-proteobacterial origin . These data suggest that the majority of genes encoding yeast mitochondrial proteins are descendants of two different genomic lineages that have evolved in different modes . First, the ancestral free-living alpha-proteobacterium evolved into an endosymbiont of an anaerobic host . Most of the ancestral bacterial genes were lost, but a small fraction of genes supporting bioenergetic and translational processes were retained and eventually transferred to what became the host nuclear genome . In a second, parallel mode, a larger number of novel mitochondrial genes were recruited from the nuclear genome to complement the remaining genes from the bacterial ancestor . These eukaryotic genes, which are primarily involved in transport and regulatory functions, transformed the endosymbiont into an ATP-exporting organelle . Mech Dev, 2000 Oct, 97(1-2), 3 - 12 Yeast two-hybrid system identifies the ubiquitin-conjugating enzyme mUbc9 as a potential partner of mouse Dac; Machon O et al.; Using a yeast two hybrid system and pull-down assays we demonstrate that mouse Dac (mDac) specifically binds to mouse ubiquitin-conjugating enzyme mUbc9 . In contrast to a direct interaction between Drosophila dachshund (dac) and eyes absent (eya)gene products, we cannot detect by the same methods that mDac binds to mEya2, a functional mouse homologue of the Drosophila Eya . Immunostaining of various cell lines that were transfected with mDac reveals that mDac protein is found predominantly in the nucleus but translocates to the cytoplasm and condensates along the nuclear membrane in a cell-cycle dependent manner . Deletion analysis of mDac show the intracellular localization and protein stability correlates with the binding to mUbc9 . The C-terminal half of mDac, which associates with mUbc9, remains cytoplasmic and is degraded in proteasome whereas the non-interacting N-terminus is exclusively nuclear and more stable than the full-length mDac or its C-terminal portion . In situ hybridization on whole-mount embryos or tissue sections detects mUbc9 transcripts in complementary and overlapping areas with mDac expression, particularly in the proliferation zone of the limb buds, the spinal cord and forebrain . Mouse embryos stained with an anti-mDac antibody document that mDac is localized both in the nucleus and the cytoplasm with a cytoplasmic predominance in migrating neural crest cells . In the proliferation zone, visible nuclear envelopes are not formed and mDac is detected throughout the cells. Nucleic Acids Res . 2000 Oct 15;28(20):E88. High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2; Abecassis V et al.; The design of a family shuffling strategy (CLERY: Combinatorial Libraries Enhanced by Recombination in Yeast) associating PCR-based and in vivo recombination and expression in yeast is described . This strategy was tested using human cytochrome P450 CYP1A1 and CYP1A2 as templates, which share 74% nucleotide sequence identity . Construction of highly shuffled libraries of mosaic structures and reduction of parental gene contamination were two major goals . Library characterization involved multiprobe hybridization on DNA macro-arrays . The statistical analysis of randomly selected clones revealed a high proportion of chimeric genes (86%) and a homogeneous representation of the parental contribution among the sequences (55.8 +/- 2.5% for parental sequence 1A2) . A microtiter plate screening system was designed to achieve colorimetric detection of polycyclic hydrocarbon hydroxylation by transformed yeast cells . Full sequences of five randomly picked and five functionally selected clones were analyzed . Results confirmed the shuffling efficiency and allowed calculation of the average length of sequence exchange and mutation rates . The efficient and statistically representative generation of mosaic structures by this type of family shuffling in a yeast expression system constitutes a novel and promising tool for structure-function studies and tuning enzymatic activities of multicomponent eucaryote complexes involving non-soluble enzymes. J Biol Chem, 2000 Dec 29, 275(52), 40961 - 6 Sds3 (suppressor of defective silencing 3) is an integral component of the yeast Sin3{middle dot}Rpd3 histone deacetylase complex and is required for histone deacetylase activity; Lechner T et al.; SDS3 (suppressor of defective silencing 3) was originally identified in a screen for mutations that cause increased silencing of a crippled HMR silencer in a rap1 mutant background . In addition, sds3 mutants have phenotypes very similar to those seen in sin3 and rpd3 mutants, suggesting that it functions in the same genetic pathway . In this manuscript we demonstrate that Sds3p is an integral subunit of a previously identified high molecular weight Rpd3p.Sin3p containing yeast histone deacetylase complex . By analyzing an sds3Delta strain we show that, in the absence of Sds3p, Sin3p can be chromatographically separated from Rpd3p, indicating that Sds3p promotes the integrity of the complex . Moreover, the remaining Rpd3p complex in the sds3Delta strain had little or no histone deacetylase activity . Thus, Sds3p plays important roles in the integrity and catalytic activity of the Rpd3p.Sin3p complex. FASEB J, 2000 Nov, 14(14), 2135 - 7 An intervention resembling caloric restriction prolongs life span and retards aging in yeast; Jiang JC et al.; The yeast Saccharomyces cerevisiae has a finite life span that is measured by the number of daughter cells an individual produces . The 20 genes known to determine yeast life span appear to function in more than one pathway, implicating a variety of physiological processes in yeast longevity . Less attention has been focused on environmental effects on yeast aging . We have examined the role that nutritional status plays in determining yeast life span . Reduction of the glucose concentration in the medium led to an increase in life span and to a delay in appearance of an aging phenotype . The increase in life span was the more extensive the lower the glucose levels . Life extension was also elicited by decreasing the amino acids content of the medium . This suggests that it is the decline in calories and not a particular nutrient that is responsible, in striking similarity to the effect on aging of caloric restriction in mammals . The caloric restriction effect did not require the induction of the retrograde response pathway, which signals the functional status of the mitochondrion and determines longevity . Furthermore, deletion of RTG3, a downstream mediator in this pathway, and caloric restriction had an additive effect, resulting in the largest increase (123%) in longevity described thus far in yeast . Thus, retrograde response and caloric restriction operate along distinct pathways in determining yeast longevity . These pathways may be exclusive, at least in part . This provides evidence for multiple mechanisms of metabolic control in yeast aging . Inasmuch as caloric restriction lowers blood glucose levels, this study raises the possibility that reduced glucose alters aging at the cellular level in mammals. Methods, 2000 Oct, 22(2), 116 - 9 Mutagenesis assays in yeast; Crouse GF; Analyzing mutation spectra is a very powerful method to determine the effects of various types of DNA damage and to understand the workings of various DNA repair pathways . However, compiling sequence-specific mutation spectra is laborious; even with modern sequencing technology, it is rare to obtain spectra with more than several hundred data points . Two assay systems are described for yeast, one for insertion/deletion mutations and one for base substitution mutations, that allow determination of specific mutations without the necessity of DNA sequencing . The assay for insertion/deletion mutations uses a variety of different simple repeats placed in frame with URA3 such that insertions or deletions lead to a selectable Ura(-) phenotype; essentially all such mutations are in the simple repeat sequence . The assay for base substitution mutations uses a series of six strains with different mutations in one essential codon of the CYC1 gene . Because only true reversions lead to a selectable phenotype, the bases mutated in any reversion event are known . The advantage of these assays is that they can quantitatively determine over several orders of magnitude the types of mutations that occur under a given set of conditions, without DNA sequencing . Arch Biochem Biophys, 2000 Sep 1, 381(1), 164 - 70 Coexpression of genetically engineered fused enzyme between yeast NADPH-P450 reductase and human cytochrome P450 3A4 and human cytochrome b5 in yeast; Hayashi K et al.; Human hepatic cytochrome P450 3A4 (CYP3A4) was expressed in yeast Saccharomyces cerevisiae . While the expression level was high as compared with other human hepatic cytochrome P450s, CYP3A4 showed almost no catalytic activity toward testosterone . Coexpression of CYP3A4 with yeast NADPH-P450 reductase did not give a full activity . Low monooxygenase activity of CYP3A4 was attributed to the insufficient reduction of heme iron of CYP3A4 by NADPH-P450 reductase . To enhance the efficiency of electron transfer from NADPH-P450 reductase to CYP3A4, a fused enzyme was constructed between CYP3A4 and yeast NADPH-P450 reductase . The rapid reduction of the heme iron of the fused enzyme by NADPH was observed . The fused enzyme showed a high testosterone 6beta-hydroxylation activity with a sigmoidal velocity saturation curve . However, the coupling efficiency between NADPH utilization and testosterone 6beta-hydroxylation was only 10% . Finally, coexpression of the fused enzyme and human cytochrome b5 was examined . A significant decrease in the Km value and a remarkable increase in the coupling efficiency were observed . Substrate-induced spectra revealed that the dissociation constant of the fused enzyme for testosterone significantly decreased with coexpression of human cytochrome b5 . These results strongly suggest that human cytochrome b5 directly interacts with the CYP3A4 domain of the fused enzyme and modifies the tertiary structure of substrate binding pocket, resulting in tight binding of the substrate and high coupling efficiency. Biochim Biophys Acta, 2000 Sep 29, 1481(2), 265 - 72 Biphasic kinetic behavior of rat cytochrome P-4501A1-dependent monooxygenation in recombinant yeast microsomes; Inouye K et al.; Rat cytochrome P-4501A1-dependent monooxygenase activities were examined in detail using recombinant yeast microsomes containing rat cytochrome P-4501A1 and yeast NADPH-P-450 reductase . On 7-ethoxycoumarin, which is one of the most popular substrates of P-4501A1, the relationship between the initial velocity (v) and the substrate concentration ({S}) exhibited non-linear Michaelis-Menten kinetics . Hanes-Woolf plots ({S}/v vs . {S}) clearly showed a biphasic kinetic behavior . Aminopyrine N-demethylation also showed a biphasic kinetics . The regression analyses on the basis of the two-substrate binding model proposed by Korzekwa et al . (Biochemistry 37 (1998) 4137-4147) strongly suggest the presence of the two substrate-binding sites in P-4501A1 molecules for those substrates . An Arrhenius plot with high 7-ethoxycoumarin concentration showed a breakpoint at around 28 degrees C probably due to the change of the rate-limiting step of P-4501A1-dependent 7-ethoxycoumarin O-deethylation . However, the addition of 30% glycerol to the reaction mixture prevented observation of the breakpoint . The methanol used as a solvent of 7-ethoxycoumarin was found to be a non-competitive inhibitor . Based on the inhibition kinetics, the real V(max) value in the absence of methanol was calculated . These results strongly suggest that the recombinant yeast microsomal membrane containing a single P-450 isoform and yeast NADPH-P-450 reductase is quite useful for kinetic studies on P-450-dependent monooxygenation including an exact evaluation of inhibitory effects of organic solvents. Biochim Biophys Acta, 2000 Sep 29, 1468(1-2), 99 - 106 Genetic characterization of the (534)DPPR motif of the yeast plasma membrane H(+)-ATPase; Portillo F; The highly conserved motif +(534)DPPR of Saccharomyces cerevisiae H(+)-ATPase, located in the putative ATP binding site, has been mutagenized and the resulting 23 mutant genes conditionally expressed in secretory vesicles . Fourteen mutant ATPases (D534A, D534V, D534L, D534N, D534G, D534T, P535A, P535V, P535L, P535G, P535T, P535E, P535K and R537T) failed to reach the secretory vesicles . Of these mutants, nine (D534N, D534T, P535A, P535V, P535L, P535G, P535T, P535E and P535K) were not detected in total cellular membranes, and five (D534A, D534V, D534G, D