Yeasts

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, D534L and R537T) were retained at the endoplasmic reticulum and exhibited a dominant lethal phenotype . The remaining mutants (D534E, R537A, R537V, R537L, R537N, R537G, R537E, R537K and R537H) reached the secretory vesicles at levels similar to that of the wild type . Of these, six (R537A, R537V, R537L, R537N, R537G, and R537E) showed severely decreased ATPase activity compared to the wild type enzyme, and three (D534E, R537K and R537H) rendered an enzyme with an altered K(m) for ATP.

Mutat Res, 2000 Oct 16, 461(2), 133 - 43
Alteration of ultraviolet-induced mutagenesis in yeast through molecular modulation of the REV3 and REV7 gene expression; Rajpal DK et al.; DNA damage can lead to mutations during replication . The damage-induced mutagenesis pathway is an important mechanism that fixes DNA lesions into mutations . DNA polymerase zeta (Pol zeta), formed by Rev3 and Rev7 protein complex, and Rev1 are components of the damage-induced mutagenesis pathway . Since mutagenesis is an important factor during the initiation and progression of human cancer, we postulate that this mutagenesis pathway may provide an inhibiting target for cancer prevention and therapy . In this study, we tested if UV-induced mutagenesis can be altered by molecular modulation of Rev3 enzyme levels using the yeast Saccharomyces cerevisiae as a eukaryotic model system . Reducing the REV3 expression in yeast cells through molecular techniques was employed to mimic Pol zeta inhibition . Lower levels of Pol zeta significantly decreased UV-induced mutation frequency, thus achieving inhibition of mutagenesis . In contrast, elevating the Pol zeta level by enhanced expression of both REV3 and REV7 genes led to a approximately 3-fold increase in UV-induced mutagenesis as determined by the arg4-17 mutation reversion assays . In vivo, UV lesion bypass by Pol zeta requires the Rev1 protein . Even overexpression of Pol zeta could not alleviate the defective UV mutagenesis in the rev1 mutant cells . These observations provide evidence that the mutagenesis pathway could be used as a target for inhibiting damage-induced mutations.

Biochimie, 2000 Aug, 82(8), 733 - 8
The role of ERG20 gene (encoding yeast farnesyl diphosphate synthase) mutation in long dolichol formation . Molecular modeling of FPP synthase; Plochocka D et al.; The yeast Saccharomyces cerevisiae strain LB332 bearing a mutation in the ERG20 gene encoding farnesyl diphosphate synthase (FPPS) synthesizes significantly longer dolichols than the wild type strain FL100 (14-31 and 14-19 isoprene units, respectively) . The measurement of the short chain prenyl alcohols excreted into the medium shows that increased amounts of geraniol, dimethylallyl and isopentenyl alcohols but not farnesol are synthesized by the mutant strain . The wild type FPPS synthesizes farnesyl diphosphate (FPP) as the only product . The K197E substitution, as opposed to F112A/F113S in avian FPPS, does not change product specificity . Consequently, the possibility that mutated yeast FPPS synthesizes longer polyprenols is unlikely . This is supported by additional evidence such as in vitro analysis of the mutated FPPS products and molecular modeling . We suggest that formation of longer dolichols in vivo is the result of a change in the isopentenyl diphosphate/farnesyl diphosphate ratio caused by the erg20 mutation which in turn affects the activity of cis-prenyltransferase.

J Colloid Interface Sci, 2000 Oct 15, 230(2), 328 - 333
Activity and Conformation of Yeast Alcohol Dehydrogenase (YADH) Entrapped in Reverse Micelles; Das S et al.; Yeast alcohol dehydrogenase (YADH) solubilized in reverse micelles of aerosol OT (i.e., AOT or sodium bis (2-ethyl hexyl) sulfosuccinate) in isooctane has been shown to be catalytically more active than that in aqueous buffer under optimum conditions of pH, temperature, and water content in reverse micelles . Studies of the secondary structure conformational changes of the enzyme in reverse micelles have been made from circular dichroism spectroscopy . It has been seen that the conformation of YADH in reverse micelles is extremely sensitive to pH, temperature, and water content . A comparison has been made between the catalytic activity of the enzyme and the alpha-helix content in the conformation and it has been observed that the enzyme is most active at the maximum alpha-helix content . While the beta-sheet content in the conformation of the entrapped enzyme was found to be dependent on the enzyme-micelle interface interaction, the alpha-helix and random coil conformations are governed by the degree of entrapment and the extent of rigidity provided by the micelle core to the enzyme structure .

Nat Struct Biol, 2000 Oct, 7(10), 894 - 902
Interactions within the yeast t-SNARE Sso1p that control SNARE complex assembly; Munson M et al.; In the eukaryotic secretory and endocytic pathways, transport vesicles shuttle cargo among intracellular organelles and to and from the plasma membrane . Cargo delivery entails fusion of the transport vesicle with its target, a process thought to be mediated by membrane bridging SNARE protein complexes . Temporal and spatial control of intracellular trafficking depends in part on regulating the assembly of these complexes . In vitro, SNARE assembly is inhibited by the closed conformation adopted by the syntaxin family of SNAREs . To visualize this closed conformation directly, the X-ray crystal structure of a yeast syntaxin, Sso1p, has been determined and refined to 2.1 A resolution . Mutants designed to destabilize the closed conformation exhibit accelerated rates of SNARE assembly . Our results provide insight into the mechanism of SNARE assembly and its intramolecular and intermolecular regulation.

Nat Biotechnol, 2000 Oct, 18(10), 1075 - 9
Use of G-protein fusions to monitor integral membrane protein-protein interactions in yeast; Ehrhard KN et al.; The control of protein-protein interactions is a fundamental aspect of cell regulation . Here we describe a new approach to detect the interaction of two proteins in vivo . By this method, one binding partner is an integral membrane protein whereas the other is soluble but fused to a G-protein gamma-subunit . If the binding partners interact, G-protein signaling is disrupted . We demonstrate interaction between known binding partners, syntaxin 1a with neuronal Sec1 (nSec1), and the fibroblast-derived growth factor receptor 3 (FGFR3) with SNT-1 . In addition, we describe a genetic screen to identify nSec1 mutants that are expressed normally, but are no longer able to bind to syntaxin 1a . This provides a convenient method to study interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods.

Biometals, 2000 Jun, 13(2), 153 - 6
Copper-release from yeast Cu(I)-metallothionein by nitric oxide (NO); Hartmann HJ et al.; The reaction of yeast Cu-MT with nitric oxide (NO) was examined . A release of copper from the Cu(I)-thiolate clusters of the protein by this remarkably important reagent was observed in vitro . The characteristic spectroscopic signals of the Cu(I)-thiolate chromophores levelled off in the presence of a two-fold molar excess of NO expressed per equivalent of thionein-copper as monitored by UV-electronic absorption, circular dichroism and luminescence emission . At the same time all of the copper became EPR detectable . The oxidized metal ions could easily be removed from the protein moiety by gelfiltration . The reversibility of the copper releasing process is of special interest . The specific fluorescence and dichroic properties of the previously demetallized protein could be recovered up to 85% under reductive conditions . Moreover, no difference in the electrophoretic behaviour was seen compared to the untreated Cu-MT . Thus, NO may act as a potent metabolic source for the transient copper release from Cu-MT . In the course of an oxidative burst this highly Fenton active copper is able to improve the efficacy of biological defence mechanisms.

Yeast, 2000 Oct, 16(14), 1313 - 23
Destabilized green fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry; Mateus C et al.; Green fluorescent protein (GFP) has many advantages as a reporter molecule, but its stability makes it unsuitable for monitoring dynamic changes in gene expression, among other applications . Destabilized GFPs have been developed for bacterial and mammalian systems to counter this problem . Here, we extend such advances to the yeast model . We fused the PEST-rich 178 carboxyl-terminal residues of the G(1) cyclin Cln2 to the C terminus of yEGFP3 (a yeast- and FACS-optimized GFP variant), creating yEGFP3-Cln2(PEST) . We tested the hybrid protein after integrating modules harbouring the yEGFP3 or yEGFP3-CLN2(PEST) ORFs into the Saccharomyces cerevisiae genome . yEGFP3- Cln2(PEST) had a markedly shorter half-life (t(1/2)) than yEGFP3; inhibition of protein synthesis with cycloheximide lead to a rapid decline in GFP content and fluorescence (t(1/2) approximately 30 min) in cells expressing yEGFP3-Cln2(PEST), whereas these parameters were quite stable in yEGFP3-expressing cells (t(1/2) approximately 7 h) . We placed yEGFP3-CLN2(PEST) under the control of the CUP1 promoter, which is induced only transiently by copper . This transience was readily discernible with yEGFP3-Cln2(PEST), whereas yEGFP3 reported only on CUP1 switch-on, albeit more slowly than yEGFP3-Cln2(PEST) . Cell cycle-regulated transcriptional activation/inactivation of the CLN2 promoter was also discernible with yEGFP3- Cln2(PEST), using cultures that were previously synchronized with nocodazole . In comparison to CLN2, expression from the ACT1 promoter was stable after release from nocodazole . We also applied a novel flow-cytometric technique for cell cycle analysis with asynchronous cultures . The marked periodicities of CLN2 and CLB2 (mitotic cyclin) transcription were readily evident from cellular yEGFP3-Cln2(PEST) levels with this non-perturbing approach . The results represent the first reported successful destabilization of a yeast-GFP . This new construct expands the range of GFP applications open to yeast workers .

Yeast, 2000 Oct, 16(14), 1299 - 312
The yeast Ura2 protein that catalyses the first two steps of pyrimidines biosynthesis accumulates not in the nucleus but in the cytoplasm, as shown by immunocytochemistry and Ura2-green fluorescent protein mapping; Benoist P et al.; The Ura2 multidomain protein catalyses the first two steps of pyrimidines biosynthesis in Saccharomyces cerevisiae . It consists of a 240 kDa polypeptide which contains carbamyl phosphate synthetase and aspartate transcarbamylase domains . The Ura2 protein was believed to be nucleoplasmic, since one of the aspartate transcarbamylase reaction products, monophosphate, was reported to be precipitated by lead ions inside nuclei . However, this ultracytochemical approach was recently shown to give artifactual lead polyphosphate precipitates, and the use of cerium instead of lead failed to reveal this nucleoplasmic localization . Ura2 localization has therefore been undertaken by means of three alternative approaches based on the detection of the protein itself: (a) indirect immunofluorescence of yeast protoplasts; (b) immunogold labelling of ultrathin sections of embedded yeast cells (both approaches using affinity purified primary antibodies directed against the 240 kDa Ura2 polypeptide chain, or against a 22 residue peptide specific of the carbamyl phosphate synthetase domain); and (c) direct fluorescence of cells expressing an Ura2-green fluorescent protein hybrid . All three approaches localize the bulk of Ura2 to the cytoplasm, whereas the signals associated with the nucleus, mitochondria or vacuoles are close to or at the background level .

Physiol Genomics, 2000 Aug 09, 3(2), 83 - 92
Global expression profiling of yeast treated with an inhibitor of amino acid biosynthesis, sulfometuron methyl; Jia MH et al.; The expression pattern of 1,529 yeast genes in response to sulfometuron methyl (SM) was analyzed by DNA microarray technology . SM, a potent herbicide, inhibits acetolactate synthase, a branched-chain amino acid biosynthetic enzyme . Exposure of yeast cells to 0.2 microg/ml SM resulted in 40% growth inhibition, a Gcn4p-mediated induction of genes involved in amino acid and cofactor biosynthesis, and starvation response . The accumulation of intermediates led to the induction of stress response genes and the repression of genes involved in carbohydrate metabolism, nucleotide biosynthesis, and sulfur assimilation . Extended exposure to SM led to a relaxation of the initial response and induction of sugar transporter and ergosterol biosynthetic genes, as well as repression of histone and lipid metabolic genes . Exposure to 5 microg/ml SM resulted in >98% growth inhibition and stimulated a similar initial expression change, but with no relaxation after extended exposure . Instead, more stress response and DNA damage repair genes become induced, suggesting a serious cellular consequence . Other salient features of metabolic regulation, such as the coordinated expression of cofactor biosynthetic genes with amino acid biosynthetic ones, were evident from our data . A potential link between SM sensitivity and ergosterol metabolism was uncovered by expression profiling and confirmed by genetic analysis.

Genetics, 2000 Oct, 156(2), 559 - 70
A role for cytosolic hsp70 in yeast {PSI(+)} prion propagation and {PSI(+)} as a cellular stress; Jung G et al.; {PSI(+)} is a prion (infectious protein) of Sup35p, a subunit of the Saccharomyces cerevisiae translation termination factor . We isolated a dominant allele, SSA1-21, of a gene encoding an Hsp70 chaperone that impairs {PSI(+)} mitotic stability and weakens allosuppression caused by {PSI(+)} . While {PSI(+)} stability is normal in strains lacking SSA1, SSA2, or both, SSA1-21 strains with a deletion of SSA2 cannot propagate {PSI(+)} . SSA1-21 {PSI(+)} strains are hypersensitive to curing of {PSI(+)} by guanidine-hydrochloride and partially cured of {PSI(+)} by rapid induction of the heat-shock response but not by growth at 37 degrees . The number of inheritable {PSI(+)} particles is significantly reduced in SSA1-21 cells . SSA1-21 effects on {PSI(+)} appear to be independent of Hsp104, another stress-inducible protein chaperone known to be involved in {PSI(+)} propagation . We propose that cytosolic Hsp70 is important for the formation of Sup35p polymers characteristic of {PSI(+)} from preexisting material and that Ssa1-21p both lacks and interferes with this activity . We further demonstrate that the negative effect of heat stress on {PSI(+)} phenotype directly correlates with solubility of Sup35p and find that in wild-type strains the presence of {PSI(+)} causes a stress that elevates basal expression of Hsp104 and SSA1.

Genetics, 2000 Oct, 156(2), 523 - 34
Suppressors of mdm20 in yeast identify new alleles of ACT1 and TPM1 predicted to enhance actin-tropomyosin interactions; Singer JM et al.; The actin cytoskeleton is required for many aspects of cell division in yeast, including mitochondrial partitioning into growing buds (mitochondrial inheritance) . Yeast cells lacking MDM20 function display defects in both mitochondrial inheritance and actin organization, specifically, a lack of visible actin cables and enhanced sensitivity to Latrunculin A . mdm20 mutants also exhibit a temperature-sensitive growth phenotype, which we exploited to isolate second-site suppressor mutations . Nine dominant suppressors selected in an mdm20/mdm20 background rescue temperature-sensitive growth defects and mitochondrial inheritance defects and partially restore actin cables in haploid and diploid mdm20 strains . The suppressor mutations define new alleles of ACT1 and TPM1, which encode actin and the major form of tropomyosin in yeast, respectively . The ACT1 mutations cluster in a region of the actin protein predicted to contact tropomyosin, suggesting that they stabilize actin cables by enhancing actin-tropomyosin interactions . The characteristics of the mutant ACT1 and TPM1 alleles and their potential effects on protein structure and binding are discussed.

Genetics, 2000 Oct, 156(2), 501 - 12
Mutation of the ATP-binding pocket of SSA1 indicates that a functional interaction between Ssa1p and Ydj1p is required for post-translational translocation into the yeast endoplasmic reticulum; McClellan AJ et al.; The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action of DnaK (hsp70) and DnaJ homologues . In Saccharomyces cerevisiae the cytosolic hsp70s that promote post-translational translocation are the products of the Ssa gene family . Ssa1p maintains secretory precursors in a translocation-competent state and interacts with Ydj1p, a DnaJ homologue . Although it has been proposed that Ydj1p stimulates the ATPase activity of Ssa1p to release preproteins and engineer translocation, support for this model is incomplete . To this end, mutations in the ATP-binding pocket of SSA1 were constructed and examined both in vivo and in vitro . Expression of the mutant Ssa1p's slows wild-type cell growth, is insufficient to support life in the absence of functional Ssa1p, and results in a dominant effect on post-translational translocation . The ATPase activity of the purified mutant proteins was not enhanced by Ydj1p and the mutant proteins could not bind an unfolded polypeptide substrate . Our data suggest that a productive interaction between Ssa1p and Ydj1p is required to promote protein translocation.

Int J Food Microbiol, 2000 Sep 15, 60(1), 99 - 105
Phenotypic adaptation to freeze-thaw stress of the yeast-like fungus Geotrichum candidum; Thammavongs B et al.; The effect of cold stress on Geotrichum candidum was investigated at chill and freezing temperatures . Specific growth rates were determined at various temperatures and plotted according to the Ratkowsky and Arrhenius equations . The obtained profiles led to the determination of characteristics including the activation energy and notional minimum temperatures . At temperatures below the optimum single linear slopes were observed . At freezing temperatures, the loss of viability of cell populations was proportional to the number of freezing-thawing cycles . Nevertheless, the ability of G . candidum to survive this challenge depended on the physiological conditions prior to the freezing stress . The loss of viability was growth phase specific . Cells harvested in stationary phase showed a higher resistance compared to those obtained with cells in exponential phase . Furthermore, the cells of G . candidum could be adapted to the freeze-thaw challenge by pre-treatment at chill temperatures . This phenomenon known as cryotolerance was a function of the duration of the preincubation exposure.

J Biol Chem, 2001 Jan 12, 276(2), 861 - 6
Intrinsically bent DNA in the promoter regions of the yeast GAAL1-10 and GAL80 genes; Bash RC et al.; Circular permutation analysis has detected fairly strong sites of intrinsic DNA bending on the promoter regions of the yeast GAL1-10 and GAL80 genes . These bends lie in functionally suggestive locations . On the promoter of the GAL1-10 structural genes, strong bends bracket nucleosome B, which lies between the UAS(G) and the GAL1 TATA . These intrinsic bends could help position nucleosome B . Nucleosome B plus two other promoter nucleosomes protect the TATA and start site elements in the inactive state of expression but are completely disrupted (removed) when GAL1-10 expression is induced . The strongest intrinsic bend ( approximately 70 degrees ) lies at the downstream edge of nucleosome B; this places it approximately 30 base pairs upstream of the GAL1 TATA, a position that could allow it to be involved in GAL1 activation in several ways, including the recruitment of a yeast HMG protein that is required for the normally robust level of GAL1 expression in the induced state (Paull, T., Carey, M., and Johnson, R . (1996) Genes Dev . 10, 2769-2781) . On the regulatory gene GAL80, the single bend lies in the non-nucleosomal hypersensitive region, between a GAL80-specific far upstream promoter element and the more gene-proximal promoter elements . GAL80 promoter region nucleosomes contain no intrinsically bent DNA.

Genomics, 2000 Oct 1, 69(1), 86 - 94
Characterization of mRAD18Sc, a mouse homolog of the yeast postreplication repair gene RAD18; van der Laan R et al.; The RAD18 gene of the yeast Saccharomyces cerevisiae encodes a protein with ssDNA binding activity that interacts with the ubiquitin-conjugating enzyme RAD6 and plays an important role in postreplication repair . We identified and characterized the putative mouse homolog of RAD18, designated mRAD18Sc . The mRAD18Sc open reading frame encodes a 509-amino-acid polypeptide that is strongly conserved in size and sequence between yeast and mammals, with specific conservation of the RING-zinc-finger and the classic zinc-finger domain . The degree of sequence conservation between mRAD18Sc, RAD18, and homologous sequences identified in other species (NuvA from Aspergillus nidulans and Uvs-2 from Neurospora crassa) is entirely consistent with the evolutionary relationship of these organisms, strongly arguing that these genes are one another's homologs . Consistent with the presence of a nuclear translocation signal in the amino acid sequence, we observed the nuclear localization of GFP-tagged mRAD18Sc after stable transfection to HeLa cells . mRNA expression of mRAD18Sc in the mouse was observed in thymus, spleen, brain, and ovary, but was most pronounced in testis, with the highest level of expression in pachytene-stage primary spermatocytes, suggesting that mRAD18Sc plays a role in meiosis of spermatogenesis . Finally, we mapped the mRAD18Sc gene on mouse chromosome 6F .

Int J Pharm, 2000 Aug 25, 204(1-2), 53 - 9
Application of acid-treated yeast cell wall (AYC) as a pharmaceutical additive I . AYC as a novel coating material; Kasai T et al.; Acid-treated yeast cell wall (AYC) was newly prepared by acidifying the cell wall of brewer's yeast and the potential to use AYC as a novel coating material was studied . AYC had an oval shape with the diameter of several microm . The rheogram of AYC aqueous dispersion showed the plastic fluid property that is generally observed in the suspension . Core tablets containing 3% of acetaminophen (AAP) were coated with the AYC aqueous dispersion containing 5% (w/v) of AYC and 0.35% (w/v) of glycerol at various coating percents . The AAP release profile from the AYC-coated tablets was studied by the JP13 paddle method using solutions at various pH . Tensile strength and permeability of oxygen and water vapor of AYC cast film were measured . The AAP release from the AYC-coated tablets showed sigmoidal release profile with an initial lag time and the duration of the lag time depended on the coating percent of AYC . The pH of the dissolution fluid or the storage at room temperature for 120 days had little affect on AAP release from the AYC-coated tablets . These results suggest that it is possible to control the start time of medicine release independent of the pH by coating of AYC, that is the time-controlled release . The AYC cast film showed a large tensile strength and an extremely small oxygen permeability coefficient and a sufficient level of water permeability coefficient in order to protect from moisture . These results present that AYC has the high utility as a novel aqueous coating material for DDS preparations.

J Biochem (Tokyo), 2000 Oct, 128(4), 553 - 9
Binding of an intrinsic ATPase inhibitor to the F(1)FoATPase in phosphorylating conditions of yeast mitochondria; Iwatsuki H et al.; Yeast mitochondrial ATP synthase has three regulatory proteins; ATPase inhibitor, 9K protein, and 15K protein . A mutant yeast lacking these three regulatory factors was constructed by gene disruption . Rates of ATP synthesis of both wild-type and the mutant yeast mitochondria decreased with decrease of respiration, while their membrane potential was maintained at 170-160 mV under various respiration rates . When mitochondrial respiration was blocked by antimycin A, the membrane potential of both types of mitochondria was maintained at about 160 mV by ATP hydrolysis . ATP hydrolyzing activity of F(1)FoATPase solubilized from normal mitochondria decreased in proportion to the rate of ATP synthesis, while the activity of the mutant F(1)FoATPase was constant regardless of changes in the rate of phosphorylation . These observations strongly suggest that F(1)FoATPase in the phosphorylating mitochondria is a mixture of two types of enzyme, phosphorylating and non-phosphorylating enzymes, whose ratio is determined by the rate of respiration and that the ATPase inhibitor binds preferentially to the non-phosphorylating enzyme.

Prog Nucleic Acid Res Mol Biol, 2001, 65, 197 - 259
Yeast chromatin structure and regulation of GAL gene expression; Bash R et al.; Yeast genomic DNA is covered by nucleosome cores spaced by short, discrete length linkers . The short linkers, reinforced by novel histone properties, create a number of unique and dynamic nucleosome structural features in vivo: permanent unpeeling of DNA from the ends of the core, an inability to bind even full 147 bp core DNA lengths, and facility to undergo a conformational transition that resembles the changes found in active chromatin . These features probably explain how yeast can maintain most of its genome in a transcribable state and avoid large-scale packaging away of inactive genes . The GAL genes provide a closely regulated system in which to study gene-specific chromatin structure . GAL structural genes are inactive without galactose but are highly transcribed in its presence; the expression patterns of the regulatory genes can account for many of the features of GAL structural gene control . In the inactive state, GAL genes demonstrate a characteristic promoter chromosomal organization; the major upstream activation sequence (UASG) elements lie in open, hypersensitive regions, whereas the TATA and transcription start sites are in nucleosomes . This organization helps implement gene regulation in this state and may benefit the organism . Induction of GAL expression triggers Gal4p-dependent upstream nucleosome disruption . Disruption is transient and can readily be reversed by a Gal80p-dependent nucleosome deposition process . Both are sensitive to the metabolic state of the cell . Induction triggers different kinds of nucleosome changes on the coding sequences, perhaps reflecting the differing roles of nucleosomes on coding versus promoter regions . GAL gene activation is a complex process involving multiple Gal4p activities, numerous positive and negative cofactors, and the histone tails . DNA bending and chromosomal architecture of the promoter regions may also play a role in GAL regulation . Regulator-mediated competition between nucleosomes and the TATA binding protein complex for the TATA region is probably a central aspect of GAL regulation and a focal point for the numerous factors and processes that contribute to it.

FEBS Lett, 2000 Sep 22, 481(3), 277 - 80
Cleavage of yeast tRNAPhe with complementary oligonucleotide conjugated to a small ribonuclease mimic; Beloglazova NG et al.; An oligonucleotide conjugate bearing a chemical construct mimicking the catalytic center of ribonuclease A has been designed and studied . The conjugate efficiently cleaves yeast tRNAPhe at a single site adjacent to the target complementary sequence.

J Biol Chem, 2000 Dec 22, 275(51), 40088 - 95
Altered distribution of the yeast plasma membrane H+-ATPase as a feature of vacuolar H+-ATPase null mutants; Perzov N et al.; The effect of vacuolar H(+)-ATPase (V-ATPase) null mutations on the targeting of the plasma membrane H(+)-ATPase (Pma1p) through the secretory pathway was analyzed . Gas1p, which is another plasma membrane component, was used as a control for the experiments with Pma1p . Contrary to Gas1p, which is not affected by the deletion of the V-ATPase complex in the V-ATPase null mutants, the amount of Pma1p in the plasma membrane is markedly reduced, and there is a large accumulation of the protein in the endoplasmic reticulum . Kex2p and Gef1p, which are considered to reside in the post-Golgi vesicles, were suggested as required for the V-ATPase function; hence, their null mutant phenotype should have been similar to the V-ATPase null mutants . We show that, in addition to the known differences between those yeast phenotypes, deletions of KEX2 or GEF1 in yeast do not affect the distribution of Pma1p as the V-ATPase null mutant does . The possible location of the vital site of acidification by V-ATPase along the secretory pathway is discussed.

Cell, 2000 Sep 1, 102(5), 695 - 704
CLIP170-like tip1p spatially organizes microtubular dynamics in fission yeast; Brunner D et al.; Rod-shaped fission yeast cells grow in a polarized manner, and unlike budding yeast, the correct positioning of the growth sites at cell ends requires interphase microtubules . Here we describe a microtubule guidance mechanism that orients microtubules in the intracellular space along the long axis of the cell, guiding them to their target region at the cell ends . This mechanism involves tip1p, a CLIP170-like protein that localizes to distal tips of cytoplasmic microtubules . In the absence of tip1p, microtubular catastrophe is no longer restricted to cell ends but occurs when microtubules reach any region of the cellular cortex . Thus, tip1p enables microtubules to discriminate different cortical regions and regulates their dynamics accordingly.

Mech Ageing Dev, 2000 Sep 29, 118(3), 115 - 27
Sponge homologue to human and yeast gene encoding the longevity assurance polypeptide: differential expression in telomerase-positive and telomerase-negative cells of Suberites domuncula; Kruse M et al.; Porifera show a characteristic Bauplan in spite of the fact that (almost) all cells are telomerase-positive and presumably provided with an unlimited potency for cell proliferation . Studies revealed that telomerase-positive cells can be triggered to telomerase-negative cells by dissociating them into single cells . Single cells from the demosponge Suberites domuncula, in contrast to cells present in primmorphs (a special form of cell aggregates), lack the property to proliferate and they undergo apoptosis . One gene, SDLAGL, was identified in primmorphs that showed high sequence similarity to the longevity assurance genes from other Metazoa . In single cells no transcripts of SDLAGL could be identified, while high expression was seen after re-aggregation of single cells and in proliferating cells of primmorphs . We concluded that SDLAGL is involved in the shift of telomerase-positive, proliferating cells to telomerase-negative, non-proliferating cells.

Biochim Biophys Acta, 2000 Aug 15, 1459(2-3), 489 - 98
Cloning and expression of cDNAs encoding plant V-ATPase subunits in the corresponding yeast null mutants; Aviezer-Hagai K et al.; Complementation of yeast null mutants is widely used for cloning of homologous genes from heterologous sources . We have used this method to clone the relevant V-ATPase genes from lemon fruit and Arabidopsis thaliana cDNA libraries . The pH levels are very different in the vacuoles of the lemon fruit and the A . thaliana, yet both are the result of the activity of the same enzyme complex, namely the V-ATPase . In order to investigate the mechanism that enables the enzyme to maintain such differences in pH values, we have compared the subunit composition of the V-ATPase complex from both sources . Towards this end, we have constructed a cDNA library from lemon fruit and cloned it into a similar shuttle vector to the one of the A . thaliana cDNA library, which is commercially available . In this work, we report the cloning and expression of VMA10 from both sources, two isoforms of the lemon proteolipid (VMA3) and the lemon homologue of yeast VPH1/STV1 subunit, LEMAC.

Biochim Biophys Acta, 2000 Aug 15, 1459(2-3), 258 - 65
Application of the obligate aerobic yeast Yarrowia lipolytica as a eucaryotic model to analyse Leigh syndrome mutations in the complex I core subunits PSST and TYKY; Ahlers PM et al.; We have used the obligate aerobic yeast Yarrowia lipolytica to reconstruct and analyse three missense mutations in the nuclear coded subunits homologous to bovine TYKY and PSST of mitochondrial complex I (proton translocating NADH:ubiquinone oxidoreductase) that have been shown to cause Leigh syndrome (MIM 25600), a severe progressive neurodegenerative disorder . While homozygosity for a V122M substitution in NDUFS7 (PSST) has been found in two siblings with neuropathologically proven Leigh syndrome (R . Triepels et al., Ann . Neurol . 45 (1999) 787), heterozygosity for a P79L and a R102H substitution in NDUFS8 (TYKY) has been found in another patient (J . Loeffen et al., Am . J . Hum . Genet . 63 (1998) 1598) . Mitochondrial membranes from Y . lipolytica strains carrying any of the three point mutations exhibited similar complex I defects, with V(max) being reduced by about 50% . This suggests that complex I mutations that clinically present as Leigh syndrome may share common characteristics . In addition changes in the K(m) for n-decyl-ubiquinone and I(50) for hydrophobic complex I inhibitors were observed, which provides further evidence that not only the hydrophobic, mitochondrially coded subunits, but also some of the nuclear coded subunits of complex I are involved in its reaction with ubiquinone.

Mol Cell Biol, 2000 Oct, 20(20), 7654 - 61
Regulation of yeast H(+)-ATPase by protein kinases belonging to a family dedicated to activation of plasma membrane transporters; Goossens A et al.; The regulation of electrical membrane potential is a fundamental property of living cells . This biophysical parameter determines nutrient uptake, intracellular potassium and turgor, uptake of toxic cations, and stress responses . In fungi and plants, an important determinant of membrane potential is the electrogenic proton-pumping ATPase, but the systems that modulate its activity remain largely unknown . We have characterized two genes from Saccharomyces cerevisiae, PTK2 and HRK1 (YOR267c), that encode protein kinases implicated in activation of the yeast plasma membrane H(+)-ATPase (Pma1) in response to glucose metabolism . These kinases mediate, directly or indirectly, an increase in affinity of Pma1 for ATP, which probably involves Ser-899 phosphorylation . Ptk2 has the strongest effect on Pma1, and ptk2 mutants exhibit a pleiotropic phenotype of tolerance to toxic cations, including sodium, lithium, manganese, tetramethylammonium, hygromycin B, and norspermidine . A plausible interpretation is that ptk2 mutants have a decreased membrane potential and that diverse cation transporters are voltage dependent . Accordingly, ptk2 mutants exhibited reduced uptake of lithium and methylammonium . Ptk2 and Hrk1 belong to a subgroup of yeast protein kinases dedicated to the regulation of plasma membrane transporters, which include Npr1 (regulator of Gap1 and Tat2 amino acid transporters) and Hal4 and Hal5 (regulators of Trk1 and Trk2 potassium transporters).

Mol Cell Biol, 2000 Oct, 20(20), 7490 - 504
Increased rates of genomic deletions generated by mutations in the yeast gene encoding DNA polymerase delta or by decreases in the cellular levels of DNA polymerase delta; Kokoska RJ et al.; In Saccharomyces cerevisiae, POL3 encodes the catalytic subunit of DNA polymerase delta . While yeast POL3 mutant strains that lack the proofreading exonuclease activity of the polymerase have a strong mutator phenotype, little is known regarding the role of other Pol3p domains in mutation avoidance . We identified a number of pol3 mutations in regions outside of the exonuclease domain that have a mutator phenotype, substantially elevating the frequency of deletions . These deletions appear to reflect an increased frequency of DNA polymerase slippage . In addition, we demonstrate that reduction in the level of wild-type DNA polymerase results in a similar mutator phenotype . Lowered levels of DNA polymerase also result in increased sensitivity to the DNA-damaging agent methyl methane sulfonate . We conclude that both the quantity and the quality of DNA polymerase delta is important in ensuring genome stability.

J Biol Chem, 2000 Dec 22, 275(51), 40594 - 600
Tropomyosin-dependent filament formation by a polymerization-defective mutant yeast actin (V266G,L267G); Wen KK et al.; A major function of tropomyosin (TPM) in nonmuscle cells may be stabilization of F-actin by binding longitudinally along the actin filament axis . However, no clear evidence exists in vitro that TPM can significantly affect the critical concentration of actin . We previously made a polymerization-defective mutant actin, GG (V266G, L267G) . This actin will not polymerize alone at 25 degrees C but will in the presence of phalloidin or beryllium fluoride . With beryllium fluoride, but not phalloidin, this polymerization rescue is cold-sensitive . We show here that GG-actin polymerizability was restored by cardiac tropomyosin and yeast TPM1 and TPM2 at 25 degrees C with rescue efficiency inversely proportional to TPM length (TPM2 > TPM1 > cardiac tropomyosin), indicating the importance of the ends in polymerization rescue . In the presence of TPM, the apparent critical concentration of actin is 5.5 microm, 10-15-fold higher than that of wild type actin but well below that of the GG-actin alone (>20 microm) . Non N-acetylated TPMs did not rescue GG-actin polymerization . The TPMs did not prevent cold-induced depolymerization of GG F-actin . TPM-dependent GG-actin polymerization did not occur at temperatures below 20 degrees C . Polymerization rescue may depend initially on the capture of unstable GG-F-actin oligomers by the TPM, resulting in the strengthening of actin monomer-monomer contacts along the filament axis.

Structure Fold Des, 2000 Aug 15, 8(8), 799 - 807
The crystal structure of the peptide-binding fragment from the yeast Hsp40 protein Sis1; Sha B et al.; BACKGROUND: Molecular chaperone Hsp40 can bind non-native polypeptide and facilitate Hsp70 in protein refolding . How Hsp40 and other chaperones distinguish between the folded and unfolded states of proteins to bind nonnative polypeptides is a fundamental issue . RESULTS: To investigate this mechanism, we determined the crystal structure of the peptide-binding fragment of Sis1, an essential member of the Hsp40 family from Saccharomyces cerevisiae . The 2.7 A structure reveals that Sis1 forms a homodimer in the crystal by a crystallographic twofold axis . Sis1 monomers are elongated and consist of two domains with similar folds . Sis1 dimerizes through a short C-terminal stretch . The Sis1 dimer has a U-shaped architecture and a large cleft is formed between the two elongated monomers . Domain I in each monomer contains a hydrophobic depression that might be involved in binding the sidechains of hydrophobic amino acids . CONCLUSIONS: Sis1 (1-337), which lacks the dimerization motif, exhibited severe defects in chaperone activity, but could regulate Hsp70 ATPase activity . Thus, dimer formation is critical for Sis1 chaperone function . We propose that the Sis1 cleft functions as a docking site for the Hsp70 peptide-binding domain and that Sis1-Hsp70 interaction serves to facilitate the efficient transfer of peptides from Sis1 to Hsp70.

Biotechniques, 2000 Sep, 29(3), 524 - 6, 528
Yeast recombinase FLP functions effectively in human cells for construction of adenovirus vectors; Ng P et al.; We have recently developed a high-efficiency method of constructing adenovirus vectors based on Cre-mediated recombination between two plasmids co-transfected into 293 cells . The simplicity and efficiency of this method should greatly expedite the construction of most recombinant vectors . However, this system would not be suitable for constructing vectors bearing loxP sites elsewhere in the genome because of undesirable Cre-mediated vector rearrangements . To address this, we have developed a similar system using FLP-mediated site-specific recombination for the construction of adenovirus vectors.

Plant Sci, 2000 Sep 8, 158(1-2), 155 - 161
Members of the Arabidopsis 14-3-3 gene family trans-complement two types of defects in fission yeast; Kuromori T et al.; 14-3-3 proteins are highly conserved among eukaryotes and perform diverse biochemical activities . We isolated five types of Arabidopsis 14-3-3 cDNAs in a screen for clones that could block ectopic meiosis driven by the pat1 mutation in fission yeast . Overexpression of fission yeast rad24, which encodes a 14-3-3 protein, also suppressed pat1 . All Arabidopsis clones isolated could rescue the deformed morphology and elevated UV sensitivity of the rad24 mutant . Thus, it appears that Arabidopsis 14-3-3 proteins can generally substitute for their fission yeast counterpart in function . Expression of an Arabidopsis 14-3-3 clone, GF14micro, was shown to be rather ubiquitous among plant organs.

Biochemistry, 2000 Sep 26, 39(38), 11788 - 800
Yeast orotidine-5'-phosphate decarboxylase: steady-state and pre-steady-state analysis of the kinetic mechanism of substrate decarboxylation; Porter DJ et al.; The catalytically active form of monofunctional yeast orotidine-5'-phosphate decarboxylase was a dimer (E(2)) . The dimer equilibrium dissociation constant was 0.25 microM in 0.01 M MOPS Na(+) at pH 7.2 . The bimolecular rate constant for dimer formation was 1.56 microM(-1) s(-1) . The dimeric form of the enzyme was stabilized by NaCl such that the enzyme was E(2) in 100 mM NaCl at all concentrations of enzyme tested . The kinetics of binding of OMP to E(2) was governed by two ionizations (pK(1) = 6.1 and pK(2) = 7.7) . From studies with substrate analogues, the higher pK was assigned to a group on the enzyme that interacted with the pyrimidinyl moiety . The value of the lower pK was dependent on the substrate analogue, which suggested that it was not exclusively the result of ionization of the phosphoryl moiety . During the decarboxylation of OMP, the fluorescence of E(2) was quenched over 20% . The enzymatic species with reduced fluorescence was a catalytically competent intermediate that had kinetic properties consistent with it being the initial enzyme-substrate complex . The stoichiometry for binding of OMP to E(2) was one OMP per enzyme monomer . The value of the first-order rate constant for conversion of the enzyme-substrate complex to free enzyme (36 s(-1)) calculated from a single turnover experiment ({E} >> {S}) was slightly greater than the value of k(cat), 20 s(-1) (corrected for stoichiometry), calculated from steady-state data . In the single turnover experiments, the enzyme was E(2)*S, whereas in the steady-state turnover the experiment enzyme was E(2)*S(2) . The similarity of these values suggested that the subunits were catalytically independent such that E(2)*S(2) could be treated as E*S and that conversion of the enzyme-substrate complex to E was k(cat) . Kinetic data for the approach to the steady-state with OMP and E(2) yield a bimolecular association rate complex of 62 microM(-1) s(-1)and a dissociation rate constant for E*S of 60 s(-1) . The commitment to catalysis was 0.25 . By monitoring the effect of carbonic anhydrase on {H(+)} changes during a single turnover experiment, the initial product of the decarboxylation reaction was shown to be CO(2) not HCO(3-) . UMP was released from the enzyme concomitantly with CO(2) during the conversion of E*S to E . Furthermore, the enzyme removed an enzyme equivalent of H(+) from solvent during this step of the reaction . The bimolecular rate constants for association of 6-AzaUMP and 8-AzaXMP, substrate analogues with markedly different nucleobases, had association rate constants of 112 and 130 microM(-1) s(-1), respectively . These results suggested that the nucleobase did not contribute significantly to the success of formation of the initial enzyme-substrate complex.

Yeast, 2000 Sep 30, 16(13), 1253 - 60
Current awareness on yeast; Kim K 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 . (3 weeks journals - search completed 21st June 2000)

Yeast, 2000 Sep 30, 16(13), 1229 - 41
A network of proteins around Rvs167p and Rvs161p, two proteins related to the yeast actin cytoskeleton; Bon E et al.; The Rvs161p and Rvs167p proteins of Saccharomyces cerevisiae, homologues of higher eukaryotes' amphiphysins, associate with actin and appear to be involved in several functions related to the actin cytoskeleton . In order to identify partners of the Rvsp proteins, yeast libraries constructed in two-hybrid vectors were screened using either Rvs167p or Rvs161p as a bait . The selected candidates, representing 34 ORFs, were then tested against both Rvsp proteins, as well as domains of Rvs167p or Rvs161p . Among the most significant ones, 24 ORFs were specific preys of Rvs167p only and two gave interactions with Rvs161p only . Interestingly, five ORFs were preys of both Rvs161p and Rvs167p (RVS167, LAS17, YNL094w, YMR192w and YPL249c) . Analysis of putative functions of the candidates confirm involvement of the Rvsp in endocytosis/vesicle traffic, but also opens possible new fields, such as nuclear functions .

Yeast, 2000 Sep 30, 16(13), 1217 - 27
Physiological role of the D-amino acid oxidase gene, DAO1, in carbon and nitrogen metabolism in the methylotrophic yeast Candida boidinii; Yurimoto H et al.; A methylotrophic yeast, Candida boidinii, exhibits D-amino acid oxidase activity (DAO, EC 1.4.3.3) during its growth on D-alanine as a sole carbon or a nitrogen source . The structural gene (DAO1), encoding DAO, was cloned from a genomic library of C . boidinii . The 1035-bp gene encoded 345 amino acids and the predicted amino acid sequence showed significant similarity to those of DAOs from other organisms . The DAO1 gene was disrupted in the C . boidinii genome by one-step gene disruption . The DAO1-deleted strain did not grow on D-alanine as a carbon source but did grow on D-alanine as a sole nitrogen source (with glucose as the carbon source) . These results suggested that, while DAO is critically involved in growth on D-alanine as a carbon source, there should be another enzyme system which metabolizes D-alanine as a nitrogen source in C . boidinii . We also showed that the three C-terminal amino acid sequence of DAO, -AKL was necessary and sufficient for the import of DAO into peroxisomes .

J Biol Chem, 2000 Dec 8, 275(49), 38929 - 37
Yeast Ran-binding protein Yrb1p is required for efficient proteolysis of cell cycle regulatory proteins Pds1p and Sic1p; Baumer M et al.; Ubiquitin-dependent proteolysis of specific target proteins is required for several important steps during the cell cycle . Degradation of such proteins is strictly cell cycle-regulated and triggered by two large ubiquitin ligases, termed anaphase-promoting complex (APC) and Skp1/Cullin/F-box complex (SCF) . Here we show that yeast Ran-binding protein 1 (Yrb1p), a predominantly cytoplasmic protein implicated in nucleocytoplasmic transport, is required for cell cycle regulated protein degradation . Depletion of Yrb1p results in the accumulation of unbudded G(1) cells and of cells arrested in mitosis implying a function of Yrb1p in the G(1)/S transition and in the progression through mitosis . Temperature-sensitive yrb1-51 mutants are defective in APC-mediated degradation of the anaphase inhibitor protein Pds1p and in degradation of the cyclin-dependent kinase inhibitor Sic1p, a target of SCF . Thus, Yrb1p is crucial for efficient APC- and SCF-mediated proteolysis of important cell cycle regulatory proteins . We have identified the UBS1 gene as a multicopy suppressor of yrb1-51 mutants . Ubs1p is a nuclear protein, and its deletion is synthetic lethal with a yrb1-51 mutation . Interestingly, UBS1 was previously identified as a multicopy suppressor of cdc34-2 mutants, which are defective in SCF activity . We suggest that Ubs1p may represent a link between nucleocytoplasmic transport and ubiquitin ligase activity.

J Biotechnol, 2000 Aug 25, 81(2-3), 141 - 50
Thermal stability of Rhizopus niveus lipase expressed in a kex2 mutant yeast; Kohno M et al.; Lipase from Rhizopus niveus (RNL) has a complex structure, and recombinant RNL, has even more complex structural properties in the yeast, Saccharomyces cerevisiae . These properties are due to the processing and to the size of the glycosylated sugar chain . The processing site was presumed to be that for the proteinase product of the KEX2 gene in yeast . We therefore, constructed an expression system in which the KEX2 gene was disrupted to produce a non-processed type of lipase with high thermal stability . This type of lipase was thermally stable to a temperature 15 degrees C higher than that of each processed type of lipase . This non-processed lipase had 50% residual activity after 2 h at 50 degrees C, while the residual activity of the processed lipases was only 10% after 30-45 min of incubation at 50 degrees C . The CD spectrum of the non-processed type of lipase at 222 nm was almost unchanged by heating, suggesting that this group of lipases had a very rigid structure and that the peptide bond between the A- and B-chain contributed to maintain this rigid structure . On the other hand, the length of the sugar chain bound to the lipase had no effect on the thermal stability.

Planta, 2000 Aug, 211(3), 325 - 34
Sugar levels altered by ectopic expression of a yeast-derived invertase affect cellular differentiation of developing cotyledons of Vicia narbonensis L; Neubohn B et al.; In order to change the sugar status during seed development a yeast-derived invertase gene was expressed in cotyledons of Vicia narbonensis . As a result, sucrose decreased whereas hexoses accumulated . We analysed cell structure and cellular differentiation in cotyledons expressing the yeast-invertase . Transgenic cells contained large and long-persisting vacuoles apparently serving as storage compartments for hexoses and clusters of storage-protein aggregates . In the wild-type, large vacuoles did not persist but were replaced by smaller protein bodies . During maturation and desiccation, the transgenic cells showed plasmolysis and vesiculation of the endo-membrane system . Immunogold-labelling revealed that the storage proteins vicilin and legumin were present within the cytoplasm and the extraprotoplasmic space and were attached to membranes of the endoplasmic reticulum and the nuclei . Protein storage vacuoles in mature seeds appeared heterogeneous and only partially filled . The data suggest that sugars control the subcellular organisation of the vacuolar system . Transcript levels encoding a tonoplast intrinsic protein, a marker for membranes of protein storage vacuoles, remained unchanged whereas mRNA levels of a hexose and a sucrose transporter increased . Generally, transgenic seeds appeared to be physiologically younger than wild-type seeds of the same age . The data underline the important role of sugars in legume seed development.

J Basic Microbiol, 2000, 40(4), 251 - 60
Yeast protein phosphatase active with acidic ribosomal proteins; Pilecki M et al.; A protein phosphatase dephosphorylating acidic ribosomal proteins was purified from Saccharomyces cerevisiae ribosome-free extract . It was shown that phosphoproteins from both P1 and P2 subfamilies as well as 60S "core" P0 protein were substrates for the enzyme . The phosphatase can dephosphorylate ribosomes as well as histones and casein but the two last substrates with significantly lower efficiency . It was found that the enzyme activity is Mn(2+)-dependent and inhibited by okadaic acid, tautomycin, cantharidin and nodularin at concentrations typical for protein phosphatase type 2A . The possible implications of those findings in the control of ribosome phosphorylation and therefore in the control of translation is discussed.

J Mol Biol, 2000 Sep 22, 302(3), 581 - 92
Structural analysis of yeast HSF by site-specific crosslinking; Bonner JJ et al.; We have introduced cysteine substitutions into the yeast HSF1 gene at a variety of locations . Most have no phenotypic effect, and therefore provide site-specific probes for thiol-specific reagents . Crosslinking of single mutants identifies locations where equivalent regions of individual monomers can approach each other in the HSF trimer . Crosslinking of double mutants indicates regions that can approach closely within a single subunit . Results for the DNA binding domain and trimerization domain are consistent with known structural information, and provide essential controls on the validity of the technique . In contrast to these two domains, the N-terminal and C-terminal domains, wherein lie the transcriptional activators, are highly flexible, and do not appear to be in stable contact with any other portions of the protein . None of these patterns are affected by the conformational change that is induced by superoxide or heat shock . We suggest a new model for the mechanism of HSF regulation that accomodates the structural information provided by these studies .

Biochemistry, 2000 Sep 19, 39(37), 11477 - 87
Two distinct regions of the yeast mitochondrial ADP/ATP carrier are photolabeled by a new ADP analogue: 2-azido-3'-O-naphthoyl-{beta-32P}ADP . Identification of the binding segments by mass spectrometry; Dianoux AC et al.; A novel photoactivatable radioactive ADP derivative, namely, 2-azido-3'-O-naphthoyl-{beta-(32)P}ADP (2-azido-N-{(32)P}ADP), was synthesized with the aim at mapping the substrate binding site(s) of the yeast mitochondrial ADP/ATP carrier . It was used with mitochondria isolated from genetically modified strains of Saccharomyces cerevisiae, producing the native or the His-tagged Anc2p isoform of the carrier . In darkness, 2-azido-N-{(32)P}ADP was reversibly bound to the carrier in mitochondria, without being transported . Upon photoirradiation, only the ADP/ATP carrier was covalently radiolabeled among all mitochondrial proteins . Specificity of labeling was demonstrated since carboxyatractyloside (CATR), a potent inhibitor of ADP/ATP transport, totally prevented the incorporation of the photoprobe . To localize the radioactive region(s), the purified photolabeled carrier was submitted to CNBr or hydroxylamine cleavage . The resulting fragments were characterized and identified by SDS-PAGE, Western blotting, amino acid sequencing, and MALDI-MS and ESI-MS analyses . Two short photolabeled distinct segments, eight and nine residues long, were identified: S183-R191, located in the central part of the ADP/ATP carrier; and I311-K318, belonging to its C-terminal end . Plausible models of organization of the nucleotide binding site(s) of the carrier involving the two regions specifically labeled by 2-azido-N-{(32)P}ADP are proposed.

Curr Biol, 2000 Aug 24, 10(16), 964 - 73
A cyclase-associated protein regulates actin and cell polarity during Drosophila oogenesis and in yeast; Baum B et al.; BACKGROUND: A polarised cytoskeleton is required to pattern cellular space, and for many aspects of cell behaviour . While the mechanisms ordering the actin cytoskeleton have been extensively studied in yeast, little is known about the analogous processes in other organisms . We have used Drosophila oogenesis as a model genetic system in which to investigate control of cytoskeletal organisation and cell polarity in multicellular eukaryotes . RESULTS: In a screen to identify genes required for Drosophila oocyte polarity, we isolated a Drosophila homologue of the yeast cyclase-associated protein, CAP . Here we show that CAP preferentially accumulates in the oocyte, where it inhibits actin polymerisation . CAP also has a role in oocyte polarity, as cap mutants fail to establish the proper, asymmetric distribution of mRNA determinants within the oocyte . Similarly in yeast, loss of CAP causes analogous polarity defects, altering the distribution of actin filaments and mRNA determinants . CONCLUSIONS: This study identifies CAP as a new effector of actin dynamics in Drosophila . As CAP controls the spatial distribution of actin filaments and mRNA determinants in both yeast and Drosophila, we conclude that CAP has an evolutionarily conserved function in the genesis of eukaryotic cell polarity.

FEBS Lett, 2000 Sep 8, 481(1), 77 - 80
Heterologous expression of a mammalian epithelial sodium channel in yeast; Gupta SS et al.; The alpha and beta subunits of the amiloride-sensitive rat epithelial sodium channel (alpha beta ENaC) were expressed in the yeast Saccharomyces cerevisiae . We used a combination of yeast strains, including a mutant in the secretory pathway (sec6), and Western blotting techniques, to show that alpha beta ENaC was synthesized and targeted through the secretory system to the plasma membrane . Yeasts expressing alpha beta ENaC were more sensitive to salt than the parent strain . In addition, amiloride, a specific blocker of ENaC, was found to suppress salt sensitivity in the yeast strain expressing alpha beta ENaC.

FEBS Lett, 2000 Sep 8, 481(1), 8 - 12
Stable plasma membrane expression of the soluble domain of the human insulin receptor in yeast; Angermayr M et al.; The soluble cytoplasmic kinase domain of the human insulin receptor was N-terminally equipped with either an N-acetylation or a dual-acylation motif (MGC box, to allow myristoylation/palmitoylation) and expressed in yeast cells under the control of the inducible CUP1 promoter . Although the cellular concentration was about the same in both instances (reflecting similar stability against proteolysis), only the myristoylated protein was capable of autophosphorylation to a significant extent and was active to phosphorylate endogenous yeast proteins at tyrosine residues in vivo . Cellular subfractionation showed that the insulin receptor was associated with plasma membranes, from where it was not extractable with high salt or alkali, but a significant fraction was also localized in the nuclear fraction . The myristoylated protein is absent from the cytoplasm . No effect of expression of either the acetylated or the myristoylated version on growth and respiration on various carbon sources was detected, suggesting a failure of the active insulin receptor kinase domain to couple to yeast (glucose) signalling cascades.

Proc Natl Acad Sci U S A, 2000 Sep 26, 97(20), 10745 - 50
An initiation element in the yeast CUP1 promoter is recognized by RNA polymerase II in the absence of TATA box-binding protein if the DNA is negatively supercoiled; Leblanc BP et al.; Purified RNA polymerase II initiated transcription from the yeast CUP1 promoter fused to a C-less cassette if the DNA was negatively supercoiled . Relaxed plasmid was not transcribed . Transcription did not require addition of any other transcription factors . TATA box-binding protein (TBP) was not detectable in the polymerase preparation and the TATA box was not required . Deletion analysis of the CUP1 promoter revealed that a 25-bp element containing the initiation region was sufficient for recognition by polymerase . Two transcription start sites were mapped, one of which is identical to one of the two major start sites observed in vivo . Our observations can be accounted for by using a theoretical analysis of the probability of DNA melting within the plasmid as a function of superhelix density: the CUP1 initiation element is intrinsically unstable to superhelical stress, permitting entry of the polymerase, which then scans the DNA to locate the start site . In support of this analysis, the CUP1 promoter was sensitive to mung bean nuclease . These observations and a previous theoretical analysis of yeast genes support the idea that promoters are stress points within the DNA superhelix . The role of transcription factors might be to mark the promoter and to regulate specific melting of promoter DNA.

J Biol Chem, 2000 Dec 1, 275(48), 38022 - 31
Characterization of the enzymatic properties of the yeast dna2 Helicase/endonuclease suggests a new model for Okazaki fragment processing; Bae SH et al.; The Saccharomyces cerevisiae Dna2, which contains single-stranded DNA-specific endonuclease activity, interacts genetically and physically with Fen-1, a structure-specific endonuclease implicated in Okazaki fragment maturation during lagging strand synthesis . In this report, we investigated the properties of the Dna2 helicase/endonuclease activities in search of their in vivo physiological functions in eukaryotes . We found that the Dna2 helicase activity translocates in the 5' to 3' direction and uses DNA with free ends as the preferred substrate . Furthermore, the endonucleolytic cleavage activity of Dna2 was markedly stimulated by the presence of an RNA segment at the 5'-end of single-stranded DNA and occurred within the DNA, ensuring the complete removal of the initiator RNA segment on the Okazaki fragment . In addition, we demonstrated that the removal of pre-existing initiator 5'-terminal RNA segments depended on a displacement reaction carried out during the DNA polymerase delta-catalyzed elongation of the upstream Okazaki fragments . These properties indicate that Dna2 is well suited to remove the primer RNA on the Okazaki fragment . Based op this information, we propose a new model in which Dna2 plays a direct role in Okazaki fragment maturation in conjunction with Fen-1.

J Cell Sci, 2000 Oct, 113 Pt 19, 3399 - 408
The budding yeast Dbf2 protein kinase localises to the centrosome and moves to the bud neck in late mitosis; Frenz LM et al.; Dbf2 is a multifunctional protein kinase in Saccharomyces cerevisiae that functions in transcription, the stress response and as part of a network of genes in exit from mitosis . By analogy with fission yeast it seemed likely that these mitotic exit genes would be involved in cytokinesis . As a preliminary investigation of this we have used Dbf2 tagged with GFP to examine intracellular localisation of the protein in living cells . Dbf2 is found on the centrosomes/spindle pole bodies (SPBs) and also at the bud neck where it forms a double ring . The localisation of Dbf2 is cell cycle regulated . It is on the SPBs for much of the cell cycle and migrates from there to the bud neck in late mitosis, consistent with a role in cytokinesis . Dbf2 partly co-localises with septins at the bud neck . A temperature-sensitive mutant of dbf2 also blocks progression of cytokinesis at 37 degrees C . Following cytokinesis some Dbf2 moves into the nascent bud . Localisation to the bud neck depends upon the septins and also the mitotic exit network proteins Mob1, Cdc5, Cdc14 and Cdc15 . The above data are consistent with Dbf2 acting downstream in a pathway controlling cytokinesis.

Nature, 2000 Aug 31, 406(6799), 1013 - 5
Myosin V orientates the mitotic spindle in yeast; Yin H et al.; Coordination of spindle orientation with the axis of cell division is an essential process in all eukaryotes . In addition to ensuring accurate chromosomal segregation, proper spindle orientation also establishes differential cell fates and proper morphogenesis . In both animal and yeast cells, this process is dependent on cytoplasmic microtubules interacting with the cortical actin-based cytoskeleton, although the motive force was unknown . Here we show that yeast Myo2, a myosin V that translocates along polarized actin cables into the bud, orientates the spindle early in the cell cycle by binding and polarizing the microtubule-associated protein Kar9 (refs 7-9) . The tail domain of Myo2 that binds Kar9 also interacts with secretory vesicles and vacuolar elements, making it a pivotal component of yeast cell polarization.

Mol Cell, 2000 Aug, 6(2), 487 - 92
The DNA damage checkpoint signal in budding yeast is nuclear limited; Demeter J et al.; The nature of the DNA damage-induced checkpoint signal that causes the arrest of cells prior to mitosis is unknown . To determine if this signal is transmitted through the cytoplasm or is confined to the nucleus, we created binucleate heterokaryon yeast cells in which one nucleus suffered an unrepairable double-strand break, and the second nucleus was undamaged . In most of these binucleate cells, the damaged nucleus arrested prior to spindle elongation, while the undamaged nucleus completed mitosis, even when the strength of the damage signal was increased . The arrest of the damaged nucleus was dependent upon the function of the RAD9 checkpoint gene . Thus, the DNA damage checkpoint causing G2/M arrest is regulated by a signal that is nuclear limited.

Mol Cell Biol, 2000 Oct, 20(19), 7088 - 98
Roles of transcription factor Mot3 and chromatin in repression of the hypoxic gene ANB1 in yeast; Kastaniotis AJ et al.; The hypoxic genes of Saccharomyces cerevisiae are repressed by a complex consisting of the aerobically expressed, sequence-specific DNA-binding protein Rox1 and the Tup1-Ssn6 general repressors . The regulatory region of one well-studied hypoxic gene, ANB1, is comprised of two operators, OpA and OpB, each of which has two strong Rox1 binding sites, yet OpA represses transcription almost 10 times more effectively than OpB . We show here that this difference is due to the presence of a Mot3 binding site in OpA . Mutations in this site reduced OpA repression to OpB levels, and the addition of a Mot3 binding site to OpB enhanced repression . Deletion of the mot3 gene also resulted in reduced repression of ANB1 . Repression of two other hypoxic genes in which Mot3 sites were associated with Rox1 sites was reduced in the deletion strain, but other hypoxic genes were unaffected . In addition, the mot3Delta mutation caused a partial derepression of the Mig1-Tup1-Ssn6-repressed SUC2 gene, but not the alpha2-Mcm1-Tup1-Ssn6-repressed STE2 gene . The Mot3 protein was demonstrated to bind to the ANB1 OpA in vitro . Competition experiments indicated that there was no interaction between Rox1 and Mot3, indicating that Mot3 functions either in Tup1-Ssn6 recruitment or directly in repression . A great deal of evidence has accumulated suggesting that the Tup1-Ssn6 complex represses transcription through both nucleosome positioning and a direct interaction with the basal transcriptional machinery . We demonstrate here that under repressed conditions a nucleosome is positioned over the TATA box in the wild-type ANB1 promoter . This nucleosome was absent in cells carrying a rox1, tup1, or mot3 deletion, all of which cause some degree of derepression . Interestingly, however, this positioned nucleosome was also lost in a cell carrying a deletion of the N-terminal coding region of histone H4, yet ANB1 expression remained fully repressed . A similar deletion in the gene for histone H3, which had no effect on repression, had only a minor effect on the positioned nucleosome . These results indicate that the nucleosome phasing on the ANB1 promoter caused by the Rox1-Mot3-Tup1-Ssn6 complex is either completely redundant with a chromatin-independent repression mechanism or, less likely, plays no role in repression at all.

J Biol Chem, 2000 Dec 1, 275(48), 37533 - 41
Endoproteolytic processing of Sst2, a multidomain regulator of G protein signaling in yeast; Hoffman GA et al.; Regulators of G protein signaling (RGS proteins) constitute a large family of G protein-binding proteins . All RGS proteins contain a conserved core domain that can accelerate G protein GTPase activity . In addition, many family members contain a unique N-terminal domain of unknown function . Here, we demonstrate that the RGS protein in yeast, Sst2, is proteolytically processed in vivo to yield separate but functional N-terminal and RGS core domain fragments . In whole cell lysates, the full-length SST2 product (82 kDa) as well as a prominent 36-kDa species are specifically recognized by antibodies against the C terminus of the Sst2 protein . Purification and chemical sequencing of the 36-kDa species revealed cleavage sites after Ser-414 and Ser-416, just preceding the region of RGS homology . Expression of a mutationally truncated form of the protein (C-Sst2) could not restore function to an sst2Delta mutant strain . In contrast, co-expression of C-Sst2 with the N-terminal domain (N-Sst2) partially restored the ability to regulate the growth arrest response but not the transcription induction response . Whereas the full-length protein was localized to the microsomal and plasma membrane fractions, the N-Sst2 species was predominantly in the microsomal fraction, and C-Sst2 was in the soluble fraction . Mutations that block proteasome or vacuolar protease function, or mutations in the cleavage site Ser residues of Sst2, did not alter processing . However, Sst2 processing did require expression of other components of the pheromone response pathway, including the receptor and the G protein . These results indicate that Sst2 is proteolytically processed, that this event is regulated by the signaling pathway, and that processing can profoundly alter the function and subcellular localization of the protein.

Nucleic Acids Res, 2000 Sep 15, 28(18), 3524 - 34
Complete deletion of yeast chromosomal rDNA repeats and integration of a new rDNA repeat: use of rDNA deletion strains for functional analysis of rDNA promoter elements in vivo; Wai HH et al.; Strains of Saccharomyces cerevisiae were constructed in which chromosomal rDNA repeats are completely deleted and their growth is supported by a plasmid carrying a single rDNA repeat, either a plasmid carrying the 35S rRNA gene transcribed from the native promoter by RNA polymerase I or a plasmid carrying the 35S rRNA gene fused to the GAL7 promoter for transcription by RNA polymerase II . This system has made it possible to assess the expression of rDNA by measuring the ability of synthesized rRNA to support cell growth as well as by measuring the actual rRNA synthesized rather than by the use of reporter mini-rDNA genes . Using this system, deletion analysis of the rDNA promoter confirmed the presence of two elements, the upstream element and the core promoter, and showed that basal transcription from the core promoter, if it takes place in vivo as was observed in vitro, is not sufficient to allow cell growth . We have also succeeded in integration of a rDNA repeat and its copy number expansion at the original chromosomal locus, which will allow future mutational analysis not only of rRNA but also other DNA elements involved in rRNA transcription, rDNA replication and recombination within a repeated rDNA structure.

Mol Biol Cell, 2000 Sep, 11(9), 2873 - 84
Homo-oligomeric complexes of the yeast alpha-factor pheromone receptor are functional units of endocytosis; Yesilaltay A et al.; alpha-Factor receptors from Saccharomyces cerevisiae are G-protein-coupled receptors containing seven transmembrane segments . Receptors solubilized with the detergent n-dodecyl beta-D-maltoside were found to sediment as a single 8S species in glycerol density gradients . When the membranes from cells coexpressing two differentially tagged receptors were solubilized with detergent and subjected to immunoprecipitation, we found that the antibodies specific for either epitope tag resulted in precipitation of both tagged species . Coprecipitation was not a consequence of incomplete detergent extraction because the abundant plasma membrane protein Pma1 did not coprecipitate with the receptors . Moreover, the receptor complexes were present prior to detergent extraction because coimmunoprecipitation was not observed when cells expressing the single tagged species were mixed prior to membrane preparation . Treatment of cultures with alpha-factor had little effect on the extent of oligomerization as judged by the sedimentation behavior of the receptor complexes and by the efficiency of coimmunoprecipitation . The ability of receptor complexes to undergo ligand-mediated endocytosis was evaluated by using membrane fractionation and fluorescence microscopy . Mutant receptors that fail to bind alpha-factor (Ste2-S184R) or lack the endocytosis signal (Ste2-T326) became competent for ligand-mediated endocytosis when they were expressed in cells containing wild-type receptors . Coimmunoprecipitation experiments indicated that the C-terminal cytoplasmic domain and intermolecular disulfide bonds were unnecessary for oligomer formation . We conclude that alpha-factor receptors form homo-oligomers and that these complexes are subject to ligand-mediated endocytosis . Furthermore, we show for the first time that unoccupied receptors participate in these endocytosis-competent complexes.

J Virol, 2000 Oct, 74(19), 9167 - 74
Yeast three-hybrid screening of rous sarcoma virus mutants with randomly mutagenized minimal packaging signals reveals regions important for gag interactions; Lee EG et al.; We previously showed that the yeast three-hybrid system provides a genetic assay of both RNA and protein components for avian retroviral RNA encapsidation . In the current study, we used this assay to precisely define cis-acting determinants involved in avian leukosis sarcoma virus packaging RNA binding to Gag protein . In vivo screening of Rous sarcoma virus mutants was performed with randomly mutated minimal packaging sequences (MPsi) made using PCR amplification after cotransformation with GagDeltaPR protein into yeast cells . Colonies with low beta-galactosidase activity were analyzed to locate mutations in MPsi sequences affecting binding to Gag proteins . This genetic assay delineated secondary structural elements that are important for efficient RNA binding, including a single-stranded small bulge containing the initiation codon for uORF3, as well as adjacent stem structures . This implies a possible tertiary structure favoring the high-affinity binding sites for Gag . In most cases, results from the three-hybrid assay were well correlated with those from the viral RNA packaging assays . The results from random mutagenesis using the rapid three-hybrid binding assay are consistent with those from site-directed mutagenesis using in vivo packaging assays.

J Protein Chem, 2000 Apr, 19(3), 199 - 208
Analysis of aluminum-yeast hexokinase interaction: modifications on protein structure and functionality; Socorro JM et al.; The aluminum and yeast hexokinase interaction was studied . Structural changes were correlated with variations in protein functionality . Results show two different behaviors: At low metal concentrations preferential adsorption of metal (and water exclusion) induces aggregate formation . No significant changes in the protein structure occur, but there is a continuous loss of activity (from the first concentration) . At large salt concentrations a monomerization process and a conformational change in the secondary structure as well as in the three-dimensional structure take place . This change reduces the percentage of alpha-helix conformation, gives thermal stability to the protein, and allows the exposure of some tryptophan residue and hydrophobic regions . The protein inhibition increases . Conformational change and monomerization may allow access of the metal to the substrate site, mainly the ATP site . The inhibition in any case is of mixed type with a competitive component.

FEMS Microbiol Lett, 2000 Sep 1, 190(1), 9 - 12
Serum-induced hypha formation in the dimorphic yeast Yarrowia lipolytica; Kim J et al.; The dimorphic yeast Yarrowia lipolytica forms true hyphae in a medium containing N-acetylglucosamine . We made a new finding that serum is a very effective inducer of hypha formation of Y . lipolytica: serum induced its hyphal growth very quickly compared to N-acetylglucosamine (4 h vs . 10 h) . Osmotic and oxidative stresses (0.2 M NaCl and 20 mM H2O2) inhibited the hypha formation induced by N-acetylglucosamine, but did not suppress the hypha formation triggered by serum . Serum-specific morphological mutants, which formed hyphae in the N-acetylglucosamine medium but not in serum medium, could be isolated . These results suggest that the signal triggered by serum may be transduced through a different pathway, at least in part, from that used for the N-acetylglucosamine signal in Y . lipolytica.

Nat Cell Biol, 2000 Sep, 2(9), 620 - 7
Polarized localization of yeast Pbs2 depends on osmostress, the membrane protein Sho1 and Cdc42; Reiser V et al.; In Saccharomyces cerevisiae cells, high external osmolarity leads to the activation of a p38-related mitogen-activated protein (MAP) kinase though Pbs2 . Pbs2 tagged with green fluorescent protein (Pbs2-GFP) is evenly distributed in the cytoplasm but excluded from the nucleus before and after exposure to stress . Here we show that a catalytically inactive form of Pbs2 attains a highly polarised localization during osmostress . This phenomenon depends of the osmosensor Sho1 and on a functional Cdc42 GTPase . Cdc42, but not the actin cytoskeleton, influences Sho1-dependent activation of the MAP kinase . Sho1 itself accumulates at sites of polar growth, but independently of stress conditions and Cdc42 . These observations allow us to define the sequence of events that occurs during propogation of osmostress signals.

Oncogene, 2000 Sep 7, 19(38), 4346 - 53
Detection of PTEN nonsense mutation and psiPTEN expression in central nervous system high-grade astrocytic tumors by a yeast-based stop codon assay; Zhang CL et al.; We have developed a new yeast-based assay for the detection of PTEN nonsense mutation, and applied it to a total of 42 astrocytic tumors . The assay utilizes homologous recombination of PCR-amplified PTEN cDNA samples to a yeast vector which expresses an in-frame PTEN::ADE2 chimera protein . An allele of nonsense mutation in the sample PTEN mRNA gives a truncated chimera protein in a yeast cell, resulting in the formation of a red colony . The assay and subsequent sequence analysis demonstrated nonsense mutations as red colonies of more than 10% in one of 10 anaplastic astrocytomas and six of 18 glioblastomas, but none in six pilocytic astrocytomas or in eight astrocytomas . Sequence analysis of white colonies showed one missense mutation in a glioblastoma . Interestingly, four of seven nonsense mutations were frame-shifts due to exon skipping . In addition, pink colonies were found in one of six pilocytic astrocytomas, three of eight astrocytomas, two of 10 anaplastic astrocytomas, and 10 of 18 glioblastomas . Sequence analysis of the pink colonies revealed a sequence similar to those reported as psiPTEN/PTH2 . By testing mRNA and genomic DNA, it was found to be a processed pseudogene which was transcribed . The psiPTEN expression was complementary to PTEN mutation, for 14 of 18 glioblastomas showed either PTEN mutation or psiPTEN expression and only one case showed both PTEN mutation and psiPTEN expression (P<0.046), suggesting a pathological role of psiPTEN expression as an alternative to PTEN mutation in glioblastomas.

Oncogene, 2000 Sep 7, 19(38), 4309 - 18
Bax and Bcl-xL independently regulate apoptotic changes of yeast mitochondria that require VDAC but not adenine nucleotide translocator; Shimizu S et al.; Mitochondria play an essential role in apoptosis by releasing apoptogenic molecules such as cytochrome c and AIF, and some caspases, which are all regulated by Bcl-2 family proteins . Pro-apoptotic Bax and Bak have been shown to induce cytochrome c release and loss of membrane potential (Deltapsi) leading to AIF release in the isolated mitochondria . We have previously shown that Bax and Bak open the voltage-dependent anion channel (VDAC) allowing cytochrome c to pass through the channel, and Bcl-xL closes the channel . However, it has been reported that it is adenine nucleotide translocator (ANT) with which Bax/Bcl-xL interacts that modulate the channel activity . Here, we investigated the role of ANT and VDAC in the changes of isolated mitochondria triggered by Bax and by chemicals that induce permeability transition (PT) . In rat and yeast mitochondria, Bax did not affect the ADP/ATP exchange activity of ANT . VDAC-deficient but not ANT-deficient yeast mitochondria showed resistance to cytochrome c release, Deltapsi loss, and swelling caused by Bax and PT inducers . Bcl-xL showed similar inhibition of all these changes in ANT-deficient and wild type yeast mitochondria . Furthermore, Bax induces cytochrome c release in wild type yeast cells but not VDAC1-deficient yeast cells . These data indicate that VDAC, but not ANT, is essential for apoptotic mitochondrial changes . The data also indicate that Bcl-xL and Bax possess an ability to regulate mitochondrial membrane permeability independently of other Bcl-2 family members.

FEMS Microbiol Rev, 2000 Oct, 24(4), 469 - 86
Stress-controlled transcription factors, stress-induced genes and stress tolerance in budding yeast; Estruch F; The transcriptional response to environmental changes is a major topic in both basic and applied research . From a basic point of view, to understand this response includes unravelling how the stress signal is sensed and transduced to the nucleus, to identify which genes are induced under each stress condition and, finally, to establish the phenotypic consequences of this induction in stress tolerance . The possibility of using genetic approaches has made the yeast Saccharomyces cerevisiae a compelling model to study stress response at a molecular level . Moreover, this information can be used to isolate and characterise stress-related proteins in higher eukaryotes and to design strategies to increase stress resistance in organisms of industrial interest . In this review the progress made in recent years is discussed.

Biochim Biophys Acta, 2000 Sep 7, 1493(1-2), 56 - 63
Regulation and evaluation of five methanol-inducible promoters in the methylotrophic yeast Candida boidinii; Yurimoto H et al.; We isolated the promoter regions of five methanol-inducible genes (P(AOD1), alcohol oxidase; P(DAS1), dihydroxyacetone synthase; P(FDH1), formate dehydrogenase; P(PMP20), Pmp20; and P(PMP47), Pmp47) from the Candida boidinii genome, and evaluated their strength and studied their regulation using the acid phosphatase gene of Saccharomyces cerevisiae (ScPHO5) as the reporter . Of the five promoters, P(DAS1) was the strongest methanol-inducible promoter whose strength was approximately 1.5 times higher than that of the commonly used P(AOD1) in methanol-induced cells . Although the expression of P(AOD1) and P(DAS1) was completely repressed by the presence of glucose, formate-induced expression of P(FDH1) was not repressed by glucose . Expression under P(PMP47), another methanol-inducible promoter, was highly induced by oleate . The induction kinetics of P(PMP47) and P(DAS1) revealed that methanol induces the expression of peroxisome membrane protein Pmp47, earlier than the expression of matrix enzyme dihydroxyacetone synthase (Das1p), and that this information is contained in the promoter region of the respective gene . This is the first report which evaluates several methanol-inducible promoters in parallel in the methylotrophic yeast.

Genetics, 2000 Sep, 156(1), 93 - 103
The cofactor-dependent pathways for alpha- and beta-tubulins in microtubule biogenesis are functionally different in fission yeast; Radcliffe PA et al.; The biogenesis of microtubules in the cell comprises a series of complex steps, including protein-folding reactions catalyzed by chaperonins . In addition a group of evolutionarily conserved proteins, called cofactors (A to E), is required for the production of assembly-competent alpha-/beta-tubulin heterodimers . Using fission yeast, in which alp11(+), alp1(+), and alp21(+), encoding the homologs for cofactors B, D, and E, respectively, are essential for cell viability, we have undertaken the genetic analysis of alp31(+), the homolog of cofactor A . Gene disruption analysis shows that, unlike the three genes mentioned above, alp31(+) is dispensable for cell growth and division . Nonetheless, detailed analysis of alp31-deleted cells demonstrates that Alp31(A) is required for the maintenance of microtubule structures and, consequently, the proper control of growth polarity . alp31-deleted cells show genetic interactions with mutations in beta-tubulin, but not in alpha-tubulin . Budding yeast cofactor A homolog RBL2 is capable of suppressing the polarity defects of alp31-deleted cells . We conclude that the cofactor-dependent biogenesis of microtubules comprises an essential and a nonessential pathway, both of which are required for microtubule integrity.

Genetics, 2000 Sep, 156(1), 7 - 20
Minisatellite variants generated in yeast meiosis involve DNA removal during gene conversion; Bishop AJ et al.; Two yeast minisatellite alleles were cloned and inserted into a genetically defined interval in Saccharomyces cerevisiae . Analysis of flanking markers in combination with sequencing allowed the determination of the meiotic events that produced minisatellites with altered lengths . Tetrad analysis revealed that gene conversions, deletions, or complex combinations of both were involved in producing minisatellite variants . Similar changes were obtained following selection for nearby gene conversions or crossovers among random spores . The largest class of events involving the minisatellite was a 3:1 segregation of parental-size alleles, a class that would have been missed in all previous studies of minisatellites . Comparison of the sequences of the parental and novel alleles revealed that DNA must have been removed from the recipient array while a newly synthesized copy of donor array sequences was inserted . The length of inserted sequences did not appear to be constrained by the length of DNA that was removed . In cases where one or both sides of the insertion could be determined, the insertion endpoints were consistent with the suggestion that the event was mediated by alignment of homologous stretches of donor/recipient DNA.

Proc Int Conf Intell Syst Mol Biol, 2000, 8, 384 - 94
Mining for putative regulatory elements in the yeast genome using gene expression data; Vilo J et al.; We have developed a set of methods and tools for automatic discovery of putative regulatory signals in genome sequences . The analysis pipeline consists of gene expression data clustering, sequence pattern discovery from upstream sequences of genes, a control experiment for pattern significance threshold limit detection, selection of interesting patterns, grouping of these patterns, representing the pattern groups in a concise form and evaluating the discovered putative signals against existing databases of regulatory signals . The pattern discovery is computationally the most expensive and crucial step . Our tool performs a rapid exhaustive search for a priori unknown statistically significant sequence patterns of unrestricted length . The statistical significance is determined for a set of sequences in each cluster with respect to a set of background sequences allowing the detection of subtle regulatory signals specific for each cluster . The potentially large number of significant patterns is reduced to a small number of groups by clustering them by mutual similarity . Automatically derived consensus patterns of these groups represent the results in a comprehensive way for a human investigator . We have performed a systematic analysis for the yeast Saccharomyces cerevisiae . We created a large number of independent clusterings of expression data simultaneously assessing the "goodness" of each cluster . For each of the over 52,000 clusters acquired in this way we discovered significant patterns in the upstream sequences of respective genes . We selected nearly 1,500 significant patterns by formal criteria and matched them against the experimentally mapped transcription factor binding sites in the SCPD database . We clustered the 1,500 patterns to 62 groups for which we derived automatically alignments and consensus patterns . Of these 62 groups 48 had patterns that have matching sites in SCPD database.

Sheng Wu Gong Cheng Xue Bao, 2000 Mar, 16(2), 124 - 8
{Effects of gene copy number and chromosomal position on the expression of a modified HBsAg gene SA-28 in yeast}; Pan H et al.; The effect of gene copy number and chromosomal position on heterologous gene expression in Saccharomyces cerevisae has been tentatively investigated using a modified hepatitis B virus surface antigen SA-28 gene . The plasmids containing different copies of SA-28 gene expression cassette were integrated into different chromosomal loci via FLP recombinase mediated targeted integration . The expression of SA-28 gene in the resultant transformants was then measured . The result indicated that the expression of SA-28 gene was positively related to the gene dosage at HIS3 locus and had chromosomal polarity at several chromosomal loci.

Cell, 2000 Aug 4, 102(3), 279 - 91
Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes; Hsu JY et al.; Phosphorylation of histone H3 at serine 10 occurs during mitosis and meiosis in a wide range of eukaryotes and has been shown to be required for proper chromosome transmission in Tetrahymena . Here we report that Ipl1/aurora kinase and its genetically interacting phosphatase, Glc7/PP1, are responsible for the balance of H3 phosphorylation during mitosis in Saccharomyces cerevisiae and Caenorhabditis elegans . In these models, both enzymes are required for H3 phosphorylation and chromosome segregation, although a causal link between the two processes has not been demonstrated . Deregulation of human aurora kinases has been implicated in oncogenesis as a consequence of chromosome missegregation . Our findings reveal an enzyme system that regulates chromosome dynamics and controls histone phosphorylation that is conserved among diverse eukaryotes.

Microbiology, 2000 Sep, 146 ( Pt 9), 2133 - 46
Suppression of sorbitol dependence in a strain bearing a mutation in the SRB1/PSA1/VIG9 gene encoding GDP-mannose pyrophosphorylase by PDE2 overexpression suggests a role for the Ras/cAMP signal-transduction pathway in the control of yeast cell-wall biogenesis; Tomlin GC et al.; Complementation studies and allele replacement in Saccharomyces cerevisiae revealed that PSA1/VIG9, an essential gene that encodes GDP-mannose pyrophosphorylase, is the wild-type SRB1 gene . Cloning and sequencing of the srb1-1 allele showed that it determines a single amino acid change from glycine to aspartic acid at residue 276 (srb1(D276)) . Genetic evidence is presented showing that at least one further mutation is required for the sorbitol dependence of srb1(D276) . A previously reported complementing gene, which this study has now identified as PDE2, is a multi-copy suppressor of sorbitol dependence and is not, as was previously suggested, the SRB1 gene . srb and pde2 mutants share a number of phenotypes, including lysis upon hypotonic shock and enhanced transformability . These data are consistent with the idea that the Ras/cAMP pathway might modulate cell-wall construction.

Microbiology, 2000 Sep, 146 ( Pt 9), 2121 - 32
Cell wall perturbation in yeast results in dual phosphorylation of the Slt2/Mpk1 MAP kinase and in an Slt2-mediated increase in FKS2-lacZ expression, glucanase resistance and thermotolerance; de Nobel H et al.; The protein kinase C (PKC1) pathway is essential for maintaining cell integrity in yeast . Here it is shown that various forms of cell wall damage result in activation of the downstream MAP kinase Slt2/Mpk1 . Several cell wall mutants displayed enhanced FKS2-lacZ expression, a known output of Slt2 activation . A similar response was obtained with wild-type cells grown in the presence of the cell wall perturbants Calcofluor white and Zymolyase . Upregulation of FKS2-lacZ in response to sublethal concentrations of these agents fully depended on the presence of Slt2 . The same cell wall stress conditions resulted in dual threonine and tyrosine phosphorylation of Slt2 . Both Slt2 phosphorylation and FKS2-lacZ induction could be largely prevented by providing osmotic support to the plasma membrane . Interestingly, Slt2 phosphorylation in response to cell wall damage required the putative plasma-membrane-located sensor Mid2 but not Hcs77/Wsc1 . Finally, cell wall perturbation gave rise to cells with increased resistance to glucanase digestion and heat shock . These responses depended on the presence of Slt2 . These results indicate that weakening of the cell wall activates the Slt2/Mpk1 MAP kinase pathway and results in compensatory changes in the cell wall.

J Biol Chem, 2000 Nov 24, 275(47), 37011 - 20
HEAT repeats mediate plasma membrane localization of Tor2p in yeast; Kunz J et al.; The subcellular distribution of Tor1p and Tor2p, two phosphatidylinositol kinase homologs and targets of the immunosuppressive drug rapamycin in Saccharomyces cerevisiae, was analyzed . We found that Tor protein is peripherally associated with membranes . Subcellular fractionation and immunofluorescence studies showed that Tor1p and Tor2p associate with the plasma membrane and a second fraction that is distinct from Golgi, vacuoles, mitochondria, and nucleus and may represent vesicular structures . Pulse-chase experiments showed that association of Tor protein with plasma membrane and the second compartment is fast, does not appear to involve components of endocytic, secretory, or Golgi to vacuole transport pathways, and is not affected by the immunosuppressive drug rapamycin . Deletion analysis reveals that two domains within Tor2p independently mediate localization to both compartments . These domains are composed of HEAT repeats that are thought to act as protein-protein interaction surfaces . Our studies therefore place Tor proteins at the site of action of their known downstream effectors and suggest that they may be part of a multiprotein complex.

J Biol Chem, 2000 Nov 24, 275(47), 37150 - 8
A novel post-translational modification of yeast elongation factor 1A . Methylesterification at the C terminus; Zobel-Thropp P et al.; Protein methylation reactions can play important roles in cell physiology . After labeling intact Saccharomyces cerevisiae cells with S-adenosyl-l-{methyl-(3)H}methionine, we identified a major methylated 49-kDa polypeptide containing {(3)H}methyl groups in two distinct types of linkages . Peptide sequence analysis of the purified methylated protein revealed that it is eukaryotic elongation factor 1A (eEF1A, formerly EF-1alpha), the protein that forms a complex with GTP and aminoacyl-tRNAs for binding to the ribosomal A site during protein translation . Previous studies have shown that eEF1A is methylated on several internal lysine residues to give mono-, di-, and tri-N-epsilon-methyl-lysine derivatives . We confirm this finding but also detect methylation that is released as volatile methyl groups after base hydrolysis, characteristic of ester linkages . In cycloheximide-treated cells, methyl esterified eEF1A was detected largely in the ribosome and polysome fractions; little or no methylated protein was found in the soluble fraction . Because the base-labile, volatile {methyl-(3)H}radioactivity of eEF1A could be released by trypsin treatment but not by carboxypeptidase Y or chymotrypsin treatment, we suggest that the methyl ester is present on the alpha-carboxyl group of its C-terminal lysine residue . From the results of pulse-chase experiments using radiolabeled intact yeast cells, we find that the N-methylated lysine residues of eEF1A are stable over 4 h, whereas the eEF1A carboxyl methyl ester has a half-life of less than 10 min . The rapid turnover of the methyl ester suggests that the methylation/demethylation of eEF1A at the C-terminal carboxyl group may represent a novel mode of regulation of the activity of this protein in yeast.

Mol Microbiol, 2000 Aug, 37(4), 926 - 40
Endocytotic uptake and retrograde transport of a virally encoded killer toxin in yeast; Eisfeld K et al.; We demonstrate that a virally encoded yeast 'killer' toxin is entering its eukaryotic target cell by endocytosis, subsequently travelling the yeast secretory pathway in reverse to exhibit its lethal effect . The K28 killer toxin is a secreted alpha/beta heterodimer that kills sensitive yeasts in a receptor-mediated fashion by blocking DNA synthesis in the nucleus . In vivo processing of the toxin precursor results in a protein whose beta-C-terminus carries the endoplasmic reticulum (ER) retention signal HDEL, which, as we show here, is essential for retrograde toxin transport . Yeast end3/4 mutants as well as cells lacking the HDEL receptor (Deltaerd2) or mutants defective in Golgi-to-ER protein recycling (erd1) are toxin resistant because the toxin can no longer enter and/or retrograde pass the cell . Site-directed mutagenesis further indicated that the toxin's beta-HDEL motif ensures retrograde transport, although in a toxin-secreting yeast the beta-C-terminus is initially masked by an R residue (beta-HDELR) until Kex1p cleavage uncovers the toxin's targeting signal in a late Golgi compartment . Prevention of Kex1p processing results in high-level secretion of a biologically inactive protein incapable of re-entering the secretory pathway . Finally, we present evidence that ER-to-cytosol toxin export is mediated by the Sec61p translocon and requires functional copies of the lumenal ER chaperones Kar2p and Cne1p.

EMBO J, 2000 Sep 1, 19(17), 4623 - 31
Yeast Cdc42 GTPase and Ste20 PAK-like kinase regulate Sho1-dependent activation of the Hog1 MAPK pathway; Raitt DC et al.; The adaptive response to hyperosmotic stress in yeast, termed the high osmolarity glycerol (HOG) response, is mediated by two independent upstream pathways that converge on the Pbs2 MAP kinase kinase (MAPKK), leading to the activation of the Hog1 MAP kinase . One branch is dependent on the Sho1 transmembrane protein, whose primary role was found to be the binding and translocation of the Pbs2 MAPKK to the plasma membrane, and specifically to sites of polarized growth . The yeast PAK homolog Ste20 is essential for the Sho1-dependent activation of the Hog1 MAP kinase in response to severe osmotic stress . This function of Ste20 in the HOG pathway requires binding of the small GTPase Cdc42 . Overexpression of Cdc42 partially complements the osmosensitivity of ste20Delta mutants, perhaps by activating another PAK-like kinase, while a dominant-negative Cdc42 mutant inhibited signaling through the SHO1 branch of the HOG pathway . Since activated Cdc42 translocates Ste20 to sites of polarized growth, the upstream and downstream elements of the HOG pathway are brought together through the membrane targeting function of Sho1 and Cdc42.

EMBO J, 2000 Sep 1, 19(17), 4577 - 88
Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells; Gillooly DJ et al.; Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking . In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3-phosphate {PI(3)P}, we constructed a probe consisting of two PI(3)P-binding FYVE domains . The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo . When expressed in fibroblasts, a tagged probe localized to endosomes, as detected by fluorescence microscopy . Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes . While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast . vps27Delta yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling . Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.

J Mol Biol, 2000 Sep 15, 302(2), 327 - 38
Analysis of yeast MSH2-MSH6 suggests that the initiation of mismatch repair can be separated into discrete steps; Bowers J et al.; The yeast MSH2-MSH6 complex is required to repair both base-pair and single base insertion/deletion mismatches . MSH2-MSH6 binds to mismatch substrates and displays an ATPase activity that is modulated by mispairs that are repaired in vivo . To understand early steps in mismatch repair, we analyzed mismatch repair (MMR) defective MSH2-msh6-F337A and MSH2-msh6-340 complexes that contained amino acid substitutions in the MSH6 mismatch recognition domain . While both heterodimers were defective in forming stable complexes with mismatch substrates, only MSH2-msh6-340 bound to homoduplex DNA with an affinity that was similar to that observed for MSH2-MSH6 . Additional analyses suggested that stable binding to a mispair is not sufficient to initiate recruitment of downstream repair factors . Previously, we observed that MSH2-MSH6 forms a stable complex with a palindromic insertion mismatch that escapes correction by MMR in vivo . Here we show that this binding is not accompanied by either a modulation in MSH2-MSH6 ATPase activity or an ATP-dependent recruitment of the MLH1-PMS1 complex . Together, these observations suggest that early stages in MMR can be divided into distinct recognition, stable binding, and downstream factor recruitment steps .

J Biol Chem, 2000 Nov 24, 275(47), 37232 - 9
Mutational analysis of subunit G (Vma10p) of the yeast vacuolar H+-ATPase; Charsky CM et al.; The G subunit of V-ATPases is a soluble subunit that shows homology with the b subunit of F-ATPases and may be part of the "stator" stalk connecting the peripheral V(1) and membrane V(0) sectors . When the N-terminal half of the G subunit is modeled as an alpha helix, most of the conserved residues fall on one face of the helix (Hunt, I . E., and Bowman, B . J . (1997) J . Bioenerg . Biomembr . 29, 533-540) . We probed the function of this region by site-directed mutagenesis of the yeast VMA10 gene . Stable G subunits were produced in the presence of Y46A and K55A mutations, but subunit E was destabilized, resulting in loss of the V-ATPase assembly . Mutations E14A and K50A allowed wild-type growth and assembly of V-ATPase complexes, but the complexes formed were unstable . Mutations R25A and R25L stabilized V-ATPase complexes relative to wild-type and partially inhibited disassembly of V(1) from V(0) in response to glucose deprivation even though the mutant enzymes were fully active . A 2-amino acid deletion in the middle of the predicted N-terminal helix (DeltaQ29D30) allowed assembly of a functional V-ATPase . The results indicate that, although the N-terminal half of the G subunit is essential for V-ATPase activity, either this region is not a rigid helix or the presence of a continuous, conserved face of the helix is not essential.

Biophys J, 2000 Sep, 79(3), 1621 - 8
Effects of high pressure on solvent isotope effects of yeast alcohol dehydrogenase; Northrop DB et al.; The effect of pressure on the capture of a substrate alcohol by yeast alcohol dehydrogenase is biphasic . Solvent isotope effects accompany both phases and are expressed differently at different pressures . These differences allow the extraction of an inverse intrinsic kinetic solvent isotope effect of 1.1 (i.e., (D(2(O)))V/K = 0.9) accompanying hydride transfer and an inverse equilibrium solvent isotope effect of 2.6 (i.e., (D(2(O)))K(s) = 0.4) accompanying the binding of nucleotide, NAD(+) . The value of the kinetic effect is consistent with a reactant-state E-NAD(+)-Zn-OH(2) having a fractionation factor of phi approximately 0.5 for the zinc-bound water in conjunction with a transition-state proton exiting a low-barrier hydrogen bond with a fractionation factor between 0.6 and 0.9 . The value of the equilibrium effect is consistent with restrictions of torsional motions of multiple hydrogens of the enzyme protein during the conformational change that accompanies the binding of NAD(+) . The absence of significant commitments to catalysis accompanying the kinetic solvent isotope effect means that this portion of the proton transfer occurs in the same reactive step as hydride transfer in a concerted chemical mechanism . The success of this analysis suggests that future measurements of solvent isotope effects as a function of pressure, in the presence of moderate commitments to catalysis, may yield precise estimates of intrinsic solvent isotope effects that are not fully expressed on capture at atmospheric pressure.

FEBS Lett, 2000 Aug 25, 480(1), 37 - 41
Four years of post-genomic life with 6,000 yeast genes; Goffeau A; Four years after disclosure of the full yeast genome sequence, a series of resources including tens of thousands of mutant strains, plasmids bearing isolated genes and disruption cassettes are becoming publicly available . Deletions of each of the 6,000 putative yeast genes are being screened systematically for dozens of phenotypic traits . In addition, new global approaches such as DNA hybridization arrays, quantitative proteomics and two-hybrid interactions are being steadily improved . They progressively build up an immense computation network of billions of data points which will, within the next decade, characterize all molecular interactions occurring in a simple eukaryotic cell . In this process of acquisition of new basic knowledge, an international community of over 1,000 laboratories cooperates with a remarkable willingness to share projects and results.

J Mol Biol, 2000 Aug 25, 301(4), 1059 - 75
A Bayesian system integrating expression data with sequence patterns for localizing proteins: comprehensive application to the yeast genome; Drawid A et al.; We develop a probabilistic system for predicting the subcellular localization of proteins and estimating the relative population of the various compartments in yeast . Our system employs a Bayesian approach, updating a protein's probability of being in a compartment, based on a diverse range of 30 features . These range from specific motifs (e.g . signal sequences or the HDEL motif) to overall properties of a sequence (e.g . surface composition or isoelectric point) to whole-genome data (e.g . absolute mRNA expression levels or their fluctuations) . The strength of our approach is the easy integration of many features, particularly the whole-genome expression data . We construct a training and testing set of approximately 1300 yeast proteins with an experimentally known localization from merging, filtering, and standardizing the annotation in the MIPS, Swiss-Prot and YPD databases, and we achieve 75 % accuracy on individual protein predictions using this dataset . Moreover, we are able to estimate the relative protein population of the various compartments without requiring a definite localization for every protein . This approach, which is based on an analogy to formalism in quantum mechanics, gives better accuracy in determining relative compartment populations than that obtained by simply tallying the localization predictions for individual proteins (on the yeast proteins with known localization, 92% versus 74%) . Our training and testing also highlights which of the 30 features are informative and which are redundant (19 being particularly useful) . After developing our system, we apply it to the 4700 yeast proteins with currently unknown localization and estimate the relative population of the various compartments in the entire yeast genome . An unbiased prior is essential to this extrapolated estimate; for this, we use the MIPS localization catalogue, and adapt recent results on the localization of yeast proteins obtained by Snyder and colleagues using a minitransposon system . Our final localizations for all approximately 6000 proteins in the yeast genome are available over the web at: edu/genome/localize .

Annu Rev Biochem, 2000, 69, 303 - 42
Autophagy, cytoplasm-to-vacuole targeting pathway, and pexophagy in yeast and mammalian cells; Kim J et al.; The sequestration and delivery of cytoplasmic material to the yeast vacuole and mammalian lysosome require the dynamic mobilization of cellular membranes and specialized protein machinery . Under nutrient deprivation conditions, double-membrane vesicles form around bulk cytoplasmic cargo destined for degradation and recycling in the vacuole/lysosome . A similar process functions to remove excess organelles under vegetative conditions in which they are no longer needed . Biochemical, morphological, and molecular genetic studies in yeasts and mammalian cells have begun to elucidate the molecular details of this autophagy process . In addition, the overlap of macroautophagy with the process of pexophagy and with the biosynthetic cytoplasm-to-vacuole targeting pathway, which delivers the resident vacuolar hydrolase aminopeptidase I, indicates that these three pathways are related mechanistically . Identification and characterization of the autophagic/cytoplasm-to-vacuole protein-targeting components have revealed the essential roles for various functional classes of proteins, including a novel protein conjugation system and the machinery for vesicle formation and fusion.

Annu Rev Biochem, 2000, 69, 247 - 75
Yeast homotypic vacuole fusion: a window on organelle trafficking mechanisms; Wickner W et al.; Homotypic (self) fusion of yeast vacuoles, which is essential for the low copy number of this organelle, uses catalytic elements similar to those used in heterotypic vesicular trafficking reactions between different organelles throughout nature . The study of vacuole inheritance has benefited from the ease of vacuole isolation, the availability of the yeast genome sequence and numerous mutants, and from a rapid, quantitative in vitro assay of fusion . The soluble proteins and small molecules that support fusion are being defined, conserved membrane proteins that catalyze the reaction have been identified, and the vacuole membrane has been solubilized and reconstituted into fusion-competent proteoliposomes, allowing the eventual purification of all needed factors . Studies of homotypic vacuole fusion have suggested a modified paradigm of membrane fusion in which integral membrane proteins termed "SNAREs" can form stable complexes in cis (when on the same membrane) as well as in trans (when anchored to opposing membranes) . Chaperones (NSF/Sec18p, LMA1, and -SNAP/Sec17p) disassemble cis-SNARE complexes to prepare for the docking of organelles rather than to drive fusion . The specificity of organelle docking resides in a cascade of trans-interactions (involving Rab-like GTPases), "tethering factors," and trans-SNARE pairing . Fusion itself, the mixing of the membrane bilayers and the organelle contents, is triggered by calcium signaling.

Anal Biochem, 2000 Sep 10, 284(2), 394 - 400
Use of bioluminometry for determination of active yeast biomass immobilized in ionotropic hydrogels; Navratil M et al.; The technique of bioluminometry was used to determine the biomass concentration of yeast cells immobilized in ionotropic hydrogel beads, including alginate, pectate, and kappa-carrageenan . The method uses determination of ATP extracted from viable cells, the concentration of which is then expressed as the active biomass concentration . Seven yeast strains divided into three categories (brewing, wine-making, and ethanol-producing yeasts) were tested, and different biomass concentrations were determined in all three immobilization materials . The described method is characterized by a good correlation (up to 99%) to classical dry biomass determination . The method is quicker, easier, and not so laborious, providing sufficient determination accuracy, and can be used for a rapid estimation of viable biomass in most biotechnological processes using immobilized living cells .

Proc Natl Acad Sci U S A, 2000 Aug 29, 97(18), 10077 - 82
Functions of the DNA damage response pathway target Ho endonuclease of yeast for degradation via the ubiquitin-26S proteasome system; Kaplun L et al.; Ho endonuclease of Saccharomyces cerevisiae is a homing endonuclease that makes a site-specific double-strand break in the MAT gene in late G(1) . Here we show that Ho is rapidly degraded via the ubiquitin-26S proteasome system through two ubiquitin-conjugating enzymes UBC2(Rad6) and UBC3(Cdc34) . UBC2(Rad6) is complexed with the ring finger DNA-binding protein Rad18, and we find that Ho is stabilized in rad18 mutants . We show that the Ho degradation pathway involving UBC3(Cdc34) goes through the Skp1/Cdc53/F-box (SCF) ubiquitin ligase complex and identify a F-box protein, Yml088w, that is required for Ho degradation . Components of a defined pathway of the DNA damage response, MEC1, RAD9, and CHK1, are also necessary for Ho degradation, whereas functions of the RAD24 epistasis group and the downstream effector RAD53 have no role in degradation of Ho . Our results indicate a link between the endonuclease function of Ho and its destruction.

Biochem Soc Trans, 2000, 28(4), 495 - 9
Membrane protein import in yeast mitochondria; Tokatlidis K et al.; The protein import pathway that targets proteins to the mitochondrial matrix has been extensively characterized in the past 15 years . Variations of this import pathway account for the sorting of proteins to other compartments as well, but the insertion of integral inner membrane proteins lacking a presequence is mediated by distinct translocation machinery . This consists of a complex of Tim9 and Tim10, two homologous, Zn(2+)-binding proteins that chaperone the passage of the hydrophobic precursor across the aqueous intermembrane space . The precursor is then targeted to another, inner-membrane-bound, complex of at least five subunits that facilitates insertion . Biochemical and genetic experiments have identified the key components of this process; we are now starting to understand the molecular mechanism . This review highlights recent advances in this new membrane protein insertion pathway.

Acta Biochim Pol, 2000, 47(1), 201 - 7
Reactive oxygen species as second messengers? Induction of the expression of yeast catalase T gene by heat and hyperosmotic stress does not require oxygen; Krawiec Z et al.; It is shown that oxygen is not absolutely needed for stress-induced synthesis of catalase T in the yeast Saccharomyces cerevisiae . Yeast cells develop heat resistance after exposure to elevated temperatures in anoxia . The levels of catalase activity and thermotolerance are comparable to those in aerobically stressed cells . While these results obviously do not exclude a stress signaling role of reactive oxygen species in some systems, as postulated by other authors, they suggest that the question of the obligatory requirement for reactive oxygen species in other stress signaling systems should be rigorously re-investigated.

Curr Biol, 2000 Jul 27-Aug 10, 10(15), 947 - 50
Cyk3, a novel SH3-domain protein, affects cytokinesis in yeast; Korinek WS et al.; Cytokinesis requires the wholesale reorganization of the cytoskeleton and secretion to complete the division of one cell into two . In the budding yeast Saccharomyces cerevisiae, the IQGAP-related protein Iqg1 (Cyk1) promotes cytokinetic actin ring formation and is required for cytokinesis and viability {1-3} . As the actin ring is not essential for cytokinesis or viability, Iqg1 must act by another mechanism {4} . To uncover this mechanism, a screen for high-copy suppressors of the iqg1 lethal phenotype was performed . CYK3 suppressed the requirement for IQG1 in viability and cytokinesis without restoration of the actin ring, demonstrating that CYK3 promotes cytokinesis through an actomyosin-ring-independent pathway . CYK3 encodes a novel SH3-domain protein that was found in association with the actin ring and the mother-bud neck . cyk3 null cells had misshapen mother-bud necks and were deficient in cytokinesis . In the cyk3 null strain, actin rearrangements associated with cytokinesis appeared normal, suggesting that the phenotype reflects a defect in secretory targeting or septal synthesis . Deletion of either cyk3 or hof1 alone results in a mild cytokinetic phenotype {5-7}, but deletion of both genes resulted in lethality and a complete cytokinetic block, suggesting overlapping function . Thus, Cyk3 appears to be important for cytokinesis and acts potentially downstream of Iqg1.

Antonie Van Leeuwenhoek, 2000 May, 77(4), 401 - 6
A novel oxylipin-associated 'ghosting' phenomenon in yeast flocculation; Kock JL et al.; Research on the distribution of oxylipins (3-hydroxy fatty acids) in flocculant strains of the yeast Saccharomyces cerevisiae led to the uncovering of a novel 'ghosting' phenomenon observed during assumed lectin-mediated aggregation . We found that intracellular oxylipin-containing osmiophilic layers migrate through yeast cell walls in a 'ghostlike' fashion without visually affecting the cell wall structure or the layers . This migration resulted in the binding of these layers to cell walls of adjacent cells . Consequently, 'ghosting' seems a prerequisite for flocculation to occur . However, 'ghosting' alone may not be sufficient to ensure flocculation.

Antonie Van Leeuwenhoek, 2000 May, 77(4), 389 - 92
Ascospore aggregation and oxylipin distribution in the yeast Dipodascopsis tothii; Smith DP et al.; Upon cultivation of the yeast Dipodascopsis tothii in its sexual stage, small ascospores are released individually from the ascus tip, which then assemble in sheathed cluster balls . In contrast to Dipodascopsis uninucleata, this yeast produced smooth bean shaped ascospores with sheath-like appendages that assemble in a disordered sheathed ball of ascospores outside the ascus . Strikingly, upon release, the ascus tip contained 3-hydroxy oxylipins, while the released ascospore clusters contained little or no 3-hydroxy oxylipins as indicated by immunofluorescence microscopy . In D . uninucleata, these oxylipins are concentrated on the spore surface and interspore matrix, but not on the ascus tip.

Antonie Van Leeuwenhoek, 2000 May, 77(4), 303 - 11
Halotolerance of the yeast Arxula adeninivorans LS3; Yang XX et al.; The non-pathogenic, dimorphic, ascomycetous yeast Arxula adeninivorans LS3 is halotolerant . It can grow in a minimal medium containing up to 20% NaCl . The growth parameters are only weakly influenced by 10% NaCl . However, NaCl in a concentration higher than 10% causes a decrease in the specific growth rate, a longer adaptation phase and a lower cell count in the stationary growth phase . Concentrations of glycerol and trehalose, which differed 100-fold in magnitude in a salt free medium, are also influenced differently by salt . NaCl induces accumulation of intracellular glycerol in exponentially growing cells but a reduced concentration of intracellular trehalose in stationary cells . Transcripts of the genes ARFC3, encoding a component of the replication factor C, and GAA, encoding a secretory glucoamylase, can be detected only in cells cultured in media with NaCl concentrations below 10% . Furthermore, NaCl in high concentration reduces the level of secreted proteins including glucoamylase end invertase.

Science, 2000 Aug 25, 289(5483), 1346 - 9
Structure of yeast poly(A) polymerase alone and in complex with 3'-dATP; Bard J et al.; Polyadenylate {poly(A)} polymerase (PAP) catalyzes the addition of a polyadenosine tail to almost all eukaryotic messenger RNAs (mRNAs) . The crystal structure of the PAP from Saccharomyces cerevisiae (Pap1) has been solved to 2.6 angstroms, both alone and in complex with 3'-deoxyadenosine triphosphate (3'-dATP) . Like other nucleic acid polymerases, Pap1 is composed of three domains that encircle the active site . The arrangement of these domains, however, is quite different from that seen in polymerases that use a template to select and position their incoming nucleotides . The first two domains are functionally analogous to polymerase palm and fingers domains . The third domain is attached to the fingers domain and is known to interact with the single-stranded RNA primer . In the nucleotide complex, two molecules of 3'-dATP are bound to Pap1 . One occupies the position of the incoming base, prior to its addition to the mRNA chain . The other is believed to occupy the position of the 3' end of the mRNA primer.

Mol Cell Biol, 2000 Sep, 20(18), 6806 - 15
Characterization of the interaction between the nuclease and reverse transcriptase activity of the yeast telomerase complex; Niu H et al.; Telomerase is a ribonucleoprotein that mediates extension of the dG-rich strand of telomeres in most eukaryotes . Like telomerase derived from ciliated protozoa, yeast telomerase is found to possess a tightly associated endonuclease activity that copurifies with the polymerization activity over different affinity-chromatographic steps . As is the case for ciliate telomerase, primers containing sequences that are not complementary to the RNA template can be efficiently cleaved by the yeast enzyme . More interestingly, we found that for the yeast enzyme, cleavage site selection is not stringent, since blocking cleavage at one site by the introduction of a nonhydrolyzable linkage can lead to the utilization of other sites . In addition, the reverse transcriptase activity of yeast telomerase can extend either the 5'- or 3'-end fragment following cleavage . Two general models that are consistent with the biochemical properties of the enzyme are presented: one model postulates two distinct active sites for the nuclease and reverse transcriptase, and the other invokes a multimeric enzyme with each protomer containing a single active site capable of mediating both cleavage and extension.

Mol Cell Biol, 2000 Sep, 20(18), 6712 - 20
Yeast glycogen synthase kinase 3 is involved in protein degradation in cooperation with Bul1, Bul2, and Rsp5; Andoh T et al.; The yeast Saccharomyces cerevisiae has four genes, MCK1, MDS1 (RIM11), MRK1, and YOL128c, that encode glycogen synthase kinase 3 (GSK-3) homologs . The gsk-3 null mutant, in which these four genes are disrupted, shows temperature sensitivity, which is suppressed by the expression of mammalian GSK-3beta and by an osmotic stabilizer . Suppression of temperature sensitivity by an osmotic stabilizer is also observed in the bul1 bul2 double null mutant, and the temperature sensitivity of the bul1 bul2 double null mutant is suppressed by multiple copies of MCK1 . We have screened rog mutants (revertants of gsk-3) which suppress the temperature sensitivity of the mck1 mds1 double null mutant and found that two of them, rog1 and rog2, also suppress the temperature sensitivity of the bul1 bul2 double null mutant . Bul1 and Bul2 have been reported to bind to Rsp5, a hect (for homologous to E6-associated-protein carboxyl terminus)-type ubiquitin ligase, but involvement of Bul1 and Bul2 in protein degradation has not been demonstrated . We find that Rog1, but not Rog2, is stabilized in the gsk-3 null and the bul1 bul2 double null mutants . Rog1 binds directly to Rsp5, and their interaction is dependent on GSK-3 . Furthermore, Rog1 is stabilized in the npi1 mutant, in which RSP5 expression levels are reduced . These results suggest that yeast GSK-3 regulates the stability of Rog1 in cooperation with Bul1, Bul2, and Rsp5.

Mol Cell Biol, 2000 Sep, 20(18), 6686 - 94
A homolog of voltage-gated Ca(2+) channels stimulated by depletion of secretory Ca(2+) in yeast; Locke EG et al.; In animal cells, capacitative calcium entry (CCE) mechanisms become activated specifically in response to depletion of calcium ions (Ca(2+)) from secretory organelles . CCE serves to replenish those organelles and to enhance signaling pathways that respond to elevated free Ca(2+) concentrations in the cytoplasm . The mechanism of CCE regulation is not understood because few of its essential components have been identified . We show here for the first time that the budding yeast Saccharomyces cerevisiae employs a CCE-like mechanism to refill Ca(2+) stores within the secretory pathway . Mutants lacking Pmr1p, a conserved Ca(2+) pump in the secretory pathway, exhibit higher rates of Ca(2+) influx relative to wild-type cells due to the stimulation of a high-affinity Ca(2+) uptake system . Stimulation of this Ca(2+) uptake system was blocked in pmr1 mutants by expression of mammalian SERCA pumps . The high-affinity Ca(2+) uptake system was also stimulated in wild-type cells overexpressing vacuolar Ca(2+) transporters that competed with Pmr1p for substrate . A screen for yeast mutants specifically defective in the high-affinity Ca(2+) uptake system revealed two genes, CCH1 and MID1, previously implicated in Ca(2+) influx in response to mating pheromones . Cch1p and Mid1p were localized to the plasma membrane, coimmunoprecipitated from solubilized membranes, and shown to function together within a single pathway that ensures that adequate levels of Ca(2+) are supplied to Pmr1p to sustain secretion and growth . Expression of Cch1p and Mid1p was not affected in pmr1 mutants . The evidence supports the hypothesis that yeast maintains a homeostatic mechanism related to CCE in mammalian cells . The homology between Cch1p and the catalytic subunit of voltage-gated Ca(2+) channels raises the possibility that in some circumstances CCE in animal cells may involve homologs of Cch1p and a conserved regulatory mechanism.

Mol Cell Biol, 2000 Sep, 20(18), 6668 - 76
Preferential accessibility of the yeast his3 promoter is determined by a general property of the DNA sequence, not by specific elements; Mai X et al.; Yeast promoter regions are often more accessible to nuclear proteins than are nonpromoter regions . As assayed by HinfI endonuclease cleavage in living yeast cells, HinfI sites located in the promoters of all seven genes tested were 5- to 20-fold more accessible than sites in adjacent nonpromoter regions . HinfI hypersensitivity within the his3 promoter region is locally determined, since it was observed when this region was translocated to the middle of the ade2 structural gene . Detailed analysis of the his3 promoter indicated that preferential accessibility is not determined by specific elements such as the Gcn4 binding site, poly(dA-dT) sequences, TATA elements, or initiator elements or by transcriptional activity . However, progressive deletion of the promoter region in either direction resulted in a progressive loss of HinfI accessibility . Preferential accessibility is independent of the Swi-Snf chromatin remodeling complex, Gcn5 histone acetylase complexes Ada and SAGA, and Rad6, which ubiquitinates histone H2B . These results suggest that preferential accessibility of the his3 (and presumably other) promoter regions is determined by a general property of the DNA sequence (e.g., base composition or a related feature) rather than by defined sequence elements . The organization of the compact yeast genome into inherently distinct promoter and nonpromoter regions may ensure that transcription factors bind preferentially to appropriate sites in promoters rather than to the excess of irrelevant but equally high-affinity sites in nonpromoter regions.

Biochemistry, 2000 Aug 29, 39(34), 10548 - 56
Domain architecture of the heme-independent yeast cystathionine beta-synthase provides insights into mechanisms of catalysis and regulation; Jhee KH et al.; Cystathionine beta-synthase from yeast (Saccharomyces cerevisiae) provides a model system for understanding some of the effects of disease-causing mutations in the human enzyme . The mutations, which lead to accumulation of L-homocysteine, are linked to homocystinuria and cardiovascular diseases . Here we characterize the domain architecture of the heme-independent yeast cystathionine beta-synthase . Our finding that the homogeneous recombinant truncated enzyme (residues 1-353) is catalytically active and binds pyridoxal phosphate stoichiometrically establishes that the N-terminal residues 1-353 compose a catalytic domain . Removal of the C-terminal residues 354-507 increases the specific activity and alters the steady-state kinetic parameters including the K(d) for pyridoxal phosphate, suggesting that the C-terminal residues 354-507 compose a regulatory domain . The yeast enzyme, unlike the human enzyme, is not activated by S-adenosyl-L-methionine . The truncated yeast enzyme is a dimer, whereas the full-length enzyme is a mixture of tetramer and octamer, suggesting that the C-terminal domain plays a role in the interaction of the subunits to form higher oligomeric structures . The N-terminal catalytic domain is more stable and less prone to aggregate than full-length enzyme and is thus potentially more suitable for structure determination by X-ray crystallography . Comparisons of the yeast and human enzymes reveal significant differences in catalytic and regulatory properties.

J Biol Chem, 2000 Nov 10, 275(45), 35592 - 9
Regulation of yeast ectoapyrase ynd1p activity by activator subunit Vma13p of vacuolar H+-ATPase; Zhong X et al.; CD39-like ectoapyrases are involved in protein and lipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae . By using a two-hybrid screen, we found that an activator subunit (Vma13p) of yeast vacuolar H(+)-ATPase (V-ATPase) binds to the cytoplasmic domain of Ynd1p, a yeast ectoapyrase . Interaction of Ynd1p with Vma13p was demonstrated by direct binding and co-immunoprecipitation . Surprisingly, the membrane-bound ADPase activity of Ynd1p in a vma13Delta mutant was drastically increased compared with that of Ynd1p in VMA13 cells . A similar increase in the apyrase activity of Ynd1p was found in a vma1Delta mutant, in which the catalytic subunit A of V-ATPase is missing, and the membrane peripheral subunits including Vma13p are dissociated from the membranes . However, the E286Q mutant of VMA1, which assembles inactive V-ATPase complex including Vma13p in the membrane, retained wild type levels of Ynd1p activity, demonstrating that the presence of Vma13p rather than the function of V-ATPase in the membrane represses Ynd1p activity . These results suggest that association of Vma13p with the cytoplasmic domain of Ynd1p regulates its apyrase activity in the Golgi lumen.

J Biol Chem, 2000 Nov 24, 275(47), 37118 - 26
Recognition of RNA encapsidation signal by the yeast L-A double-stranded RNA virus; Fujimura T et al.; The encapsidation signal of the yeast L-A virus contains a 24-nucleotide stem-loop structure with a 5-nucleotide loop and an A bulged at the 5' side of the stem . The Pol part of the Gag-Pol fusion protein is responsible for encapsidation of viral RNA . Opened empty viral particles containing Gag-Pol specifically bind to this encapsidation signal in vitro . We found that binding to empty particles protected the bulged A and the flanking-two nucleotides from cleavage by Fe(II)-EDTA-generated hydroxyl radicals . The five nucleotides of the loop sequence ((4190)GAUCC(4194)) were not protected . However, T1 RNase protection and in vitro mutagenesis experiments indicated that G(4190) is essential for binding . Although the sequence of the other four nucleotides of the loop is not essential, data from RNase protection and chemical modification experiments suggested that C(4194) was also directly involved in binding to empty particles rather than indirectly through its potential base pairing with G(4190) . These results suggest that the Pol domain of Gag-Pol contacts the encapsidation signal at two sites: one, the bulged A, and the other, G and C bases at the opening of the loop . These two sites are conserved in the encapsidation signal of M1, a satellite RNA of the L-A virus.

Yeast, 2000 Sep 15, 16(12), 1147 - 59
Inheritance of suppressors of the drug sensitivity of a NSR1 deleted yeast strain; Zabetakis D; The NSR1 gene product is involved in ribosomal RNA production and ribosome assembly in Saccharomyces cerevisiae . Yeast strains carrying a deletion of the NSR1 gene have a defect in rRNA processing, an aberrant ribosome profile and are sensitive to the drug paromomycin . This paper reports the isolation and characterization of spontaneous suppressors of the paromomycin sensitivity . Such suppressors could be isolated at very high frequency and do not exhibit straightforward single-gene inheritance patterns . The suppressors are not influenced by non-Mendelian factors such as psi or rho . Through a replacement of chromosomal rDNA with a plasmid rDNA system, I show that suppression of paromomycin sensitivity is mediated by rDNA . Swapping wild-type plasmid rDNA for chromosomal rDNA can reverse the suppression, but the effect does not appear to be due to amplification of rDNA or amplification of a pre-existing mutant rDNA copy .

Yeast, 2000 Sep 15, 16(12), 1099 - 105
Cloning, sequencing, and expression of H.a.YNR1 and H.a.YNI1, encoding nitrate and nitrite reductases in the yeast Hansenula anomala; Garcia-Lugo P et al.; A single Hansenula anomala genomic DNA fragment containing the genes H.a.YNR1 (yeast nitrate reductase) and H.a.YNI1 (yeast nitrite reductase) encoding nitrate and nitrite reductase, respectively, was isolated from a lambda EMBL3 genomic DNA library . As probe, a 3.2 kb DNA fragment isolated from a lambda gt11 H . anomala genomic DNA library screened with antiserum anti-NR from H . anomala was used . H . a.YNR1 and H.a.YNI1 genes are separated by 473 bp and encode putative proteins of 870 and 1077 amino acids, respectively, with great similarity to nitrate and nitrite reductases from other organisms . Northern blot analysis revealed that both genes are highly expressed in nitrate, very low in nitrate plus ammonium, and no expression was detected in ammonium or nitrogen-free media . Levels of nitrate reductase and nitrite reductase were very low or undetectable by Western blot analysis in nitrogen-free and ammonium media, whereas both proteins were present in nitrate and ammonium plus nitrate media . The nucleotide sequence Accession No . is AF123281 .

Yeast, 2000 Sep 15, 16(12), 1077 - 87
The cytochrome P450ALK multigene family of an n-alkane-assimilating yeast, Yarrowia lipolytica: cloning and characterization of genes coding for new CYP52 family members; Iida T et al.; Genes encoding cytochromes P450 of the CYP52 family in the n-alkane-assimilating yeast Yarrowia lipolytica have been cloned and analyzed . Degenerate PCR primers which were designed for the conserved amino acid sequences of cytochromes P450ALK of Candida species were used for amplification and isolation of genes encoding P450ALK from a genomic DNA library of Y . lipolytica CX161-1B . Seven new genes (YlALK2-YlALK8) were isolated . Five of the seven YlALK genes were induced by n-alkane under the culture conditions used in this study, whereas their expression was strictly repressed by glycerol but not by glucose, similar to the case of YlALK1, reported previously . Disruption of YlALK2, YlALK3, YlALK4 or YlALK6 did not change the growth of Y . lipolytica on medium containing n-alkanes of various chain lengths . A mutant with disruptions in both YlALK1 and YlALK2 did not grow well on n-hexadecane, whereas one with disruptions in both YlALK1 and YlALK3, which has the same phenotype as the YlALK1 singly disrupted mutant, grew well on n-hexadecane . These results suggest that the presence of multiple P450ALK species is a rather common phenomenon among the n-alkane-assimilating yeasts and that in the n-alkane assimilation of Y . lipolytica, YlALK1 functions to assimilate n-decane and longer molecules, whereas YlALK2 is involved in the assimilation of molecules longer than n-dodecane; other YlALKs are not significantly involved in the assimilation of C10-C16 n-alkanes .

Appl Microbiol Biotechnol, 2000 Jul, 54(1), 97 - 103
Construction of a flocculent brewer's yeast strain secreting Aspergillus niger beta-galactosidase; Domingues L et al.; One way of improving heterologous protein production is to use high cell density systems, one of the most attractive being the flocculating yeast production system . Also, lactose is available in large amounts as a waste product from cheese production processes . The construction of flocculent and non-flocculent brewer's yeast strains secreting beta-galactosidase and growing on lactose is presented . A plasmid was constructed coding for an extracellular beta-galactosidase of Aspergillus niger and having, as selective marker, the yeast CUP1 gene conferring resistance to copper . This selective marker allows for the transformation of wild-type yeasts . This work represents an important step towards the study of heterologous protein secretion by flocculent cells.

Eur J Biochem, 2000 Sep, 267(17), 5313 - 29
Can yeast glycolysis be understood in terms of in vitro kinetics of the constituent enzymes? Testing biochemistry; Teusink B et al.; This paper examines whether the in vivo behavior of yeast glycolysis can be understood in terms of the in vitro kinetic properties of the constituent enzymes . In nongrowing, anaerobic, compressed Saccharomyces cerevisiae the values of the kinetic parameters of most glycolytic enzymes were determined . For the other enzymes appropriate literature values were collected . By inserting these values into a kinetic model for glycolysis, fluxes and metabolites were calculated . Under the same conditions fluxes and metabolite levels were measured . In our first model, branch reactions were ignored . This model failed to reach the stable steady state that was observed in the experimental flux measurements . Introduction of branches towards trehalose, glycogen, glycerol and succinate did allow such a steady state . The predictions of this branched model were compared with the empirical behavior . Half of the enzymes matched their predicted flux in vivo within a factor of 2 . For the other enzymes it was calculated what deviation between in vivo and in vitro kinetic characteristics could explain the discrepancy between in vitro rate and in vivo flux.

J Gen Virol, 2000 Sep, 81(Pt 9), 2307 - 11
The 3a cell-to-cell movement gene is dispensable for cell-to-cell transmission of brome mosaic virus RNA replicons in yeast but retained over 10(45)-fold amplification; Ishikawa M et al.; In yeast expressing the RNA replication proteins encoded by brome mosaic virus (BMV), B3URA3, a BMV RNA3 derivative that harbours the 3a cell-to-cell movement protein gene and the yeast uracil biosynthesis gene URA3, was replicated and maintained in 85-95% of progeny at each cell division . Transmission of the B3URA3 RNA replicon from mother to daughter yeast did not require the 3a gene . Nevertheless, even after passaging for 165 cycles of RNA replication and yeast cell division, each of 40 independent Ura(+) colonies tested retained B3URA3 RNAs whose electrophoretic mobilities and accumulation levels were indistinguishable from those of the original B3URA3 . These and other results suggest that unselected genes in many positive-strand RNA virus replicons can be stably retained if the presence of the gene does not confer a selective disadvantage in RNA replication.

J Biol Chem, 2000 Nov 10, 275(45), 35600 - 6
Phosphatidylinositol 4-phosphate 5-kinase Its3 and calcineurin Ppb1 coordinately regulate cytokinesis in fission yeast; Zhang Y et al.; The ppb1(+) gene encodes a fission yeast homologue of the mammalian calcineurin . We have recently shown that Ppb1 is essential for chloride ion homeostasis, and acts antagonistically with Pmk1 mitogen-activated protein kinase pathway . In an attempt to identify genes that share an essential function with calcineurin, we screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and high temperature, and isolated a mutant, its3-1 . its3(+) was shown to be an essential gene encoding a functional homologue of phosphatidylinositol-4-phosphate 5-kinase (PI(4)P5K) . The temperature upshift or addition of FK506 induced marked disorganization of actin patches and dramatic increase in the frequency of septation in the its3-1 mutants but not in the wild-type cells . Expression of a green fluorescent protein-tagged Its3 and the phospholipase Cdelta pleckstrin homology domain indicated plasma membrane localization of PI(4)P5K and phosphatidylinositol 4,5-bisphosphate . These green fluorescent protein-tagged proteins were concentrated at the septum of dividing cells, and the mutant Its3 was no longer localized to the plasma membrane . These data suggest that fission yeast PI(4)P5K Its3 functions coordinately with calcineurin and plays a key role in cytokinesis, and that the plasma membrane localization of Its3 is the crucial event in cytokinesis.

Rev Panam Salud Publica, 2000 Jun, 7(6), 384 - 8
Performance of yeast-baited traps with Triatoma sordida, Triatoma brasiliensis, Triatoma pseudomaculata, and Panstrongylus megistus in laboratory assays; Pires HH et al.; The effectiveness of a trap for triatomines baited with yeast cultures has been previously demonstrated for Triatoma infestans in laboratory assays . We report here results from laboratory assays testing yeast traps for Triatoma sordida, Triatoma brasiliensis, Triatoma pseudomaculata, and Panstrongylus megistus . All assays were conducted in an open experimental arena 100 cm x 100 cm, with two traps placed at opposite sides of the arena . One of the traps contained a yeast culture, and the other trap contained a saccharose solution as a control . Two series of experiments were done, one without a refuge for the insects and one with a refuge . The results obtained clearly demonstrated that the yeast-baited traps were effective in the laboratory in capturing both T . sordida and P . megistus . For T . sordida, yeast-baited traps captured significantly more bugs than did the control traps (t test P value = 0.03) . For P . megistus, when a refuge was provided during the assay, yeast-baited traps also captured significantly more bugs than did the control traps (t test P value = 0.006) . In the experiments with T . brasiliensis and T . pseudomaculata, both traps captured some insects, but the yeast traps captured many fewer bugs than was true with the T . sordida and P . megistus bugs . These results indicate that, in the laboratory, yeast traps can capture considerable numbers of T . sordida and P . megistus in one night . We discuss the potential use of yeast traps for detecting and capturing both triatomine species.

Mol Cell, 2000 Jul, 6(1), 191 - 6
The yeast nuclear cap binding complex can interact with translation factor eIF4G and mediate translation initiation; Fortes P et al.; The mRNA cap structure is bound by either the nuclear (CBC) or the cytoplasmic (eIF4F) cap binding complex . Following mRNA export, CBC must be exchanged for eIF4F in the cytoplasm . It is not known how this exchange occurs or how this RNP remodeling event is integrated with mRNA function . Here we report genetic and biochemical evidence that the yeast translation initiation factor eIF4G associates with CBC, and that eIF4E, the eIF4F component that binds both the cap and eIF4G, antagonizes this interaction . Furthermore, we find that CBC can stimulate translation in extracts containing an eIF4G protein deficient for eIF4E binding . These data suggest that eIF4E binding to the eIF4G-CBC complex on newly exported mRNA displaces CBC, and that the first round of translation on mRNA may occur via a different mechanism than subsequent rounds.

Biotechniques, 2000 Aug, 29(2), 278 - 9, 282-4, 286-8
Streamlined yeast colorimetric reporter activity assays using scanners and plate readers; Serebriiskii IG et al.; Two-hybrid systems have become favored tools for detection and analysis of protein interactions because of their low cost and ease of use compared to biochemical or biophysical interaction technologies . It is possible to augment the utility of two-hybrid systems and derivative systems such as dual-bait two-hybrid systems by adapting strategies that speed the analysis of the relative strength of a series of protein-protein associations . This report describes two simple techniques that employ either a flatbed scanner or a plate reader to quantitate the activity of colorimetric reporters such as LacZ or GusA commonly used in two-hybrid approaches.

J Exp Bot, 2000 May, 51(346), 955 - 60
A method for expression cloning of transporter genes by screening yeast for uptake of radiolabelled substrate; Wittstock U et al.; A method has been developed for the cloning of plasma membrane transporters by screening yeast transformed with a cDNA library for the accumulation of radiolabelled substrate . The applicability of the method is demonstrated by cloning the amino acid permease AAP1 . A yeast mutant defective in proline uptake was transformed with an Arabidopsis thaliana cDNA library and plated on medium supplemented with L-{U-(14)C}proline . Yeast colonies accumulating radiolabelled proline were identified by autoradiography . The plasmids of these colonies were reintroduced into the yeast mutant and restoration of proline uptake was confirmed by L-{U-(14)C}proline uptake measurements . Whereas cloning of transporters by functional complementation requires that the substrate taken up is metabolized by yeast to promote growth, the method described here can be used to isolate transporters of substrates which are not metabolized . The method has great potential for the isolation of transporters of various substrates such as secondary plant products.

Genes Cells, 2000 Jul, 5(7), 543 - 53
Ebp2p, yeast homologue of a human protein that interacts with Epstein-Barr virus nuclear antigen 1, is required for pre-rRNA processing and ribosomal subunit assembly; Tsujii R et al.; BACKGROUND: A defect in the secretory pathway causes the transcriptional repression of both rRNA and ribosomal protein genes in Saccharomyces cerevisiae, suggesting a coupling of ribosome synthesis and plasma membrane synthesis . Rrs1p, an essential nuclear protein, is required for the secretory response . RESULTS: EBP2, encoding the yeast homologue of a human protein that interacts with Epstein-Barr virus Nuclear Antigen 1, was cloned in a two-hybrid screen using RRS1 as a bait . The rrs1-1 mutation, which produces Rrs1p without the C-terminal half and causes a defect in the secretory response, almost abolished the interaction with Ebp2p . Ebp2p is essential for growth and is mainly localized in the nucleolus . The effects of Ebp2p depletion on ribosome biogenesis is quite similar to that of Rrs1p depletion; in the Ebp2p-depleted cells, the rate of pre-rRNA processing is slower, and significantly less mature 25S rRNA is produced compared to those in wild-type cells . The polysome pattern indicates that Ebp2p-depletion causes a decrease of 80S monosomes and polysomes, an accumulation of 40S subunits, and the appearance of half-mer polysomes . CONCLUSIONS: Ebp2p is required for the maturation of 25S rRNA and 60S subunit assembly . Ebp2p may be one of the target proteins of Rrs1p for executing the signal to regulate ribosome biogenesis.

J Appl Microbiol, 2000 Jul, 89(1), 107 - 15
Incorporation of eicosapentaenoic and docosahexaenoic acids by a yeast (FO726A); Guo X et al.; An eicosapentaenoic acid (EPA)- and docosahexaenoic acid (DHA)-incorporating yeast, FO726A, was putatively identified as Candida guilliermondii on the basis of morphological, physiological and biochemical characteristics . Culture conditions for FO726A were investigated with respect to cell mass productivity, cellular accumulation of total lipid, triglyceride (TG), EPA and DHA . When grown at 20 degrees C for 24 h in an optimal medium containing 1 g scrap fish oil, the yeast yielded 820 mg dry cells which consisted of 40.7% lipid, 40.2% protein and 14.1% carbohydrate . The lipid (334mg) consisted of 300 mg TG (36.6% of dry cells), 23.2 mg EPA (2.8%) and 54.8 mg DHA (6.7%), and the recovery rates of EPA and DHA from the fish oil were 27.1 and 43.6%, respectively . The positional distributions of fatty acids in the TG from the yeast were then investigated and compared with those in the TG from the fish oil . The EPA and DHA in the fish oil were concentrated more in the sn-1,3 positions (8.8 and 13.7%, respectively) than in the sn-2 position (3.7 and 10.8%, respectively) . In the case of the TG from the yeast, EPA was present to a greater extent in the sn-1,3 positions than in the sn-2 position . In contrast, DHA was preferentially present in the sn-2 position, approximately twice that in the sn-1,3 positions.

Biosci Biotechnol Biochem, 2000 Jul, 64(7), 1542 - 6
Expression and secretion of scytalidopepsin B, an acid protease from Scytalidium lignicolum, in yeast; Shimuta K et al.; An expression and secretion system for scytalidopepsin B, an acid protease from Scytalidium lignicolum, was constructed in yeast . Saccharomyces cerevisiae AH22 was transformed with an yeast-E . coli shuttle vector, pAM82, in which an yeast invertase signal segment and the cDNA encoding the pro- and mature enzyme regions were inserted . The transformant was found to secret a pepstatin-insensitive acid protease, when cultured aerobically in a low phosphate (Pi) medium . Amino terminal amino acid sequencing analysis indicated that the recombinant acid protease was accurately processed and secreted as a mature form.

Pharmazie, 2000 Jul, 55(7), 533 - 7
Treatment of adjuvant arthritis in mice with yeast superoxide dismutase; Ratcheva I et al.; Yeast Cu/Zn superoxide dismutase (SODy) was used for treatment of adjuvant-induced arthritis in mice . SODy was applied intraperitoneally (i.p.) in doses of 10 mg/kg (30,000 U/kg) and 30 mg/kg (90,000 U/kg) one or three times daily on consecutive days . It was very effective in reducing the paw swelling whether administered before or immediately after induction or when the treatment began at the onset of inflammation or at the peak of the arthritic process . The effect of yeast SOD was compared to that of commercial SOD from bovine erythrocytes (SODb), as well as with indomethacin treatment . Histological data confirmed the antiinflammatory effect of yeast SOD . The schedules and doses tested did not elicit anti-SOD antibodies in serum.

RNA, 2000 Aug, 6(8), 1106 - 19
Dominant negative mutants of the yeast splicing factor Prp2 map to a putative cleft region in the helicase domain of DExD/H-box proteins; Edwalds-Gilbert G et al.; The Prp2 protein of Saccharomyces cerevisiae is an RNA-dependent ATPase required before the first transesterification reaction in pre-mRNA splicing . Prp2 binds to the spliceosome in the absence of ATP and is released following ATP hydrolysis . We determined what regions in Prp2 are essential for release from the spliceosome by analyzing dominant negative mutants in vivo and in vitro . We made mutations in conserved motif II (DExH) and motif VI (QRxGR) of the helicase (H) domain . Mutations that inactivated PRP2 had a dominant negative phenotype when overexpressed in vivo . To test whether mutations outside of the H domain could confer a dominant negative phenotype, we mutagenized a GAL1-PRP2 construct and screened for mutants unable to grow on galactose-containing media . Five dominant negative mutants were characterized; three mapped within the H domain and two mapped downstream of motif VI, indicating that an extended helicase domain is required for release of Prp2 from the spliceosome . Most mutants stalled in the spliceosome in vitro . However, not all mutants that were dominant negative in vivo were dominant negative in vitro, indicating that multiple mechanisms may cause a dominant negative phenotype . Structural modeling of the H domain of Prp2 suggests that mutants map to a cleft region found in helicases of known structure.

RNA, 2000 Aug, 6(8), 1091 - 105
The crystal structure of yeast phenylalanine tRNA at 1.93 A resolution: a classic structure revisited; Shi H et al.; The crystal structure of the monoclinic form of yeast phenylalanine tRNA has been redetermined at a resolution of 1.93 A . The structure of yeast tRNAphe described here is more accurate than its predecessors not only because it incorporates higher resolution data, but also because it has been refined using techniques that had not been developed when its predecessors were determined more than 20 years ago . The 1.93 A resolution version of this structure differs interestingly from its predecessors in its details . In loop regions particularly, the backbone torsion angles in the new structure are not the same as those reported earlier . Several new divalent cation binding sites have been identified, and the water structure that has emerged is also different.

J Mol Microbiol Biotechnol, 1999 Aug, 1(1), 135 - 40
Automation of yeast two-hybrid screening; Buckholz RG et al.; We have developed an automated format for screening yeast two-hybrid libraries for protein-protein interactions . The format consists of a liquid array in which pooled library subsets of yeast, expressing up to 1000 different cDNAs, are mated to a yeast strain of the opposite mating type, expressing a protein of interest . Interactors are detected by a liquid assay for beta-galacsidase following prototrophic selection . The method is demonstrated by the detection of interactions between two encoded yeast RNA polymerase subunits in simulated libraries of varied complexity . To demonstrate its utility for large scale screening of complex cDNA libraries, two nuclear receptor ligand-binding domains were screened through two cDNA libraries arrayed in pooled subsets . Screening these libraries yielded clones which had previously been identified in traditional yeast two hybrid screens, as well as several new putative interacting proteins . The formatting of the cDNA library into pooled subsets lends itself to functional subtraction of the promiscuous positive class of interactor from the library . Also, the liquid arrayed format enables electronic handling of the data derived from interaction screening, which, together with the automated handling of samples, should promote large-scale proteome analysis.

J Mol Microbiol Biotechnol, 1999 Aug, 1(1), 129 - 34
High level production of thermostable alpha-amylase from Sulfolobus solfataricus in high-cell density culture of the food yeast Candida utilis; Miura Y et al.; The alpha-amylase from Sulfolobus solfataricus has the commercially important ability to hydrolyze glycosyltrehalose and can be used for the production of trehalose from soluble starch . We have produced this enzyme in the food yeast Candida utilis at extremely high levels . Because the S . solfataricus gene was previously shown to be very poorly expressed, the gene was resynthesized based on codons preferentially found in the highly expressed C . utilis glyceraldehyde-3-phosphate dehydrogenase (GAP) gene . Expression of this synthetic gene under the control of the GAP promoter yielded biologically active alpha-amylase, accounting for more than 50% of the soluble protein . Comparison of the expression levels of various chimeric constructs of the synthetic and native genes indicated that the production level of the alpha-amylase was improved more than 2x10(4)-fold by substituting the native gene with the synthesized one . Northern analysis revealed the formation of short mRNAs in transformants with constructs containing native gene fragments, suggesting that premature termination of the transcripts is responsible for the low production level . The alpha-amylase-producing C . utilis cells were grown up to 92 grams dry cell weight per liter in a synthetic medium, yielding 12.3 g/l alpha-amylase which accounts for up to 27% of total cell proteins.

J Struct Biol, 2000 Jun, 130(2-3), 310 - 22
Prions of yeast as heritable amyloidoses; Wickner RB et al.; Two infectious proteins (prions) of Saccharomyces cerevisiae have been identified by their unusual genetic properties: (1) reversible curability, (2) de novo induction of the infectious prion form by overproduction of the protein, and (3) similar phenotype of the prion and mutation in the chromosomal gene encoding the protein . {URE3} is an altered infectious form of the Ure2 protein, a regulator of nitrogen catabolism, while {PSI} is a prion of the Sup35 protein, a subunit of the translation termination factor . The altered form of each is inactive in its normal function, but is able to convert the corresponding normal protein into the same altered inactive state . The N-terminal parts of Ure2p and Sup35p (the "prion domains") are responsible for prion formation and propagation and are rich in asparagine and glutamine residues . Ure2p and Sup35p are aggregated in vivo in {URE3}- and {PSI}-containing cells, respectively . The prion domains can form amyloid in vitro, suggesting that amyloid formation is the basis of these two prion diseases . Yeast prions can be cured by growth on millimolar concentrations of guanidine . An excess or deficiency of the chaperone Hsp104 cures the {PSI} prion . Overexpression of fragments of Ure2p or certain fusion proteins leads to curing of {URE3} .

J Mol Microbiol Biotechnol, 2000 Apr, 2(2), 207 - 16
Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica; Madzak C et al.; The industrial yeast Yarrowia lipolytica secretes high amounts of an alkaline extracellular protease encoded by the XPR2 gene . The industrial use of the XPR2 promoter was however hindered by its complex regulation . We designed hybrid promoters, based on tandem copies of the XPR2 promoter UAS1 region . In contrast to native XPR2 promoter, these hybrid promoters were not repressed by the preferred carbon and nitrogen sources, nor by acidic conditions, and they did not require the presence of peptones in the culture medium . They exhibited a strong quasi-constitutive activity, similar when carried on either integrative or replicative plasmids . We used these hybrid promoters to direct the production of bovine prochymosin, using XPR2 secretion signals . The production of active chymosin was several fold higher than with previously available Y . lipolytica promoters (up to 160 mg/l) . Integrative vectors based on the hybrid promoters, allowing the easy insertion of a heterologous gene and its expression or expression/secretion in Y . lipolytica, were designed . We also designed new Y . lipolytica recipient strains with good secreting abilities, able to grow on sucrose, and devoid of extracellular proteases . These new tools will add to the interest of Y . lipolytica as a host for heterologous protein production.

Mol Cell Biol, 2000 Sep, 20(17), 6435 - 48
Cell cycle-dependent binding of yeast heat shock factor to nucleosomes; Venturi CB et al.; In the nucleus, transcription factors must contend with the presence of chromatin in order to gain access to their cognate regulatory sequences . As most nuclear DNA is assembled into nucleosomes, activators must either invade a stable, preassembled nucleosome or preempt the formation of nucleosomes on newly replicated DNA, which is transiently free of histones . We have investigated the mechanism by which heat shock factor (HSF) binds to target nucleosomal heat shock elements (HSEs), using as our model a dinucleosomal heat shock promoter (hsp82-DeltaHSE1) . We find that activated HSF cannot bind a stable, sequence-positioned nucleosome in G(1)-arrested cells . It can do so readily, however, following release from G(1) arrest or after the imposition of either an early S- or late G(2)-phase arrest . Surprisingly, despite the S-phase requirement, HSF nucleosomal binding activity is restored in the absence of hsp82 replication . These results contrast with the prevailing paradigm for activator-nucleosome interactions and implicate a nonreplicative, S-phase-specific event as a prerequisite for HSF binding to nucleosomal sites in vivo.

Med Tr Prom Ekol, 2000, (6), 15 - 7
{Morbidity among the workers of the hydrolysis yeast production}; Mukhacheva EA et al.; Influence of protein-containing dust on the workers results in respiratory diseases . Individuals working in the shops with high level of protein-containing dust in the air of workplace more frequently demonstrate chronic bronchitis and exogenous allergic alveolitis, workers of the shops with low level of the dust in the air of workplace more commonly suffer from bronchial asthma . The authors necessitate adaptation of engineering, technical and medical prophylactic means to lower the morbidity level.

J Biol Chem, 2000 Oct 27, 275(43), 33267 - 71
Kinetic analysis of the cyclin-dependent kinase-activating kinase (Cak1p) from budding yeast; Enke DA et al.; Cak1p, the Cyclin-dependent kinase-activating kinase from budding yeast, is an unusual protein kinase that lacks many of the highly conserved motifs observed among members of the protein kinase superfamily . Cak1p phosphorylates and activates Cdc28p, the major cyclin-dependent kinase (CDK) in yeast, and is thereby required for passage through the yeast cell cycle . In this paper, we explore the kinetics of CDK phosphorylation by Cak1p, and we examine the role of the catalytic step in the reaction mechanism . Cak1p proceeds by a sequential reaction mechanism, binding to both ATP and CDK2 with reasonable affinities, exhibiting K(d) values of 7.2 and 0.6 microm, respectively . Interestingly, these values are approximately the same as the K(M) values, indicating that the binding of substrates is fast with respect to catalysis and that the most likely reaction mechanism is rapid equilibrium random . Cak1p is a slow enzyme, with a catalytic rate of only 4.3 min(-)(1) . The absence of a burst phase indicates that product release is not rate-limiting . This result, and a solvent isotope effect, suggests that a catalytic step is rate-limiting.

J Virol, 2000 Sep, 74(17), 8053 - 64
Functional interaction between pleiotropic transactivator pUL69 of human cytomegalovirus and the human homolog of yeast chromatin regulatory protein SPT6; Winkler M et al.; The phosphoprotein pUL69 of human cytomegalovirus (HCMV), which is a herpesvirus of considerable medical importance in immunosuppressed patients and newborns, has previously been identified as an early-late viral protein that can stimulate several viral and cellular promoters and thus exerts a rather broad activation pattern . To gain insight into the mechanism of this transactivation process, we looked for cellular factors interacting with pUL69 in a yeast two-hybrid screen . Using a B-lymphocyte cDNA library fused to the GAL4 activation domain, we identified 34 clones, 11 of which comprised one distinct gene . Interaction with this gene turned out to be very strong, producing beta-galactosidase levels 100-fold greater than the background as measured in an ONPG (o-nitrophenyl-beta-D-galactopyranoside) assay . Sequencing identified this gene as the human homolog of the yeast factor SPT6, which is thought to be involved in the regulation of chromatin structure . A direct interaction of pUL69 and the carboxy terminus of hSPT6 could be demonstrated using in vitro pull-down experiments . After having generated a specific antiserum that is able to detect the endogenous hSPT6 protein, we were able to observe an in vivo interaction of both proteins by coimmunoprecipitation analysis . The interaction domain within pUL69 was mapped to a central domain of this viral protein that is conserved within the homologous proteins of other herpesviruses such as the ICP27 protein of herpes simplex virus . Internal deletions within this central domain, as well as a single amino acid exchange at position C495, resulted in a loss of interaction . This correlated with a loss of the transactivation potential of the respective mutants, suggesting that the hSPT6 interaction of pUL69 is essential for stimulating gene expression . Furthermore, we demonstrate that the carboxy terminus of hSPT6 also binds to histon H3 and that this interaction can be antagonized by pUL69 . This allows the deduction of a model by which pUL69 acts as an antirepressor by competing for binding of histones to hSPT6, thereby antagonizing the chromatin remodeling function of this cellular protein.

J Cell Biol, 2000 Aug 7, 150(3), 513 - 26
Two distinct regions in a yeast myosin-V tail domain are required for the movement of different cargoes; Catlett NL et al.; The Saccharomyces cerevisiae myosin-V, Myo2p, is essential for polarized growth, most likely through transport of secretory vesicles to the developing bud . Myo2p is also required for vacuole movement, a process not essential for growth . The globular region of the myosin-V COOH-terminal tail domain is proposed to bind cargo . Through random mutagenesis of this globular tail, we isolated six new single point mutants defective in vacuole inheritance, but not polarized growth . These point mutations cluster to four amino acids in an 11-amino acid span, suggesting that this region is important for vacuole movement . In addition, through characterization of myo2-DeltaAflII, a deletion of amino acids 1,459-1,491, we identified a second region of the globular tail specifically required for polarized growth . Whereas this mutant does not support growth, it complements the vacuole inheritance defect in myo2-2 (G1248D) cells . Moreover, overexpression of the myo2-DeltaAflII globular tail interferes with vacuole movement, but not polarized growth . These data indicate that this second region is dispensable for vacuole movement . The identification of these distinct subdomains in the cargo-binding domain suggests how myosin-Vs can move multiple cargoes . Moreover, these studies suggest that the vacuole receptor for Myo2p differs from the receptor for the essential cargo.

Mol Microbiol, 2000 Jul, 37(1), 136 - 44
Functional roles of conserved amino acid residues surrounding the phosphorylatable histidine of the yeast phosphorelay protein YPD1; Janiak-Spens F et al.; The histidine-containing phosphotransfer (HPt) protein YPD1 is an osmoregulatory protein in yeast that facilitates phosphoryl transfer between the two response regulator domains associated with SLN1 and SSK1 . Based on the crystal structure of YPD1 and the sequence alignment of YPD1 with other HPt domains, we site-specifically engineered and purified several YPD1 mutants in order to examine the role of conserved residues surrounding the phosphorylatable histidine (H64) . Substitution of the positively charged residues K67 and R90 destabilized the phospho-imidazole linkage, whereas substitution of G68 apparently reduces accessibility of H64 . These findings, together with the effect of other mutations, provide biochemical support of the proposed functional roles of conserved amino acid residues of HPt domains.

Mol Microbiol, 2000 Jul, 37(1), 42 - 53
Trehalose is required for conformational repair of heat-denatured proteins in the yeast endoplasmic reticulum but not for maintenance of membrane traffic functions after severe heat stress; Simola M et al.; Saccharomyces cerevisiae cells grown at physiological temperature 24 degrees C require preconditioning at 37 degrees C to acquire tolerance towards brief exposure to 48-50 degrees C . During preconditioning, the cytosolic trehalose content increases remarkably and in the absence of trehalose synthesis yeast cannot acquire thermotolerance . It has been speculated that trehalose protects proteins and membranes under environmental stress conditions, but recently it was shown to assist the Hsp104 chaperone in refolding of heat-damaged proteins in the yeast cytosol . We have demonstrated that heat-denatured proteins residing in the endoplasmic reticulum (ER) also can be refolded once the cells are returned to physiological temperature . Unexpectedly, not only ER chaperones but also the cytosolic Hsp104 chaperone is required for conformational repair events in the ER lumen . Here we show that trehalose facilitates refolding of glycoproteins in the ER after severe heat stress . In the absence of Tps1p, a subunit of trehalose synthase, refolding of heat-damaged glycoproteins to bioactive and secretion-competent forms failed or was retarded . In contrast, membrane traffic operated many hours after severe heat stress even in the absence of the TPS1 gene, demonstrating that trehalose had no role in thermoprotection of membranes engaged in vesicular traffic . However, cytosolic proteins were aggregated and protein synthesis abolished, resulting finally in cell death.

Mol Microbiol, 2000 Jun, 36(6), 1460 - 9
Redox regulation of AMP synthesis in yeast: a role of the Bas1p and Bas2p transcription factors; Pinson B et al.; Expression of yeast AMP synthesis genes (ADE genes) was severely affected when cells were grown under oxidative stress conditions . To get an insight into the molecular mechanisms of this new transcriptional regulation, the role of the Bas1p and Bas2p transcription factors, known to activate expression of the ADE genes, was investigated . In vitro, DNA-binding of Bas1p was sensitive to oxidation . However, this sensitivity could not account for the regulation of the ADE genes because we showed, using a BAS1-VP16 chimera, that Bas1p DNA-binding activity was not sensitive to oxidation in vivo . Consistently, a triple cysteine mutant of Bas1p (fully resistant to oxidation in vitro) was unable to restore transcription of the ADE genes under oxidative conditions . We then investigated the possibility that Bas2p could be the oxidative stress responsive factor . Interestingly, transcription of the PHO5 gene, which is dependent on Bas2p but not on Bas1p, was found to be severely impaired by oxidative stress . Nevertheless, a Bas2p cysteine-free mutant was not sufficient to confer resistance to oxidative stress . Finally, we found that a Bas1p-Bas2p fusion protein restored ADE gene expression under oxidative conditions, thus suggesting that redox sensitivity of ADE gene expression could be due to an impairment of Bas1p/Bas2p interaction . This hypothesis was further substantiated in a two hybrid experiment showing that Bas1p/Bas2p interaction is affected by oxidative stress.

Eur J Biochem, 2000 Aug, 267(16), 5041 - 6
Kinetics of the coupled reaction catalysed by a fusion protein of yeast mitochondrial malate dehydrogenase and citrate synthase; Pettersson H et al.; The mechanistic implications of the kinetic behaviour of a fusion protein of mitochondrial malate dehydrogenase and citrate synthase have been reanalysed in view of predictions based on experimentally determined kinetic parameter values for the dehydrogenase and synthase activities of the protein . The results show that the time-course of citrate formation from malate in the coupled reaction catalysed by the fusion protein can be most satisfactorily accounted for in terms of a free-diffusion mechanism when consideration is taken to the inhibitory effects of NADH and oxaloacetate on the malate dehydrogenase activity . The effect of aspartate aminotransferase on the coupled reaction is likewise fully consistent with that expected for a free-diffusion mechanism . It is concluded that no tenable kinetic evidence is available to support the proposal that the fusion protein catalyses citrate formation from malate by a mechanism involving channelling of the intermediate oxaloacetate.

FEBS Lett, 2000 Aug 4, 478(3), 281 - 9
Production of polyomavirus structural protein VP1 in yeast cells and its interaction with cell structures; Palkova Z et al.; The gene for mouse polyomavirus major structural protein VP1 was expressed in Saccharomyces cerevisiae from the inducible GAL7 promoter . VP1 pseudocapsids were purified from cell lysates . Their subpopulation contained fragments of host DNA, which, in contrast to those of VP1 pseudocapsids produced in insect cells, did not assemble with cellular histones into pseudonucleocores . VP1 pseudocapsids accumulated in the yeast cell nuclei . A strong interaction of VP1 with tubulin fibres of the mitotic spindle was observed . The fibres of spindles were larger in diameter, apparently due to tight VP1 binding . Substantial growth inhibition of yeast cells producing VP1 was observed.

FEBS Lett, 2000 Aug 4, 478(3), 267 - 70
The human homologue of the yeast mitochondrial AAA metalloprotease Yme1p complements a yeast yme1 disruptant; Shah ZH et al.; In yeast, three AAA superfamily metalloproteases (Yme1p, Afg3p and Rca1p) are localized to the mitochondrial inner membrane where they perform roles in the assembly and turnover of the respiratory chain complexes . We have investigated the function of the proposed human orthologue of yeast Yme1p, encoded by the YME1L gene on chromosome 10p . Transfection of both HEK-293EBNA and yeast cells with a green fluorescent protein-tagged YME1L cDNA confirmed mitochondrial targeting . When expressed in a yme1 disruptant yeast strain, YME1L restored growth on glycerol at 37 degrees C . We propose that YME1L plays a phylogenetically conserved role in mitochondrial protein metabolism and could be involved in mitochondrial pathologies.

Mol Biol Cell, 2000 Aug, 11(8), 2757 - 73
Analysis of mid1p, a protein required for placement of the cell division site, reveals a link between the nucleus and the cell surface in fission yeast; Paoletti A et al.; mid1 is required for the proper placement of the contractile actin ring for cytokinesis at a medial site overlying the nucleus . Here we find that mid1 protein (mid1p) shuttles between the nucleus and a cortical medial broad band during interphase and early mitosis . The position of this broad band, which overlies the nucleus, is linked to nuclear position even in cells with displaced or multiple nuclei . We identified and created mutations in an NLS and in two crm1-dependent NES sequences in mid1p . NES mutations caused mid1p accumulation in the nucleus and loss of function . An NLS mutations greatly reduced nuclear localization but did not perturb cytoplasmic localization or function . mid1p localization to the medial broad band was also not dependent on mid1p PH domain or microtubule and actin cytoskeletons . Overexpression of mid1p produced ectopic cell growth at this band during interphase and abnormal karmellae-like nuclear membrane structures . In plo1-1, mid1p formed a medial broad band but did not incorporate into a tight ring, suggesting that polo kinase plo1p is required for activation of mid1p function . Thus, the mid1p broad band defines a compartment at the medial cell surface, whose localization is linked to the position of the nucleus, and whose function may be to position the plane of cell division.

Mol Biol Cell, 2000 Aug, 11(8), 2673 - 89
Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics; Audhya A et al.; The yeast Saccharomyces cerevisiae possesses two genes that encode phosphatidylinositol (PtdIns) 4-kinases, STT4 and PIK1 . Both gene products phosphorylate PtdIns at the D-4 position of the inositol ring to generate PtdIns(4)P, which plays an essential role in yeast viability because deletion of either STT4 or PIK1 is lethal . Furthermore, although both enzymes have the same biochemical activity, increased expression of either kinase cannot compensate for the loss of the other, suggesting that these kinases regulate distinct intracellular functions, each of which is required for yeast cell growth . By the construction of temperature-conditional single and double mutants, we have found that Stt4p activity is required for the maintenance of vacuole morphology, cell wall integrity, and actin cytoskeleton organization . In contrast, Pik1p is essential for normal secretion, Golgi and vacuole membrane dynamics, and endocytosis . Strikingly, pik1(ts) cells exhibit a rapid defect in secretion of Golgi-modified secretory pathway cargos, Hsp150p and invertase, whereas stt4(ts) cells exhibit no detectable secretory defects . Both single mutants reduce PtdIns(4)P by approximately 50%; however, stt4(ts)/pik1(ts) double mutant cells produce more than 10-fold less PtdIns(4)P as well as PtdIns(4,5)P(2) . The aberrant Golgi morphology found in pik1(ts) mutants is strikingly similar to that found in cells lacking the function of Arf1p, a small GTPase that is known to regulate multiple membrane trafficking events throughout the cell . Consistent with this observation, arf1 mutants exhibit reduced PtdIns(4)P levels . In contrast, diminished levels of PtdIns(4)P observed in stt4(ts) cells at restrictive temperature result in a dramatic change in vacuole size compared with pik1(ts) cells and persistent actin delocalization . Based on these results, we propose that Stt4p and Pik1p act as the major, if not the only, PtdIns 4-kinases in yeast and produce distinct pools of PtdIns(4)P and PtdIns(4,5)P(2) that act on different intracellular membranes to recruit or activate as yet uncharacterized effector proteins.

Mol Biol Cell, 2000 Aug, 11(8), 2631 - 42
Thioredoxin peroxidase is required for the transcriptional response to oxidative stress in budding yeast; Ross SJ et al.; A genetic screen was performed in Saccharomyces cerevisiae to identify mechanisms important for the transcriptional activation of genes encoding antioxidant proteins . Thioredoxin peroxidase, Tsa1p, of the thioredoxin system, was found to be essential for the transcriptional induction of other components of the thioredoxin system, TRX2 (thioredoxin) and TRR1 (thioredoxin reductase), in response to H(2)O(2) . The expression of TRX2 and TRR1 is known to be regulated by the transcription factors Yap1p and Skn7p in response to H(2)O(2), and the Tsa1p-dependent regulation of TRX2 requires the Yap1p/Skn7p pathway . The data suggest that expression of components of the thioredoxin system is dependent on the activity of Tsa1p in response to H(2)O(2) in a Yap1p/Skn7p-dependent pathway.

Mol Biol Cell, 2000 Aug, 11(8), 2605 - 16
The yeast TEL1 gene partially substitutes for human ATM in suppressing hyperrecombination, radiation-induced apoptosis and telomere shortening in A-T cells; Fritz E et al.; Homozygous mutations in the human ATM gene lead to a pleiotropic clinical phenotype of ataxia-telangiectasia (A-T) patients and correlating cellular deficiencies in cells derived from A-T donors . Saccharomyces cerevisiae tel1 mutants lacking Tel1p, which is the closest sequence homologue to the ATM protein, share some of the cellular defects with A-T . Through genetic complementation of A-T cells with the yeast TEL1 gene, we provide evidence that Tel1p can partially compensate for ATM in suppressing hyperrecombination, radiation-induced apoptosis, and telomere shortening . Complementation appears to be independent of p53 activation . The data provided suggest that TEL1 is a functional homologue of human ATM in yeast, and they help to elucidate different cellular and biochemical pathways in human cells regulated by the ATM protein.

J Biol Chem, 2000 Oct 20, 275(42), 33158 - 66
Poly(A) tail-dependent exonuclease AtRrp41p from Arabidopsis thaliana rescues 5.8 S rRNA processing and mRNA decay defects of the yeast ski6 mutant and is found in an exosome-sized complex in plant and yeast cells; Chekanova JA et al.; Eukaryotic 3'-->5' exonucleolytic activities are essential for a wide variety of reactions of RNA maturation and metabolism, including processing of rRNA, small nuclear RNA, and small nucleolar RNA, and mRNA decay . Two related but distinct forms of a complex containing 10 3'-->5' exonucleases, the exosome, are found in yeast nucleus and cytoplasm, respectively, and related complexes exist in human cells . Here we report on the characterization of the AtRrp41p, an Arabidopsis thaliana homolog of the Saccharomyces cerevisiae exosome subunit Rrp41p (Ski6p) . Purified recombinant AtRrp41p displays a processive phosphorolytic exonuclease activity and requires a single-stranded poly(A) tail on a substrate RNA as a "loading pad." The expression of the Arabidopsis RRP41 cDNA in yeast rescues the 5.8 S rRNA processing and 3'-->5' mRNA degradation defects of the yeast ski6-100 mutant . However, neither of these defects can explain the conditional lethal phenotype of the ski6-100 strain . Importantly, AtRrp41p shares additional function(s) with the yeast Rrp41p which are essential for cell viability because it also rescues the rrp41 (ski6) null mutant . AtRrp41p is found predominantly in a high molecular mass complex in Arabidopsis and in yeast cells, and it interacts in vitro with the yeast Rrp44p and Rrp4p exosome subunits, suggesting that it can participate in evolutionarily conserved interactions that could be essential for the integrity of the exosome complex.

J Biol Chem, 2000 Oct 27, 275(43), 33388 - 94
The family of SMF metal ion transporters in yeast cells; Cohen A et al.; Metal ions are vital for all organisms, and metal ion transporters play a crucial role in maintaining their homeostasis . The yeast (Saccharomyces cerevisiae) Smf transporters and their homologs in other organisms have a central role in the accumulation of metal ions and their distribution in different tissues and cellular organelles . In this work we generated null mutations in each individual SMF gene in yeast as well as in all combinations of the genes . Each null mutation exhibited sensitivity to metal ion chelators at different concentrations . The combination of null mutants DeltaSMF1 + DeltaSMF2 and the triple null mutant Delta3SMF failed to grow on medium buffered at pH 8 and 7.5, respectively . Addition of 5 microm copper or 25 microm manganese alleviated the growth arrest at the high pH or in the presence of the chelating agent . The transport of manganese was analyzed in the triple null mutant and in this mutant expressing each Smf protein . Although overexpression of Smf1p and Smf2p resulted in uptake that was higher than wild type cells, the expression of Smf3p gave no significant uptake above that of the triple mutant Delta3SMF . Western analysis with antibody against Smf3p indicated that this transporter does not reach the plasma membrane and may function at the Golgi or post-Golgi complexes . The iron uptake resulting from expression of Smf1p and Smf2p was analyzed in a mutant in which its iron transporters FET3 and FET4 were inactivated . Overexpression of Smf1p gave rise to a significant iron uptake that was sensitive to the sodium concentrations in the medium . We conclude that the Smf proteins play a major role in copper and manganese homeostasis and, under certain circumstances, Smf1p may function in iron transport into the cells.

Plant J, 2000 Jul, 23(2), 159 - 70
In vivo targeting of a sunflower oil body protein in yeast secretory (sec) mutants; Beaudoin F et al.; A sunflower oleosin was expressed in yeast to study the in vivo insertion of the protein into the endoplasmic reticulum (ER) and subsequent transfer to lipid bodies . The oleosin cDNA was expressed in a range of yeast secretory (sec) mutants to determine the precise targeting pathway of the oleosin to the ER . Subcellular fractionation experiments indicated that the signal recognition particle (SRP) is required for oleosin targeting to the ER and hence subsequent deposition on the lipid bodies in vivo . The expression of oleosin in a range of sec61 mutant alleles confirmed the role of the SEC61 translocon in insertion of oleosin into the ER membrane, as well as indicating an unusual substrate/translocon interaction for one particular allele (sec61-3) . Mistargeting of the oleosin due to impaired SRP function resulted in enhanced proteolysis of the plant protein in the transformed yeast, as determined by pulse-chase analysis . These data therefore provide the first in vivo evidence for the SRP-dependent targeting of the oleosin to the ER, and the subsequent requirement for a functional SEC61 translocon to mediate the correct insertion of the protein into the membrane.

Arch Neurol, 2000 Aug, 57(8), 1129 - 34
Applicability of yeast genetics to neurologic disease; Walberg MW; As advances in gene mapping technology reveal genes associated with neurologic diseases, the need to identify a gene's normal function arises often . Experimental genetics is very useful in identifying a gene's function . It relies on model organisms both because it is not appropriate in humans, and because many processes are remarkably similar among eukaryotes . Many cellular processes have evolved once, and species differences are variations on a theme . Molecular genetic tools available in the yeast Saccharomyces cerevisiae provide a means to more rapidly reach an understanding of gene function, yielding substantial insight into the same process in humans . Yeast will never complain of headache or "spells," but do have expansions of trinucleotide repeats, prions, and other processes very much analogous to those underlying many neurologic diseases . In spite of the absence of a nervous system in yeast, yeast genetics has contributed substantial insight into neurologic diseases mechanisms . The real strength of yeast in studying human disease is in genetic analysis of gene function and in providing genetically powerful functional assays . Arch Neurol . 2000;57:1129-1134

Science, 2000 Aug 4, 289(5480), 771 - 4
Pif1p helicase, a catalytic inhibitor of telomerase in yeast; Zhou J et al.; Mutations in the yeast Saccharomyces cerevisiae PIF1 gene, which encodes a 5'-to-3' DNA helicase, cause telomere lengthening and a large increase in the formation rate of new telomeres . Here, we show that Pif1p acts by inhibiting telomerase rather than telomere-telomere recombination, and this inhibition requires the helicase activity of Pif1p . Overexpression of enzymatically active Pif1p causes telomere shortening . Thus, Pif1p is a catalytic inhibitor of telomerase-mediated telomere lengthening . Because Pif1p is associated with telomeric DNA in vivo, its effects on telomeres are likely direct . Pif1p-like helicases are found in diverse organisms, including humans . We propose that Pif1p-mediated inhibition of telomerase promotes genetic stability by suppressing telomerase-mediated healing of double-strand breaks.

J Mol Biol, 2000 Aug 11, 301(2), 401 - 14
The crystal structure of yeast phenylalanine tRNA at 2.0 A resolution: cleavage by Mg(2+) in 15-year old crystals; Jovine L et al.; We have re-determined the crystal structure of yeast tRNA(Phe) to 2 . 0 A resolution using 15 year old crystals . The accuracy of the new structure, due both to higher resolution data and formerly unavailable refinement methods, consolidates the previous structural information, but also reveals novel details . In particular, the water structure around the tightly bound Mg(2+) is now clearly resolved, and hence provides more accurate information on the geometry of the magnesium-binding sites and the role of water molecules in coordinating the metal ions to the tRNA . We have assigned a total of ten magnesium ions and identified a partly conserved geometry for high-affinity Mg(2+ )binding . In the electron density map there is also clear density for a spermine molecule binding in the major groove of the TPsiC arm and also contacting a symmetry-related tRNA molecule . Interestingly, we have also found that two specific regions of the tRNA in the crystals are partially cleaved . The sites of hydrolysis are within the D and anticodon loops in the vicinity of Mg(2+) .

Somat Cell Mol Genet, 1999 Jan, 25(1), 41 - 8
Determination of the genotype of a panel of human tumor cell lines for the human homologues of yeast cell cycle checkpoint control genes: identification of cell lines carrying homoallelic missense base substitutions; Ejima Y et al.; A number of human homologues of yeast cell cycle checkpoint control genes have been identified recently . In this study, the sequence alterations in six of such novel human genes (hRAD1, hRAD9, hRAD17, hHUS1, CHK1 and CHES1) were analyzed by PCR-single-strand conformational polymorphism (PCR-SSCP) method on a panel of 25 human tumor cell lines in an attempt to search for possible in vivo cases where any of the checkpoint-related genes are altered in human systems . For hRAD9, hHUS1 or CHK1, no SSCP variant was detected in any of the cell lines tested, indicating a high stability of these genes in human cancer . Most of the SSCP variants found in the other three genes were due to single nucleotide base substitutions . Two cell lines were found to be homozygous for missense-type base substitutions, i.e., Saos-2 was homoallelic for 1637T-->G in hRAD17; and COLO320DM for 1189G-->A in CHES1, indicating a possible use of these cell lines for further study . The former nucleotide change in hRAD17, which causes a change of amino acid from arginine to lysine at codon 546, was supposed to be polymorphic . Considering that lysine, but not arginine, is the amino acid that is well conserved among fission yeast, mouse and monkey at the corresponding position, coexistence of both alleles in human may have a functional or selectional implication.

Biochim Biophys Acta, 2000 Jul 20, 1459(1), 230 - 8
Biophysical and structural characterization of proton-translocating NADH-dehydrogenase (complex I) from the strictly aerobic yeast Yarrowia lipolytica; Djafarzadeh R et al.; Mitochondrial proton-translocating NADH-dehydrogenase (complex I) is one of the largest and most complicated membrane bound protein complexes . Despite its central role in eukaryotic oxidative phosphorylation and its involvement in a broad range of human disorders, little is known about its structure and function . Therefore, we have started to use the powerful genetic tools available for the strictly aerobic yeast Yarrowia lipolytica to study this respiratory chain enzyme . To establish Y . lipolytica as a model system for complex I, we purified and characterized the multisubunit enzyme from Y lipolytica and sequenced the nuclear genes coding for the seven central subunits of its peripheral part . Complex I from Y lipolytica is quite stable and could be isolated in a highly pure and monodisperse state . One binuclear and four tetranuclear iron-sulfur clusters, including N5, which was previously known only from mammalian mitochondria, were detected by EPR spectroscopy . Initial structural analysis by single particle electron microscopy in negative stain and ice shows complex I from Y . lipolytica as an L-shaped particle that does not exhibit a thin stalk between the peripheral and the membrane parts that has been observed in other systems.

Genetics, 2000 Aug, 155(4), 1667 - 82
Suppressors of a cold-sensitive mutation in yeast U4 RNA define five domains in the splicing factor Prp8 that influence spliceosome activation; Kuhn AN et al.; The highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction . However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs . We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation . Genetic interactions between two of these regions reveal a potential long-range intramolecular fold . Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP . In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1 . On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.

Genetics, 2000 Aug, 155(4), 1657 - 65
The impact of lagging strand replication mutations on the stability of CAG repeat tracts in yeast; Ireland MJ et al.; We have examined the stability of long tracts of CAG repeats in yeast mutants defective in enzymes suspected to be involved in lagging strand replication . Alleles of DNA ligase (cdc9-1 and cdc9-2) destabilize CAG tracts in the stable tract orientation, i.e., when CAG serves as the lagging strand template . In this orientation nearly two-thirds of the events recorded in the cdc9-1 mutant were tract expansions . While neither DNA ligase allele significantly increases the frequency of tract-length changes in the unstable orientation, the cdc9-1 mutant produced a significant number of expansions in tracts of this orientation . A mutation in primase (pri2-1) destabilizes tracts in both the stable and the unstable orientations . Mutations in a DNA helicase/deoxyribonuclease (dna2-1) or in two RNase H activities (rnh1Delta and rnh35Delta) do not have a significant effect on CAG repeat tract stability . We interpret our results in terms of the steps of replication that are likely to lead to expansion and to contraction of CAG repeat tracts.

Genetics, 2000 Aug, 155(4), 1623 - 32
A DNA polymerase epsilon mutant that specifically causes +1 frameshift mutations within homonucleotide runs in yeast; Kirchner JM et al.; The DNA polymerases delta and epsilon are the major replicative polymerases in the yeast Saccharomyces cerevisiae that possess 3' --> 5' exonuclease proofreading activity . Many errors arising during replication are corrected by these exonuclease activities . We have investigated the contributions of regions of Polepsilon other than the proofreading motifs to replication accuracy . An allele, pol2-C1089Y, was identified in a screen of Polepsilon mutants that in combination with an exonuclease I (exo1) mutation could cause a synergistic increase in mutations within homonucleotide runs . In contrast to other polymerase mutators, this allele specifically results in insertion frameshifts . When pol2-C1089Y was combined with deletions of EXO1 or RAD27 (homologue of human FEN1), mutation rates were increased for +1 frameshifts while there was almost no effect on -1 frameshifts . On the basis of genetic analysis, the pol2-C1089Y mutation did not cause a defect in proofreading . In combination with a deletion of the mismatch repair gene MSH2, the +1 frameshift mutation rate for a short homonucleotide run was increased nearly 100-fold whereas the -1 frameshift rate was unchanged . This suggests that the Pol2-C1089Y protein makes +1 frameshift errors during replication of homonucleotide runs and that these errors can be corrected by either mismatch repair (MMR) or proofreading (in short runs) . This is the first report of a +1-specific mutator for homonucleotide runs in vivo . The pol2-C1089Y mutation defines a functionally important residue in Polepsilon.

Genetics, 2000 Aug, 155(4), 1607 - 21
Recombination can partially substitute for SPO13 in regulating meiosis I in budding yeast; Rutkowski LH et al.; Recombination and chromosome synapsis bring homologous chromosomes together, creating chiasmata that ensure accurate disjunction during reductional division . SPO13 is a key gene required for meiosis I (MI) reductional segregation, but dispensable for recombination, in Saccharomyces cerevisiae . Absence of SPO13 leads to single-division meiosis where reductional segregation is largely eliminated, but other meiotic events occur relatively normally . This phenotype allows haploids to produce viable meiotic products . Spo13p is thought to act by delaying nuclear division until sister centromeres/chromatids undergo proper cohesion for segregation to the same pole at MI . In the present study, a search for new spo13-like mutations that allow haploid meiosis recovered only new spo13 alleles . Unexpectedly, an unusual reduced-expression allele (spo13-23) was recovered that behaves similarly to a null mutant in haploids but to a wild-type allele in diploids, dependent on the presence of recombining homologs rather than on a diploid genome . This finding demonstrates that in addition to promoting accurate homolog disjunction, recombination can also function to partially substitute for SPO13 in promoting sister cohesion . Analysis of various recombination-defective mutants indicates that this contribution of recombination to reductional segregation requires full levels of crossing over . The implications of these results regarding SPO13 function are discussed.

Genetics, 2000 Aug, 155(4), 1577 - 91
Checkpoint proteins influence telomeric silencing and length maintenance in budding yeast; Longhese MP et al.; A complex network of surveillance mechanisms, called checkpoints, interrupts cell cycle progression when damage to the genome is detected or when cells fail to complete DNA replication, thus ensuring genetic integrity . In budding yeast, components of the DNA damage checkpoint regulatory network include the RAD9, RAD17, RAD24, MEC3, DDC1, RAD53, and MEC1 genes that are proposed to be involved in different aspects of DNA metabolism . We provide evidence that some DNA damage checkpoint components play a role in maintaining telomere integrity . In fact, rad53 mutants specifically enhance repression of telomere-proximal transcription via the Sir-mediated pathway, suggesting that Rad53 might be required for proper chromatin structure at telomeres . Moreover, Rad53, Mec1, Ddc1, and Rad17 are necessary for telomere length maintenance, since mutations in all of these genes cause a decrease in telomere size . The telomeric shortening in rad53 and mec1 mutants is further enhanced in the absence of SIR genes, suggesting that Rad53/Mec1 and Sir proteins contribute to chromosome end protection by different pathways . The finding that telomere shortening, but not increased telomeric repression of gene expression in rad53 mutants, can be suppressed by increasing dNTP synthetic capacity in these strains suggests that transcriptional silencing and telomere integrity involve separable functions of Rad53.

Genetics, 2000 Aug, 155(4), 1561 - 76
Structure-function analysis of yeast mRNA cap methyltransferase and high-copy suppression of conditional mutants by AdoMet synthase and the ubiquitin conjugating enzyme Cdc34p; Schwer B et al.; Here we present a genetic analysis of the yeast cap-methylating enzyme Abd1p . To identify individual amino acids required for Abd1p function, we introduced alanine mutations at 35 positions of the 436-amino acid yeast protein . Two new recessive lethal mutations, F256A and Y330A, were identified . Alleles F256L and Y256L were viable, suggesting that hydrophobic residues at these positions sufficed for Abd1p function . Conservative mutations of Asp-178 established that an acidic moiety is essential at this position (i.e . , D178E was viable whereas D178N was not) . Phe-256, Tyr-330, and Asp-178 are conserved in all known cellular cap methyltransferases . We isolated temperature-sensitive abd1 alleles and found that abd1-ts cells display a rapid shut-off of protein synthesis upon shift to the restrictive temperature, without wholesale reduction in steady-state mRNA levels . These in vivo results are consistent with classical biochemical studies showing a requirement for the cap methyl group in cap-dependent translation . We explored the issue of how cap methylation might be regulated in vivo by conducting a genetic screen for high-copy suppressors of the ts growth defect of abd1 mutants . The identification of the yeast genes SAM2 and SAM1, which encode AdoMet synthase, as abd1 suppressors suggests that Abd1p function can be modulated by changes in the concentration of its substrate AdoMet . We also identified the ubiquitin conjugating enzyme Cdc34p as a high-copy abd1 suppressor . We show that mutations of Cdc34p that affect its ubiquitin conjugation activity or its capacity to interact with the E3-SCF complex abrogate its abd1 suppressor function . Moreover, the growth defect of abd1 mutants is exacerbated by cdc34-2 . These findings suggest a novel role for Cdc34p in gene expression and engender a model whereby cap methylation or cap utilization is negatively regulated by a factor that is degraded when Cdc34p is overexpressed.

Genetics, 2000 Aug, 155(4), 1521 - 34
A new genetic method for isolating functionally interacting genes: high plo1(+)-dependent mutants and their suppressors define genes in mitotic and septation pathways in fission yeast; Cullen CF et al.; We describe a general genetic method to identify genes encoding proteins that functionally interact with and/or are good candidates for downstream targets of a particular gene product . The screen identifies mutants whose growth depends on high levels of expression of that gene . We apply this to the plo1(+) gene that encodes a fission yeast homologue of the polo-like kinases . plo1(+) regulates both spindle formation and septation . We have isolated 17 high plo1(+)-dependent (pld) mutants that show defects in mitosis or septation . Three mutants show a mitotic arrest phenotype . Among the 14 pld mutants with septation defects, 12 mapped to known loci: cdc7, cdc15, cdc11 spg1, and sid2 . One of the pld mutants, cdc7-PD1, was selected for suppressor analysis . As multicopy suppressors, we isolated four known genes involved in septation in fission yeast: spg1(+), sce3(+), cdc8(+), and rho1(+), and two previously uncharacterized genes, mpd1(+) and mpd2(+) . mpd1(+) exhibits high homology to phosphatidylinositol 4-phosphate 5-kinase, while mpd2(+) resembles Saccharomyces cerevisiae SMY2; both proteins are involved in the regulation of actin-mediated processes . As chromosomal suppressors of cdc7-PD1, we isolated mutations of cdc16 that resulted in multiseptation without nuclear division . cdc16(+), dma1(+), byr3(+), byr4(+) and a truncated form of the cdc7 gene were isolated by complementation of one of these cdc16 mutations . These results demonstrate that screening for high dose-dependent mutants and their suppressors is an effective approach to identify functionally interacting genes.

Biochemistry, 2000 Aug 8, 39(31), 9351 - 7
Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast; Grollman EF et al.; Monocarboxylate transporters (MCTs) comprise a group of highly homologous proteins that reside in the plasma membrane of almost all cells and which mediate the 1:1 electroneutral transport of a proton and a lactate ion . The isoform MCT3 is restricted to the basal membrane of the retinal pigment epithelium where it regulates lactate levels in the neural retina . Kinetic analysis of this transporter poses formidable difficulties due to the presence of multiple lactate transporters and their complex interaction with MCTs in adjacent cells . To circumvent these problems, we expressed both the MCT3 gene and a green fluorescent protein-tagged MCT3 construct in Saccharomyces cerevisiae . Since L-lactate metabolism in yeast depends on the CYB2 gene, we disrupted CYB2 to study the MCT3 transporter activity free from the complications of metabolism . Under these conditions L-lactate uptake varied inversely with pH, greater uptake being associated with lower pH . Whereas the V(max) was invariant, the K(m) increased severalfold as the pH rose from 6 to 8 . In addition, MCT3 was highly resistant to a number of "classical" inhibitors of lactate transport . Last, studies with diethyl pyrocarbonate and p-chloromercuribenzenesulfonate set limitations on the locus of potential residues involved in the critical site of lactate translocation.

Biochemistry, 2000 Aug 8, 39(31), 9157 - 63
The yeast mitochondrial citrate transport protein: determination of secondary structure and solvent accessibility of transmembrane domain IV using site-directed spin labeling; Kaplan RS et al.; To explore the spatial organization and functional dynamics of the citrate transport protein (CTP), a nitroxide scan was carried out along 22 consecutive residues within the fourth transmembrane domain (TMDIV) . This domain has been implicated as being of unique importance to the CTP mechanism due to (i) the presence of two intramembranous positive charges that are essential for CTP function and (ii) the existence of a transmembrane aqueous surface within this domain which likely corresponds to a portion of the citrate translocation pathway . The sequence-specific variation in the mobilities of the introduced nitroxides and their accessibilities to molecular O(2) reveal an alpha-helical conformation along the sequence . The accessibilities to NiEDDA are out of phase with accessibilites to O(2), indicating that one face of the helix is solvated by the lipid bilayer while the other is solvated by an aqueous environment . A gradient of NiEDDA accessibility is observed along the helix surface facing the aqueous phase, and the EPR spectral line shapes at these sites indicate considerable motional restriction . In the context of the model where TMDIV lines the translocation pathway, these data suggest a barrier to passive diffusion through the pathway . This paper reports the first use of site-directed spin labeling to study mitochondrial transporter structure.

Cancer Res, 2000 Jul 15, 60(14), 3813 - 22
In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene; Erbs P et al.; Direct transfer of prodrug activation systems into tumors was demonstrated to be an attractive method for the selective in vivo elimination of tumor cells . However, most current suicide gene therapy strategies are still handicapped by a poor efficiency of in vivo gene transfer and a limited bystander cell killing effect . In this study, we describe a novel and highly potent suicide gene derived from the Saccharomyces cerevisiae cytosine deaminase (FCY1) and uracil phosphoribosyltransferase genes (FUR1) . This suicide gene, designated FCU1, encodes a bifunctional chimeric protein that combines the enzymatic activities of FCY1 and FUR1 and efficiently catalyzes the direct conversion of 5-FC, a nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine-5'monophosphate, thus bypassing the natural resistance of certain human tumor cells to 5-fluorouracil . Unexpectedly, although the uracil phosphoribosyltransferase activity of FCU1 was equivalent to that encoded by FUR1, its cytosine deaminase activity was 100-fold higher than the one encoded by FCY1 . As a consequence, tumor cells transduced with an adenovirus expressing FCU1 (Ad-FCU1) were sensitive to concentrations of 5-FC 1000-fold lower than the ones used for cells transduced with a vector expressing FCY1 (Ad-FCY1) . Furthermore, bystander cell killing was also more effective in cells transduced with Ad-FCU1 than in cultures infected with Ad-FCY1 or Ad-FUR1, alone or in combination . Finally, intratumoral injections of Ad-FCU1 into allo- or xenogeneic tumors implanted s.c . into mice, with concomitant systemic administration of 5-FC, led to substantial delays in tumor growth . These unique properties make of the FCU1/5-FC prodrug activation system a novel and powerful candidate for cancer gene therapy strategies.

Yeast, 2000 Aug, 16(11), 1069 - 76
Current awareness on yeast; Xiang Z 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 . (5 weeks journals - search completed 31st May 2000)

Yeast, 2000 Aug, 16(11), 1035 - 43
The pYC plasmids, a series of cassette-based yeast plasmid vectors providing means of counter-selection; Olesen K et al.; A series of 24 general-purpose yeast plasmid vectors has been constructed . The plasmid series is composed of inter-replaceable cassettes, allowing for easy interconversion of plasmid types . In addition to the usual replication origins, selectable markers and multiple cloning sites (MCS), cassettes dedicated to counter-selection have been constructed . A pair of unique 8 bp restriction enzyme recognition sites flank each type of cassette, FseI in the case of yeast replication origins, AscI in the case of selectable markers, PacI in the case of counter-selectable markers and NotI in the case of the MCS . Thus, any given cassette can be replaced by another cassette of the same type, facilitating interconversion of any given plasmid from one type to another, even after the insertion of DNA into the MCS . Hence, the plasmids have been named pYC for 'yeast cassettes' . The cassettes consist of either NONE, CEN4/ARS or 2micro as replication origin, either URA3, MET2-CA (Lg-MET2) or the G418 resistance gene (the apt1 gene from bacterial transposon Tn903, encoding aminoglycoside phosphotransferase) as selectable markers, either NONE, PMET25-PKA3 or PCHA1-PKA3 as counter-selectable marker, and the MCS, containing recognition sites for AflII, AvrII, BspEI, PmeI, SacII, SalI, SunI, BamHI, EcoRI, HindIII, KpnI, MluI, NarI and SacI (of which the seven first are unique in all plasmids) . The counter-selectable markers consist of the PKA3 gene under control of the conditional MET25 or CHA1 promoters . At activating conditions these promoters express the PKA3 gene at toxic levels, facilitating easy selection for loss of plasmid or 'loop-out' of plasmid DNA sequence after genomic integration .

J Biol Chem, 2000 Oct 20, 275(42), 32611 - 6
Cadmium-inducible expression of the yeast GSH1 gene requires a functional sulfur-amino acid regulatory network; Dormer UH et al.; Glutathione (gamma-l-glutamyl-l-cysteinylglycine) is an important antioxidant molecule, helping to buffer the cell against free radicals and toxic electrophiles . Expression of the yeast GSH1 gene, encoding the first enzyme involved in glutathione biosynthesis, gamma-glutamylcysteine synthetase, is regulated by oxidants and the heavy metal cadmium at the level of transcription . We present evidence that the transcription factors involved in controlling the network of sulfur amino acid metabolism genes are also responsible for regulating GSH1 expression in response to cadmium . In particular the transcription factors Met-4, Met-31, and Met-32 are essential for cadmium-mediated regulation of gene expression, whereas the DNA-binding protein Cbf1 appears to play a negative role in controlling GSH1 expression.

EMBO J, 2000 Aug 1, 19(15), 4091 - 100
Derepression of DNA damage-regulated genes requires yeast TAF(II)s; Li B et al.; The general transcription factor TFIID and its individual subunits (TAF(II)s) have been the focus of many studies, yet their functions in vivo are not well established . Here we characterize the requirement of yeast TAF(II)s for the derepression of the ribonucleotide reductase (RNR) genes . Promoter mapping studies revealed that the upstream repressing sequences, the damage-responsive elements (DREs), rendered these genes dependent upon TAF(II)s . DREs are the binding sites for the sequence-specific DNA binding-protein Crt1 that represses transcription by recruiting the Ssn6-Tup1 co-repressor complex to the promoter . We demonstrate that deletion of SSN6, TUP1 or CRT1 alleviated the TAF(II) dependence of the RNR genes, indicating that TAF(II) dependence requires the co-repressor complex . Furthermore, we provide evidence that Crt1 specifies the TAF(II) dependence of these genes . Our studies show that TFIID interacts with the repression domain of Crt1, suggesting that the derepression mechanism involves an antagonism between TFIID and the co-repressor complex . Our results indicate that yeast TAF(II)s have other functions in addition to core promoter selectivity, and describe a novel activity: the derepression of promoters.

EMBO J, 2000 Aug 1, 19(15), 3968 - 77
Fission yeast Fizzy-related protein srw1p is a G(1)-specific promoter of mitotic cyclin B degradation; Yamaguchi S et al.; Downregulation of cyclin-dependent kinase (Cdk)-mitotic cyclin complexes is important during cell cycle progression and in G(1) arrested cells undergoing differentiation . srw1p, a member of the Fizzy-related protein family in fission yeast, is required for the degradation of cdc13p mitotic cyclin B during G(1) arrest . Here we show that srw1p is not required for the degradation of cdc13p during mitotic exit demonstrating that there are two systems operative at different stages of the cell cycle for cdc13p degradation, and that srw1p is phosphorylated by Cdk-cdc13p only becoming dephosphorylated during G(1) arrest . We propose that this phosphorylation targets srw1p for proteolysis and inhibits its activity to promote cdc13p turnover.

Rozhl Chir, 2000 Apr, 79(4), 179 - 85
{Yeast infections in patients in a surgical intensive care unit}; Sakra L et al.; The authors evaluate importance of candida infections in a surgical ICU . They present recent survey of aetiology, pathogenesis, diagnostic methods, clinical manifestations and therapy of invasive candida infections . The aim of this article is especially to summarize risk factors of the candida infection development.

Mutat Res, 2000 Jun 30, 451(1-2), 13 - 24
Nucleotide excision repair in yeast; Prakash S et al.; In nucleotide excision repair (NER) in eukaryotes, DNA is incised on both sides of the lesion, resulting in the removal of a fragment approximately 25-30 nucleotides long . This is followed by repair synthesis and ligation . The proteins encoded by the various yeast NER genes have been purified, and the incision reaction reconstituted in vitro . This reaction requires the damage binding factors Rad14, RPA, and the Rad4-Rad23 complex, the transcription factor TFIIH which contains the two DNA helicases Rad3 and Rad25, essential for creating a bubble structure, and the two endonucleases, the Rad1-Rad10 complex and Rad2, which incise the damaged DNA strand on the 5'- and 3'-side of the lesion, respectively . Addition of the Rad7-Rad16 complex to this reconstituted system stimulates the incision reaction many fold . The various NER proteins exist in vivo as part of multiprotein subassemblies which have been named NEFs (nucleotide excision repair factors) . Rad14 and Rad1-Rad10 form one subassembly called NEF1, the Rad4-Rad23 complex is named NEF2, Rad2 and TFIIH constitute NEF3, and the Rad7-Rad16 complex is called NEF4 . Although much has been learned from yeast about the function of NER genes and proteins in eukaryotes, the underlying mechanisms by which damage is recognized, NEFs are assembled at the damage site, and the DNA is unwound and incised, remain to be elucidated.

Hum Mol Genet, 2000 Jul 22, 9(12), 1795 - 803
Ataxin-3, the MJD1 gene product, interacts with the two human homologs of yeast DNA repair protein RAD23, HHR23A and HHR23B; Wang G et al.; Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder caused by an expansion of the polyglutamine tract near the C-terminus of the MJD1 gene product, ataxin-3 . The mutant ataxin-3 forms intranuclear inclusions in cultured cells as well as in diseased human brain and also causes cell death in transfected cells . However, the normal function of ataxin-3 remains unknown . To explore the function of ataxin-3, we used the two-hybrid system to screen for the protein(s) that interacts with ataxin-3 . We found that ataxin-3 interacts with two human homologs of the yeast DNA repair protein RAD23, HHR23A and HHR23B . Furthermore, we confirmed that ataxin-3 interacts with the -ubiquitin-like domain at the N-terminus of the HHR23 proteins, which is important for nucleotide excision repair; however, ataxin-3 does not interact with -ubiquitin, implying that ataxin-3 might be functionally associated with the HHR23 proteins through this specific interaction . The normal and mutant ataxin-3 proteins show no difference in their ability to bind to the HHR23 proteins . However, in 293 cells HHR23A is recruited to intranuclear inclusions formed by the mutant ataxin-3 through its interaction with ataxin-3 . These results suggest that this interaction is associated with the normal function of ataxin-3 and that some functional abnormality of the HHR23 proteins might exist in MJD.

Hum Mol Genet, 2000 Jul 22, 9(12), 1745 - 51
A cre-lox recombination system for the targeted integration of circular yeast artificial chromosomes into embryonic stem cells; Call LM et al.; The ability to produce embryonic stem (ES) cell lines containing different yeast artificial chromosomes (YACs) integrated into the same location in the genome provides a system for comparing the bio-logical effects of YAC transgenes without the confounding influences of integration site and copy number . A targeting system was developed for the directed integration of circular YACs into mouse ES cells . The system combines Cre-lox recombination technology, specifically a positive-selection integration system, with circular YAC lipofection technology to achieve single copy targeted integration of a transgene . Three independent germline competent ES cell lines {lox-containing ES lines (designated LES)} were created that contain a '-neo-lox' cassette integrated at different sites within the ES genome . A plasmid containing YAC vector sequences and a complementary '-neo-lox' cassette was used to circularize two linear YACs containing genomic DNA from human chromosome 21 . The circularized YACs were then targeted to the lox sites of the LES cell lines . Polymerase chain reaction and Southern analysis demonstrated that 21% (5 of 24) of lox-recombinants contain a full-length intact YAC . This system will make the study of YAC transgenic mice more reliable and reproducible, allowing the potential for direct comparison of different transgenes expressed from the same site within the genome.

Science, 2000 Jul 28, 289(5479), 595 - 9
Evidence for the prion hypothesis: induction of the yeast {PSI+} factor by in vitro- converted Sup35 protein; Sparrer HE et al.; Starting with purified, bacterially produced protein, we have created a {PSI(+)}-inducing agent based on an altered (prion) conformation of the yeast Sup35 protein . After converting Sup35p to its prion conformation in vitro, we introduced it into the cytoplasm of living yeast using a liposome transformation protocol . Introduction of substoichiometric quantities of converted Sup35p greatly increased the rate of appearance of the well-characterized epigenetic factor {PSI+}, which results from self-propagating aggregates of cellular Sup35p . Thus, as predicted by the prion hypothesis, proteins can act as infectious agents by causing self-propagating conformational changes.

Virology, 2000 Aug 1, 273(2), 300 - 6
Strain-specific interaction of the tobacco etch virus NIa protein with the translation initiation factor eIF4E in the yeast two-hybrid system; Schaad MC et al.; The NIa protein of potyviruses provides VPg and proteolytic functions during virus replication . It has also been shown to confer host genotype-specific movement functions in plants . Specifically, NIa from tobacco etch virus (TEV)-Oxnard, but not from most other strains, confers the ability to move long distances in Nicotiana tabacum cultivar "V-20." This led to the hypothesis that all or part of NIa may interact with one or more cellular factors . To identify cellular proteins that interact with NIa in a host- or strain-specific manner, a yeast two-hybrid search of a tomato cDNA library was done . Ten proteins that interacted with NIa were recovered, with translation initiation factor eIF4E being by far the most common protein identified . Interaction of eIF4E with NIa was shown to be TEV strain-specific . eIF4E from both tomato and tobacco interacted well with NIa from the HAT strain, but not from the Oxnard strain . However, using chimeric NIa proteins, the determinant for systemic infection of V20 plants was found to be genetically distinct from the determinant controlling eIF4E interaction . In TEV-eIF4E coexpression experiments, evidence suggesting that eIF4E provides a positive effect on genome amplification was obtained .

Mutat Res, 2000 Jun 30, 451(1-2), 39 - 51
Base excision repair in yeast and mammals; Memisoglu A et al.; Base excision repair (BER), as initiated by at least seven different DNA glycosylases or by enzymes that cleave DNA at abasic sites, executes the repair of a wide variety of DNA damages . Many of these damages arise spontaneously because DNA interacts with the cellular milieu, and so BER profoundly influences spontaneous mutation rates . In addition, BER provides significant protection against the toxic and mutagenic effects of DNA damaging agents present in the external environment, and as such is likely to prevent the adverse health effects of such agents . BER pathways have been studied in a wide variety of organisms (including yeasts) and here we review how these varied studies have shaped our current view of human BER.

FEBS Lett, 2000 Jul 7, 476(3), 277 - 81
Reconstitution of yeast microsomal lipid flip-flop using endogenous aminophospholipids; Nicolson T et al.; The molecular basis of transbilayer movement or flipping of phospholipids in the endoplasmic reticulum is largely unknown . To circumvent the problems inherent to studies with artificial phospholipid analogs, we studied microsomal flip-flop of endogenous phosphatidylethanolamine in yeast . The transbilayer transport of phosphatidylethanolamine was measured in reconstituted proteoliposomes derived from microsomal detergent extracts . Our results demonstrate that flipping is protease sensitive but does not require metabolic energy . Our assay is the first to use the endogenous substrate of the so-called 'flippase' to study phospholipid translocation in endomembranes and may therefore be crucial for the understanding of the catalytic properties of this elusive enzyme.

FEBS Lett, 2000 Jul 7, 476(3), 150 - 4
Primary structure and expression of peroxisomal acetylspermidine oxidase in the methylotrophic yeast Candida boidinii; Nishikawa M et al.; Acetylspermidine oxidase (ASOD) belongs to a family of FAD-containing amine oxidases and catalyzes the oxidation of N-acetylated spermidine in polyamine metabolism . ASOD was purified to apparent homogeneity from cells of the methylotrophic yeast Candida boidinii grown on spermidine as the sole nitrogen source . C . boidinii ASOD catalyzed the oxidation of only N(1)-acetylspermidine . Based on partial amino acid sequences, oligonucleotide primers were designed for polymerase chain reaction, and the ASOD-encoding gene, ASO1, was cloned . The open reading frame encoding ASO1 was 1530 bp long and corresponded to a protein of 509 amino acid residues (calculated molecular mass=57167 Da) . ASO1 contained a FAD-binding motif of G-A-G-I-A-G in the N-terminal region and carried an amino acid sequence of -S-K-L at the C-terminal, representing a typical peroxisome targeting signal 1 . ASOD was localized in the peroxisomes in overexpressed C . boidinii . To our knowledge, this is the first report on the gene coding for ASOD that can catalyze the oxidation of N-acetylated polyamine as a substrate, from any type of organism.

Biochem Biophys Res Commun, 2000 Aug 2, 274(2), 548 - 52
Interaction of yeast importin alpha with the NLS of prothymosin alpha is insufficient to trigger nuclear uptake of cargos; Shakulov VR et al.; A proliferation-related human protein prothymosin alpha displays exclusively nuclear localization when produced in human and Saccharomyces cerevisiae cells, whereas its isolated bipartite NLS confers nuclear targeting of the GFP reporter in human but not in yeast cells . To test whether this observation is indicative of the existence of specific requirements for nuclear targeting of proteins in yeast, a set of prothymosin alpha deletion mutants was constructed . Subcellular localization of these mutants fused to GFP was determined in yeast and compared with their ability to bind yeast importin alpha (Srp1p) in vitro . The NLS of prothymosin alpha turned out to be both necessary and sufficient to provide protein recognition by importin alpha . However, the NLS-importin alpha interaction did not ensure nuclear targeting of prothymosin alpha derivatives . This defect could be complemented by adding distinct prothymosin alpha sequences to the NLS-containing import substrate, possibly by providing binding site(s) for additional components of the yeast nuclear import machinery .

Biochemistry, 2000 Aug 1, 39(30), 9047 - 54
Characterization of an alkaline transition intermediate stabilized in the Phe82Trp variant of yeast iso-1-cytochrome c; Rosell FI et al.; In general, mutation of the phylogenetically conserved residue Phe82 in yeast iso-1-cytochrome c destabilizes the native conformation of the protein by facilitating the ligand exchange reactions that are associated with the alkaline conformational transitions of the ferricytochrome . Of the Phe82 variants surveyed thus far, Phe82Trp is unique in that it adopts a thermodynamically stable, high-spin conformation at mildly alkaline pH . This species exhibits spectroscopic features that can only be detected transiently in other ferricytochromes c within the first 100 ms immediately after a pH-jump from neutrality to pH >10 . Spectroscopic characterization of this high-spin reaction intermediate suggests that in addition to an obligatory pentacoordinate heme iron, a group within the heme pocket coordinates the heme iron but is then replaced either by Met80, to revert to the native conformation, or by Lys73 or Lys79, to yield one of the conventional alkaline conformers . Evidence is presented to suggest that this group is either a hydroxide ion or Tyr67 rather than a loosely bound Met80.

Mol Cell Biol, 2000 Aug, 20(16), 6062 - 73
Evidence for separable functions of Srp1p, the yeast homolog of importin alpha (Karyopherin alpha): role for Srp1p and Sts1p in protein degradation; Tabb MM et al.; Srp1p (importin alpha) functions as the nuclear localization signal (NLS) receptor in Saccharomyces cerevisiae . The srp1-31 mutant is defective in this nuclear localization function, whereas an srp1-49 mutant exhibits defects that are unrelated to this localization function, as was confirmed by intragenic complementation between the two mutants . RPN11 and STS1 (DBF8) were identified as high-dosage suppressors of the srp1-49 mutation but not of the srp1-31 mutation . We found that Sts1p interacts directly with Srp1p in vitro and also in vivo, as judged by coimmunoprecipitation and two-hybrid analyses . Mutants of Sts1p that cannot interact with Srp1p are incapable of suppressing srp1-49 defects, strongly suggesting that Sts1p functions in a complex with Srp1p . STS1 also interacted with the second suppressor, RPN11, a subunit of the 26S proteasome, in the two-hybrid system . Further, degradation of Ub-Pro-beta-galactosidase, a test substrate for the ubiquitin-proteasome system, was defective in srp1-49 but not in srp1-31 . This defect in protein degradation was alleviated by overexpression of either RPN11 or STS1 in srp1-49 . These results suggest a role for Srp1p in regulation of protein degradation separate from its well-established role as the NLS receptor.

Mol Cell Biol, 2000 Aug, 20(16), 5858 - 64
Cks1 is required for G(1) cyclin-cyclin-dependent kinase activity in budding yeast; Reynard GJ et al.; p13(suc1) (Cks) proteins have been implicated in the regulation of cyclin-dependent kinase (CDK) activity . However, the mechanism by which Cks influences the function of cyclin-CDK complexes has remained elusive . We show here that Cks1 is required for the protein kinase activity of budding yeast G(1) cyclin-CDK complexes . Cln2 and Cdc28 subunits coexpressed in baculovirus-infected insect cells fail to exhibit protein kinase activity towards multiple substrates in the absence of Cks1 . Cks1 can both stabilize Cln2-Cdc28 complexes and activate intact complexes in vitro, suggesting that it plays multiple roles in the biogenesis of active G(1) cyclin-CDK complexes . In contrast, Cdc28 forms stable, active complexes with the B-type cyclins Clb4 and Clb5 regardless of whether Cks1 is present . The levels of Cln2-Cdc28 and Cln3-Cdc28 protein kinase activity are severely reduced in cks1-38 cell extracts . Moreover, phosphorylation of G(1) cyclins, which depends on Cdc28 activity, is reduced in cks1-38 cells . The role of Cks1 in promoting G(1) cyclin-CDK protein kinase activity both in vitro and in vivo provides a simple molecular rationale for the essential role of CKS1 in progression through G(1) phase in budding yeast.

Ann N Y Acad Sci, 2000 Jun, 908, 21 - 30
Metabolic control and gene dysregulation in yeast aging; Jazwinski SM; Life span in the yeast Saccharomyces cerevisiae is usually measured by the number of divisions individual cells complete . Four broad physiologic processes that determine yeast life span have been identified: metabolic control, resistance to stress, chromatin-dependent gene regulation, and genetic stability . A pathway of interorganelle communication involving mitochondria, the nucleus, and peroxisomes has provided a molecular mechanism of aging based on metabolic control . This pathway functions continuously, rather than as an on-off switch, in determining life span . The longevity gene RAS2 modulates this pathway . RAS2 also modulates a variety of other cellular processes, including stress responses and chromatin-dependent gene regulation . An optimal level of Ras2p activity is required for maximum longevity . This may be due to the integration of life maintenance processes by RAS2, which functions as a homeostatic device in yeast longevity . Loss of transcriptional silencing of heterochromatic regions of the genome is a mark of yeast aging . It is now clear that the functional status of chromatin plays an important role in aging . Changes in this functional status result in gene dysregulation, which can be altered by manipulation of the histone deacetylase genes . Silencing of ribosomal DNA appears to be of particular importance . Extrachromosomal ribosomal DNA circles are neither sufficient nor necessary for yeast aging.

Mol Biotechnol, 2000 May, 15(1), 29 - 37
A yeast genetic assay for caspase cleavage of the amyloid-beta precursor protein; Gunyuzlu PL et al.; A functional assay for proteolytic processing of the amyloid precursor protein (APP) was set up in yeast . This consisted of a membrane-bound chimeric protein containing the beta-secretase cleaved C-terminal fragment of APP fused to the Ga14 transcription factor . Using this chimera in a GAL-reporter yeast strain, an expression library of human cDNAs was screened for clones that could activate the GAL-reporter genes by proteolytic processing of the membrane-bound APP-Gal4 . Two human proteases, caspase-3 and caspase-8, were identified and confirmed to act by a mechanism that involved proteolysis at the site in the APP-Gal4 chimera that corresponded to the natural caspase cleavage site in APP, thus linking a readily scorable phenotype to proteolytic processing of APP . The activation of caspase-3 involved a mechanism that was independent of aspartic acid residue 175 at the cleavage site normally required for processing of caspase-3.

J Cell Physiol, 2000 Sep, 184(3), 311 - 8
Transcriptional silencing in fission yeast; Grewal SI; In eukaryotes, epigenetic events govern diverse processes, ranging from gene expression to other aspects of global chromosome architecture essential for preserving the integrity of the genome . Transcriptional silencing at the mating-type locus, centromeres, and telomeres of the fission yeast is regulated by epigenetic mechanisms . Epigenetic states are inherited in cis during mitosis and, remarkably, even through meiosis . Several trans-acting genes that affect silencing are found to encode either chromatin proteins such as chromodomain proteins Swi6 and Clr4 or the factors that affect chromatin assembly, including histone deacetylase homologs Clr3 and Clr6 . A recent study showed that Swi6 is involved in imprinting at the mating-type locus and contributes to the cellular memory responsible for maintenance of the silenced state . The "gene" in this instance thus comprises DNA plus the associated Swi6-containing protein complex .

Glycobiology, 2000 Jul, 10(7), 737 - 44
Evidence for interaction of yeast protein kinase C with several subunits of oligosaccharyl transferase; Park H et al.; Oligosaccharyltransferase (OT) in Saccharomyces cerevisiae is an enzyme complex consisting of 8 transmembrane proteins located in the endoplasmic reticulum (ER) . Studies on potential protein-protein interactions in OT using a two-hybrid library screen revealed that protein kinase C (Pkc1p) interacted with the lumenal domains of several OT subunits . Additional genetic experiments revealed that overexpression of two OT subunits rescued the growth defect caused by overexpression of a Pkc1 active site mutant, implying that there are specific genetic interactions between PKC1 and OT . These in vivo findings were complemented by in vitro studies that showed that several of the OT subunits bound to a fusion protein consisting of glutathione S-transferase linked via its C-terminus to Pkc1p . Assays of OT activity, in which glycosylation of a simple acceptor peptide was assayed in microsomes from wild-type and a pkc1 null revealed a 50% reduction in activity in the microsomes from the null strain . In contrast, strains containing null mutations of two other genes known to be downstream of Pkc1p in the PKC1-MAP kinase pathway had a level of OT activity comparable to that of wild-type cells . These in vivo and in vitro experiments suggest that in yeast cells Pkc1p may be involved in regulation of the N-glycosylation of proteins.

J Biotechnol, 2000 Jun 23, 80(2), 189 - 93
Continuous ethanol production from pineapple cannery waste using immobilized yeast cells; Nigam JN; The cells of Saccharomyces cerevisiae ATCC 24553, were immobilized in k-carrageenan and packed in a tapered glass column reactor for ethanol production from pineapple cannery waste at temperature 30 degrees C and pH 4.5 . The maximum productivity was 42.8 g ethanol 1(-1) h(-1) at a dilution rate of 1.5 h(-1) . The volumetric ethanol productivity of the immobilized cells was ca . 11.5 times higher than the free cells . The immobilized cell reactor was operated over a period of 87 days at a dilution rate of 1.0 h(-1), without any loss in the immobilized cell activity . The maximum specific ethanol productivity and specific sugar uptake rate of the immobilized cells were 1.2 g ethanol g(-1) dry wt . cell h(-1) and 2.6 g sugar g(-1) dry wt . cell h(-1), respectively, at a dilution rate of 1.5 h(-1).

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