Yeasts

Curr Genet, 2004 May, 45(5), 273 - 82 Epub 2004 Mar 13.
The impact of manipulations with cytoplasmically inherited factors on nuclear transmission and degradation in yeast heterokaryons; Nevzglyadova OV et al.; Heterokaryotic zygotes in yeast provide a unique possibility to study the survival and transmission of two genetically diverse nuclei in one cell . Using partial pedigree analysis, we show that various treatments used to change cytoplasmic hereditary determinants can essentially affect nuclear transmission in yeast heterokaryons . This includes choice of nucleus to enter the first bud and incidence of various classes of mother/daughter pairs demonstrating nuclear degradation patterns in heterokaryotic zygotes . These treatments include guanidine hydrochloride, a prion-curing agent, ethidium bromide, an agent causing elimination of mitochondrial DNA, and cytoplasm replacement by cytoduction, which leads to mtDNA replacement and transfer of some other cytoplasmically inherited determinants . The genetic and cytological evidence obtained favors prion involvement in nuclear transmission and suggests apoptotic features in nuclear degradation in yeast heterokaryotic zygotes.

PLoS Biol . 2004 Mar;2(3):E79 . Epub 2004 Mar 16.
Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast; Gerber AP et al.; Genes encoding RNA-binding proteins are diverse and abundant in eukaryotic genomes . Although some have been shown to have roles in post-transcriptional regulation of the expression of specific genes, few of these proteins have been studied systematically . We have used an affinity tag to isolate each of the five members of the Puf family of RNA-binding proteins in Saccharomyces cerevisiae and DNA microarrays to comprehensively identify the associated mRNAs . Distinct groups of 40-220 different mRNAs with striking common themes in the functions and subcellular localization of the proteins they encode are associated with each of the five Puf proteins: Puf3p binds nearly exclusively to cytoplasmic mRNAs that encode mitochondrial proteins; Puf1p and Puf2p interact preferentially with mRNAs encoding membrane-associated proteins; Puf4p preferentially binds mRNAs encoding nucleolar ribosomal RNA-processing factors; and Puf5p is associated with mRNAs encoding chromatin modifiers and components of the spindle pole body . We identified distinct sequence motifs in the 3'-untranslated regions of the mRNAs bound by Puf3p, Puf4p, and Puf5p . Three-hybrid assays confirmed the role of these motifs in specific RNA-protein interactions in vivo . The results suggest that combinatorial tagging of transcripts by specific RNA-binding proteins may be a general mechanism for coordinated control of the localization, translation, and decay of mRNAs and thus an integral part of the global gene expression program.

Mol Cell Biol, 2004 Apr, 24(7), 2998 - 3010
Loss of translational control in yeast compromised for the major mRNA decay pathway; Holmes LE et al.; The cytoplasmic fate of mRNAs is dictated by the relative activities of the intimately connected mRNA decay and translation initiation pathways . In this study, we have found that yeast strains compromised for stages downstream of deadenylation in the major mRNA decay pathway are incapable of inhibiting global translation initiation in response to stress . In the past, the paradigm of the eIF2alpha kinase-dependent amino acid starvation pathway in yeast has been used to evaluate this highly conserved stress response in all eukaryotic cells . Using a similar approach we have found that even though the mRNA decay mutants maintain high levels of general translation, they exhibit many of the hallmarks of amino acid starvation, including increased eIF2alpha phosphorylation and activated GCN4 mRNA translation . Therefore, these mutants appear translationally oblivious to decreased ternary complex abundance, and we propose that this is due to higher rates of mRNA recruitment to the 40S ribosomal subunit.

Mol Cell Biol, 2004 Apr, 24(7), 2767 - 78
The yeast split-ubiquitin membrane protein two-hybrid screen identifies BAP31 as a regulator of the turnover of endoplasmic reticulum-associated protein tyrosine phosphatase-like B; Wang B et al.; In the past decade, traditional yeast two-hybrid techniques have identified a plethora of interactions among soluble proteins operating within diverse cellular pathways . The discovery of associations between membrane proteins by genetic approaches, on the other hand, is less well established due to technical limitations . Recently, a split-ubiquitin system was developed to overcome this barrier, but so far, this system has been limited to the analysis of known membrane protein interactions . Here, we constructed unique split-ubiquitin-linked cDNA libraries and provide details for implementing this system to screen for binding partners of a bait protein, in this case BAP31 . BAP31 is a resident integral protein of the endoplasmic reticulum, where it operates as a chaperone or cargo receptor and regulator of apoptosis . Here we describe a novel human member of the protein tyrosine phosphatase-like B (PTPLB) family, an integral protein of the endoplasmic reticulum membrane with four membrane-spanning alpha helices, as a BAP31-interacting protein . PTPLB turns over rapidly through degradation by the proteasome system . Comparisons of mouse cells with a deletion of Bap31 or reconstituted with human BAP31 indicate that BAP31 is required to maintain PTPLB, consistent with a chaperone or quality control function for BAP31 in the endoplasmic reticulum membrane.

J Cell Biol, 2004 Mar 15, 164(6), 803 - 9
A role for the actin cytoskeleton in cell death and aging in yeast; Gourlay CW et al.; Several determinants of aging, including metabolic capacity and genetic stability, are recognized in both yeast and humans . However, many aspects of the pathways leading to cell death remain to be elucidated . Here we report a role for the actin cytoskeleton both in cell death and in promoting longevity . We have analyzed yeast strains expressing mutants with either increased or decreased actin dynamics . We show that decreased actin dynamics causes depolarization of the mitochondrial membrane and an increase in reactive oxygen species (ROS) production, resulting in cell death . Important, however, is the demonstration that increasing actin dynamics, either by a specific actin allele or by deletion of a gene encoding the actin-bundling protein Scp1p, can increase lifespan by over 65% . Increased longevity appears to be due to these cells producing lower than wild-type levels of ROS . Homology between Scp1p and mammalian SM22/transgelin, which itself has been isolated in senescence screens, suggests a conserved mechanism linking aging to actin stability.

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 623 - 7
Malassezia nana sp . nov., a novel lipid-dependent yeast species isolated from animals; Hirai A et al.; Five isolates of a novel species of the yeast genus Malassezia were isolated from animals in Japan and Brazil . Phylogenetic trees based on the D1/D2 domains of the large-subunit (26S) rDNA sequences and nucleotide sequences of the internal transcribed spacer 1 region showed that the isolates were conspecific and belonged to the genus Malassezia . They were related closely to Malassezia dermatis and Malassezia sympodialis, but were clearly distinct from these two species and the other six species of Malassezia that have been reported, indicating that they should be classified as a novel species, Malassezia nana sp . nov . Morphologically and physiologically, M . nana resembles M . dermatis and M . sympodialis, but can be distinguished from these species by its inability to use Cremophor EL (Sigma) as the sole lipid source and to hydrolyse aesculin . The type strain of M . nana is NUSV 1003(T) (=CBS 9557(T)=JCM 12085(T)).

Mol Biol Cell, 2004 May, 15(5), 2401 - 9 Epub 2004 Mar 12.
Ubiquitin-mediated targeting of a mutant plasma membrane ATPase, Pma1-7, to the endosomal/vacuolar system in yeast; Pizzirusso M et al.; Pma1-7 is a mutant plasma membrane ATPase that is impaired in targeting to the cell surface at 37 degrees C and is delivered instead to the endosomal/vacuolar pathway for degradation . We have proposed that Pma1-7 is a substrate for a Golgibased quality control mechanism . By contrast with wild-type Pma1, Pma1-7 is ubiquitinated . Ubiquitination and endosomal targeting of Pma1-7 is dependent on the Rsp5-Bul1-Bul2 ubiquitin ligase protein complex but not the transmembrane ubiquitin ligase Tul1 . Analysis of Pma1-7 ubiquitination in mutants blocked in protein transport at various steps of the secretory pathway suggests that ubiquitination occurs after ER exit but before endosomal entry . In the absence of ubiquitination in rsp5-1 cells, Pma1-7 is delivered to the cell surface and remains stable . Nevertheless, Pma1-7 remains impaired in association with detergent-insoluble glycolipid-enriched complexes in rsp5-1 cells, suggesting that ubiquitination is not the cause of Pma1-7 exclusion from rafts . In vps1 cells in which protein transport into the endosomal pathway is blocked, Pma1-7 is routed to the cell surface . On arrival at the plasma membrane in vps1 cells, Pma1-7 remains stable and its ubiquitination disappears, suggesting deubiquitination activity at the cell surface . We suggest that Pma1-7 sorting and fate are regulated by ubiquitination.

J Biol Chem, 2004 May 28, 279(22), 23447 - 52 Epub 2004 Mar 12.
Crystal structure of yeast allantoicase reveals a repeated jelly roll motif; Leulliot N et al.; Allantoicase (EC 3.5.3.4) catalyzes the conversion of allantoate into ureidoglycolate and urea, one of the final steps in the degradation of purines to urea . The mechanism of most enzymes involved in this pathway, which has been known for a long time, is unknown . In this paper we describe the three-dimensional crystal structure of the yeast allantoicase determined at a resolution of 2.6 A by single anomalous diffraction . This constitutes the first structure for an enzyme of this pathway . The structure reveals a repeated jelly roll beta-sheet motif, also present in proteins of unrelated biochemical function . Allantoicase has a hexameric arrangement in the crystal (dimer of trimers) . Analysis of the protein sequence against the structural data reveals the presence of two totally conserved surface patches, one on each jelly roll motif . The hexameric packing concentrates these patches into conserved pockets that probably constitute the active site.

Genetics, 2004 Feb, 166(2), 765 - 77
Rtg2 protein links metabolism and genome stability in yeast longevity; Borghouts C et al.; Mitochondrial dysfunction induces a signaling pathway, which culminates in changes in the expression of many nuclear genes . This retrograde response, as it is called, extends yeast replicative life span . It also results in a marked increase in the cellular content of extrachromosomal ribosomal DNA circles (ERCs), which can cause the demise of the cell . We have resolved the conundrum of how these two molecular mechanisms of yeast longevity operate in tandem . About 50% of the life-span extension elicited by the retrograde response involves processes other than those that counteract the deleterious effects of ERCs . Deletion of RTG2, a gene that plays a central role in relaying the retrograde response signal to the nucleus, enhances the generation of ERCs in cells with (grande) or in cells without (petite) fully functional mitochondria, and it curtails the life span of each . In contrast, overexpression of RTG2 diminishes ERC formation in both grandes and petites . The excess Rtg2p did not augment the retrograde response, indicating that it was not engaged in retrograde signaling . FOB1, which is known to be required for ERC formation, and RTG2 were found to be in converging pathways for ERC production . RTG2 did not affect silencing of ribosomal DNA in either grandes or petites, which were similar to each other in the extent of silencing at this locus . Silencing of ribosomal DNA increased with replicative age in either the presence or the absence of Rtg2p, distinguishing silencing and ERC accumulation . Our results indicate that the suppression of ERC production by Rtg2p requires that it not be in the process of transducing the retrograde signal from the mitochondrion . Thus, RTG2 lies at the nexus of cellular metabolism and genome stability, coordinating two pathways that have opposite effects on yeast longevity.

Genetics, 2004 Feb, 166(2), 721 - 8
Homology modeling and mutational analysis of Ho endonuclease of yeast; Bakhrat A et al.; Ho endonuclease is a LAGLIDADG homing endonuclease that initiates mating-type interconversion in yeast . Ho is encoded by a free-standing gene but shows 50% primary sequence similarity to the intein (protein-intron encoded) PI-SceI . Ho is unique among LAGLIDADG endonucleases in having a 120-residue C-terminal putative zinc finger domain . The crystal structure of PI-SceI revealed a bipartite enzyme with a protein-splicing domain (Hint) and intervening endonuclease domain . We made a homology model for Ho on the basis of the PI-SceI structure and performed mutational analysis of putative critical residues, using a mating-type switch as a bioassay for activity and GFP-fusion proteins to detect nuclear localization . We found that residues of the N-terminal sequence of the Hint domain are important for Ho activity, in particular the DNA recognition region . C-terminal residues of the Hint domain are dispensable for Ho activity; however, the C-terminal putative zinc finger domain is essential . Mutational analysis indicated that residues in Ho that are conserved relative to catalytic, active-site residues in PI-SceI and other related homing endonucleases are essential for Ho activity . Our results indicate that in addition to the conserved catalytic residues, Hint domain residues and the zinc finger domain have evolved a critical role in Ho activity.

Genetics, 2004 Jan, 166(1), 67 - 77
Fus1p interacts with components of the Hog1p mitogen-activated protein kinase and Cdc42p morphogenesis signaling pathways to control cell fusion during yeast mating; Nelson B et al.; Cell fusion in the budding yeast Saccharomyces cerevisiae is a temporally and spatially regulated process that involves degradation of the septum, which is composed of cell wall material, and occurs between conjugating cells within a prezygote, followed by plasma membrane fusion . The plasma membrane protein Fus1p is known to be required for septum degradation during cell fusion, yet its role at the molecular level is not understood . We identified Sho1p, an osmosensor for the HOG MAPK pathway, as a binding partner for Fus1 in a two-hybrid screen . The Sho1p-Fus1p interaction occurs directly and is mediated through the Sho1p-SH3 domain and a proline-rich peptide ligand on the Fus1p COOH-terminal cytoplasmic region . The cell fusion defect associated with fus1Delta mutants is suppressed by a sho1Delta deletion allele, suggesting that Fus1p negatively regulates Sho1p signaling to ensure efficient cell fusion . A two-hybrid matrix containing fusion proteins and pheromone response pathway signaling molecules reveals that Fus1p may participate in a complex network of interactions . In particular, the Fus1p cytoplasmic domain interacts with Chs5p, a protein required for secretion of specialized Chs3p-containing vesicles during bud development, and chs5Delta mutants were defective in cell surface localization of Fus1p . The Fus1p cytoplasmic domain also interacts with the activated GTP-bound form of Cdc42p and the Fus1p-SH3 domain interacts with Bni1p, a yeast formin that participates in cell fusion and controls the assembly of actin cables to polarize secretion in response to Cdc42p signaling . Taken together, our results suggest that Fus1p acts as a scaffold for the assembly of a cell surface complex involved in polarized secretion of septum-degrading enzymes and inhibition of HOG pathway signaling to promote cell fusion.

Genetics, 2004 Jan, 166(1), 53 - 65
The yeast splicing factor Prp40p contains functional leucine-rich nuclear export signals that are essential for splicing; Murphy MW et al.; To investigate the function of the essential U1 snRNP protein Prp40p, we performed a synthetic lethal screen in Saccharomyces cerevisiae . Using an allele of PRP40 that deletes 47 internal residues and causes only a slight growth defect, we identified aphenotypic mutations in three distinct complementation groups that conferred synthetic lethality . The synthetic phenotypes caused by these mutations were suppressed by wild-type copies of CRM1 (XPO1), YNL187w, and SME1, respectively . The strains whose synthetic phenotypes were suppressed by CRM1 contained no mutations in the CRM1 coding sequence or promoter . This indicates that overexpression of CRM1 confers dosage suppression of the synthetic lethality . Interestingly, PRP40 and YNL187w encode proteins with putative leucine-rich nuclear export signal (NES) sequences that fit the consensus sequence recognized by Crm1p . One of Prp40p's two NESs lies within the internal deletion . We demonstrate here that the NES sequences of Prp40p are functional for nuclear export in a leptomycin B-sensitive manner . Furthermore, mutation of these NES sequences confers temperature-sensitive growth and a pre-mRNA splicing defect . Although we do not expect that yeast snRNPs undergo compartmentalized biogenesis like their metazoan counterparts, our results suggest that Prp40p and Ynl187wp contain redundant NESs that aid in an important, Crm1p-mediated nuclear export event.

Genetics, 2004 Jan, 166(1), 33 - 42
Chl1p, a DNA helicase-like protein in budding yeast, functions in sister-chromatid cohesion; Skibbens RV; From the time of DNA replication until anaphase onset, sister chromatids remain tightly paired along their length . Ctf7p/Eco1p is essential to establish sister-chromatid pairing during S-phase and associates with DNA replication components . DNA helicases precede the DNA replication fork and thus will first encounter chromatin sites destined for cohesion . In this study, I provide the first evidence that a DNA helicase is required for proper sister-chromatid cohesion . Characterizations of chl1 mutant cells reveal that CHL1 interacts genetically with both CTF7/ECO1 and CTF18/CHL12, two genes that function in sister-chromatid cohesion . Consistent with genetic interactions, Chl1p physically associates with Ctf7p/Eco1p both in vivo and in vitro . Finally, a functional assay reveals that Chl1p is critical for sister-chromatid cohesion . Within the budding yeast genome, Chl1p exhibits the highest degree of sequence similarity to human CHL1 isoforms and BACH1 . Previous studies revealed that human CHLR1 exhibits DNA helicase-like activities and that BACH1 is a helicase-like protein that associates with the tumor suppressor BRCA1 to maintain genome integrity . Our findings document a novel role for Chl1p in sister-chromatid cohesion and provide new insights into the possible mechanisms through which DNA helicases may contribute to cancer progression when mutated.

Infect Genet Evol, 2004 Mar, 4(1), 37 - 43
Evidence for aneuploidy and recombination in the human commensal yeast Candida parapsilosis; Fundyga RE et al.; Isolates of Candida parapsilosis, including representatives of the three major sub-species groups, were screened for single nucleotide polymorphisms (SNPs) by sequencing five independent loci totaling 4kb per isolate . Group I isolates were highly conserved and in some cases, group I alleles were found in group II and III strains . Unique alleles were also associated with groups II and III, consistent with earlier observations of intergroup divergence . There was no heterozygosity in any strain, and a FACS analysis demonstrated that for all three groups nuclei are variant in size, ranging from 0.5 to 1.0 x the size of other diploid yeast genomes . This suggests that natural isolates of C . parapsilosis are aneuploid, with some isolates being essentially haploid . Taken collectively with the observation of group I alleles within group II and III strains, we propose that some form of recombination is occurring between groups.

Biosens Bioelectron, 2004 Apr 15, 19(9), 953 - 62
The use of yeast and moulds as sensing elements in biosensors; Baronian KH; Whole cell biosensors are able to provide information that sensors based on single and multiple types of molecules are unable to do . For example, broad-spectrum catabolite analysis, cell toxicity and genotoxicity are best detected in the context of a functioning cell . Most whole cell sensors have used bacterial cells as the sensing element . Fungal cells, however, can provide all of the advantages bacterial cells offer but in addition they can provide information that is more relevant to other eukaryote organisms . These cells are easy to cultivate, manipulate for sensor configurations and are amenable to a wide range of transducer methodologies . An overview of the use of yeast and filamentous fungi as the sensing element of some biosensors is presented here.

Viral Immunol, 2004, 17(1), 115 - 22
Yeast-expressed hantavirus Dobrava nucleocapsid protein induces a strong, long-lasting, and highly cross-reactive immune response in mice; Geldmacher A et al.; In Europe, Dobrava virus (DOBV) carried by the yellow-necked field mouse Apodemus flavicollis is one of the hantaviruses that can cause severe hemorrhagic fever with renal syndrome in humans . For several hantaviruses, the nucleocapsid (N) protein has proven to be very immunogenic in humans and rodents and even can protect rodents against a virus challenge . To investigate the immunogenicity of DOBV N protein, BALB/c and C57BL/6 mice were immunized three times with a DOBV recombinant N (rN) protein expressed in yeast Saccharomyces cerevisiae together with complete Freund's, with incomplete Freund's, and without adjuvant, respectively . Mice of both strains elicited N-specific antibodies with end-point titers being as high as 1:1,000,000 in C57BL/6 mice . The antibodies induced by DOBV rN protein were highly cross-reactive to the rN proteins of hantaviruses Puumala and Hantaan . In both mice strains, DOBV rN protein induced N-specific antibodies of all IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3), suggesting a mixed Th1/Th2 immune response . Taken together, yeast-expressed DOBV rN protein represents a promising vaccine candidate.

Hum Pathol, 2004 Mar, 35(3), 350 - 6
Loss of p53 transcriptional activity in hepatocellular carcinoma evaluated by yeast-based functional assay: comparison with p53 immunohistochemistry; Mitsumoto Y et al.; We studied the transcriptional activity of p53 protein in 50 tissues of hepatocellular carcinoma (HCC) using a yeast functional assay . In this assay, red yeast colonies indicate that p53 protein cannot bind to its specific domain and has lost its transcriptional activity . We also clarified whether mutant p53 protein could inactivate wild-type p53 protein in a transdominant manner using a modified yeast assay . In addition, we examined whether immunohistochemically detectable p53 protein was functionally inactive . The incidence of p53 inactivation was significantly higher in tumors with capsular invasion . Out of 21 tumors diagnosed with p53 mutations, 11 exhibited >75% red colonies, and all contained missense mutations . In these tumors, p53 function was lost because there was supposedly no intact p53 gene on either allele . One missense mutant produced <60% red colonies, but it was also considered inactive as a p53 protein heterotetramer because of its transdominant activity . In 7 of the remaining 9 tumors, p53 was considered to be mutated on one allele and intact on the other . All of these 7 tumors contained nonsense or frameshift mutations and had no transdominant activity, which suggested that p53 function remained intact . Alternately, immunohistochemical analysis demonstrated that all of the tumors with missense mutations were positively immunostained, whereas those that contained nonsense or frameshift mutations were negatively stained . Consequently, positively immunostaining tumors mostly coincided with p53-inactive tumors . These yeast-based assays suggested that p53 function was retained in some mutant cases . Immunohistochemistry was helpful in screening functionally inactive p53 protein in HCCs.

J Virol, 2004 Apr, 78(7), 3502 - 13
High-throughput screening of the yeast kinome: identification of human serine/threonine protein kinases that phosphorylate the hepatitis C virus NS5A protein; Coito C et al.; The hepatitis C virus NS5A protein plays a critical role in virus replication, conferring interferon resistance to the virus through perturbation of multiple intracellular signaling pathways . Since NS5A is a phosphoprotein, it is of considerable interest to understand the role of phosphorylation in NS5A function . In this report, we investigated the phosphorylation of NS5A by taking advantage of 119 glutathione S-transferase-tagged protein kinases purified from Saccharomyces cerevisiae to perform a global screening of yeast kinases capable of phosphorylating NS5A in vitro . A database BLAST search was subsequently performed by using the sequences of the yeast kinases that phosphorylated NS5A in order to identify human kinases with the highest sequence homologies . Subsequent in vitro kinase assays and phosphopeptide mapping studies confirmed that several of the homologous human protein kinases were capable of phosphorylating NS5A . In vivo phosphopeptide mapping revealed phosphopeptides common to those generated in vitro by AKT, p70S6K, MEK1, and MKK6, suggesting that these kinases may phosphorylate NS5A in mammalian cells . Significantly, rapamycin, an inhibitor commonly used to investigate the mTOR/p70S6K pathway, reduced the in vivo phosphorylation of specific NS5A phosphopeptides, strongly suggesting that p70S6 kinase and potentially related members of this group phosphorylate NS5A inside the cell . Curiously, certain of these kinases also play a major role in mRNA translation and antiapoptotic pathways, some of which are already known to be regulated by NS5A . The findings presented here demonstrate the use of high-throughput screening of the yeast kinome to facilitate the major task of identifying human NS5A protein kinases for further characterization of phosphorylation events in vivo . Our results suggest that this novel approach may be generally applicable to the screening of other protein biochemical activities by mechanistic class.

Cell, 2004 Feb 6, 116(3), 405 - 15
Genetic and epigenetic regulation of the FLO gene family generates cell-surface variation in yeast; Halme A et al.; The FLO gene family of Saccharomyces cerevisiae includes an expressed gene, FLO11, and a set of silent, telomere-adjacent FLO genes . This gene family encodes cell-wall glycoproteins that regulate cell-cell and cell-surface adhesion . Epigenetic silencing of FLO11 regulates a key developmental switch: when FLO11 is expressed, diploid cells form pseudohyphal filaments; when FLO11 is silent, the cells grow in yeast form . The epigenetic state of FLO11 is heritable for many generations and regulated by the histone deacetylase (HDAC) Hda1p . The silent FLO10 gene is activated by high-frequency loss-of-function mutations at either IRA1 or IRA2 . FLO10 is regulated by the same transcription factors that control FLO11: Sfl1p and Flo8p, but is silenced by a distinct set of HDACs: Hst1p and Hst2p . These sources of epigenetic and genetic variation explain the observed heterogeneity of cell-surface protein expression within a population of cells derived from a single clone.

EMBO J, 2004 Mar 24, 23(6), 1301 - 12 Epub 2004 Mar 11.
Separation of silencing from perinuclear anchoring functions in yeast Ku80, Sir4 and Esc1 proteins; Taddei A et al.; In budding yeast, the nuclear periphery forms a subcompartment in which telomeres cluster and SIR proteins concentrate . To identify the proteins that mediate chromatin anchorage to the nuclear envelope, candidates were fused to LexA and targeted to an internal GFP-tagged chromosomal locus . Their ability to shift the locus from a random to a peripheral subnuclear position was monitored in living cells . Using fusions that cannot silence, we identify YKu80 and a 312-aa domain of Sir4 (Sir4(PAD)) as minimal anchoring elements, each able to relocalize an internal chromosomal locus to the nuclear periphery . Sir4(PAD)-mediated tethering requires either the Ku complex or Esc1, an acidic protein that is localized to the inner face of the nuclear envelope even in the absence of Ku, Sir4 or Nup133 . Finally, we demonstrate that Ku- and Esc1-dependent pathways mediate natural telomere anchoring in vivo . These data provide the first unambiguous identification of protein interactions that are both necessary and sufficient to localize chromatin to the nuclear envelope.

EMBO J, 2004 Mar 24, 23(6), 1289 - 300 Epub 2004 Mar 11.
Mechanism controlling perpendicular alignment of the spindle to the axis of cell division in fission yeast; Gachet Y et al.; In animal cells, the mitotic spindle is aligned perpendicular to the axis of cell division . This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring (CAR) . We show that, in fission yeast, spindle rotation is dependent on the interaction of astral microtubules with the cortical actin cytoskeleton . Interaction initially occurs with a region surrounding the nucleus, which we term the astral microtubule interaction zone (AMIZ) . Simultaneous contact of astral microtubules from both poles with the AMIZ directs spindle rotation and this requires both actin and two type V myosins, Myo51 and Myo52 . Astral microtubules from one pole only then contact the CAR, which is located at the centre of the AMIZ . We demonstrate that the anillin homologue Mid1, which dictates correct placement of the CAR, is necessary to stabilise the mitotic spindle perpendicular to the axis of cell division . Finally, we show that the position of the mitotic spindle is monitored by a checkpoint that regulates the timing of sister chromatid separation.

Clin Diagn Lab Immunol, 2004 Mar, 11(2), 426 - 9
Extracellular calcium and magnesium, but not iron, are needed for optimal growth of Blastomyces dermatitidis yeast form cells in vitro; Giles SS et al.; In the present study, we demonstrate that the yeast form of Blastomyces dermatitidis can proliferate for short periods of time in the absence of ferric iron but not in the absence of calcium or magnesium . The results of this study shed light on the resistance of B . dermatitidis to chelating agents, such as deferoxamine, and may explain how B . dermatitidis resists the iron-binding activity of serum transferrin.

FEBS Lett, 2004 Mar 12, 561(1-3), 22 - 8
AtHMA3, a plant P1B-ATPase, functions as a Cd/Pb transporter in yeast; Gravot A et al.; The Arabidopsis thaliana AtHMA3 protein belongs to the P(1B)-adenosine triphosphatase (ATPase) transporter family, involved in heavy metal transport . Functional expression of AtHMA3 phenotypically complements the Cd/Pb-hypersensitive yeast strain Deltaycf1, but not the Zn-hypersensitive mutant Deltazrc1 . AtHMA3-complemented Deltaycf1 cells accumulate the same amount of cadmium as YCF1-complemented Deltaycf1 cells or wild-type cells, suggesting that AtHMA3 carries out an intracellular sequestration of Cd . A mutant of AtHMA3 altered in the P-ATPase phosphorylation domain did not complement Deltaycf1, suggesting that metal transport rather than chelation is involved . The fusion protein AtHMA3::green fluorescent protein (GFP) is localized at the vacuole, consistent with a role in the influx of cadmium into the vacuolar compartment . In A . thaliana, the mRNA of AtHMA3 was detected mainly in roots, old rosette leaves and cauline leaves . The expression levels were not affected by cadmium or zinc treatments.

Sci STKE . 2004 Mar 02;2004(223):PL8.
Rapid depletion of budding yeast proteins by fusion to a heat-inducible degron; Sanchez-Diaz A et al.; One effective way to study the biological function of a protein in vivo is to inactivate it and see what happens to the cell . For proteins that are dispensable for cell viability, the corresponding gene can simply be deleted from its chromosomal locus . The study of essential proteins is more challenging, however, because the function of the protein must be inactivated conditionally . Here, we describe a method that allows the target protein to be depleted rapidly and conditionally, so that the immediate effects on the cell can be examined . The chromosomal locus of a budding yeast gene is modified so that a "heat-inducible degron cassette" is added to the N terminus of the encoded protein, causing it to be degraded by a specific ubiquitin-mediated pathway when cells are shifted from 24 degrees to 37 degrees C . Degradation requires recognition of the degron cassette by the evolutionarily conserved Ubr1 protein, which is associated with a ubiquitin-conjugating enzyme . To promote rapid and conditional depletion of the target protein, we use a yeast strain in which expression of the UBR1 gene can be either repressed or strongly induced . Degron strains are constructed by a simple "one-step" approach using the polymerase chain reaction.

DNA Repair (Amst), 2004 Apr 1, 3(4), 395 - 402
Polymerase zeta dependency of increased adaptive mutation frequencies in nucleotide excision repair-deficient yeast strains; Heidenreich E et al.; Reversions of an auxotrophy-causing frameshift allele during prolonged starvation of yeast cells were used as a means to elucidate the mechanisms concerned with the generation of spontaneous adaptive mutations in cell cycle-arrested cells . Whereas about 50% of these reversions were previously shown to depend on the non-homologous end joining pathway of DNA double-strand break repair, the origin of the residual 50% remains unknown . In search for a mechanism for generation of the latter fraction of reversions we examined the role of the translesion synthesis (TLS) polymerases zeta, eta and Rev1p in cells with wild-type or impaired nucleotide excision repair (NER) capacity . The basal level of adaptive mutations in the repair-proficient wild type was not influenced by disruptions of the genes coding for these three TLS polymerases . Intriguingly, a deficiency in NER by disruption of RAD14, RAD16 or RAD26 resulted in a significantly higher frequency of adaptive mutation, yet this increase was strictly dependent on an intact REV3 gene, coding for the catalytic subunit of polymerase zeta . Furthermore, we observed that intact REV3 was also required for the occurrence of increased frequencies of adaptive mutants in the NER-proficient wild type following UV irradiation . While in proliferating cells the translesion synthesis function of polymerase zeta is connected to DNA replication, our data suggest that in cell cycle-arrested cells this enzyme is able to carry out either TLS or error-prone polymerization along an undamaged template in the course of repair processes . Such a hitherto unappreciated activity of polymerase zeta in non-replicating cells may contribute to the incidence of mutations in evolution, aging and cancer.

Mol Microbiol, 2004 Mar, 51(6), 1649 - 59
The Sup35 domains required for maintenance of weak, strong or undifferentiated yeast {PSI+} prions; Bradley ME et al.; The Sup35 protein can exist in a non-infectious form or in various infectious forms called {PSI+} prion variants (or prion strains) . Each of the different {PSI+} prion variants converts non-infectious Sup35 molecules into that prion variant's infectious form . One definition of a 'prion domain' is the minimal fragment of a prion protein that is necessary and sufficient to maintain the prion form . We now demonstrate that the Sup35 N region (residues 1-123), which is frequently referred to as the 'prion domain', is insufficient to maintain the weak or strong {PSI+} variants per se, but appears to maintain them in an 'undifferentiated' {PSI+} state that can differentiate into weak or strong {PSI+} variants when transferred to the full-length Sup35 protein . In contrast, Sup35 residues 1-137 are necessary and sufficient to faithfully maintain weak or strong {PSI+} variants . This implicates Sup35 residues 124-137 in the variant-specific maintenance of the weak or strong {PSI+} forms . Structure predictions indicate that the residues in the 124-137 region form an alpha-helix and that the 1-123 region may have beta structure . In view of these findings, we discuss a plausible molecular basis for the {PSI+} prion variants as well as the inherent difficulties in defining a 'prion domain'.

Eur J Biochem, 2004 Mar, 271(6), 1209 - 18
Further insights into the assembly of the yeast cytochrome bc1 complex based on analysis of single and double deletion mutants lacking supernumerary subunits and cytochrome b; Zara V et al.; The cytochrome bc1 complex of the yeast Saccharomyces cerevisiae is composed of 10 different subunits that are assembled as a symmetrical dimer in the inner mitochondrial membrane . Three of the subunits contain redox centers and participate in catalysis, whereas little is known about the function of the seven supernumerary subunits . To gain further insight into the function of the supernumerary subunits in the assembly process, we have examined the subunit composition of mitochondrial membranes isolated from yeast mutants in which the genes for supernumerary subunits and cytochrome b were deleted and from yeast mutants containing double deletions of supernumerary subunits . Deletion of any one of the genes encoding cytochrome b, subunit 7 or subunit 8 caused the loss of the other two subunits . This is consistent with the crystal structure of the cytochrome bc1 complex that shows that these three subunits comprise its core, around which the remaining subunits are assembled . Absence of the cytochrome b/subunit 7/subunit 8 core led to the loss of subunit 6, whereas cytochrome c1, iron-sulfur protein, core protein 1, core protein 2 and subunit 9 were still assembled in the membrane, although in reduced amounts . Parallel changes in the amounts of core protein 1 and core protein 2 in the mitochondrial membranes of all of the deletion mutants suggest that these can be assembled as a subcomplex in the mitochondrial membrane, independent of the presence of any other subunits . Likewise, evidence of interactions between subunit 6, subunit 9 and cytochrome c1 suggests that a subcomplex between these two supernumerary subunits and the cytochrome might exist.

Genes Cells, 2004 Feb, 9(2), 95 - 104
Autoregulation of the HAC1 gene is required for sustained activation of the yeast unfolded protein response; Ogawa N et al.; Eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) by activating a transcriptional induction program termed the unfolded protein response (UPR) . The transcription factor Hac1p responsible for the UPR in Saccharomyces cerevisiae is tightly regulated by a post-transcriptional mechanism . HAC1 mRNA must be spliced in response to ER stress to produce Hac1p, which then activates transcription via direct binding to the cis-acting UPR element (UPRE) present in the promoter regions of its target genes . Here, we show that the HAC1 promoter itself responds to ER stress to induce transcription of its downstream gene, similarly to the KAR2 promoter; the KAR2 gene represents a major target of the UPR . Consistent with this observation, the HAC1 promoter contains an UPRE-like sequence, which is necessary and sufficient for the induction and to which Hac1p binds directly . Cells expressing the HAC1 gene from a mutant HAC1 promoter lacking the HAC1 UPRE could not maintain high levels of either unspliced or spliced HAC1 mRNA and became sensitive to ER stress when insulted for hours . Based on these results, we concluded that autoregulation of the HAC1 genes is required for sustained activation of the UPR and sustained resistance to ER stress.

Pharmacogenomics J, 2004, 4(3), 175 - 83
Gene editing of a human gene in yeast artificial chromosomes using modified single-stranded DNA and dual targeting; van Brabant AJ et al.; A single-nucleotide polymorphism (SNP) in a human gene can alter the behavior of the corresponding protein, and thereby affect an individual's response to drug therapy . Here, we describe a novel dual-targeting approach for introducing an SNP of choice into virtually any gene, through the use of modified single-stranded oligonucleotides (MSSOs) . We use this strategy to create SNPs in a human gene contained in a yeast artificial chromosome (YAC) . In the dual-targeting protocol, two different MSSOs are designed to edit two different bases in the same cell . A change in one of these genes is selective while the other is non-selective . We show that the population identified by selective pressure is enriched for cells that bear an edited base at the nonselective site . YACs with human genomic inserts containing particular SNPs or haplotypes can be used for pharmacogenomic applications, in cell lines and in transgenic animals.

Int J Biochem Cell Biol, 2004 May, 36(5), 900 - 8
Inhibition of yeast glutathione reductase by trehalose: possible implications in yeast survival and recovery from stress; Sebollela A et al.; Accumulation of trehalose has been implicated in the tolerance of yeast cells to several forms of stress, including heat-shock and high ethanol levels . However, yeast lacking trehalase, the enzyme that degrades trehalose, exhibit poor survival after exposure to stress conditions . This suggests that optimal cell viability also depends on the capacity to rapidly degrade the high levels of trehalose that build up under stress . Here, we initially examined the effects of trehalose on the activity of an important antioxidant enzyme, glutathione reductase (GR), from Saccharomyces cerevisiae . At 25 degrees C, GR was inhibited by trehalose in a dose-dependent manner, with 70% inhibition at 1.5M trehalose . The inhibition was practically abolished at 40 degrees C, a temperature that induces a physiological response of trehalose accumulation in yeast . The inhibition of GR by trehalose was additive to the inhibition caused by ethanol, indicating that enzyme function is drastically affected upon ethanol-induced stress . Moreover, two other yeast enzymes, cytosolic pyrophosphatase and glucose 6-phosphate dehydrogenase, showed temperature dependences on inhibition by trehalose that were similar to the temperature dependence of GR inhibition . These results are discussed in terms of the apparent paradox represented by the induction of enzymes involved in both synthesis and degradation of trehalose under stress, and suggest that the persistence of high levels of trehalose after recovery from stress could lead to the inactivation of important yeast enzymes.

Cell, 2004 Mar 5, 116(5), 699 - 709
Identification and distinct regulation of yeast TATA box-containing genes; Basehoar AD et al.; Despite being one of the first eukaryotic transcriptional regulatory elements identified, the sequence of a native TATA box and its significance remain elusive . Applying criteria associated with TATA boxes we queried several Saccharomyces genomes and arrived at the consensus TATA(A/T)A(A/T)(A/G) . Approximately 20% of yeast genes contain a TATA box . Strikingly, TATA box-containing genes are associated with responses to stress, are highly regulated, and preferentially utilize SAGA rather than TFIID when compared to TATA-less promoters . Transcriptional regulation in yeast appears to be mechanistically bipolar, possibly reflecting a need to balance inducible stress-related responses with constitutive housekeeping functions.

Cancer Biol Ther, 2004 May, 3(5), 453 - 7 Epub 2004 May 18.
Functional Characterization of BRCA1 Sequence Variants Using a Yeast Small Colony Phenotype Assay; Coyne RS et al.; Germline mutations that inactivate the tumor suppressor gene BRCA1 are associated with an increased risk of cancers of the breast and other tissues, but the functional consequence of many missense variants found in the human population is uncertain . Several predictive methods have been proposed to distinguish cancer-predisposing missense mutations from harmless polymorphisms, including a small colony phenotype (SCP) assay performed in the model organism, yeast (Saccharomyces cerevisiae) . The goal of this study was to further evaluate this colony size assay . We constructed 28 missense mutations throughout the C-terminal 305 amino acid residues of BRCA1 . Mutated proteins were expressed in yeast and evaluated using the SCP assay . We conclude there is as yet no evidence the assay can identify inactivating mutations upstream of the BRCT repeats . However, within and between the BRCT repeats, results of the assay are in general agreement with predictions based on structural modeling, other in vitro and in vivo assays, and cross-species sequence conservation . Thus, the yeast assay appears to provide confirmatory in vivo evidence to aid in characterizing some BRCA1 missense variants.

Methods, 2004 Apr, 32(4), 363 - 70
Increasing specificity in high-throughput yeast two-hybrid experiments; Vidalain PO et al.; Since its inception, the yeast two-hybrid (Y2H) system has proven to be an efficient system to identify novel protein-protein interactions . However, Y2H screens are sometimes criticized for generating high rates of false-positives . Minimizing false-positive interactions is especially important in proteome wide high-throughput (HT) Y2H . Here, we summarize various approaches that reduce false-positives in HT-Y2H projects . We evaluated the potential of examining putative positives after removing the prey encoding plasmid by negative selection . We found that this method reliably identifies false-positives caused by spontaneous conversion of baits into auto-activators and provides significant time-savings in HT screens . In addition, we present a method to eliminate an important source of false-positives: contaminating prey plasmids . Y2H interactors can be wrongly identified due to the presence of two or more different plasmids in the cells of a single yeast colony . Of these independent plasmids, only one encodes a genuine interactor . Contaminating plasmids are eliminated by extended culture of yeast cells under positive selection for the interaction, allowing the identification of the true interaction partner.

J Mol Biol, 2004 Mar 19, 337(2), 295 - 305
Enhanced CPT sensitivity of yeast cells and selective relaxation of Ga14 motif-containing DNA by novel Gal4-topoisomerase I fusion proteins; Alessandri M et al.; Human topoisomerase I-B (Top1) efficiently relaxes DNA supercoils during basic cellular processes, and can be transformed into a DNA-damaging agent by antitumour drugs, enzyme mutations and DNA lesions . Here, we describe Gal4-Top1 chimeric proteins (GalTop) with an N-terminal truncation of Top1, and mutations of the Gal4 Zn-cluster and/or Top1 domains that impair their respective DNA-binding activities . Expression levels of chimeras were similar in yeast cells, however, GalTop conferred an increased CPT sensitivity to RAD52- yeast cells as compared to a GalTop with mutations of the Gal4 domain, showing that a functional Gal4 domain can alter in vivo functions of Top1 . In vitro enzyme activity was tested with a DNA relaxation assay using negatively supercoiled plasmids with 0 to 5 Gal4 consensus motifs . Only GalTop with a functional Gal4 domain could direct DNA relaxation activity of Top1 specifically to DNA molecules containing Gal4 motifs . By using a substrate competition assay, we could demonstrate that the Gal4-anchored Top1 remains functional and efficiently relax DNA substrates in cis . The enhanced CPT sensitivity of GalTop in yeast cells may then be due to alterations of the chromatin-binding activity of Top1 . The GalTop chimeras may indeed mimic a normal mechanism by which Top1 is recruited to chromatin sites in living cells . Such hybrid Top1s may be helpful in further dissecting enzyme functions, and constitute a prototype of a site-specific DNA cutter endowed with high cell lethality.

J Biol Chem, 2004 May 7, 279(19), 19775 - 80 Epub 2004 Mar 03.
Atp10p assists assembly of Atp6p into the F0 unit of the yeast mitochondrial ATPase; Tzagoloff A et al.; The F(0)F(1)-ATPase complex of yeast mitochondria contains three mitochondrial and at least 17 nuclear gene products . The coordinate assembly of mitochondrial and cytosolic translation products relies on chaperones and specific factors that stabilize the pools of some unassembled subunits . Atp10p was identified as a mitochondrial inner membrane component necessary for the biogenesis of the hydrophobic F(0) sector of the ATPase . Here we show that, following its synthesis on mitochondrial ribosomes, subunit 6 of the ATPase (Atp6p) can be cross-linked to Atp10p . This interaction is required for the integration of Atp6p into a partially assembled subcomplex of the ATPase . Pulse labeling and chase of mitochondrial translation products in vivo indicate that Atp6p is less stable and more rapidly degraded in an atp10 null mutant than in wild type . Based on these observations, we propose Atp10p to be an Atp6p-specific chaperone that facilitates the incorporation of Atp6p into an intermediate subcomplex of ATPase subunits.

Fungal Genet Biol, 2004 Apr, 41(4), 391 - 400
Polarity in filamentous fungi: moving beyond the yeast paradigm; Harris SD et al.; Filamentous fungi grow by the polar extension of hyphae . This polar growth requires the specification of sites of germ tube or branch emergence, followed by the recruitment of the morphogenetic machinery to those sites for localized cell wall deposition . Researchers attempting to understand hyphal morphogenesis have relied upon the powerful paradigm of bud emergence in the yeast Saccharomyces cerevisiae . The yeast paradigm has provided a useful framework, however several features of hyphal morphogenesis, such as the ability to maintain multiple axes of polarity and an extremely rapid extension rate, cannot be explained by simple extrapolation from yeast models . We discuss recent polarity research from filamentous fungi focusing on the position of germ tube emergence, the relaying of positional information via RhoGTPase modules, and the recruitment of morphogenetic machinery components including cytoskeleton, polarisome and ARP2/3 complexes, and the vesicle trafficking system.

Mutat Res, 1976 Jun, 35(2), 207 - 12
Dependence of the expression of the radiation-induced gene conversion to arginine independence in diploid yeast on the amino acid concentration: effect on allelic mapping; Murthy MS et al.; The yield of radiation-induced gene conversion to arginine independence in diploid yeast depended on the concentration of the amino acid both in the plating medium and in the intracellular pool . By depletion of the level of arginine in the intracellular pool of amino acid or by provision of arginine at 0.4 mg/l of the plating medium the yield was varied by a factor as high as 20 . This may be important in studies of the genetic mapping of alleles based on the slope of conversion frequency versus dose line.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 606 - 9 Epub 2004 Feb 25.
Production and preliminary analysis of perdeuterated yeast inorganic pyrophosphatase crystals suitable for neutron diffraction; Tuominen VU et al.; Yeast inorganic pyrophosphatase (Y-PPase) is a model system for studying phosphoryl-transfer reactions catalysed by multiple metal ions . To understand the process requires knowledge of the positions of the protons in the active site, which can be best achieved by neutron diffraction analysis . In order to reduce the hydrogen incoherent-scattering background and to improve the signal-to-noise ratio of the neutron reflections, deuterated protein was produced . Deuterated protein 96% enriched with deuterium was produced in high yield and crystals as large as 2 mm on one side were obtained . These crystals have unit-cell parameters a = 58.9, b = 103.9, c = 117.0 A, alpha = beta = gamma = 90 degrees at 273 K and diffract neutrons to resolutions of 2.5-3 A . The X-ray structure of the perdeuterated protein has also been refined at 273 K to 1.9 A resolution.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 601 - 5 Epub 2004 Feb 25.
Microseed matrix screening to improve crystals of yeast cytosine deaminase; Ireton GC et al.; A crystallization strategy termed 'microseed matrix screening' is described where the optimal conditions for nucleation versus extended lattice growth are not compatible . This method is an extension of conventional seeding techniques in which microseeds from the nucleation step are systematically seeded into new conditions where all components of the drop are allowed to vary to screen for subsequent growth of well ordered specimens . The structure of a crystal form of yeast cytosine deaminase produced by streak-seeding using a single condition for both nucleation and growth is compared with the structure of a related crystal form produced by separating nucleation and growth conditions . The resulting structural comparison demonstrates that differential chelation patterns of cations by acidic surface residues of proteins within crystal lattice contacts is a critical parameter of crystal nucleation and growth.

Mol Cell Biol, 2004 Mar, 24(6), 2344 - 51
RAD51-dependent break-induced replication in yeast; Davis AP et al.; A chromosome fragmentation assay was used to measure the efficiency and genetic control of break-induced replication (BIR) in Saccharomyces cerevisiae . Formation of a chromosome fragment by de novo telomere generation at one end of the linear vector and recombination-dependent replication of 100 kb of chromosomal sequences at the other end of the vector occurred at high frequency in wild-type strains . RAD51 was required for more than 95% of BIR events involving a single-end invasion and was essential when two BIR events were required for generation of a chromosome fragment . The similar genetic requirements for BIR and gene conversion suggest a common strand invasion intermediate in these two recombinational repair processes . Mutation of RAD50 or RAD59 conferred no significant defect in BIR in either RAD51 or rad51 strains . RAD52 was shown to be essential for BIR at unique chromosomal sequences, although rare recombination events were detected between the subtelomeric Y' repeats.

Biochem J, 2004 Jun 1, 380(Pt 2), 487 - 96
The Ftr1p iron permease in the yeast plasma membrane: orientation, topology and structure-function relationships; Severance S et al.; Ftr1p is the permease component of the Fet3p-Ftr1p high affinity iron-uptake complex, in the plasma membrane of Saccharomyces cerevisiae, that transports the Fe3+ produced by the Fet3p ferroxidase into the cell . In this study we show that Ftr1p probably has seven transmembrane domains with an orientation of N-terminal outside, and C-terminal inside the cell . Within the context of this topology of the Fet3p-Ftr1p complex, we have identified several sequence elements in Ftr1p that are required for wild-type uptake function . First to be identified were two REXLE (Arg-Glu-Xaa-Leu-Glu) motifs in transmembrane domains 1 and 4 . Alanine substitutions at any one of these combined six arginine or glutamic acid residues inactivated Ftr1p in iron uptake, indicating that both motifs were essential to iron permeation . R-->K and E-->D substitutions in these two motifs led to a variable loss of activity, suggesting that while all six residues were essential, their contributions to uptake were quantitatively and/or mechanistically distinct . The terminal glutamate in an EDLWE89 element, associated with transmembrane domain 3, and a DASE motif, located in extracellular loop 6, were also required . The double substitution to AASA in the latter, inactivated Ftr1p in iron uptake while the Ftr1p(E89A) mutant had only 20% of wild-type activity . The two REXLE and the EDLWE and DASE motifs are strongly conserved among fungal Ftr1p homologues, suggesting that these motifs are essential to iron permeation . Finally another important residue, Ile369, was identified in the Ftr1p cytoplasmic C-terminal domain . Deletion or substitution of this residue led to a 70% loss of iron-uptake activity . Ile369 was the only residue identified in this domain that made such a major contribution to iron uptake by the Fet3p-Ftr1p complex.

Pac Symp Biocomput . 2004;:300-11.
Kernel-based data fusion and its application to protein function prediction in yeast; Lanckriet GR et al.; Kernel methods provide a principled framework in which to represent many types of data, including vectors, strings, trees and graphs . As such, these methods are useful for drawing inferences about biological phenomena . We describe a method for combining multiple kernel representations in an optimal fashion, by formulating the problem as a convex optimization problem that can be solved using semidefinite programming techniques . The method is applied to the problem of predicting yeast protein functional classifications using a support vector machine (SVM) trained on five types of data . For this problem, the new method performs better than a previously-described Markov random field method, and better than the SVM trained on any single type of data.

Biotechnol Bioeng, 2004 Mar 30, 85(7), 776 - 89
Yeast plasma membrane Ena1p ATPase alters alkali-cation homeostasis and confers increased salt tolerance in tobacco cultured cells; Nakayama H et al.; In plants, the plasma membrane Na(+)/H(+) antiporter is the only key enzyme that extrudes cytosolic Na(+) and contributes to salt tolerance . But in fungi, the plasma membrane Na(+)/H(+) antiporter and Na(+)-ATPase are known to be key enzymes for salt tolerance . Saccharomyces cerevisiae Ena1p ATPase encoded by the ENA1/PMR2A gene is primarily responsible for Na(+) and Li(+) efflux across the plasma membrane during salt stress and for K(+) efflux at high pH and high K(+) . To test if the yeast ATPase would improve salt tolerance in plants, we expressed a triple hemagglutinin (HA)-tagged Ena1p (Ena1p-3HA) in cultured tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY2) cells . The Ena1p-3HA proteins were correctly localized to the plasma membrane of transgenic BY2 cells and conferred increased NaCl and LiCl tolerance to the cells . Under moderate salt stress conditions, the Ena1p-3HA-expressing BY2 clones accumulated lower levels of Na(+) and Li(+) than nonexpressing BY2 clones . Moreover, the Ena1p-3HA expressing BY2 clones accumulated lower levels of K(+) than nonexpressing cells under no-stress conditions . These results suggest that the yeast Ena1p can also function as an alkali-cation (Na(+), Li(+), and K(+)) ATPase and alter alkali-cation homeostasis in plant cells . We conclude that, even with K(+)-ATPase activity, Na(+)-ATPase activity of the yeast Ena1p confers increased salt tolerance to plant cells during salt stress .

Biophys J, 2004 Mar, 86(3), 1632 - 9
Mechanism of DNA compaction by yeast mitochondrial protein Abf2p; Friddle RW et al.; We used high-resolution atomic force microscopy to image the compaction of linear and circular DNA by the yeast mitochondrial protein Abf2p, which plays a major role in packaging mitochondrial DNA . Atomic force microscopy images show that protein binding induces drastic bends in the DNA backbone for both linear and circular DNA . At a high concentration of Abf2p DNA collapses into a tight nucleoprotein complex . We quantified the compaction of linear DNA by measuring the end-to-end distance of the DNA molecule at increasing concentrations of Abf2p . We also derived a polymer statistical mechanics model that provides a quantitative description of compaction observed in our experiments . This model shows that sharp bends in the DNA backbone are often sufficient to cause DNA compaction . Comparison of our model with the experimental data showed excellent quantitative correlation and allowed us to determine binding characteristics for Abf2p . These studies indicate that Abf2p compacts DNA through a simple mechanism that involves bending of the DNA backbone . We discuss the implications of such a mechanism for mitochondrial DNA maintenance and organization.

EMBO J, 2004 Mar 10, 23(5), 1063 - 74 Epub 2004 Feb 26.
Site-specific regulation of the GEF Cdc24p by the scaffold protein Far1p during yeast mating; Wiget P et al.; Receptor-mediated cell polarization via heterotrimeric G-proteins induces cytoskeletal rearrangements in a variety of organisms . In yeast, Far1p is required for orienting cell growth towards the mating partner by linking activated Gbetagamma to the guanine-nucleotide exchange factor (GEF) Cdc24p, which activates the Rho-type GTPase Cdc42p . Here we investigated the role of Far1p in the regulation of Cdc24p in vivo . Using time-lapse microscopy of mating cells and artificial membrane targeting of Far1p, we show that Far1p is necessary and sufficient to recruit Cdc24p to the plasma membrane . Wild-type Far1p contains a PH-like domain, which is required for its membrane localization in vivo . Interestingly, expression of membrane-targeted Far1p causes toxicity, most likely by activating Cdc42p uniformly at the cell cortex . The ability of full-length Far1p to function as an activator of Cdc24p in vivo requires its interaction with Cdc24p and Gbetagamma . Our results imply that Gbetagamma not only targets Far1p to the correct site but may also trigger a conformational change in Far1p that is required for its ability to activate Cdc24p in vivo.

Proc Natl Acad Sci U S A, 2004 Mar 9, 101(10), 3370 - 5 Epub 2004 Feb 26.
Site-specific Srb10-dependent phosphorylation of the yeast Mediator subunit Med2 regulates gene expression from the 2-microm plasmid; Hallberg M et al.; The yeast Mediator complex is required for transcriptional regulation both in vivo and in vitro, and its function is conserved in all eukaryotes . Mediator interacts with both transcriptional activators and RNA polymerase II, but little is known about the mechanisms by which it operates at the molecular level . Here, we show that the cyclin-dependent kinase Srb10 interacts with, and phosphorylates, the Med2 subunit of Mediator both in vivo and in vitro . A point mutation of the single phosphorylation site in Med2 results in a strongly reduced expression of the REP1, REP2, FLP1, and RAF1 genes, which are all located on the endogenous 2-microm plasmid . Combined with previous studies on the effects of SRB10/SRB11 deletions, our data suggest that posttranslational modifications of Mediator subunits are important for regulation of gene expression.

Proc Natl Acad Sci U S A, 2004 Feb 24, 101(8), 2287 - 92
The elongation of yeast prion fibers involves separable steps of association and conversion; Scheibel T et al.; A self-perpetuating change in the conformation of the translation termination factor Sup35p is the basis for the prion {PSI+}, a protein-based genetic element of Saccharomyces cerevisiae . In a process closely allied to in vivo conversion, the purified soluble, prion-determining region of Sup35p (NM) converts to amyloid fibers by means of nucleated conformational conversion . First, oligomeric species convert to nuclei, and these nuclei then promote polymerization of soluble protein into amyloid fibers . To elucidate the nature of the polymerization step, we created single-cysteine substitution mutants at different positions in NM to provide unique attachment sites for various probes . In vivo, the mutants behaved like wild-type protein in both the {psi-} and {PSI+} states . In vitro, they assembled with wild-type kinetics and formed fibers with the same morphologies . When labeled with fluorescent probes, two mutants, NMT158C and NME167C, exhibited a change in fluorescence coincident with amyloid assembly . These mutants provided a sensitive measure for the kinetics of fiber elongation, and the lag phase in conversion . The cysteine in the mutant NMK184C remained exposed after assembly . When labeled with biotin and bound to streptavidin beads, it was used to capture radiolabeled soluble NM in the process of conversion . This process established the existence of a detergent-susceptible intermediate in fiber elongation . Thus, the second stage of nucleated conformational conversion, fiber elongation, itself contains at least two steps: the association of soluble protein with preformed fibers to form an assembly intermediate, followed by conformational conversion into amyloid.

Nucleic Acids Res, 2004 Feb 24, 32(4), 1345 - 53 Print 2004.
The DNA-binding properties of the ARID-containing subunits of yeast and mammalian SWI/SNF complexes; Wilsker D et al.; SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that are highly conserved from yeast to human . From yeast to human the complexes contain a subunit with an ARID (A-T-rich interaction domain) DNA-binding domain . In yeast this subunit is SWI1 and in human there are two closely related alternative subunits, p270 and ARID1B . We describe here a comparison of the DNA-binding properties of the yeast and human SWI/SNF ARID-containing subunits . We have determined that SWI1 is an unusual member of the ARID family in both its ARID sequence and in the fact that its DNA-binding affinity is weaker than that of other ARID family members, including its human counterparts, p270 and ARID1B . Sequence analysis and substitution mutagenesis reveals that the weak DNA-binding affinity of the SWI1 ARID is an intrinsic feature of its sequence, arising from specific variations in the major groove interaction site . In addition, this work confirms the finding that p270 binds DNA without regard to sequence specificity, excluding the possibility that the intrinsic role of the ARID is to recruit SWI/SNF complexes to specific promoter sequences . These results emphasize that care must be taken when comparing yeast and higher eukaryotic SWI/SNF complexes in terms of DNA-binding mechanisms.

Mol Microbiol, 2004 Mar, 51(5), 1375 - 87
Smt3/SUMO and Ubc9 are required for efficient APC/C-mediated proteolysis in budding yeast; Dieckhoff P et al.; Ubiquitin-mediated proteolysis triggered by the anaphase-promoting complex/cyclosome (APC/C) is essential for sister chromatid separation and the mitotic exit . Like ubiquitylation, protein modification with the small ubiquitin-related modifier SUMO appears to be important during mitosis, because yeast cells impaired in the SUMO-conjugating enzyme Ubc9 were found to be blocked in mitosis and defective in cyclin degradation . Here, we analysed the role of SUMOylation in the metaphase/anaphase transition and in APC/C-mediated proteolysis in Saccharomyces cerevisiae . We show that cells depleted of Ubc9 or Smt3, the yeast SUMO protein, mostly arrested with undivided nuclei and with high levels of securin Pds1 . This metaphase block was partially relieved by a deletion of PDS1 . The absence of Ubc9 or Smt3 also resulted in defects in chromosome segregation . Temperature-sensitive ubc9-2 mutants were delayed in proteolysis of Pds1 and of cyclin Clb2 during mitosis . The requirement of SUMOylation for APC/C-mediated degradation was tested more directly in G1-arrested cells . Both ubc9-2 and smt3-331 mutants were defective in efficient degradation of Pds1 and mitotic cyclins, whereas proteolysis of unstable proteins that are not APC/C substrates was unaffected . We conclude that SUMOylation is needed for efficient proteolysis mediated by APC/C in budding yeast.

J Cell Biol, 2004 Mar 1, 164(5), 677 - 88 Epub 2004 Feb 23.
Mmm2p, a mitochondrial outer membrane protein required for yeast mitochondrial shape and maintenance of mtDNA nucleoids; Youngman MJ et al.; The mitochondrial outer membrane protein, Mmm1p, is required for normal mitochondrial shape in yeast . To identify new morphology proteins, we isolated mutations incompatible with the mmm1-1 mutant . One of these mutants, mmm2-1, is defective in a novel outer membrane protein . Lack of Mmm2p causes a defect in mitochondrial shape and loss of mitochondrial DNA (mtDNA) nucleoids . Like the Mmm1 protein (Aiken Hobbs, A.E., M . Srinivasan, J.M . McCaffery, and R.E . Jensen . 2001 . J . Cell Biol . 152:401-410.), Mmm2p is located in dot-like particles on the mitochondrial surface, many of which are adjacent to mtDNA nucleoids . While some of the Mmm2p-containing spots colocalize with those containing Mmm1p, at least some of Mmm2p is separate from Mmm1p . Moreover, while Mmm2p and Mmm1p both appear to be part of large complexes, we find that Mmm2p and Mmm1p do not stably interact and appear to be members of two different structures . We speculate that Mmm2p and Mmm1p are components of independent machinery, whose dynamic interactions are required to maintain mitochondrial shape and mtDNA structure.

Carbohydr Res, 2004 Mar 15, 339(4), 873 - 9
Synthesis of cluster mannosides via a Ugi four-component reaction and their inhibition against the binding of yeast mannan to concanavalin A; Li Y et al.; The Ugi four-component reaction (U-4CR) was utilized to prepare divalent and trivalent cluster mannosides with different scaffolds . The glycoclusters obtained were tested for their relative inhibitory potency against the binding of yeast mannan to concanavalin A by solid-phase enzyme-linked lectin assays (ELLA) using methyl alpha-D-mannopyranoside as a standard . Among them, a divalent mannoside containing aromatic groups showed the strongest binding affinity to concanavalin A.

Gene, 2004 Mar 3, 327(2), 161 - 9
Rax1, a protein required for the establishment of the bipolar budding pattern in yeast; Fujita A et al.; In Saccharomyces cerevisiae, cell type determines two distinct spatial budding patterns . Haploid cells exhibit an axial pattern, whereas diploid cells exhibit a bipolar pattern . Axl1, a member of the insulin-degrading enzyme (IDE) family, is the key morphological determinant for the haploid axial pattern . Here we identified a novel gene, RAX1, specifically required for the bipolar budding pattern . Loss of RAX1 alters the bipolar pattern of axl1 haploids resulting in reversion to the axial pattern, and also alters the bipolar patterns of bud3 and bud4 haploids . However, bud10 rax1 haploids exhibit a random budding pattern, suggesting Bud10 acts as the key proximal landmark in axial budding . Rax1 is required for the localization of Bud8, the distal bipolar budding landmark . Interestingly, Rax1 contains a C-terminal domain possessing some similarity to insulin-related peptides . Our results suggest that Rax1 is necessary for the establishment of the bipolar budding landmark.

Redox Rep, 2003, 8(6), 371 - 7
Oxidative damage to proteins in yeast cells exposed to adaptive levels of H(2)O(2); Poljak A et al.; When yeast cells are exposed to sublethal concentrations of oxidants, they adapt to tolerate subsequent lethal treatments . Here, we show that this adaptation involves tolerance of oxidative damage, rather than protection of cellular constituents . o- and m-tyrosine levels are used as a sensitive measure of protein oxidative damage and we show that such damage accumulates in yeast cells exposed to H(2)O(2) at low adaptive levels . Glutathione represents one of the main cellular protections against free radical attack and has a role in adaptation to oxidative stress . Yeast mutants defective in glutathione metabolism are shown to accumulate significant levels of o- and m-tyrosine during normal aerobic growth conditions.

Methods Enzymol, 2004, 377, 55 - 60
Genetic analysis of chromatin remodeling using budding yeast as a model; Steger DJ et al.; Novel discoveries result from genetic analyses of transcription and chromatin remodeling because these methods identify activities in an unbiased manner . By describing our genetic approaches to identify regulators of PHO5 transcription and chromatin remodeling, we hope to encourage others to apply similar strategies to their genes of interest.

Mol Pharmacol, 2004 Mar, 65(3), 702 - 10
Random mutagenesis of the human adenosine A2B receptor followed by growth selection in yeast . Identification of constitutively active and gain of function mutations; Beukers MW et al.; To gain insight in spontaneous as well as agonist-induced activation of the human adenosine A2B receptor, we applied a random mutagenesis approach in yeast to create a large number of receptor mutants and selected mutants of interest with a robust screening assay based on growth . The amino acid sequence of 14 mutated receptors was determined . All these mutated receptors displayed constitutive activity . In particular, single-point mutations at T42A, V54L, and F84S and a triple-point mutation at N36S, T42A, and T66A resulted in high constitutive activity . In addition, a C-terminally truncated (after Lys269) mutant, Q214L I230N V240M V250M N254Y T257S K269stop, was highly constitutively active . The T42A, V54L, and F84S mutants showed a considerable decrease, 4.9- to 6.9-fold, in the EC50 value of 5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analog . Combined mutation of I242T, K269R, V284A, and H302Q, as well as F84L together with S95G, resulted in an even greater potency of NECA of 10- and 18-fold, respectively . In fact, all constitutively active mutants had an increased potency for NECA . This suggests that the wild-type (wt) human A2B receptor itself is rather silent, which may explain the low affinity of agonists for this receptor . To verify the ability of the mutant receptors to activate mammalian second messenger systems, cAMP experiments were performed in CHO cells stably expressing the wt and T42A receptors . These experiments confirmed the increased sensitivity of T42A for NECA, because the EC50 values of T42A and the wt receptor were 0.15 +/- 0.04 and 1.3 +/- 0.4 microM, respectively.

Biometals, 2004 Feb, 17(1), 25 - 33
Uptake of chromium(III) and chromium(VI) compounds in the yeast cell structure; Pas M et al.; The study presented in this article investigated the influence of different Cr(III) and Cr(VI) compounds in the cultivation medium on the uptake and localization of chromium in the cell structure of the yeast Candida intermedia . The morphology of the yeast cell surface was observed by the scanning electron microscopy . Results demonstrated that the growth inhibitory concentration of Cr(III) in the cultivation medium induced changes in the yeast cell shape and affected the budding pattern, while inhibitory concentration of Cr(VI) did not cause any visible effects on morphological properties of the yeast cells . The amount of total accumulated chromium in yeast cells and the distribution of chromium between the yeast cell walls and spheroplasts were determined by atomic absorption spectroscopy . No significant differences were found neither in total chromium accumulation nor in the distribution of chromium in yeast cell walls and spheroplasts between the two of Cr(VI) compounds . Conversely, substantial differences between Cr(III) compounds were demonstrated in the total uptake as well as the localization of chromium in yeast cells.

Folia Microbiol (Praha), 2003, 48(5), 589 - 96
Role of strategic cysteine residues in oxidative damage to the yeast plasma membrane H(+)-ATPase caused by Fe- and Cu-containing Fenton reagents; Stadler N et al.; Damage caused to Saccharomyces cerevisiae SY4 plasma membrane H(+)-ATPase by Fe- and Cu-Fenton reagents was determined in secretory vesicles containing enzyme in which Cys residues were replaced singly or in pairs by Ala . Cys-221 situated in a beta-sheet domain between M2 and M3 segments, phosphorylation domain-located Cys-409 and Cys-532 situated at the ATP-binding site play a role in the inactivation . In the presence of all three residues the enzyme exhibited a certain basic inactivation, which did not change when Cys-532 was replaced with Ala . In mutants having intact Cys-532 but lacking one or both other cysteines, replacement of Cys-221 with Ala led to lower inactivation, suggesting that Cys-221 may serve as a target for metal-catalyzed oxidation and intact Cys-532 promotes this target role of Cys-221 . In contrast, the absence of Cys-409 caused higher inactivation by Fe-Fenton . Cys-532 thus seems to serve as a target for Fe-Fenton, intact Cys-409 causing a conformational change that makes Cys-532 less accessible to oxidation . The mutant lacking both Cys-221 and Cys-409 is more sensitive to Fe-Fenton than to Cu-Fenton and the absence of both Cys residues thus seems to expose presumable extra Fe-binding sites . These data and those on protection by ATP, ADP, 1,4-dithiothreitol and deferrioxamine B point to complex interactions between individual parts of the enzyme molecule that determine its sensitivity towards Fenton reagents . ATPase fragmentation caused by the two reagents differed in that the Fe-Fenton reagent produced in Western blot "smears" whereas the Cu-Fenton reagent produced defined fragments.

Cell Cycle, 2004 Apr, 3(4), 486 - 90 Epub 2004 Apr 01.
Expression and localization studies of hSDA, the human ortholog of the yeast SDA1 gene; Babbio F et al.; The yeast SDA1 gene was reported to play a critical role in G(1) events and to be involved in 60S ribosome biogenesis . Although the basic cellular mechanisms appear conserved from yeast to man, the human genes may have more diversified functions . In this view we obtained the first experimental evidences about the human ortholog of the yeast SDA1, i.e., hSDA . The gene is localized at the chromosomal region 4q21 and encodes for a 627a.a . long protein highly homologous to the yeast Sda1 . Subcellular localization experiments indicate that the human protein behaves similarly to nucleolar proteins involved in rRNA processing machinery but not in RNA PolI transcriptional events . hSda appears localized in the granular component of the nucleolus and in the nucleoplasm, which is consistent with a role in early-intermediate steps of ribosome biogenesis . hSDA appears preferentially expressed in fetal tissues, pinpointing its role during development . Different expression levels in different tumor cell lines might suggest that the gene is involved also in tumorigenesis . However our preliminary results indicate that hSDA does not behave like a proapoptotic gene and its involvement in tumorigenesis is still to be clarified.

Methods Mol Biol, 2004, 263, 311 - 32
Flow cytometric screening of yeast surface display libraries; Feldhaus M et al.; A method to screen and isolate antigen specific clones from a library of single-chain antibodies expressed on the surface of yeast cells is presented . Two rounds of magnetic bead enrichment before flow cytometric sorting enables one to screen libraries of far greater diversity than can be screened by just flow cytometry . The strength of flow cytometric sorting is the ability to follow the selection in real time and to isolate easily the highest affinity antigen-specific clones . A major strength of yeast display as a discovery platform is the ability to characterize the binding properties, the affinity of a clone without the need for subcloning, expression, and purification of the scFv . The methodology for directed evolution of single-chain antibodies to increase the affinity of a clone is also described.

Biochem Biophys Res Commun, 2004 Mar 12, 315(3), 739 - 45
beta-Helix is a likely core structure of yeast prion Sup35 amyloid fibers; Kishimoto A et al.; We have studied the core structure of amyloid fibers of yeast prion protein Sup35 . We developed procedures to prepare straight fibers of relatively uniform diameters from three kinds of fragments; N (1-123), NMp (1-189), and NM (1-253) . X-ray fiber diffraction patterns from dried oriented fibers gave common reflections in all three cases; a sharp meridional reflection at 4.7A, and a diffuse equatorial peak at around 9A, apparently supporting the typical "cross-beta" structure with stacked beta-sheets proposed for many different amyloid fibers . However, X-ray fiber diffraction from hydrated fibers showed the meridional reflection at 4.7A but no equatorial reflections at 9A in all three cases, indicating that the stack of beta-sheets in dried fibers is an artifact produced by drying process . Thus, the core structure of these amyloid fibers made of the N domain is likely to be beta-helix nanotube as proposed by Perutz et al.

Proc Natl Acad Sci U S A, 2004 Mar 2, 101(9), 2981 - 6 Epub 2004 Feb 18.
Revealing modularity and organization in the yeast molecular network by integrated analysis of highly heterogeneous genomewide data; Tanay A et al.; The dissection of complex biological systems is a challenging task, made difficult by the size of the underlying molecular network and the heterogeneous nature of the control mechanisms involved . Novel high-throughput techniques are generating massive data sets on various aspects of such systems . Here, we perform analysis of a highly diverse collection of genomewide data sets, including gene expression, protein interactions, growth phenotype data, and transcription factor binding, to reveal the modular organization of the yeast system . By integrating experimental data of heterogeneous sources and types, we are able to perform analysis on a much broader scope than previous studies . At the core of our methodology is the ability to identify modules, namely, groups of genes with statistically significant correlated behavior across diverse data sources . Numerous biological processes are revealed through these modules, which also obey global hierarchical organization . We use the identified modules to study the yeast transcriptional network and predict the function of >800 uncharacterized genes . Our analysis framework, SAMBA (Statistical-Algorithmic Method for Bicluster Analysis), enables the processing of current and future sources of biological information and is readily extendable to experimental techniques and higher organisms.

Curr Biol, 2004 Feb 17, 14(4), 287 - 301
Two fission yeast homologs of Drosophila Mei-S332 are required for chromosome segregation during meiosis I and II; Rabitsch KP et al.; BACKGROUND: Meiosis produces haploid gametes from diploid progenitor cells . This reduction is achieved by two successive nuclear divisions after one round of DNA replication . Correct chromosome segregation during the first division depends on sister kinetochores being oriented toward the same spindle pole while homologous kinetochores must face opposite poles . Segregation during the second division depends on retention of sister chromatid cohesion between centromeres until the onset of anaphase II, which in Drosophila melanogaster depends on a protein called Mei-S332 that binds to centromeres . RESULTS: We report the identification of two homologs of Mei-S332 in fission yeast using a knockout screen . Together with their fly ortholog they define a protein family conserved from fungi to mammals . The two identified genes, sgo1 and sgo2, are required for retention of sister centromere cohesion between meiotic divisions and kinetochore orientation during meiosis I, respectively . The amount of meiotic cohesin's Rec8 subunit retained at centromeres after meiosis I is reduced in Deltasgo1, but not in Deltasgo2, cells, and Sgo1 appears to regulate cleavage of Rec8 by separase . Both Sgo1 and Sgo2 proteins localize to centromere regions . The abundance of Sgo1 protein normally declines after the first meiotic division, but extending its expression by altering its 3'UTR sequences does not greatly affect meiosis II . Its mere presence within the cell might therefore be insufficient to protect centromeric cohesion . CONCLUSIONS: A conserved protein family based on Mei-S332 has been identified . The two fission yeast homologs are implicated in meiosis I kinetochore orientation and retention of centromeric sister chromatid cohesion until meiosis II.

Acta Biochim Biophys Sin (Shanghai), 2004 Feb, 36(2), 128 - 32
An engineered PrPsc-like molecule from the chimera of mammalian prion protein and yeast Ure2p prion-inducing domain; Yin SM et al.; Production of the pathogenic prion isoform PrPsc-like molecules is thought to be useful for understanding the mysterious mechanism of conformational conversion process of prion diseases and proving the "protein-only" hypothesis . In this report, an engineered PrPsc-like conformation was produced from a chimera of mammalian bovine prion protein (bPrP) and yeast Ure2p prion-inducing domain (UPrD) . Compared with the normal form of bPrP, the engineered recombinant protein, termed bPrP-UPrD, spontaneously aggregated into ordered fibrils under physiological condition, displaying amyloid-like characteristics, such as fibrillar morphology, birefringence upon binding to Congo red and increased fluorescence intensity with Thioflavine T . Limited resistance to protease K digestion and CD spectroscopy experiments suggested that the structure of bPrP-UPrD had been changed, and adopted a new, high content beta-sheet conformation during the fibrils formation . Moreover, bPrP-UPrD amyloid fibrils could recruit more soluble forms into the aggregates . Therefore, the engineered molecules could mimic significant behaviors of PrPsc and will be helpful for further understanding the mechanism of conformational conversion process.

RNA, 2004 Mar, 10(3), 369 - 77
Internal loop mutations in the ribosomal protein L30 binding site of the yeast L30 RNA transcript; White SA et al.; Yeast ribosomal protein L30 binds to an asymmetric, purine-rich internal loop in its transcript to repress its own splicing and translation . The protein-bound form of the stem-internal loop-stem RNA is an example of a kink-turn RNA structural motif . Analysis of kink-turn motifs reveals that in (2 + 5) internal loops, the identities of five nucleotides are very important, while the remaining two may be varied . Previous SELEX experiments on the L30 binding site showed an identical pattern of sequence variation with five nucleotides highly conserved and two positions variable . In this work, internal loop residues were mutated and tested for protein binding in vitro and in vivo . The two sheared G-A pairs, which cannot be mutated without severely weakening L30 binding, make sequence specific contacts with other portions of the RNA and L30 protein . In contrast, the lone nucleotide that protrudes into the protein and an unpaired adenosine make no sequence-specific contacts, and may be mutated without compromising L30 binding . The internal loop allows the formation of a very tight bend that brings the two stems together with cross-strand stacking of two adenines and an interhelical ribose contact . Replacement of a ribonucleotide with a deoxynucleotide adjacent to the internal loop weakens protein binding significantly . In the absence of L30, some of the internal loop residues involved in the formation of the kink-turn motif are protected from chemical modification, indicating that some elements of kink-turn structure may form in the free L30 RNA.

J Biol Chem, 2004 Apr 30, 279(18), 18974 - 80 Epub 2004 Feb 16.
Requirement of the SCFPop1/Pop2 Ubiquitin Ligase for Degradation of the Fission Yeast S Phase Cyclin Cig2; Yamano H et al.; Two multiprotein E3 (ubiquitin-protein ligase) ubiquitin ligases, the SCF (Skp1-Cullin-1-F-box) and the APC/C (anaphase promoting complex/cyclosome), are vital in ensuring the temporal order of the cell cycle . Particularly, timely destruction of cyclins via these two E3s is essential for down-regulation of cyclin-dependent kinase . In general, G(1) and S phase cyclins are ubiquitylated by the SCF, whereas ubiquitylation of mitotic cyclins is catalyzed by the APC/C . Here we show that fission yeast S phase cyclin Cig2 is ubiquitylated and degraded via both the SCF and the APC/C . Cig2 instability during G(2) and M phase is dependent upon the SCF complex, whereas the APC/C is responsible for Cig2 destruction during anaphase and G(1), thereby ensuring a spike pattern of Cig2 levels, peaking only at S phase . Two F-box/WD proteins Pop1 and Pop2, homologues of budding yeast Cdc4 and human Fbw7, are responsible for Cig2 instability . Pop1 binds Cig2 in vivo . An in vitro binding assay shows that an internal 93 amino acid residues comprising a part of the cyclin box are necessary and sufficient for this binding . Cig2 phosphorylation is also required for interaction with Pop1 . We previously showed that transcriptional oscillation of cig2(+) requires Pop1 and Pop2 function . SCF(Pop1/Pop2) therefore regulates Cig2 levels in a dual manner, transcriptionally and post-translationally . Our results also highlight a collaborative action of the APC/C and the SCF toward the common substrate Cig2 . This type of composite degradation control may be more general as the regulatory mechanism in other complex systems.

J Biol Chem, 2004 Apr 23, 279(17), 17361 - 5 Epub 2004 Feb 16.
The yeast vacuolar proton-translocating ATPase contains a subunit homologous to the Manduca sexta and bovine e subunits that is essential for function; Sambade M et al.; The yeast cwh36Delta mutant was identified in a screen for yeast mutants exhibiting a Vma(-) phenotype suggestive of loss of vacuolar proton-translocating ATPase (V-ATPase) activity . The mutation disrupts two genes, CWH36 and a recently identified open reading frame on the opposite strand, YCL005W-A . We demonstrate that disruption of YCL005W-A is entirely responsible for the Vma(-) growth phenotype of the cwh36Delta mutant . YCL005W-A encodes a homolog of proteins associated with the Manduca sexta and bovine chromaffin granule V-ATPase . The functional significance of these proteins for V-ATPase activity had not been tested, but we show that the protein encoded by YCL005W-A, which we call Vma9p, is essential for V-ATPase activity in yeast . Vma9p is localized to the vacuole but fails to reach the vacuole in a mutant lacking one of the integral membrane subunits of the V-ATPase . Vma9p is associated with the yeast V-ATPase complex in vacuolar membranes, as demonstrated by co-immunoprecipitation with known V-ATPase subunits and glycerol gradient fractionation of solubilized vacuolar membranes . Based on this evidence, we propose that Vma9p is a genuine subunit of the yeast V-ATPase and that e subunits may be a functionally essential part of all eukaryotic V-ATPases.

J Cell Biol, 2004 Feb 16, 164(4), 501 - 7
Chronological aging leads to apoptosis in yeast; Herker E et al.; During the past years, yeast has been successfully established as a model to study mechanisms of apoptotic regulation . However, the beneficial effects of such a cell suicide program for a unicellular organism remained obscure . Here, we demonstrate that chronologically aged yeast cultures die exhibiting typical markers of apoptosis, accumulate oxygen radicals, and show caspase activation . Age-induced cell death is strongly delayed by overexpressing YAP1, a key transcriptional regulator in oxygen stress response . Disruption of apoptosis through deletion of yeast caspase YCA1 initially results in better survival of aged cultures . However, surviving cells lose the ability of regrowth, indicating that predamaged cells accumulate in the absence of apoptotic cell removal . Moreover, wild-type cells outlast yca1 disruptants in direct competition assays during long-term aging . We suggest that apoptosis in yeast confers a selective advantage for this unicellular organism, and demonstrate that old yeast cells release substances into the medium that stimulate survival of the clone . Copyright The Rockefeller University Press

Yeast, 2004 Feb, 21(3), 265 - 76
The role of Ynt1 in nitrate and nitrite transport in the yeast Hansenula polymorpha; Machin F et al.; Ynt1 is the only high-affinity nitrate uptake system in Hansenula polymorpha . Nitrate uptake was directly correlated with the Ynt1 levels and shown to be independent of nitrate reductase (NR) activity levels . Ynt1 failed to transport chlorate and, as a result, strains lacking YNT1 were sensitive to chlorate, as is the wild-type . Nitrite uptake in a wild-type strain was partially inhibited by nitrate to levels shown by a YNT1-disrupted strain in which, in turn, nitrite transport was not inhibited by nitrate . It is concluded that nitrite uptake takes place by two different transport systems: Ynt1 and a nitrite-specific transporter(s) . The nitrite-specific transport system was induced by nitrate; consistently, no induction was observed in strains lacking the transcription factor YNA1, which is involved in nitrate and nitrite induction of the nitrate assimilatory structural genes . Ynt1 presents its optimal rate for nitrite uptake at pH 6, while pH 4 was optimal for the specific nitrite uptake system(s) . At pH 5.5, the contribution of Ynt1 to high-affinity nitrate and nitrite uptake was around 95% and 60%, respectively . The apparent Km of Ynt1 for nitrate and nitrite is in the microM range, as is the specific nitrite uptake system for nitrite . The analysis of the effect of the reduced nitrogen sources on nitrate assimilation revealed that glutamine inactivates nitrate and nitrite transport, dependent on Ynt1, but not the nitrite-specific system .

Yeast, 2004 Feb, 21(3), 193 - 9
Four permeases import proline and the toxic proline analogue azetidine-2-carboxylate into yeast; Andreasson C et al.; We have found that proline and the toxic proline analogue azetidine-2-carboxylate (AzC) are efficiently imported into Saccharomyces cerevisiae cells by four amino acid permeases, including two nitrogen-regulated permeases (PUT4 and GAP1) and two permeases that are regulated by the SPS sensor of extracellular amino acids (AGP1 and GNP1) . In contrast to Agp1p, Gnp1p is not functionally expressed when cells are grown on media containing proline as sole nitrogen source . These findings have implications for the interpretation of studies using AzC to characterize nitrogen source-dependent regulation of amino acid uptake and of post-Golgi targeting and localization of amino acid permeases in yeast .

EMBO Rep, 2004 Mar, 5(3), 280 - 4 Epub 2004 Feb 13.
The yeast coexpression network has a small-world, scale-free architecture and can be explained by a simple model; van Noort V et al.; We investigated the gene coexpression network in Saccharomyces cerevisiae, in which genes are linked when they are coregulated . This network is shown to have a scale-free, small-world architecture . Such architecture is typical of biological networks in which the nodes are connected when they are involved in the same biological process . Current models for the evolution of intracellular networks do not adequately reproduce the features that we observe in the network . We therefore derive a new model for its evolution based on the observation that there is a positive correlation between the sequence similarity of paralogues and their probability of coexpression or sharing of transcription factor binding sites (TFBSs) . The simple, neutralist's model consists of (1) coduplication of genes with their TFBSs, (2) deletion and duplication of individual TFBSs and (3) gene loss . A network is constructed by connecting genes that share multiple TFBSs . Our model reproduces the scale-free, small-world architecture of the coregulation network and the homology relations between coregulated genes without the need for selection either at the level of the network structure or at the level of gene regulation.

Mol Cell, 2004 Feb 13, 13(3), 389 - 401
Association of Mre11p with double-strand break sites during yeast meiosis; Borde V et al.; The repair of DNA double-strand breaks (DSBs) requires the activity of the Mre11/Rad50/Xrs2(Nbs1) complex . In Saccharomyces cerevisiae, this complex is required for both the initiation of meiotic recombination by Spo11p-catalyzed programmed DSBs and for break end resection, which is necessary for repair by homologous recombination . We report that Mre11p transiently associates with the chromatin of Spo11-dependent DSB regions throughout the genome . Mutant analyses show that Mre11p binding requires the function of all genes required for DSB formation, with the exception of RAD50 . However, Mre11p binding does not require DSB formation itself, since Mre11p transiently associates with DSB regions in the catalysis-negative mutant spo11-Y135F . Mre11p release from chromatin is blocked in mutants that accumulate unresected DSBs . We propose that Mre11p is a component of a pre-DSB complex that assembles on the DSB sites, thus ensuring a tight coupling between DSB formation by Spo11p and the processing of break ends.

Mol Cell, 2004 Feb 13, 13(3), 377 - 87
A function of yeast mRNA cap methyltransferase, Abd1, in transcription by RNA polymerase II; Schroeder SC et al.; Capping enzymes bind the phosphorylated pol II CTD permitting cotranscriptional capping of nascent pre-mRNAs . We asked whether these interactions influence pol II function using ChIP in ts mutants of yeast capping enzymes . Pol II occupancy at the 5' ends of PGK1, ENO2, GAL1, and GAL10 was reduced by inactivation of the methyltransferase, Abd1, but not the guanylyltransferase, Ceg1, suggesting that Abd1 contributes to stable promoter binding . At other genes, Abd1 inactivation increased the 5':3' ratio of pol II density in the promoter-proximal region and caused Ser5 hyperphosphorylation of the pol II CTD . These results suggest an additional role for Abd1 in the promoter clearance and/or promoter-proximal elongation steps of transcription . The transcriptional functions of Abd1 are independent of methyltransferase activity . Manipulation of transcription by Abd1 may enhance cotranscriptional capping and also act as a checkpoint to ensure that a nascent transcript has a cap before it can be completed.

Biochemistry, 2004 Feb 24, 43(7), 1963 - 71
Role of active-site residues Thr81, Ser82, Thr85, Gln157, and Tyr158 in yeast cystathionine beta-synthase catalysis and reaction specificity; Aitken SM et al.; Cystathionine beta-synthase (CBS) effects the condensation of l-serine with l-homocysteine to form l-cystathionine . A series of active-site mutants, T81A, S82A, T85A, Q157A/E/H, and Y158F, was constructed to investigate effects on catalysis and reaction specificity in yeast CBS (yCBS) . The effects of these mutations on the k(cat)/K(m)(L-Ser) for the beta-replacement reaction range from a reduction of only 3-fold for Y158F to below detectable levels for the Q157A and Q157E mutants . The order of importance of these residues to the beta-replacement reaction is Gln157 >or= Thr81 > Ser82 > Thr85 approximately Tyr158 . All seven of the mutant enzymes catalyze a competing beta-elimination reaction, in which L-Ser is hydrolyzed to NH(3) and pyruvate . The ping-pong mechanism of CBS was thus expanded to include the latter reaction for these mutants . This activity is not detectable for wild-type yCBS, suggesting that the mutations result in a shift in the equilibrium between the open and the closed conformations of the active site of yCBS-substrate complexes . The Q157H and Y158F mutants additionally suffer suicide inhibition via a mechanism in which the released aminoacrylate intermediate covalently attacks the internal aldimine of the enzyme.

Mol Cell Biol, 2004 Mar, 24(5), 1884 - 96
Structural and functional conservation of the NuA4 histone acetyltransferase complex from yeast to humans; Doyon Y et al.; The NuA4 histone acetyltransferase (HAT) multisubunit complex is responsible for acetylation of histone H4 and H2A N-terminal tails in yeast . Its catalytic component, Esa1, is essential for cell cycle progression, gene-specific regulation and has been implicated in DNA repair . Almost all NuA4 subunits have clear homologues in higher eukaryotes, suggesting that the complex is conserved throughout evolution to metazoans . We demonstrate here that NuA4 complexes are indeed present in human cells . Tip60 and its splice variant Tip60b/PLIP were purified as stable HAT complexes associated with identical polypeptides, with 11 of the 12 proteins being homologs of yeast NuA4 subunits . This indicates a highly conserved subunit composition and the identified human proteins underline the role of NuA4 in the control of mammalian cell proliferation . ING3, a member of the ING family of growth regulators, links NuA4 to p53 function which we confirmed in vivo . Proteins specific to the human NuA4 complexes include ruvB-like helicases and a bromodomain-containing subunit linked to ligand-dependent transcription activation by the thyroid hormone receptor . We also demonstrate that subunits MRG15 and DMAP1 are present in distinct protein complexes harboring histone deacetylase and SWI2-related ATPase activities, respectively . Finally, analogous to yeast, a recombinant trimeric complex formed by Tip60, EPC1, and ING3 is sufficient to reconstitute robust nucleosomal HAT activity in vitro . In conclusion, the NuA4 HAT complex is highly conserved in eukaryotes, in which it plays primary roles in transcription, cellular response to DNA damage, and cell cycle control.

J Biol Chem, 2004 Apr 23, 279(17), 17443 - 8 Epub 2004 Feb 13.
Mechanism of genetic complementation of ammonium transport in yeast by human erythrocyte Rh-associated glycoprotein; Westhoff CM et al.; The Rh blood group proteins are erythrocyte proteins important in neonatal and transfusion medicine . Recent studies have shed new light on the possible biological function of Rh proteins as members of a conserved family of proteins involved in ammonium transport . The erythrocyte Rh-associated glycoprotein (RhAG) mediates uptake of ammonium when expressed in Xenopus laevis oocytes, and functional studies indicate that RhAG might function as an NH(4)(+)-H(+)-exchanger . To further delineate the functional properties of RhAG, in this study we have expressed RhAG in both a Saccharomyces cerevisiae ammonium-transport mutant (mep1Delta mep2Delta mep3Delta) and a wild-type strain . RhAG was able to complement the transport mutant, with complementation strictly pH-dependent, requiring pH 6.2-6.5 . RhAG also conferred resistance to methylamine (MA), a toxic analog of ammonium, and expression in wild-type cells revealed that resistance was correlated with efflux of MA . RhAG-mediated resistance was pH-dependent, being optimal at acid pH . The opposite pH dependence of ammonium complementation (uptake) and MA resistance (efflux) is consistent with bidirectional movement of substrate counter to the direction of the proton gradient . This report clarifies and expands previous observations of RhAG-mediated transport in yeast and supports the hypothesis that ammonium transport is coupled to the H(+) gradient and that RhAG functions as a NH(4)(+)/H(+) exchanger.

Cell Cycle . 2004 Apr;3(4) {Epub ahead of print}
The Novel Yeast PAS Kinase Rim15 Orchestrates G(0)-Associated Antioxidant Defense Mechanisms; Cameroni E et al.; The highly conserved PKA and TOR proteins define key signaling pathways that control cell proliferation in response to growth factors and/or nutrients . In yeast, inactivation of PKA and/or TOR causes cells to arrest growth in early G(1) and induces a program that is characteristic of G(0) cells . We have recently shown that the protein kinase Rim15 integrates both PKA- and TOR-mediated signals . In this work, we demonstrate that the Rim15-activated genomic expression program following glucose limitation at the diauxic shift is mediated by the three transcription factors Gis1, Msn2, and Msn4 . The Rim15 regulon comprises several gene clusters implicated in the adaptation to respiratory growth, including classical oxidative stress genes such as SOD1 and SOD2, suggesting that the reduced life span of rim15 cells may be due to their deficiency in oxidative damage prevention . Interestingly, we found that the primary amino acid sequence of Rim15 includes in its amino-terminal part a conserved PAS domain, known to act as a sensor for a variety of stimuli . We propose that Rim15 has evolved to integrate nutrient signals (transduced via TOR and PKA) and redox and/or oxidative stress signals to appropriately induce a transcriptional program that ensures survival in G(0).

J Virol, 2004 Mar, 78(5), 2405 - 13
The DNA-A component of a plant geminivirus (Indian mung bean yellow mosaic virus) replicates in budding yeast cells; Raghavan V et al.; Understanding the biochemistry of DNA replication of the plant DNA viruses is important for the development of antiviral strategies . Since DNA replication is little studied in plants, a genetically tractable, easily culturable, eukaryotic model system is required to pursue such studies in a facile manner . Here we report the development of a yeast model system that supports DNA replication of a chosen geminivirus strain, Indian mung bean yellow mosaic virus . The replication of plasmid DNA in the model system relies specifically on the virus-derived elements and factors . Usage of this model system revealed the role of at least one hitherto unknown viral factor for viral DNA replication . The episomal characteristic of single-strandedness of replicated plasmid DNA was shown, and the expression of viral genes was also confirmed . This model system is expected to shed light on the machinery and mechanism involved in geminiviral DNA replication in plants.

J Appl Microbiol, 2004, 96(3), 588 - 92
Monoacylglycerols: glycolipid biosurfactants produced by a thermotolerant yeast, Candida ishiwadae; Thanomsub B et al.; AIMS: To isolate and characterize biosurfactants produced by a thermotolerant yeast isolated in Thailand . MATERIALS AND RESULTS: Yeast strains isolated from plant material in Thailand were first screened for the ability to produce lipase and biosurfactant . A strain Y12, identified as Candida ishiwadae by physiological tests, survived at 45 degrees C and produced relatively large amounts of biosurfactants . From the culture filtrate of this strain, two glycolipid biosurfactants, a and b, were purified by solvent fractionation, silica gel and ODS column chromatographies . Compounds a and b were determined to be monoacylglycerols; 1-linoleylglycerol and 1-oleylglycerol, respectively . Both compounds exhibited higher surfactant activities tested by the drop collapse test than several artificial surfactants such as sodium dodecyl sulphate . CONCLUSIONS: Glycolipid biosurfactants produced by a thermotolerant yeast, C . ishiwadae were characterized to be monoacylglycerols which exhibited high surfactant activities . SIGNIFICANCE AND IMPACT OF THE STUDY: A thermotolerant yeast strain, C . ishiwadae, could be a potential candidate for producing monoacylglycerols which are useful in industrial applications.

Mar Biotechnol (NY), 2001 Jul, 3(4), 316 - 21
Subunit C of the vacuolar-type ATPase from the vanadium-rich ascidian Ascidia sydneiensis samea rescued the pH sensitivity of yeast vma5 mutants; Ueki T et al.; A vanadium-accumulating ascidian, Ascidia sydneiensis samea, expresses vacuolar-type H(+)-ATPases (V-ATPases) on the vacuole membrane of the vanadium-containing blood cells known as vanadocytes . Previously, we showed that the contents of their vacuoles are extremely acidic and that a V-ATPase-specific inhibitor, bafilomycin A(1), neutralized the contents of the vacuoles . To understand the function of V-ATPase in vanadocytes, we isolated complementary DNA encoding subunit C of V-ATPase from vanadocytes because this subunit has been known to be responsible for the assembly of V-ATPases and to regulate the ATPase activity of V-ATPases . The cloned cDNA was 1443 nucleotides in length, and encoded a putative 384 amino acid protein . By expressing the ascidian cDNA for subunit C under the control of a galactose-inducible promoter, the pH-sensitive phenotype of the corresponding vma5 mutant of a budding yeast was rescued . This result showed that the ascidian cDNA for subunit C functioned in yeast cells.

Nucleic Acids Res, 2004 Feb 11, 32(3), 1122 - 30 Print 2004.
Translesion synthesis of acetylaminofluorene-dG adducts by DNA polymerase zeta is stimulated by yeast Rev1 protein; Guo D et al.; Translesion synthesis is an important mechanism in response to unrepaired DNA lesions during replication . The DNA polymerase zeta (Polzeta) mutagenesis pathway is a major error-prone translesion synthesis mechanism requiring Polzeta and Rev1 . In addition to its dCMP transferase, a non-catalytic function of Rev1 is suspected in cellular response to certain types of DNA lesions . However, it is not well understood about the non-catalytic function of Rev1 in translesion synthesis . We have analyzed the role of Rev1 in translesion synthesis of an acetylaminofluorene (AAF)-dG DNA adduct . Purified yeast Rev1 was essentially unresponsive to a template AAF-dG DNA adduct, in contrast to its efficient C insertion opposite a template 1,N6-ethenoadenine adduct . Purified yeast Polzeta was very inefficient in the bypass of the AAF-dG adduct . Combining Rev1 and Polzeta, however, led to a synergistic effect on translesion synthesis . Rev1 protein enhanced Polzeta-catalyzed nucleotide insertion opposite the AAF-dG adduct and strongly stimulated Polzeta-catalyzed extension from opposite the lesion . Rev1 also stimulated the deficient synthesis by Polzeta at the very end of undamaged DNA templates . Deleting the C-terminal 205 aa of Rev1 did not affect its dCMP transferase activity, but abolished its stimulatory activity on Polzeta-catalyzed extension from opposite the AAF-dG adduct . These results suggest that translesion synthesis of AAF-dG adducts by Polzeta is stimulated by Rev1 protein in yeast . Consistent with the in vitro results, both Polzeta and Rev1 were found to be equally important for error-prone translesion synthesis across from AAF-dG DNA adducts in yeast cells.

J Struct Biol, 2004 Mar, 145(3), 272 - 88
Yeast nuclear pore complexes have a cytoplasmic ring and internal filaments; Kiseleva E et al.; The nuclear pore complex (NPC) controls transport of macromolecules across the nuclear envelope . It is large and complex but appears to consist of only approximately 30 different proteins despite its mass of > 60MDa . Vertebrate NPC structure has been analyzed by several methods giving a comprehensive architectural model . Despite our knowledge of yeast nucleoporins, structural data is more limited and suggests the basic organization is similar to vertebrates, but may lack some peripheral and other components . Using field emission scanning electron microscopy to probe NPC structure we found that the yeast, like higher eukaryotic, NPCs contain similar peripheral components . We can detect cytoplasmic rings and evidence of nucleoplasmic rings in yeasts . A filamentous basket is present on the nucleoplasmic face and evidence for cytoplasmic filaments is shown . We observed a central structure, possibly the transporter, that which may be linked to the cytoplasmic ring by internal filaments . Immuno-gold labeling suggested that Nup159p may be attached to the cytoplasmic ring, whereas Nup116p may be associated, partly, with the cytoplasmic filaments . Analysis of a Nup57p mutant suggested a role in maintaining the stability of cytoplasmic components of the NPC . We conclude that peripheral NPC components appear similar in yeasts compared to higher organisms and present a revised model for yeast NPC structural composition.

FEBS Lett, 2004 Feb 13, 559(1-3), 111 - 7
Expression regulation of the yeast PDR5 ATP-binding cassette (ABC) transporter suggests a role in cellular detoxification during the exponential growth phase; Mamnun YM et al.; The yeast ATP-binding cassette transporter Pdr5p mediates pleiotropic drug resistance (PDR) by effluxing a variety of xenobiotics . Immunoblotting demonstrates that Pdr5p levels are high in the logarithmic growth phase, while its levels decrease sharply when cells exit exponential growth . Here, we show that PDR5 promoter activity is dramatically reduced when cells stop growing due to a limitation of glucose or nitrogen or when they approach stationary phase . Interestingly, Pdr3p, a major transcriptional regulator of PDR5, shows the same regulatory pattern . Feeding glucose to starved cells rapidly re-induces both PDR5 and PDR3 transcription . Importantly, diminished Pdr5p levels, as present after starvation, are rapidly restored in response to xenobiotic challenges that activate the transcription factors Pdr1p and Pdr3p . Our data indicate a role for yeast Pdr5p in cellular detoxification during exponential growth.

Nat Genet, 2004 Mar, 36(3), 225 - 7 Epub 2004 Feb 08.
Senataxin, the ortholog of a yeast RNA helicase, is mutant in ataxia-ocular apraxia 2; Moreira MC et al.; Ataxia-ocular apraxia 2 (AOA2) was recently identified as a new autosomal recessive ataxia . We have now identified causative mutations in 15 families, which allows us to clinically define this entity by onset between 10 and 22 years, cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia and elevated alpha-fetoprotein (AFP) . Ten of the fifteen mutations cause premature termination of a large DEAxQ-box helicase, the human ortholog of yeast Sen1p, involved in RNA maturation and termination.

J Cell Biol, 2004 Feb 16, 164(4), 535 - 46 Epub 2004 Feb 09.
Spindle checkpoint proteins and chromosome-microtubule attachment in budding yeast; Gillett ES et al.; Accurate chromosome segregation depends on precise regulation of mitosis by the spindle checkpoint . This checkpoint monitors the status of kinetochore-microtubule attachment and delays the metaphase to anaphase transition until all kinetochores have formed stable bipolar connections to the mitotic spindle . Components of the spindle checkpoint include the mitotic arrest defective (MAD) genes MAD1-3, and the budding uninhibited by benzimidazole (BUB) genes BUB1 and BUB3 . In animal cells, all known spindle checkpoint proteins are recruited to kinetochores during normal mitoses . In contrast, we show that whereas Saccharomyces cerevisiae Bub1p and Bub3p are bound to kinetochores early in mitosis as part of the normal cell cycle, Mad1p and Mad2p are kinetochore bound only in the presence of spindle damage or kinetochore lesions that interfere with chromosome-microtubule attachment . Moreover, although Mad1p and Mad2p perform essential mitotic functions during every division cycle in mammalian cells, they are required in budding yeast only when mitosis goes awry . We propose that differences in the behavior of spindle checkpoint proteins in animal cells and budding yeast result primarily from evolutionary divergence in spindle assembly pathways . Copyright The Rockefeller University Press

J Cell Biol, 2004 Feb 16, 164(4), 567 - 80 Epub 2004 Feb 09.
Capping protein binding to actin in yeast: biochemical mechanism and physiological relevance; Kim K et al.; The mechanism by which capping protein (CP) binds barbed ends of actin filaments is not understood, and the physiological significance of CP binding to actin is not defined . The CP crystal structure suggests that the COOH-terminal regions of the CP alpha and beta subunits bind to the barbed end . Using purified recombinant mutant yeast CP, we tested this model . CP lacking both COOH-terminal regions did not bind actin . The alpha COOH-terminal region was more important than that of beta . The significance of CP's actin-binding activity in vivo was tested by determining how well CP actin-binding mutants rescued null mutant phenotypes . Rescue correlated well with capping activity, as did localization of CP to actin patches, indicating that capping is a physiological function for CP . Actin filaments of patches appear to be nucleated first, then capped with CP . The binding constants of yeast CP for actin suggest that actin capping in yeast is more dynamic than in vertebrates . Copyright The Rockefeller University Press

FEMS Microbiol Lett, 2004 Feb 9, 231(1), 19 - 26
Survey of molecular methods for the typing of wine yeast strains; Schuller D et al.; A survey of the genetic polymorphisms produced by distinct methods was performed in 23 commercial winery yeast strains . Microsatellite typing, using six different loci, an optimized interdelta sequence analysis and restriction fragment length polymorphism of mitochondrial DNA generated by the enzyme HinfI had the same discriminatory power: among the 23 commercial yeast strains, 21 distinct patterns were obtained . Karyotype analysis gave 22 patterns, thereby allowing the discrimination of one of the three strains that were not distinguished by the other methods . Due to the equivalence of the results obtained in this survey, any of the methods can be applied at the industrial scale.

Biochemistry, 2004 Feb 17, 43(6), 1425 - 31
Crystal structure of yeast acetyl-coenzyme A synthetase in complex with AMP; Jogl G et al.; Acetyl-coenzyme A synthetase (ACS) belongs to the family of AMP-forming enzymes that also includes acyl-CoA synthetases, firefly luciferase, and nonribosomal peptide synthetases . ACS catalyzes the two-step activation of acetate to acetyl-CoA: formation of an acetyl-AMP intermediate from acetate and ATP and the transfer of the acetyl group to CoA . In mammals, the acetyl-CoA product is used for biosynthesis of long chain fatty acids as well as energy production . We have determined the crystal structure of yeast ACS in a binary complex with AMP at 2.3 A resolution . The structure contains a large, N-terminal domain and a small, C-terminal domain . AMP is bound at the interface between the two domains . This structure represents a new conformation for the ACS enzyme, which may be competent for catalyzing the first step of the reaction . A Lys residue that is critical for this step is located in the active site . A rotation of 140 degrees in the small domain is needed for the binding of CoA and the catalysis of the second step . In contrast to the monomeric bacterial enzyme, yeast ACS is a stable trimer.

Mol Genet Genomics, 2004 Mar, 271(2), 237 - 48 Epub 2004 Feb 07.
Effects of mutations in the N terminal region of the yeast G protein alpha-subunit Gpa1p on signaling by pheromone receptors; Roginskaya M et al.; The sites and modes of interaction between G protein-coupled receptors and their cognate heterotrimeric G proteins remain poorly defined . The C-terminus of the Galpha subunit is the best established site of contact of G proteins with receptors, but structural analyses and crosslinking studies suggest the possibility of interactions at the N-terminus of Galpha as well . We screened for mutations in the N-terminal region of the Galpha subunit encoded by the yeast GPA1 gene that specifically affect the ability of the G protein to be activated by the yeast alpha-mating factor receptor . The screen led to identification of substitutions of glutamine or proline for Leu18 of Gpa1p that reduce the response to the pheromones alpha-factor and a-factor without affecting cellular levels of the subunit or its ability to interact with beta and gamma subunits . The mutations do not appear to affect the intrinsic ability of the G protein to be converted to the activated state . The low yield of different mutations with this phenotype indicates either that the N-terminal segment of the yeast Galpha subunit does not undergo extensive interactions with the alpha-factor receptor, or that this region can not be altered without detrimental effects upon the formation of G protein trimers.

Mol Cell Proteomics, 2004 May, 3(5), 478 - 89 Epub 2004 Feb 06.
Gene expression analyzed by high-resolution state array analysis and quantitative proteomics: response of yeast to mating pheromone; MacKay VL et al.; The transcriptome provides the database from which a cell assembles its collection of proteins . Translation of individual mRNA species into their encoded proteins is regulated, producing discrepancies between mRNA and protein levels . Using a new modeling approach to data analysis, a striking diversity is revealed in association of the transcriptome with the translational machinery . Each mRNA has its own pattern of ribosome loading, a circumstance that provides an extraordinary dynamic range of regulation, above and beyond actual transcript levels . Using this approach together with quantitative proteomics, we explored the immediate changes in gene expression in response to activation of a mitogen-activated protein kinase pathway in yeast by mating pheromone . Interestingly, in 26% of those transcripts where the predicted protein synthesis rate changed by at least 3-fold, more than half of these changes resulted from altered translational efficiencies . These observations underscore that analysis of transcript level, albeit extremely important, is insufficient by itself to describe completely the phenotypes of cells under different conditions.

Biochem J, 2004 May 15, 380(Pt 1), 139 - 45
Secondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast; Douette P et al.; The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production . We expressed UCP1 carrying a His6 epitope at its C-terminus in Saccharomyces cerevisiae mitochondria . The recombinant-tagged UCP1 was purified by immobilized metal-ion affinity chromatography to homogeneity (>95%) . This made it suitable for subsequent biophysical characterization . Fluorescence resonance energy transfer experiments showed that n-dodecyl-beta-D-maltoside-solubilized UCP1-His6 retained its PN (purine nucleotide)-binding capacity . The far-UV CD spectrum of the functional protein clearly indicated the predominance of alpha-helices in the UCP1 secondary structure . The UCP1 secondary structure exhibited an alpha-helical degree of approx . 68%, which is at least 25% higher than the previously reported estimations based on computational predictions . Moreover, the helical content remained unchanged in free and PN-loaded UCP1 . A homology model of the first repeat of UCP1, built on the basis of X-ray-solved close parent, the ADP/ATP carrier, strengthened the CD experimental results . Our experimental and computational results indicate that (i) alpha-helices are the major component of UCP1 secondary structure; (ii) PN-binding mechanism does not involve significant secondary-structure rearrangement; and (iii) UCP1 shares similar secondary-structure characteristics with the ADP/ATP carrier, at least for the first repeat.

EMBO J, 2004 Feb 25, 23(4), 719 - 27 Epub 2004 Feb 12.
Mapping key functional sites within yeast TFIID; Leurent C et al.; The transcription factor TFIID, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), plays a key role in the regulation of gene expression by RNA polymerase II . The structure of yeast TFIID, as determined by electron microscopy and digital image analysis, is formed by three lobes, labelled A-C, connected by thin linking domains . Immunomapping revealed that TFIID contains two copies of the WD-40 repeat-containing TAF5 and that TAF5 contributes to the linkers since its C- and N-termini were found in different lobes . This property was confirmed by the finding that a recombinant complex containing TAF5 complexed with six histone fold containing TAFs was able to form a trilobed structure . Moreover, the N-terminal domain of TAF1 was mapped in lobe C, whereas the histone acetyltransferase domain resides in lobe A along with TAF7 . TBP was found in the linker domain between lobes A and C in a way that the N-terminal 100 residues of TAF1 are spanned over it . The implications of these data with regard to TFIID function are discussed.

Science, 2004 Feb 6, 303(5659), 808 - 13
Global mapping of the yeast genetic interaction network; Tong AH et al.; A genetic interaction network containing approximately 1000 genes and approximately 4000 interactions was mapped by crossing mutations in 132 different query genes into a set of approximately 4700 viable gene yeast deletion mutants and scoring the double mutant progeny for fitness defects . Network connectivity was predictive of function because interactions often occurred among functionally related genes, and similar patterns of interactions tended to identify components of the same pathway . The genetic network exhibited dense local neighborhoods; therefore, the position of a gene on a partially mapped network is predictive of other genetic interactions . Because digenic interactions are common in yeast, similar networks may underlie the complex genetics associated with inherited phenotypes in other organisms.

J Biol Chem, 2004 Apr 16, 279(16), 16091 - 100 Epub 2004 Feb 05.
Subunits of a yeast oligomeric G protein-coupled receptor are activated independently by agonist but function in concert to activate G protein heterotrimers; Chinault SL et al.; G protein-coupled receptors (GPCRs) form dimeric or oligomeric complexes in vivo . However, the function of oligomerization in receptor-mediated G protein activation is unclear . Previous studies of the yeast alpha-factor receptor (STE2 gene product) have indicated that oligomerization promotes signaling . Here we have addressed the mechanism by which oligomerization facilitates G protein signaling by examining the ability of ligand binding- and G protein coupling-defective alpha-factor receptors to form complexes in vivo and to correct their signaling defects when co-expressed (trans complementation) . Newly and previously identified receptor mutants indicated that ligand binding involves the exofacial end of transmembrane domain (TM) 4, whereas G protein coupling involves ic1, ic3, the C-terminal tail, and the intracellular ends of TM2 and TM3 . Mutant receptors bearing substitutions in these domains formed homo-oligomeric or hetero-oligomeric complexes in vivo, as indicated by results of fluorescence resonance energy transfer experiments . Co-expression of ligand binding- and G protein coupling-defective mutant receptors did not significantly improve signaling . In contrast, co-expression of ic1 and ic3 mutations in trans but not in cis significantly increased signaling efficiency . Therefore, we suggest that subunits of the alpha-factor receptor: 1) are activated independently rather than cooperatively by agonist, and 2) function in a concerted fashion to promote G protein activation, possibly by contacting different subunits or regions of the G protein heterotrimer.

Eur J Biochem, 2004 Feb, 271(4), 771 - 9
Identification of residues controlling transport through the yeast aquaglyceroporin Fps1 using a genetic screen; Karlgren S et al.; Aquaporins and aquaglyceroporins mediate the transport of water and solutes across biological membranes . Saccharomyces cerevisiae Fps1 is an aquaglyceroporin that mediates controlled glycerol export during osmoregulation . The transport function of Fps1 is rapidly regulated by osmotic changes in an apparently unique way and distinct regions within the long N- and C-terminal extensions are needed for this regulation . In order to learn more about the mechanisms that control Fps1 we have set up a genetic screen for hyperactive Fps1 and isolated mutations in 14 distinct residues, all facing the inside of the cell . Five of the residues lie within the previously characterized N-terminal regulatory domain and two mutations are located within the approach to the first transmembrane domain . Three mutations cause truncation of the C-terminus, confirming previous studies on the importance of this region for channel control . Furthermore, the novel mutations identify two conserved residues in the channel-forming B-loop as critical for channel control . Structural modelling-based rationalization of the observed mutations supports the notion that the N-terminal regulatory domain and the B-loop could interact in channel control . Our findings provide a framework for further genetic and structural analysis to better understand the mechanism that controls Fps1 function by osmotic changes.

Mol Microbiol, 2004 Feb, 51(4), 987 - 1001
Dynamics and processivity of 40S ribosome scanning on mRNA in yeast; Berthelot K et al.; The eukaryotic 40S ribosomal subunit locates the translation initiation codon on an mRNA via the so-called scanning process that follows 40S binding to the capped 5' end . This key step in translation is required for the expression of almost all eukaryotic genes, yet the mechanism and dynamics of scanning are unknown . We have performed quantitative studies in vivo and in vitro of the movement of yeast 40S ribosomes along 5' untranslated regions (UTRs) of different lengths . 40S subunits perform cap-dependent scanning with high processivity for more than 1700 nucleotides in cells of Saccharomyces cerevisiae . Moreover, the observed rates of expression indicate that scanning is performed by an untethered 40S subunit that has been released from the 5' cap complex . Unexpectedly, the capability to maintain scanning competence on a long 5' UTR is more dependent on the Ded1/Dbp1 type of helicase than on eIF4A or eIF4B . In a yeast cell-free extract, scanning shows reduced processivity, with an estimated net 5'-->3' rate of approximately 10 nucleotides per second at 26 degrees C . We have developed a biased bidirectional walking model of ribosomal scanning that provides a framework for understanding the above observations as well as other known quantitative and qualitative features of this process.

J Antibiot (Tokyo), 2003 Nov, 56(11), 917 - 22
New quinones and hydroquinones from Malbranchea cinnamomea HKI 286 and HKI 296 and interaction with Tax/CREB expression system in yeast; Schlegel B et al.; In addition to malbranicin (1) and dihydromalbranicin (5), new substituted quinones 2, 3, 6 and hydroquinone 4 were isolated from the culture brothes of two strains of Malbranchea cinnamomea . The chemical constitutions of new metabolites 2, 3, 4 and 6 were elucidated by optical spectroscopy, mass spectrometry and 1D/2D NMR spectroscopy . 2 (7-methoxymalbranicin) at a concentration of 42 microM inhibited by 67% Tax/CREB-mediated expression of beta-galactosidase in a recombinant strain of Saccharomyces cerevisiae.

Nucleic Acids Res, 2004 Feb 03, 32(2), 855 - 62 Print 2004.
Fission yeast global repressors regulate the specificity of chromatin alteration in response to distinct environmental stresses; Hirota K et al.; The specific induction of genes in response to distinct environmental stress is vital for all eukaryotes . To study the mechanisms that result in selective gene responses, we examined the role of the fission yeast Tup1 family repressors in chromatin regulation . We found that chromatin structure around a cAMP-responsive element (CRE)-like sequence in ade6-M26 that is bound by Atf1.Pcr1 transcriptional activation was altered in response to osmotic stress but not to heat and oxidative stresses . Such chromatin structure alteration occurred later than the Atf1 phosphorylation but correlated well with stress-induced transcriptional activation at ade6-M26 . This chromatin structure alteration required components for the stress-activated protein kinase (SAPK) cascade and both subunits of the M26-binding CREB/ATF-type protein Atf1.Pcr1 . Cation stress and glucose starvation selectively caused chromatin structure alteration around CRE-like sequences in cta3(+) and fbp1(+) promoters, respectively, in correlation with transcriptional activation . However, the tup11Delta tup12Delta double deletion mutants lost the selectivity of stress responses of chromatin structure and transcriptional regulation of cta3(+) and fbp1(+) . These data indicate that the Tup1-like repressors regulate the chromatin structure to ensure the specificity of gene activation in response to particular stresses . Such a role for these proteins may serve as a paradigm for the regulation of stress response in higher eukaryotes.

J Biol Chem, 2004 Apr 9, 279(15), 15040 - 9 Epub 2004 Feb 03.
Anti-cooperative oxidation of ubiquinol by the yeast cytochrome bc1 complex; Covian R et al.; We have investigated the interaction between monomers of the dimeric yeast cytochrome bc(1) complex by analyzing the pre-steady and steady state activities of the isolated enzyme in the presence of antimycin under conditions that allow the first turnover of ubiquinol oxidation to be observable in cytochrome c(1) reduction . At pH 8.8, where the redox potential of the iron-sulfur protein is approximately 200 mV and in a bc(1) complex with a mutated iron-sulfur protein of equally low redox potential, the amount of cytochrome c(1) reduced by several equivalents of decyl-ubiquinol in the presence of antimycin corresponded to only half of that present in the bc(1) complex . Similar experiments in the presence of several equivalents of cytochrome c also showed only half of the bc(1) complex participating in quinol oxidation . The extent of cytochrome b reduced corresponded to two b(H) hemes undergoing reduction through one center P per dimer, indicating electron transfer between the two cytochrome b subunits . Antimycin stimulated the ubiquinol-cytochrome c reductase activity of the bc(1) complex at low inhibitor/enzyme ratios . This stimulation could only be fitted to a model in which half of the bc(1) dimer is inactive when both center N sites are free, becoming active upon binding of one center N inhibitor molecule per dimer, and there is electron transfer between the cytochrome b subunits of the dimer . These results are consistent with an alternating half-of-the-sites mechanism of ubiquinol oxidation in the bc(1) complex dimer.

J Biol Chem, 2004 Apr 16, 279(16), 16033 - 43 Epub 2004 Feb 03.
Hif1 is a component of yeast histone acetyltransferase B, a complex mainly localized in the nucleus; Poveda A et al.; Hat1 is the catalytic subunit of the only type B histone acetyltransferase known (HAT-B) . The enzyme specifically acetylates lysine 12, and to a lesser extent lysine 5, of free, non-chromatin-bound histone H4 . The complex is usually isolated with cytosolic fractions and is thought to be involved in chromatin assembly . The Saccharomyces cerevisiae HAT-B complex also contains Hat2, a protein stimulating Hat1 catalytic activity . We have now identified by two-hybrid experiments Hif1 as both a Hat1- and a histone H4-interacting protein . These interactions were dependent on HAT2, indicating a mediating role for Hat2 . Biochemical fractionation and co-immunoprecipitation assays demonstrated that Hif1 is a component of a yeast heterotrimeric HAT-B complex, in which Hat2 bridges Hat1 and Hif1 proteins . In contrast to Hat2, this novel subunit does not appear to regulate Hat1 enzymatic activity . Nevertheless, similarly to Hat1, Hif1 influences telomeric silencing . In a localization analysis by immunofluorescence microscopy on yeast strains expressing tagged versions of Hat1, Hat2, and Hif1, we have found that all three HAT-B proteins are mainly localized in the nucleus . Thus, we propose that the distinction between A- and B-type enzymes should henceforth be based on their capacity to acetylate histones bound to nucleosomes and not on their location within the cell . Finally, by Western blotting assays, we have not detected differences in the in vivo acetylation of H4 lysine 12 (acK12H4) between wild-type and hat1Delta, hat2Delta, or hif1Delta mutant strains, suggesting that the level of HAT-B-dependent acK12H4 may be very low under normal growth conditions.

J Biol Chem, 2004 Apr 16, 279(16), 16017 - 25 Epub 2004 Feb 03.
The Rsp5 ubiquitin ligase binds to and ubiquitinates members of the yeast CIN85-endophilin complex, Sla1-Rvs167; Stamenova SD et al.; Sla1 and Rvs167 are yeast proteins required for receptor internalization and organization of the actin cytoskeleton . Here we provide evidence that Sla1 and Rvs167 are orthologues of the mammalian CIN85 and endophilin proteins, respectively, which are required for ligand-stimulated growth factor receptor internalization . Sla1 is similar in domain structure to CIN85 and binds directly to the endophilin-like Rvs167 . Akin to CIN85, Sla1 interacts with synaptojanins and a ubiquitin ligase that regulates endocytosis . This ubiquitin ligase, Rsp5, binds directly to both Sla1 and Rvs167 . The interaction between Rsp5 and Rvs167 is mediated through Rsp5 WW domains and PXY motifs in the central Gly-Pro-Ala-rich domain of Rvs167 . Rvs167 PXY motifs are required for Rsp5-dependent monoubiquitination of Rvs167 on Lys481 in the Src homology 3 (SH3) domain . Mutation of Lys481 --> Arg causes cells to grow slowly on medium containing 1 M NaCl, although this phenotype is not due to the defect in ubiquitination caused by the K481R mutation . We propose that Rsp5 interaction with Sla1-Rvs167 promotes Rvs167 ubiquitination and regulates activity of this protein complex . Rvs167 ubiquitination is not required for general function of Rvs167, but may control specific Rvs167 SH3 domain-protein interactions or negatively regulate SH3 domain activity.

Blood Cells Mol Dis, 2004 Jan-Feb, 32(1), 124 - 30
A novel mutation in the glucose-6-phosphate dehydrogenase gene in a subject with chronic nonspherocytic hemolytic anemia--characterization of enzyme using yeast expression system and molecular modeling; Grabowska D et al.; Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy . Human G6PD gene is highly polymorphic, with over 130 mutations identified, many of which cause hemolytic anemia . We studied a novel point mutation in the G6PD gene 1226 C-->G, predicting the proline 409 to arginine substitution (G6PD Suwalki) . We expressed the human wild-type and mutated G6PD gene in yeast Saccharomyces cerevisiae which allowed the characterization of the Suwalki variant . We showed that human wild-type, as well as the mutated (1226 C-->G) G6PD gene, functionally complemented the phenotype displayed by the yeast strain with disruption of the ZWF1 gene (homologue of the human G6PD gene) . Comparison of wild-type (wt) human G6PD purified from yeast and from blood shows no significant differences in the Km values for G6P and in the utilization rate for the substrate analogue, 2-deoxyG6P . The P409R substitution leads to drastic changes in G6PD kinetics . The specific activity as well as stability of mutated G6PD is also significantly reduced . Besides this, the effect of this mutation was analyzed using a model of the tertiary structure of the human enzyme . The localization of the P409R mutation suggests that it may influence the stability of the whole protein by changing tetramer interactions and disturbing the binding of structural NADP+.

FEMS Microbiol Lett, 2004 Jan 30, 230(2), 227 - 34
The N-terminal domain of yeast Bap2 permease is phosphorylated dependently on the Npr1 kinase in response to starvation; Omura F et al.; The Saccharomyces cerevisiae branched-chain amino acid permease Bap2p plays a major role in leucine, isoleucine, and valine transport, and its synthesis is regulated transcriptionally . Bap2p undergoes a starvation-induced degradation depending upon ubiquitination and the functions of N- and C-terminal domains of Bap2p . Here we show that the N-terminal domain of Bap2p is phosphorylated in response to rapamycin treatment when both the N- and C-termini of Bap2p are fused to glutathione S-transferase . The phosphorylation is dependent on Ser/Thr kinase Npr1p . In npr1 cells, Bap2p becomes slightly more susceptible to the rapamycin-induced degradation, suggesting that Npr1p counteracts the degradation system for Bap2p.

Arch Biochem Biophys, 2004 Feb 15, 422(2), 125 - 36
Azide binding to yeast cytochrome c peroxidase and horse metmyoglobin: comparative thermodynamic investigation using isothermal titration calorimetry; Jacobson T et al.; Yeast cytochrome c peroxidase (CcP) and horse metmyoglobin (Mb) bind HN3 with similar affinities at 25 degrees C . The pH-independent equilibrium association constants for formation of the CcP.HN3 and Mb.HN3 complexes are (1.05 +/- 0.06)x10(5) and (1.6 +/- 0.8)x10(5) M(-1), respectively . However, the thermodynamic parameters for formation of the two complexes are quite different . The DeltaH0 values for formation of CcP.HN3 and Mb.HN3 are -16.4 +/- 0.7 and -9.0 +/- 0.5 kcal/mol, respectively, and the Delta S0 values are -32 +/- 2 and -16 +/- 2 cal/deg mol, respectively . The proton associated with HN3 is retained in both protein complexes at low pH but dissociates with apparent pKA values of 5.5 +/- 0.2 and > or =8.2 for the Mb.HN3 and CcP.HN3 complexes, respectively . CcP and Mb differ significantly in their reactivity toward the azide anion, N3- . CcP binds N3- very weakly, if at all, and only an upper-limit of 18 +/-5 M(-1) for the pH-independent equilibrium association constant for the CcP.N3- complex can be determined . Mb binds N3- with an association constant of (1.8 +/- 0.1)x10(4) M(-1) . The ratio of the equilibrium association constants for HN3 and N3- binding provides a discrimination factor between the neutral and charged forms of the ligand . The discrimination factor is greater than 5800 for CcP but only nine for Mb . Protonation of the distal histidines in the two proteins influences binding of HN3 . Protonation of His-64 in Mb enhances HN3 binding due to a gating mechanism while protonation of His-52 in CcP decreases the affinity for HN3 due to loss of base-assisted association of the ligand to the heme iron.

Mol Cell, 2004 Jan 30, 13(2), 225 - 39
High-definition macromolecular composition of yeast RNA-processing complexes; Krogan NJ et al.; A remarkably large collection of evolutionarily conserved proteins has been implicated in processing of noncoding RNAs and biogenesis of ribonucleoproteins . To better define the physical and functional relationships among these proteins and their cognate RNAs, we performed 165 highly stringent affinity purifications of known or predicted RNA-related proteins from Saccharomyces cerevisiae . We systematically identified and estimated the relative abundance of stably associated polypeptides and RNA species using a combination of gel densitometry, protein mass spectrometry, and oligonucleotide microarray hybridization . Ninety-two discrete proteins or protein complexes were identified comprising 489 different polypeptides, many associated with one or more specific RNA molecules . Some of the pre-rRNA-processing complexes that were obtained are discrete sub-complexes of those previously described . Among these, we identified the IPI complex required for proper processing of the ITS2 region of the ribosomal RNA primary transcript . This study provides a high-resolution overview of the modular topology of noncoding RNA-processing machinery.

Yeast, 2004 Jan 30, 21(2), 127 - 39
Binding of Cdc48p to a ubiquitin-related UBX domain from novel yeast proteins involved in intracellular proteolysis and sporulation; Decottignies A et al.; The Cdc48/p97 AAA-ATPase functions in membrane fusion and ubiquitin-dependent protein degradation . Here, we show that, in yeast, Cdc48p interacts with three novel proteins, Cuil-3p, which contain a conserved ubiquitin-related (UBX) domain . Cui2p and Cui3p are closely related, interact with each other, and are localized at the perinuclear membrane . Cdc48p binds directly the UBX domain of Cui3p in vitro . Multiple deletions of the CUI1, CUI2 and CUI3 genes confer deficiency in sporulation and degradation of model ubiquitin-protein fusions . The Cuil-3 proteins were also found to interact with Ufd3p, a WD repeat protein known to associate with Cdc48p . Together, these results indicate that the Cuil-3 proteins form complexes that are components of the ubiquitin-proteasome system .

Yeast, 2004 Jan 30, 21(2), 107 - 17
{URE3} prion propagation is abolished by a mutation of the primary cytosolic Hsp70 of budding yeast; Roberts BT et al.; {URE3} and {PSI(+)} are infectious protein forms of the Saccharomyces cerevisiae Ure2p and Sup35p, respectively . We isolated an allele of SSA2, the primary cytosolic Hsp70, in a screen for mutants unable to maintain {URE3} . Designated ssa2-10, the mutation results in a leucine substitution for proline 395, a conserved residue of the peptide-binding domain . This allele also unexpectedly destabilizes {URE3} in newly formed heterozygotes: {URE3} is either absent in heterozygotes formed by crossing wild-type {URE3} cells with ssa2-10 mutants, or present and fully stable . SSA2 deletion mutants are weakly capable of maintaining {URE3} . The ssa2-10 allele is compatible with propagation of {PSI(+)} . However, in combination with a deletion of SSA1, ssa2-10 eliminates the nonsense-suppression phenotype of {PSI(+)} cells .

Yeast, 2004 Jan 30, 21(2), 95 - 106
Development of an integrative transformation system for the opportunistic pathogenic yeast Candida lusitaniae using URA3 as a selection marker; Francois F et al.; The nucleotide sequence of the URA3 gene encoding orotidine-5'-phosphate decarboxylase (OMP DCase) of the human opportunistic pathogen yeast Candida lusitaniae was determined by degenerate PCR and chromosome walking . Deduced amino acid sequence showed strong homologies (59-85% identity) with OMP DCases of different Saccharomycetales and allowed identification of the known conserved domains . Very close upstream from the URA3 gene, the 3'-end of a gene encoding a Gea2-like protein was identified . A non-revertible C . lusitaniae ura3 mutant was selected on the basis of 5-fluoroorotic acid resistance . The mutation was a single point mutation resulting in the amino acid substitution D95V in a highly conserved domain, and in a concomitant EcoRV restriction site polymorphism . The mutant strain was successfully transformed to prototrophy following electroporation with the URA3 gene cloned in an integrative vector, with frequencies of 100-200 transformants per micro g of DNA . Southern blot analysis revealed that almost all transformants were derived from homologous recombination events at the resident locus . The GeneBank Accession No . for C . lusitaniae URA3 gene is AF450297 .

J Biol Chem, 2004 Apr 9, 279(15), 14954 - 60 Epub 2004 Jan 28.
A regulatory domain in the C-terminal extension of the yeast glycerol channel Fps1p; Hedfalk K et al.; The Saccharomyces cerevisiae gene FPS1 encodes an aquaglyceroporin of the major intrinsic protein (MIP) family . The main function of Fps1p seems to be the efflux of glycerol in the adaptation of the yeast cell to lower external osmolarity . Fps1p is an atypical member of the family, because the protein is much larger (669 amino acids) than most MIPs due to long hydrophilic extensions in both termini . We have shown previously that a short domain in the N-terminal extension of the protein is required for restricting glycerol transport through the channel (Tamas, M . J., Karlgren, S., Bill, R . M., Hedfalk, K., Allegri, L., Ferreira, M., Thevelein, J . M., Rydstrom, J., Mullins, J . G . L., and Hohmann, S . (2003) J . Biol . Chem . 278, 6337-6345) . Deletion of the N-terminal domain results in an unregulated channel, loss of glycerol, and osmosensitivity . In this work we have investigated the role of the Fps1p C terminus (139 amino acids) . A set of eight truncations has been constructed and tested in vivo in a yeast fps1Delta strain . We have performed growth tests, membrane localization following cell fractionation, and glycerol accumulation measurements as well as an investigation of the osmotic stress response . Our results show that the C-terminal extension is also involved in restricting transport through Fps1p . We have identified a sequence of 12 amino acids, residues 535-546, close to the sixth transmembrane domain . This element seems to be important for controlling Fps1p function . Similar to the N-terminal domain, the C-terminal domain is amphiphilic and has a potential to dip into the membrane.

Ann N Y Acad Sci, 2003 Dec, 1002, 43 - 55
The effect of hydroxyurea and trichostatin a on targeted nucleotide exchange in yeast and Mammalian cells; Parekh-Olmedo H et al.; Targeted nucleotide exchange (TNE) is a process by which a synthetic DNA oligonucleotide, partially complementary to a site in a chromosomal or an episomal gene directs the reversal of a single nucleotide at a specific site . To protect against nuclease digestion, the oligonucleotide is modified with derivative linkages among the terminal bases . We have termed these molecules modified single-stranded oligonucleotides (MSOs) . Current models suggest that the reaction occurs in two steps . The first, DNA pairing, involves the alignment of the MSO with the target site and its assimilation into the target helix forming a D-loop . The second phase centers around the repair of a single base mismatch formed between the MSO and its complementary strand in the D-loop . Nucleotide exchange is promoted in all likelihood by the mismatch repair system . A critical feature of successful TNE is the accessibility of the target site for the MSO and the factors that increase the dynamic nature of the chromatin that will likely increase the frequency . Here, we report that two factors, trichostatin A and hydroxyurea, elevate gene repair of a mutant hygromycin gene in Saccharomyces cerevisiae and a mutant eGFP gene in a mammalian cell line, MCF-10AT1 cells . Trichostatin A (TSA) acts by preventing the deacetylation of histones while hydroxyurea (HU) reduces the rate of replication . Both of these activities, by their very nature, create a more open configuration of the MSO into the target site.

Genes Cells, 2003 Dec, 8(12), 951 - 61
Carboxy-terminal region of the yeast heat shock factor contains two domains that make transcription independent of the TFIIH protein kinase; Sakurai H et al.; BACKGROUND: Phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II is implicated in transition from initiation to elongation in the transcription cycle . In yeast cells, Kin28, a subunit of the general transcription factor TFIIH, is responsible for the CTD phosphorylation . Although Kin28 is indispensable for transcription of many genes, its requirement is bypassed in certain genes such as SSA4 or CUP1, whose transcription is activated by the heat shock factor Hsf1 . RESULTS: We show that C-terminal region of Hsf1, which consists of an activation domain AR2 and a regulatory domain CTM, mediates the Kin28-independent transcription . The AR2 domain, when fused to the DNA-binding domain of Gal4 and recruited to the GAL7 gene via the Gal4-binding sequence, is sufficient for activating GAL7 in the absence of Kin28 . We have further found that AR2 has an ability to recruit TATA box-binding protein-associated factors (TAFs) to the promoter . Consistently, transcription from promoters occupied naturally or artificially with TAFs is sustained in the absence of Kin28 function . CONCLUSIONS: These results show that CTM modulates activation function of AR2 in the Hsf1 molecule . We also suggest that recruitment of TAFs to a promoter is involved in the Kin28-independent transcription.

Biotechnol Bioeng, 2004 Feb 5, 85(3), 340 - 50
Protein disulfide isomerase, but not binding protein, overexpression enhances secretion of a non-disulfide-bonded protein in yeast; Smith JD et al.; In eukaryotes, secretory proteins are folded and assembled in the endoplasmic reticulum (ER) . Many heterologous proteins are retained in the ER due to suboptimal folding conditions . We previously reported that heterologous secretion of Pyrococcus furiosus beta-glucosidase in Saccharomyces cerevisiae resulted in the accumulation of a large fraction of inactive beta-glucosidase in the ER . In this work, we determine the effect of introducing additional genes of ER-resident yeast proteins, Kar2p (binding protein {BiP}) and protein disulfide isomerase (PDI), on relieving this bottleneck . Single-copy expression of BiP and PDI worked synergistically to improve secretion by reverse similar 60% . In an effort to optimize BiP and PDI interactions, we created a library of beta-glucosidase expression strains that incorporated four combinations of constitutively or inducibly-expressed BiP and PDI genes integrated to random gene copynumbers in the yeast chromosome . Approximately 15% of the transformants screened had secretion level improvements higher than that seen with single BiP/PDI gene overexpression, and the highest secreting strain had threefold higher beta-glucosidase levels than the control . Nineteen of the improved strains were re-examined for beta-glucosidase secretion as well as BiP and PDI levels . Within the improved transformants BiP and PDI levels ranged sevenfold and tenfold over the control, respectively . Interestingly, increasing BiP levels decreased beta-glucosidase secretion, whereas increasing PDI levels increased beta-glucosidase secretion . The action of PDI was unexpected because beta-glucosidase is not a disulfide-bonded protein . We suggest that PDI may be acting in a chaperone-like capacity or possibly creating mixed disulfides with the beta-glucosidase's lone cysteine residue during the folding and assembly process .

Proteins, 2004 Feb 15, 54(3), 455 - 67
Functional interaction of 13 yeast SCF complexes with a set of yeast E2 enzymes in vitro; Kus BM et al.; SCF complexes are multi-subunit ubiquitin ligases that, in concert with the E1 and E2 ubiquitination enzymes, catalyze the ubiquination of specific target proteins . Only three yeast SCFs have been reconstituted and characterized to date; each of these ubiquitinates its target protein with the E2 Cdc34 . We have reconstituted and purified 1 known and 12 novel yeast SCF complexes, and explored the ability of these complexes to function with 5 different purified E2 enzymes; Ubc1, Cdc34, Ubc4, Ubc8 and Ubc11 . We have found that the ubiquitination of Sic1 by the reconstituted SCF(Cdc4) complex was specifically catalyzed by two of the five E2 enzymes tested in vitro; Cdc34 and Ubc4 . We also show that at least eight of the purified SCF complexes clearly ubiquitinated their F-box proteins in vitro, lending support for a regulatory mechanism in which F-box proteins catalyze their own destruction . The autoubiquitination of each F-box was in some cases catalyzed only by Cdc34, and in other cases preferentially catalyzed by Ubc4 . Ubc4 thus interacts with multiple SCFs in vitro, and the interactions among SCF and E2 components of the ubiquitination machinery may allow further diversification of the roles of SCFs in vivo .

J Gen Appl Microbiol, 2003 Dec, 49(6), 337 - 44
Sporobolomyces magnisporus sp . nov., a new yeast species in the Erythrobasidium cluster isolated from plants in Taiwan; Nakase T et al.; Two strains of yeasts that contain Q-10 as the major ubiquinone, lack cellular xylose and produce large bilaterally symmetrical ballistoconidia were isolated from plants collected in a protected subtropical rain forest in Taiwan and were found to represent a new species . The taxonomic properties of this species coincide with the genus Sporobolomyces, so it is described as Sporobolomyces magnisporus sp . nov . In phylogenetic trees based on the nucleotide sequences of 18S ribosomal DNA and D1/D2 domain of 26S ribosomal DNA, this species is located in the Erythrobasidium cluster.

Yeast, 2004 Jan 15, 21(1), 25 - 39
A DNA microarray for fission yeast: minimal changes in global gene expression after temperature shift; Xue Y et al.; Completion of the fission yeast genome sequence has opened up possibilities for post-genomic approaches . We have constructed a DNA microarray for genome-wide gene expression analysis in fission yeast . The microarray contains DNA fragments, PCR-amplified from a genomic DNA template, that represent > 99% of the 5000 or so annotated fission yeast genes, as well as a number of control sequences . The GenomePRIDE software used attempts to design similarly sized DNA fragments corresponding to gene regions within single exons, near the 3'-end of genes that lack homology to other fission yeast genes . To validate the design and utility of the array, we studied expression changes after a 2 h temperature shift from 25 degrees C to 36 degrees C, conditions widely used when studying temperature-sensitive mutants . Obligingly, the vast majority of genes do not change more than two-fold, supporting the widely held view that temperature-shift experiments specifically reveal phenotypes associated with temperature-sensitive mutants . However, we did identify a small group of genes that showed a reproducible change in expression . Importantly, most of these corresponded to previously characterized heat-shock genes, whose expression has been reported to change after more extreme temperature shifts than those used here . We conclude that the DNA microarray represents a useful resource for fission yeast researchers as well as the broader yeast community, since it will facilitate comparison with the distantly related budding yeast, Saccharomyces cerevisiae . To maximize the utility of this resource, the array and its component parts are fully described in On-line Supplementary Information and are also available commercially .

Yeast, 2004 Jan 15, 21(1), 11 - 24
Identification of an UDP-Glc:glycoprotein glucosyltransferase in the yeast Yarrowia lipolytica; Babour A et al.; The UDP-Glc:glycoprotein glucosyltransferase (UGT) is a soluble protein of the endoplasmic reticulum (ER) that plays a determining part in the mechanism by which unfolded, partially folded or misfolded glycoproteins are retained into the ER . We have identified an UGT in the yeast Yarrowia lipolytica . This protein, of a predicted molecular weight of 165.7 kDa, is encoded by a 5054 bp coding sequence containing a 643 bp intron at position 682-1323 . The N-terminal part of the protein displays a signal sequence whereas its C-terminal part carries an ER retrieval signal HDEL . An interruption of the gene that removes the 1075 last nucleotides of its sequence did not lead to any evident phenotype except for a slight increased sensitivity to tunicamycin . YlUGT1 mRNA levels respond to tunicamycin treatment by an induction factor of 2-4, which indicates that the gene product participates in the quality control mechanism in this yeast . Finally, an immunofluorescence study of the protein localization, shows that the protein distribution is different from that of previously studied ER resident proteins . This could indicate that UGT distribution in the secretory pathway is not confined to the ER .

Cell Mol Life Sci, 2004 Jan, 61(2), 186 - 91
Yeast Golgi apparatus--dynamics and sorting; Nakano A; The yeast Saccharomyces cerevisiae is one of the best-studied organisms to understand molecular mechanisms of membrane traffic, but as far as the organization of the Golgi apparatus is concerned, yeast is only just beginning to yield clues about how dynamic and flexible the organelle is.

Biosci Biotechnol Biochem, 2004 Jan, 68(1), 266 - 70
Truncated Sla1 induces haploid meiosis through the Pat1-Mei2 system in fission yeast; Tanabe K et al.; We previously reported that expression of Sla1DeltaC, a truncated form of Sla1, induces ectopic meiosis in heterothallic fission yeast and this was possibly due to the inhibition of Pat1 kinase by Sla1DeltaC . Here we found mei2 mRNA and the Mei2 protein accumulated and stability of the Mei2 protein increased when Sla1DeltaC was expressed . The former two results are considered to be the consequence of de-repression of Ste11, which is the transcription factor of mei2 and negatively regulated by Pat1 kinase . The latter result reflects the consequence of deregulation of Mei2 by Pat1 kinase . In addition, Ste11 accumulated in the nucleus when Sla1DeltaC was expressed . All these data consistently support the idea that the action of Sla1DeltaC is to inactivate Pat1 kinase.

Biosci Biotechnol Biochem, 2004 Jan, 68(1), 206 - 14
Self-cloning yeast strains containing novel FAS2 mutations produce a higher amount of ethyl caproate in Japanese sake; Aritomi K et al.; Point mutation of Gly1250Ser (1250S) of the yeast fatty acid synthase gene FAS2 confers cerulenin resistance . This mutation also results in a higher production of the apple-like flavor component ethyl caproate in Japanese sake . We mutated the 1250th codon by in vitro site-directed mutagenesis to encode Ala (1250A) or Cys (1250C) and examined cerulenin resistance and ethyl caproate production . The mutated FAS2 genes were inserted into a binary plasmid vector containing a drug-resistance marker and a counter-selectable marker, GALp-GIN11M86 . The plasmids were integrated into the wild-type FAS2 locus of a sake yeast strain, and the loss of the plasmid sequences from the integrants was done by growth on galactose plates, which is permissive for loss of GALp-GIN11M86 . These counter-selected strains contained either the wild type or the mutated FAS2 allele but not the plasmid sequences, from which FAS2 mutant strains were selected by allele-specific PCR . The FAS2-1250C mutant produced a higher amount of ethyl caproate in sake than FAS2-1250S, while FAS2-1250A produced an ethyl caproate level intermediate between FAS2-1250S and the parental Kyokai no . 7 strain . Interestingly, these mutants only showed detectable cerulenin resistance . These 'self-cloning' yeast strains should be acceptable to the public because they can improve sake quality without the presence of extraneous DNA sequences.

Biosci Biotechnol Biochem, 2004 Jan, 68(1), 79 - 84
Catalytic Reaction of Basidiomycete Lentinula edodes Cytochrome P450, Le.CYP1 Enzyme Produced in Yeast; Akiyama R et al.; The basidiomycete Lentinula edodes (Le.) cytochrome P450, Le.CYP1 was functionally expressed in Saccharomyces cerevisiae . The microsomal fraction containing Le.CYP1 was prepared from the recombinant yeast and the Le.CYP1 was analyzed . The 7-ethoxycoumarin and benzo(a)pyrene were found to be the substrates of Le.CYP1 enzyme . Le.CYP1 converted 7-ethoxycoumarin to 7-hydroxycoumarin.

J Cell Biol, 2004 Feb 2, 164(3), 361 - 71 Epub 2004 Jan 26.
Mating type-dependent constraints on the mobility of the left arm of yeast chromosome III; Bressan DA et al.; Mating-type gene (MAT) switching in budding yeast exhibits donor preference . MATa preferentially recombines with HML near the left telomere of chromosome III, whereas MATalpha prefers HMR near the right telomere . Donor preference is controlled by the recombination enhancer (RE) located proximal to HML . To test if HML is constrained in pairing with MATalpha, we examined live-cell mobility of LacI-GFP-bound lactose operator (lacO) arrays inserted at different chromosomal sites . Without induction of recombination, lacO sequences adjacent to HML are strongly constrained in both MATalpha and RE-deleted MATa strains, compared with MATa . In contrast, chromosome movement at HMR or near a telomere of chromosome V is mating-type independent . HML is more constrained in MATa Deltare and less constrained in MATa RE+ compared with other sites . Although HML and MATa are not prealigned before inducing recombination, the three-dimensional configuration of MAT, HML, and HMR is mating-type dependent . These data suggest there is constitutive tethering of HML, which is relieved in MATa cells through the action of RE . Copyright The Rockefeller University Press

J Biol Chem, 2004 Apr 9, 279(15), 15289 - 97 Epub 2004 Jan 26.
Degradation of mutated bovine pancreatic trypsin inhibitor in the yeast vacuole suggests post-endoplasmic reticulum protein quality control; Coughlan CM et al.; The rate-limiting step in protein secretion is folding, which occurs in the endoplasmic reticulum (ER) lumen, and almost all secreted proteins contain disulfide bonds that form in the ER and stabilize the native state . Secreted proteins unable to fold may aggregate or they may be subject to ER-associated protein degradation . To examine the fate of aberrant forms of a well characterized, disulfide-bonded secreted protein, we expressed bovine pancreatic trypsin inhibitor in yeast . Bovine pancreatic trypsin inhibitor is a single domain, 58-amino acid polypeptide containing three disulfide bonds, and yeast cells secrete the wild type protein . In contrast, the Y35L mutant, which folds rapidly but is unstable, remains soluble and is not secreted . Surprisingly, the proteolysis of Y35L is unaffected in yeast containing mutations in genes encoding factors required for ER-associated protein degradation and is stable if artificially retained in the ER . Rather, Y35L is diverted from the Golgi to the vacuole and degraded . Because only the mutant protein is quantitatively proteolyzed these data suggest that a post-ER quality control check-point diverts unstable proteins to the vacuole for degradation.

Adv Gerontol, 2003, 12, 46 - 56
How the analysis of genetic mutations can help us to solve basic problems in gerontology? II . Life extending genetic modifications in budding yeast S . cereviseae, fruit fly D . melanogaster and laboratory mice M . musculus; Khalyavkin AV et al.; Most studies of aging are conducted in humans and domestic or laboratory animals, i.e . in conditions where artificial environment protection is applied, This yields changes in physiology and behavior, which set up organism's state unobserved in wild life . This state may be less adequate to the evolutionary adjusted genetic construction of an organism, which generates a hypothesis that in natural niches the aging rate can be lower and stress resistance can be higher than in captivity despite the fact that life expectancy in habitat is essentially lower than that in laboratory conditions due to high external mortality . Direct test of this hypothesis is difficult because of problems related to reconstruction of natural environment conditions in the laboratory . Substantial life-extending effect of some mutated genes can serve as indirect test of the hypothesis . We propose that in some cases genetic mutations can distort reaction of an organism on environmental cues and change control parameters of its life cycle . As a result such mutants in laboratory may partly demonstrate life traits similar to those observed in natural environment, e.g . associated with high stress resistance and low rate of aging . These features combined with low external mortality in laboratory conditions may lead to significant extension in the life span of mutants . Recently we considered 56 life-extending gene modifications in nematode C . elegans (Adv . Gerontol., 2003, Vol . 11), scattered in many publications . In this paper we consider pertinent life-extending gene modifications corresponded to the budding yeast S . cerevisease (29 genes), fruit fly D . melanogaster (22 genes) and laboratory mice M . musculus (8 genes).

Environ Mol Mutagen, 2004, 43(1), 28 - 35
Mutation induction in haploid yeast after split-dose radiation exposure . II . Combination of UV-irradiation and X-rays; Keller B et al.; Split-dose protocols can be used to investigate the kinetics of recovery from radiation damage and to elucidate the mechanisms of cell inactivation and mutation induction . In this study, a haploid strain of the yeast, Saccharomyces cerevisiae, wild-type with regard to radiation sensitivity, was irradiated with 254-nm ultraviolet (UV) light and then exposed to X-rays after incubation for 0-6 hr . The cells were incubated either on nutrient medium or salt agar between the treatments . Loss of reproductive ability and mutation to canavanine resistance were measured . When the X-ray exposure immediately followed UV-irradiation, the X-ray survival curves had the same slope irrespective of the pretreatment, while the X-ray mutation induction curves were changed from linear to linear quadratic with increasing UV fluence . Incubations up to about 3 hr on nutrient medium between the treatments led to synergism with respect to cell inactivation and antagonism with respect to mutation, but after 4-6 hr the two treatments acted independently . Incubation on salt agar did not cause any change in the survival curves, but there was a strong suppression of X-ray-induced mutation with increasing UV fluence . On the basis of these results, we suggest that mutation after combined UV and X-ray exposure is affected not only by the induction and suppression of DNA repair processes, but also by radiation-induced modifications of cell-cycle progression and changes in the expression of the mutant phenotype .

Mol Biol Cell, 2004 Apr, 15(4), 1533 - 43 Epub 2004 Jan 23.
O-glycosylation as a sorting determinant for cell surface delivery in yeast; Proszynski TJ et al.; Little is known about the mechanisms that determine localization of proteins to the plasma membrane in Saccharomyces cerevisiae . The length of the transmembrane domains and association of proteins with lipid rafts have been proposed to play a role in sorting to the cell surface . Here, we report that Fus1p, an O-glycosylated integral membrane protein involved in cell fusion during yeast mating, requires O-glycosylation for cell surface delivery . In cells lacking PMT4, encoding a mannosyltransferase involved in the initial step of O-glycosylation, Fus1p was not glycosylated and accumulated in late Golgi structures . A chimeric protein lacking O-glycosylation motif was missorted to the vacuole and accumulated in late Golgi in wild-type cells . Exocytosis of this protein could be restored by addition of a 33-amino acid portion of an O-glycosylated sequence from Fus1p . Our data suggest that O-glycosylation functions as a sorting determinant for cell surface delivery of Fus1p.

Mol Biol Cell, 2004 Apr, 15(4), 1609 - 22 Epub 2004 Jan 23.
Interdependency of fission yeast Alp14/TOG and coiled coil protein Alp7 in microtubule localization and bipolar spindle formation; Sato M et al.; The Dis1/TOG family plays a pivotal role in microtubule organization . In fission yeast, Alp14 and Dis1 share an essential function in bipolar spindle formation . Here, we characterize Alp7, a novel coiled-coil protein that is required for organization of bipolar spindles . Both Alp7 and Alp14 colocalize to the spindle pole body (SPB) and mitotic spindles . Alp14 localization to these sites is fully dependent upon Alp7 . Conversely, in the absence of Alp14, Alp7 localizes to the SPBs, but not mitotic spindles . Alp7 forms a complex with Alp14, where the C-terminal region of Alp14 interacts with the coiled-coil domain of Alp7 . Intriguingly, this Alp14 C terminus is necessary and sufficient for mitotic spindle localization . Overproduction of either full-length or coiled-coil region of Alp7 results in abnormal V-shaped spindles and stabilization of interphase microtubules, which is induced independent of Alp14 . Alp7 may be a functional homologue of animal TACC . Our results shed light on an interdependent relationship between Alp14/TOG and Alp7 . We propose a two-step model that accounts for the recruitment of Alp7 and Alp14 to the SPB and microtubules.

Appl Microbiol Biotechnol, 2004 Apr, 64(3), 416 - 20 Epub 2004 Jan 23.
Cerebrosides of Candida lipolytica yeast; Rupcic J et al.; Candida lipolytica yeast was grown batchwise on glucose medium . Cerebrosides were isolated from the sphingolipid fraction of total lipids using column chromatography and separated into two compounds by high-performance thin-layer chromatography . Glucose was detected as the sole sugar constituent in cerebrosides . The fatty acid composition of cerebrosides was characterised by a predominance of saturated fatty acids and by a high proportion of fatty acids with 16 carbon atoms . The dominant fatty acid was h16:0 . The principal long-chain base components of both cerebroside species were trihydroxy bases, 18- and 20-phytosphinosine . The unique characteristic of cerebrosides was the presence of a high proportion of sphingosine (one-fourth of the total long-chain bases), which is a common characteristic of mammalian sphingolipids and rarely occurs in yeast cerebrosides . The ceramide moiety profile of cerebrosides is similar to that of epidermal ceramides, which implies a possibility for their application in care cosmetics.

Acta Biochim Pol, 2003, 50(4), 1039 - 56
Prohibitins and Ras2 protein cooperate in the maintenance of mitochondrial function during yeast aging; Kirchman PA et al.; The yeast Saccharomyces cerevisiae has a finite replicative life span . Yeasts possess two prohibitins, Phb1p and Phb2p, in similarity to mammalian cells . These proteins are located in the inner mitochondrial membrane, where they are involved in the processing of newly-synthesized membrane proteins . We demonstrate that the elimination of one or both of the prohibitin genes in yeast markedly diminished the replicative life span of cells that lack fully-functional mitochondria, while having no effect on cells with functioning mitochondria . This deleterious effect was suppressed by the deletion of the RAS2 gene . The expression of PHB1 and PHB2 declined gradually up to 5-fold during the life span . Cells in which PHB1 was deleted in conjunction with the absence of a mitochondrial genome displayed remarkable changes in mitochondrial morphology, distribution, and inheritance . This loss of mitochondrial integrity was not seen in cells devoid of PHB1 but possessing an intact mitochondrial genome . In a subset of the cells, the changes in mitochondrial integrity were associated with increased production of reactive oxygen species, which co-localized with the altered mitochondria . The mitochondrial deficits described above were all suppressed by deletion of RAS2 . Our data, together with published information, are interpreted to provide a unified view of the role of the prohibitins in yeast aging . This model posits that the key initiating event is a decline in mitochondrial function, which leads to progressive oxidative damage that is exacerbated in the absence of the prohibitins . This aggravation of the initial damage is ameliorated by the suppression of the production of mitochondrial proteins in the absence of Ras2p signaling of mitochondrial biogenesis.

EMBO J, 2004 Feb 11, 23(3), 500 - 10 Epub 2004 Jan 22.
Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast; Velichutina I et al.; The 20S proteasome is made up of four stacked heptameric rings, which in eucaryotes assemble from 14 different but related subunits . The rules governing subunit assembly and placement are not understood . We show that a different kind of proteasome forms in yeast when the Pre9/alpha3 subunit is deleted . Purified pre9Delta proteasomes show a two-fold enrichment for the Pre6/alpha4 subunit, consistent with the presence of an extra copy of Pre6 in each outer ring . Based on disulfide engineering and structure-guided suppressor analyses, Pre6 takes the position normally occupied by Pre9, a substitution that depends on a network of intersubunit salt bridges . When Arabidopsis PAD1/alpha4 is expressed in yeast, it complements not only pre6Delta but also pre6Delta pre9Delta mutants; therefore, the plant alpha4 subunit also can occupy multiple positions in a functional yeast proteasome . Importantly, biogenesis of proteasomes is delayed at an early stage in pre9Delta cells, suggesting an advantage for Pre9 over Pre6 incorporation at the alpha3 position that facilitates correct assembly.

J Biol Chem, 2004 Mar 26, 279(13), 12081 - 7 Epub 2004 Jan 21.
Regulation of phospholipid synthesis in the yeast cki1Delta eki1Delta mutant defective in the Kennedy pathway . The Cho1-encoded phosphatidylserine synthase is regulated by mRNA stability; Choi HS et al.; In the yeast Saccharomyces cerevisiae, the most abundant phospholipid phosphatidylcholine is synthesized by the complementary CDP-diacylglycerol and Kennedy pathways . Using a cki1Delta eki1Delta mutant defective in choline kinase and ethanolamine kinase, we examined the consequences of a block in the Kennedy pathway on the regulation of phosphatidylcholine synthesis by the CDP-diacylglycerol pathway . The cki1Delta eki1Delta mutant exhibited increases in the synthesis of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine via the CDP-diacylglycerol pathway . The increase in phospholipid synthesis correlated with increased activity levels of the CDP-diacylglycerol pathway enzymes phosphatidylserine synthase, phosphatidylserine decarboxylase, phosphatidylethanolamine methyltransferase, and phospholipid methyltransferase . However, other enzyme activities, including phosphatidylinositol synthase and phosphatidate phosphatase, were not affected in the cki1Delta eki1Delta mutant . For phosphatidylserine synthase, the enzyme catalyzing the committed step in the pathway, activity was regulated by increases in the levels of mRNA and protein . Decay analysis of CHO1 mRNA indicated that a dramatic increase in transcript stability was a major component responsible for the elevated level of phosphatidylserine synthase . These results revealed a novel mechanism that controls phospholipid synthesis in yeast.

J Environ Monit, 2004 Jan, 6(1), 71 - 9 Epub 2003 Nov 14.
A yeast-based cytotoxicity and genotoxicity assay for environmental monitoring using novel portable instrumentation; Knight AW et al.; An assay capable of simultaneously measuring both general toxicity and more subtle genotoxicity, in aqueous environmental samples, is described . The assay uses eukaryotic (yeast) cells, genetically modified to express a green fluorescent protein (GFP) whenever DNA damage, as a result of exposure to genotoxic agents, is repaired . A measure of the reduction in cell proliferation is used to characterise general toxicity producing familiar EC(50) and LOEC data . The assay protocol has been developed for proposed use in the field and hence employs dedicated, portable instrumentation, the development of which is described . A range of environmentally relevant substances has been evaluated using the assay, including solutions of metal ions, solvents and pesticides . Preliminary data comparing the yeast assay's response to that of a standard Daphnia test in the analysis of the toxicity of 34 varied industrial waste effluents are also presented . The sensitivity to a wide range of substances and effluents suggests the assay should be useful for environmental toxicity monitoring.

Bioelectromagnetics, 2004 Feb, 25(2), 127 - 33
Apoptosis induced by ultraviolet radiation is enhanced by amplitude modulated radiofrequency radiation in mutant yeast cells; Markkanen A et al.; The aim of this study was to investigate whether radiofrequency (RF) electromagnetic field (EMF) exposure affects cell death processes of yeast cells . Saccharomyces cerevisiae yeast cells of the strains KFy417 (wild-type) and KFy437 (cdc48-mutant) were exposed to 900 or 872 MHz RF fields, with or without exposure to ultraviolet (UV) radiation, and incubated simultaneously with elevated temperature (+37 degrees C) to induce apoptosis in the cdc48-mutated strain . The RF exposure was carried out in a special waveguide exposure chamber where the temperature of the cell cultures can be precisely controlled . Apoptosis was analyzed using the annexin V-FITC method utilizing flow cytometry . Amplitude modulated (217 pulses per second) RF exposure significantly enhanced UV induced apoptosis in cdc48-mutated cells, but no effect was observed in cells exposed to unmodulated fields at identical time-average specfic absorption rates (SAR, 0.4 or 3.0 W/kg) . The findings suggest that amplitude modulated RF fields, together with known damaging agents, can affect the cell death process in mutated yeast cells . Bioelectromagnetics 25:127-133, 2004 .

J Biol Chem, 2004 Apr 2, 279(14), 14096 - 103 Epub 2004 Jan 19.
Mutations in the yeast mrf1 gene encoding mitochondrial release factor inhibit translation on mitochondrial ribosomes; Towpik J et al.; Although the control of mitochondrial translation in the yeast Saccharomyces cerevisiae has been studied extensively, the mechanism of termination remains obscure . Ten mutations isolated in a genetic screen for read-through of premature stop codons in mitochondrial genes were localized in the chromosomal gene encoding the mitochondrial release factor mRF1 . The mrf1-13 and mrf1-780 mutant genes, in contrast to other alleles, caused a non-respiratory phenotype that correlated with decreased expression of mitochondrial genes as well as a reporter ARG8(m) gene inserted into mitochondrial DNA . The steady-state levels of several mitochondrially encoded proteins, but not their mRNAs, were dramatically decreased in mrf1-13 and mrf1-780 cells . Structural models of mRF1 were constructed, allowing localization of residues substituted in the mrf1 mutants and offering an insight into the possible mechanism by which these mutations change the mitochondrial translation termination fidelity . Inhibition of mitochondrial translation in mrf1-13 and mrf1-780 correlated with the three-dimensional localization of the mutated residues close to the PST motif presumably involved in the recognition of stop codons in mitochondrial mRNA.

J Biol Chem, 2004 Apr 2, 279(14), 14418 - 26 Epub 2004 Jan 19.
Dissecting transcription-coupled and global genomic repair in the chromatin of yeast GAL1-10 genes; Li S et al.; Transcription-coupled repair (TCR) and global genomic repair (GGR) of UV-induced cyclobutane pyrimidine dimers were investigated in the yeast GAL1-10 genes . Both Rpb9- and Rad26-mediated TCR are confined to the transcribed strands, initiating at upstream sites approximately 100 nucleotides from the upstream activating sequence shared by the two genes . However, TCR initiation sites do not correlate with either transcription start sites or TATA boxes . Rad16-mediated GGR tightly correlates with nucleosome positioning when the genes are repressed and are slow in the nucleosome core and fast in linker DNA . Induction of transcription enhanced GGR in nucleosome core DNA, especially in the nucleosomes around and upstream of the transcription start sites . Furthermore, when the genes were induced, GGR was slower in the transcribed regions than in the upstream regions . Finally, simultaneous deletion of RAD16, RAD26, and RPB9 resulted in no detectable repair in all sites along the region analyzed . Our results suggest that (a) . TCR may be initiated by a transcription activator, presumably through the loading of RNA polymerase II, rather than by transcription initiation or elongation per se; (b) . TCR and nucleosome disruption-enhanced GGR are the major causes of rapid repair in regions around and upstream of transcription start sites; (c) . transcription machinery may hinder access of NER factors to a DNA lesion in the absence of a transcription-repair coupling factor; and (d) . other than GGR mediated by Rad16 and TCR mediated by Rad26 and Rpb9, no other nucleotide excision repair pathway exists in these RNA polymerase II-transcribed genes.

J Biol Chem, 2004 Apr 2, 279(14), 14256 - 63 Epub 2004 Jan 20.
Inositol deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian PGAP1 and yeast Bst1p; Tanaka S et al.; The inositol moiety of mammalian glycosylphosphatidylinositol (GPI) is acylated at an early step in GPI biosynthesis . The inositol acylation is essential for the generation of mature GPI capable of attachment to proteins . However, the acyl group is usually absent from GPI-anchored proteins (GPI-APs) on the cell surface due to inositol deacylation that occurs in the endoplasmic reticulum (ER) soon after GPI-anchor attachment . Mammalian GPI inositol-deacylase has not been cloned, and the biological significance of the deacylation has been unclear . Here we report a GPI inositol-deacylase-deficient Chinese hamster ovary cell line established by taking advantage of resistance to phosphatidylinositol-specific phospholipase C and the gene responsible, which was termed PGAP1 for Post GPI Attachment to Proteins 1 . PGAP1 encoded an ER-associated, 922-amino acid membrane protein bearing a lipase consensus motif . Substitution of a conserved putative catalytic serine with alanine resulted in a complete loss of function, indicating that PGAP1 is the GPI inositol-deacylase . The mutant cells showed a clear delay in the maturation of GPI-APs in the Golgi and accumulation of GPI-APs in the ER . Thus, the GPI inositol deacylation is important for efficient transport of GPI-APs from the ER to the Golgi.

J Cell Biol, 2004 Jan 19, 164(2), 267 - 77
A conserved protein interaction network involving the yeast MAP kinases Fus3 and Kss1; Kusari AB et al.; The Saccharomyces cerevisiae mitogen-activated protein kinases (MAPKs) Fus3 and Kss1 bind to multiple regulators and substrates . We show that mutations in a conserved docking site in these MAPKs (the CD/7m region) disrupt binding to an important subset of their binding partners, including the Ste7 MAPK kinase, the Ste5 adaptor/scaffold protein, and the Dig1 and Dig2 transcriptional repressors . Supporting the possibility that Ste5 and Ste7 bind to the same region of the MAPKs, they partially competed for Fus3 binding . In vivo, some of the MAPK mutants displayed reduced Ste7-dependent phosphorylation, and all of them exhibited multiple defects in mating and pheromone response . The Kss1 mutants were also defective in Kss1-imposed repression of Ste12 . We conclude that MAPKs contain a structurally and functionally conserved docking site that mediates an overall positively acting network of interactions with cognate docking sites on their regulators and substrates . Key features of this interaction network appear to have been conserved from yeast to humans.

J Cell Biol, 2004 Jan 19, 164(2), 195 - 206
Trans-SNARE interactions elicit Ca2+ efflux from the yeast vacuole lumen; Merz AJ et al.; Ca2+ transients trigger many SNARE-dependent membrane fusion events . The homotypic fusion of yeast vacuoles occurs after a release of lumenal Ca2+ . Here, we show that trans-SNARE interactions promote the release of Ca2+ from the vacuole lumen . Ypt7p-GTP, the Sec1p/Munc18-protein Vps33p, and Rho GTPases, all of which function during docking, are required for Ca2+ release . Inhibitors of SNARE function prevent Ca2+ release . Recombinant Vam7p, a soluble Q-SNARE, stimulates Ca2+ release . Vacuoles lacking either of two complementary SNAREs, Vam3p or Nyv1p, fail to release Ca2+ upon tethering . Mixing these two vacuole populations together allows Vam3p and Nyv1p to interact in trans and rescues Ca2+ release . Sec17/18p promote sustained Ca2+ release by recycling SNAREs (and perhaps other limiting factors), but are not required at the release step itself . We conclude that trans-SNARE assembly events during docking promote Ca2+ release from the vacuole lumen.

Bioinformatics, 2004 Jan 22, 20(2), 284 - 5
YETI: Yeast Exploration Tool Integrator; Orton RJ et al.; Yeast Exploration Tool Integrator (YETI) is a novel bioinformatics tool for the integrated visualization and analysis of functional genomic data sets from the budding yeast Saccharomyces cerevisiae . AVAILABILITY: YETI is freely available for use over the WWW, or download under license, at http://www.bru.ed.ac.uk/~orton/yeti.html

FEMS Yeast Res, 2004 Jan, 4(4-5), 527 - 40
Correlations among measures of phenotypic and genetic variation within an oligotrophic asexual yeast, Candida sonorensis, collected from Opuntia; Ganter PF et al.; Many descriptions of yeast species are based on a limited number of strains collected at one time from a single locale . Often, little is known of phenotypic or genotypic variation and covariation within species . We compare 36 strains of an asexual cactophilic yeast, Candida sonorensis, collected from Opuntia cacti . Comparisons were based on geographical distances between collection locales, responses to physiological assimilation and stress tests, random amplified polymorphic DNA (RAPD) profiles, partial Lage Subunit (LSU) rDNA sequences, relative DNA-DNA hybridization values, and electrokaryotypes . There was significant variation among strains in all types of data collected . Comparisons among the different data types found significant positive associations between RAPD profiles, geographical distances, physiologies, reassociation values, and electrokaryotypes . No significant associations were found between rDNA sequences and any other type of variation measured . Based on RAPD, reassociation, electrokaryotype, and physiological data, the 36 strains could be divided into two groups: those collected in West Texas (nine strains) and all others . RAPD data indicated that 10 (of 12) Australian strains also formed a distinct clade . The taxonomic and phylogenetic status of these clades is discussed . Evidence that new genotypes can sweep through large geographic areas is also discussed.

FEMS Yeast Res, 2004 Jan, 4(4-5), 417 - 25
Yeast cells harboring human alpha-1,3-fucosyltransferase at the cell surface engineered using Pir, a cell wall-anchored protein; Abe H et al.; Human alpha-1,3-fucosyltansferase (FucT) encoded by the FUT6 gene was displayed at the cell surface of yeast cells engineered using the yeast cell wall protein Pir1 or Pir2, and the FucT activity was detected at the surface of cells producing the Pir1-HA-FUT6 or Pir2-FLAG-FUT6 fusion proteins . To obtain higher activity, we engineered the host yeast cells in which endogenous PIR genes of the PIR1-4 gene family were disrupted . Among the disruptants, the pir1Delta pir2Delta pir3Delta strain with the PIR1-HA-FUT6 fusion gene showed the highest FucT activity, which was about three-fold higher than that of the wild-type strain . Furthermore, the co-expression of both the Pir1-HA-FUT6 and the Pir2-FLAG-FUT6 fusions showed an approximately 1.5-fold higher activity than that in the cell wall displaying Pir1-HA-FUT6 alone . The present method was thus effective for producing yeast cells that can easily synthesize various oligosaccharides, such as Le(x) and sLe(x), using Pir-glycosyltransferase fusions in combination with the deletion of endogenous PIR genes.

Trends Cell Biol, 1992 Dec, 2(12), 353 - 7
Cell cycle control of gene expression in yeast; Johnston LH; Increasing evidence suggests that cell-cycle-regulated gene expression plays a crucial role in cell cycle control . In building yeast, as many as 250 genes (3-4% of all genes in this yeast) may be regulated in this way . One large group is expressed at the G1-S transition and includes cyclin genes, whose products control the p34(CDC28) protein kinase, as well as many genes essential for DNA synthesis . Two separate systems control the expression of these genes in the late G1 phase, but these systems have in common the SW16 protein, which may be a cell cycle stage-specific transcription factor.

Trends Cell Biol, 1994 Feb, 4(2), 56 - 60
The unfolded-protein-response pathway in yeast; Shamu CE et al.; The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the increased production of several ER-resident proteins . This signalling pathway exists in organisms as divergent as mammals and yeast, and is the only known example of an intracellular signalling system that links the ER and the nucleus . Recently, a transmembrane kinase similar in structure to growth-factor receptor kinases has been identified as a key component of the unfolded-protein-response pathway in yeast.

Trends Cell Biol, 1994 Feb, 4(2), 42 - 5
Is there a role for GPIs in yeast cell-wall assembly?
de Nobel H, Lipke PN.
Glycosylphosphatidylinositol (GPI) membrane anchors are essential for the integration of yeast cell adhesion proteins into the cell wall, but mature cell-wall proteins are unlikely to be attached directly to the membrane . We thus propose that GPI-anchored glycoprotein forms are intermediates in a process that crosslinks the major components of the cell wall by transglycosylation . This mechanism may be critical for both the biosynthesis and overall architecture of the cell wall.

Trends Cell Biol, 1991 Dec, 1(6), 160 - 4
Organelle inheritance in the yeast cell cycle; Yaffe MP; Cell proliferation requires the inheritance of subcellular organelles, yet little is known of the molecular basis of this essential process . Recent microscopy studies of the yeast Saccharomyces cerevisiae have characterized the cellular distribution of mitochondria, vacuoles and elements of the endoplasmic reticulum and Golgi complex . In addition, genetic and microscopical approaches have allowed the isolation and analysis of mutants defective in the inheritance of mitochondria and vacuoles . These investigations are leading to the identification of molecular components mediating the movement of organelles into daughter cells and have revealed that the inheritance of organelles is coordinated with other events of the cell division cycle.

Trends Cell Biol, 1992 Jan, 2(1), 10 - 4
Telomeric position effect in yeast; Sandell LL et al.; Telomeres are the physical ends of chromosomes . In yeast, when a gene is placed near a telomere, its transcription is repressed . Genes under the influence of this telomeric position effects switch between a repressed state and a transcriptionally active state, each of which is stable for many cell generations . Telomeric position effect may provide a model system for the study of heritable gene regulation in other, more complex organisms.

RNA, 2004 Feb, 10(2), 210 - 20
Definition of a spliceosome interaction domain in yeast Prp2 ATPase; Edwalds-Gilbert G et al.; The Saccharomyces cerevisiae splicing factor Prp2 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 . It contains three domains: a unique N-terminal domain, a helicase domain that is highly conserved in the DExD/H protein family, and a C-terminal domain that is conserved in spliceosomal DEAH proteins Prp2, Prp16, Prp22, and Prp43 . We examined the role of each domain of Prp2 by deletion mutagenesis . Whereas deletions of either the helicase or C-terminal domain are lethal, deletions in the N-terminal domain have no detectable effect on Prp2 activity . Overexpression of the C-terminal domain of Prp2 exacerbates the temperature-sensitive phenotype of a prp2(Ts) strain, suggesting that the C-domain interferes with the activity of the Prp2(Ts) protein . A genetic approach was then taken to study interactions between Prp2 and the spliceosome . Previously, we isolated dominant negative mutants in the helicase domain of Prp2 that inhibit the activity of wild-type Prp2 when the mutant protein is overexpressed . We mutagenized one prp2 release mutant gene and screened for loss of dominant negative function . Several weak binding mutants were isolated and mapped to the C terminus of Prp2, further indicating the importance of the C terminus in spliceosome binding . This study is the first to indicate that amino acid substitutions outside the helicase domain can abolish spliceosome contact and splicing activity of a spliceosomal DEAH protein.

Mol Cell Biol, 2004 Feb, 24(3), 1232 - 44
The ctf13-30/CTF13 genomic haploinsufficiency modifier screen identifies the yeast chromatin remodeling complex RSC, which is required for the establishment of sister chromatid cohesion; Baetz KK et al.; The budding yeast centromere-kinetochore complex ensures high-fidelity chromosome segregation in mitosis and meiosis by mediating the attachment and movement of chromosomes along spindle microtubules . To identify new genes and pathways whose function impinges on chromosome transmission, we developed a genomic haploinsufficiency modifier screen and used ctf13-30, encoding a mutant core kinetochore protein, as the reference point . We demonstrate through a series of secondary screens that the genomic modifier screen is a successful method for identifying genes that encode nonessential proteins required for the fidelity of chromosome segregation . One gene isolated in our screen was RSC2, a nonessential subunit of the RSC chromatin remodeling complex . rsc2 mutants have defects in both chromosome segregation and cohesion, but the localization of kinetochore proteins to centromeres is not affected . We determined that, in the absence of RSC2, cohesin could still associate with chromosomes but fails to achieve proper cohesion between sister chromatids, indicating that RSC has a role in the establishment of cohesion . In addition, numerous subunits of RSC were affinity purified and a new component of RSC, Rtt102, was identified . Our work indicates that only a subset of the nonessential RSC subunits function in maintaining chromosome transmission fidelity.

Exp Cell Res, 2004 Feb 15, 293(2), 185 - 95
Subcellular localization and oligomeric structure of the yeast putative stretch-activated Ca2+ channel component Mid1; Yoshimura H et al.; The yeast Mid1 protein with an apparent molecular mass of 100 kDa is required for Ca2+ influx stimulated by the mating pheromone and by a capacitative calcium entrylike mechanism acting in response to Ca2+ depletion from the endoplasmic reticulum (ER) and functions as a stretch-activated Ca2+ -permeable channel when expressed in mammalian cells . Our previous work with protease protection experiments has indicated that Mid1 is present in the plasma membrane . In this study, we examined a possible intracellular localization of this protein by indirect fluorescence microscopy and found that Mid1 is present in the ER membrane as well as the plasma membrane . Intracellular fluorescence images for Mid1 were the same as those for the ER marker protein Sec71 but quite different from those of the Golgi protein Ypt1 . The results were confirmed by membrane fractionation using Angiografin density gradient analysis . We also investigated the oligomeric structures and protein levels of Mid1 and found that Mid1 forms a 200-kDa oligomer by disulfide bonding . The protein level and modification of Mid1 in the plasma membrane and the ER membrane were unchanged by the mating pheromone . These findings provide new insight into the function of Mid1 in relation to localization, modification, and activation mechanisms.

Curr Genet, 2004 May, 45(5), 283 - 8 Epub 2004 Jan 15.
A fission yeast strain expressing human CDC25A phosphatase: a tool for selectivity studies of pharmacological inhibitors of CDC25; Mondesert O et al.; Fission yeast is a simple eukaryotic model organism in which many aspects of cell cycle control can be explored . We examined by homologous recombination whether the human CDC25A phosphatase could substitute for the function of the fission yeast Cdc25 . We first show: (a) . that CDC25A efficiently replaces the endogenous Cdc25 mitotic inducer for vegetative growth and (b) . that CDC25A is able to partially restore a functional checkpoint in response to both ionising and UV irradiation, but not a DNA replication checkpoint . We then describe a simple assay in which we demonstrate that growth of the humanised CDC25A strain is strongly repressed in a CDC25-dependent manner by BN2003, a potent chemical inhibitor of CDC25 belonging to the benzothiazoledione family . The ease of manipulation of fission yeast humanised CDC25 cells and the simplicity of the above assay offer a powerful tool with which to investigate the specificity of pharmacological inhibitors of CDC25.

EMBO Rep, 2004 Feb, 5(2), 201 - 6 Epub 2004 Jan 09.
Yeast N-glycanase distinguishes between native and non-native glycoproteins; Hirsch C et al.; N-glycanase from Saccharomyces cerevisiae (Png1) preferentially removes N-glycans from misfolded proteins . The ability of Png1 to distinguish between folded and misfolded glycoproteins is reminiscent of substrate recognition by UDP-glucose glycoprotein glucosyl transferase, an enzyme that possesses this trait . The only known in vivo substrates of Png1 are aberrant glycoproteins that originate in the endoplasmic reticulum, and arrive in the cytoplasm for proteasomal degradation . The substrate specificity of Png1 is admirably suited for this task.

Genes Dev, 2004 Jan 1, 18(1), 12 - 6
Calorie restriction extends yeast life span by lowering the level of NADH; Lin SJ et al.; Calorie restriction (CR) extends life span in a wide variety of species . Previously, we showed that calorie restriction increases the replicative life span in yeast by activating Sir2, a highly conserved NAD-dependent deacetylase . Here we test whether CR activates Sir2 by increasing the NAD/NADH ratio or by regulating the level of nicotinamide, a known inhibitor of Sir2 . We show that CR decreases NADH levels, and that NADH is a competitive inhibitor of Sir2 . A genetic intervention that specifically decreases NADH levels increases life span, validating the model that NADH regulates yeast longevity in response to CR.

Mol Pharmacol, 2004 Jan, 65(1), 214 - 9
Novel neuroprotective K+ channel inhibitor identified by high-throughput screening in yeast; Zaks-Makhina E et al.; Discovery of K+ channel modulators is limited by low-throughput capacity of existing K+ channel assays . To enable high-throughput screening for novel pharmacological modulators of K+ channels, we developed an assay based on growth of yeast that functionally expresses mammalian Kir2.1 channels . Screening of 10,000 small molecules from a combinatorial chemical library yielded 42 potential Kir2.1 inhibitors . One compound, 3-bicyclo{2.2.1}hept-2-yl-benzene-1,2-diol, was confirmed to inhibit K+ channels in patch-clamp measurements in mammalian cells with EC50 values of 60 and 1 microM for Kir2.1 and Kv2.1 channels, respectively . Inhibition of Kv2.1 channels decreased in the presence of the external pore blocker tetraethylammonium (TEA) and depended on a residue required for extracellular TEA action, suggesting that the identified compound targets the external mouth of the channel . Furthermore, at the nontoxic concentration of 3 microM, the identified compound completely abolished in vitro neuronal apoptosis mediated by Kv2.1 channels . Therefore, yeast-based screening has identified a novel uncharged neuroprotective mammalian K+ channel inhibitor.

J Biol Chem, 2004 Mar 26, 279(13), 13215 - 23 Epub 2004 Jan 13.
Bipartite 3'-cis-acting signal for replication in yeast 23 S RNA virus and its repair; Fujimura T et al.; 23 S RNA narnavirus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae . The viral genome is small (2.9 kb) and only encodes its RNA-dependent RNA polymerase . Recently, we have succeeded in generating 23 S RNA virus from an expression vector containing the entire viral cDNA sequence . Using this in vivo launching system, we analyzed the 3'-cis-acting signals for replication . The 3'-non-coding region of 23 S RNA contains two cis-elements . One is a stretch of 4 Cs at the 3' end, and the other is a mismatched pair in a stem-loop structure that partially overlaps the terminal 4 Cs . In the latter element, the loop or stem sequence is not important but the stem structure with the mismatch pair is essential . The mismatched bases should be purines . Any combination of purines at the mismatch pair bestowed capability of replication on the RNA, whereas converting it to a single bulge at either side of the stem abolished the activity . The terminal and penultimate Cs at the 3' end could be eliminated or modified to other nucleotides in the launching plasmid without affecting virus generation . However, the viruses generated regained or restored these Cs at the 3' terminus . Considering the importance of the viral 3' ends in RNA replication, these results suggest that this 3' end repair may contribute to the persistence of 23 S RNA virus in yeast by maintaining the genomic RNA termini intact . We discuss possible mechanisms for this 3' end repair in vivo.

Biotechnol Lett, 2003 Nov, 25(22), 1887 - 91
Changes in gluconic acid, polyols and major volatile compounds in sherry wine during aging with submerged flor yeast cultures; Peinado RA et al.; The traditional biological process by which sherry wines are aged can be accelerated by using submerged Saccharomyces cerevisiae var . capensis (G1) strain cultures previously grown in glycerol . The used controlled shaking conditions raise the acetaldehyde, acetoin, and meso 2,3-butanediol contents in the wine, and increases the consumption of gluconic acid by flor yeast relative to traditional biological aging under flor yeast velum.

Proc Natl Acad Sci U S A, 2004 Jan 20, 101(3), 793 - 8 Epub 2004 Jan 12.
Chemogenomic profiling: identifying the functional interactions of small molecules in yeast; Giaever G et al.; We demonstrate the efficacy of a genome-wide protocol in yeast that allows the identification of those gene products that functionally interact with small molecules and result in the inhibition of cellular proliferation . Here we present results from screening 10 diverse compounds in 80 genome-wide experiments against the complete collection of heterozygous yeast deletion strains . These compounds include anticancer and antifungal agents, statins, alverine citrate, and dyclonine . In several cases, we identified previously known interactions; furthermore, in each case, our analysis revealed novel cellular interactions, even when the relationship between a compound and its cellular target had been well established . In addition, we identified a chemical core structure shared among three therapeutically distinct compounds that inhibit the ERG24 heterozygous deletion strain, demonstrating that cells may respond similarly to compounds of related structure . The ability to identify on-and-off target effects in vivo is fundamental to understanding the cellular response to small-molecule perturbants.

Mol Biol Cell, 2004 Mar, 15(3), 1436 - 44 Epub 2004 Jan 12.
Yeast lacking the SRO7/SOP1-encoded tumor suppressor homologue show increased susceptibility to apoptosis-like cell death on exposure to NaCl stress; Wadskog I et al.; Yeast cells deleted for the SRO7/SOP1 encoded tumor suppressor homologue show increased sensitivity to NaCl stress . On exposure to growth-inhibiting NaCl concentrations, sro7Delta mutants display a rapid loss in viability that is associated with markers of apoptosis: accumulation of reactive oxygen species, DNA breakage, and nuclear fragmentation . Additional deletion of the yeast metacaspase gene YCA1 prevents the primary fast drop in viability and diminishes nuclear fragmentation and DNA breakage . We also observed that NaCl induced loss in viability of wild-type cells is Yca1p dependent . However, a yeast strain deleted for both SRO7 and its homologue SRO77 exhibits NaCl-induced cell death that is independent on YCA1 . Likewise, sro77Delta single mutants do not survive better after additional deletion of the YCA1 gene, and both sro77Delta and sro77Deltayca1Delta mutants display apoptotic characteristics when exposed to growth-inhibiting salinity, suggesting that yeast possesses Yca1p-independent pathway(s) for apoptosis-like cell death . The activity of Yca1p increases with increasing NaCl stress and sro7Delta mutants achieve levels that are higher than in wild-type cells . However, mutants lacking SRO77 do not enhance caspase activity when subject to NaCl stress, suggesting that Sro7p and Sro77p exert opposing effects on the cellular activity of Yca1p.

Mol Biol Cell, 2004 Apr, 15(4), 1519 - 32 Epub 2004 Jan 12.
The differential roles of budding yeast Tem1p, Cdc15p, and Bub2p protein dynamics in mitotic exit; Molk JN et al.; In the budding yeast Saccharomyces cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the mitotic exit network (MEN) in anaphase . To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo . Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud . Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB . In contrast, Bub2p-GFP fluorescence intensity decreased in mid-to-late anaphase on the dSPB . Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit . The mechanism that elevates Tem1p-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization . Finally, fluorescence recovery after photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase . These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade.

Mol Biol Cell, 2004 Mar, 15(3), 1297 - 312 Epub 2004 Jan 12.
The transcriptome of prematurely aging yeast cells is similar to that of telomerase-deficient cells; Lesur I et al.; To help define the pathologies associated with yeast cells as they age, we analyzed the transcriptome of young and old cells isolated by elutriation, which allows isolation of biochemical quantities of old cells much further advanced in their life span than old cells prepared by the biotin-streptavidin method . Both 18-generation-old wild-type yeast and 8-generation-old cells from a prematurely aging mutant (dna2-1), with a defect in DNA replication, were evaluated . Genes involved in gluconeogenesis, the glyoxylate cycle, lipid metabolism, and glycogen production are induced in old cells, signifying a shift toward energy storage . We observed a much more extensive generalized stress response known as the environmental stress response (ESR), than observed previously in biotin-streptavidin-isolated cells, perhaps because the elutriated cells were further advanced in their life span . In addition, there was induction of DNA repair genes that fall in the so-called DNA damage "signature" set . In the dna2-1 mutant, energy production genes were also induced . The response in the dna2-1 strain is similar to the telomerase delete response, genes whose expression changes during cellular senescence in telomerase-deficient cells . We propose that these results suggest, albeit indirectly, that old cells are responding to genome instability.

J Biol Chem, 2004 Mar 26, 279(13), 12951 - 8 Epub 2004 Jan 12.
Human disease-related mutations in cytochrome b studied in yeast; Fisher N et al.; Several mutations in the mitochondrially encoded cytochrome b have been reported in patients . To characterize their effect, we introduced six "human" mutations, namely G33S, S152P, G252D, Y279C, G291D, and Delta252-259 in the highly similar yeast cytochrome b . G252D showed wild type behavior in standard conditions . However, Asp-252 may interfere with structural lipid and, in consequence, destabilize the enzyme assembly, which could explain the pathogenicity of the mutation . The mutations G33S, S152P, G291D, and Delta252-259 were clearly pathogenic . They caused a severe decrease of the respiratory function and altered the assembly of the iron-sulfur protein in the bc(1) complex, as observed by immunodetection . Suppressor mutations that partially restored the respiratory function impaired by S152P or G291D were found in or close to the hinge region of the iron-sulfur protein, suggesting that this region may play a role in the stable binding of the subunit to the bc(1) complex . Y279C caused a significant decrease of the bc(1) function and perturbed the quinol binding . The EPR spectra showed an altered signal, indicative of a lower occupancy of the Q(o) site . The effect of human mutation of residue 279 was confirmed by another change, Y279A, which had a more severe effect on Q(o) site properties . Thus by using yeast as a model system, we identified the molecular basis of the respiratory defect caused by the disease mutations in cytochrome b.

Cell, 2004 Jan 9, 116(1), 121 - 37
Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes; Lum PY et al.; Modern medicine faces the challenge of developing safer and more effective therapies to treat human diseases . Many drugs currently in use were discovered without knowledge of their underlying molecular mechanisms . Understanding their biological targets and modes of action will be essential to design improved second-generation compounds . Here, we describe the use of a genome-wide pool of tagged heterozygotes to assess the cellular effects of 78 compounds in Saccharomyces cerevisiae . Specifically, lanosterol synthase in the sterol biosynthetic pathway was identified as a target of the antianginal drug molsidomine, which may explain its cholesterol-lowering effects . Further, the rRNA processing exosome was identified as a potential target of the cell growth inhibitor 5-fluorouracil . This genome-wide screen validated previously characterized targets or helped identify potentially new modes of action for over half of the compounds tested, providing proof of this principle for analyzing the modes of action of clinically relevant compounds.

Cell, 2004 Jan 9, 116(1), 63 - 73
Nuclear retention of unspliced mRNAs in yeast is mediated by perinuclear Mlp1; Galy V et al.; The molecular mechanism underlying the retention of intron-containing mRNAs in the nucleus is not understood . Here, we show that retention of intron-containing mRNAs in yeast is mediated by perinuclearly located Mlp1 . Deletion of MLP1 impairs retention while having no effect on mRNA splicing . The Mlp1-dependent leakage of intron-containing RNAs is increased in presence of ts-prp18 delta, a splicing mutant . When overall pre-mRNA levels are increased by deletion of RRP6, a nuclear exosome component, MLP1 deletion augments leakage of only the intron-containing portion of mRNAs . Our data suggest, moreover, that Mlp1-dependent retention is mediated via the 5' splice site . Intriguingly, we found Mlp-proteins to be present only on sections of the NE adjacent to chromatin . We propose that at this confined site the perinuclear Mlp1 implements a quality control step prior to export, physically retaining faulty pre-mRNAs.

Cell, 2004 Jan 9, 116(1), 5 - 7
Old drugs, new tricks: using genetically sensitized yeast to reveal drug targets; Hughes T et al.; A study in this issue of Cell illustrates the power of applying genomic approaches with model systems to characterize the biological activity of small molecules and to identify their cellular targets, which can clarify the mode of action of human therapeutics.

Appl Microbiol Biotechnol, 2004 Mar, 64(1), 28 - 40 Epub 2004 Jan 10.
Yeast cell-surface display--applications of molecular display; Kondo A et al.; In a cell-surface engineering system established using the yeast Saccharomyces cerevisiae, novel, so-called arming yeasts are constructed that are armed with biocatalysts in the form of enzymes, functional proteins, antibodies, and combinatorial protein libraries . Among the many advantages of the system, in which proteins are genetically displayed on the cell surface, are easy reproduction of the displayed biocatalysts and easy separation of product from catalyst . As proteins and peptides of various kinds can be displayed on the yeast cell surface, the system is expected to allow the preparation of tailor-made functional proteins . With its ability to express many of the functional proteins necessary for post-translational modification and in a range of different sizes, the yeast-based molecular display system appears uniquely useful among the various display systems so far developed . Capable of conferring novel additional abilities upon living cells, cell-surface engineering heralds a new era of combinatorial bioengineering in the field of biotechnology . This mini-review describes molecular display using yeast and its various applications.

Science, 2004 Jan 9, 303(5655), 240 - 3
RNA branching and debranching in the yeast retrovirus-like element Ty1; Cheng Z et al.; Ty elements of Saccharomyces cerevisiae are long terminal repeat (LTR) retroelements related to retroviruses . Normal levels of Ty1 transposition require Dbr1p, a cellular enzyme that cleaves 2'-5' RNA bonds . We show that Ty1 RNAs lacking identifiable 5' ends accumulate in virus-like particles (VLPs) in dbr1 mutants . Debranching this RNA in vitro with Dbr1p creates an uncapped version of the normal Ty1 RNA 5' end . We show that the 5' nucleotide (nt) of Ty1 RNA forms a 2'-5' bond with a nt near the 3' end of the same RNA, creating a lariat . The properties of the lariat suggest it forms by a novel mechanism and that branching and debranching may play roles in Ty1 reverse transcription at the minus-strand transfer step.

EMBO Rep, 2004 Jan, 5(1), 47 - 53
Molecular evidence indicating that the yeast PAF complex is required for transcription elongation; Rondon AG et al.; PAF is a five-subunit protein complex composed of Paf1, Cdc73, Leo1, Rtf1 and Ctr9, which was purified from yeast in association with RNA polymerase II and which is believed to function in transcription elongation . However, no direct proof exists for this yet . To assay whether PAF is required in elongation, we determined the in vitro transcription-elongation efficiencies of mutant cell extracts using a DNA template containing two G-less cassettes . paf1Delta or cdc73Delta cell extracts showed reduced transcription-elongation efficiencies (16-18% of the wild-type levels), whereas leo1Delta and rtf1Delta showed wild-type levels . In vivo transcription efficiency was diminished in the four mutants analysed, as determined by their abilities to transcribe lacZ . Our work provides molecular evidence that PAF has a positive role in transcription elongation and is composed of at least two functionally different types of subunits (Paf1-Cdc73 and Leo1-Rtf1).

J Cell Sci, 2004 Feb 1, 117(Pt 4), 601 - 8 Epub 2004 Jan 06.
A domain of Rad9 specifically required for activation of Chk1 in budding yeast; Blankley RT et al.; The Rad9 protein is a key adaptor protein in Saccharomyces cerevisiae DNA damage checkpoint pathways . Its adaptor function is to link the activity of the Mec1 kinase to the activation of two parallel signalling pathways dependent on the Rad53 and Chk1 kinases . The mechanisms by which Rad9 interacts with, and activates, Rad53 are well understood . However, little was known about how Rad9 facilitates the activation of Chk1 . We show here that the N-terminus of Rad9 is specifically important for phosphorylation and activation of the Chk1 kinase but not for the phosphorylation and activation of the Rad53 kinase . The Chk1 activation domain (CAD) of Rad9 is specifically important for signalling cell-cycle arrest after cdc13-1- and yku70Delta-induced telomere damage but not for tolerating ultraviolet-induced damage or inhibiting nuclease activity at telomeres . This work extends data showing that separable domains within the Rad9 adaptor protein allow it to activate two distinct kinase signalling pathways independently of each other.

Biotechnol Bioeng, 2003 Dec 30, 84(7), 864 - 86
Theoretical and computational studies of the glucose signaling pathways in yeast using global gene expression data; Lin X et al.; We have combined DNA microarray experiments with novel computational methods as a means of defining the topology of a biological signal transduction pathway . By DNA microarray techniques, we previously acquired data on expression over time of all genes in the yeast Saccharomyces following addition of glucose to wild-type cells and to cells mutated in one or more components of the Ras signaling network . In addition, we examined the time course of expression following activation of components of the Ras signaling network in the absence of glucose addition . In this current study, we have applied a novel theoretical and computational framework to these data to identify the network topology of the glucose signaling pathway in yeast and the role of Ras components in that network . The computational approach involves clustering genes by expression pattern, postulating a signaling network topology superstructure that includes all possible component interconnections and then evaluating the feasibility of the superstructure interconnections by optimization methods using Mixed Integer Linear Programming techniques . This approach is the first rigorous mathematical framework for addressing the biological network topology issue, and the novel formulation features the introduction of discrete variables for the connectivity and logical expressions that connect the experimental observations to the network structure . This analysis yields a topology for the glucose signaling pathway that is consistent with, and an extension of, known biological interactions in glucose signaling .

J Biochem Mol Toxicol, 2003, 17(6), 316 - 23
Stress response of yeast candida intermedia to Cr(VI); Jamnik P et al.; Stress response of yeast Candida intermedia ZIM 156 exposed to chromium(VI) was investigated . Yeast cells were treated with Cr(VI) in concentrations of 50, 100, 300 and 500 microM in the mid-exponential growth phase . Monitoring of some bioprocess parameters during growth, specifically pO(2), showed that Cr(VI) addition, specifically in concentration of 100 and partially 50 micromol/L, increased metabolism intensity, which is connected to induced stress responses . Furthermore, oxidation of 2',7'-dichlorofluorescin indicated increased intracellular oxidant level, specifically at 100 microM Cr(VI) concentration . Antioxidant defense systems were further investigated . Catalase and superoxide dismutase activity was not increased in the cells exposed to the both Cr(VI) concentrations, which indicate that catalase and superoxide dismutase do not participate in cell defense systems . In contrast intracellular glutathione content in reduced form increased significantly in the cells exposed to 100 micromol Cr(VI)/L . Therefore, we demonstrated that glutathione plays an important role in the stress response of C . intermedia to Cr(VI) .

J Immunol, 2004 Jan 15, 172(2), 1157 - 62
The role of SIGNR1 and the beta-glucan receptor (dectin-1) in the nonopsonic recognition of yeast by specific macrophages; Taylor PR et al.; We recently demonstrated that the beta-glucan receptor Dectin-1 (betaGR) was the major nonopsonic beta-glucan receptor on macrophages (Mphi) for the yeast-derived particle zymosan . However, on resident peritoneal Mphi, we identified an additional mannan-inhibitable receptor for zymosan that was distinct from the Mphi mannose receptor (MR) . In this study, we have studied the mannose-binding potential of murine Mphi and identified the dendritic cell-specific ICAM-3-grabbing nonintegrin homolog, SIGN-related 1 (SIGNR1), as a major MR on murine resident peritoneal Mphi . Both SIGNR1 and betaGR cooperated in the nonopsonic recognition of zymosan by these Mphi . When SIGNR1 was introduced into NIH3T3 fibroblasts or RAW 264.7 Mphi, it conferred marked zymosan-binding potential on these cells . However, in the nonprofessional phagocytes (NIH3T3), SIGNR1 was found to be poorly phagocytic, suggesting that other receptors such as betaGR may play a more dominant role in particle internalization on professional phagocytes . Binding of zymosan to RAW 264.7 Mphi expressing SIGNR1 resulted in TNF-alpha production . Treatment of RAW 264.7 Mphi expressing SIGNR1, which express low levels of betaGR, with beta-glucans had little effect on binding or TNF-alpha production, indicating that there was no absolute requirement for betaGR in this process . These studies have identified SIGNR1 as a major MR for fungal and other pathogens present on specific subsets of Mphi.

FEBS Lett, 2004 Jan 2, 556(1-3), 265 - 70
Critical residues for RNA discrimination of the histone hairpin binding protein (HBP) investigated by the yeast three-hybrid system; Jaeger S et al.; The histone hairpin binding protein (HBP, also called SLBP, which stands for stem-loop binding protein) binds specifically to a highly conserved hairpin structure located in the 3' UTR of the cell-cycle-dependent histone mRNAs . HBP consists of a minimal central RNA binding domain (RBD) flanked by an N- and C-terminal domain . The yeast three-hybrid system has been used to investigate the critical residues of the human HBP involved in the binding of its target hairpin structure . By means of negative selections followed by positive selections, we isolated mutant HBP species . Our results indicate tight relationships between the RBD and the N- and C-terminal domains.

FEBS Lett, 2004 Jan 2, 556(1-3), 7 - 12
Results and prospects of the yeast three-hybrid system; Jaeger S et al.; In 1996, a new method, termed the yeast three-hybrid system, dedicated to selection of RNA binding proteins using a hybrid RNA molecule as bait was described . In this minireview, we summarize the results that have been obtained using this method . Indeed, approximately 20 unknown proteins have been characterized so far . The three-hybrid strategy has also been used as a tool to dissect RNA-protein interactions . The example of such a study on human histone HBP interaction with its target mRNA is described . Problems that can be encountered are addressed in a troubleshooting section . Especially, our results with tRNA binding proteins are discussed.

Biochem Biophys Res Commun, 2004 Jan 23, 313(4), 1058 - 64
Identification of novel initiation sites for human DNA replication around ARSH1, a previously characterized yeast replicator; Hu L et al.; Replication of mammalian chromosomes depends on the activation of a large number of origins of DNA replication distributed along the chromosomes . We have focused our attention on a human DNA region, named ARSH1, localized to chromosome 2, that had been previously shown to act as an episomal origin in the yeast Saccharomyces cerevisiae . In the present study we have used a nascent strand DNA abundance assay to map initiation sites for DNA replication in in vivo human chromosomes around a 5 kb region encompassing ARSH1 . This analysis applied to a 1-1.4 kb nascent DNA strand fraction isolated from normal skin fibroblasts revealed the presence of two major initiations sites surrounding the ARSH1 region . With an equivalent DNA fraction obtained from HeLa cells, in addition to these sites, a broad initiation profile was observed which included the ARSH1 region . This DNA region however was not sufficient to support episomal replication of an ARSH1-containing plasmid transfected into HeLa cells.

Biochem Biophys Res Commun, 2004 Jan 23, 313(4), 922 - 5
A yeast one-hybrid system to detect methylation-dependent DNA-protein interactions; Feng SY et al.; We developed a method for site-selective CpG methylation of the budding yeast genome . The method recruits LexA-fused M.SssI DNA methyltransferase to LexA operator sequences integrated adjacent to the target site . Microarray analysis of methylated DNAs indicated that the tethered enzyme selectively methylates the region around the target site . Exploiting this method to methylate bait DNA in the one-hybrid system, we demonstrated methylation-dependent DNA binding of methyl-CpG binding proteins, MBD1 and Kaiso, in vivo . This methylation-dependent one-hybrid system would provide a versatile tool for the search and analysis of proteins that recognize methylated DNA to participate in epigenetic regulation.

Nucleic Acids Res, 2004 Jan 02, 32(1), 93 - 101 Print 2004.
Yeast telomerase is capable of limited repeat addition processivity; Bosoy D et al.; Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of telomere terminal repeats . Telomerase-mediated sequence addition is dictated by a short 'template' region of the RNA component . Despite the short template segment, telomerases from many organisms have been shown to mediate the synthesis of long extension products . This synthesis presumably depends on two types of translocation events: simultaneous translocation of the RNA-DNA duplex relative to the active site after each nucleotide incorporation (type I or nucleotide addition processivity), and translocation of the RNA relative to the DNA product after each round of repeat synthesis (type II or repeat addition processivity) . In contrast, telomerases from yeasts have been shown to synthesize mostly short products, implying a defect in one or both types of translocation . In this report, we analyzed the processivity of yeast telomerase in vitro, and identified two position-specific elongation barriers within the 5' region of the RNA template that can account for the synthesis of incomplete first round products . These barriers respond differently to variations in nucleotide concentration, primer sequence and mutations in the catalytic protein subunit, consistent with their having distinct mechanistic bases . In addition, by using optimal primers and high concentrations of dGTP, we were able to detect significant type II translocation by the yeast enzyme . Thus, the difference between the elongation property of yeast and other telomerases appears to be quantitative rather than qualitative . Our results suggest that yeast may be a useful system for investigating the physiologic significance of repeat addition processivity.

Genetics, 2003 Dec, 165(4), 1675 - 85
Destabilizing interactions among {PSI(+)} and {PIN(+)} yeast prion variants; Bradley ME et al.; The yeast Sup35 and Rnq1 proteins can exist in either the noninfectious soluble forms, {psi-} or {pin-}, respectively, or the multiple infectious amyloid-like forms called {PSI+} or {PIN+} prion variants (or prion strains) . It was previously shown that {PSI+} and {PIN+} prions enhance one another's de novo appearance . Here we show that specific prion variants of {PSI+} and {PIN+} disrupt each other's stable inheritance . Acquiring {PSI+} often impedes the inheritance of particular {PIN+} variants . Conversely, the presence of some {PIN+} variants impairs the inheritance of weak {PSI+} but not strong {PSI+} variants . These same {PIN+} variants generate a single-dot fluorescence pattern when a fusion of Rnq1 and green fluorescent protein is expressed . Another {PIN+} variant, which forms a distinctly different multiple-dot fluorescence pattern, does not impair {PSI+} inheritance . Thus, destabilization of prions by heterologous prions depends upon the variants involved . These findings may have implications for understanding interactions among other amyloid-forming proteins, including those associated with certain human diseases.

J Biol Chem, 2004 Mar 19, 279(12), 11027 - 34 Epub 2003 Dec 31.
Reciprocal regulation between Slt2 MAPK and isoforms of Msg5 dual-specificity protein phosphatase modulates the yeast cell integrity pathway; Flandez M et al.; Dual-specificity protein phosphatases (DSPs) are involved in the negative regulation of mitogen-activated protein kinases (MAPKs) by dephosphorylating both threonine- and tyrosine-conserved residues located at the activation loop . Here we show that Msg5 DSP activity is essential for maintaining a low level of signaling through the cell integrity pathway in Saccharomyces cerevisiae . Consistent with a role of this phosphatase on cell wall physiology, cells lacking Msg5 displayed an increased sensitivity to the cell wall-interfering compound Congo Red . We have observed that the N-terminal non-catalytic region of this phosphatase was responsible for binding to the kinase domain of Slt2, the MAPK that operates in this pathway . In vivo and in vitro experiments revealed that both proteins act on each other . Msg5 bound and dephosphorylated activated Slt2 . Reciprocally, Slt2 phosphorylated Msg5 as a consequence of the activation of the cell integrity pathway . In addition, alternative use of translation initiation sites at MSG5 resulted in two protein forms that are functional on Slt2 and became equally phosphorylated following activation of this MAPK . Under activating conditions, a decrease in the affinity between Msg5 and Slt2 was observed, leading us to suggest that the mechanism by which Slt2 controls the action of Msg5 was via the modulation of protein-protein interactions . Our results indicate the existence of posttranscriptional mechanisms of regulation of DSPs in yeast and provide new insights into the negative control of the cell integrity pathway.

Mol Cell Biol, 2004 Jan, 24(2), 837 - 45
Telomere cap components influence the rate of senescence in telomerase-deficient yeast cells; Enomoto S et al.; Cells lacking telomerase undergo senescence, a progressive reduction in cell division that involves a cell cycle delay and culminates in "crisis," a period when most cells become inviable . In telomerase-deficient Saccharomyces cerevisiae cells lacking components of the nonsense-mediated mRNA decay (NMD) pathway (Upf1,Upf2, or Upf3 proteins), senescence is delayed, with crisis occurring approximately 10 to 25 population doublings later than in Upf+ cells . Delayed senescence is seen in upfDelta cells lacking the telomerase holoenzyme components Est2p and TLC1 RNA, as well as in cells lacking the telomerase regulators Est1p and Est3p . The delay of senescence in upfDelta cells is not due to an increased rate of survivor formation . Rather, it is caused by alterations in the telomere cap, composed of Cdc13p, Stn1p, and Ten1p . In upfDelta mutants, STN1 and TEN1 levels are increased . Increasing the levels of Stn1p and Ten1p in Upf+ cells is sufficient to delay senescence . In addition, cdc13-2 mutants exhibit delayed senescence rates similar to those of upfDelta cells . Thus, changes in the telomere cap structure are sufficient to affect the rate of senescence in the absence of telomerase . Furthermore, the NMD pathway affects the rate of senescence in telomerase-deficient cells by altering the stoichiometry of telomere cap components.

Mol Cell Biol, 2004 Jan, 24(2), 774 - 86
The origin recognition complex and Sir4 protein recruit Sir1p to yeast silent chromatin through independent interactions requiring a common Sir1p domain; Bose ME et al.; Sir1p is one of four SIR (silent information regulator) proteins required for silencing the cryptic mating-type locus HMRa in the budding yeast Saccharomyces cerevisiae . A Sir1p interaction with Orc1p, the largest subunit of the origin recognition complex (ORC), is critical for Sir1p's ability to bind HMRa and function in the formation of silent chromatin . Here we show that a discrete domain within Sir1p, the ORC interaction region (OIR), was necessary and sufficient for a Sir1p-ORC interaction . The OIR contains the originally defined silencer recognition-defective region as well as additional amino acids . In addition, a Sir1p-Sir4p interaction required a larger region of Sir1p that included the OIR . Amino acid substitutions causing defects in either a Sir1p-Orc1p or a Sir1p-Sir4p interaction reduced HMRa silencing and Sir1p binding to HMRa in chromatin . These data support a model in which Sir1p's association with HMRa is mediated by separable Sir1p-ORC and Sir1p-Sir4p interactions requiring a common Sir1p domain, and they indicate that a Sir1p-ORC interaction is restricted to silencers, at least in part, through interactions with Sir4p.

J Biol Chem, 2004 Mar 19, 279(12), 11593 - 9 Epub 2003 Dec 29.
The yeast Pdr15p ATP-binding cassette (ABC) protein is a general stress response factor implicated in cellular detoxification; Wolfger H et al.; ATP-binding cassette (ABC) transporters play important roles in drug efflux, but some may also function in cellular detoxification . The Pdr15p ABC protein is the closest homologue of the multidrug efflux transporter Pdr5p, which mediates pleiotropic drug resistance to hundreds of unrelated compounds . In this study, we show that the plasma membrane protein Pdr15p displays limited drug transport capacity, mediating chloramphenicol and detergent tolerance . Interestingly, Pdr15p becomes most abundant when cells exit the exponential growth phase, whereas its closest homologue, Pdr5p, disappears after exponential growth . Furthermore, in contrast to Pdr5p, Pdr15p is strongly induced by various stress conditions including heat shock, low pH, weak acids, or high osmolarity . PDR15 induction bypasses the Pdr1p/Pdr3p regulators but requires the general stress regulator Msn2p, which directly decorates the stress response elements in the PDR15 promoter . Remarkably, however, Pdr15p induction bypasses upstream components of the high osmolarity glycerol (HOG) pathway including the Hog1p and Pbs2p kinases as well as the dedicated HOG cell surface sensors . Our data provide evidence for a novel upstream branch of the general stress response pathway activating Msn2p . In addition, the results demonstrate a cross-talk between stress response and the pleiotropic drug resistance network.

Int J Radiat Biol, 2003 Nov, 79(11), 919 - 24
Inhibition of UV-induced G1 arrest by exposure to 50 Hz magnetic fields in repair-proficient and -deficient yeast strains; Takashima Y et al.; PURPOSE: To assess the possibility that extremely low frequency (ELF) magnetic fields obstruct the damage repair process, the gene conversion frequency and cell cycle kinetics in a DNA repair-proficient and nucleotide excision repair (NER)-deficient strain of diploid yeast Saccharomyces cerevisiae . MATERIALS AND METHODS: DNA repair- or NER-deficient cells were irradiated with sublethal doses of ultraviolet light (UV) radiation followed by exposure to 50 Hz magnetic fields up to 30 mT for 48 h . After exposure, colony-forming ability was scored as revertants in which gene conversion had restored the functional allele of the ARG4 gene conversion hotspot . Cell cycle analysis was performed using flow cytometry . RESULTS: Gene conversion rate was increased by the combined exposure in DNA repair-proficient cells, whereas it remained unchanged between UV alone and the combined exposure in NER-deficient cells . The UV-induced G1 arrest was inhibited by exposure to 30 mT ELF magnetic fields in both repair-proficient and -deficient cells . CONCLUSIONS: The results suggest that exposure to high-density (30 mT) ELF magnetic fields decreases the efficiency of NER by suppressing G1 arrest, which in turn led to enhancement of the UV-induced gene conversion.

J Steroid Biochem Mol Biol, 2003 Dec, 87(4-5), 223 - 31
Use of a modified yeast one-hybrid screen to identify BAF60a interactions with the Vitamin D receptor heterodimer; Koszewski NJ et al.; A modified yeast one-hybrid screen was used to isolate proteins capable of interacting with the Vitamin D receptor (VDR) heterodimer complex while driving expression from a repressor Vitamin D response element (VDRE) . Four of nine independent colonies recovered in the screen coded for full-length BAF60a, a component of the mammalian SWI/SNF complex . Deletion studies in yeast were unable to localize a unique region of BAF60a responsible for interaction with the heterodimer complex, as only the full-length protein would support reporter gene expression . Pull-down analyses revealed that BAF60a displayed strong interactions with either the unliganded or liganded heterodimer complex, but neither individual receptor component alone . Transient transfection analysis in opossum kidney (OK) cells indicated that BAF60a decreased basal transcriptional activity from the negative VDRE, but had no effect on hormone-induced repression . Transcriptional activity from an enhancer VDRE also exhibited decreased basal transcriptional activity, but also augmented hormone-dependent enhancer activity, resulting in an overall increased sensitivity to hormone . In summary, BAF60a has been identified as a factor that specifically interacts with the VDR heterodimer complex using a modified yeast one-hybrid selection strategy . This suggests that BAF60a may be a link between mammalian SWI/SNF-like chromatin remodeling complexes and the VDR heterodimer.

Cryobiology, 2003 Dec, 47(3), 236 - 41
The role of glutathione in yeast dehydration tolerance; Espindola Ade S et al.; Among the factors that affect cell resistance against dehydration, oxidation is considered to be of great importance . In this work, we verified that both control and glutathione deficient mutant strains were much more oxidized after dehydration . Moreover, cells lacking glutathione showed a twofold higher increase in oxidation and lipid peroxidation than the control strain . While glucose 6-phosphate dehydrogenase and glutathione reductase activities did not change in response to dehydration in the control strain, the mutant strain gsh1 (glutathione deficient) showed a reduction of 50% in both activities, which could explain the high levels of oxidation shown by gsh1 cells . In conformity with these results, the mutant lacking GSH1 showed a high sensitivity to dehydration . Furthermore, the addition of glutathione to gsh1 cells restored survival rates to the levels of the control strain . We conclude that glutathione plays a significant role in the maintenance of intracellular redox balance during dehydration.

J Colloid Interface Sci, 2004 Feb 15, 270(2), 304 - 8
Adsorption of egg albumin onto methylated yeast biomass; Seki H et al.; A new biosorbent, methylated yeast (MeYE), was prepared for the adsorptive separation of proteins from aqueous solutions . Yeast was methylated in a 0.1 M HCl methyl alcohol solution at room temperature . About 80% of the carboxylic groups of yeast could be methylated within 9 h . The adsorption of egg albumin onto MeYE was studied to evaluate the protein adsorption ability of MeYE . At near neutral pH, egg albumin was scarcely adsorbed onto unmethylated yeast and the adsorbed amount of egg albumin increased with increasing methylation degree . The amount of egg albumin adsorbed onto MeYE increased with increasing pH from 4 to 7 and steeply decreased above pH 7 . The Langmuir isotherm was applied to determine the apparent adsorption constant and the saturated adsorbed amount of egg albumin on MeYE . Both the apparent adsorption constant and the saturated adsorbed amount increased with the degree of methylation . The saturated adsorbed amount of egg albumin onto MeYE having methylation degree 77% was 8.41 x 10(-6) mol g(-1) or 0.378 gg(-1) at near neutral pH.

Gene, 2004 Jan 21, 325, 187 - 200
Development of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein; Bovee TF et al.; The aim of this study was to develop an estrogen transcription activation assay that is sensitive, fast and easy to use in the routine screening of estrogen activity in complex matrices such as agricultural products . Recombinant yeast cells were constructed that express the human estrogen receptor alpha (ER alpha) and beta-Galactosidase (beta Gal), Luciferase (Luc) or yeast Enhanced Green Fluorescence Protein (yEGFP) as a reporter protein . Compared to other yeast assays, these new cells contain both the receptor construct as well as the reporter construct stably integrated in the genome with only one copy of the reporter construct . Dose-response curves for 17beta-estradiol (E2) obtained with the beta Gal assay were similar to those reported and the calculated EC(50) of 0.2 nM was even slightly better . However, 5 days of incubation were required before the chlorophenol red product could be measured . The Luc assay was as sensitive as the beta Gal assay and gave an EC(50) of 0.2 nM, but the signals were rather low and, although the assay can be performed within 1 day, the procedure is laborious and caused variability . The yEGFP revealed an EC(50) of 0.4 nM, but compared to the beta Gal and the Luc assay, the response was much better . This yEGFP assay can be performed completely in 96 well plates within 4 h and does not need cell wall disruption nor does it need the addition of a substrate . This makes the test sensitive, rapid and convenient with high reproducibility and small variation . These qualities make that this yEGFP assay is suited to be used as a high throughput system.

Biochem Biophys Res Commun, 2004 Jan 16, 313(3), 654 - 65
Chromosome 13q12 encoded Rho GTPase activating protein suppresses growth of breast carcinoma cells, and yeast two-hybrid screen shows its interaction with several proteins; Nagaraja GM et al.; We have characterized the cDNA for a Rho GTPase activating protein (GAP) mapping to chromosome 13q12 . The cDNA was characterized by determining the complete sequence of a 4.8 kb cDNA clone that represents the 5' untranslated region (UTR), the translated region, and the 3' UTR . The protein has a sterile alpha-motif (SAM), a distinct GAP domain, and a conserved START (StAR related lipid transfer) domain . The cDNA has 5 instability motifs (ATTTA) in the 3' UTR and one motif in the translated region between GAP and START domains . The RhoGAP transcript is truncated in some breast carcinoma cell lines and it has low expression in other breast cancer cell lines as compared to a normal breast cell line . We have previously observed the absence of RhoGAP transcript in a breast tumor specimen . A GST-fusion of the RhoGAP was tested for its specificity on RhoA, Cdc42, and Rac1 . The protein was most active for RhoA . Transfection of RhoGAP into MCF7 cells significantly inhibited cell growth . The introduction of the RhoGAP construct into MDAMB231 cells that had previously been transfected with a p21 construct did not affect cell proliferation, indicating the involvement of p21 in Rho-mediated proliferation of cancer cells . NIH3T3 cells overexpressing RhoGAP showed considerable inhibition of stress fiber formation . Several cDNAs were identified as RhoGAP interactors by using the yeast two-hybrid assay system . These cDNAs correspond to SWI/SNF, alpha-tubulin, HMG CoA reductase, and TAX1 binding protein (TAX1BP1) . The interaction with HMG CoA reductase may partially explain the growth inhibition of breast carcinoma cells by statin class of cholesterol lowering drugs . The biological significance of the interacting proteins is discussed in the context of their involvement in tumorigenesis . Our results indicate that loss of RhoGAP or its altered activity suppresses the growth of breast tumor cells . The presence of various motifs in RhoGAP and its interaction with several other proteins suggest that the protein may regulate Rho signaling in multiple ways and possibly function in a Rho-independent manner.

Anal Chem, 2004 Jan 1, 76(1), 233 - 7
Enzymatic probe sonication: enhancement of protease-catalyzed hydrolysis of selenium bound to proteins in yeast; Capelo JL et al.; This paper describes the dramatic activity enhancement of two proteolytic enzymes (protease XIV and subtilisin) when treated with an ultrasonic probe and their application to total Se determination and Se speciation in biological samples . Total Se extraction from enriched yeast is performed with 10 mg of yeast plus 1 mg of protease with a sonication time of 5 s, whereas 30 s is needed for extracting selenomethionine . In both cases, aqueous media was used . This spectacular finding is important because the enzymatic procedure usually requires a long treatment period at 37 degrees C . In addition to this major advantage, no control temperature is needed and the risk of species interconversion is drastically reduced or inhibited (the same Se species were detected after different sonication times) . Moreover, the extraction is performed in water, minimizing contamination risk and without further pH adjustment . The new sample treatment proposed has been successfully applied to selenium speciation in yeast using chromatographic separation (HPLC) coupled to inductively coupled plasma-mass spectrometry.

Biofactors, 2003, 18(1-4), 229 - 35
Pleiotropic phenotypes of fission yeast defective in ubiquinone-10 production . A study from the abc1Sp (coq8Sp) mutant; Saiki R et al.; We previously constructed two Schizosaccahromyces pombe ubiquinone-10 (or Coenzyme Q10) less mutants, which are either defective for decaprenyl diphosphate synthase or p-hydroxybenzoate polyprenyl diphosphate transferase . To further confirm the roles of ubiquinone in S . pombe, we examined the phenotype of the abc1Sp (coq8Sp) mutant, which is highly speculated to be defective in ubiquinone biosynthesis . We show here that the abc1Sp defective strain did not produce UQ-10 and could not grow on minimal medium . The abc1Sp-deficient strain required supplementation with antioxidants such as cysteine or glutathione to grow on minimal medium . In support of the antioxidant function of ubiquinone, the abc1Sp-deficient strain is sensitive to H2O2 and Cu2+ . In addition, expression of the stress inducible ctt1 gene was much induced in the ubiquinone less mutant than wild type . Interestingly, we also found that the abc1-deficient strain as well as other ubiquinone less mutants produced a significant amount of H2S, which suggests that oxidation of sulfide by ubiquinone may be an important pathway for sulfur metabolism in S . pombe . Thus, analysis of the phenotypes of S . pombe ubiquinone less mutants clearly demonstrate that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system.

Glycobiology, 2004 Mar, 14(3), 243 - 51 Epub 2003 Dec 23.
Characterization of N-linked oligosaccharides assembled on secretory recombinant glucose oxidase and cell wall mannoproteins from the methylotrophic yeast Hansenula polymorpha; Kim MW et al.; Presently almost no information is available on the oligosaccharide structure of the glycoproteins secreted from the methylotrophic yeast Hansenula polymorpha, a promising host for the production of recombinant proteins . In this study, we analyze the size distribution and structure of N-linked oligosaccharides attached to the recombinant glycoprotein glucose oxidase (GOD) and the cell wall mannoproteins obtained from H . polymorpha . Oligosaccharide profiling showed that the major oligosaccharide species derived from the H . polymorpha-secreted recombinant GOD (rGOD) had core-type structures (Man(8-12)GlcNAc(2)) . Analyses using anti-alpha 1,3-mannose antibody and exoglycosidases specific for alpha 1,2- or alpha 1,6-mannose linkages revealed that the mannose outer chains of N-glycans on the rGOD have very short alpha 1,6 extensions and are mainly elongated in alpha 1,2-linkages without a terminal alpha 1,3-linked mannose addition . The N-glycans released from the H . polymorpha mannoproteins were shown to contain mostly mannose in their outer chains, which displayed almost identical size distribution and structure to those of H . polymorpha-derived rGOD . These results strongly indicate that the outer chain processing of N-glycans by H . polymorpha significantly differs from that by Saccharomyces cerevisiae, thus generating much shorter mannose outer chains devoid of terminal alpha 1,3-linked mannoses.

Gene, 2004 Jan 7, 324, 97 - 104
The European flounder (Platichthys flesus) TP53 functions as a temperature-sensitive transcription factor which inhibits cell growth in yeast; Cachot J et al.; Numerous studies focus on biological roles of the TP53 tumor suppressor gene in mammals but little is known about the actual function of TP53 in lower vertebrates . In this study, we used an in vivo functional assay in yeast to address the transactivation capacity of the flounder TP53 protein . We showed that the flounder TP53 acts as a sequence-specific transcription factor which is able to transactivate various human promoters containing a p53-responsive element (RE) . This transcriptional activity was completely abrogated in the Val147Glu TP53 mutant previously identified in two flounder hepatic hyperplasia . In addition, we showed that the wild-type (wt) flounder TP53 but not the Val147Glu mutant inhibits cell growth when expressed in yeast . We finally reported that transcription regulation and growth inhibition by the wild-type flounder TP53 is temperature-dependent . The flounder TP53 optimal temperature appeared lower than those reported for the Xenopus and human homologues.

J Proteome Res, 2003 Nov-Dec, 2(6), 643 - 9
Changes in the protein expression of yeast as a function of carbon source; Gao J et al.; Protein expression trends in yeast were monitored as a function of carbon source (glucose versus galactose) using multidimensional high performance liquid chromatography (HPLC) coupled to gas-phase fractionation, using relative intensity triggering (GPFri) . Size exclusion HPLC was used to separate whole cell lysates, and following proteolysis of these fractions, each was separated by reversed phase HPLC, which was coupled on-line via electrospray to an ion trap mass spectrometer . The GPFri technique increased the dynamic range of proteins detected by increasing the number of peptide ions subjected to low energy collision induced dissociation to the 24 most intense ions in each of the survey scans . No protein or peptide labeling was used; instead, the number of SEQUEST identifications for each peptide (previously termed "hits") were used as a semiquantitative means of assessing both the direction (increase vs decrease) and significance of change in protein abundance . None of the traditional SEQUEST filters, e.g., Xcorr, DelCn, Sp, Rsp, etc., were employed in this study . Instead, a Student's t-test was used to distinguish those proteins that significantly and reproducibly changed between carbon sources from those that did not . This relied on the SEQUEST misassignments occurring in equal proportion between treatments and thereby negating each other; statistically significant changes in SEQUEST assignments were nonrandom events by definition and therefore reflective of correct identifications . This method of data analysis showed a large degree of concordance with results reported by other groups in similar transcriptional profiling and proteomic experiments . In all, 176 and 231 (fold-change > or = 1.1; p < or = 0.05) proteins were identified as being increased in peptide hit number when the yeast cells' source of carbon was changed between glucose and galactose, respectively.

J Cell Biol, 2003 Dec 22, 163(6), 1243 - 54
Mathematical model of the morphogenesis checkpoint in budding yeast; Ciliberto A et al.; The morphogenesis checkpoint in budding yeast delays progression through the cell cycle in response to stimuli that prevent bud formation . Central to the checkpoint mechanism is Swe1 kinase: normally inactive, its activation halts cell cycle progression in G2 . We propose a molecular network for Swe1 control, based on published observations of budding yeast and analogous control signals in fission yeast . The proposed Swe1 network is merged with a model of cyclin-dependent kinase regulation, converted into a set of differential equations and studied by numerical simulation . The simulations accurately reproduce the phenotypes of a dozen checkpoint mutants . Among other predictions, the model attributes a new role to Hsl1, a kinase known to play a role in Swe1 degradation: Hsl1 must also be indirectly responsible for potent inhibition of Swe1 activity . The model supports the idea that the morphogenesis checkpoint, like other checkpoints, raises the cell size threshold for progression from one phase of the cell cycle to the next.

Nutr Cancer, 2003, 46(2), 179 - 85
Comparison of the chemopreventive efficacies of 1,4-phenylenebis(methylene)selenocyanate and selenium-enriched yeast on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induced lung tumorigenesis in A/J mouse; Das A et al.; Epidemiological studies, clinical intervention trials (including the trial with selenium-enriched yeast by Clark et al . JAMA 276, 1957, 1996) and assays in laboratory animals provide evidence for a protective role of selenium against the development of several cancers, including lung cancer . We have demonstrated that selenium in the form of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) is a promising chemopreventive agent in the A/J mouse lung tumor model induced with the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK); under identical conditions, selenomethionine (SM), a component of selenium-enriched yeast, had no effect . The lack of an effect of SM suggests that other forms of selenium, or selenium-enriched yeast as a whole, are essential for lung cancer prevention; moreover, various species may respond differently to a given form of selenium . Therefore, in this study, we compared the chemopreventive efficacies of p-XSC with selenium-enriched yeast . Groups of 5-wk-old mice were fed either control diet or experimental diet containing p-XSC (5 or 10 ppm as selenium, equivalent to 20% and 40% maximum tolerated dose {MTD}, respectively) or selenium-enriched yeast (5 or 10 ppm) . Beginning at Wk 7, each mouse received NNK (3 mmol) in 0.1 ml cottonseed oil by intragastric intubation, once weekly for 8 wk . Twenty-six weeks after the first NNK administration, mice were killed and tumors in lung and forestomach were counted . p-XSC at 5 and 10 ppm doses significantly reduced lung tumor induction by NNK from 10.4 -/+ 6.0 (multiplicity) to 2.7 -/+ 1.5 (P < 0.001) and 1.8 -/+ 2.0 (P < 0.0001) respectively, whereas selenium-enriched yeast had no effect . p-XSC at 10 ppm also significantly reduced the incidence level from 96% to 68% (P < 0.01) . The amounts of selenium that reach the target organ (lung) after dietary administration of p-XSC (326 -/+ 69 ng Se/g lung tissue) were significantly higher than that from selenium-enriched yeast (34 -/+ 8.5 ng Se/g lung tissue) . However, the levels of selenium in plasma from selenium-enriched yeast (620 -/+ 54 ng Se/g plasma) were twofold higher than those from p-XSC (355 -/+ 85 ng Se/g plasma) . In biochemical studies, p-XSC was shown to significantly inhibit formation of O6-methylguanine (O6-MG) and 7-methylguanine (7-MG) in the lungs and livers of mice treated with NNK . The lack of effect of selenium-enriched yeast on these lesions agrees with the results of the bioassay . Collectively, the results of this study clearly indicate that as a chemopreventive agent, p-XSC is superior to selenium-enriched yeast under the conditions of the present protocol . The inhibition of DNA methylation and the significantly higher retention of selenium from p-XSC as compared with selenium-enriched yeast in the target organ may in part account for the inhibition of lung tumorigenesis.

Anal Biochem, 2004 Jan 15, 324(2), 258 - 68
Ca2+ transport in mitochondria from yeast expressing recombinant aequorin; Jung DW et al.; We have expressed aequorin in mitochondria of the yeast Saccharomyces cerevisiae and characterized the resulting strain with respect to mitochondrial Ca(2+) transport in vivo and in vitro . When intact cells are suspended in water containing 1.4 mM ethanol and 14 mM CaCl(2), the matrix free Ca(2+) concentration is 200 nM, similar to the values expected in cytoplasm . Addition of ionophore ETH 129 allows an active accumulation of Ca(2+) and promptly increases the value to 1.2 microM . Elevated Ca(2+) concentrations are maintained for periods of 6 min or longer under these conditions . Isolated yeast mitochondria oxidizing ethanol also accumulate Ca(2+) when ETH 129 is present, but the cation is not retained depending on the medium conditions . This finding confirms the presence of a Ca(2+) release mechanism that requires free fatty acids as previously described {P.C . Bradshaw et al . (2001) J . Biol . Chem . 276, 40502-40509} . When a respiratory substrate is not present, Ca(2+) enters and leaves yeast mitochondria slowly, at a specific activity near 0.2 nmol/min/mg protein . Transport under these conditions equilibrates the internal and external concentrations of Ca(2+) and is not affected by ruthenium red, uncouplers, or ionophores that perturb transmembrane gradients of charge and pH . This activity displays sigmoid kinetics and a K(1/2) value for Ca(2+) that is near to 900 nM, in the absence of ethanol or when it is present . It is furthermore shown that the activity coefficient of Ca(2+) in yeast mitochondria is a function of the matrix Ca(2+) content and is substantially larger than that in mammalian mitochondria . Characteristics of the aequorin-expressing strain appear suitable for its use in expression-based methods directed at cloning Ca(2+) transporters from mammalian mitochondria and for further examining the interrelationships between mitochondrial and cytoplasmic Ca(2+) in yeast.

Mol Cell, 2003 Dec, 12(6), 1439 - 52
Genome-wide analysis of mRNAs regulated by the nonsense-mediated and 5' to 3' mRNA decay pathways in yeast; He F et al.; Transcripts regulated by the yeast nonsense-mediated and 5' to 3' mRNA decay pathways were identified by expression profiling of wild-type, upf1Delta, nmd2Delta, upf3Delta, dcp1Delta, and xrn1Delta cells . This analysis revealed that inactivation of Upf1p, Nmd2p, or Upf3p has identical effects on global RNA accumulation; inactivation of Dcp1p or Xrn1p exhibits both common and unique effects on global RNA accumulation but causes upregulation of only a small fraction of transcripts; and the majority of transcripts upregulated in upf/nmd strains are also upregulated to similar extents in dcp1Delta and xrn1Delta strains . Our results define the core transcripts regulated by NMD, identify several novel structural classes of NMD substrates, demonstrate that nonsense-containing mRNAs are primarily degraded by the 5' to 3' decay pathway even in the absence of functional NMD, and indicate that 3' to 5' decay, not 5' to 3' decay, may be the major mRNA decay activity in yeast cells.

Mol Cell, 2003 Dec, 12(6), 1353 - 65
Assigning function to yeast proteins by integration of technologies; Hazbun TR et al.; Interpreting genome sequences requires the functional analysis of thousands of predicted proteins, many of which are uncharacterized and without obvious homologs . To assess whether the roles of large sets of uncharacterized genes can be assigned by targeted application of a suite of technologies, we used four complementary protein-based methods to analyze a set of 100 uncharacterized but essential open reading frames (ORFs) of the yeast Saccharomyces cerevisiae . These proteins were subjected to affinity purification and mass spectrometry analysis to identify copurifying proteins, two-hybrid analysis to identify interacting proteins, fluorescence microscopy to localize the proteins, and structure prediction methodology to predict structural domains or identify remote homologies . Integration of the data assigned function to 48 ORFs using at least two of the Gene Ontology (GO) categories of biological process, molecular function, and cellular component; 77 ORFs were annotated by at least one method . This combination of technologies, coupled with annotation using GO, is a powerful approach to classifying genes.

Biochem J, 2004 Apr 1, 379(Pt 1), 65 - 70
Pyridoxine biosynthesis in yeast: participation of ribose 5-phosphate ketol-isomerase; Kondo H et al.; To identify the genes involved in pyridoxine synthesis in yeast, auxotrophic mutants were prepared . After transformation with a yeast genomic library, a transformant (A22t1) was obtained from one of the auxotrophs, A22, which lost the pyridoxine auxotrophy . From an analysis of the plasmid harboured in A22t1, the RKI1 gene coding for ribose 5-phosphate ketol-isomerase and residing on chromosome no . 15 was identified as the responsible gene . This notion was confirmed by gene disruption and tetrad analysis on a diploid prepared from the wild-type and the auxotroph . The site of mutation on the RKI1 gene was identified as position 566 with a transition from guanine to adenine, resulting in amino acid substitution of Arg-189 with lysine . The enzymic activity of the Arg189-->Lys (R189K) mutant of ribose 5-phosphate ketolisomerase was 0.6% when compared with the wild-type enzyme . Loss of the structural integrity of the protein seems to be responsible for the greatly diminished activity, which eventually leads to a shortage of either ribose 5-phosphate or ribulose 5-phosphate as the starting or intermediary material for pyridoxine synthesis.

Appl Microbiol Biotechnol, 2004 May, 64(4), 481 - 5 Epub 2003 Dec 20.
Enantioselective transesterification using lipase-displaying yeast whole-cell biocatalyst; Matsumoto T et al.; An enantioselective transesterification in non-aqueous organic solvent was developed by utilizing a lipase-displaying yeast whole cell biocatalyst constructed in our previous study . As a model reaction, optical resolution of (RS)-1-phenylethanol, which serves as one of chiral building blocks, was carried out by enantioselective transesterification with vinyl acetate . Recombinant Rhizopus oryzae lipase displayed on the yeast cell surface retained its activity in hexane, heptane, cyclohexane and octane . The effective amount of whole-cell biocatalyst in the reaction mixture was 10 mg/ml solvent . In a reaction mixture incubated for 36 h with molecular sieves 4A, the concentration of (R)-1-phenylethyl acetate reached 39.8 mM (97.3% yield) with high enantiomeric excess (93.3%ee) . In contrast, a reaction mixture incubated without molecular sieves 4A produced little (R)- and (S)-1-phenylethyl acetate . The results obtained in this study demonstrate the applicability of the lipase-displaying yeast whole cell biocatalyst to bioconversion processes in non-aqueous organic solvents .

Nat Cell Biol, 2004 Jan, 6(1), 59 - 66 Epub 2003 Dec 14.
Late-G1 cyclin-CDK activity is essential for control of cell morphogenesis in budding yeast; Moffat J et al.; The accurate spatial and temporal coordination of cell polarization with DNA replication and segregation guarantees the fidelity of genetic transmission . Here we report that in Saccharomyces cerevisiae, a build-up or burst of G1 cyclin-dependent kinase (CDK) activity through activation of the cyclin genes CLN1,2 and PCL1,2 is essential for cell morphogenesis, but not for other events associated with the G1-S-phase transition, including DNA replication . Strains lacking a burst of late-G1 cyclin-CDK activity (LG1C(-)) undergo a catastrophic morphogenesis and halt the nuclear cycle at the morphogenesis checkpoint in G2 phase . Consistent with a role in morphogenesis, the Pho85 G1 cyclins Pcl1 and Pcl2 show a unique pattern of localization to sites of polarized cell growth, and strains lacking PCL1 and PCL2 show genetic interactions with the cell polarity GTPase Cdc42, its regulators and downstream effectors . Our data suggest that inability to assemble a septin ring and localize the GTP exchange factor Cdc24 at the incipient bud site may be the primary morphogenetic defects in LG1C-depleted cells . We conclude that a burst of late G1 cyclin-CDK activity is essential for establishing cell polarity and development of the cleavage apparatus.

Virus Res, 2004 Jan, 99(1), 69 - 80
Simian retrovirus serogroup 2 constitutive transport element recognizes the ribosomal L10-like protein and translocon gamma subunit-like protein in a yeast three-hybrid assay; Li B et al.; The simian retrovirus (SRV) serogroup 2 genome contains a constitutive transport element (CTE) within its 3' intergenic region (IR) that mediates the nuclear export of unspliced SRV RNA . In a previous report {Virology 264 (1999) 37}, CTE RNA-protein complexes were detected using UV-crosslinking/sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) . To identify these CTE-interacting cellular proteins, we utilized yeast three-hybrid interaction approaches using the complete IR as bait, modified to eliminate transcriptional termination signals recognized by RNA polymerase III, and identified several interactive clones from a Hela cell cDNA activation domain (AD) library . UV-crosslinking of RNA-protein complexes, using Hela cell extracts and the modified IR bait, were conducted prior to library screening, to verify appropriate interaction of CTE RNA-protein complexes . Over one million recombinants were screened, and our yeast hybrid results indicate that the CTE interacts with several molecules involved in cellular translational and translocation machinery, including the ribosomal L10-like protein and the tranlocon protein gamma subunit-like protein . UV-crosslinking/immunoblot assays have verified the interaction of the CTE region with molecules immunologically reactive to antibodies recognizing the ribosomal L10-like protein.

EMBO J, 2004 Jan 14, 23(1), 127 - 37 Epub 2003 Dec 18.
Roles of SWI/SNF and HATs throughout the dynamic transcription of a yeast glucose-repressible gene; Geng F et al.; Eucaryotic gene expression requires chromatin-remodeling activities . We show by time-course studies that transcriptional induction of the yeast glucose-regulated SUC2 gene is rapid and shows a striking biphasic pattern, the first phase of which is partly mediated by the general stress transcription factors Msn2p/Msn4p . The SWI/SNF ATP-dependent chromatin-remodeling complex associates with the promoter in a similar biphasic manner and is essential for both phases of transcription . Two different histone acetyltransferases, Gcn5p and Esa1p, enhance the binding of SWI/SNF to the promoter during early transcription and are required for optimal SUC2 induction . Gcn5p is recruited to SUC2 simultaneously with SWI/SNF, whereas Esa1p associates constitutively with the promoter . This study reveals an unusual transcription pattern of a metabolic gene and suggests a novel strategy by which distinct chromatin remodelers cooperate for the dynamic activation of transcription.

J Biol Chem, 2004 Mar 19, 279(12), 11293 - 303 Epub 2003 Dec 18.
Identification of phosphorylation sites on the yeast ribonucleotide reductase inhibitor Sml1; Uchiki T et al.; Sml1 is a small protein in Saccharomyces cerevisiae which inhibits the activity of ribonucleotide reductase (RNR) . RNR catalyzes the rate-limiting step of de novo dNTP synthesis . Sml1 is a downstream effector of the Mec1/Rad53 cell cycle checkpoint pathway . The phosphorylation by Dun1 kinase during S phase or in response to DNA damage leads to diminished levels of Sml1 . Removal of Sml1 increases the population of active RNR, which raises cellular dNTP levels . In this study using mass spectrometry and site-directed mutagenesis, we have identified the region of Sml1 phosphorylation to be between residues 52 and 64 containing the sequence GSSASASASSLEM . This is the first identification of a phosphorylation sequence of a Dun1 biological substrate . This sequence is quite different from the consensus Dun1 phosphorylation sequence reported previously from peptide library studies . The specific phosphoserines were identified to be Ser(56), Ser(58), and Ser(60) by chemical modification of these residues to S-ethylcysteines followed by collision activated dissociation . To investigate further Sml1 phosphorylation, we constructed the single mutants S56A, S58A, S60A, and the triple mutant S56A/S58A/S60A and compared their degrees of phosphorylation with that of wild type Sml1 . We observed a 90% decrease in the relative phosphorylation of S60A compared with that of wild type, a 25% decrease in S58A, and little or no decrease in the S56A mutant . There was no observed phosphate incorporation in the triple mutant, suggesting that Ser(56), Ser(58), and Ser(60) in Sml1 are the sites of phosphorylation . Further mutagenesis studies reveal that Dun1 kinase requires an acidic residue at the +3 position, and there is cooperativity between the phosphorylation sites . These results show that Dun1 has a unique phosphorylation motif.

Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D459 - 62
MitoP2, an integrated database on mitochondrial proteins in yeast and man; Andreoli C et al.; The aim of the MitoP2 database is to provide a comprehensive list of mitochondrial proteins of yeast and man . Based on the current literature we created an annotated reference set of yeast and human proteins . In addition, data sets relevant to the study of the mitochondrial proteome are integrated and accessible via search tools and links . They include computational predictions of signalling sequences, and summarize results from proteome mapping, mutant screening, expression profiling, protein-protein interaction and cellular sublocalization studies . For each individual approach, specificity and sensitivity for allocating mitochondrial proteins was calculated . By providing the evidence for mitochondrial candidate proteins the MitoP2 database lends itself to the genetic characterization of human mitochondriopathies.

Protein Expr Purif, 2003 Nov, 32(1), 151 - 60
kappa-Carrageenan as a carrier in affinity precipitation of yeast alcohol dehydrogenase; Mondal K et al.; kappa-Carrageenan is a non-toxic polymer from seaweeds, which becomes reversibly insoluble upon the addition of K(+) . Its conjugate with the dye, Cibacron Blue 3GA, was used to purify alcohol dehydrogenase from crude yeast extract by affinity precipitation . Response surface methodology was used to optimize conditions for affinity precipitation of the enzyme with the polymer-dye conjugate . Recovery of 58% of the enzyme activity with 13.6-fold purification was obtained.

Biochem J, 2004 Apr 15, 379(Pt 2), 309 - 15
Ubiquinone is not required for proton conductance by uncoupling protein 1 in yeast mitochondria; Esteves TC et al.; Q (coenzyme Q or ubiquinone) is reported to be a cofactor obligatory for proton transport by UCPs (uncoupling proteins) in liposomes {Echtay, Winkler and Klingenberg (2000) Nature (London) 408, 609-613} and for increasing the binding of the activator retinoic acid to UCP1 {Tomas, Ledesma and Rial (2002) FEBS Lett . 526, 63-65} . In the present study, yeast ( Saccharomyces cerevisiae ) mutant strains lacking Q and expressing UCP1 were used to determine whether Q was required for UCP function in mitochondria . Wild-type yeast strain and two mutant strains (CENDeltaCOQ3 and CENDeltaCOQ2), both not capable of synthesizing Q, were transformed with the mouse UCP1 gene . UCP1 activity was measured as fatty acid-dependent, GDP-sensitive proton conductance in mitochondria isolated from the cells . The activity of UCP1 was similar in both Q-containing and -deficient yeast mitochondria . We conclude that Q is neither an obligatory cofactor nor an activator of proton transport by UCP1 when it is expressed in yeast mitochondria.

Radiat Res, 2004 Jan, 161(1), 56 - 63
Survival and recovery of yeast cells after combined treatment with ionizing radiation and heat; Petin VG et al.; Cell survival, recovery kinetics and inactivation forms after successive and simultaneous treatments with gamma rays (60Co) and high temperatures were studied in diploid yeast cells capable of recovery . Both the extent and the rate of the recovery were shown to be greatly decreased with increase in the duration of heat treatment (60 degrees C) followed by radiation and with increase in exposure temperature after simultaneous treatment with heat and radiation . A quantitative approach describing the recovery process was used to estimate the probability of recovery per unit time and the irreversible component of damage after the combined treatment with heat and radiation . It was shown that the probability of recovery was independent of the conditions of the treatment with heat and radiation, while the irreversible component gradually increased as a function of the duration of heat treatment (60 degrees C) after sequential treatment with heat and radiation and as a function of the exposure temperature after simultaneous treatment with heat and radiation . The increase in the irreversible component was accompanied by an increase in cell death without postirradiation division . It is concluded on this basis that the synergistic interaction of ionizing radiation and hyperthermia in yeast cells is not related to the impairment of the recovery capacity itself and that it may be attributed to an increased yield of irreversible damage.

Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15724 - 9 Epub 2003 Dec 15.
High-resolution yeast phenomics resolves different physiological features in the saline response; Warringer J et al.; We present a methodology for gene functional prediction based on extraction of physiologically relevant growth variables from all viable haploid yeast knockout mutants . This quantitative phenomics approach, here applied to saline cultivation, identified marginal but functionally important phenotypes and allowed the precise determination of time to adapt to an environmental challenge, rate of growth, and efficiency of growth . We identified approximately 500 salt-sensitive gene deletions, the majority of which were previously uncharacterized and displayed salt sensitivity for only one of the three physiological features . We also report a high correlation to protein-protein interaction data; in particular, several salt-sensitive subcellular networks indicating functional modules were revealed . In contrast, no correlation was found between gene dispensability and gene expression . It is proposed that high-resolution phenomics will be instrumental in systemwide descriptions of intragenomic functional networks.

J Cell Sci, 2004 Jan 15, 117(Pt 2), 351 - 8
Activity of recycling Golgi mannosyltransferases in the yeast endoplasmic reticulum; Karhinen L et al.; In yeast primary N- and O-glycans are attached to proteins in the endoplasmic reticulum (ER), and they are elongated in the Golgi . Thus, glycan extension by Golgi enzymes has been taken as evidence for arrival of a protein in the Golgi . Two alpha 1,6-mannosyltransferase activity-containing multiprotein complexes have been reported to recycle between the Golgi and the ER, but since resident ER proteins are not Golgi-modified, Golgi enzymes were not thought to function in the ER . Here we show that when protein exit from the ER was blocked in COPII-defective yeast mutants, the N-glycans of vacuolar carboxypeptidase Y and a set of unidentified glycoproteins were decorated with an alpha 1,6-mannose residue, normally added in the Golgi by Och1p . Immunofluorescent staining demonstrated that Och1p accumulated in the ER under these conditions . Concomitantly, primary O-glycans of a secretory protein were extended, apparently by the medial Golgi transferase Mnt1p . Similar O-glycan extension occurred in wild-type cells when an HDEL-tagged protein was allowed to encounter glycosyltransferases in the Golgi during recycling between ER and Golgi . Golgi-specific glycosylation in the ER was reduced when Golgi-to-ER traffic was blocked, confirming that glycan extension in the ER was mainly due to recycling, rather than newly synthesized transferases.

Traffic, 2004 Jan, 5(1), 45 - 52
A complete set of SNAREs in yeast; Burri L et al.; Trafficking of cargo molecules through the secretory pathway relies on packaging and delivery of membrane vesicles . These vesicles, laden with cargo, carry integral membrane proteins that can determine with which target membrane the vesicle might productively fuse . The membrane fusion process is highly conserved in all eukaryotes and the central components driving membrane fusion events involved in vesicle delivery to target membranes are a set of integral membrane proteins called SNAREs . The yeast Saccharomyces cerevisiae has served as an extremely useful model for characterizing components of membrane fusion through genetics, biochemistry and bioinformatics, and it is now likely that the complete set of SNAREs is at hand . Here, we present the details from the searches for SNAREs, summarize the domain structures of the complete set, review what is known about localization of SNAREs to discrete membranes, and highlight some of the surprises that have come from the search.

Mol Cell Biol, 2004 Jan, 24(1), 338 - 51
Activation of the RAS/cyclic AMP pathway suppresses a TOR deficiency in yeast; Schmelzle T et al.; The TOR (target of rapamycin) and RAS/cyclic AMP (cAMP) signaling pathways are the two major pathways controlling cell growth in response to nutrients in yeast . In this study we examine the functional interaction between TOR and the RAS/cAMP pathway . First, activation of the RAS/cAMP signaling pathway confers pronounced resistance to rapamycin . Second, constitutive activation of the RAS/cAMP pathway prevents several rapamycin-induced responses, such as the nuclear translocation of the transcription factor MSN2 and induction of stress genes, the accumulation of glycogen, the induction of autophagy, the down-regulation of ribosome biogenesis (ribosomal protein gene transcription and RNA polymerase I and III activity), and the down-regulation of the glucose transporter HXT1 . Third, many of these TOR-mediated responses are independent of the previously described TOR effectors TAP42 and the type 2A-related protein phosphatase SIT4 . Conversely, TOR-controlled TAP42/SIT4-dependent events are not affected by the RAS/cAMP pathway . Finally, and importantly, TOR controls the subcellular localization of both the protein kinase A catalytic subunit TPK1 and the RAS/cAMP signaling-related kinase YAK1 . Our findings suggest that TOR signals through the RAS/cAMP pathway, independently of TAP42/SIT4 . Therefore, the RAS/cAMP pathway may be a novel TOR effector branch.

Am J Clin Pathol, 2003 Dec, 120(6), 882 - 5
Spurious automated platelet count . Enumeration of yeast forms as platelets by the cell-DYN 4000; Latif S et al.; We recently encountered a patient with thrombocytopenia secondary to multiple drug therapy, disseminated prostatic adenocarcinoma, and sepsis who had a sudden decrease in his platelet count as enumerated by the Cell-DYN 4000 hematology analyzer (Abbott Diagnostics, Santa Clara, CA) . A manual platelet count performed thereafter was even lower . The etiology of the spurious platelet count was clarified when numerous yeast forms were observed on routine microscopy of the peripheral blood smear . Subsequently, these organisms were identified as Candida glabrata from a positive blood culture (BACTEC 9240, Becton Dickinson, Cockeysville, MD) . To our knowledge, this is the first report of spurious enumeration of yeast forms as platelets in an automated hematology system . The principle underlying platelet enumeration by the Cell-DYN 4000 system and other hematology analyzers and the value of microscopy on peripheral smears with unexpected CBC count results are discussed.

Biochim Biophys Acta, 2003 Dec 8, 1607(2-3), 181 - 9
Each yeast mitochondrial F1F0-ATP synthase complex contains a single copy of subunit 8; Stephens AN et al.; The stoichiometry of subunit 8 in yeast mitochondrial F(1)F(0)-ATP synthase (mtATPase) has been evaluated using an immunoprecipitation approach . Single HA or FLAG epitopes were introduced at the N-terminus of subunit 8 . Expression of each tagged subunit 8 variant in yeast cells lacking endogenous subunit 8 restored a respiratory phenotype and had little measurable effect on ATP hydrolase activity of the isolated enzyme . Moreover, the two epitope-tagged subunit 8 variants could be stably co-expressed in the same host cells and both of HA-Y8 and FLAG-Y8 could be detected in ATP synthase complexes isolated by native gel electrophoresis . Mitochondria isolated from each yeast strain were solubilized to release ATP synthase complexes in either the monomeric or dimeric forms . In each case, monoclonal antibodies directed against either the FLAG or HA epitope could immunoprecipitate intact ATP synthase complexes . When both HA-Y8 and FLAG-Y8 were co-expressed in cells, monomeric ATP synthases contained only a single subunit 8 variant after immunoprecipitation, corresponding to the particular antibody used (HA or FLAG) . By contrast, both subunit 8 variants were recovered in samples of immunoprecipitated dimeric ATP synthase complexes, irrespective of the antibody used . We conclude that each monomeric yeast mitochondrial ATP synthase complex contains a single copy of subunit 8.

Biochim Biophys Acta, 2003 Dec 8, 1607(2-3), 65 - 78
Aspartate-186 in the head group of the yeast iron-sulfur protein of the cytochrome bc1 complex contributes to the protein conformation required for efficient electron transfer; Ebert CE et al.; Two conserved charged amino acids, aspartate-186 and arginine-190, localized in the aqueous head region of the iron-sulfur protein of the cytochrome bc(1) complex of yeast mitochondria, were mutated to alanine, glutamate, or asparagine and isoleucine, respectively . The R190I mutation resulted in the complete loss of antimycin- and myxothiazol-sensitive cytochrome c reductase activity due to loss of more than 60% of the iron-sulfur protein in the complex . Mitochondria isolated from the D186A mutant had a 50% decrease in cytochrome c reductase activity but no loss of the iron-sulfur protein or the {2Fe-2S} cluster . The midpoint potential of the {2Fe-2S} cluster of the D186A mutant was decreased from 281 to 178 mV . The D186E and D186N mutations did not result in a loss of cytochrome c reductase activity or content of iron-sulfur protein; however, the redox potential of the {2Fe-2S} cluster of D186N was decreased from 281 to 241 mV . Molecular modeling/dynamics studies predicted that substituting an alanine for Asp-186 causes global structural changes in the head group of the iron-sulfur protein resulting in changes in the orientation of the {2Fe-2S} cluster and consequently a lowered redox potential . The rate of electrogenic proton pumping in the bc(1) complex isolated from mutant D186A reconstituted into proteoliposomes decreased 64%; however, the H(+)/2e(-) ratio of 1.9 was identical in the mutant and the wild-type complexes . The carboxyl binding reagent, N-(ethoxycarbonyl)-2-ethoxyl-1,2-dihydroquinoline (EEDQ) blocked electrogenic proton pumping in the bc(1) complex reconstituted into proteoliposomes without affecting electron transfer resulting in a decrease in the H(+)/2e(-) ratio to 1.2 and 1.1, respectively . EEDQ was bound to the iron-sulfur protein and core protein II in both the wild type and the D186A mutant, indicating that Asp-186 of the iron-sulfur protein is not required for proton translocation in the bc(1) complex.

Mol Biol Cell, 2004 Mar, 15(3), 1397 - 406 Epub 2003 Dec 10.
The yeast casein kinase Yck3p is palmitoylated, then sorted to the vacuolar membrane with AP-3-dependent recognition of a YXXPhi adaptin sorting signal; Sun B et al.; Our previous work found the two yeast plasma membrane-localized casein kinases Yck1p and Yck2p to be palmitoylated on C-terminal Cys-Cys sequences by the palmitoyl transferase Akr1p . The present work examines a third casein kinase, Yck3p, which ends with the C-terminal sequence Cys-Cys-Cys-Cys-Phe-Cys-Cys-Cys . Yck3p is palmitoylated and localized to the vacuolar membrane . While the C-terminal cysteines are required for this palmitoylation, Akr1p is not . Palmitoylation requires the C-terminal Yck3p residues 463-524, whereas information for vacuolar sorting maps to the 409-462 interval . Vacuolar sorting is disrupted in cis through deletion of the 409-462 sequences and in trans through mutation of the AP-3 adaptin complex; both cis- and trans-mutations result in Yck3p missorting to the plasma membrane . This missorted Yck3p restores 37 degrees C viability to yck1Delta yck2-ts cells . yck1Delta yck2-ts suppressor mutations isolated within the YCK3 gene identify the Yck3p vacuolar sorting signal-the tetrapeptide YDSI, a perfect fit to the YXXPhi adaptin-binding consensus . Although YXXPhi signals have a well-appreciated role in the adaptin-mediated sorting of mammalian cells, this is the first signal of this class to be identified in yeast.

Genetics, 2003 Nov, 165(3), 997 - 1015
Phenotypic and transcriptional plasticity directed by a yeast mitogen-activated protein kinase network; Breitkreutz A et al.; The yeast pheromone/filamentous growth MAPK pathway mediates both mating and invasive-growth responses . The interface between this MAPK module and the transcriptional machinery consists of a network of two MAPKs, Fus3 and Kss1; two regulators, Rst1 and Rst2 (a.k.a . Dig1 and Dig2); and two transcription factors, Ste12 and Tec1 . Of 16 possible combinations of gene deletions in FUS3, KSS1, RST1, and RST2 in the sigma1278 background, 10 display constitutive invasive growth . Rst1 was the primary negative regulator of invasive growth, while other components either attenuated or enhanced invasive growth, depending on the genetic context . Despite activation of the invasive response by lesions at the same level in the MAPK pathway, transcriptional profiles of different invasive mutant combinations did not exhibit a unified program of gene expression . The distal MAPK regulatory network is thus capable of generating phenotypically similar invasive-growth states (an attractor) from different molecular architectures (trajectories) that can functionally compensate for one another . This systems-level robustness may also account for the observed diversity of signals that trigger invasive growth.

Genomics, 2004 Jan, 83(1), 153 - 67
Analysis of a high-throughput yeast two-hybrid system and its use to predict the function of intracellular proteins encoded within the human MHC class III region; Lehner B et al.; High-throughput (HTP) protein-interaction assays, such as the yeast two-hybrid (Y2H) system, are enormously useful in predicting the functions of novel gene-products . HTP-Y2H screens typically do not include all of the reconfirmation and specificity tests used in small-scale studies, but the effects of omitting these steps have not been assessed . We performed HTP-Y2H screens that included all standard controls, using the predicted intracellular proteins expressed from the human MHC class III region, a region of the genome associated with many autoimmune diseases . The 91 novel interactions identified provide insight into the potential functions of many MHC genes, including C6orf47, LSM2, NELF-E (RDBP), DOM3Z, STK19, PBX2, RNF5, UAP56 (BAT1), ATP6G2, LST1/f, BAT2, Scythe (BAT3), CSNK2B, BAT5, and CLIC1 . Surprisingly, our results predict that 1/3 of the proteins may have a role in mRNA processing, which suggests clustering of functionally related genes within the human genome . Most importantly, our analysis shows that omitting standard controls in HTP-Y2H screens could significantly compromise data quality.

Eukaryot Cell, 2003 Dec, 2(6), 1288 - 303
Recruitment of Tup1-Ssn6 by yeast hypoxic genes and chromatin-independent exclusion of TATA binding protein; Mennella TA et al.; The Tup1-Ssn6 general repression complex in Saccharomyces cerevisiae represses a wide variety of regulons . Regulon-specific DNA binding proteins recruit the repression complex, and their synthesis, activity, or localization controls the conditions for repression . Rox1 is the hypoxic regulon-specific protein, and a second DNA binding protein, Mot3, augments repression at tightly controlled genes . We addressed the requirements for Tup1-Ssn6 recruitment to two hypoxic genes, ANB1 and HEM13, by using chromatin immunoprecipitation assays . Either Rox1 or Mot3 could recruit Ssn6, but Tup1 recruitment required Ssn6 and Rox1 . We also monitored events during derepression . Rox1 and Mot3 dissociated from DNA quickly, accounting for the rapid accumulation of ANB1 and HEM13 RNAs, suggesting a simple explanation for induction . However, Tup1 remained associated with these genes, suggesting that the localization of Tup1-Ssn6 is not the sole determinant of repression . We could not reproduce the observation that deletion of the Tup1-Ssn6-interacting protein Cti6 was required for induction . Finally, Tup1 is capable of repression through a chromatin-dependent mechanism, the positioning of a nucleosome over the TATA box, or a chromatin-independent mechanism . We found that the rate of derepression was independent of the positioned nucleosome and that the TATA binding protein was excluded from ANB1 even in the absence of the positioned nucleosome . The mediator factor Srb7 has been shown to interact with Tup1 and to play a role in repression at several regulons, but we found that significant levels of repression remained in srb7 mutants even when the chromatin-dependent repression mechanism was eliminated . These findings suggest that the repression of different regulons or genes may invoke different mechanisms.

Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15770 - 5 Epub 2003 Dec 08.
Two different Swi5-containing protein complexes are involved in mating-type switching and recombination repair in fission yeast; Akamatsu Y et al.; Homologous recombination is an important biological process that occurs in all organisms and facilitates genome rearrangements and repair of DNA double-strand breaks . Eukaryotic Rad51 proteins (Rad51sp or Rhp51 in fission yeast) are functional and structural homologs of bacterial RecA protein, an evolutionarily conserved protein that plays a key role in homologous pairing and strand exchange between homologous DNA molecules in vitro . Here we show that the fission yeast swi5+ gene, which was originally identified as a gene required for normal mating-type switching, encodes a protein conserved among eukaryotes and is involved in a previously uncharacterized Rhp51 (Rad51sp)-dependent recombination repair pathway that does not require the Rhp55/57 (Rad55/57sp) function . Protein interactions with both Swi5 and Rhp51 were found to be mediated by a domain common to Swi2 and Sfr1 (Swi five-dependent recombination repair protein 1, a previously uncharacterized protein with sequence similarity to the C-terminal part of Swi2) . Genetic epistasis analyses suggest that the Swi5-Sfr1-Rhp51 interactions function specifically in DNA recombination repair, whereas the Swi5-Swi2-Rhp51 interactions may function, together with chromodomain protein Swi6 (HP1 homolog), in mating-type switching.

J Cell Biol, 2003 Dec 8, 163(5), 973 - 85
Yeast homotypic vacuole fusion requires the Ccz1-Mon1 complex during the tethering/docking stage; Wang CW et al.; The function of the yeast lysosome/vacuole is critically linked with the morphology of the organelle . Accordingly, highly regulated processes control vacuolar fission and fusion events . Analysis of homotypic vacuole fusion demonstrated that vacuoles from strains defective in the CCZ1 and MON1 genes could not fuse . Morphological evidence suggested that these mutant vacuoles could not proceed to the tethering/docking stage . Ccz1 and Mon1 form a stable protein complex that binds the vacuole membrane . In the absence of the Ccz1-Mon1 complex, the integrity of vacuole SNARE pairing and the unpaired SNARE class C Vps/HOPS complex interaction were both impaired . The Ccz1-Mon1 complex colocalized with other fusion components on the vacuole as part of the cis-SNARE complex, and the association of the Ccz1-Mon1 complex with the vacuole appeared to be regulated by the class C Vps/HOPS complex proteins . Accordingly, we propose that the Ccz1-Mon1 complex is critical for the Ypt7-dependent tethering/docking stage leading to the formation of a trans-SNARE complex and subsequent vacuole fusion.

Curr Opin Microbiol, 2003 Dec, 6(6), 614 - 20
RNA asymmetric distribution and daughter/mother differentiation in yeast; Darzacq X et al.; Active transport and localized translation of the ASH1 mRNA at the bud tip of the budding yeast Saccharomyces cerevisiae is an essential process that is required for the regulation of the mating type switching . ASH1 mRNA localization has been extensively studied over the past few years and the core components of the translocation machinery have been identified . It is composed of four localization elements (zipcodes), within the ASH1 mRNA, and at least three proteins, She1p/Myo4p, She2p and She3p . Whereas the movement of the RNA can be attributed to direct interaction with myosin, the regulation of the RNA expression is less well understood . Recent insights have revealed a role for translation that might have a key function in the regulation of Ash1 protein sorting.

Biochemistry, 2003 Dec 16, 42(49), 14566 - 75
Identification of sequences responsible for intracellular targeting and membrane binding of rat CYP2E1 in yeast; Neve EP et al.; The role of the hydrophobic NH(2)-terminal domain of rat CYP2E1 for intracellular targeting and membrane binding was investigated in Saccharomyces cerevisiae as a model system . Several different CYP2E1 variants with deletions and mutations were expressed in yeast, and their intracellular localization and membrane-binding properties were analyzed . We found that an amino acid stretch including the B-helix from glycine 82 to asparagine 95 is responsible for mitochondrial association of CYP2E1 in yeast . Furthermore, we investigated the membrane-binding properties of the variants and concluded that the same region in the B-helix is responsible for membrane interactions of CYP2E1 by electrostatic interactions . A soluble variant of CYP2E1 lacking the first 82 amino acids and containing leucine to aspartate amino acid exchanges at positions 90 and 91, which disrupted the amphipathic nature of the B-helix, was expressed at relatively high levels in the yeast and was found to be catalytically active toward chlorzoxazone in cumene hydroperoxide-supported reactions . We suggest that these amino acid changes at positions 90 and 91 abolish the electrostatic interaction between the negatively charged membrane and the positively charged B-helix, thereby producing a soluble product.

Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15458 - 62 Epub 2003 Dec 05.
A role in vacuolar arginine transport for yeast Btn1p and for human CLN3, the protein defective in Batten disease; Kim Y et al.; In Saccharomyces cerevisiae, transport of arginine into the vacuole has previously been shown to be facilitated by a putative H+/arginine antiport . We confirm that transport of arginine into isolated yeast vacuoles requires ATP and we demonstrate a requirement for a functional vacuolar H+-ATPase . We previously reported that deletion of BTN1 (btn1-delta), an ortholog of the human Batten disease gene CLN3, resulted in a decrease in vacuolar pH during early growth . We report that this altered vacuolar pH in btn1-delta strains underlies a lack of arginine transport into the vacuole, which results in a depletion of endogenous vacuolar arginine levels . This arginine transport defect in btn1-delta is complemented by expression of either BTN1 or the human CLN3 gene and strongly suggests a function for transport of, or regulation of the transport of, basic amino acids into the vacuole or lysosome for yeast Btn1p, and human CLN3 protein, respectively . We propose that defective transport at the lysosomal membrane caused by an absence of functional CLN3 is the primary biochemical defect that results in Batten disease.

Mol Cell Proteomics, 2004 Mar, 3(3), 226 - 37 Epub 2003 Dec 05.
Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast; Graumann J et al.; A combined multidimensional chromatography-mass spectrometry approach known as "MudPIT" enables rapid identification of proteins that interact with a tagged bait while bypassing some of the problems associated with analysis of polypeptides excised from SDS-polyacrylamide gels . However, the reproducibility, success rate, and applicability of MudPIT to the rapid characterization of dozens of proteins have not been reported . We show here that MudPIT reproducibly identified bona fide partners for budding yeast Gcn5p . Additionally, we successfully applied MudPIT to rapidly screen through a collection of tagged polypeptides to identify new protein interactions . Twenty-five proteins involved in transcription and progression through mitosis were modified with a new tandem affinity purification (TAP) tag . TAP-MudPIT analysis of 22 yeast strains that expressed these tagged proteins uncovered known or likely interacting partners for 21 of the baits, a figure that compares favorably with traditional approaches . The proteins identified here comprised 102 previously known and 279 potential physical interactions . Even for the intensively studied Swi2p/Snf2p, the catalytic subunit of the Swi/Snf chromatin remodeling complex, our analysis uncovered a new interacting protein, Rtt102p . Reciprocal tagging and TAP-MudPIT analysis of Rtt102p revealed subunits of both the Swi/Snf and RSC complexes, identifying Rtt102p as a common interactor with, and possible integral component of, these chromatin remodeling machines . Our experience indicates it is feasible for an investigator working with a single ion trap instrument in a conventional molecular/cellular biology laboratory to carry out proteomic characterization of a pathway, organelle, or process (i.e . "pathway proteomics") by systematic application of TAP-MudPIT.

J Biol Chem, 2004 Jan 16, 279(3), 1577 - 80 Epub 2003 Dec 04.
Carbohydrates induce mono-ubiquitination of H2B in yeast; Dong L et al.; Histone modifications have emerged to be a major regulatory mechanism for gene expression (1-4) . However, it is not clear how histone modifications are physiologically regulated . Here, we show that mono-ubiquitinated H2B at lysine 123 (uH2B) in the yeast (Saccharomyces cerevisiae) is present in exponential phase and absent in stationary phase . A wide array of carbohydrates or sugars, including glucose, fructose, mannose, and sucrose, are capable of inducing uH2B in stationary phase yeast . In contrast, non-metabolic glucose analogs are defective in inducing uH2B . Furthermore, uH2B induction is inhibited by iodoacetate, an inhibitor of glyceraldehyde-3-phosphate dehydrogenase in glycolysis . Moreover, uH2B induction is markedly impaired in yeast mutants, in which glycolytic genes are deleted . These data indicate that glycolysis is required for the carbohydrate-induced mono-ubiquitination of H2B at lysine 123 . Therefore, our study reveals a novel paradigm of metabolic regulation of histone modifications.

J Biol Chem, 2004 Jan 16, 279(3), 1867 - 71 Epub 2003 Dec 03.
Deubiquitination of histone H2B by a yeast acetyltransferase complex regulates transcription; Daniel JA et al.; Post-translational modifications of the histone protein components of eukaryotic chromatin play an important role in the regulation of chromatin structure and gene expression (1) . Given the requirement of Rad6/Bre1-dependent ubiquitination of histone H2B for H3 dimethylation (at lysines 4 and 79) and gene silencing (2-7), removal of ubiquitin from H2B may have a significant regulatory effect on transcription . Here we show that a putative deubiquitinating enzyme, Ubp8, is a structurally nonessential component of both the Spt-Ada-Gcn5-acetyltransferase (SAGA) and SAGA-like (SLIK) histone acetyltransferase (HAT) complexes in yeast . Disruption of this gene dramatically increases the cellular level of ubiquitinated-H2B, and SAGA and SLIK are shown to have H2B deubiquitinase activity . These findings demonstrate, for the first time, how the ubiquitin moiety can be removed from histone H2B in a regulated fashion . Ubp8 is required for full expression of the SAGA- and SLIK-dependent gene GAL10 and is recruited to the upstream activation sequence (UAS) of this gene under activating conditions, while Rad6 dissociates . Furthermore, trimethylation of H3 at lysine 4 within the UAS increases significantly under activating conditions, and remarkably, Ubp8 is shown to have a role in regulating the methylation status of this residue . Collectively, these data suggest that the SAGA and SLIK HAT complexes can regulate an integrated set of multiple histone modifications, counteracting repressive effects that alter chromatin and regulate gene expression.

Appl Environ Microbiol, 2003 Dec, 69(12), 7558 - 62
Construction of a genetically modified wine yeast strain expressing the Aspergillus aculeatus rhaA gene, encoding an alpha-L-rhamnosidase of enological interest; Manzanares P et al.; The Aspergillus aculeatus rhaA gene encoding an alpha-L-rhamnosidase has been expressed in both laboratory and industrial wine yeast strains . Wines produced in microvinifications, conducted using a combination of the genetically modified industrial strain expressing rhaA and another strain expressing a beta-glucosidase, show increased content mainly of the aromatic compound linalool.

Gene, 2003 Dec 24, 323, 101 - 13
Evolutionarily conserved cytoprotection provided by Bax Inhibitor-1 homologs from animals, plants, and yeast; Chae HJ et al.; Programmed cell death (PCD) plays important roles in the development and physiology of both animals and plants, but it is unclear whether similar mechanisms are employed . Bax Inhibitor-1 (BI-1) is an intracellular multi-membrane-spanning protein and cell death inhibitor, originally identified by a function-based screen for mammalian cDNAs capable of suppressing cell death in yeast engineered to ectopically express the pro-apoptotic protein Bax . Using this yeast assay, we screened expression libraries for cDNAs from the plant, Lycopersicon esculentum (tomato), and the invertebrate animal Drosophila melanogaster (fruit fly), identifying close homologs of BI-1 as Bax-suppressors . We studied the fly and tomato homologs of BI-1, as well as BI-1 homologs identified in Arabidopsis thaliana, Oryza sativa (rice), and Saccharomyces cerevisiae (budding yeast) . All eukaryotic homologs of BI-1 blocked Bax-induced cell death when expressed in yeast . Eukaryotic BI-1 homologs also partially rescued yeast from cell death induced by oxidative stress (H(2)O(2)) and heat shock . Deletion of a C-terminal domain from BI-1 homologs abrogated their cytoprotective function in yeast, demonstrating conserved structure-function relations among these proteins . Expression of tomato BI-1 by agroinfiltration of intact plant leaves provided protection from damage induced by heat-shock and cold-shock stress . Altogether, these findings indicate that BI-1 homologs exist in multiple eukaryotic species, providing cytoprotection against diverse stimuli, thus implying that BI-1 regulates evolutionary conserved mechanisms of stress resistance that are germane to both plants and animals.

J Mol Biol, 2004 Jan 2, 335(1), 71 - 85
Reinitiation and recycling are distinct processes occurring downstream of translation termination in yeast; Rajkowitsch L et al.; The circularisation model of the polysome suggests that ribosome recycling is facilitated by 5'-3' interactions mediated by the cap-binding complex eIF4F and the poly(A)-binding protein, Pab1 . Alternatively, downstream of a short upstream open reading frame (uORF) in the 5' untranslated region of a gene, posttermination ribosomes can maintain the competence to (re)initiate translation . Our data show that recycling and reinitiation must be distinct processes in Saccharomyces cerevisiae . The role of the 3'UTR in recycling was assessed by restricting ribosome movement along the mRNA using a poly(G) stretch or the mammalian iron regulatory protein bound to the iron responsive element . We find that although 3'UTR structure can influence translation, the main pathway of ribosome recycling does not depend on scanning-like movement through the 3'UTR . Changes in termination kinetics or disruption of the Pab1-eIF4F interaction do not affect recycling, yet the maintenance of normal in vivo mRNP structure is important to this process . Using bicistronic ACT1-LUC constructs, elongating yeast ribosomes were found to maintain the competence to (re)initiate over only short distances . Thus, as the first ORF to be translated is progressively truncated, reinitiation downstream of an uORF of 105nt is found to be just detectable, and increases markedly in efficiency as uORF length is reduced to 15nt . Experiments using a strain mutated in the Cca1 nucleotidyltransferase suggest that the uORF length-dependence of changes in reinitiation competence is affected by peptide elongation kinetics, but that ORF length per se may also be relevant.

Virus Res, 2003 Dec, 98(2), 123 - 9
Measles virus protein interactions in yeast: new findings and caveats; Chen M et al.; Complementary DNA clones of measles virus N, N (S228Q; L229D), Ncore (N1-400), Ntail (N401-525), P, PNT (P1-230), PCT (P231-507), L, MEL (L800-2183) and EL (L1300-2183) were fused in frame downstream of the Gal4 binding domain (BD) or activating domain (AD) . All but BD-L, BD-MEL and BD-EL, were detected by western blot, with additional C- and/or N-terminal truncated products in the case of BD-N, and BD-P . BD-P and BD-PNT directly activated the reporter genes, indicating that the PNT domain displays transactivating properties . In yeast two-hybrid assays, PNT and PCT domains bind to Ncore and Ntail domains, respectively, indicating that N and P interact in a head to tail orientation via two independent binding sites . BD-N (S228Q; L229D) and AD-N displayed no or poor interaction with P proteins possibly because they may not be properly folded . L binding site on P lies within the PCT domain, and two PCT binding sites lie within the L1-799 and L800-1300 regions . Thus, N to P and P to L protein interactions in measles virus shared many features with other related Paramyxoviridae . From a human cDNA library, several candidate partners of N protein were identified which all reacted with BD-Ncore, and RNA was found to bridge the N protein with one partner.

Anal Bioanal Chem, 2004 Feb, 378(3), 697 - 708 Epub 2003 Dec 05.
Integrated procedure for determination of endocrine-disrupting activity in surface waters and sediments by use of the biological technique recombinant yeast assay and chemical analysis by LC-ESI-MS; Cespedes R et al.; An integrated procedure using mass spectrometry and molecular biology for determination of estrogenicity in natural waters and sediments is reported . Solid-phase extraction (SPE) and pressurized-liquid extraction (PLE), respectively, were used for isolation of endocrine-disrupting compounds (EDC) from surface waters and sediments, followed by liquid chromatography-mass spectrometry using an electrospray interface (LC-ESI-MS) . Twenty seven EDC were determined: non-ionic surfactants (nonylphenol ethoxylate), alkylphenols (e.g . nonylphenol and octylphenol), bisphenol A, phthalates, and natural and synthetic steroid sex hormones . Limits of detection varied from 0.02 to 0.22 microg L(-1) and from 1 to 10 microg kg(-1) in water and sediments, respectively . Recoveries ranged from 65 to 125% and 73 to 97% for waters and sediments, respectively . In addition to LC-ESI-MS determination, extracts obtained by SPE and PLE were analyzed by the recombinant yeast assay (RYA) to assess total estrogenic activity . This bioassay detects natural estrogens and xenoestrogens, producing a quantitative measurement of EDC irrespective of the identity of the chemical responsible for the activity . As a novelty, a relative estrogenicity factor was determined for 19 analytes with EC(50) values ranging from 10(-10) to 10(-9) mol L(-1) for synthetic estrogens, from 10(-7) to 10(-5) mol L(-1) for alkylphenol derivatives, and from 10(-5) to 10(-4) mol L(-1) for phthalates and benzothiazoles . By use of this integrated chemical-ecotoxicological approach good correlation was usually established between chemical composition and estrogenic effects for surface water and sediment samples from Portugal . Estrogenic activity observed was mainly attributed to the presence of nonylphenolic compounds (with concentrations of NP ranging from 0.1 up to 44 microg L(-1) in waters and up to 1172 microg kg(-1) in sediments), and to the sporadic presence of estrogens, detected at ng L(-1) levels.

Science, 2003 Dec 5, 302(5651), 1772 - 5
Yeast cells provide insight into alpha-synuclein biology and pathobiology; Outeiro TF et al.; Alpha-synuclein is implicated in several neurodegenerative disorders, such as Parkinson's disease and multiple system atrophy, yet its functions remain obscure . When expressed in yeast, alpha-synuclein associated with the plasma membrane in a highly selective manner, before forming cytoplasmic inclusions through a concentration-dependent, nucleated process . Alpha-synuclein inhibited phospholipase D, induced lipid droplet accumulation, and affected vesicle trafficking . This readily manipulable system provides an opportunity to dissect the molecular pathways underlying normal alpha-synuclein biology and the pathogenic consequences of its misfolding.

Science, 2003 Dec 5, 302(5651), 1769 - 72
Yeast genes that enhance the toxicity of a mutant huntingtin fragment or alpha-synuclein; Willingham S et al.; Genome-wide screens were performed in yeast to identify genes that enhance the toxicity of a mutant huntingtin fragment or of alpha-synuclein . Of 4850 haploid mutants containing deletions of nonessential genes, 52 were identified that were sensitive to a mutant huntingtin fragment, 86 that were sensitive to alpha-synuclein, and only one mutant that was sensitive to both . Genes that enhanced toxicity of the mutant huntingtin fragment clustered in the functionally related cellular processes of response to stress, protein folding, and ubiquitin-dependent protein catabolism, whereas genes that modified alpha-synuclein toxicity clustered in the processes of lipid metabolism and vesicle-mediated transport . Genes with human orthologs were overrepresented in our screens, suggesting that we may have discovered conserved and nonoverlapping sets of cell-autonomous genes and pathways that are relevant to Huntington's disease and Parkinson's disease.

Proc Natl Acad Sci U S A, 2003 Dec 9, 100(25), 14796 - 9 Epub 2003 Dec 01.
Random Boolean network models and the yeast transcriptional network; Kauffman S et al.; The recently measured yeast transcriptional network is analyzed in terms of simplified Boolean network models, with the aim of determining feasible rule structures, given the requirement of stable solutions of the generated Boolean networks . We find that, for ensembles of generated models, those with canalyzing Boolean rules are remarkably stable, whereas those with random Boolean rules are only marginally stable . Furthermore, substantial parts of the generated networks are frozen, in the sense that they reach the same state, regardless of initial state . Thus, our ensemble approach suggests that the yeast network shows highly ordered dynamics.

Mol Biol Cell, 2004 Feb, 15(2), 883 - 95 Epub 2003 Dec 02.
Direct sorting of the yeast uracil permease to the endosomal system is controlled by uracil binding and Rsp5p-dependent ubiquitylation; Blondel MO et al.; The yeast uracil permease, Fur4p, is downregulated by uracil, which is toxic to cells with high permease activity . Uracil promotes cell surface Rsp5p-dependent ubiquitylation of the permease, signaling its endocytosis and further vacuolar degradation . We show here that uracil also triggers the direct routing of its cognate permease from the Golgi apparatus to the endosomal system for degradation, without passage via the plasma membrane . This early sorting was not observed for a variant permease with a much lower affinity for uracil, suggesting that uracil binding is the signal for the diverted pathway . The FUI1-encoded uridine permease is similarly sorted for early vacuolar degradation in cells exposed to a toxic level of uridine uptake . Membrane proteins destined for vacuolar degradation require sorting at the endosome level to the intraluminal vesicles of the multivesicular bodies . In cells with low levels of Rsp5p, Fur4p can be still diverted from the Golgi apparatus but does not reach the vacuolar lumen, being instead missorted to the vacuolar membrane . Correct luminal delivery is restored by the biosynthetic addition of a single ubiquitin, suggesting that the ubiquitylation of Fur4p serves as a specific signal for sorting to the luminal vesicles of the multivesicular bodies . A fused ubiquitin is also able to sort some Fur4p from the Golgi to the degradative pathway in the absence of added uracil but the low efficiency of this sorting indicates that ubiquitin does not itself act as a dominant signal for Golgi-to-endosome trafficking . Our results are consistent with a model in which the binding of intracellular uracil to the permease signals its sorting from the Golgi apparatus and subsequent ubiquitylation ensures its delivery to the vacuolar lumen.

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 2091 - 3
Sporobolomyces bannaensis, a novel ballistoconidium-forming yeast species in the Sporidiobolus lineage; Zhao JH et al.; Among ballistoconidium-forming yeast strains isolated from various plant leaves collected in Banna, Yunnan Province, China, five strains that formed pink-coloured colonies and asymmetric ballistoconidia were classified in a single group and assigned to the genus Sporobolomyces by conventional and chemotaxonomic studies . Analyses of the internal transcribed spacer region and 26S rDNA D1/D2 domain sequences indicated that these strains represent a novel species with a close phylogenetic relationship to Sporobolomyces blumeae in the Sporidiobolus lineage, for which the name Sporobolomyces bannaensis sp . nov . is proposed (type strain Y41(T)=AS 2.2285(T)=CBS 9204(T)).

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 2085 - 9
Bensingtonia changbaiensis sp . nov . and Bensingtonia sorbi sp . nov., novel ballistoconidium-forming yeast species from plant leaves; Wang QM et al.; Six ballistoconidium-forming yeast strains that were isolated from plant leaves collected on Changbai Mountain, north-east China, were assigned to the genus Bensingtonia Ingold emend . Nakase & Boekhout due to the formation of asymmetrical ballistoconidia, cream-coloured colonies and Q-9 as the major ubiquinone . Two separate groups, representing two novel Bensingtonia species, were recognized among these yeasts by 26S rDNA D1/D2 domain, internal transcribed spacer (ITS) region and 18S rDNA sequence analyses . The names Bensingtonia changbaiensis sp . nov . (type strain, CB 346(T)=AS 2.2310(T)=CBS 9497(T)) and Bensingtonia sorbi sp . nov . (type strain, CB 286(T)=AS 2.2303(T)=CBS 9498(T)) are proposed for these two species.

EMBO J, 2003 Dec 15, 22(24), 6573 - 83
Yeast Nop15p is an RNA-binding protein required for pre-rRNA processing and cytokinesis; Oeffinger M et al.; Nop15p is an essential protein that contains an RNA recognition motif (RRM) and localizes to the nucleoplasm and nucleolus . Cells depleted of Nop15p failed to synthesize the 25S and 5.8S rRNA components of the 60S ribosomal subunit, and exonucleolytic 5' processing of 5.8S rRNA was strongly inhibited . Pre-rRNAs co-precipitated with tagged Nop15p confirmed its association with early pre-60S particles and Nop15p bound a pre-rRNA transcript in vitro . Nop15p-depleted cells show an unusually abrupt growth arrest prior to substantial depletion of ribosomal subunits . Following cell synchronization in mitosis, Nop15p-depleted cells undergo nuclear division with wild-type kinetics, activate the mitotic exit network and disassemble their mitotic spindle . However, they uniformly arrest at cytokinesis and fail to assemble a contractile actin ring at the bud neck . In dividing wild-type cells, segregation of nucleolar proteins to the daughter nuclei occurs after separation of the nucleoplasm . In these late mitotic cells, Nop15p was partially delocalized from the nucleolus to the nucleoplasm, consistent with a specific function in cell division in addition to its role in ribosome synthesis.

EMBO J, 2003 Dec 15, 22(24), 6438 - 47
Yeast Oxa1 interacts with mitochondrial ribosomes: the importance of the C-terminal region of Oxa1; Jia L et al.; The yeast mitochondrial Oxa1 protein is a member of the conserved Oxa1/YidC/Alb3 protein family involved in the membrane insertion of proteins . Oxa1 mediates the insertion of proteins (nuclearly and mitochondrially encoded) into the inner membrane . The mitochondrially encoded substrates interact directly with Oxa1 during their synthesis as nascent chains and in a manner that is supported by the associated ribosome . We have investigated if the Oxa1 complex interacts with the mitochondrial ribosome . Evidence to support a physical association between Oxa1 and the large ribosomal subunit is presented . Our data indicate that the matrix-exposed C-terminal region of Oxa1 plays an important role supporting the ribosomal-Oxa1 interaction . Truncation of this C-terminal segment compromises the ability of Oxa1 to support insertion of substrate proteins into the inner membrane . Oxa1 can be cross-linked to Mrp20, a component of the large ribosomal subunit . Mrp20 is homologous to L23, a subunit located next to the peptide exit tunnel of the ribosome . We propose that the interaction of Oxa1 with the ribosome serves to enhance a coupling of translation and membrane insertion events.

Structure (Camb), 2003 Dec, 11(12), 1475 - 83
The crystal structure of the yeast Hsp40 Ydj1 complexed with its peptide substrate; Li J et al.; The mechanisms by which Hsp40 functions as a molecular chaperone to recognize and bind non-native polypeptides is not understood . We have identified a peptide substrate for Ydj1, a member of the type I Hsp40 from yeast . The structure of the Ydj1 peptide binding fragment and its peptide substrate complex was determined to 2.7 A resolution . The complex structure reveals that Ydj1 peptide binding fragment forms an L-shaped molecule constituted by three domains . The domain I exhibits a similar protein folds as domain III while the domain II contains two Zinc finger motifs . The peptide substrate binds Ydj1 by forming an extra beta strand with domain I of Ydj1 . The Leucine residue in the middle of the peptide substrate GWLYEIS inserts its side chain into a hydrophobic pocket formed on the molecular surface of Ydj1 domain I . The Zinc finger motifs located in the Ydj1 domain II are not in the vicinity of peptide substrate binding site.

Mol Genet Genomics, 2004 Jan, 270(6), 449 - 61 Epub 2003 Dec 04.
Two-hybrid search for proteins that interact with Sad1 and Kms1, two membrane-bound components of the spindle pole body in fission yeast; Miki F et al.; In interphase cells of fission yeast, the spindle pole body (SPB) is thought to be connected with chromosomal centromeres by an as yet unknown mechanism that spans the nuclear membrane . To elucidate this mechanism, we performed two-hybrid screens for proteins that interact with Kms1 and Sad1, which are constitutive membrane-bound components of the SPB that interact with each other . Seven and 26 genes were identified whose products potentially interact with Kms1 and Sad1, respectively . With the exception of Dlc1 (a homolog of the 14-kDa dynein light chain), all of the Kms1 interactors also interacted with Sad1 . Among the genes identified were the previously known genes rhp9+ / crb2+, cut6+, ags1+ / mok1+, gst3+, kms2+, and sid4+ . The products of kms2+ and sid4+ localize to the SPB . The novel genes were characterized by constructing disruption mutations and by localization of the gene products . Two of them, putative homologues of budding yeast UFE1 (which encodes a t-SNARE) and SFH1 (an essential component of a chromatin-remodeling complex), were essential for viability . Two further genes, which were only conditionally essential, genetically interact with sad1+ . One of these was named sif1+ (for Sad1-interacting factor) and is required for proper septum formation at high temperature . Cells in which this gene was overexpressed displayed a wee -like phenotype . The product of the other gene, apm1+, is very similar to the medium chain of an adaptor protein complex in clathrin-coated vesicles . Apm1 appears to be required for SPB separation and spindle formation, and tended to accumulate at the SPB when it was overproduced . It was functionally distinct from its homologues Apm2 and Apm4 . Other novel genes identified in this study included one for a nucleoporin and genes encoding novel membrane-bound proteins that were genetically related to Sad1 . We found that none of the newly identified genes tested were necessary for centromere/telomere clustering.

FEMS Yeast Res, 2003 Dec, 4(3), 271 - 5
Yeast communities associated with stingless bees; Rosa CA et al.; The yeast communities associated with the stingless bees Tetragonisca angustula, Melipona quadrifasciata and Frieseomelitta varia were studied . The bees T . angustula and F . varia showed a strong association with the yeast Starmerella meliponinorum . M . quadrifasciata more frequently carried a species related to Candida apicola, but also vectored low numbers of S . meliponinorum . Some of the yeasts isolated from adult bees were typical of species known to occur in flowers . Other yeast species found in adult bees were more typical of those found in the phylloplane . S . meliponinorum and the species in the C . apicola complex, also part of the Starmerella clade, may have a mutualistic relationship with the bees studied . Many yeasts in that group are often found in bees or substrates visited by bees, suggesting that a mutually beneficial interaction exists between them.

FEMS Yeast Res, 2003 Dec, 4(3), 253 - 8
The D1/D2 domain of the large-subunit rDNA of the yeast species Clavispora lusitaniae is unusually polymorphic; Lachance MA et al.; Ten different versions of the D1/D2 divergent domain of the large-subunit ribosomal DNA were identified among interbreeding members of the yeast species Clavispora lusitaniae . One major polymorphism, located in a 90-bp structural motif of the D2 domain, exists in two versions that differ by 32 base substitutions . Three other polymorphisms consist of a two-base substitution, a two-base deletion, and a single-base deletion, respectively . The polymorphisms are independent of one another and of the two mating types, indicating that the strains studied belong to a single, sexually active Mendelian population . Several strains were heterogeneous for one or more of the polymorphisms, and one strain was found to be automictic and capable of producing asci on its own by isogamous conjugation or by bud-parent autogamy . These observations suggest circumspection in the use of sequence divergence as the principal criterion for delimiting yeast species.

Chemphyschem, 2003 Nov 14, 4(11), 1183 - 8
Topological and electron-transfer properties of yeast cytochrome c adsorbed on bare gold electrodes; Bonanni B et al.; The redox metalloprotein yeast cytochrome c was directly self-chemisorbed on "bare" gold electrodes through the free sulfur-containing group Cys102 . Topological, spectroscopic, and electron transfer properties of the immobilised molecules were investigated by in situ scanning probe microscopy and cyclic voltammetry . Atomic force and scanning tunnelling microscopy revealed individual protein molecules adsorbed on the gold substrate, with no evidence of aggregates . The adsorbed proteins appear to be firmly bound to gold and display dimensions in good agreement with crystallographic data . Cyclic voltammetric analysis showed that up to 84% of the electrode surface is functionalised with electroactive proteins whose measured redox midpoint potential is in good agreement with the formal potential . Our results clearly indicate that this variant of cytochrome c is adsorbed on bare gold electrodes with preservation of morphological properties and redox functionality.

Curr Genet, 2004 Feb, 45(2), 76 - 82 Epub 2003 Dec 02.
Mapping of sporulation-specific functions in the yeast syntaxin gene SSO1; Oyen M et al.; The yeast Saccharomyces cerevisiae has two closely related plasma membrane syntaxins, Sso1p and Sso2p, which together provide an essential function in vegetative cells . However, Sso1p is also specifically needed during sporulation; and this function cannot be provided by Sso2p . We used fusions between SSO1 and SSO2 to map the sporulation-specific function of SSO1 . We found that the two N-terminal alpha-helices Ha and Hb of Sso1p are important for sporulation, since it is reduced 8-fold for fusions where Ha and Hb are derived from Sso2p . In contrast, the C-terminal half of Sso1p does not seem to be specifically required for sporulation . Surprisingly, we further found that the 3' untranslated region (3'UTR) of SSO1 is essential for sporulation . Western blots failed to reveal a preferential expression of Sso1p in sporulating cells, indicating that effects on gene expression are unlikely to explain why the SSO1 3'UTR is needed for sporulation.

Mol Microbiol, 2004 Jan, 51(1), 149 - 58
Aberrant cardiolipin metabolism in the yeast taz1 mutant: a model for Barth syndrome; Gu Z et al.; In eukaryotic cells, the acyl species of the phospholipid cardiolipin (CL) are more highly unsaturated than those of the other membrane phospholipids . Defective acylation of CL with unsaturated fatty acids and decreased total CL are associated with Barth syndrome, an X-linked cardio- and skeletal myopathy attributed to a defect in the gene G4.5 (also known as tafazzin) . We constructed a yeast mutant (taz1) containing a null mutation in the homologue of the human G4.5 gene . The yeast taz1Delta mutant was temperature sensitive for growth in ethanol as sole carbon source, but grew normally on glucose or glycerol plus ethanol . Total CL content was reduced in the taz1Delta mutant, and monolyso-CL accumulated . The predominant CL acyl species found in wild-type cells, C18:1 and C16:1, were markedly reduced in the mutant, whereas CL molecules containing saturated fatty acids were present . Interestingly, CL synthesis increased in the mutant, whereas expression of the CL structural genes CRD1 and PGS1 did not, suggesting that de novo biosynthetic enzyme activities are regulated by CL acylation . These results indicate that the taz1Delta mutant is an excellent genetic tool for the study of CL remodelling and may serve as a model system for the study of Barth syndrome.

J Synchrotron Radiat, 2004 Jan 1, 11(Pt 1), 109 - 12 Epub 2003 Nov 28.
Protein splicing of yeast VMA1-derived endonuclease via thiazolidine intermediates; Mizutani R et al.; Protein splicing precisely excises out an internal intein segment from a protein precursor, and concomitantly ligates the N- and C-terminal extein polypeptides flanking the intein . A recombinant X10SNS bearing N- and C-extein polypeptides has been prepared for the intein endonuclease derived from the Saccharomyces cerevisiae VMA1 gene . X10SNS has replacements of C284S, H362N and C738S, and forms the intein and extein segments in the crystal lattice . The crystal structure of X10SNS revealed a linkage between the N- and C-extein segments, and showed that the C284 amino group of the resultant intein segment is in interaction with the G283 O atom of the N-extein segment . A mechanism for the final S --> N acyl shift step proposes that a tetrahedral intermediate involves a five-membered thiazolidine ring at G283-C738 junction . An oxyanion of the thiazolidine intermediate is to be stabilized by the C284 N atom.

Mol Cell Biol, 2003 Dec, 23(24), 9275 - 82
Affinity purification of specific chromatin segments from chromosomal loci in yeast; Griesenbeck J et al.; Single-copy gene and promoter regions have been excised from yeast chromosomes and have been purified as chromatin by conventional and affinity methods . Promoter regions isolated in transcriptionally repressed and activated states maintain their characteristic chromatin structures . Gel filtration analysis establishes the uniformity of the transcriptionally activated state . Activator proteins interact in the manner anticipated from previous studies in vivo . This work opens the way to the direct study of specific gene regions of eukaryotic chromosomes in diverse functional and structural states.

Mol Cell Biol, 2003 Dec, 23(24), 9162 - 77
Mitotic cyclins regulate telomeric recombination in telomerase-deficient yeast cells; Grandin N et al.; Telomerase-deficient mutants of Saccharomyces cerevisiae can survive death by senescence by using one of two homologous recombination pathways . The Rad51 pathway amplifies the subtelomeric Y' sequences, while the Rad50 pathway amplifies the telomeric TG(1-3) repeats . Here we show that telomerase-negative cells require Clb2 (the major B-type cyclin in this organism), in association with Cdc28 (Cdk1), to generate postsenescence survivors at a normal rate . The Rad50 pathway was more sensitive to the absence of Clb2 than the Rad51 pathway . We also report that telomerase RAD50 RAD51 triple mutants still generated postsenescence survivors . This novel Rad50- and Rad51-independent pathway of telomeric recombination also appeared to be controlled by Clb2 . In telomerase-positive cells, a synthetic growth defect between mutations in CLB2 and RAD50 or in its partners in the conserved MRX complex, MRE11 and XRS2, was observed . This genetic interaction was independent of Mre11 nuclease activity but was dependent on a DNA repair function . The present data reveal an unexpected role of Cdc28/Clb2 in telomeric recombination during telomerase-independent maintenance of telomeres . They also uncover a functional interaction between Cdc28/Clb2 and MRX during the control of the mitotic cell cycle.

Biophys J, 2003 Dec, 85(6), 3730 - 8
Electrophysiological analysis of the yeast V-type proton pump: variable coupling ratio and proton shunt; Kettner C et al.; Isolated vacuoles from the yeast Saccharomyces cerevisiae were examined in the whole-vacuole mode of patch recording, to get a detailed functional description of the vacuolar proton pump, the V-ATPase . Functioning of the V-ATPase was characterized by its current-voltage (I-V) relationship, obtained for various levels of vacuolar and cytosolic pH . I-V curves for the V-ATPase were computed as the difference between I-V curves obtained with the pump switched on (ATP, ADP, and Pi present) or off (no ATP) . These difference current-voltage relationships usually crossed the voltage axis within the experimental range (from -80 to +80 mV), thus measuring the reversal voltage (ER) for the V-ATPase, which could be compared with the standing ion gradients and free energy of ATP hydrolysis, to calculate the apparent pump stoichiometry or coupling ratio: the number of protons transported for each ATP molecule hydrolyzed . This ratio was found to depend strongly upon the pH difference (DeltapH) across the vacuolar membrane, being approximately 2H+/ATP at high DeltapH (4 pH units) and increasing to >4H+/ATP for small or zero DeltapH . That result is in quantitative agreement with previous determinations on plant vacuoles . Considerations of purely electrical behavior, together with the physical properties of a recent detailed structural model for V-ATPases, led to a linear equivalent circuit--which quantitatively accounts for all observations of variable coupling ratios in fungal and plant V-ATPases by variations of the conductance for bona fide proton pumping (GP) through the ATPase relative to independent proton shunting (GS) through the same protein.

Gene, 2003 Dec 11, 322, 157 - 68
The lethal phenotype observed after HIV-1 integrase expression in yeast cells is related to DNA repair and recombination events; Parissi V et al.; Human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalyzes the insertion of the viral genome into the host cell DNA, an essential reaction during the retroviral cycle . We described previously that expression of HIV-1 IN in some yeast strains may lead to the emergence of a lethal phenotype which was not observed when the catalytically crucial residues D, D, (35)E were mutated . The lethal effect in yeast seems to be related to the mutagenic effect of the recombinant HIV-1 IN, most probably via the non-sequence-specific endonucleolytic activity carried by this enzyme . This non-sequence-specific endonuclease activity was further characterized . Although the enzyme was active on DNA substrates devoid of viral long terminal repeat (LTR) sequences, the presence of LTR regions stimulated significantly this activity . Genetic experiments were designed to show that both the mutagenic effect and the level of recombination events were affected in cells expressing the active retroviral enzyme, while expression of the mutated inactive IN D116A has no significant effect . A close interaction was demonstrated between integrase activity and in vivo/in vitro recombination process, suggesting that retroviral integration and recombination mechanism are linked in the infected cell . Our results show that the yeast system is a powerful cellular model to study the non-sequence-specific endonucleolytic activity of IN . Its characterization is essential since this activity might represent a very important step in the retroviral infectious cycle and would provide further insights into the function of IN . Indeed, effectors of this activity should be sought as potential antiviral agents since stimulation of this enzymatic activity would induce the destruction of early synthesized proviral DNA.

Curr Opin Cell Biol, 2003 Dec, 15(6), 648 - 53
The morphogenesis checkpoint: how yeast cells watch their figures; Lew DJ; The morphogenesis checkpoint maintains coordination between the process of bud formation and the nuclear events of the cell cycle in yeast . This checkpoint regulates the Wee1 homolog, Swe1p, to induce cell-cycle delay or arrest when aspects of bud formation are defective . A variety of studies have suggested that this checkpoint can monitor actin organization, septin organization, the presence of a bud and even the size of a bud . The evidence for these proposals is reviewed, highlighting recent findings indicating that Swe1p degradation is controlled by the cell shape change that accompanies bud emergencePublication Types:

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