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<