J Cell Sci, 2002 Sep 15, 115(Pt 18), 3683 - 91 Binding of Sly1 to Sed5 enhances formation of the yeast early Golgi SNARE complex; Kosodo Y et al.; SLY1 is an essential gene for vesicular transport between the ER and the early Golgi apparatus in Saccharomyces cerevisiae . It encodes a hydrophilic Sec1/Munc18 family protein that binds to the t-SNAREs . The amount of Sly1 protein that coprecipitated with the t-SNARE Sed5 was much reduced in a temperature-sensitive sly1(ts) mutant yeast compared with the wildtype . The mutant Sly1(ts) protein was shown to have a reduced binding activity to Sed5 . In the wildtype, a detectable amount of Sly1 was found in the complex between Sed5 and the v-SNARE Bet1 . In vitro formation of this complex on different membranes in yeast lysate was enhanced by the addition of recombinant Sly1 . These results indicate that binding of Sly1 to Sed5 enhances trans-SNARE complex formation. J Cell Sci, 2002 Sep 15, 115(Pt 18), 3609 - 18 A novel chk1-dependent G1/M checkpoint in fission yeast; Synnes M et al.; Fission yeast cells with a temperature-sensitive Orp1 protein, a component of the origin recognition complex, cannot perform DNA replication at the restrictive temperature . Seventy percent of orp1-4 cells arrest with a 1C DNA content, whereas 30% proceed to mitosis ('cut') . The arrest depends upon the checkpoint Rad proteins and, surprisingly, the Chk1 protein, which is thought to act only from late S phase . The arrested cells maintain a 1C DNA content, as judged by flow cytometry, and the early origin ars3001 has not been initiated, as judged by 2D gel analysis . We show that in G1-arrested orp1-4 cells, Wee1 phosphorylates and inactivates Cdc2 . Activation of Chk1 occurs earlier than Cdc2 phosphorylation, indicating a novel role for Chk1, namely to induce and/or maintain Cdc2 phosphorylation upon checkpoint activation in G1 . We also show that commitment to cutting occurs already in early G1 phase. BMC Genet . 2002 Aug 20;3(1):15. Conservation of the COP9/signalosome in budding yeast; Wee S et al.; BACKGROUND: The COP9/signalosome (CSN), a multiprotein complex consisting of eight subunits, is implicated in a wide variety of regulatory processes including cell cycle control, signal transduction, transcriptional activation, and plant photomorphogenesis . Some of these functions have been linked to CSN-associated enzymes, including kinases and an activity that removes the ubiquitin-like protein NEDD8/Rub1p from the cullin subunit of E3 ligases . CSN is highly conserved across species from fission yeast to humans, but sequence comparison has failed to identify the complex in budding yeast, except for a putative CSN5 subunit called Rri1p . RESULTS: We show that disruption of four budding yeast genes, PCI8 and three previously uncharacterized ORFs, which encode proteins interacting with Rrr1p/Csn5p, each results in the accumulation of the cullin Cdc53p exclusively in the Rub1p-modified state . This phenotype, which resembles that of fission yeast csn mutants, is due to a biochemical defect in deneddylation that is complemented by wild-type cell lysate and by purified human CSN in vitro . Although three of the four genes encode proteins with PCI domains conserved in metazoan CSN proteins, their disruption does not confer the DNA damage sensitivity described in some fission yeast csn mutants . CONCLUSIONS: Our studies present unexpected evidence for the conservation of a functional homologue of the metazoan CSN, which mediates control of cullin neddylation in budding yeast. Curr Genet, 2002 Aug, 41(5), 333 - 41 Epub 2002 Jul 11. The human minisatellites MS1, MS32, MS205 and CEB1 integrated into the yeast genome exhibit different degrees of mitotic instability but are all stabilised by RAD27; Maleki S et al.; The yeast Rad27 protein is homologous to mammalian Fen1 and is involved in the processing of replication intermediates . Enhanced instability of various artificial repetitive DNA sequences in RAD27-deficient yeast strains has been observed previously and shown to involve preferentially expansion mutations . In the present investigation, we characterised the mitotic instability of alleles of the naturally occurring human minisatellites MS1, MS32, MS205 and CEB1 and the modified MS1 alleles containing more highly homogeneous repeat regions than the original alleles . These minisatellites demonstrated more pronounced instability in rad27 Delta strains, with increases in the frequencies of both expansion and contraction mutants . In RAD27 strains, MS32 and MS205 were relatively stable, while MS1 and CEB1 were unstable, indicating that the effect of RAD27 on stability is influenced by intrinsic properties of the repeat array . This conclusion received further support from the remarkably high frequency of length-mutants observed for the modified allele of MS1 . Thus, our findings emphasise the importance of: (1) comparing results obtained with various naturally occurring minisatellites and (2) manipulating their sequences in attempts to understand the molecular basis for mitotic stability/instability of minisatellite DNA. Antimicrob Agents Chemother, 2002 Sep, 46(9), 3101 - 3 Development of a yeast assay for rapid screening of inhibitors of human-derived Pneumocystis carinii dihydrofolate reductase; Ma L et al.; Human-derived Pneumocystis carinii dihydrofolate reductase (DHFR) was expressed in a Saccharomyces cerevisiae strain whose growth depends on complementation by this enzyme . We utilized a quantitative assay to measure the sensitivity of this yeast strain to DHFR inhibitors . This assay should be useful for identifying new inhibitors of human-derived P . carinii DHFR. Biochim Biophys Acta, 2002 Aug 19, 1591(1-3), 157 - 162 Import of assembled PTS1 proteins into peroxisomes of the yeast Hansenula polymorpha: yes and no! Faber KN, van Dijk R, Keizer-Gunnink I, Koek A, van der Klei IJ, Veenhuis M. Previously, Waterham et al . {EMBO J . 12 (1993) 4785} reported that cytosolic oligomeric alcohol oxidase (AO) is not incorporated into peroxisomes after reassembly of the organelles in the temperature-sensitive peroxisome-deficient mutant pex1-6(ts) of Hansenula polymorpha shifted to permissive growth conditions . Here, we show that the failure to import assembled AO protein is not exemplary for other folded proteins because both an artificial peroxisomal matrix protein, PTS1-tagged GFP (GFP.SKL), and the endogenous dimeric PTS1 protein dihydroxyacetone synthase (DHAS) were imported under identical conditions . In vitro receptor-ligand binding studies using immobilised H . polymorpha Pex5p and crude extracts of methanol-induced pex1-6(ts) cells, showed that AO octamers did not interact with the recombinant PTS1 receptor, at conditions that allowed binding of folded GFP.SKL and dimeric DHAS . This shows that import of oligomeric proteins is not a universal pathway for peroxisomal matrix proteins. Mol Biol Cell, 2002 Aug, 13(8), 2963 - 76 Role of the Rab GTP-binding protein Ypt3 in the fission yeast exocytic pathway and its connection to calcineurin function; Cheng H et al.; A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein . A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin . Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton . It was also detected as a punctate staining in an actin-independent manner . Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature . Consistently, the secretion of GFP fused with the pho1(+) leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants . FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature . These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes. Mol Biol Cell, 2002 Aug, 13(8), 2919 - 32 beta-Tubulin C354 mutations that severely decrease microtubule dynamics do not prevent nuclear migration in yeast; Gupta ML Jr et al.; Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule . Mutations of yeast beta-tubulin C354, which is located near the binding site of some antimitotic compounds, reduce microtubule dynamicity greater than 90% in vivo and in vitro . The resulting intrinsically stable microtubules allowed us to determine which, if any, cellular processes are dependent on dynamic microtubules . The average number of cytoplasmic microtubules decreased from 3 in wild-type to 1 in mutant cells . The single microtubule effectively located the bud site before bud emergence . Although spindles were positioned near the bud neck at the onset of anaphase, the mutant cells were deficient in preanaphase spindle alignment along the mother-bud axis . Spindle microtubule dynamics and spindle elongation rates were also severely depressed in the mutants . The pattern and extent of cytoplasmic microtubule dynamics modulation through the cell cycle may reveal the minimum dynamic properties required to support growth . The ability to alter intrinsic microtubule dynamics and determine the in vivo phenotype of cells expressing the mutant tubulin provides a critical advance in assessing the dynamic requirements of an essential gene function. Mol Biol Cell, 2002 Aug, 13(8), 2826 - 40 Identification of the functional domains of yeast sorting nexins Vps5p and Vps17p; Seaman MN et al.; Sorting nexins (Snxs) are a recently discovered family of conserved hydrophilic cytoplasmic proteins that have been found associated with membranes of the endocytic system and that are implicated in the trafficking of many endosomal membrane proteins, including the epidermal growth factor receptor and transferrin receptor . Snx proteins are partly defined by the presence of a p40 phox homology domain that has recently been shown to bind phosphatidylinositol 3-phosphate . Most Snx proteins also contain a predicted coiled-coils domain in the carboxyl-terminal half of the protein and have been shown to form dimers with other members of the Snx family . The yeast sorting nexins Vps5p and Vps17p form a dimer and are also components of the retromer complex that mediates endosome-to-Golgi transport of the carboxypeptidase Y receptor Vps10p . To functionally define the different domains of the yeast sorting nexins Vps5p and Vps17p, we have generated various truncations to examine the role that the different domains of Vps5p/Vps17p play in their respective functions . Herein, we show that the C-terminal halves of Vps5p and Vps17p, which contain the coiled-coils domains, are necessary and sufficient for their interaction . We have also mapped the retromer assembly domain to the N-terminal half of Vps5p and found that binding of Vps5p by Vps17p synergizes the interaction between Vps5p and other retromer components . Additionally, we have examined which domain(s) of Vps5p is necessary for membrane association. Mol Biol Cell, 2002 Aug, 13(8), 2760 - 70 Overexpression of yeast Hsp110 homolog Sse1p suppresses ydj1-151 thermosensitivity and restores Hsp90-dependent activity; Goeckeler JL et al.; The Saccharomyces cerevisiae heat-shock protein (Hsp)40, Ydj1p, is involved in a variety of cellular activities that control polypeptide fate, such as folding and translocation across intracellular membranes . To elucidate the mechanism of Ydj1p action, and to identify functional partners, we screened for multicopy suppressors of the temperature-sensitive ydj1-151 mutant and identified a yeast Hsp110, SSE1 . Overexpression of Sse1p also suppressed the folding defect of v-Src kinase in the ydj1-151 mutant and partially reversed the alpha-factor translocation defect . SSE1-dependent suppression of ydj1-151 thermosensitivity required the wild-type ATP-binding domain of Sse1p . However, the Sse1p mutants maintained heat-denatured firefly luciferase in a folding-competent state in vitro and restored human androgen receptor folding in sse1 mutant cells . Because the folding of both v-Src kinase and human androgen receptor in yeast requires the Hsp90 complex, these data suggest that Ydj1p and Sse1p are interacting cochaperones in the Hsp90 complex and facilitate Hsp90-dependent activity. Mol Biol Cell, 2002 Aug, 13(8), 2747 - 59 The septation apparatus, an autonomous system in budding yeast; Roh DH et al.; Actomyosin ring contraction and chitin primary septum deposition are interdependent processes in cell division of budding yeast . By fusing Myo1p, as representative of the contractile ring, and Chs2p for the primary septum, to different fluorescent proteins we show herein that the two processes proceed essentially at the same location and simultaneously . Chs2p differs from Myo1p in that it reflects the changes in shape of the plasma membrane to which it is attached and in that it is packed after its action into visible endocytic vesicles for its disposal . To ascertain whether this highly coordinated system could function independently of other cell cycle events, we reexamined the septum-like structures made by the septin mutant cdc3 at various sites on the cell cortex at the nonpermissive temperature . With the fluorescent fusion proteins mentioned above, we observed that in cdc3 at 37 degrees C both Myo1p and Chs2p colocalize at different spots of the cell cortex . A contraction of the Myo1p patch could also be detected, as well as that of a Chs2p patch, with subsequent appearance of vesicles . Furthermore, the septin Cdc12p, fused with yellow or cyan fluorescent protein, also colocalized with Myo1p and Chs2p at the aberrant locations . The formation of delocalized septa did not require nuclear division . We conclude that the septation apparatus, composed of septins, contractile ring, and the chitin synthase II system, can function at ectopic locations autonomously and independently of cell division, and that it can recruit the other elements necessary for the formation of secondary septa. Mol Biol Cell, 2002 Aug, 13(8), 2664 - 80 Multiple functions of sterols in yeast endocytosis; Heese-Peck A et al.; Sterols are essential factors for endocytosis in animals and yeast . To investigate the sterol structural requirements for yeast endocytosis, we created a variety of ergDelta mutants, each accumulating a distinct set of sterols different from ergosterol . Mutant erg2Deltaerg6Delta and erg3Deltaerg6Delta cells exhibit a strong internalization defect of the alpha-factor receptor (Ste2p) . Specific sterol structures are necessary for pheromone-dependent receptor hyperphosphorylation, a prerequisite for internalization . The lack of phosphorylation is not due to a defect in Ste2p localization or in ligand-receptor interaction . Contrary to most known endocytic factors, sterols seem to function in internalization independently of actin . Furthermore, sterol structures are required at a postinternalization step of endocytosis . ergDelta cells were able to take up the membrane marker FM4-64, but exhibited defects in FM4-64 movement through endosomal compartments to the vacuole . Therefore, there are at least two roles for sterols in endocytosis . Based on sterol analysis, the sterol structural requirements for these two processes were different, suggesting that sterols may have distinct functions at different places in the endocytic pathway . Interestingly, sterol structures unable to support endocytosis allowed transport of the glycosylphosphatidylinositol-anchored protein Gas1p from the endoplasmic reticulum to Golgi compartment. Mol Biol Cell, 2002 Aug, 13(8), 2651 - 63 Cytoplasmic localization of Wis1 MAPKK by nuclear export signal is important for nuclear targeting of Spc1/Sty1 MAPK in fission yeast; Nguyen AN et al.; Mitogen-activated protein kinase (MAPK) cascade is a ubiquitous signaling module that transmits extracellular stimuli through the cytoplasm to the nucleus; in response to activating stimuli, MAPKs translocate into the nucleus . Mammalian MEK MAPK kinases (MAPKKs) have in their N termini an MAPK-docking site and a nuclear export signal (NES) sequence, which are known to play critical roles in maintaining ERK MAPKs in the cytoplasm of unstimulated cells . Herein, we show that the Wis1 MAPKK of the stress-activated Spc1 MAPK cascade in fission yeast also has a MAPK-docking site and an NES sequence in its N-terminal domain . Unexpectedly, an inactivating mutation to the NES of chromosomal wis1(+) does not affect the subcellular localization of Spc1 MAPK, whereas this NES mutation disturbs the cytoplasmic localization of Wis1 . However, when Wis1 is targeted to the nucleus by fusing to a nuclear localization signal sequence, stress-induced nuclear translocation of Spc1 is abrogated, indicating that cytoplasmic Wis1 is required for nuclear transport of Spc1 upon stress . Moreover, we have observed that a fraction of Wis1 translocates into the nucleus in response to stress . These results suggest that cytoplasmic localization of Wis1 MAPKK by its NES is important for stress signaling to the nucleus. Mol Biol Cell, 2002 Aug, 13(8), 2607 - 25 Scd5p and clathrin function are important for cortical actin organization, endocytosis, and localization of sla2p in yeast; Henry KR et al.; SCD5 was identified as a multicopy suppressor of clathrin HC-deficient yeast . SCD5 is essential, but an scd5-Delta338 mutant, expressing Scd5p with a C-terminal truncation of 338 amino acids, is temperature sensitive for growth . Further studies here demonstrate that scd5-Delta338 affects receptor-mediated and fluid-phase endocytosis and normal actin organization . The scd5-Delta338 mutant contains larger and depolarized cortical actin patches and a prevalence of G-actin bars . scd5-Delta338 also displays synthetic negative genetic interactions with mutations in several other proteins important for cortical actin organization and endocytosis . Moreover, Scd5p colocalizes with cortical actin . Analysis has revealed that clathrin-deficient yeast also have a major defect in cortical actin organization and accumulate G-actin . Overexpression of SCD5 partially suppresses the actin defect of clathrin mutants, whereas combining scd5-Delta338 with a clathrin mutation exacerbates the actin and endocytic phenotypes . Both Scd5p and yeast clathrin physically associate with Sla2p, a homologue of the mammalian huntingtin interacting protein HIP1 and the related HIP1R . Furthermore, Sla2p localization at the cell cortex is dependent on Scd5p and clathrin function . Therefore, Scd5p and clathrin are important for actin organization and endocytosis, and Sla2p may provide a critical link between clathrin and the actin cytoskeleton in yeast, similar to HIP1(R) in animal cells. Plant Physiol, 2002 Aug, 129(4), 1852 - 7 AtCOX17, an Arabidopsis homolog of the yeast copper chaperone COX17; Balandin T et al.; We have identified a new plant gene, AtCOX17, encoding a protein that shares sequence similarity to COX17, a Cu-binding protein from yeast (Saccharomyces cerevisiae) and vertebrates that mediates the delivery of Cu to the mitochondria for the assembly of a functional cytochrome oxidase complex . The newly characterized Arabidopsis protein has six Cys residues at positions corresponding to those known to coordinate Cu binding in the yeast homolog . Moreover, we show that the Arabidopsis COX17 cDNA complements a COX17 mutant of yeast restoring the respiratory deficiency associated with that mutation . These two lines of evidence indicate that the plant protein identified here is a functional equivalent of yeast COX17 and might serve as a Cu delivery protein for the plant mitochondria . COX17 was identified by investigating the hypersensitive response-like necrotic response provoked in tobacco (Nicotiana tabacum) leaves after harpin inoculation . AtCOX17 expression was activated by high concentrations of Cu, bacterial inoculation, salicylic acid treatment, and treatments that generated NO and hydrogen peroxide . All of the conditions inducing COX17 are known to inhibit mitochondrial respiration and to produce an increase of reactive oxygen species, suggesting that gene induction occurs in response to stress situations that interfere with mitochondrial function. Nucleic Acids Res, 2002 Aug 15, 30(16), 3540 - 7 Transcription and nuclear transport of CAG/CTG trinucleotide repeats in yeast; Fabre E et al.; Trinucleotide repeats are involved in several neurological disorders in humans . DNA sequences containing CAG/CTG repeats are prone to slippage during replication and double-strand break repair . The effects of trinucleotide repeats on transcription and on nuclear export were analyzed in vivo in yeast . Transcription of a CAG/CTG trinucleotide repeat in the 3'-untranslated region of a URA3 reporter gene leads to transcription of messenger RNAs several kilobases longer than the expected size . These long mRNAs form more readily when CAG rather than CTG repeats are transcribed . CAG- or CUG-containing transcripts show a non-homogeneous cellular localization . We propose that long mRNAs result from transcription slippage, and discuss the possible implications for human diseases. J Cell Biol, 2002 Aug 19, 158(4), 669 - 79 Epub 2002 Aug 12. Remodeling of organelle-bound actin is required for yeast vacuole fusion; Eitzen G et al.; Actin participates in several intracellular trafficking pathways . We now find that actin, bound to the surface of purified yeast vacuoles in the absence of cytosol or cytoskeleton, regulates the last compartment mixing stage of homotypic vacuole fusion . The Cdc42p GTPase is known to be required for vacuole fusion . We now show that proteins of the Cdc42p-regulated actin remodeling cascade (Cdc42p --> Cla4p --> Las17p/Vrp1p --> Arp2/3 complex --> actin) are enriched on isolated vacuoles . Vacuole fusion is dramatically altered by perturbation of the vacuole-bound actin, either by mutation of the ACT1 gene, addition of specific actin ligands such as latrunculin B or jasplakinolide, antibody to the actin regulatory proteins Las17p (yeast Wiskott-Aldrich syndrome protein) or Arp2/3, or deletion of actin regulatory genes . On docked vacuoles, actin is enriched at the "vertex ring" membrane microdomain where fusion occurs and is required for the terminal steps leading to membrane fusion . This role for actin may extend to other trafficking systems. J Biol Chem, 2002 Oct 18, 277(42), 39409 - 16 Epub 2002 Aug 09. Mutations in the RING domain of TFB3, a subunit of yeast transcription factor IIH, reveal a role in cell cycle progression; Jona G et al.; The RNA polymerase II general transcription factor TFIIH is composed of 9 known subunits and possesses DNA helicase and protein kinase activities . The kinase subunits of TFIIH in animal cells, Cdk7, cyclin H, and MAT1, were independently isolated as an activity termed CAK (Cdk-activating kinase), which phosphorylates and activates cell cycle kinases . However, CAK activity of TFIIH subunits could not be demonstrated in budding yeast . TFB3, the 38-kDa subunit of yeast TFIIH, is the homolog of mammalian MAT1 . By random mutagenesis we have isolated a temperature-sensitive mutation in the conserved RING domain . The mutant Tfb3 protein associates less efficiently with the kinase moiety of TFIIH than the wild type protein . In contrast to lethal mutants in other subunits of TFIIH, this mutation does not impair general transcription . Transcription of CLB2, and possibly other genes, is reduced in the mutant . At the restrictive temperature, the cells display a defect in cell cycle progression, which is manifest at more than one phase of the cycle . To conclude, in the present study we bring another demonstration of the multifunctional nature of TFIIH. Structure (Camb), 2002 Aug, 10(8), 1117 - 25 Structure of yeast RNA polymerase II in solution: implications for enzyme regulation and interaction with promoter DNA; Craighead JL et al.; An 18 A resolution structure of the 12-subunit yeast RNA polymerase II (RNAPII) calculated from electron microscope images of single particles preserved in amorphous ice reveals the conformation of the enzyme in solution . The Rpb4/Rpb7 polymerase subunit complex was localized and found to be ideally positioned to determine the path of the nascent RNA transcript . The RNAPII structure suggests a revised mode of interaction with promoter DNA and demonstrates that regulation of RNAPII must involve structural changes that render the enzyme competent for initiation. Curr Biol, 2002 Jul 23, 12(14), 1240 - 4 Selective recruitment of TAFs by yeast upstream activating sequences . Implications for eukaryotic promoter structure; Li XY et al.; The general transcription factor TFIID is composed of the TATA box binding protein (TBP) and multiple TBP-associated factors (TAFs) . In yeast, promoters can be grouped into two classes based on the involvement of TAFs . TAF-dependent (TAF(dep)) promoters require TAFs for transcription, and TBP and TAFs are present at comparable levels on these promoters . TAF-independent (TAF(ind)) promoters do not require TAFs for activity, and TAFs are either absent or present at levels far below those of TBP on these promoters . Here, we demonstrate that the upstream activating sequence (UAS) mediates the selective recruitment of TAFs to TAF(dep) promoters . A TAF(ind) UAS fails to recruit TAFs and to direct efficient transcription when inserted upstream of a TAF(dep) core promoter . This transcriptional defect can be overcome by a potent activator, indicating that a strong activation domain can compensate for the absence of TAFs on a TAF(dep) core promoter . Our results reveal a requirement for compatibility between the UAS and core promoter and thus help explain previous reports that only certain yeast UAS-core promoter combinations and mammalian enhancer-promoter combinations are efficiently transcribed . The differential recruitment of TAFs by UASs provides strong evidence for the proposal that in vivo TAFs are the targets of some, but not all, activators. Arch Biochem Biophys, 2002 Sep 1, 405(1), 38 - 43 ATP synthase of yeast: structural insight into the different inhibitory potencies of two regulatory peptides and identification of a new potential regulator; Hong S et al.; Mitochondrial ATP synthases, the major producers of ATP in higher eukaryotic cells, are known to be regulated by a peptide designated IF(1) . In contrast, in yeast three such peptides have been identified, IF(1) and STF(1), which inhibit the reverse ATPase reaction, and STF(2), a modulator of the action of these inhibitors . Despite significant homology to IF(1), STF(1) exhibits less than half ( approximately 40%) its inhibitory potency . The two-fold purpose of this bioinformatic study was to gain structural insight into the different inhibitory potencies of IF(1) and STF(1) and to determine to what extent yeast are unique in employing multiple peptides to regulate the ATP synthase . Sequence and secondary structural analyses and comparison with the known structure of bovine IF(1) predicted a dimeric structure for yeast STF(1) in which the C-terminal regions form a coiled-coil . Moreover, sequence comparisons showed that within this C-terminal region a conserved acidic residue (Asp 59) in yeast IF(1) is replaced by Asn in STF(1) . In the known structure of bovine IF(1), predicted to be very similar to that of yeast IF(1), the residue Glu 68 corresponding to Asp 59 participates in the formation of a four-residue conserved acidic cluster in the middle of the coiled-coil in the C-terminal region . It is deduced here that this acidic cluster is likely to be important in the regulation of IF(1)'s inhibitory capacity and that replacement of conserved Asp 59 by Asn in STF(1) may reduce its potency . Although other homologs to the inhibitors IF(1) and STF(1) were not found in searches of available eukaryotic genomes, including human, a new homolog, named STF(3), with 65% identity to the modulator STF(2), was discovered within the yeast genome and identified to be expressed by searching the yeast EST database . Thus, yeast appears unique in regulating the ATP synthase by involving multiple peptides (IF(1), STF(1), STF(2), and perhaps STF(3)). Trends Genet, 2002 Sep, 18(9), 479 - 85 Checking cell size in yeast; Rupes I; To remain viable, cells have to coordinate cell growth with cell division . In yeast, this occurs at two control points: the boundaries between G1 and S phases, also known as Start, and between G2 and M phases . Theoretically, coordination can be achieved by independent regulation of growth and division, or by participation of surveillance mechanisms in which cell size feeds back into cell-cycle control . This article discusses recent advances in the identification of sizing mechanisms in budding and in fission yeast, and how these mechanisms integrate with environmental stimuli . A comparison of the G1-S and G2-M size-control modules in the two species reveals a degree of conservation higher than previously thought . This reinforces the notion that internal sizing could be a conserved feature of cell-cycle control throughout eukaryotes. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(2), 147 - 153 The Autonomous Replication Function (ARS) of the Scaffold-associated Region (SAR) of Silkworm Attacus ricini rDNA in Yeast; He ML et al.; The one kilo-base scaffold-associated region (SAR) of silkworm Attacus ricini rRNA gene (rDNA) has been identified previously({1}) . To investigate the critical sequence and the relative activity of ARS (autonomously replicating sequence), a set of restriction from covered the whole rRNA gene unit were subcloned into the nonreplicative pSKY vector . Among the seven plasmids constructed, the plasmid pSEY, having SAR, gave obvious positive replication activity in yeast as determined by the transformation efficiency . Further dissection of SAR demonstrated that plasmid pAAY, having a 0.26 kb small fragment of SAR was 40-50 fold more active then the whole SAR, while pSAY, having the remaining part of SAR, showed no activity . There were fifteen ARS consensus sequences (ACSs) within SAR being identified through sequenced alignment . It was found that only three ACSs in pAAY, located at the 3' end of SAR displayed a positive ARS activity and the remaining sequence having the other twelve ACSs functioned as a repressor . The in vitro binding assay showed that SAR from the silkworm rRNA gene bound to the yeast nuclear scaffold . These results suggest that SAR is evolutionarily conserved, and there is a close correlation between SAR and ARS activity . Detailed analysis of the positive and negative regulatory elements in SAR can be carried out based on these results. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(3), 217 - 219 Molecular Mechanism of Yeast Phosphatase Gene Expression; Ao SZ; No Abstract Analyst, 2002 Jul, 127(7), 967 - 71 Chemical responses of single yeast cells studied by fluorescence microspectroscopy under solution-flow conditions; Kogi O et al.; A microspectroscopy system combined with a fluid manifold was developed to manipulate and analyze "single" living cells . A sample buffer solution containing living cells was introduced into a flow cell set on a thermostated microscope stage and a few cells were allowed to attach to the bottom wall of the flow cell . With these living cells being attached to the wall, other floating cells were pumped out by flowing a buffer solution . These procedures made it possible to keep a few cells in the flow cell and to analyze single cells by fluorescence microspectroscopy . The technique was applied to study the time course of staining processes of single living yeast (Saccharomyces cerevisiae) cells by using two types of a fluorescent probe . The present methodology was shown to be of primary importance for obtaining biochemical/physiological information on single living cells and also for studying cell-to-cell variations in several characteristics. Curr Genet, 2002 Jul, 41(4), 208 - 16 Epub 2002 Jul 05. Apoptosis in yeast: a new model system with applications in cell biology and medicine; Madeo F et al.; Apoptosis is a highly coordinated cellular suicide program crucial for metazoan health and diseases . Although its increasing importance in cancer, neurodegenerative disorders and AIDS led to intense research and a better understanding of apoptosis, many details of its regulation or the apoptotic phenotypes are poorly understood . The complex regulatory network and the often contradictory results obtained with human cell lines made application of an easier model system desirable . Apoptosis in yeast promises to provide a better understanding of the genetics of apoptosis . During the past 2 years, scientists were successful in identifying new cell-death regulators of humans, plants and fungi using Saccharomyces cerevisiae . The finding of apoptotic phenotypes, even in protists, suggests that apoptosis developed in unicellular organisms long before the evolutionary separation between fungi, plants and metazoan animals occurred. J Biol Chem, 2002 Nov 8, 277(45), 43495 - 504 Epub 2002 Aug 08. Regulation of the cell integrity pathway by rapamycin-sensitive TOR function in budding yeast; Torres J et al.; The TOR (target of rapamycin) pathway controls cell growth in response to nutrient availability in eukaryotic cells . Inactivation of TOR function by rapamycin or nutrient exhaustion is accompanied by triggering various cellular mechanisms aimed at overcoming the nutrient stress . Here we report that in Saccharomyces cerevisiae the protein kinase C (PKC)-mediated mitogen-activated protein kinase pathway is regulated by TOR function because upon specific Tor1 and Tor2 inhibition by rapamycin, Mpk1 is activated rapidly in a process mediated by Sit4 and Tap42 . Osmotic stabilization of the plasma membrane prevents both Mpk1 activation by rapamycin and the growth defect that occurs upon the simultaneous absence of Tor1 and Mpk1 function, suggesting that, at least partially, TOR inhibition is sensed by the PKC pathway at the cell envelope . This process involves activation of cell surface sensors, Rom2, and downstream elements of the mitogen-activated protein kinase cascade . Rapamycin also induces depolarization of the actin cytoskeleton through the TOR proteins, Sit4 and Tap42, in an osmotically suppressible manner . Finally, we show that entry into stationary phase, a physiological situation of nutrient depletion, also leads to the activation of the PKC pathway, and we provide further evidence demonstrating that Mpk1 is essential for viability once cells enter G(0). J Bioenerg Biomembr, 2002 Jun, 34(3), 165 - 76 Does any yeast mitochondrial carrier have a native uncoupling protein function? Roussel D, Harding M, Runswick MJ, Walker JE, Brand MD. In this study, we explore the hypothesis that some member of the mitochondrial carrier family has specific uncoupling activity that is responsible for the basal proton conductance of mitochondria . Twenty-seven of the 35 yeast mitochondrial carrier genes were independently disrupted in Saccharomyces cerevisiae . Six knockout strains did not grow on nonfermentable carbon sources such as lactate . Mitochondria were isolated from the remaining 21 strains, and their proton conductances were measured . None of the 21 carriers contributed significantly to the basal proton leak of yeast mitochondria . A possible exception was the succinate/fumarate carrier encoded by the Xc2 gene, but deletion of this gene also affected yeast growth and respiratory chain activity, suggesting a more general alteration in mitochondrial function . If a specific protein is responsible for the basal proton conductance of yeast mitochondria, its identity remains unknown. J Biol Chem, 2002 Oct 25, 277(43), 40981 - 8 Epub 2002 Aug 06. Stalk segment 5 of the yeast plasma membrane H(+)-ATPase . Labeling with a fluorescent maleimide reveals a conformational change during glucose activation; Miranda M et al.; Glucose is well known to cause a rapid, reversible activation of the yeast plasma membrane H(+)-ATPase, very likely mediated by phosphorylation of two or more Ser/Thr residues near the C terminus . Recent mutagenesis studies have shown that glucose-dependent activation can be mimicked constitutively by amino acid substitutions in stalk segment 5 (S5), an alpha-helical stretch connecting the catalytic part of the ATPase with transmembrane segment 5 (Miranda, M., Allen, K . E., Pardo, J . P., and Slayman, C . W . (2001) J . Biol . Chem . 276, 22485-22490) . In the present work, the fluorescent maleimide Alexa-488 has served as a probe for glucose-dependent changes in the conformation of S5 . Experiments were carried out in a "3C" version of the ATPase, from which six of nine native cysteines had been removed by site-directed mutagenesis to eliminate background labeling by Alexa-488 . In this construct, three of twelve cysteines introduced at various positions along S5 (A668C, S672C, and D676C) reacted with the Alexa dye in a glucose-independent manner, as shown by fluorescent labeling of the 100 kDa Pma1 polypeptide and by isolation and identification of the corresponding tryptic peptides . Especially significant was the fact that three additional cysteines reacted with Alexa-488 more rapidly (Y689C) or only (V665C and L678C) in plasma membranes from glucose-metabolizing cells . The results support a model in which the S5 alpha-helix undergoes a significant change in conformation to expose positions 665, 678, and 689 during glucose-dependent activation of the ATPase. Cell Mol Life Sci, 2002 Jun, 59(6), 903 - 8 Regulation of longevity and stress resistance: a molecular strategy conserved from yeast to humans? Longo VD, Fabrizio P. Recent studies implicate similar proteins in the regulation of longevity in organisms ranging from yeast to mice . Studies in yeast and worms suggest that inactivation of glucose or insulin/insulin-like growth factor-l (IGF-1) signaling pathways extends longevity by causing a shift from a reproductive phase to a non-reproductive maintenance phase involving the expression of many genes . These stress resistance pathways appear to have evolved to induce maintenance systems and promote longevity during periods of starvation . In yeast, mutations that decrease the activity of glucose signaling pathways extend longevity by activating stress resistance transcription factors that regulate the expression of genes involved in antioxidant and heat protection, glycogen storage, protein degradation, DNA repair, and metabolism . A remarkably similar set of proteins regulated by growth factors that control glucose metabolism is implicated in life span extension in worms, and possibly in flies and mice . Studies in worms and flies point to secondary hormones as mediators of the effect of insulin/ IGF-1 signaling on longevity, whereas studies in yeast and mammalian cells indicate that glucose or insulin/ IGF-1 may decrease longevity by directly down-regulating stress resistance genes . In yeast, longevity mutations postpone superoxide toxicity and mitochondrial damage . However, the small life span extension caused by the overexpression of superoxide dismutases and catalase in yeast and flies indicates that increased antioxidant protection alone cannot be responsible for the major life span extension caused by signal transduction mutations . Although we are only beginning to understand the molecular mechanisms that mediate life span extension, the similarities between longevity regulatory pathways in organisms ranging from yeast to mice suggest that insulin/ IGF-1 signaling pathways may also regulate cell damage and longevity in humans. J Protein Chem, 2002 May, 21(4), 265 - 77 Structure of triphosphoryl nucleotide bound at the active site of yeast hexokinase: 1H-nuclear magnetic resonance study; Maity H et al.; Conformation of a nonhydrolyzable adenosine triphosphate (ATP) analogue, adenylyl-(beta,gamma-methylene)-diphosphonate (AMPPCP) bound at the active site of yeast hexokinase-PII was determined by proton two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY) and molecular dynamics simulations . The effect of the glucose-induced domain closure on the conformation of the nucleotide was evaluated by making measurements on two different complexes: PII AMPPCPMg(II) and PII-Glc.AMPPCPMg(Il) . TRNOE measurements were made at 500 MHz, 10 degress C, as a function of several mixing times varying in the range of 40 to 200 ms . Interproton distances derived from the analysis of NOE buildup curves were used as restraints in molecular dynamics simulations to determine the conformation of the enzyme bound nucleotide . The adenosine moiety was found to bind in high anti conformation with a glycosidic torsion angle chi = 48 +/- 5 degrees in both complexes . However, significant differences in the conformations of the ribose and triphosphoryl chain of the nucleotide are observed between the two complexes . The phase angles of pseudorotation P in PII.AMPPCPMg(II) and PII.Glc.AMPPCPMg(II) are 87 degrees and 77 degrees, describing a OE and OT4 sugar pucker and the amplitudes of the sugar pucker (tau) are 37 degrees and 61 degrees, respectively. Int J Oncol, 2002 Sep, 21(3), 637 - 41 p53 null mutations detected by a p53 yeast functional assay predict a poor outcome in young esophageal carcinoma patients; Osugi H et al.; Esophageal carcinoma is one of the most common gastrointestinal malignant neoplasms in the world . Recent advances in treatment modalities as well as surgical resection techniques have improved the changes of survival of patients with esophageal carcinoma, although the prognosis is worse than for the other gastrointestinal carcinomas . A more precise stratification beyond clinicopathological classification may help determine optimal treatment . The importance of p53 gene mutations in the pathogenesis of human esophageal carcinoma is well established, but it is still controversial whether the presence of p53 mutations adversely affects individual patient prognosis . In this study, we investigated the p53 mutations of esophageal carcinomas and their correlation with clinicopathologic factors . We employed a p53 yeast functional assay because it is highly sensitive and can detect mutations based on the actual function of the p53 gene, clarifying more precisely the role of this gene in esophageal carcinomas . We also studied young patients (< or =65 years old), because our previous study raised the possibility of differences in the importances in esophageal carcinogenesis in young and old patients . Of 43 young esophageal carcinoma patients (42 squamous cell and 1 undifferentiated carcinoma), 38 (88.4%) harbored p53 mutations . Twenty-seven missense and 11 null mutations were detected, but the presence of p53 mutations did not correlate with any clinicopathologic factor . However, the null mutation was a significant indicator of a poor outcome (P=0.0278) . All except one patient who harbored null mutation died within 3 years after a macroscopically curative resection . These data suggest that the type of p53 gene mutation may be predictive of outcome in young esophageal carcinoma patients . Furthermore, null mutations causing loss of function of the gene product may play a more important role than missense mutations in tumor progression. Mol Cell Biol, 2002 Sep, 22(17), 6046 - 55 Biogenesis of yeast telomerase depends on the importin mtr10; Ferrezuelo F et al.; Telomerase is a ribonucleoprotein particle (RNP) involved in chromosome end replication, but its biogenesis is poorly understood . The RNA component of yeast telomerase (Tlc1) is synthesized as a polyadenylated precursor and then processed to a mature poly(A)- form . We report here that the karyopherin Mtr10p is required for the normal accumulation of mature Tlc1 and its proper localization to the nucleus . Neither TLC1 transcription nor the stability of poly(A)- Tlc1 is significantly affected in mtr10delta cells . Tlc1 was mostly nuclear in a wild-type background, and this localization was not affected by mutations in other telomerase components . Strikingly, in the absence of Mtr10p, Tlc1 was found dispersed throughout the entire cell . Our results are compatible with two alternative models . First, Mtr10p may import a cytoplasmic complex containing Tlc1 and perhaps other components of telomerase, and shuttling of Tlc1 from the nucleus to the cytoplasm and back may be necessary for the biogenesis of telomerase (the "shuttling" model) . Second, Mtr10p may be necessary for the nuclear import of some enzyme needed for the nuclear processing and maturation of Tlc1, and in the absence of this maturation, poly(A)+ Tlc1 is aberrantly exported to the cytoplasm (the "processing enzyme" model). J Biol Chem, 2002 Oct 18, 277(42), 39289 - 95 Epub 2002 Aug 06. Formation of the yeast F1F0-ATP synthase dimeric complex does not require the ATPase inhibitor protein, Inh1; Dienhart M et al.; The yeast F1F0-ATP synthase forms dimeric complexes in the mitochondrial inner membrane and in a manner that is supported by the F0-sector subunits, Su e and Su g . Furthermore, it has recently been demonstrated that the binding of the F1F0-ATPase natural inhibitor protein to purified bovine F1-sectors can promote their dimerization in solution (Cabezon, E., Arechaga, I., Jonathan P., Butler, G., and Walker J . E . (2000) J . Biol . Chem . 275, 28353-28355) . It was unclear until now whether the binding of the inhibitor protein to the F1 domains contributes to the process of F1F0-ATP synthase dimerization in intact mitochondria . Here we have directly addressed the involvement of the yeast inhibitor protein, Inh1, and its known accessory proteins, Stf1 and Stf2, in the formation of the yeast F1F0-ATP synthase dimer . Using mitochondria isolated from null mutants deficient in Inh1, Stf1, and Stf2, we demonstrate that formation of the F(1)F(0)-ATP synthase dimers is not adversely affected by the absence of these proteins . Furthermore, we demonstrate that the F1F0-ATPase monomers present in su e null mutant mitochondria can be as effectively inhibited by Inh1, as its dimeric counterpart in wild-type mitochondria . We conclude that dimerization of the F1F0-ATP synthase complexes involves a physical interaction of the membrane-embedded F0 sectors from two monomeric complexes and in a manner that is independent of inhibitory activity of the Inh1 and accessory proteins. J Biol Chem, 2002 Oct 18, 277(42), 39634 - 41 Epub 2002 Aug 06. The core protein of hepatitis C virus is imported into the nucleus by transport receptor Kap123p but inhibits Kap121p-dependent nuclear import of yeast AP1-like transcription factor in yeast cells; Isoyama T et al.; The core protein of hepatitis C virus (HCV) is a major component of the viral nucleocapsid . The HCV core protein includes nuclear localization signal-like sequences and has various effects on cellular metabolism, playing roles, for example, in the regulation of transcription, apoptosis, and transformation . To examine the possibility of an effect of the core protein on nucleocytoplasmic transport, we used the yeast Saccharomyces cerevisiae as a model system . The core protein (p23) is processed to p21 and is localized in both the cytoplasm and nucleus in yeast cells, similar to that observed in mammalian cells in several cases . The nuclear import of the core protein requires the activity of small GTPase Ran/Gsp1p and is mediated by Kap123p in yeast cells . When the core protein was expressed in yeast cells, the import of the yeast AP1-like transcription factor Yap1p into the nucleus was inhibited . Experiments in vitro involving Kap121p, also known as Pse1p, a receptor for the nuclear import of Yap1p, indicated that the amount of Yap1p bound to Kap121p was reduced in the presence of core protein . These results suggest that the HCV core protein affects cellular metabolism by disturbing transport of proteins to the nucleus. BMC Biochem . 2002 Aug 07;3(1):22. Combinatorial diversity of fission yeast SCF ubiquitin ligases by homo- and heterooligomeric assemblies of the F-box proteins Pop1p and Pop2p; Seibert V et al.; BACKGROUND: SCF ubiquitin ligases share the core subunits cullin 1, SKP1, and HRT1/RBX1/ROC1, which associate with different F-box proteins . F-box proteins bind substrates following their phosphorylation upon stimulation of various signaling pathways . Ubiquitin-mediated destruction of the fission yeast cyclin-dependent kinase inhibitor Rum1p depends on two heterooligomerizing F-box proteins, Pop1p and Pop2p . Both proteins interact with the cullin Pcu1p when overexpressed, but it is unknown whether this reflects their co-assembly into bona fide SCF complexes . RESULTS: We have identified Psh1p and Pip1p, the fission yeast homologues of human SKP1 and HRT1/RBX1/ROC1, and show that both associate with Pop1p, Pop2p, and Pcu1p into a ~500 kDa SCFPop1p-Pop2p complex, which supports polyubiquitylation of Rum1p . Only the F-box of Pop1p is required for SCFPop1p-Pop2p function, while Pop2p seems to be attracted into the complex through binding to Pop1p . Since all SCFPop1p-Pop2p subunits, except for Pop1p, which is exclusively nuclear, localize to both the nucleus and the cytoplasm, the F-box of Pop2p may be critical for the assembly of cytoplasmic SCFPop2p complexes . In support of this notion, we demonstrate individual SCFPop1p and SCFPop2p complexes bearing ubiquitin ligase activity . CONCLUSION: Our data suggest that distinct homo- and heterooligomeric assemblies of Pop1p and Pop2p generate combinatorial diversity of SCFPop function in fission yeast . Whereas a heterooligomeric SCFPop1p-Pop2p complex mediates polyubiquitylation of Rum1p, homooligomeric SCFPop1p and SCFPop2p complexes may target unknown nuclear and cytoplasmic substrates. Genes Cells, 2002 Aug, 7(8), 781 - 9 Mapping of early firing origins on a replication profile of budding yeast; Yabuki N et al.; BACKGROUND: Understanding of the firing time determination of replication origins in the entire genome will require a genome-wide survey of replication origins and their mapping on chromosomes . A microarray technology was applied to obtain a genome-wide profile of DNA replication and to classify early firing origins . RESULTS: A total of 260 potential replication origins (PROs) were identified in the entire budding yeast genome: 247 as defined peaks on the replication profile and 13 as regions located in the chromosomal termini . Based on the firing time, the 247 PROs were classified into 143 early PROs and 104 late PROs, that were not randomly distributed on chromosomes but formed separated clusters . Most of the early PROs were found to fire in the presence of hydroxyurea, indicating that they were free from the control of the intra-S-checkpoint mediated by Mec1 and Rad53 . CONCLUSIONS: The monitoring method of DNA replication and the analysis method of microarray data used in this study proved powerful for obtaining a genome-wide view of the initiation and progression of DNA replication. Can J Microbiol, 2002 Jun, 48(6), 522 - 9 The effects of fungicides on the phylloplane yeast populations of creeping bentgrass; Buck JW et al.; The effects of fungicides on population size and the development of fungicide resistance in the phylloplane yeast flora of bentgrass was investigated . In the spring of 2001, azoxystrobin, chlorothalonil, flutolanil, and propiconazole were applied separately over a 6-week period to creeping bentgrass (Agrostis palustris Huds.) . Total and fungicide-resistant yeast populations were assessed by dilution plating onto either potato dextrose agar or potato dextrose agar amended with the test fungicides . Total yeast populations in the fungicide-treated plots were significantly lower than the check plots on three out of four sample dates . In the fall, azoxystrobin or propiconazole were applied twice to the bentgrass over 3 weeks . Significantly larger total yeast populations were observed compared with resistant or highly resistant populations for each treatment on every sample date . Total yeast populations were significantly higher in the check plots compared with either the propiconazole- or azoxystrobin-treated plots on the first three of five sample dates . A collection of yeasts (N = 114) with no prior exposure to fungicides were more sensitive to chlorothalonil, propiconazole, flutolanil, and iprodione than a second group (N = 115) isolated from fungicide-treated turfgrass . These results suggest that fungicide resistance among phylloplane yeasts is widespread and could be an important factor in the development of biological control agents for turfgrass diseases. RNA, 2002 Jul, 8(7), 948 - 58 One-step affinity purification of the yeast ribosome and its associated proteins and mRNAs; Inada T et al.; We describe a one-step affinity method for purifying ribosomes from the budding yeast Saccharomyces cerevisiae . Extracts from yeast strains expressing only C-terminally tagged Rpl25 protein or overexpressing this protein in the presence of endogenous Rpl25p were used as the starling materials . The purification was specific for tagged 60S subunits, and resulted in the copurification of 80S subunits and polysomes, as well as ribosome-associated proteins and mRNAs . Two of these associated proteins, Mpt4p and Asc1p, were nearly stoichiometrically bound to the ribosome . In addition, the degree of mRNA association with the purified ribosomes was found to reflect the mRNA's translational status within the cell . The one-step purification of ribosome and its associated components from a crude extract should provide an important tool for future structural and biochemical studies of the ribosome, as well as for expression profiling of translated mRNAs. J Mol Evol, 2002 Jul, 55(1), 14 - 23 Characterization of the gene conversions between the multigene family members of the yeast genome; Drouin G; Stanley Sawyer's gene conversion detection method, implemented in his GENECONV computer program, was used to detect and characterize the gene conversions between the multigene family members of the yeast genome . This method gave different gene conversion frequencies and size distribution for gene families with two members and multigene families with more than two members . The 69 gene conversions detected in multigene families with more than two members occur at a frequency of 7.8% gene conversion/pair of genes compared and have an average size of 173+/-220 nucleotides . Larger gene conversions are found only between more similar genes, the genes involved in gene conversions are distributed almost randomly among the 16 yeast chromosomes, and the frequency of gene conversions increases as the distance between repeated genes decreases . In contrast to previous studies, no relationship was observed between the level of expression of a gene and its involvement in gene conversions . These analyses also suggest that gene conversions might occur by different mechanisms in closely linked genes and unlinked genes . The excess of converted regions at the 3? end of unlinked genes suggests that recombination with incomplete cDNA molecules is the main mechanism responsible for gene conversions between such genes. Hum Mol Genet, 2002 Aug 15, 11(17), 2025 - 36 The yeast frataxin homolog Yfh1p plays a specific role in the maturation of cellular Fe/S proteins; Muhlenhoff U et al.; The mitochondrial matrix protein frataxin is depleted in patients with Friedreich's ataxia, the most common autosomal recessive ataxia . While frataxin is important for intracellular iron homeostasis, its exact cellular role is unknown . Deletion of the yeast frataxin homolog YFH1 yields mutants ((Delta)yfh1) that, depending on the genetic background, display various degrees of phenotypic defects . This renders it difficult to distinguish primary (early) from secondary (late) consequences of Yfh1p deficiency . We have constructed a yeast strain (Gal-YFH1) that carries the YFH1 gene under the control of a galactose-regulated promoter . Yfh1p-deficient Gal-YFH1 cells are far less sensitive to oxidative stress than (Delta)yfh1 mutants, maintain mitochondrial DNA, and synthesize heme at wild-type rates . Yfh1p depletion causes a strong reduction in the assembly of mitochondrial Fe/S proteins both in vivo and in detergent extracts of mitochondria . Impaired Fe/S protein biogenesis explains the respiratory deficiency of Gal-YFH1 cells . Furthermore, Yfh1p-depleted Gal-YFH1 cells show decreased maturation of cytosolic Fe/S proteins and accumulation of mitochondrial iron . This latter phenotype is common for defects in cytosolic Fe/S protein assembly . Together, our data demonstrate a specific role of frataxin in the biosynthesis of cellular Fe/S proteins and exclude most of the previously suggested functions . Friedreich's ataxia may therefore represent a disorder caused by defects in Fe/S protein maturation. FEMS Microbiol Rev, 2002 Aug, 26(3), 257 - 76 The viral killer system in yeast: from molecular biology to application; Schmitt MJ et al.; Since the initial discovery of the yeast killer system almost 40 years ago, intensive studies have substantially strengthened our knowledge in many areas of biology and provided deeper insights into basic aspects of eukaryotic cell biology as well as into virus-host cell interactions and general yeast virology . Analysis of killer toxin structure, synthesis and secretion has fostered understanding of essential cellular mechanisms such as post-translational prepro-protein processing in the secretory pathway . Furthermore, investigation of the receptor-mediated mode of toxin action proved to be an effective means for dissecting the molecular structure and in vivo assembly of yeast and fungal cell walls, providing important insights relevant to combating infections by human pathogenic yeasts . Besides their general importance in understanding eukaryotic cell biology, killer yeasts, killer toxins and killer viruses are also becoming increasingly interesting with respect to possible applications in biomedicine and gene technology . This review will try to address all these aspects. Cell Calcium, 2002 Aug, 32(2), 83 - 91 Genome-wide analysis of yeast transcription upon calcium shortage; Lombardia LJ et al.; Several regulatory circuits related to important functions, like membrane excitation, immunoresponse, replication, control of the cell cycle and differentiation, among others, cause an increase in intracellular calcium level that finally has a consequence upon transcription of specific genes . The sequencing of the whole genome of eukaryotic cells enables genome-wide analysis of gene expression under many conditions not yet assessed by conventional methods . Using the array technology, the effect of calcium shortage in yeast cells was studied . Correspondence analysis of data showed that there is a response in transcription that is correlated to calcium shortage . The distribution of up-regulated-genes in functional categories suggests a regulatory connection between the cell-cycle progression and the energetic metabolic requirements for growth and division . In silico analysis of promoters reveals the frequent appearance of the Mlu I cell cycle box (MCB) cis element that binds the transcriptional regulatory factor Mcm1. Genes Dev, 2002 Aug 1, 16(15), 1919 - 33 EXO1-dependent single-stranded DNA at telomeres activates subsets of DNA damage and spindle checkpoint pathways in budding yeast yku70Delta mutants; Maringele L et al.; We have examined the role of checkpoint pathways in responding to a yku70Delta defect in budding yeast . We show that CHK1, MEC1, and RAD9 checkpoint genes are required for efficient cell cycle arrest of yku70Delta mutants cultured at 37 degrees C, whereas RAD17, RAD24, MEC3, DDC1, and DUN1 play insignificant roles . We establish that cell cycle arrest of yku70Delta mutants is associated with increasing levels of single-stranded DNA in subtelomeric Y' regions, and find that the mismatch repair-associated EXO1 gene is required for both ssDNA generation and cell cycle arrest of yku70Delta mutants . In contrast, MRE11 is not required for ssDNA generation . The behavior of yku70Delta exo1Delta double mutants strongly indicates that ssDNA is an important component of the arrest signal in yku70Delta mutants and demonstrates a link between damaged telomeres and mismatch repair-associated exonucleases . This link is confirmed by our demonstration that EXO1 also plays a role in ssDNA generation in cdc13-1 mutants . We have also found that the MAD2 but not the BUB2 spindle checkpoint gene is required for efficient arrest of yku70Delta mutants . Therefore, subsets of both DNA-damage and spindle checkpoint pathways cooperate to regulate cell division of yku70Delta mutants. Eur J Biochem, 2002 Aug, 269(15), 3821 - 30 Characterization and regulation of yeast Ca2+-dependent phosphatidylethanolamine-phospholipase D activity; Tang X et al.; An unconventional phospholipase D (PLD) activity was identified recently in Saccharomyces cerevisiae which is Ca2+-dependent, preferentially hydrolyses phosphatidylethanolamine (PtdEtn) and phosphatidylserine and does not catalyse a transphosphatidylation with primary short-chain alcohols . We have characterized the cytosolic and membrane-bound forms of the yeast PtdEtn-PLD and examined the regulation of its activity under certain growth, nutritional and stress conditions . Both forms of PtdEtn-PLD activity were similarly activated by Ca2+ ions in a biphasic manner . Likewise, other divalent cations affected both cytosolic and membrane-bound forms to the same extent . The yeast PtdEtn-PLD activity was found to interact with immobilized PtdEtn in a Ca2+-dependent manner . The partially purified cytosolic form and the salt-extracted membrane-bound form of yeast PtdEtn-PLD exhibited a similar elution pattern on size-exclusion chromatography, coeluting as low apparent molecular weight peaks . PtdEtn-PLD activity was stimulated, along with Spo14p/Pld1p activity, upon dilution of stationary phase cultures in glucose, acetate and galactose media, but PtdEtn-PLD activation was less pronounced . Interestingly, PtdEtn-PLD activity was found to be elevated by approximately 40% in sec14ts mutants at the restrictive temperature, whereas in other sec mutants it remained unaffected . The activity of PtdEtn-PLD was reduced by 30-40% upon addition to the medium of inositol (75 micro m) in either wild-type yeast or spo14Delta mutants and this effect was seen regardless of the presence of choline, suggesting that transcription of the PtdEtn-PLD gene is down-regulated by inositol . Finally, exposure of yeast cells to H2O2 resulted in a transient increase in PtdEtn-PLD activity followed by a profound, nearly 90% decrease in activity . In conclusion, our results indicate that yeast PtdEtn-PLD activity is highly regulated: the enzyme is acutely activated upon entry into the cell cycle and following inactivation of sec14ts, and is inhibited under oxidative stress conditions . The implications of these findings are discussed. Cell, 2002 Jul 26, 110(2), 143 - 51 Evidence for a mediator of RNA polymerase II transcriptional regulation conserved from yeast to man; Boube M et al.; Mediator complexes (MED) link transcriptional regulators to RNA polymerase II . Here, we summarize the latest advances on the functional organization of yeast Mediator . We argue for the existence of a "universal" Mediator structurally conserved from yeast to man, based on an extensive analysis of sequence databases . Finally, we examine the implications of these observations for the physiological roles of metazoan MED subunits. Mol Cell, 2002 Jul, 10(1), 207 - 13 Yeast origins establish a strand bias for replicational mutagenesis; Pavlov YI et al.; To determine whether replicational mutagenesis in the yeast genome is influenced by the positions of active origins, a reporter gene was placed in two orientations at multiple locations within a 39,000 bp region of chromosome III possessing two strong origins . The frequency of mutations resulting from misincorporation of adenine opposite 8-hydroxyguanine in one strand and 6-hydroxylaminopurine opposite cytosine in the other strand differed by 3- to 10-fold, depending on the gene orientation and its distance from the origins . The observed patterns indicate that active origins establish a strand bias for mutations that is maintained over thousands of base pairs and results from lower nucleotide selectivity and/or less efficient proofreading or mismatch repair during leading strand DNA replication. Mol Cell, 2002 Jul, 10(1), 163 - 73 The Galpha protein Gpa2 controls yeast differentiation by interacting with kelch repeat proteins that mimic Gbeta subunits; Harashima T et al.; G protein coupled receptors (GPCR) sense diverse ligands and signal via heterotrimeric G proteins . The Saccharomyces cerevisiae GPCR Gpr1 senses glucose and controls filamentous growth via an unusual Galpha protein, Gpa2, which lacks any known Gbetagamma subunits . Our genetic and biochemical studies identify Gpa2 interaction partners (Gpb1/2, Gpg1) and provide evidence that these proteins function as G protein subunit mimics and signaling effectors . Gpb1 and Gpb2 lack the seven WD-40 repeats found in Gbeta subunits and instead contain seven kelch repeats implicated in protein-protein interactions . Gbeta subunits and the kelch repeat protein galactose oxidase fold into strikingly similar seven-bladed beta propellers . Our studies demonstrate that Gpa2 signals in conjunction with Gbeta structural mimics and that homologous G protein subunits or effectors may be conserved in multicellular eukaryotes. Proc Natl Acad Sci U S A, 2002 Dec 10, 99 Suppl 4, 16392 - 9 Epub 2002 Jul 30. Interactions among prions and prion "strains" in yeast; Bradley ME et al.; Prions are "infectious" proteins . When Sup35, a yeast translation termination factor, is aggregated in its {PSI(+)} prion form its function is compromised . When Rnq1 is aggregated in its {PIN(+)} prion form, it promotes the de novo appearance of {PSI(+)} . Heritable variants (strains) of {PSI(+)} with distinct phenotypes have been isolated and are analogous to mammalian prion strains with different pathologies . Here, we describe heritable variants of the {PIN(+)} prion that are distinguished by the efficiency with which they enhance the de novo appearance of {PSI(+)} . Unlike {PSI(+)} variants, where the strength of translation termination corresponds to the level of soluble Sup35, the phenotypes of these {PIN(+)} variants do not correspond to levels of soluble Rnq1 . However, diploids and meiotic progeny from crosses between either different {PSI(+)}, or different {PIN(+)} variants, always have the phenotype of the parental variant with the least soluble Sup35 or Rnq1, respectively . Apparently faster growing prion variants cure cells of slower growing or less stable variants of the same prion . We also find that YDJ1 overexpression eliminates some but not other {PIN(+)} variants and that prions are destabilized by meiosis . Finally, we show that, like its affect on {PSI(+)} appearance, {PIN(+)} enhances the de novo appearance of {URE3} . Surprisingly, {PSI(+)} inhibited {URE3} appearance . These results reinforce earlier reports that heterologous prions interact, but suggest that such interactions can not only positively, but also negatively, influence the de novo generation of prions. Proc Natl Acad Sci U S A, 2002 Aug 6, 99(16), 10605 - 10 Epub 2002 Jul 29. Previously uncharacterized genes in the UV- and MMS-induced DNA damage response in yeast; Hanway D et al.; A competitive growth assay has been used to identify yeast genes involved in the repair of UV- or MMS-induced DNA damage . A collection of 2,827 yeast strains was analyzed in which each strain has a single ORF replaced with a cassette containing two unique sequence tags, allowing for its detection by hybridization to a high-density oligonucleotide array . The hybridization data identify a high percentage of the deletion strains present in the collection that were previously characterized as being sensitive to the DNA-damaging agents . The assay, and subsequent analysis, has been used to identify six genes not formerly known to be involved in the damage response, whose deletion renders the yeast sensitive to UV or MMS treatment . The recently identified genes include three uncharacterized ORFs, as well as genes that encode protein products implicated in ubiquitination, gene silencing, and transport across the mitochondrial membrane . Epistatsis analysis of four of the genes was performed to determine the DNA damage repair pathways in which the protein products function. J Biol Chem, 2002 Oct 11, 277(41), 38589 - 95 Epub 2002 Jul 30. The ferroxidase activity of yeast frataxin; Park S et al.; Frataxin is required for maintenance of normal mitochondrial iron levels and respiration . The mature form of yeast frataxin (mYfh1p) assembles stepwise into a multimer of 840 kDa (alpha(48)) that accumulates iron in a water-soluble form . Here, two distinct iron oxidation reactions are shown to take place during the initial assembly step (alpha --> alpha(3)) . A ferroxidase reaction with a stoichiometry of 2 Fe(II)/O(2) is detected at Fe(II)/mYfh1p ratios of < or = 0.5 . Ferroxidation is progressively overcome by autoxidation at Fe(II)/mYfh1p ratios of >0.5 . Gel filtration analysis indicates that an oligomer of mYfh1p, alpha(3), is responsible for both reactions . The observed 2 Fe(II)/O(2) stoichiometry implies production of H(2)O(2) during the ferroxidase reaction . However, only a fraction of the expected total H(2)O(2) is detected in solution . Oxidative degradation of mYfh1p during the ferroxidase reaction suggests that most H(2)O(2) reacts with the protein . Accordingly, the addition of mYfh1p to a mixture of Fe(II) and H(2)O(2) results in significant attenuation of Fenton chemistry . Multimer assembly is fully inhibited under anaerobic conditions, indicating that mYfh1p is activated by Fe(II) in the presence of O(2) . This combination induces oligomerization and mYfh1p-catalyzed Fe(II) oxidation, starting a process that ultimately leads to the sequestration of as many as 50 Fe(II)/subunit inside the multimer. J Biol Chem, 2002 Oct 11, 277(41), 38781 - 90 Epub 2002 Jul 30. Forkhead-associated domains of the tobacco NtFHA1 transcription activator and the yeast Fhl1 forkhead transcription factor are functionally conserved; Kim M et al.; NtFHA1 encodes a novel protein containing the forkhead-associated (FHA) domain and the acidic domain in Nicotiana tabacum . NtFHA1 functions as a transactivator and is targeted to the nucleus . The sequence of the FHA domain of NtFHA1 is significantly homologous to that of the Fhl1 forkhead transcription factor of yeast . FHL1 was previously identified as a suppressor of RNA polymerase III mutations, and the fhl1 deletion mutant exhibited severe growth defects and impaired rRNA processing . Ectopic expression of the FHA domain of NtFHA1 (but not its mutant form) resulted in severe growth retardation in yeast . Similarly, expression of Fhl1, its FHA domain, or chimeric Fhl1 containing the NtFHA1 FHA domain also inhibited yeast growth . Yeast cells overexpressing the FHA domains of NtFHA1 and Fhl1 contained lower levels of mature rRNAs and exhibited rRNA-processing defects, similar to the fhl1 null mutant . Chimeric Fhl1 (but not the mutant form with a small deletion in its FHA domain) fully complemented the growth and rRNA-processing defects of the fhl1 null mutant, demonstrating that the FHA domain of NtFHA1 can functionally substitute for the FHA domain of Fhl1 . These results demonstrate that the FHA domains of NtFHA1 and Fhl1 are conserved in their structure and function and that the FHA domain of Fhl1 is critically involved in regulation of rRNA processing in yeast . NtFHA1 function in plants may be analogous to Fhl1 function in yeast. BMC Genet . 2002 Jul 30;3(1):13. Setting of graded levels of a protein in yeast by a t-degron technique as applied to phosphoglycerate mutase; Heidrich K et al.; BACKGROUND: Setting of graded levels of a protein for in vivo studies by controlled gene expression has inconveniences, and we here explore the use of the t-degron technique instead . RESULTS: In a yeast t-degron (ubiquitin-argDHFR(ts))- phosphoglycerate mutase (GPM1) fusion strain, increasing periods of exposure to the non-permissive temperature 37 degrees C, even in the presence of cycloheximide, gave decreasing function, as assessed at 23 degrees C in vivo by glucose metabolism and confirmed by immunoblot . CONCLUSION: An ideal system would set a range of lower levels of a protein, do so without compensating protein synthesis, and give stable activity for in vitro comparisons . Although the first two aims appear obtainable, the third was not in this example of the application, limiting its uses for some but not all purposes. Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1423 - 33 Conflicting results obtained by RAPD-PCR and large-subunit rDNA sequences in determining and comparing yeast strains isolated from flowers: a comparison of two methods; Herzberg M et al.; Sixty-six yeast strains isolated from the nectar of various plant species in Central Europe were characterized by randomly amplified polymorphic DNA PCR (RAPD-PCR) and by sequencing of the variable D1/D2 domain of large-subunit (26S) rDNA . The usefulness of both methods for the determination and comparison of unknown ascomycetous and basidiomycetous yeast strains was compared and evaluated . The reproducibility of RAPD-PCR was shown to be low and the information obtained by this method was clearly not as precise as that obtained from sequence analysis . Numerous imponderables make RAPD-PCR analysis unreliable, at least as a means of identifying yeasts in ecological studies . The lack of standard protocols for RAPD-PCR analysis and the absence of a general database of banding patterns made it impossible to identify unknown yeast strains or to recognize new species . In contrast to RAPD-PCR, sequence analysis of the D1/D2 domain was found to be a fast and reliable method for the rapid identification of yeast species and was also shown to be an invaluable tool for the discovery of new species. Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1369 - 75 Candida davenportii sp . nov., a potential soft-drinks spoilage yeast isolated from a wasp; Stratford M et al.; During a survey of yeast ecology in a soft-drinks production facility, a dead wasp was removed from the sampling tap of an external sugar-syrup storage tank . A yeast isolated from the dead wasp was found to be similar, although not identical, in its physiological characteristics to Candida lactis-condensi and Candida stellata . Sequence analysis of the 26S rDNA D1/D2 variable domain revealed that this isolate was most closely related to C stellata, but differed sufficiently in its D1/D2 sequence to indicate that it belonged to a separate species . The yeast species has been named Candida davenportii sp . nov.; the type strain is NCYC 3013T (= CBS 9069T) . C davenportii sp . nov . was osmotolerant, moderately preservative-resistant and able to grow in very acidic conditions, i.e . pH 14 . This yeast grew well in fruit-containing soft drinks, cola-type beverages and a synthetic soft drink and is therefore a potential cause of spoilage of soft drinks and other sugary food products . Other related yeast species in the same taxonomic clade as C davenportii sp . nov . are also osmotolerant, growing in < 50% (w/v) sugar . Many of these species are associated with insects, specifically bees, bumblebees and leafcutter bees, and many have been reported as the causative agent of spoilage of sugary foods, such as condensed milk, fruit juices and concentrates . It is proposed that C davenportii sp . nov . and other closely related yeasts are primarily associated with Aculeates (bees and wasps) . In turn, bees and wasps are attracted by sugary residues in foods such as fruit juices and concentrates, forming the source of infection of these yeasts and thus instigating spoilage. Arch Biochem Biophys, 2002 Aug 15, 404(2), 279 - 84 Increased degradation of oxidized proteins in yeast defective in 26 S proteasome assembly; Inai Y et al.; An Rpn9-disrupted yeast strain, Delta rpn9, whose growth is temperature sensitive with defective assembly of the 26 S proteasome complex, was studied . This mutant yeast was more resistant to hydrogen peroxide treatment and able to degrade carbonylated proteins more efficiently than wild type . Nondenaturing gel electrophoresis followed by activity staining revealed that Delta rpn9 yeast cells had a higher activity of 20 S proteasome than wild type and that in both Delta rpn9 and wild-type cells treated with hydrogen peroxide, 20 S proteasome activity was increased with a concomitant decrease in 26 S proteasome activity . Protein multiubiquitination was not observed in the hydrogen peroxide-treated cells . Taken together, these results suggest that the 20 S proteasome degrades oxidized proteins without ubiquitination of target proteins . Biochemistry, 2002 Aug 6, 41(31), 10002 - 9 Mechanistic insights into Sky1p, a yeast homologue of the mammalian SR protein kinases; Aubol BE et al.; The SRPK family is distinguished from typical eukaryotic protein kinases by several unique structural features recently elucidated by X-ray diffraction methods {Nolen et al . (2001) Nat . Struct . Biol . 8, 176-183} . To determine whether these features impart unique catalytic function, the phosphorylation of the physiological Sky1p substrate, Npl3p, was monitored using steady-state and pre-steady-state kinetic techniques . While Sky1p has a low apparent affinity for ATP compared to other protein kinases, it binds Npl3p with very high affinity . The latter is achieved through a combination of local and distal factors in the protein substrate . The phosphoryl donor ATP has access to the nucleotide pocket in the absence or presence of Npl3p, indicating that a large protein substrate does not enforce an ordered addition of ligands . Sky1p binds two Mg(2+)-the first is essential whereas the second further enhances catalysis . While the turnover number is low (0.5 s(-1)), Npl3p is rapidly phosphorylated in the active site (40 s(-1)) based on single turnover experiments . These results indicate that Sky1p employs a catalytic pathway involving fast phosphoryl transfer followed by slow net release of products . These studies represent the first kinetic investigation of a member of the SRPK family and the first pre-steady-state kinetic study of a protein kinase using a natural protein substrate. Bioresour Technol, 2002 Oct, 85(1), 35 - 7 Palm oil mill effluent treatment by a tropical marine yeast; Oswal N et al.; Palm oil mill effluent (POME), from a factory site in India contained about 250,000 mg l(-1) chemical oxygen demand (COD), 11,000 mg l(-1) biochemical oxygen demand, 65 mg l(-1) total dissolved solids and 9000 mg l(-1) of chloroform-soluble material . Treatment of this effluent using Yarrowia lipolytica NCIM 3589, a marine hydrocarbon-degrading yeast isolated from Mumbai, India, gave a COD reduction of about 95% with a retention time of two days . Treatment with a chemical coagulant further reduced the COD and a consortium developed from garden soil clarified the effluent and adjusted the pH to between 6 and 7 . The complete treatment reduced the COD content to 1500 mg l(-1) which is a 99% reduction from the original. J Biol Chem, 2002 Sep 27, 277(39), 36152 - 60 Epub 2002 Jul 26. Yeast cells lacking the ARV1 gene harbor defects in sphingolipid metabolism . Complementation by human ARV1; Swain E et al.; arv1Delta mutant cells have an altered sterol distribution within cell membranes (Tinkelenberg, A.H., Liu, Y., Alcantara, F., Khan, S., Guo, Z., Bard, M., and Sturley, S . L . (2000) J . Biol . Chem . 275, 40667-40670), and thus it has been suggested that Arv1p may be involved in the trafficking of sterol in the yeast Saccharomyces cerevisiae and also in humans . Here we present data showing that arv1Delta mutants also harbor defects in sphingolipid metabolism . {(3)H}inositol and {(3)H}dihydrosphingosine radiolabeling studies demonstrated that mutant cells had reduced rates of biosynthesis and lower steady-state levels of complex sphingolipids while accumulating certain hydroxylated ceramide species . Phospholipid radiolabeling studies showed that arv1Delta cells harbored defects in the rates of biosynthesis and steady-state levels of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol . Neutral lipid radiolabeling studies indicated that the rate of biosynthesis and steady-state levels of sterol ester were increased in arv1Delta cells . Moreover, these same studies demonstrated that arv1Delta cells had decreased rates of biosynthesis and steady-state levels of total fatty acid and fatty acid alcohols . Gas chromatography/mass spectrometry analyses examining different fatty acid species showed that arv1Delta cells had decreased levels of C18:1 fatty acid . Additional gas chromatography/mass spectrometry analyses determining the levels of various molecular sterol species in arv1Delta cells showed that mutant cells accumulated early sterol intermediates . Using fluorescence microscopy we found that GFP-Arv1p localizes to the endoplasmic reticulum and Golgi . Interestingly, the heterologous expression of the human ARV1 cDNA suppressed the sphingolipid metabolic defects of arv1Delta cells . We hypothesize that in eukaryotic cells, Arv1p functions in the sphingolipid metabolic pathway perhaps as a transporter of ceramides between the endoplasmic reticulum and Golgi. EMBO J, 2002 Aug 1, 21(15), 4136 - 44 Localization of the yeast RNA polymerase I-specific subunits; Bischler N et al.; The spatial distribution of four subunits specifically associated to the yeast DNA-dependent RNA polymerase I (RNA pol I) was studied by electron microscopy . A structural model of the native enzyme was determined by cryo-electron microscopy from isolated molecules and was compared with the atomic structure of RNA pol II Delta 4/7, which lacks the specific polypeptides . The two models were aligned and a difference map revealed four additional protein densities present in RNA pol I, which were characterized by immunolabelling . A protruding protein density named stalk was found to contain the RNA pol I-specific subunits A43 and A14 . The docking with the atomic structure showed that the stalk protruded from the structure at the same site as the C-terminal domain (CTD) of the largest subunit of RNA pol II . Subunit A49 was placed on top of the clamp whereas subunit A34.5 bound at the entrance of the DNA binding cleft, where it could contact the downstream DNA . The location of the RNA pol I-specific subunits is correlated with their biological activity. Trends Genet, 2002 Aug, 18(8), 405 - 12 Yeast go the whole HOG for the hyperosmotic response; O'Rourke SM et al.; An evolutionarily conserved mitogen-activated protein kinase pathway--the high osmolarity glycerol (HOG) pathway--mediates the hyperosmotic response in Saccharomyces cerevisiae . A variety of powerful approaches has generated a comprehensive picture of how cells respond to this stress condition . Several presumptive osmosensors on the cell surface recruit and activate downstream signaling components, which regulate the activity of transcription factors to control gene expression. Nucleic Acids Res, 2002 Aug 1, 30(15), 3412 - 21 AtSWI3B, an Arabidopsis homolog of SWI3, a core subunit of yeast Swi/Snf chromatin remodeling complex, interacts with FCA, a regulator of flowering time; Sarnowski TJ et al.; ATP-dependent nucleosome remodeling plays a central role in the regulation of access to chromatin DNA . Swi/Snf remodeling complexes characterized in yeast, Drosophila and mammals all contain a conserved set of core subunits composed of homologs of yeast SNF2-type DNA-dependent ATPase, SNF5 and SWI3 proteins . So far, no complete Swi/Snf-type complex has been characterized in plants . Arabidopsis contains a single SNF5-type gene, BSH, which has been shown to complement the yeast snf5 mutation . Here we describe the characterization of AtSWI3B, the smallest of the four Arabidopsis homologs of SWI3 . The gene encoding AtSWI3B is expressed ubiquitously in the plant . AtSWI3B is localized to nuclei and is associated mostly with the chromatin and soluble protein fractions . When expressed in Saccharomyces cerevisiae, the cDNA encoding AtSWI3B partially complements the swi3 mutant phenotype . However, like BSH, AtSWI3B is unable to activate transcription in yeast when tethered to DNA . The analysis by yeast two-hybrid indicates that AtSWI3B is capable of forming homodimers and interacts with BSH as well as with two other members of the Arabidopsis SWI3 family: AtSWI3A and AtSWI3C . The results of phage display screen using recombinant protein, confirmed by direct yeast two-hybrid analyses, indicate that AtSWI3B interacts with FCA, a regulator of flowering time in ARABIDOPSIS: This interaction is through the C-terminal region of FCA, located outside the conserved RNA- and protein-binding domains of this protein. Mikrobiologiia, 2002 May-Jun, 71(3), 387 - 90 {A new yeast species, Candida aurita sp . nov., from oligotrophic bogs of Western Siberia}; Poliakova AV et al.; Five anamorphous yeast strains of ascomycetous affinity with a specific mode of budding were isolated from raised bog soils of Bakcharskoe Bog (Tomsk oblast) . According to their morphological and physiological properties, these strains belong to the genus Candida but differ from all species described previously . The level of DNA-DNA homology with species similar in the assimilation spectrum was as low as 7% . Based on these data, the new species Candida aurita sp . nov . is described. Gene, 2002 Jun 26, 293(1-2), 199 - 204 Metal resistance in yeast mediated by the expression of a maize 20S proteasome alpha subunit; Forzani C et al.; Transformation of yeast cells with a maize cDNA ZmPAA, encoding a 20S proteasome alpha-subunit, conferred resistance to nickel, cadmium and cobalt . This resistance is not linked to a modification of the intracellular nickel content, as no accumulation of nickel was measured between yeast cells transformed with a void vector or the ZmPAA cDNA . The abundance of the ZmPAA mRNA was increased in the shoots of maize plants upon nickel treatment . These results suggest that the proteasome might be involved in nickel resistance by scavenging metal oxidized proteins both in plants and yeast. Semin Cell Dev Biol, 2002 Jun, 13(3), 179 - 84 Internalization and trafficking of fluorescent-labeled phospholipids in yeast; Nichols JW; Phospholipid reporter molecules, containing a fluorescent group attached to a short, acyl chain, spontaneously insert into the plasma membrane of yeast cells allowing retrograde trafficking to intracellular organelles as well as their metabolic fates to be monitored . This approach provides the framework for determining the dependence of particular phospholipid trafficking and metabolic steps on a wide range of genes known to be required for related membrane transport functions as well as for developing genetic screens to identify novel genes required for these processes . This review presents an overview of insights gained into phospholipid trafficking and metabolism using this approach. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1999, 31(3), 221 - 225 Yeast Two-hybrid System and Its Application on Proteomics; Yang QH et al.; Proteomics is a new research area of the post-genomic era that aims at the analysis and identification of the entire proteins present in the cell, tissue or organism, and of the functions and the linkages of these proteins . Protein-protein interactions are characteristic of cellular activities and of course an important part of proteomics . One of the effective techniques to detect protein-protein interaction is yeast two-hybrid system . This system was successfully employed to analyze the protein linkage map of the yeast mRNA splicing machinery, which boded well for its application on human proteome research. Nucleic Acids Res, 2002 Jul 15, 30(14), 3130 - 40 Mammalian metal response element-binding transcription factor-1 functions as a zinc sensor in yeast, but not as a sensor of cadmium or oxidative stress; Daniels PJ et al.; The zinc finger protein, metal response element-binding transcription factor-1 (MTF-1) regulates the expression of genes in response to metal ions and oxidative stress . The precise mechanisms by which this occurs are not understood . To further examine this problem, mouse MTF-1 was expressed in Saccharomyces cerevisiae and tested for the ability to activate metal response element-driven reporter gene expression . Zinc was an effective inducer of reporter gene expression . In general, the magnitude of zinc induction was dependent on the concentration of zinc in the culture medium, but independent of the amount of MTF-1 expression . Zinc induction also occurred with either integrated or episomal reporter plasmids containing the native mouse metallothionein-I proximal promoter . Deletion of fingers 5 and 6 of MTF-1, which function in a zinc-dependent manner to stabilize the DNA-binding activity of the protein in vitro, did not diminish the zinc induction of either episomal or integrated promoters . However, a Gal4 DNA-binding domain- MTF-1 fusion protein, which binds constitutively to the Gal4-responsive promoter, was not zinc inducible but caused constitutive activation of reporter gene expression . This suggests that zinc activation of the DNA-binding activity of MTF-1 is the rate limiting step in its metalloregulatory function in yeast . In contrast, MTF-1 was not responsive to either cadmium or hydrogen peroxide, suggesting that distinct co-activators or signal transduction cascades not found in yeast are required to mediate MTF-1 activation of gene expression by this toxic metal and by oxidative stress. Nucleic Acids Res, 2002 Jul 15, 30(14), 3078 - 85 The role of TFIIB-RNA polymerase II interaction in start site selection in yeast cells; Zhang DY et al.; Previous studies have established a critical role of both TFIIB and RNA polymerase II (RNAPII) in start site selection in the yeast Saccharomyces cerevisiae . However, it remains unclear how the TFIIB-RNAPII interaction impacts on this process since such an interaction can potentially influence both preinitiation complex (PIC) stability and conformation . In this study, we further investigate the role of TFIIB in start site selection by characterizing our newly generated TFIIB mutants, two of which exhibit a novel upstream shift of start sites in vivo . We took advantage of an artificial recruitment system in which an RNAPII holoenzyme component is covalently linked to a DNA-binding domain for more direct and stable recruitment . We show that TFIIB mutations can exert their effects on start site selection in such an artificial recruitment system even though it has a relaxed requirement for TFIIB . We further show that these TFIIB mutants have normal affinity for RNAPII and do not alter the promoter melting/scanning step . Finally, we show that overexpressing the genetically isolated TFIIB mutant E62K, which has a reduced affinity for RNAPII, can correct its start site selection defect . We discuss a model in which the TFIIB-RNAPII interaction controls the start site selection process by influencing the conformation of PIC prior to or during PIC assembly, as opposed to PIC stability. Genetics, 2002 Jul, 161(3), 971 - 81 Genetic interaction between calcineurin and type 2 myosin and their involvement in the regulation of cytokinesis and chloride ion homeostasis in fission yeast; Fujita M et al.; Calcineurin plays a critical role in Ca(2+) signaling in various cell types . In fission yeast, calcineurin is required for cytokinesis and chloride ion homeostasis . However, most of its physiological functions remain obscure . A genetic screen was performed to identify genes that share an essential function with calcineurin . We screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and to a high concentration of chloride ion and isolated a mutant, cis2-1/myp2-c2, which contains a novel allele of the myp2(+)/myo3(+) gene that encodes a type 2 myosin heavy chain . The myp2-c2 mutant showed morphological defects similar to those associated with a calcineurin deletion mutant, such as multiseptated and branched cells . Consistently, myp2-null cells were hypersensitive to chloride ion and showed the multiseptated phenotype in the presence of immunosuppressants or at high chloride concentrations . Overexpression of constitutively active calcineurin suppressed the chloride ion-sensitive growth defect and cytokinesis abnormality of the myp2-c2 mutant and myp2-null cells . Interestingly, the essential myosin light chain mutant cdc4-8 failed to grow and could not form a normal contractile ring in the presence of immunosuppressants . Furthermore, calcineurin-null cells exhibited aberrant contractile rings, suggesting impaired contraction of the rings . These results indicate that calcineurin is involved in the regulation of cytokinesis and that chloride ion homeostasis is mediated by type 2 myosin. Genetics, 2002 Jul, 161(3), 957 - 69 Functional dissection of the global repressor Tup1 in yeast: dominant role of the C-terminal repression domain; Zhang Z et al.; In the yeast Saccharomyces cerevisiae, Tup1, in association with Cyc8 (Ssn6), functions as a general repressor of transcription . Tup1 and Cyc8 are required for repression of diverse families of genes coordinately controlled by glucose repression, mating type, and other mechanisms . This repression is mediated by recruitment of the Cyc8-Tup1 complex to target promoters by sequence-specific DNA-binding proteins . We created a library of XhoI linker insertions and internal in-frame deletion mutations within the TUP1 coding region . Insertion mutations outside of the WD domains were wild type, while insertions within the WD domains induced mutant phenotypes with differential effects on the target genes SUC2, MFA2, RNR2, and HEM13 . Deletion mutations confirmed previous findings of two separate repression domains in the N and C termini . The cumulative data suggest that the C-terminal repression domain, located near the first WD repeat, plays the dominant role in repression . Although the N-terminal repression domain is sufficient for partial repression, deletion of this region does not compromise repression . Surprisingly, deletion of the majority of the histone-binding domain of Tup1 also does not significantly reduce repression . The N-terminal region containing potential alpha-helical coiled coils is required for Tup1 oligomerization and association with Cyc8 . Association with Cyc8 is required for repression of SUC2, HEM13, and RNR2 but not MFA2 and STE2. Eur J Biochem, 2002 Jul, 269(14), 3511 - 21 Rum1, an inhibitor of cyclin-dependent kinase in fission yeast, is negatively regulated by mitogen-activated protein kinase-mediated phosphorylation at Ser and Thr residues; Matsuoka K et al.; The p25(rum1) is an inhibitor of Cdc2 kinase expressed in fission yeast and plays an important role in cell-cycle control . As its amino-acid sequence suggests that p25(rum1) has putative phosphorylation sites for mitogen-activated protein kinase (MAPK), we investigated the ability of MAPK to phosphorylate p25(rum1) . Direct in vitro kinase assay using GST-fusion proteins of wild-type as well as various mutants of p25(rum1) demonstrated that MAPK phosphorylates the N-terminal portion of p25(rum1) and residues Thr13 and Ser19 are major phosphorylation sites for MAPK . In addition, phosphorylation of p25(rum1) by MAPK revealed markedly reduced Cdc2 kinase inhibitor ability of the protein . Together with the fact that replacement of both Thr13 and Ser19 with Glu, which mimics the phosphorylated state of these residues, also significantly reduces the activity of p25(rum1) as a Cdc2 inhibitor, it was suggested that the phosphorylation of Thr13 and Ser19 negatively regulates the function of p25(rum1) . Further evidence indicates that phosphorylation of Thr13 and Ser19 may retain a negative effect on the function of p25(rum1) even in vivo . Therefore, MAPK may regulate the function of p25(rum1) via phosphorylation of its Thr and Ser residues and thus participate in cell cycle control in fission yeast. Nat Genet, 2002 Aug, 31(4), 370 - 7 Epub 2002 Jul 22. Revealing modular organization in the yeast transcriptional network; Ihmels J et al.; Standard clustering methods can classify genes successfully when applied to relatively small data sets, but have limited use in the analysis of large-scale expression data, mainly owing to their assignment of a gene to a single cluster . Here we propose an alternative method for the global analysis of genome-wide expression data . Our approach assigns genes to context-dependent and potentially overlapping 'transcription modules', thus overcoming the main limitations of traditional clustering methods . We use our method to elucidate regulatory properties of cellular pathways and to characterize cis-regulatory elements . By applying our algorithm systematically to all of the available expression data on Saccharomyces cerevisiae, we identify a comprehensive set of overlapping transcriptional modules . Our results provide functional predictions for numerous genes, identify relations between modules and present a global view on the transcriptional network. Nat Genet, 2002 Aug, 31(4), 400 - 4 Epub 2002 Jul 22. Systematic screen for human disease genes in yeast; Steinmetz LM et al.; High similarity between yeast and human mitochondria allows functional genomic study of Saccharomyces cerevisiae to be used to identify human genes involved in disease . So far, 102 heritable disorders have been attributed to defects in a quarter of the known nuclear-encoded mitochondrial proteins in humans . Many mitochondrial diseases remain unexplained, however, in part because only 40-60% of the presumed 700-1,000 proteins involved in mitochondrial function and biogenesis have been identified . Here we apply a systematic functional screen using the pre-existing whole-genome pool of yeast deletion mutants to identify mitochondrial proteins . Three million measurements of strain fitness identified 466 genes whose deletions impaired mitochondrial respiration, of which 265 were new . Our approach gave higher selection than other systematic approaches, including fivefold greater selection than gene expression analysis . To apply these advantages to human disorders involving mitochondria, human orthologs were identified and linked to heritable diseases using genomic map positions. Mol Biol Cell, 2002 Jul, 13(7), 2360 - 73 A fourth component of the fission yeast gamma-tubulin complex, Alp16, is required for cytoplasmic microtubule integrity and becomes indispensable when gamma-tubulin function is compromised; Fujita A et al.; gamma-Tubulin functions as a multiprotein complex, called the gamma-tubulin complex (gamma-TuC), and composes the microtubule organizing center (MTOC) . Fission yeast Alp4 and Alp6 are homologues of two conserved gamma-TuC proteins, hGCP2 and hGCP3, respectively . We isolated a novel gene, alp16(+), as a multicopy suppressor of temperature-sensitive alp6-719 mutants . alp16(+) encodes a 759-amino-acid protein with two conserved regions found in all other members of gamma-TuC components . In addition, Alp16 contains an additional motif, which shows homology to hGCP6/Xgrip210 . Gene disruption shows that alp16(+) is not essential for cell viability . However, alp16 deletion displays abnormally long cytoplasmic microtubules, which curve around the cell tip . Furthermore, alp16-deleted mutants are hypersensitive to microtubule-depolymerizing drugs and synthetically lethal with either temperature-sensitive alp4-225, alp4-1891, or alp6-719 mutants . Overproduction of Alp16 is lethal, with defective phenotypes very similar to loss of Alp4 or Alp6 . Alp16 localizes to the spindle pole body throughout the cell cycle and to the equatorial MTOC at postanaphase . Alp16 coimmunoprecipitates with gamma-tubulin and cosediments with the gamma-TuC in a large complex (>20 S) . Alp16 is, however, not required for the formation of this large complex . We discuss evolutional conservation and divergence of structure and function of the gamma-TuC between yeast and higher eukaryotes. Mol Biol Cell, 2002 Jul, 13(7), 2193 - 206 Activity of specific lipid-regulated ADP ribosylation factor-GTPase-activating proteins is required for Sec14p-dependent Golgi secretory function in yeast; Yanagisawa LL et al.; Yeast phosphatidylinositol transfer protein (Sec14p) coordinates lipid metabolism with protein-trafficking events . This essential Sec14p requirement for Golgi function is bypassed by mutations in any one of seven genes that control phosphatidylcholine or phosphoinositide metabolism . In addition to these "bypass Sec14p" mutations, Sec14p-independent Golgi function requires phospholipase D activity . The identities of lipids that mediate Sec14p-dependent Golgi function, and the identity of the proteins that respond to Sec14p-mediated regulation of lipid metabolism, remain elusive . We now report genetic evidence to suggest that two ADP ribosylation factor-GTPase-activating proteins (ARFGAPs), Gcs1p and Age2p, may represent these lipid-responsive elements, and that Gcs1p/Age2p act downstream of Sec14p and phospholipase D in both Sec14p-dependent and Sec14p-independent pathways for yeast Golgi function . In support, biochemical data indicate that Gcs1p and Age2p ARFGAP activities are both modulated by lipids implicated in regulation of Sec14p pathway function . These results suggest ARFGAPs are stimulatory factors required for regulation of Golgi function by the Sec14p pathway, and that Sec14p-mediated regulation of lipid metabolism interfaces with the activity of proteins involved in control of the ARF cycle. Genes Dev, 2002 Jul 15, 16(14), 1766 - 78 Fission yeast CENP-B homologs nucleate centromeric heterochromatin by promoting heterochromatin-specific histone tail modifications; Nakagawa H et al.; Heterochromatin is a functionally important chromosomal component, especially at centromeres . In fission yeast, conserved heterochromatin-specific modifications of the histone H3 tail, involving deacetylation of Lys 9 and Lys 14 and subsequent methylation of Lys 9, promote the recruitment of a heterochromatin protein, Swi6, a homolog of the Drosophila heterochromatin protein 1 . However, the primary determinants of the positioning of heterochromatin are still unclear . The fission yeast proteins Abp1, Cbh1, and Cbh2 are homologs of the human protein CENP-B that bind to centromeric alpha-satellite DNA and associate with centromeric heterochromatin . We show that the CENP-B homologs are functionally redundant at centromeres, and that Abp1 binds specifically to centromeric heterochromatin . In the absence of Abp1 or Cbh1, the centromeric association of Swi6 is diminished, resulting in a decrease in silencing of the region . CENP-B-homolog double disruptants show a synergistic reduction of Swi6 at centromeric heterochromatin, indicating that the three proteins are functionally redundant in the recruitment of Swi6 . Furthermore, using chromatin immunoprecipitation assays, we show that disruption of CENP-B homologs causes a decrease in heterochromatin-specific modifications of histone H3 . These results indicate that the CENP-B homologs act as site-specific nucleation factors for the formation of centromeric heterochromatin by heterochromatin-specific modifications of histone tails. Biophys Chem, 2002 Jul 10, 98(1-2), 65 - 77 Production and characterisation of Met80X mutants of yeast iso-1-cytochrome c: spectral, photochemical and binding studies on the ferrous derivatives; Silkstone G et al.; The iron ligand, Met80, of yeast iso-1-cytochrome c has been mutated to residues that are unable to bind to the iron . The resultant proteins, Met80Ala, Ser, Asp, Glu, have been expressed and purified . All mutant proteins exhibit well defined pH dependent spectral transitions that report the binding, at high pH, of an intrinsic ligand (probably the nitrogen of an epsilon-NH(2) of a lysine) that drives the heme low-spin . The pK values are mutant dependent . All the mutant proteins bind extrinsic ligands, such as CO, in their ferrous states and we report the apparent quantum yield (phi) for CO photo-dissociation . The values of phi range from 0.004 for Met80Ala to 0.04 for Met80Asp . We also report values for the rate constant for binding the intrinsic lysine residue . The values for this constant, for phi and for the pK values are discussed in terms of the rigidity of the cytochrome structure . We also show that the mutant proteins bind with high affinity to cytochrome c oxidase, both in the ferric and ferrous states . The potential of these proteins to act as light activated electron donors for the study of electron transfer is discussed. Biochem Biophys Res Commun, 2002 Jul 26, 295(4), 978 - 84 Uptake of lead and iron by divalent metal transporter 1 in yeast and mammalian cells; I Bannon D et al.; Although the divalent metal transporter (DMT1) was suggested to transport a wide range of metals in Xenopus oocytes, recent studies in other models have provided contrasting results . Here, we provide direct evidence demonstrating that DMT1 expressed in yeast mutants defective for high affinity iron transport facilitates the transport of iron with an 'apparent K(m)' of approximately 1.2 microM, and transport of lead with an 'apparent K(m)' of approximately 1.8 microM . DMT1-dependent lead transport was H(+)-dependent and was inhibited by iron . Human embryonic kidney fibroblasts (HEK293 cells) overexpressing DMT1 also showed a higher uptake of lead than HEK293 cells without overexpressing DMT1 . These results show that DMT1 transports lead and iron with similar affinity in a yeast model suggesting that DMT1 is a transporter for lead. Ann Chim, 2002 May-Jun, 92(5-6), 587 - 94 Preconcentration of trace silver with yeast for river water analysis; Ohta K et al.; A new preconcentration method with yeast is presented . The method was evaluated for the determination of trace silver in river waters by graphite furnace atomic absorption spectrometry (GFAAS) . A suitable cultivation bed for preconcentration of silver was 1.75 mg ml-1 2-ammonium hydrogen phosphate . The optimal cultivation time and temperature were 2 h and 25 degrees C . Under optimal conditions, silver in aqueous sample was concentrated to 6.9-fold by yeast . The detection limit was 4.6 pg ml-1 (3S/N) for silver in river water . The yeast preconcentration method was applied to the determination of silver in river waters . The recovery of spiked silver was in the range of 89 to 110% . By the preconcentration, it was found that ul