Yeasts

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 ultra trace silver in river waters could be determined without interferences of matrix elements, after only the cultivation and with no chemical treatment.

Bull Exp Biol Med, 2002 Apr, 133(4), 377 - 9
Photoprotective activity of melanin preparations from black yeast-like fungus during UV irradiation of human skin: dependence on the concentration; Paramonov BA et al.; The effect of melanin solutions on the skin exposed to UV irradiation (1,050 kJ/m(2)) depended on its dose and varied from photoprotection (0.005 mg/ml) to photosensitization and phototoxicity (burn, 0.1 mg/ml) . These results suggest that doses of melanin preparations should be empirically selected to achieve optimum photoprotective effect.

FEBS Lett, 2002 Jul 17, 523(1-3), 73 - 8
Interaction of hnRNP-C1/C2 proteins with RNA: analysis using the yeast three-hybrid system; Koloteva-Levine N et al.; Three-hybrid assays for the analysis of RNA-protein interactions in vivo are usually used, due to technical limitations, only for RNA baits that do not contain runs of four or more consecutive uridines . The present study provides the first example of a three-hybrid analysis of synthetic and natural uridine-rich RNA sequences . The use of the three-hybrid assay enabled us to demonstrate a functional difference between two closely related proteins, heterogeneous nuclear ribonucleoprotein C1 (hnRNP-C1) and hnRNP-C2 . The hnRNP-C2 protein, an alternatively spliced variant of hnRNP-C1, contains an additional 13 amino acids between an RNA binding domain (RBD) and a basic leucine zipper-like motif (bZLM), also implied in RNA binding . This study shows that (i) for efficient binding of hnRNP-C1/C2 to RNA, the context of the U-stretch is more important than the stretch itself; (ii) both the RBD and the bZLM bind RNA independently; and (iii) the C2-related 13-amino acid insert enhances the specificity of either the RBD, the bZLM, or the full-length protein towards its ligand, allowing it to bind only the most high-affinity sequences while discriminating against those that do not perfectly match this category . The three-hybrid system is a powerful tool to work out the functional significance of peptide 'modules' within RNA binding proteins generated by alternative splicing.

Mol Microbiol, 2002 Jul, 45(2), 307 - 19
A protein kinase specifically associated with proliferative forms of Trypanosoma brucei is functionally related to a yeast kinase involved in the co-ordination of cell shape and division; Garcia-Salcedo JA et al.; The life cycle of African trypanosomes is characterized by the alternation of proliferative and quiescent stages but the molecular details of this process remain unknown . Here, we describe a new cytoplasmic protein kinase from Trypanosoma brucei, termed TBPK50, that belongs to a family of protein kinases involved in the regulation of the cell cycle, cell shape and proliferation . TBPK50 is expressed only in proliferative forms but is totally absent in quiescent cells despite the fact that the gene is constitutively transcribed at the same level throughout the life cycle . It is probable that TBPK50 has very specific substrate requirements as it was unable to transphosphorylate a range of classical phosphoacceptor substrates in vitro, although an autophosphorylation activity was readily detectable in the same assays . Complementation studies using a fission yeast mutant demonstrated that TBPK50 is a functional homologue of Orb6, a protein kinase involved in the regulation of cellular morphology and cell cycle progression in yeast . These results link the expression of TBPK50 and the growth status of trypanosomes and support the view that this protein kinase is likely to be involved in the control of life cycle progression and cell division of these parasites.

Curr Biol, 2002 Jul 9, 12(13), 1100 - 5
A CDK-activating kinase network is required in cell cycle control and transcription in fission yeast; Saiz JE et al.; Cyclin-dependent kinases (CDKs) involved in cell cycle control require activation by phosphorylation, but CDK-activating kinase (CAK) has diverged between metazoans and budding yeast . Fission yeast has two CAKs: the essential Mcs6 complex, homologous to the metazoan CDK7 complex implicated in cell cycle control and transcription; and Csk1, a nonessential ortholog of budding yeast Cak1 . Both can activate the major CDK, Cdc2, but Csk1 can also activate Mcs6, so it was unclear whether the pathway is a linear cascade or a network . Here, we show that a mutation, mcs6-13, which selectively abrogates CDK activation, blocks both G1/S and G2/M transitions, but only when csk1(+) is absent . In contrast, gradual depletion or rapid inactivation of Mcs6 in csk1(+) cells causes cell separation defects or growth arrest, respectively, accompanied by decreased phosphorylation of RNA polymerase II (RNAP II), but not of Cdc2 . Finally, neither cell cycle arrest nor CAK failure is recapitulated by a second mutation in mcs6-13 that prevents Mcs6 activation by Csk1, indicating that Csk1 activates Cdc2 directly in vivo . Thus, Mcs6 acts in concert with Csk1 to activate Cdc2 and independently to support transcription and facilitate cell separation . Csk1 likewise has multiple physiologic targets, including Mcs6 and Cdc2.

Proc Natl Acad Sci U S A, 2002 Jul 23, 99(15), 9739 - 44 Epub 2002 Jul 15.
Protein-protein interactions among C-4 demethylation enzymes involved in yeast sterol biosynthesis; Mo C et al.; A Saccharomyces cerevisae microarray expression study indicated that an ORF, YER044C, now designated ERG28, was strongly coregulated with ergosterol biosynthesis . Disruption of the ERG28 gene results in slow growth and accumulation of sterol intermediates similar to those observed in erg26 and erg27 null strains, suggesting that the Erg28p may interact with Erg26p and/or Erg27p . In this study, a peptide from human hemagglutinin protein (HA) epitope tag was added to ERG26 and ERG27 genes, and a Myc tag was added to the ERG28 gene to detect interactions between Erg28p and Erg26p/Erg27p . Differential centrifugation showed that Erg26p, Erg27p, and Erg28p are all membrane-associated proteins . Green fluorescent protein-fusion protein localization studies showed that Erg26p, Erg27p, and Erg28p are all located in the endoplasmic reticulum . Solubilized membrane protein coimmunoprecipitation studies using rabbit anti-Erg25p indicated that Erg25p coimmunoprecipitates with both Erg27p and Erg28p . Erg28p was also shown to reciprocally coimmunoprecipitate with Erg27p . However, no coimmunoprecipitation was observed with Erg26p, most likely because of the poor solubilization of this protein . Sucrose gradient ultracentrifugation studies suggested that Erg25p/Erg26p/Erg27p/Erg28p, along with other proteins in sterol biosynthesis, might form a complex between 66 and 200 kDa . Using an anti-HA column with Erg27p-HA and Erg26p-HA as target proteins, a complex containing Erg25p/Erg26p/Erg27p/Erg28p was identified . Thus, we suggest that Erg28p works as a transmembrane scaffold to tether Erg27p and possibly other C-4 demethylation proteins (Erg25p, Erg26p), forming a demethylation complex in the endoplasmic reticulum.

J Cell Sci, 2002 Aug 1, 115(Pt 15), 3139 - 48
Rho5p downregulates the yeast cell integrity pathway; Schmitz HP et al.; The Rho family of proteins and their effectors are key regulators involved in many eukaryotic cell functions . In Saccharomyces cerevisiae the family consists of six members, Rho1p to Rho5p and Cdc42p . With the exception of Rho5p, these enzymes have been assigned different biological functions, including the regulation of polar growth, morphogenesis, actin cytoskeleton, budding and secretion . Here we show that a rho5 deletion results in an increased activity of the protein kinase C (Pkc1p)-dependent signal transduction pathway . Accordingly, the deletion shows an increased resistance to drugs such as caffeine, Calcofluor white and Congo red, which indicates activation of the pathway . In contrast, overexpression of an activated RHO5Q91H mutant renders cells more sensitive to these drugs . We conclude that Rho5p acts as an off-switch for the MAP-kinase cascade, which differentiates between MAP-kinase-dependent and -independent functions of Pkc1p . Kinetics of actin depolarisation and repolarisation after heat treatment of rho5 deletions as well as strains overexpressing the activated RHO5Q91H allele provide further evidence for such a function.

Bioinformatics, 2002 Jul, 18(7), 1004 - 10
Determining a unique defining DNA sequence for yeast species using hashing techniques; Wesselink JJ et al.; MOTIVATION: Yeasts are often still identified with physiological growth tests, which are both time consuming and unsuitable for detection of a mixture of organisms . Hence, there is a need for molecular methods to identify yeast species . RESULTS: A hashing technique has been developed to search for unique DNA sequences in 702 26S rRNA genes . A unique DNA sequence has been found for almost every yeast species described to date . The locations of the unique defining sequences are in accordance with the variability map of large subunit ribosomal RNA and provide detail of the evolution of the D1/D2 region . This approach will be applicable to the rapid identification of unique sequences in other DNA sequence sets . AVAILABILITY: Freely available upon request from the authors . Supplementary information: Results are available at http://www.sys.uea.ac.uk/~jjw/project/paper

Plant Physiol, 2002 Jul, 129(3), 1032 - 44
Effect of yeast CTA1 gene expression on response of tobacco plants to tobacco mosaic virus infection; Talarczyk A et al.; The response of tobacco (Nicotiana tabacum L . cv Xanthi-nc) plants with elevated catalase activity was studied after infection by tobacco mosaic virus (TMV) . These plants contain the yeast (Saccharomyces cerevisiae) peroxisomal catalase gene CTA1 under the control of the cauliflower mosaic virus 35S promoter . The transgenic lines exhibited 2- to 4-fold higher total in vitro catalase activity than untransformed control plants under normal growth conditions . Cellular localization of the CTA1 protein was established using immunocytochemical analysis . Gold particles were detected mainly inside peroxisomes, whereas no significant labeling was detected in other cellular compartments or in the intercellular space . The physiological state of the transgenic plants was evaluated in respect to growth rate, general appearance, carbohydrate content, and dry weight . No significant differences were recorded in comparison with non-transgenic tobacco plants . The 3,3'-diaminobenzidine-stain method was applied to visualize hydrogen peroxide (H(2)O(2)) in the TMV infected tissue . Presence of H(2)O(2) could be detected around necrotic lesions caused by TMV infection in non-transgenic plants but to a much lesser extent in the CTA1 transgenic plants . In addition, the size of necrotic lesions was significantly bigger in the infected leaves of the transgenic plants . Changes in the distribution of H(2)O(2) and in lesion formation were not reflected by changes in salicylic acid production . In contrast to the local response, the systemic response in upper noninoculated leaves of both CTA1 transgenic and control plants was similar . This suggests that increased cellular catalase activity influences local but not systemic response to TMV infection.

Cell Motil Cytoskeleton, 2002 Jul, 52(3), 161 - 73
Yeast polypeptide chain release factors eRF1 and eRF3 are involved in cytoskeleton organization and cell cycle regulation; Valouev IA et al.; Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3 . eRF1 recognizes nonsense codons UAA, UAG, and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRF1-dependent manner . In the yeast Saccharomyces cerevisiae, eRF1 and eRF3 are encoded by the SUP45 and SUP35 genes, respectively . Here we show that in yeast shortage of any one of the release factors was accompanied by a reduction in the levels of the other release factor and resulted in a substantial increase of nonsense codon readthrough . Besides, repression of the genes encoding these factors caused different effects on cell morphology . Repression of the SUP35 gene caused accumulation of cells of increased size with large buds . This was accompanied by the disappearance of actin cytoskeletal structures, impairment of the mitotic spindle structure, and defects in nuclei division and segregation in mitosis . The evolutionary conserved C-terminal domain of eRF3 similar to the elongation factor EF-1alpha was responsible for these effects . Repression of the SUP45 gene caused accumulation of unbudded cells with 2C and higher DNA content, indicating that DNA replication is uncoupled from budding . The data obtained suggest that eRF1 and eRF3 play additional, nontranslational roles in the yeast cell .

J Cell Biochem, 2002, 86(2), 224 - 38
Helix 12 in the human estrogen receptor (hER) is essential for the hER function by overcoming nucleosome repression in yeast; Gu X; When exogenous human estrogen receptor (hER) binds with estrogen, it can activate transcription of target genes in yeast cells . The estrogen dose-response expression patterns in yeast are very similar to those in human cells . This implies that hER may function in yeast cells via mechanisms similar to those in human cells . In this study, Saccharomyces cerevisiae was used to dissect mechanisms of hER-activated transcription in yeast . The hER contains two transcription activation domains: ER-AF-1 and ER-AF-2 (LBD or HBD) . In both human and wild-type yeast cells, hER must bind with estrogen in order to activate transcription . In those cells, ER-AF-2 is independently active upon hormone binding, but ER-AF-1 by itself is inactive . In a mutagenesis screen, we found a mutant strain in which the ER-AF-1 was independently active . It was determined that this mutant strain carried a Tup1 mutation . More interestingly, a small hER fragment ER-AF-0, containing neither ER-AF-1 nor ER-AF-2, was also fully active in the DeltaTup1 cells . This suggests that in this strain, hormone binding is not required for transcription activation by hER . It is known that the Tup1/Ssn6 complex plays an important role in general transcription repression by protecting histone acetylation sites thus stabilizing nucleosomes . In the DeltaTup1 cells, nucleosomes are known to be unstable because histones can be easily accessed by acetylase and cause nucleosome disassociation . Two point mutations in helix 12 (H12) in ER-AF-2, which abolished hER function in human cells, also completely abolished hER function in the wild-type yeast cells . This suggested that H12 is essential for hER transcription activation function . However, hER with the H12 mutation is able to activate transcription in DeltaTup1 cells . This indicates that the normal function of H12 is required for transcription activation by hER only if nucleosomes are not acetylated and are therefore stable . The results of this work suggest that there is a close relationship between hER function and nucleosome remodeling . It also provides insight about H12 activity and its functional relationship with other domains in hER . We propose here that H12 is essential for hER function by recruiting strong nucleosome remodeling proteins to the promoter region thus overcoming nucleosome repression .

Bioessays, 2002 Jul, 24(7), 659 - 66
MEN, destruction and separation: mechanistic links between mitotic exit and cytokinesis in budding yeast; Yeong FM et al.; Cellular events must be executed in a certain sequence during the cell division in order to maintain genome integrity and hence ensure a cell's survival . In M phase, for instance, chromosome segregation always precedes mitotic exit (characterized by mitotic kinase inactivation via cyclin destruction); this is then followed by cytokinesis . How do cells impose this strict order? Recent findings in budding yeast have suggested a mechanism whereby partitioning of chromosomes into the daughter cell is a prerequisite for the activation of mitotic exit network (MEN) . So far, however, a regulatory scheme that would temporally link the initiation of cytokinesis to the execution of mitotic exit has not been determined . We propose that the requirement of MEN components for cytokinesis, their translocation to the mother-daughter neck and triggering of this translocation by inactivation of the mitotic kinase may be the three crucial elements that render initiation of cytokinesis dependent on mitotic exit .

Mol Genet Genomics, 2002 Jun, 267(4), 515 - 25 Epub 2002 May 23.
Expression analysis of RNA14, a gene involved in mRNA 3' end maturation in yeast: characterization of the rna14-5 mutant strain; Brendolise C et al.; In Saccharomyces cerevisiae, Rna14 protein is involved in both cleavage and polyadenylation of mRNA in the nucleus . Previous work has demonstrated that this protein is also localized in mitochondria . Moreover, all known rna14 mutants can be separated into two distinct classes: the poly(A)-negative class, which contains mutants that are deficient in mRNA 3'-end processing, and the poly(A)-positive class, which includes those mutants that are not impaired in any of the steps in mRNA metabolism investigated . This suggests that in addition to its involvement in mRNA polyadenylation, Rna14p could have a second function related to mitochondrial metabolism . Here we investigated the regulation of RNA14 by characterizing the rna14-5 mutant, which is the only poly(A)-positive allele that also overproduces the RNA14 mRNA . We showed that both deregulation of RNA14 transcription and modification of RNA14 mRNA stability contribute to the strong accumulation of the transcripts in this mutant . Surprisingly, the RNA14 promoter itself is not essential for this phenotype of the rna14-5 mutant . However, the 3' UTR of the mRNA is necessary for overproduction of the transcripts, although it is not sufficient to deregulate a reporter gene by itself . Site-directed mutagenesis experiments provided additional data suggesting that the rna14-5 mutation acts at the protein level rather than modifying the properties of the RNA14 transcripts themselves . A tentative model accounting for the data is discussed, in light of the proposed extranuclear function of the Rna14p and its mitochondrial localization.

Appl Microbiol Biotechnol, 2002 Jul, 59(2-3), 329 - 31 Epub 2002 Jun 01.
An arming yeast with the ability to entrap fluorescent 17beta-estradiol on the cell surface; Yasui M et al.; We constructed a novel surface-engineered yeast displaying the ligand-binding domain of the rat estrogen receptor (ERLBD) . ERLBD, display of which on the yeast cell surface was confirmed by immunofluorescence, possessed strong binding activity to fluorescent 17beta-estradiol - an analogue of the natural ligand of the estrogen receptor - that was comparable to the activity of the native receptor . Environmental homeostasis has recently been disturbed by endocrine disruptors, which cause confusion in the hormone secretion system . It is therefore very important to identify chemical compounds with hormone-like activity and remove them from the environment . The present results demonstrate that the new arming yeast displaying ERLBD on its cell surface will be capable of screening, entrapping, and removing estradiol-like compounds from the environment.

Appl Microbiol Biotechnol, 2002 Jul, 59(2-3), 259 - 64 Epub 2002 May 09.
Cell surface-engineered yeast with ability to bind, and self-aggregate in response to, copper ion; Kuroda K et al.; In order to construct a cell surface-engineered yeast Saccharomyces cerevisiae that facilitates adsorption and recovery of heavy metal ions, we endowed it with the ability to self-aggregate in response to binding and accumulation of copper ion . A fusion gene for the expression of GTS1, which encodes a putative zinc-finger transcription factor related to occurrence of cell-aggregation, was constructed under the control of the copper ion-inducible CUP1 promoter from the yeast metallothionein gene . The multicopy plasmid carrying the fusion gene was introduced into a cell surface-engineered yeast displaying histidine hexa-peptide, which can chelate copper ion . This transformant strain aggregated in medium only in the presence of copper ion, with aggregation induced by as little as 1 mM copper ion . The copper ion-induced aggregation did not interfere with the copper ion-adsorbing function of the cell surface-engineered yeast, indicating that this transformant strain has the twin features of enhanced cell surface adsorption of copper ion and self-aggregation in response to environmental copper ion.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1999, 31(6), 718 - 722
The Relationship between Yeast Coexpressed Gene Clusters and Their Upstream cis-acting Elements; Xie T et al.; DNA microarrays bring biology a new approach to study functions of genes and genomes from their expression pattern on a genomic scale . With its fully sequenced genome and newly published expression patterns available, budding yeast (Saccharomyces cerevisiae) was chosen to carry out an investigation of the relationship between gene 5' upstream cis-acting elements and expression patterns using bioinformatic tools . Results show that genes in the same cluster share common cis-acting element candidates and can be regulated by same transfactors . In the sites found by this study, some sites are corresponding to known cis-acting elements, while others may indicate new ones that can be tested by experiments . The results are helpful to understand more about gene functions, metabolic pathways and genetic networks.

EMBO J, 2002 Jul 15, 21(14), 3881 - 7
Lesion bypass in yeast cells: Pol eta participates in a multi-DNA polymerase process; Bresson A et al.; Replication through (6-4)TT and G-AAF lesions was compared in Saccharomyces cerevisiae strains proficient and deficient for the RAD30-encoded DNA polymerase eta (Pol eta) . In the RAD30 strain, the (6-4)TT lesion is replicated both inaccurately and accurately 60 and 40% of the time, respectively . Surprisingly, in a rad30 Delta strain, the level of mutagenic bypass is essentially suppressed, while error-free bypass remains unchanged . Therefore, Pol eta is responsible for mutagenic replication through the (6-4)TT photoproduct, while another polymerase mediates its error-free bypass . Deletion of the RAD30 gene also reduces the levels of both accurate and inaccurate bypass of AAF lesions within two different sequence contexts up to 8-fold . These data show that, in contrast to the accurate bypass by Pol eta of TT cyclobutane dimers, it is responsible for the mutagenic bypass of other lesions . In conclusion, this paper shows that, in yeast, translesion synthesis involves the combined action of several polymerases.

Cell, 2002 Jun 28, 109(7), 849 - 60
Noc3p, a bHLH protein, plays an integral role in the initiation of DNA replication in budding yeast; Zhang Y et al.; Initiation of eukaryotic DNA replication requires many proteins that interact with one another and with replicators . Using a yeast genetic screen, we have identified Noc3p (nucleolar complex-associated protein) as a novel replication-initiation protein . Noc3p interacts with MCM proteins and ORC and binds to chromatin and replicators throughout the cell cycle . It functions as a critical link between ORC and other initiation proteins to effect chromatin association of Cdc6p and MCM proteins for the establishment and maintenance of prereplication complexes . Noc3p is highly conserved in eukaryotes and is the first identified bHLH (basic helix-loop-helix) protein required for replication initiation . As Noc3p is also required for pre-rRNA processing, Noc3p is a multifunctional protein that plays essential roles in two vital cellular processes.

J Biol Chem, 2002 Sep 20, 277(38), 35712 - 9 Epub 2002 Jul 09.
The yeast homolog of human PinX1 is involved in rRNA and small nucleolar RNA maturation, not in telomere elongation inhibition; Guglielmi B et al.; In human cells, PinX1 protein has recently been shown to regulate telomere length by repressing the telomerase . In this work, we show that the putative yeast homolog of PinX1, encoded by the YGR280c open reading frame (ORF), is a new component of the ribosomal RNA processing machinery . The protein has a KK(E/D) C-terminal domain typical of nucleolar proteins and bears a putative RNA interacting domain widespread in eukaryotes called the G-patch . The protein was hence renamed Gno1p (G-patch nucleolar protein) . GNO1 deletion results in a large growth defect due to the inhibition of the pre-ribosomal RNA processing first cleavage steps at sites A(0), A(1), and A(2) . Furthermore, Gno1p is involved in the final 3'-end trimming of U18 and U24 small nucleolar RNAs . A mutational analysis showed that the G-patch of Gno1p is essential for both functions, whereas the KK(E/D) repeats are only required for U18 small nucleolar RNA maturation . We found that PinX1 complemented the gno1-Delta mutation, suggesting that it has a dual function in telomere length regulation and ribosomal RNA maturation in agreement with its telomeric and nucleolar localization in human cells . Conversely, we found that Gno1p does not exhibit the in vivo telomerase inhibitor activity of PinX1.

Biochimie, 2002 Apr, 84(4), 279 - 89
Participation of yeast inosine 5'-monophosphate dehydrogenase in an in vitro complex with a fragment of the C-rich telomeric strand; Cornuel JF et al.; As part of our investigation of the i-motif, an intercalated structure formed by C-rich nucleic acid sequences, we searched for proteins of Saccharomyces cerevisiae which could associate with a sequence of the C-rich telomeric strand, d((CCCACA)(3)CCC) . A gel retardation assay of yeast protein extract, in conditions where the DNA fragment folds into an intramolecular i-motif, shows formation of one major retarded band . The retarding factor was further characterized by a differential affinity procedure using streptavidin beads coated (or not coated) with the biotin-labeled DNA fragment . Differentially bound proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identified by mass spectroscopy and Edman degradation as Imd2p, Imd3p and Imd4p . These highly similar (>95%) proteins are analogs of the two human NAD-dependent inosine 5'-monophosphate dehydrogenases (IMPDH) which occur as tetramers . The mass of the protein, as determined by gel exclusion chromatography, is about 250 kDa and is compatible with an IMPDH tetramer, but other compositions, involving non-IMPDH components, are not excluded . We note that the genes coding for Imd2p and Imd3p are located close to the telomere, and could therefore be subject to silencing by the telomere position effect.

Biochimie, 2002 Apr, 84(4), 265 - 72
Kinetic analysis of yeast galactokinase: implications for transcriptional activation of the GAL genes; Timson DJ et al.; Galactokinase (EC 2.7.1.6) catalyses the first step in the catabolism of galactose . Yeast galactokinase, Gal1p, and the closely related but catalytically inactive Gal3p, also function as ligand sensors in the GAL genetic switch . In the presence of galactose and ATP (the substrates of the reaction catalysed by Gal1p) Gal1p or Gal3p can bind to Gal80p, a transcriptional repressor . This relieves the inhibition of a transcriptional activator, Gal4p, and permits expression of the GAL genes . In order to learn more about the mechanism of ligand sensing by Gal3p and Gal1p, we studied the kinetics of the reaction catalysed by Gal1p . Galactose-1-phosphate, a product of the reaction, is a mixed inhibitor both with respect to galactose and to ATP suggesting that the reaction proceeds via a compulsory, ordered, ternary complex mechanism . There is little variation in either the turnover number or the specificity constants in the pH range 6.0-9.5, implying that no catalytic base is required in the reaction . These data are discussed both in the context of galactokinase enzymology and their implications for the mechanism of transcriptional induction.

Radiat Res, 2002 Aug, 158(2), 195 - 201
Detection of ATM gene mutation in human glioma cell line M059J by a rapid frameshift/stop codon assay in yeast; Tsuchida R et al.; A yeast-based frameshift/stop codon assay for examining ATM (ataxia telangiectasia mutated) mutations was established . Each of six fragments of a PCR-amplified coding sequence for ATM is inserted in frame by homologous recombination into a yeast URA3 fusion protein gene, and the transformants are assayed for growth in the absence of uracil . The usefulness of this assay was verified in a panel of cell lines derived from individuals with homozygous and heterozygous ATM mutations . The assay was also shown to distinguish between specimens with wild-type alleles and those with truncating mutations: a frameshift mutation or an inserted stop codon . Using this assay M059J cells, which fail to express the catalytic subunit of DNA-dependent protein kinase (PRKDC, also known as DNA-PKcs) and are hypersensitive to ionizing radiation, were found to express two different aberrant ATM transcripts: one characterized by 4776 del 133, which corresponds to the deletion of exon 33, and the other by 4909 ins 116 . Subsequent analysis of the intron sequences revealed that 4909 ins 116 is comprised of a nucleotide sequence corresponding to 84013-84128 in intron 33 with a cryptic splice site . Thus the radiosensitive phenotype of M059J cells appears to be due to a defect in PRKDC and a truncating ATM mutation.

Proc Natl Acad Sci U S A, 2002 Jul 23, 99(15), 9936 - 41 Epub 2002 Jul 08.
Amino acid residue 184 of yeast Hsp104 chaperone is critical for prion-curing by guanidine, prion propagation, and thermotolerance; Jung G et al.; Inactivation of Hsp104 by guanidine is contended to be the mechanism by which guanidine cures yeast prions . We now find an Hsp104 mutation (D184N) that confers resistance to guanidine-curing of the yeast {PSI(+)} prion . In an independent screen we isolated an HSP104 allele altered in the same residue (D184Y) that dramatically impairs {PSI(+)} propagation in a temperature-dependent manner . Directed mutagenesis of HSP104 produced additional alleles that conferred varying degrees of resistance to guanidine-curing or impaired {PSI(+)} propagation . The mutations similarly affected propagation of the {URE3} prion . Basal and induced abundance of all mutant proteins was normal . Thermotolerance of cells expressing mutant proteins was variably resistant to guanidine, and the degree of thermotolerance did not correlate with {PSI(+)} stability . We thus show that guanidine cures yeast prions by inactivating Hsp104 and identify a highly conserved Hsp104 residue that is critical for yeast prion propagation . Our data suggest that Hsp104 activity can be reduced substantially without affecting {PSI(+)} stability, and that Hsp104 interacts differently with prion aggregates than with aggregates of thermally denatured protein.

J Biol Chem, 2002 Sep 20, 277(38), 35642 - 9 Epub 2002 Jun 24.
Upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene 1 (LAG1), regulates N-stearoyl-sphinganine (C18-(dihydro)ceramide) synthesis in a fumonisin B1-independent manner in mammalian cells; Venkataraman K et al.; The longevity assurance gene (LAG1) and its homolog (LAC1) are required for acyl-CoA-dependent synthesis of ceramides containing very long acyl chain (e.g . C26) fatty acids in yeast, and a homolog of LAG1, ASC1, confers resistance in plants to fumonisin B(1), an inhibitor of ceramide synthesis . To understand further the mechanism of regulation of ceramide synthesis, we now characterize a mammalian homolog of LAG1, upstream of growth and differentiation factor-1 (uog1) . cDNA clones of uog1 were obtained from expression sequence-tagged clones and sub-cloned into a mammalian expression vector . Transient transfection of human embryonic kidney 293T cells with uog1 followed by metabolic labeling with {4,5-(3)H}sphinganine or L-3-{(3)H}serine demonstrated that uog1 conferred fumonisin B(1) resistance with respect to the ability of the cells to continue to produce ceramide . Surprisingly, this ceramide was channeled into neutral glycosphingolipids but not into gangliosides . Electrospray tandem mass spectrometry confirmed the elevation in sphingolipids and revealed that the ceramides and neutral glycosphingolipids of uog1-transfected cells contain primarily stearic acid (C18), that this enrichment was further increased by FB(1), and that the amount of stearic acid in sphingomyelin was also increased . UOG1 was localized to the endoplasmic reticulum, demonstrating that the fatty acid selectivity and the fumonisin B(1) resistance are not due to a subcellular localization different from that found previously for ceramide synthase activity . Furthermore, in vitro assays of uog1-transfected cells demonstrated elevated ceramide synthase activity when stearoyl-CoA but not palmitoyl-CoA was used as substrate . We propose a role for UOG1 in regulating C18-ceramide (N-stearoyl-sphinganine) synthesis, and we note that not only is this the first case of ceramide formation in mammalian cells with such a high degree of fatty acid specificity, but also that the N-stearoyl-sphinganine produced by UOG1 most significantly impacts neutral glycosphingolipid synthesis.

Genes Dev, 2002 Jul 1, 16(13), 1682 - 95
Close, stable homolog juxtaposition during meiosis in budding yeast is dependent on meiotic recombination, occurs independently of synapsis, and is distinct from DSB-independent pairing contacts; Peoples TL et al.; A site-specific recombination system that probes the relative probabilities that pairs of chromosomal loci collide with one another in living cells of budding yeast was used to explore the relative contributions of pairing, recombination, synaptonemal complex formation, and telomere clustering to the close juxtaposition of homologous chromosome pairs during meiosis . The level of Cre-mediated recombination between a pair of loxP sites located at an allelic position on homologous chromosomes was 13-fold greater than that between a pair of loxP sites located at ectopic positions on nonhomologous chromosomes . Mutations affecting meiotic recombination initiation and the processing of DNA double-strand breaks (DSBs) into single-end invasions (SEIs) reduced the levels of allelic Cre-mediated recombination levels by three- to sixfold . The severity of Cre/loxP phenotypes is presented in contrast to relatively weak DSB-independent pairing defects as assayed using fluorescence in situ hybridization for these mutants . Mutations affecting synaptonemal complex (SC) formation or crossover control gave wild-type levels of allelic Cre-mediated recombination . A delay in attaining maximum levels of allelic Cre-mediated recombination was observed for a mutant defective in telomere clustering . None of the mutants affected ectopic levels of recombination . These data suggest that stable, close homolog juxtaposition in yeast is distinct from pre-DSB pairing interactions, requires both DSB and SEI formation, but does not depend on crossovers or SC.

Genes Dev, 2002 Jul 1, 16(13), 1627 - 39
Microtubule capture by the cleavage apparatus is required for proper spindle positioning in yeast; Kusch J et al.; Cell division is the result of two major cytoskeletal events: partition of the chromatids by the mitotic spindle and cleavage of the cell by the cytokinetic apparatus . Spatial coordination of these events ensures that each daughter cell inherits a nucleus . Here we show that, in budding yeast, capture and shrinkage of astral microtubules at the bud neck is required to position the spindle relative to the cleavage apparatus . Capture required the septins and the microtubule-associated protein Kar9 . Like Kar9-defective cells, cells lacking the septin ring failed to position their spindle correctly and showed an increased frequency of nuclear missegregation . Microtubule attachment at the bud neck was followed by shrinkage and a pulling action on the spindle . Enhancement of microtubule shrinkage at the bud neck required the Par-1-related, septin-dependent kinases (SDK) Hsl1 and Gin4 . Neither the formin Bnr1 nor the actomyosin contractile ring was required for either microtubule capture or microtubule shrinkage . Together, our results indicate that septins and septin-dependent kinases may coordinate microtubule and actin functions in cell division.

EMBO Rep, 2002 Jul, 3(7), 652 - 9
Intracellular trafficking of yeast telomerase components; Teixeira MT et al.; Telomerase uses an internal RNA moiety as template for the synthesis of telomere repeats . In Saccharomyces cerevisiae, the telomerase holoenzyme contains the telomerase reverse transcriptase subunit Est2p, the telomerase RNA moiety TLC1, the telomerase associated proteins Est1p and Est3p, and Sm proteins . Here we assess telomerase assembly by determining the localization of telomerase components . We found that Est1p, Est2p and TLC1 can migrate independently of each other to the nucleus . With limiting amounts of TLC1, overexpressed Est1p and Est2p accumulated in the nucleolus, whereas enzymatically active Est2p-TLC1 complexes are distributed over the entire nucleus . The distribution to the nucleoplasm depended on the specific interaction between Est2p and TLC1 but was independent of Est1p and Est3p . Altogether, our results suggest a role of the nucleolus in telomerase biogenesis . We also describe experiments that support a transient cytoplasmic localization of TLC1 RNA.

EMBO Rep, 2002 Jul, 3(7), 628 - 35
A complex prediction: three-dimensional model of the yeast exosome; Aloy P et al.; We present a model of the yeast exosome based on the bacterial degradosome component polynucleotide phosphorylase (PNPase) . Electron microscopy shows the exosome to resemble PNPase but with key differences likely related to the position of RNA binding domains, and to the location of domains unique to the exosome . We use various techniques to reduce the many possible models of exosome subunits based on PNPase to just one . The model suggests numerous experiments to probe exosome function, particularly with respect to subunits making direct atomic contacts and conserved, possibly functional residues within the predicted central pore of the complex.

Biochim Biophys Acta, 2002 Aug 19, 1564(1), 9 - 13
Phosphatidyl ethanolamine is essential for targeting the arginine transporter Can1p to the plasma membrane of yeast; Opekarova M et al.; In continuation of our previous study, we show that phosphatidyl ethanolamine (PE) depletion affects, in addition to amino acid transporters, activities of at least two other proton motive force (pmf)-driven transporters (Ura4p and Mal6p) . For Can1p, we demonstrate that the lack of PE results in a failure of the permease targeting to plasma membrane . Despite the pleiotropic effect of PE depletion, a specific role of PE in secretion of a defined group of permeases can be distinguished . Pmf-driven transporters are more sensitive to the lack of PE than other plasma membrane proteins.

Mol Microbiol, 2002 Jul, 45(1), 233 - 41
The control of the yeast H2O2 response by the Msn2/4 transcription factors; Hasan R et al.; We have analysed the contribution of the Msn2/4 transcription factors and the Ras-cAMP-protein kinase A (PKA) pathway to the control of the yeast H2O2 response . Strains deleted for MSN2 and MSN4 are hypersensitive to H2O2, although they can still adapt to this oxidant . They are also unable to induce 27 proteins of the H2O2 stimulon as shown by quantitative two-dimensional gel analysis . This peculiar H2O2 tolerance defect, the nature of the proteins of the Msn2/4 regulon, and the partial overlap of this regulon with the Yap1 H2O2-response regulon, suggest an independent and distinctive role of these two H2O2 stress response pathways . A strain lacking PDE2, and therefore carrying high intracellular cAMP levels, is also hypersensitive to H2O2 . In the presence of exogenous cAMP, this strain does not induce the entire H2O2 Msn2/4 regulon and some other proteins . This, and the normal H2O2 induction of a gene reporter under control of the Yap1 regulator when intracellular cAMP level are high, demonstrate that the Ras-cAMP pathway negatively affects the H2O2 stress response through Msn2/4 . However, the high H2O2 sensitivity of a strain lacking the PKA-negative regulatory subunit Bcy1, is not only the consequence of the inhibition of Msn2/4 but also of Yap1 through a yet undefined mechanism.

Biochem Biophys Res Commun, 2002 Jul 19, 295(3), 724 - 9
Functional expression and analysis of the pancreatic transcription factor PDX-1 in yeast; Ozcan S et al.; The pancreas-specific transcription factor Pdx-1 is important for pancreas development and beta-cell specific gene expression in insulin-producing cells . We have expressed the mouse PDX-1 gene in the yeast Saccharomyces cerevisiae and characterized its functional domains . Pdx-1 functions as a strong activator in yeast and stimulates gene expression by more than 80-fold . The transcriptional activation domain of Pdx-1 is located within the first 144 amino-terminal amino acids . Pdx-1 is also able to bind and activate transcription from the A3 element of the human insulin gene promoter in yeast . Analysis of the effects of two-point mutations (Q59L and R197H) in the PDX-1 gene found in type II diabetes patients showed that both point mutations interfere with the ability of Pdx-1 to bind to DNA and to activate transcription in yeast . (c) 2002 Elsevier Science (USA).

Biochem Biophys Res Commun, 2002 Jul 19, 295(3), 673 - 7
Human CDC25B and CDC25C differ by their ability to restore a functional checkpoint response after gene replacement in fission yeast; Mondesert O et al.; In fission yeast, inactivation of the Cdc25 phosphatase by checkpoint kinases participates in the signaling cascade that temporarily stops cell cycle progression after DNA damage . In human, CDC25B and C are also known to be targeted by a similar checkpoint machinery . We have examined by homologous recombination, whether CDC25B and CDC25C were able to substitute for the function of fission yeast Cdc25 . We demonstrate that (i) CDC25B and C efficiently replace Cdc25 for vegetative growth, (ii) CDC25C is able to restore a functional checkpoint in response to ionizing radiation in both a Chk1- and Cds1-dependent manner, (iii) CDC25B and C are equally efficient in the response to UV irradiation, CDC25B being only dependent on Chk1, while CDC25C depends on both Chk1 and Cds1, and (iv) CDC25C is able to restore a functional DNA replication checkpoint induced by hydroxyurea in a Cds1-dependent manner . The consequences of these findings on our current view of the checkpoint cascade are discussed . (c) 2002 Elsevier Science (USA).

Proc Natl Acad Sci U S A, 2002 Jul 23, 99(15), 9846 - 51 Epub 2002 Jul 03.
Visualization of maltose uptake in living yeast cells by fluorescent nanosensors; Fehr M et al.; Compartmentation of metabolic reactions and thus transport within and between cells can be understood only if we know subcellular distribution based on nondestructive dynamic monitoring . Currently, methods are not available for in vivo metabolite imaging at cellular or subcellular levels . Limited information derives from methods requiring fixation or fractionation of tissue (1, 2) . We thus developed a flexible strategy for designing protein-based nanosensors for a wide spectrum of solutes, allowing analysis of changes in solute concentration in living cells . We made use of bacterial periplasmic binding proteins (PBPs), where we show that, on binding of the substrate, PBPs transform their hinge-bend movement into increased fluorescence resonance energy transfer (FRET) between two coupled green fluorescent proteins . By using the maltose-binding protein as a prototype, nanosensors were constructed allowing in vitro determination of FRET changes in a concentration-dependent fashion . For physiological applications, mutants with different binding affinities were generated, allowing dynamic in vivo imaging of the increase in cytosolic maltose concentration in single yeast cells . Control sensors allow the exclusion of the effect from other cellular or environmental parameters on ratio imaging . Thus the myriad of PBPs recognizing a wide spectrum of different substrates is suitable for FRET-based in vivo detection, providing numerous scientific, medical, and environmental applications.

Mol Cell Proteomics, 2002 Jan, 1(1), 3 - 10
Charting the protein complexome in yeast by mass spectrometry; Deshaies RJ et al.; It has become evident over the past few years that many complex cellular processes, including control of the cell cycle and ubiquitin-dependent proteolysis, are carried out by sophisticated multisubunit protein machines that are dynamic in abundance, post-translational modification state, and composition . To understand better the nature of the macromolecular assemblages that carry out the cell cycle and ubiquitin-dependent proteolysis, we have used mass spectrometry extensively over the past few years to characterize both the composition of various protein complexes and the modification states of their subunits . In this article we review some of our recent efforts, and describe a promising new approach for using mass spectrometry to dissect protein interaction networks.

Mol Cell Proteomics, 2002 Mar, 1(3), 186 - 96
Mass spectrometry-based methods for phosphorylation site mapping of hyperphosphorylated proteins applied to Net1, a regulator of exit from mitosis in yeast; Loughrey Chen S et al.; Prior to anaphase in Saccharomyces cerevisiae, Cdc14 protein phosphatase is sequestered within the nucleolus and inhibited by Net1, a component of the RENT complex in budding yeast . During anaphase the RENT complex disassembles, allowing Cdc14 to migrate to the nucleus and cytoplasm where it catalyzes exit from mitosis . The mechanism of Cdc14 release appears to involve the polo-like kinase Cdc5, which is capable of promoting the dissociation of a recombinant Net1.Cdc14 complex in vitro by phosphorylation of Net1 . We report here the phosphorylation site mapping of recombinant Net1 (Net1N) and a mutant Net1N allele (Net1N-19m) with 19 serines or threonines mutated to alanine . A variety of chromatographic and mass spectrometric-based strategies were used, including immobilized metal-affinity chromatography, alkaline phosphatase treatment, matrix-assisted laser-desorption post-source decay, and a multidimensional electrospray mass spectrometry-based approach . No one approach was able to identify all phosphopeptides in the tryptic digests of these proteins . Most notably, the presence of a basic residue near the phosphorylated residue significantly hampered the ability of alkaline phosphatase to hydrolyze the phosphate moiety . A major goal of research in proteomics is to identify all proteins and their interactions and post-translational modification states . The failure of any single method to identify all sites in highly phosphorylated Net1N, however, raises significant concerns about how feasible it is to map phosphorylation sites throughout the proteome using existing technologies.

J Biol Chem, 2002 Sep 13, 277(37), 33749 - 57 Epub 2002 Jul 02.
Combinatorial control of yeast FET4 gene expression by iron, zinc, and oxygen; Waters BM et al.; Acquisition of metals such as iron, copper, and zinc by the yeast Saccharomyces cerevisiae is tightly regulated . High affinity uptake systems are induced under metal-limiting conditions to maintain an adequate supply of these essential nutrients . Low affinity uptake systems function when their substrates are in greater supply . The FET4 gene encodes a low affinity iron and copper uptake transporter . FET4 expression is regulated by several environmental factors . In this report, we describe the molecular mechanisms underlying this regulation . First, we found that FET4 expression is induced in iron-limited cells by the Aft1 iron-responsive transcriptional activator . Second, FET4 is regulated by zinc status via the Zap1 transcription factor . We present evidence that FET4 is a physiologically relevant zinc transporter and this provides a rationale for its regulation by Zap1 . Finally, FET4 expression is regulated in response to oxygen by the Rox1 repressor . Rox1 attenuates activation by Aft1 and Zap1 in aerobic cells . Derepression of FET4 may allow the Fet4 transporter to play an even greater role in metal acquisition under anaerobic conditions . Thus, Fet4 is a multisubstrate metal ion transporter under combinatorial control by iron, zinc, and oxygen.

Gene, 2002 May 29, 291(1-2), 251 - 7
Effect of the DNA topoisomerase II inhibitor VP-16 on illegitimate recombination in yeast chromosomes; Asami Y et al.; Etoposide and teniposide, derivatives of podophyllotoxin, are inhibitors of DNA topoisomerase II and are potent anticancer agents . An adverse effect linked to the use of these drugs is the development of acute myeloid leukemia, a disorder usually associated with chromosomal translocation . To examine podophyllotoxin-induced DNA rearrangement, we developed an assay system to measure illegitimate recombination in Saccharomyces cerevisiae chromosomes . This approach uses juxtaposed CAN1-CYH2 negative selection markers that are introduced into the LEU2 locus, which is located on chromosome III, in a yeast strain carrying the mutated can1 and cyh2 genes . Upon formation of a deletion over the active CAN1-CYH2 genes, a cell becomes resistant to both canavanine and cycloheximide . To introduce drugs into the cell, we used a yeast strain carrying an ISE2 mutation, thereby making the cell drug-permeable . Here we show that treatment of cells with etoposide (VP-16) increases the rate of illegitimate recombination in yeast, indicating that VP-16 stimulates DNA topoisomerase-mediated illegitimate recombination . Structural analysis of the resulting recombinants indicate that most are formed by deletion mutations on chromosome III, which take place between short homologous regions of DNA . We propose a model for illegitimate recombination, in which VP-16 facilitates formation of a cleavable complex between DNA topoisomerase II and DNA, thus promoting DNA double-strand breakage with the resulting DNA ends joined by a non-homologous mechanism.

Cell Biol Int, 2002, 26(5), 393 - 405
DNA plasmid transmission in yeast is associated with specific sub-nuclear localisation during cell division; Scott-Drew S et al.; Circular plasmids in yeast carrying only an origin of DNA replication (ARS) exhibit maternal inheritance bias (MIB) and are poorly transmitted from mother to daughter cell during division . A variety of different sequences that overcome MIB have been described, including centromeric sequences (CEN), telomere-associated repeats, silencer sequences and a specific system encoded by the endogenous 2 micron circle plasmid requiring the cis-acting locus STB and the proteins Rep1 and Rep2 . In each case, DNA segregation between mother and daughter cells is dependent on DNA-protein interactions . Using plasmids carrying multiple copies of a lac repressor binding sequence, we have localised DNA molecules in the yeast nucleus using a green fluorescent protein (GFP)-lac repressor fusion protein . We compared GFP localised plasmids carrying a centromere sequence with plasmids based on 2 micron circle carrying or lacking the STB sequences required for their segregation . We show that GFP localised plasmid carrying the complete STB locus co-localises with the plasmid proteins Rep1 and Rep2 to discrete chromatin sites . These sites are distinct from both the telomeres and from sites of cohesin binding . Deletion of the region of STB essential for the stability of the plasmid, leads to a loss of plasmid association with chromatin, relocalisation of plasmids towards the nuclear periphery, and a decrease in the Rep1 protein associated with the plasmid . We conclude that specific plasmid localisation is likely to be important in the overcoming of MIB in yeast .

Int Immunopharmacol, 2002 May, 2(6), 775 - 81
Increased efficiency of Lewis lung carcinoma chemotherapy with a macrophage stimulator--yeast carboxymethyl glucan; Kogan G et al.; The efficiency of chemotherapy of Lewis lung carcinoma with cyclophosphamide was affected by administration of the water-soluble yeast polysaccharide derivative--carboxymethylated (1 --> 3)-beta-D-glucan (CMG)-a well-known macrophage stimulator . It was found that while cyclophosphamide showed 57% growth inhibition of the intramuscular tumor implants in comparison with the control group, its combined administration with CMG led to 75-90% inhibition . Similarly, increased inhibition of occurrence of lung metastases (up to 92-94%) was observed using the combined application of the two compounds . The stimulatory effect of CMG is not associated with the changed cellularity of peripheral blood, but is rather due to the obviously increased concentration of the intracellular inhibitor of cysteine proteases-stefin A and cystatin C in tumor tissue.

EMBO J, 2002 Jul 1, 21(13), 3526 - 35
The yeast THO complex and mRNA export factors link RNA metabolism with transcription and genome instability; Jimeno S et al.; The THO complex is a multimeric factor containing four polypeptides, Tho2, Hpr1, Mft1 and Thp2 . Mutations in any of the genes encoding THO confer impairment of transcription and a transcription-dependent hyper-recombination phenotype, suggesting that THO has a functional role in gene expression . Using an in vivo assay developed to study expression of long and G+C-rich DNA sequences, we have isolated SUB2, a gene involved in mRNA splicing and export, as a multicopy suppressor of the gene expression defect of hpr1 Delta . Further investigation of a putative functional relationship between mRNA metabolism and THO revealed that mRNA export mutants sub2, yra1, mex67 and mtr2 have similar defective transcription and hyper-recombination phenotypes as THO mutants . In addition, THO becomes essential in cells with a defective Mex67 mRNA export er . Finally, we have shown that THO has the ability to associate with RNA and DNA in vitro . These results indicate a functional link between the processes of elongation and metabolism of nascent mRNA mediated by THO and mRNA export proteins, which have important consequences for the maintenance of genome stability.

EMBO J, 2002 Jul 1, 21(13), 3424 - 33
Mapping histone fold TAFs within yeast TFIID; Leurent C et al.; The transcription factor TFIID is a large multiprotein complex, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), which plays a key role in the regulation of gene expression by RNA polymerase II . The three-dimensional structure of yeast (y) TFIID, determined at approximately 3 nm resolution by electron microscopy and image analysis, resembles a molecular clamp formed by three major lobes connected by thin linking domains . The yTFIID is structurally similar to the human factor although the clamp appears more closed in the yeast complex, probably reflecting the conformational flexibility of the structure . Immunolabelling experiments showed that nine TAFs that contain the histone fold structural motif were located in three distinct substructures of TFIID . The distribution of these TAFs showed that the previously reported pair-wise interactions between histone fold domain (HFD)-containing TAFs are likely to occur in the native yTFIID complex . Most of the HFD-containing TAFs have been found in two distinct lobes, thus revealing an unexpected and novel molecular organization of TFIID.

Biosci Biotechnol Biochem, 2002 May, 66(5), 978 - 85
Purification and characterization of a lipase from the glycolipid-producing yeast Kurtzmanomyces sp . I-11; Kakugawa K et al.; An extracellular lipase produced by the glycolipid-producing yeast Kurtzmanomyces sp . I-11 was purified by ammonium sulfate precipitation and column chromatographies on DEAE-Sephadex A-25, SP-Sephadex C-50, and Sephadex G-100 . Based on the analysis of the purified lipase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified lipase was judged to be homogeneous and its molecular mass was estimated to be approximately 49 kDa . The optimum temperature for the activity was 75 degrees C, and the activity was very stable at temperatures below 70 degrees C . The active pH range of this lipase was 1.9-7.2, and the activity was stable at pH below 7.1 . The lipase showed a preference for C18 acyl groups by measurements with p-nitrophenyl esters and triglycerides as substrates . The lipase was very stable in the presence of various organic solvents at a concentration of 40% . Although the N-terminal sequence of the Kurtzmanomyces lipase was very similar to that of lipase A from Candida antarctica, the pH profiles of the two lipases were significantly different.

Biosci Biotechnol Biochem, 2002 May, 66(5), 1105 - 7
Analysis of transactivation region of Elf-1 by using a yeast one-hybrid system; Nishiyama C et al.; The transcriptional regulatory region of Elf-1 was analyzed by the combination of a yeast one-hybrid system and site-directed mutagenesis . This analysis enabled us to map an activation region between 85-175 of Elf-1.

Biosci Biotechnol Biochem, 2002 May, 66(5), 1069 - 74
Phenylethylamine induces an increase in cytosolic Ca2+ in yeast; Pinontoan R et al.; Beta-phenylethylamine (PEA) induced an increase in cytosolic free calcium ion concentration ({Ca2+}c) in Saccharomyces cerevisiae cells monitored with transgenic aequorin, a Ca2+-dependent photoprotein . The PEA-induced {Ca2+}c increase was dependent on the concentrations of PEA applied, and the Ca2+ mostly originated from an extracellular source . Preceding the Ca2+ influx, H2O2 was generated in the cells by the addition of PEA . Externally added H2O2 also induced a {Ca2+}c increase . These results suggest that PEA induces the {Ca2+}c increase via H2O2 generation . The PEA-induced {Ca2+}c increase occurred in the mid1 mutant with a slightly smaller peak than in the wild-type strain, indicating that Mid1, a stretch-activated nonselective cation channel, may not be mainly involved in the PEA-induced Ca2+ influx . When PEA was applied, the MATa mid1 mutant was rescued from alpha-factor-induced death in a Ca2+-limited medium, suggesting that the PEA-induced {Ca2+}c increase can reinforce calcium signaling in the mating pheromone response pathway.

Genes Cells, 2002 Jun, 7(6), 543 - 52
Yeast Whi2 and Psr1-phosphatase form a complex and regulate STRE-mediated gene expression; Kaida D et al.; BACKGROUND: In response to various stressful situations, including diauxic conditions, the Msn2 and Msn4 transcription factors induce STRE-mediated gene expression of many stress responsive genes in Saccharomyces cerevisiae . This is called the general stress response . The whi2 cells in the stationary phase are smaller than wild-type cells.RESULTS: Here we demonstrate that STRE-mediated gene expression in whi2 cells is reduced to half of that in the wild-type cells under various stress conditions . It is also delayed for several hours when the mutant cells enter the stationary phase . Using the two-hybrid system, we isolated a WHI2-interacting gene, PSR1, which is one of the redundant genes encoding plasma membrane phosphatases . whi2 and psr1 psr2 mutants had similar phenotypes, including reduced STRE-mediated gene expression, higher sensitivity to sodium ions and heat shock, and hyper-phosphorylation of Msn2 . The phosphatase activity of Psr1 was necessary for the full activation of STRE-mediated gene expression . Furthermore, both Psr1 and Msn2 were co-immunoprecipitated with Whi2.CONCLUSIONS: Thus, Whi2 and its binding partner, Psr1-phosphatase, are required for a full activation of the general stress response, possibly through the dephosphorylation of Msn2 . These results may explain why stationary phase whi2 cells are small.

Nat Genet, 2002 Jul, 31(3), 248 - 54 Epub 2002 Jun 24.
Genome-wide binding map of the histone deacetylase Rpd3 in yeast; Kurdistani SK et al.; We describe the genome-wide distribution of the histone deacetylase and repressor Rpd3 and its associated proteins Ume1 and Ume6 in Saccharomyces cerevisiae . Using a new cross-linking protocol, we found that Rpd3 binds upstream of many individual genes and upstream of members of gene classes with similar functions in anabolic processes . In addition, Rpd3 is preferentially associated with promoters that direct high transcriptional activity . We also found that Rpd3 was absent from large sub-telomeric domains . We show by co-immunoprecipitation and by the high similarity of their binding maps that Ume1 interacts with Rpd3 . In contrast, despite the known role of Ume6 in Rpd3 recruitment, only a limited number of the genes targeted by Rpd3 are also enriched for (or targeted by) Ume6 . This suggests that Rpd3 is brought to many promoters by alternative recruiters, some of which may bind the putative cis-regulatory DNA elements that we have identified in sets of Rpd3 target genes . Finally, we show that comparing the genome-wide pattern of Rpd3 binding with gene expression and histone acetylation in the rpd3 Delta mutant strain reveals new sites of Rpd3 function.

Science, 2002 Jul 19, 297(5580), 395 - 400 Epub 2002 Jun 27.
Systematic identification of pathways that couple cell growth and division in yeast; Jorgensen P et al.; Size homeostasis in budding yeast requires that cells grow to a critical size before commitment to division in the late prereplicative growth phase of the cell cycle, an event termed Start . We determined cell size distributions for the complete set of approximately 6000 Saccharomyces cerevisiae gene deletion strains and identified approximately 500 abnormally small (whi) or large (lge) mutants . Genetic analysis revealed a complex network of newly found factors that govern critical cell size at Start, the most potent of which were Sfp1, Sch9, Cdh1, Prs3, and Whi5 . Ribosome biogenesis is intimately linked to cell size through Sfp1, a transcription factor that controls the expression of at least 60 genes implicated in ribosome assembly . Cell growth and division appear to be coupled by multiple conserved mechanisms.

J Biol Chem, 2002 Sep 20, 277(38), 34773 - 84 Epub 2002 Jun 27.
Exposure of yeast cells to anoxia induces transient oxidative stress . Implications for the induction of hypoxic genes; Dirmeier R et al.; The mitochondrial respiratory chain is required for the induction of some yeast hypoxic nuclear genes . Because the respiratory chain produces reactive oxygen species (ROS), which can mediate intracellular signal cascades, we addressed the possibility that ROS are involved in hypoxic gene induction . Recent studies with mammalian cells have produced conflicting results concerning this question . These studies have relied almost exclusively on fluorescent dyes to measure ROS levels . Insofar as ROS are very reactive and inherently unstable, a more reliable method for measuring changes in their intracellular levels is to measure their damage (e.g . the accumulation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in DNA, and oxidative protein carbonylation) or to measure the expression of an oxidative stress-induced gene, e.g . SOD1 . Here we used these approaches as well as a fluorescent dye, carboxy-H(2)-dichloro-dihydrofluorescein diacetate (carboxy-H(2)-DCFDA), to determine whether ROS levels change in yeast cells exposed to anoxia . These studies reveal that the level of mitochondrial and cytosolic protein carbonylation, the level of 8-OH-dG in mitochondrial and nuclear DNA, and the expression of SOD1 all increase transiently during a shift to anoxia . These studies also reveal that carboxy-H(2)-DCFDA is an unreliable reporter of ROS levels in yeast cells shifted to anoxia . By using two-dimensional electrophoresis and mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), we have found that specific proteins become carbonylated during a shift to anoxia and that some of these proteins are the same proteins that become carbonylated during peroxidative stress . The mitochondrial respiratory chain is responsible for much of this carbonylation . Together, these findings indicate that yeast cells exposed to anoxia experience transient oxidative stress and raise the possibility that this initiates the induction of hypoxic genes.

Appl Environ Microbiol, 2002 Jul, 68(7), 3339 - 44
Wine yeast strains engineered for glycogen overproduction display enhanced viability under glucose deprivation conditions; Perez-Torrado R et al.; We used metabolic engineering to produce wine yeasts with enhanced resistance to glucose deprivation conditions . Glycogen metabolism was genetically modified to overproduce glycogen by increasing the glycogen synthase activity and eliminating glycogen phosphorylase activity . All of the modified strains had a higher glycogen content at the stationary phase, but accumulation was still regulated during growth . Strains lacking GPH1, which encodes glycogen phosphorylase, are unable to mobilize glycogen . Enhanced viability under glucose deprivation conditions occurs when glycogen accumulates in the strain that overexpresses GSY2, which encodes glycogen synthase and maintains normal glycogen phosphorylase activity . This enhanced viability is observed under laboratory growth conditions and under vinification conditions in synthetic and natural musts . Wines obtained from this modified strain and from the parental wild-type strain don't differ significantly in the analyzed enological parameters . The engineered strain might better resist some stages of nutrient depletion during industrial use.

RNA, 2002 Jun, 8(6), 786 - 97
Dynamic conformational model for the role of ITS2 in pre-rRNA processing in yeast; Cote CA et al.; Maturation of the large subunit rRNAs includes a series of cleavages that result in removal of the internal transcribed spacer (ITS2) that separates mature 5.8S and 25/28S rRNAs . Previous work demonstrated that formation of higher order secondary structure within the assembling pre-ribosomal particle is a prerequisite for accurate and efficient pre-rRNA processing . To date, it is not clear which specific sequences or secondary structures are required for processing . Two alternative secondary structure models exist for Saccharomyces cerevisiae ITS2 . Chemical and enzymatic structure probing and phylogenetic comparisons resulted in one structure (Yeh & Lee, J Mol Biol, 1990, 211:699-712) referred to here as the "hairpin model." More recently, an alternate folded structure was proposed (Joseph et al., Nucleic Acids Res, 1999, 27:4533-4540), called here the "ring model." We have used a functional genetic assay to examine the potential significance of these predicted structures in processing . Our data indicate that elements of both structural models are important in efficient processing . Mutations that prevent formation of ring-specific structures completely blocked production of mature 25S rRNA, whereas those that primarily disrupt hairpin elements resulted in reduced levels of mature product . Based on these results, we propose a dynamic conformational model for the role of ITS2 in processing: Initial formation of the ring structure may be required for essential, early events in processing complex assembly and may be followed by an induced transition to the hairpin structure that facilitates subsequent processing events . In this model, yeast ITS2 elements may provide in cis certain of the functions proposed for vertebrate U8 snoRNA acting in trans.

Nucleic Acids Res, 2002 Jul 1, 30(13), 2727 - 35
Spontaneous and double-strand break-induced recombination, and gene conversion tract lengths, are differentially affected by overexpression of wild-type or ATPase-defective yeast Rad54; Kim PM et al.; Rad54 plays key roles in homologous recombination (HR) and double-strand break (DSB) repair in yeast, along with Rad51, Rad52, Rad55 and Rad57 . Rad54 belongs to the Swi2/Snf2 family of DNA-stimulated ATPases . Rad51 nucleoprotein filaments catalyze DNA strand exchange and Rad54 augments this activity of Rad51 . Mutations in the Rad54 ATPase domain (ATPase(-)) impair Rad54 function in vitro, sensitize yeast to killing by methylmethane sulfonate and reduce spontaneous gene conversion . We found that overexpression of ATPase(-) Rad54 reduced spontaneous direct repeat gene conversion and increased both spontaneous direct repeat deletion and spontaneous allelic conversion . Overexpression of ATPase(-) Rad54 decreased DSB-induced allelic conversion, but increased chromosome loss and DSB-dependent lethality . Thus, ATP hydrolysis by Rad54 contributes to genome stability by promoting high-fidelity DSB repair and suppressing spontaneous deletions . Overexpression of wild-type Rad54 did not alter DSB-induced HR levels, but conversion tract lengths were reduced . Interestingly, ATPase(-) Rad54 decreased overall HR levels and increased tract lengths . These tract length changes provide new in vivo evidence that Rad54 functions in the post-synaptic phase during recombinational repair of DSBs.

Cell, 2002 May 17, 109(4), 437 - 46
Microarray deacetylation maps determine genome-wide functions for yeast histone deacetylases; Robyr D et al.; Yeast contains a family of five related histone deacetylases (HDACs) whose functions are known at few genes . Therefore, we used chromatin immunoprecipitation and intergenic microarrays to generate genome-wide HDAC enzyme activity maps . Rpd3 and Hda1 deacetylate mainly distinct promoters and gene classes where they are recruited largely by novel mechanisms . Hda1 also deacetylates subtelomeric domains containing normally repressed genes that are used instead for gluconeogenesis, growth on carbon sources other than glucose, and adverse growth conditions . These domains have certain features of heterochromatin but are distinct from subtelomeric heterochromatin repressed by the deacetylase Sir2 . Finally, Hos1/Hos3 and Hos2 preferentially affect ribosomal DNA and ribosomal protein genes, respectively . Thus, acetylation microarrays uncover the "division of labor" for yeast histone deacetylases.

Proc Natl Acad Sci U S A, 2002 Jun 25, 99(13), 8591 - 6
A target essential for the activity of a nonacidic yeast transcriptional activator; Lu Z et al.; P201 is a short (eight-residue) nonacidic peptide that comprises a strong transcriptional activating region when tethered to DNA in yeast . Here we identify the mediator protein Gal11 as an essential target of P201 . Deletion of Gal11, which modestly decreases activation elicited by the typical acidic yeast activator, abolishes activation by DNA-tethered P201 . A point mutation in Gal11, which has no effect on other Gal11 functions, also greatly diminishes activation by DNA-tethered P201 . P201 binds to a fragment of Gal11 in vivo and in vitro, and the interaction is diminished by mutations in either component that decrease activation in vivo . P201, unlike the typical yeast acidic activating region, does not work in mammalian cells, which is consistent with the notion that the unique target of P201 (Gal11) is absent from mammalian cells.

Proc Natl Acad Sci U S A, 2002 Jun 25, 99(13), 8585 - 90
Hsp90 enables Ctf13p/Skp1p to nucleate the budding yeast kinetochore; Stemmann O et al.; Binding of CBF3, a protein complex consisting of Ndc10p, Cep3p, Ctf13p, and Skp1p, to the centromere DNA nucleates kinetochore formation in budding yeast . Here, we investigate how the Ctf13p/Skp1p complex becomes competent to form the CBF3-centromere DNA complex . As revealed by mass spectrometry, Ctf13p and Skp1p carry two and four phosphate groups, respectively . Complete dephosphorylation of Ctf13p and Skp1p does not interfere with the formation of CBF3-centromere DNA complexes in vitro . Furthermore, deletion of corresponding phosphorylation sites results in viable cells . Thus, in contrast to the current view, phosphorylation of Ctf13p and Skp1p is not essential for the formation of CBF3-centromere DNA complexes . Instead, the formation of active Ctf13p/Skp1p requires Hsp90 . Several lines of evidence support this conclusion: activation of heterologous Ctf13p/Skp1p by reticulocyte lysate is inhibited by geldanamycin and Hsp90 depletion . skp1 mutants exhibit growth defects on media containing geldanamycin . A skp1 mutation together with Hsp90 mutations exhibits synthetic lethality . An Hsp90 mutant contains decreased levels of active Ctf13p/Skp1p.

J Agric Food Chem, 2002 Jul 3, 50(14), 3999 - 4002
New monascus metabolite isolated from red yeast rice (angkak, red koji); Wild D et al.; Red yeast rice (angkak, red koji) obtained as cultures of Monascus purpureus on rice was extracted and analyzed by HPLC . In addition to the known red, orange, and yellow pigments and the mycotoxin citrinin, a new Monascus metabolite was detected . It is present in the original red yeast rice and formed in higher amounts when red yeast rice is heated . High-resolution mass spectrometry indicated the molecular formula C(15)H(12)O(4) . The chemical structure was elucidated by analysis of NMR data . The new compound, named monascodilone, is characterized by a propenyl group on a pyrone ring, an aromatic ring, and a gamma-lactone group.

Genes Cells, 2002 Jul, 7(7), 663 - 73
Fission yeast chk1 mutants show distinct responses to different types of DNA damaging treatments; Francesconi S et al.; BACKGROUND: Chk1 kinase is activated by phosphorylation at serine-345 by Rad3 checkpoint kinase and is required for DNA damage checkpoint in late S and G2 phase of S . pombe cell cycle . We studied the ability of two chk1 mutants, chk1-1 and chk1-2, to undergo phosphorylation and to delay cell cycle progression in response to different types of DNA lesions . RESULTS: Both the Chk1-1 and Chk1-2 mutant proteins are phosphorylated to various extents when DNA is damaged in early G2 phase of cell cycle by either UV irradiation or gamma irradiation . However, chk1-2 mutant does not delay cell cycle progression in a dose dependent manner specifically upon gamma irradiations . This defect is not associated with an important loss of survival . Furthermore, both chk1 mutants survive to Camptothecin treatment despite undetectable Chk1-1 or Chk1-2 phosphorylated forms . We show that both mutant proteins are not phosphorylated in cds1 devoid cells treated with ribonucleotide reductase inhibitor hydroxyurea or when the replisome is affected by a thermosensitive mutation in DNA polymerase delta . This inability is associated with the loss of checkpoint function . We found that an increased level of Crb2/Rhp9 protein specifically complements the defect of the chk1-1 mutant allowing Chk1-1 phosphorylation upon treatment with hydroxyurea of dcds1 cells . CONCLUSIONS: Mutants chk1-1 and chk1-2 behave differently according to the type of lesion generated on DNA.

J Magn Reson, 2002 May, 156(1), 52 - 63
Deconvolution of compartmental water diffusion coefficients in yeast-cell suspensions using combined T(1) and diffusion measurements; Silva MD et al.; An NMR method is presented for measuring compartment-specific water diffusion coefficient (D) values . It uses relaxography, employing an extracellular contrast reagent (CR) to distinguish intracellular (IC) and extracellular (EC) (1)H(2)O signals by differences in their respective longitudinal (T(1)) relaxation times . A diffusion-weighted inversion-recovery spin-echo (DW-IRSE) pulse sequence was used to acquire IR data sets with systematically and independently varying inversion time (TI) and diffusion-attenuation gradient amplitude (g) values . Implementation of the DW-IRSE technique was demonstrated and validated using yeast cells suspended in 3 mM Gd-DTPA(2-) with a wet/dry mass ratio of 3.25:1.0 . Two-dimensional (2D) NMR data were acquired at 2.0 T and analyzed using numerical inverse Laplace transformation (2D- and sequential 1D-ILT) and sequential exponential fitting to yield T(1) and water D values . All three methods gave substantial agreement . Exponential fitting, deemed the most accurate and time efficient, yielded T(1):D (relative contribution) values of 304 ms:0.023x10(-5) cm(2)/s (47%) and 65 ms:1.24x10(-5) cm(2)/s (53%) for the IC and EC components, respectively . The compartment-specific D values derived from direct biexponential fitting of diffusion-attenuation data were also in good agreement . Extension of the DW-IRSE method to in vivo models should provide valuable insights into compartment-specific water D changes in response to injury or disease . (c) 2002 Elsevier Science (USA).

Genes Dev, 2002 Jun 15, 16(12), 1528 - 39
Rap1-Sir4 binding independent of other Sir, yKu, or histone interactions initiates the assembly of telomeric heterochromatin in yeast; Luo K et al.; In Saccharomyces cerevisiae, heterochromatin-like regions are found near telomeres and at the silent mating-type loci, where they can repress genes in an epigenetic manner . Several proteins are involved in telomeric heterochromatin structure including Rap1, Sir2, Sir3, Sir4, yKu70 (Hdf1), yKu80 (Hdf2), and the N termini of histones H3 and H4 . By recognizing cis-acting DNA-binding sites, Rap1 is believed to recruit Sir and other silencing proteins and determine where heterochromatin forms . The integrity of heterochromatin also requires the binding of Sir proteins to histones that may form a scaffold for Sir protein interactions with chromatin . In this study we describe how the heterochromatin complex may form initially and how it differs from the complex that spreads along the chromosome . We found that close to the telomere end, Sir4 can bind Rap1 independently of Sir2, Sir3, yKu70/yKu80, and the intact H4 N terminus . In contrast, Sir4 binding requires all of the silencing factors further along telomeric heterochromatin . These data indicate that Sir4 binding to Rap1 initiates the sequential association of Sir and other proteins, allowing the subsequent spreading of the heterochromatin proteins along the chromosome.

Nature, 2002 Jul 4, 418(6893), 104 - 8 Epub 2002 Jun 23.
Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast; Sun ZW et al.; In eukaryotes, the DNA of the genome is packaged with histone proteins to form nucleosomal filaments, which are, in turn, folded into a series of less well understood chromatin structures . Post-translational modifications of histone tail domains modulate chromatin structure and gene expression . Of these, histone ubiquitination is poorly understood . Here we show that the ubiquitin-conjugating enzyme Rad6 (Ubc2) mediates methylation of histone H3 at lysine 4 (Lys 4) through ubiquitination of H2B at Lys 123 in yeast (Saccharomyces cerevisiae) . Moreover, H3 (Lys 4) methylation is abolished in the H2B-K123R mutant, whereas H3-K4R retains H2B (Lys 123) ubiquitination . These data indicate a unidirectional regulatory pathway in which ubiquitination of H2B (Lys 123) is a prerequisite for H3 (Lys 4) methylation . We also show that an H2B-K123R mutation perturbs silencing at the telomere, providing functional links between Rad6-mediated H2B (Lys 123) ubiquitination, Set1-mediated H3 (Lys 4) methylation, and transcriptional silencing . Thus, these data reveal a pathway leading to gene regulation through concerted histone modifications on distinct histone tails . We refer to this as 'trans-tail' regulation of histone modification, a stated prediction of the histone code hypothesis.

Mol Cell Biol, 2002 Jul, 22(14), 5047 - 53
Yng1p modulates the activity of Sas3p as a component of the yeast NuA3 Hhistone acetyltransferase complex; Howe L et al.; The mammalian ING1 gene encodes a tumor suppressor required for the function of p53 . In this study we report a novel function for YNG1, a yeast homolog of ING1 . Yng1p is a stable component of the NuA3 histone acetyltransferase complex, which contains Sas3p, the yeast homolog of the mammalian MOZ proto-oncogene product, as its catalytic subunit . Yng1p is required for NuA3 function in vivo but surprisingly is not required for the integrity of the complex . Instead, we find that Yng1p mediates the interaction of Sas3p with nucleosomes and is thus required for the ability of NuA3 to modify histone tails . These data, and the observations that other ING1 homologs are found in additional yeast complexes that posttranslationally modify histones, suggest that members of the ING1 class of proteins may have broad roles in enhancing or modifying the activities of chromatin-modifying complexes, thereby regulating their activities in transcription control.

J Biol Chem, 2002 Sep 6, 277(36), 32466 - 72 Epub 2002 Jun 20.
Transcriptional profiling identifies two members of the ATP-binding cassette transporter superfamily required for sterol uptake in yeast; Wilcox LJ et al.; In contrast to lipoprotein-mediated sterol uptake, free sterol influx by eukaryotic cells is poorly understood . To identify components of non-lipoprotein-mediated sterol uptake, we utilized strains of Saccharomyces cerevisiae that accumulate exogenous sterol due to a neomorphic mutation in the transcription factor, UPC2 . Two congenic upc2-1 strains, differing quantitatively in aerobic sterol uptake due to a modifying mutation in the HAP1 transcription factor, were compared using DNA microarrays . We identified 9 genes as responsive to UPC2 that were also induced under anaerobiosis, when sterol uptake is essential . Deletion mutants in these genes were assessed for sterol influx in the upc2-1 background . UPC2 itself was up-regulated under these conditions and was required for aerobic sterol influx . Deletion of the ATP-binding cassette transporters YOR011w (AUS1) or PDR11, or a putative cell wall protein encoded by DAN1, significantly reduced sterol influx . Sodium azide and vanadate inhibited sterol uptake, consistent with the participation of ATP-binding cassette transporters . We hypothesized that the physiological role of Aus1p and Pdr11p is to mediate sterol uptake when sterol biosynthesis is compromised . Accordingly, expression of AUS1 or PDR11 was required for anaerobic growth and sterol uptake . We proposed similar molecules may be important components of sterol uptake in all eukaryotes.

J Biol Chem, 2002 Aug 30, 277(35), 31715 - 21 Epub 2002 Jun 20.
Electrostatic control of the isoalloxazine environment in the two-electron reduced states of yeast glutathione reductase; Picaud T et al.; The resonance Raman spectra of the oxidized and two-electron reduced forms of yeast glutathione reductase are reported . The spectra of the oxidized enzyme indicate a low electron density for the isoalloxazine ring . As far as the two-electron reduced species are concerned, the spectral comparison of the NADPH-reduced enzyme with the glutathione- or dithiothreitol-reduced enzyme shows significant frequency differences for the flavin bands II, III, and VII . The shift of band VII was correlated with a change in steric or electronic interaction of the hydroxyl group of a conserved Tyr with the N(10)-C(10a) portion of the isoalloxazine ring . Upward shifts of bands II and III observed for the glutathione- or dithiothreitol-reduced enzyme indicate both a slight change in isoalloxazine conformation and a hydrogen bond strengthening at the N(1) and/or N(5) site(s) . The formation of a mixed disulfide intermediate tends to slightly decrease the frequency of bands II, III, X, XI, and XIV . To account for the different spectral features observed for the NADPH- and glutathione-reduced species, several possibilities have been examined . In particular, we propose a hydrogen bonding modulation at the N(5) site of FAD through a variable conformation of an ammonium group of a conserved Lys residue . Changes in N(5)(flavin)-protein interaction in the two-electron reduced forms of glutathione reductase are discussed in relation to a plausible mechanism of the regulation of the enzyme activity via a variable redox potential of FAD.

J Biol Chem, 2002 Sep 20, 277(38), 35274 - 81 Epub 2002 Jun 20.
YOS9, the putative yeast homolog of a gene amplified in osteosarcomas, is involved in the endoplasmic reticulum (ER)-Golgi transport of GPI-anchored proteins; Friedmann E et al.; The OS-9 gene maps to a region (q13-15) of chromosome 12 that is highly amplified in human osteosarcomas and encodes a protein of unknown function . Here we have characterized a homolog designated as YOS9 (YDR057w) from Saccharomyces cerevisiae . The yeast protein (Yos9) is a membrane-associated glycoprotein that localizes to the endoplasmic reticulum (ER) . YOS9 interacts genetically with genes involved in ER-Golgi transport, particularly SEC34, whose temperature-sensitive mutant is rescued by YOS9 overexpression . Interestingly, Yos9 appears to play a direct role in the transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi apparatus . Yos9 binds directly to Gas1 and Mkc7 and accelerates Gas1 transport and processing in cells overexpressing YOS9 . Correspondingly, Gas1 processing is slowed in cells bearing a deletion in YOS9 . No effect upon the transport and processing of non-GPI-anchored proteins (e.g . invertase and carboxypeptidase Y) was detected in cells either lacking or overexpressing Yos9 . As Yos9 is not a component of the Emp24 complex, it may act as a novel escort factor for GPI-anchored proteins in ER-Golgi transport in yeast and possibly in mammals.

Biochem Biophys Res Commun, 2002 Jun 28, 294(5), 1184 - 90
Fibrillarin binds to a 3' cis-regulatory element in pre-mRNA of uvi15+ in fission yeast; Jang YK et al.; uvi15+ is induced by various stresses including exposure to UV-light . Previously, we demonstrated that the UV-induction is mainly regulated at the post-transcriptional level through a cis-acting element in the pre-mRNA . Here we show that deletion analyses define an 18-nt element responsible for the UV-induction . RNA gel mobility shift assay showed that a specific protein(s) could form a complex with the 54-nt element but its binding ability is moderately decreased in response to UV-light . Using yeast three-hybrid screen, we isolated a homolog of fibrillarin as a protein interacting with the 54-nt element, which is a key nucleolar protein for pre-rRNA processing . We further showed that the recombinant fibrillarin specifically binds to the element in a sequence-specific manner . Thus, the data suggest that fission yeast fibrillarin might regulate uvi15+ mRNA stability via binding with the 54-nt element in the pre-mRNA, implying that fibrillarin is involved in both pre-mRNA and pre-rRNA processing.

J Ind Microbiol Biotechnol, 2002 Mar, 28(3), 186 - 91
The yeast lifecycle and DNA array technology; Williams RM; The genome variability and meiotic gene expression patterns in two unrelated laboratory yeast strains, SK1 and W303, have been characterized using high-density oligonucleotide arrays . The statistical analysis and comparison of the data has allowed identification of: (1) genes with functional importance to meiosis and sporulation in yeast and (2) genes expressed in a strain-specific manner . The genome-wide data also reveal potential reasons why these strains display significant differences in the ability to make fertile spores . Molecularly tagged yeast deletion strains have been used to determine the contribution of each gene to the execution of the sporulation/germination pathway . The application of genetics and the new genomic technologies have allowed a quantum jump in our understanding of yeast molecular biology.

Methods Enzymol, 2002, 350, 87 - 96
Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method; Gietz RD et al.; In this chapter we have provided instructions for transforming yeast by a number of variations of the LiAc/SS-DNA/PEG method for a number of different applications . The rapid transformation protocol is used when small numbers of transformants are required . The high efficiency transformation protocol is used to generate large numbers of transformants or to deliver DNA constructs or oligonucleotides into the yeast cell . The large-scale transformation protocol is primarily applicable to the analysis of complex plasmid DNA libraries, such as those required for the yeast two-hybrid system . The microtiter plate versions of the rapid and high efficiency transformation protocols can be applied to high-throughput screening technologies.

Curr Genet, 2002 Apr, 41(1), 20 - 4 Epub 2002 Mar 29.
Isolation of genes coding for Ade2 and Ura3 homologues from the multinuclear yeast Dipodascus magnusii; Kucej M et al.; Dipodascus magnusii is a nonconventional yeast species with giant multinuclear cells . We constructed two genomic DNA libraries in plasmid vectors and isolated the first two D . magnusii protein-encoding genes, DmADE2 and DmURA3, coding for phosphoribosylaminoimidazole carboxylase and orotidine-5'-phosphate decarboxylase, respectively . Both genes represent functional orthologues, since they complement ade2 and ura3 mutations in Saccharomyces cerevisiae and their putative products possess conserved sequences important for enzymatic activities . Moreover, the results of Southern blot analysis indicate that the genome of D . magnusii contains additional, paralogous sequences of the DmADE2 and DmURA3 genes.

Genetics, 2002 Jun, 161(2), 611 - 22
Expression-state boundaries in the mating-type region of fission yeast; Thon G et al.; A transcriptionally silent chromosomal domain is found in the mating-type region of fission yeast . Here we show that this domain is delimited by 2-kb inverted repeats, IR-L and IR-R . IR-L and IR-R prevent the expansion of transcription-permissive chromatin into the silenced region and that of silenced chromatin into the expressed region . Their insulator activity is partially orientation dependent . The silencing defects that follow deletion or inversion of IR-R are suppressed by high dosage of the chromodomain protein Swi6 . Combining chromosomal deletions and Swi6 overexpression shows that IR-L and IR-R provide firm borders in a region where competition between silencing and transcriptional competence occurs . IR-R possesses autonomously replicating sequence (ARS) activity, leading to a model where replication factors, or replication itself, participate in boundary formation.

Genetics, 2002 Jun, 161(2), 575 - 84
Yeast RSC function is required for organization of the cellular cytoskeleton via an alternative PKC1 pathway; Chai B et al.; RSC is a 15-protein ATP-dependent chromatin-remodeling complex related to Snf-Swi, the prototypical ATP-dependent nucleosome remodeler in budding yeast . Despite insight into the mechanism by which purified RSC remodels nucleosomes, little is known about the chromosomal targets or cellular pathways in which RSC acts . To better understand the cellular function of RSC, a screen was undertaken for gene dosage suppressors of sth1-3ts, a temperature-sensitive mutation in STH1, which encodes the essential ATPase subunit . Slg1p and Mid2p, two type I transmembrane stress sensors of cell wall integrity that function upstream of protein kinase C (Pkc1p), were identified as multicopy suppressors of sth1-3ts cells . Although the sth1-3ts mutant exhibits defects characteristic of PKC1 pathway mutants (caffeine and staurosporine sensitivities and an osmoremedial phenotype), only upstream components and not downstream effectors of the PKC1-MAP kinase pathway can suppress defects conferred by sth1-3ts, suggesting that RSC functions in an alternative PKC1-dependent pathway . Moreover, sth1-3ts cells display defects in actin cytoskeletal rearrangements and are hypersensitive to the microtubule depolymerizing drug, TBZ; both of these defects can be corrected by the high-copy suppressors . Together, these data reveal an important functional connection between the RSC remodeler and PKC1-dependent signaling in regulating the cellular architecture.

Genetics, 2002 Jun, 161(2), 563 - 74
Volatile anesthetics affect nutrient availability in yeast; Palmer LK et al.; Volatile anesthetics affect all cells and tissues tested, but their mechanisms and sites of action remain unknown . To gain insight into the cellular activities of anesthetics, we have isolated genes that, when overexpressed, render Saccharomyces cerevisiae resistant to the volatile anesthetic isoflurane . One of these genes, WAK3/TAT1, encodes a permease that transports amino acids including leucine and tryptophan, for which our wild-type strain is auxotrophic . This suggests that availability of amino acids may play a key role in anesthetic response . Multiple lines of evidence support this proposal: (i) Deletion or overexpression of permeases that transport leucine and/or tryptophan alters anesthetic response; (ii) prototrophic strains are anesthetic resistant; (iii) altered concentrations of leucine and tryptophan in the medium affect anesthetic response; and (iv) uptake of leucine and tryptophan is inhibited during anesthetic exposure . Not all amino acids are critical for this response since we find that overexpression of the lysine permease does not affect anesthetic sensitivity . These findings are consistent with models in which anesthetics have a physiologically important effect on availability of specific amino acids by altering function of their permeases . In addition, we show that there is a relationship between nutrient availability and ubiquitin metabolism in this response.

Genetics, 2002 Jun, 161(2), 549 - 62
A molecular genetic dissection of the evolutionarily conserved N terminus of yeast Rad52; Mortensen UH et al.; Rad52 is a DNA-binding protein that stimulates the annealing of complementary single-stranded DNA . Only the N terminus of Rad52 is evolutionarily conserved; it contains the core activity of the protein, including its DNA-binding activity . To identify amino acid residues that are important for Rad52 function(s), we systematically replaced 76 of 165 amino acid residues in the N terminus with alanine . These substitutions were examined for their effects on the repair of gamma-ray-induced DNA damage and on both interchromosomal and direct repeat heteroallelic recombination . This analysis identified five regions that are required for efficient gamma-ray damage repair or mitotic recombination . Two regions, I and II, also contain the classic mutations, rad52-2 and rad52-1, respectively . Interestingly, four of the five regions contain mutations that impair the ability to repair gamma-ray-induced DNA damage yet still allow mitotic recombinants to be produced at rates that are similar to or higher than those obtained with wild-type strains . In addition, a new class of separation-of-function mutation that is only partially deficient in the repair of gamma-ray damage, but exhibits decreased mitotic recombination similar to rad52 null strains, was identified . These results suggest that Rad52 protein acts differently on lesions that occur spontaneously during the cell cycle than on those induced by gamma-irradiation.

Genetics, 2002 Jun, 161(2), 493 - 507
Regulation of genome stability by TEL1 and MEC1, yeast homologs of the mammalian ATM and ATR genes; Craven RJ et al.; In eukaryotes, a family of related protein kinases (the ATM family) is involved in regulating cellular responses to DNA damage and telomere length . In the yeast Saccharomyces cerevisiae, two members of this family, TEL1 and MEC1, have functionally redundant roles in both DNA damage repair and telomere length regulation . Strains with mutations in both genes are very sensitive to DNA damaging agents, have very short telomeres, and undergo cellular senescence . We find that strains with the double mutant genotype also have approximately 80-fold increased rates of mitotic recombination and chromosome loss . In addition, the tel1 mec1 strains have high rates of telomeric fusions, resulting in translocations, dicentrics, and circular chromosomes . Similar chromosome rearrangements have been detected in mammalian cells with mutations in ATM (related to TEL1) and ATR (related to MEC1) and in mammalian cells that approach cell crisis.

Proc Natl Acad Sci U S A, 2002 Jun 25, 99(13), 9061 - 6 Epub 2002 Jun 17.
Reconstitution in yeast of the Arabidopsis SOS signaling pathway for Na+ homeostasis; Quintero FJ et al.; The Arabidopsis thaliana SOS1 protein is a putative Na+/H+ antiporter that functions in Na+ extrusion and is essential for the NaCl tolerance of plants . sos1 mutant plants share phenotypic similarities with mutants lacking the protein kinase SOS2 and the Ca2+ sensor SOS3 . To investigate whether the three SOS proteins function in the same response pathway, we have reconstituted the SOS system in yeast cells . Expression of SOS1 improved the Na+ tolerance of yeast mutants lacking endogenous Na+ transporters . Coexpression of SOS2 and SOS3 dramatically increased SOS1-dependent Na+ tolerance, whereas SOS2 or SOS3 individually had no effect . The SOS2/SOS3 kinase complex promoted the phosphorylation of SOS1 . A constitutively active form of SOS2 phosphorylated SOS1 in vitro independently of SOS3, but could not fully substitute for the SOS2/SOS3 kinase complex for activation of SOS1 in vivo . Further, we show that SOS3 recruits SOS2 to the plasma membrane . Although sos1 mutant plants display defective K+ uptake at low external concentrations, neither the unmodified nor the SOS2/SOS3-activated SOS1 protein showed K+ transport capacity in vivo, suggesting that the role of SOS1 on K+ uptake is indirect . Our results provide an example of functional reconstitution of a plant response pathway in a heterologous system and demonstrate that the SOS1 ion transporter, the SOS2 protein kinase, and its associated Ca2+ sensor SOS3 constitute a functional module . We propose a model in which SOS3 activates and directs SOS2 to the plasma membrane for the stimulatory phosphorylation of the Na+ transporter SOS1.

J Biol Chem, 2002 Sep 13, 277(37), 33624 - 31 Epub 2002 Jun 17.
Regulation of the Gts1p level by the ubiquitination system to maintain metabolic oscillations in the continuous culture of yeast; Saito T et al.; Yeast cells exhibit sustained ultradian oscillations of energy metabolism in coupling with cell cycle and stress resistance oscillations in continuous culture . We have reported that the rhythmic expression of Gts1p is important for the maintenance of ultradian rhythms . Structurally, Gts1p contains sequence motifs similar to N-degron and the ubiquitin association domain, raising the possibility that the Gts1p level is regulated by degradation via ubiquitination . When the lysine residue at the putative ubiquitination site of the N-degron was substituted with arginine, both the protein level and half-life of mutant Gts1p increased . During continuous culture, the protein level of the mutant Gts1p was elevated and did not fluctuate, leading to the disappearance of metabolic oscillation within a day . Furthermore, using three Gts1ps containing mutations in the ubiquitin association domain, we showed that the lower the binding activity of the mutant Gts1ps for polyubiquitin in vitro, the higher the protein level in vivo . Expression of the mutant Gts1ps in the continuous culture resulted in an increase in Gts1p and early loss of the oscillation . Therefore, Gts1p is degraded through conjugation with ubiquitin, and the UBA domain promoted the degradation of ubiquitinated Gts1p, causing a fluctuation in protein level, which is required for the maintenance of metabolic oscillations.

Mol Microbiol, 2002 Jun, 44(5), 1167 - 83
Rho1p mutations specific for regulation of beta(1-->3)glucan synthesis and the order of assembly of the yeast cell wall; Roh DH et al.; In the yeast Saccharomyces cerevisiae, the GTP-binding protein Rho1 is required for beta(1-->3)glucan synthase activity, for activation of protein kinase C and the cell integrity pathway and for progression in G1, cell polarization and exocytosis . A genetic screen for cells that become permeabilized at non-permissive temperature was used to isolate in vitro-generated mutants of Rho1p . After undergoing a battery of tests, several of them appeared to be specifically defective in the beta(1-->3) glucan synthesis function of Rho1p . At the non-permissive temperature (37 degrees C), the mutants developed defects in the cell wall, especially at the tip of new buds . In the yeast cell wall, beta(1-->6)glucan is linked to both beta(1-->3)glucan and mannoprotein, as well as occasionally to chitin . We have used the rho1 mutants to study the order of assembly of the cell wall components . The incorporation of {(14)C}-glucose into beta(1-->3)glucan at 37 degrees C was decreased or abolished in the mutants . Concomitantly, a partial defect in the incorporation of label into cell wall mannoproteins and beta(1-->6)glucan was observed . In contrast, YW3458, an inhibitor of glycosylphosphatidylinositol anchor formation, prevented mannoprotein incorporation, whereas the beta(1-->3)-beta(1-->6)glucan complex was synthesized at almost normal levels . As beta(1-->3)glucan can be synthesized in vitro or in vivo independently, we conclude that the order of addition in vivo is beta(1-->3)glucan, beta(1-->6)glucan, mannoprotein . Previous observations indicate that chitin is the last component to be incorporated into the complex.

Mol Biol (Mosk), 2002 May-Jun, 36(3), 491 - 5
{The use of the two-hybrid cloning in yeast for functional characterization of protein kinase MAK-V}; Korobko IV et al.; Identification of interaction partners opens a way to direct functional characterization of proteins . Several cDNAs coding for potential partners of protein kinase MAK-V/Hunk were isolated using two-hybrid cloning in yeast . Based on the partner properties, MAK-V/Hunk was assumed to play a role in tumorigenesis and tumor progression . With the previous results of two-hybrid cloning, MAK-V/Hunk was shown to participate in vesicular transport.

FEBS Lett, 2002 Jun 19, 521(1-3), 57 - 61
ERG6 and PDR5 regulate small lipophilic drug accumulation in yeast cells via distinct mechanisms; Emter R et al.; Diagnosis and circumvention of multi-drug resistance requires an understanding of the underlying cellular mechanisms . In the model organism Saccharomyces cerevisiae, deletions of PDR5 or ERG6 increase sensitivity to many small lipophilic drugs . Pdr5p is a plasma membrane ATP-binding cassette transporter that actively exports drugs, thereby lowering their intracellular levels . The mechanism by which ERG6, an enzyme in sterol biosynthesis, affects drug accumulation is less clear . We show here that ERG6 limits the rate of passive drug diffusion across the membrane, without affecting Pdr5p-mediated drug export . Consistent with their action by distinct mechanisms, PDR5 and ERG6 effects on drug accumulation are additive.

FEBS Lett, 2002 Jun 19, 521(1-3), 47 - 52
Bax expression protects yeast plasma membrane against ethanol-induced permeabilization; Marza E et al.; The mechanism by which the expression of pro-apoptotic protein Bax is able to kill yeast was investigated . Ethanol stress induces a permeabilization of the plasma membrane revealed by propidium iodide accumulation . Bax expression, although killing yeast cells, prevents this permeabilization . These effects are modulated by aeration, by manipulation of the unsaturation index of fatty acids and by addition of resveratrol, a known inhibitor of lipid oxidation . These data suggest that lipid oxidation is involved in Bax effects . Taken together, these data show for the first time a direct effect of Bax on plasma membrane permeability properties and suggest that yeast is a powerful tool for investigating the molecular mechanisms underlying this process.

J Biotechnol, 2002 Aug 7, 97(2), 183 - 90
Enzymatic characterization of a recombinant isoform hybrid of glutamic acid decarboxylase (rGAD67/65) expressed in yeast; Tong JC et al.; BACKGROUND AND AIMS: Glutamic acid decarboxylase (GAD, EC 4.1.1.15) catalyses the conversion of glutamate to gamma-aminobutyric acid (GABA) . The 65 kDa isoform, GAD65 is a potent autoantigen in type 1 diabetes, whereas GAD67 is not . A hybrid cDNA was created by fusing a human cDNA for amino acids 1-101 of GAD67 to a human cDNA for amino acids 96-585 of GAD65; the recombinant (r) protein was expressed in yeast and was shown to have equivalent immunoreactivity to mammalian brain GAD with diabetes sera . We here report on enzymatic and molecular properties of rGAD67/65 . METHODS: Studies were performed on enzymatic activity of rGAD67/65 by production of 3H-GABA from 3H-glutamate, enzyme kinetics, binding to the enzyme cofactor pyridoxal phosphate (PLP), stability according to differences in pH, temperature and duration of storage, and antigenic reactivity with various GAD-specific antisera . RESULTS: The properties of rGAD67/65 were compared with published data for mammalian brain GAD (brackets) . These included a specific enzyme activity of 22.7 (16.7) nKat, optimal pH for enzymatic activity 7.4 (6.8), K(m) of 1.3 (1.3) mM, efficient non-covalent binding to the cofactor PLP, and high autoantigenic potency . The stability of rGAD67/65 was optimal over 3 months at -80 degrees C, or in lyophilized form at -20 degrees C . CONCLUSIONS: Hybrid rGAD67/65 has enzymatic and other properties similar to those of the mixed isoforms of GAD in preparations from mammalian brain as described elsewhere, in addition to its previously described similar immunoreactivity.

EMBO J, 2002 Jun 17, 21(12), 3201 - 11
Instability of the human minisatellite CEB1 in rad27Delta and dna2-1 replication-deficient yeast cells; Lopes J et al.; Convergent studies in human and yeast model systems have shown that some minisatellite loci are relatively stable in somatic cells but not in the germline, and little is known about the mechanism(s) that can destabilize them . Unlike microsatellite sequences, mini satellites are not destabilized by mismatch repair mutations . We report here that the absence of Rad27 and Dna2 functions but not RNase H(35) or Exo1, which play an essential role in the processing of Okazaki fragments during replication, destabilize the human minisatellite CEB1 in mitotically growing Saccharomyces cerevisiae cells, up to 14% per generation in rad27Delta cells . Analysis using minisatellite variant repeat mapping by polymerase chain reaction of the internal structure of 17 variants reveals that the majority of rearrangements in rad27Delta cells are extremely complex contraction events that contain deletions, often accompanied by duplications of motif unit . Altogether, these results suggest that the improperly processed 5' flap structures that accumulate when replication is impaired can act as a potent stimulator of minisatellite destabilization and can provoke an unexpectedly broad range of mutagenic events . This replication-dependent phenomenon differs from the recombination-induced instability in yeast meiotic cells.

EMBO J, 2002 Jun 17, 21(12), 3160 - 70
Mutations in yeast Rad51 that partially bypass the requirement for Rad55 and Rad57 in DNA repair by increasing the stability of Rad51-DNA complexes; Fortin GS et al.; Yeast Rad51 promotes homologous pairing and strand exchange in vitro, but this activity is inefficient in the absence of the accessory proteins, RPA, Rad52, Rad54 and the Rad55-Rad57 heterodimer . A class of rad51 alleles was isolated that suppresses the requirement for RAD55 and RAD57 in DNA repair, but not the other accessory factors . Five of the six mutations isolated map to the region of Rad51 that by modeling with RecA corresponds to one of the DNA-binding sites . The other mutation is in the N-terminus of Rad51 in a domain implicated in protein-protein interactions and DNA binding . The Rad51-I345T mutant protein shows increased binding to single- and double-stranded DNA, and is proficient in displacement of replication protein A (RPA) from single-stranded DNA, suggesting that the normal function of Rad55-Rad57 is promotion and stabilization of Rad51-ssDNA complexes.

EMBO J, 2002 Jun 17, 21(12), 2903 - 11
The yeast prion Ure2p retains its native alpha-helical conformation upon assembly into protein fibrils in vitro; Bousset L et al.; The yeast inheritable phenotype {URE3} is thought to result from conformational changes in the normally soluble and highly helical protein Ure2p . In vitro, the protein spontaneously forms long, straight, insoluble protein fibrils at neutral pH . Here we show that fibrils of intact Ure2p assembled in vitro do not possess the cross beta-structure of amyloid, but instead are formed by the polymerization of native-like helical subunits that retain the ability to bind substrate analogues . We further show that dissociation of the normally dimeric protein to its constituent monomers is a prerequisite for assembly into fibrils . By analysing the nature of early assembly intermediates, as well as fully assembled Ure2p fibrils using atomic force microscopy, and combining the results with experiments that probe the fidelity of the native fold in protein fibrils, we present a model for fibril formation, based on assembly of native-like monomers, driven by interactions between the N-terminal glutamine and asparagine-rich region and the C-terminal functional domain . The results provide a rationale for the effect of mutagenesis on prion formation and new insights into the mechanism by which this, and possibly other inheritable factors, can be propagated.

J Biol Chem, 2002 Aug 23, 277(34), 31237 - 42 Epub 2002 Jun 12.
Yeast Cox11, a protein essential for cytochrome c oxidase assembly, is a Cu(I)-binding protein; Carr HS et al.; Cox11 is a protein essential for respiratory growth and has been implicated in the assembly of the Cu(B) site of cytochrome c oxidase . In the present study, we demonstrate that Cox11 is a copper-binding protein . The soluble C-terminal domain of Cox11 forms a dimer that coordinates one Cu(I) per monomer via three thiolate ligands . The two Cu(I) ions in the dimer exist in a binuclear cluster and appear to be ligated by three conserved Cys residues . Mutation of any of these Cys residues reduces Cu(I) binding and confers respiratory incompetence . Cytochrome c oxidase activity is reduced in these mutants . Thus, the residues important for Cu(I) binding correlate with in vivo function, suggesting that Cu(I) binding is important in Cox11 function.

J Biol Chem, 2002 Aug 23, 277(34), 30477 - 87 Epub 2002 Jun 12.
In vivo and in vitro phosphorylation of two isoforms of yeast pyruvate kinase by protein kinase A; Portela P et al.; Saccharomyces cerevisiae pyruvate kinase 1 (Pyk1) was demonstrated to be associated to an immunoprecipitate of yeast protein kinase A holoenzyme (HA-Tpk1.Bcy1) and to be phosphorylated in a cAMP-dependent process . Both glutathione S-transferase (GST)-Pyk1 and GST-Pyk2 were phosphorylated in vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1 . The specificity constant for the phosphorylation of GST-Pyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) . Both fusion proteins were phosphorylated in vivo, in intact cells overexpressing the protein, or in vitro using crude extracts, as source of protein kinase A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK gene with an attenuated mutation (tpk1(w1)) . The effect of phosphorylation on Pyk activity was assayed in partially purified preparations from three strains, containing different endogenous protein kinase A activity levels . Pyk1 activity was measured at different phosphoenolpyruvate concentrations in the absence or in the presence of the activator fructose 1,6-bisphosphate at 1.5 mm . Preliminary kinetic results derived from the comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6-bisphosphate it shows an n(H) value of 1.4, as compared with an n(H) of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity.

Biochim Biophys Acta, 2002 Jun 12, 1590(1-3), 27 - 40
Characterisation of two 14-3-3 genes from Trichoderma reesei: interactions with yeast secretory pathway components; Vasara T et al.; The 14-3-3 proteins are highly conserved, ubiquitously expressed proteins taking part in numerous cellular processes . Two genes encoding 14-3-3 proteins, ftt1 and ftt2, were isolated and characterised from the filamentous fungus Trichoderma reesei . FTTI showed the highest sequence identity (98% at the amino acid level) to the Trichoderma harzianum protein Th1433 . FTTII is relatively distinct from FTTI, showing approximately 75% identity to other fungal 14-3-3 proteins . Despite their sequence divergence, both of the T . reesei ftt genes were equally able to complement the yeast bmh1 bmh2 double disruption . The T . reesei ftt genes were also found to be quite closely linked in the genomic DNA . A C-terminally truncated version of ftt1 (ftt1DeltaC) was first isolated as a multicopy suppressor of the growth defect of the temperature-sensitive yeast secretory mutant sec15-1 . Overexpression of ftt1DeltaC also suppressed the growth defect of sec2-41, sec3-101, and sec7-1 strains . Overexpression of ftt1DeltaC in sec2-41 and sec15-1 strains could also rescue the secretion of invertase at the restrictive temperatures, and overexpression of full-length ftt1 enhanced invertase secretion by wild-type yeast cells . These findings strongly suggest that the T . reesei ftt1 has a role in protein secretion.

FEBS Lett, 2002 Apr 24, 517(1-3), 103 - 9
Yeast Npi3/Bro1 is involved in ubiquitin-dependent control of permease trafficking; Springael JY et al.; The membrane traffic and stability of the general amino acid permease Gap1 of Saccharomyces cerevisiae are under nitrogen control . Addition of a preferential nitrogen source such as ammonium to cells growing on a poor nitrogen source induces internalization of the permease and its subsequent degradation in the vacuole . This down-regulation requires ubiquitination of Gap1 through a process involving ubiquitin ligase Npi1/Rsp5, ubiquitin hydrolase Npi2/Doa4, and Bul1/2, two Npi1/Rsp5 interacting proteins . Here we report that yet another protein, Npi3, is involved in the regulation of Gap1 trafficking . We show that Npi3 is required for NH4+-induced down-regulation of Gap1, and particularly for efficient ubiquitination of the permease . Npi3 plays a pleiotropic role in permease down-regulation, since it is also involved in ubiquitination and stress-induced down-regulation of the uracil permease Fur4 and in glucose-induced degradation of hexose transporters Hxt6/7 . We further provide evidence that Npi3 is required for direct vacuolar sorting of neosynthesized Gap1 permease as it occurs in npr1 mutant cells . NPI3 is identical to BRO1, a gene encoding a protein of unknown biochemical function and recently proposed to be involved in protein turnover . Npi3/Bro1 homologues include fungal proteins required for proteolytic cleavage of zinc finger proteins and the mouse Aip1 protein involved in apoptosis . We propose that proteins of the Npi3/Bro1 family, including homologues from higher species, may play a conserved role in ubiquitin-dependent control of membrane protein trafficking.

Trends Cell Biol, 2002 May, 12(5), 231 - 5
Yeast autophagosomes: de novo formation of a membrane structure; Noda T et al.; Autophagy - the degradation of organelles and cytoplasmic material - occurs through dynamic rearrangements of cellular membrane structures . Following the induction of autophagy, newly formed autophagosomes transfer cytosolic materials to the lysosome or vacuole for degradation . The autophagosome is an organelle destined for degradation, suggesting that the membrane structure is formed de novo many times . The autophagosome is formed through the nucleation, assembly and elongation of membrane structures . The concerted action of several Apg/Aut/Cvt proteins around a characteristic subcellular structure (the preautophagosomal structure) is the key to understanding this novel type of membrane-formation process.

Cell, 2002 May 31, 109(5), 551 - 62
Chromatin boundaries in budding yeast: the nuclear pore connection; Ishii K et al.; Chromatin boundary activities (BAs) were identified in Saccharomyces cerevisiae by genetic screening . Such BAs bound to sites flanking a reporter gene establish a nonsilenced domain within the silent mating-type locus HML . Interestingly, various proteins involved in nuclear-cytoplasmic traffic, such as exportins Cse1p, Mex67p, and Los1p, exhibit a robust BA . Genetic studies, immunolocalization, live imaging, and chromatin immunoprecipitation experiments show that these transport proteins block spreading of heterochromatin by physical tethering of the HML locus to the Nup2p receptor of the nuclear pore complex . Genetic deletion of NUP2 abolishes the BA of all transport proteins, while direct targeting of Nup2p to the bracketing DNA elements restores activity . The data demonstrate that physical tethering of genomic loci to the NPC can dramatically alter their epigenetic activity.

Curr Biol, 2002 Jun 4, 12(11), 930 - 3
Telomere looping permits repression "at a distance" in yeast; Zaman Z et al.; In yeast, unlike in higher eukaryotes, transcriptional activators and repressors do not normally work when bound to DNA at large distances (over 500 base pairs) from the gene and, in particular, when positioned downstream of the gene . This restriction is relieved for a transcriptional activator if a gene bearing an activator binding site is placed near a yeast telomere . The explanation proposed is that the folded structure found at the telomere helps appose the DNA-bound activator with proteins binding to the promoter so that recruitment of the transcriptional machinery can be effected "at a distance" . Here, we show that a repressor, Tup1, works when tethered to DNA downstream of, and some 1.5-kb from, the gene when the construct is placed near a yeast telomere . The effect, observed with activated as well as basal transcription, is eliminated by deletion of Sir3 . These and other results indicate that DNA-tethered Tup1 represses by interacting with some component of the transcriptional machinery binding to the promoter, an interaction that is facilitated by the preformed loop at the telomere.

Med Mycol, 2002 Apr, 40(2), 185 - 99
Characterization of a protein kinase C gene in Sporothrix schenckii and its expression during the yeast-to-mycelium transition; Aquino-Pinero E et al.; The yeast-to-mycelium transition in Sporothrix schenckii has been shown to respond to protein kinase C (PKC) effectors, indicating the involvement of PKC in this regulation . In this study, we identified the presence of two pkcl-like genes in S . schenckii . Using fungal genomic DNA as template and primers targeted to conserved sequences in the Saccharomyces cerevisiae pkc1 gene, two partially overlapping extra long polymerase chain reaction (XL-PCR) products were obtained . These XL-PCR products were sequenced and found to encode part of the C3/C4 domains of two different PKC-like proteins . The presence of two different genes was confirmed by Southern blot analysis . These two genes were named pkcSs-1 and pkcSs-2 . The sequence of the pkcSs-2 gene was completed and revealed an open reading frame of 3942 nucleotides interrupted by five introns . A transcript of 8.7 kb was detected in northern blot analysis of poly A+ RNA . The pkcSs-2 gene encodes a protein of 1194 amino acids and 132.84 kDa that contains the characteristic structure and domains of other fungal PKCs reported to date . Using reverse transcription-PCR (RT-PCR), the pkcSs-2 gene was found to be expressed at all intervals tested during the yeast-to-mycelium transition.

Folia Microbiol (Praha), 2002, 47(2), 157 - 60
Induction of morphological alterations by antineoplastic agents in yeast; Stavrinidis E et al.; Saccharomyces cerevisiae was used as an alternative experimental model in order to investigate the effects of antineoplastic agents on eukaryotic cells . After being exposed to the most common clinically used antineoplastic agents, yeast cells were examined under the light microscope . Folate and pyrimidine antagonists, platinum derivatives, mitomycin C, actinomycin D and bleomycin induced alterations in yeast cellular morphology, which were not observed following treatment with drugs belonging to any category other than the antineoplastics, leading to the suggestion that these alterations could potentially be used as an experimental tool in pre-screening for new chemotherapeutic leads.

Mol Biol Cell, 2002 Jun, 13(6), 2080 - 90
The G1/S cyclin Cig2p during meiosis in fission yeast; Borgne A et al.; Cyclin-dependent kinases (CDKs) are important for both mitotic and meiotic cell cycles . In fission yeast, the major CDK, Cdc2p is involved in premeiotic DNA replication and in meiosis II . One of its partners, the mitotic cyclin Cdc13p is known to be required for meiosis, whereas there are no studies on the G1/S cyclin Cig2p . In this article, we have studied the regulation of the Cdc2p/Cdc13p and Cdc2p/Cig2p complexes during synchronous meiosis . We observed that Cdc2p/Cig2p kinase is activated in an unexpected biphasic manner, first at onset of premeiotic S phase and again during meiotic nuclear divisions . The role of Cig2p during meiosis was investigated using cig2-deleted strains that exhibit delays in onset of both S phase and meiotic divisions as well as an inefficient completion of MII . Furthermore, analysis of cig2 transcripts revealed a meiosis-specific regulation of cig2 expression during MI/MII dependent upon the Mei4p transcription factor leading to a different transcription start site at this stage of meiosis.

J Biol Chem, 2002 Aug 23, 277(34), 30445 - 53 Epub 2002 Jun 10.
Lack of pseudouridine 38/39 in the anticodon arm of yeast cytoplasmic tRNA decreases in vivo recoding efficiency; Lecointe F et al.; Many different modified nucleotides are found in naturally occurring tRNA, especially in the anticodon region . Their importance for the efficiency of the translational process begins to be well documented . Here we have analyzed the in vivo effect of deleting genes coding for yeast tRNA-modifying enzymes, namely Pus1p, Pus3p, Pus4p, or Trm4p, on termination readthrough and +1 frameshift events . To this end, we have transformed each of the yeast deletion strains with a lacZ-luc dual-reporter vector harboring selected programmed recoding sites . We have found that only deletion of the PUS3 gene, encoding the enzyme that introduces pseudouridines at position 38 or 39 in tRNA, has an effect on the efficiency of the translation process . In this mutant, we have observed a reduced readthrough efficiency of each stop codon by natural nonsense suppressor tRNAs . This effect is solely due to the absence of pseudouridine 38 or 39 in tRNA because the inactive mutant protein Pus3{D151A}p did not restore the level of natural readthrough . Our results also show that absence of pseudouridine 39 in the slippery tRNA(UAG)(Leu) reduces +1 frameshift efficiency . Therefore, the presence of pseudouridine 38 or 39 in the tRNA anticodon arm enhances misreading of certain codons by natural nonsense tRNAs as well as promotes frameshifting on slippery sequences in yeast.

J Biol Chem, 2002 Jul 26, 277(30), 26721 - 4 Epub 2002 Jun 10.
Localization of the Rsr1/Bud1 GTPase involved in selection of a proper growth site in yeast; Park HO et al.; Yeast cells organize their actin cytoskeleton in a highly polarized manner during vegetative growth . The Ras-like GTPase Rsr1/Bud1 and its regulators are required for selection of a specific site for growth . Here we showed that Rsr1/Bud1 was broadly distributed on the plasma membrane and highly concentrated at the incipient bud site and polarized growth sites . We also showed that localization of Cdc24, a guanine nucleotide exchange factor for the Cdc42 GTPase, to the proper bud site was dependent on Rsr1/Bud1 . Surprisingly, Rsr1/Bud1 also localized to intracellular membranes . A mutation in the lysine repeat in the hypervariable region of Rsr1/Bud1 specifically abolished its plasma membrane localization, whereas a mutation at the CAAX motif eliminated both plasma membrane and internal membrane association of Rsr1/Bud1 . Thus the lysine repeat and the CAAX motif of Rsr1/Bud1 are important for its localization to the plasma membrane and to the polarized growth sites . This localization of Rsr1/Bud1 is essential for its function in proper bud site selection because both mutations resulted in random bud site selection.

J Cell Biol, 2002 Jun 10, 157(6), 997 - 1004 Epub 2002 Jun 10.
Huntington toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1; Meriin AB et al.; The cause of Huntington's disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone . Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity . Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1-151 mutations inhibited expansion of aggregates . The latter three mutants strongly suppressed the polyQ toxicity . Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved . Aggregation defects in these mutants and in sis1-85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance . Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity . We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ . Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ . We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity.

Biochem Biophys Res Commun, 2002 Jun 14, 294(3), 687 - 91
Budding yeast Cdc5 phosphorylates Net1 and assists Cdc14 release from the nucleolus; Yoshida S et al.; Polo-like kinase Cdc5 and Cdc14 phosphatase are essential for mitotic exit in budding yeast . Cdc14 sequestered in the nucleolus by forming a complex with Net1, a nucleolar inhibitor of Cdc14, is activated after the release from the nucleolus and Cdc5 is essential for this release . Here we show that Cdc5 affects the phosphorylation state of Net1 . Tab6 is a dominant active form of Cdc14 . We found that Tab6 was released from the nucleolus of cdc5 mutant cells in a cell cycle dependent manner and that the release of Tab6 (or Cdc14) was not sufficient for the cdc5 mutant to grow at a higher temperature . Altogether, we propose that Cdc5 acts to reduce affinity between Cdc14 and Net1 and that the timing of Cdc14 release is independent of Cdc5 . We also provide evidence that the critical function of Cdc5, other than Cdc14 liberation, exists in late mitotic events.

Methods, 2002 Feb, 26(2), 123 - 41
Analyzing mRNA-protein complexes using a yeast three-hybrid system; Bernstein DS et al.; RNA-protein interactions are essential for the proper execution and regulation of every step in the life of a eukaryotic mRNA . Here we describe a three-hybrid system in which RNA-protein interactions can be analyzed using simple phenotypic or enzymatic assays in Saccharomyces cerevisiae . The system can be used to detect or confirm an RNA-protein interaction, to analyze RNA-protein interactions genetically, and to discover new protein or RNA partners when only one is known . Multicomponent complexes containing more than one protein can be detected, identified, and analyzed . We describe the method and how to use it, and discuss applications that bear particularly on eukaryotic mRNAs . (c) 2002 Elsevier Science (USA).

Biochem Biophys Res Commun, 2002 May 24, 293(5), 1383 - 8
Activation of Ras cascade increases the mitochondrial enzyme content of respiratory competent yeast; Dejean L et al.; We investigated the effects of genetic and physiological modulations of the cAMP-protein kinase A pathway on mitochondrial biogenesis of yeast cells grown on lactate . Yeast mutants with over-activated Ras/adenylate cyclase pathway (i.e., Ras2(val19), ira1Delta(ira2)Delta) or with a constitutive downstream activation of protein kinases A (i.e., bcyDelta) showed an increase in the mitochondrial enzyme content . In contrast, loss of Ras activity (i.e., Ras2 mutant) resulted in a slight decrease . The treatment by cAMP of a responsive mutant increased the oxidative phosphorylation capacity of cells and increased the transcript level of nuclear genes encoding for mitochondrial proteins . In contrast, the transcript level of mitochondrial DNA genes was unchanged . It is concluded that the Ras/cAMP/protein kinase A pathway is part of the regulatory circuit controlling biogenesis of the oxidative phosphorylation complexes in yeast cells.

Biochem Biophys Res Commun, 2002 May 3, 293(2), 733 - 40
Two-hybrid cloning and characterization of OSH3, a yeast oxysterol-binding protein homolog; Park YU et al.; We identify Osh3p, one of seven yeast oxysterol-binding protein (OSBP) homologs, by its protein-protein interactions with a DEAD-box RNA helicase, Rok1p . The ROK1 gene was initially identified by its ability on a high-copy number plasmid to suppress the nuclear fusion defect caused by the kem1 null mutation . Our results show that OSH3 also affects nuclear fusion in a kem1-specific manner; the nuclear fusion defect of kem1 was intensified by the multicopy expression of OSH3 . The Osh3p synthesis was highly induced by alpha-mating pheromone . We also found that OSH3 overexpression promoted filamentation growth of the Sigma1278b wild-type strain and suppressed the filamentation growth defect of the ste12 mutation . These results lead us to a new understanding of cellular functions of the yeast OSBPs .

Mol Cell Biol, 2002 Jul, 22(13), 4914 - 28
Yeast Ysl2p, homologous to Sec7 domain guanine nucleotide exchange factors, functions in endocytosis and maintenance of vacuole integrity and interacts with the Arf-Like small GTPase Arl1p; Jochum A et al.; We previously described the isolation of ysl2-1 due to its genetic interaction with Delta ypt51/vps21, a mutant with a deletion of the coding sequence for the yeast Rab5 homolog, which regulates endocytic traffic between early and late endosomes . Here we report that Ysl2p is a novel 186.8-kDa peripheral membrane protein homologous to members of the Sec7 family . We provide multiple genetic and biochemical evidence for an interaction between Ysl12p and the Arf-like protein Arl1p, consistent with a potential function as an Arf guanine nucleotide exchange factor (GEF) . The temperature-sensitive alleles ysl2-307 and ysl2-316 are specifically defective in ligand-induced degradation of Ste2p and alpha-factor and exhibit vacuole fragmentation directly upon a shift to 37 degrees C . In living cells, green fluorescent protein (GFP)-Ysl2p colocalizes with endocytic elements that accumulate FM4-64 . The GFP-Ysl2p staining is sensitive to a mutation in VPS27 resulting in the formation of an aberrant class E compartment, but it is not affected by a sec7 mutation . Consistent with the idea that Ysl2p and Arl1p have closely related functions, Delta arl1 cells are defective in endocytic transport and in vacuolar protein sorting.

Mol Cell Biol, 2002 Jul, 22(13), 4723 - 38
Proteomics of the eukaryotic transcription machinery: identification of proteins associated with components of yeast TFIID by multidimensional mass spectrometry; Sanders SL et al.; The general transcription factor TFIID is a multisubunit complex of TATA-binding protein (TBP) and 14 distinct TBP-associated factors (TAFs) . Although TFIID constituents are required for transcription initiation of most mRNA encoding genes, the mechanism of TFIID action remains unclear . To gain insight into TFIID function, we sought to generate a proteomic catalogue of proteins specifically interacting with TFIID subunits . Toward this end, TFIID was systematically immunopurified by using polyclonal antibodies directed against each subunit, and the constellation of TBP- and TAF-associated proteins was directly identified by coupled multidimensional liquid chromatography and tandem mass spectrometry . A number of novel protein-protein associations were observed, and several were characterized in detail . These interactions include association between TBP and the RSC chromatin remodeling complex, the TAF17p-dependent association of the Swi6p transactivator protein with TFIID, and the identification of three novel subunits of the SAGA acetyltransferase complex, including a putative ubiquitin-specific protease component . Our results provide important new insights into the mechanisms of mRNA gene transcription and demonstrate the feasibility of constructing a complete proteomic interaction map of the eukaryotic transcription apparatus.

Mol Cell Biol, 2002 Jul, 22(13), 4607 - 21
Interactions of the Mcm1 MADS box protein with cofactors that regulate mating in yeast; Mead J et al.; The yeast Mcm1 protein is a member of the MADS box family of transcriptional regulatory factors, a class of DNA-binding proteins that control numerous cellular and developmental processes in yeast, Drosophila melanogaster, plants, and mammals . Although these proteins bind DNA on their own, they often combine with different cofactors to bind with increased affinity and specificity to their target sites . To understand how this class of proteins functions, we have made a series of alanine substitutions in the MADS box domain of Mcm1 and examined the effects of these mutations in combination with its cofactors that regulate mating in yeast . Our results indicate which residues of Mcm1 are essential for viability and transcriptional regulation with its cofactors in vivo . Most of the mutations in Mcm1 that are lethal affect DNA-binding affinity . Interestingly, the lethality of many of these mutations can be suppressed if the MCM1 gene is expressed from a high-copy-number plasmid . Although many of the alanine substitutions affect the ability of Mcm1 to activate transcription alone or in combination with the alpha 1 and Ste12 cofactors, most mutations have little or no effect on Mcm1-mediated repression in combination with the alpha 2 cofactor . Even nonconservative amino acid substitutions of residues in Mcm1 that directly contact alpha 2 do not significantly affect repression . These results suggest that within the same region of the Mcm1 MADS box domain, there are different requirements for interaction with alpha 2 than for interaction with either alpha1 or Ste12 . Our results suggest how a small domain, the MADS box, interacts with multiple cofactors to achieve specificity in transcriptional regulation and how subtle differences in the sequences of different MADS box proteins can influence the interactions with specific cofactors while not affecting the interactions with common cofactors.

Mol Genet Metab, 2002 Apr, 75(4), 335 - 43
S-adenosylhomocysteine, but not homocysteine, is toxic to yeast lacking cystathionine beta-synthase; Christopher SA et al.; Elevated plasma homocysteine is associated with a variety of diseases in humans including coronary heart disease, stroke, peripheral vascular disease, and birth defects . However, the mechanism by which plasma homocysteine affects cells is unknown . We have examined the growth of isogenic wild-type and cystathionine beta-synthase (CBS) deficient yeast in response to homocysteine and its immediate metabolic precursor, S-adenosylhomocysteine (SAH) . CBS deficient yeast export significantly more homocysteine into the media than wild-type yeast and have elevated internal pools of homocysteine and SAH . We found that 5 mM homocysteine added to the media had very little effect on the growth of wild-type or CBS deficient yeast, although intracellular homocysteine concentrations increased five- to tenfold . In contrast, as little as 25 microM S-adenosylhomocysteine inhibited the growth of CBS deficient yeast, but had no effect on wild-type yeast . Measurements of the intracellular S-adenosylmethionine (SAM) and SAH indicate that CBS deficient yeast contain reduced SAM/SAH ratios relative to wild-type, and this ratio is further reduced by adding SAH to the media . Growth inhibition by SAH in CBS deficient yeast can be totally reversed by addition of SAM to the media, indicating that the ratio and not absolute level is critical for cell growth . These results suggest that CBS plays a key role in the regulation of the SAM/SAH ratio inside cells and that excessive perturbations of this ratio can inhibit growth . We hypothesize that elevated extracellular homocysteine present in humans may reflect an altered intracellular SAM/SAH ratio and that this may be related to disease pathogenesis.

J Mol Biol, 2002 May 31, 319(2), 315 - 27
The influence of sequence divergence between alleles of the human MS205 minisatellite incorporated into the yeast genome on length-mutation rates and lethal recombination events during meiosis; He Q et al.; Certain minisatellites exhibit hypervariability with respect to the number of repeat units and, thus, allele length . Such polymorphism is generated by germline-specific recombinational events that occur at high frequencies and lead to the gain or loss of repeat units . In order to elucidate the molecular details of mutagenesis in minisatellites, we have integrated human minisatellites into the yeast genome in the vicinity of a hotspot for meiotic double-strand breaks (DSBs) . Here, we describe the results of tetrad analyses of mutations in the human MS205 minisatellite in yeast strains heterozygous for alleles composed of 51 and 31 repeat units, as well as in a strain homozygous for the same 51 repeat unit allele . The length-mutation rate was twice as high in the heterozygous strain as in the homozygous strain, suggesting that sequence divergence between alleles enhances the generation of length mutations . In the case of heterozygotes, the frequency of length mutants resulting from inter-allelic exchange was significantly higher in tetrads with three viable spores than in tetrads with four viable spores, indicating that there is a higher probability for spore mortality in tetrads originating from meioses during which inter-allelic exchange of repeat units occurs . In an attempt to explain these findings, we propose a model for minisatellite mutation involving recombination, in which sequence divergence between alleles results in a heteroduplex containing numerous mismatches . We suggest that convergent mismatch-repair tracts in this heteroduplex give rise to a DSB that may be repaired by an additional round of recombination resulting in mutation of a third allele, or be lethal if such recombination fails . It appears probable that the formation of such additional mutants is the major explanation for the difference in meiotic length-mutation rates between the heterozygous and homozygous yeast strains, and that this phenomenon contributes to high germline length-mutation frequencies at minisatellites in humans .

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(2), 246 - 250
Screening of Genes Related to the Expression of Human epsilon-globin Gene by Using Yeast One-hybrid System; Huang J et al.; The developmental stage-specific silencing of the human epsilon-globin gene during embryonic period is controlled, in part, by a silencer (from -392 to -177 bp) upstream of this gene . In our previous work, by using DNase I footprinting assay, a major protected region from -278 to -235 bp within this silencer DNA was identified . In order to isolate genes encoding proteins that bind to this target element, a human fetal liver cDNA library was screened by using yeast one-hybrid system . The candidate clones were sequenced, and the ribosomal protein L3 (RPL3) was preliminarily isolated . Electrophoretic mobility shift assay (EMSA) and competition tests also demonstrated its binding activity to the target element . These imply that RPL3 might play an important role in silencing the human embryonic epsilon-globin gene expression through the interaction with this silencer.

Mol Cell, 2002 May, 9(5), 1101 - 11
Functional interaction of yeast pre-mRNA 3' end processing factors with RNA polymerase II; Licatalosi DD et al.; The RNA polymerase II CTD is essential for 3' end cleavage of metazoan pre-mRNAs and binds 3' end processing factors in vitro . We show genetic and biochemical interactions between the CTD and the Pcf11 subunit of the yeast cleavage/polyadenylation factor, CFIA . In vitro binding to Pcf11 required phosphorylation of the CTD on Ser2 in the YSPTSPS heptad repeats . Deletion of the yeast CTD reduced the efficiency of cleavage at poly(A) sites, and the length of poly(A) tails suggesting that it helps couple 3' end formation with transcription . Consistent with this model, the 3' end processing factors CFIA, CFIB, and PFI were recruited to genes progressively, starting at the 5' end, in a process that required ongoing transcription.

Mol Cell, 2002 May, 9(5), 1067 - 78
The yeast CDK inhibitor Sic1 prevents genomic instability by promoting replication origin licensing in late G(1); Lengronne A et al.; G(1) cell cycle regulators are often mutated in cancer, but how this causes genomic instability is unclear . Here we show that yeast lacking the CDK inhibitor Sic1 initiate DNA replication from fewer origins, have an extended S phase, and inefficiently separate sister chromatids during anaphase . This leads to double-strand breaks (DSBs) in a fraction of sic1 cells as evidenced by the accumulation of Ddc1 foci and a 575-fold increase in gross chromosomal rearrangements . Both S and M phase defects are rescued by delaying S-CDK activation, indicating that Sic1 promotes origin licensing in late G(1) by preventing the untimely activation of CDKs . We propose that precocious CDK activation causes genomic instability by altering the dynamics of S phase, which then hinders normal chromosome segregation.

J Biol Chem, 2002 Aug 16, 277(33), 29608 - 16 Epub 2002 Jun 04.
Characterization of a hyperthermophilic P-type ATPase from Methanococcus jannaschii expressed in yeast; Morsomme P et al.; We report on the biochemical and structural properties of a putative P-type H(+)-ATPase, MJ1226p, from the anaerobic hyperthermophilic Archaea Methanococcus jannaschii . An efficient heterologous expression system was developed in Saccharomyces cerevisiae and a four-step purification protocol, using n-dodecyl beta-d-maltoside, led to a homogeneous detergent-solubilized protein fraction with a yield of over 2 mg of protein per liter of culture . The three-dimensional structure of the purified detergent-solubilized protein obtained at 2.4 nm resolution by electron microscopy showed a dimeric organization in which the size and the shape of each monomer was compatible with the reported structures of P-type ATPases . The purified MJ1226p ATPase was inactive at 40 degrees C and was active at elevated temperature reaching high specific activity, up to 180 micromol of P(i) x min(-1) x mg(-1) at 95 degrees C . Maximum ATPase activity was observed at pH 4.2 and required up to 200 mm monovalent salts . The ATPase activity was stable for several days upon storage at 65 degrees C and was highly resistant to urea and guanidine hydrochloride . The protein formed catalytic phosphoenzyme intermediates from MgATP or P(i), a functional characteristic specific of P-type ATPases . The highly purified, homogeneous, stable, and active MJ1226p ATPase provides a new model for further structure-function studies of P-type ATPases.

J Biol Chem, 2002 Aug 9, 277(32), 28439 - 45 Epub 2002 Jun 04.
Control of mitotic exit in budding yeast . In vitro regulation of Tem1 GTPase by Bub2 and Bfa1; Geymonat M et al.; The elimination of mitotic kinase activity at the end of mitosis is essential for progression to the next stage of the eukaryotic cell cycle . In budding yeast, this process is controlled by a regulatory cascade called the mitotic exit network . Extensive genetic data indicate that mitotic exit network activity is determined by a GTP-binding protein, Tem1, and its putative regulators, Bub2, Bfa1, and Lte1 . Here we describe the purification and in vitro activities of Tem1, Bub2, and Bfa1 . We describe the nucleotide binding properties of Tem1 and characterize its intrinsic GTPase activity . The combination of Bfa1 and Bub2 acts as a two-component GTPase-activating protein for Tem1 . In the absence of Bub2, Bfa1 inhibits the GTPase and GTP exchange activities of Tem1 . This inhibition is elicited by either the N- or C-terminal regions of Bfa1, which also retain some ability to co-activate GTPase activity in the presence of Bub2 . Although the C-terminal region of Bfa1 binds to Bub2, no interaction of the N-terminal half of Bfa1 with Bub2 was detected despite their combined GAP activity . Therefore, we propose that Bfa1 acts both as an adaptor to connect Bub2 and Tem1 and as an allosteric effector that facilitates this interaction.

Plant J, 2002 Jun, 30(5), 511 - 9
Expression of Arabidopsis SR-like splicing proteins confers salt tolerance to yeast and transgenic plants; Forment J et al.; Searching for novel targets of salt toxicity in eukaryotic cells, we have screened an Arabidopsis thaliana cDNA library to isolate genes conferring increased tolerance to salt stress when expressed in the yeast Saccharomyces cerevisiae . Here we show that expression of the 'alternating arginine-rich' (or RS) domains of two different SR-like, putative splicing proteins from Arabidopsis allows yeast cells to tolerate higher lithium and sodium concentrations . Protection against salt stress appears to require the in vivo phosphorylation of these plant polypeptides, since the yeast SR protein kinase Sky1p, which was able to phosphorylate in vitro at least one of them, also proved to be essential for the observed salt tolerance phenotype . In addition, a clone encoding the U1A protein, a previously characterised Arabidopsis splicing factor, was also isolated in the screening . No significant decrease in the intracellular concentration of lithium was observed in yeast cells incubated in the presence of LiCl upon expression of any of the Arabidopsis proteins, suggesting that their effects are not mediated by the stimulation of ion transport . In support of the general significance of these data, we also show that the expression of the RS domain of one of the SR-like proteins in transgenic Arabidopsis plants increases their tolerance to LiCl and NaCl . These results point to an important role of pre-mRNA splicing and SR-like proteins in the salt tolerance of eukaryotic cells, offering a novel route to improve this important trait in crop plants.

Biomed Environ Sci, 2002 Mar, 15(1), 36 - 40
Yeast one-hybrid system used to identify the binding proteins for rat glutathione S-transferase P enhancer I; Liao MX et al.; OBJECTIVE: To detect the trans-factors specifically binding to the strong enhancer element (GPEI) in the upstream of rat glutathione S-transferase P (GST-P) gene . METHODS: Yeast one-hybrid system was used to screen rat lung MATCHMAKER cDNA library to identify potential trans-factors that can interact with core sequence of GPEI(cGPEI) . Electrophoresis mobility shift assay (EMSA) was used to analyze the binding of transfactors to cGPEI . RESULTS: cDNA fragments coding for the C-terminal part of the transcription factor c-Jun and rat adenine nucleotide translocator (ANT) were isolated . The binding of c-Jun and ANT to GPEI core sequence were confirmed . CONCLUSIONS: Rat c-jun transcriptional factor and ANT may interact with cGPEI . They could play an important role in the induced expression of GST-P gene.

J Biol Chem, 2002 Aug 9, 277(32), 28757 - 64 Epub 2002 Jun 03.
Redundant mitochondrial targeting signals in yeast adenylate kinase; Schricker R et al.; Yeast adenylate kinase (Aky2p, Adk1p) occurs simultaneously in cytoplasm and mitochondrial intermembrane space . It has no cleavable mitochondrial targeting sequence, and the signal for mitochondrial import and submitochondrial sorting is largely unknown . The extreme N terminus of Aky2p is able to direct cytoplasmic passengers to mitochondria . However, an Aky2 mutant lacking this sequence is imported with about the same efficiency as the wild type . To identify possible import-relevant information in the interior, parts of Aky2p were exchanged by homologous in vitro recombination for the respective segments of the purely cytoplasmic isozyme, Ura6p . Import studies revealed an internal region of about 40 amino acids, which was sufficient to direct the chimera to mitochondria but not for correct submitochondrial sorting . The respective Ura6p hybrid was arrested in the mitochondrial membrane at a position where it was inaccessible to protease but was released by alkaline extraction, suggesting that it had entered an import channel and passed the initial steps of recognition and uptake . Site-specific mutations within the presumptive address-specifying segment identified the amphipathic helix 5 . A Ura6 mutant protein in which helix 5 had been replaced with the respective sequence from Aky2p was imported, and this address sequence cooperates with the N terminus in the respective double mutant in a synergistic fashion.

J Cell Biol, 2002 Jun 10, 157(6), 1005 - 15 Epub 2002 Jun 03.
Ypt32 recruits the Sec4p guanine nucleotide exchange factor, Sec2p, to secretory vesicles; evidence for a Rab cascade in yeast; Ortiz D et al.; SEC2 is an essential gene required for polarized growth of the yeast Saccharomyces cerevisiae . It encodes a protein of 759 amino acids that functions as a guanine nucleotide exchange factor for the small GTPase Sec4p, a regulator of Golgi to plasma membrane transport . Activation of Sec4p by Sec2p is needed for polarized transport of vesicles to exocytic sites . Temperature-sensitive (ts) mutations in sec2 and sec4 result in a tight block in secretion and the accumulation of secretory vesicles randomly distributed in the cell . The proper localization of Sec2p to secretory vesicles is essential for its function and is largely independent of Sec4p . Although the ts mutation sec2-78 does not affect nucleotide exchange activity, the protein is mislocalized . Here we present evidence that Ypt31/32p, members of Rab family of GTPases, regulate Sec2p function . First, YPT31/YPT32 suppress the sec2-78 mutation . Second, overexpression of Ypt31/32p restores localization of Sec2-78p . Third, Ypt32p and Sec2p interact biochemically, but Sec2p has no exchange activity on Ypt32p . We propose that Ypt32p and Sec4p act as part of a signaling cascade in which Ypt32p recruits Sec2p to secretory vesicles; once on the vesicle, Sec2p activates Sec4p, enabling the polarized transport of vesicles to the plasma membrane.

Mech Ageing Dev, 2002 Apr 30, 123(8), 857 - 67
Paradigms and pitfalls of yeast longevity research; Sinclair DA; Over the past 10 years, considerable progress has been made in the yeast aging field . Multiple lines of evidence indicate that a cause of yeast aging stems from the inherent instability of repeated ribosomal DNA (rDNA) . Over 16 yeast longevity genes have now been identified and the majority of these have been found to affect rDNA silencing or stability . Environmental conditions such as calorie restriction have been shown to modulate this mode of aging via Sir2, an NAD-dependent histone deacetylase (HDAC) that binds at the rDNA locus . Although this mechanism of aging appears to be yeast-specific, the longevity function of Sir2 is conserved in at least one multicellular organism, Caenorhabditis elegans (C . elegans) . These findings are consistent with the idea that aging is a by-product of natural selection but longevity regulation is a highly adaptive trait . Characterizing this and other mechanisms of yeast aging should help identify additional components of longevity pathways in higher organisms.

Biochim Biophys Acta, 2002 Jun 3, 1597(2), 193 - 220
Yeast cytochrome c peroxidase: mechanistic studies via protein engineering; Erman JE et al.; Cytochrome c peroxidase (CcP) is a yeast mitochondrial enzyme that catalyzes the reduction of hydrogen peroxide to water by ferrocytochrome c . It was the first heme enzyme to have its crystallographic structure determined and, as a consequence, has played a pivotal role in developing ideas about structural control of heme protein reactivity . Genetic engineering of the active site of CcP, along with structural, spectroscopic, and kinetic characterization of the mutant proteins has provided considerable insight into the mechanism of hydrogen peroxide activation, oxygen-oxygen bond cleavage, and formation of the higher-oxidation state intermediates in heme enzymes . The catalytic mechanism involves complex formation between cytochrome c and CcP . The cytochrome c/CcP system has been very useful in elucidating the complexities of long-range electron transfer in biological systems, including protein-protein recognition, complex formation, and intracomplex electron transfer processes.

FEBS Lett, 2002 Jun 5, 520(1-3), 133 - 8
Phospholipase C is required for glucose-induced calcium influx in budding yeast; Tisi R et al.; Intracellular calcium is a second messenger involved in several processes in yeast, such as mating, nutrient sensing, stress response and cell cycle events . It was reported that glucose addition stimulates a rapid increase in free calcium level in yeast . To investigate the calcium level variations induced by different stimuli we used a reporter system based on the photoprotein aequorin . Glucose addition (50 mM) to nutrient-starved cells induced an increase in free intracellular calcium concentration, mainly due to an influx from external medium . The increase of calcium reached its maximum 100-120 s after the stimulus . A concentration of about 20 mM glucose was required for a 50% increase in intracellular calcium . This response was completely abolished in strain plc1 Delta and in the isogenic wild-type strain treated with 3-nitrocoumarin, a phosphatidylinositol-specific phospholipase C inhibitor, suggesting that Plc1p is essential for glucose-induced calcium increase . This suggests that Plc1p should have a significant role in transducing glucose signal . The calcium influx induced by addition of high glucose on cells previously stimulated with low glucose levels was inhibited in strains with a deletion in the GPR1 or GPA2 genes, which suggests that glucose would be detected through the Gpr1p/Gpa2p receptor/G protein-coupled (GPCR) complex . Moreover, the signal was completely abolished in a strain unable to phosphorylate glucose, which is consistent with the reported requirement of glucose phosphorylation for GPCR complex activation.

FEBS Lett, 2002 Jun 5, 520(1-3), 63 - 7
The ATP-binding cassette (ABC) transporter Bpt1p mediates vacuolar sequestration of glutathione conjugates in yeast; Klein M et al.; Vacuolar sequestration or cellular extrusion of glutathione-conjugated xenobiotics and catabolites by ATP-binding cassette (ABC) transporters is an important detoxification mechanism operating in many species . In this study, we show that the yeast ABC transporter Bpt1p, a paralogue of Ycf1p, acts as an ATP-dependent vacuolar pump for glutathione conjugates . Bpt1p, which is inhibited by vanadate and glibenclamide, accounts for one third of the total vacuolar transport of glutathione conjugates . Furthermore, immunoblot analyses show that Bpt1p levels are strongly elevated in early stationary phase, consistent with a function of Bpt1p in vacuolar detoxification.

Neurosci Lett, 2002 Jun 7, 325(2), 119 - 23
Analysis of synphilin-1 and synuclein interactions by yeast two-hybrid beta-galactosidase liquid assay; Neystat M et al.; Synphilin-1 interacts with alpha-synuclein, which has been implicated in the pathogenesis of Parkinson's disease (PD) . By examination of their interactions quantitatively, with the use of the yeast two-hybrid beta-galactosidase assay, we find that the synuclein amino acid (aa) 1-65 region is sufficient for an interaction . A central domain of synphilin-1, aa 349-555, is both necessary and sufficient for an interaction with alpha-synuclein . We did not observe an effect of the synuclein A53T mutation, which causes one familial form of PD, on interactions with synphilin-1 . However, the A30P mutation caused an increase in the interaction between the synuclein aa 1-65 fragment and the synphilin-1 central domain.

Eur J Histochem, 2002, 46(1), 23 - 9
Hydrophobins in ectomycorrhizas: heterologous transcription of the Pisolithus HydPt-1 gene in yeast and Hebeloma cylindrosporum; Tagu D et al.; Hydrophobins are fungal cell wall proteins involved in aggregation of hyphae . Upon the development of the ectomycorrhizal symbiosis between tree roots and fungal hyphae, the transcripts of hydrophobin genes markedly accumulated . As the precise role of these proteins in symbiosis is not yet known, we develop heterologous expression system of the Pisolithus hydrophobin HYDPt-1 . This gene has been introduced in Saccharomyces cerevisiae and in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum . Introns were required for hydPt-1 transcript accumulation in the basidiomycete H . cylindrosporum . Heterologous transcript accumulation did not alter the phenotype of either species . The lack of altered phenotype resulted from the absence of HYDPt-1 polypeptide accumulation in transformed strains.

Nat Rev Mol Cell Biol, 2002 Jun, 3(6), 453 - 9
Yeast and apoptosis; Jin C et al.; Even though yeast lack much of the molecular machinery that is responsible for apoptosis in metazoans, they can be a powerful tool in apoptosis research . The ectopic expression of several animal apoptosis proteins in yeast can help us to discover new genes -- and chemical compounds -- that modulate the cell-death pathways of higher eukaryotes.

J Nutr, 2002 Jun, 132(6), 1141 - 8
Yeast extract stimulates glucose metabolism and inhibits lipolysis in rat adipocytes in vitro; Edens NK et al.; Foods contain bioactive components that contribute to optimal health . Food-grade yeast may contain components that enhance cellular glucose metabolism . We tested the effect of brewer's yeast (Saccharomyces cerevisiae) extract (YE), in vitro on rat fat cell glucose transport, glucose metabolism to lipid, and lipolysis . YE was fractionated by reverse-phase chromatography on a C18 open column using ammonium acetate (0.05 mol/L, pH 5.8), with acetonitrile (40%) elution solvent into fraction 1 (Fx1), fraction 2 (Fx2) and fraction 3 (Fx3) . Isolated rat adipocytes were preincubated with insulin (51 pmol/L), YE (10 mg/L) or both; transport of U-(14)C-glucose was measured . Adipocytes were incubated with insulin and YE fractions (10 mg/L); glucose metabolism to lipid was measured by incorporation of U-(14)C-glucose into total lipids . Lipolysis was measured by glycerol release . Insulin stimulated glucose transport to sevenfold the basal value (P < 0.05) . YE did not affect glucose transport . Insulin stimulated glucose metabolism to 2.6-fold the basal value (P < 0.001); YE stimulated glucose metabolism 14% (P < 0.005) . YE potentiated the action of insulin 30% (P < 0.002) . YE Fx2 and Fx3 stimulated glucose metabolism 25-40% (P < 0.05) . Insulin inhibited lipolysis 47% (P < 0.001) . YE alone inhibited lipolysis 63% (P < 0.001) . YE and insulin inhibited lipolysis 81% (P < 0.001) . Fractions of YE inhibited lipolysis in the presence of insulin (P < 0.05); the order of potency was Fx2 = Fx3 >> Fx1 . A novel yeast extract (YE) and its fractions affect pathways of adipocyte metabolism differentially . YE and its fractions are good candidates for in vivo study.

J Biol Chem, 2002 Aug 9, 277(32), 29125 - 31 Epub 2002 May 31.
A cis-acting sequence homologous to the yeast filamentation and invasion response element regulates expression of a pectinase gene from the bean pathogen Colletotrichum lindemuthianum; Herbert C et al.; Phytopathogenic fungi secrete hydrolytic enzymes that degrade plant cell walls, notably pectinases . The signaling pathway(s) that control pectinase gene expression are currently unknown in filamentous fungi . Recently, the green fluorescent protein coding sequence was used as a reporter gene to study the expression of CLPG2, a gene encoding an endopolygalacturonase of the bean pathogen Colletotrichum lindemuthianum . CLPG2 is transcriptionally induced by pectin in the axenic culture of the fungus and during formation of the appressorium, an infection structure specialized in plant tissue penetration . In the present study, promoter deletion and mutagenesis, as well as gel shift mobility assays, allowed for the first time identification of cis-acting elements that bind protein factors and are essential for the regulation of a pectinase gene . We found that two different adjacent DNA motifs are combined to form an active element that shows a strong sequence homology with the yeast filamentation and invasion response element . The same element is required for the transcriptional activation of CLPG2 by pectin and during appressorium development . This study strongly suggests that the control of virulence genes of fungal plant pathogens, such as pectinases, involves the formation of a complex of transcriptional activators similar to those regulating the invasive growth in yeast.

Gene, 2002 Feb 20, 285(1-2), 213 - 20
Identification, characterization and cytogenetic mapping of a yeast Vps54 homolog in rat and mouse; Walter L et al.; A novel gene, VPS54-like (Vps54l), is described in the rat that is homologous to the yeast Vps54 gene which is known to be involved in intracellular protein sorting . Furthermore, Vps54-related sequences of human, mouse, Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana could be identified in the EMBL/GenBank/DDBJ database . Each of the deduced amino acid sequences of the Vps54 genes in these species contain a coiled-coil region and eight to 13 dileucine motifs . The rat Vps54l gene could be mapped to the end of chromosome 14 by radiation hybrid analysis 7 cR(3000) from the D14Rat22 marker and to 14q22 by fluorescence in situ hybridization . Using a rat Vps54l-containing P1-derived artificial chromosome (PAC) clone the respective ortholog was mapped to chromosome 11A3 in the mouse . In addition, the rat genome contains a processed pseudogene of Vps54l on chromosome 7q22 . PAC clone analysis shows that the rat Vps54l gene maps close to the UDP-glucose-pyrophosphorylase 2 gene . The two genes are in tail to tail orientation with their polyadenylation sites 497 bp apart . Rat Vps54l appears to be expressed ubiquitously, but at a relatively low level . Alternatively spliced transcripts could be isolated which lack the sequence coding for the coiled-coil region.

Int J Food Microbiol, 2002 Jun 5, 76(1-2), 47 - 53
Rapid viability assessment of yeast cells using vital staining with 2-NBDG, a fluorescent derivative of glucose; Oh KB et al.; A fluorescent glucose analogue, 2-{N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino}-2-deoxy-D-glucose (2-NBDG), which had been developed previously for the analysis of glucose uptake activity by living cells, was investigated to evaluate its applicability for assaying the viability of yeasts . Fluorescence intensities of the yeast population were measured by fluorescence spectrophotometry upon exposure to antifungal agents after staining with 2-NBDG and were compared to the number of colony forming units (CFU) . A good correlation was obtained between the yeast viability, determined by the CFU, and the accumulation of 2-NBDG by yeast cells (correlation constant: r=0.98) . Susceptibility testing of amphotericin B and miconazole against yeast strains by plate count and 2-NBDG fluorescence method yielded corresponding results . In conclusion, we found that staining with 2-NBDG is a rapid and sensitive method for the assessment of yeast cell viability.

J Clin Microbiol, 2002 Jun, 40(6), 2240 - 3
Automated extraction of genomic DNA from medically important yeast species and filamentous fungi by using the MagNA Pure LC system; Loeffler J et al.; A fully automated assay was established for the extraction of DNA from clinically important fungi by using the MagNA Pure LC instrument . The test was evaluated by DNA isolation from 23 species of yeast and filamentous fungi and by extractions (n = 28) of serially diluted Aspergillus fumigatus conidia (10(5) to 0 CFU/ml) . Additionally, DNA from 67 clinical specimens was extracted and compared to the manual protocol . The detection limit of the MagNA Pure LC assay of 10 CFU corresponded to the sensitivity when DNA was extracted manually; in 9 of 28 runs, we could achieve a higher sensitivity of 1 CFU/ml blood, which was found to be significant (p <or= 0.004) . DNA from all fungal species analyzed could be extracted and amplified by real-time PCR . Negative controls from all MagNA Pure isolations remained negative . Sixty-three clinical samples showed identical results by both methods, whereas in 4 of 67 samples, discordant results were obtained . Thus, the MagNA Pure LC technique offers a fast protocol for automated DNA isolation from numerous fungi, revealing high sensitivity and purity.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(6), 719 - 722
Identification of A Protein Interacting with Type 2 Methionine Aminopeptidase by Yeast Two-hybrid System; Liu WF et al.; Type 2 methionine aminopeptidase (MetAP2) is the molecular target for the fumagillin inhibitors against angiogenesis . Making use of the yeast two-hybrid system with GAL4 DBD-fused MetAP2 as a bait, a human brain cDNA library was screened to isolate protein factors that might interact with MetAP2 . Among the 2x10(6) transformants obtained, five positive clones were picked out . Sequence analysis revealed that three of them contained cDNA fragments from flotillin and encode a carboxy terminus (starting from amino acids 145--233, respectively) of flotillin protein . The interaction between MetAP2 and flotillin detected by yeast two-hybrid system suggests that MetAP2 might play a role in some biological processes where flotillin is involved.

Nucleic Acids Res, 2002 Jun 1, 30(11), 2358 - 64
The yeast protein Xtc1 functions as a direct transcriptional repressor; Traven A et al.; The yeast protein Xtc1 was identified as a protein that binds directly and specifically to the activation domains of acidic activators such as E2F-1, Gal4 and VP16 . Additionally, it was shown to co-purify with the RNA polymerase II holoenzyme complex and it was suggested that Xtc1 functions as a regulator of transcription that modulates the response of RNA polymerase II to transcriptional activators . We have further analyzed the transcription function of Xtc1 and show that its fusion to a heterologous DNA binding domain can repress transcription of a reporter gene in vivo in an Srb10/11-dependent manner . We suggest that the presence of Xtc1 in the RNA polymerase II holoenzyme could help to recruit an Srb10-active form of the holoenzyme to target promoters . This same protein has also been implicated in mitochondrial DNA recombination, maintenance and repair . Determination of the subcellular localization using a GFP-Xtc1 fusion shows that it localizes to both the nucleus and the mitochondria in vivo, which is consistent with Xtc1 having a function in both cellular compartments.

J Cell Biol, 2002 May 27, 157(5), 783 - 93 Epub 2002 May 28.
Roles of fission yeast tea1p in the localization of polarity factors and in organizing the microtubular cytoskeleton; Behrens R et al.; The cylindrical shape of the fission yeast cell is generated by linear polarized growth from its cell ends . Using immunofluorescence and live imaging microscopy, we have investigated the roles of the cell end marker tea1p in generating linear polarized growth . We found that tea1p is primarily transported on plus ends of microtubules from the vicinity of the nucleus to the cell ends, and that its movement near the nucleus is independent of the kinesin tea2p . Deletion analysis identified a coiled-coil domain in tea1p essential for its retention at cell ends, and demonstrated that tea1p exerts different functions dependent on its location . On the tips of microtubules, tea1p prevents the curling of microtubules around the cell ends, whereas it is required for maintaining linear cell growth and for retention of polarity factors such as the Dyrk kinase pom1p, the CLIP170-like tip1p, and tea2p at the cell ends . We propose that tea1p has roles in organizing the microtubule cytoskeleton on the tips of microtubules, and in the retention of factors at the cell ends necessary for the cell to grow in a straight line.

Biochemistry, 2002 Jun 4, 41(22), 7065 - 73
Effect of AMP on mRNA binding by yeast NAD+-specific isocitrate dehydrogenase; Anderson SL et al.; Yeast mitochondrial NAD+-specific isocitrate dehydrogenase (IDH) has previously been shown to bind specifically to 5'-untranslated regions of yeast mitochondrial mRNAs, and transcripts containing these regions have been found to allosterically inhibit activity of the enzyme . This inhibition is relieved by AMP, an allosteric activator of this regulatory enzyme of the tricarboxylic acid cycle . We further investigated these enzyme/ligand interactions to determine if binding of RNA and AMP by IDH is competitive or independent . Gel mobility shift experiments indicated no effect of AMP on formation of an IDH/RNA complex . Similarly, sedimentation velocity ultracentrifugation experiments used to analyze interactions in solution indicated that AMP alone had little effect on the formation or stability of an RNA/IDH complex . However, when these sedimentation experiments were conducted in the presence of isocitrate, which has been shown to be essential for binding of AMP by IDH, the proportion of RNA sedimenting in a complex with IDH was significantly reduced by AMP . These results suggest that AMP can affect the binding of RNA by IDH but that this effect is apparent only in the presence of substrate . They also suggest that the catalytic activity of IDH in vivo may be subject to complex allosteric control determined by relative mitochondrial concentrations of mRNA, isocitrate, and AMP . We also found evidence for binding of 5'-untranslated regions of mitochondrial mRNAs by yeast mitochondrial NADP+-specific isocitrate dehydrogenase (IDP1) but not by the corresponding cytosolic isozyme (IDP2) . However, this appears to be a nonspecific interaction since no evidence was obtained for any effect on the catalytic activity of IDP1.

EMBO J, 2002 Jun 3, 21(11), 2736 - 45
Mammalian and yeast U3 snoRNPs are matured in specific and related nuclear compartments; Verheggen C et al.; Nucleolar localization of vertebrate box C/D snoRNA involves transit through Cajal bodies, but the significance of this event is unknown . To define better the function of this compartment, we analyzed here the maturation pathway of mammalian U3 . We show that 3'-extended U3 precursors possess a mono-methylated cap, and are not associated with fibrillarin and hNop58 . Importantly, these precursors are detected at both their transcription sites and in Cajal bodies . In addition, mature U3, the core box C/D proteins and the human homolog of the methyltransferase responsible for U3 cap tri-methylation, hTgs1, are all present in Cajal bodies . In yeast, U3 follows a similar maturation pathway, and equivalent 3'-extended precursors are enriched in the nucleolus and in the nucleolar body, a nucleolar domain that concentrates Tgs1p under certain growth conditions . Thus, spatial organization of U3 maturation appears to be conserved across evolution, and involves specialized and related nuclear compartments, the nucleolus/nucleolar body in yeast and Cajal bodies in higher eukaryotes . These are likely places for snoRNP assembly, 3' end maturation and cap modification.

Chem Biol, 2002 May, 9(5), 607 - 18
Transcriptional response pathways in a yeast strain sensitive to saframycin a and a more potent analog: evidence for a common basis of activity; Plowright AT et al.; Saframycin A (SafA) is a natural product that inhibits human cancer cell proliferation . Its synthetic analog, QAD, is a more potent inhibitor of these cells . SafA does not affect wild-type yeast, but it does inhibit growth of the strain CCY333 (DeltaPDR1/PDR3/ERG6) (IC50 = 0.9 microM) . QAD is also a more effective inhibitor of CCY333 growth (IC50 = 0.4 microM) . Transcription profiling of SafA- and QAD-treated CCY333 cultures showed that both drugs generated nearly identical profiles, with altered expression levels (> or =2-fold) of more than 240 genes . Both agents induced the overexpression of genes involved in glycolysis, oxidative stress, and protein degradation and repressed genes encoding histones, biosynthetic enzymes, and the cellular import machinery . Significantly, neither drug affected the expression of known DNA-damage repair genes, as might have been expected if their primary mechanism of action involved the covalent modification of DNA.

Lipids, 2002 Apr, 37(4), 417 - 26
Cloning and molecular characterization of the delta6-desaturase from two echium plant species: production of GLA by heterologous expression in yeast and tobacco; Garcia-Maroto F et al.; The synthesis of GLA (delta6,9,12-1-8:3) is carried out in a number of plant taxa by introducing a double bond at the delta6 position of its precursor, linoleic acid (delta9,12-18:2), through a reaction catalyzed by a delta6-desaturase enzyme . We have cloned genes encoding the delta6-desaturase (D6DES) from two different Macaronesian Echium species, E . pitardii and E . gentianoides (Boraginaceae), which are characterized by the accumulation of high amounts of GLA in their seeds . The Echium D6DES genes encode proteins of 438 amino acids bearing the prototypical cytochrome b(5) domain at the N-terminus . Cladistic analysis of desaturases from higher plants groups the Echium D6DES proteins together with other delta6-desaturases in a different cluster from that of the highly related delta8-desaturases . Expression analysis carried out in E . pitardii shows a positive correlation between the D6DES transcript level and GLA accumulation in different tissues of the plant . Although a ubiquitous expression in all organs is observed, the transcript is particularly abundant in developing fruits, whereas a much lower level is present in mature leaves . Functional characterization of the D6DES gene from E . gentianoides has been achieved by heterologous expression in tobacco plants and in the yeast Saccharomyces cerevisiae . In both cases, overexpression of the gene led to the synthesis of GLA . Biotechnological application of these results can be envisaged as an initial step toward the generation of transgenic oleaginous plants producing GLA.

Science, 2002 May 24, 296(5572), 1483 - 6
Regulation of MAPK function by direct interaction with the mating-specific Galpha in yeast; Metodiev MV et al.; The mating response of the budding yeast Saccharomyces cerevisiae is mediated by a prototypical heterotrimeric GTP-binding protein (G protein) and mitogen-activated protein kinase (MAPK) cascade . Although signal transmission by such pathways has been modeled in detail, postreceptor down-regulation is less well understood . The pheromone-responsive G protein alpha subunit (Galpha) of yeast down-regulates the mating signal, but its targets are unknown . We have found that Galpha binds directly to the mating-specific MAPK in yeast cells responding to pheromone . This interaction contributes both to modulation of the mating signal and to the chemotropic response, and it demonstrates direct communication between the top and bottom of a Galpha-MAPK pathway.

Biochem J, 2002 Sep 1, 366(Pt 2), 549 - 56
The human homologue of yeast ArgRIII protein is an inositol phosphate multikinase with predominantly nuclear localization; Nalaskowski MM et al.; The function of the transcription regulator ArgRIII in the expression of several genes involved in the metabolism of arginine in yeast has been well studied . It was previously reported that it is also an inositol phosphate multikinase and an important factor of the mRNA export pathway {reviewed by Shears (2000) Bioessays 22, 786-789} . In the present study we report the cloning of a full-length 1248-bp cDNA encoding a human inositol phosphate multikinase (IPMK) . This protein has a calculated molecular mass of 47.219 kDa . Functionally important motifs {inositol phosphate-binding site, ATP-binding site, catalytically important SSLL (Ser-Ser-Leu-Leu) domain} are conserved between the human IPMK and yeast ArgRIII . Bacterially expressed protein demonstrated an inositol phosphate multikinase activity similar to that of yeast ArgRIII . Ins(1,4,5)P3 is phosphorylated at positions 3 and 6 up to Ins(1,3,4,5,6)P5 . The human IPMK fused with a fluorescent protein tag is localized predominantly in the nucleus when transiently expressed in mammalian cells . A basic cluster in the protein's C-terminus is positively involved in nuclear targeting . These findings are consistent with the concept of a nuclear inositol phosphate signalling and phosphorylation pathway in mammalian cells.

Plant Cell Rep, 2001 Mar, 20(3), 227 - 34
Characterization of the yeast copper-inducible promoter system in Arabidopsis thaliana; Granger CL et al.; Inducible promoters or gene-switches are used to both spatially and temporally regulate gene expression . Such regulation can provide information concerning the function of a gene in a developmental context as well as avoid potential harmful effects due to overexpression . A gfp construct under the control of a copper-inducible promoter was introduced into Arabidopsis thaliana (L.) Heynh . and the regulatory parameters of this inducible promoter were determined . Here, we describe the time-course of up- and down-regulation of GFP expression in response to copper level, the optimal regulatory levels of copper, and the tissue specificity of expression in three transgenic lines . We conclude that the copper-inducible promoter system may be useful in regulating the time and location of gene expression in A . thaliana.

Mol Cell Biol, 2002 Jun, 22(12), 4383 - 9
Yeast RAD26, a homolog of the human CSB gene, functions independently of nucleotide excision repair and base excision repair in promoting transcription through damaged bases; Lee SK et al.; RAD26 in the yeast Saccharomyces cerevisiae is the counterpart of the human Cockayne syndrome group B (CSB) gene . Both RAD26 and CSB act in the preferential repair of UV lesions on the transcribed strand, and in this process, they function together with the components of nucleotide excision repair (NER) . Here, we examine the role of RAD26 in the repair of DNA lesions induced upon treatment with the alkylating agent methyl methanesulfonate (MMS) . MMS-induced DNA lesions include base damages such as 3-methyl adenine and 7-methyl guanine, and these lesions are removed in yeast by the alternate competing pathways of base excision repair (BER), which is initiated by the action of MAG1-encoded N-methyl purine DNA glycosylase, and NER . Interestingly, a synergistic increase in MMS sensitivity was observed in the rad26 Delta strain upon inactivation of NER or BER, indicating that RAD26 promotes the survival of MMS-treated cells by a mechanism that acts independently of either of these repair pathways . The galactose-inducible transcription of the GAL2, GAL7, and GAL10 genes is reduced in MMS-treated rad26 Delta cells and also in mag1 Delta rad14 Delta cells, whereas a very severe reduction in transcription occurs in MMS-treated mag1 Delta rad14 Delta rad26 Delta cells . From these observations, we infer that RAD26 plays a role in promoting transcription by RNA polymerase II through damaged bases . The implications of these observations are discussed in this paper.

Mol Cell Biol, 2002 Jun, 22(12), 4167 - 80
Steps in assembly of silent chromatin in yeast: Sir3-independent binding of a Sir2/Sir4 complex to silencers and role for Sir2-dependent deacetylation; Hoppe GJ et al.; Transcriptional silencing at the budding yeast silent mating type (HM) loci and telomeric DNA regions requires Sir2, a conserved NAD-dependent histone deacetylase, Sir3, Sir4, histones H3 and H4, and several DNA-binding proteins . Silencing at the yeast ribosomal DNA (rDNA) repeats requires a complex containing Sir2, Net1, and Cdc14 . Here we show that the native Sir2/Sir4 complex is composed solely of Sir2 and Sir4 and that native Sir3 is not associated with other proteins . We further show that the initial binding of the Sir2/Sir4 complex to DNA sites that nucleate silencing, accompanied by partial Sir2-dependent histone deacetylation, occurs independently of Sir3 and is likely to be the first step in assembly of silent chromatin at the HM loci and telomeres . The enzymatic activity of Sir2 is not required for this initial binding, but is required for the association of silencing proteins with regions distal from nucleation sites . At the rDNA repeats, we show that histone H3 and H4 tails are required for silencing and rDNA-associated H4 is hypoacetylated in a Sir2-dependent manner . However, the binding of Sir2 to rDNA is independent of its histone deacetylase activity . Together, these results support a stepwise model for the assembly of silent chromatin domains in Saccharomyces cerevisiae.

Mol Cell Biol, 2002 Jun, 22(12), 4136 - 46
Mutations in DNA replication genes reduce yeast life span; Hoopes LL et al.; Surprisingly, the contribution of defects in DNA replication to the determination of yeast life span has never been directly investigated . We show that a replicative yeast helicase/nuclease, encoded by DNA2 and a member of the same helicase subfamily as the RecQ helicases, is required for normal life span . All of the phenotypes of old wild-type cells, for example, extended cell cycle time, age-related transcriptional silencing defects, and nucleolar reorganization, occur after fewer generations in dna2 mutants than in the wild type . In addition, the life span of dna2 mutants is extended by expression of an additional copy of SIR2 or by deletion of FOB1, which also increase wild-type life span . The ribosomal DNA locus and the nucleolus seem to be particularly sensitive to defects in dna2 mutants, although in dna2 mutants extrachromosomal ribosomal circles do not accumulate during the aging of a mother cell . Several other replication mutations, such as rad27 Delta, encoding the FEN-1 nuclease involved in several aspects of genomic stability, also show premature aging . We propose that replication fork failure due to spontaneous, endogenous DNA damage and attendant genomic instability may contribute to replicative senescence . This may imply that the genomic instability, segmental premature aging symptoms, and cancer predisposition associated with the human RecQ helicase diseases, such as Werner, Bloom, and Rothmund-Thomson syndromes, are also related to replicative stress.

J Immunol, 2002 Jun 1, 168(11), 5659 - 66
A three-megabase yeast artificial chromosome contig spanning the C57BL mouse Igh locus; Chevillard C et al.; The mouse Ig H chain (Igh) complex locus is composed of >100 gene segments encoding the variable, diversity, joining, and constant portions of the Ab H chain protein . To advance the characterization of this locus and to identify all the V(H) genes, we have isolated the entire region from C57BL/6 and C57BL/10 as a yeast artificial chromosome contig . The mouse Igh locus extends approximately three megabases and contains at least 134 V(H) genes classified in 15 partially interspersed families . Two non-Igh pseudogenes (Odc-rs8 and Rpl32-rs14) were localized in the distal part of the locus . This physical yeast artificial chromosome map will provide important structure and guidance for the sequencing of this large, complex, and highly repetitive locus.

FEBS Lett, 2002 May 22, 519(1-3), 41 - 4
Respiratory oscillations in yeast: clock-driven mitochondrial cycles of energization; Lloyd D et al.; Respiratory oscillations in continuous yeast cultures can be accounted for by cyclic energization of mitochondria, dictated by the demands of a temperature-compensated ultradian clock with a period of 50 min . Inner mitochondrial membranes show both ultrastructural modifications and electrochemical potential changes . Electron transport components (NADH and cytochromes c and c oxidase) show redox state changes as the organisms cycle between their energized and de-energized phases . These regular cycles are transiently perturbed by uncouplers of energy conservation, with amplitudes more affected than period; that the characteristic period is restored after only one prolonged cycle, indicates that mitochondrial energy generation is not part of the clock mechanism itself, but is responding to energetic requirement.

Biochemistry, 2002 May 28, 41(21), 6798 - 804
Physical evidence that yeast frataxin is an iron storage protein; Gakh O et al.; Frataxin is a conserved mitochondrial protein required for iron homeostasis . We showed previously that in the presence of ferrous iron recombinant yeast frataxin (mYfh1p) assembles into a regular multimer of approximately 1.1 MDa storing approximately 3000 iron atoms . Here, we further demonstrate that mYfh1p and iron form a stable hydrophilic complex that can be detected by either protein or iron staining on nondenaturing polyacrylamide gels, and by either interference or absorbance measurements at sedimentation equilibrium . The molecular mass of this complex has been refined to 840 kDa corresponding to 48 protein subunits and 2400 iron atoms . Solution density measurements have determined a partial specific volume of 0.58 cm(3)/g, consistent with the amino acid composition of mYfh1p and the presence of 50 Fe-O equivalents per subunit . By dynamic light scattering, we show that the complex has a radius of approximately 11 nm and assembles within 2 min at 30 degrees C when ferrous iron, not ferric iron or other divalent cations, is added to mYfh1p monomer at pH between 6 and 8 . Iron-rich granules with diameter of 2-4 nm are detected in the complex by scanning transmission electron microscopy and energy-dispersive X-ray spectroscopy . These findings support the hypothesis that frataxin is an iron storage protein, which could explain the mitochondrial iron accumulation and oxidative damage associated with frataxin defects in yeast, mouse, and humans.

RNA, 2002 May, 8(5), 626 - 36
Yeast Pescadillo is required for multiple activities during 60S ribosomal subunit synthesis; Oeffinger M et al.; The Pescadillo protein was identified via a developmental defect and implicated in cell cycle progression . Here we report that human Pescadillo and its yeast homolog (Yph1p or Nop7p) are localized to the nucleolus . Depletion of Nop7p leads to nuclear accumulation of pre-60S particles, indicating a defect in subunit export, and it interacts genetically with a tagged form of the ribosomal protein Rpl25p, consistent with a role in subunit assembly . Two pre-rRNA processing pathways generate alternative forms of the 5.8S rRNA, designated 5.8S(L) and 5.8Ss . In cells depleted for Nop7p, the 27SA3 pre-rRNA accumulated, whereas later processing intermediates and the mature 5.8Ss rRNA were depleted . Less depletion was seen for the 5.8S(L) pathway . TAP-tagged Nop7p coprecipitated precursors to both 5.8S(L) and 5.8Ss but not the mature rRNAs . We conclude that Nop7p is required for efficient exonucleolytic processing of the 27SA3 pre-rRNA and has additional functions in 60S subunit assembly and transport . Nop7p is a component of at least three different pre-60S particles, and we propose that it carries out distinct functions in each of these complexes.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 May, 34(3), 369 - 72
{Identification of a protein interacting with apoptin from human leucocyte cDNA library by using yeast two-hybrid screening}; Sun GJ et al.; To screen the protein interacting with apoptin from human leucocyte cDNA library by using yeast two-hybrid system, four clones interacting with apoptin were identified . One of them was homologous with Nmi (N-Myc interaction protein) . Cell co-immunoprecipitation showed that apoptin could bind to Nmi in mammalian cells . Apoptin mutants T1, T2 and T3 lacked the C-terminal 11 AA,33-46 AA and both,respectively . Apoptin mutants T2 and T3 failed to interact with Nmi, suggesting that its 33-46 AA was pivotal for the interaction . Apoptin mutant T1 still interacted with Nmi, suggesting that its C-terminal 11 AA was not essential for the interaction.

Mol Cells, 2002 Apr 30, 13(2), 327 - 33
Fission yeast Rap1 homolog is a telomere-specific silencing factor and interacts with Taz1p; Park MJ et al.; Taz1p is the fission yeast orthologue of human TRF2, a telomeric repeat-binding protein . Delta(taz1) mutants are defective in telomeric silencing, telomere length control, and meiotic recombination events . A recent report demonstrated that the human Rap1p homolog (hRap1) is recruited to telomere by interaction with TRF2, arguing that the telomere control mechanism of higher eukaryotes is distinct from that of the budding yeast . Taz1p showed a significant similarity to human TRF2, but not with the budding yeast Rap1p (scRap1p) . This suggests that Taz1p and TRF2 share common features in telomere regulation . To assess the roles of Taz1p in telomere-related functions in detail, we attempted to identify a protein(s) that interacts with Taz1p by using two-hybrid screening . Interestingly, the sequence analysis of a positive clone revealed a perfect match with a Rap1 homolog in S . pombe (spRap1), which showed a significant homology with scRap1p and hRap1p . Here we show that the spRap1 deficiency in haploid cells is viable, which results in increased telomere length regulation, disruption of telomere silencing, and aberrant meiosis (like the delta(taz1) mutant) . This suggests that spRap1p might be recruited to the telomere by Taz1p and play crucial roles in telomere function . Interestingly, the delta(rap1) mutants in fission yeast are defective only for telomere silencing . Therefore, the role of spRap1p may be distinct from that of scRap1p, which is involved in the silencing at both the telomere and mating type locus . Our data, therefore, suggest that the regulation mechanisms of telomere in fission yeast resemble that of higher eukaryotic cells rather than the budding yeast.

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