J Biol Chem, 2001 Mar 16, 276(11), 7705 - 8 Epub 2001 Jan 19. A novel family of phosphatidylinositol 4-kinases conserved from yeast to humans; Barylko B et al.; Phosphatidylinositolpolyphosphates (PIPs) are centrally involved in many biological processes, ranging from cell growth and organization of the actin cytoskeleton to endo- and exocytosis . Phosphorylation of phosphatidylinositol at the D-4 position, an essential step in the biosynthesis of PIPs, appears to be catalyzed by two biochemically distinct enzymes . However, only one of these two enzymes has been molecularly characterized . We now describe a novel class of phosphatidylinositol 4-kinases that probably corresponds to the missing element in phosphatidylinositol metabolism . These kinases are highly conserved evolutionarily, but unrelated to previously characterized phosphatidylinositol kinases, and thus represent the founding members of a new family . The novel phosphatidylinositol 4-kinases, which are widely expressed in cells, only phosphorylate phosphatidylinositol, are potently inhibited by adenosine, but are insensitive to wortmannin or phenylarsine oxide . Although they lack an obvious transmembrane domain, they are strongly attached to membranes by palmitoylation . Our data suggest that independent pathways for phosphatidylinositol 4-phosphate synthesis emerged during evolution, possibly to allow tight temporal and spatial control over the production of this key signaling molecule. J Bacteriol, 2001 Apr, 183(7), 2306 - 15 Functional domains of yeast plasmid-encoded Rep proteins; Sengupta A et al.; Both of the Saccharomyces cerevisiae 2 microm circle-encoded Rep1 and Rep2 proteins are required for efficient distribution of the plasmid to daughter cells during cellular division . In this study two-hybrid and in vitro protein interaction assays demonstrate that the first 129 amino acids of Rep1 are sufficient for self-association and for interaction with Rep2 . Deletion of the first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still allowed some self-association, suggesting that different but overlapping domains specify these interactions . Amino- or carboxy-terminally truncated Rep1 fusion proteins were unable to complement defective segregation of a 2 microm-based stability vector with rep1 deleted, supporting the idea of the requirement of Rep protein interaction for plasmid segregation but indicating a separate required function for the carboxy-terminal portion of Rep1 . The results of in vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both of which are capable of mediating Rep2 self-association . The amino-terminal domain interacts with Rep1, while the carboxy-terminal domain was shown by Southwestern analysis to have DNA-binding activity . The overlapping Rep1 and Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1, Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2 microm circle plasmid stability locus, forming a structure that mediates plasmid segregation . In this model, competition between Rep1 and Rep2 for association with Rep1 determines the formation or disassembly of the segregation complex. Biochem Biophys Res Commun, 2001 Mar, 281(5), 1170 - 5 Analysis of molecular interactions of the p53-family p51(p63) gene products in a yeast two-hybrid system: homotypic and heterotypic interactions and association with p53-regulatory factors; Kojima T et al.; p51 in the p53 tumor suppressor family, also referred to as p63, encodes multiple isoforms including p51A (TAp63gamma) and p51B (TAp63alpha) . The p53 protein forms a tetramer, and its stability and activity are regulated by molecular association with viral and cellular proteins and by biochemical modifications . Using a yeast two-hybrid system, the p51A and p51B isoforms were examined for homotypic and heterotypic interactions in the p53 family proteins and for their affinity to the p53-regulatory factors . Results indicate a homotypic interaction dependent on the presumed oligomerization domain of the p51 proteins . The possibility of a weak heterotypic interaction between p51 and p73 proteins was suggested, while association between p51 and p53 appeared improbable . Furthermore, unlike p53, the p51 proteins failed to display an affinity to SV40 large T antigen or MDM2-family proteins . Having several features in common with p53, the p51 proteins may function in biological processes apart from p53 . J Mol Biol, 2001 Mar 16, 307(1), 247 - 57 Crystal structure of unligated guanylate kinase from yeast reveals GMP-induced conformational changes; Blaszczyk J et al.; The crystal structure of guanylate kinase (GK) from yeast (Saccharomyces cerevisiae) with a non-acetylated N terminus has been determined in its unligated form (apo-GK) as well as in complex with GMP (GK.GMP) . The structure of apo-GK was solved with multiwavelength anomalous diffraction data and refined to an R-factor of 0.164 (R(free)=0.199) at 2.3 A resolution . The structure of GK.GMP was determined using the crystal structure of GK with an acetylated N terminus as the search model and refined to an R-factor of 0.156 (R(free)=0.245) at 1.9 A . GK belongs to the family of nucleoside monophosphate (NMP) kinases and catalyzes the reversible phosphoryl transfer from ATP to GMP . Like other NMP kinases, GK consists of three dynamic domains: the CORE, LID, and NMP-binding domains . Dramatic movements of the GMP-binding domain and smaller but significant movements of the LID domain have been revealed by comparing the structures of apo-GK and GK.GMP . apo-GK has a much more open conformation than the GK.GMP complex . Systematic analysis of the domain movements using the program DynDom shows that the large movements of the GMP-binding domain involve a rotation around an effective hinge axis approximately parallel with helix 3, which connects the GMP-binding and CORE domains . The C-terminal portion of helix 3, which connects to the CORE domain, has strikingly higher temperature factors in GK.GMP than in apo-GK, indicating that these residues become more mobile upon GMP binding . The results suggest that helix 3 plays an important role in domain movement . Unlike the GMP-binding domain, which moves toward the active center of the enzyme upon GMP binding, the LID domain moves away from the active center and makes the presumed ATP-binding site more open . Therefore, the LID domain movement may facilitate the binding of MgATP . The structure of the recombinant GK.GMP complex superimposes very well with that of the native GK.GMP complex, indicating that N-terminal acetylation does not have significant impact on the three-dimensional structure of GK . Gene Expr, 2000, 9(3), 145 - 56 An antiprion effect of the anticytoskeletal drug latrunculin A in yeast; Bailleul-Winslett PA et al.; Prions are infectious aggregation-prone isoforms of the normal proteins, supposedly able to seed aggregation of the normal cellular counterparts . In vitro, prion proteins form amyloid fibers, resembling cytoskeletal structures . Yeast prion {PSI}, which is a cytoplasmically inherited aggregated isoform of the translation termination factor Sup35p (eRF3), serves as a useful model for studying mechanisms of prion diseases and other amyloidoses . The previously described interaction between Sup35p and cytoskeletal assembly protein Sla1p points to the possible relationships between prions and cytoskeletal networks . Although the Sup35PSI+ aggregates do not colocalize with actin patches, we have shown that yeast cells are efficiently cured of the {PSI} prion by prolonged incubation with latrunculin A, a drug disrupting the actin cytoskeleton . On the other hand, treatments with sodium azide or cycloheximide, agents blocking yeast protein synthesis and cell proliferation but not disrupting the cytoskeleton, do not cause a significant loss of {PSI} . Moreover, simultaneous treatment with sodium azide or cycloheximide blocks {PSI} curing by latrunculin A, indicating that prion loss in the presence of latrunculin A requires a continuation of protein synthesis during cytoskeleton disruption . The sodium azide treatment also decreases the toxic effect of latrunculin A . Latrunculin A influences neither the levels of total cellular Sup35p nor the levels of chaperone proteins, such as Hsp104 and Hsp70, which were previously shown to affect {PSI} . This makes an indirect effect of latrunculin A on {PSI} via induction of Hsps unlikely . Fluorescence microscopy detects changes in the structure and/or localization of the Sup35PSI+ aggregates in latrunculin A-treated cells . We conclude that the stable maintenance of the {PSI} prion aggregates in the protein-synthesizing yeast cells partly depends on an intact actin cytoskeleton, suggesting that anticytoskeletal treatments could be used to counteract some aggregation-related disorders. Biochemistry (Mosc), 2001 Jan, 66(1), 47 - 54 Effect of zinc ions on conformational stability of yeast alcohol dehydrogenase; Yang Y et al.; Yeast alcohol dehydrogenase preparations were prepared with the conformational zinc ion removed (Apo-I YADH) and with both the conformational and catalytic zinc ions removed (Apo-II YADH) . The unfolding of Apo-I YADH and Apo-II YADH during denaturation in urea solutions was then followed by fluorescence emission, circular dichroism, and second-derivative optical spectroscopies . Compared with the native enzyme, Apo-I YADH incurred some slight unfolding, and its stability against urea was markedly decreased, while Apo-II YADH incurred marked unfolding but contained residual ordered structure even at high urea concentrations . The results show that native YADH is more conformationally stable against urea denaturation than Apo-I YADH, indicating that the conformational Zn(2+) plays an important role in stabilizing the conformation of the YADH molecule . However, unfolding of the region around the conformational zinc ion is shown not to be the rate limited step in the unfolding of the molecule by the fact that the unfolding and inactivation rate constants of native and Apo-I YADH are the same . It is suggested that the catalytic zinc ion is more important in maintaining the structure of YADH . YADH lost its cooperative unfolding ability after the zinc ions were removed . The shape of the transition curves of Apo-I YADH suggests the existence of an unfolding intermediate . For both native and Apo-I YADH, inactivation occurs at much lower urea concentrations than that needed to produce significant conformational changes of the enzyme molecule . At urea concentration above 4 M, the inactivation rate constants are much higher than those of the fast phase of the reaction of unfolding . These results support the suggestion of flexibility at the active site of the enzyme (C . L . Tsou (1986) Trends Biochem . Sci., 11, 427-429; (1993) Science, 262, 308-381). Mol Cell Biol, 2001 Mar, 21(6), 2098 - 106 Interactions of Isw2 chromatin remodeling complex with nucleosomal arrays: analyses using recombinant yeast histones and immobilized templates; Gelbart ME et al.; To facilitate the biochemical characterization of chromatin-associated proteins in the budding yeast Saccharomyces cerevisiae, we have developed a system to assemble nucleosomal arrays on immobilized templates using recombinant yeast core histones . This system enabled us to analyze the interaction of Isw2 ATP-dependent chromatin remodeling complex with nucleosomal arrays . We found that Isw2 complex interacts efficiently with both naked DNA and nucleosomal arrays in an ATP-independent manner, suggesting that ATP is required at steps subsequent to this physical interaction . We identified the second subunit of Isw2 complex, encoded by open reading frame YGL 133w (herein named ITC1), and found that both subunits of the complex, Isw2p and Itc1p, are essential for efficient interaction with DNA and nucleosomal arrays . Both subunits are also required for nucleosome-stimulated ATPase activity and chromatin remodeling activity of the complex . Finally, we found that ITC1 is essential for function of Isw2 complex in vivo, since isw2 and itc1 deletion mutants exhibit virtually identical phenotypes . These results demonstrate the utility of our in vitro system in studying interactions between chromatin-associated proteins and nucleosomal arrays. Mol Cell Biol, 2001 Mar, 21(6), 2057 - 69 Identification of the Sin3-binding site in Ume6 defines a two-step process for conversion of Ume6 from a transcriptional repressor to an activator in yeast; Washburn BK et al.; The DNA-binding protein Ume6 is required for both repression and activation of meiosis-specific genes, through interaction with the Sin3 corepressor and Rpd3 histone deacetylase and the meiotic activator Ime1 . Here we show that fusion of a heterologous activation domain to Ume6 is unable to convert it into a constitutive activator of early meiotic gene transcription, indicating that an additional function is needed to overcome repression at these promoters . Mutations in UME6 allowing the fusion to activate lie in a predicted amphipathic alpha helix and specifically disrupt interaction with Sin3 but not with Teal, an activator of Ty transcription also found to interact with Ume6 in a two-hybrid screen . The mutations cause a loss of repression by Ume6 and precisely identify the Ume6 Sin3-binding domain, which we show interacts with the paired amphipathic helix 2 region of Sin3 . Analysis of these mutants indicates that conversion of Ume6 to an activator involves two genetically distinct steps that act to relieve Sin3-mediated repression and provide an activation domain to Ume6 . The mutants further demonstrate that premature expression and lack of subsequent rerepression of Ume6-Sin3-regulated genes are not deleterious to meiotic progression and suggest that the essential role of Sin3 in meiosis is independent of Ume6 . The model for Ume6 function arising from these studies indicates that Ume6 is similar in many respects to metazoan regulators that utilize Sin3, such as the Myc-Mad-Max system and nuclear hormone receptors, and provides new insights into the control of transcriptional repression and activation by the Ume6-URS1 regulatory complex in yeast. Mol Cell Biol, 2001 Mar, 21(6), 2018 - 25 Acidic residues critical for the activity and biological function of yeast DNA polymerase eta; Kondratick CM et al.; Rad30 is a member of the newly discovered UmuC/DinB/Rad30 family of DNA polymerases . The N-terminal regions of these proteins are highly homologous, and they contain five conserved motifs, I to V, while their C-terminal regions are quite divergent . We examined the contributions of the C-terminal and N-terminal regions of Rad30 to its activity and biological function . Although deletion of the last 54 amino acids has no effect on DNA polymerase or thymine-thymine (T-T) dimer bypass activity, this C-terminal deletion-containing protein is unable to perform its biological function in vivo . The presence of a bipartite nuclear targeting sequence within this region suggests that at least one function of this portion of Rad30 is nuclear targeting . To identify the active-site residues of Rad30 important for catalysis, we generated mutations of nine acidic residues that are invariant or highly conserved among Rad30 proteins from different eukaryotic species . Mutations of the Asp30 and Glu39 residues present in motif I and of the Asp155 residue present in motif III to alanine completely inactivated the DNA polymerase and T-T dimer bypass activities, and these mutations did not complement the UV sensitivity of the rad30Delta mutation . Mutation of Glu156 in motif III to alanine confers a large reduction in the efficiency of nucleotide incorporation, whereas the remaining five Rad30 mutant proteins retain wild-type levels of DNA polymerase and T-T dimer bypass activities . From these observations, we suggest a role for the Asp30, Glu39, and Asp155 residues in the binding of two metal ions required for the reaction of the incoming deoxynucleoside 5'-triphosphate with the 3'-hydroxyl in the primer terminus, while Glu156 may participate in nucleotide binding. Mol Cell Biol, 2001 Mar, 21(5), 1854 - 65 HIRA, the human homologue of yeast Hir1p and Hir2p, is a novel cyclin-cdk2 substrate whose expression blocks S-phase progression; Hall C et al.; Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP) . To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs . One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p . Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase . First, HIRA bound to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner . Second, HIRA was phosphorylated in vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin A-cdk2 in vitro . Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21(cip1) . Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active . Fifth, HIRA was localized preferentially to the nucleus, where active cyclin A- and E-cdk2 are located . Finally, ectopic expression of HIRA in cells caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control of the cell cycle. Mol Cell Biol, 2001 Mar, 21(5), 1841 - 53 Histone folds mediate selective heterodimerization of yeast TAF(II)25 with TFIID components yTAF(II)47 and yTAF(II)65 and with SAGA component ySPT7; Gangloff YG et al.; We show that the yeast TFIID (yTFIID) component yTAF(II)47 contains a histone fold domain (HFD) with homology to that previously described for hTAF(II)135 . Complementation in vivo indicates that the yTAF(II)47 HFD is necessary and sufficient for vegetative growth . Mutation of highly conserved residues in the alpha1 helix of the yTAF(II)47 HFD results in a temperature-sensitive phenotype which can be suppressed by overexpression of yTAF(II)25, as well as by yTAF(II)40, yTAF(II)19, and yTAF(II)60 . In yeast two-hybrid and bacterial coexpression assays, the yTAF(II)47 HFD selectively heterodimerizes with yTAF(II)25, which we show contains an HFD with homology to the hTAF(II)28 family We additionally demonstrate that yTAF(II)65 contains a functional HFD which also selectively heterodimerizes with yTAF(II)25 . These results reveal the existence of two novel histone-like pairs in yTFIID . The physical and genetic interactions described here show that the histone-like yTAF(II)s are organized in at least two substructures within TFIID rather than in a single octamer-like structure as previously suggested . Furthermore, our results indicate that ySPT7 has an HFD homologous to that of yTAF(II)47 which selectively heterodimerizes with yTAF(II)25, defining a novel histone-like pair in the SAGA complex. Mol Cell Biol, 2001 Mar, 21(5), 1710 - 8 Checkpoint adaptation precedes spontaneous and damage-induced genomic instability in yeast; Galgoczy DJ et al.; Despite the fact that eukaryotic cells enlist checkpoints to block cell cycle progression when their DNA is damaged, cells still undergo frequent genetic rearrangements, both spontaneously and in response to genotoxic agents . We and others have previously characterized a phenomenon (adaptation) in which yeast cells that are arrested at a DNA damage checkpoint eventually override this arrest and reenter the cell cycle, despite the fact that they have not repaired the DNA damage that elicited the arrest . Here, we use mutants that are defective in checkpoint adaptation to show that adaptation is important for achieving the highest possible viability after exposure to DNA-damaging agents, but it also acts as an entree into some forms of genomic instability . Specifically, the spontaneous and X-ray-induced frequencies of chromosome loss, translocations, and a repair process called break-induced replication occur at significantly reduced rates in adaptation-defective mutants . This indicates that these events occur after a cell has first arrested at the checkpoint and then adapted to that arrest . Because malignant progression frequently involves loss of genes that function in DNA repair, adaptation may promote tumorigenesis by allowing genomic instability to occur in the absence of repair. Mol Cell Biol, 2001 Mar, 21(5), 1656 - 61 3'-phosphodiesterase and 3'-->5' exonuclease activities of yeast Apn2 protein and requirement of these activities for repair of oxidative DNA damage; Unk I et al.; In Saccharomyces cerevisiae, the AP endonucleases encoded by the APN1 and APN2 genes provide alternate pathways for the removal of abasic sites . Oxidative DNA-damaging agents, such as H(2)O(2), produce DNA strand breaks which contain 3'-phosphate or 3'-phosphoglycolate termini . Such 3' termini are inhibitory to synthesis by DNA polymerases . Here, we show that purified yeast Apn2 protein contains 3'-phosphodiesterase and 3'-->5' exonuclease activities, and mutation of the active-site residue Glu59 to Ala in Apn2 inactivates both these activities . Consistent with these biochemical observations, genetic studies indicate the involvement of APN2 in the repair of H(2)O(2)-induced DNA damage in a pathway alternate to APN1, and the Ala59 mutation inactivates this function of Apn2 . From these results, we conclude that the ability of Apn2 to remove 3'-end groups from DNA is paramount for the repair of strand breaks arising from the reaction of DNA with reactive oxygen species. Mol Cell Biol, 2001 Mar, 21(5), 1647 - 55 Pir1p mediates translocation of the yeast Apn1p endonuclease into the mitochondria to maintain genomic stability; Vongsamphanh R et al.; The mitochondrial genome is continuously subject to attack by reactive oxygen species generated through aerobic metabolism . This leads to the formation of a variety of highly genotoxic DNA lesions, including abasic sites . Yeast Apn1p is localized to the nucleus, where it functions to cleave abasic sites, and apn1 Delta mutants are hypersensitive to agents such as methyl methanesulfonate (MMS) that induce abasic sites . Here we demonstrate for the first time that yeast Apn1p is also localized to the mitochondria . We found that Pir1p, initially isolated as a cell wall constituent of unknown function, interacts with the C-terminal end of Apn1p, which bears a bipartite nuclear localization signal . Further analysis revealed that Pir1p is required to cause Apn1p mitochondrial localization, presumably by competing with the nuclear transport machinery . pir1 Delta mutants displayed a striking (approximately 3-fold) increase of Apn1p in the nucleus, which coincided with drastically reduced levels in the mitochondria . To explore the functional consequences of the Apn1p-Pir1p interaction, we measured the rate of mitochondrial mutations in the wild type and pir1 Delta and apn1 Delta mutants . pir1 Delta and apn1 Delta mutants exposed to MMS exhibited 3.6- and 5.8-fold increases, respectively, in the rate of mitochondrial mutations, underscoring the importance of Apn1p in repair of the mitochondrial genome . We conclude that Pir1p interacts with Apn1p, at the level of either the cytoplasm or nucleus, and facilitates Apn1p transport into the mitochondria to repair damaged DNA. Genetics, 2001 Mar, 157(3), 1159 - 68 The git5 Gbeta and git11 Ggamma form an atypical Gbetagamma dimer acting in the fission yeast glucose/cAMP pathway; Landry S et al.; Fission yeast adenylate cyclase, like mammalian adenylate cyclases, is regulated by a heterotrimeric G protein . The gpa2 Galpha and git5 Gbeta are both required for glucose-triggered cAMP signaling . The git5 Gbeta is a unique member of the Gbeta family in that it lacks an amino-terminal coiled-coil domain shown to be essential for mammalian Gbeta folding and interaction with Ggamma subunits . Using a git5 bait in a two-hybrid screen, we identified the git11 Ggamma gene . Co-immunoprecipitation studies confirm the composition of this Gbetagamma dimer . Cells deleted for git11 are defective in glucose repression of both fbp1 transcription and sexual development, resembling cells lacking either the gpa2 Galpha or the git5 Gbeta . Overexpression of the gpa2 Galpha partially suppresses loss of either the git5 Gbeta or the git11 Ggamma, while mutational activation of the Galpha fully suppresses loss of either Gbeta or Ggamma . Deletion of gpa2 (Galpha), git5 (Gbeta), or git11 (Ggamma) confer quantitatively distinct effects on fbp1 repression, indicating that the gpa2 Galpha subunit remains partially active in the absence of the Gbetagamma dimer and that the git5 Gbeta subunit remains partially active in the absence of the git11 Ggamma subunit . The addition of the CAAX box from the git11 Ggamma to the carboxy-terminus of the git5 Gbeta partially suppresses the loss of the Ggamma . Thus the Ggamma in this system is presumably required for localization of the Gbetagamma dimer but not for folding of the Gbeta subunit . In mammalian cells, the essential roles of the Gbeta amino-terminal coiled-coil domains and Ggamma partners in Gbeta folding may therefore reflect a mechanism used by cells that express multiple forms of both Gbeta and Ggamma subunits to regulate the composition and activity of its G proteins. Genetics, 2001 Mar, 157(3), 1141 - 58 Yeast frameshift suppressor mutations in the genes coding for transcription factor Mbf1p and ribosomal protein S3: evidence for autoregulation of S3 synthesis; Hendrick JL et al.; The SUF13 and SUF14 genes were identified among extragenic suppressors of +1 frameshift mutations . SUF13 is synonymous with MBF1, a single-copy nonessential gene coding for a POLII transcription factor . The suf13-1 mutation is a two-nucleotide deletion in the SUF13/MBF1 coding region . A suf13::TRP1 null mutant suppresses +1 frameshift mutations, indicating that suppression is caused by loss of SUF13 function . The suf13-1 suppressor alters sensitivity to aminoglycoside antibiotics and reduces the accumulation of his4-713 mRNA, suggesting that suppression is mediated at the translational level . The SUF14 gene is synonymous with RPS3, a single-copy essential gene that codes for the ribosomal protein S3 . The suf14-1 mutation is a missense substitution in the coding region . Increased expression of S3 limits the accumulation of SUF14 mRNA, suggesting that expression is autoregulated . A frameshift mutation in SUF14 that prevents full-length translation eliminated regulation, indicating that S3 is required for regulation . Using CUP1-SUF14 and SUF14-lacZ fusions, run-on transcription assays, and estimates of mRNA half-life, our results show that transcription plays a minor role if any in regulation and that the 5'-UTR is necessary but not sufficient for regulation . A change in mRNA decay rate may be the primary mechanism for regulation. Genetics, 2001 Mar, 157(3), 1117 - 40 Overlapping functions of the yeast oxysterol-binding protein homologues; Beh CT et al.; The Saccharomyces cerevisiae genome encodes seven homologues of the mammalian oxysterol-binding protein (OSBP), a protein implicated in lipid trafficking and sterol homeostasis . To determine the functions of the yeast OSBP gene family (OSH1-OSH7), we used a combination of genetics, genomics, and sterol lipid analysis to characterize OSH deletion mutants . All 127 combinations and permutations of OSH deletion alleles were constructed . Individual OSH genes were not essential for yeast viability, but the elimination of the entire gene family was lethal . Thus, the family members shared an essential function . In addition, the in vivo depletion of all Osh proteins disrupted sterol homeostasis . Like mutants that affect ergosterol production, the viable combinations of OSH deletion alleles exhibited specific sterol-related defects . Although none of the single OSH deletion mutants was defective for growth, gene expression profiles revealed that each mutant had a characteristic molecular phenotype . Therefore, each gene performed distinct nonessential functions and contributed to a common essential function . Our findings indicated that OSH genes performed a multitude of nonessential roles defined by specific subsets of the genes and that most shared at least one essential role potentially linked to changes in sterol lipid levels. Genetics, 2001 Mar, 157(3), 1089 - 105 Mutations in the YRB1 gene encoding yeast ran-binding-protein-1 that impair nucleocytoplasmic transport and suppress yeast mating defects; Kunzler M et al.; We identified two temperature-sensitive (ts) mutations in the essential gene, YRB1, which encodes the yeast homolog of Ran-binding-protein-1 (RanBP1), a known coregulator of the Ran GTPase cycle . Both mutations result in single amino acid substitutions of evolutionarily conserved residues (A91D and R127K, respectively) in the Ran-binding domain of Yrb1 . The altered proteins have reduced affinity for Ran (Gsp1) in vivo . After shift to restrictive temperature, both mutants display impaired nuclear protein import and one also reduces poly(A)+ RNA export, suggesting a primary defect in nucleocytoplasmic trafficking . Consistent with this conclusion, both yrb1ts mutations display deleterious genetic interactions with mutations in many other genes involved in nucleocytoplasmic transport, including SRP1 (alpha-importin) and several beta-importin family members . These yrb1ts alleles were isolated by their ability to suppress two different types of mating-defective mutants (respectively, fus1Delta and ste5ts), indicating that reduction in nucleocytoplasmic transport enhances mating proficiency . Indeed, in both yrb1ts mutants, Ste5 (scaffold protein for the pheromone response MAPK cascade) is mislocalized to the cytosol, even in the absence of pheromone . Also, both yrb1ts mutations suppress the mating defect of a null mutation in MSN5, which encodes the receptor for pheromone-stimulated nuclear export of Ste5 . Our results suggest that reimport of Ste5 into the nucleus is important in downregulating mating response. Genes Dev, 2001 Mar 1, 15(5), 619 - 26 In vivo chromatin remodeling by yeast ISWI homologs Isw1p and Isw2p; Kent NA et al.; Isw1p and Isw2p are budding yeast homologs of the Drosophila ISWI chromatin-remodeling ATPase . Using indirect-end-label and chromatin immunoprecipitation analysis, we show both independent and cooperative Isw1p- and Isw2p-mediated positioning of short nucleosome arrays in gene-regulatory elements at a variety of transcription units in vivo . We present evidence that both yeast ISWI complexes regulate developmental responses to starvation and that for Isw2p, recruitment by different DNA-binding proteins controls meiosis and haploid invasive growth. Biochem J, 2001 Mar 15, 354(Pt 3), 655 - 61 Identification of Cdc6 protein domains involved in interaction with Mcm2 protein and Cdc4 protein in budding yeast cells; Jang SW et al.; The Cdc6 protein (Cdc6p) has essential roles in regulating initiation of DNA replication . Cdc6p is recruited to origins of replication by the origin recognition complex (ORC) late in mitosis; Cdc6p in turn recruits minichromosome maintenance (Mcm) proteins to form the pre-replicative complex . Cdc6p is thought to interact with one or more Mcm proteins but this point has not yet been demonstrated . In the present study we observed that Cdc6p interacted significantly only with Mcm2p out of six Mcm proteins in yeast two-hybrid cells . Our results indicate that the interaction of Cdc6p with Mcm2p is specific, although we cannot exclude the possibility that the interaction might not be direct . In attempts to identify domains of Cdc6p important for interaction with Mcm2p, we tested interactions of various deleted versions of Cdc6p with Mcm2p and also with Cdc4p, which was previously known to interact with Cdc6p . The portion of Cdc6p from amino acid residues 51 to 394 was able to interact with Mcm2p . During the course of the studies we also discovered a previously undetected Cdc4p interaction domain between residues 51 and 394 . Interestingly, when all six putative Cdc28 phosphorylation sites in Cdc6p were changed to alanine, a 6-7-fold increase in binding to Mcm2p was observed . This result suggests that unphosphorylated Cdc6p has higher affinity than phosphorylated Cdc6p for Mcm2p; this might partly explain the previous observation that Cdc6p failed to load Mcm proteins on replication origins during S phase when the cyclin-dependent protein kinase was active, thus helping to prevent the reinitiation of activated replicons. Prikl Biokhim Mikrobiol, 2001 Jan-Feb, 37(1), 29 - 35 {Nucleoprotein complexes of yeast and their biological effect on T-lymphocytes}; Sinitskaia NS et al.; Complexes of nucleic acids and acid nuclear proteins that are active toward human T-lymphocytes were isolated from cells of bakers' yeast Saccharomyces cerevisiae . The conditions of isolation of nucleoprotein complexes by acid extraction followed by microfiltration for concentration of macromolecular components were optimized . Gel filtration and electrophoresis were used to study the composition and molecular weights of components of the preparations obtained . It was shown that nucleoprotein complex had a molecular weight of 1430 kDa . However, only one zone was determined by electrophoresis of the protein component with a molecular weight of 30 kDa. Nat Biotechnol, 2001 Mar, 19(3), 242 - 7 Large-scale analysis of the yeast proteome by multidimensional protein identification technology; Washburn MP et al.; We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT) . MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date . A total of 1,484 proteins were detected and identified . Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases . Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins . MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins. Eur J Biochem, 2001 Mar, 268(5), 1155 - 62 Effects of mutations in mitochondrial cytochrome b in yeast and man . Deficiency, compensation and disease; Fisher N et al.; The mitochondrial cytochrome bc(1) complex is a key protonmotive component of eukaryotic respiratory chains . The mitochondrially encoded cytochrome b forms, with cytochrome c(1) and the iron--sulfur protein, the catalytic core of this multimeric enzyme . Mutations of cytochrome b have been reported in association with human diseases . In the highly homologous yeast cytochrome b, several mutations that impair the respiratory function, and reversions that correct the defect, have been described . In this paper, we re-examine the mutations in the light of the atomic structure of the complex, and discuss the possible effect, at enzyme level, of the human cytochrome b mutations and the correcting effect of the reversions. Curr Biol, 2001 Jan 23, 11(2), 88 - 97 B-type cyclins CLB5 and CLB6 control the initiation of recombination and synaptonemal complex formation in yeast meiosis; Smith KN et al.; BACKGROUND: The life cycle of most eukaryotic organisms includes a meiotic phase, in which diploid parental cells produce haploid gametes . During meiosis a single round of DNA replication is followed by two rounds of chromosome segregation . In the first, or reductional, division (meiosis I), which is unique to meiotic cells, homologous chromosomes segregate from one another, whereas in the second, or equational, division (Meiosis II) sister centromeres disjoin . Meiotic DNA replication precedes the initiation of recombination by programmed Spo11-dependent DNA double-strand breaks . Recent reports that meiosis-specific cohesion is established during meiotic S phase and that the length of S phase is modified by recombination factors (Spo11 and Rec8) raise the possibility that replication plays a fundamental role in the recombination process . RESULTS: To address how replication influences the initiation of recombination, we have used mutations in the B-type cyclin genes CLB5 and CLB6, which specifically prevent premeiotic replication in the yeast Saccharomyces cerevisiae . We find that clb5 and clb5 clb6 but not clb6 mutants are defective in DSB induction and prior associated changes in chromatin accessibility, heteroallelic recombination, and SC formation . The severity of these phenotypes in each mutant reflects the extent of replication impairment . CONCLUSIONS: This assemblage of phenotypes reveals roles for CLB5 and CLB6 not only in DNA replication but also in other key events of meiotic prophase . Links between the function of CLB5 and CLB6 in activating meiotic DNA replication and their effects on subsequent events are discussed. Plant Cell Physiol, 2001 Feb, 42(2), 245 - 9 Improved salt tolerance of transgenic tobacco expressing apoplastic yeast-derived invertase; Fukushima E et al.; We investigated the salt tolerance of transgenic tobacco, in which yeast invertase is expressed in the apoplastic (Apo-Inv) spaces . Whereas photosynthetic activities in wild-type tobacco in light were inhibited under salt stress, transgenic Apo-Inv tobacco maintained constant photosynthetic activities . The physical appearance of plants under salt stress also indicates that yeast invertase expression in the apoplastic space is beneficial for inducing salt tolerance . Apo-Inv tobacco had a much higher osmotic pressure increase in the cell sap than did wild-type tobacco under this type of stress . The physiological importance of sucrose metabolism under salt stress is discussed. EMBO J, 2001 Mar 1, 20(5), 1074 - 85 GSK-3 kinase Mck1 and calcineurin coordinately mediate Hsl1 down-regulation by Ca2+ in budding yeast; Mizunuma M et al.; The Ca2+-activated pathways of Saccharomyces cerevisiae induce a delay in the onset of mitosis through the activation of Swe1, a negative regulatory kinase that inhibits the Cdc28-Clb complex . Calcineurin and Mpk1 activate Swe1 at the transcriptional and post-translational level, respectively, and both pathways are essential for the cell cycle delay . Our genetic screening identified the MCK1 gene, which encodes a glycogen synthetase kinase-3 family protein kinase, as a component of the Ca2+ signaling pathway . Genetic analyses indicated that Mck1 functions downstream of the Mpk1 pathway and down-regulates Hsl1, an inhibitory kinase of Swe1 . In medium with a high concentration of Ca2+, Hsl1 was delocalized from the bud neck and destabilized in a manner dependent on both calcineurin and Mck1 . Calcineurin was required for the dephosphorylation of autophosphorylated Hsl1 . The E3 ubiquitin ligase complex SCF(Cdc4), but not the anaphase-promoting complex (APC), was essential for Hsl1 destabilization . The Ca2+-activated pathway may play a role in the rapid inactivation of Hsl1 at the cell cycle stage(s) when APC activity is low. Fresenius J Anal Chem, 2000 Nov, 368(5), 471 - 4 Stability of inorganic mercury and methylmercury on yeast-silica gel microcolumns: field sampling capabilities; Perez-Corona MT et al.; The stability of methylmercury and inorganic mercury retained on yeast-silica gel microcolumns was established and compared with the stability of these species in solution . Yeast-silica gel columns with the retained analytes were stored for two months at three different temperatures: -20 degrees C, 4 degrees C and room temperature . At regular time intervals, both mercury species were eluted and quantified by cold vapor atomic absorption spectrometry (CVAAS) . Methylmercury was found stable in the columns over the two-month period at the three different temperatures tested while the concentration of inorganic mercury decreased after one week's storage even at -20 degrees C . These results are of great interest since the use of these microcolumns allows the preconcentration and storage of mercury species until analysis, thus saving laboratory space and avoiding the problems associated with maintaining species integrity in aqueous solution. Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2375 - 80 Epub 2001 Feb 20. An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated beta-sheet structure for amyloid; Balbirnie M et al.; X-ray diffraction and other biophysical tools reveal features of the atomic structure of an amyloid-like crystal . Sup35, a prion-like protein in yeast, forms fibrillar amyloid assemblies intrinsic to its prion function . We have identified a polar peptide from the N-terminal prion-determining domain of Sup35 that exhibits the amyloid properties of full-length Sup35, including cooperative kinetics of aggregation, fibril formation, binding of the dye Congo red, and the characteristic cross-beta x-ray diffraction pattern . Microcrystals of this peptide also share the principal properties of the fibrillar amyloid, including a highly stable, beta-sheet-rich structure and the binding of Congo red . The x-ray powder pattern of the microcrystals, extending to 0.9-A resolution, yields the unit cell dimensions of the well-ordered structure . These dimensions restrict possible atomic models of this amyloid-like structure and demonstrate that it forms packed, parallel-stranded beta-sheets . The unusually high density of the crystals shows that the packed beta-sheets are dehydrated, despite the polar character of the side chains . These results suggest that amyloid is a highly intermolecularly bonded, dehydrated array of densely packed beta-sheets . This dry beta-sheet could form as Sup35 partially unfolds to expose the peptide, permitting it to hydrogen-bond to the same peptide of other Sup35 molecules . The implication is that amyloid-forming units may be short segments of proteins, exposed for interactions by partial unfolding. EMBO J, 2001 Jan 15, 20(1-2), 262 - 71 Sec63p and Kar2p are required for the translocation of SRP-dependent precursors into the yeast endoplasmic reticulum in vivo; Young BP et al.; The translocation of secretory polypeptides into the endoplasmic reticulum (ER) occurs at the translocon, a pore-forming structure that orchestrates the transport and maturation of polypeptides at the ER membrane . In yeast, targeting of secretory precursors to the translocon can occur by two distinct pathways that are distinguished by their dependence upon the signal recognition particle (SRP) . The SRP-dependent pathway requires SRP and its membrane-bound receptor, whereas the SRP-independent pathway requires a separate receptor complex consisting of Sec62p, Sec63p, Sec71p, Sec72p plus lumenal Kar2p/BiP . Here we demonstrate that Sec63p and Kar2p are also required for the SRP-dependent targeting pathway in vivo . Furthermore, we demonstrate multiple roles for Sec63p, at least one of which is exclusive to the SRP-independent pathway. EMBO J, 2001 Jan 15, 20(1-2), 222 - 30 Reconstitution of Epstein-Barr virus-based plasmid partitioning in budding yeast; Kapoor P et al.; The EBNA1 protein of Epstein-Barr virus (EBV) mediates the partitioning of EBV episomes and EBV-based plasmids during cell division by a mechanism that appears to involve binding to the cellular EBP2 protein on human chromosomes . We have investigated the ability of EBNA1 and the EBV segregation element (FR) to mediate plasmid partitioning in Saccharomyces cerevisiae . EBNA1 expression alone did not enable the stable segregation of FR-containing plasmids in yeast, but segregation was rescued by human EBP2 . The reconstituted segregation system required EBNA1, human EBP2 and the FR element, and functionally replaced a CEN element . An EBP2 binding mutant of EBNA1 and an EBNA1 binding mutant of EBP2 each failed to support FR-plasmid partitioning, indicating that an EBNA1-EBP2 interaction is required . The results provide direct evidence of the role of hEBP2 in EBNA1-mediated segregation and demonstrate that heterologous segregation systems can be reconstituted in yeast. EMBO J, 2001 Jan 15, 20(1-2), 197 - 209 A cytosolic NAD-dependent deacetylase, Hst2p, can modulate nucleolar and telomeric silencing in yeast; Perrod S et al.; In budding yeast, the silent information regulator Sir2p is a nuclear NAD-dependent deacetylase that is essential for both telomeric and rDNA silencing . All eukaryotic species examined to date have multiple homologues of Sir two (HSTs), which share a highly conserved globular core domain . Here we report that yeast Hst2p and a mammalian Hst2p homologue, hSirT2p, are cytoplasmic in yeast and human cells, in contrast to yHst1p and ySir2p which are exclusively nuclear . Although yHst2p cannot restore silencing in a sir2 deletion, overexpression of yHst2p influences nuclear silencing events in a SIR2 strain, derepressing subtelomeric silencing while increasing repression in the rDNA . In contrast, a form of ySir2p carrying a point mutation in the conserved core domain disrupts both telomeric position effect (TPE) and rDNA repression at low expression levels . This argues that non-nuclear yHst2p can compete for a substrate or ligand specifically required for telomeric, and not rDNA repression. EMBO J, 2001 Jan 15, 20(1-2), 82 - 90 Specificity of Cdk activation in vivo by the two Caks Mcs6 and Csk1 in fission yeast; Hermand D et al.; Activating phosphorylation of cyclin-dependent kinases (Cdks) is mediated by at least two structurally distinct types of Cdk-activating kinases (Caks): the trimeric Cdk7-cyclin H-Mat1 complex in metazoans and the single-subunit Cak1 in budding yeast . Fission yeast has both Cak types: Mcs6 is a Cdk7 ortholog and Csk1 a single-subunit kinase . Both phosphorylate Cdks in vitro and rescue a thermosensitive budding yeast CAK1 strain . However, this apparent redundancy is not observed in fission yeast in vivo . We have identified mutants that exhibit phenotypes attributable to defects in either Mcs6-activating phosphorylation or in Cdc2-activating phosphorylation . Mcs6, human Cdk7 and budding yeast Cak1 were all active as Caks for Cdc2 when expressed in fission yeast . Although Csk1 could activate Mcs6, it was unable to activate Cdc2 . Biochemical experiments supported these genetic results: budding yeast Cak1 could bind and phosphorylate Cdc2 from fission yeast lysates, whereas fission yeast Csk1 could not . These results indicate that Mcs6 is the direct activator of Cdc2, and Csk1 only activates Mcs6 . This demonstrates in vivo specificity in Cdk activation by Caks. J Membr Biol, 2001 Jan 15, 179(2), 143 - 53 Functional asymmetry of the sodium-D-glucose cotransporter expressed in yeast secretory vesicles; Firnges MA et al.; The sodium-D-glucose cotransporter (SGLT1) was expressed in a yeast mutant strain NY 17 (sec6-4) that accumulates secretory vesicles at a nonpermissive temperature because of a block in the delivery of these vesicles to the plasma membrane . By differential centrifugation a microsomal fraction enriched in secretory vesicles was prepared with a high specific activity of the vanadate-sensitive H+-ATPase and invertase . In this membrane fraction one protein band of an apparent molecular weight of 55 kDa representing the nonglycosylated SGLT1 protein could be detected by immunochemical analysis . In addition, higher molecular weight protein bands probably representing dimers and aggregates were found . In transport studies with the microsomes D-glucose fluxes showed asymmetric properties: efflux experiments revealed the typical properties of the SGLT1 such as sodium dependence, inhibition by phlorizin and potential dependence . Influx of D-glucose showed no dependence on sodium and was not inhibited by phlorizin . Furthermore, the transporter exhibited a striking asymmetry with regard to the D-glucose affinity and the sugar specificity . These results suggest that the orientation of the SGLT1 expressed in yeast secretory vesicles is, indeed, inverted with regard to its configuration in the plasma membrane of epithelial cells . Moreover, there are striking functional differences between the periplasmic and cytoplasmic face of the transporter. Acta Crystallogr D Biol Crystallogr, 2001 Mar, 57(Pt 3), 441 - 4 Structural studies of a yeast quaternary transcription-initiation complex; Wang SM et al.; A 96.7 kDa quaternary transcription-factor complex consisting of the conserved core domains of yeast TBP, TFIIA, TFIIB and TATA-box DNA has been assembled from purified components . Crystals of the complex were obtained by the hanging-drop vapor-diffusion method . Native data sets were collected at synchrotron sources . Crystal form I belongs to space group R32, with unit-cell parameters a = b = 173.2, c = 164.1 A, and diffracts to 2.5 resolution, but contains substoichiometric amounts of TFIIB . Crystal form II was assembled with a longer piece of DNA than that used for form I crystals and contains the complete quaternary complex . These crystals belong to space group P42(1)2, with unit-cell parameters a = b = 141.7, c = 112.0 A, and diffract to 3.6 A resolution. Nucleic Acids Res . 2001 Mar 1;29(5):E28. Identification of partial loss of function p53 gene mutations utilizing a yeast-based functional assay; Kovvali GK et al.; Missense mutations within the central DNA binding region of p53 are the most prevalent mutations found in human cancer . Numerous studies indicate that 'hot-spot' p53 mutants (which comprise approximately 30% of human p53 gene mutations) are largely devoid of transcriptional activity . However, a growing body of evidence indicates that some non-hot-spot p53 mutants retain some degree of transcriptional activity in vivo, particularly against strong p53 binding sites . We have modified a previously described yeast-based p53 functional assay to readily identify such partial loss of function p53 mutants . We demonstrate the utility of this modified p53 functional assay using a diverse panel of p53 mutants. Nucleic Acids Res, 2001 Mar 1, 29(5), 1222 - 7 Translational control of human p53 expression in yeast mediated by 5'-UTR-ORF structural interaction; Mokdad-Gargouri R et al.; We have expressed human p53 cDNA in the yeast Saccharomyces cerevisiae and shown that the level of production and the length of the p53 protein depends on the presence of untranslated mRNA regions (UTRs) . The expression of the ORF alone leads to a p53 protein of correct size (53 kDa) that accumulates to high levels, concomitantly with the presence of a small amount of a p40 protein (40 kDa) . However, when either the entire 5'-UTR and a part of the 3'- or 5'-UTR alone is used, this leads to the production of small amounts of the 40 kDa truncated form only . The p40 protein corresponds to a truncated form of p53 at the C-terminal extremity since it reacts only with a monoclonal antibody recognising the N-terminal epitope . This effect on the amount and length of p53 protein had no correlation at the mRNA level, suggesting that translational control probably occurs through the 5'-UTR . We propose a model of structural interaction between this UTR and a part of the ORF mRNA for the regulation of p53 expression in this heterologous context. Nucleic Acids Res, 2001 Mar 1, 29(5), 1144 - 55 Characterization and mutational analysis of yeast Dbp8p, a putative RNA helicase involved in ribosome biogenesis; Daugeron MC et al.; RNA helicases of the DEAD box family are involved in almost all cellular processes involving RNA molecules . Here we describe functional characterization of the yeast RNA helicase Dbp8p (YHR169w) . Our results show that Dbp8p is an essential nucleolar protein required for biogenesis of the small ribosomal subunit . In vivo depletion of Dbp8p resulted in a ribosomal subunit imbalance due to a deficit in 40S ribosomal subunits . Subsequent analyses of pre-rRNA processing by pulse-chase labeling, northern hybridization and primer extension revealed that the early steps of cleavage of the 35S precursor at sites A(1) and A(2) are inhibited and delayed at site A(0) . Synthesis of 18S rRNA, the RNA moiety of the 40S subunit, is thereby blocked in the absence of Dbp8p . The involvement of Dbp8p as a bona fide RNA helicase in ribosome biogenesis is strongly supported by the loss of Dbp8p in vivo function obtained by site-directed mutagenesis of some conserved motifs carrying the enzymatic properties of the protein family. Nucleic Acids Res, 2001 Mar 1, 29(5), 1107 - 13 Excision of 8-oxoguanine within clustered damage by the yeast OGG1 protein; David-Cordonnier MH et al.; Clustered damages are formed in DNA by ionising radiation and radiomimetic anticancer agents and are thought to be biologically severe . 7,8-dihydro-8-oxoguanine (8-oxoG), a major DNA damage resulting from oxidative attack, is highly mutagenic leading to a high level of G.C-->T.A transversions if not previously excised by OGG1 DNA glycosylase/AP lyase proteins in eukaryotes . However, 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell . The ability of yeast OGG1 to excise 8-oxoG was determined when another type of damage {dihydrothymine, uracil, 8-oxoG, abasic (AP) site or various types of single-strand breaks (SSBs)} is present on the complementary strand 1, 3 or 5 bases 5' or 3' opposite to 8-oxoG . Base damages have little or no influence on the excision of 8-oxoG by yeast OGG1 (yOGG1) whereas an AP site has a strong inhibitory effect . Various types of SSBs, obtained using either oligonucleotides with 3'- and 5'-phosphate termini around a gap or through conversion of an AP site with either endonuclease III or human AP endonuclease 1, strongly inhibit excision of 8-oxoG by yOGG1 . Therefore, this large inhibitory effect of an AP site or a SSB may minimise the probability of formation of a double-strand break in the processing of 8-oxoG within clustered damages. J Exp Biol, 2001 Mar, 204(Pt 6), 1053 - 61 Properties of the mammalian and yeast metal-ion transporters DCT1 and Smf1p expressed in Xenopus laevis oocytes; Sacher A et al.; Transition metals are essential for many metabolic processes, and their homeostasis is crucial for life . Metal-ion transporters play a major role in maintaining the correct concentrations of the various metal ions in living cells . Little is known about the transport mechanism of metal ions by eukaryotic cells . Some insight has been gained from studies of the mammalian transporter DCT1 and the yeast transporter Smf1p by following the uptake of various metal ions and from electrophysiological experiments using Xenopus laevis oocytes injected with RNA copies (c-RNA) of the genes for these transporters . Both transporters catalyze the proton-dependent uptake of divalent cations accompanied by a 'slippage' phenomenon of different monovalent cations unique to each transporter . Here, we further characterize the transport activity of DCT1 and Smf1p, their substrate specificity and their transport properties . We observed that Zn(2+) is not transported through the membrane of Xenopus laevis oocytes by either transporter, even though it inhibits the transport of the other metal ions and enables protons to 'slip' through the DCT1 transporter . A special construct (Smf1p-s) was made to enhance Smf1p activity in oocytes to enable electrophysiological studies of Smf1p-s-expressing cells . 54Mn(2+) uptake by Smf1p-s was measured at various holding potentials . In the absence of Na(+) and at pH 5.5, metal-ion uptake was not affected by changes in negative holding potentials . Elevating the pH of the medium to 6.5 caused metal-ion uptake to be influenced by the holding potential: ion uptake increased when the potential was lowered . Na(+) inhibited metal-ion uptake in accordance with the elevation of the holding potential . A novel clutch mechanism of ion slippage that operates via continuously variable stoichiometry between the driving-force pathway (H(+)) and the transport pathway (divalent metal ions) is proposed . The possible physiological advantages of proton slippage through DCT1 and of Na(+) slippage through Smf1p are discussed. Naunyn Schmiedebergs Arch Pharmacol, 2001 Feb, 363(2), 203 - 8 Sanguinarine induces K+ outflow from yeast cells expressing mammalian sodium pumps; Scheiner-Bobis G; Sanguinarine, an alkaloid from Sanguinaria canadensis, has no effect on the yeast Saccharomyces cerevisiae at concentrations of up to 225 microM . Yeast cells become sensitive to sanguinarine and lose cytosolic K+ in a time- and concentration-dependent manner when they express the mammalian Na+,K+-ATPase (sodium pump) . Dose-response studies show that sanguinarine induces K+ outflow from cells expressing wild-type sodium pumps with an EC50 of 29.3+/-1.2 microM . A similar effect with a comparable EC50) of 26.8+/-1.3 microM is obtained with cells expressing an Asp369Ala mutant of the sodium pump alpha1 subunit . Since this sodium pump mutant does not hydrolyze ATP, it can be excluded that the observed sanguinarine-induced outflow of K+ is an active ion transport process . Ouabain inhibits the sanguinarine effect at concentrations higher than 1 mM . In contrast, proscillaridin A inhibits the sanguinarine-induced K+ outflow from cells expressing the wild-type sodium pump with an IC50 of 48.9+/-1.3 microM . A similar IC50 of 52.2+/-3.0 microM is obtained with cells expressing the Asp369Ala mutant . These data, together with the fact that sanguinarine inhibits the binding of {3H}ouabain to microsomes prepared from yeast cells expressing the sodium pump with an IC50 of 94.5+/-4.3 microM, all indicate that sanguinarine specifically targets the sodium pump, and that the observed K+ outflow is tightly associated with the presence of the enzyme. Indian J Pathol Microbiol, 2000 Apr, 43(2), 143 - 5 Comparison of Vitek Yeast Biochemical Card with conventional methods for speciation of Candida; Sood P et al.; The ability of the Vitek Yeast Biochemical Card to identify yeast isolates was compared with conventional methods . Of the fifty yeast isolates tested same species identification was obtained in thirty-four isolates . The Vitek yeast biochemical card identified 13 isolates which could not be identified by the conventional tests . Though the Vitek Yeast biochemical card gave a good rapid identification the high cost of each test severely limits its routine use in most of the laboratories. J Environ Pathol Toxicol Oncol, 2001, 20(1), 39 - 45 Effect of caffeine on the genotoxic effects of gamma radiation and 4-NQO in diploid yeast; Anjaria KB et al.; Caffeine is an environmental agent to which people are commonly exposed through medicines, drinks, food items, etc . It has been shown to be mutagenic in a number of test systems . In addition, it has also been shown to modify the mutagenic response of ionizing radiation, UV, and several chemical mutagens in a number of test systems . We have studied the effect of caffeine on gamma radiation and 4-Nitroquinoline 1-oxide (4-NQO)-induced gene conversion in the yeast Saccharomyces cerevisiae D7 . Stationary phase cells were either exposed to 100-600 Gy of 60Co gamma radiation or treated with 0.15-0.3 microM 4-NQO (30 degrees C, 1 hour), after which they were plated on synthetic complete or minimal media with or without caffeine . Caffeine concentrations ranged from 5 to 15 mM . The results indicated that caffeine at 5 and 10 mM decreased gamma radiation-induced gene conversion frequencies significantly at 400 and 600 Gy . At 600 Gy, the decrease was about 30% and 50% with caffeine concentrations of 5 and 10 mM, respectively . In contrast, caffeine was found to increase the induced gene conversion frequency when cells treated with 0.15, 0.225, and 0.3 microM 4-NQO were plated on media containing caffeine . The increase with 5, 10, and 15 mM caffeine was approximately 1.5, 2, and 2.5, respectively, times the value of 4-NQO alone . The results indicate that the posttreatment repair processes following gamma irradiation or 4-NQO treatment are modified via different pathways. RNA, 2001 Jan, 7(1), 133 - 42 Yeast U1 snRNP-pre-mRNA complex formation without U1snRNA-pre-mRNA base pairing; Du H et al.; Base pairing between the 5' end of U1 snRNA and the conserved 5' splice site of pre-mRNA is important for commitment complex formation in vitro . However, the biochemical mechanisms by which pre-mRNA is initially recognized by the splicing machinery is not well understood . To evaluate the role of this base pairing interaction, we truncated U1 snRNA to eliminate the RNA-RNA interaction and surprisingly found that U1 snRNP can still form a nearly normal RNA-protein complex and maintain sequence specificity . We propose that some feature of U1 snRNP, perhaps one or more protein factors, is more important than the base pairing for initial 5' splice site recognition . In addition, at least five sets of interactions contribute to complex formation or stability . Only one of these is base pairing between the 5' splice site and the 5' end of U1 snRNA, without which the U1 snRNP-pre-mRNA complex is less stable and has a somewhat altered conformation. Cell Mol Life Sci, 1999 Nov 30, 56(9-10), 807 - 16 Yeast aging research: recent advances and medical relevance; Sinclair DA; The molecular mechanisms of aging are most fully understood for the budding yeast Saccharomyces cerevisiae . Recent advances in our understanding of aging in this organism have enabled researchers to answer some fundamental questions about the aging process . Is aging due to a multitude of 'mechanisms' or can there be a key few? Can we design single-gene mutations that will prolong life? Can we prolong life whilst maintaining health and fecundity? The various contributing factors to yeast longevity, uncovered thus far, fall into three classes: DNA metabolism, heterochromatin, and metabolic activity . However, these separate classes may actually represent different aspects of the same aging mechanism based on genome stability . This review examines the recent advances in our understanding of yeast aging and discusses their relevance, if any, to the human condition. Cell Mol Life Sci, 1999 Oct 30, 56(5-6), 415 - 26 Molecular mechanism of heme signaling in yeast: the transcriptional activator Hap1 serves as the key mediator; Zhang L et al.; Heme is a key molecule in mediating the effects of oxygen on various molecular and cellular processes in many living organisms . In the yeast Saccharomyces cerevisiae, heme serves as a secondary signal for oxygen; intracellular heme synthesis directly correlates with oxygen tension in the environment . In yeast, oxygen sensing and heme signaling are primarily mediated by the heme activator protein Hap1, which, in response to heme, activates the transcription of genes required for respiration and for controlling oxidative damage . Heme regulation of many genes required for anaerobic growth is mediated by the aerobic repressor Rox1, whose expression is controlled by heme . In this review, we summarize recent knowledge about (i) how heme synthesis may be controlled by oxygen tension, (ii) how heme precisely and stringently controls Hap1 activity and (iii) whether other transcriptional activators can also mediate heme action. Int J Syst Evol Microbiol, 2001 Jan, 51(Pt 1), 231 - 5 Sporobolomyces yunnanensis sp . nov., a Q-10(H2)-containing yeast species with a close phylogenetic relationship to Erythrobasidium hasegawianum; Bai FY et al.; A ballistoconidia-forming yeast strain, CH 2.141T, isolated from a semi-dried leaf sample collected in Yunnan, China, was found to have Q-10(H2) as its major ubiquinone . Molecular phylogenetic analysis based on the nucleotide sequences of small subunit (18S) rDNA and the internal transcribed spacer region (including 5.8S rDNA) indicated that the strain was closely related to the two described Q-10(H2)-containing yeast species, Erythrobasidium hasegawianum and Sporobolomyces elongatus, with a closer relationship to the former . A DNA-DNA reassociation experiment showed that strain CH 2.141T represents a new yeast species, for which the name Sporobolomyces yunnanensis sp . nov . is proposed. J Dairy Sci, 2001 Jan, 84(1), 204 - 12 Effects of forage neutral detergent fiber and yeast culture on performance of cows during early lactation; Wang Z et al.; Sixty Holstein cows were assigned to two treatments at 21 d before calving and were group-fed a prepartum diet with or without yeast culture . After parturition, cows were individually fed one of five treatments for 140 d: 1) 21% forage neutral detergent fiber (NDF) without yeast culture, 2) 21% forage NDF with yeast culture, 3) 17% forage NDF without yeast culture, 4) 17% forage NDF with yeast culture, and 5) 25% forage NDF with yeast culture for 30 d and then switched to diet 4 for 110 d . Cows fed yeast culture prepartum were also fed yeast culture postpartum (60 g/d) . A quadratic increase to 25, 21, and 17% forage NDF occurred during the first 30 d in milk (DIM) for dry matter intake, milk yield, and milk protein yield . No differences were observed for yeast culture or interaction of yeast culture and forage NDF for the first 30 DIM . Feeding 17 versus 21% forage NDF increased milk protein percentage and tended to increase dry matter intake as a percentage of body weight from 31 to 140 DIM . During this period, yeast culture tended to increase milk fat percentage and appeared to have positive effects on dry matter intake, milk yield, and milk fat yield when supplemented to diets with 21% forage NDF but not with 17% forage NDF . Feeding 17% forage NDF may be too low for the first 30 DIM but may improve animal performance after 30 DIM compared to 21% forage NDF. Biol Chem, 2000 Dec, 381(12), 1175 - 83 Yeast translational activator Cbs2p: mitochondrial targeting and effect of overexpression; Tzschoppe K et al.; The yeast translational activator protein Cbs2p is imported into mitochondria without obvious proteolytic processing . To test the importance of amino-terminal amino acids for mitochondrial targeting we fused varying portions of the N-terminus with green fluorescent protein and examined the intracellular distribution of the reporter protein . We show that the 25 N-terminal amino acids are sufficient to direct the majority of the fusion protein into mitochondria . Cbs2p derivatives lacking 9 to 35 amino acids from the N-terminus fail to complement the respiratory deficiency of a deltacbs2 strain, but are still imported into mitochondria . Therefore Cbs2p contains at least one independent mitochondrial targeting information in addition to the N-terminal signal . We further analyzed the effect of over-expression of Cbs2p on mitochondrial function . Elevated concentrations of Cbs2p lead to slightly impaired mitochondrial gene expression, probably as the result of the formation of inactive Cbs2p aggregates. Biochem Cell Biol, 2000, 78(6), 683 - 90 A comparative hybridization analysis of yeast DNA with Paramecium parafusin- and different phosphoglucomutase-specific probes; Wyroba E et al.; Molecular probes designed for the parafusin (PFUS), the Paramecium exocytic-sensitive phosphoglycoprotein, gave distinct hybridization patterns in Saccharomyces cerevisiae genomic DNA when compared with different phosphoglucomutase specific probes . These include two probes identical to segments of yeast phosphoglucomutase (PGM) genes 1 and 2 . Neither of the PGM probes revealed the 7.4 and 5.9 kb fragments in Bgl II-cut yeast DNA digest detected with the 1.6 kb cloned PFUS cDNA and oligonucleotide constructed to the PFUS region (insertion 3--I-3) not found in other species . PCR amplification with PFUS-specific primers generated yeast DNA-species of the predicted molecular size which hybridized to the I-3 probe . A search of the yeast genome database produced an unassigned nucleotide sequence that showed 55% identity to parafusin gene and 37% identity to PGM2 (the major isoform of yeast phosphoglucomutase) within the amplified region. Nature, 2001 Jan 25, 409(6819), 533 - 8 Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF; Iyer VR et al.; Proteins interact with genomic DNA to bring the genome to life; and these interactions also define many functional features of the genome . SBF and MBF are sequence-specific transcription factors that activate gene expression during the G1/S transition of the cell cycle in yeast . SBF is a heterodimer of Swi4 and Swi6, and MBF is a heterodimer of Mbpl and Swi6 (refs 1, 3) . The related Swi4 and Mbp1 proteins are the DNA-binding components of the respective factors, and Swi6 mayhave a regulatory function . A small number of SBF and MBF target genes have been identified . Here we define the genomic binding sites of the SBF and MBF transcription factors in vivo, by using DNA microarrays . In addition to the previously characterized targets, we have identified about 200 new putative targets . Our results support the hypothesis that SBF activated genes are predominantly involved in budding, and in membrane and cell-wall biosynthesis, whereas DNA replication and repair are the dominant functions among MBF activated genes . The functional specialization of these factors may provide a mechanism for independent regulation of distinct molecular processes that normally occur in synchrony during the mitotic cell cycle. Protein Sci, 2000 Dec, 9(12), 2354 - 65 Backbone dynamics of sequence specific recognition and binding by the yeast Pho4 bHLH domain probed by NMR; Cave JW et al.; Backbone dynamics of the basic/helix-loop-helix domain of Pho4 from Saccharomyces cerevisae have been probed by NMR techniques, in the absence of DNA, nonspecifically bound to DNA and bound to cognate DNA . Alpha proton chemical shift indices and nuclear Overhauser effect patterns were used to elucidate the secondary structure in these states . These secondary structures are compared to the co-crystal complex of Pho4 bound to a cognate DNA sequence (Shimizu T . Toumoto A, Ihara K, Shimizu M, Kyogou Y, Ogawa N, Oshima Y, Hakoshima T, 1997, EMBO J 15: 4689-4697) . The dynamic information provides insight into the nature of this DNA binding domain as it progresses from free in solution to a specifically bound DNA complex . Relative to the unbound form, we show that formation of either the nonspecific and cognate DNA bound complexes involves a large change in conformation and backbone dynamics of the basic region . The nonspecific and cognate complexes, however, have nearly identical secondary structure and backbone dynamics . We also present evidence for conformational flexibility at a highly conserved glutamate basic region residue . These results are discussed in relation to the mechanism of sequence specific recognition and binding. Ukr Biokhim Zh, 2000 May-Jun, 72(3), 25 - 30 {Riboflavin biosynthesis in yeast as a model for estimating mechanisms of regulating synthesis of biologically active substances}; Fedorovych DV et al.; General principles of regulation of transcription in eucaryotic cells are characterized . Investigations on riboflavin biosynthesis and regulation of this process in yeasts are summarized . The data obtained permit to consider riboflavin biosynthesis control as an interesting and convenient model capable to estimate the mechanisms various transcription factors action . These investigations are required in order to reveal general features of vitamins and relative compounds biosynthesis regulation. Bioresour Technol, 2001 Feb, 76(3), 253 - 8 Utilization of brewer's yeast cells for the production of food-grade yeast extract . Part 1: Effects of different enzymatic treatments on solid and protein recovery and flavor characteristics; Chae HJ et al.; Yeast extract was produced from brewer's yeast of a beer factory by combined enzymatic treatments using endoprotease, exoprotease, 5'-phosphodiesterase, and adenosine monophosphate (AMP)-deaminase . Effects of enzyme combination, enzyme dosages and treatment sequence on the recovery of solid and protein, flavor and compositional characteristics were investigated . Exoprotease dosage strongly affected the recovery of protein and degree of hydrolysis (DH) and sensory characteristics . When the yeast cells were treated using optimal combination of endoprotease and exoprotease (0.6% Protamex and 0.6% Flavourzyme), high solid recovery (48.3-53.1%) and the best flavor profile were obtained . Among various treatment sequences using multiple enzymes, treatment with protease followed by nuclease resulted in the highest 5'-guanosine monophosphate (5'-GMP) content . The optimal concentrations of both 5'-phosphodiesterase and AMP-deaminase were found to be 0.03% . After treatments using optimal combination of enzyme, enzyme dosages and treatment sequence for four enzymes, a high solid yield of 55.1% and 5'-nucleotides content of 3.67% were obtained. Cancer Res, 2001 Jan 1, 61(1), 64 - 70 Human homologue of yeast Rad23 protein A interacts with p300/cyclic AMP-responsive element binding (CREB)-binding protein to down-regulate transcriptional activity of p53; Zhu Q et al.; The tumor suppressor protein p53 regulates various cellular responses to DNA damage and plays a significant role in DNA repair . The nuclear p300/cyclic AMP-responsive element binding (CREB)-binding protein (CBP) proteins act as coactivators in supporting the transcription function of p53 . We examined the role of the human homologue of yeast Rad23 protein A (hHR23A), one of the two human homologues of the Saccharomyces cerevisiae nucleotide excision repair gene product Rad23, in the p300/CBP-associated regulation of p53 activity . Overexpression of wild-type hHR23A inhibits the p53 transcriptional activity and results in a decreased steady-state protein level of cellular p53 . The inhibitory effect of hHR23A can be overcome by the concomitant expression of p300, CBP, and p300 segments harboring C/H1 domain and neutralized by the coexpression of HIV accessory protein Vpr, which binds COOH terminus of hHR23A/B . Additionally, hHR23A was shown to interact in vitro and in vivo with p300 segments harboring C/H1 domain . These studies provide evidence for the involvement of hHR23A in the regulation of p53 activity through p300/CBP . Although the precise direct role of hHR23 proteins in regulation of p53 and DNA repair remains to be elucidated, our data suggest that the interaction between hHR23A and p300/CBP has important implications in cross-talk between the p53 pathway and DNA repair. Mikrobiologiia, 2000 Nov-Dec, 69(6), 801 - 4 {A new species of psychrophilic basidiomycetes yeast Leucosporidium fasciculatum sp . Nov.}; Bab'eva IP et al.; A psychrophilic yeast with a basidiomycetous developmental cycle and properties corresponding to the genus Leucosporidium Fell et al . was isolated from the fruiting body of the edible spring mushroom Gyromitra esculenta Pers . picked near Moscow . However, the isolate differed from all Leucosporidium species described to date in a number of characteristics . The results of the study of the developmental cycle and of the cultural, morphological, physiological, and biochemical properties of the new isolate, strain KBP Y-3696, allow it to be assigned to a new species of the genus Leucosporidium. Mikrobiologiia, 2000 Nov-Dec, 69(6), 790 - 5 {Composition of lipids from yeast-like and mycelial cells of the fungus Mucor hiemalis, grown in the presence of 4-chloroaniline}; Mysiakina IS et al.; The fungus Mucor hiemalis, which is commonly thought to be monomorphic, produced two types of cells, yeastlike and mycelial, during growth in a medium containing 4-chloroaniline . Among the polar lipids of yeastlike cells, diphosphatidylglycerol was dominant, while phosphatidylcholine and phosphatidylethanolamine were present in minor amounts . Conversely, mycelial cells mainly contained phosphatidylcholine and phosphatidylethanolamine, whereas the content of diphosphatidylglycerol was low . The neutral lipids of yeastlike cells were dominated by diacylglycerides, sterols, and fatty acids . The content of triacylglycerides and sterol esters was low . Yeastlike cells contained higher amounts of saturated fatty acids and lower amounts of unsaturated fatty acids than the mycelium . The content of stearic acid in the fatty acids of the mycelium grown in the presence of 4-chloroaniline was as high as 25.3-29.9%. Hokkaido Igaku Zasshi, 2000 Nov, 75(6), 385 - 97 {Development of a detection system (APC yeast color assay) of APC mutations by color change of yeast}; Furuuchi K; The author developed a sensitive yeast-based color assay which expresses APC-ADE2 (reporter) fusion protein in yeast and can screen almost the entire coding region of the APC gene . In this assay, the wild-type APC coding sequence of 8.5 kb is divided into 5 overlapping regions which are respectively ligated in-frame with an ADE2 open reading frame . The resulting five constructs containing a part of wild-type APC gene preserve the ADE2(+) phenotype (white yeast colony) when introduced into the yeast, whereas the yeast transfected with plasmids containing frameshift mutations of the APC gene shows an ADE2(-) phenotype (red yeast colony) . Six human colon cancer cell lines were analyzed by this yeast color assay . HCT116 cells with wild-type APC and normal colonic mucosa gave low percentages of red colonies (0-9.9%) in all the regions . On the other hand, more than 96% red colonies were observed in one of the five regions in SW480, Colo201 and Colo320DM cells . Sequence analysis demonstrated the clonal APC mutations at codon 1,338 in SW480, 1,554 in Colo201 and 811 in Colo320DM . Moreover, the assay detected a germline mutation of the APC gene in polyps of a familial adenomatous polyposis (FAP) patient which gave about 50% red colonies . For testing the assay for clinical utilization, 18 colon cancer tissues were subjected to the assay . Eleven cancers (61%) gave more than 10% red colonies (17-57%) and clonal mutations were detected in all these samples . The same mutations were demonstrated in both DNA and RNA samples derived from idendical tissues . These results suggest that this APC yeast color assay is powerful means for detection of APC mutations in clinical samples. Biologist (London), 2000 Feb, 47(1), 15 - 8 Yeast as factory and factotum; Dixon B; After centuries of vigorous activity in making fine wines, beers and breads, Saccharomyces cerevisiae is now acquiring a rich new portfolio of skills, bestowed by genetic manipulation . As shown in a recent shop-window of research supported by the European Commission, yeasts will soon be benefiting industries as diverse as fish farming, pharmaceuticals and laundering. IUBMB Life, 2000 Aug, 50(2), 105 - 13 A CD2-based model of yeast alpha-agglutinin elucidates solution properties and binding characteristics; Grigorescu A et al.; We have previously shown that the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin has sequence characteristics of immunoglobulin-like proteins and have successfully modeled residues 200-325, based on the structure of immunoglobulin variable-type domains . Alignments matching residues 20-200 of alpha-agglutinin with domains I and II of members of the CD2/CD4 subfamily of the immunoglobulin superfamily showed > 80% conservation of key residues despite low sequence similarity overall . Three-dimensional models of two alpha-agglutinin domains constructed on the basis of these alignments were shown to conform to peptide mapping data and biophysical properties of alpha-agglutinin . In addition, the residue volume and surface accessibility characteristics of these models resembled those of the well-packed structures of related proteins . Residue-by-residue analysis showed that packing and accessibility anomalies were largely confined to glycosylated and protease-susceptible loop regions of the domains . Surface accessibility of hydrophobic residues was typical of proteins with extensive domain interactions, a finding compatible with the hydrodynamic properties of alpha -agglutinin and the hydrophobic nature of binding to its peptide ligand alpha-agglutinin . The procedures used to align the alpha-agglutinin sequence and test the quality of the model may be applicable to other proteins, especially those that resist crystallization because of extensive glycosylation. Traffic, 2001 Jan, 2(1), 37 - 50 NBD-labeled phosphatidylcholine and phosphatidylethanolamine are internalized by transbilayer transport across the yeast plasma membrane; Grant AM et al.; The internalization and distribution of fluorescent analogs of phosphatidylcholine (M-C6-NBD-PC) and phosphatidylethanolamine (M-C6-NBD-PE) were studied in Saccharomyces cerevisiae . At normal growth temperatures, M-C6-NBD-PC was internalized predominantly to the vacuole and degraded . M-C6-NBD-PE was internalized to the nuclear envelope/ER and mitochondria, was not transported to the vacuole, and was not degraded . At 2 degrees C, both were internalized to the nuclear envelope/ER and mitochondria by an energy-dependent, N-ethylmaleimide-sensitive process, and transport of M-C6-NBD-PC to and degradation in the vacuole was blocked . Internalization of neither phospholipid was reduced in the endocytosis-defective mutant, end4-1 . However, following pre-incubation at 37 degrees C, internalization of both phospholipids was inhibited at 2 degrees C and 37 degrees C in sec mutants defective in vesicular traffic . The sec18/NSF mutation was unique among the sec mutations in further blocking M-C6-NBD-PC translocation to the vacuole suggesting a dependence on membrane fusion . Based on these and previous observations, we propose that M-C6-NBD-PC and M-C6-NBD-PE are transported across the plasma membrane to the cytosolic leaflet by a protein-mediated, energy-dependent mechanism . From the cytosolic leaflet, both phospholipids are spontaneously distributed to the nuclear envelope/ER and mitochondria . Subsequently, M-C6-NBD-PC, but not M-C6-NBD-PE, is sorted by vesicular transport to the vacuole where it is degraded by lumenal hydrolases. Traffic, 2000 May, 1(5), 425 - 34 A novel pathway for transport and metabolism of a fluorescent phosphatidic acid analog in yeast; Trotter PJ; Phosphatidic acid is a central intermediate of biosynthetic lipid metabolism as well as an important signaling molecule in the cell . These studies assess the internalization, or retrograde transport, and metabolism of phosphatidic acid in yeast using a fluorescent analog . An analog of phosphatidic acid fluorescently labeled at the sn-2 position with N-4-nitrobenz-2-oxa-1, 3-diazole-aminocaproic acid (NBD-phosphatidic acid) was introduced to yeast cells by spontaneous transfer from phospholipid vesicles . Transport and metabolism of the NBD-phosphatidic acid were then monitored by fluorescence spectrophotometry, fluorescence microscopy and routine biochemical methods . Primary metabolites of the NBD-phosphatidic acid in yeast were found to be NBD-diacylgycerol and NBD-phosphatidylinositol . Experiments in cells possessing different levels of phosphatidate phosphatase activity suggest that conversion of the NBD-phosphatidic acid to NBD-diacylglycerol is not a pre-requisite for internalization in yeast . Internalization is sensitive to decreased temperature, but neither ATP depletion nor a sec6-4 mutation, which interrupts endocytosis, has an affect . Thus, internalization of NBD-phosphatidic acid apparently occurs via a non-endocytic route . These characteristics of retrograde transport of NBD-phosphatidic acid in yeast differ significantly from transport of other NBD-phospholipids in yeast as well as NBD-phosphatidic acid transport in mammalian fibroblasts. Traffic, 2000 Mar, 1(3), 259 - 69 Pep12p is a multifunctional yeast syntaxin that controls entry of biosynthetic, endocytic and retrograde traffic into the prevacuolar compartment; Gerrard SR et al.; Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of the endosomal syntaxin, Pep12p . Many vacuolar proteins, such as the soluble vacuolar hydrolase, carboxypeptidase Y (CPY), traverse the prevacuolar compartment (PVC) en route to the vacuole . Here we show that deletion of the carboxy-terminal transmembrane domain of Pep12p results in a temperature-conditional block in transport of CPY to the PVC . The PVC also receives traffic from the early endosome and the vacuole, and mutation in PEP12 also blocks these other trafficking pathways into the PVC . Therefore, Pep12p is a multifunctional syntaxin that is required for all known trafficking pathways into the yeast PVC . Finally, we found that the internalized pheromone receptor, Ste3p, can cycle out of the PVC in a VPS27-independent fashion. Traffic, 2000 Mar, 1(3), 248 - 58 Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking; Babst M et al.; The mammalian tumor susceptibility gene tsg101 encodes the homologue of Vps23p, a class E Vps protein essential for normal membrane trafficking in the late endosome/multivesicular body of yeast . Both proteins assemble into large (approximately 350 kDa) cytosolic protein complexes and we show that the yeast complex contains another class E Vps protein, Vps28p . tsg101 mutant cells exhibit defects in sorting and proteolytic maturation of the lysosomal hydrolase cathepsin D, as well as in the steady-state distribution of the mannose-6-phosphate receptor . Additionally, endocytosed EGF receptors that are normally sorted to the lysosome are instead rapidly recycled back to the cell surface in tsg101 mutant cells . We propose that tsg101 mutant cells are defective in the delivery of cargo proteins to late endosomal compartments . One consequence of this endosomal trafficking defect is the delayed down-regulation/degradation of activated cell surface receptors, resulting in prolonged signaling . This may contribute to the tumorigenic phenotype exhibited by the tsg101 mutant fibroblasts. Traffic, 2000 Feb, 1(2), 172 - 83 Sec7p directs the transitions required for yeast Golgi biogenesis; Deitz SB et al.; Endoplasmic reticulum (ER)-to-Golgi traffic in yeast proceeds by the maturation of membrane compartments from post-ER vesicles to intermediate small vesicle tubular clusters (VTCs) to Golgi nodular membrane networks (Morin-Ganet et al., Traffic 2000; 1: 56-68) . The balance between ER and Golgi compartments is maintained by COPII- and COPI-mediated anterograde and retrograde traffic, which are dependent on Sec7p and ARF function . The sec7-4 temperature-sensitive allele is a mutation in the highly conserved Sec7 domain (Sec7d) found in all ARF-guanine nucleotide exchange factor proteins . Post-ER trafficking is rapidly inactivated in sec7-4 mutant yeast at the restrictive temperature . This conditional defect prevented the normal production of VTCs and instead generated Golgi-like tubes emanating from the ER exit sites . These tubes progressively developed into stacked cisternae defining the landmark sec7 mutant phenotype . Consistent with the in vivo results, a Sec7d peptide inhibited ER-to-Golgi transport and displaced Sec7p from its membrane anchor in vitro . The similarities in the consequences of inactivating Sec7p or ARFs in vivo was revealed by genetic disruption of yeast ARFs or by addition of brefeldin A (BFA) to whole cells . These treatments, as in sec7-4 yeast, affected the morphology of membrane compartments in the ER-Golgi transition . Further evidence for Sec7p involvement in the transition for Golgi biogenesis was revealed by in vitro binding between distinct domains of Sec7p with ARFs, COPI and COPII coat proteins . These results suggest that Sec7p coordinates membrane transitions in Golgi biogenesis by directing and scaffolding the binding and disassembly of coat protein complexes to membranes, both at the VTC transition from ER exit sites to form Golgi elements and for later events in Golgi maturation. Traffic, 2000 Oct, 1(10), 763 - 8 The use of yeast two-hybrid screens in studies of protein:protein interactions involved in trafficking; Stephens DJ et al.; The yeast two-hybrid system has provided a convenient means to both screen for proteins that interact with a protein of interest and to characterise the known interaction between two proteins . Several groups with an interest in the molecular mechanisms that underlie discrete steps along trafficking pathways have exploited the yeast two-hybrid system . Here, we provide a brief background to the technology, attempt to point out some of the pitfalls and benefits of the different systems that can be employed, and mention some of the areas (within the trafficking field) where yeast two-hybrid interaction assays have been particularly informative. Traffic, 2000 Jan, 1(1), 56 - 68 Morphogenesis and dynamics of the yeast Golgi apparatus; Morin-Ganet MN et al.; A kinetic and morphometric study was conducted with the electron microscope to clarify the biogenesis and structural diversity of the Golgi apparatus in the yeast Saccharomyces cerevisiae . Secretion was synchronized by inhibiting protein synthesis and/or by subjecting thermosensitive secretory mutants to double temperature shifts . Five membrane-bounded structures disappeared or reappeared in an orderly manner at approximately the rate of secretory protein flow . 1) The first detectable post-ER intermediates were very short-lived clusters of small vesicles that appeared next to the endoplasmic reticulum (ER) . 2) Their constituent small vesicles were rapidly bridged by membrane tubules in a SEC18-dependent manner, giving short-lived tubular clusters of small vesicles, analogous to mammalian vesicular-tubular clusters . 3) Fine and 4) large nodular networks (coated with the Golgi protein Sec7), and 5) secretory granules . Upon relieving a secretory block, each structure successively reappeared, seemingly by transformation of the previous one . When no secretory cargo was to be transported, these structures were not renewed . They disappeared more than five times faster than some Golgi enzymes such as Och1p, implying that the latter are recycled and perhaps partially retained . Retention could arise from intra-compartmental flow of cargo/carrier, hinted at by the varying calibers within a single nodular network. Traffic, 2000 Jan, 1(1), 45 - 55 The yeast endosomal t-SNARE, Pep12p, functions in the absence of its transmembrane domain; Gerrard SR et al.; Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of two distinct SNARE complexes . Pep12p and Vam3p are both t-SNAREs of the syntaxin family that are components of these SNARE complexes . We have used a genetic approach to address the role of Pep12p in vacuolar protein transport . Our screen for temperature-sensitive pep12 mutants yielded six alleles that were rapidly inactivated upon exposure to the non-permissive temperature . Surprisingly, the proteins encoded by these alleles were all truncated immediately prior to the transmembrane domain . Here we demonstrate that Pep12p requires its transmembrane domain for proper localization, but not for its role in vesicle fusion . In addition, we show that although Pep12p can replace Vam3p in the vacuolar SNARE complex, its transmembrane domain is required to function at this step . Therefore, the transmembrane domain of Pep12p performs different roles in the prevacuolar and vacuolar SNARE complexes. Bull Exp Biol Med, 2000 Nov, 130(11), 1084 - 6 Allergen-containing drug from Malassezia spp . yeast; Arzumanyan VG et al.; Malassezia spp . yeast habituating the skin of healthy humans can be a source of allergens for patients with atopic dermatitis . We proposed a method for obtaining allergen-containing preparation by trimming outer cell wall proteins with 1% sodium dodecyl sulfate . The resultant preparation contained 36 and 67 kD proteins known as Malassezia allergens . IgE antibodies to these proteins were detected in the sera of young people with atopic dermatitis. Yeast, 2001 Feb, 18(3), 239 - 49 Pho85 kinase, a yeast cyclin-dependent kinase, regulates the expression of UGP1 encoding UDP-glucose pyrophosphorylase; Nishizawa M et al.; The PHO85 gene is a negative regulator of the PHO system in the yeast Saccharomyces cerevisiae and encodes a protein kinase (Pho85) highly homologous to the Cdc28 kinase (Cdc28) . Ten cyclin-like proteins are known to interact with Pho85, and combination with different cyclins is believed to be responsible for distinct Pho85 functions, including phosphate metabolism, carbon source utilization and cell cycle regulation . However, only a limited number of substrates of Pho85 kinase, including Pho4, Gsy2 and Sicl, have so far been identified . To search for more targets of Pho85 and to clarify the genetic control mechanisms by Pho85 kinase in these cellular functions, we carried out a genome-wide analysis of the effect of a pho85Delta mutation on gene expression . We found that expression of various genes involved in carbon metabolism are affected by the mutation and that among them, UGP1 promoter activity was increased in the absence of Pho85 kinase . This increase in the promoter activity was not observed in a pho4Delta mutant or with a mutant UGP1 promoter that is devoid of putative Pho4 and Bas2 binding sites, suggesting that UGP1 expression is modulated by Pho85 through Pho4 . We also found that expression of several Pho85-cyclin genes were altered by the carbon source, the growth phase and Pho85 kinase itself. Mol Biol Cell, 2001 Feb, 12(2), 323 - 37 Remodeling of yeast genome expression in response to environmental changes; Causton HC et al.; We used genome-wide expression analysis to explore how gene expression in Saccharomyces cerevisiae is remodeled in response to various changes in extracellular environment, including changes in temperature, oxidation, nutrients, pH, and osmolarity . The results demonstrate that more than half of the genome is involved in various responses to environmental change and identify the global set of genes induced and repressed by each condition . These data implicate a substantial number of previously uncharacterized genes in these responses and reveal a signature common to environmental responses that involves approximately 10% of yeast genes . The results of expression analysis with MSN2/MSN4 mutants support the model that the Msn2/Msn4 activators induce the common response to environmental change . These results provide a global description of the transcriptional response to environmental change and extend our understanding of the role of activators in effecting this response. EMBO J, 2001 Feb 15, 20(4), 777 - 91 The budding yeast proteins Spc24p and Spc25p interact with Ndc80p and Nuf2p at the kinetochore and are important for kinetochore clustering and checkpoint control; Janke C et al.; Here, we show that the budding yeast proteins Ndc80p, Nuf2p, Spc24p and Spc25p interact at the kinetochore . Consistently, Ndc80p, Nuf2p, Spc24p and Spc25p associate with centromere DNA in chromatin immunoprecipitation experiments, and SPC24 interacts genetically with MCM21 encoding a kinetochore component . Moreover, although conditional lethal spc24-2 and spc25-7 cells form a mitotic spindle, the kinetochores remain in the mother cell body and fail to segregate the chromosomes . Despite this defect in chromosome segregation, spc24-2 and spc25-7 cells do not arrest in metaphase in response to checkpoint control . Furthermore, spc24-2 cells showed a mitotic checkpoint defect when microtubules were depolymerized with nocodazole, indicating that Spc24p has a function in checkpoint control . Since Ndc80p, Nuf2p and Spc24p are conserved proteins, it is likely that similar complexes are part of the kinetochore in other organisms. J Biochem (Tokyo), 2001 Feb, 129(2), 297 - 302 Study on the biosynthesis of dolichol in yeast: recognition of the prenyl chain length in polyprenol reduction; Tateyama S et al.; We synthesized three water-soluble biotin-tagged compounds with different prenyl chain lengths, biotinylated farnesal (BF), biotinylated C(55)-polyprenal (BP55), and biotinylated C(80)-polyprenal (BP80), and examined their effects on in vitro dolichol synthesis from farnesyl diphosphate . BF and BP55 did not affect the dolichol synthesis, whereas BP80 inhibited the reduction pathway from polyprenol to dolichol, accompanied by a decrease in the entire polyprenol and dolichol synthesis . Comparison of BP80 with eighteen detergents, including Triton X-100, CHAPS, octylglucoside, deoxycholate, and Tween 80, revealed the specific effect of BP80 on the reduction pathway . On SDS-polyacrylamide gel electrophoresis, BP80 was detected in an associated form with a 50 kDa protein . These results suggest that the reduction of polyprenol to dolichol in the dolichol biosynthetic pathway proceeds with the recognition of the polyprenol chain length by a 50 kDa protein. Biochemistry (Mosc), 2000 Dec, 65(12), 1362 - 6 Interaction of catalytic domains in cytochrome P450scc--adrenodoxin reductase--adrenodoxin fusion protein imported into yeast mitochondria; Novikova LA et al.; We have constructed plasmids for yeast expression of the fusion protein pre-cytochrome P450scc--adrenodoxin reductase-adrenodoxin (F2) and a variant of F2 with the yeast CoxIV targeting presequence . Mitochondria isolated from transformed yeast cells contained the F2 fusion protein at about 0.5% of total protein and showed cholesterol hydroxylase activity with 22(R)-hydroxycholesterol . The activity increased 17- or 25-fold when sonicated mitochondria were supplemented with an excess of purified P450scc or a mixture of adrenodoxin (Adx) and adrenodoxin reductase (AdxRed), respectively . These data suggest that, at least in yeast mitochondria, the interactions of the catalytic domains of P450scc, Adx, and AdxRed in the common polypeptide chain are restricted. Biochemistry (Mosc), 2000 Dec, 65(12), 1352 - 6 The Ca(2+)-transport system of yeast (Endomyces magnusii) mitochondria: independent pathways for Ca(2+) uptake and release; Deryabina YI et al.; Some features of the Ca(2+)-transport system in mitochondria of the yeast Endomyces magnusii are considered . The Ca(2+) uniporter was shown to be specifically activated by low concentrations of physiological modulators such as ADP, NADH, spermine, and Ca(2+) itself . The Na(+)-independent system responsible for Ca(2+) release from Ca(2+)-preloaded yeast mitochondria was characterized . The rate of the Ca(2+) release was proportional to the Ca(2+) load, insensitive to cyclosporin A and to Na(+), inhibited by La(3+), TPP(+), P(i), and nigericin, while being activated by spermine . We conclude that Ca(2+) release from preloaded E . magnusii yeast mitochondria is mediated by a Na(+)-independent pathway, very similar to that in mitochondria from nonexcitable mammalian tissues . A scheme describing an arrangement of the Ca(2+) transport system of yeast mitochondria is proposed. J Gen Virol, 2001 Mar, 82(Pt 3), 677 - 86 Identification of an N-terminal domain of the plum pox potyvirus CI RNA helicase involved in self-interaction in a yeast two-hybrid system; Lopez L et al.; Potyvirus CI RNA helicase is a protein involved in RNA genome replication and virus movement . The protein aggregates in the cytoplasm of infected cells to form typical cylindrical inclusions . A yeast two-hybrid system was used to analyse interactions of the CI RNA helicase from plum pox potyvirus (PPV) with itself and with other viral proteins . No interactions could be detected of full-length CI protein with itself or with PPV P3/6K1, NIa, NIb or CP proteins . However, positive self-interactions were detected for N-terminal fragments of the CI protein, allowing the mapping of a CI-CI binding domain to the N-terminal 177 aa of the protein . Further deletion analysis suggested that several regions of this domain contribute to the interaction . Moreover, pull-down experiments demonstrate that, at least in vitro, full-length PPV CI protein is able to self-interact in the absence of other virus or plant factors. Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1501 - 6 Molecular cloning of a cytochrome P450 taxane 10 beta-hydroxylase cDNA from Taxus and functional expression in yeast; Schoendorf A et al.; The early steps in the biosynthesis of Taxol involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation at C5, acetylation of this intermediate, and a second cytochrome P450-dependent hydroxylation at C10 to yield taxadien-5 alpha-acetoxy-10 beta-ol . Subsequent steps of the pathway involve additional cytochrome P450 catalyzed oxygenations and CoA-dependent acylations . The limited feasibility of reverse genetic cloning of cytochrome P450 oxygenases led to the use of Taxus cell cultures induced for Taxol production and the development of an approach based on differential display of mRNA-reverse transcription-PCR, which ultimately provided full-length forms of 13 unique but closely related cytochrome P450 sequences . Functional expression of these enzymes in yeast was monitored by in situ spectrophotometry coupled to in vivo screening of oxygenase activity by feeding taxoid substrates . This strategy yielded a family of taxoid-metabolizing enzymes and revealed the taxane 10 beta-hydroxylase as a 1494-bp cDNA that encodes a 498-residue cytochrome P450 capable of transforming taxadienyl acetate to the 10 beta-hydroxy derivative; the identity of this latter pathway intermediate was confirmed by chromatographic and spectrometric means . The 10 beta-hydroxylase represents the initial cytochrome P450 gene of Taxol biosynthesis to be isolated by an approach that should provide access to the remaining oxygenases of the pathway. Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1459 - 64 Epub 2001 Feb 06. The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p; Umland TC et al.; The yeast nonchromosomal gene {URE3} is due to a prion form of the nitrogen regulatory protein Ure2p . It is a negative regulator of nitrogen catabolism and acts by inhibiting the transcription factor Gln3p . Ure2p residues 1--80 are necessary for prion generation and propagation . The C-terminal fragment retains nitrogen regulatory activity, albeit somewhat less efficiently than the full-length protein, and it also lowers the frequency of prion generation . The crystal structure of this C-terminal fragment, Ure2p(97--354), at 2.3 A resolution is described here . It adopts the same fold as the glutathione S-transferase superfamily, consistent with their sequence similarity . However, Ure2p(97--354) lacks a properly positioned catalytic residue that is required for S-transferase activity . Residues within this regulatory fragment that have been indicated by mutational studies to influence prion generation have been mapped onto the three-dimensional structure, and possible implications for prion activity are discussed. Biochem Soc Trans, 2000 Dec, 28(6), 935 - 7 Utility of the Arabidopsis FAE1 and yeast SLC1-1 genes for improvements in erucic acid and oil content in rapeseed; Katavic V et al.; High-erucic acid (HEA) Brassica napus cultivars are regaining interest in industrial contexts . Erucic acid and its derivatives are important renewable raw materials utilized in the manufacture of plastic films, in the synthesis of Nylon 13,13, and in the lubricant and emollient industries . Theoretically, the highest level of erucic acid that can be achieved by means of classical breeding is 66 mol%; however, using new approaches on the basis of genetic engineering, it might be possible to develop a B . napus cultivar containing levels of erucic acid significantly above 66 mol% (>80 mol%) . In an attempt to increase the amounts of very-long-chain fatty acids (VLCFAs), and erucic acid in particular, in Canadian HEA B . napus cultivars, we have focused on two targets using a transgenic approach . We examined both the role/function of the Arabidopsis thaliana FAE1 (fatty acid elongase) gene by expressing it under the control of the seed-specific napin promoter in B . napus germplasm with analysis of the changes in VLCFA content in the seed oil of transgenic lines, and the performance of the yeast SLC1-1 (sphingolipid compensation mutant) in B . napus cv . Hero transgenic progeny in the field . Here, we report analyses of the contents of 22:1, total VLCFAand oil in the seed oil, as well as seed yield of the field-grown FAE1 and SLC1-1 B . napus cv . Hero progeny. Biochem Soc Trans, 2000 Dec, 28(6), 692 - 5 Expression in yeast of an acyl-CoA:diacylglycerol acyltransferase cDNA from Caenorhabditis elegans; Bouvier-Nave P et al.; We have identified a cDNA from the nematode worm Caenorhabditis elegans that encodes an acyl-CoA:diacylglycerol acyltransferase (DGAT) . Its expression in Saccharomyces cerevisiae resulted in an increase both in triacylglycerol content and in microsomal oleyl-CoA:diacylglycerol acyltransferase activity . Such effects were similar to those of characterized plant DGAT genes . This is the first DGAT gene isolated from an invertebrate . The phylogenetic relationships between DGATs and animal and yeast acyl-CoA:sterol acyltransferases are illustrated. Biochem J, 2001 Mar 1, 354(Pt 2), 407 - 12 Site-directed mutations in the mitochondrially encoded subunits I and III of yeast cytochrome oxidase; Meunier B; Since yeast is amenable to mitochondrial transformation, designed mutations can be introduced in the mitochondrially encoded subunits of the respiratory complexes . In the present work, six mutations have been introduced by the biolistic method into yeast (Saccharomyces cerevisiae) cytochrome oxidase subunits I and III . The effects of these mutations on respiratory growth competence, cytochrome oxidase activity and optical properties were then characterized . Firstly, the conserved glutamate Glu-243 in the D-channel of subunit I was replaced by an asparagine or an aspartate residue . The effects of the mutations showed that Glu-243, which is essential for proton movement in bacterial oxidases, is also required for the activity of the eukaryotic enzyme . Secondly, four mutations associated with human disease were introduced in yeast, allowing detailed analysis of their deleterious effects on cytochrome oxidase function: Met-273-->Thr, Ile-280-->Thr and Gly-317-->Ser, affecting residues located in or near the K-channel in subunit I, and a short in-frame deletion comprising residues Phe-102 to Phe-106 in subunit III (DeltaF102-F106) . The subunit III mutation was highly deleterious and abolished enzyme assembly . The change Gly-317-->Ser had no effect on respiratory function . However, mutations Met-273-->Thr and Ile-280-->Thr were mildly deleterious, decreased cytochrome oxidase activity and slightly perturbed the properties of the binuclear centre. Biochem J, 2001 Feb 1, 353(Pt 3), 681 - 8 Multiple polyamine transport systems on the vacuolar membrane in yeast; Tomitori H et al.; We recently identified a gene (TPO1, YLL028w) that encodes a polyamine transport protein on the vacuolar membrane in yeast {Tomitori, Kashiwagi, Sakata, Kakinuma and Igarashi (1999) J . Biol . Chem . 274, 3265-3267} . Because the existence of one or more other genes for a polyamine transport protein on the vacuolar membrane was expected, we searched sequence databases for homologues of the protein encoded by TPO1 . Membrane proteins encoded by the open reading frames YGR138c (TPO2), YPR156c (TPO3) and YOR273c (TPO4) were postulated to be polyamine transporters and, indeed, were subsequently shown to be polyamine transport proteins on the vacuolar membrane . Cells overexpressing these genes were resistant to polyamine toxicity and showed an increase in polyamine uptake activity and poly