Cell Mol Life Sci, 1999 Mar, 55(3), 437 - 49 Roles of the DNA mismatch repair and nucleotide excision repair proteins during meiosis; Kirkpatrick DT; Numerous proteins are involved in the nucleotide excision repair (NER) and DNA mismatch repair (MMR) pathways . The function and specificity of these proteins during the mitotic cell cycle has been actively investigated, in large part due to the involvement of these systems in human diseases . In contrast, comparatively little is known about their functioning during meiosis . At least three repair pathways operate during meiosis in the yeast Saccharomyces cerevisiae to repair mismatches that occur as a consequence of heteroduplex formation in recombination . The first pathway is similar to the one acting during postreplicative mismatch repair in mitotically dividing cells, while two pathways are responsible for the repair of large loops during meiosis, using proteins from MMR and NER systems . Some MMR proteins also help prevent recombination between diverged sequences during meiosis, and act late in recombination to affect the resolution of crossovers . This review will discuss the current status of DNA mismatch repair and nucleotide excision repair proteins during meiosis, especially in the yeast S . cerevisiae. EMBO J, 1999 May 4, 18(9), 2648 - 58 Sister chromatid-based DNA repair is mediated by RAD54, not by DMC1 or TID1; Arbel A et al.; In the mitotic cell cycle of the yeast Saccharomyces cerevisiae, the sister chromatid is preferred over the homologous chromosome (non-sister chromatid) as a substrate for DNA double-strand break repair . However, no genes have yet been shown to be preferentially involved in sister chromatid-mediated repair . We developed a novel method to identify genes that are required for repair by the sister chromatid, using a haploid strain that can embark on meiosis . We show that the recombinational repair gene RAD54 is required primarily for sister chromatid-based repair, whereas TID1, a yeast RAD54 homologue, and the meiotic gene DMC1, are dispensable for this type of repair . Our observations suggest that the sister chromatid repair pathway, which involves RAD54, and the homologous chromosome repair pathway, which involves DMC1, can substitute for one another under some circumstances . Deletion of RAD54 in S.cerevisiae results in a phenotype similar to that found in mammalian cells, namely impaired DNA repair and reduced recombination during mitotic growth, with no apparent effect on meiosis . The principal role of RAD54 in sister chromatid-based repair may also be shared by mammalian and yeast cells. Genetics, 1999 May, 152(1), 179 - 90 Interorganelle signaling is a determinant of longevity in Saccharomyces cerevisiae; Kirchman PA et al.; Replicative capacity, which is the number of times an individual cell divides, is the measure of longevity in the yeast Saccharomyces cerevisiae . In this study, a process that involves signaling from the mitochondrion to the nucleus, called retrograde regulation, is shown to determine yeast longevity, and its induction resulted in postponed senescence . Activation of retrograde regulation, by genetic and environmental means, correlated with increased replicative capacity in four different S . cerevisiae strains . Deletion of a gene required for the retrograde response, RTG2, eliminated the increased replicative capacity . RAS2, a gene previously shown to influence longevity in yeast, interacts with retrograde regulation in setting yeast longevity . The molecular mechanism of aging elucidated here parallels the results of genetic studies of aging in nematodes and fruit flies, as well as the caloric restriction paradigm in mammals, and it underscores the importance of metabolic regulation in aging, suggesting a general applicability. J Biol Chem, 1999 May 7, 274(19), 12979 - 83 SEC14-dependent secretion in Saccharomyces cerevisiae . Nondependence on sphingolipid synthesis-coupled diacylglycerol production; Stock SD et al.; The SEC14 gene in Saccharomyces cerevisiae encodes a phosphatidylinositol transfer protein required for secretory protein movement from the Golgi . Mutation of SAC1, a gene of unknown function, restores secretory flow in sec14-1(ts) strains . The existing model for the bypass of the sec14-1(ts) defect by sac1-22 involves stimulation of sphingolipid biosynthesis and, in particular, the synthesis of mannosyl-diinositolphosphoryl-ceramide with concomitant increases in Golgi diacylglycerol levels . To test this model, we disrupted IPT1, the mannosyl-diinositolphosphoryl-ceramide synthase of S . cerevisiae . Disruption of the IPT1 gene had no effect on the ability of sac1-22 to bypass sec14-1(ts) . Furthermore, sphingolipid analysis of sec14-1(ts) and sec14-1(ts) sac1-22 strains showed that mannosyl-diinositolphosphoryl-ceramide synthesis was not stimulated in the bypass mutant . However, the sec14-1(ts) strain had elevated mannosyl-monoinositolphosphoryl-ceramide levels, and the sec14-1(ts) sac1-22 strain showed an 8-fold increase in phosphatidylinositol 4-phosphate along with a decrease in phosphatidylinositol 4,5-bisphosphate . Cellular diacylglycerol levels, measured by {14C}acetate incorporation, did not differ between the sec14-1(ts) and the sec14-1 sac1-22 bypass strains, although disruption of IPT1 in the bypass strain resulted in reduced levels . These data indicate that phosphatidylinositol 4-phosphate, rather than mannosyl-diinositolphosphoryl-ceramide, accumulates in the sec14-1(ts) sac1-22 bypass strain, and that Golgi diacylglycerol accumulation is not required for bypass of the sec14-1(ts) growth and secretory phenotypes. Appl Environ Microbiol, 1999 May, 65(5), 1915 - 8 Expression of an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae; Han Y et al.; Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste . Our objectives were to express an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae and to determine the effects of glycosylation on the phytase's activity and thermostability . A 1.4-kb DNA fragment containing the coding region of the phyA gene was inserted into the expression vector pYES2 and was expressed in S . cerevisiae as an active, extracellular phytase . The yield of total extracellular phytase activity was affected by the signal peptide and the medium composition . The expressed phytase had two pH optima (2 to 2.5 and 5 to 5.5) and a temperature optimum between 55 and 60 degrees C, and it cross-reacted with a rabbit polyclonal antibody against the wild-type enzyme . Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 120 kDa and appeared to be more thermostable than the commercial enzyme . Deglycosylation of the phytase resulted in losses of 9% of its activity and 40% of its thermostability . The recombinant phytase was effective in hydrolyzing phytate phosphorus from corn or soybean meal in vitro . In conclusion, the phyA gene was expressed as an active, extracellular phytase in S . cerevisiae, and its thermostability was affected by glycosylation. FEMS Microbiol Lett, 1999 Apr 1, 173(1), 63 - 8 Cell-cell recognition and pheromone response of the yeast Saccharomyces globosus; Fujimura H; Sexual agglutination and pheromone interaction between cells of two mating types, a and alpha, in the yeast Saccharomyces globosus were studied . S . globosus was shown to produce mating-type-specific factors analogs to a- and alpha-mating pheromones of Saccharomyces cerevisiae and to undergo the sexual agglutination reaction between cells of two mating types . While the sexual agglutination of cells of different species was not observed, mating type a cells of each species were shown to respond to alpha-factors produced by the other species . Thus, the mating response of S . globosus was shown to be identical to what has been observed in two other species of the same genera: S . cerevisiae and Saccharomyces kluyveri. Proc Natl Acad Sci U S A, 1999 Apr 27, 96(9), 4780 - 5 Progress toward the evolution of an organism with an expanded genetic code; Liu DR et al.; Several significant steps have been completed toward a general method for the site-specific incorporation of unnatural amino acids into proteins in vivo . An "orthogonal" suppressor tRNA was derived from Saccharomyces cerevisiae tRNA2Gln . This yeast orthogonal tRNA is not a substrate in vitro or in vivo for any Escherichia coli aminoacyl-tRNA synthetase, including E . coli glutaminyl-tRNA synthetase (GlnRS), yet functions with the E . coli translational machinery . Importantly, S . cerevisiae GlnRS aminoacylates the yeast orthogonal tRNA in vitro and in E . coli, but does not charge E . coli tRNAGln . This yeast-derived suppressor tRNA together with yeast GlnRS thus represents a completely orthogonal tRNA/synthetase pair in E . coli suitable for the delivery of unnatural amino acids into proteins in vivo . A general method was developed to select for mutant aminoacyl-tRNA synthetases capable of charging any ribosomally accepted molecule onto an orthogonal suppressor tRNA . Finally, a rapid nonradioactive screen for unnatural amino acid uptake was developed and applied to a collection of 138 amino acids . The majority of glutamine and glutamic acid analogs under examination were found to be uptaken by E . coli . Implications of these results are discussed. Nucleic Acids Res, 1999 May 15, 27(10), 2181 - 8 A Saccharomyces cerevisiae RNA 5'-triphosphatase related to mRNA capping enzyme; Rodriguez CR et al.; The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: the RNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1) . Using computer homology searching, a S . cerevisiae gene was identified that encodes a protein resembling the C-terminal region of Cet1 . Accordingly, we designated this gene CTL1 (capping enzyme RNAtriphosphatase-like 1) . CTL1 is not essential for cell viability and no genetic or physical interactions with the capping enzyme genes were observed . The protein is found in both the nucleus and cytoplasm . Recombinant Ctl1 protein releases gamma-phosphate from the 5'-end of RNA to produce a diphosphate terminus . The enzyme is specific for polynucleotide RNA in the presence of magnesium, but becomes specific for nucleotide triphosphates in the presence of manganese . Ctl1 is the second member of the yeast RNA triphosphatase family, but is probably involved in an RNA processing event other than mRNA capping. FEBS Lett, 1999 Mar 19, 447(1), 65 - 70 Human members of the SCO1 gene family: complementation analysis in yeast and intracellular localization; Paret C et al.; Cytochrome c oxidase is a multiprotein complex in the mitochondrial membrane whose biogenesis requires a number of proteins besides the structural subunits . Several yeast proteins as well as a human disease-related protein have been reported which are involved in cytochrome c oxidase assembly . The S . cerevisiae Sco1p protein has been implicated in the transfer of copper to cytochrome c oxidase subunits Cox1p and/or Cox2p . Here we report on the complementation behavior in yeast of two recently identified ScSco1p homologs of chromosome 17 and chromosome 22 from human . When allotropically expressed in yeast, both genes fail to complement the lack of the ScSCO1 gene . However, a chimera of the N-terminal half of ScSco1p and the C-terminal half of the chromosome 17 homolog does substitute for the ScSco1p function . Interestingly, the respective chimera with the human homolog of chromosome 22 is not able to complement . Expression of EGFP fusions in HeLa cells shows that both human ScSco1p homologs are located in the mitochondria of human cells. Eur J Biochem, 1999 May, 261(3), 812 - 20 Identification of Delta12-fatty acid desaturase from arachidonic acid-producing mortierella fungus by heterologous expression in the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae; Sakuradani E et al.; Based on the sequence information for the omega3-desaturase genes (from Brassica napus and Caenorhabditis elegans), which are involved in the desaturation of linoleic acid (Delta9, Delta12-18 : 2) to alpha-linolenic acid (Delta9, Delta12, Delta15-18 : 3), a cDNA was cloned from the filamentous fungal strain, Mortierella alpina 1S-4, which is used industrially to produce arachidonic acid . Homology analysis with protein databases revealed that the amino acid sequence showed 43.7% identity as the highest match with the microsomal omega6-desaturase (from Glycine max, soybean), whereas it exhibited 38.9% identity with the microsomal omega3-desaturase (from soybean) . The evolutionary implications of these enzymes will be discussed . The cloned cDNA was confirmed to encode a Delta12-desaturase, which was involved in the desaturation of oleic acid (Delta9-18 : 1) to linoleic acid, by its expression in both the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae . Analysis of the fatty acid composition of yeast and fungus transformants demonstrated that linoleic acid (which was not contained in the control strain of S . cerevisiae) was accumulated in the yeast transformant and that the fungal transformant contained a large amount of linoleic acid (71.9%) . Genomic Southern blot analysis of the transformants with the Mortierella Delta12-desaturase gene as a probe confirmed integration of this gene into the genome of A . oryzae . The M . alpina 1S-4 Delta12-desaturase is the first example of a cloned nonplant Delta12-desaturase. J Cell Biol, 1999 Apr 19, 145(2), 291 - 304 Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA; Meeusen S et al.; Maintenance of mitochondrial DNA (mtDNA) during cell division is required for progeny to be respiratory competent . Maintenance involves the replication, repair, assembly, segregation, and partitioning of the mitochondrial nucleoid . MGM101 has been identified as a gene essential for mtDNA maintenance in S . cerevisiae, but its role is unknown . Using liquid chromatography coupled with tandem mass spectrometry, we identified Mgm101p as a component of highly enriched nucleoids, suggesting that it plays a nucleoid-specific role in maintenance . Subcellular fractionation, indirect immunofluorescence and GFP tagging show that Mgm101p is exclusively associated with the mitochondrial nucleoid structure in cells . Furthermore, DNA affinity chromatography of nucleoid extracts indicates that Mgm101p binds to DNA, suggesting that its nucleoid localization is in part due to this activity . Phenotypic analysis of cells containing a temperature sensitive mgm101 allele suggests that Mgm101p is not involved in mtDNA packaging, segregation, partitioning or required for ongoing mtDNA replication . We examined Mgm101p's role in mtDNA repair . As compared with wild-type cells, mgm101 cells were more sensitive to mtDNA damage induced by UV irradiation and were hypersensitive to mtDNA damage induced by gamma rays and H2O2 treatment . Thus, we propose that Mgm101p performs an essential function in the repair of oxidatively damaged mtDNA that is required for the maintenance of the mitochondrial genome. Biochem Biophys Res Commun, 1999 Apr 21, 257(3), 835 - 8 Involvement of Spt7p in vacuolar polyphosphate level of Saccharomyces cerevisiae; Nishimura K et al.; Saccharomyces cerevisiae became less sensitive to nickel by a defect of the SPT7 gene encoding a transcription factor . Initial rate of nickel uptake by whole cells of a SPT7-negative mutant FY963 was nearly equal to that of the parent strain FY61, and FY963 accumulated nickel about 1.7-fold of the value of FY61 when cultured in medium containing 0.1 mM NiCl2; most of which was sequestered into vacuoles . The pH gradient-driven nickel uptake by vacuolar membrane vesicles was not altered in FY963, but the amount of polyphosphate in vacuoles was highly elevated . Involvement of Spt7p in nickel detoxification through regulation of vacuolar polyphosphate level in S . cerevisiae was discussed . Biochem Soc Symp, 1999, 64, 13 - 27 Roles of the AMP-activated/SNF1 protein kinase family in the response to cellular stress; Hardie DG; The AMP-activated protein kinase (AMPK) in mammals, and its homologue in Saccharomyces cerevisiae, are activated by cellular stresses associated with ATP depletion . AMPK is a heterotrimer comprising a catalytic alpha subunit with associated beta and gamma subunits, these being homologous with the products of the SNF1, SIP1/SIP2/GAL83 and SNF4 genes in S . cerevisiae . The alpha subunit has at least two isoforms (alpha 1 and alpha 2), which differ in their AMP-dependence and subcellular localization, with alpha 2 complexes being partly nuclear . AMPK is activated allosterically by 5'-AMP, which also promotes phosphorylation and activation by an upstream kinase, and inhibits dephosphorylation and inactivation . Elevation of AMP always accompanies depletion of ATP due to the action of adenylate kinase . Since high ATP antagonizes the activating effects of AMP, the system behaves like a cellular 'fuel gauge' . It is activated by various types of stress associated with ATP depletion, such as hypoxia, heat shock, metabolic poisoning and, in muscle, exercise . AMPK phosphorylates multiple targets which switch off anabolic pathways and switch on alternative catabolic pathways . The yeast SNF1 complex is switched on by glucose starvation, and its targets include transcription factors that repress transcription of genes required for catabolism of alternative carbon sources. Yeast, 1999 Mar 15, 15(4), 295 - 309 Identification and characterization of individual cyclin-dependent kinase complexes from Saccharomyces cerevisiae; Shellman YG et al.; In S . cerevisiae, regulation of cell cycle progression is known to be carried out by a single cyclin-dependent kinase homologue, Cdc28p, acting at different stages of the cell cycle in association with various cyclins and other regulatory subunits . However, a still unsolved problem is the identification of the physiologically relevant substrates of the different Cdc28p kinase complexes which participate in this regulation . Purification and characterization of the subunit composition and enzymological properties of these Cdc28p complexes would therefore contribute substantially to our understanding of the molecular mechanisms controlling the cell cycle . We have used a combination of ammonium sulphate fractionation, nickel nitrilotriacetate affinity purification, ATP Sepharose affinity chromatography and Resource Q ion exchange chromatography to purify two different Cdc28p kinase complexes . Using specific clb deletion mutants and plasmid or genomic HA epitope-tagged CLBs, we show that one of these complexes is composed almost exclusively (93% or greater) of Clb2p-Cdc28p, whereas the other is mainly (75% or greater) Clb3p-Cdc28p . These procedures provide the basis for the analysis of regulatory, enzymatic and functional properties of individual Cdc28p kinase complexes. Mol Cell Biol, 1999 May, 19(5), 3580 - 7 Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70; Robinson NG et al.; The Rho3 protein plays a critical role in the budding yeast Saccharomyces cerevisiae by directing proper cell growth . Rho3 appears to influence cell growth by regulating polarized secretion and the actin cytoskeleton, since rho3 mutants exhibit large rounded cells with an aberrant actin cytoskeleton . To gain insights into how Rho3 influences these events, we have carried out a yeast two-hybrid screen using an S . cerevisiae cDNA library to identify proteins interacting with Rho3 . Two proteins, Exo70 and Myo2, were identified in this screen . Interactions with these two proteins are greatly reduced or abolished when mutations are introduced into the Rho3 effector domain . In addition, a type of mutation known to produce dominant negative mutants of Rho proteins abolished the interaction with both of these proteins . In contrast, Rho3 did not interact with protein kinase C (Pkc1), an effector of another Rho family protein, Rho1, nor did Rho1 interact with Exo70 or Myo2 . Rho3 did interact with Bni1, another effector of Rho1, but less efficiently than with Rho1 . The interaction between Rho3 and Exo70 and between Rho3 and Myo2 was also demonstrated with purified proteins . The interaction between Exo70 and Rho3 in vitro was dependent on the presence of GTP, since Rho3 complexed with guanosine 5'-O-(3-thiotriphosphate) interacted more efficiently with Exo70 than Rho3 complexed with guanosine 5'-O-(3-thiodiphosphate) . Overlapping subcellular localization of the Rho3 and Exo70 proteins was demonstrated by indirect immunofluorescence . In addition, patterns of localization of both Exo70 and Rho3 were altered when a dominant active allele of RHO3, RHO3(E129,A131), which causes a morphological abnormality, was expressed . These results provide a direct molecular basis for the action of Rho3 on exocytosis and the actin cytoskeleton. EMBO J, 1999 Apr 1, 18(7), 1974 - 81 Translation termination efficiency can be regulated in Saccharomyces cerevisiae by environmental stress through a prion-mediated mechanism; Eaglestone SS et al.; {PSI+} is a protein-based heritable phenotype of the yeast Saccharomyces cerevisiae which reflects the prion-like behaviour of the endogenous Sup35p protein release factor . {PSI+} strains exhibit a marked decrease in translation termination efficiency, which permits decoding of translation termination signals and, presumably, the production of abnormally extended polypeptides . We have examined whether the {PSI+}-induced expression of such an altered proteome might confer some selective growth advantage over {psi-} strains . Although otherwise isogenic {PSI+} and {psi-} strains show no difference in growth rates under normal laboratory conditions, we demonstrate that {PSI+} strains do exhibit enhanced tolerance to heat and chemical stress, compared with {psi-} strains . Moreover, we also show that the prion-like determinant {PSI+} is able to regulate translation termination efficiency in response to environmental stress, since growth in the presence of ethanol results in a transient increase in the efficiency of translation termination and a loss of the {PSI+} phenotype . We present a model to describe the prion-mediated regulation of translation termination efficiency and discuss its implications in relation to the potential physiological role of prions in S.cerevisiae and other fungi. Cell Death Differ, 1999 Feb, 6(2), 124 - 9 Alix, a novel mouse protein undergoing calcium-dependent interaction with the apoptosis-linked-gene 2 (ALG-2) protein; Missotten M et al.; ALG-2 is a EF hand calcium binding protein with sequence homologies to calmodulin . Vito et al have shown that ALG-2 expression is required for apoptosis following a number of death stimuli,1 although nothing is known about the effectors which underlie ALG-2 function . Here we have used ALG-2 as bait in a yeast two hybrid screen of a mouse brain cDNA library . We found that ALG-2 binds to itself and to a novel protein that we call ALG-2 interacting protein X, Alix . Using co-immunoprecipitation experiments, we confirmed ALG-2/ALG-2 binding and demonstrated that this interaction is calcium independent . ALG-2/Alix interaction was also validated by co-immunoprecipitation, but in this case, the binding was found to be strictly calcium dependent . Alix seems highly conserved throughout evolution since it shows significant homologies to a putative C . elegans protein (YNK-1) and to proteins of A . nidulans (PalA) and S . cerevisiae (BRO1) . Alix is a potential regulator or downstream effector of ALG-2 action. Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4390 - 5 RNA polymerase switch in transcription of yeast rDNA: role of transcription factor UAF (upstream activation factor) in silencing rDNA transcription by RNA polymerase II; Vu L et al.; Transcription factor UAF (upstream activation factor) is required for a high level of transcription, but not for basal transcription, of rDNA by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae . RRN9 encodes one of the UAF subunits . We have found that rrn9 deletion mutants grow extremely slowly but give rise to faster growing variants that can grow without intact Pol I, synthesizing rRNA by using RNA polymerase II (Pol II) . This change is reversible and does not involve a simple mutation . The two alternative states, one suitable for rDNA transcription by Pol I and the other favoring rDNA transcription by Pol II, are heritable not only in mitosis, but also in meiosis . Thus, S . cerevisiae has an inherent ability to transcribe rDNA by Pol II, but this transcription activity is silenced in normal cells, and UAF plays a key role in this silencing by stabilizing the first state. J Bacteriol, 1999 Apr, 181(8), 2620 - 3 The lactate-proton symport of Saccharomyces cerevisiae is encoded by JEN1; Casal M et al.; A mutant of Saccharomyces cerevisiae deficient in the lactate-proton symport was isolated . Transformation of the mutant with a yeast genomic library allowed the isolation of the gene JEN1 that restored lactate transport . Disruption of JEN1 abolished uptake of lactate . The results indicate that, under the experimental conditions tested, no other monocarboxylate permease is able to efficiently transport lactate in S . cerevisiae. J Biol Chem, 1999 Apr 16, 274(16), 11296 - 302 Disruption of YHC8, a member of the TSR1 gene family, reveals its direct involvement in yeast protein translocation; Mamoun CB et al.; Genetic studies of Saccharomyces cerevisiae have identified many components acting to deliver specific proteins to their cellular locations . Genome analysis, however, has indicated that additional genes may also participate in such protein trafficking . The product of the yeast Yarrowia lipolytica TSR1 gene promotes the signal recognition particle-dependent translocation of secretory proteins through the endoplasmic reticulum . Here we describe the identification of a new gene family of proteins that is well conserved among different yeast species . The TSR1 genes encode polypeptides that share the same protein domain distribution and, like Tsr1p, may play an important role in the early steps of the signal recognition particle-dependent translocation pathway . We have identified five homologues of the TSR1 gene, four of them from the yeast Saccharomyces cerevisiae and the other from Hansenula polymorpha . We generated a null mutation in the S . cerevisiae YHC8 gene, the closest homologue to Y . lipolytica TSR1, and used different soluble (carboxypeptidase Y, alpha-factor, invertase) and membrane (dipeptidyl-aminopeptidase) secretory proteins to study its phenotype . A large accumulation of soluble protein precursors was detected in the mutant strain . Immunofluorescence experiments show that Yhc8p is localized in the endoplasmic reticulum . We propose that the YHC8 gene is a new and important component of the S . cerevisiae endoplasmic reticulum membrane and that it functions in protein translocation/insertion of secretory proteins through or into this compartment. Biochem J, 1999 Apr 15, 339 ( Pt 2), 291 - 8 Cloning and expression of a human choline/ethanolaminephosphotransferase: synthesis of phosphatidylcholine and phosphatidylethanolamine; Henneberry AL et al.; Cholinephosphotransferase catalyses the final step in the synthesis of phosphatidylcholine (PtdCho) via the Kennedy pathway by the transfer of phosphocholine from CDP-choline to diacylglycerol . Ethanolaminephosphotransferase catalyses an analogous reaction with CDP-ethanolamine as the phosphobase donor for the synthesis of phosphatidylethanolamine (PtdEtn) . Together these two enzyme activities determine both the site of synthesis and the fatty acyl composition of PtdCho and PtdEtn synthesized de novo . A human choline/ethanolaminephosphotransferase cDNA (hCEPT1) was cloned, expressed and characterized . Northern blot analysis revealed one hCEPT1 2.3 kb transcript that was ubiquitous and not enriched, with respect to actin, in any particular cell type . The open reading frame predicts a protein (hCEPT1p) of 416 amino acid residues with a molecular mass of 46550 Da containing seven membrane-spanning domains . A predicted amphipathic helix resides within the active site of the enzyme with the final two aspartic residues of the CDP-alcohol phosphotransferase motif, DG(X)2AR(X)8G(X)3D(X)3D, positioned within this helix . hCEPT1p was successfully expressed in a full-length, active form in Saccharomyces cerevisiae cells devoid of endogenous cholinephosphotransferase or ethanolaminephosphotransferase activities (HJ091, cpt1::LEU2 ept1-) . In vitro, hCEPT1p displayed broad substrate specificity, utilizing both CDP-choline and CDP-ethanolamine as phosphobase donors to a broad range of diacylglycerols, resulting in the synthesis of both PtdCho and PtdEtn . In vivo, S . cerevisiae cells (HJ091, cpt1::LEU2 ept1-) expressing hCEPT1 efficiently incorporated both radiolabelled choline and ethanolamine into phospholipids, demonstrating that hCEPT1p has the ability to synthesize both choline- and ethanolamine- containing phospholipids in vitro and in vivo. Syst Appl Microbiol, 1999 Feb, 22(1), 145 - 55 Characterization of Saccharomyces cerevisiae CBS 7764 isolated from rainbow trout intestine; Andlid T et al.; A wild-type Saccharomyces cerevisiae, strain CBS 7764, isolated from the intestine of rainbow trout, was analyzed with respect to general growth parameters and global protein expression . Characterization of this strain was of interest since previous data show non-typical S . cerevisiae cell surface properties and because data suggest a probiotic potential of CBS 7764 . The heat production rate (dQ/dt), monitored by microcalorimetry, showed that the typical growth phases resulting from diauxic growth on glucose were present in the fish isolate . However, CBS 7764 differentiated from a reference strain by becoming limited in the respiratory phase as demonstrated by a plateau in the dQ/dt signal . The global protein expression, as studied by two-dimensional gel electrophoresis (2D-PAGE), revealed a large degree of resemblance of the fish isolate to the reference strain, however, also clear qualitative and quantitative expression differences were detected; e.g . 14% of the proteins differed in expression level by a factor of at least 2 . In addition, the fish isolate expressed 12 unique proteins . The heat shock proteins, which for other organisms have been identified as important in mucosal colonization, were generally expressed to a higher level in CBS 7764. Eur J Biochem, 1999 Apr, 261(1), 317 - 24 Pregnenolone esterification in Saccharomyces cerevisiae . A potential detoxification mechanism; Cauet G et al.; While studying the effect of steroids on the growth of the yeast Saccharomyces cerevisiae, we found that pregnenolone was converted into the acetate ester . This reaction was identified as a transfer of the acetyl group from acetyl-CoA to the 3beta-hydroxyl group of pregnenolone . The corresponding enzyme, acetyl-CoA:pregnenolone acetyltransferase (APAT) is specific for Delta5- or Delta4-3beta-hydroxysteroids and short-chain acyl-CoAs . The apparent Km for pregnenolone is approximately 0.5 microm . The protein associated with APAT activity was partially purified and finally isolated from an SDS/polyacrylamide gel . Tryptic peptides were generated and N-terminally sequenced . Two peptide sequences allowed the identification of an open reading frame (YGR177c, in the S . cerevisiae genome database) translating into a 62-kDa protein of hitherto unknown function . This protein encoded by a gene known as ATF2 displays 37% identity with an alcohol acetyltransferase encoded by the yeast gene ATF1 . Disruption of ATF2 led to the complete elimination of APAT activity and consequently abolished the esterification of pregnenolone . In addition, a toxic effect of pregnenolone linked to the disruption of ATF2 was observed . Pregnenolone toxicity is more pronounced when the atf2-Delta mutation is introduced in a yeast strain devoid of the ATP-binding cassette transporters, PDR5 and SNQ2 . Our results suggest that Atf2p (APAT) plays an active role in the detoxification of 3beta-hydroxysteroids in association with the efflux pumps Pdr5p and Snq2p. Genetics, 1999 Apr, 151(4), 1341 - 51 Tempo and mode of Ty element evolution in Saccharomyces cerevisiae; Jordan IK et al.; The Saccharomyces cerevisiae genome contains five families of long terminal repeat (LTR) retrotransposons, Ty1-Ty5 . The sequencing of the S . cerevisiae genome provides an unprecedented opportunity to examine the patterns of molecular variation existing among the entire genomic complement of Ty retrotransposons . We report the results of an analysis of the nucleotide and amino acid sequence variation within and between the five Ty element families of the S . cerevisiae genome . Our results indicate that individual Ty element families tend to be highly homogenous in both sequence and size variation . Comparisons of within-element 5' and 3' LTR sequences indicate that the vast majority of Ty elements have recently transposed . Furthermore, intrafamily Ty sequence comparisons reveal the action of negative selection on Ty element coding sequences . These results taken together suggest that there is a high level of genomic turnover of S . cerevisiae Ty elements, which is presumably in response to selective pressure to escape host-mediated repression and elimination mechanisms. Biotechnol Bioeng, 1999 May 5, 63(3), 290 - 7 Virus-like particle analysis in yeast homogenate using a laser light-scattering assay; Tsoka S et al.; Virus-like particles (VLPs) expressed intracellularly by the yeast S . cerevisiae have helped set the framework of a wide range of biologicals, particularly as carriers for viral antigens . This article investigates the use of dynamic light scattering (DLS) for the rapid evaluation of the concentration and purity of VLPs to aid the complex purification strategy . Development of the assay was performed in a high background process stream (yeast homogenate) and involved a change in the signal proportional to the VLP concentration by addition of antibodies that bind on the VLP surface and detection of that size change by DLS . Overall, the assay was found to provide a significant improvement of rapid monitoring alternatives for VLPs, exhibiting good sensitivity and speed of measurement . Data are given for the use of the DLS-based assay for optimization of VLP release during a yeast cell disruption treatment . Biotechnol Bioeng, 1998 Sep 20, 59(6), 754 - 61 Optimization of polyphosphate degradation and phosphate secretion using hybrid metabolic pathways and engineered host strains Van Dien SJ, Keasling JD. Polyphosphate degradation and phosphate secretion were optimized in Escherichia coli strains overexpressing the E . coli polyphosphate kinase gene (ppk) and either the E . coli polyphosphatase gene (ppx) or the Saccharomyces cerevisiae polyphosphatase gene (scPPX1) from different inducible promoters on medium- and high-copy plasmids . The use of a host strain without functional ppk or ppx genes on the chromosome yielded the highest levels of polyphosphate, as well as the fastest degradation of polyphosphate when the gene for polyphosphatase was induced . The introduction of a hybrid metabolic pathway consisting of the E . coli ppk gene and the S . cerevisiae polyphosphatase gene resulted in lower polyphosphate concentrations than when using both the ppk and ppx genes from E . coli, and did not significantly improve the degradation rate . It was also found that the rate of polyphosphate degradation was highest when ppx was induced late in growth, most likely due to the high intracellular polyphosphate concentration . The phosphate released from polyphosphate allowed the growth of phosphate-starved cells; excess phosphate was secreted into the medium, leading to a down-regulation of the phosphate-starvation (Pho) response . The production of alkaline phosphatase, an indicator of the Pho response, can be precisely controlled by manipulating the degree of ppx induction . Mol Cell Biochem, 1999 Jan, 191(1-2), 161 - 7 Transcriptional regulation of the S . cerevisiae ENA1 gene by casein kinase II; Tenney KA et al.; The regulatory subunit of S . cerevisiae casein kinase II (CKII) is encoded of two genes, CKB1 and CKB2 . Strains harboring deletions of either or both genes exhibit specific sensitivity to high concentrations of Na+ or Li+ . Na+ tolerance in S . cerevisiae is mediated primarily by transcriptional induction of ENA1, which encodes the plasma membrane sodium pump, and by conversion of the potassium uptake system to a higher affinity form that discriminates more efficiently against Na+ . To determine whether reduced ENA1 expression plays a role in the salt sensitivity of ckb mutants, we integrated an ENA1-lacZ reporter gene into isogenic wild-type, ckb1, ckb2, and ckb1 ckb2 strains and monitored beta-galactosidase activity at different salt concentrations . In all three mutants transcription from the ENA1 promoter remained salt-inducible, but both basal and salt-induced expression was depressed approximately 3- to 4-fold . The degree of reduction in ENA1 expression was comparable to that observed in an isogenic strain carrying a null mutation in protein phosphatase 2B (calcineurin), which is also required for salt tolerance . These results suggest that reduced expression ofENA1 contributes to the salt sensitivity of ckb strains . Consistent with this conclusion, overexpression of ENA1 from a heterologous promoter (GAL1) completely suppressed the salt sensitivity of ckb mutants . Induction of ENA1 expression by alkaline pH is also depressed in ckb mutants, but unlike calcineurin mutants, ckb strains are not growth inhibited by alkaline pH. Biochem Biophys Res Commun, 1999 Apr 2, 257(1), 139 - 44 Molecular and functional study of AQY1 from Saccharomyces cerevisiae: role of the C-terminal domain; Laize V et al.; The yeast YPR192w gene, which encodes a protein (Aqy1p) with strong homology to aquaporins (AQPs), was cloned from nine S . cerevisiae strains . The osmotic water permeability coefficient (Pf) of X . laevis oocytes expressing the gene cloned from the Sigma1278b strain (AQY1-1) was 5.7 times higher than the Pf of oocytes expressing the gene cloned from other strains (AQY1-2) . Aqy1-1p, initially cloned without its C-terminus (Aqy1-1DeltaCp), mediated an approximately 3 times higher water permeability than the full-length protein . This corresponds to a 3-fold higher protein density in the oocyte plasma membrane, as shown by freeze-fracture electron microscopy . Pf measurements in yeast spheroplasts confirmed the presence of functional water channels in Sigma1278b and a pharmacological study indicated that this strain contains at least a second functional aquaporin . Biosystems, 1999 Jan, 49(1), 63 - 70 Fractal properties of DNA walks; Abramson G et al.; We describe two dimensional DNA walks, and analyze their fractal properties . We show results for the complete genome of S . cerevisiae . We find that the mean square deviation of the walks is superdifussive, corresponding to a fractal structure of dimension lower than two . Furthermore, the coding part of the genome seems to have smaller fractal dimension, and longer correlations, than noncoding parts. J Cell Biol, 1999 Mar 8, 144(5), 823 - 37 The beta4 integrin interactor p27(BBP/eIF6) is an essential nuclear matrix protein involved in 60S ribosomal subunit assembly; Sanvito F et al.; p27(BBP/eIF6) is an evolutionarily conserved protein that was originally identified as p27(BBP), an interactor of the cytoplasmic domain of integrin beta4 and, independently, as the putative translation initiation factor eIF6 . To establish the in vivo function of p27(BBP/eIF6), its topographical distribution was investigated in mammalian cells and the effects of disrupting the corresponding gene was studied in the budding yeast, Saccharomyces cerevisiae . In epithelial cells containing beta4 integrin, p27(BBP/eIF6) is present in the cytoplasm and enriched at hemidesmosomes with a pattern similar to that of beta4 integrin . Surprisingly, in the absence and in the presence of the beta4 integrin subunit, p27(BBP/eIF6) is in the nucleolus and associated with the nuclear matrix . Deletion of the IIH S . cerevisiae gene, encoding the yeast p27(BBP/eIF6) homologue, is lethal, and depletion of the corresponding gene product is associated with a dramatic decrease of the level of free ribosomal 60S subunit . Furthermore, human p27(BBP/eIF6) can rescue the lethal effect of the iihDelta yeast mutation . The data obtained in vivo suggest an evolutionarily conserved function of p27(BBP/eIF6) in ribosome biogenesis or assembly rather than in translation . A further function related to the beta4 integrin subunit may have evolved specifically in higher eukaryotic cells. Biochem J, 1999 Apr 1, 339 ( Pt 1), 185 - 92 Mammalian PIG-L and its yeast homologue Gpi12p are N-acetylglucosaminylphosphatidylinositol de-N-acetylases essential in glycosylphosphatidylinositol biosynthesis; Watanabe R et al.; Glycosylphosphatidylinositol (GPI) is used as a membrane anchor by many eukaryotic cell-surface proteins . The second step of GPI biosynthesis is de-N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) . We have previously cloned the rat PIG-L gene by expression cloning that complemented a mutant Chinese hamster ovary cell line defective in this step . Here we show that recombinant rat PIG-L protein purified from Escherichia coli as a complex with GroEL has GlcNAc-PI de-N-acetylase activity in vitro . The activity was not enhanced by GTP, which is known to enhance the de-N-acetylase activity of mammalian cell microsomes . As with other de-N-acetylases that act on the GlcNAc moiety, metal ions, in particular Mn2+ and Ni2+, enhanced the enzyme activity of PIG-L . The Saccharomyces cerevisiae YMR281W open reading frame encodes a protein (termed Gpi12p) with 24% amino acid identity with rat PIG-L . On transfection into mammalian PIG-L-deficient cells, this gene, GPI12, restored the cell-surface expression of GPI-anchored proteins and GlcNAc-PI de-N-acetylase activity . The disruption of the gene caused lethality in S . cerevisiae . These results indicate that GlcNAc-PI de-N-acetylase is conserved between mammals and yeasts and that the de-N-acetylation step is also indispensable in yeasts. J Biol Chem, 1999 Mar 26, 274(13), 8850 - 7 Mitochondrial telomere-binding protein from Candida parapsilosis suggests an evolutionary adaptation of a nonspecific single-stranded DNA-binding protein; Nosek J et al.; The mitochondrial genome in a number of organisms is represented by linear DNA molecules with defined terminal structures . The telomeres of linear mitochondrial DNA (mtDNA) of yeast Candida parapsilosis consist of tandem arrays of large repetitive units possessing single-stranded 5' extension of about 110 nucleotides . Recently we identified the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of C . parapsilosis linear mtDNA and protects it from attack by various DNA-modifying enzymes (Tomaska, L'., Nosek, J., and Fukuhara, H . (1997) J . Biol . Chem . 272, 3049-3059) . Here we report the isolation of MTP1, the gene encoding mtTBP of C . parapsilosis . Sequence analysis revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded DNA-binding proteins that nonspecifically bind to single-stranded DNA with high affinity . Recombinant mtTBP displays a preference for the telomeric 5' overhang of C . parapsilosis mtDNA . The heterologous expression of a mtTBP-GFP fusion protein resulted in its localization to the mitochondria but was unable to functionally substitute for the loss of the S . cerevisiae homologue Rimlp . Analysis of the MTP1 gene and its translation product mtTBP may provide an insight into the evolutionary origin of linear mitochondrial genomes and the role it plays in their replication and maintenance. J Biol Chem, 1999 Mar 26, 274(13), 8604 - 10 The Saccharomyces cerevisiae YOR163w gene encodes a diadenosine 5', 5"'-P1,P6-hexaphosphate (Ap6A) hydrolase member of the MutT motif (Nudix hydrolase) family; Cartwright JL et al.; The YOR163w open reading frame on chromosome XV of the Saccharomyces cerevisiae genome encodes a member of the MutT motif (nudix hydrolase) family of enzymes of Mr 21,443 . By cloning and expressing this gene in Escherichia coli and S . cerevisiae, we have shown the product to be a (di)adenosine polyphosphate hydrolase with a previously undescribed substrate specificity . Diadenosine 5',5"'-P1, P6-hexaphosphate is the preferred substrate, and hydrolysis in H218O shows that ADP and adenosine 5'-tetraphosphate are produced by attack at Pbeta and AMP and adenosine 5'-pentaphosphate are produced by attack at Palpha with a Km of 56 microM and kcat of 0.4 s-1 . Diadenosine 5',5"'-P1,P5-pentaphosphate, adenosine 5'-pentaphosphate, and adenosine 5'-tetraphosphate are also substrates, but not diadenosine 5',5"'-P1,P4-tetraphosphate or other dinucleotides, mononucleotides, nucleotide sugars, or nucleotide alcohols . The enzyme, which was shown to be expressed in log phase yeast cells by immunoblotting, displays optimal activity at pH 6.9, 50 degrees C, and 4-10 mM Mg2+ (or 200 microM Mn2+) . It has an absolute requirement for a reducing agent, such as dithiothreitol (1 mM), and is inhibited by Ca2+ with an IC50 of 3.3 mM and F- (noncompetitively) with a Ki of 80 microM . Its function may be to eliminate potentially toxic dinucleoside polyphosphates during sporulation. Mol Cell Biol, 1999 Apr, 19(4), 2967 - 76 An activator binding module of yeast RNA polymerase II holoenzyme; Lee YC et al.; The Mediator complex of Saccharomyces cerevisiae is required for both general and regulated transcription of RNA polymerase II (PolII) and is composed of two stable subcomplexes (Srb4 and Rgr1 subcomplexes) . To decipher the function of each Mediator subcomplex and to delineate the functional relationship between the subcomplexes, we characterized the compositions and biochemical activities of PolII-Mediator complexes (holoenzymes) prepared from several Mediator mutant strains of S . cerevisiae . We found that holoenzymes devoid of a functional Gal11 module were defective for activated but not basal transcription in a reconstituted in vitro system . This activation-specific defect was correlated with a crippled physical interaction to transcriptional activator proteins, which could be bypassed by artificial recruitment of a mutant holoenzyme to a promoter . Consistent with this observation, a direct interaction between Gal11 and gene-specific transcriptional activator proteins was detected by far-Western analyses and column binding assays . In contrast, the srb5 deletion mutant holoenzyme was defective for both basal and activated transcription, despite its capacity for activator binding that is comparable to that of the wild-type holoenzyme . These results demonstrate that the Gal11 module of the Rgr1 subcomplex is required for the efficient recruitment of PolII holoenzyme to a promoter via activator-specific interactions, while the Srb4 subcomplex functions in the modulation of general polymerase activity. Mol Cell Biol, 1999 Apr, 19(4), 2929 - 35 Overlapping specificities of base excision repair, nucleotide excision repair, recombination, and translesion synthesis pathways for DNA base damage in Saccharomyces cerevisiae; Swanson RL et al.; The removal of oxidative damage from Saccharomyces cerevisiae DNA is thought to be conducted primarily through the base excision repair pathway . The Escherichia coli endonuclease III homologs Ntg1p and Ntg2p are S . cerevisiae N-glycosylase-associated apurinic/apyrimidinic (AP) lyases that recognize a wide variety of damaged pyrimidines (H . J . You, R . L . Swanson, and P . W . Doetsch, Biochemistry 37:6033-6040, 1998) . The biological relevance of the N-glycosylase-associated AP lyase activity in the repair of abasic sites is not well understood, and the majority of AP sites in vivo are thought to be processed by Apn1p, the major AP endonuclease in yeast . We have found that yeast cells simultaneously lacking Ntg1p, Ntg2p, and Apn1p are hyperrecombinogenic (hyper-rec) and exhibit a mutator phenotype but are not sensitive to the oxidizing agents H2O2 and menadione . The additional disruption of the RAD52 gene in the ntg1 ntg2 apn1 triple mutant confers a high degree of sensitivity to these agents . The hyper-rec and mutator phenotypes of the ntg1 ntg2 apn1 triple mutant are further enhanced by the elimination of the nucleotide excision repair pathway . In addition, removal of either the lesion bypass (Rev3p-dependent) or recombination (Rad52p-dependent) pathway specifically enhances the hyper-rec or mutator phenotype, respectively . These data suggest that multiple pathways with overlapping specificities are involved in the removal of, or tolerance to, spontaneous DNA damage in S . cerevisiae . In addition, the fact that these responses to induced and spontaneous damage depend upon the simultaneous loss of Ntg1p, Ntg2p, and Apn1p suggests a physiological role for the AP lyase activity of Ntg1p and Ntg2p in vivo. Adv Exp Med Biol, 1999, 448, 247 - 54 Intracellular pathways of copper trafficking in yeast and humans; Culotta VC et al.; In the bakers yeast S . cerevisiae, there at least four intracellular targets requiring copper ions-1) Ccc2p and Fet3p in the secretory pathway (homologues to Menkes/Wilson proteins and ceruloplasmin); 2) cytochrome oxidase in the mitochondria; 3) copper transcription factors in the nucleus; and 4) Cu/Zn superoxide dismutase (SOD1) in the cytosol . We have discovered a small soluble copper carrier that specifically delivers copper ions to the secretory pathway . This 8.2 kDa factor known as Atx1p, exhibits striking homology to the MERp mercury carrier of bacteria and contains a single MTCXXC metal binding site also found in the Menkes/Wilson family of copper transporting ATPases . Our studies show that Atx1p is cytosolic and facilitates the delivery of copper ions from the cell surface copper transporter to Ccc2p and Fet3p in the secretory pathway; furthermore, it is not involved in the delivery of copper ions to the mitochondria, the nucleus or cytosolic SOD1, implicating specific signals directing Atx1p to the secretory pathway . Homologues to Atx1p have been found in invertebrates, plants and humans, and the human gene is abundantly expressed in all tissues . In addition to Atx1p, we have recently uncovered an additional metal trafficking protein that appears to specifically deliver copper ions to SOD1 . Mutants in the corresponding gene (lys7) are defective for SOD1 activity, and are unable to incorporate copper into SOD1, while there is no obvious impairment in copper delivery to cytochrome oxidase of Fet3p . The encoded 27 kDa protein contains a single MHCXXC consensus copper binding sequence and close homologues have been identified in a wide array of eukaryotic species including humans. Curr Genet, 1999 Mar, 35(2), 103 - 8 Mitochondrial copper metabolism in yeast: mutational analysis of Sco1p involved in the biogenesis of cytochrome c oxidase; Rentzsch A et al.; Saccharomyces cerevisiae Sco1p is believed to be involved in the transfer of copper from the carrier Cox17p to the mitochondrial cytochrome c oxidase subunits 1 and 2 . We here report on the results of a mutational analysis of Sco1p . The two cysteine residues of a potential metal-binding motif (CxxxC) are essential for protein function as shown by their substitution by alanines . Chimeras consisting of Sco1p and its homolog S . cerevisiae Sco2p restrict the specificity of Sco1p function to the N-terminal half of the protein . A candidate region for conferring specificity on Sco1p is a stretch of hydrophobic amino acids, which act as a membrane anchor . In line with this suggestion is the result that alterations of individual amino acids within this region impair Sco1p function. Mol Gen Genet, 1999 Feb, 261(1), 11 - 20 Functional analysis of multiple AUG codons in the transcripts of the STA2 glucoamylase gene from Saccharomyces cerevisiae; Vivier MA et al.; A scanning ribosome will usually initiate translation as soon as it encounters the first favourable AUG codon and only a few eukaryotic transcripts have more complex arrangements . These relatively few complex transcripts are normally characterized by structural features such as multiple AUGs and significant secondary structure . However, the functional relevance of these features has rarely been established . We present here a study of the functional significance of the multiple AUGs in the leader of STA2 transcripts of the budding yeast Saccharomyces cerevisiae, and extrapolate, where applicable, these results to a co-regulated gene, MUC1 . The STA2 gene (a representative member of the polymorphic STA1-3 gene family), encodes an extracellular glucoamylase, and is evolutionarily linked to, and transcriptionally co-regulated with, the MUC1 gene, which encodes a mucin-like protein essential for pseudohyphal/invasive growth and cell-adhesion in S . cerevisiae . Each of these genes contains a putative upstream ORF, while STA2 has two additional in-frame AUG codons 5' to the major cistron . We show that utilization of the alternative translational start-sites of STA2 results in glucoamylases that differ at their N-termini, which are associated with differences in their localization patterns . Analysis of mutants revealed the presence of a putative secretion-enhancing signal that might prove to be relevant to the alternative targeting mechanism recently uncovered in S . cerevisiae . We show that a short up-stream ORF present in the leaders of STA1-3 and MUC1 is probably bypassed by a process of leaky scanning. Mol Biol Cell, 1999 Mar, 10(3), 581 - 96 GCS1, an Arf guanosine triphosphatase-activating protein in Saccharomyces cerevisiae, is required for normal actin cytoskeletal organization in vivo and stimulates actin polymerization in vitro; Blader IJ et al.; Recent cloning of a rat brain phosphatidylinositol 3,4, 5-trisphosphate binding protein, centaurin alpha, identified a novel gene family based on homology to an amino-terminal zinc-binding domain . In Saccharomyces cerevisiae, the protein with the highest homology to centaurin alpha is Gcs1p, the product of the GCS1 gene . GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth . Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects . Here, we have shown that similar to centaurin alpha, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity . A novel GCS1 disruption strain (gcs1Delta) exhibited morphological defects, as well as mislocalization of cortical actin patches . gcs1Delta was hypersensitive to the actin monomer-sequestering drug, latrunculin-B . Synthetic lethality was observed between null alleles of GCS1 and SLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton . In addition, synthetic growth defects were observed between null alleles of GCS1 and SAC6, the gene encoding the yeast fimbrin homologue . Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro . These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S . cerevisiae. J Mol Biol, 1999 Mar 12, 286(5), 1507 - 17 Sulphate ions observed in the 2.12 A structure of a new crystal form of S . cerevisiae phosphoglycerate mutase provide insights into understanding the catalytic mechanism; Rigden DJ et al.; The structure of a new crystal form of Saccharomyces cerevisiae phosphoglycerate mutase has been solved and refined to 2.12 A with working and free R-factors of 19.7 and 22.9 %, respectively . Higher-resolution data and greater non-crystallographic symmetry have produced a more accurate protein structure than previously . Prominent among the differences from the previous structure is the presence of two sulphate ions within each active site cleft . The separation of the sulphates suggests that they may occupy the same sites as phospho groups of the bisphosphate ligands of the enzyme . Plausible binding modes for 2,3-bisphosphoglycerate and 1, 3-bisphosphoglycerate are thereby suggested . These results support previous conclusions from mutant studies, highlight interesting new targets for mutagenesis and suggest a possible mechanism of enzyme phosphorylation . Biochemistry, 1999 Mar 2, 38(9), 2628 - 35 DNA ligation during excision repair in yeast cell-free extracts is specifically catalyzed by the CDC9 gene product; Wu X et al.; Excision repair of DNA is an important cellular response to DNA damage induced by radiation and many chemicals . In eukaryotes, base excision repair (BER) and nucleotide excision repair (NER) are two major excision repair pathways which are completed by a DNA ligation step . Using a cell-free system, we have determined the DNA ligase requirement during BER and NER of the yeast S . cerevisiae . Under nonpermissive conditions in extracts of the cdc9-2 temperature-sensitive mutant, DNA ligation in both BER and NER pathways was defective, and the repair patches were enlarged . At the permissive temperature (23 degrees C), DNA ligation during excision repair was only partially functional in the mutant extracts . In contrast, deleting the DNA ligase IV gene did not affect DNA ligation of BER or NER . Defective DNA ligation of BER and NER in cdc9-2 mutant extracts was complemented in vitro by purified yeast Cdc9 protein, but not by DNA ligase IV even when overexpressed . These results demonstrate that the ligation step of excision repair in yeast cell-free extracts is catalyzed specifically by the Cdc9 protein, the homologue of mammalian DNA ligase I. Appl Environ Microbiol, 1999 Mar, 65(3), 1191 - 7 Stimulation of strontium accumulation in linoleate-enriched Saccharomyces cerevisiae is a result of reduced Sr2+ efflux; Avery SV et al.; The influence of modified plasma membrane fatty acid composition on cellular strontium accumulation in Saccharomyces cerevisiae was investigated . Growth of S . cerevisiae in the presence of 1 mM linoleate (18:2) (which results in 18:2 incorporation to approximately 70% of total cellular and plasma membrane fatty acids, with no effect on growth rate) yielded cells that accumulated Sr2+ intracellularly at approximately twice the rate of S . cerevisiae grown without a fatty acid supplement . This effect was evident over a wide range of external Sr2+ concentrations (25 microM to 5 mM) and increased with the extent of cellular 18:2 incorporation . Stimulation of Sr2+ accumulation was not evident following enrichment of S . cerevisiae with either palmitoleate (16:1), linolenate (18:3) (n-3 and n-6 isomers), or eicosadienoate (20:2) (n-6 and n-9 isomers) . Competition experiments revealed that Ca2+- and Mg2+-induced inhibition of Sr2+ accumulation did not differ between unsupplemented and 18:2-supplemented cells . Treatment with trifluoperazine (TFP) (which can act as a calmodulin antagonist and Ca2+-ATPase inhibitor), at a low concentration that precluded nonspecific K+ efflux, increased intracellular Sr2+ accumulation by approximately 3.6- and 1.4-fold in unsupplemented and 18:2-supplemented cells, respectively . Thus, TFP abolished the enhanced Sr2+ accumulation ability of 18:2-supplemented cells . Moreover, the rate of Sr2+ release from Sr2+-loaded fatty acid-unsupplemented cells was found to be at least twice as great as that from Sr2+-loaded 18:2-enriched cells . The influence of enrichment with other fatty acids on Sr2+ efflux was variable . The results reveal an enhanced Sr2+ accumulation ability of S . cerevisiae following 18:2-enrichment, which is attributed to diminished Sr2+ efflux activity in these cells. J Cell Physiol, 1999 Feb, 178(2), 216 - 26 Carbon source-dependent regulation of cell growth by murine protein kinase C epsilon expression in Saccharomyces cerevisiae; Parissenti AM et al.; Protein kinase C is known to play a role in cell cycle regulation in both lower and higher eucaryotic cells . Since mutations in yeast proteins involved in cell cycle regulation can often be rescued by the mammalian homolog and since significant conservation exists between PKC-signalling pathways in yeast and mammalian cells, cell cycle regulation by mammalian PKC isoforms may be effectively studied in a simpler genetically-accessible model system such as Saccharomyces cerevisiae . With this objective in mind, we transfected S . cerevisiae cells with a plasmid (pYECepsilon) coding for the expression of murine protein kinase C epsilon (PKCepsilon) under the control of a galactose-inducible promoter . Unlike mock-transfected cells, yeast cells transformed with pYECepsilon expressed, in a galactose-dependent manner, an 89 kDa protein that was recognized by a human PKCepsilon antibody . Extracts from these pYECepsilon-transfected cells could phosphorylate a PKCepsilon substrate peptide in a phospholipid/phorbol ester-dependent manner . Moreover, this catalytic activity could be inhibited by a fusion protein in which the regulatory domain of murine PKCepsilon was fused in frame with GST (GST-Repsilon), further confirming the successful expression of murine PKCepsilon . Induction of PKCepsilon expression by galactose in cells transformed with pYECepsilon increased Ca++ uptake by the cells approximately 5-fold and resulted in a dramatic inhibition of cell growth in glycerol . However, when glucose was used as the carbon source, PKCepsilon expression had no effect on cell growth . This was in contrast to what was observed upon bovine PKCalpha or PKCbeta-I expression in yeast, where expression of these PKC isoforms strongly and moderately inhibited growth in glucose, respectively . Visualization of the cells by phase contrast microscopy indicated that murine PKCepsilon expression in the presence of glycerol resulted in a significant increase in the number of yeast cells exhibiting very small buds . Since overall growth of the cells was dramatically decreased, the data suggests that PKCepsilon expression potently inhibits the progression of yeast cells through the cell cycle after the initiation of budding . In addition, a small amount of the PKCepsilon-expressing yeast cells (1-2%) exhibited gross alterations in cell morphology and defects in both chromosome segregation and septum formation . This suggests that for those cells which do complete DNA synthesis, murine PKCepsilon expression may nevertheless inhibit yeast cell growth by retarding and/or imparing cell division . Taken together, the data suggests murine PKCepsilon expression potently reduces the growth of yeast cells in a carbon source-dependent fashion by affecting progression through multiple points within the cell cycle . This murine PKCepsilon-expressing yeast strain may serve as a very useful tool in the elucidation of mechanism(s) by which external environmental signals (possibly through specific PKC isoforms) regulate cell cycle progression in both yeast and mammalian cells. Cell Signal, 1999 Feb, 11(2), 127 - 35 The SH3 domain of the S . cerevisiae Cdc25p binds adenylyl cyclase and facilitates Ras regulation of cAMP signalling; Mintzer KA et al.; Cdc25 and Ras are two proteins required for cAMP signalling in the budding yeast Saccharomyces cerevisiae . Cdc25 is the guanine nucleotide exchange protein that activates Ras . Ras, in turn, activates adenylyl cyclase . Cdc25 has a Src homology 3 (SH3) domain near the N-terminus and a catalytic domain in the C-terminal region . We find that a point mutation in the SH3 domain attenuates cAMP signalling in response to glucose feeding . Furthermore, we demonstrate, by using recombinant adenylyl cyclase and Cdc25, that the SH3 domain of Cdc25 can bind directly to adenylyl cyclase . Binding was specific, because the SH3 domain of Abp1p (actin-binding protein 1), which binds the 70,000 Mr subunit of adenylyl cyclase, CAP/Srv2, failed to bind adenylyl cyclase . A binding site for Cdc25-SH3 localised to the C-terminal catalytic region of adenylyl cyclase . Finally, pre-incubation with Ras enhanced the SH3-bound adenylyl cyclase activity . These studies suggest that a direct interaction between Cdc25 and adenylyl cyclase promotes efficient assembly of the adenylyl cyclase complex. J Biotechnol, 1999 Feb 5, 68(1), 15 - 28 Non-linear optimization of biotechnological processes by stochastic algorithms: application to the maximization of the production rate of ethanol, glycerol and carbohydrates by Saccharomyces cerevisiae; Rodriguez-Acosta F et al.; A non-linear optimization, based on an stochastic multi-start search algorithm, has been applied to the maximization of the production rates of ethanol, glycerol and carbohydrates by Saccharomyces cerevisiae . This optimization is applied to two alternative (non-linear) model representations of the same system, namely the Michaelis-Menten and the generalized mass action forms . We find a complete agreement between the results obtained using both representations . This is, maximization of the ethanol production rate requires modulation of up to six enzymes, while modification of only one enzyme is sufficient to obtain a significant improvement in the production rate of glycerol and carbohydrates . When the results are compared with those previously obtained using an indirect linear optimization method (Torres, N.V., Voit, E.O., Gonzalez-Alcon, C., Rodriguez, F . 1997 . An integrated optimization method for biochemical systems . Description of method and application to ethanol, glycerol and carbohydrate production in S . cerevisiae . Biotechnol . Bioeng . 55(5), 758-772.), we find close agreement between both optimization techniques . Qualitatively, both optimization approaches render the same profile of enzymes to be modulated, while quantitatively, discrepancies arise when the objective function is the maximization of the ethanol production rate . Reasons for such discrepancies and an evaluation of the advantages of each method (linear vs non-linear) are given. Curr Genet, 1999 Feb, 35(1), 51 - 7 Transformation of Candida oleophila and survival of a transformant on orange fruit under field conditions; Chand-Goyal T et al.; Histidine auxotrophs of wild-type strain I-182 of Candida oleophila, produced using ethyl methanesulfonate, were transformed with plasmids containing the HIS3, HIS4 and HIS5 genes of Saccharomyces cerevisiae . Histidine auxotrophy was complemented by the HIS5 gene of S . cerevisiae . Stability of the transformants under non-selective conditions and DNA gel-blot analysis suggested that the transforming DNA had integrated into the C . oleophila genome . There were no detectable physiological differences between the wild-type and the transformants . The biological control ability of C . oleophila was not affected by the transformation . A genetically marked transformant (with a beta-glucuronidase gene) colonized wounds on oranges, and its population increased under field conditions . The identity of the genetically marked transformant was established by PCR-amplification of a portion of the beta-glucuronidase gene. Mol Cell Biol, 1999 Mar, 19(3), 2142 - 54 Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA; Massenet S et al.; Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method . In contrast to vertebrate UsnRNAs, S . cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions . At these positions, UsnRNAs are universally modified . When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs . Indeed, Psi44 formation in U2 snRNA was abolished . By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S . cerevisiae U2 snRNA . Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA . As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S . cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism. Genes Cells, 1998 Nov, 3(11), 737 - 49 Cell cycle dependent topological changes of chromosomal replication origins in Saccharomyces cerevisiae; Fujita M et al.; BACKGROUND: The ORC (Origin Recognition Complex) of Saccharomyces cerevisiae is a protein complex for the initiation of replication which interacts with a cis-element, ACS (ARS Consensus Sequence), essential for DNA replication . The protein-DNA complex detected by the DNase I genomic footprinting method has been shown to vary depending on cell cycle progression . Further studies on topological changes of replication origin in vivo caused by ORC association are crucial for an understanding of chromosomal DNA replication in S . cerevisiae . RESULTS: Topological changes in the replication origins of the S . cerevisiae chromosome were studied by an in vivo UV photofootprinting method which is capable of detecting the change in the flexibility of DNA caused by protein binding . The footprinting method detected the inhibition and enhancement of UV-induced pyrimidine dimer formation in A and B1 elements of a chromosomal origin, ARS1, depending on the activity of native ORC subunits . Furthermore, footprint patterns were reproduced in vitro with purified ORC . The inhibition regarding the A element was stronger during the S to late M phase than that during the progression through the G1 phase . Functional CDC6 and MCM5 were required for maintaining the weaker inhibition state in G1-arrested cells . CONCLUSION: The application of in vivo UV photofootprinting in studies of topological changes of S . cerevisiae replication origins revealed the presence of two modes of topological ORC-ACS interaction . The weaker footprint in the G1 phase represents a specific topology of ACS, resulting from an alteration of the ORC-ACS interaction aided by CDC6 and MCM5, and this topological change may make the replication origin competent for initiating DNA replication. Eur J Biochem, 1998 Dec 15, 258(3), 941 - 7 Mutational analysis of chitin synthase 2 of Saccharomyces cerevisiae . Identification of additional amino acid residues involved in its catalytic activity; Yabe T et al.; Saccharomyces cerevisiae harbors three chitin synthases termed Chs1p, Chs2p and Chs3p . Previously, we demonstrated that con1, a region that is highly conserved among all chitin synthases, contains amino acids essential for the catalytic activity of the enzyme and that Asp562, Gln601, Arg604, and Trp605 found in con1 together with Asp441 were probable catalytic sites of the enzyme . Here we report that another region, con2, in the C-terminal half of Chs2p is also conserved exclusively in chitin synthases that resemble S . cerevisiae Chs1p and Chs2p . Alanine substitutions for the conserved amino acids in con2 identified five amino acids, Asn797, His799, Asp800, Trp803, and Thr805, the mutation of which severely diminished enzymatic activity and the enzyme's ability to rescue the yeast chs2 delta chs3 delta null mutant strain . Although the activities of some of the mutant enzymes were too low to measure enzyme kinetics, most of the alanine mutations in con2 affected the kcat values rather than the K(m) values . Whereas a conservative mutation of Asn797 restored the activity, those of His799, Asp800, Trp803, and Thr805 did not . A fine alignment of the amino acid sequences of con2 and Chs3p revealed that Asp800, Trp803 and Thr805 are completely conserved near the C-terminal ends of Chs3p and its homologs in other fungi . On the basis of these findings, we propose that Asp800, Trp803, and Thr805 in con2 are additional residues involved in catalysis, and hypothesise that Asp800 together with the previously identified Asp441 and Asp562 serve as polar residues necessary for the acid-based catalytic reaction of chitin synthase. J Biol Chem, 1999 Feb 19, 274(8), 4869 - 75 Mutations in ribosomal protein L10e confer resistance to the fungal-specific eukaryotic elongation factor 2 inhibitor sordarin; Justice MC et al.; The natural product sordarin, a tetracyclic diterpene glycoside, selectively inhibits fungal protein synthesis by impairing the function of eukaryotic elongation factor 2 (eEF2) . Sordarin and its derivatives bind to the eEF2-ribosome-nucleotide complex in sensitive fungi, stabilizing the post-translocational GDP form . We have previously described a class of Saccharomyces cerevisiae mutants that exhibit resistance to varying levels of sordarin and have identified amino acid substitutions in yeast eEF2 that confer sordarin resistance . We now report on a second class of sordarin-resistant mutants . Biochemical and molecular genetic analysis of these mutants demonstrates that sordarin resistance is dependent on the essential large ribosomal subunit protein L10e in S . cerevisiae . Five unique L10e alleles were characterized and sequenced, and several nucleotide changes that differ from the wild-type sequence were identified . Changes that result in the resistance phenotype map to 4 amino acid substitutions and 1 amino acid deletion clustered in a conserved 10-amino acid region of L10e . Like the previously identified eEF2 mutations, the mutant ribosomes show reduced sordarin-conferred stabilization of the eEF2-nucleotide-ribosome complex . To our knowledge, this report provides the first description of ribosomal protein mutations affecting translocation . These results and our previous observations with eEF2 suggest a functional linkage between L10e and eEF2. Mol Microbiol, 1999 Jan, 31(1), 103 - 16 Msn1p/Mss10p, Mss11p and Muc1p/Flo11p are part of a signal transduction pathway downstream of Mep2p regulating invasive growth and pseudohyphal differentiation in Saccharomyces cerevisiae; Gagiano M et al.; In Saccharomyces cerevisiae, a network of signal transduction pathways governs the switch from yeast-type growth to pseudohyphal and invasive growth that occurs in response to nutrient limitation . Important elements of this network have been identified, including nutrient signal receptors, GTP-binding proteins, components of the pheromone-dependent MAP kinase cascade and several transcription factors . However, the structural and functional mapping of these pathways is far from complete . Here, we present data regarding three genes, MSN1/MSS10, MSS11 and MUC1/FLO11, which form an essential part of the signal transduction network establishing invasive growth . Both MSN1 and MSS11 are involved in the co-regulation of starch degradation and invasive growth . Msn1p and Mss11p act downstream of Mep2p and Ras2p and regulate the transcription of both STA2 and MUC1 . We show that MUC1 mediates the effect of Msn1p and Mss11p on invasive growth . In addition, our results suggest that the activity of Msn1p is independent of the invasive growth MAP kinase cascade, but the Mss11p is required for the activation of pseudohyphal and invasive growth by Ste12p . We also show that starch metabolism in S . cerevisiae is subject to regulation by components of the MAP kinase cascade. Biosci Biotechnol Biochem, 1998 Dec, 62(12), 2425 - 7 Cloning of genomic DNA of Rhizopus niveus lipase and expression in the yeast Saccharomyces cerevisiae; Kohno M et al.; Genomic DNA encoding Lipase I was cloned from Rhizopus niveus strain IFO4759 . For expression of this gene in S . cerevisiae, DBY746 was transformed with YEp352PLipS, which had the cloned lipase gene under the control of a PGK promoter . This strain secreted the lipase at a high level (350 U/ml) . The strain ND-12B was produced by a mating of DBY746, harboring YEp352PLipS, and NA74-3A, and dissection of asci . This new strain secreted the lipase up to 530 U/ml . Moreover, the lipase was produced most effectively in a medium containing Bacto-yeast extract, soy-peptide, and sucrose. Biosci Biotechnol Biochem, 1998 Nov, 62(11), 2280 - 2 Identification of the orotidine-5'-phosphate decarboxylase gene and development of a transformation system in the yeast Saccharomyces exiguus Yp74L-3; Hisatomi T et al.; To investigate the uracil biosynthetic pathway of the yeast Saccharomyces exiguus Yp74L-3, uracil auxotrophic mutants were isolated . Using conventional genetic techniques, four mutant genes concerned in uracil biosynthesis were identified and denoted as ura1, ura2, ura3, and ura4 . Mutations in the URA3 and URA4 genes were specifically selected with 5-fluoroorotic acid (5-FOA) . Vector plasmids containing the URA3 gene and an autonomously replicating sequence (ARS) of S . cerevisiae produced sufficient amounts of Ura+ transformants from the ura4 mutant of S . exiguus . This fact indicates that the S . exiguus URA4 gene encodes orotidine-5'-phosphate decarboxylase (OMP decarboxylase) and demonstrates that vector plasmids for S . cerevisiae are also usable in S . exiguus. Lipids, 1998 Dec, 33(12), 1229 - 34 Lysophosphatidylcholine acyltransferase activity in Saccharomyces cerevisiae: regulation by a high-affinity Zn2+ binding site; Richard MG et al.; Saccharomyces cerevisiae cells were demonstrated to contain lysophosphatidylcholine (lysoPtdCho) acyltransferase (E.C . 2.3.1.23) activity . The enzyme displayed Km(app) of 69 microM for lysoPtdCho and 152 microM for oleoyl CoA . Enzyme activity was not affected by the addition of 1 mM Mg2+, Mn2+, Ca2+, or 200 mM EDTA . However, Zn2+ inhibited lysoPtdCho acyltransferase activity to 33% control values at 0.1 mM and to 7% at 1.0 mM Zn2+ . To further explore the possibility that lysoPtdCho acyltransferase may contain a high-affinity Zn2+ binding site, we tested the strong Zn2+ chelator o-phenanthroline for its ability to inhibit enzyme activity . LysoPtdCho acyltransferase activity was inhibited to 18 and 27%, respectively, those of control values in the presence of 2 and 1 mM o-phenanthroline, implying that a high-affinity Zn2+ binding site exists in lysoPtdCho acyltransferase or in an accessory protein that is essential for protein stability and/or activity . Saccharomyces cerevisiae lysoPtdCho acyltransferase activity displayed a broad lysoPtdCho fatty acyl chain substrate specificity utilizing lysoPtdCho molecules ranging in length from C10-C20 (the entire range tested) . In addition, the enzyme was capable of using the ether-linked analog of lysoPtdCho, 1-O-alkyl-2-hydroxy-sn-3-glycerophosphocholine, as a substrate . The ability of S . cerevisiae to incorporate radiolabeled 1-O-alkyl-2-hydroxy-sn-3-glycerophosphocholine into phosphatidylcholine in vitro was exploited to demonstrate a direct precursor-product relationship between lysoPtdCho molecules and their incorporation into phosphatidylcholine in vivo . Identical labeling results were obtained in S . cerevisiae cells disrupted for their major transacylase activity, PLB1, demonstrating that the incorporation of lysolipid was via acyltransferase, and not transacylase, activity. J Mol Evol, 1999 Feb, 48(2), 133 - 41 Relationships between transcriptional and translational control of gene expression in Saccharomyces cerevisiae: a multiple regression analysis; Pavesi A; Natural selection for an increased translation efficiency has been proposed as the main determinant for the bias in codon usage observed in many genes of Saccharomyces cerevisiae . Recently, the efficiency of transcription of a large number of yeast genes has been determined, based on the cellular content of the respective mRNAs: this provides an additional dimension to the study of the multisep process of gene expression . Using a representative set of yeast genes with a known level of transcription, the relationship between transcriptional and translational steps was evaluated by a multiple linear regression model . This analysis demonstrated a positive correlation between the amount of transcript, given as the number of mRNA copies per cell for each individual gene, and indices evaluating the effects of translational selection on the corresponding codon usage pattern . This finding suggests a close association of the cellular mRNA content, regulated also at the transcriptional level, to its efficiency of translation, mediated by a fine-tuning of codon usage strategy . Moreover, multiple regression analysis demonstrated that the transcription level of a gene can be approximately predicted using indices of bias deriving from its nucleotide sequence . This allowed for an extensive investigation of uncharacterized regions of the complete genome sequence of S . cerevisiae, to detect new potential short protein coding genes that were not considered by previous searching procedures . Several small open reading frames exhibiting a statistically significant coding potential were thus identified as good candidates for functional analysis. Mol Gen Genet, 1999 Jan, 260(6), 510 - 21 Molecular characterization of HymA, an evolutionarily highly conserved and highly expressed protein of Aspergillus nidulans; Karos M et al.; Aspergillus nidulans reproduces asexually via uninucleate, haploid spores, which are produced on morphologically differentiated aerial structures, called conidiophores . These consist of four distinct cell types, a foot with a terminally swollen stalk, metulae, phialides and conidiospores . The molecular mechanisms underlying the morphological changes that occur during conidiophore development have been studied by mutant analysis . We have isolated the hym A mutant, in which conidiophore development is affected at the metula stage . In the mutant metulae do not differentiate properly but come to resemble hyphae (hym = hypha-like metulae) . In this paper we have analyzed the corresponding gene . It encodes a highly expressed 44 kDa protein which resides in the cytoplasm and has homologues in yeast, plants, fly, worm, fish, mice and man . We constructed hym deletion strains of Saccharomyces cerevisiae and of A . nidulans and found that the gene is essential in S . cerevisiae but is dispensable in the filamentous fungus . A cellular function for the Hym protein has not yet been defined in any organism . To demonstrate functional conservation we constructed a chimeric protein comprised of the N-terminal half of the A . nidulans and the C-terminal half of the mouse homologue MO25 . This hybrid protein could fully substitute for HymA function in A . nidulans . In addition, the mouse protein itself partially rescued the hym A mutation in the fungus . HymA is thus highly conserved in evolution and probably serves similar functions . The fact that hym A is required for conidiophore development in A . nidulans suggests that homologous genes in other organisms might also be involved in morphogenesis. Biochim Biophys Acta, 1998 Dec 8, 1429(1), 93 - 101 Interaction of adenosine nucleotide analogs with Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase; Carrasco L et al.; The substrate characteristics and interactions of different adenosine nucleotide analogs with Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase were investigated by steady-state kinetic analysis and calculations of interaction energies . Comparison of Vmax/Km values showed that analogs substituted at C8 in the adenine ring (8-Br-ATP, 8-N3-ATP, 8-N3-ADP) gave almost the same kinetic values as ATP and ADP, whereas those substituted in the ribose hydroxyls (3'(2')-O-(N-methylanthraniloyl)-ATP (MANT-ATP), 3'(2')-O-(N-methylanthraniloyl)-ADP (MANT-ADP), 2'(3')-O-(2,4,6-trinitrophenyl)-ADP (TNP-ADP), 2'(3')-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP)) showed 1-8% the value for the corresponding physiological substrate . A comparison between the experimental results and molecular mechanics calculations was performed, employing a model for the S . cerevisiae PEP carboxykinase-ATP-Mn2+ complex . The calculated interaction energies of S . cerevisiae PEP carboxykinase with ATP, MANT-ATP, TNP-ATP, 8-Br-ATP, and 8-N3-ATP were linearly related (correlation coefficient 0.92) with -ln(Vmax/Km) . This good correlation supports the proposal that the interaction of the substituent with the enzyme affects the interaction of the common region of ATP with the active site, thus leading to effects in Vmax. Biochim Biophys Acta, 1998 Dec 8, 1429(1), 29 - 39 D-arabinose dehydrogenase and its gene from Saccharomyces cerevisiae; Kim ST et al.; D-Arabinose dehydrogenase was purified 843-fold from the cytosolic fraction of Saccharomyces cerevisiae with a recovery of 9% . The purified enzyme gave two bands with a molecular mass of 40 and 39 kDa on SDS-PAGE . The native enzyme had a molecular mass of 74 kDa as estimated by Sephacryl S-200 chromatography . Therefore, this enzyme was considered to be a heterodimer . The purified enzyme exhibited maximum activity at pH 10.0 and around 30 degrees C . The enzyme catalysed the oxidation of D-arabinose, L-xylose, L-fucose and L-galactose in the presence of NADP+ . The apparent Km values at pH 10.0 with 50 microM NADP+ for D-arabinose, L-xylose, L-fucose, and L-galactose were 161, 24, 98 and 180 mM, respectively . The pH profile of Vmax and kcat/Km showed one ionisable groups around pH 8.3 . D-Erythroascorbic acid was formed in vitro from D-arabinose by D-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase . The N-terminal amino acid sequence of the heavy subunit was Ser-Thr-Glu-Asn-Ile-Val-Glu-Asn-Met-Leu-His-Pro-Lys-Thr- . The N-terminus of the light subunit was blocked . The obtained peptide sequence was identical to the translational product of an unknown open reading frame, YBR149W, in chromosome II of S . cerevisiae . When compared with the translational product of this open reading frame, the peptide sequence was identical to the amino acid sequences of residues 7 to 20 . The first six amino acids of this open reading frame were lost in protein sequence, which may be modified post-translationally . The heavy subunit was composed of 344 amino acid residues and its deduced amino acid sequence contained the motifs I, II, and III of aldo-keto reductase and also leucine zipper motif . This enzyme is the first heterodimeric protein of aldo-keto reductase family . In the deletion mutant of this gene, D-arabinose dehydrogenase activity and D-erythroascorbic acid were not detected. Biochem Biophys Res Commun, 1999 Jan 19, 254(2), 345 - 50 Med8, a subunit of the mediator CTD complex of RNA polymerase II, directly binds to regulatory elements of SUC2 and HXK2 genes; Chaves RS et al.; In a search to identify new factors required for expression of SUC2 gene in Saccharomyces cerevisiae, we have partially purified a 27 kDa protein (p27) that bound both the DRSs of the HXK2 gene and the UASs of SUC2 gene . The amino terminal sequence of p27 identified the MED8 gene (open reading frame YBR193C), located in chromosome II of S . cerevisiae, as the gene coding for the protein . Disruption of this gene has demonstrated that is an essential gene for yeast growth . To determine whether the p27 protein represents the Med8 product, we expressed MED8 gene in E . coli and demonstrated that the heterologous synthesized protein specifically binds to both UASSUC2 and DRS2HXK2 . This observation suggests that Med8 may be important for the coupling of the glucose repression pathway of SUC2 gene to the HXK2 gene expression . Med8 has been described as a mediator protein interacting with the CTD of the RNA polymerase II . Thus, the role of Med8 could be to act as coupling factor by linking activating and repressing transcription complexes to the RNA polymerase II holoenzyme transcriptional machinery . J Biol Chem, 1999 Jan 29, 274(5), 2609 - 12 A built-in arginine finger triggers the self-stimulatory GTPase-activating activity of rho family GTPases; Zhang B et al.; Signal transduction through the Rho family GTPases requires regulated cycling of the GTPases between the active GTP-bound state and the inactive GDP-bound state . Rho family members containing an arginine residue at position 186 in the C-terminal polybasic region were found to possess a self-stimulatory GTPase-activating protein (GAP) activity through homophilic interaction, resulting in significantly enhanced intrinsic GTPase activities . This arginine residue functions effectively as an "arginine finger" in the GTPase activating reaction to confer the catalytic GAP activity but is not essential for the homophilic binding interactions of Rho family proteins . The arginine 186-mediated negative regulation seems to be absent from Cdc42, a Rho family member important for cell-division cycle regulation, of lower eukaryotes, yet appears to be a part of the turn-off machinery of Cdc42 from higher eukaryotes . Introduction of the arginine 186 mutation into S . cerevisiae CDC42 led to phenotypes consistent with down-regulated CDC42 function . Thus, specific Rho family GTPases may utilize a built-in arginine finger, in addition to RhoGAPs, for negative regulation. Eur J Biochem, 1999 Jan, 259(1-2), 244 - 52 Analysis of a conditional degradation signal in yeast and mammalian cells; Levy F et al.; The N-end rule pathway is a ubiquitin-dependent proteolytic system, the targets of which include proteins that bear destabilizing N-terminal residues . The latter are a part of the degradation signal called the N-degron . Arg-DHFRts, an engineered N-end rule substrate, bears N-terminal arginine (a destabilizing residue) and DHFRts {a temperature-sensitive mouse dihydrofolate reductase (DHFR) moiety} . Previous work has shown that Arg-DHFRts is long-lived at 23 degreesC but short-lived at 37 degreesC in the yeast Saccharomyces cerevisiae . In the present work, we extended this analysis, and found that the degradation of Arg-DHFRts can be nearly completely inhibited in vivo by methotrexate (MTX), a low-Mr ligand of DHFR . In S . cerevisiae, Arg-DHFRts is degraded at 37 degreesC exclusively by the N-end rule pathway, whereas in mouse cells the same protein at the same temperature is also targeted by another proteolytic system, through a degron in the conformationally perturbed DHFRts moiety . In mouse cells, MTX completely inhibits the degradation of Arg-DHFRts through its degron within the DHFRts moiety, but only partially inhibits degradation through the N-degron . When the N-terminus of Arg-DHFRts was extended with a 42-residue lysine-lacking extension, termed eDeltaK, the resulting Arg-eDeltaK-DHFRts was rapidly degraded at both 23 degreesC and 37 degreesC . Moreover, the degradation of Arg-eDeltaK-DHFRts, in contrast with that of Arg-DHFRts, could not be inhibited by MTX, suggesting that the metabolic stability of Arg-DHFRts at 23 degreesC results, at least in part, from steric inaccessibility of its N-terminal arginine . The N-degron of Arg-DHFRts is the first example of a portable degradation signal the activity of which can be modulated in vivo by a cell-penetrating compound . We discuss implications of this advance and the mechanics of targeting by the ubiquitin system. Mol Cell Biol, 1999 Feb, 19(2), 1369 - 80 Control of Saccharomyces cerevisiae filamentous growth by cyclin-dependent kinase Cdc28; Edgington NP et al.; The ascomycete Saccharomyces cerevisiae exhibits alternative vegetative growth states referred to as the yeast form and the filamentous form, and it switches between the two morphologies depending on specific environmental signals . To identify molecules involved in control of morphologic differentiation, this study characterized mutant S . cerevisiae strains that exhibit filamentous growth in the absence of the normal external signals . A specific amino acid substitution in the cyclin-dependent protein kinase Cdc28 was found to cause constitutive expression of most filamentous growth characteristics . These effects include specifically modified cell polarity characteristics in addition to the defined shape and division cycle alterations typical of the filamentous form . Several other mutations affecting Cdc28 function also had specific effects on filamentous growth . Constitutive filamentous growth resulting from deletion of the protein kinase Elm1 was prevented by modification of Cdc28 such that it could not be phosphorylated on tyrosine residue 19 . In addition, various mutations affecting Hsl1 or Swe1, known or presumed components of a protein kinase cascade that mediates Cdc28 phosphorylation on Y19, either prevented or enhanced filamentous growth . The data suggest that a protein kinase cascade involving Elm1, Hsl1, and Swe1 can modulate Cdc28 activity and that Cdc28 in turn exerts global effects that cause filamentous growth. J Cell Sci, 1999 Feb, 112 ( Pt 3), 339 - 47 Antagonistic effects of NES and NLS motifs determine S . cerevisiae Rna1p subcellular distribution; Feng W et al.; Nucleus/cytosol exchange requires a GTPase, Ran . In yeast Rna1p is the GTPase activating protein for Ran (RanGAP) and Prp20p is the Ran GDP/GTP exchange factor (GEF) . RanGAP is primarily cytosolic and GEF is nuclear . Their subcellular distributions led to the prediction that Ran-GTP hydrolysis takes place solely in the cytosol and GDP/GTP exchange solely in the nucleus . Current models propose that the Ran-GTP/Ran-GDP gradient across the nuclear membrane determines the direction of exchange . We provide three lines of evidence that Rna1p enters and leaves the nuclear interior . (1) Rna1p possesses leucine-rich nuclear export sequences (NES) that are able to relocate a passenger karyophilic protein to the cytosol; alterations of consensus residues re-establish nuclear location . (2) Rna1p possesses other sequences that function as a novel nuclear localization sequence able to deliver a passenger cytosolic protein to the nucleus . (3) Endogenous Rna1p location is dependent upon Xpo1p/Crm1p, the yeast exportin for leucine-rich NES-containing proteins . The data support the hypothesis that Rna1p exists on both sides of the nuclear membrane, perhaps regulating the Ran-GTP/Ran-GDP gradient, participating in a complete RanGTPase nuclear cycle or serving a novel function. Yeast, 1998 Dec, 14(16), 1471 - 510 Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae; Daum G et al.; The yeast Saccharomyces cerevisiae is a powerful experimental system to study biochemical, cell biological and molecular biological aspects of lipid synthesis . Most but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of this unicellular eukaryote have been cloned, and many gene products have been functionally characterized . Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes, turnover and degradation of complex lipids, regulation of lipid biosynthesis, and linkage of lipid metabolism to other cellular processes . Here we summarize current knowledge about lipid biosynthetic pathways in S . cerevisiae and describe the characteristic features of the gene products involved . We focus on recent discoveries in these fields and address questions on the regulation of lipid synthesis, subcellular localization of lipid biosynthetic steps, cross-talk between organelles during lipid synthesis and subcellular distribution of lipids . Finally, we discuss distinct functions of certain key lipids and their possible roles in cellular processes. Cell Signal, 1998 Nov, 10(10), 699 - 711 Casein kinase I: spatial organization and positioning of a multifunctional protein kinase family; Gross SD et al.; The casein kinase I family of serine/threonine protein kinases is highly conserved from yeast to humans . Until only recently, both the function and regulation of these enzymes remained poorly uncharacterised in that they appeared to be constitutively active and were capable of phosphorylating an untold number of other proteins . While relatively little was known regarding the exact function of the higher eukaryotic isoforms, the casein kinase I (CKI) isoforms from yeast have been genetically linked to vesicular trafficking, DNA repair, cell cycle progression and cytokinesis . All five S . cerevisiae isoforms are known to be associated with discrete cellular compartments and this localization has been shown to be absolutely essential for their respective functions . New evidence now suggests that the CKI isoforms in more complex systems also exhibit non-homogeneous subcellular distributions that may prove vital to defining the function and regulation of these enzymes . In particular, CKIalpha, the most-characterized vertebrate isoform, is associated with cytosolic vesicles, the mitotic spindle and structures within the nucleus . Functions associated with these localizations coincide with those previously reported in yeast, suggesting a conservation of function . Other reports have indicated that each of the remaining CKI isoforms have the capacity to make associations with components of several signal transduction pathways, thereby channeling CKI function toward specific regulatory events . This review will examine what is now known about the higher eukaryotic CKI family members from the perspective localization as a means of gaining a better understanding of the function and regulation of these kinases. J Biol Chem, 1999 Jan 15, 274(3), 1783 - 90 Red1p, a MEK1-dependent phosphoprotein that physically interacts with Hop1p during meiosis in yeast; de los Santos T et al.; The synaptonemal complex (SC) is a proteinaceous structure formed between pairs of homologous chromosomes during prophase I of meiosis . The proper assembly of axial elements (AEs), lateral components of the SC, during meiosis in the yeast, Saccharomyces cerevisiae, is essential for wild-type levels of recombination and for the accurate segregation of chromosomes at the first meiotic division . Genetic experiments have indicated that the stoichiometry between two meiosis-specific components of AEs in S . cerevisiae, HOP1 and RED1, is critical for proper assembly and function of the SC . A third meiosis-specific gene, MEK1, which encodes a putative serine/threonine protein kinase, is also important for proper AE function, suggesting that AE formation is regulated by phosphorylation . In this paper, we demonstrate that Mek1p is a functional kinase in vitro and that catalytic activity is an essential part of the meiotic function of Mek1 in vivo . Immunoblot analysis revealed that Red1p is a MEK1-dependent phosphoprotein . Co-immunoprecipitation experiments demonstrated that the interaction between Hop1p and Red1p is enhanced by the presence of MEK1 . Thus, MEK1-dependent phosphorylation of Red1p facilitates the formation of Hop1p/Red1p hetero-oligomers, thereby enabling the formation of functional AEs. Biochim Biophys Acta, 1999 Jan 6, 1426(2), 385 - 400 The Gas1 glycoprotein, a putative wall polymer cross-linker; Popolo L et al.; The yeast cell wall, which for years has been regarded as a static cellular component, has been revealed to be dynamic in its structure and composition and complex in its enzymatic activity . The S . cerevisiae cell wall is composed of beta-1,3/beta-1,6-glucans, mannoproteins, and chitin, which are assembled into an extracellular matrix essential for maintenance of cell integrity . Gas1p, a glycoprotein anchored to the outer leaflet of the plasma membrane through a glycosylphosphatidylinositol, plays a key role in cell wall assembly . Loss of Gas1p leads to several morphogenetic defects and to a decrease in the amount of cross-links between the cell wall glucans . These defects in turn trigger a compensatory response that guarantees cell viability . Several Gas1p homologs have been isolated from Candida species and S . pombe . The Gas1p family also includes two plant proteins with endo-beta-1,3-glucanase activity . Sequence comparisons reveal that Gas1p family proteins have a modular organization of domains . The genetic and molecular analyses reviewed here suggest that Gas1p could play a role as a polymer cross-linker, presumably by catalyzing a transglycosylation reaction. Biochim Biophys Acta, 1999 Jan 6, 1426(2), 335 - 45 Mannosylphosphate transfer to yeast mannan; Jigami Y et al.; Mannoproteins located in the outermost layer of yeast cell wall determine the wall's porosity and thereby regulate leakage of proteins from the periplasmic space and entrance of macromolecules from the environment . In several yeasts, including Saccharomyces cerevisiae, the glycan portion of mannoproteins is composed not only of neutral oligosaccharides containing mannose and N-acetylglucosamine, but also of acidic oligosaccharides containing mannosylphosphate . The mannosylphosphate residues confer a net negative charge on the cell wall, and so change the properties and environment of the cell surface . Progress on mannosylphosphorylation and its regulation in S . cerevisiae is summarized . Two genes required for mannosylphosphate transfer, MNN4 and MNN6, have been cloned, and a functional analysis of these genes suggests a mechanism for mannosylphosphate transfer . Possible functions for mannosylphosphate transfer in yeast are also discussed . These include supply of GMP for sugar nucleotide transport in the Golgi, cross-linking of mannoproteins to beta-glucan, and a cellular stress response to environmental changes . Glycans in pathogenic yeast and protozoa are also modified with mannosylphosphate, and the potential contribution of this modification to the pathogenicity of these organisms is evaluated. Eur J Biochem, 1998 Dec 1, 258(2), 402 - 10 Isolation of a cDNA from Arabidopsis thaliana that complements the sec14 mutant of yeast; Jouannic N et al.; The SEC14 gene of Saccharomyces cerevisiae codes for a phosphatidylinositol-transfer protein (Sec14p(sc)) which is capable of transferring both phosphatidylinositol and phosphatidylcholine between membranes in vitro . Genetic and biochemical studies conducted in S . cerevisiae have shown that this protein acts as an inhibitor of phosphatidylcholine biosynthesis via the so-called Kennedy pathway only . This inhibition is controlled by the binding of phospholipids to the Sec14p(sc) protein . Here we describe the isolation of a cDNA from Arabidopsis thaliana by functional complementation of a sec14(ts) mutant of S . cerevisiae . This cDNA, designated AtSEC14, is capable of restoring the growth of the sec14(ts) mutant at the restrictive temperature of 37 degrees C . Extracellular invertase measurements indicated that the cDNA can partly restore protein secretion . In addition, the phosphatidylinositol-transfer activity measured in protein extracts is greatly enhanced in the complemented mutant strain when compared with the sec14(ts) mutant . The best sequence similarity at the amino acid level is found with the Sec14p protein of S . cerevisiae (36.5% similarity), and most of the amino acids that are thought to be involved in the binding of phospholipids in the yeast protein are conserved in the AtSEC14 gene product . Southern analysis suggests the presence of a single gene in the Arabidopsis genome, although the existence of distantly related sequences cannot be excluded . This gene is expressed in roots, leaves, flowers and siliques of Arabidopsis. Curr Microbiol, 1999 Feb, 38(2), 122 - 5 The autonomously replicating sequence (ARS) of the yeast Saccharomyces exiguus Yp74L-3; Hisatomi T et al.; Fragments containing ARSes were cloned from the genomic DNA of the yeast Saccharomyces exiguus Yp74L-3, and the essential regions for ARSes were restricted for these fragments . Mapping studies of ARS-acting sequences in one of these fragments suggested that S . exiguus recognizes a sequence as an ARS that is different from that recognized by Saccharomyces cerevisiae . Two ARS essential regions of S . exiguus were sequenced, and an ARS core consensus sequence of S . exiguus was deduced to be MATTAMWAWWTK . This sequence differs significantly from that of S . cerevisiae in two positions, suggesting that these nucleotide substitutions cause the difference in the ARS-recognition modes between S . exiguus and S . cerevisiae. Glycoconj J, 1998 Aug, 15(8), 815 - 22 Characterization of oligosaccharides from an antigenic mannan of Saccharomyces cerevisiae; Young M et al.; Mannans of the yeast Saccharomyces cerevisiae have been implicated as containing the allergens to which bakers and brewers are sensitive and also the antigen recognized by patients with Crohn's disease . A fraction of S . cerevisiae mannan, Sc500, having high affinity for antibodies in Crohn's patients has been characterized by NMR spectroscopy followed by fragmentation using alkaline elimination, partial acid hydrolysis and acetolysis . The released oligosaccharides were separated by gel filtration on a Biogel P4 column and analyzed by fluorescence labeling, HPLC and methylation analysis . The relationship between structure and antigen activity was measured by competitive ELISA . The antigenic activity of the original high molecular weight mannan could be ascribed to terminal Manalpha1-->3Manalpha1-->2 sequences which are rarely found in human glycoproteins but were over-represented in Sc500 compared to other yeast mannans. J Eukaryot Microbiol, 1998 Nov-Dec, 45(6), 589 - 99 The HSP70 gene family in Pneumocystis carinii: molecular and phylogenetic characterization of cytoplasmic members; Stedman TT et al.; Pneumocystis carinii, a major opportunistic lung pathogen of AIDS patients, is found in a number of mammals and is proposed to be a member of the fungi . In this work, several members of the highly conserved HSP70 multigene family were characterized from rat-derived P . carinii . Previously, we reported characterization of the ER resident HSP70 homolog known as BiP from prototype (P.c . carinii) and variant (P . c . rattus) strains of the organism . We report here, from P . c . carinii, characterization of Pcsa1, an HSP70 homolog that encodes a cognate/stress-induced HSP70 homolog of the SSA subfamily in Saccharomyces cerevisiae . We also identify, from both rat strains and from a human isolate of P . carinii (P.c . hominis), a third set of HSP70 homologs that apparently encode a ribosome-associated cytoplasmic HSP70 homologous to the S . cerevisiae SSB subfamily . Our data indicate that Pcsal mRNA, like Pcbip mRNA, bears an intron in the 5' untranslated region, is induced by heat shock, and suggest that this gene undergoes alternative transcription and splicing . The SSB homologs display significant sequence heterogeneity between P . carinii source strains, supporting the genetic divergence and likely speciation of P . carinii isolates within and between host species . Phylogenetic analysis with the PcSA1 protein supports inclusion of P . carinii among the higher fungi. J Cell Biol, 1998 Dec 28, 143(7), 1859 - 69 Pex18p and Pex21p, a novel pair of related peroxins essential for peroxisomal targeting by the PTS2 pathway; Purdue PE et al.; We have identified ScPex18p and ScPex21p, two novel S . cerevisiae peroxins required for protein targeting via the PTS2 branch of peroxisomal biogenesis . Targeting by this pathway is known to involve the interaction of oligopeptide PTS2 signals with Pex7p, the PTS2 receptor . Pex7p function is conserved between yeasts and humans, with defects in the human protein causing rhizomelic chondrodysplasia punctata (RCDP), a severe, lethal peroxisome biogenesis disorder characterized by aberrant targeting of several PTS2 peroxisomal proteins, but uncertainty remains about the subcellular localization of this receptor . Previously, we have reported that ScPex7p resides predominantly in the peroxisomal matrix, suggesting that it may function as a highly unusual intraorganellar import receptor, and the data presented in this paper identify Pex18p and Pex21p as key components in the targeting of Pex7p to peroxisomes . They each interact specifically with Pex7p both in two-hybrid analyses and in vitro . In cells lacking both Pex18p and Pex21p, Pex7p remains cytosolic and PTS2 targeting is completely abolished . Pex18p and Pex21p are weakly homologous to each other and display partial functional redundancy, indicating that they constitute a two-member peroxin family specifically required for Pex7p and PTS2 targeting. J Bacteriol, 1999 Jan, 181(1), 347 - 52 APT1, but not APT2, codes for a functional adenine phosphoribosyltransferase in Saccharomyces cerevisiae; Alfonzo JD et al.; The yeast Saccharomyces cerevisiae has two separate genes (APT1 and APT2) that encode two potentially different forms of adenine phosphoribosyltransferase (APRT) . However, genetic analysis indicated that only APT1 could code for a complementing activity . Cloning and expression of both the APT1 and APT2 genes in Escherichia coli showed that although discrete proteins (APRT1 and APRT2) were made by these genes, only APRT1 had detectable APRT activity . Northern and Western blot analyses demonstrated that only APT1 was transcribed and translated under normal physiological conditions in yeast . Phylogenetic analysis revealed that APRT1 and APRT2 are evolutionary closely related and that they arise from a gene duplication event . We conclude that APT1 is the functional gene in S . cerevisiae and that APT2 is a pseudogene. Mol Gen Genet, 1998 Nov, 260(2-3), 232 - 41 Efficient initiation of S-phase in yeast requires Cdc40p, a protein involved in pre-mRNA splicing; Boger-Nadjar E et al.; The S . cerevisiae CDC40 gene was originally identified as a cell-division-specific gene that is essential only at elevated temperatures . Cells carrying mutations in this gene arrest with a large bud and a single nucleus with duplicated DNA content . Cdc40p is also required for spindle establishment or maintenance . Sequence analysis reveals that CDC40 is identical to PRP17, a gene involved in pre-mRNA splicing . In this paper, we show that Cdc40p is required at all temperatures for efficient entry into S-phase and that cell cycle arrest associated with cdc40 mutations is independent of all the known checkpoint mechanisms . Using immunofluorescence, we show that Cdc40p is localized to the nuclear membrane, weakly associated with the nuclear pore . Our results point to a link between cell cycle progression, pre-mRNA splicing, and mRNA export. Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15388 - 93 A protein required for nuclear-protein import, Mog1p, directly interacts with GTP-Gsp1p, the Saccharomyces cerevisiae ran homologue; Oki M et al.; We previously isolated 25 temperature-sensitive gsp1 alleles of Saccharomyces cerevisiae Ran homologue, each of which possesses amino acid changes that differ from each other . We report here isolation of three multicopy suppressors-PDE2, NTF2, and a gene designated MOG1-all of which rescued a growth defect of these gsp1 strains . The gsp1 suppression occurred even in the absence of GSP2, another S . cerevisiae GSP1-like gene . Previously, NTF2 was reported to suppress gsp1 but not PDE2 . Mog1p, with a calculated molecular mass of 24 kDa, was found to be encoded by the yeast ORF YJR074W . Both MOG1 and NTF2 suppressed a series of gsp1 alleles with similar efficiency, and both suppressed gsp1 even with a single gene dose . Consistent with the high efficiency of gsp1 suppression, Mog1p directly bound to GTP, but not to GDP-Gsp1p . The disruption of MOG1 made yeast temperature-sensitive for growth . Deltamog1, which was suppressed by overexpression of NTF2, was found to have a defect in both classic and nonclassic nuclear localization signal-dependent nuclear-protein imports, but not in mRNA export . Thus, Mog1p, which was localized in the nucleus, is a Gsp1p-binding protein involved in nuclear-protein import and that functionally interacts with Ntf2p . Furthermore, the finding that PDE2 suppressed both gsp1 and rna1-1 indicates that the Ran GTPase cycle is regulated by the Ras-cAMP pathway. Gene, 199