J Biol Chem, 1998 Mar 27, 273(13), 7235 - 43 Heat-induced elevation of ceramide in Saccharomyces cerevisiae via de novo synthesis; Wells GB et al.; Sphingolipid-related metabolites have been implicated as potential signaling molecules in many studies with mammalian cells as well as in some studies with yeast . Our previous work showed that sphingolipid-deficient strains of Saccharomyces cerevisiae are unable to resist a heat shock, indicating that sphingolipids are necessary for surviving heat stress . Recent evidence suggests that one role for the sphingolipid intermediate ceramide may be to act as a second messenger to signal accumulation of the thermoprotectant trehalose . We examine here the mechanism for generating the severalfold increase in ceramide observed during heat shock . As judged by compositional analysis and mass spectrometry, the major ceramides produced during heat shock are similar to those found in complex sphingolipids, a mixture of N-hydroxyhexacosanoyl C18 and C20 phytosphingosines . Since the most studied mechanism for ceramide generation in animal cells is via a phospholipase C-type sphingomyelin hydrolysis, we examined S . cerevisiae for an analogous enzyme . Using {3H}phytosphingosine and {3H}inositol-labeled yeast sphingolipids, a novel membrane-associated phospholipase C-type activity that generated ceramide from inositol-P-ceramide, mannosylinositol-P-ceramide, and mannose(inositol-P)2-ceramide was demonstrated . The sphingolipid head groups were concomitantly liberated with the expected stoichiometry . However, other data demonstrate that the ceramide generated during heat shock is not likely to be derived by breakdown of complex sphingolipids . For example, the water-soluble fraction of heat-shocked cells showed no increase in any of the sphingolipid head groups, which is inconsistent with complex sphingolipid hydrolysis . Rather, we find that de novo ceramide synthesis involving ceramide synthase appears to be responsible for heat-induced ceramide elevation . In support of this hypothesis, we find that the potent ceramide synthase inhibitor, australifungin, completely inhibits both the heat-induced increase in incorporation of {3H}sphinganine into ceramide as well as the heat-induced increase in ceramide as measured by mass . Thus, heat-induced ceramide most likely arises by temperature activation of the enzymes that generate ceramide precursors, activation of ceramide synthase itself, or both. Int Endod J, 1997 Sep, 30(5), 313 - 7 Observation of Saccharomyces cerevisiae in blood of patient undergoing root canal treatment; Debelian GJ et al.; In this study, an unusual observation of Saccharomyces cerevisiae isolated from an infected root canal and from the blood of a patient undergoing endodontic therapy of a tooth with asymptomatic apical periodontitis is reported . Phenotypic (biochemical tests, antifungal susceptibility tests and SDS-PAGE of cellular proteins) and genetic (ribotyping) methods were used to characterize the strains . By using these methods it was found that the blood and root canal isolates were identical but differed from S . cerevisiae strains of other sources . It was therefore more than likely that the root canal was the source of the blood isolate and that it had been transferred unintentionally to the bloodstream during root canal treatment. Eur J Biochem, 1998 Mar 15, 252(3), 391 - 9 Functional expression of recombinant N-methyl-D-aspartate receptors in the yeast Saccharomyces cerevisiae--localization and pharmacological characterization; Li Z et al.; The yeast Saccharomyces cerevisiae was used for expressing the genes encoding the ionotropic N-methyl-D-aspartate (NMDA) receptor subunits from rats (NR1a, NR2A, NR2C) and mice (NR2B) . Four plasmids were constructed by cloning the different NMDA receptor genes in the two multi-copy yeast-Escherichia coli shuttle vectors pMB01 (--> NR1a gene) and pMB02 (--> NR2A-2C genes) . The protease-deficient S . cerevisiae strain cI3-ABYS-86 (leu-, ura-) was transformed or co-transformed with the resulting plasmids pMBNR1a (leu+) or pMBNR1a/pMBNR2A-C (ura+) respectively . Western blotting analysis with antibodies raised against amino acid sequences at the C-termini of the respective subunits revealed that the recombinant receptor proteins were differently expressed and only partially glycosylated in the cell membranes of the recombinant yeast strains . The expression and localization of the recombinant NMDA receptor proteins were also proved by immunofluorescence microscopy which indicated a distinct expression of the different NMDA receptor subunits in the plasma membrane of the transformed yeast cells . Pharmacological characterization of crude membrane preparations of the recombinant yeast cells showed saturable binding of the glycine antagonist {3H}MDL105,519 with Kd values of 56.9 +/- 6.19 nM (NR1a/NR2A), 26.72 +/- 2.13 nM (NR1a/NR2B), and 21.22 +/- 1.64 nM (NR1a/NR2C), and bound capacities of 17.94 +/- 1.24 pmol/mg membrane protein (NR1a/NR2A), 11.45 +/- 0.67 pmol/mg (NR1a/NR2B), and 16.15 +/- 0.86 (NR1a/NR2C) pmol/mg . The {3H}MDL105,519 binding was inhibited by the glycine antagonist 5,7-dichlorokynurenate, ethyl-2-carboxy-4,6-dichloro-3-indoleacetate, and itself, but not by glycine, D-serine or 1-amino-cyclopropanecarboxylic acid . Specific binding of {3H}glycine or the NMDA channel blocker {3H}dizolcipine were not observed. Appl Environ Microbiol, 1998 Apr, 64(4), 1303 - 7 Pyruvate decarboxylase catalyzes decarboxylation of branched-chain 2-oxo acids but is not essential for fusel alcohol production by Saccharomyces cerevisiae; ter Schure EG et al.; The fusel alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-propanol are important flavor compounds in yeast-derived food products and beverages . The formation of these compounds from branched-chain amino acids is generally assumed to occur via the Ehrlich pathway, which involves the concerted action of a branched-chain transaminase, a decarboxylase, and an alcohol dehydrogenase . Partially purified preparations of pyruvate decarboxylase (EC 4.1.1.1) have been reported to catalyze the decarboxylation of the branched-chain 2-oxo acids formed upon transamination of leucine, isoleucine, and valine . Indeed, in a coupled enzymatic assay with horse liver alcohol dehydrogenase, cell extracts of a wild-type Saccharomyces cerevisiae strain exhibited significant decarboxylation rates with these branched-chain 2-oxo acids . Decarboxylation of branched-chain 2-oxo acids was not detectable in cell extracts of an isogenic strain in which all three PDC genes had been disrupted . Experiments with cell extracts from S . cerevisiae mutants expressing a single PDC gene demonstrated that both PDC1- and PDC5-encoded isoenzymes can decarboxylate branched-chain 2-oxo acids . To investigate whether pyruvate decarboxylase is essential for fusel alcohol production by whole cells, wild-type S . cerevisiae and an isogenic pyruvate decarboxylase-negative strain were grown on ethanol with a mixture of leucine, isoleucine, and valine as the nitrogen source . Surprisingly, the three corresponding fusel alcohols were produced in both strains . This result proves that decarboxylation of branched-chain 2-oxo acids via pyruvate decarboxylase is not an essential step in fusel alcohol production. J Biol Chem, 1998 Mar 20, 273(12), 6844 - 52 Expression, purification, and characterization of choline kinase, product of the CKI gene from Saccharomyces cerevisiae; Kim KH et al.; In the yeast Saccharomyces cerevisiae, choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) is the product of the CKI gene . Choline kinase catalyzes the committed step in the synthesis of phosphatidylcholine by the CDP-choline pathway . The yeast enzyme was overexpressed 106-fold in Sf-9 insect cells and purified 71.2-fold to homogeneity from the cytosolic fraction by chromatography with concanavalin A, Affi-Gel Blue, and Mono Q . The N-terminal amino acid sequence of purified choline kinase matched perfectly with the deduced sequence of the CKI gene . The minimum subunit molecular mass (73 kDa) of purified choline kinase was in good agreement with the predicted size (66.3 kDa) of the CKI gene product . Native choline kinase existed in oligomeric structures of dimers, tetramers, and octomers . The amounts of the tetrameric and octomeric forms increased in the presence of the substrate ATP . Antibodies were raised against the purified enzyme and were used to identify choline kinase in insect cells and in S . cerevisiae . Maximum choline kinase activity was dependent on Mg2+ ions (10 mM) at pH 9.5 and at 30 degrees C . The equilibrium constant (0.2) for the reaction indicated that the reverse reaction was favored in vitro . The activation energy for the reaction was 6.26 kcal/mol, and the enzyme was labile above 30 degrees C . Choline kinase exhibited saturation kinetics with respect to choline and positive cooperative kinetics with respect to ATP (n = 1.4-2.3) . Results of the kinetic experiments indicated that the enzyme catalyzes a sequential Bi Bi reaction . The Vmax for the reaction was 138.7 micromol/min/mg, and the Km values for choline and ATP were 0.27 mM and 90 microM, respectively . The turnover number per choline kinase subunit was 153 s-1 . Ethanolamine was a poor substrate for the purified choline kinase, and it was also poor inhibitor of choline kinase activity . ADP inhibited choline kinase activity (IC50 = 0.32 mM) in a positive cooperative manner (n = 1.5), and the mechanism of inhibition with respect to ATP and choline was complex . The regulation of choline kinase activity by ATP and ADP may be physiologically relevant. Biochem J, 1998 Mar 1, 330 ( Pt 2), 811 - 7 Importance of glucose-6-phosphate dehydrogenase in the adaptive response to hydrogen peroxide in Saccharomyces cerevisiae; Izawa S et al.; Glucose-6-phosphate dehydrogenase (G6PDH)-deficient cells of Saccharomyces cerevisiae showed increased susceptibility and were unable to induce adaptation to oxidative stress . Historically, mainly in human erythrocytes, it has been suggested and accepted that decreased cellular GSH, due to loss of the NADPH-dependent activity of glutathione reductase (GR), is responsible for the increased sensitivity to oxidative stress in G6PDH-deficient cells . In the present study we investigated whether the increased susceptibility and the inability to induce adaptation to H2O2 stress of G6PDH-deficient yeast is caused by incompleteness of glutathione recycling . We constructed G6PDH- and GR-deficient mutants and analysed their adaptive response to H2O2 stress . Although G6PDH-deficient cells contained comparable amounts of GSH and GR activity to wild-type cells, GSSG was not reduced efficiently, and intracellular GSSG levels and the ratio of GSSG to total glutathione (GSSG/tGSH) were higher in G6PDH-deficient cells than in wild-type . On the other hand, GR-deficient cells showed a susceptibility identical with that of wild-type cells and induced adaptation to H2O2 stress, even though the GSSG/tGSH ratio in GR-deficient cells was higher than in G6PDH-deficient cells . These results indicate that incompleteness of glutathione recycling alone is not sufficient to account for the increased sensitivity and inability to induce adaptation to H2O2 stress of G6PDH-deficient yeast cells . In S . cerevisiae, G6PDH appears to play other important roles in the adaptive response to H2O2 stress besides supplying NADPH to the GR reaction. Genes Cells, 1997 Dec, 2(12), 753 - 70 Gin4 of S . cerevisiae is a bud neck protein that interacts with the Cdc28 complex; Okuzaki D et al.; BACKGROUND: A number of proteins make up the Cdc28 complex in Saccharomyces cerevisiae, and regulate CDK activity . The cell cycle regulator Nik1 (Hsl1) is a protein kinase that interacts with the Cdc28 complex . The growth inhibitor Gin4 is structurally similar to Nik1 and may play a redundant role in the regulation of the cell cycle . We investigated the functions of Gin4 with respect to those of Nik1 . RESULTS: GIN4 was not essential for growth, and cells deficient in the GIN4 gene displayed no obvious defects in cell cycle regulation . The delta(gin)4 delta(nik)1 strain was temperature sensitive and showed an abnormal cell shape and FACS profile at permissive temperatures . GFP-fused Gin4 was localized at the bud-neck from late G1 to the M phase . Over-production of the C-terminal portion of Gin4 was toxic for cell growth, and this domain was required for the bud-neck localization of Gin4-GFP . High copy expression of Gin4-GFP disturbed the bud-neck localization of Gin4 in the abnormally elongated cells . Cytokinesis was defective in the delta(gin)4 cdc28 double mutants . The GST-Gin4 fusion protein physically associates with the Cdc28 complex . CONCLUSIONS: Gin4 is a bud-neck protein . GIN4 and NIK1 have distinct but partially overlapping functions . The major function of GIN4 is to ensure proper mitotic progression and cytokinesis. Biochim Biophys Acta, 1998 Mar 5, 1401(3), 235 - 8 Correlation between stationary phase survival and acid trehalase activity in yeast; Roy A et al.; The levels of two trehalose hydrolysing enzymes, acid trehalase (AT) and neutral trehalase (NT), have been investigated in Candida utilis at different stages of growth; in complete contrast to Saccharomyces cerevisiae, significant AT activity appears to be absent at all stages of growth studied in C . utilis . In addition, presence of only very low amounts of iso-aspartyl methyl transferase (IMT) activity at the onset of stationary phase and lower survival ability in early stationary phase in contrast to that of S . cerevisiae lend support to the ideas that (a) lower degree of survival of C . utilis in the stationary phase may be a direct consequence of inability to mobilise stored trehalose due to absence of intracellular AT and reduced levels of IMT activities and (b) trehalose may have a dual role vis-a-vis stress resistance in yeasts. Microbiology, 1998 Mar, 144 ( Pt 3), 671 - 80 Role of trehalose in survival of Saccharomyces cerevisiae under osmotic stress; Hounsa CG et al.; Trehalose is an enigmatic compound that accumulates in Saccharomyces cerevisiae and has been implicated in survival under various stress conditions by acting as membrane protectant, as a supplementary compatible solute or as a reserve carbohydrate that may be mobilized during stress . In this study, specific mutants in trehalose metabolism were used to evaluate whether trehalose contributes to survival under severe osmotic stress and generates the compatible solute glycerol under moderate osmotic stress . The survival under severe osmotic stress (0.866 aw' NaCl or sorbitol) of mutants was compared to that of the wild-type strain when cultivated to either the mid-exponential or the stationary growth phase on glucose, galactose or ethanol . Stationary-phase cells survived better than exponential-phase cells . The death rates of ethanol-grown cells were lower than those of galactose-grown cells, which in turn survived better than glucose-grown cells . There was a strong relationship between intracellular trehalose levels and resistance to osmotic stress . The mutant strains unable to produce trehalose (tps1 delta tps2 delta and tps1 delta hxk2 delta) were more sensitive to severe osmotic stress (0.866 aw) than the isogenic wild-type strain, confirming a role for trehalose in survival . Hyperaccumulation of trehalose found in the nth1 delta and the nth1 delta gpd1 delta mutant strains, however, did not improve survival rates compared to the wild-type strain . When wild-type, nth1 delta and nth1 delta gpd1 delta cells were exposed to moderate osmotic stress (0.98 and 0.97 aw' NaCl), which permits growth, glycerol production did not appear to be related to the intracellular trehalose levels although glycerol levels increased more rapidly in nth1 delta cells than in wild-type cells during the initial response to osmotic stress . These data indicate that trehalose does not act as a reserve compound for glycerol synthesis under these conditions . No evidence was found for solutes other than glycerol and trehalose being significant for the survival of or growth by S . cerevisiae under osmotic stress conditions. Biochem J, 1998 Feb 15, 330 ( Pt 1), 421 - 7 The small GTPase Gsp1p binds to the repeat domain of the nucleoporin Nsp1p; Stochaj U et al.; The small GTPase Gsp1p of Saccharomyces cerevisiae and its homologue Ran play essential roles in several nuclear processes, such as cell-cycle progression, nuclear organization and nucleocytoplasmic traffic of RNA and proteins . Gsp1p/Ran is an abundant nuclear protein that interacts with different cytoplasmic and nuclear factors . Several of the previously identified Ran-binding proteins located at the nuclear-pore complex carry a specific Ran-binding domain . So far, direct interactions between the GTPase and other proteins have been mostly characterized in higher eukaryotes . Here we report that the yeast protein Gsp1p can directly bind to the nucleoporin Nsp1p in vitro . Nsp1p does not contain a Ran-binding domain and therefore represents a distinct type of nucleoporin that associates with Gsp1p . We demonstrate that the middle domain of Nsp1p is sufficient to mediate this interaction . Importantly, we show that a conserved cluster of positively charged amino acid residues of Gsp1p located at positions 142-144 is essential for the binding reaction . Thus we have identified Nsp1p as a new candidate protein located at the nuclear pore complex of the yeast S . cerevisiae that interacts directly with Gsp1p . We further demonstrate that both Gsp1p and Nsp1p are components of larger protein complexes in vivo, supporting the idea that the association between both proteins takes place in growing cells. Microbiol Mol Biol Rev, 1998 Mar, 62(1), 230 - 47 Vacuole biogenesis in Saccharomyces cerevisiae: protein transport pathways to the yeast vacuole; Bryant NJ et al.; Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae provides an excellent model system in which to study vacuole and lysosome biogenesis and membrane traffic . This organelle receives proteins from a number of different routes, including proteins sorted away from the secretory pathway at the Golgi apparatus and endocytic traffic arising from the plasma membrane . Genetic analysis has revealed at least 60 genes involved in vacuolar protein sorting, numerous components of a novel cytoplasm-to-vacuole transport pathway, and a large number of proteins required for autophagy . Cell biological and biochemical studies have provided important molecular insights into the various protein delivery pathways to the yeast vacuole . This review describes the various pathways to the vacuole and illustrates how they are related to one another in the vacuolar network of S . cerevisiae. Mol Cell Biol, 1998 Apr, 18(4), 2309 - 23 Ca2+ content and expression of an acidocalcisomal calcium pump are elevated in intracellular forms of Trypanosoma cruzi; Lu HG et al.; The survival of a eukaryotic protozoan as an obligate parasite in the interior of a eukaryotic host cell implies its adaptation to an environment with a very different ionic composition from that of its extracellular habitat . This is particularly important in the case of Ca2+, the intracellular concentration of which is 3 orders of magnitude lower than the extracellular value . Ca2+ entry across the plasma membrane is a widely recognized mechanism for Ca2+ signaling, needed for a number of intracellular processes, and obviously, it would be restricted in the case of intracellular parasites . Here we show that Trypanosoma cruzi amastigotes possess a higher Ca2+ content than the extracellular stages of the parasite . This correlates with the higher expression of a calcium pump, the gene for which was cloned and sequenced . The deduced protein product (Tca1) of this gene has a calculated molecular mass of 121,141 Da and exhibits 34 to 38% identity with vacuolar Ca2+-ATPases of Saccharomyces cerevisiae and Dictyostelium discoideum, respectively . The tca1 gene suppresses the Ca2+ hypersensitivity of a mutant of S . cerevisiae that has a defect in vacuolar Ca2+ accumulation . Indirect immunofluorescence and immunoelectron microscopy analysis indicate that Tca1 colocalizes with the vacuolar H+-ATPase to the plasma membrane and to intracellular vacuoles of T . cruzi . These vacuoles were shown to have the same size and distribution as the calcium-containing vacuoles identified by the potassium pyroantimoniate-osmium technique and as the electron-dense vacuoles observed in whole unfixed parasites by transmission electron microscopy and identified in a previous work (D . A . Scott, R . Docampo, J . A . Dvorak, S . Shi, and R . D . Leapman, J . Biol . Chem . 272:28020-28029, 1997) as being acidic and possessing a high calcium content (i.e., acidocalcisomes) . Together, these results suggest that acidocalcisomes are distinct from other previously recognized organelles present in these parasites and underscore the ability of intracellular parasites to adapt to the hostile environment of their hosts. Nucleic Acids Symp Ser, 1997, (36), 42 - 4 Mitochondrial RNase P: the RNA family grows; Martin NC et al.; Recent results of biochemical approaches and genome sequencing approaches has extended the members of the family of mitochondrial RNase P RNA genes . So far all of them are AU rich, and most of their secondary structures are easier to predict than was the structure of the first mitochondrial RNase P RNA from S . cerevisiae . The recently sequenced protozoan R . americana mitochondrial gene displays many of the evolutionarily conserved primary sequence and secondary structure attributes of the well characterized bacterial RNase P RNAs . Continued addition of RNAs to this data base should allow increasingly informative alignments and an understanding of what structural elements are dispensable in the smallest mitochondrial RNAs . The only protein subunits identified to date are Rpm2p from S.cerevisiae and the homologous protein from S . douglasii. Curr Genet, 1998 Jan, 33(1), 52 - 9 Evolution of mitochondrial DNA in yeast: gene order and structural organization of the mitochondrial genome of Saccharomyces uvarum; Cardazzo B et al.; We have determined the size, the restriction map and the gene order of the mitochondrial genome of the yeast Saccharomyces uvarum . Sequence analysis of the mitochondrial COXII gene confirmed the position of this yeast in the Saccharomyces cerevisiae-like group, near Saccharomyces cerevisiae and Saccharomyces douglasii . Most mitochondrial genes have been positioned on this approximately 57-kb long genome and three regions containing putative replication origins have been identified . The gene order of S . uvarum suggests that the mitochondrial genome of the S.cerevisiae-like yeasts could have evolved from an ancestral molecule, similar to that of S . uvarum, through specific genome rearrangements. Curr Genet, 1998 Jan, 33(1), 1 - 3 A rapid method to monitor repair and mis-repair of DNA double-strand breaks by using cell extracts of the yeast Saccharomyces cerevisiae; Jha B et al.; We present a rapid in vitro method to scan the repair of DNA double-strand breaks (DSBs) . A DSB was introduced at the EcoRI site within the lacZ gene of the plasmid pUC18 and the plasmid was exposed to cellular extracts from a wild-type repair-competent (RAD) and a mutant (rad52Delta) strain of the yeast Saccharomyces cerevisiae . The fidelity of rejoining was determined by the expression of the lacZ gene after bacterial transformation with the treated plasmid . A cellular extract from the yeast S . cerevisiae was found to be capable of rejoining DNA DSBs . Breaks at the EcoRI site were rejoined by extracts from both wild-type and mutant strains to form circular plasmids with almost equal efficiency . However, the fidelity of rejoining was lower for the rad52Delta extract than for normal wild-type. Gene, 1998 Jan 12, 206(2), 165 - 74 The DUG gene of Drosophila melanogaster encodes a structural and functional homolog of the S . cerevisiae SUG1 predicted ATPase associated with the 26S proteasome; Mounkes LC et al.; The DUG gene of Drosophila encodes a putative ATPase that is a structural and functional homolog of the yeast SUG1 product . When introduced into S . cerevisiae, the Drosophila DUG gene rescued the lethality associated with a SUG1 mutant . Anti-DUG antibodies recognized a protein that migrated in high molecular weight complexes, along with components of the 26S proteasome, and also immunoprecipitated components of the 26S proteasome from embryonic extracts . Proteins recognized by the affinity-purified antibody raised against DUG were localized in either a punctate cytoplasmic distribution or in the nucleus, depending on the cell type, consistent with the subcellular localization of the 26S proteasome in various cell types. Oncogene, 1998 Feb 26, 16(8), 1085 - 9 The CXXC Zn binding motifs of the human papillomavirus type 16 E7 oncoprotein are not required for its in vitro transforming activity in rodent cells; Braspenning J et al.; The conserved region 3 (CR3) of the E7 protein of human papillomaviruses contains two CXXC motifs involved in zinc binding and in the homodimerization of the molecule . Studies have suggested that the intact CXXC motifs in the CR3 of HPV16 and HPV18 E7 are required for the in vitro transforming activity of these proteins . CR3 also contains a low affinity pRb binding site and is involved in the disruption of the E2F/Rb1 complex . E7 is structurally and functionally related to Adenovirus E1A protein, which also has two CXXC motifs in CR3 . However, the Ad E1A transforming activity appears to be independent of the presence of such domains . In fact, this viral protein exists in vivo as two different forms of 289 and 243 amino acids . The shorter Ad E1A form (Ad E1A243), where both CXXC motifs are deleted by internal splicing, retains its in vitro transforming activity . We have investigated if the HPV16 E7 CR3 can be functionally replaced by the Ad E1A243 CR3, which lacks both CXXC motifs . A chimeric protein (E7/E1A243) containing the CR1 and CR2 of HPV16 E7 fused to the CR3 of Ad E1A 243 was constructed . The E7/E1A243 while not able to homodimerize in the S . cerevisiae two-hybrid system retains several of the properties of the parental proteins, HPV16 E7 and Ad E1A . It associates with the 'pocket' proteins, induces growth in soft agar of NIH3T3 cells and immortalizes rat embryo fibroblasts . These data suggest that the CXXC motifs in CR3 of E7 do not play a direct role in the transforming properties of this viral protein but probably are important for maintaining the correct protein configuration. Biochem Biophys Res Commun, 1998 Mar 17, 244(2), 505 - 13 A novel Dictyostelium discoideum gene required for cAMP-dependent cell aggregation; Nagasaki A et al.; Using a method of random insertional mutagenesis called REMI (restriction enzyme-mediated integration), we isolated two mutant strains of Dictyostelium discoideum with a defect in cAMP-dependent cell aggregation . On bacterial lawns, both of the cells formed large and smooth plaques . When starved in a non-nutrient medium, they became elongated and extended pseudopods very frequently like starved wild type cells . However, they never formed streams toward an aggregation center . Genomic DNA fragments flanking the sites of insertion of the REMI tag were rescued from the mutant cells . The fragments contained one common open reading frame encoding a protein of 1148 amino acid residues . The protein's sequence is homologous to those of two hypothetical proteins of S . cerevisiae and S . pombe. Biochem Biophys Res Commun, 1998 Mar 6, 244(1), 102 - 9 Cloning and developmental expression of a nuclear ubiquitin-conjugating enzyme (DmUbc9) that interacts with small heat shock proteins in Drosophila melanogaster; Joanisse DR et al.; In a two hybrid screen designed to identify proteins that interact with small heat shock proteins (sHsps), a Drosophila melanogaster homologue of yeast and human ubc9 (Dmubc9) was found to interact with Drosophila Hsp23 . Further, two-hybrid system analysis reveals DmUbc9 interaction with Drosophila and mammalian Hsp27 . In situ hybridization localizes Dmubc9 as a doublet at locus 21D on chromosome 2L, and genomic cloning of the gene reveals a single open reading frame without introns . The predicted Dmubc9 protein sequence shares a very high level of homology with mouse (85.4%) and human (> or = 82.9%) Ubc9 . Genetic complementation analysis show that Dmubc9 functionally rescues a temperature-sensitive S . cerevisiae ubc9ts mutant . Co-immunoprecipitation with antibody raised against DmUbc9 confirms the interaction with Drosophila Hsp23 and Hsp26 and preferentially with Hsp27 . The DmUbc9 protein, which localizes primarily to the nucleus in Drosophila S2 cells, is found at high levels in embryos but is also present at lower levels throughout development . The significance of the sHsp-Ubc9 interaction is discussed. J Mol Biol, 1998 Feb 20, 276(2), 449 - 59 The 2.3 A X-ray crystal structure of S . cerevisiae phosphoglycerate mutase; Rigden DJ et al.; The high resolution crystal structure of Saccharomyces cerevisiae phosphoglycerate mutase has been determined . This structure shows important differences from the lower resolution structure deposited in 1982 . The crystal used to determine the new structure was of a different form, having spacegroup P2(1) . The model was refined to a crystallographic R-factor of 18.9% and a free R-factor of 28.4% using all data between 25 and 2.3 A and employing a bulk solvent correction . The enzyme is a tetramer of identical, 246 amino acid subunits, whose structure is revealed to be a dimer of dimers, with four independent active sites located well away from the subunit contacts . Each subunit contains two domains, the larger with a typical nucleotide binding fold, although phosphoglycerate mutase has no physiological requirement to bind nucleotides . The catalytic-site histidine residues are no longer in a "clapping-hands" conformation, but more resemble the conformation seen in the distantly related enzymes prostatic acid phosphatase and fructose-2,6-bisphosphatase . However, the catalytic histidine residues in the mutase are found to be much closer to each other than in the phosphatase structures, perhaps due to the absence of bound ligands in the mutase crystal . An intricate web of H-bonds is found around the catalytic histidine residues, high-lighting residues probably important for maintaining their correct orientation and charge . The positions of certain other residues, including some found near the catalytic site and some lining the catalytic-site cleft, have been changed by the correction of registration errors between sequence and electron density in the original structure . Electron density was apparent for a portion of the functionally important C-terminal tail, which was absent from the earlier structure, showing it to adopt a mainly helical conformation. Biochim Biophys Acta, 1998 Feb 11, 1395(3), 329 - 44 Correct targeting of a vacuolar tobacco chitinase in Saccharomyces cerevisiae--post-translational modifications are dependent on the host strain; Kunze I et al.; The chitinase gene FB7-1 of Nicotiana tabacum cv . samsun line 5 was expressed in the two Saccharomyces cerevisiae strains, INVSC2 and H4, under the control of the GAL1 promoter from S . cerevisiae and a multicopy plasmid vector . Both yeast strains express the plant gene as enzymatic active proteins . In transformants of the strain INVSC2, 94% of the total plant chitinase is contained inside the cells, probably within the vacuole which has been confirmed by subcellular fractionation as well as immunohistochemical experiments . This retention inside the cells is due to the C-terminally located 7 amino acids long vacuolar targeting peptide of the prochitinase . When this sequence was removed, chitinase was transported into the culture medium . Pulse-chase experiments revealed that during translation in transformants of both yeast strains one chitinase polypeptide can be immunoadsorbed with specific antibodies . In the case of INVSC2-transformants, newly formed chitinase is modified in a 60 min chase to slightly increase its molecular mass, whereas in H4-transformants the molecular mass constantly remained 32 kDa . By Western blot analysis two chitinase corresponding polypeptides of 32 and 37 kDa were accumulated in the culture medium of both transformants carrying the chitinase gene without the vacuolar targeting sequence . The larger one was very likely O-glycosylated . Whereas, both polypepitdes were also detected in cell extracts of the H4-transformant, only the smaller one was found in the INVSC2-transformant . The plant chitinase passed through the endoplasmic reticulum on its way to the vacuole . The N-terminal signal peptide responsible for the uptake into the endoplasmic reticulum is cleaved correctly . However, cleavage of the vacuolar targeting peptide located at the C-terminus, to give the mature chitinase is obviously influenced by the genetic background of the host strain . In INVSC2-transformants chitinase accumulates in its mature form whereas both the polypeptides of H4-transformants retain their vacuolar targeting peptide . Our results demonstrate that in the case of plant class I chitinase, the plant sorting signal is recognized in yeast cells but post-translational modifications are influenced by the host strain. Bioessays, 1998 Jan, 20(1), 30 - 40 Silent chromatin in yeast: an orchestrated medley featuring Sir3p {corrected}; Stone EM et al.; Extensive regions of chromosomes can be transcriptionally repressed through silencing mechanisms mediated by complex chromatin structures . One of the most refined molecular portraits of silenced chromatin comes from studies of the silent mating-type loci and telomeres of S . cerevisiae . In this budding yeast, the Sir3p silent information regulator emerges as a critically important silencing component that interacts with nucleosomes and other silencing proteins . Not only is it essential for silencing, but Sir3p is also capable of spreading silenced chromatin when its dosage is increased . Sir3p is a target of mitogen-activated protein (MAP) kinase cascade regulation and has significant similarity to the Orc1p subunit of the DNA replication origin recognition complex . Thus, in concert with other silencing proteins, Sir3p appears poised to respond to cellular signals and reprogram silencing through replication-associated assembly of repressive chromatin structures. Fungal Genet Biol, 1998 Feb, 23(1), 1 - 17 Cytochrome P450 enzyme systems in fungi; van den Brink HM et al.; The involvement of cytochrome P450 enzymes in many complex fungal bioconversion processes has been characterized in recent years . Accordingly, there is now considerable scientific interest in fungal cytochrome P450 enzyme systems . In contrast to S . cerevisiae, where surprisingly few P450 genes have been identified, biochemical data suggest that many fungi possess numerous P450 genes . This review summarizes the current information pertaining to these fungal cytochrome P450 systems, with emphasis on the molecular genetics . The use of molecular techniques to improve cytochrome P450 activities in fungi is also discussed. Glycobiology, 1998 Feb, 8(2), 155 - 64 Genetic tailoring of N-linked oligosaccharides: the role of glucose residues in glycoprotein processing of Saccharomyces cerevisiae in vivo; Jakob CA et al.; In higher eukaryotes a quality control system monitoring the folding state of glycoproteins is located in the ER and is composed of the proteins calnexin, calreticulin, glucosidase II, and UDP-glucose: glycoprotein glucosyltransferase . It is believed that the innermost glucose residue of the N- linked oligosaccharide of a glycoprotein serves as a tag in this control system and therefore performs an important function in the protein folding pathway . To address this function, we constructed Saccharomyces cerevisiae strains which contain nonglucosylated (G0), monoglucosylated (G1), or diglucosylated (G2) glycoproteins in the ER and used these strains to study the role of glucose residues in the ER processing of glycoproteins . These alterations of the oligosaccharide structure did not result in a growth phenotype, but the induction of the unfolded protein response upon treatment with DTT was much higher in G0 and G2 strains as compared to wild-type and G1 strains . Our results provide in vivo evidence that the G1 oligosaccharide is an active oligosaccharide structure in the ER glycoprotein processing pathway of S.cerevisiae . Furthermore, by analyzing N- linked oligosaccharides of the constructed strains we can directly show that no general glycoprotein glucosyltransferase exists in S . cerevisiae. Essays Biochem, 1997, 32, 113 - 25 The co-ordination of nuclear and organellar genome expression in eukaryotic cells; Surpin M et al.; Mitochondria and chloroplasts contain their own genetic complement . Eukaryotic cells must establish mechanisms by which the organellar and nuclear genomes are co-ordinately expressed . In the yeast S . cerevisiae, mitochondrial mutants that have respiratory deficiencies have altered patterns of nuclear gene expression . In strains that are deficient in the mitochondrial isoform of citrate synthase there is an up-regulation of the peroxisomal isoform . There is evidence that the mitochondria send a signal to the nucleus which results in the increased expression of the peroxisomal form of citrate synthase . Oxygen induces the biosynthesis of haem . When yeast are grown in aerobic culture, mitochondrial haem is transduced to the nucleus where it induces the expression of nuclear genes which encode proteins important for cellular respiration . Plant cells in which the chloroplasts have suffered photo-oxidative damage do not express nuclear genes that encode photosynthetic protein . Genetic studies in Arabidopsis have resulted in the characterization of mutants that do express nuclear photosynthetic genes when the development of the chloroplast is inhibited . These mutants will be instrumental in identifying the components of the chloroplast-to-nucleus signal-transduction pathways in plant cells . The identity of the signal that informs the nucleus of the developmental site of the chloroplast is unknown . Chlorophyll precursors, haem and porphyrin are considered candidate signal molecules . Recent studies have implicated that the redox state of the plastoquinone pool in chloroplasts may be involved in regulating nuclear gene expression . These studies have also shown that kinases and phosphatases may participate in relaying signals to the nucleus about the functional state of the chloroplast. Cell, 1998 Feb 20, 92(4), 489 - 99 Ump1p is required for proper maturation of the 20S proteasome and becomes its substrate upon completion of the assembly; Ramos PC et al.; We report the discovery of a short-lived chaperone that is required for the correct maturation of the eukaryotic 20S proteasome and is destroyed at a specific stage of the assembly process . The S . cerevisiae Ump1p protein is a component of proteasome precursor complexes containing unprocessed beta subunits but is not detected in the mature 20S proteasome . Upon the association of two precursor complexes, Ump1p is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated . Cells lacking Ump1p exhibit a lack of coordination between the processing of beta subunits and proteasome assembly, resulting in functionally impaired proteasomes . We also show that the propeptide of the Pre2p/Doa3p beta subunit is required for Ump1p's function in proteasome maturation. Genes Cells, 1997 Nov, 2(11), 655 - 65 The efficiency and timing of initiation of replication of multiple replicons of Saccharomyces cerevisiae chromosome VI; Yamashita M et al.; BACKGROUND: A complete set of nine ARSs was identified (the tenth ARS in this paper), mapped on chromosome VI of Saccharomyces cerevisiae, and characterized for functional elements . RESULTS: The level of activity of all ARSs as chromosomal replication origins was determined by neutral/neutral 2D gel-electrophoresis . These origins were classified into three groups: (i) three high frequency origins used once nearly every cell cycle, (ii) four intermediate frequency origins used once in two to three cycles and (iii) two low frequency origins used in fewer than 5% of cell cycles . These variations in initiation frequency among origins of chromosome VI are present in three common laboratory wild-type strains . Each origin is initiated at a fixed time and passively replicated by incoming replication forks at a fixed time during a synchronous S phase . Replication of each arm of the chromosome starts from one major origin located one-fifth (left arm) and one-third (right arm) of the distance from the centromere, and expands sequentially in both directions . Two telomere vicinity origins are replicated last . Time of initiation and replication of the last replicating origin, Ori609, was remarkably variable from cell to cell . CONCLUSIONS: Chromosome VI of S . cerevisiae contains nine replication origins that comprise five active replicons under normal cell growth conditions . A clear correlation was found between the efficiency of initiation and the order of replication . The timing of initiation of most origins, except for the first and last, is coincident with the time of passive replication by incoming forks from neighbouring origins. Mol Gen Genet, 1998 Jan, 257(2), 149 - 56 The RHC21 gene of budding yeast, a homologue of the fission yeast rad21+ gene, is essential for chromosome segregation; Heo SJ et al.; The Saccharomyces cerevisiae gene RHC21 is a homologue of the fission yeast rad21+ gene, which affects the sensitivity of cells to gamma-irradiation and is essential for cell growth in S . pombe . Disruption of the RHC21 gene showed that it is also essential in S . cerevisiae . To examine its function in cell growth further, we have isolated temperature-sensitive mutants for the RHC21 gene and characterized one of them, termed rhc21-sk16 . When this mutant was incubated at 36 degrees C, the percentage of large-budded cells was increased . Most of the large-budded cells had aberrant nuclear structures, such as unequally extended nuclear DNA with incompletely elongated spindles across the mother-daughter neck or only in a mother cell . Furthermore, a circular minichromosome is more unstable in the mutant than in the wild-type, even at 25 degrees C . Flow cytometry showed that the bulk of DNA replication takes place normally at the restrictive temperature in the mutant . These results indicated that the RHC21 gene is required for proper segregation of the chromosomes . In addition, we found that the mutant is sensitive not only to UV radiation and gamma-rays but also to the antimicrotubule agent nocodazole at 25 degrees C . This suggests that the RHC21 gene is involved in the microtubule function . We discuss how the RHC21 gene product may be involved in chromosome segregation and microtubule function. J Biol Chem, 1998 Feb 6, 273(6), 3702 - 11 IDP3 encodes a peroxisomal NADP-dependent isocitrate dehydrogenase required for the beta-oxidation of unsaturated fatty acids; Henke B et al.; In Saccharomyces cerevisiae the metabolic degradation of saturated fatty acids is exclusively confined to peroxisomes . In addition to a functional beta-oxidation system, the degradation of unsaturated fatty acids requires auxiliary enzymes, including a Delta2, Delta3-enoyl-CoA isomerase and an NADPH-dependent 2,4-dienoyl-CoA reductase . We found both enzymes to be present in yeast peroxisomes . The impermeability of the peroxisomal membrane for pyrimidine nucleotides led to the question of how the NADPH needed by the reductase is regenerated in the peroxisomal lumen . We report the identification and functional analysis of the IDP3 gene product, which is a yeast peroxisomal NADP-dependent isocitrate dehydrogenase . The newly identified peroxisomal protein is homologous to the mitochondrial Idp1p and cytosolic Idp2p, which both are yeast NADP-dependent isocitrate dehydrogenases . Yeast cells lacking Idp3p grow normally on saturated fatty acids, but growth is impaired on unsaturated fatty acids, indicating that the peroxisomal Idp3p is involved in their metabolic utilization . The data presented are consistent with the assumption that peroxisomes of S . cerevisiae contain the enzyme equipment needed for the degradation of unsaturated fatty acids, including an NADP-dependent isocitrate dehydrogenase, a putative constituent of a peroxisomal NADPH-regenerating redox system. J Biol Chem, 1998 Feb 6, 273(6), 3278 - 84 Isolation and characterization of the Saccharomyces cerevisiae DPP1 gene encoding diacylglycerol pyrophosphate phosphatase; Toke DA et al.; Diacylglycerol pyrophosphate (DGPP) is involved in a putative novel lipid signaling pathway . DGPP phosphatase (DGPP phosphohydrolase) is a membrane-associated 34-kDa enzyme from Saccharomyces cerevisiae which catalyzes the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyzes the dephosphorylation of PA to yield diacylglycerol . Amino acid sequence information derived from DGPP phosphatase was used to identify and isolate the DPP1 (diacylglycerol pyrophosphate phosphatase) gene encoding the enzyme . Multicopy plasmids containing the DPP1 gene directed a 10-fold overexpression of DGPP phosphatase activity in S . cerevisiae . The heterologous expression of the S . cerevisiae DPP1 gene in Sf-9 insect cells resulted in a 500-fold overexpression of DGPP phosphatase activity over that expressed in wild-type S . cerevisiae . DGPP phosphatase possesses a Mg2+-independent PA phosphatase activity, and its expression correlated with the overexpression of DGPP phosphatase activity in S . cerevisiae and in insect cells . DGPP phosphatase was predicted to be an integral membrane protein with six transmembrane-spanning domains . The enzyme contains a novel phosphatase sequence motif found in a superfamily of phosphatases . A dpp1Delta mutant was constructed by deletion of the chromosomal copy of the DPP1 gene . The dpp1Delta mutant was viable and did not exhibit any obvious growth defects . The mutant was devoid of DGPP phosphatase activity and accumulated (4-fold) DGPP . Analysis of the mutant showed that the DPP1 gene was not responsible for all of the Mg2+-independent PA phosphatase activity in S . cerevisiae. Yeast, 1998 Jan 15, 14(1), 67 - 76 Engineering yeast for efficient cellulose degradation; Van Rensburg P et al.; Saccharomyces cerevisiae produces several beta-1,3-glucanases, but lacks the multicomponent cellulase complexes that hydrolyse the beta-1,4-linked glucose polymers present in cellulose-rich biomass as well as in haze-forming glucans in certain wines and beers . We have introduced into S . cerevisiae a functional cellulase complex for efficient cellulose degradation by cloning the Endomyces fibuliger cellobiase (BGL1) gene and co-expressing it with the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase (END1), the Phanerochaete chrysosporium cellobiohydrolase (CBH1) and the Ruminococcus flavefacies cellodextrinase (CEL1) gene constructs in this yeast . The END1, CBH1 and CEL1 genes were inserted into yeast expression/secretion cassettes . Expression of END1, CBH1 and CEL1 was directed by the promoter sequences derived from the alcohol dehydrogenase II (ADH2), the phosphoglycerate kinase I (PKG1) and the alcohol dehydrogenase I (ADH1) genes, respectively . In contrast, BGL1 was expressed under the control of its native promoter . Secretion of End1p and Cel1p was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1), whereas Cbh1p and Bgl1p were secreted using their authentic leader peptides . The construction of a fur1 ura3 S . cerevisiae strain allowed for the autoselection of this multicopy URA3-based plasmid in rich medium . S . cerevisiae transformants secreting biologically active endo-beta-1,4-glucanase, cellobiohydrolase, cellodextrinase and cellobiase were able to degrade various substrates including carboxymethylcellulose, hydroxyethylcellulose, laminarin, barley glucan, cellobiose, polypectate, birchwood xylan and methyl-beta-D-glucopyranoside . This study could lead to the development of industrial strains of S . cerevisiae capable of converting cellulose in a one-step process into commercially important commodities. J Biol Chem, 1998 Jan 30, 273(5), 2977 - 83 Expression of the glyoxalase I gene of Saccharomyces cerevisiae is regulated by high osmolarity glycerol mitogen-activated protein kinase pathway in osmotic stress response; Inoue Y et al.; Methylglyoxal is a cytotoxic metabolite derived from dihydroxyacetone phosphate, an intermediate of glycolysis . Detoxification of methylglyoxal is performed by glyoxalase I . Expression of the structural gene of glyoxalase I (GLO1) of Saccharomyces cerevisiae under several stress conditions was investigated using the GLO1-lacZ fusion gene, and expression of the GLO1 gene was found to be specifically induced by osmotic stress . The Hog1p is one of the mitogen-activated protein kinases (MAPKs) in S . cerevisiae, and both Msn2p and Msn4p are the transcriptional regulators that are thought to be under the control of Hog1p-MAPK . Expression of the GLO1 gene under osmotic stress was completely repressed in hog1Delta disruptant and was repressed approximately 80 and 50% in msn2Delta and msn4Delta disruptants, respectively . A double mutant of the MSN2 and MSN4 gene was unable to induce expression of the GLO1 gene under highly osmotic conditions . Glucose consumption increased approximately 30% during the adaptive period in osmotic stress in the wild type strain . On the contrary, it was reduced by 15% in the hog1Delta mutant . When the yeast cell is exposed to highly osmotic conditions, glycerol is synthesized as a compatible solute . Glycerol is synthesized from glucose, and a rate-limiting enzyme in glycerol biosynthesis is glycerol-3-phosphate dehydrogenase (GPD1 gene product), which catalyzes reduction of dihydroxyacetone phosphate to glycerol 3-phosphate . Expression of the GPD1 gene is also under the control of Hog1p-MAPK . Methylglyoxal is also synthesized from dihydroxyacetone phosphate; therefore, induction of the GLO1 gene expression by osmotic stress was thought to scavenge methylglyoxal, which increased during glycerol production for adaptation to osmotic stress. EMBO J, 1998 Jan 15, 17(2), 396 - 405 Cell wall 1,6-beta-glucan synthesis in Saccharomyces cerevisiae depends on ER glucosidases I and II, and the molecular chaperone BiP/Kar2p; Simons JF et al.; The role of glucose trimming in the endoplasmic reticulum of Saccharomyces cerevisiae was investigated using glucosidase inhibitors and mutant strains devoid of glucosidases I and II . These glucosidases are responsible for removing glucose residues from the N-linked core oligosaccharides attached to newly synthesized polypeptide chains . In mammalian cells they participate together with calnexin, calreticulin and UDP-glucose:glycoprotein glucosyltransferase in the folding and quality control of newly synthesized glycoproteins . In S.cerevisiae, glucosidase II is encoded by the GLS2 gene, and glucosidase I, as suggested here, by the CWH41 gene . Using castanospermine (an alpha-glucosidase inhibitor) and yeast strains defective in glucosidase I, glucosidase II and BiP/Kar2p, it was demonstrated that cell wall synthesis depends on the two glucosidases and BiP/Kar2p . In double mutants with defects in both BiP/Kar2p and either of the glucosidases the phenotype was particularly clear: synthesis of 1,6-beta-glucan_a cell wall component_was reduced; the cell wall displayed abnormal morphology; the cells aggregated; and their growth was severely inhibited . No defects in protein folding or secretion could be detected . We concluded that glucose trimming in S.cerevisiae is necessary for proper cell wall synthesis, and that the glucosidases function synergistically with BiP/Kar2p in this process. EMBO J, 1998 Jan 2, 17(1), 269 - 77 The N-end rule pathway controls the import of peptides through degradation of a transcriptional repressor; Byrd C et al.; Ubiquitin-dependent proteolytic systems underlie many processes, including the cell cycle, cell differentiation and responses to stress . One such system is the N-end rule pathway, which targets proteins bearing destabilizing N-terminal residues . Here we report that Ubr1p, the main recognition component of this pathway, regulates peptide import in the yeast Saccharomyces cerevisiae through degradation of Cup9p, a 35 kDa homeodomain protein . Cup9p was identified using a screen for mutants that bypass the previously observed requirement for Ubr1p in peptide import . We show that Cup9p is a short-lived protein (t1/2 approximately 5 min) whose degradation requires Ubr1p . Cup9p acts as a repressor of PTR2, a gene encoding the transmembrane peptide transporter . In contrast to engineered N-end rule substrates, which are recognized by Ubr1p through their destabilizing N-terminal residues, Cup9p is targeted by Ubr1p through an internal degradation signal . The Ubr1p-Cup9p-Ptr2p circuit is the first example of a physiological process controlled by the N-end rule pathway . An earlier study identified Cup9p as a protein required for an aspect of resistance to copper toxicity in S.cerevisiae . Thus, one physiological substrate of the N-end rule pathway functions as both a repressor of peptide import and a regulator of copper homeostasis. Nucleic Acids Res, 1998 Jan 15, 26(2), 521 - 4 Human cytosolic asparaginyl-tRNA synthetase: cDNA sequence, functional expression in Escherichia coli and characterization as human autoantigen; Beaulande M et al.; The cDNA for human cytosolic asparaginyl-tRNA synthetase (hsAsnRSc) has been cloned and sequenced . The 1874 bp cDNA contains an open reading frame encoding 548 amino acids with a predicted M r of 62 938 . The protein sequence has 58 and 53% identity with the homologous enzymes from Brugia malayi and Saccharomyces cerevisiae respectively . The human enzyme was expressed in Escherichia coli as a fusion protein with an N-terminal 4 kDa calmodulin-binding peptide . A bacterial extract containing the fusion protein catalyzed the aminoacylation reaction of S.cerevisiae tRNA with {14C}asparagine at a 20-fold efficiency level above the control value confirming that this cDNA encodes a human AsnRS . The affinity chromatography purified fusion protein efficiently aminoacylated unfractionated calf liver and yeast tRNA but not E.coli tRNA, suggesting that the recombinant protein is the cytosolic AsnRS . Several human anti-synthetase sera were tested for their ability to neutralize hsAsnRSc activity . A human autoimmune serum (anti-KS) neutralized hsAsnRSc activity and this reaction was confirmed by western blot analysis . The human asparaginyl-tRNA synthetase appears to be like the alanyl- and histidyl-tRNA synthetases another example of a human Class II aminoacyl-tRNA synthetase involved in autoimmune reactions. Nucleic Acids Res, 1998 Jan 15, 26(2), 477 - 85 The second subunit of DNA polymerase III (delta) is encoded by the HYS2 gene in Saccharomyces cerevisiae; Hashimoto K et al.; DNA polymerase III (delta) of Saccharomyces cerevisiae is purified as a complex of at least two polypeptides with molecular masses of 125 and 55 kDa as judged by SDS-PAGE . In this paper we determine partial amino acid sequences of the 125 and 55 kDa polypeptides and find that they match parts of the amino acid sequences predicted from the nucleotide sequence of the CDC2 and HYS2 genes respectively . We also show by Western blotting that Hys2 protein co-purifies with DNA polymerase III activity as well as Cdc2 polypeptide . The complex form of DNA polymerase III activity could not be detected in thermosensitive hys2 mutant cell extracts, although another form of DNA polymerase III was found . This form of DNA polymerase III, which could also be detected in wild-type extracts, was not associated with Hys2 protein and was not stimulated by addition of proliferating cell nuclear antigen (PCNA), replication factor A (RF-A) or replication factor C (RF-C) . The temperature-sensitive growth phenotype of hys2-1 and hys2-2 mutations could be suppressed by the CDC2 gene on a multicopy plasmid . These data suggest that the 55 kDa polypeptide encoded by the HYS2 gene is one of the subunits of DNA polymerase III complex in S.cerevisiae and is required for highly processive DNA synthesis catalyzed by DNA polymerase III in the presence of PCNA, RF-A and RF-C. J Cell Sci, 1998 Jan, 111 ( Pt 1), 11 - 21 RagA is a functional homologue of S . cerevisiae Gtr1p involved in the Ran/Gsp1-GTPase pathway; Hirose E et al.; Human RagA and RagB is reported to be 52% identical to a putative GTPase of Saccharomyces cerevisiae, Gtr1p . According to the reported nucleotide sequence, we amplified human RagA and RagBs cDNAs from the human B cell cDNA library with PCR . Both cDNAs rescued a cold sensitivity of S . cerevisiae, gtr1-11 . Furthermore, we introduced into the cloned human RagA cDNA, the mutation 'T21L' corresponding to the gtr1-11 mutation which has been reported to suppress not only all of rcc1-, temperature-sensitive mutants of Ran/Gsp1p GTPase GDP/GTP-exchanging factor, but also rna1-1, a temperature-sensitive mutant of Ran/Gsp1p GTPase-activating protein . The resulting RagAgtr1-11 cDNA partially, but significantly, suppressed both rcc1- and rna1-1 mutations . These results indicated that RagA and RagBs are functional homologues of S . cervisiae Gtr1p . Interestingly, while wild-type human RagA and RagBs were localized within the cytoplasm, similar to S . cerevisiae Gtr1p, the mutated human RagAgtr1-11 corresponding to a dominant negative form of RagA was distributed in discrete speckles in the nucleus, being localized side by side with SC-35, a non-snRNP of the splicing complex . In contrast, a dominant positive form of RagA, Q66L was localized in the cytoplasm . Thus, RagA was suggested to shuttle between the cytoplasm and the nucleus, depending on the bound nucleotide state. Biochem Biophys Res Commun, 1998 Feb 4, 243(1), 205 - 9 TAT1 encodes a low-affinity histidine transporter in Saccharomyces cerevisiae; Bajmoczi M et al.; Previous studies have revealed the presence of at least two histidine uptake systems in S . cerevisiae; one with high affinity and the other with low affinity for histidine . The HIP1 gene is known to encode the high affinity permease . The purpose of this study was to identify the gene that encodes the low affinity permease . A mutant strain of S . cerevisiae that is both a histidine auxotroph and a hip1 deletion mutant is unable to grow on low histidine media . This strain was transformed with a yeast cDNA library constructed in a yeast expression vector . Transformants with increased histidine transport were selected by their ability to grow on a low histidine media . Sequencing of the inserts revealed the presence of the HIP1 gene and also the presence of the TAT1 gene . Estimated Km and Vmax values for histidine transport by each system were determined . In a hip1 tat1 double mutant, the level of histidine required for growth increased eight-fold in comparison to the hip1 single mutant . Our results suggest that the TAT1-encoded protein, previously characterized as the high-affinity tyrosine permease, also acts as the low affinity histidine permease. EMBO J, 1997 Dec 15, 16(24), 7326 - 41 Overexpression of Pex15p, a phosphorylated peroxisomal integral membrane protein required for peroxisome assembly in S.cerevisiae, causes proliferation of the endoplasmic reticulum membrane; Elgersma Y et al.; We have cloned PEX15 which is required for peroxisome biogenesis in Saccharomyces cerevisiae . pex15Delta cells are characterized by the cytosolic accumulation of peroxisomal matrix proteins containing a PTS1 or PTS2 import signal, whereas peroxisomal membrane proteins are present in peroxisomal remnants . PEX15 encodes a phosphorylated, integral peroxisomal membrane protein (Pex15p) . Using multiple in vivo methods to determine the topology, Pex15p was found to be a tail-anchored type II (Ncyt-Clumen) peroxisomal membrane protein with a single transmembrane domain near its carboxy-terminus . Overexpression of Pex15p resulted in impaired peroxisome assembly, and caused profound proliferation of the endoplasmic reticulum (ER) membrane . The lumenal carboxy-terminal tail of Pex15p protrudes into the lumen of these ER membranes, as demonstrated by its O-glycosylation . Accumulation in the ER was also observed at an endogenous expression level when Pex15p was fused to the N-terminus of mature invertase . This resulted in core N-glycosylation of the hybrid protein . The lumenal C-terminal tail of Pex15p is essential for targeting to the peroxisomal membrane . Furthermore, the peroxisomal membrane targeting signal of Pex15p overlaps with an ER targeting signal on this protein . These results indicate that Pex15p may be targeted to peroxisomes via the ER, or to both organelles. Nucleic Acids Res, 1998 Jan 1, 26(1), 73 - 9 SGD: Saccharomyces Genome Database; Cherry JM et al.; The Saccharomyces Genome Database (SGD) provides Internet access to the complete Saccharomyces cerevisiae genomic sequence, its genes and their products, the phenotypes of its mutants, and the literature supporting these data . The amount of information and the number of features provided by SGD have increased greatly following the release of the S.cerevisiae genomic sequence, which is currently the only complete sequence of a eukaryotic genome . SGD aids researchers by providing not only basic information, but also tools such as sequence similarity searching that lead to detailed information about features of the genome and relationships between genes . SGD presents information using a variety of user-friendly, dynamically created graphical displays illustrating physical, genetic and sequence feature maps . SGD can be accessed via the World Wide Web at http://genome-www.stanford.edu/Saccharomyces/ Biochim Biophys Acta, 1998 Jan 8, 1379(1), 118 - 28 Expression of high-affinity trehalose-H+ symport in Saccharomyces cerevisiae; Stambuk BU et al.; The expression of the high-affinity trehalose-H+ symport was investigated in various Saccharomyces cerevisiae strains and culture conditions . Previous kinetic studies of trehalose transport in yeast have revealed the existence of at least two different uptake mechanisms: a high-affinity trehalose-H+ symport activity repressed by glucose, and a constitutive low-affinity transport activity, a putative facilitated diffusion process . Exogenously added trehalose was not an inducer of the high-affinity transport activity, and a correlation between trehalose and maltose uptake by yeast cells was found . Our results indicate that the maltose-H+ symporters encoded by MAL11, MAL21, and MAL41 are not responsible for the trehalose transport activity . The analysis of both trehalose and maltose transport activities in wild-type and in laboratory strains with defined MAL genes showed that the trehalose-H+ symporter was under control of MAL regulatory genes . Our results also suggest that the recently characterized AGT1 gene of S . cerevisiae may encode the high-affinity trehalose-H+ symporter . During diauxic growth on glucose the transport activity was low during the first exponential phase of growth, increased as glucose was exhausted from the medium, and decreased again as the cells reached the late stationary phase . This pattern was coincident with that of the intracellular levels of trehalose . The strong correlation between these two parameters may be of physiological significance during adaptation of yeast cells to stress conditions. J Clin Microbiol, 1998 Feb, 36(2), 557 - 62 Epidemiological investigation of vaginal Saccharomyces cerevisiae isolates by a genotypic method; McCullough MJ et al.; Saccharomyces cerevisiae is a ubiquitous, ascomycetous yeast, and vaginitis caused by this organism has been reported only very rarely . The aim of the present investigation was to assess the epidemiological relatedness of a group of vaginal and commercial S . cerevisiae isolates by a previously reported genetic typing method, which divided the isolates into two broad groups with numerous subtypes . Nineteen S . cerevisiae isolates obtained from patients suffering from vaginitis and four isolates from commercial products in the same city were analyzed . The cellular DNA from each isolate was digested with the restriction endonuclease EcoRI, and restriction fragment length polymorphisms were generated by horizontal gel electrophoresis . The results showed that although vaginal isolates did not cluster in any particular genetic subtype, multiple patients were infected with indistinguishable strains (there were nine distinct strains among 23 isolates) . For two of three patients, all three with two episodes of S . cerevisiae vaginitis, different strains were isolated during the recurrence of this disease . Three other patients with indistinguishable isolates were epidemiologically related in that two were practitioners in the same clinic and the third was a patient at this clinic . We also found that one commercial strain was indistinguishable from the strain isolated from three different women at the time that they were suffering from vaginitis . The findings of the present study suggest that some S . cerevisiae strains may possess properties permitting persistence in the human host . Furthermore, person-to-person contact and the proliferation of the use of S . cerevisiae as a health-food product, in home baking, and in home brewing may be a contributing factor in human colonization and infection with this organism. Appl Environ Microbiol, 1998 Feb, 64(2), 665 - 8 Effects of ethanol and other alkanols on transport of acetic acid in Saccharomyces cerevisiae; Casal M et al.; In glucose-grown cells of Saccharomyces cerevisiae IGC 4072, acetic acid enters only by simple diffusion of the undissociated acid . In these cells, ethanol and other alkanols enhanced the passive influx of labelled acetic acid . The influx of the acid followed first-order kinetics with a rate constant that increased exponentially with the alcohol concentration, and an exponential enhancement constant for each alkanol was estimated . The intracellular concentration of labelled acetic acid was also enhanced by alkanols, and the effect increased exponentially with alcohol concentration . Acetic acid is transported across the plasma membrane of acetic acid-, lactic acid-, and ethanol-grown cells by acetate-proton symports . We found that in these cells ethanol and butanol inhibited the transport of labelled acetic acid in a noncompetitive way; the maximum transport velocity decreased with alcohol concentration, while the affinity of the system for acetate was not significantly affected by the alcohol . Semilog plots of Vmax versus alcohol concentration yielded straight lines with negative slopes from which estimates of the inhibition constant for each alkanol could be obtained . The intracellular concentration of labelled acid was significantly reduced in the presence of ethanol or butanol, and the effect increased with the alcohol concentration . We postulate that the absence of an operational carrier for acetate in glucose-grown cells of S . cerevisiae, combined with the relatively high permeability of the plasma membrane for the undissociated acid and the inability of the organism to metabolize acetic acid, could be one of the reasons why this species exhibits low tolerance to acidic environments containing ethanol. Appl Environ Microbiol, 1998 Feb, 64(2), 564 - 8 Intracellular signal triggered by cholera toxin in Saccharomyces boulardii and Saccharomyces cerevisiae; Brandao RL et al.; As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT) . The addition of glucose or dinitrophenol to cells of S . boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S.cerevisiae . The addition of CT to the same cells also resulted in trehalase activation . Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation . Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S . boulardii and S . cerevisiae . These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade . The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells . This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells . The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts. Appl Environ Microbiol, 1998 Feb, 64(2), 530 - 4 Measurement of the effects of acetic acid and extracellular pH on intracellular pH of nonfermenting, individual Saccharomyces cerevisiae cells by fluorescence microscopy; Guldfeldt LU et al.; The effects of acetic acid and extracellular pH (pHex) on the intracellular pH (pHi) of nonfermenting, individual Saccharomyces cerevisiae cells were studied by using a new experimental setup comprising a fluorescence microscope and a perfusion system . S . cerevisiae cells grown in brewer's wort to the stationary phase were stained with fluorescein diacetate and transferred to a perfusion chamber . The extracellular concentration of undissociated acetic acid at various pHex values was controlled by perfusion with 2 g of total acetic acid per liter at pHex 3.5, 4.5, 5.6, and 6.5 through the chamber by using a high-precision pump . The pHi of individual S . cerevisiae cells during perfusion was measured by fluorescence microscopy and ratio imaging . Potential artifacts, such as fading and efflux of fluorescein, could be neglected within the experimental time used . At pHex 6.5, the pHi of individual S . cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.035 g/liter, whereas at pHex 3.5, 4.5, and 5.6, the pHi of individual S . cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.10 g/liter . At concentrations of undissociated acetic acid of more than 0.10 g/liter, the pHi remained constant . The decreases in pHi were dependent on the pHex; i.e., the decreases in pHi at pHex 5.6 and 6.5 were significantly smaller than the decreases in pHi at pHex 3.5 and 4.5. FEBS Lett, 1998 Jan 2, 421(1), 37 - 40 Rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria: the enzyme expressed in Escherichia coli acts as a member of the respiratory chain in the host cells; Kitajima-Ihara T et al.; The NDI1 gene encodes the internal rotenone-insensitive NADH-quinone oxidoreductase localized in the inner mitochondrial membranes of Saccharomyces cerevisiae . The T7 tag-fused mature NDI1 was overexpressed in Escherichia coli . The overexpressed NDI1 was exclusively found in the membrane fraction . The NDI1-overexpressed membranes showed significantly increased activities of NADH oxidase and NADH-ubiquinone-1 (UQ1) reductase when compared with the control membranes . Flavone, which is a specific inhibitor of the S . cerevisiae NDI1, inhibited almost completely NADH oxidase and NADH-UQ1 reductase activities of NDI1-overexpressed membranes but scarcely inhibited these activities of the control membranes . In addition, the NADH oxidase activity of the NDI1-overexpressed membranes was also inhibited by KCN as well as the control membranes . These results indicate that the overexpressed NDI1 worked as a member of the respiratory chain in the host cells, even though E . coli membranes are different from S . cerevisiae inner mitochondrial membranes in terms of quinones and lipid composition. Biochemistry (Mosc), 1997 Oct, 62(10), 1146 - 51 Detection and some properties of membrane-bound and soluble polyphosphatases in mitochondria of the yeast Saccharomyces cerevisiae; Lichko LP et al.; Saccharomyces cerevisiae mitochondria possess polyphosphatases that are tightly bound to the membranes and differ from soluble polyphosphatase of these organelles in a number of properties . Molecular weights of the membrane-bound polyphosphatases are 120 and 76 kD, and the molecular weight of the soluble polyphosphatase is about 36 kD . All three enzymes are evidently monomers, since antibodies against purified cell-envelope polyphosphatase of S . cerevisiae reacted with 115, 78, and 37 kD polypeptides in immunoblotting . The activities of membrane-bound and soluble polyphosphatase are maximal at neutral pH . The soluble polyphosphatase activity is stimulated by divalent cations, unlike the membrane-bound enzymes which are inhibited by the same cations including Mg2+ . Monovalent cations do not affect the activity of the soluble enzyme but stimulate polyphosphatases in the membrane preparation . The specific activities for hydrolysis of polyphosphates with average chain lengths of 9 to 188 phosphate residues are enhanced by increasing the degree of substrate polymerization in the case of the membrane preparation and are unchanged in case of the soluble enzyme . Affinity of the soluble enzyme to polyphosphates is 5-10 times higher than that of the membrane-bound polyphosphatases . In the soluble fraction of mitochondria, high tripolyphosphatase activity is detected which is approximately 80% of that in isolated mitochondria. Gene, 1998 Jan 5, 206(1), 107 - 16 Molecular cloning of YlPMR1, a S . cerevisiae PMR1 homologue encoding a novel P-type secretory pathway Ca2+ -ATPase, in the yeast Yarrowia lipolytica; Park CS et al.; A novel P-type ATPase gene, Saccharomyces cerevisiae PMR1 homologue (YlPMR1), has been cloned and sequenced in the yeast, Yarrowia lipolytica . The putative gene product has 928 amino acids with a calculated molecular mass of 100050 Da and a pI of 5.15 . The deduced amino-acid sequence analysis demonstrated that the cloned gene product contains all 10 of the conserved regions in P-type ATPases and exhibits 55% amino-acid identity to the S . cerevisiae PMR1 gene product; however, it shows a relatively lower homology to PMCA (24%) and SERCA (33%), confirming the presence of a third class of Ca2+-ATPase (secretory pathway Ca2+-ATPase, SPCA) . The YlPMR1-disrupted strain shows defective growth in low Ca2+ or EGTA-containing medium . In fact, a longer lag time (60 h) was observed in YlPMR1-defective mutant cells during cultivation in EGTA-containing YPD medium . These growth defects were overcome by adding Ca2+ and Mn2+ into the medium . Interestingly, whereas Mn2+ inhibits growth of the control strain, it significantly improves the growth of YlPMR1-disrupted cells . These results suggest an involvement of the YlPMR1 gene product in Ca2+ and Mn2+ ion homeostasis in Y . lipolytica. J Biol Chem, 1997 Dec 12, 272(50), 31382 - 8 The carboxyl terminus of the Saccharomyces cerevisiae succinate dehydrogenase membrane subunit, SDH4p, is necessary for ubiquinone reduction and enzyme stability; Oyedotun KS et al.; The succinate dehydrogenase (SDH) of Saccharomyces cerevisiae is composed of four nonidentical subunits encoded by the nuclear genes SDH1, SDH2, SDH3, and SDH4 . The hydrophilic subunits, SDH1p and SDH2p, comprise the catalytic domain involved in succinate oxidation . They are anchored to the inner mitochondrial membrane by two small, hydrophobic subunits, SDH3p and SDH4p, which are required for electron transfer and ubiquinone reduction . Comparison of the deduced primary sequence of the yeast SDH4p subunit to SDH4p subunits from other species reveals the presence of an unusual 25-30 amino acid carboxyl-terminal extension following the last predicted transmembrane domain . The extension is predicted to be on the cytoplasmic side of the inner mitochondrial membrane . To investigate the extension's function, three truncations were created and characterized . The results reveal that the carboxyl-terminal extension is necessary for respiration and growth on nonfermentable carbon sources, for ubiquinone reduction, and for enzyme stability . Combined with inhibitor studies using a ubiquinone analog, our results suggest that the extension and more specifically, residues 128-135 are involved in the formation of a ubiquinone binding site . Our findings support a two-ubiquinone binding site model for the S . cerevisiae SDH. Genes Dev, 1997 Dec 1, 11(23), 3218 - 31 A novel cytosolic regulator, Pianissimo, is required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase in Dictyostelium; Chen MY et al.; Genetic analysis was applied to identify novel genes involved in G protein-linked pathways controlling development . Using restriction enzyme-mediated integration (REMI), we have identified a new gene, Pianissimo (PiaA), involved in cAMP signaling in Dictyostelium discoideum . PiaA encodes a 130-kD cytosolic protein required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase . In piaA- null mutants, neither chemoattractant stimulation of intact cells nor GTPgammaS treatment of lysates activates the enzyme; constitutive expression of PiaA reverses these defects . Cytosols of wild-type cells that contain Pia protein reconstitute the GTPgammaS stimulation of adenylyl cyclase activity in piaA- lysates, indicating that Pia is directly involved in the activation . Pia and CRAC, a previously identified cytosolic regulator, are both essential for activation of the enzyme as lysates of crac- piaA- double mutants require both proteins for reconstitution . Homologs of PiaA are found in Saccharomyces cerevisiae and Schizosaccaromyces pombe; disruption of the S . cerevisiae homolog results in lethality . We propose that homologs of Pia and similar modes of regulation of these ubiquitous G protein-linked pathways are likely to exist in higher eukaryotes. Proc Natl Acad Sci U S A, 1997 Dec 23, 94(26), 14361 - 6 The anaphase inhibitor of Saccharomyces cerevisiae Pds1p is a target of the DNA damage checkpoint pathway; Cohen-Fix O et al.; Inhibition of DNA replication and physical DNA damage induce checkpoint responses that arrest cell cycle progression at two different stages . In Saccharomyces cerevisiae, the execution of both checkpoint responses requires the Mec1 and Rad53 proteins . This observation led to the suggestion that these checkpoint responses are mediated through a common signal transduction pathway . However, because the checkpoint-induced arrests occur at different cell cycle stages, the downstream effectors mediating these arrests are likely to be distinct . We have previously shown that the S . cerevisiae protein Pds1p is an anaphase inhibitor and is essential for cell cycle arrest in mitosis in the presence DNA damage . Herein we show that DNA damage, but not inhibition of DNA replication, induces the phosphorylation of Pds1p . Analyses of Pds1p phosphorylation in different checkpoint mutants reveal that in the presence of DNA damage, Pds1p is phosphorylated in a Mec1p- and Rad9p-dependent but Rad53p-independent manner . Our data place Pds1p and Rad53p on parallel branches of the DNA damage checkpoint pathway . We suggest that Pds1p is a downstream target of the DNA damage checkpoint pathway and that it is involved in implementing the DNA damage checkpoint arrest specifically in mitosis. Curr Genet, 1997 Nov, 32(5), 309 - 14 Arabidopsis thaliana RAD6 homolog AtUBC2 complements UV sensitivity, but not N-end rule degradation deficiency, of Saccharomyces cerevisiae rad6 mutants; Zwirn P et al.; AtUBC2 of Arabidopsis thaliana encodes a structural homolog of the RAD6 gene of Saccharomyces cerevisiae with approximately 65% identical amino acids . Like structural homologs from other organisms, AtUBC2 lacks the carboxyl-terminal extension of mostly acidic amino acids which is present in Rad6p . AtUBC2 was expressed in S . cerevisiae rad6 mutants . It was found to partially complement the UV sensitivity and reduced growth rate of rad6 mutants at elevated temperatures . AtUBC2 however, has no apparent influence on the degradation of N-end rule substrates in the heterologous host. FEMS Microbiol Rev, 1997 Nov, 21(3), 231 - 41 Osmoregulation and glycerol metabolism in the yeast Saccharomyces cerevisiae; Nevoigt E et al.; Glycerol is the main compatible solute in Saccharomyces cerevisiae . It is accumulated intracellularly when cells are exposed to decreased extracellular water activity . In general, increased intracellular accumulation of a solute may be caused by enhanced production, restricted dissimilation, increased retention by the plasma membrane and increased uptake from the medium . In this review, we evaluate current knowledge concerning mechanisms leading to the accumulation of glycerol in osmotically stressed cells of S . cerevisiae at the molecular and metabolic levels . An overview of glycerol metabolism in S . cerevisiae is provided. Cell Mol Biol (Noisy-le-grand), 1997 Nov, 43(7), 1007 - 18 Cloning, characterization and identification of the gene encoding phosphatidylinositol 4-kinase; Pramanik A et al.; The vast majority of AIDS-related deaths are associated with opportunistic infections . For fungal infections, there are few effective antifungals, particularly for systemic use . The discovery that very low doses of the bleomycin family of anticancer chemical congeners compromise the integrity of fungal cell walls led to our approach to identify genes that complement-cell wall defects, and develop methods to facilitate the identification of new antifungals targeted to fungal cell walls . This report describes one of the genes cloned by complementation of the blm1-1 mutation of S . cerevisiae using a YCp50-based yeast genomic library . Characterization and identification of the gene were carried out using drug screening tests, Southern hybridization analyses, DNA sequencing and DNA sequence similarity searches in databases . The gene STT4, is essential for viability and encodes a phosphatidylinositol 4-kinase that plays an important role in the phosphatidylinositol-mediated signal transduction pathway required for cell wall integrity . Like blm1-1 mutant strains, stt4 cells arrest mostly in the G2/M phase of the cell cycle . Further studies using this approach should help us understand the role of PI4-K in maintaining fungal cell-wall integrity, identify additional genes affecting potential target structures in cell walls of opportunistic fungal pathogens in AIDS patients, and assist in drug discovery and antifungal drug design. Antonie Van Leeuwenhoek, 1997 Nov, 72(4), 283 - 90 Modulation of sporulation and metabolic fluxes in Saccharomyces cerevisiae by 2 deoxy glucose; Aon JC et al.; Quantitative studies of metabolic fluxes during Saccharomyces cerevisiae sporulation on acetate in the presence of the glucose analog, 2-deoxy glucose (2dG) are reported . We have studied the inhibition of sporulation and associated catabolic or anabolic fluxes by 2dG . Sporulation frequencies decreased from 50% to 2% asci per cell at 2dG concentrations in the range of 0.03 to 0.30 g l-1, respectively . Under the same conditions, the acetate consumption flux was inhibited up to 60% and the glyoxylate cycle and gluconeogenic fluxes decreased from 0.7 and 0.3 mmol h-1 g-1 dw, respectively, to negligible values . We observed a linear correlation of the acetate consumption rate with the sporulation frequency by varying the 2dG concentration . The linear correlation was also verified between the frequency of sporulation and the fluxes through glyoxylate cycle and gluconeogenic pathways . In addition, the same association of inhibition of sporulation and metabolic fluxes was found in other S . cerevisiae strains displaying different potentials of sporulation . The results presented suggest that inhibition of sporulation in the presence of the glucose analog may be attributed, at least in part, to the inhibition of anabolic fluxes and might be associated with catabolite repression. J Biol Chem, 1998 Jan 23, 273(4), 2402 - 8 Cardiolipin synthase is associated with a large complex in yeast mitochondria; Zhao M et al.; The phospholipid cardiolipin (CL) is ubiquitous in eucaryotes and is unique in structure, subcellular localization, and potential function . Previous studies have shown that CL is associated with major respiratory complexes in the mitochondrial membrane . To determine whether CL biosynthesis requires the presence of intact respiratory complexes, we measured activity of CL synthase, which catalyzes the synthesis of CL from cytidine diphosphate diacylglycerol and phosphatidylglycerol, in Saccharomyces cerevisiae strains with genetic defects in the oxidative phosphorylation system . Assembly mutants of cytochrome oxidase had significantly reduced CL synthase activity, while assembly mutants of respiratory complex III and the F0F1-ATPase were less inhibited . To obtain further information on the activity of CL synthase, we purified the enzyme and compared the size of the catalytic protein with the functional molecular mass . The enzyme was solubilized by Triton X-100 from KSCN-extracted mitochondrial membranes of S . cerevisiae . The functional molecular mass of Triton-solubilized CL synthase, determined by radiation inactivation, was 150-240 kDa, indicating that the functional enzyme was a large complex . After partial purification, the enzyme eluted from a Superose 12 gel filtration column with an apparent molecular mass of 70 kDa . CL synthase was further purified by hydroxylapatite and cytidine diphosphate diacylglycerol affinity chromatographies, Mono Q anion exchange FPLC, and preparative gel electrophoresis . These steps led to identification of a 28-kDa protein, which had catalytic activity when eluted from an SDS-polyacrylamide gel . This 28-kDa protein also reacted with an antiserum that inactivated the enzyme . We conclude that yeast CL synthase is a 28-kDa protein, which forms an oligomeric complex whose biogenesis and/or activity is influenced by the assembly of cyto-chrome oxidase. J Biol Chem, 1998 Jan 16, 273(3), 1677 - 83 Deoxyhypusine synthase activity is essential for cell viability in the yeast Saccharomyces cerevisiae; Park MH et al.; Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual amino acid, hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine), in the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein . The null mutation in the single copy gene, yDHS, encoding deoxyhypusine synthase results in the loss of viability in the yeast Saccharomyces cerevisiae . Upon depletion of deoxyhypusine synthase, and consequently of eIF-5A, cessation of growth was accompanied by a marked enlargement of cells, suggesting a defect in cell cycle progression or in cell division . Two residues of the yeast enzyme, Lys308 and Lys350, corresponding to Lys287 and Lys329, respectively, known to be critical for the activity of the human enzyme, were targeted for site-directed mutagenesis . The chromosomal ydhs null mutation was complemented by the plasmid-borne yDHS wild-type gene, but not by mutated genes encoding inactive proteins, including that with Lys350-->Arg substitution or with substitutions at both Lys308 and Lys350 . The mutated gene ydhs (K308R) encoding a protein with diminished activities (< 1% of wild type) could support growth but only to a very limited extent . These findings provide strong evidence that the hypusine modification is indeed essential for the survival of S . cerevisiae and imply a vital function for eIF-5A in all eukaryotes. J Biol Chem, 1998 Jan 16, 273(3), 1506 - 10 Edg-2/Vzg-1 couples to the yeast pheromone response pathway selectively in response to lysophosphatidic acid; Erickson JR et al.; We have functionally expressed the human cDNA encoding the putative lysophosphatidic acid (LPA) receptor Edg-2 (Vzg-1) in Saccharomyces cerevisiae in an attempt to determine the agonist specificity of this G-protein-coupled receptor . LPA activated the pheromone response pathway in S . cerevisiae expressing Edg-2 in a time- and dose-dependent manner as determined by induction of a pheromone-responsive FUS1::lacZ reporter gene . LPA-mediated activation of the pheromone response pathway was dependent on mutational inactivation of the SST2 gene, the GTPase-activating protein for the yeast G alpha protein (the GPA1 gene product) . This indicates that, in sst2 delta yeast cells, Edg-2 can efficiently couple to the yeast heterotrimeric G-protein in response to LPA and activate the yeast mitogen-activated protein kinase pathway . The Edg-2 receptor showed a high degree of specificity for LPA; other lyso-glycerophospholipids, sphingosine 1-phosphate, and diacyl-glycerophospholipids did not activate FUS1::lacZ . LPA analogs including a cyclic phosphoester form and ether-linked forms of LPA activated FUS1::lacZ, although fatty acid chains of 6 and 10 carbons did not activate FUS1::lacZ, suggesting a role for the side chain in ligand binding or receptor activation . These results indicate that Edg-2 encodes a highly specific LPA receptor. Chemosphere, 1997 Nov, 35(10), 2277 - 83 Comparative heavy metal biosorption study of brewery yeast and Myxococcus xanthus biomass; Ben Omar N et al.; The biosorption for La2+, Co2+, Mn2+, UO2(2+), Pb2+, Ag+, Zn2+, Cd2+ and Cr2+ by wet and dry biomass form Myxococcus xanthus obtained from laboratory cultures and Saccharomyces cerevisiae from the brewing industry has been studied . M . xanthus biomass was found to be the most efficient biosorbent for all of the metals assayed . However, due to the fact that S . cerevisiae is a low cost residual by-product from the brewing industry, and at the same time yields good levels of biosorption, it is considered in this work to be of great interest for use as a detoxifier of heavy metals contaminated waters . In addition, the use of sodium carbonate as a desorbent agent is discussed where it was possible to recover up to 94,53% of UO2(2+) by both M . xanthus and S . cerevisiae biomass. Yeast, 1997 Dec, 13(15), 1409 - 21 Saccharomyces carlsbergensis contains two functional genes encoding the acyl-CoA binding protein, one similar to the ACB1 gene from S . cerevisiae and one identical to the ACB1 gene from S . monacensis; Borsting C et al.; Saccharomyces carlsbergensis is an amphiploid, and it has previously been suggested that the genomes of S . carlsbergensis originate from S . cerevisiae and S . monacensis . We have cloned the ACB1 genes encoding the acyl-CoA binding protein (ACBP) from S . carlsbergensis, S . cerevisiae and S . monacensis . Two genes were found in S . carlsbergensis and named ACB1 type 1 and type 2, respectively . The type 1 gene is identical to the S . cerevisiae ACB1 gene except for three substitutions, one single base pair deletion and one double base pair insertion, all located in the promoter region . The type 2 gene is completely identical to the S . monacensis ACB1 gene . These findings substantiate the notion that S . carlsbergensis is a hybrid between S . cerevisiae and S . monacensis . Both ACB1 type 1 and type 2 are actively transcribed in S . carlsbergensis and transcription is initiated at sites identical to those used for transcriptional initiation of the ACB1 genes in S . cerevisiae and S . monacensis, respectively . Two polyadenylation sites, spaced 225 bp apart, are present in the S . cerevisiae ACB1 gene . The upstream polyadenylation site is used exclusively during exponential growth, whereas both sites are utilized during later stages of growth. Gene, 1997 Dec 19, 204(1-2), 251 - 8 The Dictyostelium discoideum beta-1,4-mannosyltransferase gene, mntA, has two periods of developmental expression; Lee SK et al.; The precise roles of protein glycosylation in multicellular development are poorly understood . We have characterized the mntA gene from Dictyostelium discoideum which encodes the beta-1,4-mannosyltransferase enzyme that catalyzes the reaction: GDP-Man + dolichol-PP-GlcNAc2 --> dolichol-PP-GlcNAc2-Man + GDP . This gene has a central role in the synthesis of the lipid-linked oligosaccharide precursor which becomes the core of all asparagine-linked (N-linked) glycans . The mntA gene contains a single small intron and encodes a 493 aa protein with a predicted molecular size of 56 kDa . It is located 5' to the repE gene on chromosome IV and is transcribed in the opposite orientation to repE with which it shares a 585 bp of upstream intergenic region . The predicted mntA gene product shares 38% homology with the S . cerevisiae ALG1 gene product . The MntA protein has a region homologous to the putative dolichol-binding region in the yeast ALG1 protein, but it is located in a different part of the molecule . Northern analysis revealed that the expression of the mntA gene is regulated during multicellular development with two periods of mRNA accumulation . The mntA gene product has a classical endoplasmic reticulum retention motif, and is the first Dictyostelium gene encoding a protein that is active in this organelle . The identification of this gene will allow expanded studies of the role of N-linked glycans in multicellular development. Prog Nucleic Acid Res Mol Biol, 1998, 59, 95 - 133 On the physiological role of casein kinase II in Saccharomyces cerevisiae; Glover CV 3rd; Casein kinase II (CKII) is a highly conserved serine/threonine protein kinase that is ubiquitous in eukaryotic organisms . This review summarizes available data on CKII of the budding yeast Saccharomyces cerevisiae, with a view toward defining the possible physiological role of the enzyme . Saccharomyces cerevisiae CKII is composed of two catalytic and two regulatory subunits encoded by the CKA1, CKA2, CKB1, and CKB2 genes, respectively . Analysis of null and conditional alleles of these genes identifies a requirement for CKII in at least four biological processes: flocculation (which may reflect an effect on gene expression), cell cycle progression, cell polarity, and ion homeostasis . Consistent with this, isolation of multicopy suppressors of conditional cka mutations has identified three genes that have a known or potential role in either the cell cycle or cell polarity: CDC37, which is required for cell cycle progression in both G1 and G2/M; ZDS1 and 2, which appear to have a function in cell polarity; and SUN2, which encodes a protein of the regulatory component of the 26S protease . The identity and properties of known CKII substrates in S . cerevisiae are also reviewed, and advantage is taken of the complete genomic sequence to predict globally the substrates of CKII in this organism . Although the combined data do not yield a definitive picture of the physiological role of CKII, it is proposed that CKII serves a signal transduction function in sensing and/or communicating information about the ionic status of the cell to the cell cycle machinery. Gene, 1997 Dec 5, 203(1), 75 - 84 Isolation and cloning of the Yarrowia lipolytica SEC65 gene, a component of the yeast signal recognition particle displaying homology with the human SRP19 gene; Sanchez M et al.; The signal recognition particle (SRP) is a ribonucleoprotein composed of a 7SL RNA and six polypeptides . Here we report the results of a series of experiments carried out to define the function of the Yarrowia lipolytica homologue of the 19 kDa subunit of mammalian SRP . The YlSEC65 gene product is a 310 amino acid protein . Coimmuneprecipitation of Sec65p and 7SL RNA in Y . lipolytica revealed that these components are stable associated in a complex . Deletion of the YlSEC65 gene is lethal, in contrast with the results described for the Saccharomyces cerevisiae SEC65 gene, which is not essential for cell growth and whose deletion results in slowly growing strains . Using site-directed mutagenesis we demonstrate that the two arginine residues of the EGRR motif conserved in all SRP19 homologues are essential for SRP activity . By random mutagenesis of YlSEC65, we have isolated a temperature-sensitive mutant and shown that it was affected in protein secretion at the non-permissive temperature . We also show that the YlSEC65 gene is able to functionally complement the temperature-sensitive growth of S . cerevisiae sec65 mutants . Our results suggest that SRP-dependent targeting may be the main secretory pathway in Y lipolytica, as has been described for higher eukaryotes. Cell Biochem Funct, 1997 Dec, 15(4), 265 - 9 The effects of selenium, vitamin E and their combination on the composition of fatty acids and proteins in Saccharomyces cerevisiae; Dilsiz N et al.; The aim of our studies was to test the effect and role of vitamin E and selenium supplements on yeast cell . In this study, the effects of selenium (Se), vitamin E (Vit . E), and their combination (Se plus Vit . E) on the composition of fatty acids and proteins were examined in Saccharomyces cerevisiae strains WET136 and 522 . S . cerevisiae cells were grown up in YEPD medium supplemented with Se, Vit . E or their combination . It was found that the level of stearic acid was increased in all supplemented groups (p < 0.05; p < 0.001) . The content of saturated and unsaturated fatty acids was decreased (p < 0.05; p < 0.01; p < 0.001) in Vit . E and Vit . E plus Se supplemented S . cerevisiae . On the other hand, Se alone caused an increase (p < 0.001) in the saturated fatty acids but a decrease (p < 0.05; p < 0.001) in the unsaturated fatty acids . Total proteins in S . cerevisiae were significantly increased (p < 0.001) by Vit . E supplement . There was no significant change observed in S . cerevisiae supplemented with Se . These findings indicate that membrane composition of S . cerevisiae is affected by both Vit . E and Se supplements. Mol Cell Biol, 1998 Jan, 18(1), 353 - 60 Yeast pre-mRNA splicing requires a pair of U1 snRNP-associated tetratricopeptide repeat proteins; McLean MR et al.; The U1 snRNP functions to nucleate spliceosome assembly on newly transcribed pre-mRNA . Saccharomyces cerevisiae is unusual among eukaryotes in the greatly extended length of its U1 snRNA and the apparent increased polypeptide complexity of the corresponding U1 snRNP . In this paper, we report the identification of a novel U1 snRNP protein, Prp42p, with unexpected properties . Prp42p was identified by its surprising structural similarity to the essential U1 snRNP protein, Prp39p . Both Prp39p and Prp42p possess multiple copies of a variant tetratricopeptide repeat, an element implicated in a wide range of protein assembly events . Yeast strains depleted of Prp42p by transcriptional repression of a GAL1::PRP42 fusion gene arrest for splicing prior to pre-mRNA 5' splice site cleavage . Prp42p was not observed in a recent biochemical analysis of purified U1 snRNPs from S . cerevisiae (28) . Nevertheless, antibodies directed against an epitope-tagged version of Prp42p specifically precipitate U1 snRNA from yeast extracts . Furthermore, Prp42p is required for U1 snRNP biogenesis, because yeast strains depleted of Prp42p formed incomplete U1 snRNPs that failed to produce stable complexes with pre-mRNA in vitro . The evidence shows that Prp39p and Prp42p are both required to configure the atypical yeast U1 snRNP into a structure compatible with its evolutionarily conserved role in pre-mRNA splicing. Genetics, 1997 Dec, 147(4), 1643 - 52 A MADS-box homologue in Ustilago maydis regulates the expression of pheromone-inducible genes but is nonessential; Kruger J et al.; Mating and pathogenic development in the smut fungus Ustilago maydis are controlled by a pheromone/receptor system and two homeodomain proteins, bEp and bWp, which form heterodimers in nonallelic combinations . We describe the isolation of a gene, umc1, encoding a MADS-box protein, which displays significant similarity to the Saccharomyces cerevisiae MCM1 gene . umc1 complemented the viability defect of yeast mcm1 mutants . In U . maydis, umc1 deletion mutants were viable and pathogenic development was unaffected . Nevertheless, the basal expression levels of several pheromone-inducible genes were significantly reduced leading to an attenuated mating reaction . In contrast to S . cerevisiae, where Mcm1p plays a crucial role in the cell-type specific expression of a- and alpha-specific genes, the U . maydis umc1 gene appears to have only a modulatory effect on the expression of mating type-specific genes. Microbiol Mol Biol Rev, 1997 Dec, 61(4), 503 - 32 Metabolism of sulfur amino acids in Saccharomyces cerevisiae; Thomas D et al.; Sulfur amino acid biosynthesis in Saccharomyces cerevisiae involves a large number of enzymes required for the de novo biosynthesis of methionine and cysteine and the recycling of organic sulfur metabolites . This review summarizes the details of these processes and analyzes the molecular data which have been acquired in this metabolic area . Sulfur biochemistry appears not to be unique through terrestrial life, and S . cerevisiae is one of the species of sulfate-assimilatory organisms possessing a larger set of enzymes for sulfur metabolism . The review also deals with several enzyme deficiencies that lead to a nutritional requirement for organic sulfur, although they do not correspond to defects within the biosynthetic pathway . In S . cerevisiae, the sulfur amino acid biosynthetic pathway is tightly controlled: in response to an increase in the amount of intracellular S-adenosylmethionine (AdoMet), transcription of the coregulated genes is turned off . The second part of the review is devoted to the molecular mechanisms underlying this regulation . The coordinated response to AdoMet requires two cis-acting promoter elements . One centers on the sequence TCACGTG, which also constitutes a component of all S . cerevisiae centromeres . Situated upstream of the sulfur genes, this element is the binding site of a transcription activation complex consisting of a basic helix-loop-helix factor, Cbf1p, and two basic leucine zipper factors, Met4p and Met28p . Molecular studies have unraveled the specific functions for each subunit of the Cbf1p-Met4p-Met28p complex as well as the modalities of its assembly on the DNA . The Cbf1p-Met4p-Met28p complex contains only one transcription activation module, the Met4p subunit . Detailed mutational analysis of Met4p has elucidated its functional organization . In addition to its activation and bZIP domains, Met4p contains two regulatory domains, called the inhibitory region and the auxiliary domain . When the level of intracellular AdoMet increases, the transcription activation function of Met4 is prevented by Met30p, which binds to the Met4 inhibitory region . In addition to the Cbf1p-Met4p-Met28p complex, transcriptional regulation involves two zinc finger-containing proteins, Met31p and Met32p . The AdoMet-mediated control of the sulfur amino acid pathway illustrates the molecular strategies used by eucaryotic cells to couple gene expression to metabolic changes. J Biol Chem, 1997 Nov 28, 272(48), 30196 - 200 Sphingolipids are potential heat stress signals in Saccharomyces; Dickson RC et al.; The ability of organisms to quickly respond to stresses requires the activation of many intracellular signal transduction pathways . The sphingolipid intermediate ceramide is thought to be particularly important for activating and coordinating signaling pathways during mammalian stress responses . Here we present the first evidence that ceramide and other sphingolipid intermediates are signaling molecules in the Saccharomyces cerevisiae heat stress response . Our data show a 2-3-fold transient increase in the concentration of C18-dihydrosphingosine and C18-phytosphingosine, more than a 100-fold transient increase in C20-dihydrosphingosine and C20-phytosphingosine, and a more stable 2-fold increase in ceramide containing C18-phytosphingosine and a 5-fold increase in ceramide containing C20-phytosphingosine following heat stress . Treatment of cells with dihydrosphingosine activates transcription of the TPS2 gene encoding a subunit of trehalose synthase and causes trehalose, a known thermoprotectant, to accumulate . Dihydrosphingosine induces expression of a STRE-LacZ reporter gene, showing that the global stress response element, STRE, found in many yeast promoter sequences can be activated by sphingolipid signals . The TPS2 promoter contains four STREs that may mediate dihydrosphingosine responsiveness . Using genetic and other approaches it should be possible to identify sphingolipid signaling pathways in S . cerevisiae and quantify the importance of each during heat stress. J Biol Chem, 1997 Nov 21, 272(47), 29620 - 5 Synthesis of mannose-(inositol-P)2-ceramide, the major sphingolipid in Saccharomyces cerevisiae, requires the IPT1 (YDR072c) gene; Dickson RC et al.; Knowledge of the Saccharomyces cerevisiae genes and proteins necessary for sphingolipid biosynthesis is far from complete . Such information should expedite studies of pathway regulation and sphingolipid functions . Using the Aur1 protein sequence, recently identified as necessary for synthesis of the sphingolipid inositol-P-ceramide (IPC), we show that a homolog (open reading frame YDR072c), termed Ipt1 (inositolphosphotransferase 1) is necessary for synthesis of mannose-(inositol-P)2-ceramide (M(IP)2C), the most abundant and complex sphingolipid in S . cerevisiae . This conclusion is based upon analysis of an ipt1-deletion strain, which fails to accumulate M(IP)2C and instead accumulates increased amounts of the precursor mannose-inositol-P-ceramide . The mutant also fails to incorporate radioactive precursors into M(IP)2C, and membranes prepared from it do not incorporate {3H-inositol}phosphatidylinositol into M(IP)2C, indicating a lack of M(IP)2C synthase activity (putatively phosphatidylinositol:mannose-inositol-P-ceramide phosphoinositol transferase) . M(IP)2C synthase activity is inhibited in the micromolar range by aureobasidin A, but drug sensitivity is over 1000-fold lower than reported for IPC synthase activity . An ipt1-deletion mutant has no severe phenotypic effects but is slightly more resistant to growth inhibition by calcium ions . Identification of the IPT1 gene should be helpful in determining the function of the M(IP)2C sphingolipid and in determining the catalytic mechanism of IPC and M(IP)2C synthases. Fungal Genet Biol, 1997 Oct, 22(2), 77 - 83 Expression of YWP1, a gene that encodes a specific Yarrowia lipolytica mycelial cell wall protein, in Saccharomyces cerevisiae; Ramon AM et al.; The YWP1 gene encoding a specific mycelial cell wall protein of Yarrowia lipolytica has been cloned and expressed in Saccharomyces cerevisiae using different episomal plasmids . Because the plasmids pYAE35BB and pYAE35ES carrying the YWP1 gene (including the 5' noncoding promoter sequences) failed to express it, the YWP1 gene was cloned under the control of GAL/CYC or ACT S . cerevisiae promoters . A main band with an apparent molecular mass of 70 kDa was detected by immunoblotting in the cell wall fraction of transformants . Ywp1 processing and incorporation to the cell wall were similar in both Y . lipolytica and S . cerevisiae but not in its final localization in the cell wall . In Y . lipolytica Ywp1 is covalently bound to the cell wall (it is released only by Zymolyase digestion), whereas in S . cerevisiae it was not (it was released by boiling SDS solutions) . These results suggest that the sequences involved in recognition, anchoring of a protein to the cell wall, or the catalytic activities implicated are different, at least for Ywp1, in Y . lipolytica and S . cerevisiae . Another possibility is that the target for attachment of Ywp1 is missing or cryptic in the cell wall of S . cerevisiae . Arch Biochem Biophys, 1997 Nov 15, 347(2), 193 - 200 A dominant negative mutation in Saccharomyces cerevisiae methionine aminopeptidase-1 affects catalysis and interferes with the function of methionine aminopeptidase-2; Klinkenberg M et al.; Methionine aminopeptidase (MetAP) enzymes require the metal ion cobalt, but little is known about the role of cobalt in the structural stability or catalysis of these enzymes . In Escherichia coli MetAP, for which a crystal structure is available, the five amino acid residues liganding the two cobalt ions are Asp97, Asp108, His171, Glu204, and Glu235 . These five amino acids are conserved in all MetAPs sequenced to date . The C-terminal domain of the yeast Saccharomyces cerevisiae MetAP1 is 41% identical to E . coli MetAP and contains these cobalt coordinating residues . Using site-directed mutagenesis on the gene coding for yeast MetAP1, we replaced Asp219 (corresponding to Asp97 in E . coli MetAP) with Asn . The yeast D219N mutant enzyme has 10(3)-fold lower catalytic activity and a different substrate specificity when compared to wild-type yeast MetAP1 . These results indicate that the side-chain of Asp219 is important for catalysis . Expression of D219N-MetAP1 in yeast causes a slow-growth phenotype and interferes with wild-type MetAP1 in a dominant manner . Expression of D219N-MetAP1 also affects the function of S . cerevisiae MetAP2 . RNA, 1997 Dec, 3(12), 1374 - 87 A new cyclophilin and the human homologues of yeast Prp3 and Prp4 form a complex associated with U4/U6 snRNPs; Horowitz DS et al.; We have purified three new human U4/U6-snRNP proteins from HeLa cells . The three proteins formed a tightly bound complex and behaved as a single species throughout the purification . All three proteins have been identified by peptide sequencing, and full-length cDNA sequences have been obtained for all of them . Two of the proteins are homologues of the Saccharomyces cerevisiae splicing factors Prp3 and Prp4, and the third protein is a cyclophilin . Both the human and S . cerevisiae Prp4 proteins have seven repeats of the WD motif and likely fold into structures very similar to those of the beta subunits of G proteins . The human Prp3 protein is highly basic and is closely related to S . cerevisiae Prp3 only in its carboxyl-terminal half . The human homologues of Prp3 and Prp4 are part of a stable complex in the absence of RNA . The third protein in the complex is a new cyclophilin . Cyclophilins have been proposed to act as chaperones in a variety of cellular processes, and we discuss some possible roles of this U4/U6 snRNP-associated cyclophilin. Antonie Van Leeuwenhoek, 1997 Oct, 72(3), 229 - 37 The incorporation of mannoproteins in the cell wall of S . cerevisiae and filamentous Ascomycetes; Brul S et al.; In yeast, glucanase extractable cell wall proteins are anchored to the plasma membrane at an intermediate stage in their biogenesis via a glycosylphosphatidylinositol (GPI) moiety before they become anchored to the wall glucan via a beta 1,6-glucan linkage . The mechanism of the membrane processing step of cell wall proteins is not known . Here, we report that Ascomycete filamentous fungi involved in food spoilage such as Aspergillus, Paecilomyces and Penicillium, also contain GPI membrane-anchored proteins some of which are processed by an endogenous phospholipase C activity . Furthermore, similar to the situation in yeast, their cell walls contain mannoproteins which are linked to the glucan backbone through a beta 1,6-glucan linkage . Interestingly, one mould which contains a significant amount of non covalently linked beta 1,6-glucosylated cell wall proteins, is much more sensitive towards beta 1,3-glucanases and membrane perturbing peptides than the others. J Bacteriol, 1997 Dec, 179(24), 7790 - 5 Characteristics of Fps1-dependent and -independent glycerol transport in Saccharomyces cerevisiae; Sutherland FC et al.; Eadie-Hofstee plots of glycerol uptake in wild-type Saccharomyces cerevisiae W303-1A grown on glucose showed the presence of both saturable transport and simple diffusion, whereas an fps1delta mutant displayed on