J Biol Chem, 1993 Apr 25, 268(12), 8665 - 8 Saccharomyces cerevisiae elongation factor 2 . Mutagenesis of the histidine precursor of diphthamide yields a functional protein that is resistant to diphtheria toxin; Phan LD et al.; Protein synthesis elongation factor 2 (EF-2) is the target of the ADP-ribosylating activity of diphtheria toxin which is responsible for cell killing . Diphthamide, an unique post-translationally modified histidine residue, is both required for and the site of this ADP-ribosylation . Although present in the EF-2 of all eukaryotes and archaebacteria, the function of diphthamide is unknown . Here we describe the site-specific mutagenesis of the histidine precursor of diphthamide, histidine 699, in yeast EF-2 . Plasmid-borne EFT was randomly mutagenized at the histidine 699 codon, and the technique of plasmid shuffling was utilized to select strains that were maintained by the mutant EFT . These mutants were screened for diphtheria toxin resistance . Sequence analysis of the EFT in 49 toxin-resistant isolates showed that histidine 699 had been replaced by 1 of 4 amino acids: asparagine, glutamine, leucine, or methionine . All 11 of the possible codons corresponding to these 4 amino acids were found . The growth rates of cells sustained by the mutant forms of EF-2 were slightly slower than those of isogenic wild-type cells . We conclude that despite its strict conservation and universal post-translational modification, the histidine precursor of diphthamide is not essential to the function of yeast EF-2 in protein synthesis. Biochem Biophys Res Commun, 1993 Apr 15, 192(1), 143 - 50 Biochemical characterisation of the isolated Anc2 adenine nucleotide carrier from Saccharomyces cerevisiae mitochondria; Brandolin G et al.; The yeast mitochondrial adenine nucleotide carrier isoform encoded by the ANC2 gene has been specifically expressed in a yeast strain disrupted for the two other genes, ANC1 and ANC3 . Isolation of the carrier in a functional form was achieved by utilisation of a mixture of two detergents, dodecylmaltoside and Emulphogen . The intrinsic fluorescence of the Anc2 protein was specifically and rapidly enhanced upon addition of the transportable nucleotides ADP and ATP . Fluorescence enhancement was prevented or reversed by the addition of a stoichiometric amount of CATR . Addition of CATR alone elicited a dose-dependent decrease of fluorescence . The ANC2-specific yeast stain offers the means to study a single ADP/ATP carrier, with a well-defined amino acid sequence, suitable for analysis of substrate- or inhibitor-induced conformational changes. J Biol Chem, 1993 Apr 15, 268(11), 8341 - 9 Phosphoglucomutase in Saccharomyces cerevisiae is a cytoplasmic glycoprotein and the acceptor for a Glc-phosphotransferase; Marchase RB et al.; UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins . The predominant acceptor in vertebrates and Paramecium tetraurelia is a cytoplasmic 62-kDa glycoprotein . To determine if the yeast Saccharomyces cerevisiae also possesses Glc-phosphotransferase activity, a crude cellular lysate was incubated with {beta-32P}UDP-Glc and analyzed . A phosphoglycoprotein having an apparent molecular mass of 62 kDa (pgp62) was found to be the predominant labeled macromolecule . Reconstitution experiments determined that both a soluble and membrane fraction were required for labeling, and suggested that the Glc-phosphotransferase is membrane-associated while pgp62 is cytoplasmic . The reaction is evolutionarily conserved to the extent that rat liver Glc-phosphotransferase was capable of recognizing the yeast acceptor and vice versa . The yeast 62-kDa acceptor was purified, and partial amino acid sequences showed a high level of identity with rabbit muscle phosphoglucomutase . Subsequently, both yeast and rabbit muscle phosphoglucomutase were found to be acceptors in the Glc-phosphotransferase reaction . The label was found on a tryptic peptide distinct from that containing the enzyme's active site serine . When phosphoglucomutase was overexpressed, an increase was seen in Glc-phosphotransferase acceptor activity and in specific metabolic labeling of the acceptor by glucose and mannose. J Biol Chem, 1993 Apr 15, 268(11), 7728 - 32 Functional replacement of the Saccharomyces cerevisiae Trg1/Pdi1 protein by members of the mammalian protein disulfide isomerase family; Gunther R et al.; The TRG1/PDI1 gene of Saccharomyces cerevisiae is essential for growth and encodes a lumenal endoplasmic reticulum (ER) glycoprotein that is structurally related to thioredoxin and is involved in the secretory pathway . We have tested whether the yeast Trg1/Pdi1 protein can be replaced in vivo by three members of the mammalian thioredoxin-related protein family, protein disulfide isomerase (PDI), ERp72, and ERp61 . Multicopy plasmids containing galactose-inducible rodent PDI and ERp72 genes support germination and growth of haploid trg1 null mutants in galactose-containing media, whereas the ERp61 gene is inactive . Strains expressing PDI or ERp72 instead of Trg1 are thermosensitive . An overproduced mutant Trg1 protein lacking the HDEL retention signal supports growth, whereas a truncated version of the protein containing only one thioredoxin-like domain is inactive . The mammalian proteins were localized to both the soluble and microsomal membrane fraction of yeast cells . Our observations indicate that the two unglycosylated mammalian proteins PDI and ERp72 are capable of replacing at least some of the critical functions of Trg1, in spite of the fact that the three proteins diverge considerably in sequences surrounding the thioredoxin-related domains. FEBS Lett, 1993 Apr 12, 320(3), 271 - 5 Two structural genes are encoding malate synthase isoenzymes in Saccharomyces cerevisiae; Fernandez E et al.; We report on the isolation of a gene encoding yeast malate synthase . A yeast genomic library was screened using a probe homologous to the yeast enzyme obtained by the polymerase chain reaction . The nucleotide sequence of the cloned gene was determined . Computer analysis showed that the isolated gene is identical to the one previously described as DAL7, which is involved in allantoin metabolism {Mol . Cel . Biol . 9 (1989) 3231-3243} . Enzymatic activities of multicopy transformants, Southern analysis and disruption mutants predict the existence of two genes encoding malate synthases that are differentially regulated at the transcriptional level. Biochemistry, 1993 Apr 6, 32(13), 3261 - 70 Similarities and differences between yeast and vertebrate calmodulin: an examination of the calcium-binding and structural properties of calmodulin from the yeast Saccharomyces cerevisiae; Starovasnik MA et al.; The Ca(2+)-binding and structural properties of calmodulin (CaM) from the yeast Saccharomyces cerevisiae (yCaM) were analyzed by flow dialysis and NMR spectroscopy . Full-length yCaM and two truncated versions of yCaM were expressed in Escherichia coli and purified . yTR1 (residues 1-76) and yTR2 (residues 75-147) are similar to the vertebrate CaM fragments TR1 and TR2, which are generated by limited proteolysis with trypsin . As was found for the fragments of vertebrate CaM, the yCaM fragments retain native conformation and are useful for examining structure and metal-binding properties by NMR . Evidence for a short beta-sheet in each domain, as well as characteristic NOEs to aromatic residues, suggests that yCaM folds similarly to vertebrate CaM . Furthermore, although the previously considered "invariant" glycine at position 6 is replaced by a histidine in site II of yCaM, the far downfield chemical shift of His-61's amide proton suggests that this site adopts a conformation similar to that found in other EF-hand sites . Macroscopic Ca(2+)-binding constants were determined for yCaM by flow dialysis, revealing three high-affinity sites (dissociation constants were 5.2, 3.3, and 2.3 microM in the presence of 1 mM MgCl2 and 100 mM KCl) . Positive cooperativity was observed among all sites . Ca2+ binding was also monitored indirectly by one-dimensional NMR . Titrations of the fragment molecules reveal that two binding sites reside in the N-terminal domain (sites I and II) and one in the C-terminal domain (site III) . All three sites exhibit slow-exchange behavior in the intact protein, but site III exhibits fast-exchange behavior in the isolated C-terminal domain fragment (yTR2) . Thus, an interaction between the two domains of intact yCaM affects the behavior of site III . These results with yCaM differ from those of vertebrate CaM in terms of Ca(2+)-binding stoichiometry, affinity of sites I and II, relative affinity of sites in the N- and C-terminal domains, and the exchange behaviors observed. J Biol Chem, 1993 Apr 5, 268(10), 7442 - 8 A distal heat shock element promotes the rapid response to heat shock of the HSP26 gene in the yeast Saccharomyces cerevisiae; Chen J et al.; Induction of heat shock genes is mediated by heat shock factor (HSF) . Our recent genomic footprinting experiments demonstrate that HSF binds constitutively to perfect and imperfect heat shock elements (HSEs) in the HSP26 gene in yeast . Site-directed mutagenesis of the single perfect HSE, previously reported to not be involved in regulating gene expression, significantly reduces the rate of response of the gene to heat shock . However, the same mutation only slightly reduced the rate of accumulation of HSP26 mRNA during heat shock . Genomic footprinting experiments indicate that this lag in response to heat shock is due to the failure of HSF to bind efficiently to the mutated HSE . The rate of response to heat shock of synthetic promoters containing one, two, three, or seven perfect HSEs was similar to that observed for the wild-type HSP26 gene . These results suggest that the rate of response to heat shock is correlated with HSF occupancy of HSEs, rather than the number of HSEs in a promoter . As with the wild-type and mutant HSP26 genes, the rate of accumulation of mRNA from synthetic promoters increased only moderately with an increase in the number of HSEs . These results suggest that as few as two HSE-HSF complexes are sufficient to saturate HSF's target in the basal transcription apparatus. FEBS Lett, 1993 Apr 5, 320(2), 125 - 9 Precise mapping and molecular characterization of the MFT1 gene involved in import of a fusion protein into mitochondria in Saccharomyces cerevisiae; Ito M et al.; Garrett et al . {Mol . Gen . Genet . 225 (1991) 483-491} recently reported that an Atp2-lacZ fusion protein was transported into mitochondria in yeast, thus identifying the MFT1 (mitochondrial fusion targeting) gene as a genomic fragment which complements a mutation (mft1) that failed in targeting a fusion protein into mitochondria . They mapped this gene to the ORF, which we have independently identified as a gene homologous to the cyc07 gene, which is expressed specifically in the S phase during the plant cell cycle . We have mapped the MFT1 gene precisely and found that this gene should correspond to the neighboring ORF, rather than the ORF they identified. J Biol Chem, 1993 Apr 5, 268(10), 7350 - 7 Isolation and characterization of an NTP-dependent 3'-exoribonuclease from mitochondria of Saccharomyces cerevisiae; Min J et al.; RNA turnover in eukaryotes is thought to require 3'-exonuclease activity but so far no RNase with that specificity has been isolated from a eukaryote . We report here on the purification and characterization of a 3'-exoribonuclease isolated from the mitochondria of Saccharomyces cerevisiae . In vitro the purified enzyme displayed an absolute requirement of NTPs for activity . Each of the eight standard ribo- and deoxyribonucleotides supported activity with Km values ranging from 20 to 90 microM . The enzyme also displayed RNA-stimulated NTPase activity . The NTP-dependent enzyme cofractionated with three polypeptides of molecular masses 75,000, 90,000, and 110,000 daltons, although the native enzyme appears to have a molecular mass of 160,000 daltons predicted from the Stokes radius . The possible functions of this enzyme in vivo in the regulated decay of mitochondrial RNAs are discussed. Yeast, 1993 Apr, 9(4), 423 - 7 Sequence of the open reading frame of the FLO1 gene from Saccharomyces cerevisiae; Teunissen AW et al.; The cloned part of the flocculation gene FLO1 of Saccharomyces cerevisiae (Teunissen, A.W.R.H., van den Berg, J.A . and Steensma, H.Y . (1993) . Physical localization of the flocculation gene FLO1 on chromosome I of Saccharomyces cerevisiae, Yeast, in press) has been sequenced . The sequence contains a large open reading frame of 2685 bp . The amino acid sequence of the putative protein reveals a serine- and threonine-rich C-terminus (46%), the presence of repeated sequences and a possible secretion signal at the N-terminus . Although the sequence is not complete (we assume the missing fragment consists of repeat units), these data strongly suggest that the protein is located in the cell wall, and thus may be directly involved in the flocculation process. Yeast, 1993 Apr, 9(4), 339 - 49 Control of Saccharomyces cerevisiae carboxypeptidase S (CPS1) gene expression under nutrient limitation; Bordallo J et al.; Expression of the vacuolar carboxypeptidase S (CPS1) gene in Saccharomyces cerevisiae is regulated by the availability of nutrients . Enzyme production is sensitive to nitrogen catabolite repression; i.e . the presence of ammonium ions maintains expression of the gene at a low level . Transfer of ammonium-glucose pre-grown cells to a medium deprived of nitrogen causes a drastic increase in CPS1 RNA level provided that a readily usable carbon source, such as glucose or fructose, is available to the cells . Derepression of the gene by nitrogen limitation is cycloheximide-insensitive . Neither glycerol, ethanol, acetate nor galactose support derepression of CPS1 expression under nitrogen starvation conditions . Non-metabolizable sugar analogs (2-deoxyglucose, 6-methyl-glucose or glucosamine) do not allow derepression of CPS1, showing that the process is energy-dependent . Production of carboxypeptidase yscS also increases several-fold when ammonium-pregrown cells are transferred to media containing glucose and a non-readily metabolizable nitrogen source such as proline, leucine, valine or leucyl-glycine . Analysis of CPS1 expression in RAS2+ (high cAMP) and ras2 mutant (low cAMP) strains and in cells grown at low temperature (23 degrees C) and in heat-shocked cells (38 degrees C) shows that steady-state levels of CPS1 mRNA are not controlled by a low cAMP level-signalling pathway. Mol Microbiol, 1993 Apr, 8(1), 167 - 78 AUA1, a gene involved in ammonia regulation of amino acid transport in Saccharomyces cerevisiae; Sophianopoulou V et al.; In Saccharomyces cerevisiae the general amino acid (GAP1) permease catalyses active transport of apparently all amino acids across the plasma membrane . GAP1 activity is regulated by control of synthesis and control of activity in response to the nitrogen source supplied; ammonia and glutamine inactivate GAP1 function while proline and urea allow its maximum expression . We have isolated and characterized a gene, AUA1, involved in ammonia regulation of GAP1 activity . AUA1 is not essential for growth but overexpression of the AUA1 transcript in a high-copy vector or due to a regulatory mutation, aua1-1, present approximately 10 bp upstream from the start of AUA1 transcription, releases GAP1 activity from ammonia-inactivation without affecting GAP1 transcription . The aua1-1 mutation has no phenotype when ammonia is replaced by proline or glutamate as the nitrogen source or when it is present in a gap1 background . AUA1 expression is itself ammonia repressible in a wild-type strain but not in the aua1-1 mutant . The AUA1 gene sequence contains a unique short open reading frame of 94 codons corresponding to a polypeptide of 11,714 Da . This polypeptide is highly hydrophilic and extremely basic . The AUA1 product shows no significant similarity with any previously known protein sequence . Interestingly, a 10-amino acid segment of AUA1 is directly repeated in the most basic segment of the protein . Possible roles of AUA1 are discussed. Mol Gen Genet, 1993 Apr, 238(3), 444 - 54 Promoter analysis of the PHO81 gene encoding a 134 kDa protein bearing ankyrin repeats in the phosphatase regulon of Saccharomyces cerevisiae; Ogawa N et al.; The PHO81 gene encoding one of the regulators of the phosphatase regulon in Saccharomyces cerevisiae was mapped 9.8 centimorgans distal from the ser2 locus on the right arm of chromosome VII . Determination of the nucleotide sequence of cloned PHO81 DNA revealed a 3537 bp open reading frame encoding a 134 kDa protein . This protein has six repeats of a 33-amino acid sequence homologous to the ankyrin repeat and an asparagine-rich region . Transcription of PHO81 is activated by Pho4 protein in cooperation with Pho2 (i.e., Bas2/Grf10) protein under the influence of the inorganic phosphate (Pi) concentration in the medium, through the PHO regulatory system . Major transcription initiation sites of PHO81, determined by primer extension analysis, are at nucleotide positions -66 and -65 relative to the ATG codon . Deletion analysis showed that a 95 bp region from nucleotide position -385 to -291 is essential for response to the Pi signals . Purified Pho4 protein protected a 19 bp region (positions -350 to -332) in the 95 bp fragment from DNase I digestion in vitro and the protected region includes the core sequence 5'-CACGTG-3', which is also observed in other genes of phosphate metabolism. Mol Gen Genet, 1993 Apr, 238(3), 390 - 400 Expression of the Saccharomyces cerevisiae RAD50 gene during meiosis: steady-state transcript levels rise and fall while steady-state protein levels remain constant; Raymond WE et al.; In Saccharomyces cerevisiae, the RAD50 gene is required for repair of X-ray and MMS-induced DNA damage during vegetative growth, and for synaptonemal complex formation and genetic recombination during meiosis . We show below that the RAD50 gene encodes major and minor transcripts of 4.2 and 4.6 kb in length which differ primarily at their 5' ends . Steady-state levels of both RAD50 transcripts increase coordinately during meiosis, reaching maximal levels midway through meiotic prophase, about 3 or 4 h after transfer of cells to sporulation medium . The 5' ends of the major RAD50 transcript in both meiotic and vegetative cells map to the same cluster of sites approximately 20 bp upstream of the amino-terminal ATG of the RAD50 coding sequence . We conclude that the increased RAD50 transcript level observed during meiosis does not reflect utilization of a new promoter . In contrast, steady-state levels of Rad50 protein do not increase during meiosis . Thus, changes in RAD50 transcript levels are not necessarily accompanied by commensurate changes in Rad50 protein levels . Possible explanations are considered. Eur J Biochem, 1993 Apr 1, 213(1), 547 - 53 Transcriptional control of AAC3 gene encoding mitochondrial ADP/ATP translocator in Saccharomyces cerevisiae by oxygen, heme and ROX1 factor; Sabova L et al.; The AAC3 gene of Saccharomyces cerevisiae encodes a mitochondrial ADP/ATP translocator which is subject to oxygen repression . Evidence is presented here, that the repression of AAC3 expression is dependent upon heme and the ROX1 factor . The promoter region of the AAC3 gene was isolated, sequenced, and deletion analysis was performed using lacZ as a reporter gene to determine the cis-acting regions responsible for the regulation of AAC3 expression . The results of the deletion analysis show that the negative control of the AAC3 gene by oxygen and ROX1 factor is mediated by an upstream repression sequence consisting of a T-rich segment adjacent to the consensus elements that are present in the 5' flanking regions of several other yeast genes . An additional upstream repressor site was located within the AAC3 promoter which, however, is not related either to oxygen or to ROX1 factor . The data presented here delineate the main cellular factors and DNA sequences involved in the regulatory mechanism by which an essential function for anaerobic cells growth, ADP/ATP translocation, is ensured . In addition, they show that the AAC3 gene belongs to the family of yeast genes including TIF51B, COX5b, HEM13 and CYC7 that are negatively regulated by oxygen and heme. EMBO J, 1993 Apr, 12(4), 1459 - 66 The control in cis of the position and the amount of the ARG4 meiotic double-strand break of Saccharomyces cerevisiae; de Massy B et al.; During meiosis, a transient DNA double-strand break (DSB) occurs in the promoter region (positions -200/-185) of the Saccharomyces cerevisiae ARG4 gene and is a likely intermediate in the initiation of meiotic gene conversion events in this region . We report here a functional analysis of the ARG4 DSB based on the study of various deletions in this chromosomal region . We have identified several cis-acting elements located within the -465/+3 region of the ARG4 promoter that control the formation of this DSB . The -465/-317 region includes a transcription terminator and is necessary for a normal amount of ARG4 DSB, but not for its positioning . The -316/-140 region can be replaced by an unrelated DNA sequence where a meiotic DSB then occurs, suggesting that the site of DSB is not sequence-specific, but is positioned at a fixed distance from the adjacent -139/+3 region . Also, in all strains constructed, the amount of meiotic DSB is correlated with the frequency of gene conversion in ARG4, which provides a strong argument for the initiation of gene conversion by a DSB in this region of the yeast genome. Genetics, 1993 Apr, 133(4), 851 - 63 Mutational analysis of pre-mRNA splicing in Saccharomyces cerevisiae using a sensitive new reporter gene, CUP1; Lesser CF et al.; We have developed a new reporter gene fusion to monitor mRNA splicing in yeast . An intron-containing fragment from the Saccharomyces cerevisiae ACT1 gene has been fused to CUP1, the yeast metallothionein homolog . CUP1 is a nonessential gene that allows cells to grow in the presence of copper in a dosage-dependent manner . By inserting previously characterized intron mutations into the fusion construct, we have established that the efficiency of splicing correlates with the level of copper resistance of these strains . A highly sensitive assay for 5' splice site usage was designed by engineering an ACT1-CUP1 construct with duplicated 5' splice sites; mutations were introduced into the upstream splice site in order to evaluate the roles of these highly conserved nucleotides in intron recognition . Almost all mutations in the intron portion of the 5' consensus sequence abolish recognition of the mutated site, while mutations in the exon portion of the consensus sequence have variable affects on cleavage at the mutated site . Interestingly, mutations at intron position 4 demonstrate that this nucleotide plays a role in 5' splice site recognition other than by base pairing with U1 snRNA . The use of CUP1 as a reporter gene may be generally applicable for monitoring cellular processes in yeast. Genetics, 1993 Apr, 133(4), 837 - 49 A mutational analysis of killer toxin resistance in Saccharomyces cerevisiae identifies new genes involved in cell wall (1-->6)-beta-glucan synthesis; Brown JL et al.; Recessive mutations leading to killer resistance identify the KRE9, KRE10 and KRE11 genes . Mutations in both the KRE9 and KRE11 genes lead to reduced levels of (1-->6)-beta-glucan in the yeast cell wall . The KRE11 gene encodes a putative 63-kD cytoplasmic protein, and disruption of the KRE11 locus leads to a 50% reduced level of cell wall (1-->6)-glucan . Structural analysis of the (1-->6)-beta-glucan remaining in a kre11 mutant indicates a polymer smaller in size than wild type, but containing a similar proportion of (1-->6)- and (1-->3)-linkages . Genetic interactions among cells harboring mutations at the KRE11, KRE6 and KRE1 loci indicate lethality of kre11 kre6 double mutants and that kre11 is epistatic to kre1, with both gene products required to produce the mature glucan polymer at wild-type levels . Analysis of these KRE genes should extend knowledge of the beta-glucan biosynthetic pathway, and of cell wall synthesis in yeast. Genetics, 1993 Apr, 133(4), 785 - 97 A conditional allele of the Saccharomyces cerevisiae HOP1 gene is suppressed by overexpression of two other meiosis-specific genes: RED1 and REC104; Hollingsworth NM et al.; The HOP1 gene of Saccharomyces cerevisiae is believed to encode a protein component of the synaptonemal complex, the structure formed when homologous chromosomes synapse during meiotic prophase . Five new mutant alleles (three conditional, two nonconditional) of HOP1 were identified by screening EMS-mutagenized cells for a failure to complement the spore viability defect of a hop1 null allele . Two high copy plasmids were found that partially suppress the temperature-sensitive spore inviability phenotype of one of these alleles, hop1-628 . The suppression is allele-specific; no effect of the plasmids is observed in hop1 null diploids . Mutation of either of the two suppressor genes results in recessive spore lethality, indicating that these genes play important roles during meiosis . The DNA sequence of one high copy suppressor gene matched that of RED1, a previously identified meiosis-specific gene . Our data strongly support the idea that RED1 protein is also a component of the synaptonemal complex and further suggest that the RED1 and HOP1 gene products may interact . The second suppressor maps to the right arm of chromosome VIII distal to CDC12 and is REC104, a meiosis-specific gene believed to act early in meiosis. J Bacteriol, 1993 Apr, 175(7), 2102 - 6 Purification and characterization of the Saccharomyces cerevisiae BGL2 gene product, a cell wall endo-beta-1,3-glucanase; Mrsa V et al.; One of the major proteins of the Saccharomyces cerevisiae cell wall, a beta-glucanase (BGL2 gene product), has been isolated and purified to homogeneity under conditions for preserving enzyme activity . The study of enzyme properties of the protein revealed that it is an endo-beta-1,3-glucanase and not an exoglucanase as reported previously (F . Klebl and W . Tanner, J . Bacteriol . 171:6259-6264, 1989) . The examination of the glucanase structure showed that the lower apparent molecular mass of the protein (29 kDa) compared with what was calculated from the amino acid sequence of the enzyme (33.5 kDa) is due to anomalous migration in sodium dodecyl sulfate gels and not to posttranslational processing of the polypeptide chain . Of two potential N glycosylation sites at Asn-202 and Asn-284, only the latter site is glycosylated . The overproduction of the beta-glucanase from the high-copy-number plasmid brought about a significant decrease in the growth rate of transformed yeast cells. J Bacteriol, 1993 Apr, 175(7), 1879 - 85 Physiological analysis of mutants indicates involvement of the Saccharomyces cerevisiae GPI-anchored protein gp115 in morphogenesis and cell separation; Popolo L et al.; This paper reports a phenotypic characterization of ggp1 mutants . The cloned GGP1 (GAS1) gene, which encodes a major GPI-anchored glycoprotein (gp115) of Saccharomyces cerevisiae of unknown function, was used to direct the inactivation of the chromosomal gene in haploid and diploid strains by gene replacement . The analysis of the null mutants reveals a reduction in the growth rate of 15 to 40% . Cells are round, with more than one bud, and extensively vacuolized . In the stationary phase, mutant cells are very large, arrest with a high percentage of budded cells (about 54 and 70% for haploid and diploid null mutants, respectively, in comparison with about 10 to 13% for control cells), and have reduced viability . The observed phenotype suggests defects in cell separation . Flow cytometric analysis of DNA reveals an increase in the fraction of cells in the G2+M+G1* compartment during exponential growth . Conjugation and sporulation are not affected . The exocellular location of gp115 led us to examine cell wall properties . Cell wall and septum ultrastructure of abnormally budded cells was analyzed by electron microscopy analysis, and no appreciable differences from wild-type cells were found . Microscopic analysis revealed an increase in chitin content and delocalization . In comparison with control cells, ggp1 null mutants are shown to be resistant to Zymolyase during the exponential growth phase . A fivefold overexpression of gp115 does not bring about any effects on cell growth parameters and cell wall properties. Mol Cell Biol, 1993 Apr, 13(4), 2586 - 92 Genetic evidence for a role for MCM1 in the regulation of arginine metabolism in Saccharomyces cerevisiae; Messenguy F et al.; ARGRI, ARGRII, and ARGRIII regulatory proteins control the expression of arginine anabolic and catabolic genes in Saccharomyces cerevisiae . We have shown that MCM1 is part of the ARGR regulatory complex, by in vitro binding experiments, at the ARGR5,6 promoter . The participation of MCM1 in the regulation of arginine metabolism is confirmed by the behavior of an mcm1-gcn4 mutant, which is affected in the repression of arginine anabolic genes . In this mcm1 mutant, synthesis of the catabolic enzymes is rather constitutive, but this derepression requires the integrity of the ARGR system and of the target sequences of these proteins in the CAR1 promoter . Our in vitro binding experiments confirm the presence of MCM1 in the protein complex interacting with the promoters of the catabolic CAR1 and CAR2 genes . This is the first in vivo transcription role ascribed to MCM1 other than its role in the transcription of cell-type-specific genes. Mol Cell Biol, 1993 Apr, 13(4), 2554 - 63 Cell surface anchorage and ligand-binding domains of the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin, a member of the immunoglobulin superfamily; Wojciechowicz D et al.; alpha-Agglutinin is a cell adhesion glycoprotein expressed on the cell wall of Saccharomyces cerevisiae alpha cells . Binding of alpha-agglutinin to its ligand a-agglutinin, expressed by a cells, mediates cell-cell contact during mating . Analysis of truncations of the 650-amino-acid alpha-agglutinin structural gene AG alpha 1 delineated functional domains of alpha-agglutinin . Removal of the C-terminal hydrophobic sequence allowed efficient secretion of the protein and loss of cell surface attachment . This cell surface anchorage domain was necessary for linkage to a glycosyl phosphatidylinositol anchor . A construct expressing the N-terminal 350 amino acid residues retained full a-agglutinin-binding activity, localizing the binding domain to the N-terminal portion of alpha-agglutinin . A 278-residue N-terminal peptide was inactive; therefore, the binding domain includes residues between 278 and 350 . The segment of alpha-agglutinin between amino acid residues 217 and 308 showed significant structural and sequence similarity to a consensus sequence for immunoglobulin superfamily variable-type domains . The similarity of the alpha-agglutinin-binding domain to mammalian cell adhesion proteins suggests that this structure is a highly conserved feature of adhesion proteins in diverse eukaryotes. Mol Cell Biol, 1993 Apr, 13(4), 2441 - 55 Structural alterations of the nucleolus in mutants of Saccharomyces cerevisiae defective in RNA polymerase I; Oakes M et al.; We have previously constructed mutants of Saccharomyces cerevisiae in which the gene for the second-largest subunit of RNA polymerase I (Pol I) is deleted . In these mutants, rRNA is synthesized by RNA polymerase II from a hybrid gene consisting of the 35S rRNA coding region fused to the GAL7 promoter on a plasmid . These strains thus grow in galactose but not glucose media . By immunofluorescence microscopy using antibodies against the known nucleolar proteins SSB1 and fibrillarin, we found that the intact crescent-shaped nucleolar structure is absent in these mutants; instead, several granules (called mininucleolar bodies {MNBs}) that stained with these antibodies were seen in the nucleus . Conversion of the intact nucleolar structure to MNBs was also observed in Pol I temperature-sensitive mutants at nonpermissive temperatures . These MNBs may structurally resemble prenucleolar bodies observed in higher eukaryotic cells and may represent a constituent of the normal nucleolus . Furthermore, cells under certain conditions that inhibit rRNA synthesis did not cause conversion of the nucleolus to MNBs . Thus, the role of Pol I in the maintenance of the intact nucleolar structure might include a role as a structural element in addition to (or instead of) a functional role to produce rRNA transcripts . Our study also shows that the intact nucleolar structure is not absolutely required for rRNA processing, ribosome assembly, or cell growth and that MNBs are possibly functional in rRNA processing in the Pol I deletion mutants. Mol Cell Biol, 1993 Apr, 13(4), 2324 - 31 Physical detection of heteroduplexes during meiotic recombination in the yeast Saccharomyces cerevisiae; Nag DK et al.; We describe a general physical method for detecting the heteroduplex DNA that is formed as an intermediate in meiotic recombination in the yeast Saccharomyces cerevisiae . We use this method to study the kinetic relationship between the formation of heteroduplex DNA and other meiotic events . We show that strains with the rad50, but not the rad52, mutation are defective in heteroduplex formation . We also demonstrate that, although cruciform structures can be formed in vivo as a consequence of heteroduplex formation between DNA strands that contain different palindromic insertions, small palindromic sequences in homoduplex DNA are rarely extruded into the cruciform conformation. Mol Cell Biol, 1993 Apr, 13(4), 2172 - 81 Bipartite structure of an early meiotic upstream activation sequence from Saccharomyces cerevisiae; Bowdish KS et al.; Diploid a/alpha Saccharomyces cerevisiae cells cease mitotic growth and enter meiosis in response to starvation . Expression of meiotic genes depends on the IME1 gene product, which accumulates only in meiotic cells . We report here an analysis of the regulatory region of IME2, an IME1-dependent meiotic gene . Deletion and substitution studies identified a 48-bp IME1-dependent upstream activation sequence (UAS) . Activity of the UAS also requires the RIM11, RIM15, and RIM16 gene products, which are required for expression of the chromosomal IME2 promoter and for meiosis . Through a selection for suppressors that permit UAS activity in an ime1 deletion mutant, we identified recessive mutations in three genes, SIN3 (also called RPD1, UME4, and SDI1), RPD3, and UME6 (also called CAR80), that were previously known as negative regulators of other early meiotic genes . Mutational analysis of the IME2 UAS reveals two critical sequence elements: a G+C-rich sequence (called URS1), previously identified at many meiotic genes, and a newly described element, the T4C site, that we found at a subset of meiotic genes . In agreement with prior studies, URS1 mutations lead to elevated IME2 UAS activity in the absence of IME1 . However, the URS1 mutations prevent any further stimulation of UAS activity by IME1 . Repression through URS1 has been shown to require the UME6 gene product . We find that activation of the IME2 UAS by IME1 also requires the UME6 gene product . Thus, UME6 and the URS1 site both have dual negative and positive roles at the IME2 UAS . We propose that IME1 modifies UME6 to convert it from a negulator to a positive Regulor. Mol Cell Biol, 1993 Apr, 13(4), 2126 - 33 U1 small nuclear ribonucleoprotein particle-protein interactions are revealed in Saccharomyces cerevisiae by in vivo competition assays; Stutz F et al.; Two highly conserved regions of the 586-nucleotide yeast (Saccharomyces cerevisiae) U1 small nuclear RNA (snRNA) can be mutated or deleted with little or no effect on growth rate: the universally conserved loop II (corresponding to the metazoan A loop) and the yeast core region (X . Liao, L . Kretzner, B . Seraphin, and M . Rosbash, Genes Dev . 4:1766-1774, 1990) . To examine the contribution of these regions to U1 small nuclear ribonucleoprotein particle (snRNP) activity, a competitor U1 gene, encoding a nonfunctional U1 snRNA molecule, was introduced into a number of strains carrying a U1 snRNA gene with loop II or yeast core mutations . The presence of the nonfunctional U1 gene lowered the growth rate of these mutant strains but not wild-type strains, consistent with the notion that mutant U1 RNAs are less active than wild-type U1 snRNAs . A detailed analysis of the U1 snRNA levels and half-lives in a number of merodiploid strains suggests that these mutant U1 snRNAs interact with U1 snRNP proteins less well than do their wild-type counterparts . Competition for protein factors during snRNP assembly could account for a number of previous observations in both yeast and mammalian cells. Mol Cell Biol, 1993 Apr, 13(4), 2069 - 80 Pheromone-induced signal transduction in Saccharomyces cerevisiae requires the sequential function of three protein kinases; Zhou Z et al.; Protein phosphorylation plays an important role in pheromone-induced differentiation processes of haploid yeast cells . Among the components necessary for signal transduction are the STE7 and STE11 kinases and either one of the redundant FUS3 and KSS1 kinases . FUS3 and presumably KSS1 are phosphorylated and activated during pheromone induction by a STE7-dependent mechanism . Pheromone also induces the accumulation of STE7 in a hyperphosphorylated form . This modification of STE7 requires the STE11 kinase, which is proposed to act before STE7 during signal transmission . Surprisingly, STE7 hyperphosphorylation also requires a functional FUS3 (or KSS1) kinase . Using in vitro assays for FUS3 phosphorylation, we show that pheromone activates STE7 even in the absence of FUS3 and KSS1 . Therefore, STE7 activation must precede modification of FUS3 (and KSS1) . These findings suggest that STE7 hyperphosphorylation is a consequence of its activation but not the determining event. Mol Cell Biol, 1993 Apr, 13(4), 2050 - 60 Function of the ste signal transduction pathway for mating pheromones sustains MAT alpha 1 transcription in Saccharomyces cerevisiae; Mukai Y et al.; Sterile mutants of Saccharomyces cerevisiae were isolated from alpha * cells having the a/alpha aar1-6 genotype (exhibiting alpha mating ability and weak a mating ability as a result of a defect in a1-alpha 2 repression) . Among these sterile mutants, we found two ste5 mutants together with putative ste7, ste11, and ste12 mutants of the signal transduction pathway of mating pheromones . The amino acid sequence of the Ste5p protein predicted from the nucleotide sequence of a cloned STE5 DNA has a domain rich in acidic amino acids close to its C terminus, a cysteine-rich sequence, resembling part of a zinc finger structure, in its N-terminal half, and a possible target site of cyclic AMP-dependent protein kinase at its C terminus . Northern (RNA) blot analysis revealed that STE5 transcription is under a1-alpha 2-Aar1p repression . The MAT alpha 1 cistron has a single copy of the pheromone response element in its 5' upstream region, and its basal level of transcription was reduced in these ste mutant cells . However, expression of the MAT alpha 1 cistron was not enhanced appreciably by pheromone signals . One of the ste5 mutant alleles conferred a sterile phenotype to a/alpha aar1-6 cells but a mating ability to MATa cells. Mol Cell Biol, 1993 Apr, 13(4), 2041 - 9 The COT2 gene is required for glucose-dependent divalent cation transport in Saccharomyces cerevisiae; Conklin DS et al.; Eleven cobalt-tolerant mutants were found to belong to a single complementation group, cot2 . In addition to cobalt, the cot2 mutants were found to tolerate increased levels of the divalent cations Zn2+, Mn2+, and Ni2+ as well . All of the cot2 mutants exhibited a wiener-shaped cellular morphology that was exacerbated by the carbon and nitrogen source but was unaffected by metals . The rate of glucose-dependent transport of cobalt into cells was reduced in strains that carry mutations in the COT2 gene . COT2 is not essential for growth . Strains that carry a COT2 allele conferring complete loss of function are viable and exhibit phenotypes similar to those of spontaneous cot2 mutations . The sequence of the COT2 gene shows that it is identical to GRR1, which encodes a protein required for glucose repression . The glucose dependence of the transport defect implies that cot2 mutations affect the link between glucose metabolism and divalent cation active transport. Yeast, 1993 Apr, 9(4), 399 - 409 Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae; Schreuder MP et al.; The sexual adhesion protein of Saccharomyces cerevisiae MAT alpha cells, alpha-agglutinin, could not be extracted from the cell wall with hot sodium dodecyl sulfate (SDS), but became soluble after digestion of the cell wall with laminarinase . This indicates that it is intimately associated with cell wall glucan . A fusion protein was constructed consisting of the signal sequence of yeast invertase, guar alpha-galactosidase, and the C-terminal half of the alpha-agglutinin . Most of the fusion protein was incorporated in the cell wall . A small amount could be extracted with SDS, but most of it could only be extracted with laminarinase . On the other hand, cells containing a construct consisting of the signal sequence of invertase and alpha-galactosidase released most of the alpha-galactosidase into the medium and all cell wall-associated alpha-galactosidase was released by SDS . Labelling with antibodies showed that the alpha-galactosidase part of the fusion protein was exposed on the surface of the cell wall . The results demonstrate that the C-terminal half of the alpha-agglutinin contains the information needed to incorporate a protein into the cell wall. Mol Gen Genet, 1993 Apr, 238(1-2), 6 - 16 Characterization of the MKS1 gene, a new negative regulator of the Ras-cyclic AMP pathway in Saccharomyces cerevisiae; Matsuura A et al.; In order to isolate genes that function downstream of the Ras-cAMP pathway in Saccharomyces cerevisiae, a YEp13-based genomic library was screened for clones that inhibit growth of cells with diminished A-kinase activity . One such gene, MKS1, was found to encode a hydrophilic 52 kDa protein that shares weak homology with the yeast SPT2/SIN1 gene product . Three lines of evidence suggest that the MKS1 gene product is a negative regulator downstream of the Ras-cAMP pathway: (i) overexpression of MKS1 inhibits growth of cyr1 disruptant cells on YPD medium containing a low concentration of cAMP; (ii) overexpression of MKS1 does not affect TPK1 expression; and (iii) the temperature-sensitive cyr1-230 mutation is partially suppressed by mks1 disruption . The mks1 mutant shows similar phenotypes to gal11/spt13, i.e., it cannot grow on YPGal containing ethidium bromide at 25 degrees C, or on YPGly or SGal at 37 degrees C . The mks1 gal11 double mutant shows more marked phenotypic changes than the single mutants . These results suggest that MKS1 is involved in transcriptional regulation of several genes by cAMP. Eur J Biochem, 1993 Apr 1, 213(1), 641 - 8 Synthesis of human parathyroid-hormone-related protein(1-141) in Saccharomyces cerevisiae . A correct amino-terminal processing vital for the hormone's biological activity is obtained by an ubiquitin fusion protein approach; Rian E et al.; Gene fusions have been widely used in heterologous expression systems as a technique to stabilize the recombinant product against proteolysis, increase the translational initiation efficiency or to serve as an affinity handle for the purification of the protein . A further advantage is the potential to generate an authentic amino terminus of the foreign protein when this is vital for its biological activity, such as for the ability of human parathyroid-hormone-related protein (hPTHrP) to mediate activation of adenylate cyclase . We report here the construction and utility of a ubiquitin fusion protein system for production of the otherwise short-lived hPTHrP(1-141) as a carboxyl extension to ubiquitin in yeast . A hybrid gene containing the hPTHrP(1-141) cDNA coding region fused in-frame to the 3' end of the yeast ubiquitin cDNA was constructed and expressed under the control of the regulatable yeast metallothionein promoter . The recombinant protein was purified to homogeneity and finally characterized by N-terminal amino acid sequencing and amino acid composition analysis, demonstrating that the fusion protein was cleaved correctly and quantitatively in vivo by an ubiquitin-specific yeast endoprotease to generate authentic hPTHrP(1-141) . hPTHrP(1-141) stimulated adenylate cyclase in rat osteosarcoma cell membranes to the same extent as equimolar amounts of recombinant human parathyroid hormone(1-84) and {Tyr34}hPTHrP(1-34)amide . Thus, this expression cloning strategy permits the production of authentic, biologically active recombinant hPTHrP(1-141), and the procedure can easily be adapted to make PTHrP analogues for further studies of its domain-specific activities and biological roles. Eur J Biochem, 1993 Apr 1, 213(1), 137 - 45 Rapid screening of cytochromes of respiratory mutants of Saccharomyces cerevisiae . Application to the selection of strains containing novel forms of cytochrome-c oxidase; Brown S et al.; A technique has been developed for the direct analysis by visible spectrophotometry of yeast spots growing on agar plates . This allows rapid semi-quantitative estimations of cytochromes c, b and oxidase and permits the identification of strains with impaired respiratory electron flow . Results of screening of 105 mutants are presented . There appears to be a correlation between the exonic location of the mutation in COX1 of oxidase and the level of optically detectable enzyme . Mutations in cytochrome b of the bc1 complex also affect the level of expression of cytochrome oxidase and can cause either an increased or decreased level of expression of oxidase relative to the wild-type strain . Twelve strains selected by the rapid level-1 screening were grown as lawns on sections of an agar plate and resuspended for a second level of screening . Quantitative estimates have been made of the concentrations of cytochromes, the turnover number of cytochrome oxidase and the kinetics of recombination of carbon monoxide with oxidase after flash photolysis . This confirmed the validity of the rapid screening procedure, and we have identified several strains which contain high levels of a mutant form of cytochrome oxidase with properties worthy of further investigation. Eur J Biochem, 1993 Apr 1, 213(1), 129 - 35 Genetic screening in Saccharomyces cerevisiae for large numbers of mitochondrial point mutations which affect structure and function of catalytic subunits of cytochrome-c oxidase; Meunier B et al.; A new search for mitochondrial respiratory deficient mutants (Mit-) has been undertaken in order to accumulate a large number of point mutations in the coding portions of cytochrome-c-oxidase catalytic subunits and cytochrome b . Therefore, a mitochondrial DNA which retains the exons and lacks all the introns of the cytochrome oxidase subunit I and of the cytochrome-b split genes has been introduced into a strain carrying a nuclear recessive mutation affecting the adenine-nucleotide translocator, the op1 mutation, which is known to prevent the accumulation of large deletion petite mutants and this was used as the parental strain . After a moderate MnCl2 mutagenesis in order to limit multiple mutations, 105 Mit- mutants were isolated from 15,000 mutagenised clones in Saccharomyces cerevisiae . Mutations were mapped to the three catalytic subunits encoding genes (COX1, COX2 and COX3) of the cytochrome-c oxidase (70 mutations) and to the cytochrome-b gene (15 mutations) . More than 50% of the mutants tested still exhibited mitochondrial translation products (subunits I, II and III), suggesting that they carry a missense mutation, rather than a nonsense mutation which would normally have led to a truncated protein . Mutations in the COX1 gene were allocated to four different subregions corresponding to exons 4 and 8 or to groups of exons, exons 1, 2, 3 or exons 5, 6, 7 . Seven missense monosubstitution mutations and two frameshift mutations were also identified . The amino acid changes of the missense mutations were located in the vicinity of the CuB-heme alpha 3 binuclear centre ligands. EMBO J, 1993 Apr, 12(4), 1467 - 73 Pathway correcting DNA replication errors in Saccharomyces cerevisiae; Morrison A et al.; Mutation of predicted 3'-->5' exonuclease active site residues of Saccharomyces cerevisiae POL3 DNA polymerase (delta) or deletion of the PMS1 mismatch repair gene lead to relative (to wild type) spontaneous mutation rates of approximately 130 and 41, respectively, measured at a URA3 reporter gene inserted near to a defined replication origin . The POL3 exonuclease-deficient mutant pol3-01 generated most classes of single base mutation in URA3, indicating a broad specificity that generally corresponds to that of the PMS1 system . pol3-01 pms1 haploid cells ceased growth after a few divisions with no unique terminal cell morphology . A pol3-01/pol3-01 pms1/pms1 diploid was viable and displayed an estimated URA3 relative mutation rate of 2 x 10(4), which we calculate to be catastrophically high in a haploid . The relationship between the relative mutation rates of pol3-01 and pms1 was multiplicative, indicating action in series . The PMS1 transcript showed the same cell cycle periodicity as those of a set of DNA replication genes that includes POL3, suggesting PMS1 is co-regulated with these genes . We propose that the POL3 3'-->5' exonuclease and the PMS1 mismatch repair system act on a common pathway analogous to the dnaQ-->mutHLS pathway of DNA replication error correction in Escherichia coli. Mol Cell Biol, 1993 Apr, 13(4), 2091 - 103 Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae; Turkel S et al.; Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2 . The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII) . In addition, the negative region includes at least two downstream activation sites (DASs) . This paper concerns the characterization of DASI . A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation . We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell . We have termed the two sites GCN4 binding sites (GBS1 and GBS2) . GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site . GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity . Nucleotides 13 to 36 of DASI overlap DRSII . Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements . We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4) . One of these is repeated in the 12 bp immediately adjacent to DASI . Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators . The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins. Mol Cell Biol, 1993 Apr, 13(4), 2081 - 90 Three downstream sites repress transcription of a Ty2 retrotransposon in Saccharomyces cerevisiae; Farabaugh PJ et al.; Transcription of Ty1 and Ty2 retrotransposons of the yeast Saccharomyces cerevisiae is modulated by multiple downstream regulatory sites . Both transposon families include a positively acting site within the transcribed region which resembles a higher eukaryotic enhancer . We have demonstrated the existence of a repression site distal to the enhancer of the Ty2-917 element . Here we describe experiments investigating the internal structure of this site . We show that this 200-bp region includes three distinct repression sites which we term DRSI (downstream repression site I), DRSII, and DRSIII . Individually each site causes almost twofold repression, and together the sites repress eightfold . Unexpectedly, when the entire region encompassing the DRS sites is moved outside the transcription unit, it acts as a qualitatively positively acting element . In this context the DRS sites still repress transcription, since eliminating them increases transcription further . That the region can activate transcription implies that it includes activation sites in addition to the three repression sites . The change from qualitatively negatively acting to positively acting must reflect a change in the relative effects of the multiple positive and negative sites; when moved outside the transcription unit, the activators predominate . Importantly, DRSII and DRSIII repress transcription autonomously when inserted upstream of a heterologous promoter activated by the transcriptional activator GCN4, showing that they are indeed transcriptional repression sites. Yeast, 1993 Apr, 9(4), 419 - 21 Genetic and physical localization of the acetyl-coenzyme A synthetase gene ACS1 on chromosome I of Saccharomyces cerevisiae; Steensma HY et al.; The ACS1 gene, encoding acetyl-coenzyme A synthetase, was mapped genetically at the left arm of chromosome I between pURA3 and PYK1 at 19 and 28 cM respectively . Comparison with the physical map defined a recombinational 'hot-spot' in this region in addition to the one between CDC24 and PYK1. Gene, 1993 Mar 30, 125(2), 115 - 23 Effect of a pmr 1 disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba alpha-galactosidase by Saccharomyces cerevisiae; Harmsen MM et al.; We fused the yeast-derived sequences encoding the invertase, acid phosphatase and alpha-factor pre- and prepro-signal peptides (SP) to the Cyamopsis tetragonoloba (guar plant) alpha-galactosidase(alpha Gal)-encoding gene and expressed these gene fusions in yeast . Whereas the amount of fusion protein produced by each of the constructs did not vary significantly, the secretion efficiency of the fusion protein that carried the SP of the prepro-alpha-factor (MF alpha 1) was consistently found to be about 10% higher than that of the other fusions (99% vs . 90%) . Furthermore, when the secretion of alpha Gal was directed by the invertase (SUC2) SP, the intracellular enzyme localized to the endoplasmic reticulum (ER), whereas use of the MF alpha 1 SP caused the intracellular enzyme to be outer-chain-glycosylated and processed by the KEX2 endoproteinase, implying that it had passed the ER . These results suggest that the pro-peptide of MF alpha 1 stimulates the efflux of the heterologous protein from the ER . Null mutants of PMR1 (encoding a Ca(2+)-dependent ATPase) are known to give higher secretion efficiencies for a number of different heterologous proteins . Therefore, we also studied the secretion of alpha Gal in a pmr 1 disruption mutant . Structural analysis of the enzyme secreted by the mutant cells showed that it was completely processed by KEX2 and outer-chain-glycosylated, although the length of the outer-chain carbohydrate moiety was reduced when compared with the enzyme secreted by wild-type cells . These results contradict the hypothesis advanced by Rudolph et al . {Cell 58 (1989) 133-145} that disruption of PMR1 causes the secretory pathway to bypass the Golgi apparatus. Gene, 1993 Mar 30, 125(2), 185 - 9 Characterization of a lysine-to-glutamic acid mutation in a conservative sequence of farnesyl diphosphate synthase from Saccharomyces cerevisiae; Blanchard L et al.; The mutant gene erg20-2 was isolated from a yeast strain defective in farnesyl diphosphate synthase (FPPS) . This strain had the unusual property of excreting prenyl alcohols such as geraniol . The nucleotide (nt) sequence, compared with that of the wild-type gene, showed a single nt change, resulting in a Lys197-->Glu substitution in FPPS which is directly involved in terpenic alcohol formation . In addition, disruption of ERG20 revealed that in yeast no other prenyl transferase is able to synthesize the FPP molecules required for essential non-sterol metabolites. J Biol Chem, 1993 Mar 25, 268(9), 6453 - 62 Inhibition of translational initiation in the yeast Saccharomyces cerevisiae as a function of the stability and position of hairpin structures in the mRNA leader; Vega Laso MR et al.; A new modular in vivo/in vitro expression system was constructed which facilitates studies of the control and regulation of gene expression in the yeast Saccharomyces cerevisiae . We studied the influence of stem-loop structures inserted into the non-translated leader region upon the steady-state levels and translation of mRNAs bearing the cat gene from the bacterial transposon Tn9 . mRNA abundance changed relatively little in response to alterations in the leader sequence and structure, whereas stem-loop structures clearly inhibited translation to a degree that was dependent upon the predicted stability as well as the position of the inserted secondary structure . A stem-loop structure with a predicted stability greater than -28 kcal mol-1 and with a stem comprising at least 15 (mainly G/C) base pairs inhibited translation in vivo by at least 98% . A stem-loop structure with a predicted stability of approximately -14 kcal mol-1, whose stem comprised at least six G/C base pairs, inhibited translation in vivo by at least 66% . The hairpins were more inhibitory when placed close to the start codon than when positioned near the 5' end of the leader . An mRNA showing extensive complementarity between the leader and trailer regions was not only poorly translated but also had a steady-state level at least three times higher than the average for all the cat constructs examined . Translation of the various mRNAs in a yeast cell-free system followed qualitatively the same pattern as the results obtained in vivo . The stem-loop structures were far less inhibitory in a reticulocyte lysate system . Overall, the data are likely to reflect the full spectrum of translational activities of yeast mRNAs in vivo determined by secondary structure and emphasize the importance of translation as a control step in gene expression. J Biol Chem, 1993 Mar 25, 268(9), 6248 - 55 Regulation of the 45- and 55-kDa forms of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae by nucleotides; Buxeda RJ et al.; The synthesis of phosphatidylinositol (PI) 4-phosphate and PI 4,5-bisphosphate in the yeast Saccharomyces cerevisiae is stimulated by glucose . PI 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) catalyzes the committed step in the synthesis of these phosphoinositides . Previous studies have suggested that the glucose effect on phosphoinositide synthesis is mediated by cellular levels of ATP and ADP and by the RAS/cAMP pathway . Using purified preparations of the membrane-associated 45- and 55-kDa forms of PI 4-kinase, we examined the regulation of these activities by nucleotides and cAMP-dependent protein kinase . MgADP was a potent inhibitor of both forms of the enzyme . Detailed kinetic analyses of the 45- and 55-kDa enzymes using Triton X-100/PI-mixed micelles showed that MgADP was a competitive inhibitor (Ki = 0.14 and 0.25 mM, respectively) with respect to MgATP and a noncompetitive inhibitor (Ki = 1.3 and 0.9 mM, respectively) with respect to PI . The Ki values for MgADP were about 2-fold lower than the Km values the enzymes have for their substrate MgATP and about 2-fold lower than the cellular concentration of ADP . The 45- and 55-kDa forms of PI 4-kinase activity were regulated differentially by CTP, an important nucleotide involved in phospholipid biosynthesis . Whereas the 55-kDa PI 4-kinase was inhibited by CTP, the 45-kDa enzyme was unaffected by CTP . CTP was a mixed type of inhibitor (Ki = 1.5 mM) with respect to MgATP and a noncompetitive inhibitor (Ki = 4 mM) with respect to PI . The Ki value for CTP was 4-fold higher than the Km value for MgATP and 7-fold higher than the cellular concentration of CTP . The 45- and 55-kDa PI 4-kinases were neither phosphorylated nor regulated by cAMP-dependent protein kinase . These results did not support the previous conclusion that PI 4-phosphate synthesis was mediated by the RAS/cAMP pathway . Our kinetic studies supported the conclusion that the glucose effect on the synthesis of PI 4-phosphate was mediated by cellular levels of ATP and ADP through the regulation of membrane-associated PI 4-kinase activity. Biochim Biophys Acta, 1993 Mar 21, 1156(3), 263 - 6 Role of the trehalose carrier in dehydration resistance of Saccharomyces cerevisiae; Eleutherio EC et al.; Yeast cells are well known for their ability to survive complete dehydration, a phenomenon that is directly linked to the presence of the sugar trehalose in these cells . This sugar apparently endows the cells with the capacity to survive dehydration . Previous studies on in vitro models showed that trehalose must be present on both sides of the bilayer to stabilize dry membranes . The present report demonstrates that a specific trehalose carrier seems to enable the sugar to protect the yeast cell membrane by translocating trehalose from the cytosol to the extracellular environment . Saccharomyces cerevisiae mutant strains which lack the trehalose carrier did not survive after dehydration although they accumulated endogenous trehalose . Furthermore, when carrier mutants were dehydrated in the presence of exogenous trehalose the cells became more resistant showing increased survival. FEMS Microbiol Lett, 1993 Mar 15, 108(1), 23 - 6 Acetoin production in Saccharomyces cerevisiae wine yeasts; Romano P et al.; One hundred strains of Saccharomyces cerevisiae were examined for the capacity to produce acetoin in synthetic medium and in grape must . The low production of acetoin was found to be the more common pattern in this species . Most strains exhibited a similar distribution in both media, production ranging from non-detectable amounts to 12 mg l-1 . Only four strains produced high quantities of acetoin, up to 29.5 mg l-1 in synthetic medium and up to 194.6 mg l-1 in grape must . This biometric study showed the existence of two phenotypes, "low and high acetoin production", that could be selected for conferring a desirable flavour of the final product. Biochem Biophys Res Commun, 1993 Mar 15, 191(2), 750 - 8 Characterization of DNA binding properties of Yp20: an abundant nuclear protein isolated from Saccharomyces cerevisiae; Bartuv J et al.; The aim of this study was to elucidate the function of Yp20 (yeast 20 kDa protein) which is an abundant basic DNA-binding protein copurified with yeast chromatin . The work presented here shows that Yp20 is a sequence specific DNA binding protein . DNA binding activity was extremely thermostable . The affinity of binding to TRP1 was higher than the affinity of binding to the B domain of ARS1 . The dissociation half time of Yp20-DNA complexes was less than 1 min . Yp20 showed no homology to a similar abundant 21 kDa ARS binding factor II (ABFII), previously described . Competitive gel retardation assays revealed two different regions that were protected by Yp20 . One was overlapping the ABF1 binding site on ARS1 and another protected region was found upstream to the translational start codon of the TRP1 gene . It thus appears that Yp20 may have a role in DNA replication and/or transcription. J Biol Chem, 1993 Mar 15, 268(8), 5754 - 62 Isolation and immunochemical characterization of eukaryotic translation initiation factor 5 from Saccharomyces cerevisiae; Chakravarti D et al.; Eukaryotic translation initiation factor 5 (eIF-5), which catalyzes the hydrolysis of GTP bound to the 40 S ribosomal initiation complex has been purified from yeast cell lysates . The purified factor eluted from gel filtration columns as a protein of apparent M(r) = 45,000-50,000 . However, when the purified preparation was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two distinct polypeptides of apparent M(r) = 54,000 and 56,000 were observed . Each of the two polypeptides individually was found to contain eIF-5 activity, and they were immunologically related to each other . In less pure preparations of yeast eIF-5, however, a significant proportion of eIF-5 activity eluted from gel filtration columns as a protein of M(r) > 140,000 . Immunochemical methods were therefore employed to determine the molecular structure of eIF-5 in crude yeast cell lysates . Antisera against purified yeast eIF-5 were prepared in rabbits and shown to be highly potent in inhibiting eIF-5-mediated 80 S initiation complex formation . When crude eIF-5 preparations, as well as yeast cells that were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate, were analyzed by Western blots probed with affinity-purified anti-eIF-5 antibodies, a major immunoreactive polypeptide (apparent M(r) = 54,000) and a minor band (apparent M(r) = 56,000) were observed . No precursor forms of molecular weight higher than 56,000 were detected in any preparations . These results suggest that yeast eIF-5 is a monomeric protein of apparent M(r) = 50,000-56,000. J Biol Chem, 1993 Mar 15, 268(8), 5728 - 34 Functional characterization of flavin-containing monooxygenase 1B1 expressed in Saccharomyces cerevisiae and Escherichia coli and analysis of proposed FAD- and membrane-binding domains; Lawton MP et al.; A cDNA encoding the flavin-containing monooxygenase of rabbit lung (FMO 1B1) was expressed in yeast and Escherichia coli and the recombinant enzymes characterized . A high copy, isopropyl-1-thio-beta-D-galactopyranoside (IPTG)-inducible E . coli expression vector, pKKHC, was used for expression in E . coli strain JM109, and a galactose-inducible vector, YEp53, was used for expression in yeast strain 334 . Following transcriptional induction with IPTG or galactose, subcellular fractions were prepared and analyzed immunochemically and catalytically . Antibodies to rabbit FMO 1B1 were used to detect the recombinant proteins in the 100,000 x g pellet prepared from the 10,000 x g supernatant fraction of yeast homogenates and the 2,000 x g supernatant fraction of E . coli homogenates . No FMO 1B1 was detected in cytosol . Mobilities of the recombinant proteins in SDS-polyacrylamide gel electrophoresis appeared identical to that of the native microsomal enzyme . Catalytic similarity to the native FMO 1B1 was demonstrated by the ability of the expressed enzymes to metabolize methimazole, thiourea, dimethylaniline, and cysteamine, but not chlorpromazine or imipramine . In addition, the recombinant enzymes exhibited a number of the unique physical properties associated with FMO 1B1, including stability to elevated temperature and activation by sodium cholate and magnesium chloride . Based on the specific content of FAD, the level of expression was estimated to be approximately 2% of the total protein in the E . coli 100,000 x g particulate fraction and 1% in the fraction from yeast . To demonstrate the utility of the E . coli expression system for studying structure/function relationships of the flavin-containing monooxygenase, two mutant FMOs were expressed and characterized . One mutant, formed by deletion of a putative membrane-anchoring peptide (the 26 carboxyl-terminal amino acids) was tested for membrane association . No difference in the subcellular distribution was found between the truncated and unmodified proteins, suggesting that the 26-residue COOH-terminal peptide is not important in membrane association . Catalytic analysis of the truncated FMO 1B1 established its functional similarity to the full-length protein, indicating that the COOH terminus does not contribute to any of the unique properties of the lung enzyme.(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1993 Mar 5, 268(7), 4832 - 8 Functional expression of human cathepsin S in Saccharomyces cerevisiae . Purification and characterization of the recombinant enzyme; Bromme D et al.; A cDNA encoding the human lysosomal cysteine proteinase cathepsin S precursor has been expressed in yeast using the pVT100-U expression vector containing the alpha-factor promoter . The procathepsin S gene was expressed either as a fusion protein with the pre-region or with the prepro-region of the yeast alpha-factor precursor gene . Following in vitro processing both constructs gave an identical active mature enzyme with a molecular weight of 24,000 . After prolonged cultivation of the cells the recombinant protein is also found as an active proteinase in the culture supernatant . The precursor can be activated in vitro at pH 4.5 and 40 degrees C under reducing conditions . The in vitro activated enzyme has a 6-amino acid NH2-terminal extension when compared with the native bovine enzyme . The purified enzyme displays a bell-shaped pH activity profile with a pH optimum of 6.5 and pK values of 4.5 and 7.8 . The isoelectric point of the recombinant human cathepsin S is between 8.3 and 8.6 and about 1.5 pH units higher than for the bovine enzyme . The kinetic data for several synthetic substrates and inhibitors reveal a preference for smaller amino acid residues in the binding subsites S2 and S3 of cathepsin S . Like the bovine enzyme, the recombinant human cathepsin S is characterized by a broader range of pH stability (pH 5-7.5) than cathepsins B and L. J Membr Biol, 1993 Mar, 132(3), 183 - 99 Gating and conductance in an outward-rectifying K+ channel from the plasma membrane of Saccharomyces cerevisiae; Bertl A et al.; The plasma membrane of the yeast Saccharomyces cerevisiae has been investigated by patch-clamp techniques, focusing upon the most conspicuous ion channel in that membrane, a K(+)-selective channel . In simple observations on inside-out patches, the channel is predominantly closed at negative membrane voltages, but opens upon polarization towards positive voltages, typically displaying long flickery openings of several hundred milliseconds, separated by long gaps (G) . Elevating cytoplasmic calcium shortens the gaps but also introduces brief blocks (B, closures of 2-3 msec duration) . On the assumption that the flickery open intervals constitute bursts of very brief openings and closings, below the time resolution of the recording system, analysis via the beta distribution revealed typical closed durations (interrupts, I) near 0.3 msec, and similar open durations . Overall behavior of the channel is most simply described by a kinetic model with a single open state (O), and three parallel closed states with significantly different lifetimes: long (G), short (B) and very short (I) . Detailed kinetic analysis of the three open/closed transitions, particularly with varied membrane voltage and cytoplasmic calcium concentration, yielded the following stability constants for channel closure: K1 = 3.3 x e-zu in which u = eVm/kT is the reduced membrane voltage, and z is the charge number; KG = 1.9 x 10(-4) ({Ca2+}.ezu)-1; and KB = 2.7 x 10(3)({Ca2+}.ezu)2 . Because of the antagonistic effects of both membrane voltage (Vm) and cytoplasmic calcium concentration ({Ca2+}cyt) on channel opening from the B state, compared with openings from the G state, plots of net open probability (Po) vs . either Vm or {Ca2+} are bell-shaped, approaching unity at low calcium (microM) and high voltage (+150 mV), and approaching 0.25 at high calcium (10 mM) and zero voltage . Current-voltage curves of the open channel are sigmoid vs . membrane voltage, saturating at large positive or large negative voltages; but time-averaged currents, along the rising limb of Po (in the range 0 to +150 mV, for 10 microM {Ca2+}) make this channel a strong outward rectifier . The overall properties of the channel suggest that it functions in balancing charge movements during secondary active transport in Saccharomyces. Yeast, 1993 Mar, 9(3), 221 - 34 Reduced efficiency in the glycosylation of the first sequon of Saccharomyces cerevisiae exoglucanase leads to the synthesis and secretion of a new glycoform of the molecule; Basco RD et al.; In addition to exoglucanases (EXGs) I and II, old cultures of Saccharomyces cerevisiae secreted into the culture medium a new immunologically-related material that exhibited exoglucanase activity . The new exoglucanase (EXGII1/2) was purified from stationary-phase cultures . It turned out to be a glycoprotein whose protein portion was identical to that of the other two isoenzymes in terms of ionic properties, size, amino acid composition and NH2-terminal sequence (25 residues) . Disruption of the structural gene encoding EXGs I and II resulted in a strain unable to secrete all three isoenzymes . EXGII1/2 was indistinguishable in terms of molecular weight from the single intermediate detected during the deglycosylation (mediated by endo H) of EXGII by sodium dodecyl sulphate-polyacrylamide gel electrophoresis . Thus, the new isoenzyme contains only one of the two slightly elongated mannan inner cores present in enzyme II . Two intermediates were, however, detected when the deglycosylation of EXGII was monitored by ion-exchange chromatography (high-pressure liquid chromatography) . Site-directed mutagenesis indicated that the major intermediate, which eluted at about the same position as enzyme II1/2, corresponded to protein molecules carrying the oligosaccharide attached to the Asn of the second sequon, whereas the minor one carried the oligosaccharide in the first potential glycosylation site . Several lines of evidence indicate that EXGII1/2 is a biosynthetic product resulting from an imbalance between the rate of protein synthesis and the glycosylation capabilities of the glycosylation machinery. Mol Gen Genet, 1993 Mar, 237(3), 463 - 6 Characterization of the cyr1-2 UGA mutation in Saccharomyces cerevisiae; Morishita T et al.; cyr1-2 is a temperature-sensitive mutation of the yeast adenylate cyclase structural gene, CYR1 . The cyr1-2 mutation has been suggested to be a UGA mutation since a UGA suppressor SUP201 has been isolated as a suppressor of the cyr1-2 mutation . Construction of chimeric genes restricted the region containing the cyr1-2 mutation, and the cyr1-2 UGA mutation was identified at codon 1282, which lies upstream of the region coding for the catalytic domain of adenylate cyclase . Alterations in the region upstream of the cyr1-2 mutation site result in null mutations . The complete open reading frame of the cyr1-2 gene expressed under the control of the GAL1 promoter complemented cyr1-d1 in a galactose-dependent manner . These results suggest that at the permissive temperature weak readthrough occurs at the cyr1-2 mutation site to produce low levels of active adenylate cyclase . An endogenous suppressor in yeast cells is assumed to be responsible for this readthrough. Mol Gen Genet, 1993 Mar, 237(3), 449 - 59 Molecular and genetic characterization of SPT4, a gene important for transcription initiation in Saccharomyces cerevisiae; Malone EA et al.; Mutations in the SPT4 gene of Saccharomyces cerevisiae were isolated as suppressors of delta insertion mutations that interfere with adjacent gene transcription . Recent genetic evidence indicates that the SPT4 protein functions with two other proteins, SPT5 and SPT6, in some aspect of transcription initiation . In this work we have characterized the SPT4 gene and we demonstrate that spt4 mutations, like spt5 and spt6 mutations, cause changes in transcription . Using the cloned SPT4 gene, spt4 null mutations were constructed; in contrast to spt5 and spt6 null mutants, which are inviable, spt4 null mutants are viable and have an Spt- phenotype . The DNA sequence of the SPT4 gene predicts a protein product of 102 amino acids that contains four cysteine residues positioned similarly to those of zinc binding proteins . Mutational analysis suggests that at least some of these cysteines are essential for SPT4 function . Genetic mapping showed that SPT4 is a previously unidentified gene that maps to chromosome VII, between ADE6 and CLY8. Mol Gen Genet, 1993 Mar, 237(3), 375 - 84 Post-transcriptional regulation of IME1 determines initiation of meiosis in Saccharomyces cerevisiae; Sherman A et al.; The IME1 gene of Saccharomyces cerevisiae is required for initiation of meiosis . Transcription of IME1 is detected under conditions which are known to induce initiation of meiosis, namely starvation for nitrogen and glucose, and the presence of MATa1 and MAT alpha 2 gene products . In this paper we show that IME1 is also subject to translational regulation . Translation of IME1 mRNA is achieved either upon nitrogen starvation, or upon G1 arrest . In the presence of nutrients, constitutively elevated transcription of IME1 is also sufficient for the translation of IME1 RNA . Four different conditions were found to cause expression of Ime1 protein in vegetative cultures: elevated transcription levels due to the presence of IME1 on a multicopy plasmid; elevated transcription provided by a Gal-IME1 construct; G1 arrest due to alpha-factor treatment; G1 arrest following mild heat-shock treatment of cdc28 diploids . Using these conditions, we obtained evidence that starvation is required not only for transcription and efficient translation of IME1, but also for either the activation of Ime1 protein or for the induction/activation of another factor that, either alone or in combination with Ime1, induces meiosis. J Gen Microbiol, 1993 Mar, 139 ( Pt 3), 501 - 7 Saccharomyces cerevisiae has an inducible response to menadione which differs from that to hydrogen peroxide; Flattery-O'Brien J et al.; Exponential phase cells of Saccharomyces cerevisiae treated with the superoxide free-radical generating agent menadione (MD; 0.2 mM) for 60 min adapted to become resistant to the lethal effects of a higher concentration of MD (4 mM) . Inhibition of protein synthesis by treatment with cycloheximide totally prevented the adaptation to MD, indicating that this is an inducible response completely dependent on protein synthesis; this differs from the situation with peroxide in which only some of the adaptive response is cycloheximide-sensitive . Cells subjected to heat shock (23 to 37 degrees C) or treatment with hydrogen peroxide (H2O2; 0.2 mM, 60 min) became more resistant to 4 mM-MD; however, MD pretreatment did not induce any thermotolerance or resistance to peroxide . These differences between the response to MD and H2O2 were reflected in the results of L-{35S}methionine labelling studies . Using one-dimensional electrophoresis, only one polypeptide (60 kDa) was seen to be induced by 0.2 mM-MD and this was also induced by heat shock but not peroxide stress . With heat shock or peroxide treatment the induction of at least 10 polypeptides was detected using this approach . Using an isogenic petite strain, it was found that functional mitochondria were needed for conferring full resistance to MD, but that induction of the adaptive response was not dependent on mitochondrial function. Mol Microbiol, 1993 Mar, 7(5), 683 - 92 Deletion analysis of the SUP35 gene of the yeast Saccharomyces cerevisiae reveals two non-overlapping functional regions in the encoded protein; Ter-Avanesyan MD et al.; SUP35 is an omnipotent suppressor gene of Saccharomyces cerevisiae coding for a protein consisting of a C-terminal part similar to the elongation factor EF-1 alpha and a unique N-terminal sequence of 253 amino acids . Twelve truncated versions of the SUP35 gene were generated by the deletion of fragments internal to the coding sequence . Functional studies of these deletion mutants showed that: (i) only the EF-1 alpha-like C-terminal part of the Sup35 protein is essential for the cell viability; (ii) overexpression of either the N-terminal part of the Sup35 protein or the full-length Sup35 protein decreases translational fidelity, resulting in omnipotent suppression and reduced growth of {psi+} strains; (iii) expression of the C-terminal part of the Sup35 protein generates an antisuppressor phenotype; and (iv) both the N- or C-terminal segments of the Sup35 protein can bind to 80S ribosomes . Thus, the data obtained define two domains within the Sup35 protein which are responsible for different functions. EMBO J, 1993 Mar, 12(3), 1209 - 18 Evidence for an interaction between the CYP1(HAP1) activator and a cellular factor during heme-dependent transcriptional regulation in the yeast Saccharomyces cerevisiae; Fytlovich S et al.; Previously, it was shown that the CYP1(HAP1) gene product mediates the transcription of several oxygen-regulated genes through a metabolic co-effector, heme, in the yeast Saccharomyces cerevisiae . This study investigates the overproduction of the CYP1 protein when the CYP1(HAP1) gene is placed under the control of the GAL10-CYC1 hybrid promoter (either at the locus of the CYP1(HAP1) gene or cloned in a high-copy-number plasmid) . In these conditions, the CYP1 protein is detected by Western blot analysis and has a molecular mass in agreement with the open reading frame sequence . Band-shift experiments show that the CYP1(HAP1) protein is able to interact specifically with its target sequences in vitro without addition of hemin, and forms a large complex with one or several unidentified factors denoted as X . Addition of hemin allows the formation of a new complex which has a lower molecular mass . The internal deletion of the seven repeated amino acid sequences containing the KCPVDH motif in the CYP1(HAP1) protein modifies the heme responsiveness phenomenon observed in vitro in the band-shift experiments and in vivo in the transcription of the CYB2, CYC1, CYP3(CYC7) and ERG11 genes . On the basis of these data, we propose a new model for heme-induced activation of the CYP1 protein. Genetics, 1993 Mar, 133(3), 489 - 98 a/alpha-control of DNA repair in the yeast Saccharomyces cerevisiae: genetic and physiological aspects; Heude M et al.; It has long been known that diploid strains of yeast are more resistant to gamma-rays than haploid cells, and that this is in part due to heterozygosity at the mating type (MAT) locus . It is shown here that the genetic control exerted by the MAT genes on DNA repair involves the a1 and alpha 2 genes, in a RME1-independent way . In rad18 diploids, affected in the error-prone repair, the a/alpha effects are of a very large amplitude, after both UV and gamma-rays, and also depends on a1 and alpha 2 . The coexpression of a and alpha in rad18 haploids suppresses the sensitivity of a subpopulation corresponding to the G2 phase cells . Related to this, the coexpression of a and alpha in RAD+ haploids depresses UV-induced mutagenesis in G2 cells . For srs2 null diploids, also affected in the error-prone repair pathway, we show that their G1 UV sensitivity, likely due to lethal recombination events, is partly suppressed by MAT homozygosity . Taken together, these results led to the proposal that a1-alpha 2 promotes a channeling of some DNA structures from the mutagenic into the recombinational repair process. Genetics, 1993 Mar, 133(3), 469 - 87 Replication-dependent sister chromatid recombination in rad1 mutants of Saccharomyces cerevisiae; Kadyk LC et al.; Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation . In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G1 or the G2 phases of the cell cycle than was observed in RAD1/rad1-1 cells . Since sister chromatids are not present during G1, this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication . The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G2 phase, but only when they are present during DNA replication . We propose that there are two types of sister chromatid recombination in yeast . In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells . In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G2 . Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination. Eur J Biochem, 1993 Mar 1, 212(2), 521 - 8 Cloning and expression of Saccharomyces cerevisiae copper-metallothionein gene in Escherichia coli and characterization of the recombinant protein; Sayers Z et al.; The gene sequences for intact and truncated forms of copper-binding metallothionein from Saccharomyces cerevisiae were cloned and overexpressed in Escherichia coli BL21(DE3)pLysE cells . In contrast to several other genes, the intact and truncated metallothionein genes are amplified in the polymerase chain reaction when Mg2+ is replaced by Co2+ . The recombinant truncated protein binds copper in vivo and in vitro . A ratio of 8 Cu/12 cysteines was determined from atomic absorption, X-ray fluorescence and amino acid analysis . Extended X-ray absorption spectroscopy indicates that all Cu is in Cu(I) form and coordinated to three S atoms. Eur J Biochem, 1993 Mar 1, 212(2), 315 - 23 Disruption of TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae, causes accumulation of trehalose-6-phosphate and loss of trehalose-6-phosphate phosphatase activity; De Virgilio C et al.; Preparations of the trehalose-6-phosphate synthase/phosphatase complex from Saccharomyces cerevisiae contain three polypeptides with molecular masses 56, 100 and 130 kDa, respectively . Recently, we have cloned the gene for the 56-kDa subunit of this complex (TPS1) and found it to be identical with CIF1, a gene essential for growth on glucose and for the activity of trehalose-6-phosphate synthase . Peptide sequencing of the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex (TPS2) revealed one sequence to be 100% identical with the deduced amino acid sequence of the upstream region of PPH3 on the right arm of chromosome IV . This sequence was used to clone an upstream region of PPH3 containing an open reading frame of 2685 nucleotides, predicted to encode a polypeptide of 102.8 kDa . The N-terminal sequence, as well as three internal amino acid sequences, obtained from peptide sequencing of the 100-kDa subunit, were identical with specific regions of the deduced amino acid sequence . Thus, the sequence cloned represents TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex . Interestingly, a stretch of about 500 amino acids from the first part of TPS2 was 33% identical with the entire TPS1 sequence . Disruption of TPS2 had no effect on trehalose-6-phosphate synthase activity but caused complete loss of trehalose-6-phosphate phosphatase activity, measured in vitro, and accumulation of excessive amounts of trehalose-6-phosphate instead of trehalose upon heat shock or entrance into stationary phase in vivo . These results suggest that TPS2 codes for the structural gene of the trehalose-6-phosphate phosphatase . Heat shock induced an increase in trehalose-6-phosphate phosphatase activity and this was preceded by an accumulation in TPS2 mRNA, suggesting that the trehalose-6-phosphate phosphatase is subjected to transcriptional control under heat-shock conditions. Mol Cell Biol, 1993 Mar, 13(3), 1805 - 14 Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein; Wang H et al.; The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator . Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins . Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro . We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators . The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions . We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity . Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity . A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription. Mol Cell Biol, 1993 Mar, 13(3), 1489 - 96 A DNA polymerase mutation that suppresses the segregation bias of an ARS plasmid in Saccharomyces cerevisiae; Houtteman SW et al.; Yeast autonomously replicating sequence (ARS) plasmids exhibit an unusual segregation pattern during mitosis . While the nucleus divides equally into mother and daughter cells, all copies of the ARS plasmid will often remain in the mother cell . A screen was designed to isolate mutations that suppress this segregation bias . A plasmid with a weak ARS (wARS) that displayed an extremely high segregation bias was constructed . When cells were grown under selection for the wARS plasmid, the resulting colonies grew slowly and had abnormal morphology . A spontaneous recessive mutation that restored normal colony morphology was identified . This mutation suppressed plasmid segregation bias, as indicated by the increased stability of the wARS plasmid in the mutant cells even though the plasmid was present at a lower copy number . An ARS1 plasmid was also more stable in mutant cells than in wild-type cells . The wild-type allele for this mutant gene was cloned and identified as POL delta (CDC2) . This gene encodes DNA polymerase delta, which is essential for DNA replication . These results indicate that DNA polymerase delta plays some role in causing the segregation bias of ARS plasmids. Curr Genet, 1993 Mar, 23(3), 187 - 91 Saccharomyces cerevisiae phosphoglucose isomerase and fructose bisphosphate aldolase can be replaced functionally by the corresponding enzymes of Escherichia coli and Drosophila melanogaster; Boles E et al.; Two glycolytic enzymes, phosphoglucose isomerase and fructose-1,6-bisphosphate aldolase, of Saccharomyces cerevisiae could be replaced by their heterologous counterparts from Escherichia coli and Drosophila melanogaster . Both heterologous enzymes, which show respectively little and no sequence homology to the corresponding yeast enzymes, fully restored wild-type properties when their genes were expressed in yeast deletion mutants . This result does not support notions of an obligatory formation of glycolytic multi-enzyme aggregates in yeast; nor does it support possible regulatory functions of yeast phosphoglucose isomerase. Mol Microbiol, 1993 Mar, 7(6), 831 - 5 The Rad3 protein from Saccharomyces cerevisiae: a DNA and DNA:RNA helicase with putative RNA helicase activity; Deschavanne PJ et al.; The Rad3 protein from Saccharomyces cerevisiae is a DNA helicase which participates in the repair of ultraviolet-irradiated DNA and is inhibited in the presence of DNA containing thymine dimers . This protein is also involved in mitotic recombination and spontaneous mutagenesis and is essential for cell viability in the absence of DNA damage . Furthermore, the Rad3 protein also exhibits a DNA:RNA helicase activity in which there is a significant preference for a partial DNA:RNA hybrid rather than a partial duplex DNA substrate, which suggests that this protein might be involved in DNA repair within transcriptionally active genes . Finally, the Rad3 protein contains the DEAH motif and shares high amino acid sequence similarity with the DEAD family of RNA helicase proteins, suggesting that it might also possess an RNA helicase activity. Proc Natl Acad Sci U S A, 1993 Mar 1, 90(5), 1804 - 8 The putative phosphoinositide-specific phospholipase C gene, PLC1, of the yeast Saccharomyces cerevisiae is important for cell growth; Yoko-o T et al.; Using the polymerase chain reaction technique, we have isolated a gene that encodes a putative phosphoinositide-specific phospholipase C (PLC) in the yeast Saccharomyces cerevisiae . The nucleotide sequence indicates that the gene encodes a polypeptide of 869 amino acid residues with a calculated molecular mass of 101 kDa . This polypeptide has both the X and Y regions conserved among mammalian PLC-beta, -gamma, and -delta, and the structure is most similar to that of mammalian PLC-delta . This putative yeast PLC gene has been designated PLC1 . Disruption of PLC1 results in slow growth or lethality for cells, depending on their genetic background and the medium, indicating that PLC1 is important for cell growth . Expression of rat PLC-delta 1 cDNA suppressed the growth defect of plc1 disruptants, strongly suggesting that PLC1 encodes PLC. Curr Genet, 1993 Mar, 23(3), 201 - 4 Messenger RNA 3'-end formation of a DNA fragment from the human c-myc 3'-end region in Saccharomyces cerevisiae; Irniger S et al.; We have tested the functioning of the human c-myc polyadenylation signal in Saccharomyces cerevisiae . A DNA fragment containing the two AATAAA polyadenylation signals of the c-myc gene was inserted into a plasmid designed for the in-vivo testing of polyadenylation signals in yeast . The c-myc fragment had a partial capacity for directing mRNA 3'-end formation in yeast . The 3'-endpoints were 50-100 bp distant from the mRNA 3'-ends mapped in humans . This human DNA fragment is therefore unspecifically functional in yeast, indicating that other sequence elements than the human polyadenylation signal, AATAAA, are necessary for 3'-end formation. Appl Microbiol Biotechnol, 1993 Mar, 38(6), 795 - 8 Effect of n-alcohols on the electrotransformation and permeability of Saccharomyces cerevisiae; Ganeva VJ et al.; The correlation between electrotransformation and electropermeability was studied in yeast cells following the modification of their membranes by treatment with n-alcohols . It was found that the number of transformed cells decreased with increase of chain length of the alcohols used as follows: methanol < ethanol < propanol < butanol . The electropermeability was unaffected by the prepulse n-alcohol treatment . The lack of a unidirectional link between permeability and transformation leads to the assumption that the mechanism of DNA introduction into the cell could not be interpreted solely as a result of the existence of pores. Genetics, 1993 Mar, 133(3), 509 - 16 PET111 acts in the 5'-leader of the Saccharomyces cerevisiae mitochondrial COX2 mRNA to promote its translation; Mulero JJ et al.; PET111 is a yeast nuclear gene specifically required for the expression of the mitochondrial gene COX2, encoding cytochrome c oxidase subunit II (coxII) . Previous studies have shown that PET111 activates translation of the COX2 mRNA . To map the site of PET111 action we have constructed, in vitro, genes coding for chimeric mRNAs, introduced them into mitochondria by transformation and studied their expression . Translation of a chimeric mRNA with the 612-base 5'-untranslated leader of the COX3 mRNA fused precisely to the structural gene for the coxII-precursor protein is independent of PET111, but does require a COX3 mRNA-specific translational activator known to work on the COX3 5'-leader . This result demonstrates that PET111 is not required for any post-translational step . Translation of a chimeric mRNA with the 54-base 5'-leader of the COX2 mRNA fused precisely to the structural gene for cytochrome c oxidase subunit III was dependent on PET111 activity . These results demonstrate that PET111 acts specifically at a site in the short COX2 5'-leader to activate translation of downstream coding sequences. J Cell Biol, 1993 Mar, 120(5), 1203 - 15 The a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae; Kuchler K et al.; STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells . Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein . Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane-associated protein . The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function . The amino acid sequence of Ste6 predicts two ATP-binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-{alpha-32P}ATP . Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane . In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells . The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell. Eur J Biochem, 1993 Feb 15, 212(1), 113 - 20 Purification and characterization of recombinant human beta 1-4 galactosyltransferase expressed in Saccharomyces cerevisiae; Krezdorn CH et al.; A protease-defective strain of Saccharomyces cerevisiae (BT 150) was used to express full-length cDNA of HeLa cell beta-D-N-acetylglucosaminide-beta-1,4-galactosyltransferase (gal-T) . To ascertain import of the recombinant gal-T into the secretory pathway of yeast cells, metabolically labeled enzyme was immunoprecipitated from extracts of yeast transformants, analysed by SDS/PAGE/fluorography and tested for sensitivity to treatment with endoglycosidase-H . Untreated recombinant gal-T had an apparent molecular mass of 48 kDa, which was reduced to 47 kDa after treatment, indicating that the recombinant enzyme was N-glycosylated and, therefore, competent for translocation across the membranes of the endoplasmic reticulum . Using specific gal-T assays employing N-acetylglucosamine or glucose in combination with alpha-lactalbumin as exogenous acceptor substrates, recombinant gal-T enzyme activity could readily be detected in crude homogenates . Analysis of the disaccharide products by 1H-NMR spectroscopy demonstrated that only beta-1-4 linkages were formed by the recombinant gal-T . The recombinant gal-T was detergent solubilized and subsequently purified by affinity chromatography on N-acetylglucosamine-derivatized Sepharose followed by alpha-lactalbumin-Sepharose . The purified enzyme preparation had a specific activity comparable to that of the soluble gal-T isolated from human milk . Furthermore, kinetic parameters determined for both acceptor and donor substrates of both enzymes differed only slightly . This work shows that yeast provides an appropriate host system for the heterologous expression of mammalian glycosyltransferases. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1252 - 6 Characterization of PDH beta 1, the structural gene for the pyruvate dehydrogenase beta subunit from Saccharomyces cerevisiae; Miran SG et al.; The gene encoding the pyruvate dehydrogenase (PDH) beta subunit (E1 beta) of the PDH complex from Saccharomyces cerevisiae has been cloned, sequenced, disrupted, and expressed . Two overlapping DNA fragments were generated from a yeast genomic DNA library by the polymerase chain reaction with synthetic oligonucleotide primers based on amino acid sequences of the yeast and human E1 beta subunits . The DNA fragments were subcloned and sequenced . The composite sequence has an open reading frame of 1098 nucleotides encoding a putative presequence of 33 amino acid residues and a mature protein of 333 residues with a calculated M(r) = 36,486 . Yeast and human E1 beta exhibit 62% sequence identity . The size of the mRNA is approximately 1.5 kilobases . Hybridization analysis showed that the E1 beta gene (PDH beta 1) is localized to chromosome II . Disruption of PDH beta 1 is not lethal under vegetative growth conditions . The null mutant transformed with PDH beta 1 on a unit-copy plasmid produced mature E1 beta and a functional PDH complex. J Biol Chem, 1993 Feb 15, 268(5), 3594 - 603 Repression and redirection of Saccharomyces cerevisiae tRNA synthesis from upstream of the transcriptional start site; Leveillard T et al.; Derivatives of the Saccharomyces cerevisiae SUP4 tRNATyr gene with binding sites for the transcription regulatory protein GCN4 located upstream of the transcriptional start site have been constructed . The effect of GCN4 on transcription of these genes by purified RNA polymerase III and transcription factors (TF) IIIB and IIIC has been analyzed . GCN4 effectively blocks initiation of transcription only when prebound to sites that overlap with the binding site of TFIIIB . Residual GCN4-repressed transcription is significantly redirected to nearby downstream sites, the selection of which depends on the location of bound GCN4 . That prebound repressing GCN4 redirects, instead of merely blocking, the TFIIIC-dependent interaction of TFIIIB with DNA has been directly demonstrated by footprinting . The effect of GCN4 on transcription persists after it has been stripped off its DNA-binding site: once it has been redirected, DNA-bound TFIIIB remains in place, a consequence of the fact that it binds extraordinarily tightly to DNA without recognizing specific DNA sequence. J Biol Chem, 1993 Feb 15, 268(5), 3456 - 62 Stimulation of calcium uptake in Saccharomyces cerevisiae by bovine protein kinase C alpha; Riedel H et al.; Ca2+ plays essential roles as a second messenger often in synergism with the calcium- and phospholipid-dependent phorbol ester receptor, protein kinase C (PKC), which stimulates Ca2+ influx in various cell types in a potential positive feedback mechanism . To address the compatibility of these mechanisms between lower eukaryotes and mammals, we have stably expressed bovine PKC alpha in the yeast Saccharomyces cerevisiae . We find that phorbol ester binding sites are created which stimulate a specific calcium- and phospholipid-dependent catalytic activity in vitro . Phorbol ester activation in vivo stimulates PKC down-regulation, uptake of extracellular Ca2+, Ca2+ dependence of cell viability, and changes in cell morphology . This may represent activation of a putative PKC-mediated signaling pathway utilized by functional yeast homologs of mammalian PKC isoforms . These are suggested by some protein data; however, their genes have not yet been characterized (Simon, A . J., Milner, Y., Saville, S . P., Dvir, A., Mochly-Rosen, D., and Orr, E . (1991) Proc . R . Soc . Lond . B 243, 165-171) . Our findings indicate that bovine PKC alpha is functional in yeast and stimulates calcium uptake in a manner similar to some of its responses in mammalian cells, which suggests compatible aspects of higher and lower eukaryotic signaling pathways and the feasibility of dissecting parts of the action of common signaling mediators in a simple genetic model. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1290 - 4 Futile cycles in Saccharomyces cerevisiae strains expressing the gluconeogenic enzymes during growth on glucose; Navas MA et al.; The systems which control the levels of the gluconeogenic enzymes in Saccharomyces cerevisiae have been bypassed to ascertain their physiological significance . The coding regions of the genes FBP1 and PCK1, which encode fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, have been put under the control of the promoter of ADC1 (alcohol dehydrogenase I), a gene not repressed by glucose, and introduced into yeast in multicopy plasmids . The transformed yeast cells show high levels of the gluconeogenic enzymes during growth on glucose . Generation time and growth yield of yeast expressing either fructose-1,6-bisphosphatase or phosphoenolpyruvate carboxykinase are not significantly different from those of the wild-type strain . For a strain expressing both enzymes the increase in generation time is about 20% and the decrease in growth yield around 30% . The concentration of ATP is about 1.5 mM in the growing cells of the different strains . The extent of in vivo cycling was measured by 13C NMR in cell-free extracts from yeast growing on {6-13C}glucose . Cycling between fructose-6-phosphate and fructose-1,6-bisphosphate is < 2%, most likely due to the very strong inhibition of fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate . Cycling between phosphoenolpyruvate and pyruvate is low, but a precise figure could not be obtained due to poor equilibration of label between carbons 2 and 3 of oxaloacetate. Gene, 1993 Feb 14, 124(1), 141 - 2 The sequence of Saccharomyces cerevisiae cloning vector pCS19 allowing direct selection for DNA inserts; Sengstag C; The complete nucleotide sequence of plasmid pCS19 has been determined . This centromeric Saccharomyces cerevisiae cloning vector was designed for the construction of gene libraries, since it allows direct selection for DNA inserts. Biochemistry, 1993 Feb 9, 32(5), 1294 - 301 Phosphomannose isomerase from Saccharomyces cerevisiae contains two inhibitory metal ion binding sites; Wells TN et al.; Phosphomannose isomerase (PMI) from Saccharomyces cerevisiae is a zinc-dependent metalloenzyme . Besides its role in catalysis, zinc is also a potent inhibitor of the enzyme . The inhibition is competitive with the substrate mannose 6-phosphate, with Kis = 6.4 microM in 50 mM Tris-HCl buffer, pH 8.0, at 37 degrees C . This inhibition constant is 4 orders of magnitude smaller than for group II divalent cations, indicating that the binding is not primarily electrostatic . Micromolar inhibition is also observed with ions of the other metals of the electronic configuration d10 . Under identical conditions, cadmium is a predominantly competitive inhibitor with Kis = 19.5 microM . Inhibition by mercury is predominantly competitive with Kis = 6.0 microM but shows a hyperbolic Dixon plot . Theorell-Yonetani double-inhibition analysis shows that zinc and cadmium ions are mutually exclusive inhibitors against mannose 6-phosphate . However, analysis of zinc and mercury double inhibition shows that they can simultaneously bind in the mannose 6-phosphate binding pocket, with only a small mutual repulsion . Inhibition of the enzyme by cadmium and zinc ions is strongly pH dependent with pKa = 9.2 for cadmium and one pKa at 6.6 and two at 8.9 for zinc . The inhibitory species are the monohydroxide forms, Zn(OH)+ and Cd(OH)+ . However, inhibition by mercury is relatively pH-independent, consistent with the neutral Hg(OH)2 being the inhibitory species . In all three cases, the metal ion binding causes a conformational change in the enzyme as judged by tryptophan fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1993 Feb 5, 268(4), 2451 - 7 The activity-controlling phosphorylation site is not the same in the four acidic ribosomal proteins from Saccharomyces cerevisiae; Naranda T et al.; By using site-directed mutagenesis and chemical analysis of phosphopeptides, a unique phosphorylation site has been shown at serine 73 in the amino acid sequence of the Saccharomyces cerevisiae acidic ribosomal protein YP1 beta (L44') . The mutation in this position prevents in vitro phosphorylation by protein kinases that modify the wild-type polypeptide . The unphosphorylatable mutated protein is unable to bind to the ribosomes and to rescue the growth deficiency of yeast strains in which the corresponding original gene is inactivated by gene disruption . Sequencing of tryptic phosphopeptides has shown that acidic proteins YP1 alpha and YP2 alpha (L44) are also phosphorylated at positions near the carboxyl end . These results contrast with the data indicating that in the highly homologous protein YP2 beta, phosphorylation takes place at serine 19, close to the amino terminus . The results show that phosphorylation is definitely required for the biological activity of these ribosomal proteins . However, the differences in the phosphorylation sites suggest that the effect of this modification is not the same in all of them, confirming the heterologous role of these peculiar ribosomal components . In fact, the different context of the modification sites in the four polypeptides suggests the existence of more than one protein kinase specific for this set of proteins. Biochem Mol Biol Int, 1993 Feb, 29(2), 375 - 85 Effect of anisotropic inhibitors of ATP synthesis in mitochondria of Saccharomyces cerevisiae . Comparison between a wild-type and a mutant strain altered in the stoichiometry of the MT DNA encoded proteolipids of ATP synthase; Manon S et al.; The effect of a group of non-usual inhibitors of ATP synthesis was investigated in yeast mitochondria . Tetraphenylphosphonium, tetraphenylarsonium and ethidium decreased the rate of ATP synthesis but did not decrease the ATP/O ratio . They did not inhibit the ATPase activity and the translocase, and did not decrease the proton-motive force . They inhibited phos