Showing papers in "Yeast in 1990"
TL;DR: A set of plasmids was constructed that contain the yeast selectable markers HIS3, LEU2, TRP1 or URA3 embedded in the multiple cloning site of pUC18.
Abstract: A set of plasmids was constructed that contain the yeast selectable markers HIS3, LEU2, TRP1 or URA3 embedded in the multiple cloning site of pUC18.
369 citations
TL;DR: The cell porosity of batch‐grown Saccharomyces cerevisiae was maximal in the early exponential phase and fell off rapidly to lower levels in later growth phases.
Abstract: The cell wall porosity of batch-grown Saccharomyces cerevisiae was maximal in the early exponential phase and fell off rapidly to lower levels in later growth phases. Treatment of stationary-phase cells with alpha-mannosidase restored wall porosity to the level of cells in early exponential phase. When cells in the early exponential phase were treated with alpha-mannosidase, or tunicamycin, an inhibitor of N-glycosylation, even higher porosities were obtained. Mutants with truncated mannan side-chains in their wall proteins also had very porous walls. The importance of the mannan side-chains for wall porosity was also seen during sexual induction. Treatment with alpha pheromone, which leads to the formation of wall proteins with shorter mannan side-chains, enhanced wall porosity. Disulphide bridges also affect cell wall porosity. They were predominantly found in the glucanase-soluble wall proteins. Because the main part of the mannan side-chains is also found in this family of wall proteins, our results demonstrate that the glucanase-soluble mannoproteins limit cell wall porosity in yeast.
244 citations
TL;DR: La structure molecule et l'assemblage de the toxine sont egalement etudies au systeme killer de Kluyveromyces lactis.
Abstract: Article de synthese consacre au systeme killer de Kluyveromyces lactis. La structure du plasmide lineaire codant pour la toxine est analysee ainsi que sa replication, sa recombinaison et l'expression des genes portes. La structure molecule et l'assemblage de la toxine sont egalement etudies
179 citations
TL;DR: It is found that the composition of the growth medium affected cell wall porosity in S. cerevisiae and the ratio between DEAE‐dextran‐ and poly‐L‐lysine‐induced cell leakage was used as a measure of cell wall Porosity.
Abstract: We have developed a new assay to determine relative cell wall porosity in yeasts, which is based on polycation-induced leakage of UV-absorbing compounds. Polycations with a small hydrodynamic radius as measured by gel filtration (poly-L-lysine) caused cell leakage independent of cell wall porosity whereas polycations with a large hydrodynamic radius (DEAE-dextrans) caused only limited cell leakage due to limited passage through the cell wall. This allowed the ratio between DEAE-dextran- and poly-L-lysine-induced cell leakage to be used as a measure of cell wall porosity in Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe. Using this assay, we found that the composition of the growth medium affected cell wall porosity in S. cerevisiae. In addition, we could show that cell wall porosity is limited by the number of disulphide bridges in the wall and is dependent on cell turgor. It is argued that earlier methods to estimate cell wall porosity in S. cerevisiae resulted in large underestimations.
145 citations
TL;DR: The amino acid sequence deduced from the gene shows extensive homology to a number of aspartyl proteases including the PEP4 and BARI gene products from S. cerevisiae and contains a C‐terminal serine/threonine‐rich sequence and potential transmembrane domain similar to those found in the KEX2 gene product.
Abstract: Mutants of Saccharomyces cerevisiae which lack the KEX2-encoded endopeptidase are unable to process proteolytically the mating factor alpha (MF alpha) propheromone produced from the chromosomal MF alpha 1 and MF alpha 2 genes (Julius et al., 1983). Overproduction of pheromone precursor from multiple, plasmid-borne MF alpha genes did, however, lead to the production of active MF alpha peptides in the absence of the KEX2 gene product. S. cerevisiae therefore must possess an alternative processing enzyme. The cleavage site of this enzyme appeared identical to that of the KEX2-encoded endopeptidase. To identify the gene responsible for the alternative processing, we have isolated clones which allowed production of mature MF alpha in a kex2-disrupted strain even from the chromosomal MF alpha genes. The gene isolated in this way was shown also to be essential for the KEX2-independent processing of propheromone overproduced from plasmid-borne MF alpha 1. The amino acid sequence deduced from the gene shows extensive homology to a number of aspartyl proteases including the PEP4 and BAR1 gene products from S. cerevisiae. In contrast to the BAR1 gene product, the novel aspartyl protease (YAP3 for Yeast Aspartyl Protease 3) contains a C-terminal serine/threonine-rich sequence and potential transmembrane domain similar to those found in the KEX2 gene product. The corresponding gene YAP3 was located to chromosome XII. The normal physiological role of the YAP3 gene product is not known. Strains disrupted in YAP3 are both viable and able to process the mating factor a precursor.
143 citations
TL;DR: A general procedure is described for determining the chromosomal constitution of industrial strains of Saccharomyces cerevisiae based on analysis of segregation frequencies for input markers among random spore progeny of industrial‐laboratory strain hybrids.
Abstract: A general procedure is described for determining the chromosomal constitution of industrial strains of Saccharomyces cerevisiae based on analysis of segregation frequencies for input markers among random spore progeny of industrial-laboratory strain hybrids. The multiply auxotrophic haploid testers used carried a dominant erythromycin-resistance marker, allowing hybrids to be selected in mass matings with spores produced by the wild-type industrial strains. Analysis of a number of independent crosses between the haploid testers and an unselected population of spores of each wine strain distinguished between disomic, trisomic and tetrasomic chromosomal complements in the parents. Possible explanations for a significant class of aberrant segregation frequencies are discussed. Results of the analysis indicate that UCD Enology 522 (Montrachet) is diploid and possibly trisomic for chromosome VII; 522X is diploid; UCD Enology 505 (California Champagne) is disomic for chromosome XVI, trisomic for chromosomes I, II, III, VI, VIII, IX, X, XII, XV, tetrasomic for chromosomes IV, XI, XIII, XIV and either trisomic or tetrasomic for chromosomes V and VII; and that UCD Enology 595 (Pasteur Champagne) is disomic for chromosomes I, II, III, IX, XVI, trisomic for chromosomes IV, VI, X, XII, XIV, XV, tetrasomic for chromosomes V, VIII, XI, XIII and either disomic or tetrasomic for chromosome VII.
135 citations
TL;DR: In this paper, a collection of 260 methanolutilization-defective Hansenula polymorpha strains was isolated and screened for the ability to utilize a second compound, ethanol, the metabolism of which involves peroxisomes.
Abstract: As a first step in a genetic approach towards understanding peroxisome biogenesis and function, we have sought to isolate mutants of the methylotrophic yeast Hansenula polymorpha which are deficient in peroxisomes. A collection of 260 methanol-utilization-defective strains was isolated and screened for the ability to utilize a second compound, ethanol, the metabolism of which involves peroxisomes. Electron microscopical investigations of ultrathin sections of selected pleiotropic mutants revealed two strains which were completely devoid of peroxisomes. In both, different peroxisomal matrix enzymes were active but located in the cytosol; these included catalase, alcohol oxidase, malate synthase and isocitrate lyase.
Subsequent backcrossing experiments revealed that for all crosses involving both strains, the methanol- and ethanol utilizing-deficient phenotypes segregated independently of each other, indicating that different gene mutations were responsible for these phenotypes. The phenotype of the backcrossed peroxisome-deficient derivates was identical: defective in the ability to utilize methanol but capable of growth on other carbon sources, including ethanol.
The mutations complemented and therefore were recessive mutations in different genes.
122 citations
TL;DR: The DNA sequence of a segment of chromosome III extending over 8·2 kb, determined using the random clone strategy followed by oligonucleotide‐directed sequencing, endodes a ribokinase, a new function for yeast.
Abstract: We report here the DNA sequence of a segment of chromosome III extending over 8·2 kb. The sequence was determined using the random clone strategy followed by oligonucleotide-directed sequencing. The segments contains five long open reading frames, YCR521, 522, 523, 524 and 526, with only short distances between them. YCR523 (333 codons) endodes a ribokinase, a new function for yeast. YCR526 originates inside the MAT cassette, which is in continuity with the present segment, and extends over 358 codons outside of MAT. YCR524 (923 codons) codes for a putative membrane protein. YCR521, 522 and 524, have each been disrupted by insertion of a URA3 cassette and are non-essential genes. An active ARS element is located within YCR523 or its vicinity.
95 citations
TL;DR: An ordered clone bank is constructed that covers almost the whole of chromosome III with a single gap of several kilobases in length and it is found that the genes located near both termini are expressed only at low levels and that highly expressed genes are rather scattered over the chromosome.
Abstract: Using λ phage vector EMBL4, we isolated 344 clones containing segments of chromosome III of Saccharomyces cerevisiae, analysed their physical structure with eight restriction enzymes and sorted the data in contiguous groups with computer programmes. Furthermore, we performed Southern hybridizations between the sorted contiguous clone groups and interrelated them into larger groups. In this way, we constructed an ordered clone bank that covers almost the whole of chromosome III with a single gap of several kilobases in length. The consensus physical map thus obtained totals 334·6 kb, which is in good agreement with the size of this chromosome estimated by pulsed-field gel electrophoresis. Southern hybridization analysis with the DNA probes containing telomere-specific sequences showed that the bank contained a telomere at a position corresponding to the right arm terminus of chromosome III. Also, five Ty elements were found to be present. To estimate the number of genes on this chromosome and to analyse their levels of expression, we performed a series of Northern hybridization experiments using total poly(A)+ RNA from vegetatively growing cells and appropriate restriction enzyme fragments from the bank. Thus, we identified a total of 156 transcripts on chromosome III, indicating, on an average, one gene in every 2 kb on this chromosome. The transcripts were visually categorized into five groups according to their apparent levels of expression. It was found that the genes located near both termini are expressed only at low levels and that highly expressed genes are rather scattered over the chromosome.
87 citations
TL;DR: Evidence is presented here that the two gradients are linked through a sodium‐glycerol symport that uses the sodium gradient as a driving force for maintaining the glycerol gradient, and that the protonophore carbonylcyanide‐M‐chlorophenylhydrazone induced collapse of the glucose gradient, supporting earlier proposals by others that the Sodium gradient is maintained by an active sodium‐proton exchange mechanism.
Abstract: Several authors have shown that the halotolerant yeast Debaryomyces hansenii, when growing exponentially in glucose medium in the presence of sodium chloride, maintains osmotic balance by establishing sodium and glycerol gradients of opposite signs across the plasma membrane. Evidence is presented here that the two gradients are linked through a sodium-glycerol symport that uses the sodium gradient as a driving force for maintaining the glycerol gradient. The symporter also accepts potassium ions as co-substrate. The kinetic parameters at 25°C, pH 5·0 were the following: Vmax, decreasing from over 500 to less than 40 μmol g−1 per h over a concentration range of 0–3 M extracellular sodium chloride; Km (glycerol) 0·40–0·6 mM over the same range; Km (sodium ions) 16·0 ± 3·21μM; Km, (potassium ions) 10·4 ± 3·6μM. Furthermore, it was observed that glycerol uptake was accompanied by proton uptake when extracellular sodium chloride was present and that the protonophore carbonylcyanide-M-chlorophenylhydrazone induced collapse of the glycerol gradient, supporting earlier proposals by others that the sodium gradient is maintained by an active sodium-proton exchange mechanism.
71 citations
TL;DR: ADH activities resolved by gel electrophoresis revealed several ADH isozymes which are differently expressed in K. lactis cells depending on the carbon source.
Abstract: We have studied the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis. Southern hybridization to the Saccharomyces cerevisiae ADH2 gene indicates four probable structural ADH genes in K. lactis. Two of these genes have been isolated from a genomic bank by hybridization to ADH2. The nucleotide sequence of one of these genes shows 80% and 50% sequence identity to the ADH genes of S. cerevisiae and Schizosaccharomyces pombe respectively. One K. lactis ADH gene is preferentially expressed in glucose-grown cells and, in analogy to S. cerevisiae, was named K1ADH1. The other gene, homologous to K1ADH1 in sequence, shows an amino-terminal extension which displays all of the characteristics of a mitochondrial targeting presequence. We named this gene K1ADH3. The two genes have been localized on different chromosomes by Southern hybridization to an orthogonal-field-alternation gel electrophoresis-resolved K. lactis genome. ADH activities resolved by gel electrophoresis revealed several ADH isozymes which are differently expressed in K. lactis cells depending on the carbon source.
TL;DR: The aim of this paper is to provide an overview of the transcription regulators described in yeast, and the structural and functional characteristics of the proteins are given first.
Abstract: The aim of this paper is to provide an overview of the transcription regulators described in yeast. Forty-three factors are listed in alphabetical order. The structural and functional characteristics of the proteins are given first. This includes the consensus DNA-binding sequence, the effect on transcription and, if relevant, particular structures, other names for the protein and names of homologous proteins in mammalian and yeast cells
Journal Article•
TL;DR: In this article, the peroxisomal matrix (internal compartment) is composed almost exclusively of two proteins, alcohol oxidase and dihydroxyacetone synthase; catalase is present in much lower abundance.
Abstract: Peroxisomes are massively induced when methylotrophic yeasts are cultured in medium containing methanol. These organelles contain enzymes that catalyze the initial steps of methanol assimilation. In Candida boidinii, a methylotrophic yeast, the peroxisomal matrix (internal compartment) is composed almost exclusively of two proteins, alcohol oxidase and dihydroxyacetone synthase; catalase is present in much lower abundance. Monoclonal and polyclonal antibodies are available against peroxisomal matrix and membrane proteins. These were utilized to correlate the induction of specific proteins with the morphological changes occurring during peroxisomal proliferation. Cells cultured in glucose-containing medium contain two to five small microbodies, which are identifiable by catalase staining and immunoreactivity with a monoclonal antibody against PMP47, an integral peroxisomal membrane protein. Three stages of proliferation can be distinguished when cells are switched to methanol as the carbon source. (1) There is an early stage (within 1 h) in which several peroxisomes develop from a preexisting organelle. This is accompanied by an increase in catalase activity and an induction of PMP47, but no detectable induction of alcohol oxidase or dihydroxyacetone synthase is observed. (2) From 1 to 2.5 h there is further division of these microbodies until up to 30 small peroxisomes generally are present in each of one or two clusters per cell. Induction of alcohol oxidase, dihydroxyacetone synthase and PMP20, a protein that is distributed in the matrix and membrane, is detectable during this time. Serial sections reveal that some peroxisomes remain uninduced while others undergo proliferation. Such sections also show no obvious connections between peroxisomes within clusters. (3) After 2.5 h there is a decrease in the number of peroxisomes per cell (caused at least in part by the movement of organelles into buds) but an increase in volume per peroxisome, until a steady state is reached by 5-10 h.
TL;DR: SUP2(SUP35) is an omnipotent suppressor gene, coding for an EF‐1α‐like protein factor, intimately involved in the control of translational accuracy in yeast Saccharomyces cerevisiae.
Abstract: SUP2 (SUP35) is an omnipotent suppressor gene, coding for an EF-1 alpha-like protein factor, intimately involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. In the present study a SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of the temperature-sensitive sup2 mutation of S. cerevisiae. The nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82.4 kDa, exceeding the Sup2 protein of S. cerevisiae by 6 kDa. Like the SUP2 gene product of S. cerevisiae, the Sup2 protein of P. pinus represents a fusion of a unique N-terminal part and a region homologous to EF-1 alpha. The comparison of amino acid sequences of the Sup2 proteins reveals high conservation (76%) of the C-terminal region and low conservation (36%) of the N-terminal part where, in addition, the homologous correspondence is ambiguous. Proteins related to the Sup2 of S. cerevisiae were found in P. pinus and some other yeast species by the immunoblotting technique. The relation between the evolutionary conservation of different regions of the Sup2 protein and their functional significance is discussed.
TL;DR: Surprisingly, it was observed that those strains devoid of one or both TPK1 alleles were defective in glycogen storage, while strains lacking Tpk2 accumulated higher levels of the polysaccharide, indicating that T pk1 and Tk2 have opposite roles in carbohydrate metabolism.
Abstract: Fil: Giacometti, Romina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Invetigaciones Bioquimicas y Fisiologicas; Argentina
TL;DR: The effect on n‐alcohols on ATP‐dependent generation of ΔpH and Em across the plasma membrane vesicles of the yeast Saccharomyces carlsbergenesis was investigated and Mg2+ partially prevented the increase in the plasmalemma ion permeability by the alcohols investigated.
Abstract: The effect of n-alcohols on ATP-dependent generation of delta pH and Em across the plasma membrane vesicles of the yeast Saccharomyces carlsbergensis was investigated. The alcohols were shown to collapse delta pH and Em in the order C2 less than C3 less than C4 less than C5 less than or equal to C6 greater than or equal to C7 greater than C8 greater than C11, the dissipation of Em being more pronounced. Inhibition of the plasmalemma H(+)-ATPase was insignificant; at low alcohol concentrations its activity even increased. The basic reason for the toxic effect of the alcohols on the yeast cells was suggested to be due to the increase in the anion and proton permeability of the plasma membrane. Mg2+ partially prevented the increase in the plasmalemma ion permeability by the alcohols investigated.
TL;DR: When Saccharomyces cerevisiae CBS 8066 was grown under maltose limitation, two enzymes specific for maltose utilization were present: a maltose carrier, and the maltose‐hydrolysing α‐glucosidase.
Abstract: When Saccharomyces cerevisiae CBS 8066 was grown under maltose limitation, two enzymes specific for maltose utilization were present: a maltose carrier, and the maltose-hydrolysing α-glucosidase. The role of these two enzymes in the physiology of S. cerevisiae was investigated in a comparative study in which Candida utilis CBS 621 was used as a reference organism.
Maltose pulses to a maltose-limited chemostat culture of S. cerevisiae resulted in ‘substrate-accelerated death’. This was evident from: (1) enhanced protein release from cells: (2) excretion of glucose into the medium; (3) decreased viability. These effects were specific with respect to both substrate and organism: pulses of glucose to maltose-limited cultures of S. cerevisiae did not result in cell death, neither did maltose pulses to maltose-limited cultures of C. utilis. The maltose-accelerated death of S. cerevisiae is most likely explained in terms of an uncontrolled uptake of maltose into the cell, resulting in an osmotic burst. Our results also provide evidence that the aerobic alcoholic fermentation that occurs after pulsing sugars to sugar-limited cultures of S. cerevisiae (short-term Crabtree effect) cannot solely be explained in terms of the mechanism of sugar transport. Both glucose and maltose pulses to maltose-limited cultures triggered aerobic alcohol formation. However, glucose transport by S. cerevisiae occurs via facilitated diffusion, whereas maltose entry into this yeast is mediated by a maltose/proton symport system.
TL;DR: Evidence is presented, based on sequence comparisons and secondary structure prediction, of the presence of a DNA‐binding and dimerization helix‐loop‐helix motif in the yeast transcription activator PHO4.
Abstract: Evidence is presented, based on sequence comparisons and secondary structure prediction, of the presence of a DNA-binding and dimerization helix-loop-helix motif in the yeast transcription activator PHO4. Interest in the existence of this first known motif in yeast is discussed.
TL;DR: During the cell division cycle of the budding yeast Saccharomyces cerevisiae there are stage‐specific fluctuations in the intracellular concentration of adenosine 3′,5′‐cyclic monophosphate (cAMP).
Abstract: Using the technique of centrifugal elutriation it was demonstrated that during the cell division cycle of the budding yeast Saccharomyces cerevisiae there are stage-specific fluctuations in the intracellular concentration of adenosine 3',5'-cyclic monophosphate (cAMP). Results shown here indicate that the intracellular concentration of cAMP is at its highest during the division cycle, and at its lowest immediately prior to and just after cell separation. Results also show the extrusion of extracellular cAMP into the medium by Saccharomyces cerevisiae, extracellular cAMP levels being ten to one hundred times higher than intracellular levels. During the cell cycle of Saccharomyces cerevisiae the extracellular level of cAMP does not fluctuate.
TL;DR: Growth tests were defined that allow recognition of all Zygosaccharomyces species and Candida mogii, the proposed anamorph of Z. rouxii, showed low relatedness to all nine species.
Abstract: The extent of nuclear DNA complementarity was determined for members of the genus Zygosaccharomyces. From these comparisons, nine species have been identified: Z. baillii, Z. bisporus, Z. cidri, Z. fermentati, Z. florentinus, Z. mellis, Z. microellipsoides, Z. mrakii, and Z. riuxii. Candida mogii, the proposed anamorph of Z. rouxii, showed low relatedness to all nine species. The recently described Saccharomyces astigiensis and S. albasitensis were conspecific with Z. fermentati, and S. placentae showed high relatedness with Z. rouxii. Growth tests were defined that allow recognition of all Zygosaccharomyces species.
TL;DR: The taxonomic status of various species of Dekkera, Brettanomyces and Eeniella was examined by electrophoretic comparison of enzymes, by deoxyribonucleic acid homology and by physiological characterization to demonstrate that two teleomorphic DekkerA species, D. anomala and D. bruxellensis, can be recognized.
Abstract: The taxonomic status of various species of Dekkera, Brettanomyces and Eeniella was examined by electrophoretic comparison of enzymes, by deoxyribonucleic acid homology and by physiological characterization. These studies demonstrated that two teleomorphic Dekkera species, D. anomala and D. bruxellensis (synonym D. intermedia), and four anamorphic Brettanomyces species, B. anomalus (synonym B. claussenii), B. bruxellensis (synonym B. abstinens, B. custersii, B. intermedius, B. lambicus), B. custersianus and B. naardenensis, can be recognized. The anamorphic genus Eeniella remained as a separate, monotypic taxon.
TL;DR: The cdc28‐srm mutation in Saccheromyces cerevisiae decreases spontaneous and induced mitochondrial rhomutability and the mitotic stability of native chromosomes and recombinant circular minichromosomes.
Abstract: The cdc28-srm mutation in Saccharomyces cerevisiae decreases spontaneous and induced mitochondrial rho- mutability and the mitotic stability of native chromosomes and recombinant circular minichromosomes. The effects of cdc28-srm on the genetic stability of cells support the hypothesis that links cell cycle regulation in yeast to changes in chromatin organization dependent on the start gene CDC28 (Hayles and Nurse, 1986).
TL;DR: The substructure and polypeptide composition of the peroxisomal membranes in two methylotrophic yeasts in relation to different growth conditions are obtained and no significant ultrastructural differences existed between the membranes of variously grown cells.
Abstract: We have studied the substructure and polypeptide composition of the peroxisomal membranes in two methylotrophic yeasts in relation to different growth conditions. The results obtained indicated that no significant ultrastructural differences existed between the membranes of variously grown cells. The presence of specific peroxisomal membrane proteins (PMPs) was studied biochemically. On sodium dodecyl sulphate-polyacrylamide gels of purified microbody membranes isolated from methanol-grown Hansenula polymorpha, prominent protein bands were observed at 22, 31, 35, 42, 49 and 51 kD. These proteins were also present when the cells were grown in media containing ethanol and/or ethylamine. Apart from these, several other PMPs were specifically induced under these conditions, namely 24, 29, 37 and 62 kD proteins. The polypeptide composition of peroxisomal membranes from H. polymorpha was compared with that of another methylotroph, Candida biodinii. In the latter organism a specific PMP with a molecular weight of 23 kD was induced during growth on D-alanine instead of ammonium sulphate as the nitrogen source.
TL;DR: The MAS gene, encoding the microbody matrix enzyme malate synthase (EC 4.3.2.) from the methylotrophic yeast Hensenula polymorphia, is cloned and the homology of all four proteins is established.
Abstract: We have cloned the MAS gene, encoding the microbody matrix enzyme malate synthase (EC 4132) from the methylotrophic yeast Hansenula polymorpha The gene was isolated by screening of a genomic library with a mixed-sequence probe, based on the partial amino acid sequence of the purified enzyme The nucleotide sequence of a 24-kilobase stretch of DNA covering the MAS gene was determined The gene contains an open reading frame of 555 amino acids, amounting to a calculated molecular mass of 63,254 for the encoded protein Comparison of the amino acid sequence with the malate synthase sequences of Escherichia coli, Brassica napus L and Cucumis sativus L clearly establishes the homology of all four proteins Compared to the soluble enzyme from E coli, the malate synthases from H polymorpha and both plant species, which are located in the microbodies, have a short carboxy-terminal extension In the plant malate synthases, the extension is probably involved in routing to the microbodies, since it contains the potential peroxisomal targeting signal, Ser-Arg/Lys-Leu, at the carboxy terminus The H polymorpha enzyme terminates with similar amino acids, but their sequence, Ser-Leu-Lys, does not conform to any of the known peroxisomal targeting signals
TL;DR: To determine the effect of nucleotide composition of the 5′‐untranslated (leader) region on the translational efficiency of mRNA in yeast, a large part of the leader region of the phosphoglycerate kinase (PGK) gene was replaced by various deoxyoligonucleotides of defined sequence.
Abstract: In order to determine the effect of the nucleotide composition of the 5'-untranslated (leader) region on the translational efficiency of mRNA in yeast, we replaced a large part of the leader region of the phosphoglycerate kinase (PGK) gene by various deoxyoligonucleotides of defined sequence. All mutations left the context of the transcription initiation site and AUG start codon intact. The mutant genes were introduced into yeast cells on a multicopy vector and the ratio of the steady-state levels of PGK mRNA and protein were determined. We found translational efficiency to be unaffected by the presence of either an 18 nucleotides (nt) long polyA or polyC tract or by sequences consisting of mixtures of A and C residues in any proportion. In contrast, a poly U tract, as well as mixtures of U and C residues, reduced translational efficiency by a factor of two to three, presumably by long-range base-pairing between the leader and sequences elsewhere in the coding or 3'-non-coding regions of the messenger. In agreement with this hypothesis, a five-fold reduction in translational efficiency was found for an mRNA carrying a polyC tract in the leader as well as a polyG tract in the trailer, neither of which had any effect on translational efficiency by itself. Therefore, we conclude that the leader and trailer regions (including the polyA tail) of PGK mRNA are sufficiently close to base-pair when containing complementary sequences. The resulting secondary structure evidently constitutes a barrier for incoming 40S subunits on their way to the AUG start codon. The presence of an 18 nt long polyG tract in the leader completely abolished translation of the PGK mRNA in accordance with earlier observations. However, we found that leaders containing up to 40% G residues interspersed with either A or U, still allow highly efficient translation. This value is about four times as high as the average G content of leader sequences in naturally occurring yeast mRNAs. Finally, neither deletion of about 40% of the trailer sequence of PGK mRNA, nor replacement of this sequence by homopolymer tracts had any effect on translational efficiency.
TL;DR: Electron microscopy indicated that, dependent on the stage of growth, the enzymes may form large protein aggregates.
Abstract: We have studied growth of two peroxisome-deficient mutant strains of Hansenula polymorpha on glucose in the presence of different organic nitrogen sources (methylamine, ethylamine and D-alanine), the metabolism of which is mediated by peroxisome-borne oxidases in wild-type (WT) cells. Both strains grew well on each of these substrates with growth rates comparable to WT cells. Growth on both methylamine and ethylamine was associated with enhanced levels of catalase and amine oxidase in the cells; in D-alanine-grown cells D-amino acid oxidase activity and increased. In WT cells of H-polymorpha the activities of these enzymes were confined to the peroxisomal matrix; however, in both peroxisome-deficient strains their activities were localized in the cytosol. Electron microscopy indicated that, dependent on the stage of growth, the enzymes may form large protein aggregates.
The molecular masses of both amine oxidase and D-amino acid oxidase in the mutant strains were identical to their respective counterparts in WT cells, indicating that both proteins were correctly assembled and active in the cytosol.
TL;DR: During the purification process an enhancement in total δ‐aminolevulinate dehydratase activity suggested the possibility that removal of an inhibitor of the enzyme could be occurring, and the yeast enzyme was purified 193‐fold to near‐homogeneity.
Abstract: Saccharomyces cerevisiae transformed with a multicopy plasmid carrying the yeast structural gene HEM2, which codes for delta-aminolevulinate dehydratase, was enriched 20-fold in the enzyme. Beginning with cell-free extracts of transformed cells, the dehydratase was purified 193-fold to near-homogeneity. This represents a 3900-fold purification relative to the enzyme activity in normal, untransformed yeast cells. The specific activity of the purified enzyme was 16.2 mumol h-1 per mg protein at pH 9.4 and 37.5 degrees C. In most respects the yeast enzyme resembles mammalian enzymes. It is a homo-octamer with an apparent Mr of 275,000, as determined by centrifugation in glycerol density gradients, and under denaturing conditions behaved as a single subunit of Mr congruent to 37,000. The enzyme requires reduced thiol compounds to maintain full activity, and maximum activity was obtained in the presence of 1.0 mM-Zn2+. It is sensitive to inhibition by the heavy metal ions Pb2+ and Cu2+. The enzyme exhibits Michaelis-Menten kinetics and has an apparent Km of 0.359 mM. Like dehydratases from animal tissues, the yeast enzyme is rather thermostable. During the purification process an enhancement in total delta-aminolevulinate dehydratase activity suggested the possibility that removal of an inhibitor of the enzyme could be occurring.
TL;DR: It is shown that Sdo1p interacts with nucleolar rRNA processing factors and ribosomal proteins, indicating that it might bind the pre‐60S complex and remain associated with it during processing and transport to the cytoplasm.
Abstract: The Shwachman–Bodian–Diamond syndrome protein (SBDS) is a member of a highly conserved protein family of not well understood function, with putative orthologues found in different organisms ranging from Archaea, yeast and plants to vertebrate animals. The yeast orthologue of SBDS, Sdo1p, has been previously identified in association with the 60S ribosomal subunit and is proposed to participate in ribosomal recycling. Here we show that Sdo1p interacts with nucleolar rRNA processing factors and ribosomal proteins, indicating that it might bind the pre-60S complex and remain associated with it during processing and transport to the cytoplasm. Corroborating the protein interaction data, Sdo1p localizes to the nucleus and cytoplasm and co-immunoprecipitates precursors of 60S and 40S subunits, as well as the mature rRNAs. Sdo1p binds RNA directly, suggesting that it may associate with the ribosomal subunits also through RNA interaction. Copyright © 2009 John Wiley & Sons, Ltd.
TL;DR: It was found that both sequences are required for maximum expression of APase activity by using synthetic oligonucleotides, which are likely to act as USAs in Saccharomyces cerevisiae.
Abstract: The expression of acid phosphatase (APase) from PHO5 and MF alpha-PHO5 hybrid genes is regulated by inorganic phosphate and mating type locus respectively, as well as the PHO4 and MAT alpha 1 gene products respectively. When PHO5 and MF alpha-PHO5 hybrid genes were cloned in the BamHI site of the pBR322 sequence of the yeast shuttle vectors (YRp7 or YEp9T), in one orientation they were regulated normally but in the other orientation their expression was not regulated but expressed constitutively. The pBR322 sequences present upstream of the inserted genes are responsible for the constitutive expression. By replacing the PHO5 upstream activating sequences (UAS) element with pBR322 fragments, we have identified three pBR322 sequences, from nucleotides 376 to 650, 2068 to 2116 and 2136 to 2247, which were able to promote expression of APase. A comparison of these three pBR322 fragments revealed 5' ATCGCGCGAG 3' and 5' CGGTGATGNCGG 3' to be the common sequences likely to act as UASs in Saccharomyces cerevisiae. By using synthetic oligonucleotides, it was found that both sequences are required for maximum expression of APase activity.
TL;DR: The hydrophilic, highly acidic N‐terminus of PSS is required for efficient membrane insertion but does not appear to contain sequences required for a targeting to the membrane compartment.
Abstract: The product of the yeast CHO 1 gene, phosphatidylserine synthase (PSS), is an integral membrane protein that catalyses a central step in cellular phospholipid biosynthesis. A 1.2 kb fragment containing the regulatory and structural components of the CHO 1 gene was sequenced. Transcription initiation in wild-type cells was found to occur between -1 and -15 relative to the first ATG of a large open reading frame capable of encoding a 30,804 molecular weight protein. This translation initiation site was active in vivo and in vitro in a heterologous system. In both cases it supported production of a protein of approximately 30,000 molecular weight. A second potential translation initiation site was detected 225 or 228 bases downstream from the first ATG. This second site was active in vitro where it supported production of a protein of 22,400 molecular weight. A subclone, lacking the 5' regulatory region and the sequence encoding the first 12 amino acids of the large open reading frame, allowed translation in vivo starting at the second ATG. The resulting protein was 22,000 molecular weight, lacked the 74 N-terminal amino acids and was capable of complementing the choline auxotrophy of a cho 1 null-mutant. In transformants carrying this construct, PSS activity and 22 kDa protein was found to be associated with membrane fractions corresponding to mitochondria and endoplasmic reticulum. However, most of the truncated PSS protein accumulated in the cytosol in an inactive form. A hybrid-protein containing the 63 N-terminal amino acids of PSS fused to mouse dihydrofolate reductase was found exclusively in the cytosol when expressed in wild-type yeast. Thus, the hydrophilic, highly acidic N-terminus of PSS is required for efficient membrane insertion but does not appear to contain sequences required for a targeting to the membrane compartment.