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D H Wolf

Publications and source records attributed to D H Wolf.

At least 91 records · Page 5Linked to original sources
PubMed1992

Purification and characterization of proteinase yscJ, a new yeast peptidase.

A newly recognized peptidase, designated proteinase yscJ, was purified from the yeast Saccharomyces cerevisiae. The enzyme is of non-vacuolar origin and cleaves the Tyr-Lys bond of the synthetic peptide substrate Cbz-Tyr-Lys-Arg-NH-Ph (Cbz, benzyloxycarbonyl; NH-Ph, 4-nitroanilide) and the Glu-Lys bond of the substrate Boc-Glu-Lys-Lys-NH-Mec (Boc, butoxycarbonyl; Mec, 4-methylcoumarinyl) with high efficiency. Optimum pH for cleavage of Cbz-Tyr-Lys-Arg-NH-Ph is in the range 7.0-7.5. The purified enzyme has a molecular mass of approximately 58 kDa, as judged by gel filtration on a Superose 12 FPLC column. Mercury compounds and EDTA were found to be potent inhibitors of proteinase yscJ activity.

Amino Acid Sequence↗
PubMed1992

Differential proteolytic sensitivity of yeast fatty acid synthetase subunits alpha and beta contributing to a balanced ratio of both fatty acid synthetase components.

The Saccharomyces cerevisiae genes FAS1 and FAS2 encoding the beta and alpha subunit of yeast fatty acid synthetase (FAS), respectively, were individually deleted by one-step gene disruption. Northern blot analysis of RNA from the resulting fas null allele mutants indicated that deletion of FAS2 did not influence the transcription of FAS1, while FAS2 transcription was significantly reduced in the delta fas1 strain. These data suggest an activating role of subunit beta on FAS2 gene expression or, alternatively, a repression of FAS2 by an excess of its own gene product. Compared to the intact alpha 6 beta 6 complex, the individual FAS subunits synthesized in the delta fas1 or delta fas2 strains exhibit a considerably increased sensitivity towards the proteinases present in the yeast cell homogenate. Using yeast mutants specifically defective in the vacuolar proteinases yscA (PRA1/ PEP4 gene product) and/or yscB (PRB1 gene product), it was shown that in vitro, subunit alpha is efficiently degraded by proteinase yscA while for degradation of subunit beta, the combined action of proteinases yscA and yscB is necessary. In vivo, besides the vacuolar proteinases, an additional proteolytic activity specifically affecting free FAS subunit alpha becomes increasingly apparent in cells entering the stationary growth phase. In contrast, under similar conditions uncomplexed FAS subunit beta is stable in strains lacking the vacuolar proteinases yscA and yscB. The reduced FAS subunit levels, at the stationary phase, were independent of the corresponding FAS transcript concentrations. Thus, differential degradation pathways are obviously removing an excess of either FAS subunit, at least under starvation conditions. A combination of both regulation of FAS gene expression and proteolysis of free FAS polypeptides may therefore explain the equimolar amounts of both FAS subunits observed in yeast wild-type cells.

Blotting, Northern↗
PubMed1992

Biogenesis of the yeast vacuole (lysosome). Mutation in the active site of the vacuolar serine proteinase yscB abolishes proteolytic maturation of its 73-kDa precursor to the 41.5-kDa pro-enzyme and a newly detected 41-kDa peptide.

The codon of the catalytic serine in the active site of the vacuolar serine proteinase yscB (PrB) was changed to alanine, yielding the mutant gene prb1-Ala519. Following replacement of the wild-type PRB1 allele with prb1-Ala519, only a 73-kDa molecule was detected by immunoprecipitation with PrB-specific antiserum. The size of the mutant molecule corresponds to the unprocessed cytoplasmic precursor (pre-super-pro-PrB), as detected in sec61 mutants, when translocation into the endoplasmic reticulum is blocked. However, the mutant molecule is completely translocated into the secretory pathway, as indicated by protection from proteinase K digestion in spheroplast lysates in the absence of detergent. When N-glycosylation was inhibited in prb1-Ala519 mutant cells by tunicamycin, a smaller molecule of about 71 kDa appeared consistent with single N-glycosylation and signal-sequence cleavage of the translocated mutant PrB molecule in the endoplasmic reticulum. Thus, the active-site mutation prevents the wild-type processing of the N-glycosylated 73-kDa precursor of PrB to the 41.5 kDa pro-PrB in the endoplasmic reticulum. In order to characterize the processing of wild-type super-pro-PrB in more detail, we generated antibodies against the non-enzymatic superpeptide domain of the 73-kDa precursor expressed in Escherichia coli. We find that, in addition to pro-PrB, a distinct protein (superpeptide) with a mobility of about 41 kDa in SDS/PAGE is generated in the endoplasmic reticulum. Pulse-chase experiments indicate rapid degradation of the 41-kDa superpeptide in wild-type cells. Correspondingly, the superpeptide was virtually undetectable by immunoblotting wild-type cell extracts. In contrast, no degradation of radioactively labeled 41-kDa superpeptide was observed within 60 min in mutant strains deficient in the vacuolar proteinase yscA (PrA), in which maturation of vacuolar pro-PrB to active PrB is blocked. Accordingly, superpeptide antigenic material was readily detected by immunoblotting cell extracts and enriched in vacuolar preparations of PrA deficient mutant cells. These results indicate that the superpeptide and pro-PrB travel to the vacuole, where the superpeptide is rapidly degraded upon pro-PrB activation to PrB. Using purified vacuoles, rapid degradation of the superpeptide was reconstituted in vitro by addition of either mature PrA or mature PrB. However, the PrA-triggered in vitro degradation of the superpeptide required PrB activity, as this process was inhibited in the presence of the PrB inhibitor chymostatin.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1992

Stress-induced proteolysis in yeast.

Survival of cells in their natural environment is crucially dependent on their ability to adapt to constantly occurring changes. The ability of cells to respond to extremes of environmental influences is vital to survival. Proteolysis is a central cellular tool in stress response. Proteins of pathways necessary for normal growth, but harmful under stress conditions, as well as proteins damaged by stress have to be eliminated. The yeast Saccharomyces cerevisiae, a model eukaryote, has evolved two different proteolytic systems: (i) a membrane-enveloped, vacuolar (lysosomal) system, which contains a variety of non-specific peptidases and (ii) highly specific peptidases residing at different cellular locations. The best characterized peptidase of the specific system is proteinase yscE, the proteasome equivalent found in all eukaryotic cells. Both the vacuolar and the non-vacuolar systems are vital components of the stress response in yeast.

Adaptation, Physiological↗
PubMed1991

Biogenesis of the yeast vacuole (lysosome). Active site mutation in the vacuolar aspartate proteinase yscA blocks maturation of vacuolar proteinases.

The activation process of vacuolar (lysosomal) proteinases in the yeast Saccharomyces cerevisiae is initiated by the PRA1 (PEP4) gene product, proteinase yscA. To elucidate the activation process of proteinase yscA the catalytically active amino acid Asp294 of the enzyme was exchanged with Ala294 using site directed mutagenesis. The resulting proteinase yscA-Ala294 showed no proteolytic activity against the substrate hemoglobin. The mutant protein did not undergo processing in vivo. This phenomenon is in good agreement with the hypothesis that maturation of proteinase yscA is due to self-processing of pro-proteinase yscA. Furthermore, proteinase yscA-Ala294 is not able to convert the zymogens pro-proteinase yscB and pro-carboxypeptidase yscY to the mature enzymes.

Amino Acid Sequence↗
PubMed1991

The proteinase yscA-inhibitor, IA3, gene. Studies of cytoplasmic proteinase inhibitor deficiency on yeast physiology.

The gene of the proteinase yscA inhibitor IA3, PAI3, of the yeast Saccharomyces cerevisiae was isolated by oligonucleotide screening of a genomic DNA library and sequenced. The gene codes for a single protein of 68 amino acids. The structural PAI3 gene was deleted in vitro by oligonucleotide-site-directed mutagenesis. The mutated allele was introduced via homologous recombination into the genome of wild-type yeast and into the genome of a yeast mutant, which lacks the second cytoplasmic proteinase-inhibitor, IB2. The deficiency of either or of both inhibitors has no effect on the cell viability under various physiological conditions. The inhibitor mutants, however, show an increase in the general in vivo protein degradation rate. The IA3 mutant has a 2-3-fold increased protein degradation rate in the first 6 h after a shift from rich medium onto starvation-medium, whereas the IB2 mutant shows a constantly increased degradation rate of 20-50% under the same conditions. The inhibitor double null mutant has the same protein degradation rate as the IA3 null mutant. These results suggest an in vivo interaction between the vacuolor endopeptidases and their cytoplasmic inhibitors.

Amino Acid Sequence↗
PubMed1991

Carboxypeptidase yscS: gene structure and function of the vacuolar enzyme.

The gene encoding carboxypeptidase yscS in Saccharomyces cerevisiae, CPS1, was cloned by complementation of the cps1-3 mutation. The cloned CPS1 gene, which again enabled a leucine auxotrophic cps1-3 mutant to grow on the modified dipeptide Cbz-Gly-Leu (Cbz, benzyloxycarbonyl) as sole leucine source, was sequenced and found to consist of an open reading frame of 1728 bp encoding a protein of 576 amino acids. The putative protein contains a hydrophobic stretch of 20 amino acids and a putative signal sequence cleavage site. Five putative N-glycosylation sites are also in the protein sequence. This data is consistent with the previous finding of carboxypeptidase yscS being a vacuolar peptidase. Chromosomal disruption of the CPS1 gene completely abolishes carboxypeptidase yscS activity. This protein is yet another member of the peptidases in S. cerevisiae involved in nitrogen metabolism.

Amino Acid Sequence↗
PubMed1991

The proteinase yscB inhibitor (PB12) gene of yeast and studies on the function of its protein product.

The gene for proteinase yscB inhibitor I2B (PBI2) from Saccharomyces cerevisiae was isolated by oligonucleotide screening of a genomic DNA library, and was sequenced. The gene codes for a single protein of 75 amino acids. In contrast to the published amino acid sequence [Maier, K., Müller, H., Tesch, R., Trolp, T., Witt, I. & Holzer, H. (1979) J. Biol. Chem. 254, 12,555-12,561] the DNA sequence revealed a valine instead of a leucine at position 33 (32 of the mature protein). Therefore the primary sequences of the isoinhibitors I2B of S. cerevisiae and I1B of Saccharomyces carlsbergensis differ only at position 34 (glutamic acid/lysine). The open reading frame of PBI2 was replaced in vitro by the URA3 gene and a I2B null mutant of S. cerevisiae was constructed by gene replacement. The mutation resulted in an elevation of the protein degradation rate by 50% when grown under nutritional stress compared to the isogenic wild type. Growth and viability of the cells was not significantly affected by the absence of I2B.

Amino Acid Sequence↗
PubMed1991

Proteinase yscE, the yeast proteasome/multicatalytic-multifunctional proteinase: mutants unravel its function in stress induced proteolysis and uncover its necessity for cell survival.

Proteinase yscE is the yeast equivalent of the proteasome, a multicatalytic-multifunctional proteinase found in higher eukaryotic cells. We have isolated three mutants affecting the proteolytic activity of proteinase yscE. The mutants show a specific reduction in the activity of the complex against peptide substrates with hydrophobic amino acids at the cleavage site and define two complementation groups, PRE1 and PRE2. The PRE1 gene was cloned and shown to be essential. The deduced amino acid sequence encoded by the PRE1 gene reveals weak, but significant similarities to proteasome subunits of other organisms. Two-dimensional gel electrophoresis identified the yeast proteasome to be composed of 14 different subunits. Comparison of these 14 subunits with the translation product obtained from PRE1 mRNA synthesized in vitro demonstrated that PRE1 encodes the 22.6 kd subunit (numbered 11) of the yeast proteasome. Diploids homozygous for pre1-1 are defective in sporulation. Strains carrying the pre1-1 mutation show enhanced sensitivity to stresses such as incorporation of the amino acid analogue canavanine into proteins or a combination of poor growth medium and elevated temperature. Under these stress conditions pre1-1 mutant cells exhibit decreased protein degradation and accumulate ubiquitin-protein conjugates.

Amino Acid Sequence↗
PubMed1989

Lysosomal (vacuolar) proteinases of yeast are essential catalysts for protein degradation, differentiation, and cell survival.

Mutants deficient in the vacuolar (lysosomal) endopeptidases proteinase yscA and proteinase yscB of the yeast Saccharomyces cerevisiae exhibit a drastically reduced protein degradation rate under nutritional stress conditions. The differentiation process of sporulation is considerably disturbed by the absence of the two endopeptidases. Also under vegetative growth conditions and under conditions of false protein synthesis, the two vacuolar endopeptidases exhibit some effect on protein degradation, which is, however, much less pronounced as found under starvation conditions. Proteinase yscA deficiency leads to rapid cell death when glucose-grown cells starve for nitrogen or other nutrients. Whereas overall protein degradation is affected in the endopeptidase mutants, degradation of two distinct false proteins analyzed is not altered in the absence of proteinase yscA and proteinase yscB. Also catabolite inactivation and degradation of fructose-1,6-bisphosphatase is not affected to a greater extent in the endopeptidase-deficient strains.

Cell Differentiation↗
PubMed1988

Proteinase yscE of yeast shows homology with the 20 S cylinder particles of Xenopus laevis.

Proteinase yscE of the yeast Saccharomyces cerevisiae has been compared with the 20 S cylinder particles of Xenopus laevis. Both proteins are characterized by a similar group of 10-12 polypeptides with molecular masses ranging between 21 and 38 kDa. Antibodies generated against the 20 S Xenopus cylinder particles show cross-reactivity with yeast proteinase yscE subunits. The Xenopus particles and yeast proteinase yscE exhibit an identical image in electron microscopy. Both proteins appear as hollow cylinders mostly composed of four stacked annuli. The Xenopus 20 S particles exhibit proteolytic activity against the three peptide derivatives known to be substrates of proteinase yscE. The pH optimum for activity and the inhibition spectrum of the proteolytic activities of Xenopus 20 S particles and of yeast proteinase yscE are identical. The RNA content of the cylinder particles and of proteinase yscE is below 0.1 RNA chain per molecule. Our data suggest that proteinase yscE from yeast and the 20 S cylinder particles of X. laevis are homologous, highly conserved proteins carrying the catalytic character of a peptidase.

Animals↗
PubMed1988

Aminopeptidase yscII of yeast. Isolation of mutants and their biochemical and genetic analysis.

Mutant strains of the yeast Saccharomyces cerevisiae defective in aminopeptidase yscII were isolated by screening for reduced external activity against the chromogenic substrate lysine beta-naphthylamide. One of the selected mutant strains analyzed in detail showed wild-type staining activity when tested at 23 degrees C but mutant activity after exposure to 37 degrees C, suggesting a temperature-sensitive mutation. Electrophoretic separation of mutant crude extracts on non-denaturing polyacrylamide gels and subsequent activity staining using lysine and leucine beta-naphthylamides as substrates revealed that in all strains isolated the same distinct activity band was affected, which corresponded to the aminopeptidase activity identified previously as aminopeptidase yscII [Achstetter, T., Ehmann, C. & Wolf, D. H. (1983) Arch. Biochem. Biophys. 226, 292-305]. All mutants strains isolated fell into the same complementation group. Tetrad dissection of sporulated diploids heterozygous for the wild-type and mutant allele resulted in a 2:2 segregation of mutant and wild-type phenotype indicating a single gene mutation. The characteristics of the mutations analyzed point to the gene which we called APE2 as the structural gene of aminopeptidase yscII. No vital consequences of aminopeptidase yscII deficiency on cell life and differentiation could be detected. However, the enzyme seems to be involved in the cellular supply of leucine from externally offered leucine-containing dipeptide substrates.

Aminopeptidases↗
PubMed1988

Biogenesis of the yeast lysosome (vacuole): biosynthesis and maturation of proteinase yscB.

The biosynthesis and processing of the vacuolar (lysosomal) proteinase yscB was followed in vivo and in vitro. In vitro transcription--translation of the cloned proteinase yscB gene results in a high mol. wt precursor protein (HMr-precursor) of Mr = 73,000. In vivo a precursor of identical mol. wt is found in sec6l mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum (ER). In contrast to N-glycosylated intermediate mol. wt forms of proteinase yscB, the HMr-precursor of the enzyme is not N-glycosylated, indicating its appearance outside the ER. sec18 Mutant cells, wild type for translocation of proteins into the ER but blocked in the delivery step of secretory proteins to the Golgi apparatus, accumulate a lower mol. wt precursor (LMr-precursor) of proteinase yscB of Mr = 41,500. Processing of the HMr-precursor of proteinase yscB to the LMr-precursor requires proteolytic cleavage which is independent of the proteinase yscA gene product, a protein known to be involved in the processing event of the LMr-precursor of proteinase yscB to the mature enzyme of Mr = 33,000. A LMr-precursor of proteinase yscB of Mr = 42,000 accumulates in proteinase yscA-deficient mutant cells (allele pep4-3). This form is enzymatically active. Incubation in vitro of the LMr-precursor of proteinase yscB with mature proteinase yscA leads to further activation and to processing of the enzyme yielding the mature 33,000 Mr protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Fungal Proteins↗
PubMed1987

Hormone (pheromone) processing enzymes in yeast. The carboxy-terminal processing enzyme of the mating pheromone alpha-factor, carboxypeptidase ysc alpha, is absent in alpha-factor maturation-defective kex1 mutant cells.

Carboxy-terminal processing of the mating pheromone alpha-factor of the yeast Saccharomyces cerevisiae has been assumed to be due to the action of carboxypeptidase ysc alpha [(1985) EMBO J. 4, 173-177]. Here it is shown that a mutant (kex1) defective in alpha-factor maturation is defective in carboxypeptidase ysc alpha activity, indicating that the enzyme is indeed the processing catalyst. It is proposed that carboxypeptidase ysc alpha is the product of the KEX1 gene.

Carboxypeptidases↗
PubMed1987

Some characteristics of hormone (pheromone) processing enzymes in yeast.

The KEX2 gene-encoded, membrane-bound Ca2+-dependent thiol endoproteinase, proteinase yscF, responsible for processing of the precursor protein of the sex pheromone alpha-factor of the yeast Saccharomyces cerevisiae was solubilized from the membraneous fraction and partially purified. Gel filtration revealed an apparent Mr of the native protein of around 150,000. Ca2+ concentration for half-maximal activity was in the micromolar range and concentration of the substrate Cbz-Tyr-Lys-Arg-4-nitroanilide for half-maximal velocity was 0.05 mM. The enzyme able to cleave basic amino acids from the carboxy-terminus of peptides and probably involved in final maturation of the alpha-factor peptides generated by proteinase yscF is membrane-associated, active at neutral pH and responds strongly to the serine proteinase inhibitor phenyl-methylsulfonyl fluoride as well as to -SH group blocking agents.

Chromatography, Gel↗
PubMed1987

Proteinase yscD mutants of yeast. Isolation and characterization.

Mutants of the yeast Saccharomyces cerevisiae, devoid of proteinase yscD activity, were isolated by screening for the inability of mutagenized cells to hydrolyze Ac-Ala-Ala-Pro-Ala-beta-naphthylamide in situ. One of the selected mutants bears a thermolabile activity pointing to the gene called PRD1 as being the structural gene for proteinase yscD. All mutants isolated fell into one complementation group. The defect segregates 2:2 in meiotic tetrads indicating a single gene mutation, which was shown to be recessive. Diploids heterozygous for PRD1 display gene dosage. The absence of proteinase yscD did not affect mitotic growth under rich or poor growth conditions, neither mating nor ascopore formation. Also growth of mutant cells after a nutritional shift-down was not altered. Inactivation of enzymes tested which are subject to carbon-catabolite inactivation, a process proposed to be of proteolytic nature, is not affected by the absence of proteinase yscD. Protein degradation rates in growing cells, in cells under conditions of differentiation or heat shock, showed no obvious alteration in the absence of proteinase yscD activity. Also inactivation of alpha-factor pheromone was not affected by proteinase yscD absence. Normal growth of mutant cells on glycerol indicates that the enzyme is not involved in any vital event in mitochondrial biogenesis.

Cysteine Endopeptidases↗