Site of catabolite inactivation.
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Autophagy↗
Biomedical subjects
D H Wolf
Publications and source records attributed to D H Wolf.
PRE3, highly homologous to the human major histocompatibility complex-linked LMP2 (RING12) gene, codes for a yeast proteasome subunit necessary for the peptidylglutamyl-peptide hydrolyzing activity.
20S proteasomes are multifunctional proteinase complexes ubiquitous in eucaryotes. We have cloned the yeast PRE3 gene by complementation of the pre3-2 mutation, which leads to a defect in the peptidylglutamyl-peptide hydrolyzing activity of the 20S proteasome. The PRE3 gene, a beta-type member of the proteasomal gene family, is essential for cellular life and codes for a 193-amino acid proteasomal subunit with a predicted molecular mass of 21.2 kDa. The Pre3 protein shows striking homology to the human proteasome subunits Hs delta and Lmp2 (Ring12). Lmp2 is encoded in the major histocompatibility complex class II region implicating proteasomes in antigen processing.
Amino Acid Sequence↗
Proteinase yscD (oligopeptidase yscD). Structure, function and relationship of the yeast enzyme with mammalian thimet oligopeptidase (metalloendopeptidase, EP 24.15).
The yeast PRD1 gene, encoding proteinase yscD, was cloned by complementation of the prd1-6 point mutation. Sequencing of the gene revealed an open reading frame of 2.136 kb, encoding a protein of 712 amino acids with a calculated molecular mass of 81.8 kDa. The sequence HEGLG beginning at residue 501 represents the HEXXH motif, unique for the zinc metallo-peptidases. Sequence comparison revealed complete identity of the proteinase yscD gene with a recently published open reading frame of yeast chromosome III. We found 34.8% identity between proteinase yscD and rat metalloendopeptidase (thimet oligopeptidase, EP 24.15). Proteinase yscD hydrolyzes several chromogenic and fluorogenic peptides that are substrates of thimet oligopeptidase. N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoic acid, a compound designed as specific inhibitor of EP 24.15, is also a strong inhibitor of the yeast enzyme. Proteinase yscD is a nonvacuolar enzyme. 3-5% of the total enzyme activity can be detected in the intermembrane space of mitochondria. In a mutant carrying a deletion of the PRD1 gene no proteinase yscD activity is detectable in the cytoplasm and in mitochondria of these cells. They do not show any grossly altered phenotype but exhibit a decrease in the intracellular degradation of peptides. This suggests a function of proteinase yscD in the late stages of protein degradation.
Amino Acid Sequence↗
Tracing intracellular proteolytic pathways. Proteolysis of fatty acid synthase and other cytoplasmic proteins in the yeast Saccharomyces cerevisiae.
Yeast fatty acid synthase consists of two independent polypeptide strains, alpha and beta. The functional multienzyme complex, composed of six alpha- and six beta-subunits, is rather stable against proteolysis in vivo. Mutations in one of the subunits or deletion of one subunit lead to degradation of the nonmutated remaining fatty acid synthase protein. We show that the unassembled alpha-subunit of this enzyme is short-lived, and degradation depends on the presence of active cytoplasmic proteinase yscE, the yeast proteasome. The unassembled beta-subunit is degraded by a nonvacuolar proteolytic system under vegetative growth conditions. However, starvation of a vacuolar proteinase mutant strain, which lacks the alpha-subunit of fatty acid synthase, leads to appearance of the unassembled beta-subunit is isolated vacuoles. This indicates that the major vacuolar peptidases proteinase yscA and yscB are at least partly involved in degradation of the beta-subunit of fatty acid synthase. In a proteinase yscA and yscB double mutant strain wild type for fatty acid synthase both subunits of fatty acid synthase, alpha and beta, are detectable in vacuoles. In addition, under the same starvation conditions other cytoplasmic proteins are found in the vacuole of a proteinase yscA and yscB double mutant strain. The experiments in conjunction with the previous finding of the appearance of vesicles in vacuoles of starved cells (Simeon, A., van der Klei, I.J., Veenhuis, M., and Wolf, D. H. (1992) FEBS Lett. 301, 231-235) indicate that transport of these tested cytoplasmic proteins into the vacuole is an unselective bulk process induced by nutritional stress.
Cysteine Endopeptidases↗
Proteasome and cell cycle. Evidence for a regulatory role of the protease on mitotic cyclins in yeast.
The cell cycle of eukaryotic cells is strictly regulated. This regulation is performed by a serine/threonine kinase. The different functions of this kinase in the cell cycle are modulated by different cyclins, which fluctuate in concentration ('cycle') during the different stages of the cell cycle. Using yeast as a model organism we show here that the activity of the multifunctional proteinase, the proteasome, is directly connected to the function of the mitotic cyclin Clb2. Our studies indicate that the proteasome is the proteolytic regulator of this cyclin and thus a central regulator of the cell cycle.
Cell Cycle↗
Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast.
The fate of a mutant form of each of the two yeast vacuolar enzymes proteinase yscA (PrA) and carboxypeptidase yscY (CPY) has been investigated. Both mutant proteins are rapidly degraded after entering the secretory pathway. Mutant PrA is deleted in 37 amino acids spanning the processing site region of the PrA pro-peptide. The mutant enzyme shows no activity towards maturation of itself or other vacuolar hydrolases, a function of wild-type PrA. Mutant CPY carries an Arg instead of a Gly residue in a highly conserved region, two positions distant from the active-site Ser. In contrast to wild-type CPY, the mutant form was quickly degraded by trypsin in vitro, indicating an altered structure. Using antisera specific for alpha-1-->6 and alpha-1-->3 outer-chain mannose linkages, no Golgi-specific carbohydrate modification could be detected on either mutant protein. Subcellular fractionation studies located both mutant enzymes in the endoplasmic reticulum. Degradation kinetics of both proteins show the same characteristics, indicating similar degradation pathways. The degradation process was shown to be independent of a functional sec18 gene product and takes place before Golgi-specific carbohydrate modifications occur. The proteasome, the major proteolytic activity of the cytoplasm, is not involved in this degradation event. All degradation characteristics of the two mutant proteins are consistent with a degradation process within the endoplasmic reticulum ('ER degradation').
Adenosine Triphosphatases↗
Purification and characterization of the cystinyl bond cleaving yeast aminopeptidase yscXVI.
Aminopeptidase yscXVI was purified from the yeast Saccharomyces cerevisiae. By SDS-PAGE the enzyme has a molecular weight of 45,000 Da, and in chromatofocusing, elution was observed at pH 6.2. The synthetic substrate cystinyl-4-nitroanilide (Km 22.5 microM, Vmax 12.9 mU/mg) is cleaved most efficiently in the pH range 7-8. Besides cleaving this standard substrate, aminopeptidase yscXVI acts on several other 4-nitroanilide substrates with unsubstituted N-terminal L-amino acids. Highest hydrolysis rate was measured with Lys-4-nitroanilide and Leu-4-nitroanilide. The activity of aminopeptidase yscXVI is abolished by chelating agents and restored by Zn2+, Mn2+ and Co2+ ions. Bestatin and amastatin are both strong inhibitors of the enzyme, with Ki values of 0.53 microM and 0.93 microM, respectively. Aminopeptidase yscXVI is detectable in the logarithmic growth phase, stationary phase, and in starved cultures of yeast.
Amino Acid Sequence↗
BLH1 codes for a yeast thiol aminopeptidase, the equivalent of mammalian bleomycin hydrolase.
We have cloned the BLH1 gene of the yeast Saccharomyces cerevisiae coding for a peptidase with significant homology to rabbit bleomycin hydrolase. Bleomycin is a glycopeptide antibiotic used for the treatment of human cancers. The antitumor activity of the drug is limited by its metabolic inactivation caused by bleomycin hydrolase, a member of the cysteine protease family. The open reading frame of BLH1 consists of 1,449 base pairs encoding a 55.4-kDa protein consistent with the molecular mass found in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The consensus sequence within the active site region of cysteine proteases is conserved in the yeast enzyme. Biochemical studies on the purified enzyme confirm its classification as a thiol protease. The nonvacuolar enzyme has a molecular mass of 220 kDa, suggesting a homotetrameric structure. It exhibits an aminopeptidase activity with broad substrate specificity. Biochemical and genetic linkage data give evidence that the BLH1 and the LAP3 (Trumbly, R. J., and Bradley, G. (1983) J. Bacteriol. 156, 36-48) encoded aminopeptidases are identical. Deletion of the BLH1 gene is not lethal under normal growth conditions. However, blh1 mutants show hypersensitivity to bleomycin, indicating that bleomycin hydrolase is able to inactivate bleomycin in vivo and to protect cells from bleomycin-induced toxicity.
Amino Acid Sequence↗
PRE2, highly homologous to the human major histocompatibility complex-linked RING10 gene, codes for a yeast proteasome subunit necessary for chrymotryptic activity and degradation of ubiquitinated proteins.
We have cloned the yeast PRE2 gene by complementation of pre2 mutants, which are defective in the chymotrypsin-like activity of the 20 S proteasome (multicatalytic-multifunctional proteinase complex). The PRE2 gene, a beta-type member of the proteasomal gene family, is essential for life and codes for a 287-amino acid proteasomal subunit with a predicted molecular mass of 31.6 kDa. Missense mutations in two pre2 mutant alleles were identified. They led to enhanced sensitivity of yeast cells against stress. At the same time, pre2 mutants accumulated ubiquitinated proteins. The Pre2 protein shows striking homology to the human Ring10 protein (60% identity excluding the 70 amino-terminal residues), which is encoded in the major histocompatibility complex class II region. It represents a component of the low molecular mass polypeptide complex, previously shown to be a special type of the 20 S proteasome. The low molecular mass polypeptide complex is assumed to be involved in antigen presentation, generating peptides from cytosolic protein antigens, which are subsequently presented to cytotoxic T-lymphocytes on the cell surface. The high homology of Pre2 to Ring10 implies the hypothesis that Ring10 is a subunit of the low molecular mass polypeptide complex central in its chymotryptic activity. One might further suggest that replacement of constitutive proteasomal components by functionally related major histocompatibility complex-linked low molecular mass polypeptides, as is Ring10, adapts mammalian proteasomes for functions in the immune response.
Amino Acid Sequence↗
The PRE4 gene codes for a subunit of the yeast proteasome necessary for peptidylglutamyl-peptide-hydrolyzing activity. Mutations link the proteasome to stress- and ubiquitin-dependent proteolysis.
Proteinase yscE, the yeast proteasome, is a member of the nonlysosomal, high molecular mass (approximately 700 kDa) multifunctional proteinase complexes that are highly conserved from yeast to man. We have isolated mutants defective in one of the three proteolytic activities of the enzyme complex, i.e. in cleavage of peptide bonds after acidic amino acids. Using one of these mutants (pre4-1), we cloned the PRE4 gene and uncovered an open reading frame with 266 amino acids coding for a predicted protein of 29.4 kDa. The protein proved to be a subunit of proteinase yscE. The Pre4 amino acid sequence shows strong homology to the beta-subunit of the Xenopus laevis proteasome. Chromosomal deletion of the PRE4 gene is lethal. The pre4-1 mutant allele was cloned and sequenced. The mutant protein is shortened by 15 amino acids at the carboxyl terminus. Mutations (pre1-1, pre2-2) in the chymotrypsin-like activity of proteinase yscE uncovered the enzyme to be involved in ubiquitin-linked and stress-dependent proteolytic pathways. In contrast to these mutants, pre4-1 mutants did not exhibit any apparent stress-dependent phenotypes. However, pre1-1 pre4-1 double mutants showed enhanced canavanine sensitivity and increased accumulation of ubiquitin protein conjugates, as compared with pre1-1 single mutants.
Amino Acid Sequence↗
Vacuolar/lysosomal proteolysis: proteases, substrates, mechanisms.
Proteolysis is an essential post-transcriptional process. The lysosome/vacuole is the central organelle for non-specific proteolysis in eukaryotes. Most proteases that work in the lysosome enter it via the secretory pathway. The bulk of proteins to be degraded enter this proteolytic compartment via endocytosis and autophagocytosis. Our understanding of the mechanisms involved in these processes has increased considerably, and the information obtained in these studies has enabled new, specific proteolytic pathways to be investigated in other cellular compartments, particularly in the cytoplasm.
Animals↗
Studies on the yeast proteasome uncover its basic structural features and multiple in vivo functions.
Proteasomes are large multicatalytic protease complexes found in the cytoplasm and nucleus of all eukaryotic cells. 20S proteasomes are cylindrically shaped particles composed of a set of different subunits arranged in a stack of 4 rings with 7-fold symmetry. In yeast 14 different genes are known, which are proposed to code for the complete set of 20S proteasomal subunits. They can be divided in 7 alpha- and 7 beta-type subunits. 26S proteasomes are even larger proteinase complexes which contain the 20S proteasome as the functional proteolytic core. They degrade ubiquitinylated proteins in vitro. Several yeast 26S proteasome subunits have been characterized as members of a novel ATPase family. Studies with yeast 20S and 26S proteasome mutants uncovered the function of proteasomes in stress-dependent and ubiquitin-mediated proteolytic pathways. Proteasomes are important for cellular regulation, cell differentiation, adaptation to environmental changes and are involved in cell cycle control.
Animals↗
Biogenesis of the yeast vacuole (lysosome). Signal sequence deletion of the vacuolar aspartic proteinase yscA does not block 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 importance of the different domains of proteinase yscA in a first attempt the signal sequence of the enzyme was deleted using site-directed mutagenesis. Proteinase yscA delta S underwent processing to an active enzyme, although the amount of mature enzyme was considerably reduced as compared to wild type. Isolation of vacuoles of the mutant strain showed mature-sized proteinase yscA in this organelle. Pro-proteinase yscB is converted to 5-10% to the mature enzyme, whereas procarboxypeptidase yscY is matured, although with reduced kinetics. Thus, the signal sequence of proteinase yscA is not essential for delivery to the vacuole and the activation of a certain amount of the yeast vacuolar proteolytic capacity.
Aspartic Acid Endopeptidases↗
Biogenesis of the yeast vacuole (lysosome). Proteinase yscB contributes molecularly and kinetically to vacuolar hydrolase-precursor maturation.
The vacuolar proteinase yscB (PrB) has been implicated in the final maturation of procarboxypeptidase yscY (pro-CpY) to the mature wild-type form CpYb of 61 kDa. In PrB-deficient mutants, only the proteinase yscA processed form CpYa of 62 kDa is found [Mechler, B., Müller, H. & Wolf, D. H. (1987) EMBO J. 6, 2157-2163]. We report now that, akin to CpY, two forms of mature proteinase yscA (PrA) can be distinguished. In PrB-deficient mutant cells, PrAa, migrating at about 43 kDa in SDS/PAGE, is found, whereas PrAb, found in wild-type cells, had the known molecular mass of 42 kDa. In the PrB-deficient strain, pro-PrA and pro-CpY matured only to the higher-molecular-mass forms, PrAa and CpYa, and the maturation of both precursors was slower than in the isogenic wild-type strain. Pulse-labeling experiments indicated that the mature forms, PrAb or CpYb, are generated directly in the PrB-containing wild-type strain in vivo. In vitro experiments showed that PrB is able to trigger maturation of its 42-kDa pro-PrB precursor to mature PrB in the absence of PrA. Mature PrB and its proteolytic activity, however, shows a higher stability in the presence of mature PrA. The data indicate a molecular and kinetic participation of proteinase yscB in vacuolar hydrolase precursor maturation.
Blotting, Western↗
The proteasome/multicatalytic-multifunctional proteinase. In vivo function in the ubiquitin-dependent N-end rule pathway of protein degradation in eukaryotes.
Proteinase yscE, the proteasome/multicatalytic-multifunctional proteinase of yeast had been shown to function in stress response and in the degradation of ubiquitinated proteins [(1991) EMBO J. 10, 555-562]. A well-defined set of proteins degraded via ubiquitin-mediated proteolysis are the substrates of the N-end rule pathway [(1986) Science 234, 179-186; (1989) Science 243, 1576-1583]. We show that mutants defective in the chymotryptic activity of proteinase yscE fail to degrade substrates of the N-end rule pathway. This gives further proof of the proteasome being a central catalyst in ubiquitin-mediated proteolysis.
Catalysis↗
Biogenesis of the yeast vacuole (lysosome). The precursor forms of the soluble hydrolase carboxypeptidase yscS are associated with the vacuolar membrane.
We have studied the structure, biosynthesis, intracellular routing, and vacuolar localization of carboxypeptidase ysCS in the yeast Saccharomyces cerevisiae. Nondenaturing polyacrylamide gel electrophoresis revealed two forms of carboxypeptidase yscS with different electrophoretic mobility. Antibodies specific for carboxypeptidase yscS recognized two glycoproteins of 77- and 74-kDa apparent molecular mass which differ by one N-linked carbohydrate residue. Both observations suggest that carboxypeptidase yscS exists in two catalytically active forms. The enzyme was found to be synthesized as two active high molecular mass precursor forms which are converted to the mature forms with a half-time of 20 min. The mature forms of carboxypeptidase yscS appeared soluble in the vacuolar lumen, while the precursor proteins accumulated tightly associated with the vacuolar membrane. The single hydrophobic domain present at the N terminus is believed to be responsible for the membrane association of the precursor molecules. Double mutants defective in proteinase yscA and proteinase yscB synthesize solely the carboxypeptidase yscS precursor forms. Correct proteolytic cleavage of the precursor forms was performed using purified proteinase yscB in vitro. Sec61, sec18, and sec7 mutants, conditionally defective in the secretory pathway, accumulate carboxypeptidase yscS precursor protein. Thus the carboxypeptidase yscS precursor molecules are delivered to the vacuole in a membrane bound form via the secretory pathway. After assembly into the vacuolar membrane, proteinase yscB presumably cleaves the precursor molecules to release soluble carboxypeptidase yscS forms into the lumen of the vacuole. The proposed mechanism is different from the delivery mechanism found for the other soluble vacuolar hydrolases in yeast.
Carboxypeptidases↗
Ubiquitin, a central component of selective cytoplasmic proteolysis, is linked to proteins residing at the locus of non-selective proteolysis, the vacuole.
Ubiquitin, an evolutionary highly conserved protein, is known to be involved in selective proteolysis in the cytoplasm. Here we show that ubiquitin-protein conjugates are also found in the yeast vacuole. Mutants defective in the major vacuolar endopeptidases, proteinase yscA and yscB, lead to accumulation of ubiquitin-protein conjugates in this cellular organelle.
Aspartic Acid Endopeptidases↗
Proteases as biological regulators. Introductory remarks.
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Animals↗