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D H Wolf

Publications and source records attributed to D H Wolf.

At least 55 records · Page 3Linked to original sources
PubMed1997

AUT1, a gene essential for autophagocytosis in the yeast Saccharomyces cerevisiae.

Autophagocytosis is a starvation-induced process responsible for transport of cytoplasmic proteins to the vacuole. In Saccharomyces cerevisiae, autophagy is characterized by the phenotypic appearance of autophagic vesicles inside the vacuole of strains deficient in proteinase yscB. The AUT1 gene, essential for autophagy, was isolated by complementation of the sporulation deficiency of a diploid aut1-1 mutant strain by a yeast genomic library and characterized. AUT1 is located on the right arm of chromosome XIV, 10 kb from the centromere, and encodes a protein of 310 amino acids, with an estimated molecular weight of 36 kDa. Cells carrying a chromosomal deletion of AUT1 are defective in the starvation-induced bulk flow transport of cytoplasmic proteins to the vacuole. aut1 null mutant strains are completely viable but show decreased survival rates during starvation. Homozygous delta aut1 diploid cells fail to sporulate. The selective cytoplasm-to-vacuole transport of aminopeptidase I is blocked in logarithmically growing and in starved delta autl cells. Deletion of the AUT1 gene had no obvious influence on secretion, fluid phase endocytosis, or vacuolar protein sorting. This supports the idea of autophagocytosis as being a novel route transporting proteins from the cytoplasm to the vacuole.

Amino Acid Sequence↗
PubMed1997

Catabolite inactivation of the galactose transporter in the yeast Saccharomyces cerevisiae: ubiquitination, endocytosis, and degradation in the vacuole.

When Saccharomyces cerevisiae cells growing on galactose are transferred onto glucose medium containing cycloheximide, an inhibitor of protein synthesis, a rapid reduction of Gal2p-mediated galactose uptake is observed. We show that glucose-induced inactivation of Gal2p is due to its degradation. Stabilization of Gal2p in pra1 mutant cells devoid of vacuolar proteinase activity is observed. Subcellular fractionation and indirect immunofluorescence showed that the Gal2 transporter accumulates in the vacuole of the mutant cells, directly demonstrating that its degradation requires vacuolar proteolysis. In contrast, Gal2p degradation is proteasome independent since its half-life is unaffected in pre1-1 pre2-2, cim3-1, and cim5-1 mutants defective in several subunits of the protease complex. In addition, vacuolar delivery of Gal2p was shown to be blocked in conditional end3 and end4 mutants at the nonpermissive temperature, indicating that delivery of Gal2p to the vacuole occurs via the endocytic pathway. Taken together, the results presented here demonstrate that glucose-induced proteolysis of Gal2p is dependent on endocytosis and vacuolar proteolysis and is independent of the functional proteasome. Moreover, we show that Gal2p is ubiquitinated under conditions of glucose-induced inactivation.

Azides↗
PubMed1996

N-Glycosylation affects endoplasmic reticulum degradation of a mutated derivative of carboxypeptidase yscY in yeast.

The endoplasmic reticulum (ER) of eukaryotic cells contains a quality control system, that is required for the proteolytic removal of aberrantly folded proteins that accumulate in this organelle. We used genetic and biochemical methods to analyse the involvement of N-glycosylation in the degradation of a mutant derivative of carboxypeptidase yscY in the ER of the yeast Saccharomyces cerevisiae. Our results demonstrate that N-glycosylation of this protein is required for its degradation since an unglycosylated species is retained stably in the ER. Cells that were devoid of the ER-processing alpha 1,2-mannosidase showed reduced degradation of the glycosylated substrate protein. Disruption of CNE1, a gene encoding a putative yeast homologue for calnexin, did not exhibit any effects on the degradation of this substrate protein in vivo. Also, the alpha 1,2-mannosidase-dependent reduction in the degradation rate did not show any correlation with the function of the CNE1 gene product. Our results suggest that the ER of yeast contains a glycosylation-dependent quality control system, as has been shown for higher eukaryotic cells.

Calcium-Binding Proteins↗
PubMed1996

ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway.

Secretion of proteins is initiated by their uptake into the endoplasmic reticulum (ER), which possesses a proteolytic system able to degrade misfolded and nonassembled proteins. The ER degradation system was studied with yeast mutants defective in the breakdown of a mutated soluble vacuolar protein, carboxypeptidase yscY (CPY*). The ubiquitin-conjugating enzyme Ubc7p participated in the degradation process, which was mediated by the cytosolic 26S proteasome. It is likely that CPY* entered the ER, was glycosylated, and was then transported back out of the ER lumen to the cytoplasmic side of the organelle, where it was conjugated with ubiquitin and degraded.

Biological Transport↗
PubMed1996

Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast.

The endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae contains of proteolytic system able to selectively degrade misfolded lumenal secretory proteins. For examination of the components involved in this degradation process, mutants were isolated. They could be divided into four complementation groups. The mutations led to stabilization of two different substrates for this process. The mutant classes were called 'der' for 'degradation in the ER'. DER1 was cloned by complementation of the der1-2 mutation. The DER1 gene codes for a novel, hydrophobic protein, that is localized to the ER. Deletion of DER1 abolished degradation of the substrate proteins. The function of the Der1 protein seems to be specifically required for the degradation process associated with the ER. The depletion of Der1 from cells causes neither detectable growth phenotypes nor a general accumulation of unfolded proteins in the ER. In DER1-deleted cells, a substrate protein for ER degradation is retained in the ER by the same mechanism which also retains lumenal ER residents. This suggests that DER1 acts in a process that directly removes protein from the folding environment of the ER.

Amino Acid Sequence↗
PubMed1996

Proteasomes: destruction as a programme.

Proteasomes are large multi-subunit protease complexes that selectively degrade intracellular proteins. Most of the proteins removed by these proteases are tagged for destruction by ubiquitination. Proteasomes have a role to play in controlling cellular processes, such as metabolism and the cell cycle, through signal-mediated proteolysis of key enzymes and regulatory proteins. They also operate in the stress response, by removing abnormal proteins, and in the immune response, by generating antigenic peptides.

Animals↗
PubMed1995

Catabolite inactivation of fructose-1,6-bisphosphatase of Saccharomyces cerevisiae. Degradation occurs via the ubiquitin pathway.

Catabolite inactivation of fructose-1,6-bisphosphatase (FBPase), a key enzyme in gluconeogenesis, is due to phosphorylation and subsequent degradation in the yeast Saccharomyces cerevisiae. The degradation process of the enzyme had been shown to depend on the action of the proteasome. Here we report that components of the ubiquitin pathway target FBPase to proteolysis. Upon glucose addition to yeast cells cultured on nonfermentable carbon sources FBPase is ubiquitinated in vivo. A multiubiquitin chain containing isopeptide linkages at Lys48 of ubiquitin is attached to FBPase. Formation of a multiubiquitin chain is a prerequisite for the degradation of FBPase. Catabolite degradation of FBPase is dependent on the ubiquitin-conjugating enzymes Ubc1, Ubc4, and Ubc5. The 26 S proteasome is involved in the degradation process.

Amino Acid Sequence↗
PubMed1995

Biogenesis of the yeast vacuole (lysosome). The use of active-site mutants of proteinase yscA to determine the necessity of the enzyme for vacuolar proteinase maturation and proteinase yscB stability.

The activation process of vacuolar proteinases in the yeast Saccharomyces cerevisiae via precursor maturation is initiated by the PRA1/PEP4 gene product, proteinase yscA. Chromosomal deletion of the PRA1/PEP4 locus leads to accumulation of inactive pro-proteinases in the vacuole. Nine active-site mutations of proteinase yscA have been constructed in vitro. All these mutations lead to the expression of proteinase yscA species in vivo that are inactive against the in vitro substrate hemoglobin and the in vivo substrates pro-proteinase yscB and pro-carboxypeptidase yscY. However, three active-site mutations in proteinase yscA sustained the precursor maturation of proteinase yscB and carboxypeptidase yscY after exchange of the genomic wild-type allele with the respective proteinase yscA mutant alleles. In contrast to yeast strains deleted in proteinase yscA, the respective mutants carry out all cellular functions that rely on a proteolytically active vacuole. This wild-type behaviour of proteinase yscA mutant cells is dependent on the presence of active proteinase yscB. Proteinase yscA and proteinase yscB are equally able to fulfil essential cellular functions. For instance, either proteinase is able to maintain viability under starvation. However, mature proteinase yscB is not stable in the absence of proteinase yscA. The wild-type-like conformation of proteolytically inactive mutant proteinase yscA proteins stabilizes mature proteinase yscB and thus enables continuous maturation of pro-proteinase yscB by active proteinase yscB. After inhibition of the proteolytic activity of proteinase yscB in these proteinase yscA mutants with phenylmethysulfonyl fluoride or deletion of the PRB1 gene, maturation of all zymogens investigated in the vacuole, including the proteinase yscA mutant proteins, is blocked. The proteolytic activities of the vacuole in such a strain can be regained, however, by introduction of a wild-type proteinase yscA gene allowing subsequent autocatalytic maturation of wild-type pro-proteinase yscA. This indicates that an initial self-activation process of proteinase yscA is necessary for the activation of vacuolar zymogens.

Alleles↗
PubMed1995

Degradation of ornithine decarboxylase by the mammalian and yeast 26S proteasome complexes requires all the components of the protease.

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is an extremely short-lived protein. This attribute is important for the regulation of the activity of the enzyme and implies that the mechanisms involved in its degradation play an important role in the control of the cellular processes in which the enzyme is involved. Recently, it has been shown that ODC is degraded by the 26S proteasome complex in a process that requires antizyme, but not ubiquitin. With one reported exception, ODC, the 26S complex recognizes and degrades specifically ubiquitinated proteins. Their unconjugated counterparts are not targeted. The 26S complex is composed of a core catalytic unit, the 20S proteasome complex, and two additional, and apparently distinct, subcomplexes. The two additional subcomplexes are regulatory subunits that are required in order to confer specificity and control. In this study, we demonstrate that, like the degradation of ubiquitin-conjugated proteins, ubiquitin-independent degradation of ODC also requires prior assembly of the mammalian 26S proteasome from all the three subunits, the 20S proteasome and the two subcomplexes. The combination of any two subunits does not support generation of a proteolytically active complex. This is also true for the yeast 26S complex. Like the mammalian 20S proteasome, the yeast 20S complex can cleave short peptides in an ATP-independent mode, but cannot degrade ODC or ubiquitin-conjugated proteins. These proteins are degraded only following addition of the regulatory subunits and generation of the high-molecular-mass 26S complex. In a distinct, but related, set of experiments, we demonstrate that the degradation of ODC by the assembled 26S proteasome in vitro reproduces faithfully proteolysis of the enzyme in the intact cell. Namely, (a) a C-terminal-deleted mouse ODC and trypanosome ODC are stable both in vitro and in vivo, and (b) like proteolysis in the intact cell, degradation in the reconstituted cell-free system is also dependent upon the addition of antizyme.

Animals↗
PubMed1995

Proteasomes of the yeast S. cerevisiae: genes, structure and functions.

Proteasomes are large multicatalytic protease complexes which fulfil central functions in major intracellular proteolytic pathways of the eukaryotic cell. 20S proteasomes are 700 kDa cylindrically shaped particles, found in the cytoplasm and the nucleus of all eukaryotes. They are composed of a pool of 14 different subunits (MW 22-25 kDa) arranged in a stack of 4 rings with 7-fold symmetry. In the yeast Saccharomyces cerevisiae a complete set of 14 genes coding for 20S proteasome subunits have been cloned and sequenced. 26S proteasomes are even larger proteinase complexes (about 1700 kDa) which degrade ubiquitinylated proteins in an ATP-dependent fashion in vitro. The 26S proteasome is build up from the 20S proteasome as core particle and two additional 19S complexes at both ends of the 20S cylinder. Recently existence of a 26S proteasome in yeast has been demonstrated. Several 26S proteasome specific genes have been cloned and sequenced. They share similarity with a novel defined family of ATPases. 20S and 26S proteasomes are essential for functioning of the eukaryotic cell. Chromosomal deletion of 20S and 26S proteasomal genes in the yeast S. cerevisiae caused lethality of the cell. The in vivo functions of proteasomes in major proteolytic pathways have been demonstrated by the use of 20S and 26S proteasomal mutants. Proteasomes are needed for stress dependent and ubiquitin mediated proteolysis. They are involved in the degradation of short-lived and regulatory proteins. Proteasomes are important for cell differentiation and adaptation to environmental changes. Proteasomes have also been shown to function in the control of the cell cycle.

Cysteine Endopeptidases↗
PubMed1995

[Proteasomes. Complex proteases lead to a new understanding of cellular regulation through proteolysis].

Proteasomes are large multicatalytic protease complexes which fulfill central functions in major proteolytic pathways of the eukaryotic cell. Two types of proteasomes are known: the cylindrically shaped 20S proteasome (700 kDa) and the 26S proteasome (1700 kDa) which contains the 20S proteasome as a functional core. Proteasomes are needed for stress-dependent and ubiquitin-mediated proteolysis. They are involved in degradation of abnormal, short-lived, and regulatory proteins. Proteasomes are important for cell differentiation and adaptation to environmental changes. Proteasomes have been shown to function in the control of the cell cycle and are suggested to be involved in antigen presentation by processing of intracellular proteins to antigenic peptides.

Animals↗
PubMed1995

Catabolite inactivation of the yeast maltose transporter occurs in the vacuole after internalization by endocytosis.

The maltose transporter of Saccharomyces cerevisiae is rapidly degraded during fermentation in the absence of a nitrogen source. The location and mechanism of degradation of the transporter have been investigated. Using mutants defective in endocytosis, we have shown that degradation of this transporter requires internalization by endocytosis. In addition, studies of mutants defective in proteasome or vacuolar proteolysis revealed that degradation occurs in the vacuole and is independent of proteasome function. The results also revealed that degradation of the maltose transporter requires Sec18p and raised the question of whether in the absence of Sec18p activity the internalized maltose transporter is recycled back to the plasma membrane.

Adenosine Triphosphatases↗
PubMed1994

The 26S proteasome of the yeast Saccharomyces cerevisiae.

Proteasomes are large multicatalytic proteinase complexes found in all eukaryotic organisms investigated so far. They have been shown to play a central role in cytosolic and nuclear proteolysis. According to their sedimentation coefficients two types of these particles can be distinguished: 20S proteasomes and 26S proteasomes. In contrast to 20S proteasomes, which were mainly characterized on the basis of their ability to cleave small chromogenic peptide substrates and certain proteins in an ATP-independent manner, 26S proteasomes degrade ubiquitinylated proteins in an ATP-dependent reaction. 20S proteasomes have been found in all eukaryotes from yeast to man. So far 26S proteasomes have only been discovered in higher eukaryotes. We now report the existence of the 26S proteasome in a lower eukaryote, the yeast Saccharomyces cerevisiae. Formation of the 26S proteasome could most effectively be induced in crude extracts of heat stressed yeast cells by incubation with ATP and Mg2+ ions. This treatment yielded a protein complex, which eluted from gel filtration columns at molecular masses higher than 1500 kDa. Besides chromogenic peptide substrates, this complex cleaves ubiquitinylated proteins in an ATP-dependent fashion. In non-denaturing-PAGE, the purified 26S proteasome disintegrated and migrated as four protein bands. One of these bands could be identified as the 20S proteasome. On SDS-PAGE, the 26S proteasome showed a complex pattern of subunit bands with molecular masses between 15 and 100 kDa. Further evidence for the 20S proteasome being the proteolytically active core of the 26S proteasome was obtained by following peptide cleaving activities in extracts of yeast strains carrying mutations in various subunits of the 20S proteasome.

Adenosine Triphosphate↗
PubMed1994

Degradation of the yeast MAT alpha 2 transcriptional regulator is mediated by the proteasome.

Rapid degradation of specific regulatory proteins plays a role in a wide range of cellular phenomena, including cell cycle progression and the regulation of cell growth and differentiation. A major mechanism of selective protein turnover in vivo involves a large multi-subunit protease known as the proteasome or multi-catalytic proteinase. At the same time, the degradation of many cellular proteins requires their covalent ligation to the polypeptide ubiquitin. Here we show that the yeast S. cerevisiae MAT alpha 2 repressor, which is known to be ubiquitinylated in vivo, requires the proteasome for its rapid intracellular proteolysis.

Cysteine Endopeptidases↗
PubMed1994

PRE5 and PRE6, the last missing genes encoding 20S proteasome subunits from yeast? Indication for a set of 14 different subunits in the eukaryotic proteasome core.

The 20S proteasome of eukaryotes is an abundant multicatalytic/multifunctional proteinase complex composed of an array of nonidentical subunits which are encoded by alpha- or beta-type members of the proteasomal gene family. In budding yeast, 14 subunits had been detected and 12 proteasomal genes had been cloned and sequenced so far. Starting from peptide sequences of purified subunits of the yeast 20S proteasome, we cloned two additional proteasomal genes, PRE5 and PRE6, which both encode essential alpha-type subunits. Sequence comparison of all known eukaryotic proteasomal proteins show the presence of a total of 14 subgroups, which can be divided into seven alpha- and seven beta-type groups. Including the Pre5 and Pre6 proteins, every subgroup contains a single yeast member. We anticipate that the 14 genes encoding subunits of the yeast proteasome represent the complete set of proteasomal genes of this organism. The ancestral archaebacterial proteasome is composed of four stacks of rings, the two outer rings containing seven identical alpha-subunits and the inner rings containing seven identical beta-subunits. We speculate that, in analogy to the archaebacterial proteasome, every eukaryotic proteasome is made of two halves of 14 distinct subunits, each half consisting of seven different alpha-type and 7 different beta-type subunits. In higher eukaryotes, subunit isoforms may contribute to variability in the subunit composition of the 20S proteasome allowing functional modulations.

Amino Acid Sequence↗
PubMed1994

Catabolite inactivation of fructose-1,6-bisphosphatase in yeast is mediated by the proteasome.

Fructose-1,6-bisphosphatase, a key enzyme in gluconeogenesis, undergoes catabolite inactivation when glucose is added to gluconeogenetically active cells of the yeast Saccharomyces cerevisiae. Phosphorylation of the enzyme is followed by rapid degradation. To elucidate the cellular proteolytic system involved in catabolite-triggered degradation of fructose-1,6-bisphosphatase this event was followed in different protease-deficient yeast mutants. In a mutant defective in the proteolytic function of the vacuole the degradation rate of the enzyme is not diminished. In contrast mutants defective in the proteolytic activity of the proteasome exhibit a strongly reduced glucose-induced degradation of fructose-1,6-bisphosphatase as compared to their isogenic wild-type counterparts. Our studies suggest that catabolite inactivation of fructose-1,6-bisphosphatase occurs in the cytosol, the degradation event being mediated by the proteasome. An explanation is presented which tries to resolve the formerly conflicting results, which suggested glucose-triggered uptake of fructose-1,6-bisphosphatase into the vacuole followed by vacuolar proteolysis.

Cysteine Endopeptidases↗
PubMed1994

Isolation of autophagocytosis mutants of Saccharomyces cerevisiae.

Protein degradation in the vacuole (lysosome) is an important event in cellular regulation. In yeast, as in mammalian cells, a major route of protein uptake for degradation into the vacuole (lysosome) has been found to be autophagocytosis. The discovery of this process in yeast enables the elucidation of its mechanisms via genetic and molecular biological investigations. Here we report the isolation of yeast mutants defective in autophagocytosis (aut mutants), using a rapid colony screening procedure.

Microscopy, Electron↗