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W Hilt

Publications and source records attributed to W Hilt.

14 recordsLinked to original sources
PubMed1998

Mammalian Bax triggers apoptotic changes in yeast.

Apoptosis is co-regulated by the conserved family of Bcl-2-related proteins, which includes both its agonists (Bax) and antagonists (Bcl-X(L)). A mutant strain of the yeast Saccharomyces cerevisiae has been shown to express all morphological signs of apoptosis. Overexpression of Bax is lethal in S. cerevisiae, whereas simultaneous overexpression of Bcl-X(L) rescues the cells. We report that overexpression of mammalian Bax in a S. cerevisiae wild type strain triggers morphological changes similar to those of apoptotic metazoan cells: the loss of asymmetric distribution of plasma membrane phosphatidylserine, plasma membrane blebbing, chromatin condensation and margination, and DNA fragmentation. Simultaneous overexpression of Bcl-X(L) prevents these changes. We demonstrate that Bax triggers phenotypic alterations in yeast strongly resembling those it causes in metazoan apoptotic cells.

Animals↗
PubMed1998

Mutations in the yeast proteasome beta-type subunit Pre3 uncover position-dependent effects on proteasomal peptidase activity and in vivo function.

Proteasomes are highly complex proteases responsible for selective protein degradation in the eukaryotic cell. 26 S proteasomes consist of two regulatory 19 S cap complexes and the 20 S proteasome, which acts as the proteolytic core module. We isolated six mutants of the yeast Saccharomyces cerevisiae containing mutations in the 20 S proteasome beta-type subunit Pre3. Three mutations (pre3-2, pre3-3, and pre3-5) which reside at the active site cleft of the Pre3 subunit solely caused reduction of the proteasomal peptidylglutamyl peptide-hydrolyzing activity but did not lead to detectable defects in protein degradation nor to any other phenotype. However, the pre3-2 mutation strengthened phenotypes induced by other 20 S proteasomal mutations, indicating that the peptidylglutamyl peptide-hydrolyzing activity has to fulfill some rescue functions. The other three mutations (pre3-1, pre3-4, and pre3-6) are located at diverse sites of the Pre3 protein and caused multiple defects in proteasomal peptide cleaving activities. pre3-1 and pre3-6 mutants exhibited significant defects in proteasomal protein degradation; they accumulated ubiquitinated proteins and stabilized defined substrate proteins as, e.g. fructose-1,6-bisphosphatase. In addition, pre3-1 and pre3-6 mutant cells exhibited pleiotropic phenotypes as temperature sensitivity and cell cycle-related effects.

Amino Acid Sequence↗
PubMed1997

Yeast cycloheximide-resistant crl mutants are proteasome mutants defective in protein degradation.

In 1988 McCusker and Haber generated a series of mutants which are resistant to the minimum inhibitory concentration of the protein synthesis inhibitor cycloheximide. These cycloheximide-resistant, temperature-sensitive (crl) mutants, in addition, exhibited other pleiotropic phenotypes, e.g., incorrect response to starvation, hypersensitivity against amino acid analogues, and other protein synthesis inhibitors. Temperature sensitivity of one of these mutants, crl3-2, had been found to be suppressed by a mutation, SCL1-1, which resided in an alpha-type subunit of the 20S proteasome. We cloned the CRL3 gene by complementation and found CRL3 to be identical to the SUG1/CIM3 gene coding for a subunit of the 19S cap complex of the 26S proteasome. Another mutation, crl21, revealed to be allelic with the 20S proteasomal gene PRE3. crl3-2 and crl21 mutant cells show significant defects in proteasome-dependent proteolysis, whereas the SCL1-1 suppressor mutation causes partial restoration of crl3-2-induced proteolytic defects. Notably, cycloheximide resistance was also detected for other proteolytically deficient proteasome mutants (pre1-1, pre2-1, pre3-1, pre4-1). Moreover, proteasomal genes were found within genomic sequences of 9 of 13 chromosomal loci to which crl mutations had been mapped. We therefore assume that most if not all crl mutations reside in the proteasome and that phenotypes found are a result of defective protein degradation.

Adenosine Triphosphatases↗
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

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↗
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

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↗
PubMed1993

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↗
PubMed1993

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↗
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

Glucose dehydrogenase from Bacillus subtilis expressed in Escherichia coli. I: Purification, characterization and comparison with glucose dehydrogenase from Bacillus megaterium.

Escherichia coli containing the Bacillus subtilis glucose dehydrogenase gene on a plasmid (prL7) was used to produce the enzyme in high quantities. Gluc-DH-S was purified from the cell extract by (NH4)2SO4-precipitation, ion-exchange chromatography and Triazine-dye chromatography to a specific activity of 375 U/mg. The enzyme was apparently homogenous on SDS-PAGE with a subunit molecular mass of 31.5 kDa. Investigation of Gluc-DH-S was performed for comparison with the corresponding properties of Gluc-DH-M. The limiting Michaelis constant at pH 8.0 for NAD+ is Ka = 0.11 mM and for D-glucose Kb = 8.7 mM. The dissociation constant for NAD+ is Kia = 17.1 mM. Similar to Gluc-DH-M, Gluc-DH-S is inactivated by dissociation under weak alkaline conditions at pH 9.0. Complete reactivation is attained by readjustment to pH 6.5. Ultraviolet absorption, fluorescence and CD-spectra of native Gluc-DH-S, as well as fluorescence- and CD-backbone-spectra of the dissociated enzyme were nearly identical to the corresponding spectra of Gluc-DH-M. The aromatic CD-spectrum of dissociated Gluc-DH-S was different, representing a residual ellipticity of tryptophyl moieties in the 290-310 nm region. Density gradient centrifugation proved that this behaviour is due to the formation of inactive dimers in equilibrium with monomers after dissociation. In comparison to Gluc-DH-M, the kinetics of inactivation as well as the time-dependent change of fluorescence intensity at pH 9.0 of Gluc-DH-S showed a higher velocity and a changed course of the dissociation process.

Bacillus megaterium↗