Showing papers on "Ubiquitin ligase published in 1992"

Ubiquitin as a degradation signal.

Abstract: For many short-lived eukaryotic proteins, conjugation to ubiquitin, yielding a multiubiquitin chain, is an obligatory pre-degradation step. The conjugated ubiquitin moieties function as a 'secondary' signal for degradation, in that their posttranslational coupling to a substrate protein is mediated by amino acid sequences of the substrate that act as a primary degradation signal. We report that the fusion protein ubiquitin--proline--beta-galactosidase (Ub-P-beta gal) is short-lived in the yeast Saccharomyces cerevisiae because its N-terminal ubiquitin moiety functions as an autonomous, primary degradation signal. This signal mediates the formation of a multiubiquitin chain linked to Lys48 of the N-terminal ubiquitin in Ub-P-beta gal. The degradation of Ub-P-beta gal is shown to require Ubc4, one of at least seven ubiquitin-conjugating enzymes in S.cerevisiae. Our findings provide the first direct evidence that a monoubiquitin moiety can function as an autonomous degradation signal. This generally applicable, cis-acting signal can be used to manipulate the in vivo half-lives of specific intracellular proteins.

In vivo function of the proteasome in the ubiquitin pathway.

Abstract: A major eukaryotic proteolytic system is known to require the covalent attachment of ubiquitin to substrates prior to their degradation, yet the proteinase involved remains poorly defined. The proteasome, a large conserved multi-subunit protein complex of the cytosol and the nucleus, has been implicated in a variety of cellular functions. It is shown here that a yeast mutant with a defective proteasome fails to degrade proteins which are subject to ubiquitin-dependent proteolysis in wild-type cells. Thus, the proteasome is part of the ubiquitin system and mediates the degradation of ubiquitin-protein conjugates in vivo.

Identification of a portable determinant of cell cycle function within the carboxyl-terminal domain of the yeast CDC34 (UBC3) ubiquitin conjugating (E2) enzyme.

Abstract: The ubiquitin conjugating (E2) enzyme encoded by CDC34 (UBC3) in Saccharomyces cerevisiae is required for the G1 to S transition of the cell cycle. CDC34 consists of a 170 residue amino-terminal domain that is homologous to that found in other E2s, followed by a 125 residue carboxyl-terminal domain that is specific to CDC34. We found that a truncation mutant of CDC34 which lacked the CDC34 carboxyl-terminal domain could not support the essential function of CDC34 in the cell cycle in vivo. To explore further the role of the carboxyl-terminal domain in determining the cell cycle function of CDC34, we constructed and characterized genes encoding chimeric E2s incorporating sequences from CDC34 and the related but functionally distinct E2 RAD6 (UBC2). We found that a construct encoding a chimeric RAD6-CDC34 ubiquitin conjugating enzyme, in which the 21 residue acidic carboxyl-terminal domain of RAD6 has been replaced with the 125 residue carboxyl-terminal domain of CDC34, performed the essential functions of CDC34 in vivo. This chimeric E2 also complemented the growth deficiency, UV sensitivity and sporulation deficiency of rad6 mutant strains. Deletion analysis of the CDC34 carboxyl-terminal domain in both CDC34 and the RAD6-CDC34 chimeric E2 identified a region comprising residues 171-244 of CDC34 that was sufficient to confer CDC34 function on the amino-terminal domains of CDC34 and RAD6. We suggest that this region interacts with substrates of CDC34 or with trans-acting factors (such as CDC34-specific ubiquitin protein ligases) that govern the substrate selectivity of CDC34. Congruent results demonstrating a positive role for the carboxyl-terminal domain of CDC34 in the essential function of CDC34 have also been obtained by Silver et al. (1992) and are reported in the accompanying paper.

Drosophila UbcD1 encodes a highly conserved ubiquitin-conjugating enzyme involved in selective protein degradation.

Abstract: Ubiquitin-dependent selective protein degradation serves to eliminate abnormal proteins and provides controlled short half-lives to certain cellular proteins, including proteins of regulatory function such as phytochrome, yeast MAT alpha 2 repressor, p53 and cyclin. Moreover, ubiquitin-dependent proteolysis is thought to play an essential role during development and in programmed cell death. We have cloned a gene from Drosophila melanogaster, UbcD1, coding for a protein with striking sequence similarity to the yeast ubiquitin-conjugating enzymes UBC4 and UBC5. These closely related yeast enzymes are known to be central components of a major proteolytic pathway of Saccharomyces cerevisiae. By doing a precise open reading frame replacement in the yeast genome we could show that the Drosophila UbcD1 enzyme can functionally substitute for yeast UBC4. UbcD1 driven by the UBC4 promoter rescues growth defects and temperature sensitivity of yeast ubc4 ubc5 double mutant cells. Moreover, expression of UbcD1 restores proteolysis proficiency in the ubc4 ubc5 double mutant, indicating that the Drosophila enzyme also mediates protein degradation. This structural and functional conservation suggests that the UbcD1-UBC4-UBC5 class of enzymes defines a major proteolytic pathway in probably all eukaryotes.

Stress-induced alterations in autophagic pathway: relationship to ubiquitin system.

Abstract: The autophagic response of the cell to nutrient deprivation or heat stress is characterized by an increase in the rate of cellular protein degradation. Using temperature-sensitive mutant cell lines that harbor a mutation in the ubiquitin pathway, we have recently shown that this response is dependent on a functional ubiquitin-activating enzyme E1. The ubiquitin pathway is involved in a multitude of cellular events including protein degradation, the best understood of these. Herein the activation of the ubiquitin molecule via E1 is followed by its covalent conjugation to acceptor proteins followed by proteolysis. It is therefore important to study the linkage between the autophagic response and E1. Using these same cell lines, CHO E36 and CHO ts20, we demonstrate that after heat stress or nutrient deprivation there is a rapid and reversible decrease in the buoyant density of subcellular vesicles containing lysosomal hydrolases, a characteristic found to accompany autophagy. This stress-induced change is found in all cell lines examined, independent of the activity of the E1. The light-density vesicles, which comigrate with endosomes on colloidal silica gradients, are not accessible to the endocytic marker transferrin-horseradish peroxidase (HRP) after cellular uptake and subsequent HRP-mediated density shift analysis. Furthermore, morphology of the isolated fractions from control and stress-induced cells was similar. These results thus demonstrate the changes in hydrolase-containing intracellular vesicles that accompany nutritional deprivation or heat stress and support the notion that the linkage of the autophagic response to the ubiquitin system is at a step in autophagy which does not affect the formation of autophagic vesicles.

A ubiquitin conjugating enzyme encoded by African swine fever virus.

Abstract: The post-translational modification of proteins by covalent attachment of ubiquitin occurs in all eukaryotes by a multi-step process. A family of E2 or ubiquitin conjugating (UBC) enzymes catalyse one step of this process and these have been implicated in several diverse regulatory functions. We report here the sequence of a gene encoded by African swine fever virus (ASFV) which has high homology with UBC enzymes. This ASFV encoded enzyme has UBC activity when expressed in Escherichia coli since it forms thiolester bonds with [125I]ubiquitin in the presence of purified ubiquitin activating enzyme (E1) and ATP, and subsequently transfers [125I]ubiquitin to specific protein substrates. These substrates include histones, ubiquitin and the UBC enzyme itself. The ASFV encoded UBC enzyme is similar in structure and enzyme activity to the yeast ubiquitin conjugating enzymes UBC2 and UBC3. This is the first report of a virus encoding a functionally active UBC enzyme and provides an example of the exploitation of host regulatory mechanisms by viruses.

Ubiquitin-mediated protein modification and degradation.

Abstract: Ubiquitin is a small, 8 kD protein found in all eukaryotic cells. It is involved in a wide variety of regulatory roles within the cell, including gene expression, ribosome biosynthesis, receptor expression, and the stress response. The best understood of these is that of ubiquitin-mediated proteolysis, in which ubiquitin is covalently attached to specific protein target substrates that are then recognized and degraded by a high molecular weight protease.

Genetic analysis of ubiquitin-dependent protein degradation

Abstract: Selective degradation of cellular proteins serves to eliminate abnormal proteins and to mediate the turnover of certain short-lived proteins, many of which have regulatory functions. In eukaryotes a major pathway for selective protein degradation is ATP-dependent and is mediated by the ubiquitin system. This pathway involves substrate recognition by components of a ubiquitin-protein ligase system, covalent attachment of ubiquitin moieties to proteolytic substrates, and subsequent degradation of these conjugates by a multicatalytic protease complex. Recent genetic evidence suggests that the remarkable selectivity of this process is largely controlled at the level of substrate recognition by the ubiquitin ligase system. InSaccharomyces cerevisiae, ubiquitin-conjugating enzymes UBC1, UBC4 and UBC5 have been identified as key components of this highly conserved degradation pathway. Genetic analysis indicates that ubiquitin-dependent proteolysis is essential for cell viability and that UBC4 and UBC5 enzymes are essential components of the eukaryotic stress response.

10 The Yeast Ubiquitin System

Abstract: I. INTRODUCTION Ubiquitin is a small protein found in eukaryotic cells either free or covalently joined to a variety of cytoplasmic, nuclear, and integral membrane proteins. Ubiquitination, like phosphorylation, is used in a wide variety of regulatory roles; DNA repair, cell cycle control, and the stress response all require the participation of specific ubiquitinating enzymes. Whereas the phosphorylation of proteins is often used to change their functional state, Ubiquitination appears to serve as a marker that targets a protein for degradation. However, since not all ubiquitinated proteins are selectively degraded, ubiquitin may play nonproteolytic roles as well. The formation, deubiquitination, and degradation of ubiquitin-protein conjugates each involves a large number of gene products. The elucidation of this complex system requires a combination of genetics and biochemistry for which yeast is ideally suited, and yeast has become the principal model sytem for functional if not mechanistic studies of ubiquitination (for previous reviews, see Finley and Chau 1991; Jentsch 1992; Varshavsky 1992). Because the ubiquitin system is highly conserved in evolution, it has been possible to interpret genetic studies of ubiquitination in yeast in light of enzymological work carried out in mammalian extracts. Therefore, some description of the mammalian work is given in this chapter to provide a more complete framework for discussion. II. UBIQUITIN-PROTEIN CONJUGATES Ubiquitin is ligated to acceptor proteins through its carboxyl terminus following an initial activation step. In protein substrates, ɛ-amino groups of lysine residues serve as target sites. Thus, the ubiquitin-protein linkage is in the form of...