Sequence determinants for regulated degradation of yeast 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein

The degradation rate of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-R), a key enzyme of the mevalonate pathway, is regulated through a feedback mechanism by the mevalonate pathway. To discover the intrinsic determinants involved in the regulated degradation of the yeast HMG-R isozyme Hmg2p, we replaced small regions of the Hmg2p transmembrane domain with the corresponding regions from the other, stable yeast HMG-R isozyme Hmg1p. When the first 26 amino acids of Hmg2p were replaced with the same region from Hmg1p, Hmg2p was stabilized. The stability of this mutant was not due to mislocalization, but rather to an inability to be recognized for degradation. When amino acid residues 27-54 of Hmg2p were replaced with those from Hmg1p, the mutant was still degraded, but its degradation rate was poorly regulated. The degradation of this mutant was still dependent on the first 26 amino acid residues and on the function of the HRD genes. These mutants showed altered ubiquitination levels that were well correlated with their degradative phenotypes. Neither determinant was sufficient to impart regulated degradation to Hmg1p. These studies provide evidence that there are sequence determinants in Hmg2p necessary for degradation and optimal regulation, and that independent processes may be involved in Hmg2p degradation and its regulation.     

Effects of overproduction of the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase on squalene synthesis in Saccharomyces cerevisiae

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMG-R) is the major rate-limiting enzyme of the mevalonate pathway in many organisms, including yeasts. In the yeast Saccharomyces cerevisiae, there are two isoenzymes of HMG-R (Hmg1p and Hmg2p). Both consist of an anchoring transmembrane domain and a catalytic domain. We have removed the known controlling features of HMG-R by overproducing the catalytic domain of Hmg1p. This overproduction leads to an enhancement of squalene production, implying that HMG-R has been deregulated. The enhancement is apparent under semianaerobic and aerobic conditions. Despite the increase in squalene production, the amount of ergosterol produced by the HMG-R-overproducing yeast was not increased. This result suggests the presence of another regulatory step between squalene and ergosterol formation. Squalene levels generated by cells overproducing the catalytic domain of HMG-R were estimated to be up to 10 times those produced by wild-type cells. The enhancement in squalene production coincided with a reduction in growth rate. This reduction may be a direct consequence of the buildup of high concentrations of squalene and presqualene intermediates of the pathway.     

TPR proteins required for anaphase progression mediate ubiquitination of mitotic B-type cyclins in yeast

The abundance of B-type cyclin-CDK complexes is determined by regulated synthesis and degradation of cyclin subunits. Cyclin proteolysis is required for the final exit from mitosis and for the initiation of a new cell cycle. In extracts from frog or clam eggs, degradation is accompanied by ubiquitination of cyclin. Three genes, CDC16, CDC23, and CSE1 have recently been shown to be required specifically for cyclin B proteolysis in yeast. To test whether these genes are required for cyclin ubiquitination, we prepared extracts from G1-arrested yeast cells capable of conjugating ubiquitin to the B-type cyclin Clb2. The ubiquitination activity was cell cycle regulated, required Clb2's destruction box, and was low if not absent in cdc16, cdc23, cdc27, and cse1 mutants. Furthermore all these mutants were also defective in ubiquitination of another mitotic B-type cyclin, Clb3. The Cdc16, Cdc23, and Cdc27 proteins all contain several copies of the tetratricopeptide repeat and are subunits of a complex that is required for the onset of anaphase. The finding that gene products that are required for ubiquitination of Clb2 and Clb3 are also required for cyclin proteolysis in vivo provides the best evidence so far that cyclin B is degraded via the ubiquitin pathway in living cells. Xenopus homologues of Cdc16 and Cdc27 have meanwhile been shown to be associated with a 20S particle that appears to function as a cell cycle-regulated ubiquitin-protein ligase.     

The ubiquitin-like proteins SMT3 and SUMO-1 are conjugated by the UBC9 E2 enzyme

The ubiquitin-like protein SMT3 from Saccharomyces cerevisiae and SUMO-1, its mammalian homolog, can be covalently attached to other proteins posttranslationally. Conjugation of ubiquitin requires the activities of ubiquitin-activating (E1) and -conjugating (E2) enzymes and proceeds via thioester-linked enzyme-ubiquitin intermediates. Herein we show that UBC9, one of the 13 different E2 enzymes from yeast, is required for SMT3 conjugation in vivo. Moreover, recombinant yeast and mammalian UBC9 enzymes were found to form thioester complexes with SMT3 and SUMO-1, respectively. This suggests that UBC9 functions as an E2 in a SMT3/SUMO-1 conjugation pathway analogous to ubiquitin-conjugating enzymes. The role of yeast UBC9 in cell cycle progression may thus be mediated through its SMT3 conjugation activity.     

9,13-di-cis-Retinoic acid induces the production of tPA and activation of latent TGF-beta via RAR alpha in a human liver stellate cell line, LI90

Fas(Apo-1/CD95), a receptor belonging to the tumor necrosis factor receptor family, induces apoptosis when triggered by Fas ligand. Upon its activation, the cytoplasmic domain of Fas binds several proteins which transmit the death signal. We used the yeast two-hybrid screen to isolate Fas-associated proteins. Here we report that the ubiquitin-conjugating enzyme UBC9 binds to Fas at the interface between the death domain and the membrane-proximal region of Fas. This interaction is also seen in vivo. UBC9 transiently expressed in HeLa cells bound to the co-expressed cytoplasmic segment of Fas. FAF1, a Fas-associated protein that potentiates apoptosis (Chu et al. (1996) Proc. Natl. Acad. Sci. USA 92, 11894-11898), was found to contain sequences similar to ubiquitin. These results suggest that proteins related to the ubiquitination pathway may modulate the Fas signaling pathway.     

Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae

Combinations of different ubiquitin-conjugating (Ubc) enzymes and other factors constitute subsidiary pathways of the ubiquitin system, each of which ubiquitinates a specific subset of proteins. There is evidence that certain sequence elements or structural motifs of target proteins are degradation signals which mark them for ubiquitination by a particular branch of the ubiquitin system and for subsequent degradation. Our aim was to devise a way of searching systematically for degradation signals and to determine to which ubiquitin system subpathways they direct the proteins. We have constructed two reporter gene libraries based on the lacZ or URA3 genes which, in Saccharomyces cerevisiae, express fusion proteins with a wide variety of C-terminal extensions. From these, we have isolated clones producing unstable fusion proteins which are stabilized in various ubc mutants. Among these are 10 clones whose products are stabilized in ubc6, ubc7 or ubc6ubc7 double mutants. The C-terminal extensions of these clones, which vary in length from 16 to 50 amino acid residues, are presumed to contain degradation signals channeling proteins for degradation via the UBC6 and/or UBC7 subpathways of the ubiquitin system. Some of these C-terminal tails share similar sequence motifs, and a feature common to almost all of these sequences is a highly hydrophobic region such as is usually located inside globular proteins or inserted into membranes.     

Mdm2 association with p53 targets its ubiquitination

Key to p53 ability to mediate its multiple cellular functions lies in its stability. In the present study we have elucidated the mechanism by which Mdm2 regulates p53 degradation. Using in vitro and in vivo ubiquitination assays we demonstrate that Mdm2 association with p53 targets p53 ubiquitination. Exposure of cells to UV-irradiation inhibits this targeting. Mdm2 which is deficient in p53 binding failed to target p53 ubiquitination, suggesting that the association is essential for Mdm2 targeting ability. While mdm2-p53 complex is found in non-stressed cells, the amount of p53-bound mdm2 is decreased after UV-irradiation, further pointing to the relationship between mdm2 binding and p53 level. Similar to Swiss 3T3 cells, the dissociation of mdm2-p53 complex was also found in UV-treated Scid cells, lacking functional DNA-PK, suggesting that DNA-PK is not sufficient for dissociating mdm2 from p53. Together our studies point to the role of Mdm2, as one of p53-associated proteins, in targeting p53 ubiquitination.     

The ubiquitin-mediated proteolytic pathway as a therapeutic area

Ubiquitin-mediated proteolysis is involved in the turnover of many short-lived regulatory proteins. This pathway leads to the covalent attachment of one or more multiubiquitin chains to target substrates which are then degraded by the 26S multicatalytic proteasome complex. Multiple classes of regulatory enzymes have been identified that mediate either ubiquitin conjugation or ubiquitin deconjugation from target substrates. Timed destruction of cellular regulators by the ubiquitin-proteasome pathway plays a critical role in ensuring normal cellular processes. This review provides multiple examples of key growth regulatory proteins whose levels are regulated by ubiquitin-mediated proteolysis. Pharmacological intervention which alters the half-lives of these cellular proteins may have wide therapeutic potential. Specifically, prevention of p53 ubiquitination (and subsequent degradation) in human papilloma virus positive tumors, and perhaps all tumors retaining wild-type p53 but lacking the retinoblastoma gene function, should lead to programmed cell death. Specific inhibitors of p27 and cyclin B ubiquitination are predicted to be potent antiproliferative agents. Inhibitors of IkappaB ubiquitination should prevent NFkappaB activation and may have utility in a variety of autoimmune and inflammatory conditions. Finally, we present a case for deubiquitination enzymes as novel, potential drug targets.     

Cdc53 targets phosphorylated G1 cyclins for degradation by the ubiquitin proteolytic pathway

In budding yeast, cell division is initiated in late G1 phase once the Cdc28 cyclin-dependent kinase is activated by the G1 cyclins Cln1, Cln2, and Cln3. The extreme instability of the Cln proteins couples environmental signals, which regulate Cln synthesis, to cell division. We isolated Cdc53 as a Cln2-associated protein and show that Cdc53 is required for Cln2 instability and ubiquitination in vivo. The Cln2-Cdc53 interaction, Cln2 ubiquitination, and Cln2 instability all depend on phosphorylation of Cln2. Cdc53 also binds the E2 ubiquitin-conjugating enzyme, Cdc34. These findings suggest that Cdc53 is a component of a ubiquitin-protein ligase complex that targets phosphorylated G1 cyclins for degradation by the ubiquitin-proteasome pathway.     

Role of newly synthesized cholesterol or its metabolites on the regulation of bile acid biosynthesis after short-term biliary diversion in the rat

Cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in the bile acid biosynthetic pathway, is thought to be regulated by hydrophobic bile acids through negative feedback control. The role of cholesterol in the regulation of cholesterol 7 alpha-hydroxylase is more controversial, in part because of incomplete understanding of the relationship between the pathways of cholesterol synthesis and degradation. The main objective of this study was to define the interaction between these two pathways in an experimental model in which the supply of newly synthesized cholesterol was interrupted by sustained infusion of mevinolin (lovastatin), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) or accelerated by a continuous infusion of mevalonate, a cholesterol precursor. The study was carried out in rats subjected to short-term bile fistula. In one set of experiments, rats were treated postoperatively with mevinolin (5 mg/kg loading dose followed by 2 mg/kg/hr infusion), mevalonate (180 mumol/hr infusion) or both for up to 96 hr. In a separate set of experiments, rats were infused intraduodenally with taurocholate (36 mumol/100 gm/hr for up to 96 hr). We determined cholesterol 7 alpha-hydroxylase- and HMG-CoA reductase specific activities at those time intervals, whereas bile acid synthesis rates were determined throughout the study. Compared with rats not subjected to surgery, rats with short-term biliary diversion had increases in cholesterol 7 alpha-hydroxylase activity of 259% and 827% at 48 and 96 hr, respectively. The increase in bile acid biosynthesis was less pronounced. Continuous infusion of mevinolin completely prevented increases in cholesterol 7 alpha-hydroxylase specific activity and bile acid biosynthesis at both time intervals.(ABSTRACT TRUNCATED AT 250 WORDS)     

An evolutionarily conserved cysteine protease, human bleomycin hydrolase, binds to the human homologue of ubiquitin-conjugating enzyme 9

Bleomycin hydrolase (BH) is a highly conserved cysteine proteinase that deamidates and inactivates the anticancer drug bleomycin. Yeast BH self-assembles to form a homohexameric structure, which resembles a 20 S proteasome and may interact with other proteins. Therefore, we searched for potential human BH (hBH) partners using the yeast two-hybrid system with a HeLa cDNA library and identified the full-length human homologue of yeast ubiquitin-conjugating enzyme 9 (UBC9). Cotransformation assays using hBH deletion mutants revealed that the carboxyl terminus of hBH (amino acids 356-455), which contains two of the three essential catalytic amino acids, was not critical for protein binding in the yeast two-hybrid environment. In vitro translated human UBC9 was precipitated by glutathione S-transferase-hBH fusion protein but not by glutathione S-transferase. Efficient in vitro binding occurred in the absence of the first 24 amino acids of UBC9 and the catalytic Cys93 of UBC9. We confirmed that hBH and UBC9 interacted in vivo by affinity copurification of proteins overexpressed in mammalian cells. Using immunocytochemical analysis, hBH was colocalized with UBC9. Coexpression of hBH and UBC9 in mammalian cells did not markedly alter the bleomycin-hydrolyzing activity of hBH or apparent small ubiquitin-related modifier 1 addition. This is the first reported heteromeric interaction with hBH, and it suggests a role for hBH in intracellular protein processing and degradation.     

Contractile roles of the M2 and M3 muscarinic receptors in the guinea pig colon

Ubiquitin-dependent proteolytic systems underlie many processes, including the cell cycle, cell differentiation and responses to stress. One such system is the N-end rule pathway, which targets proteins bearing destabilizing N-terminal residues. Here we report that Ubr1p, the main recognition component of this pathway, regulates peptide import in the yeast Saccharomyces cerevisiae through degradation of Cup9p, a 35 kDa homeodomain protein. Cup9p was identified using a screen for mutants that bypass the previously observed requirement for Ubr1p in peptide import. We show that Cup9p is a short-lived protein (t1/2 approximately 5 min) whose degradation requires Ubr1p. Cup9p acts as a repressor of PTR2, a gene encoding the transmembrane peptide transporter. In contrast to engineered N-end rule substrates, which are recognized by Ubr1p through their destabilizing N-terminal residues, Cup9p is targeted by Ubr1p through an internal degradation signal. The Ubr1p-Cup9p-Ptr2p circuit is the first example of a physiological process controlled by the N-end rule pathway. An earlier study identified Cup9p as a protein required for an aspect of resistance to copper toxicity in S.cerevisiae. Thus, one physiological substrate of the N-end rule pathway functions as both a repressor of peptide import and a regulator of copper homeostasis.     

Cloning of human ubiquitin-conjugating enzymes UbcH6 and UbcH7 (E2-F1) and characterization of their interaction with E6-AP and RSP5

E6-AP, a 100-kDa cellular protein, was originally identified through its interaction with the E6 protein of the oncogenic human papillomavirus types 16 and 18. The complex of E6-AP and E6 specifically interacts with p53 and mediates ubiquitination of p53 in concert with the E1 ubiquitin-activating enzyme and the E2 ubiquitin-conjugating enzyme UbcH5. Recent results suggest that E6-AP is representative of a family of putative ubiquitin-protein ligases. Members of this family are characterized by a conserved C-terminal region, termed hect domain. In this paper, we describe the isolation of two human E2s, designated as UbcH6 and UbcH7, that in addition to UbcH5 can interact with E6-AP. UbcH6 is a novel member of an evolutionally conserved subfamily of E2s that includes UbcH5 and Saccharomyces cerevisiae UBC4. Although UbcH7 does not appear to be a member of this subfamily, UbcH7 efficiently substitutes for UbcH5 in E6-AP-dependent ubiquitination. Surprisingly, UbcH6 was only weakly active in this particular assay. In addition, UbcH5 but not UbcH6 or UbcH7 efficiently interacts with the heet protein RSP5. These results indicate that E6-AP can interact with at least two species of E2 and that different hect proteins may interact with different E2s.     

The breast cancer gene product TSG101: a regulator of ubiquitination?

Sequence analysis is a powerful tool to obtain structural and functional information about genes and their products. Here we show that TSG101, a gene subjected to somatic mutations in breast cancer, contains an amino terminal domain that is a homologue of ubiquitin conjugating enzymes (UBCs) and not, as previously proposed, DNA-binding domains. As the UBC active site residue is replaced in the TSG101 sequence in a similar manner to several other members of the UBC family, we propose a role for TSG101 in regulating the ubiquitination of short-lived gene products.     

Linkage of the ubiquitin-conjugating system and the endocytic pathway in ligand-induced internalization of the growth hormone receptor

The major function of the ubiquitin-conjugating system is the targeting of cytosolic and nuclear proteins for degradation by the proteasome. Recently, ubiquitin conjugation has been implicated in the downregulation of signalling receptors such as the mammalian growth hormone receptor (GHR) and the alpha-factor receptor in yeast. By examining truncated receptors, the internalization-deficient receptor mutant F327A and conditions under which clathrin-mediated GHR endocytosis is inhibited, we show here that GHR ubiquitination and ligand-induced GHR internalization are coupled events. Previously, we had shown that GHR endocytosis is dependent on an intact ubiquitination system. Here we present evidence that GHR ubiquitination depends on an intact endocytic pathway. Our data indicate that the ubiquitin-conjugating system and the endocytic pathway interact at the cytoplasmic tail of the GHR at the plasma membrane, where they cooperate to regulate internalization of the GHR.     

A novel component involved in ubiquitination is required for development of Dictyostelium discoideum

A novel component of the ubiquitination system, called NOSA, is essential for cellular differentiation in Dictyostelium discoideum. Disruption of nosA does not affect the growth rate but causes an arrest in development after the cells have aggregated. nosA contains seven exons and codes for a developmentally regulated 3.5-kb mRNA. The 125-kDa NOSA protein is present in the cytosol at constant levels during growth and development. The C-terminal region of NOSA has homology with ubiquitin fusion degradation protein-2 (UFD2) of Saccharomyces cerevisiae and putative homologs in Caenorhabditis elegans and humans. UFD2 is involved in the ubiquitin-mediated degradation of model substrates in which ubiquitin forms part of the translation product, but ufd2 mutants have no detected phenotype. In accord with the homology to UFD2, we found differences in the ubiquitination patterns between nosA mutants and their parental cell line. While general in vivo and in vitro ubiquitination is minimally affected, ubiquitination of individual proteins is altered throughout growth and development in nosA mutants. These findings suggest that events involving ubiquitination are critical for progression through the aggregate stage of the Dictyostelium life cycle.