The ubiquitin system plays an important role in endoplasmic reticulum (ER)-associated degradation of proteins that are misfolded, that fail to associate with their oligomerization partners, or whose levels are metabolically regulated. E3 ubiquitin ligases are key enzymes in the ubiquitination process as they recognize the substrate and facilitate coupling of multiple ubiquitin units to the protein that is to be degraded. The Saccharomyces cerevisiae ER-resident E3 ligase Hrd1p/Der3p functions in the metabolically regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and additionally facilitates the degradation of a number of misfolded proteins from the ER. In this study we characterized the structure and function of the putative human orthologue of yeast Hrd1p/Der3p, designated human HRD1. We show that human HRD1 is a nonglycosylated, stable ER protein with a cytosolic RING-H2 finger domain. In the presence of the ubiquitin-conjugating enzyme UBC7, the RING-H2 finger has in vitro ubiquitination activity for Lys 48 -specific polyubiquitin linkage, suggesting that human HRD1 is an E3 ubiquitin ligase involved in protein degradation. Human HRD1 appears to be involved in the basal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase but not in the degradation that is regulated by sterols. Additionally we show that human HRD1 is involved in the elimination of two model ER-associated degradation substrates, TCR-␣ and CD3-␦.When a newly synthesized protein molecule is translocated into the ER, 1 there is a fair chance that it may never reach its final destination as a functional molecule, since a significant proportion of newly synthesized proteins is degraded via the endoplasmic reticulum-associated degradation (ERAD) pathway (1). In particular, proteins that misfold along the folding pathway or cannot be appropriately folded as a result of mutations are degraded via this route. The cystic fibrosis transmembrane conductance regulator (CFTR) and its common mutation ⌬F508 in cystic fibrosis serve as an example in this context (2). In addition, proteins that lack their oligomerization partner(s) are prone to degradation, e.g. individual subunits of the T-cell receptor like TCR-␣ and CD3-␦ (3). Finally, ERAD also functions in the homeostatic regulation of metabolic pathways to degrade proteins whose activity needs to be attenuated at a certain metabolic state. Examples include 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) (4), which is further described below, and apolipoprotein B (5).Degradation of proteins from the ER requires dislocation of the substrate from the ER to the cytosol followed by proteolysis via the ubiquitin-proteasome pathway. The dislocation process is thought to require components of the translocon channel, including Sec61␣ (6 -8), as well as a complex of proteins designated CDC48/p97-Ufd1-Npl4 (9 -11). Ubiquitination also plays an essential role in dislocation as illustrated by the inhibition of protein dislocation when the ubiquitination machinery is disrupted (9...

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The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), 1 catalyzes the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A to MVA, the major regulatory step in the MVA pathway that leads to the synthesis of cholesterol and a variety of essential nonsterol isoprenoids. The content of HMGR is controlled according to the cellular demands for sterols and nonsterol products derived from MVA (see Ref. 1 for review). This metabolic control is achieved through changes in the rate of HMGR gene transcription (2-5), altered stability and translational efficiency of its mRNA (6 -10), and by accelerated degradation of the enzyme when these requirements have been satisfied (7,11,12).HMGR is a 97-kDa integral membrane glycoprotein of the ER, which consists of a C-terminal catalytic domain that faces the cytoplasm and a non-catalytic N-terminal membrane domain that anchors the enzyme in the ER (13,14). This hydrophobic domain, with its eight membrane spans (15), constitutes the cis-acting element that is necessary and sufficient to regulate the enzyme's stability (16 -19).The signaling pathway(s) and proteolytic machinery that is responsible for the degradation of mammalian HMGR in response to metabolic cues remain largely unknown. Studies by Simoni and coworkers (20,21) have revealed that degradation of HMGR takes place in a pre-Golgi compartment in a process that requires ongoing protein synthesis. Moreover, HMGR degradation is blocked by ALLN and ALLM, known inhibitors of calpains, which also inhibit proteasome activity, as well as by the more specific proteasome inhibitor lactacystin (22-24). These latter observations suggested that the proteasome, or an as yet unidentified lactacystin-sensitive protease(s), is involved either directly or indirectly in the degradation of HMGR.Genetic studies in Saccharomyces cerevisiae have identified several factors that are involved in the degradation of HMGR. Similar to the mammalian enzyme, the stability of the yeast HMGR isozyme Hmg2p is controlled by the intracellular levels of MVA-derived metabolite(s) (25,26). At least three genes, HRD1, HRD2, and HRD3 were implicated in Hmg2p degradation. Hrd1p/Der3p and Hrd3p are generally involved in the reverse translocation of ER membrane and luminal proteins for their disposal in the cytoplasmic ubiquitin-proteasome pathway (27-30), and Hrd2p was identified as a subunit of the 26 S proteasome (26). Hmg2p degradation is also strongly dependent on the activity of the ubiquitin-conjugating enzyme Ubc7p (26,31). Together, these results demonstrated that the ubiquitin-proteasome pathway mediates the degradation of Hmg2p in yeast. Indeed, regulated attachment of polyubiquitin chains to Hmg2p was directly demonstrated, with farnesol being implicated as the MVA-derived metabolic product that signals for this process (32, 33). Both ubiquitination and regulated turnover of Hmg2p required specific structural determinants within the membrane domain of the enzyme (34 -36).In the current study, we demonstrate that, similar to Hmg2p, the metabol...

The stability of the endoplasmic reticulum (ER) glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the key enzyme in cholesterol biosynthesis, is negatively regulated by sterols. HMGR is anchored in the ER via its N-terminal region, which spans the membrane eight times and contains a sterolsensing domain. We have previously established that degradation of mammalian HMGR is mediated by the ubiquitin-proteasome system (Ravid, T., Doolman, R., Avner, R., Harats, D., and Roitelman, J. (2000) 89 and Lys 248 attenuate ubiquitination at the latter residues. The ATP-dependent ubiquitination of HMGR in isolated microsomes requires E1 as the sole cytosolic protein, indicating that ER-bound E2 and E3 enzymes catalyze this modification. Polyubiquitination of HMGR is correlated with its extraction from the ER membrane, a process likely to be assisted by cytosolic p97/VCP/Cdc48p-Ufd1-Npl4 complex, as only ubiquitinated HMGR pulls down p97.The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) 1 catalyzes the rate-limiting production of mevalonate, the committed precursor for the biosynthesis of sterols and a myriad of essential nonsterol isoprenoids. The intracellular levels of HMGR are regulated by the cellular needs for sterol and nonsterol metabolites. This regulation involves changes in the transcription of the HMGR gene and, at the post-translational level, alteration of enzyme stability (1-3). Thus, when demands for sterols are high, HMGR gene is transcribed at a high rate, and the resulting HMGR protein is relatively stable. When the requirements for mevalonate-derived metabolites have been satisfied, transcription ceases, and the enzyme is rapidly degraded (1-3).