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...

Misfolded proteins are eliminated from the endoplasmic reticulum (ER) by retrotranslocation into the cytosol, a pathway hijacked by certain viruses to destroy MHC class I heavy chains. The translocation of polypeptides across the ER membrane requires their polyubiquitination and subsequent extraction from the membrane by the p97 ATPase [also called valosin-containing protein (VCP) or, in yeast, Cdc48]. In higher eukaryotes, p97 is bound to the ER membrane by a membrane protein complex containing Derlin-1 and VCP-interacting membrane protein (VIMP). How the ubiquitination machinery is recruited to the p97͞Derlin͞VIMP complex is unclear. Here, we report that p97 interacts directly with several ubiquitin ligases and facilitates their recruitment to Derlin-1. During retrotranslocation, a substrate first interacts with Derlin-1 before p97 and other factors join the complex. These data, together with the fact that Derlin-1 is a multispanning membrane protein forming homo-oligomers, support the idea that Derlin-1 is part of a retrotranslocation channel that is associated with both the polyubiquitination and p97-ATPase machineries.AAA ATPase ͉ ER-associated degradation ͉ dislocation ͉ Derlin ͉ Hrd1