The p27 mammalian cell cycle protein is an inhibitor of cyclin-dependent kinases. Both in vivo and in vitro, p27 was found to be degraded by the ubiquitin-proteasome pathway. The human ubiquitin-conjugating enzymes Ubc2 and Ubc3 were specifically involved in the ubiquitination of p27. Compared with proliferating cells, quiescent cells exhibited a smaller amount of p27 ubiquitinating activity, which accounted for the marked increase of p27 half-life measured in these cells. Thus, the abundance of p27 in cells is regulated by degradation. The specific proteolysis of p27 may represent a mechanism for regulating the activity of cyclin-dependent kinases.

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that is characterized by the development of multiple vascular tumors and is caused by inactivation of the von Hippel-Lindau protein (pVHL). Here we show that pVHL, through its beta-domain, binds directly to hypoxia-inducible factor (HIF), thereby targeting HIF for ubiquitination in an alpha-domain-dependent manner. This is the first function to be ascribed to the pVHL beta-domain. Furthermore, we provide the first direct evidence that pVHL has a function analogous to that of an F-box protein, namely, to recruit substrates to a ubiquitination machine. These results strengthen the link between overaccumulation of HIF and development of VHL disease.

The ubiquitin-dependent degradation of a test protein beta-galactosidase (beta gal) is preceded by ubiquitination of beta gal. The many (from 1 to more than 20) ubiquitin moieties attached to a molecule of beta gal occur as an ordered chain of branched ubiquitin-ubiquitin conjugates in which the carboxyl-terminal Gly76 of one ubiquitin is jointed to the internal Lys48 of an adjacent ubiquitin. This multiubiquitin chain is linked to one of two specific Lys residues in beta gal. These same Lys residues have been identified by molecular genetic analysis as components of the aminoterminal degradation signal in beta gal. The experiments with ubiquitin mutated at its Lys48 residue indicate that the multiubiquitin chain in a targeted protein is essential for the degradation of the protein.

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...