Substrate Targeting in the Ubiquitin System

As we have seen from the examples discussed above, the recognition mechanisms of many ubiquitin system substrates may be keyed to a fairly limited set of substrate features. Proteins whose degradation rates must be tightly coupled to environmental status, developmental state, or cell cycle stage are often phosphorylated via specific signal transduction cascades. These phosphorylated substrates are targets for particular variants of a large family of multisubunit ubiquitin–protein ligases, the SCF complexes. Interestingly, some of these substrates contain a rather short, closely related phosphopeptide motif.A very different type of degradation determinant, exemplified by the yeast α2 repressor, is a solvent–exposed hydrophobic protein surface. Such exposure usually only occurs in nonnative proteins or in the absence of a protein partner that contacts and buries the surface. Modulation of protein degradation rates by regulated folding or protein–protein interaction will in general not have the temporal precision of a transiently activated phosphorylation cascade, so the recognition of hydrophobic determinants may be utilized primarily for protein quality control and for bringing about changes in proteolytic rate that occur on somewhat longer time scales. It also seems particularly well-suited for ER-associated proteolysis, for the reasons discussed earlier.Notwithstanding these common themes, other types of protein motifs are also clearly recognized by the ubiquitin system. The first described E3 recognition determinant is a surprisingly simple one: the amino acid at the protein N terminus (Varshavsky 1997xVarshavsky, A. Genes Cells. 1997; 2: 13–28Crossref | PubMedSee all ReferencesVarshavsky 1997). In certain proteins, specific N-terminal residues stimulate polyubiquitination and rapid degradation in vivo. A different ubiquitination determinant has been found in the large subunit of yeast RNA polymerase II (Rpb1). Rpb1 is recognized through its conserved carboxy-terminal domain (CTD) by the Rsp5 ubiquitin ligase; this domain is both necessary and sufficient for Rsp5 binding and ubiquitination. The CTD contains the heptapeptide repeat sequence SPTSPSY, which is likely to be recognized by the WW domains of Rsp5. These WW domains, named for their two absolutely conserved tryptophan residues, are protein–protein interaction modules that bind directly to proline-rich sequences or phosphoserine- and phosphothreonine-containing elements in their targets (13xLu, P.J., Zhou, X.Z., Shen, M., and Lu, K.P. Science. 1999; 283: 1325–1328Crossref | PubMed | Scopus (471)See all References, 22xWang, G., Yang, J., and Huibregtse, J.M. Mol. Cell Biol. 1999; 19: 342–352Crossref | PubMedSee all References and references therein). Interestingly, a similar interaction occurs between the human epithelial sodium channel (ENaC) and Nedd4, a mammalian homolog of Rsp5. ENaC, whose impaired degradation in the heritable disorder Liddle’s syndrome is believed to cause hypertension, bears several proline-rich motifs that bind directly to the WW domains of Nedd4 (Goulet et al. 1998xGoulet, C.C., Volk, K.A., Adams, C.M., Prince, L.S., Stokes, J.B., and Snyder, P.M. J. Biol. Chem. 1998; 273: 30012–30017Crossref | PubMed | Scopus (141)See all ReferencesGoulet et al. 1998). These examples reinforce the notion that substrate discrimination can rely on very short sequence motifs, like those found in IκBα and β-catenin.Thus, a few general principles of substrate recognition in the ubiquitin system are at last beginning to emerge, and these should be useful guides for the challenges that lie ahead. These include questions about the details of the E3-substrate interaction and how a substrate can then become polyubiquitinated. Other central issues are how the activity of an E3 is modulated and what controls E2–E3 interactions. Finally, it is known that ubiquitin chains of different lengths and distinct ubiquitin–ubiquitin linkages can form on substrates, so investigation of what regulates the length and topology of these chains will certainly be of interest. There is little doubt that answers to many of these questions will not be long in coming, given the current pace of work in the field.*To whom correspondence should be addressed (e-mail: hoc1@midway.uchicago.edu).