Mechanisms and regulation of ubiquitin-mediated cyclin degradation.

Abstract

We have been studying the biochemical mechanisms of intracellular protein degradation in an ATP-dependent proteolytic system from reticulocytes. Our studies led to the identification of a pathway mediated by ubiquitin (Ub), a highly conserved 76-amino acid residue polypeptide. We found that proteins are committed to degradation by their covalent ligation to Ub. We have also identified several enzymatic reactions in the formation and breakdown of Ub-protein conjugates. Currently available information on the enzymatic reactions of the Ub proteolytic pathway is reviewed in refs. 1–3 and is summarized in Fig. 1. The initial reaction is the activation of the C-terminal Gly residue of Ub by a specific enzyme, E1 (Step 1). Next, activated Ub is further transferred by transacylation to thiol groups of a family of Ub-carrier proteins, E2s (Step 2). Multiple species of E2 were observed in reticulocytes (4) and yeasts (5). Some species of E2 may transfer Ub directly to certain proteins, such as histones (Step 3). In most cases leading to protein degradation, the ligation of Ub to specific proteins requires a third enzyme, E3. The Ub-protein ligase E3 binds suitable substrates (ref. 6, Step 4) and an E2 (7), and thus allows the transfer of Ub from E2 to the protein substrate. Two species of E3, called E3αand E3ß, have been isolated from reticulocytes. E3α binds proteins with basic or hydrophobic N-terminal amino acid residues (8, 9), while E3ß mainly acts on proteins with small, uncharged amino acid residues at the N-terminal position (10). In the ligation process, isopeptide bonds are formed between the C-terminal Gly residue of Ub end ε-amino groups of Lys residues of proteins. In all cases of E3-dependent ligation and in some E3-independent reactions, multiple Ub units are linked to proteins.