PROTEIN UBIQUITINATION INVOLVING AN E1-E2-E3 ENZYME UBIQUITIN THIOESTER CASCADE

UBIQUITINATION Of proteins involves the concerted action of the El ubi quitin-activating enzyme, E2 ubiquitin-conjugating enzymes and E3 ubiq uitin-protein ligases(1-3). It has been proposed that E3s function as 'docking proteins', specifically binding substrate proteins and specif ic E2s, and that ubiquitin is then transferred directly from E2s to su bstrates(1-5). We show here that formation of a ubiquitin thioester on E6-AP, an E3 involved in the human papillomavirus EB-induced ubiquiti nation of p53 (refs 6-10), is an intermediate step in E6-AP-dependent ubiquitination. The order of ubiquitin transfer is from E1 to E2, from E2 to E6-AP, and finally from E6-AP to a substrate. This cascade of u biquitin thioester complexes suggests that E3s have a defined enzymati c activity and do not function simply as docking proteins. The cystein e residue of E6-AP responsible for ubiquitin thioester formation was m apped to a region that is highly conserved among several proteins of u nknown function, suggesting that these proteins share the ability to f orm thioesters with ubiquitin.