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Ubiquitination of proteins involves the concerted action of the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes and E3 ubiquitin-protein ligases. It has been proposed that E3s function as 'docking proteins', specifically binding substrate proteins and specific E2s, and that ubiquitin is then transferred directly from E2s to substrates. We show here that formation of a ubiquitin thioester on E6-AP, an E3 involved in the human papillomavirus E6-induced ubiquitination 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 ubiquitin thioester complexes suggests that E3s have a defined enzymatic activity and do not function simply as docking proteins. The cysteine residue of E6-AP responsible for ubiquitin thioester formation was mapped to a region that is highly conserved among several proteins of unknown function, suggesting that these proteins share the ability to form thioesters with ubiquitin.

The E6 protein of human papillomavirus types 16 and 18 (HPV‐16 and HPV‐18) can stably associate with the p53 protein in vitro. In the presence of rabbit reticulocyte lysate, this association leads to the specific degradation of p53 through the ubiquitin‐dependent proteolysis system. We have examined the E6‐p53 complex in more detail and have found that association of E6 with p53 is mediated by an additional cellular factor. This factor is present in rabbit reticulocyte lysate, primary human keratinocytes and in each of five human cell lines examined. The factor is designated E6‐AP, for E6‐associated protein, based on the observation that the E6 proteins of HPV‐16 and 18 can form a stable complex with the factor in the absence of p53, whereas p53 association with the factor can be detected only in the presence of E6. Gel filtration and coprecipitation experiments indicate that E6‐AP is a monomeric protein of approximately 100 kDa.