A FAMILY OF PROTEINS STRUCTURALLY AND FUNCTIONALLY RELATED TO THE E6-AP UBIQUITIN PROTEIN LIGASE

E6-AP is a 100-kDa cellular protein that interacts with the E6 protein of the cancer-associated human papillomavirus types 16 and 18. The E6 /E6-AP complex binds to and targets the p53 tumor-suppressor protein f or ubiquitin-mediated proteolysis. E6-AP is an E3 ubiquitin-protein li gase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme i n the form of a thioester and then directly transfers the ubiquitin to targeted substrates, The amino acid sequence of E6-AP shows similarit y to a number of protein sequences over an approximate to 350-aa regio n corresponding to the carboxyl termini of both E6-AP and the E6-AP-re lated proteins. Of particular note is a conserved cysteine residue wit hin the last 32-34 aa, which in E6-AP is likely to be the site of ubiq uitin thioester formation. Two of the E6-AP-related proteins, a rat 10 0-kDa protein and a yeast 95-kDa protein (RSP5), both of previously un known function, are shown here to form thioesters with ubiquitin. Muta tion of the conserved cysteine residue of these proteins destroys thei r ability to accept ubiquitin. These data strongly suggest that the ra t 100-kDa protein and RSP5, as well as the other E6-AP-related protein s, belong to a class of functionally related E3 ubiquitin-protein liga ses, defined by a domain homologous to the E6-AP carboxyl terminus (he ct domain).