Fig. 5. In vitro thioester bond formation assay. (A) Comparison of the C-terminal amino acid sequences of E6-AP and HERC5 are shown. The catalytic site cysteine residues are boxed. Identical residues are marked by asterisks. (B) Wild-type or C833A mutant E6-AP proteins were synthesized in vitro in the presence of L-[35S]methionine and tested for thioester bond formation in the presence (+) or absence (-) of GST-ubiquitin. Reactions were stopped in non-reducing sample buffer for 20 minutes at room temperature and analyzed by 9% SDS-PAGE and autoradiography. Slower migrating proteins are marked by an asterisk. GST-ub, GST-ubiquitin; wt, wildtype. (C) HERC5-HECT or HERC5-HECT C994A proteins were synthesized and processed as in B. Crude bacterial lysates from cells overexpressing a particular E2 enzyme were added to the samples as indicated. Slower migrating proteins are marked by an asterisk.