The RING-H2 finger protein APC11 and the E2 enzyme UBC4 are sufficient to ubiquitinate substrates of the anaphase-promoting complex

The anaphase-promoting complex (APC) is a cell cycle-regulated ubiquitin-pr otein ligase that targets cyclin B, securin and other destruction box conta ining proteins for proteolysis. Nine APC subunits have been identified in v ertebrates and eleven in yeast, but for none of them it is known how they c ontribute to the catalysis of ubiquitination reactions. Here we report the mass spectrometric identification of CDC26 and of the RING-H2 finger protei n APC11 in the human APC. We have expressed these proteins and several othe r APC subunits in Escherichia coli and have tested their activities in vitr o. We find that APC11 alone is sufficient to allow the synthesis of multiub iquitin chains in the presence of El and UBC4. These multiubiquitin chains are partly unanchored and partly bound to APC11 itself. APC11 and UBC4 are also able to ubiquitinate securin and cyclin a, but these reactions show a decreased dependency on the destruction box. The integrity of the putative zinc binding RING-H2 finger is required for the ability of APC11 to support ubiquitination reactions. These results suggest that APC11 and UBC4 cataly ze the formation of isopeptide bonds in APC-mediated ubiquitination reactio ns.