UCSF

The anaphase-promoting complex/cyclosome (APC/C) is a large, multi-subunit E3 ubiquitin ligase that governs key mitotic events in eukaryotes. The APC/C catalyzes the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a protein substrate, building polyubiquitin signals that mark substrates for destruction by the proteasome. In yeast, the APC/C collaborates with two E2s, Ubc4 and Ubc1: APC/CUbc4 catalyzes the attachment of the initial ubiquitin to the substrate, while APC/CUbc1 elongates ubiquitin chains. Both E2s seem to interact with the same site on the APC/C, and it is not clear how their competing activities collaborate to generate a polyubiquitin chain that is sufficient for proteosomal recognition. We hypothesized that E2 synergy requires a finely tuned balance of the affinities of the two E2 proteins for the APC/C, allowing E2s to alternate on the APC/C. In this work, we uncovered new insights into this problem by studying the role of a C-terminal ubiquitin-associated (UBA) domain in Ubc1. Deletion of the UBA domain decreased the length of polyubiquitin chains and increased the concentration of Ubc1 required for half-maximal APC/C activity in vitro. Surprisingly, the stimulatory effect of the UBA domain does not depend on previous initiation of a ubiquitin chain on the substrate, suggesting that the UBA domain does not promote polyubiquitination by interacting with ubiquitin on a substrate. Instead, deletion of the UBA domain reduced Ubc1 binding to the APC/C. Finally, deletion of the UBA domain from Ubc1 decreased its ability to compete with Ubc4 and reduced polyubiquitin chain length, while attachment of the UBA domain to Ubc4 increased its ability to compete with Ubc1 and reduced polyubiquitin chain length. Thus, the extra affinity provided by the UBA domain of Ubc1 ensures efficient polyubiquitination of substrate by balancing Ubc1 affinity with that of Ubc4, resulting in an efficient collaboration between the two E2s.