The UBA-domain of Ubc1/Ube2K Facilitates the Assembly of K48-/K63-Branched Ubiquitin Chains

Assembly of polymeric ubiquitin (Ub) chains is an essential posttranslational protein modification that regulates widespread intracellular processes in eukaryotic cells. Factors and mechanisms that regulate the formation of Ub chains are key to the understanding of many cellular processes. The E2 enzyme Ubc1 (Ube2K) exclusively targets K48 in Ub for chain formation and has been linked to cell-cycle progression and proteostasis. Uniquely among E2 enzymes, it harbors a Ub binding UBA domain, which has been implicated in Ub chain formation but its function remained elusive. Through in vitro binding experiments, we unexpectedly found that the UBA domain enables preferential binding of K63-linked Ub chains. Based on structural modeling, extensive in vitro ubiquitination experiments and NMR binding studies, we propose a mechanism through which Ubc1 selectively forms K48/K63 branched Ub chains – an usual chain architecture, about whose prevalence and function little is known to date. Ultimately, we link UBA dependent activity of Ubc1 to its role in proteostasis through genetic experiments.