Monoubiquitination empowers ubiquitin chain elongation

Ubiquitination often generates lysine 48-linked polyubiquitin chains that signal proteolytic destruction of the protein target. A significant subset of ubiquitination proceeds by a priming/extending mechanism, in which a substrate is fi rst monoubiquitinated with a priming E2-conjugating enzyme or a set of E3 ARIH/E2 enzymes specific for priming. This is then followed by ubiquitin (Ub) chain extension catalyzed by an E2 enzyme capable of elongation. This report provides further insights into the priming/extending mechanism. We employed reconstituted ubiquitination systems of substrates CK1a (casein kinase 1a) and (3-catenin by Cullin-RING E3 Ub ligases (CRLs) CRL4CRBN and CRL1(3TrCP, respectively, in the presence of priming E2 UbcH5c and elongating E2 Cdc34b (cell division cycle 34b). We have established a new " apyrase chase" strategy that uncouples priming from chain elongation, which allows accurate measurement of the decay rates of the ubiquitinated substrate with a defined chain length. Our work has revealed highly robust turnover of monoubiquitinated (3-catenin that empowers efficient polyubiquitination. The results of competition experiments suggest that the interactions between the ubiquitinated (3-catenin and CRL1 (3 TrCP are highly dynamic. Moreover, ubiquitination of the Ub-modified (3-catenin appeared more resistant to inhibition by competitors than the unmodified substrate, suggesting tighter binding with CRL1(3TrCP. These fi ndings support a role for conjugated Ub in enhancing interactions with E3.