In Vitro Assembly and Recognition of Lys-63 Polyubiquitin Chains

Abstract

Polyubiquitin chains assembled through lysine 48 (Lys-48) of ubiquitin act as a signal for substrate proteolysis by 26 S proteasomes, whereas chains assembled through Lys-63 play a mechanistically undefined role in post-replicative DNA repair. We showed previously that the products of the UBC13 and MMS2 genes function in error-free post-replicative DNA repair in the yeast Saccharomyces cerevisiae and form a complex that assembles Lys-63-linked polyubiquitin chains in vitro. Here we confirm that the Mms2⅐Ubc13 complex functions as a high affinity heterodimer in the chain assembly reaction in vitro and report the results of a kinetic characterization of the polyubiquitin chain assembly reaction. To test whether a Lys-63-linked polyubiquitin chain can signal degradation, we conjugated Lys-63-linked tetraubiquitin to a model substrate of 26 S proteasomes. Although the noncanonical chain effectively signaled substrate degradation, the results of new genetic epistasis studies agree with previous genetic data in suggesting that the proteolytic activity of proteasomes is not required for error-free post-replicative repair.

Solution Conformation of Lys63-linked Di-ubiquitin Chain Provides Clues to Functional Diversity of Polyubiquitin Signaling

“…4g. In addition we observed a significant chemical shift perturbation in Phe 24 and weaker but noticeable shifts in peak positions for Gln 21 and Leu 18 , all located in helix 2, which is on the other side of the UBA molecule. These perturbations, also present in the monoUb-UBA complex (36), are probably caused by an indirect effect of the binding.…”

“…E1 was from BostonBiochem Inc., and ethyleneimine was from Chemservice. Yeast Mms2 and Ubc13 were expressed and purified as in previous studies (21,22). Ub (K63C), Ub (K63R), and Ub (Asp 77 ) were expressed and purified as described in Haldeman et al (23).…”

“…Isotope-enriched Ub moieties in Ub 2 s are labeled P for the proximal (with respect to a possible substrate) or D for the distal location in the chain. Lys 63 -linked Ub 2 molecules were synthesized using E1 and the heterodimeric complex of Ubc13/Mms2 (21) in overnight reactions at 37°C. Proximal 15 N-labeled Ub 2 (here referred to as Ub 2 -P) was synthesized by using 15 N-labeled Ub (Asp 77 ) and unlabeled Ub (K63C or K63R as required); distal 15 N-labeled Ub 2 (Ub 2 -D) was synthesized using monomers of 15 N-labeled Ub (K63C or K63R) and unlabeled Ub (Asp 77 ).…”

Innate antiviral response targets HIV-1 release by the induction of ubiquitin-like protein ISG15

“…Although we have not detected ISGylation of Gag or Tsg101, ISG15 was shown to conjugate to Ubc13 (7), Ub carrier protein E2. Ubc13 forms heterodimers with Mms2, and these catalyze assembly of K63-linked polyubiquitination (50). While this study was underway, it was shown that ISGylation of Ubc13 suppresses its ability to form thioester intermediates with Ub (51), which leads to inhibition of K63-linked ubiquitination.…”

Ubiquitination of the Epstein–Barr virus-encoded latent membrane protein 1 depends on the integrity of the TRAF binding site

“…Chains linked through Lys-48 are the principal signal for proteolysis by the proteasome (Hofmann and Pickart, 2001). In order to stabilize the ubiquitin-conjugated products, and ultimately to detect ubiquitin-conjugated LMP1, we generated an expression vector encoding ubiquitin mutated at lysine 48 (K48RUb).…”