Biophysical and functional study of CRL5Ozz, a muscle specific ubiquitin ligase complex

Ozz, a member of the SOCS-box family of proteins, is the substrate-binding component of CRL5 Ozz , a muscle-specific Cullin-RING ubiquitin ligase complex composed of Elongin B/C, Cullin 5 and Rbx1. CRL5 Ozz targets for proteasomal degradation selected pools of substrates, including sarcolemma-associated β-catenin, sarcomeric MyHC emb and Alix/PDCD6IP, which all interact with the actin cytoskeleton. Ubiquitination and degradation of these substrates are required for the remodeling of the contractile sarcomeric apparatus. However, how CRL5 Ozz assembles into an active E3 complex and interacts with its substrates remain unexplored. Here, we applied a baculovirus-based expression system to produce large quantities of two subcomplexes, Ozz–EloBC and Cul5–Rbx1. We show that these subcomplexes mixed in a 1:1 ratio reconstitutes a five-components CRL5 Ozz monomer and dimer, but that the reconstituted complex interacts with its substrates only as monomer. The in vitro assembled CRL5 Ozz complex maintains the capacity to polyubiquitinate each of its substrates, indicating that the protein production method used in these studies is well-suited to generate large amounts of a functional CRL5 Ozz . Our findings highlight a mode of assembly of the CRL5 Ozz that differs in presence or absence of its cognate substrates and grant further structural studies.