Biophysical and functional study of CRL5^Ozz, 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.