The Moonlighting Mechanisms of UNC-45 for Keeping Muscle Myosin in Shape

Abstract

Muscle development requires the coordinated activities of specific protein folding and degradation factors. UNC-45, for example, is a highly specialized chaperone that orchestrates myosin folding and assembly. Structural and biochemical data from our lab demonstrated that UNC-45 can form linear protein chains offering multiple binding sites for co-working chaperones and client proteins. Accordingly, Hsp70 and Hsp90, which bind to the TPR domain of UNC-45, can act in concert and with defined periodicity on captured myosin molecules. The identified multi-chaperone complex represents a novel type of filament assembly factor coupling myosin folding with myofilament formation. Strikingly, UNC-45 does not only team-up with Hsp70/Hsp90, but also with UFD-2, a U-box ubiquitin ligase previously implicated in regulating UNC-45 levels. To better understand the UNC-45/UFD-2 interplay, we studied the substrate-targeting mechanism of UFD-2 and its distinct mechanistic features as an E4 ubiquitination enzyme (a so-called processivity factor, extending preformed Ub chains). Using Caenorhabditis elegans as model system, we demonstrate that UFD-2 is not regulating the protein levels of UNC-45 in muscle cells, but rather shows characteristic properties of a bona-fide E3 ligase involved in protein quality control. Our data demonstrate that UFD-2 preferentially targets unfolded protein segments. Moreover, the UNC-45 chaperone can serve as an adaptor protein of UFD-2 to poly-ubiquitinate unfolded myosin, pointing to a possible role of the UFD-2/UNC-45 pair in maintaining proteostasis in muscle cells. These data highlight the moonlighting function of UNC-45 in promoting both the folding and the degradation of myosin, depending on its partner proteins.