title: Unc45b forms a cytosolic complex with Hsp90 and targets the unfolded myosin motor domain.
abstract: Myosin folding and assembly in striated muscle is mediated by the general chaperones Hsc70 and Hsp90 and a myosin specific co-chaperone, UNC45. Two UNC45 genes are found in vertebrates, including a striated muscle specific form, Unc45b. We have investigated the role of Unc45b in myosin folding. Epitope tagged murine Unc45b (Unc45b(Flag)) was expressed in muscle and non-muscle cells and bacteria, isolated and characterized. The protein is a soluble monomer in solution with a compact folded rod-shaped structure of approximately 19 nm length by electron microscopy. When over-expressed in striated muscle cells, Unc45b(Flag) fractionates as a cytosolic protein and isolates as a stable complex with Hsp90. Purified Unc45b(Flag) re-binds Hsp90 and forms a stable complex in solution. The endogenous Unc45b in muscle cell lysates is also found associated with Hsp90. The Unc45b(Flag)/Hsp90 complex binds the partially folded myosin motor domain when incubated with myosin subfragments synthesized in a reticulocyte lysate. This binding is independent of the myosin rod or light chains. Unc45b(Flag) does not bind native myosin subfragments consistent with a chaperone function. More importantly, Unc45b(Flag) enhances myosin motor domain folding during de novo motor domain synthesis indicating that it has a direct role in myosin maturation. Thus, mammalian Unc45b is a cytosolic protein that forms a stable complex with Hsp90, selectively binds the unfolded conformation of the myosin motor domain, and promotes motor domain folding.
authors.affiliation: Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey, United States of America.
authors.forename: Rajani
authors.initials: R
authors.lastname: Srikakulam
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.forename: Donald A
authors.initials: DA
authors.lastname: Winkelmann
mesh_terms: D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism*; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006868:Hydrolysis; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D051379:Mice; D018832:Molecular Chaperones; D009132:Muscles / Q000166:cytology / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D018076:DNA, Complementary; D018841:HSP90 Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; C529478:Unc45 protein, mouse; D009218:Myosins