De novo folding and assembly of striated muscle myosin was analyzed by expressing a GFP-tagged embryonic myosin heavy chain (GFP-myosin) in post-mitotic C2C12 myocytes using replication defective adenoviruses. In the early stages of muscle differentiation, the GFP-myosin accumulates in bright globular foci and short filamentous structures that are later replaced by brightly fluorescent myofibrils. Time-lapse microscopy shows that the intermediates are dynamic and are present in elongating and fusing myocytes and in multinucleated myotubes. Immunostaining reveals the co-localization of the molecular chaperones Hsc70 and Hsp90 with the GFP-myosin in the intermediates, but not in the mature myofibrils. Uninfected cells have similar intermediates suggesting a common pathway for myosin maturation. Two conformation-sensitive antibodies that bind the unfolded motor domain and the coiled-coil conformation of the rod demonstrate that in the intermediates, the myosin rod is folded but the motor domain is not folded. Electron microscopy reveals that the intermediates contain loose filament bundles surrounded by a protein rich matrix. Geldanamycin, a specific inhibitor of Hsp90, reversibly blocks myofibril assembly and triggers accumulation of myosin folding intermediates. We conclude that multimeric complexes of nascent myosin filaments associated with Hsc70 and Hsp90 are intermediates in the folding and assembly pathway of muscle myosin.

Myosin folding and assembly in striated muscle are mediated by the general chaperones Hsc70 and Hsp90 and involve a myosin-specific co-chaperone related to the Caenorhabditis elegans gene unc-45. Two unc-45 genes are found in vertebrates, a general cell isoform, unc45a, and a striated muscle-specific isoform, unc45b. We have investigated the role of both isoforms of mouse Unc45 in myosin folding using an in vitro synthesis and folding assay. A smooth muscle myosin motor domain (MD) fused to green fluorescent protein (GFP) (MD::GFP) was used as substrate, and folding was measured by native gel electrophoresis and functional assays. In the absence of Unc45, the MD::GFP chimera folds poorly. Addition of either Unc45a or Unc45b dramatically enhances the folding in a reaction that is dependent on Hsp90 ATPase activity. Unc45a is more effective than Unc45b with a higher apparent affinity and greater extent of folding. The Unc45-Hsp90 chaperone complex acts late in the folding pathway and promotes motor domain maturation after release from the ribosome. Unc45a behaves kinetically as an activator of the folding reaction by stimulating the rate of the Hsp90-dependent folding by >20-fold with an apparent K act of 33 nM. This analysis of vertebrate Unc45 isoforms clearly demonstrates a direct role for Unc45 in Hsp90-mediated myosin motor domain folding and highlights major differences between the isoforms in substrate specificity and mechanism.

The folding pathway of the heavy meromyosin subfragment (HMM) of a skeletal muscle myosin has been investigated by in vitro synthesis of the myosin heavy and light chains in a coupled transcription and translation assay. Analysis of the nascent translation products for folding intermediates has identified a major intermediate that contains all three myosin subunits in a complex with the eukaryotic cytosolic chaperonin. Partially folded HMM is released from this complex in an ATP-dependent manner. However, biochemical and functional assays reveal incomplete folding of the myosin motor domain. Dimerization of myosin heavy chains and association of heavy and light chains are accomplished early in the folding pathway. To test for other factors necessary for the complete folding of myosin, a cytoplasmic extract was prepared from myotubes produced by a mouse myogenic cell line. This extract dramatically enhanced the folding of HMM, suggesting a role for muscle-specific factors in the folding pathway. We conclude that the molecular assembly of myosin is mediated by the eukaryotic cytosolic chaperonin with folding of the motor domain as the slow step in the pathway.

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 (Unc45bFlag) 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 ∼19 nm length by electron microscopy. When over-expressed in striated muscle cells, Unc45bFlag fractionates as a cytosolic protein and isolates as a stable complex with Hsp90. Purified Unc45bFlag re-binds Hsp90 and forms a stable complex in solution. The endogenous Unc45b in muscle cell lysates is also found associated with Hsp90. The Unc45bFlag/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. Unc45bFlag does not bind native myosin subfragments consistent with a chaperone function. More importantly, Unc45bFlag 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.

We have investigated the folding of the myosin motor domain using a chimera of an embryonic striated muscle myosin II motor domain fused on its COOH terminus to a thermal stable, fast folding variant of green fluorescent protein (GFP). In in vitro expression assays, the GFP domain of the chimeric protein, S1 795 GFP, folds rapidly enabling us to monitor the folding of the motor domain using fluorescence. The myosin motor domain folds very slowly and transits through multiple intermediates that are detectable by gel filtration chromatography. The distribution of the nascent protein among these intermediates is strongly dependent upon temperature. At 25°C and above the predominant product is an aggregate of S1 795 GFP or a complex with other lysate proteins. At 0°C, the motor domain folds slowly via an energy independent pathway. The unusual temperature dependence and slow rate suggests that folding of the myosin motor is highly susceptible to off-pathway interactions and aggregation. Expression of the S1 795 GFP in the C2C12 muscle cell line yields a folded and functionally active protein that exhibits Mg 2؉ ATP-sensitive actin-binding and myosin motor activity. In contrast, expression of S1 795 GFP in kidney epithelial cell lines (human 293 and COS 7 cells) results in an inactive and aggregated protein. The results of the in vitro folding assay suggest that the myosin motor domain does not fold spontaneously under physiological conditions and probably requires cytosolic chaperones. The expression studies support this conclusion and demonstrate that these factors are optimized in muscle cells.