Myosin is the crucial motor component of muscle contraction and essential to numerous forms of cellular movement. The predominant myosin in striated muscle is a conventional class II myosin. This subfamily is unique among all other members of this large superfamily in a number of important ways. First, the striated muscle family, including skeletal and cardiac muscle myosin, are expressed at levels that far exceed all other myosin classes. Second, striated muscle myosin is incorporated into highly organized sarcomeric units of the specialized contractile cytoskeleton of muscle. Considering its abundance, it is perhaps paradoxical then that striated muscle myosin has been exceedingly difficult to produce as a recombinant protein in heterologous expression systems. We have shown that myosin folding is mediated by molecular chaperones, myosin motor domain folding is rate limiting and muscle specific factors are required for striated muscle myosin folding. We have discovered and characterized a novel maturation complex in differentiating muscle cells that contains partially folded myosin in association with Hsc70 and Hsp90. From these observations, we hypothesize that the unique demands of striated muscle require myosin folding and assembly to follow a regulated and chaperoned pathway. Consequently, myosin folding in muscle is mediated by molecular chaperones, and muscle specific co-chaperones have evolved to complement this requirement. We propose to use a novel myosin motor domain-GFP chimera (MD::GFP) to identify muscle specific chaperones required for motor domain folding. Synthesis in a translation/folding assay of cytoplasmic and muscle isoforms of the MD::GFP chimera will be used to characterize early intermediates in the pathway and identify interacting factors from muscle extracts. We have developed a novel approach for following the expression and assembly of myosin in differentiating muscle cells based on adenovirus induced expression of a GFP-myosin that will be used to analyze the cellular mechanism of folding and myosin filament assembly. Myosin can be trapped in a maturation complex using a specific inhibitor of Hsp90. The formation of that complex and release of the trapped intermediate will be analyzed. A protcomics approach will be used to define the components of the complex. Finally, a canditate muscle specific co-chaperone that may link myosin with Hsp90 in muscle has been identified (smUNC-45). We propose to determine if smUNC-45 acts with Hspg0 and Hsc70 in the myosin folding/assembly pathway and contributes to folding or regulation of myosin assembly. These experiments will provide critical new insights into the molecular events leading from myosin synthesis to folding, targeting and assembly of this crucial motor enzyme that is central to the cellular physiology of striated muscle.
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