Myosin II Folding Is Mediated by a Molecular Chaperonin

Abstract

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.

The myosin-interacting protein SMYD1 is essential for sarcomere organization

“…Remarkably, and similar to the situation in fla, myosin protein levels are severely reduced in this mutant. Another muscle chaperone, UNC-45b, interacts with Hsp90a1 to guide the assembly of myosins into sarcomeric units (Srikakulam and Winkelmann, 1999). UNC-45b-deficient zebrafish embryos and worms have strongly disorganized sarcomeres, although initial Zbody formation and the basal organization of thick filaments seems not to be altered, implicating a rather restricted role of UNC-45b in the integration of thick filaments into sarcomeres (Barral et al, 2002;Etard et al, 2007).…”

Chaperone-mediated folding and assembly of myosin in striated muscle

“…Sections were examined and photographed using a JEOL 1200EX transmission electron microscope operated at 80 kV. Chaperoning myosin folding Co-immunoprecipitation of S1 with Hsp90 and Hsc70 The S30 fraction was prepared essentially as described previously (Srikakulam and Winkelmann, 1999). The Papain MgS1 subfragment of pectoralis muscle myosin was dissolved in S30 buffer supplemented with 6.5 M guanidine hydrochloride (GuHCl).…”

“…The successful production of recombinant striated muscle myosin has been limited to expression in muscle systems (Chow et al, 2002;Kinose et al, 1996;Swank et al, 2000). The difficulty in the expression of striated muscle myosin in non-muscle cells has been attributed recently to a folding defect, suggesting that folding and assembly of components unique to muscle are needed Chow et al, 2002;Price et al, 2002;Srikakulam and Winkelmann, 1999).…”

“…In a reticulocyte lysate expression system the folding of a skeletal muscle heavy meromyosin subfragment (HMM) is mediated by the eukaryotic chaperones including the cytosolic chaperonin, CCT (Srikakulam and Winkelmann, 1999). This system supports folding and dimerization of the rod and light chain binding to the myosin heavy chain.…”

DrosophilaUNC-45 accumulates in embryonic blastoderm and in muscles, and is essential for muscle myosin stability

“…To facilitate the investigation of its function, researchers have attempted to synthesize muscle myosin in vitro. Functional cardiac (Sweeney et al, 1994) and smooth muscle (Trybus, 1994) myosin isoforms have been produced using insect cells, but in vitro expression of skeletal muscle isoforms has not been routinely performed without using a myogenic cell line (Chow et al, 2002) or its lysate (Srikakulam and Winkelmann, 1999). Because functional -helical tails of rabbit skeletal muscle myosin can be synthesized in Escherichia coli (Atkinson and Stewart, 1991), it appears that some factor(s) in the myogenic cell line facilitates the folding of skeletal muscle myosin globular heads into the correct conformation.…”