elegans UNC-45 and MHC-B (myosin II heavy chain isoform B, also known as UNC-54) in a cellular context. Using insect cells as host system, we monitor the interaction between the C. To address the myosin targeting mechanism of UNC-45, we reconstitute the chaperone-substrate interplay both in vitro and in vivo. elegans revealed that the chaperone can form a linear protein chain, which constitutes a myosin assembly line licensing Hsp70 and Hsp90 to act in a defined periodicity on myosin heads protruding from the myofilaments 19, 26. UNC-45 is composed of an N-terminal TPR (tetratricopeptide repeat) domain mediating the interaction with Hsp70/Hsp90, an elongated UCS (UNC-45/Cro1/She4p) domain at the C-terminus providing a myosin-binding site 14, 19, and a central domain aligning the two functional TPR and UCS units to each other. Mechanistic insight into the chaperone function of UNC-45 comes from structural studies revealing its 3-domain architecture 19, 25. In higher organisms, UNC-45 is particularly important for the maturation of myosin II in muscle cells 20, 21, 22 and, together with Hsp90 (heat shock protein 90) and UFD-2 (ubiquitin fusion degradation 2), maintaining the functionality of myosin filaments during stress situations 23, 24. elegans, Xenopus, Zebrafish, and Drosophila confirmed the role of UNC-45 as a myosin-specific chaperone, promoting the folding of the myosin ATPase domain as well as coordinating the assembly of thick filaments during muscle development 14, 15, 16, 17, 18, 19. elegans, where site-specific temperature-sensitive (ts) mutants revealed the importance of the chaperone for myosin function 10, 11, 12, 13. The UNC-45 protein was initially identified in C. Failure of muscle filament assembly or maintenance leads to severe myopathies 8, 9.Īmong the chaperones expressed in muscle cells, UNC-45 appears to be the key component of the myosin assembly machinery. Therefore, muscle cells express a vast number of specialized folding and assembly factors that control the expression, folding, assembly and interplay of actin and myosin molecules 3, 4, 5, 6, 7. Establishing and maintaining the intricate myosin–actin interplay, occurring at the interface of the two differently organized muscle filaments, is a great challenge for the cellular chaperone machinery. These hexamers further assemble into myosin thick filaments, from where the N-terminal myosin ATPase domains project to interact with the adjacent actin (thin) filaments. Myosin II molecules found in skeletal and cardiac muscles dimerize via their C-terminal coiled-coil domain and associate with essential and regulatory light-chains to yield the basic hexameric myosin complex. The myosin superfamily contains about 35 subtypes, among which the class II muscle proteins (myosin II) are the arguably most prominent member driving muscle contraction 2. Myosins are cytoskeletal, molecular motors promoting a variety of mechano-chemical processes in the cell 1. Moreover, our data uncover the molecular basis of temperature-sensitive UNC-45 mutations underlying one of the most prominent motility defects in C. This so-called UCS domain can adopt discrete conformations to efficiently bind and fold substrate. Data from in vitro and cellular chaperone assays, together with crystal structures of binding-deficient UNC-45 mutants, highlight the importance of utilizing a flexible myosin-binding domain. In addition to providing a cellular chaperone assay, the established system enabled us to produce large amounts of functional muscle myosin, as evidenced by a biochemical and structural characterization, and to directly monitor substrate binding to UNC-45. To address its substrate-targeting mechanism, we reconstitute the interplay between Caenorhabditis elegans UNC-45 and muscle myosin MHC-B in insect cells. Folding and assembly of myosin relies on a specific chaperone, UNC-45. Myosin is a motor protein that is essential for a variety of processes ranging from intracellular transport to muscle contraction.
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