Mouse myosin V is a two-headed unconventional myosin with an extended neck that binds six calmodulins. Double-headed (heavy meromyosin-like) and singleheaded (subfragment 1-like) fragments of mouse myosin V were expressed in Sf9 cells, and intact myosin V was purified from mouse brain. The actin-activated MgATPase of the tissue-purified myosin V, and its expressed fragments had a high V max and a low K ATPase . Calcium regulated the MgATPase of intact myosin V but not of the fragments. Both the MgATPase activity and the in vitro motility were remarkably insensitive to ionic strength. Myosin V and its fragments translocated actin at very low myosin surface densities. ADP markedly inhibited the actin-activated MgATPase activity and the in vitro motility. ADP dissociated from myosin V subfragment 1 at a rate of about 11.5 s ؊1 under conditions where the V max was 3.3 s ؊1 , indicating that, although not totally rate-limiting, ADP dissociation was close to the rate-limiting step. The high affinity for actin and the slow rate of ADP release helps the myosin head to remain attached to actin for a large fraction of each ATPase cycle and allows actin filaments to be moved by only a few myosin V molecules in vitro.Fifteen classes of myosins have been defined based on sequence analysis of the conserved motor domain of the molecule (1, 2). There has been extensive biochemical characterization of myosins from class I and II (1). In addition, some of the in vitro properties of chicken brain myosin V have been reported (3, 4). Myosin V is a two-headed molecule composed of two heavy chains weighing 212 kDa. Each heavy chain contains a typical myosin motor domain with a neck domain consisting of six IQ motifs that interact with myosin light chain subunits and calmodulin. Following this is a tail domain that contains regions of coiled-coil interspersed with nonhelical regions. Chicken myosin V copurifies with both calmodulin and essential light chain (5), but the stoichiometry of the various light chains has not been rigorously determined (5). The elongated appearance of the myosin head in rotary shadowed electron micrographic images, however, is consistent with a neck region that has many light chain subunits (4). In addition, chicken brain myosin V contains another low molecular mass subunit (8 kDa) termed the "dynein light chain," which has also been found in association with several other proteins including dynein and nitric-oxide synthase (6).Myosin V genes have been found in humans, rats, mice, chickens, Drosophila, Caenorhabditis elegans, and Saccharomyces cerevisiae (4, 7-13). There are two myosin V genes in yeast and at least two genes for myosin V in mammals, termed Va and Vb. Mutations in yeast and mouse myosin V genes reveal that this myosin likely functions in vesicle transport (see Ref. 2 for review). The dilute gene in mouse encodes myosin Va (9). dilute mutations are characterized by pale coat color, and the severe alleles also display neurological defects. The coat color phenotype is due to defective trafficking...
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