title: Mammalian myosin-18A, a highly divergent myosin.
abstract: The Mus musculus myosin-18A gene is expressed as two alternatively spliced isoforms, α and β, with reported roles in Golgi localization, in maintenance of cytoskeleton, and as receptors for immunological surfactant proteins. Both myosin-18A isoforms feature a myosin motor domain, a single predicted IQ motif, and a long coiled-coil reminiscent of myosin-2. The myosin-18Aα isoform, additionally, has an N-terminal PDZ domain. Recombinant heavy meromyosin- and subfragment-1 (S1)-like constructs for both myosin-18Aα and -18β species were purified from the baculovirus/Sf9 cell expression system. These constructs bound both essential and regulatory light chains, indicating an additional noncanonical light chain binding site in the neck. Myosin-18Aα-S1 and -18Aβ-S1 molecules bound actin weakly with Kd values of 4.9 and 54 μm, respectively. The actin binding data could be modeled by assuming an equilibrium between two myosin conformations, a competent and an incompetent form to bind actin. Actin binding was unchanged by presence of nucleotide. Both myosin-18A isoforms bound N-methylanthraniloyl-nucleotides, but the rate of ATP hydrolysis was very slow (<0.002 s(-1)) and not significantly enhanced by actin. Phosphorylation of the regulatory light chain had no effect on ATP hydrolysis, and neither did the addition of tropomyosin or of GOLPH3, a myosin-18A binding partner. Electron microscopy of myosin-18A-S1 showed that the lever is strongly angled with respect to the long axis of the motor domain, suggesting a pre-power stroke conformation regardless of the presence of ATP. These data lead us to conclude that myosin-18A does not operate as a traditional molecular motor in cells.
authors: Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, MD 20892-8015, USA.
authors: Stephanie
authors: S
authors: Guzik-Lendrum
authors: Sarah M
authors: SM
authors: Heissler
authors: Neil
authors: N
authors: Billington
authors: Yasuharu
authors: Y
authors: Takagi
authors: Yi
authors: Y
authors: Yang
authors: Peter J
authors: PJ
authors: Knight
authors: Earl
authors: E
authors: Homsher
authors: James R
authors: JR
authors: Sellers
mesh_terms: D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D002465:Cell Movement; D003001:Cloning, Molecular; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008027:Light; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011817:Rabbits; D014335:Tropomyosin / Q000737:chemistry
chemical_list: D000199:Actins; C407101:Myo18a protein, mouse; D020033:Protein Isoforms; D014335:Tropomyosin; D000255:Adenosine Triphosphate; D009218:Myosins
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