Drosophila myosin VIIA is a high duty ratio motor with a unique kinetic mechanism.
Mutations of myosin VIIA cause deafness in various species from human and mice to Zebrafish and Drosophila. We analyzed the kinetic mechanism of the ATPase cycle of Drosophila myosin VIIA by using a single-headed construct with the entire neck domain. The steady-state ATPase activity (0.06 s(-1)) was markedly activated by actin to yield V(max) and K(ATPase) of 1.72 s(-1) and 3.2 microm, respectively. The most intriguing finding is that the ATP hydrolysis predominantly takes place in the actin-bound form (actin-attached hydrolysis) for the actomyosin VIIA ATPase reaction. The ATP hydrolysis rate was much faster for the actin-attached form than the dissociated form, in contrast to other myosins reported so far. Both the ATP hydrolysis step and the phosphate release step were significantly faster than the entire ATPase cycle rate, thus not rate-determining. The rate of ADP dissociation from actomyosin VIIA was 1.86 s(-1), which was comparable with the overall ATPase cycle rate, thus assigned to be a rate-determining step. The results suggest that Drosophila myosin VIIA spends the majority of the ATPase cycle in an actomyosin.ADP form, a strong actin binding state. The duty ratio calculated from our kinetic model was approximately 0.9. Therefore, myosin VIIA is classified to be a high duty ratio motor. The present results suggested that myosin VIIA can be a processive motor to serve cargo trafficking in cells once it forms a dimer structure.
Watanabe
Shinya
S
Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA.
Ikebe
Reiko
R
Ikebe
Mitsuo
M
D000199:Actins / Q000737:chemistry; D000205:Actomyosin / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D003001:Cloning, Molecular; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D004398:Dyneins / Q000737:chemistry / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D051379:Mice; D008956:Models, Chemical; D009154:Mutation; D000081412:Myosin VIIa; D009218:Myosins / Q000737:chemistry / Q000502:physiology*; D010710:Phosphates / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D013997:Time Factors
D000199:Actins; C000631455:MYO7A protein, human; C000631456:Myo7a protein, mouse; D000081412:Myosin VIIa; D010710:Phosphates; D020033:Protein Isoforms; C000631458:myosin 7a, Drosophila; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin; D000251:Adenosine Triphosphatases; D009218:Myosins; D004398:Dyneins; D008274:Magnesium