Motor function and regulation of myosin X.
Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues. Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level. We expressed bovine myosin X containing the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells. The Mg(2+)-ATPase activity of myosin X was significantly activated by actin with low K(ATP). The actin-activated ATPase activity was reduced at Ca(2+) concentrations above pCa 5 in which 1 mol of calmodulin light chain dissociates from the heavy chain. Myosin X translocates F-actin filaments with the velocity of 0.3 microm/s with the direction toward the barbed end. The actin translocating activity was inhibited at concentrations of Ca(2+) at pCa 6 in which no calmodulin dissociation takes place, suggesting that the calmodulin dissociation is not required for the inhibition of the motility. Unlike class V myosin, which shows a high affinity for F-actin in the presence of ATP, the K(actin) of the myosin X ATPase was much higher than that of myosin V. Consistently nearly all actin dissociated from myosin X in the presence of ATP. ADP did not significantly inhibit the actin-activated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting. These results suggest that myosin X is a nonprocessive motor. Consistently myosin X failed to support the actin translocation at low density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement.
Homma
K
K
Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0127, USA.
Saito
J
J
Ikebe
R
R
Ikebe
M
M
D000199:Actins / Q000378:metabolism / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D024701:Myosin Type V; D009218:Myosins / Q000737:chemistry* / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014981:Xenopus / Q000378:metabolism
D000199:Actins; D002148:Calmodulin-Binding Proteins; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C078829:chicken brain myosin-V p190; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D024701:Myosin Type V; D009218:Myosins; D011188:Potassium; D002118:Calcium