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 acti n in cells has been shown, little is known about its biochemical and mechan oenzymatic function at the molecular level. We expressed bovine myosin X co ntaining the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells. The Mg2+-ATPase activity of myosin X was significant ly activated by actin with low K-ATP. The actin-activated ATPase activity w as reduced at Ca2+ 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 mum/s with the direction toward the bar bed end. The actin translocating activity was inhibited at concentrations o f Ca2+ 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 i n the presence of ATP, the K-actin of the myosin X ATPase was much higher t han that of myosin V. Consistently nearly all actin dissociated from myosin X in the presence of ATP. ADP did not significantly inhibit the actin-acti vated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting. These results suggest that myosin X is a nonprocessive m otor. Consistently myosin X failed to support the actin translocation at lo w density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement.