Load and Pi control flux through the branched kinetic cycle of myosin V

Abstract

Myosin V is a processive actin-based motor protein that takes multiple 36-nm steps to deliver intracellular cargo to its destination. In the laser trap, applied load slows myosin V heavy meromyosin stepping and increases the probability of backsteps. In the presence of 40 mM phosphate (Pi), both forward and backward steps become less load-dependent. From these data, we infer that Pi release commits myosin V to undergo a highly load-dependent transition from a state in which ADP is bound to both heads and its lead head trapped in a pre-powerstroke conformation. Increasing the residence time in this state by applying load increases the probability of backstepping or detachment. The kinetics of detachment indicate that myosin V can detach from actin at two distinct points in the cycle, one of which is turned off by the presence of Pi. We propose a branched kinetic model to explain these data. Our model includes Pi release prior to the most load-dependent step in the cycle, implying that Pi release and load both act as checkpoints that control the flux through two parallel pathways. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.