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J. Biol. Chem., Vol. 283, Issue 25, 17477-17484, June 20, 2008

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Load and P_i Control Flux through the Branched Kinetic Cycle of Myosin V^*

From the Department of Molecular Physiology & Biophysics, University of Vermont, Burlington, Vermont 05405

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 (P_i), both forward and backward steps become less load-dependent. From these data, we infer that P_i 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 P_i. We propose a branched kinetic model to explain these data. Our model includes P_i release prior to the most load-dependent step in the cycle, implying that P_i release and load both act as checkpoints that control the flux through two parallel pathways.

Received for publication, January 22, 2008 , and in revised form, April 3, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants HL059408, HL085489 (to D. M. W.), and GM078097 (to K. M. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1S.

¹ Present address: Department of Biological Sciences, University of Essex, Essex CO4 3SQ, UK.

² To whom correspondence should be addressed: Dept. of Molecular Physiology & Biophysics, University of Vermont, Burlington, VT 05405. Tel.: 802-656-2540; Fax: 802-656-0747; E-mail: warshaw{at}physiology.med.uvm.edu.

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