Cell organization requires motor-dependent transport of specific cargos along cytoskeletal elements. How the delivery cycle is coordinated with other events is poorly understood. Here we define the in vivo delivery cycle of myosin-V in its essential function of secretory vesicle transport along actin cables in yeast. We show that myosin-V is activated by binding a secretory vesicle and that myosin-V mutations that compromise vesicle binding render the motor constitutively active. About ten motors associate with each secretory vesicle for rapid transport to sites of cell growth. Once transported, the motors remain associated with the secretory vesicles until they undergo exocytosis. Motor release is temporally regulated by vesicle-bound Rab-GTP hydrolysis and requires vesicle tethering by the exocyst complex but does not require vesicle fusion with the plasma membrane. All components of this transport cycle are conserved in vertebrates, so these results should be generally applicable to other myosin-V delivery cycles.
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