Coordinating The Transport Cycle Of The Myosin-V Motor Myo2 With Secretory Vesicle Delivery And Exocytosis

Abstract

The polarization of proteins, lipids, and organelles within a eukaryotic cell allows for the spatial regulation of numerous biological processes. Saccharomyces cerevisiae displays exaggerated polarized growth of its plasma membrane during budding through the directed transport of secretory vesicles. In addition, several organelles are actively transported into the growing bud. These processes are accomplished using formin-nucleated actin cables extending from the bud tip and neck and the myosin-V motor Myo2p. While many of the components linking Myo2p to its various cargoes are known, the dynamic behavior of the motor and how its dynamics is regulated at the molecular level remains unclear. Here I define the in vivo delivery cycle of a myosin-V in its essential function of secretory vesicle transport, and show how that transport is coordinated with other events in exocytosis. I determined that Myo2p is activated from an inactive state by binding to competent secretory vesicles. This inactive state is caused by an autoinhibitory interaction between the head and tail of the motor. Mutations that disrupt this interaction render the motor constitutively active and compromise cargo transport functions. About 10 motors associate with each secretory vesicle during rapid transport to sites of cell growth. Motor release is temporally regulated by vesicle-bound Rab-GTP hydrolysis and requires vesicle tethering via the exocyst complex, but does not require vesicle fusion with the plasma membrane. Additionally, I developed a vesicletracking assay to study single-vesicle fusion dynamics at the cortex. This aided in the creation of a timeline of events for exocytosis, allowing for the dynamics of single-vesicle populations of the Rab Sec4p, the exocyst complex, and Myo2p to be visualized. The components of this transport cycle are highly conserved in mammalian cells, so these results should be generally applicable to other myosin-V delivery cycles.