Regulation of myosin V interaction with cargo.

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The goal of this proposal is to determine how the actin based motor, myosin V attaches to its cargo. This is a key aspect of myosin V function because myosin V moves diverse cargo to unique places at distinct times. Therefore, it is likely that myosin V attachment to cargo and/or myosin V motor activity is acutely regulated. It has been postulated that the C-terminal globular tail domain of myosin-V interacts with protein receptors, and that these receptors are critical for the regulation of myosin V-cargo interactions. Our laboratory recently discovered that the yeast myosin-V, Myo2p, moves the vacuole (lysosome) to the daughter cell during cell division. Interestingly, specific point mutations in the globular tail of Myo2p disrupt vacuole movement but not other Myo2p functions. Using these vacuole-specific mutants, a novel protein, Vac17p, was discovered. Several lines of evidence suggest that it is a component of the vascular receptor for Myo2p. The overall goals of this proposal are to understand the precise function of Vac17p, to extend preliminary observations that conformational changes in the Myo2p tail play a role in specifying cargo, and to identify the mechanisms that regulate Myo2p-vacuole association. These goals will be pursued as follows: 1) Map the sites on Vac17p that are required for its interaction with Myo2p. 2) Determine whether Vac17p levels drive the cell-cycle coordination of vacuole inheritance. 3) Determine whether conformational changes within the Myo2p globular tail regulate cargo specificity. 4) Identify additional receptor subunits and regulatory molecules that are required for Myo2p association with the vacuole. These studies should reveal how regulation of both myosin V and its receptor move cargo to the correct place at the proper time. This question is of clear relevance to human health because myosin V is required for correct positioning of synaptic vesicles, the endoplasmic reticulum, and melanosomes. Moreover, patients with Griscelli's syndrome due to mutations in myosin Va, display both neurological and pigmentation defects.