Organelle movement is a hallmark of cell division and cellular differentiation. In every cell-type, the localization, number and morphology of each type of organelle is modified to achieve specific cellular functions. Long-range movement of organelles occurs on microtubules, while positioning of organelles at their final destination relies on actin-based motors. Myosin V motors play a critical role in actin based movement in all eukaryotes, and disruption of myosin V function causes disease in humans. For example, partial defects in myosin Va based transport cause Griscelli's syndrome, characterized by neurological and pigmentation defects. Partial defects in myosin Vb cause microvillus inclusion disease, characterized by infantile, life threatening diarrhea. Currently there are no effective drug-based treatments for either disease. Determination of how myosin V based transport is achieved may provide important new insights into treating theseetach from cargoes at the correct time and place. The globular tail domain (GTD) of myosin V attaches to its cargoes through organelle-specific adaptor proteins. Regulation of the GTD and adaptor proteins contributes to specifying cargo attachment. We determined a high-resolution structure of the GTD of Myo2, a Saccharomyces cerevisiae myosin V motor, and identified two distinct cargo binding regions;one required for binding to the yeast vacuole, the other required for binding to secretory vesicles. We found that the vacuole binding site interacts with a novel protein, Vac17. The secretory vesicle binding site attaches directly to a Rab GTPase. Importantly, we found that the Rab GTPase binding site is conserved in human myosin Va and myosin Vb. Thus, our studies in yeast revealed the Rab GTPase binding site on human myosin V motors. The general conservation of the cargo binding domains of yeast Myo2 and human myosin Va and Vb, and the fact that Rab GTPases act directly to attach carns. Our major goals are to: 1) Determine whether the binding of an individual Myo2 adaptor protein enhances or inhibits binding of other adaptor proteins. 2) Identify and characterize proteins that regulate the attachment of Rab GTPases to the GTD of Myo2. 3) Determine mechanisms that regulate the detachment of myosin V from cargoes. PUBLIC HEALTH RELEVANCE: Intracellular transport of organelles by myosin V motors is crucial to normal cellular function, and animal physiology. Defects in myosin V based transport cause selected human diseases including neurological disorders. Our overall goal is to determine the mechanisms that regulate for myosin V-based transport.