PROJECT SUMMARY/ABSTRACT Molecular motors are essential for cellular organization, and drive cell-type specific architectures. Motors distribute organelles, mRNA, and other cellular components to the correct place, at the proper time, and regulate multiple pathways including secretion, and intracellular traffic. Thus, the proper control of molecular motors is essential for animal physiology. Myosin V motors (MyoV) are present in most eukaryotes and provide relatively long-range movement of cargoes on actin cables. Humans express three MyoV. Mutations in each MyoV have been linked to serious diseases. In many cases, the defects may be due to an inability of the MyoV to properly associate with cargo. We uncovered some of the mechanisms required for cargo attachment and detachment from MyoV. However, there are large gaps in knowledge of how cargo attachment and detachment from MyoV is controlled. In S. cerevisiae (hereafter referred to as yeast), long-range transport of many cargoes occurs solely on actin via MyoV. This contrasts with most organisms, where transport initiates on microtubules via kinesins with a subsequent handoff to MyoV. Thus, regulation of yeast MyoV, Myo2, is a simpler system. Strikingly, selected cargoes of yeast Myo2 have distinct trajectories. This led to our discovery that disengagement of Myo2 from its cargoes is a key property of organelle transport. Importantly, due to their sequence similarity, mechanisms discovered for yeast Myo2 have provided insights into human MyoV function. This proposal is focused on vacuole/lysosome inheritance which occurs via Myo2 and is controlled both spatially and temporally. We identified the Vac17-Vac8 vacuole adaptors, yet how Myo2 interacts with Vac17- Vac8 and the mechanisms(s) that regulate formation and disassociation of the complex are poorly understood. Our recent inroads in biochemical and genetic analyses have led to the following two aims. 1) Determine the organization of the Myo2-Vac17-Vac8 complex. This aim will use genetics, fluorescence microscopy, biochemistry, structural approaches and AlphaFold multimer predictions to determine how Vac17 associates with Myo2 and with the scaffold protein Vac8. 2) Determine roles of Vps41 and the kinase Yck3 in the regulated disassembly of the Myo2-Vac17-Vac8 complex. We discovered that Vps41 and Yck3 are critical for disruption of the Myo2-Vac17-Vac8 complex, yet their roles in this pathway are distinct from their known roles in vacuole fusion. To determine their Myo2-specific roles, we will identify additional proteins that act with Vps41 and Yck3 in the context of the Myo2-Vac17-Vac8 complex. We will use pull-down approaches, high- content microscopy screens and proximity labeling. Further analysis of the candidates will be pursued with fluorescence microscopy. Given that little is known about how molecular motors attach or detach from cargoes, the proposed studies will likely reveal new pathways that are crucial for cell function.