The organization of organelles within cells is a critical factor in normal cellular function as well as animal physiology. Many pathways contribute to the localization of organelles; one example is molecular motors that transport cargoes to a designated location to establish cellular polarity. This proposal focuses on myosin V motors. Defects in myosin V motors cause mislocalization of cargoes, which underlies several diseases including skin pigment disorders, gut diseases, and neurological disorders. The regulation of myosin V based cargo transport occurs in part via the regulation of cargo-specific adaptor proteins. This proposal focuses on the yeast vacuole/lysosome, which is inherited in coordination with the cell cycle. Early in the cell cycle, the myosin V motor, Myo2, attaches to the vacuole via the vacuole specific adaptor, Vac17. This attachment is regulated in part by the cyclin dependent kinase Cdk1. Notably, detachment from Myo2 is also highly controlled, indicating that complex mechanisms signal that the cargo has arrived at the correct location. Release of Myo2 from the vacuole requires an E3 ubiquitin ligase, Dma1, which is recruited to the PEST sequence on Vac17. Moreover, Dma1 is also essential for the release of peroxisomes, suggesting a conserved role for Dma1 in terminating Myo2 mediated cargo transport. Our preliminary studies suggest that an additional step is required to facilitate Dma1 dependent ubiquitylation of Vac17. We found that Cla4 directly phosphorylates Vac17-S222, and that this phosphorylation event is important for release of the vacuole from the bud tip, ubiquitylation of Vac17, and ultimately Vac17 turnover. Additionally, inhibiting Cla4-dependent phosphorylation of Va17 results in the accumulation of Vac17 at the bud tip, suggesting that Cla4 acts as a spatiotemporal regulator and initiates the termination of vacuole transport. My central hypothesis is that there are mechanisms that signal that myosin V has arrived at its destination, which in turn regulates the release of its cargoes. In this proposal, I aim to determine the downstream consequences of Cla4-dependent phosphorylation of Vac17. These studies will elucidate mechanisms that regulate motor-based cargo delivery and provide greater insight into diseases caused by impaired myosin V mediated cargo transport.
Our laboratory uses vacuole inheritance as a model for myosin V-mediated cargo transport and our studies have revealed the importance of regulating the detachment of cargoes from myosin. Mutations in myosin motors result in several diseases that affect polarized cell types, such as Griscelli Syndrome, Microvillus Inclusion Disease, cancer, and neurological disorders. This proposal will expand our knowledge of how cargo transport is regulated and thus help us understand the basic biology underlying these diseases.
