Mechanisms that Regulate the Termination of Myosin V Mediated Transport

Abstract

A major question in cell biology is, how are materials, such as organelles, transported within a cell? The mis-localization of organelles underlies diseases in the skin, gut, and brain. Thus, the delivery of organelles to their proper destination is important for cellular function. Molecular motor proteins are one way by which cargoes are transported to their destination within the cell. Yet, little is known about how cargo transport is regulated. Specifically, it is largely unknown how motors release cargo at the correct destination.

In this thesis, I explore mechanisms that regulate how a myosin V motor transports organelles in the budding yeast, Saccharomyces cerevisiae. These studies focus on the vacuole, an organelle similar to mammalian lysosomes, which is transported into the daughter bud by myosin V during budding/cell division. Myosin V-based transport requires that cargo-specific adaptor proteins physically link the motor to the cargo. Early in the cell cycle, the vacuole-specific adaptor, Vac17, bridges a myosin V motor, Myo2, to the vacuole. Both the attachment and the detachment of the vacuole to/from Myo2 is highly controlled.

Release of Myo2 from the vacuole is mediated through Vac17. Vac17 is regulated by post-translational modifications. Dma1, an E3 ubiquitin ligase, ubiquitylates Vac17, which targets it for degradation. Interestingly, the bud cortex is a spatial landmark that signals the successful delivery of the vacuole to the bud. Upon arrival at the bud cortex, Vac17 is phosphorylated by a p21-activated kinase (PAK), Cla4. Cla4-dependent phosphorylation is required for the ubiquitylation and subsequent degradation of Vac17. These studies reveal a critical step in the spatial regulation of myosin V–dependent organelle transport.

In addition to ubiquitylation, a second pathway is required to release the vacuole from Myo2. A vacuole-localized Casein Kinase I, Yck3, along with the homotypic fusion and protein-sorting (HOPS) subunit, Vps41, regulate the phosphorylation of Vac17 in its Myo2 binding region. Yck3 and Vps41-dependent phosphorylation results in the dissociation of ubiquitylated Vac17 from the motor-adaptor complex. Moreover, ubiquitylation of Vac17 can occur independent of Yck3 and Vps41. Conversely, Yck3 and Vps41 -dependent phosphorylation can occur without ubiquitylation. However, both signals must be present for the vacuole to be released from Myo2 and for Vac17 to be degraded. Thus, the termination of cargo transport is tightly regulated and likely critical to cellular health and function.

Overall, these studies reveal some of the mechanisms required to release cargo from their motors at the right place and time. Many of the players are conserved in mammalian cells. Thus, it is tempting to speculate that these mechanisms are conserved for other yeast myosin V cargoes, as well as conserved in mammalian cells. Further work based on these findings has the promise to provide greater insight into motor-based cargo transport, how protein complexes are dissociated, and how proteins are regulated and degraded in coordination with cellular events.