Involvement of Kinesin-related Smy1 and Rho GAP Gym1 in Regulating Vesicle Transport by Myosin V

Abstract

Every nucleated cell has a defined morphology to cater to the needs of its unique function. To generate polarity during growth, budding yeast Saccharomyces cerevisiae transports all necessary organelles into the bud. This transport requires the essential myosin V motor, Myo2. Using different cargo receptors, Myo2 transports secretory vesicles, mitochondria, vacuoles and other cargoes. A conditional mutant myo2-66 in particular fails to transport secretory vesicles. Initially discovered as a multicopy suppressor of myo2-66, Smy1 suggested the involvement of a kinesin motor for transport of secretory vesicles. However, because suppression does not need either kinesin motor activity or an interaction with microtubules, the function of Smy1 remained elusive. Here I show that kinesin-related Smy1 stabilizes the association between Myo2 and the secretory vesicle receptor, the Rab GTPase Sec4. Smy1 function is specific for secretory vesicle transport. Characterization of Smy1 individual domains indicates that full length Smy1 is required since the head domain is important for vesicle association and the tail domain for Myo2 binding. Furthermore, I used a GAL1 cDNA library to suppress a conditional myo2 smy1 mutant as a way to identify additional components that may function in secretory vesicle transport. One novel component we identified has a RhoGAP domain in its C-terminus, so I named it GYM1 (GAP with Yeast Myo2). I found that Gym1 is polarized to the bud and its localization is dependent on Myo2. Further, biochemical assays showed that Gym1specifically stimulates the GTPase activity of Rho3 and mutation of its critical arginine at residue 546 abolishes this activity. Genetic analysis also showed that the ability of Gym1 to suppress the myo2 smy1 mutant requires the GAP activity towards Rho3. In mammalian cells, TC10 Rho proteins are also reported to mediate vesicle transport. This new finding of Rho3 and its associated GAP, Gym1 in budding yeast suggests that there is an interplay between Rho and Rab GTPases in secretory vesicle transport.