Identification and Characterization of Novel Players in the Establishment of Receptor Polarity in Yeast

The formation of shmoos by S. cerevisiae during mating is a chemotropic response related to chemotaxis. Chemotropic and chemotactic cells exhibit a remarkable ability to interpret gradients and sense direction. The two haploid mating types, MATa and MATα, sense the pheromone secreted by the opposite mating type, polarize their growth towards the closest mating partner, and fuse to form diploids. How do these cells sense direction? In MATa cells, the Ste2 pheromone receptor is the primary gradient sensor. Ste2 is uniformly distributed on the plasma membrane (PM) in vegetative cells, but upon ligand binding, it is rapidly internalized. It then reappears as a polarized crescent that coincides with the incipient shmoo site. In mating mixtures, the receptor crescent orients towards the closest mating partner prior to morphogensis. Although actin-dependent directed secretion stabilizes and amplifies receptor polarity, we have shown that receptor polarization is upstream and independent of actin-directed secretion. In contrast, internalization of the receptor is essential for its polarization. Phosphorylation of the G-protein Gβ is also crucial for receptor polarization. How is receptor polarity established and what are the key players? From a directed genetic screen, I identified four haploid-specific genes, DCV1, PCL1, FIG1 and PDE1, which contribute to polarization of the receptor. Further characterization revealed that only Dcv1 and Pcl1 were required for the polarization of receptor upstream and independent of actin-directed secretion. Dcv1 and Pcl1 were also required for efficient chemotropism whose role in receptor polarity was further characterized. The yeast claudin Dcv1 was found to be important for proper lipid distribution and composition of the plasma membrane and maintaining cell intergrity. Dcv1 localized uniformly to the plasma membrane of vegetative cells and inversely to the receptor in shmooing cells. Experiment with mating mixtures also revealed that Dcv1 was required for various polarized mating function much as receptor polarization, proper polarization of fusion marker Fus1 and efficient mating. these observations support a role for Dcv1 in organizing mating-specific membrane domains essential for the polarization of the receptor and other mating functions. Characterization of Pcl1 demonstrated that only Pcl1, but not its closely related cyclin Pcl2 is required for proper receptor polarization. In contrast to PCL1 deletion, over-expression of Pcl1 causes the receptor to hyper polarize to a very narrow region on the membrane. Pcl1 localizes to sites of polarized growth during mating, similar to its localization pattern in budding cells. Pcl1 localizes as a ring to the tip of emergent shmoos and later to the neck of mature shmoos. Lastly, Gβ and Pcl1 exhibit genetic and physical interaction. Taken together, these data suggest a role of Pho85/Pcl1 in the mating pathway via regulating phosphorylation of Gβ. In summary I have identified two novel players that affect receptor polarization by distinct mechanisms.