How Phosphorylation of G Affects its Localization and Function During the Yeast Chemotropic Response.

Cells detect external chemical gradients and generate appropriate responses in a process known as gradient sensing. Chemotaxis (directed movement) and chemotropism (directed growth) are two related phenomena in eukaryotes, whereby cells interpret chemical gradients. These directed responses are necessary for biological processes across eukaryotic phyla including cell differentiation, immune response, embryogenesis, tumor metastasis, growth cone guidance, fungal life cycles, angiogenesis, and pollen tube guidance. Our lab proposed a gradient sensing model to explain how yeast cells interpret pheromone gradients: cells assembly a Gradient Tracking Machine (GTM) at the default polarity site (DS) and track the pheromone gradient to the chemotropic growth site (CS). A central postulate of our model is that yeast gradient tracking depends on differential phosphorylation of the receptor, which in turn, depends on localized phosphorylation and/or dephosphorylation of G. In Chapter 2, I developed a GP biosensor and tested this prediction. I found that in mating cells, the GP biosensor mimicked the behavior of GFP G, redistributing upgradient from the DS to the CS; in mating cells expressing both the GP biosensor and either a G reporter or a receptor reporter, GP concentrated upgradient relative to total G and total receptor, consistent with the proposed role for G phosphorylation as a determinant of GTM positioning. In Chapter 3, I investigated the functional relationship between GP and the yeast pheromone protease Bar1, during gradient sensing. I discovered that GP and Bar1 synergistically interact to promote yeast mating, consistent with the idea that they function in independent pathways to enhance the directional signal for efficient mating. In chapter 4, I examined chemotropism in MAT cells and I found that a and  cells largely differ in their chemotropic responses. Additionally, MAT cells showed novel global sensing mating phenotypes like ‘jumping’ and ‘direct polarization’. In mating mixtures,  cells were the first to stably polarize to the CS and did so largely by jumping or direct polarization. Additionally, cells were more likely to jump or directly polarize when the mating partner was close. Cells unable to phosphorylate G had a reduced ability to track and an increased tendency to undergo global sensing.