| G protein coupled signal pathways are conserved from plants to fungi to humans, and mediate the responses to a diverse array of signals including pheromones, light, odorants and neurotransmitters. In the yeast Saccharomyces cerevisiae, activation of a G protein pathway by pheromone triggers a series of events leading to mating. Traditional paradigms have assumed that the pathway originates solely from the plasma membrane, and that the purpose of the Gpa1Galpha protein is to sequester the Ste4/Ste18Gbetagamma dimer in the absence of stimulus. However, evidence has come to light showing that the activated Gpa1 Galpha protein transduces a signal, and this signal originates from the endosome. Moreover, the catalytic and regulatory subunits of the only phosphatidylinositol-3-kinase in yeast, Vps34 and Vps15, are required to transmit this endosomal signal. The endosomal G protein signal activates many of the same events as the plasma membrane derived signal, and results in activation of the MAPK Fus3 in preference to Kss1.;Here, we show that the kinase domain with the intermediate domain of Vps15 is both necessary and sufficient for binding to Gpa1 and for transmitting the G protein coupled signal. The WD domain of Vps15 most likely serves as a scaffold to assemble binding partners, and folds into a 7-bladed propeller, as demonstrated by X-ray crystallography. Moreover, our investigation of the kinase domain of Vps15 shows that fully activated Vps34 is also necessary for both the expression and phosphorylation of Vps15.;Finally, as Fus3 is preferentially activated at the endosome, we investigated the regulation of Fus3. Here, we show that Fus3 is positively and negatively regulated by PtdIns(3)P, a second messenger produced by Vps34 that localizes to the endosomes. Furthermore, we show that lysine and histidine point mutations of Fus3 in its basic patch result in a decrease of Fus3 ubiquitination at the lysine residue as well as a decrease in Fus3 phosphorylation of Far1. Together, these data further our knowledge of the differences between the endosomal and plasma membrane G protein pathways. |
