Study of the Gpa2p signaling pathway and its role in the dimorphic transition of Saccharomyces cerevisiae

A diverse set of extracellular stimuli transmits signals to the cell through G proteins by activating G protein coupled receptors (GPCRs). In yeast, the G protein a subunit Gpa2p is involved in a dimorphic transition from a yeast form to a filamentous form. This transition is crucial for virulence of fungi that are pathogenic to plants or humans. Two genes that encode proteins that interact with Gpa2p were identified in the laboratory with a yeast two-hybrid screen. Analysis of one gene product, Gpr1p, revealed that it has the features of a potential Gpa2p receptor. Characterization of the function of the second gene, GPE2, and of a highly homologous gene GPE1 , is described in this work.; The aim of this work was to study the signal transduction pathway mediated by the Gpa2p protein in S. cerevisiae and to identify other proteins involved in the dimorphic transition. We developed an assay that measures activation of the Gpa2p pathway. This assay involved measuring the transcriptional induction of the FLO11 gene, which encodes a flocculin protein that is essential for the filamentous growth. Wild type cells transformed with a constitutively active GPA2 allele showed an increase in FLO11 mRNA levels. We demonstrated that deletion of the GPE1 and GPE2 genes results in elevated FLO11 mRNA levels. This result indicates that the GPE1 and GPE2 gene products, like Gpa2p, are involved in filamentous growth and that they behave as inhibitors of FLO11 mRNA induction. Epistasis analysis revealed that the cAMP-dependent protein kinase (PKA) Tpk2p acts downstream of Gpe1p and Gpe2p. Deletion of the TPK2 gene completely abolished the elevated FLO11 mRNA levels present in the gpe1- gpe2- cells collected in logarithmic phase. We also analyzed the role of SCH9 in filamentous growth. SCH9 encodes a protein kinase that is functionally redundant with PKAs in S. cerevisiae . We found that SCH9 function is required for the effect of the constitutively active Gpa2p mutant on FLO11 mRNA levels in cells collected in saturated phase.; Altogether these data are in agreement with a model in which Gpa2p interacts with Gpe2p, in logarithmic phase cells, releasing the inhibition of Tpk2p by Gpe2p. In contrast, Gpa2p activates the Sch9p kinase in later growth phases.