Heterotrimeric (alpha-beta-gamma) G proteins are essential for many responses to environmental stimuli in eukaryotes. In the filamentous fungus Neurospora crassa, there are three known Galpha proteins (GNA-1 and GNA-3) and one Gbeta subunit (GNB-1). GNA-1 and GNA-2 have overlapping functions in regulating female fertility and vegetative growth. Only GNA-1 modulates GTP-dependent adenylyl cyclase activity (AC), while GNA-1 and GNA-2 both affect cAMP phosphodiesterase activity (PDE). GNA-3 regulates vegetative growth, but is not necessary for the sexual cycle. GNA-3 is implicated as a positive regulator of AC. These and other findings indicated that cAMP is important for vegetative, but not sexual functions. GNA-1 was the first microbial Galpha that could be classified in a mammalian family (Galpha/i). Systematic testing of deltagna-1 complementation by several mammalian Galpha genes was performed. The results also support separate signaling pathways for sexual and vegetative growth in N. crassa. Deltagnb-1 strains have greatly reduced GNA-1, but normal GNA-2 levels. It has been shown in other systems that beta-gamma regulates transcription. Beta-gamma proteins regulate phospholipase C (PLC) in mammals, leading to release of Ca/2+ from intracellular stores. The vacuole is the major Ca/2+ reservoir in N. crassa; and deltagnb-1 strains have vacuolar defects. Hence, GNB-1 may regulate gna-1 transcription and/or Ca/2+ mobilization, in N. crassa. Therefore, we hypothesize that G proteins differentially regulate 1) cAMP levels, 2) vacuolar function and 3) gene expression during vegetative and sexual development in Neurospora crassa. Our objectives are: 1) A direct interaction between GNA-1 and AC will be tested using reconstitution assays, co-immunoprecipitation, affinity chromatography and two-hybrid assays. Unknown targets of GNA-1 and GNA-2 will be identified by two- hybrid screens and genetic suppression analysis. Roles of GNA-1 and GNA-2 during the pheromone responses will be tested. 2) AC, PDE and cAMP levels will be measured in the mammalian Galpha transformants. Interactions between the mammalian Galpha's and GNB-1 will be tested in two-hybrid assays. Chimeras between complementing and non-complementing genes will be constructed to define regions important for vegetative or sexual functions. 3) AC and PDE activity will be measured to probe a role for GNA-3 in cAMP metabolism. Interactions between GNA-3 and other proteins will be determined using reconstitution, affinity chromatography and two- hybrid analysis. 4) Transcriptional versus post-translational regulation of GNA-1 levels by GNB-1 will be tested. AC, PDE and cAMP levels will be measured in deltagnb-1 strains. Vacuolar function will be assessed by measuring arginine and Ca/2+ pools and vacuolar ATPase activity PLC activity and Ca/2+ metabolism will be tested in deltagnb-1 mutants. Interactions between GNB-1 and other proteins will be defined using two- hybrid assays. These studies will elucidate G protein signaling pathways in filamentous fungi, speed the functional analysis of mammalian Galpha's and yield insight into G protein evolution. Furthermore, since homologues of N. crassa G alpha genes modulate virulence in several filamentous species, these investigations will also illuminate the role of G proteins in fungal pathogenesis, leading to new therapies for emerging fungal pathogens.
