Heterotrimeric (alpha/beta/gamma) G proteins are essential for many responses to environmental stimuli in eukaryotes. The genome of the filamentous fungus Neurospora crassa contains three G-alpha (GNA-1, GNA-2, GNA-3) one G-beta (GNB-1) and one G-gamma (GNG-1) subunits, and also predicts the existence of cAMP, pheromone and glucose-sensing G protein coupled receptors (GPCR's). GNA-1 is required for apical growth, asexual sporulation and female fertility. GNA-3 is a major regulator of asexual sporulation. GNA-2 function is redundant to GNA-1 and GNA-3. GNA-1 positively-regulates GTP-dependent adenylyl cyclase (CR-1) activity, while GNA-3 is required for normal levels of CR-1 protein. However, several phenotypes of delta-gna-I and delta-gna-3 mutants cannot be rescued by exogenous cAMP. These and other results indicate that sexual fertility is largely cAMP-independent, while other functions, such as asexual sporulation, are regulated using camp-dependent and independent pathways. GNB-1 modulates G-alpha amount via a post-transcriptional mechanism, but certain delta-gnb-I phenotypes can not be explained by low G-alpha protein levels. G-beta/gamma regulates Mitogen-Activated Protein Kinase (MAPK) pathways in other systems. Therefore, we hypothesize that G proteins differentially regulate cAMP levels, MAPK pathways and unknown effectors to modulate gene expression during vegetative and sexual development in N. crassa.
The Specific Aims are: 1) Mutate six N. crassa GPCR genes and characterize phenotypes and G-alpha subunit coupling. Phenotypic analysis will include cAMP metabolism defects. Localization and expression patterns of each GPCR will be determined using antisera. GTPase-deficient G-alpha alleles and the two-hybrid assay will be utilized to determine epistatic relationships and binding between receptors and G-alpha's. Pheromones, cAMP and other molecules will be tested as ligands. 2) Determine functional and physical relationships between G protein subunits. The mechanism of posttranscriptional regulation of G-alpha levels by GNB-1 will be determined using pulse-chase and in vitro translation experiments. Coimmunoprecipitation and two-hybrid assays will be used to test interactions between GNB-1, GNG-1, and the three G-alpha proteins. Epistatic relationships will be probed using GTPase-deficient G-alpha alleles in G protein mutant backgrounds. 3) Investigate regulation of known or suspected targets by G-alpha and G-beta/gamma subunits. Purified GNA-1 will be tested for reconstitution of AC activity in delta-gna-1 preparations. Epistasis between cr-1 and G-alpha genes will be analyzed, and the two-hybrid assay and coimmunoprecipitation will be used to test for association of G proteins and CR-I. MAPK activity will be measured in GPCR and G protein subunit mutants. 4) Identify unknown G protein signaling components. Unknown components will be identified by cloning delta-gna-1 delta-gna-3 suppressors and the cr-2, cr-3 and cr-4 genes, and through transcriptional profiling experiments. These studies will elucidate G protein signaling pathways in filamentous fungi and yield insights into G protein evolution. Furthermore, since homologues of N. crassa G-alpha genes are required for virulence in numerous fungal species, these investigations will also lead to new therapies for emerging fungal pathogens.
