Heterotrimeric G proteins are critical regulators of growth and asexual and sexual development in the filamentous fungus Neurospora crassa. Three G␣ subunits (GNA-1, GNA-2, and GNA-3), one G␤ subunit (GNB-1), and one G␥ subunit (GNG-1) have been functionally characterized, but genetic epistasis relationships between G␤ and G␣ subunit genes have not been determined. Physical association between GNB-1 and FLAG-tagged GNG-1 has been previously demonstrated by coimmunoprecipitation, but knowledge of the G␣ binding partners for the G␤␥ dimer is currently lacking. In this study, the three N. crassa G␣ subunits are analyzed for genetic epistasis with gnb-1 and for physical interaction with the G␤␥ dimer. We created double mutants lacking one G␣ gene and gnb-1 and introduced constitutively active, GTPase-deficient alleles for each G␣ gene into the ⌬gnb-1 background. Genetic analysis revealed that gna-3 is epistatic to gnb-1 with regard to negative control of submerged conidiation. gnb-1 is epistatic to gna-2 and gna-3 for aerial hyphal height, while gnb-1 appears to act upstream of gna-1 and gna-2 during aerial conidiation. None of the activated G␣ alleles restored female fertility to ⌬gnb-1 mutants, and the gna-3 Q208L allele inhibited formation of female reproductive structures, consistent with a need for G␣ proteins to cycle through the inactive GDP-bound form for these processes. Coimmunoprecipitation experiments using extracts from the gng-1-FLAG strain demonstrated that the three G␣ proteins interact with the G␤␥ dimer. The finding that the G␤␥ dimer interacts with all three G␣ proteins is supported by epistasis between gnb-1 and gna-1, gna-2, and gna-3 for at least one function.

Receptor for Activated C Kinase-1 (RACK1) is a multifunctional eukaryotic scaffolding protein with a seven WD repeat structure. Among their many cellular roles, RACK1 homologs have been shown to serve as alternative Gβ subunits during heterotrimeric G protein signaling in many systems. We investigated genetic interactions between the RACK1 homolog cpc-2, the previously characterized Gβ subunit gnb-1 and other G protein signaling components in the multicellular filamentous fungus Neurospora crassa. Results from cell fractionation studies and from fluorescent microscopy of a strain expressing a CPC-2-GFP fusion protein revealed that CPC-2 is a cytoplasmic protein. Genetic epistasis experiments between cpc-2, the three Gα genes (gna-1, gna-2 and gna-3) and gnb-1 demonstrated that cpc-2 is epistatic to gna-2 with regards to basal hyphae growth rate and aerial hyphae height, while deletion of cpc-2 mitigates the increased macroconidiation on solid medium observed in Δgnb-1 mutants. Δcpc-2 mutants inappropriately produce conidiophores during growth in submerged culture and mutational activation of gna-3 alleviates this defect. Δcpc-2 mutants are female-sterile and fertility could not be restored by mutational activation of any of the three Gα genes. With the exception of macroconidiation on solid medium, double mutants lacking cpc-2 and gnb-1 exhibited more severe defects for all phenotypic traits, supporting a largely synergistic relationship between GNB-1 and CPC-2 in N. crassa.