Genetic and Physical Interactions between Gα Subunits and Components of the Gβγ Dimer of Heterotrimeric G Proteins in Neurospora crassa

Heterotrimeric G proteins are critical regulators of growth and asexual and sexual development in the filamentous fungus Neurospora crassa. Three G alpha subunits (GNA-1, GNA-2, and GNA-3), one G beta subunit (GNB-1), and one G gamma subunit (GNG-1) have been functionally characterized, but genetic epistasis relationships between G beta and G alpha 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 alpha binding partners for the G beta gamma dimer is currently lacking. In this study, the three N. crassa G alpha subunits are analyzed for genetic epistasis with gnb-1 and for physical interaction with the G beta gamma dimer. We created double mutants lacking one G alpha gene and gnb-1 and introduced constitutively active, GTPase-deficient alleles for each G alpha gene into the Delta 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 alpha alleles restored female fertility to Delta gnb-1 mutants, and the gna-3(Q208L) allele inhibited formation of female reproductive structures, consistent with a need for G alpha 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 alpha proteins interact with the G beta gamma dimer. The finding that the G beta gamma dimer interacts with all three G alpha proteins is supported by epistasis between gnb-1 and gna-1, gna-2, and gna-3 for at least one function.