Genetic and Physical Interactions between G Subunits and Components of the G Dimer of Heterotrimeric G Proteins in

Heterotrimeric G proteins are critical regulators of growth and asexual and sexual development in the filamentous fungusNeu-rospora 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 threeN. crassaG subunits are analyzed for genetic epistasis with gnb-1 and for physical interaction with the G dimer. We created double mutants lack-ing one G gene and gnb-1 and introduced constitutively active, GTPase-deficient alleles for each G gene into thegnb-1 back-ground. Genetic analysis revealed that gna-3 is epistatic to gnb-1with 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 tognb-1mutants, and the gna-3Q208L allele inhibited for-mation 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. Eukaryotic cells sense extracellular stimuli such as hormones,neurotransmitters, odorants, chemoattractants, light, and nu