Characterization of the Arabidopsis heterotrimeric G protein

We have used fluorescence resonance energy transfer and co-immunoprecipitation to analyze the interactions among the alpha, beta, and gamma 1 subunits of the Arabidopsis heterotrimeric G protein. Using cyan and yellow fluorescent protein fusion constructs, we show that overexpressed G(gamma 1) localizes to protoplast membranes, but G beta exhibits membrane localization only when the G gamma(1) protein is co-overexpressed. Overexpressed G alpha shows membrane localization unaccompanied by overexpression of either G beta or G gamma(1). We detect fluorescence resonance energy transfer between G beta and G gamma(1) in the absence of G alpha overexpression and between G alpha and G gamma(1) but only when all three subunits are co-overexpressed. Both G alpha and G beta are associated with large macromolecular complexes of similar to 700 kDa in the plasma membrane. G alpha is present in both large complexes and as free G alpha in plasma membranes from wild type plants. In plants homozygous for a null allele of the G beta gene, G alpha is associated with smaller complexes in the 200-400-kDa range, indicating that its presence in the large complex depends on association with G beta gamma. Activation of the G alpha subunit with guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) results in partial dissociation of G alpha from the complex. Hydrogen peroxide (H2O2) promotes extensive dissociation of the G alpha complex but does not interfere with binding of GTP gamma S to purified recombinant G alpha, suggesting that reactive oxygen species affect the stability of the large complex but not the activity of G alpha itself.