Characterization of the Arabidopsis Heterotrimeric G Protein

We have used fluorescence resonance energy transfer and co-immunoprecipitation to analyze the interactions among the α, β, and γ1 subunits of the Arabidopsis heterotrimeric G protein. Using cyan and yellow fluorescent protein fusion constructs, we show that overexpressed Gγ1 localizes to protoplast membranes, but Gβ exhibits membrane localization only when the Gγ1 protein is co-overexpressed. Overexpressed Gα shows membrane localization unaccompanied by overexpression of either Gβ or Gγ1. We detect fluorescence resonance energy transfer between Gβ and Gγ1 in the absence of Gα overexpression and between Gα and Gγ1 but only when all three subunits are co-overexpressed. Both Gα and Gβ are associated with large macromolecular complexes of ∼700 kDa in the plasma membrane. Gα is present in both large complexes and as free Gα in plasma membranes from wild type plants. In plants homozygous for a null allele of the Gβ gene, Gα is associated with smaller complexes in the 200–400-kDa range, indicating that its presence in the large complex depends on association with Gβγ. Activation of the Gα subunit with guanosine 5′-3-O-(thio)triphosphate (GTPγS) results in partial dissociation of Gα from the complex. Hydrogen peroxide (H2O2) promotes extensive dissociation of the Gα complex but does not interfere with binding of GTPγS to purified recombinant Gα, suggesting that reactive oxygen species affect the stability of the large complex but not the activity of Gα itself.