Characterization of the Arabidopsis Heterotrimeric G Protein

Abstract

We have used fluorescence resonance energy transfer and co-immunoprecipitation to analyze the interactions among the alpha, beta, and gamma1 subunits of the Arabidopsis heterotrimeric G protein. Using cyan and yellow fluorescent protein fusion constructs, we show that overexpressed Ggamma1 localizes to protoplast membranes, but Gbeta exhibits membrane localization only when the Ggamma1 protein is co-overexpressed. Overexpressed Galpha shows membrane localization unaccompanied by overexpression of either Gbeta or Ggamma1. We detect fluorescence resonance energy transfer between Gbeta and Ggamma1 in the absence of Galpha overexpression and between Galpha and Ggamma1 but only when all three subunits are co-overexpressed. Both Galpha and Gbeta are associated with large macromolecular complexes of approximately 700 kDa in the plasma membrane. Galpha is present in both large complexes and as free Galpha in plasma membranes from wild type plants. In plants homozygous for a null allele of the Gbeta gene, Galpha is associated with smaller complexes in the 200-400-kDa range, indicating that its presence in the large complex depends on association with Gbetagamma. Activation of the Galpha subunit with guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) results in partial dissociation of Galpha from the complex. Hydrogen peroxide (H2O2) promotes extensive dissociation of the Galpha complex but does not interfere with binding of GTPgammaS to purified recombinant Galpha, suggesting that reactive oxygen species affect the stability of the large complex but not the activity of Galpha itself.