Nucleotide exchange-dependent and nucleotide exchange-independent functions of plant heterotrimeric GTP-binding proteins

Heterotrimeric guanine nucleotide-binding proteins (G proteins), which are composed of alpha, beta, and gamma subunits, are versatile, guanine nucleotide-dependent, molecular on-off switches. In animals and fungi, the exchange of GDP for GTP on G alpha controls G protein activation and is crucial for normal cellular responses to diverse extracellular signals. The model plant Arabidopsis thaliana has a single canonical G. subunit, AtGPA1. We found that, in planta, the constitutively active, GTP-bound AtGPA1(Q222L) mutant and the nucleotide-free AtGPA1(S52C) mutant interacted with G beta gamma 1 and G beta gamma 2 dimers with similar affinities, suggesting that G protein heterotrimer formation occurred independently of nucleotide exchange. In contrast, AtGPA1(Q222L) had a greater affinity than that of AtGPA1(S52C) for G beta gamma 3, suggesting that the GTP-bound conformation of AtGPA1(Q222L) is distinct and tightly associated with G beta gamma 3. Functional analysis of transgenic lines expressing either AtGPA1(S52C) or AtGPA1(Q222L) in the gpa1-null mutant background revealed various mutant phenotypes that were complemented by either AtGPA1(S52C) or AtGPA1(Q222L). We conclude that, in addition to the canonical GDP-GTP exchange-dependent mechanism, plant G proteins can function independently of nucleotide exchange.