GTP Binding Assays in Arabidopsis thaliana

Abstract

Signaling through G proteins constitutes an ancient mechanism that functions in the transduction of extracellular signals into intracellular responses. Activation of a proper receptor by a stimuli leads to an exchange of GDP for GTP, the activation of G proteins and the dissociation of Gα-GTP from the Gβγ dimer for the heterotrimeric G proteins. The G protein subunits remain active until the intrinsic GTPase activity or/and a GTPase activating protein result in the hydrolysis of GTP to GDP and the inactivation of the protein. Here we describe a protocol for measuring GTP binding activity from Arabidopsis plant protein extracts using GTP γ35S. GTP γ35S assay measures the level of G protein activation following a stimuli, by determining the binding activity of the non-hydrolysable analog GTP γ35S. To determine specific G protein activities specific mutants and/or overexpressors extracts should be included and measured as controls.