Biochemical characterization of TGÃ1, a plant heterotrimeric G-protein à subunit homologue

grantor: University of TorontoA cDNA encoding an $\alpha$ subunit homologue of a heterotrimeric G-protein in tomato (TGA1) was overexpressed in Escherichia coli as a GST fusion protein and purified by glutathione affinity. The molecular weight of the purified protein (TG$\alpha$1), after thrombin proteolysis to release the GST fragment, was 45 kDa. Assays using ($\gamma$-$\sp{32}$P) -GTP determined the $K\sb{cat}$ value of GTP hydrolysis (0.075-0.125 min$\sp{-1}$) while no hydrolysis was detected when ($\gamma$-$\sp{32}$P) -ATP was used as an alternative nucleotide. A conserved glutamine residue in position 223 was shown to regulate GTP hydrolysis by the enzyme. A point mutation in Q223 $\to$ L resulted in a GTPase-deficient enzyme, capable of binding GTP but with an extremely slow rate of hydrolysis. Polyclonal antibodies were raised using TG$\alpha$1 as an antigen and used for its subcellular localization. Immunoblot analysis using the affinity-purified anti-TG$\alpha$1 antibody detected a 45 kDa protein most abundantly in the plasma membrane and endoplasmic reticulum. The stimulation of the plasma membrane H$\sp+$-ATPase in response to mastoparan was inhibited by the use of synthetic peptides corresponding to the C-terminus of TG$\alpha$1. This suggests the coupling of TG$\alpha$1 to a seven-transmembrane spanning receptor in plants with its C-terminus as a receptor-G-protein contact. An immunologically-based interactive cloning screen identified an interaction between TG$\alpha$1 and a DnaJ-like protein. This interaction may represent a role for TG$\alpha$1 in regulation of polypeptide translocation into the ER; a mechanism for its processing and targeting; or a mechanism for its stabilization under stress responses.M.Sc