Characterization of the putative alpha subunit of a heterotrimeric G protein in rice.

A recombinant protein with a cDNA that encodes the putative alpha subunit of a rice heterotrimeric G protein was synthesized in Escherichia coli and purified. The recombinant protein (rGrice alpha) with an apparent molecular mass of 45 kDa was bound with guanosine 5'-(3-O-thio)triphosphate with an apparent association constant (kapp) of 0.36. The protein also hydrolyzed GTP and its kcat was 0.44. rGrice alpha was ADP-ribosylated by activated cholera toxin. Monoclonal antibodies raised against rGrice alpha reacted with a 45 kDa polypeptide localized in the plasma membrane of rice seedlings. The peptide map of this polypeptide after digestion with V8 protease was identical to that of rGrice alpha. A 45 kDa polypeptide in the plasma membrane, as well as rGrice alpha, was ADP-ribosylated by activated cholera toxin. The GTPase activity of the plasma membrane was stimulated 2.5-fold by mastoparan 7 but not mastoparan 17. These properties were similar to those of the alpha subunits of heterotrimeric G proteins in animals, suggesting that the putative alpha subunit is truly the alpha subunit itself.