CHARACTERIZATION OF THE PUTATIVE ALPHA-SUBUNIT OF A HETEROTRIMERIC G-PROTEIN IN RICE

A recombinant protein with a cDNA that encodes the putative a 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)tripho sphate with an apparent association constant (k(app)) of 0.36. The pro tein also hydrolyzed GTP and its k(cat) 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 th e plasma membrane of rice seedlings. The peptide map of this polypepti de after digestion with V8 protease was identical to that of rGrice al pha. A 45 kDa polypeptide in the plasma membrane, as well as rGrice al pha, was ADP-ribosylated by activated cholera toxin. The GTPase activi ty 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 a: subunit is truly the a subunit itself.