Characterization of heterotrimeric G protein complexes in rice plasma membrane

Abstract

Two genes in the rice genome were identified as those encoding the γ subunits, γ1 and γ2, of heterotrimeric G proteins. Using antibodies against the recombinant proteins for the α, β, γ1, and γ2 subunits of the G protein complexes, all of the subunits were proven to be localized in the plasma membrane in rice. Gel filtration of solubilized plasma membrane proteins showed that all of the α subunits were present in large protein complexes (about 400 kDa) containing the other subunits, β, γ1, and γ2, and probably also some other proteins, whereas large amounts of the β and γ (γ1 and γ2) subunits were freed from the large complexes and took a 60-kDa form. A yeast two-hybrid assay and co-immunoprecipitation experiments showed that the β subunit interacted tightly with the γ1 and γ2 subunits, and so the β and γ subunits appeared to form dimers in rice cells. Some dimers were associated with the α subunit, because few β, γ1, and γ2 subunits were present in the 400-kDa complexes in a rice mutant, d1, which was lacking in the α subunit. When a constitutively active form of the a subunit was prepared by the exchange of one amino acid residue and introduced into d1, the mutagenized subunit was localized in the plasma membrane of the transformants and took a free, and not the 400-kDa, form.