Characterization of heterotrimeric G protein complexes in rice plasma membrane

• Published in: The Plant journal : for cell and molecular biology 2004-04, Vol.38 (2), p.320-331
• Main Authors: Kato, Chiyuki, Mizutani, Tomohiro, Tamaki, Hisanori, Kumagai, Hidehiko, Kamiya, Takehiro, Hirobe, Ayumi, Fujisawa, Yukiko, Kato, Hisaharu, Iwasaki, Yukimoto
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Biological and medical sciences; Cell biochemistry; Cell Membrane - metabolism; Cell physiology; Cross Reactions; DNA, Complementary - genetics; DNA, Plant - genetics; Fundamental and applied biological sciences. Psychology; Genes, Plant; heterotrimeric G protein; Heterotrimeric GTP-Binding Proteins - chemistry; Heterotrimeric GTP-Binding Proteins - genetics; Heterotrimeric GTP-Binding Proteins - metabolism; Macromolecular Substances; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Molecular Sequence Data; Mutation; Oryza - genetics; Oryza - metabolism; Oryza sativa; Plant physiology and development; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; Protein Subunits; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Recombinant Proteins - metabolism; rice; Sequence Homology, Amino Acid; Solubility; subunit composition; Two-Hybrid System Techniques
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313X.2004.02046.x

Summary 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 α 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.