Poster: Intracellular Signaling

Abs # 824: Studies on the subunits of heterotrimeric G proteins in rice

Presenter:	Iwasaki, Yukimoto , iwasaki@fpu.ac.jp
Authors	Iwasaki, Yukimoto  (A)   Kato, Chiyuki  (A)   Mizutani, Tomohiro  (B)   Tamali, Hisanori  (B)   Kumagai, Hidehiko  (B)   Hirobe, Ayumi  (A)   Fujisawa, Yukiko  (A)   Kato, Hisaharu  (A)
Affiliations:	(A): Fukui Prefectural University (B): Kyoto University

We have analyzed the intracellular localization of the subunits and the subunit-subunit interaction for heterotrimeric G proteins in rice. A single species of cDNA has been cloned for each of the alpha (RGA1) and beta (RGB1) subunits of the rice G-proteins, whereas two were isolated for gamma subunit (RGG1 and RGG2). The recombinant proteins for these four subunits were produced in E. coli and purified by affinity chromatography. Specific antibodies against the recombinant proteins were produced in rabbits to use for Western blot analysis. The alpha, beta, gamma1 and gamma2 subunits were found to be localized mainly in plasma membrane fractions, and their stoicheiometric ratio was 1 : 2 : 0.5 : 1.5. When protein fractions solubilized from a membrane fraction with cholate were fractionated by gel filtration (Superdex 200 PC3.2/30, SMART System, Pharmacia), the alpha subunit was mainly present in fractions with about 400kDa. The beta and gamma subunits were localized mainly in the 400kDa fractions and mainly in fractions with about 60kDa. The beta subunit interacted strongly with gamma1 or gamma2 and weekly with the alpha subunit, when analyzed with yeast two-hybrid system. The results show that, in rice, a large amount of beta and gamma dimmer is present in the plasma membrane in a form free from the alpha subunit.