Identification of Heterotrimeric G Protein γ3 Subunit in Rice Plasma Membrane.

Aki Nishiyama,Takafumi Itoh,Sakura Matsuta,Kotaro Miura,Yukimoto Iwasaki,Genki Chaya

doi:10.3390/ijms19113591

Abstract

Heterotrimeric G proteins are important molecules for regulating plant architecture and transmitting external signals to intracellular target proteins in higher plants and mammals. The rice genome contains one canonical α subunit gene (RGA1), four extra-large GTP-binding protein genes (XLGs), one canonical β subunit gene (RGB1), and five γ subunit genes (tentatively named RGG1, RGG2, RGG3/GS3/Mi/OsGGC1, RGG4/DEP1/DN1/OsGGC3, and RGG5/OsGGC2). RGG1 encodes the canonical γ subunit; RGG2 encodes the plant-specific type of γ subunit with additional amino acid residues at the N-terminus; and the remaining three γ subunit genes encode the atypical γ subunits with cysteine abundance at the C-terminus. We aimed to identify the RGG3/GS3/Mi/OsGGC1 gene product, Gγ3, in rice tissues using the anti-Gγ3 domain antibody. We also analyzed the truncated protein, Gγ3∆Cys, in the RGG3/GS3/Mi/OsGGC1 mutant, Mi, using the anti-Gγ3 domain antibody. Based on nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, the immunoprecipitated Gγ3 candidates were confirmed to be Gγ3. Similar to α (Gα) and β subunits (Gβ), Gγ3 was enriched in the plasma membrane fraction, and accumulated in the flower tissues. As RGG3/GS3/Mi/OsGGC1 mutants show the characteristic phenotype in flowers and consequently in seeds, the tissues that accumulated Gγ3 corresponded to the abnormal tissues observed in RGG3/GS3/Mi/OsGGC1 mutants.

Highlights

Heterotrimeric G proteins are well known to consist of three subunits, α, β, and γ, in mammals and yeast [1,2,3,4]
When GTP binds to the α subunit (Gα-GTP), heterotrimeric G proteins dissociate into the α subunit (Gα-GTP) and βγ dimer
The interaction of rice β subunit with atypical γ subunits and the localization of these subunits in the plasma membrane were demonstrated with a bi-molecular fluorescence complementation (BiFC) assay [55,56]. These results indicated that both the canonical and atypical γ subunits can interact with the β subunit, and that βγ dimers are localized in the plasma membrane fraction, in Arabidopsis and rice

Summary

Introduction

Heterotrimeric G proteins are well known to consist of three subunits, α, β, and γ, in mammals and yeast [1,2,3,4]. The α subunit and βγ dimer can regulate respective effector molecules. Heterotrimeric G proteins are signal mediators from receptors to effector molecules. Heterotrimeric G proteins are important molecules for regulating plant architecture and transmitting external signals to intracellular target proteins [5,6,7]. The biochemical characteristics of the plant heterotrimeric G protein [5] and the signaling mechanism and effector molecules regulating the plant heterotrimeric G protein [6] have been previously reviewed. The plant morphology of heterotrimeric G protein mutants has been previously summarized [7]. There are three extra-large GTP-binding protein genes (AtXLG1~AtXLG3) [8,9], one canonical α subunit gene (GPA1) [10], one canonical β subunit gene (AGB1) [11], and three γ subunit genes (AGG1~AGG3) [12,13,14], in Arabidopsis; and four extra-large GTP-binding protein genes (prediction by in silico) [15], one canonical α subunit gene (RGA1) [16], one canonical β subunit gene (RGB1) [17], and five γ subunit genes, which we tentatively named RGG1 [18], RGG2 [18], RGG3/GS3/Mi/OsGGC1 [19], RGG4/DEP1/DN1/OsGGC3 [20], and RGG5/OsGGC2 [21], in this paper

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