Completing the heterotrimer: isolation and characterization of an Arabidopsis thaliana G protein gamma-subunit cDNA.
Heterotrimeric G proteins consist of three subunits (alpha, beta, and gamma). alpha- and beta- subunits have been previously cloned in plants, but the gamma-subunit has remained elusive. To isolate the gamma-subunit of a plant heterotrimeric G protein an Arabidopsis thaliana yeast two-hybrid library was screened by using a tobacco G-beta-subunit as the bait protein. One positive clone (AGG1) was isolated several times; it displays significant homology to the conserved domains of mammalian gamma-subunits. The predicted AGG1 protein sequence contains all of the typical characteristics of mammalian gamma-subunits such as small size (98 amino acids, 10.8 kDa), presence of a C-terminal CAAX box to direct isoprenyl modification, and an N-terminal alpha-helix region capable of forming a coiled-coil interaction with the beta-subunit. Northern and Southern analyses showed that AGG1 is a single-copy gene in Arabidopsis with a similar expression pattern to the Arabidopsis beta-subunit, AGB1 [Weiss, C. A., Garnaat, C. W., Mukai, K., Hu, Y. & Ma, H. (1994) Proc. Natl. Acad. Sci. USA 91, 9554-9558]. By using the yeast two-hybrid system, we show that AGG1 strongly interacts with tobacco and Arabidopsis beta-subunits. The in vivo results have been confirmed by using in vitro methods to prove the interaction between AGG1 and the Arabidopsis beta-subunit. As previously observed in mammalian systems, both the coiled-coil domain and the WD repeat regions of the beta-subunit are essential for AGG1 interaction. Also in agreement with previous observations, the removal of the N-terminal alpha-helix of the AGG1 greatly reduces but does not completely block the interaction.
Mason
M G
MG
Plant Genetic Engineering Laboratory, Department of Botany, University of Queensland, Brisbane, Queensland 4072, Australia.
Botella
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics*; D001483:Base Sequence; D018076:DNA, Complementary; D018744:DNA, Plant; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics*; D018628:Gene Dosage; D020869:Gene Expression Profiling; D017343:Genes, Plant; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
C521249:AGG1 protein, Arabidopsis; D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D018744:DNA, Plant; D044388:GTP-Binding Protein gamma Subunits