Heterotrimeric guanine nucleotide binding proteins (G-proteins) are involved m a myriad of different signal transduction pathways, from sexual mating in yeast to perception of low light m mammals. In plants, G-proteins have been associated with several physiological responses including pathogen defence, stomatal control and light and phytohormone response although, the cellular pathways regulated by the plant G-proteins have yet to be identified. Until recently, research into plant signal transduction through G-proteins has progressed slowly. Since the cloning of the first plant G-protein subunit, several Gα and Gβ subunits have been cloned from various plant species including Arabidopsis, tomato, rice, pea, maize, and tobacco. However, the third G-protein subunit (Gγ) has remained elusive.
The strong association between Gy and GP subunits was exploited in an attempt to clone a plant G-protein y-subunit. Initially, antibodies were raised against the tobacco GP subunit (TGB1) and used in immunoprecipitation experiments to isolate the GPγ complex from a tobacco leaf extract. However, the antibodies failed to extract tobacco GP or its associated Gγ subunit despite their ability to detect TGBl by western analysis.
In different approach, an Arabidopsis thaliana yeast 2-hybrid library was screened using the tobacco and Arabidopsis GP subunits (TGBl and AGBl, respectively) as bait proteins. The use of AGBl as bait resulted in the isolation of 26 different genes, a number of which encoded proteins involved in vesicle trafficking, transcriptional activation and lipid biosynthesis and modification. The putative role of G-proteins m several plant responses including pathogen defence, cell division and phytohormone action may be explained by G-protein regulation of these interacting proteins. Approximately half of the genes isolated by AGBl display no significant homology to any previously characterised gene in the Genbank database. Further investigations into these unidentified genes will most likely lead to the discovery of new plant pathways that are regulated by G-proteins.
Seven genes were isolated in the yeast 2-hybrid screen using TGBl as the bait protein including the chloroplast sedoheptulose-l,7-bisphosphatase gene, a putative displayed no significant DNA homology to any characterised gene in the Genbank database. Two of the unidentified genes (AGG1 and AGG2) display 47% DNA identity to each other and contain long ORFs that have several characteristics in common with heterotrimeric G-protein y-subunits. These genes became the focus of this study and were further characterised.
The predicted protein sequences of AGGl and AGG2 display 48% identity and 59% homology to each other and show significant homology to mammalian y-subunits within the three Gγ conserved domains. More importantly, both AGGl and AGG2 contain all of the defining features of y-subunits including a small size between 6-12 kDa, presence of a N-terminus coiled-coil domain, C-terminal CAAX box and strong interaction with Gp both in vivo and in vitro. Based on these results, it was concluded that AGG1 and AGG2 are Arabidopsis G-protein y-subunits.
The discovery of AGGl and AGG2 demonstrates that plant G-protein y-subunits form a small multigene family but more importantly, suggests that different heterotrimer complexes are possible and can presumably regulate different signal transduction pathways in Arabidopsis. Like their mammalian counterparts, plant G-proteins may regulate a large number of pathways through the formation of different GPγ heterodimer combinations in different tissues and developmental stages. The cloning of two Gγ subunits completes the plant heterotrimer and will provide a strong foundation for future G-protein research. Furthermore, the isolation of several GP interacting proteins provides a focal point for further research into the signal transduction pathways of plant heterotrimeric G-proteins.