Plant G protein heterotrimers require dual lipidation motifs of Gα and Gγ and do not dissociate upon activation

In plants one bona fide G alpha subunit has been identified, as well as a single G beta and two G gamma subunits. To study the roles of lipidation motifs in the regulation of subcellular location and heterotrimer formation in living plant cells, GFP-tagged versions of the Arabidopsis thaliana heterotrimeric G protein subunits were constructed. Mutational analysis showed that the Arabidopsis G alpha subunit, GP alpha 1, contains two lipidation motifs that were essential for plasma membrane localization. The Arabidopsis G beta subunit, AG beta 1, and the G gamma subunit, AGG1, were dependent upon each other for tethering to the plasma membrane. The second G gamma subunit, AGG2, did not require AG beta 1 for localization to the plasma membrane. Like AGG1, AGG2 contains two putative lipidation motifs, both of which were necessary for membrane localization. Interaction between the subunits was studied using fluorescence resonance energy transfer ( FRET) imaging by means of fluorescence lifetime imaging microscopy (FLIM). The results suggest that AG beta 1 and AGG1 or AG beta 1 and AGG2 can form heterodimers independent of lipidation. In addition, FLIM-FRET revealed the existence of GP alpha 1-AG beta 1-AGG1 heterotrimers at the plasma membrane. Importantly, rendering GP alpha 1 constitutively active did not cause a FRET decrease in the heterotrimer, suggesting no dissociation upon GP alpha 1 activation.