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

Abstract

In plants one bona fide Gα subunit has been identified, as well as a single Gβ and two Gγ 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 Gα subunit, GPα1, contains two lipidation motifs that were essential for plasma membrane localization. The Arabidopsis Gβ subunit, AGβ1, and the Gγ subunit, AGG1, were dependent upon each other for tethering to the plasma membrane. The second Gγ subunit, AGG2, did not require AGβ1 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 AGβ1 and AGG1 or AGβ1 and AGG2 can form heterodimers independent of lipidation. In addition, FLIM-FRET revealed the existence of GPα1-AGβ1-AGG1 heterotrimers at the plasma membrane. Importantly, rendering GPα1 constitutively active did not cause a FRET decrease in the heterotrimer, suggesting no dissociation upon GPα1 activation.