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.

Abscisic acid regulation of guard-cell K + and anion channels in Gβ- and RGS-deficient Arabidopsis lines

“…3 A and B) supports previous observations of their interaction in heterologous systems (30,32) as well as biochemical evidence for interaction in rice and pea (33,34). Interaction between AGG1 or AGG2 and AGB1 is seen in yeast two-hybrid and in vitro binding assays and by FRET (15,32). Collectively, these data support the notion that plant ␣, ␤, and ␥ subunits form heterotrimers.…”

“…Heterotrimer-dependent signaling would be expected to be perturbed equally in agb1 single mutants, gpa1 single mutants, and gpa1 agb1 double mutants, i.e., the phenotypes we observe. Our data do not directly speak to the question of whether such a heterotrimer would contain GDP-GPA1 or GTP-GPA1: one FRET study on plant cells indicates that a mutant, constitutively active (GTP-bound) form of GPA1 can still exhibit FRET with AGB1, consistent with retention of a GTP-GPA1 subunit in a heterotrimer (32,50,52). Alternatively, ABA might stimulate activity of an as yet unidentified GDI or RGS protein (other than RGS1) and thus shift GDP-G␣ into the heterotrimeric complex.…”

Roles of ion channels and transporters in guard cell signal transduction

“…Many channels and transport proteins have been identified on the basis of their similarity to their mammalian counterparts however, novel plant-specific proteins or plant-specific functions of known proteins may exist. For proteins known to be involved in GC signaling, FRET-based studies could be helpful in analyzing subcellular co-localization and interaction [154]. Recent development of mass spectrometry-based proteomic approaches coupled with purification of protein complexes, as well as study of membrane protein interactions using the split ubiquitin based system [155,156], should also prove useful in identification of new protein partners of known components.…”

Dissecting Arabidopsis Gβ Signal Transduction on the Protein Surface

“…1, bottom panel), thus revealing the expected AGB1 subcellular localization. Plasma membrane tethering of AGB1 depends on the formation of a heterodimer with the AGG1 subunit via its N-terminal coiled-coil motif (Obrdlik et al, 2000;Adjobo-Hermans et al, 2006). Therefore, proper plasma membrane localization of mutated AGB1 suggests a functional conformation.…”