Time-dependent, glucose-regulated Arabidopsis Regulator of G-protein Signaling 1 network ☆

摘要

Abstract Plants lack 7-transmembrane, G-protein coupled receptors (GPCRs) because the G alpha subunit of the heterotrimeric G protein complex is “self-activating”—meaning that it spontaneously exchanges bound GDP for GTP without the need of a GPCR. In lieu of GPCRs, most plants have a seven transmembrane receptor-like regulator of G-protein signaling (RGS) protein, a component of the complex that keeps G-protein signaling in its non-activated state. The addition of glucose physically uncouples AtRGS1 from the complex through specific endocytosis leaving the activated G protein at the plasma membrane. The complement of proteins in the AtRGS1/G-protein complex over time from glucose-induced endocytosis was profiled by immunoprecipitation coupled to mass spectrometry (IP-MS). A total of 119 proteins in the AtRGS1 complex were identified. Several known interactors of the complex were identified, thus validating the approach, but the vast majority (93/119) were not known previously. AtRGS1 protein interactions were dynamically modulated by d -glucose. At low glucose levels, the AtRGS1 complex is comprised of proteins involved in transport, stress and metabolism. After glucose application, the AtRGS1 complex rapidly sheds many of these proteins and recruits other proteins involved in vesicular trafficking and signal transduction. The profile of the AtRGS1 components answers several questions about the type of coat protein and vesicular trafficking GTPases used in AtRGS1 endocytosis and the function of endocytic AtRGS1. Keywords Heterotrimeric G-protein ; Membrane protein complexes ; Tandem affinity purification ; Mass spectrometry ; Glucose signaling prs.rt("abs_end"); 1. Introduction The Arabidopsis G-proteins, despite having a simpler repertoire than that in metazoans, impart many physiological and biochemical responses and affect growth and development in plants [1] . In metazoans, the duration of G-protein signal termination is dependent on (1) the residence time of the GPCR agonist, (2) the number of activating interactions between the cognate GPCR coupled to the G protein complex over time and (3) the rate of deactivation through intrinsic GTPase activity of the Gα protein. The latter is accelerated by interaction with a group of regulator of G protein signaling (RGS) proteins, accelerating hydrolysis of G_αGTP into G_αGDP and returning the G protein complex to the resting state [2] . It is now well established that plants use a distinct mechanism to regulate G-protein signaling from metazoans and differ in many aspects. First, plant cells lack G-protein coupled receptors (GPCR) that stimulate guanine nucleotide exchange. Second, plant Gα proteins exchange guanine nucleotides spontaneously in vitro . Third, all plants, except the cereals, contain a receptor-like RGS protein that deactivates until decoupled from the G protein complex. In Arabidopsis , the RGS protein (AtRGS1) has a seven transmembrane (7TM) domain at its N-terminus and a catalytic RGS box at its C-terminal domain [3] , [4] , [5] , [6] and [7] . The mechanism of glucose-induced G protein activation is known. Urano et al. demonstrated that d -glucose recruits WITH NO LYSINE (WNK) kinases to phosphorylate AtRGS1 and that this phosphorylation is necessary and sufficient for endocytosis [7] and [8] . AtRGS1 endocytosis leads to physical uncoupling of AtRGS1 from the Arabidopsis G protein α subunit (AtGPA1) and thus a release of the GAP activity and concomitant sustained activation of G-protein signaling. One of the most intriguing aspects of glucose-regulated, AtRGS1-mediated G-protein activation is that the response is receptive to both signal concentration and timing information, unlike G-protein signaling in animals which is triggered by a threshold of signal [8] . This emergent property has dose and duration reciprocity such that an acute dose of glucose ( e.g. 6%) induces complete AtRGS1 endocytosis in 30?min while a low dose induces endocytosis over many hours [8] . While AtRGS1 is considered an inhibitor of G-protein signaling as a GTPase Activating Protein (GAP), the effect of genetic ablation of AtRGS1 suggests that AtRGS1 is a positive regulator of G-protein signaling [9] . Given that trafficking of AtRGS1 is an important part of plant G protein signaling, we hypothesized that signaling through AtRGS1 is both time and sub-cellular location contingent. Specifically, AtRGS1 signaling from the endosome may be an obligatory aspect of signaling output as has been recently shown for β2-adrenoceptor-mediated signaling [10] . Studies of plant G-proteins in the last decade revealed associations with fundamental biological processes such as sugar perception [11] and [7] , organ development [12] , hormone signaling [13] and [14] , light responsiveness [15] , biotic and abiotic stress [16] , [17] , [18] , [19] and [6] , among others. Sugar-induced signal transduction pathways play significant roles in many physiological process