Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs) out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway-compatible vector. The mating-based split ubiquitin system was used to screen for potential protein–protein interactions (pPPIs) among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases (RLKs), 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions, and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 386) pPPIs between 179 proteins, yielding a scale-free network (r2 = 0.863). Eighty of 142 transmembrane RLKs tested positive, identifying 3 homomers, 63 heteromers, and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs) had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G-protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.
Keywords: protein interaction, transport, split ubiquitin system, yeast two hybrid, receptor, kinase, phosphorylation
Citation: Lalonde S, Sero A, Pratelli R, Pilot G, Chen J, Sardi MI, Parsa SA, Kim D-Y, Acharya BR, Stein EV, Hu H-C, Villiers F, Takeda K, Yang Y, Han YS, Schwacke R, Chiang W, Kato N, Loqué D, Assmann SM, Kwak JM, Schroeder JI, Rhee SY and Frommer WB (2010) A membrane protein/signaling protein interaction network for Arabidopsis version AMPv2. Front. Physio. 1:24. doi: 10.3389/fphys.2010.00024
This article was submitted to Frontiers in Plant Physiology, a specialty of Frontiers in Physiology
Received: 11 June 2010;
Paper pending published: 04 July 2010;
Accepted: 20 July 2010; Published online: 22 September 2010.
Edited by:Steven Huber, University of Illinois at Urbana-Champaign, USA
Reviewed by:Jeffrey F. Harper, University of Nevada, Reno, USA
Copyright: © 2010 Lalonde, Sero, Pratelli, Pilot, Chen, Sardi, Parsa, Kim, Acharya, Stein, Hu, Villiers, Takeda, Yang, Han, Schwacke, Chiang, Kato, Loqué, Assmann, Kwak, Schroeder, Rhee and Frommer. This is an open-access article subject to an exclusive license agreement between the authors and the Frontiers Research Foundation, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are credited.
*Correspondence: Sylvie Lalonde, Carnegie Institution for Science, 260 Panama St., Stanford, CA 94305, USA. e-mail: firstname.lastname@example.org