AUTHOR=Lalonde Sylvie , Sero Antoinette , Pratelli Réjane , Pilot Guillaume , Chen Jin , Sardi Maria I., Parsa Saman A., Kim Do-Young , Acharya Biswa R., Stein Erica V., Hu Heng-Cheng , Villiers Florent , Takeda Kouji , Yang Yingzhen , Han Yong S., Schwacke Rainer , Chiang William , Kato Naohiro , Loqué Dominique , Assmann Sarah M., Kwak June M., Schroeder Julian , Rhee Seung Y., Frommer Wolf B.

TITLE=A Membrane Protein/Signaling Protein Interaction Network for Arabidopsis Version AMPv2

JOURNAL=Frontiers in Physiology

VOLUME=Volume 1 - 2010

YEAR=2010

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2010.00024

DOI=10.3389/fphys.2010.00024

ISSN=1664-042X

ABSTRACT=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, 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 387) pPPIs between 179 proteins, yielding a scale-free network (r2=0.863). Eighty of 142 transmembrane receptor-like kinases (RLK) tested positive, identifying three 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.