PA (phosphatidic acid) is a lipid second messenger involved in an array of processes occurring during a plant's life cycle. These include development, metabolism, and both biotic and abiotic stress responses. PA levels increase in response to salt, but little is known about its function in the earliest responses to salt stress. In the present study we have combined an approach to isolate peripheral membrane proteins of Arabidopsis thaliana roots with lipid-affinity purification, to identify putative proteins that interact with PA and are recruited to the membrane in response to salt stress. Of the 42 putative PA-binding proteins identified by MS, a set of eight new candidate PA-binding proteins accumulated at the membrane fraction after 7 min of salt stress. Among these were CHC (clathrin heavy chain) isoforms, ANTH (AP180 N-terminal homology) domain clathrin-assembly proteins, a putative regulator of potassium transport, two ribosomal proteins, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and a PI (phosphatidylinositol) 4-kinase. PA binding and salt-induced membrane recruitment of GAPDH and CHC were confirmed by Western blot analysis of the cellular fractions. In conclusion, the approach of the present study is an effective way to isolate biologically relevant lipid-binding proteins and provides new leads in the study of PA-mediated salt-stress responses in roots.
Key words: cellular membrane, phospholipid signalling, phosphatidic acid, protein–lipid interaction, quantitative proteomics, salinity.
Abbreviations used: ABA, abscisic acid; ABI1, ABA-insensitive 1; ACN, acetonitrile; ANTH, AP180 N-terminal homology; CHC, clathrin heavy chain; CME, clathrin-mediated endocytosis; COXII, cytochrome c oxidase subunit II; DGK, diacylglycerol kinase; DTT, dithiothreitol; ENTH, epsin N-terminal homology; ER, endoplasmic reticulum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HILIC, hydrophilic interaction liquid chromatography; iTRAQ, isobaric tags for relative and absolute quantification; KAB1, potassium channel β subunit 1; LC, liquid chromatography; MitM, mitochondrial membrane; MSE, elevated energy MS; PA, phosphatidic acid; PDK1, phosphoinositide-dependent kinase 1; PEPC, phosphoenolpyruvate carboxylase; PI4Kγ, phosphoinositide 4-kinase γ; PIP2, phosphatidylinositol bisphosphate; PLC, phospholipase C; PLD, phospholipase D; PM, plasma membrane; PMP, peripheral membrane protein; PPI, phosphoinositide; PTEN, phosphatase and tensin homologue; Q-TOF, quadrupole time-of-flight; Rboh, respiratory burst oxidase homologue protein; ROS, reactive oxygen species; S/C, salt-treated/control; SnRK2, Snf1-related protein kinase 2; TFA, trifluoroacetic acid; UGPase, UDP-glucose pyrophosphorylase; V-ATPase, vacuolar ATPase.
1Present address: University of Massachusetts, 1003 Lederle Graduate Research Center, 710 North Pleasant Street, Amherst, MA 01003, U.S.A.
2To whom correspondence should be addressed (email c.s.testerink@uva.nl).
Received 21 November 2012/3 January 2013; accepted 17 January 2013
Published as BJ Immediate Publication 17 January 2013, doi:10.1042/BJ20121639
© The Authors Journal compilation © 2013 Biochemical Society
