Proteomics of Plant Detergent-resistant Membranes

Abstract

A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional microdomains that play important roles in protein sorting, signal transduction, or infection by pathogens. Recent reports demonstrated the presence, in plants, of detergent-resistant fractions isolated from plasma membrane. Analysis of the lipidic composition of this fraction revealed its enrichment in sphingolipids and sterols and depletion in phospho-and glycerolipids as previously observed for animal microdomains. One-dimensional gel electrophoresis experiments indicated that these detergent-resistant fractions are able to recruit a specific set of plasma membrane proteins and exclude others. In the present study, we used mass spectrometry to give an extensive description of a tobacco plasma membrane fraction resistant to solubilization with Triton X-100. This led to the identification of 145 proteins whose functional and physicochemical characteristics were analyzed in silico. Parameters such as isoelectric point, molecular weight, number and length of transmembrane segments, or global hydrophobicity were analyzed and compared with the data available concerning plant plasma membrane proteins. Post-translational modifications, such as myristoylation, palmitoylation, or presence of a glycosylphosphatidylinositol anchor, were examined in relation to the presence of the corresponding proteins in these microdomains. From a functional point of view, this analysis indicated that if a primary function of the plasma membrane, such as transport, seems under-represented in the detergent-resistant fraction, others undergo a significant increase of their relative importance. Among these are signaling and response to biotic and abiotic stress, cellular trafficking, and cell wall metabolism. This suggests that these domains are likely to constitute, as in animal cells,

Characterization of Lipid Rafts from Medicago truncatula Root Plasma Membranes: A Proteomic Study Reveals the Presence of a Raft-Associated Redox System

“…2), a percentage similar to that observed in tobacco leaves but 2-fold higher than BY2 cells (Morel et al, 2006). By using a combination of peptide extraction and sequencing by MS, we were able to identify 270 proteins in this lipid raft fraction, a much larger number than in most previous studies of plant lipid rafts (Peskan et al, 2000;Mongrand et al, 2004;Shahollari et al, 2004;Borner et al, 2005;Morel et al, 2006).…”

“…We observed that some proteins were enriched, whereas others were excluded from the DIMs when compared to the PM (data not shown). We examined the distribution of the PM proton pump ATPases (PMAs) found to be associated with other plant DIM fractions (Mongrand et al, 2004;Borner et al, 2005;Morel et al, 2006) and also in Medicago DIMs (see Table I). Results presented in Figure 2 show that PMAs are about equally distributed between the DIM fraction and the TX100 soluble fraction.…”

“…A protein and lipid characterization was also performed from Arabidopsis (Arabidopsis thaliana) callus microsomes (Borner et al, 2005). Other studies have identified proteins present in DIMs of Arabidopsis leaves and the tobacco (Nicotiana tabacum) Bright-Yellow2 (BY2) cell line (Peskan et al, 2000;Shahollari et al, 2004;Morel et al, 2006). The protein composition of the plant DIMs seems to differ from the composition of the PM (Alexandersson et al, 2004;Marmagne et al, 2004), and proteins found suggest roles of these domains in plant defense reactions (Leu-rich repeat receptor-like kinases [LRR-RLKs], remorin, kinases, NADPH oxidase), vectorial water flow (aquaporin), vesicular trafficking (syntaxin, dynamin-like protein), cell growth (proton ATPase), and cytoskeleton organization (actin, tubulin).…”

A Membrane Microdomain-Associated Protein, Arabidopsis Flot1, Is Involved in a Clathrin-Independent Endocytic Pathway and Is Required for Seedling Development

“…A number of studies have been performed on Arabidopsis thaliana (Borner et al, 2005), tobacco (Nicotiana tabacum; Mongrand et al, 2004;Morel et al, 2006), Sinapis alba (Shahollari et al, 2004), and Medicago truncatula (Lefebvre et al, 2007;Haney and Long, 2010). However, generally, these studies have focused on the analysis of protein and lipid components within membrane microdomains, using a variety of biochemical and cytological tools.…”

Modification of Plasma Membrane Organization in Tobacco Cells Elicited by Cryptogein

“…Plant DIMs with a lipid content similar to animal DIMs have been isolated from several species, including tobacco cells, and are enriched in proteins involved in signaling and stress responses (Mongrand et al, 2004;Borner et al, 2005;Morel et al, 2006;Lefebvre et al, 2007;Kierszniowska et al, 2009). Moreover, immunoelectron microscopy experiments have revealed that lateral segregation of lipids and proteins occurs at the nanoscale level at the tobacco PM, thus correlating detergent insolubility with membrane domain localization of presumptive raft proteins (Raffaele et al, 2009;Furt et al, 2010;Demir et al, 2013).…”