Proteomics of Plant Detergent-resistant Membranes

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 phosphoand 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, signaling platforms involved in these physiological functions. Molecular & Cellular Proteomics 5:1396–1411, 2006. The plasma membrane of eukaryotes delineates the interface between the cell and the environment. It thus plays a crucial role in many essential functions such as cell nutrition (involving transport of solutes in and out of the cell), endocytosis, or response to environmental modifications (including defense against pathogens). Although this renders the elucidation of the proteic composition of this membrane a hot topic for a better understanding of these cellular processes, only a minority of integral membrane proteins have been experimentally identified in plants. Indeed membrane proteins are widely known as an unsuitable materiel for classical proteomic analysis using two-dimensional gel electrophoresis separation followed by the identification of each spot by mass spectrometry: many hydrophobic proteins are not solubilized in the isoelectric focusing sample buffer and precipitate at their isoelectric point (1). Previous two-dimensional gel electrophoresis of tobacco leaf plasma membrane led to an estimation of 500 polypeptides (2) but with a clearly established under-representation of intrinsic membrane proteins. Two more recent studies performed on Arabidopsis thaliana plasma membrane using mass spectrometry directly after SDS-PAGE separation identified, respectively, 97 hydrophobic plasma membrane proteins (3) and 238 putative plasma membrane proteins (4). An in silico analysis deduced from the genome of the model plant A. thaliana has been undertaken to identify putative membrane proteins (5). However, the diversity of the types of proteins associated to the plasma membrane (inferred from studies already performed) and the lack of signal peptide or specific signature indicating the targeting to this plasma membrane make the results very uncertain. A new aspect of the plasma membrane organization has arisen from biophysical and biochemical studies performed with animal cells for several years. Evidence has been given that the various types of lipids forming this membrane are not uniformly distributed inside the bilayer but rather spatially organized (6). This leads in particular to the formation of specialized phase domains, also called lipid rafts (7, 8). These domains, enriched in sterols and sphingolipids, form a liquid ordered phase inside the membrane. This structural characteristic renders these domains resistant to solubilization by non-ionic detergents, and this property has been widely used to isolate lipid rafts for further analysis (6). The most important hypothesis to explain the function of these domains is that they provide for lateral compartmentalization of membrane proteins and thereby create a dynamic scaffold to organize certain cellular processes. This ability to temporally and spatially organize protein complexes, while excluding others, conceivably allows for synchronization efficiency and speciFrom the ‡Laboratoire de Phytopharmacie, Unite Mixte de Recherche (UMR) 692 Institut National de la Recherche Agronomique (INRA)/ Ecole Nationale d’Enseignement Superieur Agronomique de Dijon (ENESAD)/Universite de Bourgogne, BP 86510, 21065 Dijon Cedex, France, ¶Plateforme de Genomique Fonctionnelle, Universite Victor Segalen Bordeaux 2, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France, and Laboratoire de Biogenese Membranaire, UMR 5200CNRS-Universite Victor Segalen Bordeaux 2, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France Received, February 3, 2006, and in revised form, March 31, 2006 Published, MCP Papers in Press, April 28, 2006, DOI 10.1074/ mcp.M600044-MCP200 Research