Quantitative Proteomics Reveals a Dynamic Association of Proteins to Detergent-resistant Membranes upon Elicitor Signaling in Tobacco

A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional microdomains playing important roles in protein sorting, signal transduction, or infection by pathogens. In plants, as previously observed for animal microdomains, detergent-resistant fractions, enriched in sphingolipids and sterols, were isolated from plasma membrane. A characterization of their proteic content revealed their enrichment in proteins involved in signaling and response to biotic and abiotic stress and cell trafficking suggesting that these domains were likely to be involved in such physiological processes. In the present study, we used ¹⁴N/¹⁵N metabolic labeling to compare, using a global quantitative proteomics approach, the content of tobacco detergent-resistant membranes extracted from cells treated or not with cryptogein, an elicitor of defense reaction. To analyze the data, we developed a software allowing an automatic quantification of the proteins identified. The results obtained indicate that, although the association to detergent-resistant membranes of most proteins remained unchanged upon cryptogein treatment, five proteins had their relative abundance modified. Four proteins related to cell trafficking (four dynamins) were less abundant in the detergent-resistant membrane fraction after cryptogein treatment, whereas one signaling protein (a 14-3-3 protein) was enriched. This analysis indicates that plant microdomains could, like their animal counterpart, play a role in the early signaling process underlying the setup of defense reaction. Furthermore proteins identified as differentially associated to tobacco detergent-resistant membranes after cryptogein challenge are involved in signaling and vesicular trafficking as already observed in similar studies performed in animal cells upon biological stimuli. This suggests that the ways by which the dynamic association of proteins to microdomains could participate in the regulation of the signaling process may be conserved between plant and animals.The plasma membrane of eukaryotes delineates the interface between the cell and the environment. Thus it is particularly involved in environmental signal recognition and their transduction into intracellular responses, playing a crucial role in many essential functions such as cell nutrition (involving transport of solutes in and out of the cell) or response to environmental modifications (including defense against pathogens).Over the last 10 years, a new aspect of the plasma membrane organization has arisen from biophysical and biochemical studies performed with animal cells. Evidence has been given that the various types of lipids forming this membrane are not uniformly distributed inside the bilayer but rather spatially organized (1). This leads in particular to the formation of specialized phase domains, also called lipid rafts (2, 3). Recently a consensus emerged on the characteristics of these domains. Both proteins and lipids contribute to the formation and the stability of membrane domains that should be called “membrane rafts” and are envisaged as small (10–200-nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes (4). Small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions (5). Because of their particular lipidic composition (enrichment in sterol, sphingolipids, and saturated fatty acids), these domains form a liquid ordered phase inside the membrane. This structural characteristic renders them resistant to solubilization by non-ionic detergents, and this property has been widely used to isolate lipid rafts as detergent-resistant membranes (DRMs)¹ for further analysis (1). 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 (5). This ability to temporally and spatially organize protein complexes while excluding others conceivably allows for efficiency and specificity of cellular responses. In yeasts and animal cells, the association of particular proteins with these specialized microdomains has emerged as an important regulator of crucial physiological processes such as signal transduction, polarized secretion, cytoskeletal organization, generation of cell polarity, and entry of infectious organisms in living cells (6). Much of the early evidence for a functional role of lipid rafts came from studies of hematopoietic cells in which multichain immune receptors including the high affinity IgE receptor (FcεRI), the T cell receptor, and the B cell receptor (BCR) translocate to lipid rafts upon cross-linking (7). Moreover this signaling involves the relocalization of several proteins; for instance the ligation of the B cell antigen receptor with antigen induced a dissociation of the adaptor protein ezrin from lipid rafts (8). This release of ezrin acts as a critical trigger that regulates lipid raft dynamics during BCR signaling.In plants, the investigations of the presence of such microdomains are very recent and limited to a reduced number of publications (for a review, see Ref. 9). A few years ago, Peskan et al. (10) reported for the first time the isolation of Triton X-100-insoluble fractions from tobacco plasma membrane. Mongrand et al. (11) provided a detailed analysis of the lipidic composition of such a detergent-resistant fraction indicating that it was highly enriched in a particular species of sphingolipid (glycosylceramide) and in several phytosterols (stigmasterol, sitosterol, 24-methylcholesterol, and cholesterol) compared with the whole plasma membrane from which it originates. Similar results were then obtained with DRMs prepared from Arabidopsis thaliana cell cultures (12) and from Medicago truncatula roots (13). So the presence in plant plasma membrane of domains sharing with animal rafts a particular lipidic composition, namely strong enrichment in sphingolipids together with free sterols and sterol conjugates, the latter being specific to the plant kingdom (11, 13), now seems established. In plant only a few evidences suggest in vivo the role of dynamic clustering of plasma membrane proteins, and they refer to plant-pathogen interaction. A cell biology study reported the pathogen-triggered focal accumulation of components of the plant defense pathway in the plasma membrane (PM), a process reminiscent of lipid rafts (14). Consistently a proteomics study of tobacco DRMs led to the identification of 145 proteins among which a high proportion were linked to signaling in response to biotic stress, cellular trafficking, and cell wall metabolism (15). This suggests that these domains are likely to constitute, as in animal cells, signaling platforms involved in such physiological functions.Cryptogein belongs to a family of low molecular weight proteins secreted by many species of the oomycete Phytophthora named elicitins that induce a hypersensitivity-like response and an acquired resistance in tobacco (16). To understand molecular processes triggered by cryptogein, its effects on tobacco cell suspensions have been studied for several years. Early events following cryptogein treatment include fixation of a sterol molecule (17, 18); binding of the elicitor to a high affinity site located on the plasma membrane (19); alkalinization of the extracellular medium (20); efflux of potassium, chloride, and nitrate (20, 21); fast influx of calcium (22); mitogen-activated protein kinases activation (23, 24); nitric oxide production (25, 26); and development of an oxidative burst (27, 28). We previously identified NtrbohD, an NADPH oxidase located on the plasma membrane, as responsible for the reactive oxygen species (ROS) production occurring a few minutes after challenging tobacco Bright Yellow 2 (BY-2) cells with cryptogein (29). The fact that most of these very early events involve proteins located on the plasma membrane and that one of them, NtrbohD, has been demonstrated as exclusively associated to DRMs in a sterol-dependent manner (30) prompted us to analyze the modifications of DRM proteome after cryptogein treatment. In the present study, we aimed to confirm the hypothesis that, as observed in animal cells, the dynamic association to or exclusion of proteins from lipid rafts could participate in the signaling process occurring during biotic stress in plants.To achieve this goal, we had to set up a quantitative assay allowing a precise comparison of the amounts of each protein in DRMs extracted from either control or cells treated with cryptogein. Among several technologies, we excluded DIGE (31), recently used to analyze whole cell proteome variations in plants (32–34), because membrane proteins are poorly soluble in the detergents used for two-dimensional electrophoresis; this limitation is all the more marked for proteins selected on the basis of their insolubility in non-ionic detergent, the criteria for DRMs isolation. Stable isotope labeling of proteins or peptides combined with MS analysis represents alternative strategies for accurate, relative quantification of proteins on a global scale (35, 36). In these approaches, proteins or peptides of two different samples are differentially labeled with stable isotopes, combined in an equal ratio, and then jointly processed for subsequent MS analysis. Relative quantification of proteins is based on the comparison of signal intensities or peak areas of isotope-coded peptide pairs extracted from the respective mass spectra. Stable isotopes can be introduced either chemically into proteins/peptides via derivatization of distinct functional groups of amino acids or metabolically during protein biosynthesis (for a review, see Ref. 37). Metabolic labeling strategies are based on the in vivo incorporation of stable isotopes during growth of organisms. Nutrients or amino acids in a defined medium are replaced by their isotopically labeled (¹⁵N, ¹³C, or ²H) counterparts eventually resulting in uniform labeling of proteins during the processes of cell growth and protein turnover (38). As a consequence, differentially labeled cells or organisms can be combined directly after harvesting. This minimizes experimental variations due to separate sample handling and thus allows a relative protein quantification of high accuracy.¹⁴N/¹⁵N labeling has been recently proved to be suitable for comparative experiments performed with whole plants (39–42) and in plant suspension cells where the level of incorporation is equal to the isotopic purity of the salt precursor (43, 44). It has been used successfully to analyze some variations induced in A. thaliana plasma membrane proteome following heat shock (45) or cadmium exposure (46) and to compare phosphorylation levels of plasma membrane proteins after challenge of Arabidopsis cells with elicitors of defense reaction (47). In the present study, we used a mineral ¹⁴N/¹⁵N metabolic labeling of tobacco BY-2 cells before treatment with cryptogein and subsequent isolation of DRMs. The DRM proteins were further analyzed by one-dimensional SDS-PAGE and digested by trypsin, and peptides were subjected to microcapillary high performance LC-MS/MS (nano-LC-MS/MS). This metabolic method allowed a complete labeling of the proteome, and consequently a major drawback of this method is probably the difficulty to perform an exhaustive analysis of the very large amount of data generated. To solve this problem, a new quantification module of the MFPaQ software (48) was developed, allowing the automatic quantification of the identified peptides. The results derived from the program were validated through a comparison with manual quantification. Thus, we achieved the complete analysis of the DRM proteome variation and identified four proteins whose abundance in DRMs was decreased and one that was enriched in DRMs upon elicitation. The biological relevance of these results, which indicate that, in plant as in animals, the dynamic association of proteins to membrane domains is part of a signaling pathway, will be further discussed.