The lipid composition of plasma membrane (
PM) and the corresponding detergent-insoluble membrane (
DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl inositol phosphorylceramides (
GIPCs). Using tobacco (
Nicotiana tabacum) ‘Bright Yellow 2’ cell suspension and leaves, evidence is provided that
GIPCs represent up to 40 mol % of the
PM lipids. Comparative analysis of
DIMs with the
PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing
GIPCs and polyglycosylated
GIPCs in the
DIMs. Purified antibodies raised against these
GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated
GIPCs in domains of 35 ± 7 nm in the plane of the
PM. Biophysical studies also showed strong interactions between
GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two
PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed.Eukaryotic plasma membranes (
PMs) are composed of three main classes of lipids, glycerolipids, sphingolipids, and sterols, which may account for up to 100,000 different molecular species (
Yetukuri et al., 2008;
Shevchenko and Simons, 2010). Overall, all glycerolipids share the same molecular moieties in plants, animals, and fungi. By contrast, sterols and sphingolipids are different and specific to each kingdom. For instance, the plant
PM contains an important number of sterols, among which β-sitosterol, stigmasterol, and campesterol predominate (
Furt et al., 2011). In addition to free sterols, phytosterols can be conjugated to form steryl glycosides (
SG) and acyl steryl glycosides (
ASG) that represent up to approximately 15% of the tobacco (
Nicotiana tabacum)
PM (
Furt et al., 2010). As for sphingolipids, sphingomyelin, the major phosphosphingolipid in animals, which harbors a phosphocholine as a polar head, is not detected in plants. Glycosyl inositol phosphorylceramides (
GIPCs) are the major class of sphingolipids in plants, but they are absent in animals (
Sperling and Heinz, 2003;
Pata et al., 2010). Sphingolipidomic approaches identified up to 200 plant sphingolipids (for review, see
Pata et al., 2010;
Cacas et al., 2013).Although
GIPCs belong to one of the earliest classes of plant sphingolipids that were identified in the late 1950s (
Carter et al., 1958), only a few
GIPCs have been structurally characterized to date because of their high polarity and a limited solubility in typical lipid extraction solvents. For these reasons, they were systematically omitted from published plant
PM lipid composition.
GIPCs are formed by the addition of an inositol phosphate to the ceramide moiety, the inositol headgroup of which can then undergo several glycosylation steps. The dominant glycan structure, composed of a hexose-GlcA linked to the inositol, is called series A. Polar heads containing three to seven sugars, so-called series B to F, have been identified and appeared to be species specific (
Buré et al., 2011;
Cacas et al., 2013;
Mortimer et al., 2013). The ceramide moiety of
GIPCs consists of a long-chain base (
LCB), mainly t18:0 (called phytosphingosine) or t18:1 compounds (for review, see
Pata et al., 2010), to which is amidified a very-long-chain fatty acid (
VLCFA), the latter of which is mostly 2-hydroxylated (
hVLCFA) with an odd or even number of carbon atoms. In plants, little is known about the subcellular localization of
GIPCs. It is assumed, however, that they would be highly represented in the
PM (
Worrall et al., 2003;
Sperling et al., 2005), even if this remains to be experimentally proven. The main argument supporting such an assumption is the strong enrichment of trihydroxylated
LCB (t18:n) in detergent-insoluble membrane (
DIM) fractions (
Borner et al., 2005;
Lefebvre et al., 2007),
LCB being known to be predominant in
GIPC’s core structure as aforementioned.In addition to this chemical complexity, lipids are not evenly distributed within the
PM. Sphingolipids and sterols can preferentially interact with each other and segregate to form microdomains dubbed the membrane raft (
Simons and Toomre, 2000). The membrane raft hypothesis suggests that lipids play a regulatory role in mediating protein clustering within the bilayer by undergoing phase separation into liquid-disordered and liquid-ordered phases. The liquid-ordered phase, termed the membrane raft, was described as enriched in sterol and saturated sphingolipids and is characterized by tight lipid packing. Proteins, which have differential affinities for each phase, may become enriched in, or excluded from, the liquid-ordered phase domains to optimize the rate of protein-protein interactions and maximize signaling processes. In animals, rafts have been implicated in a huge range of cellular processes, such as hormone signaling, membrane trafficking in polarized epithelial cells, T cell activation, cell migration, and the life cycle of influenza and human immunodeficiency viruses (
Simons and Ikonen, 1997;
Simons and Gerl, 2010). In plants, evidence is increasing that rafts are also involved in signal transduction processes and membrane trafficking (for review, see
Mongrand et al., 2010;
Simon-Plas et al., 2011;
Cacas et al., 2012a).Moreover, lipids are not evenly distributed between the two leaflets of the
PM. Within the
PM of eukaryotic cells, sphingolipids are primarily located in the outer monolayer, whereas unsaturated phospholipids are predominantly exposed on the cytosolic leaflet. This asymmetrical distribution has been well established in human red blood cells, in which the outer leaflet contains sphingomyelin, phosphatidylcholine, and a variety of glycolipids like gangliosides. By contrast, the cytoplasmic leaflet is composed mostly of phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and their phosphorylated derivatives (
Devaux and Morris, 2004). With regard to sphingolipids and glycerolipids, the asymmetry of the former is established during their biosynthesis and that of the latter requires ATPases such as the aminophospholipid translocase that transports lipids from the outer to the inner leaflet as well as multiple drug resistance proteins that transport phosphatidylcholine in the opposite direction (
Devaux and Morris, 2004). This ubiquitous scheme encountered in animal cells could apply in plant cells as proposed (
Tjellstrom et al., 2010). Indeed, the authors showed that there is a pronounced transverse lipid asymmetry in root at the
PM. Phospholipids and galactolipids dominate the cytosolic leaflet, whereas the apoplastic leaflet is enriched in sphingolipids and sterols.From such a high diversity of the plant
PM thus arises the question of the respective contribution of lipids to membrane suborganization. Our group recently tackled this aspect by characterizing the order level of liposomes prepared from various plant lipids and labeled with the environment-sensitive probe di-4-ANEPPDHQ (
Grosjean et al., 2015). Fluorescence spectroscopy experiments showed that, among phytosterols, campesterol exhibits the strongest ability to order model membranes. In agreement with these data, spatial analysis of the membrane organization through multispectral confocal microscopy pointed to the strong ability of campesterol to promote liquid-ordered domain formation and organize their spatial distribution at the membrane surface. Conjugated sterols also exhibit a striking ability to order membranes. In addition,
GIPCs enhance the sterol-induced ordering effect by emphasizing the formation and increasing the size of sterol-dependent ordered domains.The aim of this study was to reinvestigate the lipid composition and organization of the
PM with a particular focus on
GIPCs using tobacco leaves and cv Bright Yellow 2 (
BY-2) cell cultures as models. Analyzing all membrane lipid classes at once, including sphingolipids, is challenging because they all display dramatically different chemical polarity, from very apolar (like free sterols) to highly polar (like polyglycosylated
GIPCs) molecules. Most lipid extraction techniques published thus far use a chloroform/methanol mixture and phase partition to remove contaminants, resulting in the loss
GIPCs, which remain in the aqueous phase, unextracted in the insoluble pellet, or at the interphase (
Markham et al., 2006). In order to gain access to both glycerolipid and sphingolipid species at a glance, we developed a protocol whereby the esterifed or amidified fatty acids were hydrolyzed from the glycerol backbone (glycerolipids) or the
LCB (sphingolipids) of membrane lipids, respectively. Fatty acids were then analyzed by gas chromatography-mass spectrometry (
GC-MS) with appropriate internal standards for quantification. We further proposed that the use of methyl
tert-butyl ether (
MTBE) ensures the extraction of all classes of plant polar lipids. Our results indicate that
GIPCs represent up to 40 mol % of total tobacco
PM lipids. Interestingly, polyglycolyslated
GIPCs are 5-fold enriched in
DIMs of
BY-2 cells when compared with the
PM. Further investigation led us to develop a preparative purification procedure that allowed us to obtain enough material to raise antibodies against
GIPCs. Using immunogold labeling on
PM vesicles, it was found that polyglycosylated
GIPCs cluster in membrane nanodomains, strengthening the idea that lateral nanosegregation of sphingolipids takes place at the
PM in plants. Multispectral confocal microscopy was performed on vesicles prepared using
GIPCs, phospholipids, and sterols and labeled with the environment-sensitive probe di-4-ANEPPDHQ. Our results show that, despite different fatty acid and polar head compositions,
GIPCs extracted from tobacco leaves and
BY-2 cells have a similar intrinsic propensity of enhancing vesicle global order together with sterols. Assuming that
GIPCs are mostly present in the outer leaflet of the
PM, interactions between sterols and sphingolipids were finally studied by the Langmuir monolayer technique, and the area of a single molecule of
GIPC, or in interaction with phytosterols, was calculated. Using the calculation docking method, the energy of interaction between
GIPCs and phytosterols was determined. A model was proposed in which
GIPCs and phytosterols interact together to form liquid-ordered domains and in which the
VLCFAs of
GIPCs promote the interdigitation of the two membrane leaflets. The implications of domain formation and the asymmetrical distribution of lipids at the
PM in plants are also discussed. Finally, we propose a model that reconsiders the intricate organization of the plant
PM bilayer.
相似文献