Polyphosphoinositides Are Enriched in Plant Membrane Rafts and Form Microdomains in the Plasma Membrane

Abstract

In this article, we analyzed the lipid composition of detergent-insoluble membranes (DIMs) purified from tobacco (Nicotiana tabacum) plasma membrane (PM), focusing on polyphosphoinositides, lipids known to be involved in various signal transduction events. Polyphosphoinositides were enriched in DIMs compared with whole PM, whereas all structural phospholipids were largely depleted from this fraction. Fatty acid composition analyses suggest that enrichment of polyphosphoinositides in DIMs is accompanied by their association with more saturated fatty acids. Using an immunogold-electron microscopy strategy, we were able to visualize domains of phosphatidylinositol 4,5-bisphosphate in the plane of the PM, with 60% of the epitope found in clusters of approximately 25 nm in diameter and 40% randomly distributed at the surface of the PM. Interestingly, the phosphatidylinositol 4,5-bisphosphate cluster formation was not significantly sensitive to sterol depletion induced by methyl-b-cyclodextrin. Finally, we measured the activities of various enzymes of polyphosphoinositide metabolism in DIMs and PM and showed that these activities are present in the DIM fraction but not enriched. The putative role of plant membrane rafts as signaling membrane domains or membrane-docking platforms is discussed.

Modification of Plasma Membrane Organization in Tobacco Cells Elicited by Cryptogein

“…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). Together, these data point to the existence of specialized lipid domains in plants.…”

“…Multidistance spatial cluster statistics analysis (Ripley's k function) of the immunogold-labeled ganglioside GM1 showed spatial aggregation in normal mouse fibroblasts for distances ranging from 32 to 68 nm (Fujita et al, 2007). Similarly, plant-based immunoelectron microscopy experiments have revealed partitioning of both the lipid phosphatidylinositol 4,5-bisphosphate into 25-nm clusters on the PM of tobacco BY2 cells (Furt et al, 2010) and the protein flotillin into clusters of less than 100 nm on the PM of Arabidopsis roots apices (Li et al, 2012). In addition, the protein remorin was observed by immunoelectron microscopy in domains of about 80 nm in the cytosolic leaflet of the membrane of tobacco leaves (Raffaele et al, 2009) and was more recently localized by stimulated emission depletion microscopy within domains of 97 nm in Arabidopsis, whereas the apparent size of these domains was about 250 nm, using classical confocal microscopy (Demir et al, 2013).…”

Plasma Membrane Localization of Solanum tuberosum Remorin from Group 1, Homolog 3 Is Mediated by Conformational Changes in a Novel C-Terminal Anchor and Required for the Restriction of Potato Virus X Movement

“…of lipids bound in vitro. Interestingly, these lipids are enriched in membrane rafts from plants (Vermeer et al, 2009;Furt et al, 2010) or animal cells (Hope and Pike, 1996;Pike et al, 2002), suggesting that RemCA lipidspecificity contributes to driving the segregation of StREM1.3 into membrane microdomains.…”

“…Indeed, polyphosphoinositides and sphingolipids are enriched in membrane rafts from plants (Vermeer et al, 2009;Furt et al, 2010) and animal cells (Hope and Pike, 1996), the latter being also enriched in phosphatidylserine (PS) (Pike et al, 2002). The ability to specifically target DIMs, therefore, might be related to the differential affinity of RemCA to certain negative lipids, as known for SNARE proteins (Mima and Wickner, 2009).…”

“…For PM-like liposomes, the lipid content was a phosphatidylethanolamine (PE)/ phosphatidylcholine (PC)/ phosphatidic acid/glucosylcerebrosides/tobacco mix of free sterols/acylsterylglucosides/ sterylglucosides at molar ratio 1/0.6/0.2/0.6/0.8/0.5/0.5 and for DIM-like liposomes, the same lipids at molar ratio 1/0.6/0.2/1.2/2.5/3/1 (Mongrand et al, 2004;Furt et al, 2010). Signals were quantified by ImageJ software.…”