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-beta-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.

Figure 1.

Polyphosphoinositides are enriched in DIMs. A, Specificity of the antibody to PtdIns(4,5)P₂ tested by dot blot and immunodetection. Each lipid solution was dried, and lipids were resuspended in 50 μL of TBS-T, sonicated for 5 min, and plotted on a PVDF membrane. GluCER, Glucosylceramide. B, PtdIns(4,5)P₂ was enriched in DIMs. Five or 10 μg of PM or DIMs purified from BY-2 cells was analyzed by dot blot and immunodetection using an antibody to PtdIns(4,5)P₂.

Figure 2.

Lipid composition of PM and DIMs from tobacco leaves (A) and BY-2 cells (B). Lipids from membrane fractions were extracted with organic solvent mixture, separated by TLC, and quantified by GC as described in “Materials and Methods.” The data are expressed as means of three independent experiments ± sd. SG, Steryl glucoside; ASG, acylated steryl glucoside.

Figure 3.

Fatty acid composition of phospholipids from PM and DIMs purified from tobacco leaves and BY-2 cells. Total lipid extracts were subjected to TLC. Individual phospholipids were isolated, and fatty acids were transmethylated and analyzed by GC as described in “Materials and Methods.” To compare fatty acid compositions, bars represent mol % of total fatty acids present in each lipid and are means of three independent experiments ± sd.

Figure 4.

PtdIns(4,5)P₂ locates in membrane domains in BY-2 cell PM vesicles. A, Theoretical partitioning of gold particles in the plane of the PM. B, Transmission electron micrographs of negatively stained tobacco PM vesicles immunogold labeled on grids with antibodies to REM α130, detected by goat anti-mouse gold complex of 5 nm, and observed by electron microscopy. C and D, Transmission electron micrographs of negatively stained tobacco PM vesicles treated (D) or not (C) with mbCD and immunogold labeled on grids with antibodies to PtdIns(4,5)P₂, detected by goat anti-mouse gold complex of 5 nm, and observed by electron microscopy. Circles indicate obvious membrane domains. Statistical analyses are shown for each condition on the right (−mbCD, n = 3; +mbCD, n = 2). Bars = 100 nm.

Figure 5.

Biosynthetic pathways of signaling phospholipids. Lipid signaling molecules investigated in this study are part of a network of interdependent chemical conversions. DAGK, DAG kinase; InsP₃, inositol trisphosphate; PtdGro, phosphatidylglycerol; PAK, phosphatidic acid kinase; PIK, PtdIns kinase; PI3K, PtdIns 3-kinase; PI4K, PtdIns 4-kinase; PI5K, PtdIns 5-kinase; PIPK, PtdIns phosphate kinase.

Figure 6.

Comparison of specific enzyme activities in C-PM and PM purified from tobacco leaves (A) and BY-2 cells (B). The specific activity of each enzyme was determined in both C-PM and PM as described in “Materials and Methods.” The results are expressed as percentage gain or loss of specific activities in C-PM compared with PM. The data are means of three independent experiments ± sd and two independent experiments for DAGK in tobacco leaves and BY-2 cells and for PLD in tobacco leaves.

Figure 7.

Lipid signaling enzymes are active in DIMs. Comparison of specific enzyme activities in DIMs and C-PM purified from tobacco leaves (A) and BY-2 cells (B). The specific activity of each enzyme was determined in both DIMs and C-PM as described in “Materials and Methods.” Enzyme activities in DIMs are expressed as percentages of specific activities in C-PM. The data are means of at least three independent experiments ± sd and two independent experiments for DAGK in tobacco leaves and BY-2 cells, for PLD in tobacco leaves, and for PIPK in BY-2 cells.