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Depletion of phytosterols from the plant plasma membrane provides evidence for disruption of lipid rafts

Yann Roche^*, Patricia Gerbeau-Pissot^*,1, Blandine Buhot^*, Dominique Thomas^*, Laurent Bonneau^*, Joseph Gresti, Sébastien Mongrand, Jean-Marie Perrier-Cornet and Françoise Simon-Plas^*

^* Laboratoire Plantes-Microbe-Environnement, UMR INRA 1088/CNRS 5184, and

UMR 866 Lipides, Nutrition, Cancer, Equipe Physiopathologie des Dyslipidémies, Université de Bourgogne, Dijon, France;

Laboratoire de Biogenèse Membranaire, UMR 5200 CNRS-Université Victor Segalen Bordeaux 2, Bordeaux, France; and

Laboratoire de Génie des Procédés Microbiologiques et Alimentaires, Ensbana, Dijon, France

¹Correspondence: Laboratoire Plantes-Microbe-Environnement, UMR INRA 1088/CNRS 5184/Université de Bourgogne, 17 rue Sully, BP 86510, 21065 Dijon cedex, France. E-mail: patricia.gerbeau-pissot{at}dijon.inra.fr

Involvement of sterols in membrane structural properties has been extensively studied in model systems but rarely assessed in natural membranes and never investigated for the plant plasma membrane (PM). Here, we address the question of the role of phytosterols in the organization of the plant PM. The sterol composition of tobacco BY-2 cell PM was determined by gas chromatography. The cyclic oligosaccharide methyl-β-cyclodextrin, commonly used in animal cells to decrease cholesterol levels, caused a drastic reduction (50%) in the PM total free sterol content of the plant material, without modification in amounts of steryl-conjugates. Fluorescence spectroscopy experiments using DPH, TMA-DPH, Laurdan, and di-4-ANEPPDHQ indicated that such a depletion in sterol content increased lipid acyl chain disorder and reduced the overall liquid-phase heterogeneity in correlation with the disruption of phytosterol-rich domains. Methyl-β-cyclodextrin also prevented isolation of a PM fraction resistant to solubilization by nonionic detergents, previously characterized in tobacco, and induced redistribution of the proteic marker of this fraction, NtrbohD, within the membrane. Altogether, our results support the role of phytosterols in the lateral structuring of the PM of higher plant cells and suggest that they are key compounds for the formation of plant PM microdomains.—Roche, Y., Gerbeau-Pissot, P., Buhot, B., Thomas, D., Bonneau, L., Gresti, J., Mongrand, S., Perrier-Cornet, J.-M., Simon-Plas, F. Depletion of phytosterols from the plant plasma membrane provides evidence for disruption of lipid rafts.

Key Words: cyclodextrin • detergent-resistant membranes • membrane lateral organization • microdomains • tobacco • spectrofluorimetry