Lipid rafts in higher plant cells: purification and characterization of Triton X-100-insoluble microdomains from tobacco plasma membrane.
A large body of evidence from the past decade supports the existence of functional microdomains in membranes of animal and yeast cells, which play important roles in protein sorting, signal transduction, or infection by pathogens. They are based on the dynamic clustering of sphingolipids and cholesterol or ergosterol and are characterized by their insolubility, at low temperature, in nonionic detergents. Here we show that similar microdomains also exist in plant plasma membrane isolated from both tobacco leaves and BY2 cells. Tobacco lipid rafts were found to be greatly enriched in a sphingolipid, identified as glycosylceramide, as well as in a mixture of stigmasterol, sitosterol, 24-methylcholesterol, and cholesterol. Phospho- and glycoglycerolipids of the plasma membrane were largely excluded from lipid rafts. Membrane proteins were separated by one- and two-dimensional gel electrophoresis and identified by tandem mass spectrometry or use of specific antibody. The data clearly indicate that tobacco microdomains are able to recruit a specific set of the plasma membrane proteins and exclude others. We demonstrate the recruitment of the NADPH oxidase after elicitation by cryptogein and the presence of the small G protein NtRac5, a negative regulator of NADPH oxidase, in lipid rafts.
Mongrand
Sébastien
S
Laboratoire de Biogenèse Membranaire, FRE 2694-CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France. sebastien.mongrand@biomemb.u-bordeaux2.fr
Morel
Johanne
J
Laroche
Jeanny
J
Claverol
Stéphane
S
Carde
Jean-Pierre
JP
Hartmann
Marie-Andrée
MA
Bonneu
Marc
M
Simon-Plas
Françoise
F
Lessire
René
R
Bessoule
Jean-Jacques
JJ
D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003902:Detergents / Q000494:pharmacology*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004875:Ergosterol / Q000378:metabolism; D007477:Ions; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism; D008854:Microscopy, Electron; D019255:NADPH Oxidases / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology*; D010840:Phytosterols; D018515:Plant Leaves / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D012855:Sitosterols / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013265:Stigmasterol / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology; D013696:Temperature; D014026:Nicotiana / Q000378:metabolism*
D003902:Detergents; D007477:Ions; D008055:Lipids; D010840:Phytosterols; D012855:Sitosterols; D013395:Sucrose; C021273:campesterol; C025473:gamma-sitosterol; D017830:Octoxynol; D002784:Cholesterol; D013265:Stigmasterol; D019255:NADPH Oxidases; D004875:Ergosterol