lipid rafts in higher plant cells: purification and ... · 6/9/2004 · tobacco lipid rafts were...

Purification and characterization of higher plant lipid rafts

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LIPID RAFTS IN HIGHER PLANT CELLS: PURIFICATION AND CHARACTERIZATION

OF TX100-INSOLUBLE MICRODOMAINS FROM TOBACCO PLASMA MEMBRANE

Sébastien Mongrand a§, Johanne Morel b, Jeanny Laroche a, Stéphane Claverol d,

Jean-Pierre Carde c, Marie-Andrée Hartmann e, Marc Bonneu d,

Françoise Simon-Plas b, René Lessire a, Jean-Jacques Bessoule a

a Laboratoire de Biogenèse Membranaire, FRE 2694-CNRS-Université Victor Segalen Bordeaux 2, 146,

rue Léo Saignat, 33076 Bordeaux Cedex, Franceb Laboratoire de Phytopharmacie, UMR 692 INRA-Université de Bourgogne 86510, 21065 Cédex Dijon,

Francec Institut de Biologie Végétale Moléculaire, UMR 619INRA-Université Bordeaux 1-Université Bordeaux

2. 71, Av. Edouard Bourlaux, B.P. 81, 33883 Villenave d’Ornon Cedex, Franced Plateforme Génomique Fonctionnelle, Université Victor Segalen Bordeaux 2, 146, rue Léo Saignat,

33076 Bordeaux Cedex, Francee Institut de Biologie Moléculaire des Plantes, UPR-CNRS 2357, 67083 Strasbourg Cedex, France

JBC Papers in Press. Published on June 9, 2004 as Manuscript M403440200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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SUMMARY

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 non-ionic 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 glyco-

glycerolipids of the plasma membrane were largely excluded from lipid rafts. Membrane

proteins were separated by one- and two-dimensional gel electrophoresis and identified by

MS/MS 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 (NtrbohD) after elicitation by cryptogein and the presence of

the small G protein NtRac5, a negative regulator of NtrbohD, in lipid rafts. The results are

discussed in relation to their possible involvement in some plant cell signaling events and

particularly in plant’s pathogen defense.

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INTRODUCTION

A new aspect of the lipid bilayer organization has raised from biophysical and biochemical

studies performed with animal cells for several years. Indeed, lipids are not uniformly miscible,

but lateral separation of specific lipid species leads to the formation of specialized phase domains

also called “lipid rafts”. (1). The main role in the process of domain organization is played by

sterols and sphingolipids, these latter interacting together through weak interaction between

aliphatic chains stabilized by the presence of saturated alkyl chains; voids between sphingolipids

being filled by sterols (for review 2). The cholesterol-sphingolipid-enriched domain formation is

also enhanced by the fact that sphingolipids have higher melting temperatures than

phospholipids. Regions between rafts are occupied by phospholipids with unsaturated fatty acids

forming a liquid-crystalline phase, whereas lipid rafts which contain more saturated aliphatic

chains form a liquid ordered phase. In model and biological membranes, the formation of the

liquid ordered phase correlates with resistance to solubilization by nonionic detergent such as

Triton X-100 at 4ºC and buoyancy at specific density in sucrose gradient (3). Thus, isolation of

Detergent-Insoluble Membranes (DIM) or Detergent-Insoluble Glycolipid-enriched membrane

domains (DIGs) is one of the most widely used methods for studying lipid rafts.

In animal cells, these membrane domains act, for example, as sorting devices for the

accumulation of acylated, GPI-anchored, palmitoylated signaling molecules, that selectively

locate in these domains. Tyrosine kinases of the Src-family protein, heterotrimeric and small G-

proteins as well as phosphoinositides have been proved to be located in rafts. In addition, several

transmembrane receptors are inductively recruited or stabilized within lipid rafts, including T

and B cell receptors, and IL-2/IL-15 receptors (4). This targeting of proteins to rafts might affect

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their function in two ways: the concentration of proteins in rafts could facilitate interactions

between them, and the ordered lipid environment might directly modulate their activity, possibly

by a modification of their conformation. The size of rafts seems to be a critical parameter: the

small size of an individual raft may be important for keeping raft-bound signaling proteins in an

inactive state. Upon stimulation, many rafts may have to cluster to form a larger platform where

functionally related proteins can interact (4).

In yeast and animal cells, the association of proteins with these specialized plasma membrane

microdomains has emerged as an important regulator of signal transduction, protein and

membrane polarized intracellular sorting, cytoskeletal re-organization and entry of infectious

organisms in living cells (for review 1, 5). In plants, numerous biological evidences support the

existence of functional domains in the plasma membrane. For example, the extreme polarized

growth of pollen tube and root hair could be explained by the presence of very specialized

membrane domains at the end of the cell (6). The concomitant recruitment of the membrane

phosphoinositide PIP2 and the small G-protein RAC at the tip of the tobacco pollen tube is

indeed consistent with such an hypothesis (6). More generally, the asymmetric growth of plant

cells is probably due to an asymmetric distribution of membrane components. For example, the

vectorial auxin flow has been postulated to arise from a polarized localization of efflux carriers,

that could be also located within rafts (7). Therefore, isolating and examining lipid raft in plant

membranes may provide new insights into the control of these crucial physiological processes,

but also of some others events which require the set up of a rapid and coordinate signal

transduction pathway.

While very little attention has been paid to lipid rafts in plant membranes, one previous work

evidenced that Triton X-100-insoluble membranes could be isolated from tobacco leaf plasma

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membranes (8). This work showed that membrane-bound heterotrimeric G-protein beta-subunit

and GPI-anchored proteins are located in a Triton X-100-insoluble fraction. In the present study,

we fully characterized the morphology, the lipid composition of plant Triton X-100-insoluble

fraction and initiated a proteomic study of proteins enriched in the lipid rafts to decipher the

physiological role(s) of plant membrane microdomains. We first determined the experimental

conditions to isolate Triton X-100-insoluble fractions from tobacco plasma membrane,

characterized their morphology by electron microscopy, and further analyzed their lipid

composition using HP-TLC, GC and mass spectrometry. We further studied by mono- and two-

dimensional electrophoresis the protein composition of lipid rafts vs. plasma membrane. The

results obtained confirm the presence of membrane domains with a lipid and protein composition

distinct from the whole plasma membrane. Results are discussed in relation to their possible

involvement in some plant physiological functions.

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EXPERIMENTAL PROCEDURES

Material—Leaves were obtained from 8 week-old tobacco plants (Nicotiana tabacum cv.

Xanthi) grown in a growth chamber at 25°C under 16/8 hours day /night conditions. BY2 cells

(N. tabacum cv. Bright Yellow 2) wild type or transformed with a construct corresponding to the

fusion of Ntrac5 (AJ250174) with a RGS-His tag (9) were grown as previously described in 10.

A purified mouse monoclonal RGS-6xHis antibody (0.2mg.ml-1) was obtained from Qiagen

(Courtaboeuf, France). Cryptogein was purified as described previously (9) and BY2 cells were

treated at a final concentration of 50 nM. A polyclonal rabbit serum raised against aminoacids

138-152 and 784-798 of the NtrbohD protein was obtained from Eurogentec (4102 Seraing-

Belgium). IgG were purified according to manufacturer’s instructions and resuspended in

distilled water at a concentration of 2mg.ml-1.

Preparation and purity of tobacco plasma membrane (PM)—All steps were performed at 4°C.

PM were obtained after cell fractionation according to (10) by partitioning in an aqueous

polymer two-phase system with PEG3350/DextranT-500 (6.6% each). ATPase activity was

measured according to 11. Marker activities used to evaluate the contamination of the plasma

membrane fraction were: azide sensitive ATPase activity at pH 9 for mitochondria, nitrate

sensitive ATPase activity at pH 6 for tonoplast and antimycin insensitive NADH cytochrome c

reductase for endoplasmic reticulum (12), analysis of chlorophyll (13) and lipid MGDG (14)

contents for chloroplasts (thylakoids and envelope, respectively).

Preparation of Detergent-insoluble Membrane (DIM)—PM (ca. 2mg) were washed in 4ml of TE

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buffer (50mM tris-HCl, pH7,5, 3mM EDTA) to remove residual PEG-Dextran, and then

resuspended in 1 ml of ice-cold TE buffer. Protein amount was determined according to 15 using

BSA as standard. Prechilled (4°C) 10% Triton X-100 was added to the final amount indicated in

the figures (in all case, 1% final concentration), and the membranes were solubilized at 4°C for

30min. After solubilization, the membranes were brought to a final concentration of 52% sucrose

(w/w), overlaid with 3 ml of 35, 30 and 5% sucrose in TE buffer (w/w), and then spun for 16 h at

150,000g at 4°C in TST41 rotor (SORVALL). DIM could be recovered below the 30-35% layers

and washed in 4 ml of TE buffer to remove residual sucrose. The protein concentration was

determined with the Bio-Rad protein assay kit (BCA) to avoid TX-100 interference, using BSA

as a protein standard. All buffers were ice-cold and the final recovering buffer contained the

following protease inhibitors: 0.5mg/ml leupeptin, 0.7mg/ml pepstatin, and 0.2mM

phenylmethylsulfonyl fluoride.

Electron microscopy—Purified tobacco PM and DIM of the sucrose gradient were pelleted at

100,000g. The pellets were covered with 2.5% glutaraldehyde in 100mM phosphate buffer, for

2h at 4°C, then rinsed and treated with buffered 1% osmium tetroxide for 2h at 4°C. After

several rinses, the samples were postfixed with 1% tannic acid (Mallinckrodt) for 30mn at 20°C.

The pellets were then stripped with a fine needle and embedded in droplets of low melting point

agarose (Sigma). Hardened agarose blocks were then dehydrated with ethanol and epoxypropane

and embedded in epon. Ultrathin sections, 45nm thick, were collected on bare 600 mesh copper

grids, stained with uranyl and lead and observed with a Tecnai 12 electron microscope (FEI)

operated at 80 kV.

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Analysis of Lipids by HP-TLC and quantification—Lipids were extracted according to 16. In

Fig.1b, lipids of PM and DIM were globally quantified from TLC plates by densitometric

scanning (14). For more precise quantification, individual polar lipids were purified from the

extracts by monodimensional HP-TLC using the solvent system described by 17. Lipids were

then located by spraying the plates with a solution of 0.1% (w/v) primuline in 80% acetone,

followed by visualization under UV light. Fatty acids of individual lipids were determined and

quantified by gas chromatography after conversion to the corresponding methyl esters by hot

methanolic H2SO4 according to 18. The retention times of fatty acid methyl esters were

determined by comparison with the standards. The mole percent polar lipid was determined by

quantifying the amount of each fatty acid by gas chromatography. Sphingolipids were identified

by mild alkaline hydrolysis of the total lipid extract for 1h at 80 °C in methanolic 2.5M KOH.

Neutral lipids were separated by the solvent system described by 19. For mass spectrometry

analysis preparation, the sphingolipids were purified by HP-TLC. Recovered lipids were further

purified through an Oasis C18 Waters® cartridge according to the manufacturer’s instructions

and analyzed by MALDI-MS (see below).

Sterols Analysis—Free sterols from tobacco leaf PM and DIM were first purified by

chromatography on silica gel plates developed in CH2Cl2 (2 runs), then quantified as acetate

derivatives by GC. Analysis was carried out with a Varian GC model 8300 equipped with a

flame ionization detector and a fused capillary column (WCOT 30m x 0.25mm i.d.) coated with

DB1, with the following program: an initial rise of temperature from 60 to 220°C at 30°C/min, a

rise from 220 to 280°C at 2°C/min and a plateau at 280°C for 10 min. Cholesterol was used as

internal standard. Sterol acetates were identified by GC-MS according to 20.

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MALDI-MS Analysis of lipid—MALDI-MS spectra were acquired using a TofSpec Maldi-Tof

mass spectrometer from Micromass (Manchester, UK). The instrument is equipped with a pulsed

N2 laser (337nm, 4ns pulse width) and a time-delayed extracted ion source. Spectra were

recorded in the positive-ion mode using the reflectron and with an accelerating voltage of 20 kV.

Samples were dissolved in CHCl3/MeOH (2:1, v/v) at 10 mg/ml. The THAP

(trihydroxyacetophenon) matrix solution was prepared by dissolving 10mg in 1ml of

CHCl3/MeOH (2:1, v/v). A methanolic solution of cationisation agent (NaI, 10mg/ml) was also

prepared. The solutions were combined in a 10:1:1 volume ratio of matrix-to-sample-to-

cationisation agent. One to two microliters of the obtained solution was deposited onto the

sample target and vacuum-dried.

Monodimensional gel protein electrophoresis—Proteins from PM and DIM were separated by

mono-dimensional electrophoresis at 4°C on high-resolution 100mm/140mm/1mm, 10%

polyacrylamide SDS gels. Molecular mass standards were prestained full range markers

(SIGMA).

Bidimensionnal electrophoresis—All steps were performed at room temperature. 100mg of PM

and DIM proteins were solubilized 2h under gentle shaking in 30ml of a buffer containing 7M

urea, 2M thiourea, ASB14 2% (w/v) and 0,2% TX-100 (w/v). After solubilization, 120ml of the

same buffer (7M urea, 2M thiourea) without detergents were added to reach a final volume of

150ml suitable for rehydration of the IPG gel strips (ReadyStrip IPG Strip 7cm, pH 4-7, BioRad).

Finally, 0.6% carrier ampholytes 4-7 (Bio-Lyte 4-6 and 5-7 mixed, BioRad) and 1%

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tributylphosphine (2mM TBP, BioRad) were added. After passive rehydration (2h) and active

rehydration (12h, 50V), isoelectric focusing was performed with the following running

conditions: 100V for 1h, 250V for 1h, 500V for 1h, 1000V for 1h, from 1000 to 4000V for 2h

and 4000V to reach 35000Vhrs. After IEF, the IPG gel strips were incubated at room

temperature for 2 x 7 min in an equilibration buffer containing 50mM Tris-HCl pH 8.8, 6M urea,

30% glycerol (v/v), 2% SDS (w/v), 130mM DTT. The second equilibration step was carried out

for 2 x 8min in the same buffer, except that DTT was replaced by iodoacetamide. The second

dimension (SDS-PAGE) was carried out on 12% running gels without stacking: 25V for 15min

and then 15mA/gel for 1 hour. The gels were stained with the Silver Staining Kit Protein

(Amersham Pharmacia Biotech).

Proteomic Analysis—In-gel Protein Digestion. Coomassie Blue Brilliant stained proteins

separated by mono-dimensional SDS PAGE were excised and washed twice with ultra pure

water. Spots were subsequently washed in H2O/MeOH/acetic acid (50:50:5) until complete

destaining. Solvent mixture was removed and replaced by ACN. After shrinking of the gel

pieces, ACN was removed and gel pieces were dried in a vacuum centrifuge. Gel pieces were

rehydrated in 12.5ng/µl trypsin (Roche) in 50mM NH4HCO3 and incubated overnight at 37°C.

The supernatant was removed and stored at -20 °C and the gel pieces were incubated 15min in

50mM NH4HCO3 at room temperature under rotary shaking. This second supernatant was pooled

with the previous one, and a H2O/ACN/HCOOH (50:50:5) solution was added onto the gel

pieces for 15min. This step was repeated again twice. Supernatants were pooled and

concentrated in a vacuum centrifuge to a final volume of 30µl. Digests were finally acidified by

addition of 1.8µl of acetic acid and stored at -20°C.

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On-line Capillary HPLC Nanospray Ion Trap MS/MS Analyses—Peptide mixtures were analyzed

by on-line capillary HPLC (LC Packings, Amsterdam, The Netherlands) coupled to a nanospray

LCQ ion trap mass spectrometer (ThermoFinnigan, San Jose, CA). Peptides were separated on a

75µm inner diameter x 15cm C18 PepMapTM column (LC Packings, Amsterdam, The

Netherlands). The flow rate was set at 200nl/min. Peptides were eluted using a 5-50% linear

gradient of solvent B in 30 min (solvent A was 0.1% formic acid in 5% acetonitrile, and solvent

B was 0.1% formic acid in 80% acetonitrile). The mass spectrometer was operated in positive

ion mode at a 2 kV needle voltage and a 30 V capillary voltage. Data acquisition was performed

in a data-dependent mode consisting of, alternatively in a single run, a full scan MS over the

range m/z 150ˆ2000 and a full scan MS/MS in an exclusion dynamic mode. MS/MS data were

acquired using a 2 m/z units ion isolation window, a 35% relative collision energy, and a 5min

dynamic exclusion duration. Peptides were identified using SEQUEST (ThermoFinnigan,

Torrence, CA) and the Swissprot and NCBI database.

Western blot analysis—All steps were performed at room temperature. After electrophoresis

separation (1 h, 20 mA/gel), proteins were electroblotted onto nitrocellulose membrane (20min,

15V, Trans-blot SD semi-dry transfer cell, BioRad) in a buffer containing 30mM Tris, 192mM

glycine and methanol 20%. Membranes were blocked 1h with 5% powdered milk in Tris-buffer-

saline (TBS)-Tween (2mM Tris, 15mM NaCl, 0.05% Tween 20, pH 7.6), washed 7min 3 times

in TBS-Tween and then incubated 1h with primary antibody. Antibodies were diluted in TBS-

Tween as follows: anti-6His 1:1000, antiNtrbohD 1:500. Blots were washed and then incubated

1 h with horseradish peroxidase-conjugated secondary antibodies: anti-mouse IgG (Santa Cruz

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Biotechnology, CA, USA) at a concentration of 0.4mg/ml 1:5000. After washing 2x10min in

TBS-Tween and 1x10min in TBS, probing and detection were performed as described in the

ECL Western blotting detection kit (Amersham Pharmacia Biotech).

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RESULTS

Isolation of detergent-insoluble membranes from tobacco leaf and BY2 cell plasma

membrane—Animal and yeast raft membranes can be isolated on the basis of their insolubility in

non-ionic detergents such as TritonX-100 at low temperature (see introduction). To determine

whether similar membrane domains form in tobacco membranes, we tried to isolate detergent-

insoluble membranes (DIM) from purified plasma membrane (PM). We selected the

conventional phase partitioning procedure (21) to obtain a highly enriched PM fraction either

from tobacco leaves or from tobacco BY2 cells. Typical preparations were enriched by a factor 5

to 8 in the PM marker, vanadate-sensitive ATPase activity, compared with the starting material

consisting of a crude microsomal fraction. Biochemical characterization of this PM fraction

(Table 1 in supplemental information and experimental procedure) revealed that mitochondrial

membranes were virtually absent of the PM fraction. Endoplasmic reticulum marker enzyme was

depleted by a factor 7 to 8 between microsomes and PM; tonoplast was depleted by a factor 20 in

BY2 PM fraction and by a factor 5 in the leaf PM fraction. We assayed the chlorophyll amount

for thylakoid contamination (chlorophyll not detected, data not shown) and the presence of

MGDG (less than 0.5mol% of total lipid, data not shown) for envelope contamination. These

results showed that we were able to obtain highly-enriched plasma membrane fractions from

both tobacco leaves and BY2 cells.

To isolate DIM, we first tested different ratios TX100-to-protein using leaf PM, incubated for

30 min at 4°C using 1% TX100 final concentration (Fig.1). DIM were further isolated from

solubilized material by their ability to float through a sucrose density gradient. Except for the

ratio 60, a white and opaque band was easily discernable at 1.14g/cm3 i.e. below the 30-35%

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interface (data not shown). The gradient separation was highly reproducible between

experiments. As expected, the amount of DIM recovered from the gradient decreased when the

amount of detergent increased (Fig.1A). DIM lipid composition analysis evidenced a correlated

enrichment in sphingolipids and sterols, and a decrease of phosho- and glyco- glycerolipid

amounts with respect to PM. The maximum level of enrichment of both sterols and sphingolipids

was reached when a ratio TX100-to-protein of 15 was being used (Fig.1B, C). At this ratio,

9.3%±2 of the PM proteins were recovered after detergent treatment. Beyond this ratio, the

amount of DIM recovered decreased dramatically without any further overall change in the lipid

composition (Fig.1A). We therefore established that a TX100 treatment for 30 min. at 40C with a

ratio TX100-to-protein of 15 (1% TX100 final concentration) was suitable for DIM isolation

from tobacco leaf PM. We used with success these conditions to isolate DIM from BY2 cell PM,

although less than 3.3%±1 of PM proteins were recovered after the procedure (data not shown).

Morphological analysis of tobacco Leaf Detergent-Insoluble Membranes compared to

plasma membrane—The DIM fraction recovered by high-speed centrifugation, observed by

electron microscopy (see Experimental procedure) and compared with the PM fraction used to

prepare the DIM (Fig 2). The PM fraction mainly contained closed membrane vesicles (Fig.2A).

Higher magnifications showed that the membrane leaflets were highly contrasted and

approximately 7.5 nm thick (Fig.2B), which corresponds to in situ observations of PM in intact

tobacco leaf cells (not illustrated here). DIM did not appear as vesicles but as more or less

parallel membrane sheets, with some particulate material enclosed inside (Fig.2C). The length of

the membrane profiles was very variable, ranging from a few nanometers to several micrometers.

The longer ones were often tightly associated with each other, either by small contact zones or

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by longer adhesion areas (Fig.2D). Many membrane profiles were running tilted along the

section and appeared either as cross-sectioned or parallel to the section plane. In the latter case, it

could be easily shown that isolated DIM areas were organized as a network of ribbon-like

structures (Fig.2E). The width of these flat membrane fragments did not exceed 100 nm. The

smallest DIM fragments were often clumped as dense aggregates at the ends of longer membrane

sheets (Fig.2F).

Lipid characterization of Tobacco Detergent-insoluble Membranes; Glucosylceramide and

free sterols are the major lipids—We combined HP-TLC, GC and mass spectrometry to compare

the lipid composition of the whole PM and DIM (Fig.3, 4). We first separated by HP-TLC polar

lipids and stained them with primuline (Fig.3a). The lipid composition of PM fraction from

tobacco leaf and from BY2 cells was firstly determined. Results are consistent with previous

works on PM lipid analysis (e.g. 22, 23). Briefly, phospholipids in both membranes comprised

for ca. 50% of total lipids with major phospholipids being PC and PE. We detected DGDG

(identity confirmed by mass spectrometry, data not shown) in leaf and BY2 cell PM. DGDG was

previously acknowledged to be an exclusive plastidial lipid (24). Nevertheless, experimental

evidence has been presented for the presence of this lipid outside plastids (e.g. 25) particularly in

highly purified plant PM (26). Sphingolipid was found to be the major non-glycerolipid in leaf

PM (21.1±2,3% of total lipid), but represented two-fold less in BY2 cell PM (Fig.2A). Finally,

the amount of an unknown glycerolipid, non-resistant to mild-base cleavage, named lipid X, was

determined (ca. 5.0% of total lipids). Mass spectrometry analysis revealed a single peak at

1014.7 for this lipid (data not shown).

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The polar lipid composition analysis of DIM fraction from both leaf and BY2 cell PM

showed that the sphingolipid was strongly enriched, whereas phospholipids, DGDG and lipid X

were largely excluded from these domains. A sphingolipid -resistant to mild-base cleavage (data

not shown)- constituted therefore the major polar lipid of tobacco lipid rafts (ca. 40mol% of total

lipids). Its structure was determined by MALDI-TOF mass spectrometry. The full-range positive

ion spectra of this tobacco lipid revealed that it contained a single, highly abundant lipid species

of m/z 736.6 (Fig.3B), identified as a glucosylceramide, named GluCer (see 27 as the main

reference for this identification). Tobacco GluCer was comprised primarily of ceramide with 4,8-

sphingadiene (d18:2D14, D18) and a-hydroxypalmitic acid (h16:0) : MW=736.6 (peak a and b with

Na+ or K+ as counter-ions, respectively. (Fig.3B). Additionally, ions characteristic of less

abundant GluCer molecular species were also detected with the same backbone but with h18:0

(MW=764.7, peak g), h22:0 (MW=820.7, peak e) and h24:0 (MW=848.7) as fatty acyl chains.

We next analyzed the glycerolipids still present after TX100 treatment, in comparison with

those of the PM. Glycerolipids of DIM contained more saturated fatty acids i.e. 16:0, 18:0 and

20:0 than their PM counterpart lipids (Fig.3C).

Neutral lipid content of DIM was determined in parallel by HP-TLC and showed that a very

few amount of DAG (less that 1% of total lipid) was detected, while free sterols consituted the

major neutral lipids (Fig.4A). As shown in Fig.3A, these compounds constitute therefore up to

40mol% of total lipids present in DIM isolated from either tobacco leaf or BY-2 cells. GC

analysis showed that the same molecular species i.e. stigmasterol, 24-methylcholesterol,

sitosterol and cholesterol were identified in both PM and DIM fractions in similar proportion.

Stigmasterol represented about 50 % of the sterol mixture (Fig.4B).

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Taken together, these experiments show that detergent solubilization can be used to isolate a

tobacco membrane fraction enriched in sterols and sphingolipids. It must be noted that plant

DIM have a lower ratio lipid-to-protein (ca. 0.48±0.09) than that of PM 1.09±0.1 (n=3, data not

shown).

Analysis of Tobacco leaf and BY2 cell TX100-insoluble Membrane Proteins—We carried out

mono- and bi-dimensional gel electrophoresis to analyze the protein content of DIM vs. PM from

both leaf and BY2 cells (Fig.5). Our results clearly showed that some proteins are enriched,

while others are excluded from DIM. We therefore initiated a proteomic approach to allow the

identification of proteins in tobacco lipid rafts (Table 1). MS/MS identifications were achieved

by similarity searching in NCBI or Swiss Prot. databases (see Experimental procedure). We were

aware of the difficulties to solubilize proteins present in an already detergent-insoluble fraction

to perform the first dimension of the 2D-gel; a subsequent enrichment in extrinsic or soluble

proteins, and a decrease (sometimes a complete lost) in intrinsic membrane proteins is most often

observed (28, 29). This explains the differences between the mono- and the bi-dimensionnal

electrophoresis pattern shown in Fig.5. For this reason we used for MS/MS identification mono-

dimensional SDS-PAGE in which the majority of the proteins were solubilized by SDS including

integral membrane proteins (see e.g. 30, 31). Results in Fig.5A from leaf PM and DIM fractions

showed that only some of the bands were enhanced in the DIM (band #1,7,8,10,11,12) and were

identified as H+-pumping ATPase (band 1, enrichment confirmed by western blot analysis, data

not shown), aquaporins (band 11, 12) and the oligogalacturonic acid (OGA)-binding protein

called remorin (band 7, 8). Although soluble, carbonic anhydrase CA1 (bands 9, 10) was found

to be present in the PM, but was also identified in DIM (see discussion below). Beside these

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proteins, we also identified the cell elongation factor DIMINUTO (band 4), the heat shock

cognate 70kDa protein (band 2), 14-3-3-like protein (band 9), and the elongation factor 1a (band

6) were also present. Contamination by the peripheral water-soluble catalytic subunit of the

vacuolar ATP synthase (band 3) was observed (see table 1, supplemental information).

We also analyzed the protein content of BY2 cell DIM (fig.5B). We confirmed the

enrichment of DIM in H+-pumping ATPase and aquaporins in DIM, as well as in remorin,

elongation factor 1a, heat shock cognate protein and 14-3-3 protein. Moreover, this analysis

allowed us to identify new proteins in DIM namely Syntaxin 71 (band D), a and b tubulin (band

C) and dynamin-like protein (band C). Neither carbonic anhydrase, nor DIMINUTO were

detected in BY2 cell DIM.

NADPH oxidase (after elicitation by cryptogein) and Ntrac5 associate with TX100-insoluble

membranes—As indicated in Fig.5A, B the protein remorin seems particularly abundant in DIM.

This PM-associated protein has been shown to be phosphorylated in response to addition of

oligosaccharides (OGAs), regulatory molecules released from plant cell wall by degradative

enzymes during pathogenesis and capable of triggering a variety of plant’s defense reactions (32,

33). This suggests that plant membrane microdomains might play a specific role in plant’s

defense reponses. We carried out an immunological approach to investigate the presence in these

microdomains of two proteins involved in plant’s defense signal transduction, namely the

tobacco PM-localized NADPH-oxidase, NtrbohD, responsible for active oxygen species (AOS)

production in elicited tobacco cells (10) and the small G protein Ntrac5, a negative regulator of

this oxidase (9). The fungal elicitor cryptogein induces the synthesis of NtrbohD, which is almost

undetectable in absence of cryptogein (10). The western-blot presented in Fig.6 confirmed the

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presence of NtrbohD in the PM fraction as described in 10 after cryptogein treatment, but also

clearly demonstrated its recruitment in DIM. Western-blot carried out on protein fractions from

BY2 cells expressing His::Ntrac5 also evidenced the particular location of this regulator in DIM.

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DISCUSSION

Do detergent-insoluble membranes exist in the higher plant plasma membrane?—Numerous

studies on animal and fungi membranes evidenced that functional domains of the plasma

membrane (PM) could be isolated as a low density fraction on sucrose gradient after

solubilization in 1% Triton X-100 at 4°C. The association of proteins and lipids with membrane

microdomains has emerged as an important regulator of signal transduction, protein and

membrane polarized intracellular sorting, cytoskeletal organization and entry of infectious

organisms in living cells (for reviews 1, 5). Despite numerous discussions about the presence of

such domains in plant membranes (34-36), full characterization of lipid rafts in plant PM has

never been reported. Here, we show that Triton X-100 detergent-insoluble membrane (DIM)

could be isolated from tobacco leaf and BY2 cell PM and display a lipid and protein composition

distinct form the whole PM. Nevertheless, the present results emphasized some differences

between plant and animal/yeast lipid rafts.

Firstly, the buoyant density of lipid rafts isolated from tobacco PM appeared to be much

higher than that of rafts isolated from yeast and animal cells. This higher density is likely related

to the lower ratio lipid-to-protein of plant DIM. Interestingly, high density DIMs have also been

detected in neutrophil PM together with low-density DIM (30). Proteomic analysis of such high-

density domains evidenced cytoskeleton-associated proteins, suggesting that high-density DIM

represents a membrane skeleton-associated subset of leukocyte signaling domains (30). In our

case, we failed to detect any low-density detergent-insoluble membrane in tobacco PM (data not

shown).

Secondly, tobacco DIMs appeared as a population of non-vesiculised bilayer membrane

ribbons (Fig.2), about 100 nm wide with a very variable length (up to several micrometers). The

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latter observation suggests the existence of rather long DIM areas within the PM, whereas

smaller fragments could correspond to over-fragmentation of the membranes in the course of

Triton treatment. Such an unusual organization of lipid bilayer could be due to the high rigidity

of liquid-ordered sterol/sphingolipid association, but we cannot rule out that morphology of DIM

is due to treatment with TX100. A similar modification of the morphological aspect (i.e. from

vesicles to large sheets of membranes) was already observed in ER membranes after TX100

treatment (37), although we do not know yet whether plant ER membranes contain lipid rafts.

Moreover, it is known that TX100 treatment could triggers aggregation of rafts (e.g. 38 for

review). That may explain the final micrometer-size of DIM. Finally, the presence of a number

of close contacts between isolated DIM indicates that these membranes have high self-adhesion

properties.

Thirdly, the chemical structures of sphingolipids and sterols found associated in plant lipid

rafts are different from those in animal and yeast rafts. While glucosylceramide is the unique

glycosphingolipid that plants, fungi and animals have in common, there are specific differences

in the structure of the ceramide backbone. The sphingolipid found in the tobacco lipid rafts

differs from those of animal membranes which often have a 14-double bond and an a-hydroxy

fatty acid only in a few cases (39). In plants, the most abundant amide-linked fatty acid was a a-

hydroxypalmitic acid (Fig.3c), much shorter than the lignoceric acid (C24:0) often found in

mammalian sphingolipids.

Microdomains of the tobacco PM were found to also contain sterols. Whereas mammalian

and fungal cells contain only one major sterol (cholesterol and ergosterol, respectively), higher

plant cells display a vast array of sterol molecules, with sitosterol, stigmasterol and 24-

methylcholesterol as the most represented compounds. Plant sterols mainly accumulate in the

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PM, where they play a major role in regulating the membrane fluidity and permeability as well

as the function of vectorial enzymes such as the H+-ATPase, see below (40). Here, we show that

DIM of the tobacco PM are greatly enriched in sterols. Interestingly, we found the same sterol

molecules as in the whole PM, and in similar proportions, indicating that sterol recruitment in

lipid rafts does not involve any structural selectivity. Such a result is somewhat surprising if one

considers the very different efficiencies of sitosterol and stigmasterol in ordering phospholipid

acyl chains of soybean phosphatidylcholine bilayers (40). However, the present results are

entirely consistent with a recent work demonstrating a similar ability of both sitosterol and

stigmasterol to promote domain formation (41).

Glycerolipids of tobacco PM contained the same head groups as those of animal cells, but

shorter fatty acyl chains. The longest fatty acyl chain found in tobacco glycerolipids was C20,

whereas those of mammalians contain up to C24. Moreover, glycerolipids of plant DIM contain

more saturated fatty acyl chains (Fig.3B), which would contribute to the rigidity of the liquid

ordered phase of lipid rafts (42).

Lipid composition characterization of plant rafts emphasizes that despite differences with

yeast and animal lipids, the structural properties of tobacco sphingolipids, glycerolipids and

sterols are consistent with the ability to separate into liquid ordered and disordered phases.

Plant detergent-insoluble membranes are able to specifically recruit proteins and exclude

others— Although membrane lipids and proteins were previously thought to be homogeneously

distributed in the membrane, the lipid raft concept suggests that membrane domains are able to

specifically sort proteins. This differential localization of proteins in definite areas of the PM has

been associated in animal cells with some precise physiological functions. The present study on

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plant membrane microdomains further strengthens this statement since gel electrophoresis

analysis clearly reveals that compared to the whole PM fraction, DIM are enriched in some

proteins but depleted in others (Fig.5). An interesting point concerning most of the proteins we

found associated with tobacco DIM is that their yeast or animal counterpart have been proved to

be also located in rafts (table 1). We observed that H+-ATPases, the major integral membrane

proteins of the PM, are highly enriched in lipid rafts, as described in yeast (43). Interestingly,

immunolocalisation of PM H+-ATPase in minor veins of Vicia faba showed a non-homogeneous

distribution in transfer cells, this pump being more concentrated in the regions adjacent to the

bundle sheath, phloem parenchyma and xylem vessels (44). Plant PM H+-ATPases are activated

by 14-3-3 proteins, some of which being tighly associated with membrane (45) and with rafts

(46).

We found the carbonic anhydrase strongly associated with lipid rafts. The carbonic

anhydrase located in the chloroplast plays a crucial role in the CO2-concentrating mechanism,

but the role for the isoform detected in the cytoplasm is still not clear (47, 48). Santoni et al. (49)

and Kawamura and Uemura (50) have previously found this enzyme in highly-enriched PM

fractions from Arabidopsis. We evidenced that the tobacco PM- and DIM-associated carbonic

anhydrase is the cytosolic form containing the N-term transit peptide (Western blot analysis in

collaboration with Dominique Rumeau and MS/MS peptide mapping not shown) and not a

contamination by the mature chloroplastic isoform which does not contain this transit peptide.

Moreover, the enzyme is probably tightly bound to the membrane because the binding resists to

the TX100 treatment (1%) during the lipid raft purification procedure.

Aquaporins were found associated with tobacco DIMs. Very recent work in the animal field

evidenced that Aquaporin 3 is located in caveolin-rich membrane microdomains in mouse

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keratinocytes (51). The protein elongation factor 1-alpha (called eEF1A), also present in tobacco

DIM, is a multifunctional protein that binds aminoacyl-tRNAs to the ribosome, but appears to

have several other activities (52). This factor has been shown to be anchored at the membrane in

animal cells (53, 54) and associated with plant PM (50, 55). The DIMINUTO protein catalyzes

the last step of the sterol biosynthetic pathway (56, 57). To the best of our knowledge, sterol

biosynthesis mainly occurs in the endoplasmic reticulum (40). In view of our results a

participation of the PM and its microdomains in the last step of this pathway might be

considered.

In search for physiological functions supported by rafts in plants

Very interestingly, the protein remorin seems to be particularly well represented in tobacco

DIM as attested by 1D and 2D electrophoresis experiments. Remorin is the putative receptor of

OGA, components of the extracellular pectic matrix which regulates the expression of a variety

of defense genes (58). Experiments to directly test for the physiological role(s) of remorin

showed that Nicotiana benthamiana plants display massive necrotic lesions when infected with

transgenic virus expressing remorin (59). In this context, the high enrichment of remorin in DIM

suggests a crucial role of these microdomains in sensing defense signaling molecules. We

examined the presence in tobacco DIM of two proteins already known as a components of the

plant’s defense signaling cascade: the NADPH oxidase (NtrbohD) and the small G protein

Ntrac5, recently shown as able to regulate the production of AOS triggered by the fungal elicitor

cryptogein in tobacco cells (9). The fact that NtrbohD and Ntrac5 are associated with tobacco

DIM reinforces the putative role of these microdomains in plant’s defense reactions.

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The numerous studies performed on animal rafts revealed their involvement in crucial

physiological processes as various as cell trafficking, establishment of cell polarity,

immunological response, hormone signalling or apoptosis. The results presented here

demonstrated for the first time the presence in plant PM of microdomains with a specific lipid

and protein composition. Our data suggest putative roles of these domains in plant’s defense

(remorin, NtrbohD, Ntrac5…), vectorial water flow (aquaporin), vesicular trafficking (syntaxin,

dynamin-like protein…), cell growth (H+-ATPase, carbonic anhydrase…), cytoskeleton

organization (actin, tubulin…), etc. Above all, this study opens a large field of investigations:

deciphering the different cellular processes of plant physiology which use these domains as a hub

for signaling.

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FOOT NOTES

* This work was supported by Conseil Régional d’Aquitaine (JJB, RL, SM, JLT).

§ To whom correspondence may be addressed: Sébastien Mongrand, Laboratoire de Biogenèse

Membranaire, FRE 2694-CNRS-Université Victor Segalen Bordeaux 2, 146, rue Léo Saignat,

BP 33076 Bordeaux Cedex, France. Tel: 033 5 57 57 14 35; Fax 033 5 56 51 83 61; e-mail:

[email protected]

ABBREVIATIONS—ACN, Acetonitrile; AOS, Active Oxygen Species; BSA; Bovine Serum

Albumin; DAG, Diacylglycerol; DIM, Detergent-insoluble Membrane; DGDG,

DiGalactosylDiacylGlycerol; GC, Gas Chromatography; GPI, GlycosylPhosphatidylInositol;

gluCER, glucosylCERamides; HP-TLC, High-Performance Thin Layer Chromatography; PI,

PhosphatidylInositol; PC, PhosphatidylCholine; PE, PhosphatidylEthanolamine; PM, Plasma

Membrane; TX100, Triton X100

ACKNOWLEDGMENTS—We are grateful to Pascale Pracros for her precious help with

tobacco culture and to Damien Palomo, Loic Cerf, Myriam Vallet and Jérome Fromentin for

technical assistance. We are grateful to Christelle Absalon at CESAMO (University of Bordeaux

1) for mass spectrometry analysis. We thank Jérome Joubès, Francoise Sargueil and Jean-Pierre

Blein for helpful comments on the manuscript. Electron microscope observations were

conducted at SERCOMI (Université Bordeaux 2). We thank David Slaymaker for information

about the two isoforms of tobacco carbonic anhydrase CA1 and CA2. We thank Dominique

Rumeau (CEA, Cadarache) for the western blot which allowed us to discriminate between the

cytosolic and the chloroplastic form of CA. Antibody against the H+-ATPase was a kind gift of

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Dr Ramon Serrano (IBMCP, Valencia). We thank Jan Oliver Jost for his contribution to the

development of 2D electrophoresis methods.

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FIGURE LEGENDS

FIG. 1. Isolation of Detergent-Insoluble Membranes (DIMs) from Leaf Tobacco Plasma

Membrane (PM). A, Highly-enriched fractions of tobacco leaf PM were solubilized for 30 min

with 1% TX100 with a ratio detergent-to-proteins from 0 to 60 (w/w). Insoluble membranes

were collected by flotation through a density gradient and amount of recovered membrane

proteins were determined according to Experimental procedure; B, Polar lipid analysis of DIM

was performed for each TX100-to-PM ratio by high performance thin layer chromatography; C,

Lipids of DIM were globally quantified from HP-TLC plates by densitometric scanning (14).

Data are the mean of two independent experiments with less than 2-3% of variation.

FIG. 2. Morphological study of leaf tobacco highly-enriched plasma membrane (PM) and

detergent-insoluble membranes (DIM). PM and DIM (ratio TX100/PM=15) from tobacco leaf

were purified as shown in Fig.1. High-speed centrifugation pellets of PM (A, B) or DIM (C to F)

were prepared as described in Experimental procedures and examined by transmission electron

microscopy. Arrows point out close contacts between DIMs.

FIG. 3. Polar Lipid analysis of plasma membrane (PM) and detergent-insoluble membranes

(DIM) from tobacco leaf or BY2 cells. DIM isolated from tobacco leaf or BY2 cell PM were

purified (ratio TX100/PM=15) as shown in Fig.1A. A, Lipid extracts were separated on HP-TLC

plates and stained with primuline. The positions of PC, PE, PI, PS, PA, DGDG, unknown lipid

X, sphingolipid and neutral lipids (see Fig 4) are indicated. The identities of glycerolipids were

determined by running standards. The mole percent of each lipids was calculated by GC

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according to Experimental procedure expressing as a percent of the total. The data are expressed

as the mean of three independent experiments ± SD; B, Sphingolipid were analyzed by MALDI-

TOF mass spectrometry. Ions characteristic of glucosylceramide with 4,8-sphingadiene

(d18:2D14, D18) and a-hydroxypalmitic acid (h16:0) were detected with Na+ (peak a) or K+ (peak

b) as counter-ions, respectively. Ions characteristic of less abundant GluCer molecular species

were detected with h18:0 (peak g), h22:0 (peak e) as amide-linked fatty acid. The peaks at 779

and 808 m/z are unidentified non-ceramide-related compounds; C, Fatty acid length and degree

of saturation of the glycerolipids were analyzed qualitatively by GC. The data are expressed as

the mean of three independent experiments ± SD.

FIG. 4. Neutral Lipid analysis of tobacco detergent-insoluble membranes (DIM) from leaf

or BY2 cell plasma membrane (PM). DIM from tobacco leaf or BY2 cell PM were purified

(ratio TX100/PM=15) as shown in Fig.1A. A, Lipid extracts were separated on HP-TLC plates

and stained with primuline. The positions of sterol ester, free fatty acid (FFA), tri-, di-, mono-

glyceride (TAG, DAG, MA, respectively) and free sterols are indicated. The identities of lipid

were determined by running standards. All sterols migrated indistinguishably from each other; B,

The free sterols in tobacco PM and DIM were identified and quantified by GC. The data are

expressed as the mean of three independent experiments ± SD.

FIG. 5. Comparison by mono- and bi-dimensional electrophoresis of plasma membrane

(PM) and detergent-insoluble membrane (DIM) proteins isolated from tobacco leaf and

BY2 cells. Coomassie Blue-staining proteins (50µg each) from leaf PM and DIM (A) and silver-

staining proteins (50µg each) from BY2 cell PM and DIM (B) were separated by mono-

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dimensional 10% acrylamide SDS-PAGE, with sizes of molecular mass markers in kDa on the

left and band numbers of excised bands on the right (see table 1 for identification by

LC/MS/MS). (Note that silver-staining gel electrophoresis of BY2 proteins is shown as

illustration, MS/MS identification was carried out from coomassie blue-staining gel); C, Silver-

staining proteins (100µg each) from leaf PM and DIM separated by bi-dimensional gel

electrophoresis, see experimental procedures for details.

FIGURE6. NADPH oxidase (NtrbohD) and Ntrac5 associate with detergent-insoluble

membranes (DIMs). A, BY2 cells were elicited by cryptogein (50nM) according to

Experimental procedures. Plasma membranes were further purified and solubilized with 1%

TX100 and then floated through a density gradient as shown Fig.1. Western blot was performed

according to Experimental procedures with antibody to NADPH oxidase (anti-NtrbohD).

Abbreviations are the following: C, soluble proteins precipitated with 45% ammonium sulfate

(25µg of proteins); M, microsomes (25µg of proteins); PM (25µg of proteins); DIM (15µg of

proteins); B, Proteins correspond to different subfractions of either wild type BY2 cells (WT), or

transgenic BY2 cells overexpressing Ntrac5 (AJ250174) in fusion with a RGS-His tag (9).

Western-blot was performed as described in “Experimental procedures” using a anti:6His

antibody. Each lane was loaded with 15µg proteins.

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TABLE 1 in supplemental information: Specific activities of marker enzymes from various

cellular membranes measured in microsomes (M) or plasma membrane fractions (PM)

from tobacco leaves and BY2 cells. Activities are expressed in µmol/min/mg of proteins.

BY2 cells LeavesM PM M PM

Vanadate-sensitive ATPaseactivity (pH=6.5)

0.52 4.68 0.21 1.25

Nitrate-sensitive ATPaseactivity (pH=6.5)

0.65 0.03 0.2 0.04

Azide-sensitive ATPaseactivity (pH=9)

1.35 0.02 0.46 0.01

Antimycin-insensitive NADHcytochrome c reductase

0.05 0.007 0.012 0.0015

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TABLE 1: Identification by LC/MS/MS of the major proteins of tobacco leaf (A) and BY2

cell (B) detergent-insoluble membrane. Mono-dimensional gel electrophoresis are shown

Fig.5. Proteins strongly enriched in DIM are shown in boldface. Abbreviations are the following:

#, band number, see Fig.5; MWth.: theoretical molecular mass based on deduced amino acid

sequences; MWapp.: apparent molecular mass; Species: At, Arabidopsis thaliana; As, Abies

sachalinensis; Bn, Brassica napus; Bv, Beta vulgaris; Dc, Daucus carota; Gm, Glycine max; Le,

Lycopersicon esculentum; Nt, Nicotiana tabacum; Np, Nicotiana plumbaginifolia; St, Solanum

tuberosum. Identity: based on tandem mass spectrometry (MS/MS); Accession #: accession

number in SwissProt data base; Subcellular Location: S, Stroma; ER, Endoplasmic reticulum; C,

Cytoplasm; PM, plasma membrane; V, Vacuole. Peptide #: number of different peptides.

Sequence Coverage: fraction of the protein sequence covered by the identified peptides.

A,# MW

th.

MWapp.

Sp. Identity Accession # Sub.Location

Peptide#

SequenceCoverage

1 105.1 102.3 Le Plasma mb. ATPase 1 sw|P22180 PM 8 6,8 %2 70.7 75.2 Le Heat shock cognate prot. 2 sw|P27322 C/PM 5 7.6 %3 68.5 70.3 Bv V ATPsynthase A sw|Q39442 V 3 5.3 %4 65.4 68.5 At DIMINUTO protein sw|Q39085 ER 5 8.0 %5 48.9 52.3 Ab RuBisCO large chain sw|O78262 C/S 4 10.2 %6 49.3 51.1 Dc Elongation factor 1-alpha sw|P29521 C/PM 7 11.1 %7 30.7

21.745.2 Le

StProbable aquaporin PIPRemorin

sw|Q08451sw|P93788

PMPM

42

11.9 %12.6 %

8 21.7 38.2 St Remorin sw|P93788 PM 3 18.2 %9 34.5

28.631.0 Nt

NtCarbonic anhydrase14-3-3-like protein A

sw|P27141sw|P93342

S/C/PMC/PM

56

17.4 %22.7 %

10 34.5 28.0 Nt Carbonic anhydrase sw|P27141 S/C/PM 8 17.4 %11 30.7 24.6 Le Probable aquaporin PIP sw|Q08451 PM 4 11.9 %12 30.7 23.5 Le Probable aquaporin PIP sw|Q08451 PM 3 7.7 %B,

# MWth.

MWapp.

Sp. Identity Accession# Sub.Location

Peptide#

SequenceCoverage

A 105.2 ≈114 Np Plasma mb. ATPase 4 sw|Q03194 PM 20 17.6 %B 80.1 ≈88 Le Heat shock cognate prot. 80 sw|P36181 C 3 4.7 %C 70.7

68.768.2

≈70 LeBnAt

Heat shock cognate prot. 2V ATPsynthase subunit ADynamin-like protein

sw|P27322sw|Q39291sw|P42697

CPM

PM/C

5132

9.5 %19.1 %2.1 %

D 49.949.3

≈50 GmDc

Tubulin beta-1 chainElongation factor 1-alpha

sw|P12459sw|P29521

CC/PM

66

11.9 %9.6 %

E 21.729.9

≈35 StAt

RemorinSyntaxin 71

sw|P93788sw|Q9SF29

PMPM

36

17.7 %12.0 %

F 28.9 ≈30 St 14-3-3-like protein 16R sw|P93784 C/PM 3 9.3 %G 30.7 ≈25 Le Probable aquaporin PIP sw|Q08451 PM 2 7.7 %H ≈22 Un-identified

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Mongrand et al.Figure 1

Cs

0

10

20

30

40

50

60

70

80

glycerolipid sphingolipid+sterols

Lip

id c

om

po

siti

on

(%

of

tota

l lip

id) 0

5

15 0

5

15PM

PM30

30

A

0 5 15 30 60 TX100/protein

DIM recovered after sucrose gradient

0

10

20

30

40

50

60

70

80

90%oftotalPM

B

Lipid composition of PM and DIM

Lipids equivalent to 100µg of proteins

--DGDG

==Neutral lipids

--PE+PG

--PC

--start

-- Sphingolipid

--X

0 5 15 30 60PM TX100/protein

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A B

C

E F

D1 µm

500 nm

500 nm 100 nm

100 nm

100 nm


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A

Sphingolipid--

Neutral lipids--

X?--DGDG------

PE--

PC==

PS+PI+PA

Start---

PM DIM PM DIM

Leaf BY2B a

g

eb

Sphingolipid MALDI-TOF analysis

Fatty acid composition (mol% of total)Lipid 16:0 18:0 18:1 18:2 18:3 20:0 Saturated FA

PM 31.7 ±5.7 8.3 ± 2.6 3.8 ± 1.0 29.4 ± 4.3 25.7 ± 4.7 1.2 ± 0.7 41.2±9.0PCDIM 40.4 ±7.2 15.4 ± 4.8 4.7 ± 2.1 20.8 ± 1.3 14.7 ± 1.0 - 55.8±12.0

PM 35.1 ±1.5 7.0 ± 1.1 2.9 ± 0.3 35.5 ± 3.3 19.1 ± 0.6 0.4 ± 0.1 42.5±2.7PEDIM 49.8 ±6.8 15.3 ± 0.2 - 24.2 ± 1.4 7.4 ± 2.4 2.8 ± 0.8 67.9±7.8

PM 37.9 ±2.0 7.6 ± 1.4 4.9 ± 0.6 21.8 ± 1.0 27.8 ± 2.3 - 45.5±3.4DGDGDIM 68.2 1.3 18.6 ± 1.1 - 13.2 ± 2.3 - - 86.8±2.4

PM 37.1 ±3.1 9.2 ± 2.5 3.1 ± 0.9 23.6 ± 4.7 18.1 ± 5.9 8.9 ± 2.7 55.2±8.3PS+PI+PADIM 65.0±4.0 15.5 ± 1.8 4.0 ± 1.1 6.7 ± 2.3 - 8.8 ± 3.5 89.3±9.3

C

%mol totalPC PS+PI

+PAPE DGDG X Sphingolipid Neutral

lipidsLeaf

PM 14.6±4.1 4.9±1.2 19.0±4.5 12.5±2.2 5.8±2.4 20.6±2.3 22.6±1.8DIM 5.0±1.2 1.7±1.3 10.3±1.2 4.2±0.9 0.5±0.3 38.1±4.2 40.2±5.0

BY2 cellsPM 15.5±3.9 8.3±1.1 16.5±4.6 15.1±2.4 6.6±2.4 11.3±3.1 26.7±1.7DIM 6.0±1.2 3.8±0.4 10.1±1.6 11.0±1.3 3.5±0.5 21.7±5.3 43.9±5.9

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B sterol % of total sterolPM DIM

Stigmasterol 51.7 ± 1.4 49,3 ± 0,924-methyl cholesterol 19.2 ± 2.6 22,8 ± 0,2Sitosterol 15.1 ± 0.7 12,9 ± 1,9Cholesterol 14.0 ± 1.3 15,0 ± 1,3

A

DIM

Sterol ester--

TAG--

Free sterols--DAG--MAG--

start--

FFA--

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B BY2 cells

114-88--

50.7-

35.5-

28.8-

22--

kDa

PM DIM

| A| B| C

| D| E| F| G

| H

A Tobacco leaf

kDa

84--

185-

116-

61--55--

36--31--

12111098765

4 32

1

PM DIMC Leaf PM Leaf DIMkDa

F

---114--

---73--

---50.7--

---35.5--

---28.8 -

---22 --

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A Western blot: NADPH oxidase (NtrbohD)

Western blot: anti-6His for His::NtRAC5 expressed in AJ250174 cells

C C M PM DIM

BY2 AJ250174 cells

-- 21 kDa

B

--105 kDa

C M PM DIM

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Jean-Jacques ;BessouleCarde, Marie-Andrée Hartmann, Marc Bonneu, Françoise Simon-Plas, René Lessire and

Sebastien Mongrand, Johanne Morel, Jeanny Laroche, Stéphane Claverol, Jean-Pierremicrodomains from tobacco plasma membrane

Lipid rafts in higher plant cells: Purification and characterization of TX100-insoluble

published online June 9, 2004J. Biol. Chem.

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