Purification and characterization of higher plant lipid rafts
1
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.
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
2
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.
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
3
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
4
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
5
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.
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
6
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
7
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.
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
8
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.
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
9
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%
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
10
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.
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
11
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
12
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).
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
13
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%
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
14
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
15
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).
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
16
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).
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
17
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
18
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
19
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.
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
20
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
21
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
22
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
23
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
24
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.
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
25
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.
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
26
REFERENCES
1. Simons, K. and Ikonen, E (1997) Nature. 387(6633), 569-72
2. Slotte, J.P. (1999) Chem Phys Lipids. 102(1-2), 13-27
3. Brown, D.A. and London, E. (1998) J Membr Biol 164, 103-114
4. Garcia, A. Cayla, X. Fleischer, A. Guergnon, J. Alvarez-Franco Canas, F. Rebollo, M.P.
Roncal, and F. Rebollo, A. (2003) Biochimie 85(8):727-31
5. Bagnat, M. and Simons, K. (2002) Biol Chem. 383(10), 1475-80
6. Kost, B. Lemichez, E. Spielhofer, P. Hong, Y. Tolias, K. Carpenter, C. and Chua, N.H. (1999)
J Cell Biol. 19;145(2), 317-30
7. Schindelman, G. Morikami, A. Jung, J. Baskin, T.I. Carpita, N.C. Derbyshire, P. McCann,
M.C. and Benfey, P.N. (2001) Genes Dev. 15(9), 1115-27
8. Peskan, T. Westermann, M. and Oelmuller, R. (2000) Eur J Biochem. 267(24), 6989-95
9. Morel, J. Fromentin, J. Blein, JP. Simon-Plas, F. and Elmayan, T. (2004) Plant J. 37(2), 282-
93
10. Simon-Plas, F. Elmayan, T. and Blein, J.P. (2002) Plant J. 31. 137-147
11. Ames, B.N. (1966) Methods Enzymol. 8, 115-118
12. Moore, A.L. and Proudlove, M.O. (1983) In Hall, JL, Moore AL, eds. Isolation of organelles
and membranes from plant cells. London Academic Press, 153-184
13. Wintermans, J.F., and de Mots, A. (1965). Biochim. Biophys. Acta 109, 448–453
14. Macala, L.J. Yu, R.K. and Ando, S. (1983) J Lipid Res. 24(9), 1243-50
15. Bradford, M.M. (1976) Anal Biochem. 72, 248-54
16. Bligh, E.G. and Dyer, W.J. (1959). Can J Med Sci. 37(8), 911-7
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
27
17. Vitiello, F. and Zanetta, J.P. (1978) J Chromatogr. 166(2), 637-40
18. Browse, J. McCourt, P.J. and Somerville C.R. (1986) Anal Biochem. 152(1), 141-5
19. Juguelin, H. Heape, A. Boiron, F. and Cassagne, C. (1986) Brain Res. 390(2):249-52
20. Rahier, A. Benveniste, P. (1989) In WD Nes, E Parish, eds, Analysis of Sterols and Other
Significant Steroids. Academic Press, New York, 223-250
21. Larsson, C. Sommarin, M. and Widell S. (1994) Meth. Enzymol. 228: 451-459
22. Gronewald, J. AbouKhalil, W. Weber, E and Hansin, J (1982) Phytochem. 21. 4 859-862
23. Bohn, M. Heinz, E. and Luthje, S. (2001) Arch Biochem Biophys. 387(1), 35-40
24. Douce, R. and Joyard, J. (1990) Annu Rev Cell Biol. 6, 173-216
25. Hartel, H. Dormann, P. and Benning, C. (2000) Proc Natl Acad Sc USA i 97(19), 10649-54
26. Andersson, M.X. Stridh, M.H. Larsson, K.E. Liljenberg, C. and Sandelius, A.S. (2003) FEBS
Lett. 537(1-3), 128-32
27. Sullards, M.C. Lynch, D.V. Merrill, A.H. Jr. and Adams, J. (2000) J Mass Spectrom. 35(3),
347-53
28. Luche, S. Santoni, V. and Rabilloud, T.(2003) Proteomics. 3(3), 249-53
29. Sickmann, A. Reinders, J. Wagner, Y. Joppich, C. Zahedi, R. Meyer, H.E. Schonfisch, B.
Perschil, I. Chacinska, A. Guiard, B. Rehling, P. Pfanner, N. and Meisinger, C. (2003) Proc Natl
Acad Sci USA 100(23), 13207-12
30. Nebl, T. Pestonjamasp, K.N. Leszyk, J.D. Crowley, J.L. Oh, S.W. and Luna, E.J. (2002) J
Biol Chem. 277(45), 43399-409
31. Foster, L.J. De Hoog, C.L. and Mann, M. (2003) Proc Natl Acad Sci USA 100(10), 5813-8
32. Jacinto, T. Farmer, E.E. and Ryan, CA. (1993) Plant Physiol. 103(4), 1393-1397
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
28
33. Reymond, P. Kunz, B. Paul-Pletzer, K. Grimm, R. Eckerskorn, C. and Farmer, E.E. (1996)
Plant Cell. 8(12), 2265-76
34. Berczi, A. and Horvath, G. (2003) Acta Biologica Szegediensis 47(1-4), 7-10
35. Lindsey, K. Pullen, M.L. and Topping, J.F. (2003) Trends Plant Sci. 8(11), 521-5
36. Clouse, S.D (2002) Plant Cell. 14(9), 1995-2000
37. Hartmann, M.A. Benveniste, P. and Roland, J.C. (1983) Plant Science Lett. 30, 239-248
38. Chamberlain, L.H. (2004) FEBS Lett. 13;559(1-3), 1-5
39 Sperling, P. and Heinz, E. (2003) Biochim Biophys Acta. 1632(1-3), 1-15
40. Hartmann, M.A. (1998). Trends Plant Sci. 3. 170–175
41. Xu, X. Bittman, R. Duportail, G. Heissler, D. Vilcheze, C. and London, E. (2001) J Biol
Chem. 276(36), 33540-6
42. van Duyl, B.Y. Ganchev, D. Chupin, V. de Kruijff, B. and Killian, J.A. (2003) FEBS Lett.
17;547(1-3), 101-6
43. Bagnat, M. Chang, A. and Simons, K. (2001) Mol Biol Cell. 12(12), 4129-38
44. Bouche-Pillon, S. Fleurat-Lessard, P. Fromont, J.C. Serrano, R. and Bonnemain, J.L. (1994)
Plant Physiol. 105(2), 691-697
45. Martin, H. Rostas, J. Patel, Y. and Aitken, A. (1994) J Neurochem. 63(6):2259-65.
46. Assossou, O. Besson, F. Rouault, J.P. Persat, F. Brisson, C. Duret, L. Ferrandiz, J.
Mayencon, M. Peyron, F. and Picot S. (2003) FEMS Microbiol Lett. 224(2), 161-8
47. Fett, JP. and Coleman, J.R. (1994) Plant Physiol. 105(2), 707-13
48. Rumeau, D. Cuine, S. Fina, L. Gault, N. Nicole, M. and Peltier, G. (1996) Planta. 199(1), 79-
88
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
29
49. Santoni, V. Doumas, P. Rouquie, D. Mansion, M. Rabilloud, T. and Rossignol, M. (1999)
Biochimie. 81(6), 655-61
50. Kawamura, Y. and Uemura, M. (2003) Plant J. 36(2), 141-54
51. Zheng, X. and Bollinger Bollag, W. (2003) J . Invest Dermatol. 121(6), 1487-95
52. Browning, K. (1996) Plant Mol Biol 32, 107–144
53. Rosenberry, T.L. Krall, J.A. Dever, T.E. Haas, R. Louvard, D. and Merrick, W.C. (1989) J
Biol Chem. 264(13), 7096-9
54. Whiteheart, S.W. Shenbagamurthi, P. Chen, L. Cotter, R.J. and Hart, G.W. (1989) J Biol
Chem. 264(24), 14334-41
55. Yang, W. and Boss, W.F.(1994) J Biol Chem. 269(5), 3852-7
56. Klahre, U. Noguchi, T. Fujioka, S. Takatsuto, S. Yokota, T. Nomura, T. Yoshida, S. and
Chua, N.H. (1998) Plant Cell. 10(10), 1677-90
57. Takahashi, T. Gasch, A. Nishizawa, N. and Chua, N.H. (1995) Genes Dev. 9(1), 97-107
58. Ryan, C.A. and Farmer, E.E. (1991) Ann. Rev. of Plant Phys. and Plant Mol. Biol. 42. 651-
674
59. Retelska, D. Fleming, A. and Farmer, E.E. (2000) Poster: Abs # 36, Plant Biology meeting
2000, American society of plant physiology
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
30
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:
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
31
Dr Ramon Serrano (IBMCP, Valencia). We thank Jan Oliver Jost for his contribution to the
development of 2D electrophoresis methods.
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
32
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
33
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-
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
34
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.
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
35
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
36
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
37
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
38
A B
C
E F
D1 µm
500 nm
500 nm 100 nm
100 nm
100 nm
Mongrand et al.Figure 2
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
39
Mongrand et al.Figure 3
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
40
Mongrand et al.Figure 4
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--
Leaf BY2 by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
41
Mongrand et al.Figure 5
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 --
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Purification and characterization of higher plant lipid rafts
42
Mongrand et al.Figure 6
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
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
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.
10.1074/jbc.M403440200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
Supplemental material:
http://www.jbc.org/content/suppl/2004/06/21/M403440200.DC1
by guest on Decem
ber 5, 2020http://w
ww
.jbc.org/D
ownloaded from
Top Related