Plant Physiol. (1999) 120: 245-256
ATP-Dependent Formation of Phosphatidylserine-Rich Vesicles from
the Endoplasmic Reticulum of Leek Cells
Bénédicte Sturbois-Balcerzak,
Patrick Vincent,
Lilly Maneta-Peyret,
Michel Duvert,
Béatrice Satiat-Jeunemaitre,
Claude Cassagne, and
Patrick Moreau*
Laboratoire de Biogenèse Membranaire, Unite Mixte de
Recherche-5544
Centre National de la Recherche Scientifique (CNRS)
(B.S.-B., P.V., L.M.-P., C.C., P.M.), Centre de Microscopie
Électronique (M.D.), and Ecole Supérieure de Technologie des
Biomolécules (C.C.), Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cédex, France; and Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cédex, FranceInstitut des Sciences Végétales, Unité Propre de
Recherche 40-CNRS, 91198 Gif-sur-Yvette, France (B.S.-J.)
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ABSTRACT |
Leek (Allium porrum)
plasma membrane is enriched in phosphatidylserine (PS) by the vesicular
pathway, in a way similar to that already observed in animal cells (B. Sturbois-Balcerzak, D.J. Morré, O. Loreau, J.P. Noel, P. Moreau,
C. Cassagne [1995] Plant Physiol Biochem 33: 625-637). In this paper
we document the formation of PS-rich small vesicles from leek
endoplasmic reticulum (ER) membranes upon addition of ATP and other
factors. The omission of ATP or its replacement by ATP
-S prevents
vesicle formation. These vesicles correspond to small structures
(70-80 nm) and their phospholipid composition, characterized by a PS enrichment, is compatible with a role in PS transport. Moreover, the PS
enrichment over phosphatidylinositol in the ER-derived vesicles is the
first example, to our knowledge, of phospholipid sorting from the ER to
ER-derived vesicles in plant cells.
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INTRODUCTION |
The biosynthesis of most of the phospholipid species of the plasma
membrane of plant cells, as in animal cells, takes place primarily in
the ER (Moore, 1990
). This is the case for PS for which an
intracellular transport from the ER to the plasma membrane was
postulated and demonstrated in vivo. This transport is inhibited by
monensin and low temperatures and follows the ER-Golgi-plasma membrane
pathway (Sturbois-Balcerzak et al., 1995; Moreau et al., 1998a).
Therefore, this transport is expected to be mediated by carrier
vesicles. Such structures can transport membrane and secretory proteins
in plant cells (Satiat-Jeunemaitre and Hawes, 1993; Bar-Peled et al.,
1996), and we also suspect their involvement in lipid transport (Moreau
et al., 1988, 1998a; Bertho et al., 1991; Sturbois-Balcerzak et al.,
1995).
Few attempts have concerned the isolation of putative vesicular
intermediates involved in the delivery of membrane material from the ER
in plant cells (Morré et al., 1989; Hellgren et al., 1993).
Recently, a cell-free ATP-dependent transfer of phospholipids was
obtained between the ER and the Golgi apparatus of leek
(Allium porrum) cells (Sturbois et al., 1994).
PC, PE, and particularly PS were transferred, mimicking the in vivo
situation. On the other hand, PI was not found to be transported
(Sturbois et al., 1994; Sturbois-Balcerzak et al., 1995).
Although the characterization of proteins likely to be involved in
vesicular transport is in progress in plant cells (Bar-Peled et al.,
1996; Gomord and Faye, 1996; Hawes and Satiat-Jeunemaitre, 1996), there
is still no specific marker for ER-derived vesicles in plant cells. We
have developed another strategy to monitor the isolation of putative
vesicular structures involved in the transport of phospholipids and
especially PS in leek cells.
It has been observed that ER-derived vesicles are 50- to 80-nm
vesicular structures in many eukaryotic organisms (Paulik et al., 1988;
Morré et al., 1989; Hellgren et al., 1993; Moreau et al., 1993;
Bednarek et al., 1995). Moreover, the transport vesicles isolated from
the ER of rat liver show an enrichment in PS (Moreau et al., 1992,
1993), and we have observed a selective transfer of PS in vivo
(Sturbois-Balcerzak et al., 1995) and between the ER and the Golgi
apparatus of leek cells in vitro (Sturbois et al., 1994).
We incubated an ER-enriched membrane fraction from leek cells with ATP
and other factors and observed the formation of small vesicles that
were PS enriched. Their partial isolation was performed by
sedimentation on Suc-density gradients and/or filtration through 200- and 100-nm-pore membranes (Anotop, Anotec/Whatman). Our results show
for the first time, to our knowledge, in a cell-free system from plant
cells that phospholipids can be sorted and targeted from the ER to
ER-derived vesicles, as is the case for proteins (Bar-Peled et al.,
1996).
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MATERIALS AND METHODS |
Leek (Allium porrum L.) seeds were purchased from
Vilmorin (La Ménitré, France) and stored overnight at 4°C
before being hydrated with distilled water for 2 h. The seeds were
allowed to germinate in the dark for 7 d at 24°C as described
previously (Moreau et al., 1988).
All chemicals were from Sigma. [1-4C]Acetate was obtained
from CEA (Saclay, France). [14C]Ser and
[3H]inositol were purchased from NEN.
Labeling and Isolation of ER Membranes
Twenty batches of 20 seedlings were incubated, each with 2 µCi
of [1-4C]acetate (54 Ci/mol) for 120 min at
24°C. Leek seedlings were homogenized in a mortar in the presence of
10 mM KH2PO4,
pH 8.2, with 0.5 M sorbitol, 5% (w/v) PVP 40, 0.5% (w/v)
BSA, 2 mM salicylhydroxamic acid, and 1 mM
PMSF. The homogenate was then filtered through two layers of Miracloth
(Calbiochem) and centrifuged at 1,000g for 10 min. The
supernatant was centrifuged for 10 min at 12,000g, and the
resulting supernatant was centrifuged at 150,000g for 60 min
with a rotor (model TST 2838, Kontron, Eching, Germany, or model AH
629, Sorvall). The resulting microsomal pellets were resuspended in 10 mM
KH2PO4, pH 7.8, with 0.5 M sorbitol and loaded onto a discontinuous
Suc-density gradient consisting of 2.5 mL of 37% (w/v) Suc, 3.5 mL of
25% (w/v) Suc, and 3.5 mL of 18% (w/v) Suc. After the sample was
centrifuged at 150,000g for 150 min, the ER membranes at the
18%/25% Suc interface were collected, diluted in the appropriate
buffer (see below), and centrifuged at 150,000g for 60 min.
The pellets were resuspended in the appropriate buffer for marker
enzyme assays (Moreau et al., 1988; Bertho et al., 1991).
Preparation of Golgi and Plasma Membrane-Enriched Fractions
Three to four hundred unlabeled leek seedlings were homogenized as
described above. One-half of the microsomal pellet was used to isolate
the Golgi membranes at the 25%/37% Suc interface. The membranes were
diluted with the appropriate buffer and centrifuged at
150,000g for 60 min. The pellet was resuspended in the
appropriate buffer for marker enzyme assays (Moreau et al., 1988;
Bertho et al., 1991).
Plasma membranes were isolated by phase partition using PEG 4000 and
dextran T500. The other half of the microsomal suspension was mixed
with a polymer (PEG/dextran mixture) in 0.5 M sorbitol containing 10 mM
KH2PO4 and 40 mM NaCl, pH 7.8, to obtain final PEG 4000 and dextran T500
concentrations of 6.0% (w/w). The solution (final volume, 28 mL) was
centrifuged for 15 min at 1,000g, and the PEG-enriched upper
phase (12 mL) was recovered without disturbing the interface. Membranes
were then recovered after centrifugation at 150,000g for 60 min and resuspended in the appropriate buffer for marker enzyme assays
(Moreau et al., 1988; Bertho et al., 1991).
For the assay of glucuronyltransferase, we followed the procedure of
Hobbs et al. (1991) and Baydoun and Brett (1997).
Isolation of ER-Derived Vesicles
The cell-free assay used for the formation of putative carrier
vesicles consisted of labeled donor ER membranes (600 µg of protein),
cytosol (300 µg of protein), ATP, and an ATP-regenerating system (500 µM ATP, 2.5 mM magnesium acetate, 3 mM UTP, 2 mM phosphocreatine, and 10 units/mL
phosphocreatine kinase), a "lipid mixture" composed of 12.5 µM CDP-choline, 12.5 µM CDP-ethanolamine,
2.5 µM palmitic acid, 7.5 µM oleic acid,
500 µM CoA, and 30 mM Hepes buffer (pH 6.8, total volume 1 mL). A lipid mixture was added because we found
that an ongoing biosynthesis of lipids stimulated the formation of the
ER-derived vesicles.
A cytosolic fraction, stimulating the ER-Golgi transport and prepared
as described earlier (Sturbois et al., 1994), was also added since the
formation of ER-derived vesicles has been shown to be cytosol dependent
in animal and yeast cells (Rothman and Wieland, 1996; Schekman and
Orci, 1996).
The ER membranes were incubated in glass vials at 22°C to 24°C for
20 min, either in the absence (
) or in the presence (+) of ATP.
ATP-S was used at the same concentration as ATP. We determined earlier (Sturbois et al., 1994) that the ATP-stimulated transport of
lipids between the ER and the Golgi membranes was dependent on
time, temperature, and ATP concentration. The optimal conditions for
the formation of the vesicles were chosen accordingly.
After the ER membranes were incubated according to the conditions
described above, the reaction mixtures were treated alternatively by
the following two methods: (a) The incubation mixture was loaded onto a
discontinuous Suc-density gradient consisting of 1.5 mL of 18% (w/v)
and 1.5 mL of 37% (w/v) Suc solutions in 30 mM Hepes buffer containing 30 mM KCl, pH 6.8. After centrifugation
at 100,000g for 45 min, the sample/18% Suc interface and
the 18%/37% Suc interface were collected, diluted with distilled
water, and centrifuged at 150,000g with a rotor (model RT
80, Himac CS 100, Hitachi, Tokyo) for 15 min. The putative ER-derived
vesicles and the ER membranes were resuspended in distilled water. (b)
The incubation mixture was centrifuged at 10,000g for 5 min
to discard the bulk of the ER donor membranes. The resulting
supernatant was passed successively through 200- and 100-nm filters
(Anotop membranes purchased from Anotec/Whatman). The 200-nm filters
eliminated large ER fragments that did not sediment after
centrifugation. Filters of 100 nm were chosen according to earlier
findings showing that transition vesicles arising from the ER generally
occur as 50- to 80-nm membrane structures (Paulik et al., 1988;
Morré et al., 1989; Hellgren et al., 1993; Moreau et al., 1993;
Bednarek et al., 1995). The membrane vesicles recovered after
filtration through the 100-nm filters were sedimented at
150,000g with a rotor (model RT 80) for 15 min. The pellets
were resuspended in an appropriate volume of distilled water.
The membrane material isolated from the ER incubated in the absence of
ATP was called TV() and the ER-derived vesicles obtained from the ER
incubated in the presence of ATP were named TV(+).
Protein Determination, SDS-PAGE, and Immunoblots
The quantity of proteins from the ER and Golgi membranes and from
the ER-derived vesicle fractions was determined according to the
Bradford (1976) and bicinchoninic acid procedures (Smith et al., 1985)
using BSA as a standard.
SDS-PAGE was carried out on 12% polyacrylamide gels (Bio-Rad). A
molecular mass kit was used that contained phosphorylase b (94 kD), BSA
(67 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD), and soybean
trypsin (20 kD). Membrane fractions were mixed with an equal volume of
a solution of 0.125 M Tris buffer, pH 6.8, 4% SDS, 20%
glycerol, and 10% 2-mercaptoethanol and incubated for 5 min at
100°C. After electrophoresis, the gel was subjected to
electrophoretic transfer on a PVDF membrane (NEN). Immunostaining was
carried out at room temperature. The membrane was sequentially subjected to incubation with (a) a blocking reagent for 1 h, (b) an antiserum for 16 h (both antibodies were used as undiluted culture supernatants), (c) an anti-mouse or
anti-rat-immunoglobulin-peroxidase conjugate (Sigma) for 30 min, and
(d) a chemiluminescent reagent (Renaissance kit, NEN). The
membrane was finally exposed to Reflection film (NEN).
Immunofluorescence
Root apices were treated for immunofluorescence staining as
described previously (Satiat-Jeunemaitre et al., 1996a). They were fixed for 1 h in 4% paraformaldehyde in PBS, pH 6.9. A
partial cell wall digestion was performed by a 20-min treatment with
1% cellulase (Onazuka R10) and 1% pectinase in PBS. Release of
individual cells was achieved by gently squashing the roots on coated
Vectabond (Vector Laboratories, London, UK) multiwell slides. Cells
were then permeabilized with 0.5% Triton X-100 in PBS for 20 min. For the immunoreaction procedure, cells were incubated in 1% BSA in PBS to
block the unspecific reaction sites for 20 min before incubation in
primary antibody (anti-HDEL or JIM 84) for 1 h at room
temperature. After four washes in buffer containing 1% fish gelatin,
cells were stained with an appropriate fluorescein
isothiocyanate- conjugated second antibody (anti-mouse or
anti-rat, diluted 1:40, Sigma) for 1 h at room temperature. After
the preparations were washed thoroughly, they were mounted in
Vectashield antifade agent (Vector Laboratories) and viewed with a
confocal laser microscope.
The anti-HDEL monoclonal antibody was a generous gift from Richard
Napier (Horticulture Research International, Wellesbourne, UK). It was
produced in mice against a synthetic peptide corresponding to the C
terminus of yeast Bip (Napier et al., 1992). The JIM 84 monoclonal antibody was a generous gift from Chris Hawes (Oxford Brookes University, UK). It was produced in rats and recognizes glycoproteins associated with cisternal membranes (Horsley et al.,
1993). Both antibodies were used as undiluted culture supernatants.
Lipid Analyses of the ER-Derived Vesicles and the ER Membranes
Lipids were extracted by chloroform:methanol (1:1, v/v) for 30 min
at room temperature. They were then washed three times with distilled
water. The solvent was evaporated and the lipids were resuspended in an
appropriate volume of chloroform:methanol (1:1, v/v) according to
procedures described previously (Moreau et al., 1988; Bertho et al.,
1991). Lipids (5-10 µg) were loaded onto HPTLC plates (60F254,
Merck, Darmstadt, Germany) and chromatograms were developed
by methyl acetate: n-propanol:chloroform:methanol:aqueous 0.25% KCl (25:25:25:10:9, v/v), according to the method of Heape et
al. (1985).
Calibration curves for phospholipids were established with standard
lipids of PC, PS, PI, and PE and by using monogalactosyldiacylglycerol (2 µg) as an external standard (Heape et al., 1985). After separation on HPTLC plates, the lipids were charred using the technique of Fewster
et al. (1969), as modified by Macala et al. (1983). The plates were
scanned within 1 h using a TLC/HPTLC densitometer (model 76510, Camag, Muttenz, Switzerland) coupled with a computing integrator (model
SP 4100, Spectra Physics, Mountain View, CA). The scans were carried
out at 366 nm (mercury lamp) at a speed of 0.5 mm/s. The quantities of
the various phospholipids of the different membrane fractions were
deduced from the calibration curves (Heape et al., 1985).
The radioactivity of the phospholipids was determined as follows. After
the different phospholipids were identified by comparison with
standards, they were scraped off directly into scintillation vials, and
radioactivity was determined by liquid-scintillation counting in a
scintillation counter (model 2000 CA, Packard).
Radioactivity of the phospholipids was also determined after
autoradiography of the HPTLC plates (Hyperfilm MP RPN 1675, Amersham) and scanning with a densitometer (model 76510, Camag). Both methods gave similar results and were used alternatively.
Fatty Acid Analyses of the Phospholipids
The fatty acid compositions of the phospholipids of the ER and
ER-derived vesicle fractions were determined as follows. Lipid extracts
were heated in screw-capped tubes at 80°C in 1 mL of 2.5% (v/v)
H2SO4 in methanol for 60 min. After the addition of 1.5 mL of water and 300 µL of hexane,
fatty acid methyl esters were extracted by shaking, and the tubes were
centrifuged at low speed. Samples of the organic phase were separated
by GLC on a 15-m × 0.53-mm Carbowax column (Alltech, Deerfield,
IL) and quantified using a flame-ionization detector. The gas
chromatograph was programmed for an initial temperature of 160°C for
1 min and then a 20°C/min ramp to 190°C, a secondary ramp of
5°C/min to 230°C, and a third ramp of 20°C/min to 240°C; this
final temperature was maintained for 6 min. Peak identities were
determined by comparison with fatty acid methyl ester standards.
Electron Microscopy and Morphometry
ER membranes and ER-derived vesicles were fixed at 4°C by
recovering the pellets with a fixative solution containing 2.5% glutaraldehyde in 0.1 M sodium cacodylate at pH 7.5. Fixed
pellets were then washed and postfixed in 1% osmium tetroxide in the
same buffer. After the samples were washed with distilled water, they were treated in an aqueous solution of 1% uranyl acetate for 30 min at
room temperature, washed again, dehydrated, and embedded in Epon. Each
pellet was divided into several blocks that were cut into thin
sections. The section plane was random but constant in thickness.
Sections were contrasted with a uranyl acetate solution and then with
lead citrate. They were observed and photographed using an electron
microscope (model EM 210, Philips, Cambridge, UK).
Measurement of vesicle sizes and the determination of the relative
abundance of the different vesicles were performed as follows. At least
20 photographs were taken at random from the thin sections obtained
from several blocks. Two magnifications (×8,900 and ×20,000) were
used for the analyses. Vesicle sizes and relative proportions were
determined and calculated with an analyzer (model IBAS1, Kontron).
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RESULTS |
Characterization of the ER-Enriched Membrane Fraction
The various membrane fractions were first characterized by several
enzyme markers (Table I). CDP-choline
phosphotransferase and NADPH-Cyt c reductase were used as ER
markers (Moreau et al., 1988; Bertho et al., 1991). IDPase (which can
be considered a nucleotide diphosphatase) was taken for a Golgi marker
(Goff, 1973; Morré et al., 1977; Quail, 1979). However, several
nucleotide diphosphatases have also been observed to be present in
other membranes (Goff, 1973). Therefore, we also measured the activity of glucuronyltransferase, which was found with the Golgi apparatus in
pea epicotyls (Hobbs et al., 1991; Baydoun and Brett, 1997). For the
plasma membrane, we measured the K+-stimulated
Mg2+ ATPase and the glucan synthase II (Quail,
1979). The ATPase activity measured in the plasma membrane fraction
(Table I) presented a 93% inhibition by vanadate (Moreau et al.,
1988), which corresponds well to its plasma membrane origin (Sze,
1985). The relative enrichments of the various activities in the
different membrane fractions (ER, Golgi, and plasma membrane) indicate
the extent of purification achieved. Contamination of the membrane
fractions by plastidial membranes was considered acceptable according
to the levels of chlorophyll, carotenoids, and galactolipids (Table I,
legend). The ER fraction, which will be used to form the small vesicles in vitro, has further been characterized by ER-specific antibodies raised against the C terminus (including the HDEL sequence) of yeast
Bip (Napier et al., 1992). JIM84 antibodies, which are more specific
for Golgi proteins (Horsley et al., 1993), were also used.
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Table I.
Purity of membrane fractions according to marker
enzymes
Activities of CDP-choline phosphotransferase, NADPH-Cyt c
reductase, IDPase, glucane synthetase II, and the
K+-stimulated Mg+ ATPase were measured as
previously described (Moreau et al., 1988; Bertho et al., 1991).
Activity of succinate dehydrogenase was followed as reported previously
(Moreau, 1986). Glucuronyl transferase activity was assayed according
to the methods of Hobbs et al. (1991) and Baydoun and Brett (1997).
Activities of succinate dehydrogenase in the 1,000g pellet
were 9.4 µmol/h and 0.2 µmol h 1 mg1 and
those of the 12,000g pellet were 127.2 µmol/h and 4.8 µmol h1 mg1. Numbers in parentheses
represent the enrichment factors of the ER, Golgi, and plasma membrane
markers in the corresponding membranes. These values were calculated
taking the specific activities of the homogenate as equal to 1. The
microsomal pellet contained only 2% and 4% of the total chlorophyll
and carotenoids, respectively (Moreau, 1986). Those molecules were not
detected in the ER, Golgi, or plasma membrane fractions but only in the
heavier fraction of the discontinuous gradient and the lower
(dextran-enriched) phase after phase partition. In addition, a low
contamination of the ER and Golgi fractions by plastid envelope was
determined by the presence of small amounts of galactolipids (<10% of
the total glycerolipids). Enzyme activities were determined from at
least three different fractionations. ND, Not detected.
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Immunofluorescence microscopy of leek root cells stained with the
anti-HDEL antibody revealed a fine reticulate network of ER throughout
the cells (Fig. 1, top). The nuclear
envelope was often stained as well. When leek cells were stained with
the JIM 84 antibody, a characteristic punctuated pattern was seen that corresponded to hundreds of Golgi stacks dispersed throughout the
cytoplasm (Fig. 1, bottom). The immunofluorescent staining patterns of
leek cells observed with anti-HDEL and JIM84 antibodies corresponded
very well to the ER and Golgi staining previously reported for other
plant cells (Satiat-Jeunemaitre and Hawes, 1992; Horsley et al., 1993;
Henderson et al., 1994; Satiat-Jeunemaitre et al., 1996a).
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| Figure 1.
Confocal laser scanning micrographs of the Golgi
apparatus and ER in leek root cells. Projection of optical sections
from confocal data sets. Magnification, ×1400. Top, Immunolocalization
of the ER stained with anti-HDEL. The ER appears as a fine network,
often radiating out from the nucleus through the cell. There is often
staining in the nuclear membrane as well. Bottom, Immunolocalization of
the Golgi apparatus in leek root cells stained with JIM 84. Golgi
stacks appear scattered throughout the cytoplasm.
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Immunoblot analyses revealed a strong labeling of the ER fraction with
the anti-HDEL antibody (Fig. 2), whereas
only a weak band was detected in the Golgi fraction. As expected, JIM84
labeled the Golgi fraction (Fig. 2), but no significant label was
observed for the ER fraction. The fact that several bands were revealed in the ER fraction is a result of the presence of several proteins carrying the C terminus HDEL sequence, which is an ER-targeting sequence (Napier et al., 1992; Bar-Peled et al., 1996). Together with
enzyme markers (Table I), the immunoblot staining patterns support the
obtaining of an ER-enriched membrane fraction from leek cells.
This ER fraction was then used to form ER-derived vesicles in vitro and
undertake their isolation.
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| Figure 2.
Immunoblots of ER and Golgi fractions with
anti-HDEL and JIM 84 antibodies. SDS-PAGE and immunostaining were done
as explained in ``Materials and Methods''. Lanes corresponding to the
membranes of the ER and the Golgi-enriched membrane fraction are
indicated, respectively, as ER and Golgi.
Mrs (in thousands) of classic standards
are also indicated. For each lane, 5 µg of proteins was loaded.
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Isolation of ER-Derived Vesicles on Discontinuous Suc-Density
Gradients
The buoyant density of lipid-rich vesicles involved in lipid
transport from the ER to the Golgi apparatus may differ from that of ER
from which they are derived. We used this as a first attempt to isolate
an ER-derived vesicle fraction. ER membranes were incubated in the
presence of ATP, as described in "Materials and Methods," and the
incubation medium was loaded onto a discontinuous Suc-density gradient
comprising two layers, 18% and 37% Suc. As a control, one-half of the
ER membranes was incubated in the same incubation medium deprived of
ATP. In the absence of ATP, the amount of membrane material obtained at
the sample/18% Suc interface (TV[]) represented 5.5% ± 0.7%
(32 ± 4 µg) of the amount of total ER membranes.
In the presence of ATP, the quantity of membranes recovered at the
sample/18% Suc interface (TV[+]) accounted for 10% ± 3.3% (57 ± 11 µg) of the ER starting material. The amounts of
vesicles recovered in the absence or presence of ATP were significantly different (P < 0.01). The membrane material recovered from the sample/18% Suc interface (likely to contain the ER-derived vesicles) and the ER membranes (recovered at the 18%/37% Suc interface) were
sedimented by ultracentrifugation.
Mass quantitation of the four major phospholipids, PC, PS, PI, and PE,
was performed for the three membrane fractions (ER before incubation,
vesicles isolated in the absence of ATP, and vesicles isolated in the
presence of ATP). For the vesicles isolated from the ER membranes
incubated with ATP (TV[+]), there was four times as much PS (Table
II). Similarly, after a 120-min labeling of phospholipids with [14C]acetate, we
determined the radioactivity of the phospholipids in the ER and
ER-derived vesicles (Table III). There
were two major differences between the vesicles isolated from ER
membranes incubated in the presence of ATP (TV[+]) and those isolated
from ER membranes incubated without ATP (TV[]). First, we found an
increase in labeled PS in the vesicles produced from the ER membranes
incubated with ATP. Concomitantly, a significant decrease in labeled PI was obtained. Therefore, the stimulation of the formation of the ER-derived vesicles by ATP resulted in the isolation of PS-rich vesicles that were decreased in their labeled PI content.
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Table II.
Phospholipid composition of the ER and the putative
ER-derived vesicles isolated on discontinuous Suc-density gradients
In the absence of ATP, the amount of membrane material obtained at the
sample/18% Suc interface (TV[]) represented 5.5% ± 0.7%
(32 ± 4 µg) of the amount of total ER membranes (as starting
material). In the presence of ATP, the quantity of membranes recovered
at the sample/18% Suc interface (TV[+]) accounted for 10% ± 3.3%
(57 ± 11 µg) of the ER starting material. Lipids were
extracted, separated on HPTLC plates, and revealed as mentioned in
``Materials and Methods''. Then, HPTLC plates were scanned, and the
amount of each phospholipid was determined from the calibration curves.
PS increase in the vesicles TV(+) compared with the ER was significant
(P < 0.01, according to Student's t test).
n indicates the number of fractionations and lipid analyses
performed.
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Table III.
Labeling of the various phospholipids of the ER
and the putative ER-derived carrier vesicles isolated on discontinuous
Suc-density gradients
Yield of vesicles was as in Table II. Lipids were extracted and
separated as already described. The spots corresponding to the
phospholipids were scraped off the HPTLC plates and their radioactivity
was determined by liquid-scintillation counting. The increase of PS
labeling in the vesicles TV(+) compared with the ER was significant
(P < 0.02, according to Student's t test).
n indicates the number of fractionations and lipid analyses
performed.
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Isolation of ER-Derived Vesicles by Filtration
Vesicle size was taken as another advantage for the purification
of ER-derived vesicles. For this, the incubation medium containing the
ER membranes incubated in the absence or presence of ATP was filtered
through successive Anotop filters of 200- and 100-nm-pore sizes. The
putative ER-derived vesicles were then sedimented at 200,000g for 15 min.
In the absence of ATP, the amount of membrane material recovered in the
vesicle fraction (TV[]) represented 6.1% ± 1% (39 ± 6 µg) of the amount of total ER starting material. In the presence of
ATP, the amount of membranes recovered in the vesicle fraction (TV[+]) accounted for 10.3% ± 2.7% (67 ± 10 µg) of the
initial ER membranes. The amounts of vesicles recovered in the absence or presence of ATP were significantly different (P < 0.01).
After sedimentation of the respective vesicle fractions, the lipids
were extracted and analyzed according to the experimental procedures
described in ``Materials and Methods''.
The results concerning the mass proportions of the four phospholipids
PC, PS, PI, and PE are given in Table IV.
As for the isolation on Suc-density gradients, we observed an increase
in PS in the ER-derived vesicles.
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Table IV.
Phospholipid composition of the ER and the putative
ER-derived carrier vesicles isolated by filtration through Anotop
filters
In the absence of ATP, the amount of membrane material recovered in the
vesicle fraction (TV[]) represented 6.1% ± 1% (39 ± 6 µg) of the amount of total ER starting material. In the presence of
ATP, the amount of membranes recovered in the vesicle fraction
(TV[+]) accounted for 10.3% ± 2.7% (67 ± 10 µg) of the
initial ER membranes. Lipids were extracted, separated, and revealed as
already described. Then, the HPTLC plates were scanned, and the amount
of each phospholipid was determined from the calibration curves. PS
increase in the vesicles TV(+) compared with the ER was significant
(P < 0.01, according to Student's t test).
n indicates the number of fractionations and lipid analyses
performed.
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Similarly, after a 120-min de novo labeling of phospholipids with
[14C]acetate, we determined the radioactivity
of the phospholipids in the ER and ER-derived vesicles (Table
V). PS labeling was also greatly
increased in the ER-derived vesicles obtained from the ER incubated in
the presence of ATP (TV[+] fraction) and the amount of labeled PI was
slightly decreased.
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Table V.
Labeling of the various phospholipids of the ER and
the putative ER-derived carrier vesicles isolated by filtration through
Anotop filters
Yield of vesicles was as in Table IV. Lipids were extracted and
separated as already described. The spots corresponding to the
phospholipids were scraped off the HPTLC plates and their radioactivity
was determined by liquid-scintillation counting. The increase of PS
labeling in the vesicles TV(+) compared with the ER was significant
(P < 0.01 according to Student's t test).
n indicates the number of fractionations and lipid analyses
performed.
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The proportion of labeled PS in the ER-derived vesicles (TV[+]
fraction) was increased 4 and 7 times compared with the TV[]) fraction (control) and the ER membranes, respectively.
Both methods (Suc-density gradient and filtration) gave similar
results, i.e. the isolation of an ER-derived vesicle fraction that is
PS enriched and has a decreased amount of labeled PI.
To determine whether the formation of ER-derived vesicles required ATP
hydrolysis, we also carried out these experiments in the presence of
ATP-S and compared it with that of ATP. In the presence of ATP-S,
the amount of membrane material recovered at the sample/18% Suc
interface was only 33 ± 6 µg (n = 4). In the
presence of ATP, the amount of proteins recovered in the ER-derived vesicles was 72 ± 14 µg (n = 4). The amounts of
vesicles recovered in the presence of ATP-S or ATP were
significantly different (P < 0.01). The results obtained in the
presence of ATP-S were similar to those reported in the absence of
ATP (Tables II and IV). Similarly, incubating ER membranes at 4°C in
the presence of ATP did not result in the formation of ER-derived
vesicles.
Fatty Acid Composition of the Phospholipids of the ER and the
ER-Derived Vesicles
To complete the lipid analysis of the ER-derived vesicles, we
determined the fatty acid composition of the major phospholipids PC,
PE, and PS of the vesicles by GLC and compared it with that of the
phospholipids of the ER membranes.
The fatty acid composition of PC and PE was similar in the ER membranes
and the ER-derived vesicles (Table VI).
However, a large increase in VLCFA-containing PS was observed in the
ER-derived vesicles (Table VI). VLCFA were a mixture of saturated and
unsaturated fatty acids with 20 to 24 carbon atoms, the major
species being the saturated 22 and 24 fatty acids. No or few VLCFA
molecules were observed to be esterified to PI in both membranes.
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Table VI.
Fatty acid composition of the three major
phospholipids of the putative ER-derived carrier vesicles (TV[+]) and
the ER membranes
For these analyses, the ER-derived carrier vesicles were either
isolated on density-Suc gradients or by filtration. The data are mean
values ± SD of nine experiments. For each
phospholipid, 16 is a fatty acid with 16 carbon atoms (more than 97%
of 16:0); 18 is a fatty acid with 18 carbon atoms (>75% of 18:2, the
rest corresponding to 18:1 and 18:3); VLCFA is a fatty acid with more
than 18 carbon atoms (major fatty acids representing >75% are 22:0,
24:0, and 26:0; the rest correspond to 20:0, 20:1, and 22:1).
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Since PS was enriched compared with the other phospholipids in the
ER-derived vesicles (Tables II, IV, and VIII), VLCFA-containing PS was
therefore greatly increased in the vesicles, and we calculated that its
amount (percentage of total) was 6 times higher than in the ER
membranes. Therefore, PS molecules and particularly VLCFA-containing PS
are targeted to the ER-derived vesicles, supporting the fact that these
molecules could be transported via these small vesicles.
Characterization of the ER-Derived Vesicles by Electron
Microscopy
Figure 3A shows ER-derived vesicles
formed from the ER membranes incubated with ATP and isolated on
Suc-density gradients. Most of the vesicles were small (<100 nm), but
larger structures could be observed. The pictures of the ER-derived
vesicles isolated by filtration appeared similar by electron
microscopy. The vesicle fraction obtained after incubation of ER
membranes with (B) or without ATP (C) is shown with higher
magnification in Figure 3, B and C.
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| Figure 3.
Electron micrograph of the ER-derived vesicles
isolated on discontinuous Suc-density gradients. Incubation of ER
membranes with or without ATP and isolation of ER-derived vesicles were
performed as described in ``Materials and Methods''. Membranes were
fixed and prepared for electron microscopy, as described in
``Materials and Methods''. A, ER-derived vesicles (TV[+]) obtained
from the ER membranes incubated in the presence of ATP. B, The same
fraction as in A but at a higher magnification. C, Membrane fraction
(TV[]) isolated from the ER membranes incubated in the absence of
ATP. Bars = 1 µm.
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The mean sizes of the different vesicles and their proportion were
analyzed and determined with a Kontron IBAS 1 analyzer. The results are
shown in Table VII. Two major membrane
populations are present in the ER fraction, one having a mean size
greater than 300 nm and a second of about 200 nm. Very few small
vesicles (<100 nm) were observed in the ER fraction (Table VII).
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Table VII.
Diameter and relative proportions of the different
membrane structures present in the ER, the membrane fraction TV(),
and the putative ER-derived carrier vesicles TV(+)
The amount of proteins recovered for the fractions TV() was 34 ± 4 µg (n = 8) and that obtained for the fraction
TV(+) was 63 ± 11 µg (n = 14). Therefore, we
estimated per experiment an average quantity of 2 µg of proteins
corresponding to small vesicles recovered in the absence of ATP and as
much as 25 µg of proteins for the small vesicles that were formed in
the presence of ATP. Surface areas were estimated according to perfect
spherical structures. Although this calculation is not accurate, it
allows a comparison between TV() and TV(+) that clearly highlights
the stimulation by ATP of the formation of small vesicles. a, Very few
small vesicles (<100 nm) were detectable; b, very few structures were
observed with a diameter  1 µm (Fig. 3C). These structures were
omitted from the statistics not to artificially decrease the number of
small structures in the TV() fraction as compared with the TV(+)
fraction.
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The TV() fraction contains chiefly a vesicle population of about 250 nm, accounting for 67% of the total number of membrane vesicles and
94% of the total (estimated) mem-brane surface. Smaller vesicles (mean
size of 90 nm) were also present (33% of the total number of membrane
vesicles and only 6% of the total membrane surface).
The TV(+) fraction, which corresponds to vesicles isolated from the ER
membranes incubated in the presence of ATP, also contains two membrane
populations but is characterized by a high proportion of small vesicles
(with a mean size of 76 nm). The small vesicles accounted for 74% of
the total number of vesicles and 40% of the total membrane surface
(Table VII). The other population of vesicles had a mean size of about
190 nm and represented only 26% of the total number of membrane
structures (60% of the total membrane surface). A simplified
calculation (detailed in the legend of Table VII) showed that the
amount of small ER-derived vesicles in the TV(+) fractions was 12 times
that found in the control (TV[]).
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DISCUSSION |
PS was demonstrated to be transported to the plasma membrane of
leek cells through the vesicular pathway (Sturbois-Balcerzak et al.,
1995; Moreau et al., 1998a). A first attempt to reconstitute in vitro
an ATP-dependent transfer of phospholipids and especially PS from the
ER to the Golgi apparatus of leek cells was performed previously
(Sturbois et al., 1994). However, no carrier vesicles were isolated and
shown as intermediate structures. The aim of this study was to
create a cell-free system from leek cells, making it possible to
reconstitute the formation of ER-derived vesicles in vitro, and to
address the question of phospholipid sorting and targeting (here PS) to
the ER-derived vesicles.
In this paper we have shown that an ER-enriched fraction from leek
cells (Table I; Fig. 2) is capable of producing small vesicles in vitro
when incubated with ATP and other factors. We observed that ATP
addition resulted in a significant increase and, therefore, formation
of small vesicles from the ER membranes (Table
VIII).
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Table VIII.
Comparison between the distribution of the
phospholipids transferred in vitro and the phospholipid composition of
the putative ER-derived carrier vesicles
For this comparison, the phospholipid compositions of the vesicles
isolated by both methods were combined. PS enrichment and the increase
of PS labeling in the transition vesicles compared with the ER were
significant with P values < 0.001. PS:PI ratios were also
significantly different with P values < 0.001; n = 12.
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The small vesicles were isolated and partially purified by two
different methods (ultracentrifugation on Suc-density gradients and
filtration on Anotop membranes). The small vesicles obtained using both
methods were similar according to their morphology (mean size of 70-80
nm) and phospholipid composition (Table VIII) and were significantly
different from the initial ER membranes.
These vesicles were characterized by a PS enrichment as shown by the
amount of the lipids (Tables II and IV) and their labeling (Tables III
and V). PS enrichment in the ER-derived vesicles (6.2% of the total
phospholipids compared with 1.8% in the ER, Table VIII) corresponds to
previous findings obtained for ER-derived vesicles isolated from rat
liver in which the amount of PS (among total phospholipids) was 7.2%
and only 2.5% to 3% in the ER membranes (Moreau et al., 1992, 1993;
Moreau and Cassagne, 1994).
However, it is still possible that PS, enriched in the small vesicles,
could have been synthesized locally (i.e. by enzymes located in the
small vesicles). In fact, whereas isolated ER membranes exhibited high
activities of PS (0.23 nmol mg1 protein
min1) and PI (0.15 nmol
mg1 protein min1)
synthesis from [14C]Ser and
[3H]inositol, ER-derived vesicles were
practically devoid of these activities (< 2% of the total activity of
the ER). Moreover, the specific activity (per milligram of proteins) of
PS synthesis in the ER-derived vesicles was 5 to 10 times lower than
that in the ER. The residual activity in the vesicles does not account for their PS enrichment. Isolated ER membranes (labeled with
[14C]Ser and
[3H]inositol) were also used to form and
isolate small vesicles. These were labeled and their PS:PI label ratio
was 2.0 ± 0.3 (n = 5) and of the same order of
magnitude as those calculated from Table VIII (2.6 ± 0.6).
Similar results were obtained whatever the labeling pathway of the
phospholipids, i.e. from the de novo synthesis of the fatty acids
(Table VIII) or from the incorporation of
[14C]Ser and
[3H]inositol by the isolated ER membranes (see
above).
It could be argued that the small vesicles do not originate from the ER
membranes but from contaminant plastid envelope or outer mitochondrial
membranes. However, it has been shown that the plastid envelope is
totally devoid of PS and PE (Maréchal et al., 1997) and that the
outer mitochondrial membrane is also poor in PS (Douce, 1985; Douce and
Joyard, 1990; Guillot-Salomon et al., 1997). Moreover, PS is not
synthesized by these membranes (Moore, 1990). Therefore, because ER
membranes were the major site of synthesis of PS, the labeled PS that
accumulated in the vesicles was likely to be ER derived. Our data
strongly support the conclusion that the labeled small vesicles were
formed from the ER membranes.
In addition, the phospholipid composition (percentage of total
labeling) of the ER-derived vesicles (Table VIII) showed a very good
correlation with the distribution (percentage) of the phospholipid transferred in vitro to the Golgi apparatus (Sturbois et al., 1994,
Table VIII). For the ER-derived vesicles, we calculated PS:PI ratios of
2.1 (for the amount of lipids) and 2.6 (for the radioactivity of the
lipids), whereas these ratios were only 0.51 and 0.68 for the ER
membranes (Table VIII). These calculations illustrate a PS enrichment
over PI that was observed in the ER-derived vesicles. However, the PS
enrichment over PI was more easily observed for the labeled molecules
than for their total amount (Table VIII). Our data are consistent with
a targeting of PS to the ER-derived vesicles. In addition, we observed
a high amount of VLCFA in PS (Table VI). This result clearly indicates
that there is a preferential accumulation of VLCFA-PS over the bulk PS.
This observation is highly consistent with well-known observations that
plasma membrane PS has a high VLCFA content in many plant tissues
(Murata et al., 1984) and suggests that the sorting of these PS species
starts at the level of ER budding and confirms the vesicular pathway proposed for the VLCFA (Bertho et al., 1991; Moreau and Cassagne, 1994; Moreau et al., 1998a).
In addition, we recently showed that plant sterols are likely to be
transported from the ER to the plasma membrane through the vesicular
pathway with kinetics similar to that of PS transport (Moreau et al.,
1998b). Therefore, it is tempting to speculate that these lipids could
be associated within the same membrane domains, as is the case in
animal cells for glycosphingolipids, cholesterol, and some specific
proteins that are concentrated in structures called "glycolipid
rafts" (Fiedler et al., 1993; Simons and Ikonen, 1997). The cell-free
reconstitution of phospholipid sorting from the ER to ER-derived
vesicles in leek cells suggests that the formation of membrane lipid
domains also exists in plant cells.
A goal and challenge of this study was to perform an in vitro assay,
allowing the formation and subsequently the isolation of ER-derived
vesicles. It will now be possible to analyze the protein content of
these ER-derived vesicles and to investigate the presence of specific
"cargo" proteins and/or proteins of the "vesicular transport
machinery" (Rothman and Wieland, 1996; Scheckman and Orci, 1996). It
will be of great interest to look for proteins of the "snare
family" that are involved in the targeting and fusion of transport
vesicles (Söllner et al., 1993; Kaiser and Ferro-Novick, 1998).
In addition to these perspectives, the cell-free system could also be
used to investigate some of the molecular mechanisms involved in the
budding of the vesicles from the ER membrane. Phosphoinositides (Alb et
al., 1996; De Camilli et al., 1996; Toker, 1998), as well as
phospholipid transfer proteins (Alb et al., 1996; Kearns et al., 1998;
Paul et al., 1998) and phospholipase D (Tüscher et al., 1997;
Siddhanta and Shields, 1998), have been shown to participate in the
regulation of vesicle budding from the Golgi apparatus in animal and
yeast cells. It will be of particular interest to investigate whether
specific lipids (HII-phase-forming lipids,
diacylglycerol, phosphatidic acid, lysophospholipids, or
phosphoinositides) and enzymes of lipid metabolism (synthesizing or
degrading lipids) are active components of the transport process. For
example, a PI 3-kinase was recently suggested to be implicated in the
targeting of plant vacuolar proteins (Welters et al., 1994). Lipid-binding proteins such as annexin are also expected to play critical roles in membrane traffic in plant cells (Clark and Roux, 1995; Carroll et al., 1998).
Although most of the phospholipid transfer proteins described so far
are secreted in plant cells, some can have an intracellular location
(Kader, 1996). We can therefore question whether such proteins are
involved in the regulation of membrane traffic in plant cells.
Recently, a cDNA from Arabidopsis corresponding to Sec 14p was found to
complement the Sec 14 mutant of the yeast Saccharomyces
cerevisiae (Jouannic, 1998). Sec 14p is involved in the regulation
of vesicle budding from the Golgi apparatus in yeast (Alb et al., 1996;
Kearns et al., 1998).
Therefore, future studies are expected to reveal many proteins,
enzymes, and lipids likely to be required in the molecular mechanisms
governing vesicular trafficking in plant cells; few proteins have begun
to be identified (Bar-Peled et al., 1996, 1997; Gomord and Faye, 1996;
Hawes and Satiat-Jeunemaitre, 1996; Robinson et al., 1998).
| |
FOOTNOTES |
*
Corresponding author; e-mail
pmoreau{at}biomemb.u-bordeaux2.fr; fax
33-5-56-51-83-61.
Received December 1, 1998;
accepted January 27, 1999.
1
This work was supported by grants from the
Centre National de la Recherche Scientifique and the University Victor
Segalen Bordeaux 2. B.S.-B. was the recipient of a doctoral fellowship from the Conseil Régional d'Aquitaine, and P.V. was supported by
a doctoral fellowship from the Ministère de l'Education
Nationale de la Recherche et de la Technologie.
| |
ABBREVIATIONS |
Abbreviations:
HPTLC, high-performance TLC.
PC, phosphatidylcholine.
PE, phosphatidylethanolamine.
PI, phosphatidylinositol.
PS, phosphatidylserine.
VLCFA, very-long-chain
fatty acid(s).
X:Y, a fatty acyl group containing X carbon atoms and Y
cis double bonds.
| |
Acknowledgments |
Anti-HDEL (ER marker) and JIM 84 (Golgi marker) antibodies were
kindly provided by Dr. R. Napier (Wellesbourne, UK) and Dr. C. Hawes
(Oxford, UK), respectively. The help given by Dr. M. Miquel (UMR
5544-CNRS) with the GLC analyses is appreciated. We thank Mrs
Breichenmeicher for her help in using the IBAS1 Kontron analyzer, Mrs.
Gué for technical assistance, and Mr. Senon for electron
microscopy photographs. The technical assistance of A.M. Perret with
plant cultures and figure drawings is gratefully acknowledged. We thank
A. Latour-Dantès for her excellent secretarial assistance and J. Pope for critically reading the English text.
| |
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Top
Abstract
Introduction