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Plant Physiol. (1998) 117: 931-937
Transport of Sterols to the Plasma Membrane
of Leek
Seedlings1
Patrick Moreau*,
Marie-Andrée Hartmann,
Anne-Marie Perret,
Bénédicte Sturbois-Balcerzak, and
Claude Cassagne
Laboratoire de Biogenèse Membranaire, UMR 5544 Centre
National de la Recherche Scientifique-Université Victor Segalen
Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cedex, France
(P.M., A.-M.P., B.S.-B., C.C.); Institut de Biologie Moléculaire
des Plantes (UPR Centre National de la Recherche Scientifique no. 406),
Strasbourg, France (M.-A.H.); and Lipid and Lipoprotein Research Group,
University of Alberta, Edmonton, Canada T6G252 (B.S.-B.)
 |
ABSTRACT |
To investigate the intracellular
transport of sterols in etiolated leek (Allium porrum
L.) seedlings, in vivo pulse-chase experiments with
[1-14C]acetate were performed. Then, endoplasmic
reticulum-, Golgi-, and plasma membrane (PM)-enriched fractions were
prepared and analyzed for the radioactivity incorporated into free
sterols. In leek seedlings sterols are present as a mixture in which
(24R)-24-ethylcholest-5-en-3 -ol is by far the major compound (around
60%). The other sterols are represented by cholest-5-en-3-ol,
24-methyl-cholest-5-en-3-ol, (24S)-24-ethylcholesta-5,22E-dien-3-ol, and
stigmasta-5,24(241)Z-dien-3-ol. These compounds are
shown to reside mainly in the PM. Our results clearly indicate that
free sterols are actively transported from the endoplasmic reticulum to
the PM during the first 60 min of chase, with kinetics very similar to
that of phosphatidylserine. Such a transport was found to be decreased
at low temperature (12°C) and following treatment with monensin and
brefeldin A. These data are consistent with a membrane-mediated process
for the intracellular transport of sterols to the PM, which likely involves the Golgi apparatus.
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INTRODUCTION |
Whereas mammalian and fungal cells mainly contain one major
sterol, cholesterol and ergosterol, respectively, higher plant cells
are characterized by a mixture of sterols in which sitosterol, stigmasterol, and 24-methylcholesterol often predominate. Sterol biosynthesis in plants has been extensively studied (Benveniste, 1986 ).
From the conversion of farnesyl diphosphate into squalene and end
products, this pathway represents a sequence of more than 30 enzyme-catalyzed reactions, all associated with membranes. It is now
well established that sterols are synthesized at the level of the ER,
but mainly accumulate in the PM (Hartmann and Benveniste, 1987). Thus,
the neosynthesized sterols must be transferred from the ER to the PM.
In contrast to recent advances in the understanding of intracellular
movement of membrane proteins, relatively little attention has been
paid to the intracellular transport of membrane lipids. Recent studies
have been devoted to the transport of phospholipids in etiolated leek
seedlings (Moreau et al., 1988; Bertho et al., 1991; Sturbois et al.,
1994). It has been shown that the different phospholipid classes do not
follow the same route from the ER to the PM. Moreover, low temperatures
and treatment with monensin have been shown to block the transfer of
only some molecular species (Bertho et al., 1991; Moreau and Cassagne,
1994; Sturbois-Balcerzak et al., 1995). As no information on the
mechanisms involved in the delivery of sterol molecules to the PM is so
far available, we have taken the advantage of this plant system to
investigate the intracellular transport of sterols in higher plant
cells. In vivo pulse-chase experiments with
[1-14C]acetate clearly indicate that the
sterols synthesized in the ER membranes are transferred to the PM with
kinetics similar to that of PS. The effects of low temperature and
treatment with monensin and brefeldin A on the transport of sterols
were also investigated.
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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 distillated water for 2 h. The seeds
were allowed to germinate in the dark for 7 d at 22 to 24°C, as
described previously (Moreau et al., 1988).
Chemicals
All chemicals were purchased from Sigma.
[1-14C]Acetate was obtained from CEA (Saclay,
France).
Pulse-Chase Experiments
For each experimental value, 10 batches of 20 seedlings (cut into
5- to 10-mm segments, including roots) were first incubated in 0.2 mL
of 3.5 × 105 Bq of
[1-14C]acetate (2 × 1012 Bq mol 1) for 120 min
at 24°C or 12°C, in the presence or absence of monensin (5 µM) or brefeldin A (100 or 500 µM). Chase
was made with 0.5 mL of 0.2 M unlabeled acetate for periods
of time ranging from 30 to 120 min.
Isolation of ER, Golgi, and PM Fractions
Leek seedlings were homogenized in a buffer consisting 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 submitted to differential centrifugations at
1,000g for 10 min, 10,000g for 10 min, and
150,000g for 60 min. The resulting microsomal pellet was
resuspended in 10 mM
KH2PO4 and 0.5 M sorbitol. One-half of the suspension was 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
centrifugation at 80,000g for 150 min, membranes at the
18/25% (ER fraction) and 25/37% (Golgi fraction) Suc interface were
collected, diluted with 30 mM Hepes-KCl, pH 6.8, and
centrifuged at 100,000g for 60 min.
PMs were isolated by phase partitioning 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 1000g 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 30 mM
Hepes-KCl, pH 6.8.
Specific membrane compartments were identified by assays for the
following markers: ER, NADPH-Cyt c reductase and CDP-choline phosphotransferase; Golgi apparatus, IDPase; and PM, glucan synthetase II (Moreau et al., 1988; Bertho et al., 1991). Setting the specific activities of the marker enzymes at 1 in the homogenate, we obtained the following relative enrichments in the membrane fractions: ER
fraction, 8.5 for NADPH-Cyt c reductase, 3.5 for CDP-choline phosphotransferase, 2.5 for IDPase, and 0.1 for glucan synthetase II;
Golgi fraction, 2.4 for NADPH-Cyt c reductase, 0.5 for
CDP-choline phosphotransferase, 9.5 for IDPase, and 0.6 for glucan
synthetase II; PM fraction, 0.1 for NADPH-Cyt c reductase,
0.15 for CDP-choline phosphotransferase, 0.35 for IDPase, and 4.4 for
glucan synthetase II. The specific activity of succinodeshydrogenase (a
mitochondrial marker) was < 0.1 in the microsomes compared with
the homogenate.
A low contamination of ER and Golgi fractions by plastid envelope
membranes was determined by the presence of small amounts of
galactolipids (< 10% of the total glycerolipids). Protein
concentrations were determined by the method of Bradford (1976) using
BSA as standard.
Lipid Analyses
Lipids were extracted by chloroform:methanol (1:1, v/v) for 30 min
at room temperature, and then washed three times with distilled water.
The solvent was evaporated and lipids were resuspended in an
appropriate volume of chloroform:methanol (1:1, v/v) according to
procedures already described (Moreau et al., 1988; Bertho et al.,
1991).
PS isolation was carried out on HPTLC plates (60F254, Merck,
Darmstadt, Germany) developed with
methylacetate:n-propanol:chloroform:methanol:aqueous 0.25%
(w/v) KCl (25:25:25:10:9, v/v) according to the method of Heape et al.
(1985). Neutral lipids were isolated onto HPTLC plates developed with
hexane:chloroform:methanol (100:60:10, v/v) to separate into sterols
(RF 0.54), fatty alcohols
(RF 0.64), and free fatty acids
(RF 1); with hexane:ethylether:acetic acid
(90:15:2, v/v) to give diacylglycerols (RF 0.08),
4-demethylsterols (RF 0.17), fatty alcohols
(RF 0.22), and free fatty acids
(RF 0.29).
After identification by comparison with standards, the different lipids
were scraped off directly into vials and their radioactivity was
determined by liquid scintillation counting (model 2000CA counter,
Packard Instruments, Meriden, CT). Radioactivity of the lipids was also
determined after autoradiography of the HPTLC plates (Hyperfilm MP-RPN
1675, Amersham) and scanning with a densitometer (model 76510, Camag,
Muttenz, Switzerland). Both methods gave similar results and were
alternately used.
Sterols were identified and quantified as previously reported (Hartmann
and Benveniste, 1987). After extraction from membrane fractions with
hexane, sterols were subjected to TLC with dichloromethane as the
developing solvent for two runs. The 4-demethylsterols (end products)
were eluted and acetylated before being analyzed by GC on a glass
capillary column (30 m long, 0.25-mm i.d., coated with DB-1). The
temperature program used includes a fast rise from 60°C to 230°C
(30°C/min), then a slow rise from 230°C to 280°C (2°C/min). A
cholesterol standard (not acetylated) was added to the samples prior to
analysis. Sterol identification was made by GC-MS (Rahier and
Benveniste, 1989).
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RESULTS |
Free Sterol Composition of Membrane Fractions
ER, Golgi, and PM fractions were prepared from 7-d-old etiolated
leek seedlings and characterized by enzymatic markers (Moreau et al.,
1988; Bertho et al., 1991) and labeling by anti-HDEL (Napier et al.,
1992) and JIM 84 (Horsley et al., 1993) antibodies for ER and Golgi
fractions, respectively (B. Sturbois-Balcerzak, L. Maneta-Peyret, M. Duvert, B. Satiat-Jeunemaitre, P. Vincent, C. Cassagne, and P. Moreau,
unpublished data). These fractions were analyzed for their free
4-demethylsterol content. Data are shown in Table
I. In leek seedlings, like in other plant
tissue, the PM was found to be the richest membrane in free sterols
(expressed in micrograms per milligram of protein). A few sterol
molecules were present in the ER. The Golgi fraction contained an
intermediate concentration of free sterols. PM was also
characterized by the highest sterol-to-phospholipid molar ratio.
In all of the fractions, sterols are present as a mixture in which
sitosterol is largely predominent (62%-70%). The other sterols are
represented by 24-methylcholesterol, stigmasterol, cholesterol, and
isofucosterol. Such a sterol composition and the relatively high
content of cholesterol (10%) are in agreement with published data
concerning other plants belonging to Liliaceae (Itoh et al., 1977).
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Table I.
Sterol composition of membrane fractions isolated
from 7-d-old etiolated leek seedlings
The values are from two independent lipid analyses.
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Transport Kinetics of Sterols to the PM
Leek seedlings were first incubated with
[1-14C]acetate for 120 min, then with unlabeled
acetate for 30 to 120 min (Figs. 1-3). Membrane fractions were prepared and analyzed for their radioactivity incorporated into free sterols. Figure 1 shows the comparative evolution of the radioactivity incorporated into sterols as a function
of chase time in microsomes and purified PM. A significant increase in
the sterol labeling of PM was observed whatever the chase time, whereas
the amount of radioactivity associated with sterols of microsomes
remained stable or decreased (after 120 min), indicating that the
increase in the sterol labeling of PM was likely due to a delivery of
newly synthesized sterols (made during the pulse period). To determine
the origin of labeled sterols in the PM, we analyzed the sterol
labeling of ER, Golgi, and PM fractions from leek seedlings after
similar pulse-chase experiments. Results are presented in Figure
2. At time 0, i.e. at the end of the
pulse, the radioactivity associated with sterols of ER was about 2-fold
higher than that present in the Golgi and PM fractions, as would be
expected for the involvement of ER in the synthesis of free sterols
(Hartmann and Benveniste, 1987). During the chase, an increase in the
sterol labeling of PM was correlated with a decrease in the
radioactivity associated with sterols of ER. The Golgi fraction
appeared to have an intermediate behavior, as the sterol labeling
increased after 30 and 60 min of chase and decreased after 120 min.
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| Figure 1.
Sterol labeling in the microsomes ( ) and the PM
( ) as a function of chase time. Leek seedlings were first
incubated with [14C]- acetate for 120 min, then
with unlabeled acetate for the indicated periods of time. Microsomes
and PM were prepared and the radioactivity associated with sterols
determined as explained in ``Materials and Methods''. Values are expressed as percentages of the radioactivity (dpm mg1
protein) incorporated during the 120-min labeling period
(n = 7), and correspond to an average of 4 (30- and
60-min chase) and 7 (120-min chase) determinations (±SD).
Sterol labeling in the microsomes and PM fraction at the end of the
pulse (i.e. 0-min chase) was 65,000 and 25,000 dpm mg1
protein, respectively.
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| Figure 2.
Sterol labeling in the ER (), Golgi ( ), and
PM () as a function of chase time. Pulse-chase procedures and
measurements of sterol labeling were as in Figure 1. PM/ER ratios
increase from 0.54 ± 0.06 (120-min pulse) to 5.07 ± 0.45 (120-min chase). The values are from three independent experiments
(±SD).
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The kinetics of transfer of sterols from the ER to the PM were compared
with that of PS, a phospholipid previously shown to be transported to
the PM in a vesicular pathway (Sturbois-Balcerzak et al., 1995). As
shown in Figure 3, the kinetics of
labeling of sterols and PS, as expressed as the ratio of
radioactivities associated with the PM to those associated with the ER,
were quite similar, suggesting closely related mechanisms of delivery
to the cell surface for these two classes of molecules. As a control, the evolution of the labeling of other lipid classes such as
diacylglycerols and free fatty acids was checked. No variation in their
radioactivities was observed during the chase, indicating that these
lipids were not transported.
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| Figure 3.
PM/ER ratios of sterol and PS labeling (dpm
mg1 protein) as a function of chase time. Pulse-chase
procedures and measurements of lipid labeling were as in Figure 1. The
data concerning sterols were taken from Figure 2 and compared with
those obtained for PS, which is considered as a marker for
membrane-mediated processes (Sturbois-Balcerzak et al., 1995), with
free fatty acids and diacylglycerol as negative controls. For the sake
of clarity, the SDS from three experiments with values
between 7% and 13% were omitted. Sterol labeling in the ER and PM
fractions at the end of the pulse (i.e. 0-min chase) was 50,000 and
27,000 dpm mg1 protein, respectively. , Sterol;  ,
PS; , diacylglycerol plus free fatty acids.
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Effect of Low Temperature on Sterol Transport
Leek seedlings were incubated with
[14C]acetate for 120 min at 24°C or at
12°C, a temperature that was found to block the transport of some
phospholipids (particularly PS and PE) to the PM (Sturbois-Balcerzak et
al., 1995). ER, Golgi, and PM fractions were then prepared and their
sterol and PS (used as a reporter for the vesicular transport) labeling
was determined. To focus only on the effect of low temperature on the
distribution of labeled lipids between the various cellular membranes
independently of the effect of low temperature on their synthesis, we
have calculated for each lipid L (PS or sterols) of each
membrane fraction X (ER, Golgi, or PM) the following ratio:
[L (X) 12°C/L (X)
24°C] × [L (µ) 24°C/L (µ) 12°C]
(see legend of Table II). Two situations
can be observed: (a) a value close to 1 for the lipid of the PM means
that there is no apparent temperature block, and therefore no great
change in the ratio of this lipid in the intracellular membrane
fractions is expected; and (b) a value less than 1 for the lipid of the PM indicates an effect of low temperature on the transport of this
lipid to the PM. The lipid not transferred would be expected to
accumulate intracellulary, and thus the ratio of this lipid will be
greater than 1 in one or both intracellular membrane fractions (Table
II).
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Table II.
Effect of low temperature (12°C) on the delivery
of free sterols and PS to the PM
Leek seedlings were labeled at 24°C or 12°C with
[14C]acetate for 120 min. ER, Golgi, and PM were isolated
and the radioactivity of free sterols and PS was determined.
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The value obtained for free sterols was  1 for the PM, suggesting
that sterols were less efficiently delivered to the PM at 12°C.
Moreover, a sterol accumulation was observed in the ER and Golgi
fractions (ratios > 1). In agreement with previous results (Sturbois-Balcerzak et al., 1995), the value for PS in the PM was also
1 and confirmed the arrest of the transfer of this phospholipid
to the PM at 12°C. PS was shown to accumulate in the ER and Golgi
fractions, since the values of ratios were found to be > 1 in
both fractions.
These results strongly suggest that the transport of free sterols to
the PM was either slowed down or partly blocked at 12°C in a way
similar to that observed for PS transfer. Their accumulation in the
intracellular membranes could be explained by the decrease in the
number of secretory vesicles and the increase in the surface area of
the trans-Golgi/trans-Golgi network that was
morphologically observed at 12°C (Sturbois-Balcerzak et al., 1995).
Effect of Monensin and Brefeldin A on Sterol Transport
Monensin has been shown to disturb the secretory function of the
Golgi apparatus (Mollenhauer et al., 1990) and was previously used to
show the intermediate position of this organelle in the delivery of
some phospholipids (including PS) to the PM (Bertho et al., 1991;
Sturbois-Balcerzak et al., 1995). The monensin concentration (5 µM) used led to a 25% inhibition of sterol synthesis in
the microsomes (Fig. 4). Under these
conditions, the amount of the radioactivity associated with sterols of
the PM was decreased by 75% (Fig. 4), suggesting that the delivery of
these molecules to the PM was affected.
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| Figure 4.
Effect of monensin and brefeldin A on sterol
delivery to the PM. Leek seedlings were incubated with
[14C]acetate for 120 min in the absence or presence of 5 µM monensin or 100 µM or 500 µM brefeldin A. Microsomes and PM were then prepared and
the radioactivity (dpm mg1 protein) incorporated into
sterols was determined. The ratios of treated to untreated were then
calculated for each membrane fraction. The data are from three
independent experiments and the results are expressed as arbitrary
units, with the control values equal to 1. M, Microsomes. Sterol
labeling in the crude microsomes and PM in the absence of drug were
56,000 and 31,000 dpm mg1 protein, respectively.
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Brefeldin A, a fungus-derived cyclic lactone, has been largely used to
dissect membrane-trafficking events in mammalian cells (Klausner et
al., 1992) and has only recently been implemented in plant cells
(Satiat-Jeunemaître et al., 1996). Following treatment of leek
seedlings with 100 and 500 µM of brefeldin A, the sterol synthesis in the microsomes was found to be inhibited by 37% and 62%,
respectively. Under these conditions, the decrease in the sterol
labeling of the PM reached 73% and 88%, respectively (Fig. 4),
indicating that in addition to an effect on sterol synthesis, brefeldin
A also inhibited the delivery of sterols to the PM.
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DISCUSSION |
In etiolated leek seedlings, free sterols are mainly concentrated
in the PM (Table I), as they are in other plant tissues (Hartmann and
Benveniste, 1987). A few sterol molecules are present in the ER
membranes and an intermediate amount of sterol was found in the Golgi
fraction. In all of these membrane fractions, sterols are present as a
mixture, with sitosterol as the major compound, suggesting that free
sterols are transported together to the PM. In vivo pulse-chase
experiments with [1-14C]acetate clearly
indicated that sterols are actively transferred from the ER to
the PM during the first 60 min of chase. These results are
totally in agreement with previous data obtained with maize
coleoptiles after in vivo labeling with
[5-14C]mevalonic acid (Hartmann, 1980). Such a
study indicated that a few molecules of biosynthetic precursors were
also transported to the PM. In contrast, steryl esters were found not
to be transferred. In leek seedlings kinetics of intracellular
transport of free sterols from the ER to the PM were shown to be
similar to that of PS. The transport of these two classes of lipids was
found to be decreased by low temperature and treatment with monensin and brefeldin A (Table II; Fig. 4), suggesting similar mechanisms of
delivery to the PM.
Which mechanism(s) might be responsible for the movement of free
sterols to the PM: a simple diffusion through the cytoplasm, a
protein-mediated transport, or a vesicular transfer? It is generally accepted that a spontaneous diffusion through the aqueous medium is a
slow phenomenon and therefore not a significant route for sterol
transport, so an activation-collision mechanism was suggested (Steck et
al., 1988). However, a significant sterol exchange through the aqueous
phase was recently observed in fibroblasts (Frolov et al., 1996). This
spontaneous exchange was faster from PM to intracellular membranes than
in the reverse direction (Frolov et al., 1996). These results are
therefore not in favor of a quantitative transport of sterols to the PM
by simple diffusion.
Specific carrier proteins could enhance the diffusion through the
aqueous phase. Frolov et al. (1996) have observed that sterol exchange
between biological membranes is highly enhanced by the sterol carrier
protein SCP2. In good agreement with these in vitro studies, Puglielli
et al. (1995) have shown a requirement for SCP2 in sterol transport
from the ER to the PM of cultured fibroblasts. Their results pointed to
a predominant SCP2-mediated transport of cholesterol in normal
fibroblasts but also to the occurrence of a membrane-mediated transport
revealed in the SCP2-deficient fibroblasts. Thus, a protein-stimulated
as well as a membrane-mediated transport of sterols has to be
considered. There is no indication in the literature of the existence
of specialized proteins in plant cells that are able to transfer
sterols (Kader, 1996). Although the occurrence of specific
sterol-carrier proteins in plants cannot be ruled out, a much more
likely way to transfer sterols is via a membrane-mediated process.
Our data are compatible with such a process for several reasons. First,
we estimated the t1/2 of sterol transfer to
the PM to be about 30 min (Fig. 2), which is of the same order of
magnitude as those obtained for phospholipids, which are known to
follow a vesicular pathway (Bertho et al., 1991; Sturbois et al.,
1994), and for proteins (Mitsui et al., 1985; Kappler et al., 1986). Moreover, the transport of sterols was similar to that of PS (Fig. 3),
which has been shown to be membrane mediated (Sturbois et al., 1994;
Sturbois-Balcerzak et al., 1995).
In addition, we have observed that low temperature (12°C) partly
blocked the delivery of sterols to the PM and resulted in their
intracellular accumulation (as was also the case with PS; Table II).
Similar results have been obtained in animal cells treated at 15°C
(Kaplan and Simoni, 1985), and the existence of cholesterol-rich
intracellular membranes potentially involved in cholesterol transport
has been demonstrated (Kaplan and Simoni, 1985; Lange and Steck, 1985).
Sterol-rich lipid particles have also been suggested as possible
structures for the transport of sterols from internal membranes to the
PM of yeast (Zinser et al., 1993).
Other arguments favoring a membrane-mediated process come from
experiments carried out with monensin and brefeldin A (Fig. 4).
Although the effects of monensin on the plant secretory system are
controversial (Sticher and Jones, 1988; Zhang et al., 1993; Satiat-Jeunemaître et al., 1994), it has been shown that in our system monensin led to a transport block of some phospholipid species
and to their accumulation in internal membranes, including a
Golgi-enriched fraction (Bertho et al., 1991; Sturbois-Balcerzak et
al., 1995). We found that at a monensin concentration that only
slightly affected the de novo synthesis of sterols, their delivery to
the PM was dramatically inhibited (Fig. 4). Therefore, it is possible
that monensin induces an accumulation of sterol molecules in an
intracellular compartment resembling the Golgi-derived swollen vesicles
described by Zhang et al. (1996).
The other drug used in these experiments is brefeldin A, and its effect
on the Golgi apparatus in plant cells has been extensively described
(Satiat-Jeunemaître et al., 1996). Contrary to monensin, the
effect of brefeldin A on plant cells has been widely reproduced (Satiat-Jeunemaître et al., 1996) with only few exceptions
(Robinson, 1993). The results with brefeldin A are similar to those
obtained with monensin (compare 5 µM monensin and 100 µM brefeldin A, Fig. 4). As a consequence, it is likely
that a disturbance of the Golgi apparatus was at least to some extent
at the origin of the inhibition of sterol transport to the PM.
These data clearly differ from those obtained with Chinese hamster
ovary cells, in which none of these drugs affected the intracellular
transport of cholesterol (Kaplan and Simoni, 1985; Urbani and Simoni,
1990). In this case, the delivery of newly synthesized cholesterol to
the PM would be mediated by lipid-rich vesicles not related to the
Golgi apparatus (Urbani and Simoni, 1990; Liscum and Underwood, 1995).
The potential occurrence of a direct ER-to-PM pathway for intracellular
transport of sterols in leek seedlings also has to be considered
(Kristen et al., 1987; Sturbois-Balcerzak et al., 1995).
In conclusion, the present data clearly favor a membrane-mediated
process for the transport of free sterols from the ER to the PM in leek
seedlings. Free sterols are known to play a key role in regulating the
physical properties of the PM as well as the activity of some
membrane-bound enzymes such as H+-ATPase.
Consequently, levels of these molecules within the PM have to be
tightly regulated. Mechanisms contributing to homeostasis of sterols in
higher plant cells remain to be elucidated, but certainly differ
fundamentally from those operating in mammalian cells. One argument is
the absence of the classic low-density lipoprotein pathway in plants.
It now appears crucial to isolate membrane vesicles participating in
lipid transport from plant tissues. Such experiments are currently
being performed in our laboratory and are expected to determine whether
free sterols and PS molecules are transported together in the same
vesicles, and to shed more light on sterol trafficking in plant cells.
| |
FOOTNOTES |
1
This work was supported by the Centre National
de la Recherche Scientifique and the University Victor Segalen Bordeaux
2.
*
Corresponding author; e-mail pmoreau{at}biomemb.u-bordeaux2.fr; fax
33-05-56-51-83-61.
Received January 15, 1998;
accepted March 31, 1998.
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ABBREVIATIONS |
Abbreviations:
cholesterol, cholest-5-en-3-ol.
HPTLC, high-performance TLC.
isofucosterol, stigmasta-5,24(241)Z-dien-3-ol.
PM, plasma membrane.
PS, phosphatidylserine.
sitosterol, (24R)-24-ethylcholest-5-en-3-ol.
stigmasterol, (24S)-24-ethylcholesta-5,22E-dien-3-ol.
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ACKNOWLEDGMENT |
We thank Mr. John F. Ackerson for critically reading the English
text.
| |
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Abstract
Introduction