From the Department of Membrane Cell Biology, Faculty of Medical Sciences, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands
Received for publication, August 13, 2002, and in revised form, October 15, 2002
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ABSTRACT |
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In HepG2 cells, the subapical compartment (SAC)
is involved in the biogenesis of membrane polarity. By contrast, direct
apical transport originating from the trans-Golgi network (TGN), which may contribute to polarity establishment, has been poorly defined in
these cells. Thus, although newly synthesized sphingolipids can be
directly transported from the TGN to the apical membrane, numerous
apical resident proteins are traveling via the transcytotic route.
Here, we developed an in vitro transport assay and compared the molecular sorting of 6-[N-(7-nitrobenz-2-oxa-1,3
diazol-4-yl)amino] hexanoyl-sphingomyelin (C6NBD-SM) and
C6NBD-glucosylceramide (C6NBD-GlcCer) in TGN
and SAC. SM is released from both TGN and SAC in the lumenal leaflet of
transport vesicles. This holds also for GlcCer released from the SAC
but not for a substantial fraction that departed from the Golgi.
Distinct transport vesicles, enriched in either SM or GlcCer are
released from SAC, consistent with their rigid sorting in this
compartment. Different vesicle populations could not be recovered from
TGN, although in situ experiments reveal that GlcCer is
preferentially transported to the apical membrane, reflecting different
transport mechanisms. The results indicate that in HepG2 cells
sphingolipids are mainly sorted in the SAC membrane and that the
release of SM from SAC and TGN is differentially regulated.
Polarized cells have distinct plasma membrane domains,
i.e. the apical and basolateral membranes each displaying a
specific protein and lipid composition. These differences are generated and maintained, despite a continuous membrane flux between these domains and intracellular organelles, sorting and vesicular transport being instrumental in these events (1).
Two major pathways have been implicated in polarized sorting. De
novo synthesized proteins and lipids are sorted to either plasma
membrane in the trans-Golgi network
(TGN)1 (2, 3). In addition,
molecular redistribution can also be mediated by the endosomal sorting
machinery (4). Thus, proteins and lipids internalized from the
basolateral and apical membrane meet in an apically localized common
endosome (5), defined as the subapical compartment (SAC) in HepG2 cells
(6). Here, molecules are sorted and either recycled to the original
membrane or transcytosed to the opposite membrane domain (7), thereby securing and maintaining a polarized distribution. The existence of two
such pathways raise intriguing questions concerning the relative
contributions of SAC and TGN to polarity generation and maintenance and
the molecular nature of the sorting machinery involved in either pathway.
Detergent-insoluble membrane domains, called rafts (8), have been
implicated in sorting (9), although raft formation per se is
probably not sufficient for apical targeting (10). (Glyco)sphingolipids, together with cholesterol, are the main lipid constituents of rafts and the finding that inhibition of sphingolipid synthesis, but not cholesterol depletion, interferes with
the apical sorting of detergent-insoluble GPI-anchored proteins in
Fischer rat thyroid cells (11) indicates that sphingolipids are
especially important for the sorting of apical cargo. In many epithelial cells newly synthesized proteins are sorted in the TGN and
directly transported to either the basolateral or apical membrane (3).
In contrast, hepatocytes transport most (12, 13), but not all (14, 15)
newly synthesized apical proteins first to the basolateral membrane,
prior to reaching the apical (bile canalicular (BC)) membrane via
transcytosis. Yet, in these cells, newly synthesized analogs of SM and
GlcCer can be transported directly from the TGN to the BC membrane
(16), although it has not been determined whether, similar to MDCK and
Caco-2 cells (17, 18), sphingolipids are actually sorted in the TGN. In the latter studies, it was shown that de novo synthesized
C6NBD-labeled sphingolipid analogs can be sorted in the
TGN, directing C6NBD-GlcCer preferentially to the apical
membrane, whereas SM distributes about equally over apical and
basolateral domains.
In MDCK cells, it has been demonstrated that the common recycling
endosome is enriched in raft lipids (19). This could imply that by
analogy the formation of sphingolipid-enriched membrane domains may be
important for polarized sorting in the SAC as well. In this context, we
have recently demonstrated that the SAC is intimately involved in
regulating the distribution of both of these lipids during transcytotic
transport (20, 21). At steady state, SM is transported predominantly
from the SAC to the basolateral membrane, whereas GlcCer is recycling
between the SAC and the BC membrane (20). Circumstantial evidence
suggests that the exiting of both lipid analogs from SAC is
vesicle-mediated (20). However, de novo synthesized GlcCer
analogs (22, 23) as well as the natural counterpart (24) may at least
in part be transported as monomers, likely from an early Golgi
compartment, where its synthesis occurs at the cytosolic surface (25,
26). Following arrival at the inner leaflet of the plasma membrane,
transmembrane translocation of the GlcCer analogs has been shown to
occur via ABC transporter proteins (23, 27).
To understand the mechanisms that govern these various sorting
processes, the regulation of sphingolipid transport in the biosynthetic
and endocytotic pathways, and how vesicular transport derived from SAC
and TGN contribute to the biogenesis and maintenance of polarity, the
isolation of the putative transport vesicles and the reconstitution of
vesicular transport of both proteins and lipids in permeabilized cells
are desirable. Thus far, most studies focusing on these issues have
been restricted to the analysis of vesicular protein transport
(28-30), whereas only a few in vitro studies have been
developed to characterize the vesicular transport of lipids (31, 32).
Here, we developed an in vitro assay using permeabilized
HepG2 cells to study the exit of fluorescently tagged SM and GlcCer
from the SAC and TGN. The purpose was to reveal the occurrence and
nature of sorting in either compartment, thereby improving our
knowledge of where and how membrane domains enriched in sphingolipids
may contribute to the sorting of apical and basolateral proteins. We
demonstrate that both sphingolipids can exit SAC and TGN in transport
vesicles. However, whereas SAC-derived trafficking is exclusively
vesicle-mediated, a fraction of the newly synthesized GlcCer exits from
Golgi by a mechanism that does not involve its packaging into the
lumenal side of transport vesicles. Moreover, evidence is presented
that SM and GlcCer are released in distinct vesicles from the SAC,
highlighting the prominent role of this compartment as a sorting
station in the transcytotic pathway.
Materials--
Dulbecco's modified Eagle's medium and
Hanks' balanced salt solution were from Invitrogen, and
trifluoperazine (TFP) was from Calbiochem (La Jolla, CA).
C6NBD was obtained from Molecular Probes (Eugene, OR), and
cyclosporin A (CSA) was from Alexis (Läufelfingen, Switzerland).
High performance-TLC plates and sodium dithionite were purchased
from Merck, and PDMP was from Matreya (Pleasant Gap, PA). BSA (fraction
V), Dowex 1X8-200, rhodamine 123, D-sphingosine, 1 Cell Culture--
HepG2 cells were grown in 25-cm2
cell culture flasks (Costar) in Dulbecco's modified Eagle's medium
containing 4500 mg of glucose/liter, supplemented with 10% fetal calf
serum and 2 mM L-glutamine. For the
experiments, the cells were used 3 days after plating when they were
maximally polarized (33).
Synthesis of C6NBD-labeled
Sphingolipids--
C6NBD-ceramide,
C6NBD-GlcCer, and C6NBD-SM were synthesized
from C6NBD and D-sphingosine,
1 Labeling of HepG2 Cells with
C6NBD-Sphingolipids--
To analyze sphingolipid transport
of intact HepG2 cells, they were labeled with 4 µM of
C6NBD-sphingolipids. For in vitro studies 8 µM of sphingolipids were used. To label the TGN, the cells were incubated with C6NBD-ceramide in Hanks'
balanced salt solution for 90 min at 18 °C. Noninternalized ceramide
was removed from the plasma membrane by back-exchange with 5% BSA at
4 °C in Hanks' balanced salt solution (2 × 30 min). The cells
were incubated for an additional 90 min at 18 °C in presence of 5% BSA. To label the SAC, the cells were incubated with
C6NBD-SM and -GlcCer in Hanks' balanced salt solution for
30 min at 37 °C. Noninternalized sphingolipids were removed by
back-exchange, and the sphingolipids were chased into the SAC for
2 h at 18 °C in presence of BSA. Sphingolipids that had been
transported to the BC membrane were quenched for 10 min with 30 mM sodium dithionite at 4 °C. At these conditions, the
agent does not permeate the membrane and exclusively abolishes NBD
fluorescence that is localized in the external leaflet of the BC
(20).
Transport of Sphingolipids in Intact Cells--
To study
basolateral transport after labeling of the TGN, HepG2 cells were
incubated for various time intervals at 37 °C in presence of 5%
BSA, acting as a scavenger for basolaterally arrived lipid. The
incubation medium and the cells were collected separately, and the
lipids of both fractions were extracted and analyzed by TLC as
described previously (34). To study apical transport the cells were
grown on coverslips, and the TGN was labeled as described above. The
cells were incubated for various time intervals at 37 °C in
back-exchange medium. Transport to the BC membrane was analyzed by
fluorescence microscopy. BC structures were identified in phase
contrast and characterized as C6NBD-positive or -negative after switching to epifluorescence, as has been described in detail elsewhere (33). Images were scanned and processed with Paint Shop Pro.
In Vitro Budding Assay--
After loading either TGN or SAC of
HepG2 cells with C6NBD-sphingolipids, the cells were
scraped from the culture dish into 1 ml of transport buffer (140 mM KCl, 0.5 mM KH2PO4,
20 mM Hepes-KOH, pH 7.3). The cells were permeabilized by
pressing them four times through a 25-gauge syringe, and cell debris
was removed by a brief centrifugation step (10 min, 2000 × g). The permeabilized cells were treated for 30 min at
4 °C with 5% BSA, washed once in transport buffer, and incubated at
37 °C in the presence of cytosol (2 mg/ml), ATP, an ATP-regenerating
system, and 1% BSA. After the incubation, the cells were removed by
centrifugation at 2000 × g, and the supernatant was
centrifuged for 2 h at 100,000 × g to recover membrane-associated sphingolipids, which were protected from BSA. The
lipids of all fractions were extracted and analyzed by TLC. To
calculate the release of C6NBD-sphingolipids in the lumenal leaflet of vesicles, the amount recovered in the 100,000 × g pellet was correlated to the total amount of this
sphingolipid prior to the incubation (2000 × g pellet + 100,000 × g supernatant + 100,000 × g pellet). The amount of sphingolipids that became
accessible to BSA (100,000 × g supernatant) was
determined similarly.
Fractionation of Vesicles by Gradient Centrifugation--
The
vesicular supernatant obtained from the in vitro budding
assay was immediately loaded onto a continuous gradient of 20-50% sucrose (w/w) in transport buffer. The gradient was centrifuged for
16 h at 100,000 × g in an SW50 rotor (Beckman)
and fractionated. Immediately before lipid extraction the obtained
gradient fractions were treated for 5 min with 30 mM sodium
dithionite at 4 °C to quench any NBD fluorescence associated with
sphingolipids that were extracted by BSA and/or exposed in the
outer leaflet of the membranes.
Preparation of Cytosol--
HepG2 cells were grown for 3 days on
165-cm2 dishes, washed and scraped into 0.5 ml of transport
buffer. The cells of 5-10 dishes were collected and homogenized with a
tight Dounce homogenizer. The homogenate was centrifuged for 10 min at
1000 × g to remove cell debris and nuclei, and the
post nuclear supernatant was centrifuged two times at 100,000 × g. The final supernatant was used in the in vitro experiments.
Transmission Electron Microscopy--
Gradient fractions
containing vesicles enriched in C6NBD-sphingolipids were
collected and adjusted with transport buffer to a sucrose concentration
of 10% (w/w). The vesicles were pelleted for 3 h at 100,000 × g. After fixation for 15 min with 0.5% glutaraldehyde in
transport buffer containing 10% sucrose, the pellets were preincubated for 15 min in the dark with 0.15% (w/w) diaminobenzidine (DAB). For
photoconversion of DAB by the NBD-labeled sphingolipids, the pellets
were illuminated for 30 min at a wavelength of 460 nm (for experimental
details see Ref. 36). Thereafter the pellets were incubated for 60 min
at room temperature with 1% OsO4 in cacodylate buffer (pH
7.4) and embedded in Epon. After sectioning, the structures containing
osmiophilic precipitates were analyzed using a Philips CM100 electron
microscope at 80 kV. The micrographs were scanned and processed with
Paint Shop Pro.
Determination of Galactosyltransferase Activity--
To assay
for galactosyltransferase (GTase) activity, the samples were incubated
for 1 h at 37 °C in 50 µl of a mixture containing (final
concentrations) 100 mM cacodylic acid (pH 7.3), 20 mM N-acetylglucosamine, 20 mM
MnCl2, 4 mM ATP, 0.5% Triton X-100, and 0.7 mM UDP-galactose (including 100,000 dpm
[14C]UDP-galactose). The incubation was stopped by the
addition of 450 µl of cold H2O, and
[14C]N-acetyllactosamine was separated from
[14C]UDP-galactose by a Dowex 1X8-200 column. The values
obtained in the presence of N-acetylglucosamine were
corrected for control values (containing H2O), and the
specific activity (dpm/µg protein) was determined. Finally the amount
of GTase in each sample was determined by multiplying the specific
activity with the protein content.
Both Golgi-derived transport of de novo synthesized
sphingolipids and SAC-derived glycosphingolipid transport contribute to the biogenesis and maintenance of polarized membrane domains in HepG2
cells. Although direct sphingolipid transport from Golgi to the BC
membrane can occur in liver cells (16), the mechanism of transport has
not been determined, and it is also unknown whether sorting of the
lipid species occurs as observed in other epithelial cells (17, 18).
Accordingly, we first determined the mechanism by which newly
synthesized GlcCer and SM reach the basolateral and apical
membrane in optimally polarized HepG2 cells. This flux was then
compared with the transport of both these lipids from the SAC, involved
in membrane dynamics related to endocytotic events at either membrane
surface, to appreciate the relative contribution of either pathway to
membrane polarity development in liver cells.
Direct Transport of Newly Synthesized C6NBD-SM and
-GlcCer from TGN to BC Membrane--
To analyze the exit of newly
synthesized sphingolipids from the TGN in HepG2 cells, the cells were
labeled for 90 min with C6NBD-ceramide at 18 °C. After a
wash, the sphingolipids were chased for an additional 90 min at
18 °C in the presence of 5% BSA. The latter will preclude the
re-entry of basolaterally arrived lipids into the cell. After the
chase, the perinuclear region of the cells was brightly labeled (Fig.
1, A and B), which
is typical for a Golgi localization in these cells (16). 50% of the
ceramide was metabolized to C6NBD-SM and -GlcCer (in a
molar ratio of 3:1, Fig. 1C), the only products that could
be detected. The accumulation of the newly synthesized lipids in the
Golgi is reflected by the observation that only approximately 10% of both sphingolipids were transported from the Golgi, presumably exiting
from the TGN, to the basolateral plasma membrane during the incubation
at 18 °C (Fig. 1D).
During the incubation at 18 °C, no significant labeling of the BC
could be detected (Fig. 1B), implying that sphingolipid transport from Golgi to BC was impeded, whereas evidently
C6NBD-Cer did not acquire access to the BC. However, when
the temperature was subsequently raised to 37 °C in the presence of
5% BSA, 50-60% of the BCs were C6NBD-positive after a
30-min incubation period (Fig. 2,
A-C). Treatment with sodium dithionite after the incubation reduced the amount of labeled BCs to control levels (Fig.
2C), providing evidence that the C6NBD-labeled
sphingolipids were transported directly to the exoplasmic
leaflet of the BC membrane. After longer incubation times (30-60 min)
the amount of positive BCs decreased again, indicating that both
sphingolipids are transcytosed from the apical to the basolateral
membrane (33), where they are captured by BSA (Fig. 2C).
Because the BC membrane is not accessible to BSA, the direct
quantification of the apically transported sphingolipid analogs is not
possible. To discriminate between the fate of either newly synthesized
C6NBD-GlcCer or -SM, the cells were therefore incubated with 10 µM PDMP, which completely inhibited the synthesis
of C6NBD-GlcCer but did not affect synthesis of SM (data
not shown). At these conditions, the fraction of the total pool of SM
that was transported over a 30-min time interval to the basolateral
membrane was not affected (Fig. 2D), but the number of
labeled BCs after 30 min of transport was reduced by approximately 40%
(Fig. 2D). Because only 25% of the total newly synthesized
sphingolipid fraction (C6NBD-GlcCer plus
C6NBD-SM) at control conditions consists of GlcCer,
this result indicates that, in relative amounts,
C6NBD-GlcCer is the major sphingolipid that is
transported to the BC membrane. Yet, evidently, (a fraction of)
C6NBD-SM is also transported from the Golgi to the BC membrane.
Mechanism of Sphingolipid Transport from TGN to Basolateral and
Apical Membranes--
Interestingly, C6NBD-GlcCer
transport from the Golgi to the basolateral membrane was much faster
than that of C6NBD-SM. After 15 min at 37 °C, 32% of
the pool of C6NBD-GlcCer and 18% of that of
C6NBD-SM had reached the basolateral membrane (Fig.
3, A and B). These
different transport kinetics to the basolateral membrane of HepG2 cells
are reminiscent of the fast transport of natural GlcCer compared with
other sphingolipids in Chinese hamster ovary cells (24). With
increasing incubation times the difference between SM and GlcCer with
regard to the fraction of the total lipid pools that reached the
basolateral membrane diminished, amounting to 75-80% of the pools of
both sphingolipids after 60 min. As shown above (Fig. 2C)
the percentage of labeled BCs decreased concomitantly, emphasizing the
integration of the newly synthesized sphingolipids in a dynamic
transport process between apical and basolateral membrane. This is not
the case after short term incubations (30 min; Fig. 2C).
Accordingly, this would therefore suggest that the rapid transfer of
GlcCer from the Golgi to basolateral membrane after 15 min involves a
direct transport step and, given its pool size compared with that of
SM, may be related to a mechanism that differs from that of SM.
GlcCer is synthesized at the cytoplasmic leaflet of the cis-Golgi (25)
and its transport from the Golgi surface to the cytoplasmic leaflet of
the plasma membrane, either by monomeric flow or in the cytoplasmic
leaflet of transport vesicles, has been described (24). In contrast to
its natural counterpart, the short chain fluorescent GlcCer analog can
be translocated to the exoplasmic leaflet by the basolaterally
localized multidrug resistance protein MRP1 to become accessible for
BSA (23, 37). Indeed, incubation with the MRP1 inhibitor MK571
significantly reduced the amount of C6NBD-GlcCer that
became accessible to BSA but had no effect on the basolateral transport
of C6NBD-SM (Fig. 3, A and B). The effect of MK571 on GlcCer transport was time-dependent,
showing 60% inhibition of lipid translocation after 15 min, which
diminished to approximately 35% after 30 min, whereas the effect had
largely disappeared after 60 min. These data thus indicate that early (i.e. up to 15-30 min) after the onset of sphingolipid
biosynthesis in HepG2 cells, the transport mechanism of SM and GlcCer
from Golgi to the basolateral membrane at least partly differs.
However, after longer incubation times, an increasing pool of the
C6NBD-GlcCer is transported directly to the exoplasmic
leaflet of the plasma membrane via a (slower) MRP-independent pathway
that originates from an intracellular site that differs from the
cytoplasmic surface of the Golgi. In contrast to trafficking of
de novo synthesized GlcCer from Golgi to the basolateral
membrane, GlcCer (and SM) exiting from SAC, after specific accumulation
in this compartment following endocytic internalization (see
"Experimental Procedures"), reaches the outer leaflet of the
(apical) plasma membrane, entirely independent of multidrug resistance
protein 1 (MDR1) activity (20).
To analyze how newly synthesized C6NBD-GlcCer and -SM are
transported from the Golgi to the apical membrane, we examined the effect of CSA, an inhibitor of the MDR1 protein that is localized in
the BC membrane of HepG2 cells (37). Incubation with 5 µM CSA, which completely abolished the translocation of rhodamine 123 into
the BCs (data not shown) neither affected the number of labeled BCs nor
the accessibility of the C6NBD-labeled sphingolipids to
sodium dithionite (Fig. 3C). In conjunction with the
observation that upon inhibition of GlcCer biosynthesis BC labeling is
reduced by almost 40% (see above), the results imply that the major
part of the C6NBD-GlcCer (and -SM) is transported directly
to the exoplasmic leaflet of the BC membrane, i.e. the lipid
must have been localized in the lumenal leaflet of transport vesicles.
To obtain direct evidence for this notion, we subsequently developed an
assay to isolate the putative transport vesicles, released from TGN and SAC.
C6NBD-SM and -GlcCer Can Be Recovered in the Lumenal
Leaflet of Transport Vesicles Released from the TGN and the
SAC--
HepG2 cells were mechanically permeabilized to induce holes
in the cell surface that are large enough for the release of transport vesicles. Because of cell detachment, the filter stripping method described previously (28, 32) was not suitable for HepG2 cells, and
permeabilization was therefore carried out by pressing the cells
through a 25-gauge syringe. After this treatment 80-90% of the cells
were permeable for trypan blue and had largely lost their plasma
membrane as revealed by phase contrast microscopy (not shown). Using
this protocol, 15-20% of the total cell associated C6NBD-sphingolipids (ceramide, SM, and GlcCer) were
released from the cells following the Golgi labeling procedure, whereas
25-30% of the total pool of C6NBD-SM and -GlcCer were
released from SAC-labeled cells (Fig. 4).
To remove any sphingolipids that remained exposed on the cytoplasmic
leaflet of intracellular membranes following permeabilization, the cell
pellet was washed with BSA. 30% of both C6NBD-ceramide and
-GlcCer was localized in the cytoplasmic leaflet of the Golgi membrane
(Fig. 4A). In contrast, only 10% of the newly synthesized
C6NBD-SM was accessible to BSA. After labeling the SAC of
HepG2 cells with C6NBD-SM and -GlcCer, 10-15% of both
sphingolipids were accessible to BSA after permeabilization (Fig.
4B).
After these treatments, the remaining cell-associated fractions of
C6NBD-Cer and newly synthesized GlcCer and SM, associated with the Golgi, as well as the association of both sphingolipids with
SAC, displayed a largely localized appearance, as reflected by bright
fluorescent spots within the cellular lumen (not shown). Under these
conditions, C6NBD-GlcCer and -SM should be localized solely
in the lumenal leaflet of the SAC or the TGN/Golgi (Ref. 33; see also
"Discussion"). Accordingly, if transport vesicles exclusively
mediate subsequent transport and assuming that the topology is
maintained, the lipids should now be recovered in the lumenal leaflet
of such vesicles after their release and thus must be inaccessible to
BSA and pelletable at high speed. After an incubation for 30 min at
37 °C in presence of ATP and cytosolic proteins, 8% of either newly
synthesized sphingolipid was recovered in the high speed pellet. In
contrast, at 4 °C or in the absence of ATP and cytosol only 2-4%
of C6NBD-GlcCer and -SM were recovered (Fig.
5A). Comparably low vesicle
budding was obtained when ATP or cytosol alone was omitted from the
assay (data not shown). Essentially the same requirements applied for
the release of C6NBD-SM and -GlcCer from the SAC. Again
2-3% of C6NBD-SM and -GlcCer were released at 4 °C or
at 37 °C in the absence of ATP and/or cytosol. However, in presence
of ATP and cytosol the vesicular release of both sphingolipids
increased 2-3-fold, amounting to 7-8% of the initial cell-associated
lipid fraction (Fig. 5B). The results thus show that the
release of sphingolipid-containing vesicles from TGN and SAC in this
assay is ATP- and cytosol-dependent and requires incubation
at an elevated (37 °C) temperature.
Prior to monitoring vesicle release, the permeabilized cells were
extensively washed to ascertain that only lipid release in newly formed
vesicles was monitored. Nevertheless, to exclude the possibility that
the release of sphingolipids was due to nonspecific fragmentation of
intracellular membranes, we monitored the activity of the
Golgi-resident enzyme GTase in the cell lysate and the various
fractions obtained during vesicle isolation (Table
I). The specific activity of GTase
in the membrane fractions that were released during permeabilization
and the subsequent washing steps was similar to the specific activity
in the cell lysate. 17% of the GTase was released during the first
permeabilization step, whereas only a residual release was observed
during the subsequent washing steps. More than 75% of the GTase
activity was still intracellular prior to the final incubation step.
During the incubation at 4 °C, a very minor amount of protein was
released. At 37 °C more proteins were released, but the specific
activity of GTase was much lower than in the other fractions,
indicating that the membranes that were released in a
temperature-dependent manner contained almost exclusively
cargo proteins, whereas the amount of resident Golgi proteins was very
low (Table I).
In principle, the transport vesicles that were released could have
acquired NBD-labeled lipids that were transferred to the cell surface
of intact cells, still present in the sample, as well as from plasma
membrane fragments of the permeabilized cells. Although most cells were
permeabilized during the preparation, an effort was made to examine
this possibility and to estimate the contribution. Therefore, 1% BSA
was added during the various incubation conditions. Only a minor
fraction of C6NBD-SM and -GlcCer (3%) became accessible to
BSA after labeling the SAC and a subsequent incubation at 4 °C. The
amount of sphingolipids was increased to 7-8% after incubation for 30 min at 37 °C, irrespective of the addition of ATP and cytosol (Fig.
5D, hatched versus black bars). The ATP
independence demonstrates that the sphingolipids were released from
cells that were left intact during the preparation. Although newly
synthesized C6NBD-SM became accessible to BSA in significant amounts only after incubation at 37 °C, this was not the
case for C6NBD-ceramide and C6NBD-GlcCer. Even
at 4 °C 20% of the C6NBD-GlcCer and 10% of the
C6NBD-ceramide became accessible to BSA (Fig.
5C). Incubation at 37 °C did not markedly change the
relative amount of released C6NBD-ceramide and -GlcCer.
Distinct C6NBD-SM and -GlcCer-containing Vesicles Are
Released from the SAC but Not from the TGN--
To analyze whether
C6NBD-SM and -GlcCer are sorted in the SAC- and/or TGN
membrane, which might be reflected by distinct vesicle populations
enriched in either lipid, we fractionated the vesicle-containing supernatants by sucrose gradient centrifugation. The fractions were
treated on ice for 5 min with sodium dithionite to ensure that only
C6NBD-sphingolipids in the lumenal leaflet of transport vesicles were analyzed. In SAC-derived vesicles, C6NBD-SM
was clearly enriched over C6NBD-GlcCer in vesicles obtained
at a density corresponding to 30-33% sucrose (w/w), whereas
C6NBD-GlcCer was enriched at a sucrose density between 40 and 44% (Fig. 6A). Although neither lipid was exclusively retrieved in a particular vesicle fraction, it is evident that distinct "preferential" transport vesicles from the SAC for either sphingolipid can be identified.
To further characterize the potentially different nature of the
carriers responsible for the exit of C6NBD-SM and -GlcCer from the SAC, we took advantage of the ability of NBD to photoconvert DAB into an osmiophilic precipitate that can be visualized by electron
microscopy (36). Prior to incubation with DAB and processing for
electron microscopy, the gradient fractions corresponding to 30-33 and
40-44% sucrose, respectively, were adjusted to 10% sucrose to avoid
possible morphological alterations caused by osmotic effects. The
30-33% fraction contained labeled and unlabeled spherical vesicles of
different diameters. Especially small vesicles of approximately 50 nm
in diameter were heavily labeled with DAB and are most likely those
containing C6NBD-SM (arrows in Fig. 7, A and B). Such
vesicles were only occasionally observed in the 40-44% fraction, in
which the main DAB-positive structures consisted of larger, cup-like
vesicles with a diameter of 80-100 nm (arrowheads in Fig.
7B). No DAB-positive, osmiophilic structures were obtained
when the pellets were kept in the dark, although faint vesicular
structures could be identified (Fig. 7, C and D).
The analysis of TGN-derived vesicles was hindered by the different
amounts of the sphingolipids, because HepG2 cells synthesize much more
C6NBD-SM than C6NBD-GlcCer. As observed for the
SAC-derived vesicles, C6NBD-SM was found predominantly at a
sucrose concentration of 30-33% (Fig. 6B).
C6NBD-GlcCer was recovered at slightly lower sucrose
concentrations, in contrast to its location at a higher density seen
for SAC-derived vesicles. However, the difference in localization
between SM and GlcCer, present in the TGN-derived vesicles, was usually
not large enough to support the view that sorting of the two
sphingolipids into distinct transport vesicles occurred.
The Release of C6NBD-SM-containing Vesicles from the
SAC and the TGN Is Differently Affected by the Calmodulin Antagonist
TFP--
We have previously shown that C6NBD-SM and
-GlcCer are located in different domains in the SAC. Among others, the
evidence relied on the selective regulation of their exiting to the
apical and basolateral domain upon treatment of the cells with the
calmodulin antagonist TFP (38). The preferential localization of SM and GlcCer in distinct SAC-derived transport vesicles (Fig. 6A)
is therefore fully consistent with this notion. To further corroborate these observations, the present assay system provides the possibility to investigate in a direct manner whether TFP modulates vesicular exiting of the sphingolipids.
In intact cells, TFP inhibited the transport of C6NBD-SM
and -GlcCer from the SAC to the basolateral membrane, whereas it had no
effect on the recycling of C6NBD-GlcCer between SAC and BC
membrane (38). When 20 µM TFP was included during the BSA step after the permeabilization and the budding assay, the
temperature-dependent release of C6NBD-SM from
the SAC was inhibited by 50%, whereas there was no effect on the
release of C6NBD-GlcCer (Fig.
8A). At higher concentrations
of TFP (50 µM), the release of C6NBD-SM was
inhibited by 70%, and a slight effect on the release of
C6NBD-GlcCer was observed (20% inhibition; not shown).
Surprisingly, incubation with 20 µM TFP
increased the vesicular release of C6NBD-SM from
the TGN by 30%, whereas it had no effect on the release of
C6NBD-GlcCer (Fig. 8B). It is unlikely that this
increased release of C6NBD-SM was due to nonspecific fragmentation of the TGN, because higher concentrations of TFP (100 µM) slightly inhibited the release of
C6NBD-SM and abolished the release of
C6NBD-GlcCer (Fig. 8B). Interestingly, the data thus suggest that distinct mechanistic features are involved in mediating transport vesicle assembly and/or release from SAC on the one
hand and Golgi on the other.
The present study demonstrates that the mode of sorting of SM and
GlcCer during their exit from Golgi and SAC, the major sorting compartments in the biogenesis and maintenance of polarized membrane domains, differs. This is reflected by the recovery of transport vesicles, distinctly enriched in these sphingolipids. In addition, sphingolipid sorting in HepG2 cells is mainly associated with the SAC,
highlighting its prominent role in polarity development of these cells.
In Vitro Budding System and Sphingolipid Release from SAC and
TGN--
To appreciate the involvement of SAC and TGN, including their
hierarchy, in the polarized transport itinerary of hepatocytes, the
isolation and characterization of the respective transport vesicles
will be imperative. In fact, a variety of in vitro systems have been developed to analyze vesicular transport of proteins from the
TGN to the plasma membrane (28-30). Also in hepatocytes distinct
constitutive pathways from the TGN to the basolateral plasma membrane
have been described (39, 40). However, although lipids, especially
sphingolipids, play an important role in the polarized sorting of
apical and basolateral proteins in epithelial cells (11), only very few
in vitro systems have been developed to study the polarized
sorting of de novo synthesized sphingolipids in the context
of the assembly of transport vesicles (31, 32). Therefore, as a first
approach, an in vitro assay was developed to characterize
transport and sorting of sphingolipids from either compartment. To this
end, we used mechanically permeabilized HepG2 cells that had been
labeled with C6NBD-tagged sphingolipid analogs. Our data
reveal that the release of C6NBD-SM- and -GlcCer-containing vesicles from both compartments was temperature-dependent
and required the addition of ATP and cytosolic proteins. A significant contribution of nonspecific fragmentation of the organelles to the
observed sphingolipid transport is excluded, because no significant release of the Golgi marker GTase occurred during vesicle formation, whereas the TGN-derived vesicles (Fig. 5A) did not contain
significant amounts of the Golgi-localized
C6NBD-ceramide.
From an experimental point of view it is relevant to emphasize that,
although after internalization at the plasma membrane a fraction of the
fluorescent sphingolipid analogs may be transported to the Golgi, as
occurs in undifferentiated cells (35, 41, 42), in polarized HepG2 cells
both C6NBD-GlcCer and -SM are specifically accumulated in
the SAC. As shown previously (20, 33), localization in this compartment
can be readily distinguished from the Golgi apparatus, e.g.
in its sensitivity to monensin and nocodazole. Indeed, the present
results demonstrate that, prior to their vesicular release, newly
synthesized and internalized sphingolipid analogs are located in the
intended "target" compartments, i.e. Golgi and SAC. This
is, for example, apparent from the distinct effects of TFP on the
release of SM (Fig. 8), present in either compartment, or the
differences in accessibility of GlcCer to BSA at 4 °C (Fig. 5,
compare C and D).
In the context of the latter observation, it is interesting that only
de novo synthesized C6NBD-GlcCer,
after its translocation to the lumenal leaflet in the Golgi,
became accessible to BSA at 4 °C. Because all accessible
sphingolipid analogs had been removed from the cytoplasmic leaflet of
internal membranes prior to the incubation, this implies that a
temperature-independent (and energy-independent) activity exists in the
Golgi membrane that translocates C6NBD-GlcCer but
not C6NBD-SM from the lumenal to the cytoplasmic
leaflet. Because such a translocation was not observed in the
SAC-labeled membranes, the data reveal that the compartments display
different properties concerning C6NBD-GlcCer translocation
and emphasize Golgi specificity rather than a nonspecific translocation
of the lipid caused by the C6NBD group.
Finally, although these analogs resemble the properties of natural
sphingolipids in many aspects (42, 43), a direct comparison would be
preferable. However, to specifically label SAC with sphingolipids or to
accumulate newly synthesized sphingolipids in Golgi, the use of
exchangeable or modifiable (dithionite quenching) sphingolipids is
essential. In this manner, "contamination" of SAC- or Golgi-derived transport vesicle fractions with (natural) sphingolipid-containing vesicle fractions of similar density, but derived from different sources (plasma membrane, endocytic pathway), is avoided.
Besides, a lack of suitable markers currently precludes further
purification of the recovered vesicle fractions. It is most relevant,
however, that the analogs used are sorted in SAC in a manner that
depends on the progress of polarity (20, 21). This emphasizes their validity as markers for apically and basolaterally directed transport from SAC (or TGN) and for characterization of the respective transport vesicles (see also below).
Sorting of SM and GlcCer in SAC and TGN Prior to Vesicle
Release--
The present data reveal that despite the transcytotic
processing of apical proteins (12), the Golgi of hepatic cells does display (direct) sphingolipid sorting capacity. This fact gains considerable significance by recent observations that several hepatic
apical proteins may reach the apical membrane by direct transport (14,
15, 44), which might link sphingolipid sorting to raft assembly. In
this regard, questions have been raised concerning the ability of NBD
lipids to partition into rafts, triggered in artificial liposomal
systems, given their relative water solubility (45). However, in
biological systems it has been shown that exogenous addition of
C6NBD-SM to developing neurons triggered raft domain
formation and the apical sorting of the Thy-1 receptor to axonal
domains (46). Other, but similar, short chain fluorescent (BODIPY)
sphingolipid analogs are sorted in a cholesterol-dependent, i.e. a potentially raft-mediated mechanism, in fibroblasts
derived from patients suffering from various sphingolipid storage
diseases (47). Nevertheless, further work will be needed to directly clarify these issues.
The present data are schematically summarized in Fig.
9. During the C6-NBD-ceramide
labeling at 18 °C (Fig. 9A, steps 1 and 2), not only C6NBD-SM but also
C6NBD-GlcCer accumulated in the Golgi. This differs from
LLC-Pk1 cells (23) and indicates that in liver cells,
C6NBD-GlcCer release from the TGN is also largely vesicular. Inhibition of MRP1 activity confirmed previous findings (23)
that transport of C6NBD-SM to the plasma membrane is
exclusively in the lumenal leaflet of transport vesicles (Fig.
9A, step 3a). Because C6NBD-GlcCer is
partly transported to the cytoplasmic leaflet of the basolateral
membrane, prior to its translocation to the exoplasmic leaflet (Fig.
9A, step 4), the temperature dependence of this
process (>18 °C) may suggest involvement of transport vesicles
(Fig. 9A, step 3b). Nevertheless, monomeric
transport, possibly mediated by a glycosphingolipid transfer protein
(48), cannot yet be excluded (Fig. 9A, step 3c).
Interestingly, even in presence of MK571, the majority of the
C6NBD-GlcCer became accessible to BSA after prolonged
incubation. These data indicate that only in an early phase (15-30
min) after de novo synthesis of GlcCer, may
vesicle-independent transport occur. At later stages, when the lipid
has acquired its correct topological transmembrane distribution,
transport of GlcCer is predominantly of a vesicular nature.
Interestingly, inhibition of the apically localized MDR1 had no effect
on the number of labeled BCs and the accessibility of the lipids to
sodium dithionite, indicating that the sphingolipid analogs were
transported directly to the exoplasmic leaflet of the BC membrane.
Furthermore, inhibition of GlcCer synthesis reduced the number of
labeled BCs, in spite of the fact that the pool of
C6NBD-SM, synthesized in control cells, is more than 3-fold in excess of that of GlcCer. Together, these results imply that (i)
C6NBD-GlcCer is, in relative amounts, the major
sphingolipid that is transported to the BC membrane, whereas
C6NBD-SM is predominantly transported to the basolateral
plasma membrane (indicated in Fig. 9A, steps 3a
and 3d) and (ii) the majority of apically transported C6NBD-GlcCer is localized in the lumenal leaflet
of transport vesicles. This is in contrast to studies on LLC-Pk1 cells,
where MDR1 activity was required for the transport of
C6NBD-GlcCer to the outer leaflet of the apical membrane
(23, 27), thus indicating cell type-dependent differences
in sphingolipid sorting in polarized cells.
By using the in vitro assay, direct evidence demonstrated
that C6NBD-GlcCer and -SM, after their accumulation in the
SAC, are released in distinct transport vesicles (Fig. 9B).
Thus, TFP, which blocks the exit of C6NBD-SM but not that
of C6NBD-GlcCer from the SAC in intact cells (38),
inhibited only the release of SM-containing vesicles but had no effect
on the release of C6NBD-GlcCer (Fig. 8). This shows that
the vesicular release of C6NBD-SM and -GlcCer is
differentially regulated and indicates that both sphingolipids are
localized in distinct membrane domains of the SAC, presumably
accomplished by lateral sorting in the luminal leaflet (38).
Intriguingly the sucrose fractions enriched in the apically sorted
C6NBD-GlcCer contained DAB-positive cup-shaped vesicles.
These are reminiscent of the vesicles derived from the common recycling
endosome of MDCK cells that contain predominantly the apically directed
polymeric immunoglobulin receptor (7). Because the fractions of the
30-33% sucrose contain virtually no C6NBD-GlcCer, we
assume that the small DAB-positive vesicles found there contain
exclusively C6NBD-SM, which is predominantly transported to
the basolateral membrane under our experimental conditions. Similarly,
in MDCK cells the transferrin receptor is released from recycling
endosomes predominantly in small vesicles of 60-nm diameter (7).
Further work should clarify whether the C6NBD-SM identified
in the heavier gradient fractions is localized only in the small
vesicles that were occasionally seen or whether it is also localized in
the larger cup-shaped vesicles. If so, another issue would be whether
C6NBD-GlcCer and -SM are present in the same vesicles or
whether they are sorted into distinct but morphologically similar vesicles.
Using different epithelial cells, it has been shown that de
novo synthesized sphingolipid analogs can be sorted into distinct transport vesicles at the Golgi (31, 32). However, a more pronounced
sorting capacity of these lipids in the SAC of HepG2 cells is in line
with the fact that in hepatocytes most newly synthesized apical
proteins are transported to the basolateral membrane, prior to their
transport to the apical membrane via transcytosis. In WIF-B cells,
transcytosis via an endosomal subapical compartment has been
demonstrated (49), implying that a heavy burden may be placed on the
sorting capacity of the SAC. Cell type-dependent
differences in the relative contribution of distinct sorting sites (SAC
versus TGN) may also explain that sorting of sphingolipid
analogs was not observed in the transcytotic pathway in MDCK cells
(50). Indeed the initial sorting observed for de novo
synthesized sphingolipids was abolished after prolonged incubation
times, indicating that the SAC equivalent in these cells
(i.e. the common endosome) has a lower sorting capacity than
in HepG2 cells and underlining the importance to investigate vesicular
transport in different cell types to fully understand polarity
establishment. The current approach may provide an excellent opportunity to analyze the mechanisms that mediate the sorting of
sphingolipids and proteins in the SAC (and the TGN), by careful analysis of the molecular mechanisms that underlie the formation of the
transport vesicles and the regulation of their release.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-glucosylsphingosine, and sphingosylphosphorylcholine
were from Sigma. MK571 was kindly provided by E. Vellenga (University of Groningen). All other materials were of analytical grade.
-D-glucosylsphingosine, and
sphingosylphosphorylcholine, respectively, as described elsewhere (34).
Sphingolipids were stored at
20 °C and routinely checked for purity.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (58K):
[in a new window]
Fig. 1.
C6NBD-ceramide is metabolized at
18 °C, but only a minor part of the newly synthesized sphingolipids
is transported to the cell surface. The cells were labeled with
C6NBD-ceramide as described under "Experimental
Procedures." A and B, the perinuclear area is
brightly labeled in the fluorescence microscope (B), but BC
structures (arrows) that were identified in phase contrast
microscope (A) appear unlabeled. C, analysis of
C6NBD-ceramide metabolism by TLC; 100% corresponds to the
total amount of cell-associated NBD. D, basolateral
sphingolipid transport during the 18 °C-step; 100% corresponds to
the total amount of each sphingolipid recovered in the cells and the
medium. In C and D a representative TLC analysis
as well as the mean values and standard deviations of three independent
experiments are shown. Bar, 20 µm.
View larger version (71K):
[in a new window]
Fig. 2.
Sphingolipid transport to the exoplasmic
leaflet of the BC membrane is reduced by PDMP. The cells were
labeled with C6NBD-ceramide and incubated at 37 °C in
presence of BSA. BC membranes (arrows) were identified by
phase contrast microscopy (A) and analyzed for
C6NBD labeling by switching to epifluorescence
(B). C, quantification of transport to the BC
membrane. In control cells (white bars) the amount of
labeled BCs reached a maximum after 30 min at 37 °C and decreased
after prolonged incubation. Treatment of the cells with sodium
dithionite after the transport reduced the amount of labeled BCs to
control levels (black bars). D, effect of 10 µM PDMP on basolateral transport of SM and the amount of
labeled BCs. The amount of basolaterally transported SM and of labeled
BCs in control cells (white bars), respectively, was set to
100 and compared with the values obtained in PDMP-treated cells
(black bars). In C and D the mean
values and standard deviations of three independent experiments are
shown. Bar, 20 µm.
View larger version (16K):
[in a new window]
Fig. 3.
C6NBD-GlcCer transport to the
basolateral membrane is partly MRP-dependent.
A and B, after labeling with
C6NBD-ceramide, the cells were preincubated at 4 °C with
(lanes 4-6 in A and black bars in
B) or without (lanes 1-3 in A,
white bars in B) MK571. The cells were incubated
at 37 °C for 15 (lanes 1 and 4 in
A), 30 (lanes 2 and 5 in
A), and 60 (lanes 3 and 6 in
A) min in back-exchange medium, and the basolaterally
transported sphingolipids were analyzed by TLC. C, effect of
CSA on sphingolipid transport from the Golgi to the apical membrane.
After labeling the cells with C6NBD-ceramide they were
preincubated with (black bars) or without (white
bars) 5 µM CSA and incubated further for 30 min at
37 °C in the presence of CSA. Thereafter, the cells were incubated
for 10 min with or without sodium dithionite (DT) at 4 °C
to determine the localization of the sphingolipids. The BC structures
were identified by phase contrast, and the percentage of labeled BCs at
the different conditions is shown. A representative TLC plate
(A) as well as the mean values and standard deviations of
three independent experiments (B and C) are
shown.
View larger version (20K):
[in a new window]
Fig. 4.
Distinct susceptibility of intracellular
C6NBD-sphingolipids to BSA after cell
permeabilization. The cells were incubated with
C6NBD-ceramide (A) or a mixture of
C6NBD-SM and -GlcCer (B) to accumulate lipids in
the TGN and SAC, respectively. Subsequently the cells were
permeabilized, and the sphingolipids localized in the cytoplasmic
leaflet of intracellular membranes were back-exchanged with BSA. The
sphingolipids that were released during permeabilization (lane
P and white bars) and BSA treatment (lane B
and black bars) were quantified by TLC. For each sample a
representative TLC plate and the mean values and standard deviations of
at least four independent experiments are shown.
View larger version (25K):
[in a new window]
Fig. 5.
The vesicle formation at both TGN and SAC is
temperature- and energy-dependent. HepG2 cells were
incubated with C6NBD-ceramide (A and
C) or a mixture of C6NBD-SM and -GlcCer
(B and D). After permeabilization and BSA
treatment the cells were incubated in presence of BSA at 4 °C
(lane 1 and white bars), at 37 °C without ATP
and cytosol (lane 2 and hatched bars) and at
37 °C in presence of ATP and cytosol (lane 3 and
black bars). The cells were removed, and the supernatant was
centrifuged at 100,000 × g for 2 h. The
sphingolipids in the high speed pellet (A and B)
and in the supernatant (C and D) were extracted
and analyzed by TLC. For each sample a representative TLC plate, and
the mean values and standard deviations of at least three independent
experiments are shown.
Galactosyltransferase activity in fractions obtained during
permeabilization and vesicle formation
View larger version (25K):
[in a new window]
Fig. 6.
Distinct C6NBD-SM and
C6NBD-GlcCer-containing vesicles are released from the SAC
but not from the TGN. HepG2 cells were incubated with a mixture of
C6NBD-SM and -GlcCer (A) or
C6NBD-ceramide (B). After permeabilization and
BSA treatment the cells were incubated at 37 °C in the presence of
BSA, ATP, and cytosol. The cells were removed, and the supernatant was
layered on top of a sucrose gradient consisting of 20-50% sucrose
(w/w) in transport buffer. The gradient was centrifuged for 16 h
at 100,000 × g and fractionated. Fraction 1 corresponds to the bottom of the gradient. The fractions were treated
with sodium dithionite for 5 min on ice, and the sphingolipids were
extracted and analyzed by TLC. In A the relative amount of
C6NBD-SM ( ) and C6NBD-GlcCer (
) in each
fraction is calculated to demonstrate the enrichment of one
sphingolipid over the other at a particular sucrose concentration. In
B the distribution of C6NBD-SM and -GlcCer over
the whole gradient is shown. For both TGN- and SAC-derived vesicles a
representative TLC plate as well as the mean and extreme values of two
gradients are shown.
View larger version (135K):
[in a new window]
Fig. 7.
C6NBD-containing vesicles derived
from SAC are morphologically distinct. HepG2 cells were incubated
with a mixture of C6NBD-SM and -GlcCer, permeabilized, and
incubated at 37 °C in the presence of BSA, ATP, and cytosol. After
sucrose gradient centrifugation of the vesicular supernatant, the
fractions containing 30-33% sucrose (A and C)
and 40-44% sucrose (B and D) were pooled,
adjusted to a sucrose concentration of 10%, and pelleted. The pellets
were preincubated with 0.15% DAB in the dark and then either
illuminated in presence of DAB at a wavelength of 460 nm (A
and B) or kept in the dark (C and D)
for 30 min. The pellets were then incubated with OsO4 and
processed for transmission electron microscopy. Arrows (in
A and B) indicate small DAB-positive vesicles,
whereas cup-shaped vesicles (in B) are indicated by
arrowheads. Bar, 100 nm.
View larger version (35K):
[in a new window]
Fig. 8.
TFP treatment affects the release of
C6NBD-SM from the SAC and TGN differently. HepG2 cells
were incubated with a mixture of C6NBD-SM and -GlcCer
(A) or C6NBD-ceramide (B). After
permeabilization and BSA treatment the cells were incubated at 4 or at
37 °C in presence of BSA, ATP, and cytosol. During BSA treatment and
vesicle formation the cells were incubated with (lane 2 and
hatched bars in A and B) or without 20 µM TFP (lane 1 and white bars in
A and B). In some experiments cells labeled with
C6NBD-ceramide were incubated with 100 µM TFP
(lane 3 and black bars in B). The
cells were removed, and the supernatant was centrifuged at 100,000 × g. The sphingolipids in the vesicle pellet were extracted
and analyzed by TLC. To calculate the temperature-dependent
release of sphingolipids, the values obtained at 4 °C were
subtracted from those obtained at 37 °C, and the resulting values
for the control cells were set as 100. Representative TLC plates,
showing the release at 37 °C, and the mean values and standard
deviations of at least three independent experiments are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (39K):
[in a new window]
Fig. 9.
Model for the transport of sphingolipids in
the biosynthetic (A) and transcytotic
(B) pathways of polarized HepG2 cells. Distinct
transport steps that are discussed in the text are indicated by
numbers. In addition the effect of TFP on the formation of
SM-containing vesicles is indicated. In B sphingolipids in
the BC membrane represent the situation between transport steps
2 and 3, whereas sphingolipids in the SAC membrane
indicate the situation prior to steps 4a and 4b
(i.e. after sorting of GlcCer and SM into distinct membrane
domains). The final localization of the SM and GlcCer in the exoplasmic
leaflet of the apical and basolateral plasma membrane domains is not
shown.
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ACKNOWLEDGEMENTS |
---|
The help of Ruby Kalicharan (Department of Cell Biology, Electron Microscopy Section) in the electron microscopic analyses is much appreciated. We thank Drs. Johanna van der Wouden, Jan Willem Kok, and Sven van IJzendoorn for helpful discussions and Karin Klappe for the synthesis of the C6NBD-sphingolipids.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by Grant BMBF-LPD 9801-21 from the Deutsche
Akademie der Naturforscher Leopoldina.
§ To whom correspondence should be addressed. Tel.: 31-50-363-2741; Fax: 31-50-363-2728; E-mail: d.hoekstra@med.rug.nl.
Published, JBC Papers in Press, October 28, 2002, DOI 10.1074/jbc.M208259200
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ABBREVIATIONS |
---|
The abbreviations used are: TGN, trans-Golgi network; BC, bile canaliculus; CSA, cyclosporin A; C6NBD, 6-[N-(7-nitrobenz-2-oxa-1,3 diazol-4-yl)amino] hexanoyl; DAB, diaminobenzidine, GlcCer, glucosylceramide; GTase, galactosyltransferase; MRP1, multidrug resistance-related protein 1; PDMP, D,-threo-1-phenyl-2-decanoyl amino-3-morpholino-1-propanol; SAC, subapical compartment; SM, sphingomyelin; TFP, trifluoperazine; BSA, bovine serum albumin.
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