(Received for publication, May 8, 1995; and in revised form, May 30, 1995)
From the
The cellular mechanism of cholesterol transport from the endoplasmic reticulum to the plasma membrane is currently unknown. To assess the possibility that sterol carrier protein-2 (SCP-2) is involved in this transport, we studied the time course of newly synthesized cholesterol incorporation in the plasma membrane of normal and SCP-2-deficient (Zellweger syndrome) human fibroblasts.
Cholesterol transfer was rapid, cytoskeleton-independent, and Golgi-independent in normal cells, but it was slower, cytoskeleton-dependent, and Golgi-dependent in SCP-2-deficient cells. After SCP-2 antisense oligonucleotides treatment of normal fibroblasts, the rapid transport was reduced by 81% with a simultaneous increase of the slower one.
These results suggest that in normal fibroblasts the major fraction of newly synthesized cholesterol is transported to the plasma membrane by a SCP-2-dependent mechanism. In contrast, in SCP-2-deficient cells, newly synthesized cholesterol leaves the endoplasmic reticulum by a cytoskeleton/Golgi-dependent mechanism.
Intracellular cholesterol trafficking may occur through several mechanisms, including aqueous diffusion, vesicle budding and fusion, and protein-mediated transport through the cytosol(1, 2, 3, 4) . These transport systems are ultimately responsible for the high compartmentalization of cholesterol between the intracellular organelles and the plasma membrane. In several cell types, over 80% of total unesterified cellular cholesterol is associated with the plasma membrane (2, 4) but the mechanism of this segregation is unknown. Immediately after its synthesis, cholesterol is transferred from the endoplasmic reticulum to the plasma membrane with a reported t of 10-20 min(5, 6, 7) . The exact mechanism of this transfer is presently unknown, although it has been shown to be not affected by several cytoskeleton- and Golgi-disrupting drugs(8, 9) , and to be both temperature-dependent (8, 9) and energy-dependent(5) .
The 13.2-kDa sterol carrier protein 2
(SCP-2) ()has been postulated to facilitate the movement of
sterols within cells(10, 11, 12) . However,
evidence that SCP-2 participate in cholesterol transfer to the plasma
membrane in vivo is lacking. Antibodies against rat liver
SCP-2 recognize the 13.2-kDa polypeptide, as well as 40- and 58-kDa
polypeptides(11, 12) , which are thought to be SCP-2
precursors(12) . The processing of SCP-2 precursors depends on
the presence of functional peroxisomes(12) . These precursors
are rapidly degraded with a marked decrease of SCP-2 in cells that are
deficient in peroxisomes, such as Zellweger syndrome
fibroblasts(11, 12) .
In this study, we examined the transfer of newly synthesized cholesterol to the plasma membrane in normal and SCP-2-deficient fibroblasts. Our results provide evidence for the involvement of SCP-2 in a rapid and direct transfer of cholesterol from the endoplasmic reticulum to the plasma membrane.
The possibility that SCP-2 serves as a carrier protein for
newly synthesized cholesterol to the plasma membrane was investigated
in normal and SCP-2-deficient (Zellweger syndrome) fibroblasts. After
72 h of culture in medium A, normal and SCP-2-deficient cells showed
similar rates of cholesterol synthesis. The maximal values of newly
synthesized cholesterol in the plasma membrane of normal fibroblasts
were observed 10 min after the beginning of the pulse, decreasing
rapidly thereafter (Fig.1). By contrast, the maximal values of
radiolabeled cholesterol were found only 20 min after the beginning of
the pulse in the plasma membrane of SCP-2-deficient fibroblasts. It is
noteworthy that the same amount of
[C]cholesterol was found 20 min after the pulse
in both normal and SCP-2-deficient fibroblasts (Fig.1, inset). Immunoblot analysis, using a specific antiserum
against SCP-2, confirmed the absence of SCP-2 in these Zellweger
syndrome fibroblasts (Fig.2).
Figure 1:
Time course
of newly synthesized cholesterol transport to the plasma membrane.
Confluent fibroblasts were pulsed with
[C]acetate for 7 min at 37 °C and chased for
the indicated times. [
C]Cholesterol
incorporation in the plasma membrane was determined using the
cholesterol oxidase assay. The maximal incorporation rates of
[
C]acetate into total cell cholesterol were
similar in control and SCP-2-deficient fibroblasts (2564 ± 301 versus 2832 ± 409 counts
min/10
cells, respectively). Results are the mean ± S.D. of four
different experiments and are expressed as the relative and total (inset) percentage of cell
[
C]cholesterol/10
cells that was
accessible to cholesterol oxidase. 1
10
cells
corresponded approximately to 600 µg of protein.
, normal
fibroblasts;
, SCP-2-deficient
fibroblasts.
Figure 2: Western blot analysis of normal and Zellweger syndrome fibroblasts. 50 µg of proteins of cell extracts were analyzed by Western blot. Polyclonal anti-rat SCP-2 antisera binding was visualized using the enhanced chemiluminescence procedure. Lane1, normal human fibroblasts; lane2, Zellweger syndrome fibroblasts.
Next, we evaluated the effects of colchicine, cytocalasin B, monensin, and brefeldin A, which are well known cytoskeleton- and Golgi-disrupting drugs(18, 19, 20, 21) , on newly synthesized cholesterol incorporation in the plasma membrane of normal and SCP-2-deficient cells. None of these drugs altered cholesterol transfer in normal fibroblasts (Fig.3A). In contrast, the same drugs, used at equivalent concentrations, reduced significantly (37-66%) the transport of newly synthesized cholesterol to the plasma membrane in SCP-2-deficient cells (Fig.3B).
Figure 3:
Effect of colchicine, cytocalasin B,
monensin, and brefeldin A on newly synthesized cholesterol transport.
Confluent normal (A, ) and SCP-2-deficient (B,
) fibroblasts were pulsed with
[
C]acetate and chased for the indicated times.
60 µM colchicine (
), 50 µM cytocalasin
B (
), and 15 µM monensin (
) were added 90, 30,
and 60 min before the pulse, respectively(8) . Brefeldin A
(
) was added at a final concentration of 1 µg/ml 30 min before
the pulse(9) . Cells preincubated with 0.1% ethanol or with
0.5% methanol, which were used to add the
drugs(8, 9) , served as internal controls (
).
The maximal incorporation rates of [
C]acetate
into total cell cholesterol were similar in control and SCP-2-deficient
fibroblasts and were not affected by pretreatment with these drugs.
Results are the mean ± S.D. of four different experiments and
are expressed as the relative and total (insets) percentage of
cell [
C]cholesterol/10
cells that
was accessible to cholesterol oxidase. *, p <
0.001 (versus control).
In the last series of experiments, we
treated normal fibroblasts with SCP-2 antisense oligonucleotides
corresponding to the 5` initiation region of the SCP-2 coding
sequence(17) . SCP-2 antisense treatment markedly reduced the
total amount of both SCP-2 and its 58- and 40-kDa related proteins (Fig.4), whereas treatment with control oligonucleotides (sense
and missense) did not affect SCP-2 expression. In agreement with these
results, the biosynthesis of SCP-2 and its related proteins was
significantly lowered by 95%, 80%, and 91%, respectively, at 5 days of
antisense treatment (data not shown). After 5 days of oligonucleotides
treatment, cells were pulsed with [C]acetate. As
shown in Fig.5, the relative percentage of
[
C]cholesterol transported to the plasma
membrane in the first 10 min was reduced by 81%, with a simultaneous
increase of the radiolabeled cholesterol incorporation at 20 min after
the beginning of the pulse. Such effect was not observed in sense- or
missense-treated cells. When SCP-2 antisense-treated cells were
incubated with colchicine, before the pulse with
[
C] acetate, the values of
[
C]cholesterol incorporated in the plasma
membrane 20 min after the beginning of the pulse were reduced by 45% (Fig.5).
Figure 4: Effect of SCP-2 antisense oligonucleotides on SCP-2 expression in normal fibroblasts. Confluent normal fibroblasts were cultured in serum-free DMEM. SCP-2 antisense, sense, or missense were added directly to the cultured medium at 10 µM final concentration for 5 days, without a change of medium. Cells cultured without oligonucleotides served as internal controls. Immunoblot analysis of SCP-2 content of cell lysates was carried out as described in the legend of Fig.2.
Figure 5:
Effect of SCP-2 antisense oligonucleotides
on newly synthesized cholesterol transport to the plasma membrane.
Confluent normal fibroblasts received SCP-2 antisense, sense, or
missense, for 5 days as described in the legend of Fig.4. Cells
cultured without oligonucleotides served as internal controls. The day
of the experiment, cells were pulsed and chased for the indicated
times. Experiments were conducted as described in Fig.1. The
maximal incorporation rates of [C]acetate into
total cell cholesterol of the different groups were (expressed as
counts
min/10
cells): control cells, 3682 ±
405; antisense-treated cells, 3562 ± 701; antisense +
colchicine-treated cells, 3428 ± 388; sense-treated cells, 3875
± 241. Results are the mean ± S.D. of three different
experiments. *, p < 0.0005 (antisense versus control); &, p < 0.0005 (antisense +
colchicine versus antisense).
The present results provide the first evidence in vivo for the involvement of SCP-2 in a rapid transport of newly synthesized cholesterol to the plasma membrane in normal fibroblasts. When the expression of SCP-2 is abolished, such as occurs in peroxisomes-deficient cells, or after SCP-2 antisense treatment, newly synthesized cholesterol reaches the plasma membrane through a slower, cytoskeleton- and Golgi-dependent route.
Previous reports have shown
that transfer of newly synthesized cholesterol to the plasma membrane
of animal cells is not affected by cytoskeleton- and Golgi-disrupting
agents(8, 9) . This is consistent with present results
in normal human fibroblasts. However, we found that both the
cytoskeleton and the Golgi apparatus are partially required for
cholesterol transport in SCP-2-deficient cells. These results support
the possibility that two different mechanisms are involved in the
transport of newly synthesized cholesterol to the plasma membrane of
normal and SCP-2-deficient cells. This possibility is supported by the
evidence that, in normal cells, SCP-2 antisense treatment induced a
shift of the maximal values of [C]cholesterol
transport from 10 to 20 min, and that this shift was reduced by
colchicine. These observations suggest that the endoplasmic reticulum
may modulate the two routes and, when SCP-2 is absent, cholesterol
leaves this organelle by the cytoskeleton/Golgi-dependent route. The
molecular mechanisms by which the endoplasmic reticulum may direct
cholesterol to one or the other route are unknown. It is noteworthy
that the same amount of radiolabeled cholesterol was found 20 min after
the pulse in both normal and SCP-2-deficient cells. In agreement with
this result, normal and SCP-2-deficient fibroblasts showed comparable
amounts of free cholesterol in the whole cell and in the plasma
membrane (results not shown). We do not know if SCP-2-deficient cells
remove cholesterol by increasing the number of vesicles leaving the
endoplasmic reticulum, the mass of cholesterol per vesicle, or both.
SCP-2 antisense treatment reduced the total amount of both SCP-2 and its 58- and 40-kDa related proteins. Both the 58- and 40-kDa proteins represent N-terminal extensions of SCP-2(11) , but only the 13.2-kDa moiety has the functional capacity to transfer cholesterol(2, 4) . We cannot rule out the possibility that SCP-2 is involved in cholesterol transport only for its affinity for sterols while other factors (i.e. docking proteins or cytoskeleton-independent vesicles) are carrying the cholesterol-SCP-2 complex to the final destination.
Pre-Golgi cholesterol-enriched vesicles have been isolated previously (8, 9) . Moreover, the Golgi apparatus contains substantial amounts of cholesterol, which is particularly enriched in the trans Golgi(22) . Our evidence that cholesterol may be transported by a Golgi-dependent route is consistent with these previous studies (8, 9, 22) and supports the possibility that this route may also serve as a mechanism for the sorting of proteins, as proposed recently(23) .
Plasma membrane cholesterol is not evenly distributed throughout the bilayer but is instead localized into cholesterol-rich and cholesterol-poor domains(24) , which show different exchange rates in the presence of lipoprotein acceptors(25) . We do not know if the SCP-2-dependent and the Golgi/cytoskeleton-dependent routes are carrying cholesterol to any specific plasma membrane domains, and if they are playing a specific role in reverse cholesterol transport. These possibilities would imply the presence of docking proteins, or receptors in the plasma membrane, and would give a functional role to the two different routes of intracellular cholesterol transport.