Endocrinology Vol. 139, No. 9 3903-3912
Copyright © 1998 by The Endocrine Society
Effect of Truncated Forms of the Steroidogenic Acute Regulatory Protein on Intramitochondrial Cholesterol Transfer1
Xingjia Wang,
Zhiming Liu,
Sarah Eimerl,
Rina Timberg,
Aryeh M. Weiss,
Joseph Orly and
Douglas M. Stocco
Department of Cell Biology and Biochemistry, Texas Tech University
Health Science Center (X.W., Z.L., D.M.S.), Lubbock, Texas 79430; the
Department of Biological Chemistry, The Alexander Silberman Institute
of Life Sciences, Hebrew University of Jerusalem (S.E., R.T., A.M.W.,
J.O.), Jerusalem 91904, Israel
Address all correspondence and requests for reprints to: Dr. Douglas M. Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Science Center, Lubbock, Texas 79430.
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Abstract
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It has been proposed that the steroidogenic acute regulatory (StAR)
protein controls hormone-stimulated steroid production by mediating
cholesterol transfer to the mitochondrial inner membrane. This study
was conducted to determine the effect of wild-type StAR and several
modified forms of StAR on intramitochondrial cholesterol transfer.
Forty-seven N-terminal or 28 C-terminal amino acids of the StAR protein
were removed, and COS-1 cells were transfected with pCMV vector only,
wild-type StAR, N-47, or the C-28 constructs. Lysates from the
transfected COS-1 cells were then incubated with mitochondria from
MA-10 mouse Leydig tumor cells that were preloaded with
[3H]cholesterol. After incubation, mitochondria were
collected and fractionated on sucrose gradients into outer membranes,
inner membranes, and membrane contact sites, and
[3H]cholesterol content was determined in each membrane
fraction. Incubation of MA-10 mitochondria with wild-type StAR
containing cell lysate resulted in a significant 34.9% increase in
[3H]cholesterol content in contact sites and a
significant 32.8% increase in inner mitochondrial membranes.
Incubations with cell lysate containing N-47 StAR protein also resulted
in a 16.4% increase in [3H]cholesterol in contact sites
and a significant 26.1% increase in the inner membrane fraction. In
contrast, incubation with the C-28 StAR protein had no effect on
cholesterol transfer. The cholesterol-transferring activity of the N-47
truncation, in contrast to that of the C-28 mutant, was corroborated
when COS-1 cells were cotransfected with F2 vector (containing
cytochrome P450 side-chain cleavage enzyme, ferridoxin, and ferridoxin
reductase) and either pCMV empty vector or the complementary DNAs of
wild-type StAR, N-47 StAR, or C-28 StAR. Pregnenolone production was
significantly increased in both wild-type and N-47-transfected cells,
whereas that in C-28-transfected cells was similar to the control
value. Finally, immunolocalization studies with confocal image and
electron microscopy were performed to determine the cellular location
of StAR and its truncated forms in transfected COS-1 cells. The results
showed that wild-type and most of the C-28 StAR protein were imported
into the mitochondria, whereas most of N-47 protein remained in the
cytosol. These studies demonstrate a direct effect of StAR protein on
cholesterol transfer to the inner mitochondrial membrane, that StAR
need not enter the mitochondria to produce this transfer, and the
importance of the C-terminus of StAR in this process.
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Introduction
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BIOSYNTHESIS of all steroids begins with
the conversion of cholesterol to pregnenolone, as catalyzed by the
cytochrome P450 side-chain cleavage enzyme (P450scc), which resides on
the matrix side of the inner mitochondrial membrane (1, 2). Delivery of
the substrate cholesterol to P450scc is a critical step because of the
hydrophobic nature of cholesterol and the aqueous barrier present in
the intermembrane space of mitochondria. To supply sufficient substrate
to P450scc, it is necessary to overcome this barrier, a step that
requires de novo protein synthesis (3, 4, 5). It has recently
been proposed that the protein responsible for effecting the transfer
of cholesterol to the inner mitochondrial membrane and thus acting as
the regulatory protein in this process is the steroidogenic acute
regulatory (StAR) protein (reviewed in Refs. 6, 7, 8, 9). Many studies have
shown that stimulation of various steroidogenic cells resulted in the
increased synthesis of steroids and a concomitant increase in StAR
expression (10, 11, 12, 13). Also, it has recently been demonstrated that
stimulation of bovine adrenal glomerulosa cells with Ca2+
increased StAR protein synthesis, cholesterol transfer to the inner
mitochondrial membrane, and steroid production in a temporally related
fashion (14, 15). However, definitive data indicating that the presence
of StAR protein can directly result in the transfer of cholesterol to
the inner mitochondrial membrane is still lacking, and little is known
about the mechanism by which cholesterol transfer results from StAR
expression.
Mutations in the StAR gene sequence of patients who suffer
from the potentially lethal disease, lipoid congenital adrenal
hyperplasia, indicated that the C-terminal portion of the StAR protein
was important in cholesterol delivery and steroid biosynthesis (16).
Further, it was demonstrated that constructs lacking the C-terminal
region, but not those lacking the N-terminal region, of the StAR
protein were unable to support steroid synthesis when transfected into
COS-1 cells (17). Therefore, in an effort to demonstrate a direct
link among specific regions of the StAR protein, cholesterol transfer,
and steroid biosynthesis, we constructedN- and C-terminal
truncations of StAR and examined their effects on cholesterol transfer
and steroid production. In addition, we determined their cellular
locations during the process of cholesterol transfer and steroid
biosynthesis.
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Materials and Methods
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Chemicals
DMEM, kynurenine sulfate, aminoglutethimide,
22(R)-hydroxycholesterol, low gelling temperature agarose
(A-4018), and BSA were purchased from Sigma Chemical Co. (St. Louis,
MO). Waymouths MB/752 medium, FBS, horse serum, Lipofectamine,
trypsin-EDTA, antibiotics, and PBS were obtained from Life Technologies
(Gaithersburg, MD, or Paisley, Scotland). [3H]Cholesterol
was purchased from DuPont-New England Nuclear (Boston, MA). Anti-StAR
antisera against amino acids 8898 of mouse StAR protein were produced
in rabbits by Research Genetics (Huntsville, AL). Mouse monoclonal
antibodies to cytochrome c oxidase subunit IV were purchased
from Molecular Probes (Eugene, OR). All of the restriction enzymes were
purchased from Promega (Madison, WI). Other common chemicals used in
these studies were obtained from either Sigma or Fisher Chemicals
(Fairlawn, NJ).
Cell culture
The COS-1 cell line was purchased from American Type Culture
Collection (Rockville, MD) and cultured in DMEM containing 10% FBS,
penicillin G sodium (100 U/ml), streptomycin sulfate (100 µg/ml), and
amphotericin B (250 ng/ml). MA-10 mouse Leydig tumor cells were a
generous gift from Dr. Mario Ascoli (Department of Pharmacology,
University of Iowa College of Medicine, Iowa City, IA) and were grown
in Waymouths MB/752 medium containing 15% horse serum as previously
described (18). Both COS-1 and MA-10 cells were incubated in 5%
CO2 at 37 C.
Plasmid construction of StAR truncations
To delete the first 47 amino acids at the N-terminus of the
mouse StAR protein, PCR was used, and the methionine at position 48 was
selected to initiate the N-47 truncated StAR mutant. The 5'-primer,
5'-CCA GAA TTC ACC ATG GGT CAA GTT CGA CG-3', was designed to introduce
an EcoRI restriction site. A HindIII restriction
site was introduced 17 bp downstream of the stop codon (TAA) using the
primer 5'-TAT AAG CTT AAT GTG GTG GAC AG-3'. PCR was performed for 30
cycles of 92 C for 1 min, 45 C for 30 sec, and 72 C for 30 sec, each
cycle in a buffer containing 50 mM KCl; 10 mM
Tris-HCl (pH 8.4); 0.1% Triton X-100; 2.5 mM MgCl; 0.2
mM each of deoxy (d)-ATP, dGTP, dTTP, and dCTP; 50 pmol of
each primer; 5 U Taq DNA polymerase; and 0.5 µg pCMV5/StAR
template. The PCR fragments were digested with
EcoRI/HindIII and inserted into the pCMV plasmid
(10) cut with the same enzymes.
For plasmid construction of the C-28 amino acid truncated StAR mutant,
the 5'-primer, 5'-CCA GAA TTC GTC GAC CCA CGC GTC CGC-3', was used to
introduce an EcoRI restriction site immediately before the
initiation codon (ATG) of the StAR complementary DNA (cDNA). The
3'-primer, 5'-ATA AAG CTT TTA GTT GAT GAT TGT CTT CGG-3', was designed
to introduce a HindIII restriction site and a stop codon at
the position of the C-28 amino acid. The 858-bp fragments obtained from
PCR were digested and inserted into EcoRI/HindIII
sites of the pCMV plasmid using the same procedure as that used for the
N-47 truncation.
Transfection of COS-1 cells
The procedure used for the transfection of COS-1 cells has been
previously described (19). Briefly, plasmids of pCMV, pCMV/StAR,
pCMV/N-47 StAR truncation (N-47), or pCMV/C-28 StAR truncation (C-28)
were transiently transfected into COS-1 cells for a 6-h period using
Lipofectamine (Life Technologies). Forty-eight hours after
transfection, COS-1 cells were harvested and sonicated three times for
30 sec each time in import buffer containing 0.25 M
sucrose, 10 mM MOPS buffer (pH 7.4 with KOH), 80
mM KCl, 5 mM MgCl, 3% BSA, 10 mM
isocitrate, and 2 mM NADH. The sonicates were centrifuged
at 600 x g to remove the debris, and the supernatants
were retained.
Steroid production in COS-1 cells
COS-1 cells were transfected for 6 h with plasmids
containing pCMV only, pCMV/StAR, pCMV/N-47, or pCMV/C-28 as well as
with F2 plasmid (20). The cells were cultured for an additional 48
h, and the medium was collected. The cells were washed with PBS and
cultured for an additional 2 h with 25 µM
22(R)-hydroxycholesterol to authenticate F2 transfection.
The medium was collected, and samples from both the 48- and 2-h
incubations were tested for pregnenolone production by RIA as
previously described (19).
Cholesterol transfer in MA-10 mitochondria
MA-10 cells were cultured overnight in 100-mm plates containing
1 µCi [3H]cholesterol/10 ml medium. The cells were
collected, washed in cold PBS, then suspended in import buffer
containing 0.76 mM aminoglutethimide to block
metabolism of cholesterol by the P450scc enzyme. The cells were gently
homogenized by hand using a glass on glass homogenizer. The MA-10 cell
homogenates were divided into four 1-ml aliquots. Five hundred
microliters of cell lysate from COS-1 cells transfected with pCMV,
StAR, N-47, or C-28 were then added to each aliquot of MA-10 cell
homogenate, mixed, and incubated for 2 h at 37 C. The samples were
diluted with 5 ml cold Tris buffer containing 10 mM Tris
(pH 7.4), 0.25 M sucrose, 1.0 mM EDTA, and 0.76
mM aminoglutethimide, and mitochondria were isolated as
previously described (21).
Subfractionation of mitochondria
Submitochondrial membrane fractions were separated on sucrose
gradients following a previously described procedure (14, 15, 22).
Briefly, the mitochondrial pellet containing 2 mg protein was
resuspended in cold 10 mM sodium phosphate buffer, pH 7.4,
containing 0.76 mM aminoglutethimide and kept on ice for 20
min. Sucrose (1.8 M) was then added to give a final
concentration of 0.45 M. After another 20-min period, the
mitochondria were sonicated three times for 30 sec each time. The
sonicated sample was centrifuged at 8000 x g for 15
min. The supernatant was collected and centrifuged at 150,000 x
g for 90 min. The membrane-containing pellet was resuspended
in 10 mM phosphate buffer, pH 7.4, containing 0.45
M sucrose and 0.76 mM aminoglutethimide. The
membrane suspension was layered onto a linear sucrose gradient
(1550%) and centrifuged at 100,000 x g for 20
h at 4 C. Each gradient was collected into 20 fractions. The locations
of mitochondrial membrane marker enzymes in this gradient were
determined. Fractions 38 (corresponding to high activities of outer
membrane-specific enzyme, kynurenine hydroxylase), 913 (containing
both kynurenine hydroxylase and cytochrome c oxidase), and
1418 (containing high amounts of cytochrome c oxidase, a
inner membrane marker) were pooled, respectively. The pooled fractions
were tested again to check the distribution of the marker enzymes. The
amount of [3H]cholesterol present in each fraction was
assayed using a Beckman LS 6500 scintillation counter (Beckman,
Fullerton, CA).
Marker enzyme assays
Kynurenine hydroxylase, an outer mitochondrial membrane enzyme,
was assayed following Bandlows procedure (23). The cytochrome
c oxidase content of each fraction was determined by Western
analysis using a monoclonal antibody against subunit IV and was
quantitated using a BioImage Visage 2000 (BioImage Corp., Ann Arbor,
MI) image analysis system, as previously described (21). The protein
concentration was determined using a modification of the Bradford
method (24).
Western blot analysis
The cell lysates from transfected COS-1 cells were tested for
expression of StAR and its truncations by Western analysis as described
previously (10). The samples were solubilized in sample buffer [25
mM Tris-HCl (pH 6.8), 1% SDS, 5% ß-mercaptoethanol, 1
mM EDTA, 4% glycerol, and 0.01% bromophenol blue],
boiled for 5 min, and loaded onto a 12% SDS-PAGE minigel (MiniProtean
II System, Bio-Rad, Richmond, CA). Electrophoresis was performed at 200
V for 45 min using a standard SDS-PAGE running buffer (25
mM Tris, 192 mM glycine, and 0.1% SDS, pH
8.3). The proteins were electrophoretically transferred to a
polyvinylidene difluoride membrane (Bio-Rad) at 100 V for 2 h at 4
C using a transfer buffer containing 20 mM Tris, 150
mM glycine, and 20% methanol, pH 8.3. The membrane was
incubated in blocking buffer (PBS buffer containing 4% Carnation
nonfat dry milk and 0.2% Tween-20) at room temperature for 1 h,
followed by incubation with a primary antibody against StAR for 30 min.
The membrane was washed with PBS containing 0.2% Tween-20 three times
for 10 min each time. After incubation with the second antibody, donkey
antirabbit IgG conjugated with horseradish peroxidase (Amersham,
Arlington Heights, IL), the membrane was washed five times for 10 min
each time. Specific protein bands were detected by chemiluminescence
using the Renaissance Kit (DuPont-New England Nuclear, Wilmington, DE),
and quantitated using the BioImage Visage 2000 (21).
Immunofluorescence staining and confocal microscopy analysis
For immunolocalization studies of wild-type and mutant StAR
proteins, COS-1 cells (2 x 106 cells/0.8 ml) were
transfected by electroporation (25) using 30 µg DNA StAR, N-47, or
C-28. The electroporated cells were seeded onto 13-mm round glass
slides and placed in wells of a 24-well plate (Nunc, Copenhagen,
Denmark). After 48-h incubation in DMEM containing 5% FCS (Biological
Industries, Kibbutz Beit-Haemek, Israel), the cell monolayers were
further processed for immunofluorescence and confocal microscopy.
Fixation and immunofluorescence staining procedures used to visualize
wild-type StAR and its mutants in cultured cells have been described
previously (26). Incubations of permeabilized cells (26) with antisera
to recombinant murine StAR (1:50) and lissamine-rhodamine-labeled goat
(IgG) antirabbit IgG (1:20) were performed before confocal analysis of
fluorescently labeled cells.
Confocal images were acquired using a confocal microscope (Bio-Rad
MRC-1024 scanhead attached to a Zeiss Axiovert 135M). The cells were
excited with the 514-nm line of an argon ion laser, and the emission
was detected using an emission filter with a 580-nm center wavelength
and a 32-nm band width. A x63/(NA = 1.4) objective was used, and
the iris aperture was 12 mm, resulting in optical sections between
12 µm. Intensity profiles were measured across the cells using
Image Pro Plus (Media Cybernetics, Silver Spring, MD). First, a 3
x 3 median filter was applied to the images to remove point noise.
Then, the average background value (as measured in an area in which
cells were not present) was subtracted from the images. The long
rectangle on each image denotes the area across which the profiles were
measured. For each rectangle, the intensity was averaged across the
short dimension of the rectangle, and these average values are shown as
a function of distance along the long dimension of the rectangle.
Immunoelectron microscopy of COS-1 cells
Forty-eight hours after electroporation, COS-1 cells were
harvested by brief trypsinization (2 min at 37 C) using commercial
trypsin-EDTA solution (Biological Industries). Trypsinization was
terminated by the addition of growth medium containing serum, and the
cells were collected by centrifugation and fixed for 1 h at room
temperature in a 0.05% electron microscopy grade glutaraldehyde
solution (Electron Microscopy Sciences, Fort Washington, PA) in PBS (pH
7.4), freshly prepared with 3% paraformaldehyde (BDH, Poole, UK), 0.1
mM CaCl2, and 1 mM
MgCl2. After a brief washing with PBS, excessive aldehyde
groups were neutralized by incubating the cells for 30 min in 0.1
M glycine in PBS. The cells were suspended in 50 ml melted
agarose (low gelling temperature type VII) prepared in water (2%) and
kept at 45 C. Cells were then pelleted by a brief centrifugation
(1000 x g), and the agarose was allowed to solidify at
room temperature. Small cubes (1 x 1 mm) of agarose-embedded
cells were dehydrated in graded dilutions of ethanol in water (30 min
in 30%, 50%, and 70%) and embedded in LR White resin (London Resin
Co., Bassingstoke, UK) as recently described (27). Thin sections were
incubated with 1:20 dilutions of rabbit antisera to recombinant mouse
StAR, followed by incubation with a 1:10 dilution of gold-labeled goat
antirabbit IgG, as previously described (26). Epon-embedded cells were
similarly fixed in 1% glutaraldehyde and 3% paraformaldehyde
solution. After osmication, Epon blocks were prepared using standard
procedures.
Statistical analysis
Each experiment was repeated at least three times. Statistical
analyses of the data were performed using ANOVA and Duncans multiple
range test using the Statistical Analysis System (SAS Institute, Cary,
NC)
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Results
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Characterization of submitochondrial membrane fractions
Membranes obtained from MA-10 cell mitochondria incubated with
transfected COS-1 cell lysates were separated into different fractions
by continuous sucrose density gradient centrifugation. The distribution
of marker enzymes in the fractions indicates the presence of three
distinct submitochondrial regions (Fig. 1
). Near the top of the gradient in the
least dense fractions (no. 38), a high level of the mitochondrial
outer membrane specific marker enzyme, kynurenine hydroxylase (23), was
found. In fractions close to the bottom of the tube, the more dense
fractions (no. 1418), a high concentration of cytochrome c
oxidase, a mitochondrial inner membrane enzyme, was found. In addition
to outer and inner mitochondrial membrane fractions, a third membrane
population of intermediate density was found. Fractions 913,
possessed both kynurenine hydroxylase and cytochrome c
oxidase, characteristic of mitochondrial contact sites.

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Figure 1. Separation of submitochondrial membrane fractions
on sucrose density gradients. Mitochondrial membranes suspended in 10
mM phosphate buffer, pH 7.4, containing 0.45 M
sucrose were loaded onto a 1550% linear sucrose gradient,
centrifuged at 100,000 x g for 20 h, and
collected into 20 fractions. Each fraction was analyzed for the
activity of the outer mitochondrial membrane enzyme, kynurenine
hydroxylase (KNH), which is expressed as nanometers of
hydroxykynurenine formed per mg protein. Western analysis of the inner
mitochondrial membrane protein, cytochrome c oxidase
(COX), was performed (not shown) and expressed as integrated optical
density (IOD) per mg protein in each fraction, also as shown. As a
result of these assays, fractions 38 (containing high activities of
KNH) were designated OM (outer membrane), fractions 913 (containing
both KNH and COX) were designated CS (contact sites), and fractions
1418 (containing a high content of COX) were designated IM (inner
membrane).
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Intramitochondrial cholesterol transfer
Figure 2
demonstrates the expression
of StAR and its truncations in COS-1 cells. Cells transfected with
wild-type StAR cDNA contained high levels of the 37-kDa StAR precursor
and the 30-kDa mature form as reported previously (19). N-47 and C-28
truncated proteins were also identified by Western blot analysis.
Lysates from COS-1 cells transfected with cDNA of StAR or its
truncations were incubated together with mitochondria from unstimulated
MA-10 cells that had been preloaded with [3H]cholesterol.
After incubation, the mitochondria were collected, sonicated, and
fractionated on sucrose gradients, and the various membrane fractions
were pooled and assayed for [3H]cholesterol content (Fig. 3
). Incubation of the mitochondria with
COS-1 cell lysate containing wild-type StAR protein resulted in a
highly significant 34.9% increase in [3H]cholesterol
content in the contact sites (P < 0.01) and a 32.8%
increase in the inner mitochondrial membranes (P <
0.01). Incubation with cell lysate containing N-47 protein increased
the [3H]cholesterol content of contact sites by 16.4%
(not significant) and that of the inner membranes by 26.1% (highly
significant, P < 0.01). The
[3H]cholesterol content in submitochondrial fractions
from C-28-treated mitochondria did not show any differences compared
with incubations with lysates from empty vector transfected cells.
There was no significant difference in the
[3H]cholesterol content of outer membranes among all the
treatments used in these studies.

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Figure 2. Western blot analysis of StAR and its truncations
expressed in transfected COS-1 cells. COS-1 cells were transfected with
plasmids of pCMV, pCMV/StAR, pCMV/N-47, or pCMV/C-28 using
Lipofectamine. Transfection was carried out for 6 h, followed by a
48-h incubation, at which time the COS-1 cell lysate was collected as
described in Materials and Methods and prepared for gel
electrophoresis. After electrophoresis, all samples were analyzed for
StAR content using Western analysis with a polyclonal antisera to StAR
protein as described in Materials and Methods. Lane 1,
pCMV; lane 2, pCMV/StAR; lane 3, pCMV/N-47; lane 4, pCMV/C-28.
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Figure 3. The effect of StAR protein and its truncations on
[3H]cholesterol transfer in MA-10 cell mitochondria.
MA-10 cells were incubated for 16 h with 1 µCi
[3H]cholesterol/10 ml medium. Cells were homogenized, and
the cell homogenate, containing 0.76 mM aminoglutethimide,
was incubated for 2 h with cell lysate from COS-1 cells
transfected with the cDNA pCMV empty vector (control), pCMV/wild-type
StAR, pCMV/N-47, or pCMV/C-28. The mitochondria were collected after
incubation. Mitochondrial membranes were prepared and separated into
outer membrane (OM), contact site (CS), and inner membrane (IM) on
sucrose density gradients as described, and each fraction was assayed
for [3H]cholesterol content. The
[3H]cholesterol contents (in disintegrations per min) of
OM, CS, and IM are shown in the inset. The amounts of
[3H]cholesterol in OM, CS, and IM treated with wild-type
StAR-, N-47-, or C-28-containing lysate were then expressed as a
percentage of that found using control lysate. **, Highly significant
(P < 0.01; n = 6).
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Steroid production in transfected COS-1 cells
The results of steroid production are shown in Fig. 4
. After cotransfection of COS-1 cells
with the cDNA of wild-type StAR and the F2 plasmid, the pregnenolone
concentration in the culture medium significantly increased from
161 ± 8 to 2013 ± 95 pg/ml (Fig. 4
). Cotransfection with
the N-47 truncation resulted in a similar increase (1691 ± 79
pg/ml). However, in the C-28 group, the pregnenolone concentration in
the culture medium was only 262 ± 12 pg/ml, similar to that in
controls. To test the transfection efficiency and activity of the
P450scc introduced by transfection with F2 plasmid,
22(R)-hydroxycholesterol was added as a substrate. There was
no significant difference in pregnenolone production among the groups
after the addition of 22(R)-hydroxycholesterol (Fig. 4
).

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Figure 4. The effect of StAR protein and its truncations on
steroid production in COS-1 cells. COS-1 cells were transfected with
plasmids containing pCMV empty vector, pCMV/StAR, pCMV/N-47, or
pCMV/C-28 and then cotransfected with F2 plasmid (a vector containing
P450scc, adrenodoxin, and adrenodoxin reductase) for each group. The
cells were cultured for 48 h, and the medium was collected. The
cells then were washed with PBS and cultured for an additional 2 h
with 25 µM 22(R)-hydroxycholesterol. The
media from the 48- and 2-h incubations were collected and assayed for
pregnenolone production by RIA. The absolute amount of pregnenolone
production is shown in the inset, and the values
obtained were then expressed as a percentage of wild-type steroid
production.
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Confocal and immunoelectron microscopy
Figure 5
, AC, shows the results of
immunofluorescence staining and confocal microscopy analysis of StAR
and its truncations expressed in COS-1 cells. Wild-type StAR protein
was readily imported into mitochondria, as reported previously (19),
and showed a distinct mitochondrial localization. In Fig. 5A
, StAR"
(left) depicts a low power conventional fluorescence image
of wild-type StAR. It shows a typical staining of filamentous
mitochondria (inset). As expected, only 1520% of the
cells in a typical transfection experiment expressed StAR, whereas
nonexpressing cells showed background diffuse staining. The same result
was shown by confocal image, shown in the middle panel (Fig. 5A
, StAR). The relative fluorescence intensities from quantitative
analysis of this image (see right panel) also indicated that
the majority of the StAR protein was localized in mitochondria. Figure 5B
shows the distribution of the N-47 protein in COS-1 cells, which is
quite different from that of wild-type StAR. The results from both the
confocal image (left panel) and quantitative analysis of the
confocal image (right panel) demonstrated the high
distribution of this truncated protein in the cytosol and nucleus. No
mitochondrial fluorescence signals could be detected that were above
the cytosolic labeling intensities. We also performed ultrastructural
localization of N-47 expression by immunoelectron microscopy. Figure 6
shows that, indeed, the vast majority
of the immunogold particles decorating the N-47 antigenic sites
remained in the cytosol, whereas the inner compartments of the
mitochondria were practically devoid of labeling. A control study of
COS-1 cells transfected with wild-type StAR (Fig. 6B
) showed the
reverse distribution of StAR, in which over 95% of the gold particles
were inside the mitochondria. It is noteworthy that in N-47 transfected
cells, a substantial number of the gold particles resided on the outer
mitochondrial membrane (Fig. 6A
). Also, in some of the cells expressing
high levels of the transfected construct, a massive concentration of
N-47 StAR antigen was noticed in electron-dense lysosome-like bodies
(Fig. 6A
').

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Figure 5. Immunolocalization of StAR mutants expressed
in COS-1 cells. COS-1 cells were transfected with different StAR cDNA
constructs, and 48 h later, the cellular localization of StAR was
studied by immunofluorescence analysis as described in Materials
and Methods. Confocal images (left panels) were
acquired using a confocal microscope, and the fluorescence intensity
profiles across the long rectangle denoted in each image were measured
as described in Materials and Methods. The right
panels present the average fluorescence intensity values across
the cells as a function of distance along the long dimension of the
rectangle. A, StAR" (left) depicts a low power
conventional fluorescence image of cells expressing wild-type StAR.
Note the typical staining of filamentous mitochondria
(inset) denoted by arrows. Diffuse
background staining of nontransfected cells (arrowheads)
represents a similar labeling pattern of cells transfected with empty
pCMV vector (not shown). StAR (middle panel) presents a
confocal image depicting two cells (fused as a result of
electroporation) expressing wild-type StAR. Quantitative analysis of
this image provides the relative fluorescence intensities (right
panel) in the mitochondria (m, black), the
cytosol (c, dotted), and the nuclear area (n,
gray). nc, Nucleolus. B, Confocal image of two cells
expressing the N-47 deletion construct of StAR. Image analysis was
conducted as described for StAR above. Note the high cytosolic and
nuclear staining. C, Confocal image of a single cell expressing the
C-28 deletion construct of StAR. Image analysis was conducted as
described for StAR above. Note the exclusive staining of the
mitochondria, most of which are circular in shape
(arrows). Bars represent 10 mm. D, a,
High power electron micrograph depicting two circular doughnut-shaped
mitochondria (thick arrows) in C-28-expressing cells.
Note the gold labeling (thin arrows) of C-28 StAR in the
mitochondria and an apparently "white" cavity, free of
electron-dense material (*), which confers a doughnut shape to the
immunofluorescently labeled mitochondria seen in C. Note that the
cytoplasm (Cyt) is free of gold labeling. D, b and c, The
doughnut-shaped mitochondria could also result from a curving process
of an elongated mitochondrion to form a closed circle, as demonstrated
by arrows in b (Epon-embedded cells) and c (LR
White-embedded cells). Note that this process creates a central
mitochondrial cavity that is filled with electron-dense cytoplasm (#)
entrapped by the fusing ends of the circular mitochondrion
(arrowheads in c). Magnifications: a, x50,600; b,
x40,400; c, x61,000.
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Figure 6. Ultrastructural localization of N-47 and wild-type
StAR expressed in COS-1 cells. COS-1 cells were transfected with N-47
StAR cDNA construct (A) or wild type StAR as a control (B). Forty-eight
hours later, the cells were harvested and prepared for immunogold
electron microscopy as described in Materials and
Methods. A, Note that the vast majority of N-47 StAR gold
labeling localizes to the cytosol (Cyt), and fewer particles (denoted
by circles) are observed inside the mitochondria (m).
Particles located on the outer membranes of the mitochondria are
denoted by arrows. Magnification, x50,600. A',
Lysosome-like electron dense bodies heavily labeled with StAR antibody
are frequently seen in N-47-expressing cells. Note that the size of
each vesicle (L) matches the size of the mitochondria
(arrowheads). Magnification, x40,400. B, Note
ample gold labeling of wild-type StAR inside the mitochondria
(thick arrows). By contrast, very few gold particles
(denoted by squares) are localized in the cytosol (Cyt).
Magnification: c, x61,000.
|
|
Localization of the C-28 protein in COS-1 cells is shown in Fig. 5C
.
The confocal image (left panel) shows exclusive staining of
the mitochondria, most of which are circular in shape. Quantitative
analysis also indicated that most of the C-28 protein was localized in
mitochondria. To better understand the unusual shape of the C-28
transfectants, immunoelectron microscopy and standard transmission
electron microscopy were conducted to reveal the ultrastructural
details of the aberrant mitochondria. Figure 5D
shows that either one
of two processes conferred a circular doughnut shape to the
mitochondria harboring the C-28 truncated StAR; Fig. 5D
a shows that the
C-28-containing mitochondria are devoid of electron-dense material in
their center, as if the cristae membranes collapsed to form an
apparently empty hole in the mitochondrial core. Osmicated sections of
such mitochondria (not shown) did not reveal any membrane-rich
structures, as has been documented in mitochondria of cells undergoing
necrosis (28). Alternatively, Fig. 5D
, b and c, depicting C-28
transfected cells, show a process for curving of an elongated
mitochondria to form a closed circle. Therefore, the circular
mitochondria appear to engulf the electron-dense cytoplasm observed in
the center portion of the organelles (indicated by #).
 |
Discussion
|
|---|
For steroid hormones to be synthesized, the precursor for all
steroids, cholesterol, must be transported to the inner mitochondrial
membrane where P450scc, which converts it to pregnenolone, resides. As
cholesterol is very hydrophobic, and its diffusion through water is
very slow (29, 30), it cannot traverse the aqueous intermembrane space
within a time frame consistent with the rate of steroid hormone
biosynthesis known to occur in response to trophic hormone stimulation.
Thus, the delivery of cholesterol requires some assistance if it is to
reach the P450scc enzyme rapidly, and it has long been known that this
step requires the de novo synthesis of a protein whose role
is believed to be the regulation of this transfer (31, 32, 33). A protein
known as the StAR protein has been shown to be a strong candidate in
fulfilling the role of this regulatory protein. The case for StAR as
the acute regulator has been made recently in several review articles
(6, 7, 8, 9).
Previous studies have indicated that de novo synthesis of
StAR protein resulted in steroid production. However, a direct link
between the presence of StAR protein and cholesterol transfer had not
been made. Three lines of evidence obtained in the present study
directly demonstrated the effects of StAR protein on intramitochondrial
cholesterol transfer. 1) After incubation of MA-10 mitochondria with
StAR-containing cell lysates, the [3H]cholesterol
contents of contact sites and inner mitochondrial membrane fractions
increased significantly over those in controls containing no StAR in
the lysates. These results are in agreement with previous studies that
indicated that stimulation of bovine adrenal glomerulosa cells with the
steroidogenic stimuli angiotensin II or Ca2+ resulted in an
increased content of cholesterol in both contact sites and the inner
mitochondrial membrane (14, 15). 2) The increase in cholesterol
transfer was directly correlated with an increase in steroid synthesis
when COS-1 cells were cotransfected with F2 plasmid and wild type StAR.
3) The lack of steroid production was also correlated with the
inability of a C-terminus truncated StAR protein to mobilize
[3H]cholesterol into the contact sites and inner
membranes. Conversely, the N-47 truncation mutant of StAR was capable
of conferring both high steroidogenic activity and cholesterol transfer
to the inner membranes of the mitochondria. The magnitude of the
cholesterol transfer observed in the present study in response to
wild-type and N-47 StAR is similar to that previously observed in
Ca+2-clamped bovine adrenal glomerulosa cells (14, 15).
Very little is known concerning the mechanism by which StAR protein is
involved in cholesterol transfer to the mitochondrial inner membrane.
Evidence from patients with lipoid congenital adrenal hyperplasia
strongly supports the importance of the C-terminal region of the StAR
protein in supporting steroidogenesis (16, 34, 35). One patient in
particular had a mutation in the StAR gene that resulted in the
insertion of a stop codon that removed the C-terminal 28 amino acids
from the wild-type StAR protein (16). The results from the present
study shows that the C-terminus of StAR is indeed critical in
cholesterol transfer, clearly illustrating that the C-28 truncation was
unable to transfer cholesterol to the inner mitochondrial membrane in
isolated MA-10 cell mitochondria. As further corroboration, expression
of this StAR cDNA in COS-1 cells indicated that the protein was
completely inactive in promoting steroidogenesis. Similar results were
obtained using mutated cDNAs from lipoid CAH patients in which
expression of C-terminal truncated forms of the StAR protein in COS-1
cells resulted in either partially diminished steroidogenic capacity
when 10 amino acids were removed or completely diminished capacity when
25 amino acids were removed (17).
No less curious were the results obtained with the N-47 mutant of StAR.
Very little of this truncated form of StAR lacking its signal peptide
entered the mitochondria, as indicated by Western blot analyses.
Confocal microscopy and ultrastructural visualization studies confirmed
that ample amounts of the N-47 truncated protein resided in the
cytosol, whereas the inner compartments of the mitochondria were devoid
of the antigen. Yet, a substantial, but not exclusive, immunogold
decoration of N-47 on the surface of the outer mitochondrial membrane
could suggest that truncation of the amino-terminus still allowed a
potential interaction of N-47 StAR with the organelle, even in the
visualized absence of its import. Such interaction may explain the fact
that the N-47 truncation is fully active in both cholesterol transfer
and stimulation of steroidogenesis.
Previously we have employed an affinity column constructed of a
synthetic signal sequence of the StAR protein and have used it to
purify a mitochondrial protein complex that showed specific binding to
a 35S-labeled signal peptide (36). Although the identities
of the components of this complex remain unknown, it is highly likely
that the N-terminal sequence of StAR binds to the mitochondrial
translocase machinery, which facilitates the import of the StAR
precursor into the organelle. As the N-47 truncation is fully active
biologically, whereas the transfected C-28 can neither augment
steroidogenesis nor facilitate cholesterol transfer, these observations
provide further evidence for the importance of the C-terminus for the
function of StAR. This finding also raises the question of the possible
function of the import process in steroidogenesis. An early model from
our laboratory hypothesized that cholesterol transfer may occur as a
result of the import and processing of the StAR protein into the
mitochondria (6, 7). This model was based on the well characterized
observation that import of mitochondrial proteins resulted in the
formation of contact sites between the outer and inner mitochondrial
membranes at the point of insertion of the protein (37, 38, 39). Therefore,
it was reasoned that perhaps during the formation of these contact
sites, cholesterol would be able to transfer to the inner mitochondrial
membrane, as the aqueous intermembrane barrier was essentially removed
(40). This model predicted that it was the insertion of the N-terminus
of the StAR protein that would be important for cholesterol transfer.
At this time, however, the majority of observations indicate that
import of the StAR protein into the mitochondria is not required for
its ability to stimulate steroidogenesis (16, 17). Thus, as indicated
previously, perhaps the import of the StAR protein functions as an off
switch by removing the protein from the outer membrane where its
function is expressed (17).
Like wild-type StAR, the C-28 truncated form was almost totally
imported into the mitochondria, as readily observed with confocal
microscopy analyses. Curiously, there were morphological differences in
the shape of the mitochondria after import of the C-28 form compared
with wild-type StAR import. Import of the C-28 protein resulted in the
formation of doughnut-shaped mitochondria as observed in the
immunofluorescence studies. This alteration in morphology was confirmed
in the results obtained from immunoelectron microscopy, which showed a
curving process of elongated mitochondria to form closed circular
organelles. The reasons for these morphological differences and their
possible consequences are unknown at this time. Perhaps the removal of
the C-terminal portion of the StAR protein affected the manner in which
the protein is refolded after import into mitochondria, resulting in an
improper conformation that caused the curving process.
Recent studies have shown that in addition to an increase in their
cholesterol content, the contact sites and inner mitochondrial
membranes of Ca2+-stimulated bovine adrenal glomerulosa
cells also contained StAR protein and the first two enzymes in the
steroidogenic pathway, namely P450scc and 3ß-hydroxysteroid
dehydrogenase (15, 22, 41). Therefore, the colocalization of these
three proteins raises the possibility that StAR initiates the formation
of a protein complex that could result in cholesterol transfer, then
convert cholesterol directly into progesterone. This complex may
contain 3ß-hydroxysteroid dehydrogenase and P450scc, as alluded to
above. In addition, based on the observations of Papadopoulos and
colleagues (42, 43, 44, 45), it is possible that if such a complex exists, it
may include the peripheral benzodiazepine receptor and its ligand the
diazepam binding inhibitor proteins. Interestingly, the possible
existence of a multiprotein complex in steroidogenic mitochondria,
referred to as hormonads, was predicted several years ago by Lieberman
and colleagues (46, 47). It will be of interest to determine whether an
association between any or all of these proteins exists in the
mitochondrial membrane and, perhaps even more interestingly, whether
they exist in contact sites and if this association occurs
preferentially with the C-terminal portion of the StAR protein.
 |
Acknowledgments
|
|---|
We are grateful to D. B. Hales and K. H. Hales for
providing the antiserum to recombinant murine StAR.
 |
Footnotes
|
|---|
1 This work was supported by NIH Grant HD-17481 (to D.M.S.), United
States-Israel Binational Sciences Foundation Grant 9500350, the
Israel Science Foundation founded by the Israel Academy of Sciences and
Humanities (Grant 547/97; to J.O.), and NIH Grant HD-07271 (to
Z.L.). 
Received February 18, 1998.
 |
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