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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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Introduction |
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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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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 Waymouth’s 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
(15–50%) 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 3–8 (corresponding to high activities of outer
membrane-specific enzyme, kynurenine hydroxylase), 9–13 (containing
both kynurenine hydroxylase and cytochrome c oxidase), and
14–18 (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 Bandlow’s 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 1–2 mm, resulting in optical sections between 1–2 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 Duncan’s multiple
range test using the Statistical Analysis System (SAS Institute, Cary,
NC)
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Near the top of the gradient in the
least dense fractions (no. 3–8), 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. 14–18), 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 9–13,
possessed both kynurenine hydroxylase and cytochrome c
oxidase, characteristic of mitochondrial contact sites.
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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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. 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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, A–C, 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 15–20% 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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.
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. 5Da 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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Acknowledgments |
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Footnotes |
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Received February 18, 1998.
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 A. A. Gonzalez, M. L. Reyes, C. A. Carvajal, J. A. Tobar, L. M. Mosso, P. Baquedano, A. Solar, A. Venegas, and C. E. Fardella Congenital Lipoid Adrenal Hyperplasia Caused by a Novel Splicing Mutation in the Gene for the Steroidogenic Acute Regulatory Protein J. Clin. Endocrinol. Metab., February 1, 20 |
