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J Biol Chem, Vol. 273, Issue 26, 16339-16345, June 26, 1998
From the Steroidogenic acute regulatory protein (StAR)
plays an essential role in steroidogenesis, facilitating delivery of
cholesterol to cytochrome P450scc on the inner
mitochondrial membrane. StAR is synthesized in the cytoplasm and is
subsequently imported by mitochondria and processed to a mature form by
cleavage of the NH2-terminal mitochondrial targeting
sequence. To explore the mechanism of StAR action, we produced
6-histidine-tagged N-62 StAR (His-tag StAR) constructs lacking the
NH2-terminal 62 amino acids that encode the mitochondrial
targeting sequence and examined their steroidogenic activity in intact
cells and on isolated mitochondria. His-tag StAR proteins stimulated
pregnenolone synthesis to the same extent as wild-type StAR when
expressed in COS-1 cells transfected with the cholesterol side-chain
cleavage system. His-tag StAR was diffusely distributed in the
cytoplasm of transfected COS-1 cells whereas wild-type StAR was
localized to mitochondria. There was no evidence at the light or
electron microscope levels for selective localization of His-tag StAR
protein to mitochondrial membranes. In vitro import assays
demonstrated that wild-type StAR preprotein was imported and processed
to mature protein that was protected from subsequent trypsin treatment.
In contrast, His-tag StAR was not imported and protein associated with
mitochondria was sensitive to trypsin. Using metabolically labeled
COS-1 cells transfected with wild-type or His-tag StAR constructs, we
confirmed that wild-type StAR preprotein was imported and
processed by mitochondria, whereas His-tag StAR remained largely
cytosolic and unprocessed. To determine whether cytosolic factors are
required for StAR action, we developed an assay system using washed
mitochondria isolated from bovine corpora lutea and purified
recombinant His-tag StAR proteins expressed in Escherichia
coli. Recombinant His-tag StAR stimulated pregnenolone production
in a dose- and time-dependent manner, functioning at
nanomolar concentrations. A point mutant of StAR (A218V) that causes
lipoid congenital adrenal hyperplasia was incorporated into the His-tag
protein. This mutant was steroidogenically inactive in COS-1 cells and
on isolated mitochondria. Our observations conclusively document that
StAR acts on the outside of mitochondria, independent of mitochondrial
import, and in the absence of cytosol. The ability to produce bioactive
recombinant StAR protein paves the way for refined structural studies
of StAR and StAR mutants.
Steroidogenic acute regulatory protein
(StAR)1 is essential for
efficient adrenal and gonadal steroidogenesis (1, 2). The evidence that
StAR is critical for steroid hormone production has been derived, in
part, from the demonstration that mutations in the StAR gene cause
congenital lipoid adrenal hyperplasia, a disease in which adrenal and
gonadal steroid synthesis is severely impaired at the cholesterol
side-chain cleavage step (1, 3, 4). Targeted disruption of the murine
StAR gene results in a phenotype in the homozygous null mutants similar
to that of congenital lipoid adrenal hyperplasia in humans (5).
Although these observations demonstrate that StAR plays a key role in
steroidogenesis, the mechanism of the action of the protein remains
obscure. The protein is believed to stimulate the movement of
cholesterol from the mitochondrial outer membrane to the inner membrane
where cytochrome P450scc, the enzyme that catalyzes the
first step in steroid hormone synthesis, resides.
StAR expression is acutely regulated by trophic hormones (6, 7). Cyclic
AMP influences StAR gene expression, like many proteins important for
steroidogenesis (8-10), and enhances StAR activity by triggering
posttranslational modifications (11-13).
The NH2 terminus of StAR is characteristic of proteins
destined to be imported into mitochondria (14-16). Radiolabeled
pre-StAR is incorporated into isolated mitochondria and processed
to the mature 30-kDa protein (15, 17). Immunoelectron microscopy localized StAR to the intermembranous face of cristae and the intermembranous space (17). Based on these observations it has been
suggested that contact sites form between the outer and inner membranes
during the import of StAR into mitochondria, permitting cholesterol to
move to P450scc on the inner membranes. Recent reports
identifying StAR in isolated mitochondrial membrane contact sites (18),
and the inhibition of StAR action by compounds that disrupt the
mitochondrial electrochemical gradient and protein import (19),
supported the notion that StAR import is obligatorily linked to the
stimulation of steroidogenesis (2). However, we previously reported
that NH2-terminal deletion mutants of StAR had
steroidogenic activity equivalent to wild-type StAR, despite the fact
that they could not enter into mitochondria (20).
To shed light on the mechanism of StAR action and resolve apparent
discrepancies in the models of how StAR works, we embarked upon
experiments to produce biologically active recombinant StAR and study
its activity on isolated mitochondria. We elected to produce human StAR
proteins lacking the mitochondrial import sequence in bacteria, and to
incorporate a 6-histidine-tagged N-62 StAR (His-tag StAR) to facilitate
their purification. By using a construct from which the first 62-amino
acid residues had been deleted (N-62), we could examine the action of
StAR independent of the protein import process. Here we show that the
His-tag StAR proteins have biological activity equal to wild-type StAR
and that they act without being imported into mitochondria. We further
demonstrate that purified His-tag recombinant protein acts directly on
mitochondria to stimulate pregnenolone synthesis.
StAR cDNA Constructs for Transfection--
To produce both
NH2- and C-terminal His-tag StAR proteins lacking the first
62 amino acids, a cDNA encoding human StAR sequences from amino
acid 63-285 was cloned into the pQE-30 (21) and pET24 vectors (see
below), which place 6His-tags at the NH2 and C termini of
the cDNA, respectively. His-tag sequences were subcloned into pSV-SPORT-1. The A218V mutation, which causes congenital lipoid adrenal hyperplasia (4), was introduced by site-directed mutagenesis into the His-tag StAR construct (20). DNA sequences of all constructs were verified before use (20).
Cell Culture and Evaluation of Steroidogenic Activity--
COS-1
cells were cultured to 50-80% confluence and transfected using 10 µg/ml LipofectAMINETM (Life Technologies, Inc.) with 1 µg/ml of
either an empty pSV-SPORT-1 plasmid, wild-type or His-tag StAR
cDNAs in pSV-SPORT-1 and 1 µg/ml of a plasmid-directing
expression of a fusion protein consisting of human P450scc
adrenodoxin and adrenodoxin reductase (1, 3, 22), kindly provided by
Dr. Walter L. Miller, University of California, San Francisco. Culture media were changed after 24 h, and some cultures received 5 µg/ml 22(R)-hydroxycholesterol. Media were collected
36 h later, and cells were scraped from the dishes in
homogenization buffer consisting of 0.25 M sucrose, 10 mM Tris-HCl, pH 7.4, 10 mM EDTA (20). Relative
steroidogenic activity was determined by normalizing pregnenolone
production in the absence of 22(R)-hydroxycholesterol to
pregnenolone formation in the presence of the exogenous substrate, which reflects maximal cholesterol side-chain cleavage activity (3,
20). Experiments included triplicate cultures in each treatment group
and were repeated on at least three separate occasions.
Western Blotting--
COS-1 cells collected into homogenization
buffer were sonicated for 5 s. Disrupted cells were centrifuged at
600 × g for 15 min and the supernatant was used for
the Western blot analysis (20).
Metabolic Labeling Experiments--
COS-1 cells transfected with
plasmid-directing expression of wild-type and C-His-tag StAR were
incubated in methionine/cysteine-free Dulbecco's minimal essential
medium without serum for 30 min and then labeled with
[35S]methionine/cysteine (20 µCi/ml) for 4 h.
Carbonyl cyanide m-chlorophenylhydrazone (m-CCCP;
40 µM) or 1,10-phenanthroline (2 mM) were
added to some cultures. After labeling, cells were collected into
homogenization buffer and sonicated for 5 s. Disrupted cells were
centrifuged at 600 × g for 15 min, and supernatants
were spun at 13,000 × g for an additional 20 min to
isolate the cytoplasm and the mitochondrial-enriched subcellular
fraction. In some cases the mitochondrial fraction was treated with
proteinase K (15 µg/ml) at 4 °C for 90 min. After protein assay
(Pierce), equal aliquots of protein from each fraction were pre-cleared
with 30 µl of protein A-agarose in a total volume of 1 ml of RIPA
buffer (50 mM Tris-HCl, 1% Nonidet P-40, 0.1% deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM
EDTA, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, 2 µg/ml leupeptin,
1 mM dithiothreitol, 0.1 mM PMSF) for 30 min at
4 °C. After pelleting the protein A-agarose, supernatants were
incubated with 10 µl anti-human StAR antibody and 30 µl protein
A-agarose at 4 °C on a rocking platform overnight. Immunocomplexes
were washed four times by resuspension in 500 µl of RIPA buffer and
collected by centrifugation. Pellets were resuspended in 50 µl of
2 × SDS sample buffer and then subjected to SDS-PAGE and
fluorography. In some experiments protein bands corresponding to
wild-type StAR preprotein, wild-type mature protein, and C-His-tag StAR
were excised from the gels, and radioactivity was quantified by
scintillation counting.
The Mechanism of Action of Steroidogenic Acute Regulatory
Protein (StAR)
StAR ACTS ON THE OUTSIDE OF MITOCHONDRIA TO STIMULATE
STEROIDOGENESIS*
**,**,,,, and§
Center for Research on Reproduction and
Women's Health and the Department of Obstetrics and Gynecology,
§ Physiology, and ¶ Biochemistry and Biophysics,
University of Pennsylvania Medical Center, Philadelphia,
Pennsylvania 19104
ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
Immunocytochemistry-- COS-1 cells grown on uncoated coverslips were transfected with expression plasmids for wild-type and His-tag StAR and processed for immunocytochemistry (21). Nonspecific antibody binding was blocked by incubation with 1.5% normal goat serum and then cells were incubated with primary antibody (1:500 dilution). Controls consisted of substitution of the primary antibody with preimmune serum used at a dilution equal to that of the primary antibody.
Immunoelectron Microscopy-- COS-1 cells were fixed in 0.2 M cacodylate buffer (pH 7.4) containing 2% paraformaldehyde and 0.2% glutaraldehyde for 1 h at 4 °C, dehydrated in an increasing series of ethanol treatments, and embedded in LR Gold resin (Polysciences, Fort Washington, PA). Grids containing sections were incubated in blocking buffer (Tris-buffered saline (20 mM Tris, pH 7.5, 0.5 M NaCl) containing 10% normal goat serum) for 30 min at room temperature and were subsequently incubated in blocking buffer containing a 1:400 dilution of rabbit anti-StAR antiserum or preimmune rabbit serum overnight (~16 h) at 4 °C. Following three washes in Tris-buffered saline, grids were incubated in blocking buffer containing 18 nm colloidal gold conjugated to a goat anti-rabbit IgG antibody (Jackson Immunoresearch Laboratories, West Grove, PA) diluted 1:50. After three more washes in Tris-buffered saline, grids were fixed and counterstained with 1% osmium tetroxide followed by 7% aqueous uranyl acetate and Reynold's lead citrate. Specimens were observed and photographed using a Phillips 201 transmission electron microscope.
Mitochondrial Import Assays-- Wild-type and mutant StAR proteins were synthesized using an SP6 TNT-coupled in vitro transcription/translation kit (Promega) following the manufacturer's protocol for 2 h at 30 °C. Import assays of radiolabeled protein were performed as described previously (23). Selected reactions were performed in the presence of valinomycin (5 µg/ml), an electrochemical uncoupler that blocks active import. Some mitochondria were treated with trypsin (0.1 mg/ml) in the absence or presence of 0.5% Triton X-100 following the import reaction. Trypsin was neutralized, using soybean trypsin inhibitor (5 mg/ml), and mitochondria were washed prior to sonication, followed by separation of proteins by SDS-PAGE and autoradiography.
Preparation of Purified StAR Protein--
cDNA-encoding StAR
lacking the amino-terminal 62-amino acid residues was amplified from a
StAR cDNA clone using the following primers
5'-GGGAATTCCATATGCTGGAAGAGACTCTC-3' and
5'-GCCTCTGAAGCCAGGTGTCTCGAGCGGCCC-3' and cloned into the pET-24 vector
(Novagen) using XhoI and NdeI restriction sites,
creating a C-His-tag StAR cDNA. The C-His-tag StAR A218V mutant was
constructed using identical primers and mutant cDNA template. These
constructs were expressed in BL-21(DE3) cells (Novagen) at 37 °C
using 1 mM isopropyl-1-thio-
-D-galactoside for 4 h, bacterial cell pellets were sonicated in 300 mM NaCl, 50 mM NaH2PO4,
20 mM Tris, pH 7.4, 10 mM 2-mercaptoethanol,
and 0.5 mM PMSF. Bacterial lysates were centrifuged at
13,000 × g for 30 min and incubated with
nitrilotriacetic acid-chelate resin (Qiagen) for 30 min. The resin was
washed with lysis buffer (minus PMSF) and then lysis buffer containing
20 mM imidazole, until washes demonstrated
A280 < 0.01. His-tag StAR was eluted using 250 mM imidazole, dialyzed into a solution consisting of 50 mM KCl, 10 mM HEPES, 1.0 mM
dithiothreitol, 0.1 mM PMSF, and stored frozen in this
buffer.
Assay of Steroidogenic Activity Using Isolated Mitochondria and
Purified StAR Proteins--
Bovine corpora lutea were homogenized at
4 °C using a buffer consisting of 0.25 M sucrose, 10 mM Tris, pH 7.4, 1.0 mM EDTA, 1 mM
dithiothreitol, 0.1 mM PMSF, 1.0 µg/ml aprotinin, and 10 mg/ml bovine serum albumin. Homogenates were spun at 600 × g for 10 min, and supernatants were spun at 13,000 × g for an additional 20 min to obtain the
mitochondrial-enriched subcellular fraction. The pellets were washed
with homogenization buffer and subjected to the centrifugation steps
described above a second time. The final mitochondrial pellets were
resuspended in a modified homogenization buffer that contained only
1 mg/ml bovine serum albumin and were used immediately or stored at
80 °C. Preliminary experiments established that freezing of the
mitochondria did not impair StAR protein import and processing or
steroidogenic activity.
-hydroxysteroid
dehydrogenase inhibitor trilostane, 100 µM GTP, 10 mM isocitrate, 200 µM cholesterol, and the
indicated concentrations of purified StAR protein. Incubations were
conducted at 37 °C for the indicated times. As a control, purified
StAR protein was heat-denatured at 100 °C for 5 min. Incubations
were terminated by flash freezing of samples. Pregnenolone was
quantitated by radioimmunoassay (3, 20).
In some experiments, [3H]-cholesterol (0.005 µCi/µl)
was included in the incubation reactions to assess the conversion of
exogenous substrate into pregnenolone. Samples were then flash frozen
and extracted with 1.5 ml petroleum ether. The organic phases
were dried under nitrogen gas and resuspended in chloroform/methanol (2:1, v/v) and loaded onto silica gel G thin layer plates, which were
developed in hexane/ethyl acetate (7:3, v/v). Pregnenolone bands
detected with iodine vapor were collected, and radioactivity was
quantitated using a scintillation counter (24).
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RESULTS |
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Abstract
Introduction
Procedures
Results
Discussion
References
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His-tag StAR Proteins Are Biologically Active-- Before producing recombinant protein for in vitro studies, we documented that the His-tag did not affect the activity of StAR in COS-1 cells transfected with an expression plasmid for the human cholesterol side-chain cleavage system (1, 20, 25). Both the NH2- and the C-His-tag N-62 StAR proteins stimulated pregnenolone secretion by COS-1 cells to the same extent as wild-type StAR (Fig. 1A). Moreover, the His-tag StAR proteins containing the A218V mutation, which inactivates full-length StAR, were devoid of steroidogenic activity. Expression of each of the His-tag StAR proteins was documented by Western blot analysis (Fig. 1B). Wild-type StAR preprotein and mature protein were identified in transfected COS-1 cells and only a single protein of 32 kDa was identified in COS-1 cells transfected with the His-tag StAR constructs. The apparent molecular mass of the His-tag StAR in the SDS-PAGE system (32 kDa) is greater than the calculated molecular mass of approximately 26 kDa, which may reflect posttranslational modification of the protein. The level of expression of the NH2-His-tag StAR A218V mutant was approximately one-third of that for the NH2-His-tag "wild-type" protein, probably reflecting relative instability of this mutant.
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The His-tag StAR Proteins Are Not Imported into Mitochondria-- At the light microscope level, wild-type StAR was located in vermiform organelles representing mitochondria (Fig. 2A). The C-His-tag StAR protein was diffusely distributed in the cytosol of the transfected COS-1 cells without selective mitochondrial localization (Fig. 2B). Cells transfected with empty plasmid did not stain for StAR (Fig. 2C). Immunoelectron microscopy confirmed that the C-His-tag StAR protein was distributed throughout the cytoplasm and excluded from mitochondria (Fig. 3B), whereas wild-type StAR protein accumulated inside the mitochondria (Fig. 3A). Preimmune serum demonstrated negligible staining (Fig. 3C).
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Purified C-His-tag StAR Stimulates Pregnenolone Synthesis by Isolated Mitochondria-- To determine whether StAR can act directly on mitochondria, we developed an in vitro assay system using mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in Escherichia coli (Fig. 7A). The recombinant C-His-tag StAR protein stimulated pregnenolone production more than 10-fold. This stimulation was dose- and time-dependent, with a 2.5-fold increase in pregnenolone synthesis observed at a 20 nM concentration of C-His-tag StAR (Fig. 7B). Increases in pregnenolone synthesis were seen after 15 min of incubation, the first time point assayed, and continued for 90 min of incubation. In four separate experiments, 10 µM C-His-tag StAR produced a 10.5- ± 4.2-fold (mean ± S.E.) increase in mitochondrial pregnenolone synthesis after 90 min of incubation. Denaturation of the recombinant C-His-tag StAR abrogated its steroidogenic activity (Fig. 7C). The A218V mutant C-His-tag StAR protein was incapable of increasing pregnenolone synthesis (Fig. 7D).
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DISCUSSION |
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Abstract
Introduction
Procedures
Results
Discussion
References
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Investigations into the mechanism of action of StAR have failed to reveal to date how the protein promotes steroidogenesis. Indeed, proof that StAR acts directly on mitochondria, rather than via an intermediary pathway, has not been forthcoming. King et al. (17) reported stimulation of pregnenolone synthesis by isolated mitochondria incubated with StAR-expressing COS cell lysates. Because these experiments did not employ purified protein, the possibility remained that the StAR activity in the cell lysates was dependent upon factors present in the COS cell cytosol. This potential explanation deserved serious consideration in view of the evidence that import of StAR is not required for its steroidogenic activity. Our data, using purified recombinant StAR and isolated mitochondria, strongly suggest a direct effect of StAR on the organelles. Furthermore, our studies provide a system and a rationale for a search for the mitochondrial outer membrane molecules through which StAR acts to increase cholesterol metabolism.
StAR proteins, His-tagged either at the NH2- or C termini, stimulated pregnenolone synthesis in a well characterized transient transfection system with potency equal to wild-type StAR. Our prior studies indicated that the C-terminal half of the StAR protein contains the domains that are functionally important for stimulation of steroidogenesis (20). Thus, it is notable that addition of the His-tag to the C terminus did not affect the protein's biological activity. The observation that the C-terminal 4-amino acid residues can be deleted from StAR without impacting steroidogenic activity (20) and the present findings that the addition of sequences beyond amino acid residue 285 does not interfere with StAR's ability to stimulate pregnenolone synthesis argue that the tail end of the protein is not incorporated into functionally critical domains.
Immunocytochemistry at the light and electron microscopic levels yielded no evidence for selective accumulation of the C-His-tag StAR protein on the outer mitochondrial membranes. In contrast, wild-type StAR was almost exclusively localized inside of the mitochondria. These findings were corroborated by biochemical studies on metabolically labeled transfected COS-1 cells. Collectively, these observations strongly suggest that StAR acts on the outside of the mitochondrion. In previous work (20), we examined the distribution of wild-type and N-62 StAR in transfected COS-1 cells by immunoelectron microscopy. In that study we ignored the general distribution of the proteins with the goal of confirming that removal of the mitochondrial targeting sequence prevented import of the protein into mitochondria. The present experiments suggest that random interactions between StAR and the mitochondrial surface may be sufficient to promote steroidogenesis.
Convinced that the His-tag does not interfere with the action of StAR or introduce some artificial steroidogenic activity into the recombinant protein, we produced His-tag StAR in E. coli and tested its action on bovine corpus luteum mitochondria. Purified His-tag StAR stimulated pregnenolone production within minutes and at nanomolar concentrations, demonstrating a direct effect of the protein on mitochondria. Because we do not know whether all of the recombinant protein was biologically active, it is possible that StAR functions at much lower concentrations. Moreover, the in vitro assay system we used may not be optimal for documenting the steroidogenic activity of StAR. Thus, the minimally effective concentration of StAR needed to stimulate mitochondrial pregnenolone synthesis in vivo cannot be estimated.
We recently presented evidence that phosphorylation of StAR at serine residue 195 by protein kinase A is essential for maximal steroidogenic activity (11), consistent with earlier studies demonstrating that StAR is a phosphoprotein (13, 26). The recombinant proteins we employed were not phosphorylated. However, treatment with protein kinase A catalytic subunit and ATP did not increase the steroidogenic activity of recombinant His-tag StAR.2 This finding was not unexpected, because we have found that removal of the NH2-terminal 62 amino acids overcomes the negative impact of mutating serine residue 195 to a nonphosphorylatable alanine residue (27), indicating that phosphorylation either increases the activity of wild-type StAR by retarding mitochondrial import or overcomes a negative influence of the NH2 terminus.
In the absence of cytochemical and biochemical evidence for targeting of His-tag StAR to mitochondria, we speculate that StAR stimulates delivery of cholesterol to the mitochondrial inner membranes as a result of either a few high affinity stable interactions with the cytoplasmic face of the mitochondria or as a consequence of transient interactions. What could be the nature of these interactions? First, it is notable that mitochondria from nonsteroidogenic cells (e.g. COS-1 cells) respond to StAR. Therefore, StAR's action presumably involves molecules that are not uniquely expressed in steroidogenic tissues. Recent data implicate the peripheral benzodiazepine receptor, located on the outer mitochondrial membrane, in the pathway of StAR-mediated cholesterol translocation (28). Although the peripheral benzodiazepine receptor remains an attractive candidate for the mitochondrial StAR-interacting protein, alternatives should be entertained. One interesting possibility is that StAR participates in a process involving GTP hydrolysis. GTP hydrolysis is known to be important for substrate delivery to cytochrome P450scc (25), and GTPases participate in membrane trafficking and membrane fusion events (29, 30). Remarkably, there is homology between StAR and members of the RhoGAP family of GTPase activating proteins, although the homology does not encompass the catalytic domain.3 This homology raises the possibility that StAR triggers a change in the structure of mitochondrial membranes through an effect on a GTPase.
Pulse-chase studies suggest that the short functional life of wild-type StAR, predicted from studies demonstrating rapid inhibition of steroidogenesis by drugs that inhibit protein synthesis (e.g. cycloheximide), is not because of rapid destruction of the protein. Rather, the protein's short functional half-life appears to result from its rapid import into mitochondria. Orme-Johnson et al. (26) reported that the half-life of the StAR preprotein in rat adrenal cortex cells is 3-4 min. Our studies, carried out in a system that floods COS-1 cells with StAR preprotein, arrived at a longer half-life (15 min) that may be ascribed to saturation of the import process or possibly to a more efficient import system operating in cells with endogenous steroidogenic activity. We propose that mitochondrial import terminates StAR action, an interpretation that is entirely consistent with the idea that StAR acts outside of the mitochondria.
The conclusion that StAR stimulates steroidogenesis by acting on the outside of the mitochondria conflicts with the original notion that StAR must be imported to exert its function. The more recent observation that StAR is localized to contact sites between outer and inner mitochondrial membranes (18) is likely to reflect a role of this machinery in StAR import. The relevance of this finding to the steroidogenic function of StAR is entirely speculative, although our data strongly supports the notion that StAR import and StAR-mediated cholesterol transport are distinct processes.
Our studies have several other important implications. First, we have demonstrated a biological activity of a recombinant StAR protein that can be produced in E. coli in large quantities and easily purified, paving the way for protein crystallization and refined structural studies. Second, the activity of recombinant StAR protein on isolated mitochondria will permit further exploration of its mechanism of action, which has escaped elucidation in experiments using intact cells.
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ACKNOWLEDGEMENT |
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We thank Walter L. Miller, University of California, San Francisco, for the gift of the human cholesterol side-chain cleavage system fusion protein-expression plasmid.
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FOOTNOTES |
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* This work was supported by U.S. Public Health Service Grant HD-06274, a fellowship from the Lalor Foundation (to F. A.), and the University of Pennsylvania Medical Scientist Training Program (to C. B. K.).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.
** These authors contributed equally to this work.
To whom correspondence should be addressed: University of
Pennsylvania Medical Center, 778 Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-0147; Fax: 215-573-5408; E-mail: jstrauss{at}mail.med.upenn.edu.
1 The abbreviations used are: StAR, steroidogenic acute regulatory protein; His-tag StAR, 6-histidine-tagged N-62 StAR; m-CCCP, carbonyl cyanide m-chlorophenylhydrazone; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis.
2 F. Arakane, C. B. Kallen, and J. F. Strauss, III, unpublished observations.
3 M. E. Baker and J. F. Strauss, III, unpublished observations.
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REFERENCES |
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Abstract
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Discussion
References
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F. Alpy, V. K. Latchumanan, V. Kedinger, A. Janoshazi, C. Thiele, C. Wendling, M.-C. Rio, and C. Tomasetto Functional Characterization of the MENTAL Domain J. Biol. Chem., May 6, 2005; 280(18): 17945 - 17952. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. C. Yaworsky, B. Y. Baker, H. S. Bose, K. B. Best, L. B. Jensen, J. D. Bell, M. A. Baldwin, and W. L. Miller pH-dependent Interactions of the Carboxyl-terminal Helix of Steroidogenic Acute Regulatory Protein with Synthetic Membranes J. Biol. Chem., January 21, 2005; 280(3): 2045 - 2054. [Abstract] [Full Text] [PDF]
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