Proteolysis of Normal and Mutated Steroidogenic Acute Regulatory Proteins in the Mitochondria: the Fate of Unwanted Proteins
Zvi Granot,
Ruth Geiss-Friedlander,
Naomi Melamed-Book,
Sarah Eimerl,
Rina Timberg,
Aryeh M. Weiss,
Karen H. Hales,
Dale B. Hales,
Douglas M. Stocco and
Joseph Orly
Department of Biological Chemistry (Z.G., R.G.-F., N.M.-B., S.E., R.T., J.O.), The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; Department of Electronics (A.M.W.), Jerusalem College of Technology, Jerusalem 91160, Israel; Department of Physiology (K.H.H., D.B.H.), University of Illinois at Chicago, Chicago, Illinois 60612-7342; and Department of Cell Biology and Biochemistry (D.M.S.), Texas Tech University Health Sciences Center, Lubbock, Texas 79430
Address all correspondence and requests for reprints to: Dr. Joseph Orly, Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. E-mail: orly{at}vms.huji.ac.il.
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ABSTRACT
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Steroidogenic acute regulatory protein (StAR) is a nuclear encoded mitochondrial protein that enhances steroid synthesis by facilitating the transfer of cholesterol to the inner membranes of mitochondria in hormonally regulated steroidogenic cells. It is currently assumed that StAR activity commences before or during StAR import into the mitochondrial matrix. The present study was designed to demonstrate that, once imported and becoming physiologically irrelevant, exhaustive accumulation of StAR must be limited by a rapid degradation of the protein to prevent potential damage to the organelles. The use of uncouplers and manipulation of the interior mitochondrial pH in hormone-induced ovarian granulosa cells and StAR-expressing COS cells suggests that StAR degradation is biphasic and involves two classes of proteases. During phase I, which normally lasts for the first approximately 2 h following import, StAR is rapidly degraded by a protease, or proteases, that can be arrested by a nonclassical action of proteasome inhibitors such as MG132. StAR molecules that evade phase I are subjected to a second class of protease(s), which is slower and MG132 resistant. A third proteolytic entity was revealed in studies with C-28 StAR, a loss-of-function mutant of StAR. Upon initiation of its import, C-28 StAR dissipates the inner membrane potential and causes swelling of the mitochondria. Degradation of C-28 StAR, probably by an intermembrane space protease, is extremely rapid and MG132 insensitive. Collectively, this study defines StAR as the first naturally occurring mitochondrial protein that can serve as a substrate to probe multiple proteolytic activities in mammalian mitochondria.
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INTRODUCTION
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THE KEY REACTION in steroid hormone biosynthesis converts the substrate for all steroids, cholesterol, to pregnenolone, the first steroid molecule formed. This reaction is catalyzed by an inner mitochondrial membrane protein, the cholesterol side chain cleavage cytochrome P450 (P450scc) enzyme (1, 2, 3, 4, 5, 6). Steroidogenic acute regulatory protein (StAR) is a tissue-specific mitochondrial protein that facilitates the translocation of cholesterol from the outer membrane of the mitochondria to the inner membranes where P450scc can use it for steroidogenesis (7, 8). StAR is acutely up-regulated by trophic hormones that promote steroidogenesis in the adrenal cortex and the gonads (5, 9, 10, 11, 12). StAR is also expressed in the steroidogenic cells of the rodent placenta (13), whereas it is absent from the human placenta (14).
StAR is synthesized as a 37-kDa preprotein that contains a typical N-terminal mitochondrial targeting sequence, 62 amino acids long in human and 47 residues in the rodent preprotein. The latter presequence is cleaved upon StAR import into the matrix, which yields a 30-kDa mature mitochondrial protein (4, 15, 16, 17). The exact mechanism of StAR action is still obscure. Currently, two potential models for StAR action are being considered. The more intuitive model is supported by recent crystallographic studies suggesting that a 210- to 220-amino acid domain at the carboxy end of StAR may fold to form a cholesterol-binding channel that could facilitate the shuttling of cholesterol across the intermembrane space (IMS) while StAR is being imported (18). This new information conforms well with established studies that have shown that the carboxy domain is required for StAR activity because a number of mutations in this region render StAR inactive (5, 7, 16, 18, 19) and cause postnatal death due to adrenocortical hormone insufficiencies (5, 20, 21).
The second model of StAR action hypothesizes that mitochondrial import is not necessarily required for StAR activity. It was conceived as a result of unexpected findings (16), which showed that, in transfected COS cells, N-terminally truncated StAR proteins still retain full activity in the apparent absence of their import into the mitochondria (7, 8). The steroidogenic activity of the human N-62 and murine N-47 StAR mutants, however, was still dependent on an intact mitochondrial membrane potential (22, 23) Additional studies suggested that normal StAR stimulates cholesterol transfer while acting on the outer surface of the mitochondria, before import (19, 24, 25). It was further proposed that the mitochondrial targeting sequence of StAR may have two roles: first, to increase the local effective concentration of bioactive StAR preprotein at the outer mitochondrial membrane by binding to the mitochondrial import receptor (25); second, to facilitate StAR import and thereby termination, rather than activation, of StAR bioactivity (19, 24). In both models, it seems imperative that the perpetuation of cholesterol transfer and steroidogenesis requires the constant synthesis and import of newly synthesized StAR molecules.
Whatever the mechanism of StAR action may be, the acute response of steroidogenic cells to hormones implies that their mitochondria are confronted with an accumulation of StAR protein. Furthermore, according to the current perception of StAR action, it is highly likely that, once inside the mitochondrion, StAR has no direct physiological role in steroidogenesis, and eventually the accumulation of this useless protein might damage the organ (26). Indications for potential damage to mitochondrial shape and function were also observed when we expressed a deletion mutant of StAR, C-28 StAR (7), which was the first loss-of-function mutated protein identified in patients with lipoid congenital adrenal hyperplasia (5) This study shows that C-28 StAR acts as a potent uncoupling protein. In an attempt to reveal the cellular strategy employed to minimize potential damage caused by post-, or nonfunctional StAR proteins, we studied the degradation of StAR after import. For comparison, it was necessary to also characterize the turnover of StAR preprotein in the cytosol, showing that the latter is probably degraded by the proteasome. Proteolysis of mature StAR was studied in intact cells by manipulating the inner mitochondrial environment with the use of uncouplers and protease inhibitors. These experiments suggest that proteolysis of wild-type and C-28 StAR in the mitochondria is likely to be carried out by compartmentalized proteases of the organelle.
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RESULTS
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Degradation of StAR Preprotein
To study the pattern of StAR import into the mitochondria and determine the fate of StAR preprotein, we used either primary rat granulosa cells treated with FSH or COS cells transiently expressing the murine StAR protein (Fig. 1
, A and B, respectively). Immunoprecipitation of pulse-labeled [35S]StAR revealed a typical 30-kDa mature StAR protein that accumulates in the mitochondrial matrix after being processed by the matrix peptidases (Fig. 1
, lanes 1, 5, 9, and 13). As expected, StAR import was inhibited when the cells were labeled in the presence of carbonyl cyanide m-chlorophenylhydrazone (mCCCP) that dissipates the mitochondrial membrane potential by acting as a protonophore. Consequently, the mature form of StAR did not accumulate (Fig. 1
, lanes 2, 6, 10, and 14). However, in the presence of mCCCP, the cytosolic 37-kDa preprotein form was not recovered, suggesting that the preprotein undergoes a rapid degradation. Such degradation of the preprotein could be inhibited by incubating the cells with a reversible inhibitor of the proteasome, MG132 (Fig. 1
, lanes 4 and 12), or an irreversible proteasome inhibitor, clasto-lactacystin ß-lactone (not shown). Furthermore, epoxomicin, an anticancer antibiotic shown to be an efficient and more specific inhibitor of the proteasome (27) also inhibited the turnover of StAR preprotein (Fig. 1
, lanes 8 and 16). Other than the 30- and 37-kDa protein bands, two intermediate forms of StAR were revealed in the presence of mCCCP plus MG132 or epoxomicin (Fig. 1
, lanes 8, 12, and 16). Similar intermediate forms of StAR were previously described by Artemenko and colleagues (28), who suggested that these p32 and p35 forms are processed and accumulate in the IMS in Y-1 cells treated with mCCCP or o-phenanthroline.

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Fig. 1. Import and Cytosolic Degradation of StAR Preprotein
A, Primary granulosa cells were pulse labeled for 3 h with [35S]methionine added together with FSH (100 ng/ml). The effects of mCCCP (5 µM), MG132 (20 µM), and epoxomicin (20 µM) on the cell content of StAR forms were examined as described in Materials and Methods. Immunoprecipitation of [35S]StAR was conducted using anti-StAR serum, and the resulting proteins were resolved on SDS-PAGE as shown in Materials and Methods. B, COS cells expressing the murine StAR protein were labeled with [35S]methionine and analyzed as described in panel A. C, COS cells expressing StAR were used to examine the effects of mCCCP, valinomycin, and nigericin on the import of StAR preprotein with or without MG132. p, StAR preprotein (37 kDa); m, mature StAR (30 kDa). Two StAR intermediate forms (I and II) are denoted with arrows (lanes 7, 8, 11, 12, 15, and 16).
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Accumulation of MG132-stabilized StAR preprotein was also observed when COS cells were incubated with other drugs that manipulate the electrochemical potential (
) and/or
pH across the inner mitochondrial membrane. Potassium ionophore valinomycin and the K+/H+ exchanger, nigericin, were used in such experiments. As expected, valinomycin blocked StAR import (Fig. 1C
, compare lanes 1 and 4) due to dissipation of the inner mitochondrial membrane potential 
(but retention of
pH). In contrast, nigericin allowed import (Fig. 1C
, compare lanes 1 and 6) because it abrogates
pH without affecting 
, which is necessary for StAR import. As before, the presence of MG132 inhibited the degradation of StAR preprotein (Fig. 1C
, lanes 3, 5, and 7). It should be noted that despite the apparent involvement of the proteasome in degradation of StAR preprotein, as suggested by the effect of the proteasome inhibitors, we were not able to demonstrate the presence of a ubiquitinated StAR species (not shown) using various techniques often used to identify ubiquitin adducts (29).
To better characterize the cytosolic degradation of StAR preprotein, we conducted time-dependent pulse-chase experiments using 35S-labeled COS cells expressing wild-type StAR, as well as two mutants of this protein. Figure 2
shows that the estimated half-life of the wild-type preprotein, trapped in the cytosol due to the presence of mCCCP, did not exceed 15 min (Fig. 2D
). By contrast, the half-life of a nonimportable N-47 StAR mutant, lacking its mitochondrial leader sequence, was as long as 6.5 h. Further reduction of the N terminus of StAR to N-81 (a product of the downstream ATG initiation of translation) drastically shortened the half-life back to a scale of minutes (Fig. 2B
). A relatively short half-life was also observed for a third nonimportable StAR mutant (Fig. 2C
), lacking both the N terminus and 28 amino acids of the carboxy terminus, N-47/C-28 StAR (t1/2 = 1 h), suggesting that, more than anything else, protein conformation probably determines the rate of StAR degradation by the proteasome.

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Fig. 2. Turnover of StAR Preproteins in the Cytoplasm
Pulse-chase patterns of StAR forms expressed in COS cells were analyzed as described in Materials and Methods. A, To test the turnover of the wild-type StAR preprotein (WT PreP), mCCCP was added to the cells 15 min before termination of the pulse and kept in the medium thereafter. The cytoplasmic turnover rates of the nonimportable N-47 (B) and N-47/C-28 (C) StAR mutants were evaluated in a similar fashion without the need for mCCCP treatment. At the indicated times of chase, cell monolayers were harvested for immunoprecipitation with StAR antiserum. Autoradiograms of 35S-labeled StAR proteins are shown. D, A quantitative PhosphorImager (Molecular Dynamics, Sunnyvale, CA) analysis of the results, presented as percent of maximal labeling at time 0 of chase. Similar findings were obtained in three repeats of this experiment. p, StAR pre-protein (37 kDa); m, mature StAR (30 kDa). The 28-kDa and 25-kDa StAR forms observed in cells transfected with the N-47 and N-47/C-28 cDNAs, respectively, are products of downstream ATG initiators of translation.
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Turnover of Mitochondrial StAR: Biphasic Degradation
To examine the clearance rate of StAR inside the mitochondria, we performed pulse-chase experiments in metabolically labeled primary granulosa cells treated with FSH. Figure 3A
shows a typical chase experiment comparing the turnover rates of 35S-labeled mature forms of StAR and P450scc. The time-dependent levels of the immunoprecipitated proteins suggest a t1/2 of 4.8 h for StAR turnover and 21 h for the half-life of P450scc.

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Fig. 3. Turnover Rates of Mitochondrial StAR and P450scc Proteins in Granulosa Cells
A, Chase analyses in FSH-treated granulosa cells were conducted as described in Materials and Methods. At the indicated times of chase, cell monolayers were harvested for immunoprecipitation (IP) with StAR or P450scc antisera. Depicted are representative autoradiograms of 35S-labeled mature forms of StAR and P450scc (30 and 54 kDa, respectively). The graph shows PhosphorImager analyses of 10 and three independent pulse-chase experiments testing the half-life of StAR and P450scc, respectively. The quantitative results are presented as percent of maximal labeling at time zero of chase. B, Chase analyses of StAR degradation in the mitochondria were conducted in the presence of the proteasome inhibitors (20 µM), MG132, clasto-lactacystin ß-lactone (cLßL), or epoxomicin added 15 min before the onset of the chase. This experiment was repeated twice with similar results. Phase I designates the first 2 h during which the degradation of mature StAR was fully inhibited by the proteasome inhibitors. Phase II designates the following time interval during which StAR degradation resumed despite the presence of the inhibitors.
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Surprisingly, the addition of MG132, or lactacystin, at the onset of the chase period inhibited the degradation rate of the mature StAR localized within the mitochondrial matrix (Fig. 3B
). This observation suggested that MG132 and lactacystin can affect mitochondrial nonproteasomal proteases. Importantly, although epoxomicin had an inhibitory effect on the degradation of StAR preprotein in the cytosol, this more specific inhibitor of the proteasome did not affect the degradation of the 30-kDa StAR (Fig. 3B
), consistent with the idea that mitochondria do not contain true proteasome components.
The stabilizing effect of MG132 and lactacystin was biphasic: during the initial 2 h of chase, MG132 and lactacystin did not allow any StAR degradation (Fig. 3B
). We termed this postimport interval phase I. A second phase commenced when degradation of StAR ensued despite the presence of the inhibitors (phase II). To test whether the latter pattern of StAR degradation represents a generalized phenomenon, we also examined the fate of recombinant StAR transiently expressed in COS cells. Figure 4A
shows a similar biphasic pattern of StAR degradation in StAR expressing COS cells. Furthermore, a second fresh dose of MG132 added 2 h after the onset of the chase (Fig. 4A
) failed to extend the inhibitory effect of MG132, suggesting that MG132 activity was not neutralized by cellular metabolism. Moreover, when MG132 was added for the first time 2 h after the onset of the chase period (Fig. 4B
), it did not inhibit StAR degradation at all. This result suggested that the inhibitor was effective during a limited window of time immediately after import (phase I).

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Fig. 4. Effects of MG132 on the Biphasic Degradation of StAR Expressed in COS Cells
A, COS cells were transfected with StAR plasmid and 24 h later a chase experiment was conducted as described in Materials and Methods. The inhibitor MG132 (20 µM) was added 15 min before the onset of the chase (1st), and a second fresh dose was added at 2 h of chase (2nd). Control cells were untreated. B, The experiment described in panel A was repeated while MG132 was added only once, 2 h after the onset of the chase period (1st, arrow). These experiments were repeated twice.
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Effect of mCCCP on Mitochondrial Degradation
Unexpectedly, a more rapid degradation of StAR was observed when mCCCP was added to granulosa cells at the onset of the chase period, i.e. after StAR has already been imported into the mitochondrial matrix. The 30-kDa mature StAR form was degraded at t1/2of 3 h, or less, instead of nearly 5 h as determined for control cells without mCCCP (Fig. 5A
). When MG132 was added together with mCCCP at time 0 of the chase, the protonophore extended the inhibitory effect of MG132 up to 4 h (Fig. 5A
), instead of a 2-h phase I, typically observed in the presence of MG132 alone (Fig. 3B
). As previously observed, during phase II, the levels of mitochondrial StAR began to decline despite the presence of the protease inhibitor (Fig. 5A
). The effect of mCCCP seemed specific for StAR because the protonophore did not have any effect on degradation of an integral membrane protein, such as P450scc, nor did it affect the levels of cytochrome c residing in the IMS (Fig. 5B
). Also, mCCCP had no effect on the degradation rate of green fluorescent protein targeted to the matrix of COS cell mitochondria (not shown).

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Fig. 5. Accelerated Degradation of Mitochondrial StAR in mCCCP-Treated Granulosa Cells
A, Chase experiments of StAR degradation in granulosa cells were conducted as described in Fig. 3 . In addition, mCCCP was added (5 µM), either alone or with MG132 (20 µM), 15 min before the onset of the chase. The autoradiograms depict the turnover rate of the mature mitochondrial form of StAR (30 kDa). Control cells (Cont.) were not treated with any drug. Presented are average values of four independent experiments. B, To allow expression of P450scc in granulosa cells, FSH was applied 17 h before pulse labeling with [35S]methionine, as described in Fig. 3A . Then, the chase period was conducted in the absence or presence of mCCCP. At the indicated time points the cells were extracted, and immunoprecipitations of mitochondrial P450scc (54 kDa) or cytochrome c (Cyt. c, 15 kDa) were performed as shown. This experiment was repeated twice with similar results.
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A significantly faster degradation of StAR was also observed in StAR-expressing COS cells (Fig. 6A
). Also, mCCCP potentiated the inhibitory effect of MG132 during phase I (Fig. 6A
), suggesting that the fate of StAR was similar irrespectively of the cell type expressing this protein. Comparable results were seen in testicular MA-10 cells, where treatment with mCCCP decreased the half-life of mature StAR from 6 h to approximately 3 h (Hales, K.H., and D. B. Hales, unpublished observation).

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Fig. 6. Effects of mCCCP, Valinomycin, and Nigericin on Degradation of Mitochondrial StAR in COS Cells
COS cells were transfected with StAR plasmid, and 24 h later a chase experiment was conducted as described in Materials and Methods. Fifteen minutes before the onset of the chase (time 0) the cells received either 5 µM of mCCCP (A), 10 nM of valinomycin (B), or 0.5 µM nigericin (C). Where indicated, MG132 was added once, together with the uncouplers. Cells without treatment served as controls. Presented are typical autoradiograms of each experiment, while average values (n = 4, n = 2 and n = 4 for A, B, and C, respectively) are depicted in the graphs. Other than valinomycin, the t1/2 values of StAR in mCCCP- and nigericin-treated cells were significantly shorter than in control cells. The effect of the inhibitors on the length of phase I is denoted by the shaded area.
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StAR Degradation Is Affected by the Matrix pH
We next questioned whether the effects of mCCCP on StAR degradation result from dissipation of the inner membrane potential 
. To test this possibility, we used an alternative uncoupler of the mitochondria, valinomycin, which acts as a potassium ionophore. Figure 6B
shows that valinomycin had no significant effect on the degradation rate of StAR, nor did it potentiate MG132 action during phase I. On the contrary, it is tempting to speculate that in the presence of MG132 plus valinomycin, the duration of phase I was much reduced (Fig. 6B
) when compared with MG132 alone (Fig. 4A
). By contrast, nigericin, which causes a charged neutral dissipation of
pH and intensifies 
(30), effectively accelerated the overall degradation rate of mitochondrial StAR and tripled phase I up to 6 h without StAR degradation (Fig. 6C
). These results suggested that the observed effects of mCCCP on StAR degradation did not result from dissipation of the mitochondrial inner membrane potential. Instead, we postulated that mCCCP and nigericin may have caused an acidification of the mitochondrial matrix by dissipating
pH and, thereby, generated the observed effects on StAR degradation. To validate this possibility, we treated COS cells with 125 mM sodium acetate added at the onset of chase. Staining COS cells with carboxy SNARF-1 AM acetate (4 µM, 1 h), a permeable long-wavelength fluorescent pH indicator, confirmed (not shown) that the mitochondrial pH decreased in cells treated with acetate, mCCCP, and nigericin, but not valinomycin. As shown in Fig. 7A
, acetate accurately mimicked the effects of mCCCP or nigericin, i.e. the overall mitochondrial degradation of StAR was accelerated. If acetate was added together with MG132, the inhibitory phase I was extended up to 4 h (Fig. 7A
). We could also generate an opposite effect by adding ammonium chloride to mCCCP-treated cells. As shown in Fig. 7B
, alkalination of the mitochondrial matrix clearly abrogated the protective effect of MG132 during phase I, so that StAR degradation in the presence of mCCCP and MG132 was no longer biphasic.

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Fig. 7. Effects of Sodium Acetate and Ammonium Chloride on Degradation of Mitochondrial StAR in COS Cells
Chase experiments testing the rate of StAR degradation in COS cells were conducted using the same protocol described in Fig. 6 . A, At time 0 of chase the cells received either 125 mM Na-acetate (Ac-), with or without MG132. Cells without treatment served as controls. B, Ammonium chloride (NH4+) was added to the medium (125 mM) at time 0 of chase, together with mCCCP and MG132. Presented are typical autoradiograms of each experiment, while average values (n = 3) are depicted in the graphs. The half-life value (t1/2) of StAR in acetate-treated cells was significantly shorter than control cells.
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Degradation and Fate of C-28 StAR Mutant
An additional degradation pattern was revealed when we examined the fate of the C-28 truncated mutant of StAR. Pulse-chase experiments in COS cells showed that the degradation of the C-28 protein was extremely rapid (t1/2 = 1 h). Also, mCCCP did not affect degradation and MG132 did not inhibit the turnover of the C-28 protein (Fig. 8A
). Oddly, C-28 StAR seemed to exist only as a preprotein form of approximately 31 kDa (Fig. 8A
). This observation was substantiated by pulse experiments that showed no effect of mCCCP on the single form of C-28 StAR examined in cells with or without MG132 (Fig. 8B
, lanes 14). The apparent lack of C-28 StAR processing was more clearly demonstrated after a longer exposure of the radioactive gel to x-ray film, as shown in lanes 58 of Fig. 8B
. By contrast, control experiments with cells expressing wild-type StAR showed typical mCCCP-inhibitable import of the preprotein (Fig. 8B
, lanes 910), which was detected in large amounts if examined in the presence of MG132 (Fig. 8B
, lane 12). These results suggested that the 31-kDa C-28 preprotein was never processed by the matrix peptidases to yield a mature form as expected. It should be noted that another nonfunctional mutant of StAR, the human A218V StAR, was normally imported and degraded as shown in a typical chase experiment (Fig. 8C
).

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Fig. 8. The Effects of mCCCP and MG132 on Degradation of C-28 StAR Expressed in COS Cells
A, Chase experiments performed with COS cells expressing C-28 StAR and treated with MG132 or mCCCP, as described in Materials and Methods. Control cells did not receive any treatment during the chase period. This experiment was repeated twice with similar results. B, COS cells expressing either wild-type (WT) StAR or C-28 StAR were pulse labeled with [35S]methionine in the presence of mCCCP or MG132, or the two together, as described in Fig. 1B . Shown are immunoprecipitation patterns that allow for a comparison between the different forms of StAR proteins. To provide compelling evidence for the lack of C-28 processing in the mitochondrion, the gel analyzing the C-28 protein (C-28) was exposed to x-ray film twice, once for 1 h (lanes 14) and once for 3 h (C-28*, lanes 58). Processed C-28 protein, if it existed, would have been expected to migrate in the gel as a 27-kDa protein. The gel depicting the wild-type StAR analysis (WT, lanes 912) was exposed to the x-ray film for 1 h. Arrows denote intermediate forms of StAR, as described in Fig. 1 . C, Chase experiment performed with human A218V StAR (h-A218V) expressing COS cells without any treatment. Note a normal processing of the protein and a minute-scale clearance of the preprotein.
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The C-28 preprotein was clearly imported into the mitochondria, as demonstrated by electron microscopy (Fig. 9F
) and double-immunofluorescence staining of COS cells with anti-StAR and anticytochrome c sera (Fig. 9
, B1 and B2). Furthermore, instead of the elongated shapes of mitochondria seen in cells expressing wild-type StAR (Fig. 9A
), C-28 StAR bearing mitochondria were rounded and swollen (Fig. 9
, B1 and B2). Such swelling and structural changes of the mitochondria are typical of mCCCP-treated cells (31). We confirmed that the mitochondrial swelling resulted from dissipation of their membrane potential, as monitored by JC-1, an indicator of mitochondrial membrane potential (30). Observations illustrated in Fig. 9C
shows that JC-1-stained mitochondria lost their red coloration after treatment with mCCCP, acetate, or valinomycin. By contrast, mitochondria in nigericin-treated monolayers contained red JC-1 aggregates and remained normally elongated (Fig. 9C
). Collectively, these results establish a firm correlation between membrane depolarization and changes in mitochondrial shape.

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Fig. 9. Expression of C-28 StAR Mutant Causes Mitochondrial Shape Changes and Dissipation of the Inner Membrane Potential
COS cells expressing wild-type StAR (A) and C-28 StAR (B1 and B2) proteins were analyzed by double immunofluorescence staining with anti StAR (red) and anticytochrome c (green) sera (Materials and Methods). Note typically elongated mitochondria in nontransfected cells (arrows, cytochrome c-labeled mitochondria in panels A and B2) and in a cell expressing the wild-type StAR protein (arrows, inset of panel A). In contrast, C-28-containing mitochondria are rounded and swollen (arrows, doughnut- shaped mitochondria depicted in insets of B1 and B2). n, Nucleus. C, COS cells were treated for 20 min with either mCCCP (5 µM), valinomycin (Valino., 10 nM), sodium acetate (125 mM), or nigericin (Nig., 0.5 µM) before a vital staining of the cells with JC-1 was performed. The resulting observations were analyzed by confocal microscopy (Materials and Methods). The insets depict higher power magnifications of the boxed mitochondria. Note that control cells without treatment (Cont.) and nigericin-treated cells have filamentous mitochondria with intact membrane potential, as indicated by orange aggregates along the organelles (arrows). Other treatments caused swelling of the mitochondria and resulted in doughnut-shaped mitochondria (arrows in insets). The predominant green staining of such mitochondria indicates a loss of  . n, Nucleus. D1, HEK293 cells expressing C-28 StAR were grown on CELLocate grids and stained with JC-1 as described in Materials and Methods. Loss of  (green mitochondria) was observed in the cells designated n1n5. These same cells also expressed C-28 StAR (orange mitochondria), visualized after fixation and processed for double-immunofluorescence staining using anti-StAR and anticytochrome c sera (cells n1n5, panel D2). Nontransfected cells (arrows in D1 and D2) displayed intact  demonstrated by JC-1 staining of the typically small mitochondria in the HEK293 cells (orange coloration, D1). E1E2, HEK293 cells expressing wild-type StAR (WT) were vitally stained with JC-1, followed by processing for double-immunofluorescence staining of StAR and cytochrome c, as described for D1D2. Note that all the cells in the monolayer, including transfected (n1n4) and nontransfected cells (arrows), exhibited normal membrane potential in their mitochondria (orange coloration, E1). F and G, Immunogold labeling of C-28 (F) and wild-type (WT, G) StAR proteins examined in mitochondria (mt) of COS cells prepared for electron microscopy 24 h after transfection (Materials and Methods). F, Note the gold particles that decorate the C-28 protein in the IMS (arrowheads in F). Asterisks denote protein-free swollen areas. G, Most of the wild-type protein is localized in the matrix, or next to the cristae membranes (gray-white borderlines). No labeling was observed in the cytosol (cyt). Bars, 100 nm.
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Because expression of C-28 StAR resulted in changes in mitochondrial shape similar to those observed after treatment of cells with mitochondrial uncouplers, we examined whether C-28 StAR may act as an uncoupling protein. To test this possibility, we examined individual cells in two ways; first, by JC-1 staining to assess 
, followed by fixation and second by immunofluorescence staining of the very same cells with anti-StAR serum. Figure 9
, D1 and D2, show clearly that the mitochondria in C-28-expressing cells lost their membrane potential as if treated with mCCCP, valinomycin, or acetate. No such depolarization was observed in wild-type StAR-expressing cells (Fig. 9
, E1 and E2).
It should be noted that the loss of membrane potential in C-28 StAR-expressing cells was not apoptogenic (32) because C-28-bearing mitochondria did not release their cytochrome c content (Fig. 9B
2). Also, as mentioned above, the immunoelectron micrograph shown in Fig. 9F
provided compelling evidence arguing that the C-28 protein is not localized on the outer surface of the mitochondria, but fully enters the organelle despite the loss of the electromotive force. To some extent, Fig. 9F
may suggest that the immunogold labeling of C-28 StAR is localized in the IMSs. By contrast, wild-type StAR was mostly positioned in the mitochondrial matrix (gray matter in Fig. 9G
and Ref. 17). Also, note that the amount of the gold particles labeling the C-28 StAR in the mitochondria was lower than that observed for WT StAR (Fig. 9G
), which could be explained by the high turnover of the C-28 protein.
Collectively, these findings suggest that C-28 StAR preprotein is not competent to complete import into the mitochondrial matrix. Due to its uncoupling activity, C-28 StAR can traverse only the outer mitochondrial membrane and does not cross the inner membrane; therefore, processing to a mature form was not observed. This model explains why mCCCP cannot impair import of the C-28 preprotein (Fig. 8B
, lanes 2 and 6), which is probably trapped in the IMS as a result of its ability to disrupt the 
. Importantly, these features were characteristic only of the C-28 mutant, because normal processing and degradation patterns were observed when we examined another loss-of-function mutation of StAR, the human A218V StAR protein (Fig. 8C
).
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DISCUSSION
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Rapid Degradation of StAR: the Rationale
Currently proposed models of StAR action accept the assumption that, after reaching the mitochondrial matrix, the mature form of StAR is no longer involved in steroidogenic activity. Therefore, the mitochondria of steroidogenic cells are a rare, if not the only, example of mitochondria that are acutely filled with mounting levels of a protein that, probably, does not serve any physiological purpose within the organelles. We hypothesized that such an accumulation, if not carefully controlled, could inflict potential damage to the mitochondria. In support of this assumption, our immunofluorescence and electron microscopy studies have recently shown that exaggerated expression of StAR in COS cells can cause time-dependent damage to the inner mitochondrial compartments, including cristae swelling and deformation (26) Because swelling and structural abnormalities of the mitochondria are often associated with cell death (32, 33), we hypothesized that the level of StAR in the mitochondria should be restrained, as part of a cellular defense mechanism, through the action of mitochondrial proteolysis. To test this possibility, we used hormonally regulated rat ovarian granulosa cells, which represent an authentic steroidogenic cell type, as well as COS cells transiently expressing StAR proteins. In both cell types, StAR was rapidly degraded, thus providing compelling evidence that the patterns of StAR turnover do not necessarily depend on specialized features of steroidogenic mitochondria, but is rather dictated by the structure of StAR itself.
Proteasomal Degradation in the Cytosol
To characterize StAR degradation in the mitochondria of metabolically labeled cells, the examination of StAR fate before import was inevitable. By use of pulse and pulse-chase approaches, we realized that the 37-kDa StAR preprotein was hardly detectable in the granulosa cells responding to FSH. Somewhat more of the preprotein was observable in transiently transfected COS cells, probably resulting from overexpression of the StAR cDNA. Moreover, both in COS and granulosa cells, the steady-state levels of StAR preprotein did not increase when import was efficiently blocked in the presence of mCCCP. Instead, the preprotein was degraded in a matter of minutes (t1/2 = 15 min). This suggests that, once synthesized, StAR preprotein must quickly associate with the mitochondria or is otherwise rapidly degraded in the cytosol. Similar estimates of cytosolic half-lives were previously reported for the human StAR preprotein (19) as well as other mitochondrial proteins such as aspartate aminotransferase (34). The latter study also noted that the rate of the import process of aspartate aminotransferase preprotein is 1 order of magnitude faster than the rate of its degradation in the cytosol. Collectively, these findings may explain why the faster process of StAR import was selected for termination of StAR activity, instead of the much slower alternative of degrading the preprotein while it is still positioned on the mitochondrial outer surface.
This study shows that the degradation of StAR preprotein is far from being a nonspecific process, as previously believed (34, 35) We show that StAR degradation in the cytosol can be inhibited by representatives of three classes of proteasome inhibitors, including MG132 (36, 37), as well as nonreversible inhibitors, such as clasto-lactacystin ß-lactone (38) and epoxomicin (39) Although we have not been able to provide evidence linking the degradation of StAR preprotein to the ubiquitin pathway, the utter arrest of degradation by a specific proteasomal inhibitor, such as epoxomicin, suggests that StAR preprotein is indeed removed by the proteasome. Future studies should test whether such elimination of the preprotein is unique for StAR or represents a more general mechanism by which the cells clear the cytosol of mislocated mitochondrial preproteins.
In contrast to the wild-type StAR preprotein, the markedly longer half-life of the N-47 StAR mutant, deprived of its presequence, suggests one of three alternatives. First, that N-47 StAR is not easily accessible for proteasomal degradation due to possible aggregation of the overexpressed protein, as shown before (7). However, this possibility is highly unlikely since N-47 StAR is not degraded in the presence of MG132 (26). The other two hypotheses are either that the mitochondrial presequence serves as a distinct contextual motif tagging StAR for proteasomal degradation in the cytosol or that the entire conformation of the preprotein is detrimental to its survival in the cytosol. The structural hypothesis is supported by the fact that, in contrast to the long half-life of the N-47 form (6.5 h), an additional deletion of the carboxy terminus renders the N-47/C-28 protein relatively unstable in the cytosol (t1/2 = 1 h).
Intramitochondrial Degradation of StAR
Once imported, mature StAR is degraded with an average half-life of 4.2 ± 0.7 h. As predicted, this turnover rate is several fold faster than that of cytochrome P450scc, cytochrome c, or green fluorescent protein targeted to the mitochondrial matrix. Unexpectedly, two of the proteasome inhibitors, MG132 and lactacystin, effectively inhibited the degradation of mature StAR in the mitochondria. To the best of our knowledge, our studies (Ref. 26 and present study) provide the first example of inhibitors that can modify the activity of mitochondrial proteases (40). The fact that StAR degradation inside the mitochondria was completely unaffected by a more specific inhibitor of the proteasome, epoxomicin, agrees well with the general notion that true proteasome components probably do not exist in the mitochondria. On the other hand, MG132 and lactacystin are not necessarily exclusive inhibitors of the proteasome and, therefore, may inhibit nonproteasomal protease(s) in the mitochondria. Earlier studies support this possibility by showing that MG132 (41) and lactacystin (39, 42) can inhibit other cellular proteases, in addition to their action on the proteasome. Further studies should elucidate the nature of the matrix proteases that are vulnerable to inhibition by MG132 and lactacystin. Those might be mammalian orthologs of prokaryotic proteases, such as Lon (43, 44, 45, 46), or members of the Clp protease family (47).
What structural features of StAR protein dictate its relatively short half-life inside the mitochondrion? Studies in bacteria and yeast models have shown that protein quality control systems recognize and degrade nonnative proteins, but not their normally folded counterparts (48, 49, 50). However, less is known about the parameters that determine the turnover rate of mitochondrial proteins in animal cells, or in plant chloroplasts (51). For example, there is no distinct destruction sequence (52) that would target StAR for a rapid degradation in the mitochondria. It is possible that the secondary structure of StAR could provide the means for marking this protein for a rapid turnover in the mitochondrion.
Manipulation of StAR Degradation: Effect of the Matrix pH
This study shows that the pattern of StAR degradation in the mitochondrion can be manipulated by reagents that alter the matrix pH. Acidification of the matrix pH by either mCCCP or nigericin accelerated the overall turnover rate of mitochondrial StAR. No such pattern was observed during treatment with the potassium ionophore, valinomycin, which causes even a slight increase of
pH, in addition to efficient dissipation of 
. This observation is consistent with the notion that the matrix pH modulates the pattern of StAR fate. In support of this model, direct acidification of the mitochondrial matrix by addition of acetate to the culture medium led to a 2-fold increase in the rate of StAR degradation.
Combining matrix acidification with MG132 treatment was instrumental in revealing another aspect of StAR degradation in the mitochondria. In the presence of mCCCP and nigericin, or acetate, the inhibitory effect of MG132 seemed to be biphasic. In control cells, MG132 fully arrested StAR degradation during the first 2 h. We designated this time interval as phase I, beyond which proteolysis resumed despite the presence of the inhibitor. However, in the presence of mCCCP, acetate, or nigericin, but not valinomycin, the inhibitory effect of MG132 during phase I extended up to 46 h. These results may suggest that acidification of the matrix prolongs phase I by either modifying the activity of the involved protease(s) or affecting the folding status of StAR and its consequent vulnerability to degradation.
The fact that MG132 was no longer effective if added beyond the phase I interval favors a mechanism involving more than one protease and led us to propose the interpretation of the data illustrated in Fig. 10
. We propose that in its refolded compact state, the mature form of StAR in the matrix does not remain as a soluble protein but, with time, translocates onto the surface of the inner cristae membranes, as previously noted by immunoelectron microscopy (17). In the new location, StAR degradation ensues at a somewhat slower rate by a phase II protease(s) that is apparently resistant to MG132 and lactacystin. Phase II protease(s) may be a membrane-bound member of the AAA (ATPases associated with various cellular activities)-proteases (48). Finally, it is possible that acidification of the matrix compartment retards the process of StAR translocation to the membranes and thereby extends the time interval of StAR residency in the matrix, where the protein is exposed to faster and MG132-inhibitable proteases. Future work will address this hypothesis in an attempt to identify the proteases involved in StAR degradation.

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Fig. 10. Hypothetical Illustration of StAR Degradation in the Mitochondria
This hypothetical model suggests that after importation via the mitochondrial translocase complexes TOM and TIM (A), StAR is processed in the mitochondrial matrix (B), and the mature protein undergoes a two-phase degradation process: during the first 2 h, designated phase I, the 30-kDa mature StAR is degraded by matrix proteases (C). The latter are inhibitable by MG132 and lactacystin, but not by epoxomicin. Then, it is suggested that StAR does not remain soluble and begins to adhere onto the matrix face of the cristae membranes (D). At this location, the translocated protein is probably degraded by a phase II protease, which is expected to be MG132 resistant. We also assume that acidification of the matrix by mCCCP, nigericin, or acetate slows down the translocation of StAR onto the cristae membranes. Consequently, phase I is extended because StAR remains susceptible to degradation by the matrix proteases for a longer time period (46 h), during which its degradation remains MG132 sensitive. E, It is hypothesized that once C-28 traverses the outer membrane (OM), it causes dissipation of  and the protein, therefore, is entrapped in the inter-membrane space (IMS), where it is rapidly degraded by IMS protease that is not affected by MG132 or acidification of the matrix. IM, Inner membrane; TOM, translocase OM; TIM, translocase IM.
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The Fate of C-28 StAR
A third class of mitochondrial protease(s) is probably involved in degradation of Gln258Stop (C-28) StAR, the first nonfunctional StAR mutant discovered in patients with lipoid congenital adrenal hyperplasia (20). Initially, we were intrigued by the fact that expression of C-28 StAR in COS cells caused mitochondrial swelling, a morphological change usually associated with a loss of membrane potential (7). Moreover, the study shows that normal import of C-28 StAR as a matrix protein does not occur because the addition of mCCCP does not generate the expected accumulation of the preprotein form. We have explained these results by showing that expression of C-28 StAR led to a loss of 
and, therefore, incomplete import of the protein into the mitochondrial matrix. If so, where is C-28 StAR located? First, our immunofluorescence images provide compelling evidence that C-28 StAR is exclusively associated with the mitochondria. Second, by immunoelectron microscopy studies, we excluded the possibility that C-28 StAR is positioned on the cytosolic face of the outer membrane. Third, the N-terminal sequence of C-28 StAR is not processed by the matrix peptidases. Therefore, it is tempting to speculate that, after traversing the outer membrane, an importation step which does not require 
, C-28 StAR probably remains trapped in the IMS. Our proposed model (Fig. 10
) also suggests that, in the IMS, C-28 StAR is probably recognized by a local, MG132-insensitive IMS protease that rapidly degrades this aberrant protein.
Other than mitochondrial swelling, the short-term effects of C-28 StAR in the transiently transfected cells were not deleterious. Mitochondria containing C-28 StAR did not release their cytochrome c content and did not show any signs of mitochondria agenesis. Transfected cells survived several days with no apparent signs of programmed cell death. The steady-state rate of ATP synthesis in C-28-bearing cells is, at worst, half of the normal rate (data not shown), which is most likely due to a partial impairment of their mitochondria function. Because intact membrane potential is required for cholesterol transfer activity of wild-type StAR (22, 23), we were intrigued by the apparent possibility that C-28 StAR and possibly other StAR mutants are functionally disabled due to dissipation of 
. However, mitochondria swelling, dissipation of 
, or altered processing were not observed in cells expressing loss-of-function mutants of StAR, including A218V, L275P, and
R272 (data not shown). These results suggest, therefore, that the uncoupling activity of the C-28 StAR is a unique feature of this protein, which might also account, at least in part, for its impaired function as a cholesterol transporter. On the other hand, activity loss in other StAR mutants (20, 53) seems to result from a net impairment of cholesterol binding due to direct mutations in its binding pocket, or possible perturbation of the roof covering the cholesterol binding tunnel in the StAR START domain (18).
This study revealed the C-28 StAR to be a rare example of an uncoupling protein. Other examples of such proteins are mitochondrial carriers known as uncoupling protein families in mammals and plants (54) However, because the uncoupling proteins are integral membrane proteins possibly derived from a proton/anion transporter ancestor, it is unlikely that their mechanism of action (55) bears any relevance to the action of the soluble C-28 protein. Therefore, the mechanism underlying the dramatic impact of C-28 StAR on mitochondrial structure and function remains a challenge for future studies.
Finally, in recent years, considerable progress has been made toward the characterization of proteolytic complexes essential for the removal and, thereby, the prevention of the accumulation of potentially harmful nonnative proteins in mitochondria of eukaryotic cells. Many studies on protein turnover in mitochondria were performed using yeast models and genetically engineered proteins modified to serve as substrates targeted to different compartments of the organelles (48) Much less is known about the functions of mitochondrial proteases in mammalian cells. In humans, it has been shown that loss-of-function mutations in genes encoding mitochondrial proteases are often associated with clinical disorders (43, 46, 56, 57). Therefore, molecular and biochemical characterization of such proteolytic activities is of prime importance. The present study suggests that StAR proteins can be used as authentic natural substrates to explore multiple mitochondrial proteases and provide new insights on their mode of action in healthy and diseased cells.
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MATERIALS AND METHODS
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Materials
Ovine FSH (NIDDK-oFSH-20) was kindly provided by National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Pituitary Program and A. F. Parlow (Harbor-UCLA Medical Center, Torrance, CA). Sodium orthovanadate, protease inhibitor cocktail (P 8340), carbonyl cyanide m-chlorophenylhydrazone, nigericin, and valinomycin were obtained from Sigma (St. Louis, MO). clasto-Lactacystin ß-lactone and MG132 were purchased from Calbiochem (San Diego, CA). Epoxomicin was obtained from Affinity Research Products (Manhead, Exeter, UK). JC-1 dye and carboxy SNARF-1 AM acetate (C-1272) were purchased from Molecular Probes (Eugene, OR). [35S]methionine was obtained from Amersham International (Little Chalfont, UK). Immunoreagents included a polyclonal rabbit antiserum to recombinant mature mouse StAR protein (12, 17), a polyclonal rabbit antiserum to rat P450scc (6), and mouse IgG1 antirat cytochrome c (clone 6H2.B4, PharMingen, San Diego, CA). Other antisera (obtained from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) included: peroxidase-conjugated goat antirabbit, lissamine rhodamine-conjugated affiniPure goat antirabbit IgG, peroxidase-conjugated AffiniPure goat antimouse IgG, and fluorescein-conjugated goat IgG antimouse IgG. Protein A Sepharose was from Sigma, and Protein G-Agarose was obtained from Boehringer Mannheim (Mannheim, Germany). Tissue culture media used for serum-free granulosa cell culture included DMEM and Hams F-12 nutrient media from GibcoBRL Life Technology (Paisley, Scotland, UK). The same manufacturer supplied the fetal bovine serum. COS cell culture medium (DMEM) and metabolic labeling starvation medium without cysteine and methionine (Hams F-12) were supplied by Biological Industries (Kibbutz Beit-Haemek, Israel).
Animals
Intact, immature female Sprague Dawley rats (21 d old) were obtained from Harlan (Jerusalem, Israel) and maintained under a 16-h light, 8-h dark schedule with food and water ad libitum. Animals were treated in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. All protocols had the approval of the Institutional Committee on Animal Care and Use, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem.
Cell Cultures
Primary rat ovarian granulosa cells were prepared from E2-primed animals and grown in serum-free cultures exactly as described previously (58). COS-1 and human embryonic kidney 293 (HEK293) cells were cultured in DMEM containing 10% fetal bovine serum, penicillin G sodium (100 U/ml), streptomycin sulfate (100 µg/ml), and 2 mM glutamine. COS cells were transfected with murine wild-type StAR cDNA or mutant StAR lacking the N-terminal 47 amino acids (N-47) subcloned into the expression vector PCMV-5 (7). To this end, 2 x 106 cells (0.8 ml) were electroporated (58) using 20 µg of plasmid DNA. Cells were then seeded into eight to 10 wells (24-well plate) containing medium and 10% fetal bovine serum. Transfection of HEK293 by polyethyleneimine was performed as described previously (59).
Plasmid Construction of StAR Truncations
The plasmid construction of the N-47 and C-28 amino acid truncated StAR mutants has been described previously (7). Construction of the StAR mutant truncated on both ends of the protein, designated N-47/C-28 StAR, was achieved by PCR using mouse wild-type StAR cDNA as template. The sequence encoding the first 47 amino acids at the N terminus of the mouse StAR protein was deleted, and the methionine at position 48 was used to initiate translation of the N-47 StAR mutant. The 5'-primer, 5'-CCAGAATTCACCATGGG TCAAGTTCGACG-3' was designed to introduce an EcoRI restriction site. At the carboxy terminus, a HindIII restriction site was introduced 17 bp downstream of the stop codon (TAA) using the primer 5'-ATATTCGAATTAGTTGATGATTGTCTTCGGC-3'. PCR was performed for 30 cycles, 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 dATP, dGTP, dTTP, and dCTP, 50 pmol of each primer, 5 U of Taq DNA polymerase, and 0.5 µg of pCMV5/StAR template. The PCR fragments were digested with EcoRI/HindIII and inserted into a pCMV plasmid cut with the same enzymes.
Metabolic Labeling and Immunoprecipitation
For pulse experiments, cells (
105) were metabolically labeled the day after seeding. After two washes and 1-hour incubation with cysteine-methionine-free medium (starvation medium), pulse labeling was initiated by addition of 22 µCi [35S]methionine in 0.2 ml per well (24-well plate, see above). For labeling of StAR in granulosa cells, FSH (100 ng/ml) was added concomitantly with starvation medium. In experiments designed to study both StAR and P450scc in the same cells, the addition of radioactive methionine commenced 17 h after the onset of FSH treatment to attain a linear rate of P450scc synthesis, which follows a typical lag period (60). Treatments with mCCCP or proteasome inhibitors began 15 min before addition of the [35S]methionine. Duration of the [35S]methionine pulse was 3 h. After thorough washings with growth medium, cells in each well were harvested with 0.25 ml RIPA buffer (61) containing protease inhibitor cocktail, 1 mM Na-vanadate, and 0.5 mg/ml BSA (ICN Biochemicals, Cleveland, OH). The cell lysate was transferred to a tube and kept on ice for 30 min. DNA and cellular debris were removed by centrifugation (2 min at 13,000 x g) and the supernatant was stored at -70 C until immunoprecipitation could be performed. Cells prepared for pulse-chase experiments were labeled with [35S]methionine, as above, and after washing of the radioactive medium, the onset of the chase period was initiated by further incubation of the cells in complete growth medium containing 1 mM methionine and 0.8 mM cysteine. Where indicated, mitochondrial uncouplers and/or proteasome inhibitors were added at the onset of the chase. At the indicated time points the cells were extracted by addition of RIPA buffer as described above.
For immunoprecipitation, frozen samples were quickly defrosted in a 37 C bath, and 4% of each well served to determine the total radioactivity. Antiserum was added to equal amounts of radiolabeled proteins (1:1000, 1:1000, and 1:500 dilutions for StAR, P450scc, and cytochrome c antisera, respectively) for a 2-h incubation on ice. After this incubation, 25 µl protein A-Sepharose beads (20% vol/vol), or protein G-agarose, were added to each sample, followed by a 1-h incubation in a revolving tube carousel at 4 C. Samples were centrifuged at low speed (200 x g) for 2 min, and the supernatants were transferred into new tubes and kept at -70 C for optional secondary precipitations. The pelleted beads were washed three times with PBS-Tween 20 (0.1%), and the immune complex was released with 15 µl SDS-PAGE sample buffer and 2 min boiling. The immunoprecipitated radioactivity was analyzed by SDS-PAGE using 10% gels (17). The dried gel was analyzed with a Fuji Bio-Imaging analyzer (BAS-1000, Fuji Photo Film Co., Tokyo, Japan) before further visualization by exposure (516 h at -70 C) to Kodak BioMax MS-1 film, using BioMax TranScreen low-energy intensifying screen (Eastman Kodak Co., Rochester, NY).
Immunofluorescence Staining of StAR-Expressing Cells
For immunolocalization studies of StAR proteins, COS-1 cells (2 x 106 cells/0.8 ml) were transfected by electroporation (62) using 20 µg DNA from wild-type or C-28 StAR plasmids (7). The electroporated cells were seeded onto 13-mm round glass slides placed in wells of a 24-well plate (Nunc, Roskilde, Denmark). After 48 h incubation in DMEM containing 5% fetal calf serum (Biological Industries), the cell monolayers were further processed for immunofluorescence and confocal microscopy. HEK293 cells were transfected by use of polyethyleneimine (2 µg StAR cDNA) as previously described (59). Fixation and staining procedures have also been described previously (7). COS cells were double-immunofluorescently stained by concomitant incubations with StAR and cytochrome c antisera (1:50 each), followed by incubation with lissamine-rhodamine-labeled goat (IgG) antirabbit IgG (1:20) and fluorescein-conjugated goat (IgG) antimouse IgG. Finally, cells were mounted for immunofluorescence confocal microcopy. Confocal images were acquired using a confocal microscope [Bio-Rad MRC-1024 scanhead (Bio-Rad Laboratories, Inc., Hercules, CA) attached to a Zeiss Axiovert 135M (Carl Zeiss, Inc., Thornwood, NY)]. The cells were excited with the 514-nm line of an argon ion laser, and the emission was detected using an emission filter with either 580- or 540-nm center wavelength (32-nm band width). Oil immersion objectives [x63/(numerical aperture, NA = 1.4) or x40/(NA = 1.3)] utilizing an iris aperture of 12 mm resulted in optical sections between 12 µm. All cells were scanned using the same scanning conditions (laser power, gain of photomultiplier tube, iris size, and zoom).
JC-1 Staining
For the purpose of JC-1 staining, COS cells were seeded onto a glass slide (12-mm diameter) glued onto the outside of a 35-mm tissue culture dish (Falcon 3001, Becton Dickinson, Plymouth, UK) in which a 10-mm hole was drilled at the center. This custom-made dish allowed scanning of live cells with an inverted microscope [Bio-Rad 1024 confocal workstation (Bio-Rad Laboratories, Inc.)]. To detect dissipated membrane potential resulting from the mCCCP treatment, we used 5 µM JC-1 added for a 15-min incubation (37 C) before washing and confocal scanning. The cells were scanned using a 488-nm wavelength for excitation, and the dual emission of JC-1 was collected with a 525 ± 20 filter (green) and a 585 long-pass filter (red). Loss of membrane potential results in a concomitant loss of red emission, but the mitochondria remain visible when viewed through the green filter. Therefore, JC-1-stained cells are shown in red and/or red-green overlay. In cases where immunofluorescence of the same cells was required after JC-1 images were taken, the cells were seeded on CELLocate glass grids (Eppendorf-Netherler-Hinz, Hamburg, Germany), instead of the previously described regular glass slide. To this aim, we used HEK293 cells, of which up to 7085% of the cells in a typical monolayer express the recombinant protein, as compared with 1020% transfection efficiency of COS cells.
Immunoelectron Microscopy
Electron microscopy approaches for immunolocalization of recombinant StAR proteins expressed in COS cells have been detailed elsewhere (7).
Data Presentation and Statistical Analysis
StAR degradation data are presented as average percent of maximum 35S-labeled StAR protein. Students unpaired two-tailed t test was performed using Microsoft Excel 2001 statistical analysis functions. A level of P < 0.05 was accepted as statistically significant.
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ACKNOWLEDGMENTS
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We are indebted to Dr. Paolo Bernardi (University of Padua, Padua, Italy) for his creative and most helpful advice. We thank Dr. Phillip Nagley (Monash University, Melbourne, Australia) for inspiring discussions of the experimental evidence. We also thank Dr. Jerome Strauss III (University of Pennsylvania Medical Center, Philadelphia, PA) for providing the expression plasmids of human StAR mutants (A218V, L275P, and
R272) and Noa Sher for editorial help.
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FOOTNOTES
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This work was supported by Israel Science Foundation Grant 672/00 and United States-Israel Binational Foundation Grant 1999315 (to J.O.); NIH Grant HD 17481 and funds from the Robert A. Welch Foundation (to D.M.S.); and NIH Grants HD-27571 and HD-35544 (to K.H.H. and D.B.H.).
Present address for R.G.-F.: Max-Delbrueck Center for Molecular Medicine, Robert-Roessle Strasse 10, 13092 Berlin, Germany.
Abbreviations: HEK, Human embryonic kidney; IMS, intermembrane space; mCCCP, carbonyl cyanide m-chlorophenylhydrazone; P450scc, side chain cleavage cytochrome P450; StAR, steroid acute regulatoy protein.
Received for publication March 5, 2003.
Accepted for publication August 25, 2003.
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