The SO-kDa Mitochondrial Proteins Induced by Hormone Stimulation ...

THEJOURNALOF

Vol. 266, No. 29, Issue of October 15, pp. 19731-19738,1991 Printed in U.S.A.

BIOLOGICAL CHEMISTRY

0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

The SO-kDa Mitochondrial Proteins Induced by Hormone Stimulation in MA-10 Mouse Leydig TumorCells Are Processed fromLarger Precursors* (Received for publication, April 25, 1991)

Douglas M. StoccoS and Thomas C. Sodeman From the Department of Biochemistry and Molecular Biology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

Acute regulation of steroidogenesis in steroidogenic (CSCC)’ which is located in the inner mitochondrial memtissue is controlled by the transfer of cholesterol from brane (1-6). While CSCC activity is responsible for this the outer to theinner mitochondrial membrane where conversion, acute regulation of steroidogenesis is, in fact, cleavage to produce pregnenolone occurs. Hormonal limited by the transfer of cholesterol to the inner mitochonstimulation of MA- 10 mouse Leydig tumor cells results drial membrane (7-10). One of the earliest and most imporin a large increase in steroidogenesis and the concom- tant observations in this process was that acute regulation of which steroidogenesis was dependent on de nouo protein synthesis mitant appearance of a series of 30-kDa proteins have been localized to themitochondria. In the present (3, 11-13). Thus, itis believedthat thecholesterol pool which study we have shown that the appearance of these is located in the inner mitochondrial membrane constitutes proteins occurs in a dose-responsive manner with both the rapidly metabolizable substrate pool, and hormone reguhuman chorionic gonadotropin and cyclic AMP analog. We have also shown that while steroidogenesis is in- lation of steroidogenesis involves the movement of cholesterol hibited rapidly in response to a cessation of protein from other sites in the cell into this inner membrane pool synthesis, the 30-kDa mitochondrial proteins remain (14). If CSCC activity is blocked with AG, while there is an accumulation of cholesterol in the inner mitochondrial memin the mitochondria, posing a potential dilemna for arguments favoring their role in the acute regulation brane in response to trophic hormone, no steroids are syntheof steroidogenesis. We report that the 30-kDa mito- sized. Removal of the AG results in a rapid and substantial chondrial proteins arise from two precursor proteins increase in steroid production. In addition, if cycloheximide with molecular masses of 37 and 32 kDa which are is administered along with AG, while there is no effect on also found to be associated with the mitochondria. The total mitochondrial cholesterol accumulation, the transfer of use of pulse-chase experiments and the inhibitors or- this substrate to the inner membrane is inhibited and the thophenanthrolineandcarbonylcyanide m-chloro- pool of rapidly metabolizable cholesterol is lost (15). Therephenylhydrazone demonstrated the precursor-product fore, it has been concluded that trophic hormone in steroidorelationship between the 37-, 32-, and 30-kDa progenic tissues functions by stimulating the transfer of cholesteins. We have also demonstrated that, as shown for a terol from the outer to the inner mitochondrial membrane number of other mitochondrial proteins, the 30-kDa through the action of a cycloheximide-sensitive protein(s) proteins are transferred to the inner mitochondrial (15). Despite nearly three decades of research in thisarea, the membrane by a process requiring both proteolytic re- identity and role of the required, rapidly synthesized, cyclomoval of the targeting sequences and an electrical heximide-sensitive protein(s) in the transfer of cholesterol potential across the inner mitochondrial membrane. from the outer to theinner mitochondrial membrane remains sites form We propose that during this transfer contact unknown. Since cholesterol would tendto exchange very between the two mitochondrial membranes and may offer an ideal situation for the transfer of cholesterol slowly between membranes separated by an aqueous phase, from the outer membrane to the inner membrane by such as the intermembrane space of the mitochondria (16, an as yet unknown mechanism. Following transfer, the 17), it has been proposed that trophic hormone action could 30-kDa proteins remain in the inner membrane no result in the accelerated transfer of cholesterol transport from longer able to function in the further transferof cho- outer to innermembrane through increased contacts between lesterol, andit is the continuing synthesis and process- the two membranes (18, 19). It therefore follows that such ing of more precursor proteins which provides addi- proteins would either be present in or in contact with the mitochondria at some point in their activity. tional substrate forsteroidogenesis. Many efforts have been made to identify the short-lived protein(s) responsible for this stimulation andhave attempted The rate-limiting enzymatic step in the synthesis of steroids by steroidogenic tissue is the conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage complex

* This work wassupported by National Institutes of Health Grants HD 17481, HD 00685, and RR 03948. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed.

The abbreviations used are: CSCC, cholesterol side-chain cleavage; AG, aminoglutethimide; SAP, steroidogenesis activator polypeptide; PBS-, Dulbecco’s phosphate buffered saline without calcium and magnesium; PBS+,Dulbecco’s phosphate buffered saline with calcium and magnesium; hCG, human chorionic gonadotropin; Bt2cAMP, N6, 2’-O-dibutyryladenosine-3’:5’-cyclic monophosphate; CHAPS, 3-[(3cholamidopropyl)dimethylammonio]l-propanesulfonate; CH, cycloheximide; OP, 1,lO-phenanthroline or orthophenanthroline; mCCCP, carbonyl cyanide m-chlorophenylhydrazone; RIA, radioimmunoassay; 2-D PAGE, two dimensional polyacrylamide gel electrophoresis; 1-D PAGE, one dimensional polyacrylamide gel electrophoresis.

19731

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Mitochondrial Proteins

to correlate the appearance of newly synthesized proteins with the observed increase in steroid production in various tissues. These studies have resulted in the identification of proteins in adrenal, granulosa, and testicular cells which are approximately 28 kDa in size and whose synthesis is concomitant with the increased production of steroids (20-26). Studies in our laboratory have demonstrated the presence of a series of 30-kDa proteins in the mitochondrial fraction of stimulated MA-10 mouse Leydigtumor cells as well as in the mitochondria of unstimulated, constitutive steroidproducing R& rat Leydig tumor cells (27-29). Further strengthening the model of a rapidly synthesized mitochondrial protein playing an essential role in this process, a recent study has described the mitochondrial location of a phosphoprotein which accumulates rapidly in response to adrenocorticotropin (ACTH) in adrenal cells (30). Another approach to this problem has been to test the ability of subcellular fractions as well as purified proteins to stimulate pregnenolone production in isolated mitochondria. These efforts have resulted in the description of several compounds with this capability (31-34). One of these compounds is the 3.2-kDa SAP (31, 32, 35), which can stimulate pregnenolone production in isolated mitochondria. Since the addition of SAP to isolated mitochondria promoted the accumulation of cholesterol in the inner membrane (36), it appears that SAP is able to regulate intramitochondrial cholesterol transport. Another protein candidate which appears to function in the intracellular transport of cholesterol has been purified from bovine adrenal cells (37). Lastly, it has recently been proposed that the peripheral benzodiazepine receptor located in the outer mitochondrial membrane may function in cholesterol transfer in the mitochondria of steroidogenic cells (38). While the proteins referred to above all may perform some function in the transfer of cholesterol, it remains unknown which, if any, of them may be essential for the acute regulation of side-chain cleavage. Ourrecent finding that a family of 30kDa mitochondrial proteins which are induced only through hormone treatment in MA-10 cells arepresentin large amounts in the constitutive steroidproducing R2C rat Leydig tumor cell line greatly strengthened the probability that these proteins, which are in all likelihood identical to those described by Orme-Johnson and colleagues(20-26, 30), are involved in the acute regulation of steroidogenesis. Experiments described here indicate that the30-kDa mitochondrial proteins found in the mitochondria of stimulated steroidogenic cells appear to arise from precursor proteins which are also found associated with the mitochondria of stimulated cells. We propose the hypothesis that during the insertion and subsequent processing of the precursors into the 30-kDa proteins, contact sitesmay form between the inner andouter mitochondrial membranes and allow the substrate cholesterol to be transferred, by an as yet unknown mechanism, to the inner mitochondrial membrane where it can be converted to pregnenolone by the CSCC. Studies on the biogenesis and assembly of mitochondria are replete with examples of larger molecular weight precursors being synthesized in the cytosol, transferred to the mitochondria where theyinteract with specific membrane receptors, and areultimately inserted into one of the mitochondrial compartments concomitantwith the cleavage of signal sequences (39-44). It has also been very well documented that these events occur at points of close contact and perhapsfusion of the inner andouter mitochondrial membranes (45-48). Thus cholesterol, which is hydrophobic and unable to traverse the aqueous intermembrane

i n Leydig Cells space, may find itself in an environment in which passage to the inner membrane can occur more easily. MATERIALS ANDMETHODS

Chemicals-Waymouth's MB 752/1 medium, horse serum, PBS-, lyophilized trypsin-EDTA, and tissue culture grade sodium bicarbonate were purchased from GIBCO. PBS+ was obtained from Oxoid Ltd. (Bassingstoke, United Kingdom). hCG was obtained from the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD. Bt2cAMP, CHAPS, gentamycin sulfate, CH, progesterone, OP, and mCCCP were purchased from Sigma. [36S]Methionine-cysteine ( T r a n ~ ~ ~ S - L a bapproximately el, 1000 Ci/mmol) was obtained from ICN Biomedicals Inc., Irvine CA. [7-3H)(1,2,6,7-3H)Progesterone (specific activity 115 Ci/mmol) was obtained from Du Pont-New England Nuclear. Ampholines were obtained from Pharmacia LKB Biotechnology Inc. Other chemicals used in the preparation of isoelectric focusing gels and mini-gels were obtained from Bio-Rad. 1020% gradient gels werepurchasedpre-cast from Integrated Separation Systems (Hyde Park, MA). Dextran T-70 was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Decolorizing charcoal (Norit) was obtained from Fisher. Cell Culture-The MA-10 cell line used in these experiments was a generous gift from Dr. Mario Ascoli of The University of Iowa and was maintained using standard techniques (49). For experiments in which progesterone production was measured, 50,000 cells were plated in each well of 96-well plates (Corning, Corning, NY) and grown for 24 h in medium plus 15% horse serum. In experiments designed to study mitochondrial proteins, 1.5 X lo6 cells were plated into each well of 12-wellCorning dishes and grown for 24 h. After this time the medium was removed, cells washed twice with PBS+,and Waymouth's medium lacking serum placed back on the cells. Stimulation and radiolabeling of the cells was performed as described below. In the dose-response experiments, 100p1of Waymouth's medium containing the appropriate dose of either hCG or Bt2cAMP was placed directly on the cells for the incubation periods described in the legend for Fig. 2 at 37 "C in a humidified atmosphere containing5% C02.At the end of the treatment period, the mediumwasremoved and frozen at -20 "C until RIA for progesterone could be performed. In experiments employing CH, the final concentration of the inhibitor was 1 mM, while m-CCCP was employed at 20 WM and OP at 1.0 mM final concentrations. In experiments measuring progesterone production, the cells in the wellswere solubilized with 0.01% sodium dodecyl sulfate and analyzed for protein content. RIA-Quantitation of progesterone was performed by RIA as previously described (50) directly on aliquots of the medium from control and treated cells. Antibodies to progesterone were obtained from Holly Hills Biological (Hillsboro, OR). Analysis of the RIA data was performed using a computer program specifically designed for this purpose. The data were expressed as nanograms of progesterone/ milligram of protein/unit of time. Radiolabeling of Cells-Radiolabeling of proteins was accomplished by covering the cells with Waymouth's medium containing the appropriate level of hormone as well as [35S]methionine-cysteinea t concentrations given in the figure legends. At the end of the labeling period, the medium was removed and the cells washed three times with PBS+ prior to further processing. Preparation of Protein Samples for Electrophoresis-For isolation of mitochondria, cells were washed with PBS+ and scraped from the dishes in a buffer consisting of 0.25 M sucrose, 0.01 M Tris, 0.01 M EDTA, pH 7.4, and homogenized using a Potter-Elvejhelm motordriven glass homogenizer with a Teflon pestle. Homogenates were centrifuged at 600 X g for 15 min, and the supernatantwas collected and centrifuged at 13,000 X g for 15 min. The mitochondrial pellet was washed once in sucrose-Tris-EDTA buffer and again collected by centrifugation. Mitochondrial pellets were then solubilized in a small volume of isoelectric focusing lysis buffer (IEF lysis buffer: 9.8 M urea, 2% CHAPS, 2% ampholines, and 100 mM dithiothreitol). Following centrifugation in an Eppendorf centrifuge, aliquots of the solubilized mitochondrial proteins were taken andassessed for radioactivity by liquid scintillation counting. Previous studies in our laboratory have indicated that mitochondria prepared using this method have approximately 5% contamination with plasma membranes and 23% contamination with cytosol (27). Separation of Inner and Outer Mitochondrial Membranes-Mitochondrial inner and outer membrane fractions were prepared using digitonin at aconcentration of0.6 mg/mg mitochondrial protein

k y d i g Cells

Mitochondrial in Proteins essentially as described previously (51). 2-0 PAGE-Equal counts/minute from each sample were then applied to the IEFgels and two-dimensional gel electrophoresis was performed on the mitochondrial proteins previously as described (2729, 52, 53). At the conclusion of electrophoresis, gels were prepared for fluorography (54) using Resolution (E. M. Corp., Chestnut Hill, MA), dried under moderate heat and vacuum, and exposed to x-ray film at -80 “C for the appropriate lengthof time. Radioactive molecular weight protein standardsfor the second dimension were included in each series of experiments. pH gradients of the IEF gels were determined using a surface electrode. Molecular weights assigned to the mitochondrial proteins described in this study were determined using a programin theVisage 2000 software designed forthis purpose. I-D PAGE-One-dimensional gel electrophoresis was performed using a Bio-Rad mini-gel apparatus. Samples were assayed for radioactivityand equal counts/minute placed onto the gels. Following electrophoresis, the gels were prepared for fluorography as described above and once again exposed to x-ray film forthe appropriate length of time. Quantitation of Proteins on Gels-Proteins of interest on both 1-D and 2-D gels were quantitated using a BioImage Visage 2000 (BioImage, Ann Arbor MI) computer-assisted image analysis system as previouslydescribed (28, 29). Briefly, x-ray images of 2-D PAGE protein profiles were scanned, captured, and spotlistsof the proteins automatically generated. The images were then adjusted for differences in the amountof radioactivity loaded onto thegels by selecting a number of identical proteins in eachgel which did not change with hormone treatment and determining the ratios of the total integrated intensities of these proteins in each gel in an experimental series using the Visage 2000. Proteins on the 2-D gels described in this study were then quantitated usinga comparative log software program and the results reported as total corrected integrated intensity in each spot. For analysis of 1-D gels, the images were scanned, lanes defined, andthebandsineachlaneautomaticallydetectedand quantitated by the Visage 2000. In each case, the total integrated intensity of each band was recorded, and several marker proteins in each lane were then used to adjust for differences in counts/minute loading which may have occurred.Therefore, for eachof the proteins described in this study the values reported represent the total integrated intensities following correction for differences in the loading of the gels.

19733

1 30

-

RESULTS

Effects of Hormone Stimulation on MA-10 Mitochondrial Proteins-As shown inFig. 1,stimulation of MA-10 cells with maximal doses of Bt2cAMP for 6 h results in the induction of several mitochondrial proteins. The 30-kDa proteins have isoelectric points of6.7,6.5,6.3, and 6.1 and havebeen describedpreviously (28,29). In addition,we have shown that theseproteinsareidenticalto each other,their differing isoelectric points being a result of post-translational modification (29). We have also demonstrated that the synthesis of these proteins is inhibited by cycloheximide indicating that they are encoded by nuclear, not mitochondrial genes (Table I). In the present study,we report the increased synthesis of another mitochondrial protein which is 32 kDa in molecular mass and has an isoelectric point of approximately 6.8. In several of our studies, this protein appears asa doublet, and at this timewe do notknow the relationshipbetween the two. I n all studies reported here we have included both of these proteins in the quantitation of the 32-kDa protein. Dose-response of Progesterone Production and Mitochndriul Proteins-These experiments were performed in order to determine if a relationship existed between progesterone production and the appearance of these mitochondrial proteins in MA-10 cells. As shown in Fig. 2, A and C, increasing the dose of either hCG or Bt2cAMP results in theincreased synthesis of progesterone in a typical dose-response fashion. Also shown are the resultsof the quantitationof the 32- and 30-kDa proteins from 2-D gels following stimulation at several doses of each hormone which were selected on the basis of producing low, intermediate, and maximal progesterone pro-

6.8

6.k

6.3

i.0

FIG. 1. Effect of Bt2cAMP on the synthesis of mitochondrial proteins. Cells were grown as described under “Materials and Methods.” The cells werethen incubated in the presence of maximally stimulating levels of Bt2cAMP (1 mM), and 1.0 mCi/ml (“‘Sjmethionine-cysteine Translabel a t 37 “C for a period of 6 h. Cells were harvested,mitochondria isolated, andproteins subjected to 2-D PAGE and fluorography. In the gels in this figure, molecular weight values (in thousands) aregiven on they axis and pHvalues on the x axis. It canreadily be seen that Bt2cAMP stimulationof MA-10 cells results in an increase in the synthesis of a 32-kDa protein with an isoelectric point of 6.8 and four 30-kDa mitochondrial proteins with isoelectric points of 6.7, 6.5, 6.3, and 6.1 when compared to unstimulated cultures. These proteins are notedwith arrows.

duction. As readily seen in Fig. 2, B and D, there isa strong correlation between the dose selected and the synthesis of each of the 32- and 30-kDa mitochondrial proteins. In the case of the hCG-stimulatedcells, it canbe seen thatonly two of the 30-kDa proteins appear in response to this hormone. This observation has previously beenreported by us and probably represents anexample of functional coupling of the second messenger system in Leydig cells (28,55). Itis important to note that in each case at least 15 other proteins on the gels were selected as markers to use in the correction of the gels for loading differences and that the 32- and 30-kDa proteins are the only ones which consistently increase following hormone stimulation.It should also be noted that the data shown for the quantitation of the mitochondrial proteins in this and other figures are representative of several experi-

19734

Mitochondrial Proteins inLeydig Cells TABLE I Effects of BtzcAMP, CH, m-CCCP, and OPon the 37-, 32-, and 30-kDa mitochondrial proteins In these experiments, control cells were incubated in the presence of 1.0 mCi/ml [35S]methionine-cysteine while BtZcAMP cells were incubated with 35S plus 1 mM Bt,cAMP for 5 h. Cycloheximide-treated cells were first incubated in the presence of [35S]methionine-cysteinefor 5 h followed by 5 min in 1 mM CH and finally by an additional 3 h in 1 mM dbcAMP. In addition, cells were incubated with [?3]methionine-cysteine and 1 mM BtZcAMP in the presence and absence of 20 p M m-CCCP or 1.0 mM OP for a period of 5 h. At the end of the incubations, mitochondrial proteins were prepared for 2-D PAGE and fluorography as described. The data below represent the total corrected integrated intensities of the 37-, 32-, and 30-kDa mitochondrial proteins in a typical experiment as determined with the Visage 2000. The data which appears below represent the compilation of data obtained from experiments performed at different times but are representative of the results seen with these treatments. Corrected integrated intensities

Treatment 30 kDa 32 kDa 31

Control +BtzcAMP +B&cAMP CH +Bt2cAMP m-CCCP OP +BtzcAMP 0.49

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4000 3500

3000

FIG.2. Dose-response relationship between progesterone production and the synthesisof the 32-and 30-kDa mitochondrial proteins. Progesterone production was determined as outlined under “Materials and Methods.” The synthesis of the mitochondrial proteins was determined by radiolabeling the cells with 1.0 mCi/ml [35S]methionine-cysteinefor a period of 6 h in the presence of varying concentrations of either hCG or Bt2cAMP. Mitochondria were isolated and 2-D PAGE performed on the solubilized proteins. The gels were analyzed as described under “Materials and Methods.” As readily seen in this figure, there is an excellent relationship between the production of progesterone andthe appearance of these mitochondrial proteins with both hCG and Bt2cAMP. Panel A = hCGstimulated progesterone production; panel B = hCG-stimulated synthesis of mitochondrial proteins; panel C = Bt2cAMP-stimulated progesterone production;panel D = Bt2cAMP-stimulated synthesis of mitochondrial proteins.

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hCG (ng/ml) ments which showedthe same trend. Time Course of Appearance of Mitochondrial Proteins-

Experiments were conducted to determine the temporal relationship between the synthesis of the 32- and 30-kDa mitochondrial proteins. The results of theseexperiments(not shown) indicate that the 32-kDa protein is found at a very high level after 1 h of stimulation and is maintained at approximately this level for the 4-h duration of the experiment. The 30-kDa proteins display a somewhat different pattern in that while 30-kDa 1 is also found after 1 h and does not change a great deal during the 4-h period, 30-kDa

.01

.1

1

10

Bt2cAMP (mM)

proteins 2,3, and4 appearto accumulate in the mitochondria with time. We have consistently observed this pattern and in longer incubation periods 30-kDa proteins 3 and 4 represent greater and greater amounts of the induced mitochondrial 30kDa proteins. Half-life of Progesterone Production and Mitochondrial 32and 30-kDa Proteins-In the first part of these experiments, MA-10 cells were prestimulated with Bt2cAMP for 3 h prior to treatment with CH as described in thelegend. As illustrated in Fig. 3A,the production of progesterone decays very rapidly in the absence of continuing proteinsynthesis. For the exper-

MitochondrialProteins in Leydig Cells

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in Fig. 3B. It can readily be seen that at least during the period of time chosen for these studies, while there appears to be a slight decline in the 32-kDa protein, the 30-kDa proteins, (combined for the sake of clarity), exhibit no decline whatsoever. This is in sharp contrast to the rapid and nearly complete inhibition of progesterone production which occurs in cycloheximide treated MA-10 cells during this time. Effects of Puke-chase and m-CCCP on Mitochondrial Protein+” series of studies were performed to determine if a relationship existed between the 32- and 30-kDa mitochondrial proteins.MA-10 cells were labeled with [35S]methioninecysteine for 4 h in the presence of maximally stimulating doses of Bt2cAMP. At this time the isotope was removed,and in one case the cells were harvested immediately and mitochondrial proteinsprepared for electrophoresis. In thesecond case, the medium was replaced with fresh medium containing Bt2cAMP plus 5 mM unlabeled methionine and the incubation continued for an additional hour prior to preparation of samples. In the third case the cells were treated for 5 min with 20 p~ m-CCCP followed by an additional hour in the presence of Bt2cAMP, 5 mM methionine, and m-CCCP. As shown in Fig. 4, incubation of the cells in the presence of unlabeled methionine results in a decrease in the total integrated intensity in the 32-kDa protein and a concomitant increase in the integrated intensity of the 30-kDa proteins as compared to controls. Once again, for the sake of clarity, we have combined the totalintegrated intensityof all the 30-kDa proteins. Finally, as shown in this figure, addition of m-CCCP

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Minutes After Cycloheximide FIG. 3. Decay in the production of progesterone in the presence of CH and the apparent half-lives of the 32- and 30-kDa mitochondrial proteins. In panel A , cells were grown as described and stimulated with Bt2cAMP a t 37 “C for 3 h prior to the startof the experiment. 1 mM CH was added to all the wells except for controls. Incubation was continued at 37 “C, and samples were taken after 30 min, and at30-min intervals the medium was removed from subsequent wells and replaced with fresh medium containing CH. Thus the data reflect progesterone produced during specific 30-min intervals at thevarious times following addition of CH. It can readily be seen that the rate of inhibition of progesterone producing capacity rapidly decreases in MA-10 cells. In panel B, cells were radiolabeled with 1.0 mCi/ml [35S]methionine-cysteinein the presence of maximally stimulating doses of Bt2cAMP for a period of 6 h. The medium was removed from all the cells and replaced with Waymouth’s medium containing 1 mM CH. At 0, 60, 90, and 120 min, cells were harvested and mitochondria prepared as described. The solubilized mitochondrial proteins were subjected to 2-D PAGE analyzed with the Visage 2000 and theproteins of interest quantitated. The results shown here represent a typical example of several separate experiments. It can be seen that during the time course chosen here there is no significant decrease in the presence of any of the proteins measured. The halflives of these proteins appear to be much longer than that seen for the decay of progesterone under similar conditions as shown in panel A.

iments shown in Fig. 3B, cells were incubated in the presence of dbcAMP and [35S]methionine-cysteinefor 6 h. At this time the isotope wasremoved, andthe medium replaced with medium containing 1 mM CH. At 30-min intervals for up to 2 h, one group of cells was harvested and mitochondrial proteins prepared for 2-D PAGE. The mitochondrial 32- and 30-kDa proteins were quantitated with the Visage 2000 and the results of a typical experiment of a total of three shown

19735

I: 0.8

0.0 0.2

Bt2cAMP Bt2cAMP

+ chase chase

Bt2cAMP + + CCCP

FIG. 4. Quantitation of the 32- and 30-kDa proteins during the experimental conditions of pulse-chase and treatment with m-CCCP. Cells were radiolabeled in the presence of [35S] methionine-cysteine and maximally stimulating doses of Bt2cAMP for 4 h. At this time, one group of cells was thoroughly washed and mitochondrial protein samples prepared for 2-D PAGE as described under “Materials and Methods.” Another group of cells was washed and incubation was continued for an additional hour in the presence of Waymouth’s medium containing Bt2cAMP and 5 mM cold methionine. In a third group of cells, 20 p M m-CCCP was added for 5 min prior to the startof the “chase” period. Mitochondrial proteins were again subjected to 2-D PAGE and analyzed as described. It can be seen that following adjustment of the gels for loading differences the total integrated intensity in the 32-kDa mitochondrial protein decreases to approximately half while increasing approximately 2.5-fold in the 30-kDa proteins (combined). Treatment with m-CCP resulted in no change in the 32-kDa protein and prevented the increase in the 30-kDa proteins. Analysis of a number of additional proteins on the radiofluorograms of these samples failed to show any reproducible changes. The datashown in this figure represent typical results from two separate experiments.

19736

Mitochondrial Proteins

in Leydig Cells

blocked the increase in the 30-kDa proteins which was ob- we have performed studies on theeffects of n-CCCP and OP served following treatment with unlabeled methionine alone. on steroid production in MA-10 cells and have determined Effect of OP on Mitochondrial 37-, 32-, and 30-kDa Pro- that progesterone production is completely inhibited at the teins-The compound OP inhibits the activity of the signal doses of inhibitors used in these studies (data not shown). proteases and blocks processing of the mitochondrial precur- However, since n-CCCP actsas an uncoupler, it would result sor proteins. The 2-D PAGE gels shown in Fig. 5 illustrate in a loss of the production of reducing equivalents necessary that treatment of MA-10 cells with OP in the presence of for CSCC to occur. Thus theeffect of n-CCCP onprogestermaximally stimulating Bt2cAMP concentrationsfor a period one production cannot be directly linked to its effect on the of 5 h results in a decrease in the 32-kDa protein and an transfer of the 32-kDa protein into the mitochondria. Also, almost complete loss of the 30-kDa proteins in themitochon- OP has a significant effect on protein synthesis at thiscondrial samples. Also seen in these gels is the appearance of a centration making interpretationof its effect on steroidogen37-kDa protein with an isoelectric point of approximately 7.3. esis difficult. However, these inhibitors can be used to illusThis protein hasbeen observed in three separate experimentstrate that the37- and 32-kDa proteins appear tobe processed but hasonly been demonstrated inour hands when processing in a mannersimilarto a number of other mitochondrial of the mitochondrial precursor proteins areblocked with OP. proteins. Quantitation of the levels of each of these proteins with the Localization of the 30-kDa Mitochondrial Proteins-DigiVisage 2000 can be seen in Table I. It should be noted that tonin treatmentwhich resulted in the separationof the inner and outer mitochondrial membranes followed by electrophoresis of the membrane proteins demonstrated that30-kDa the mitochondrial proteins are localized in the inner mitochondrial membrane. This result can be seen in Fig. 6 and is consistent with our observation that treatment of the cells with rn-CCCP blocked the appearanceof the 30-kDa proteins in the mitochondria. We have also used 2-D PAGE on minigels and have demonstrated that the30-kDa band seen in 1D gels does indeed consist of the four 30-kDa proteins consistently seen on 2-D gels (not shown). In several experiments we have observed that approximately 75-85% of the 30-kDa proteins arefound in the innermembrane of stimulated cells. We have also shown that the 30-kDa proteins appear to be tightly bound to the inner membrane, being insoluble in 10 mM cholate (not shown).

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FIG. 6. Localization of the 30-kDa mitochondrial proteins. Cells were stimulated with 1 mM Bt2cAMP in the presence of 1.0 mCi/ml (RSS]methionine-cysteinefor 6 h. Cells were harvested, mitochondria isolated, and separated into inner and outer membranes as described under "Materials and Methods." Membrane proteins were solubilized, electrophoresed on mini-gels, and subjected to fluorography. The resulting fluorograph is shown. The legend for the FIG. 5. Effects ofOP o n t h e36-, 32-,and 30-kDa mitochon- lanes is asfollows: 1 and 6 = molecular weight standards; 2 = control drial proteins. This compound inhibits the proteolytic removal of inner membrane; 3 = control outer membrane; 4 = stimulated inner the targeting sequences from the transported precursor mitochondrialmembrane; 5 = stimulated outer membrane. It can be seen that the proteins, thus blocking processing of the precursors. Cells were radi- major portion of the induced 30-kDa mitochondrialproteins arefound olabeled with 1.0 mCi/ml ['sS]methionine-cysteine in thepresence of on the inner membraneof stimulated cells. Analysis with the Visage maximally stimulating doses of Bt2cAMP and in the presence and 2000 indicated that following correction of the proteins in the lanes absence of 1.0 mM OP for 5 h. The cells were harvested, mitochondria for differences in loading, approximately 75% of the 30-kDa proteins isolated, solubilized, and the proteins subjected to 2-D PAGE and (arrow)were found in the inner membrane. This amount is consistent fluorography. As shown in the 2-D PAGE gels in thisfigure, treatment with the degree of contamination of outer membranewith inner of the cells with OP resulted in a large increase in the amountof the membrane as determined with marker enzyme analysis. This result 37-kDa protein and a concomitant decrease in both the 32- and 30- was also shownusing 2-D PAGE on mini-gels and illustrated that all kDa mitochondrial proteins. These gels represent the typical results four of the 30-kDa proteins are found predominantly in the inner of three separate experiments. mitochondrial membrane (not shown).

Mitochondrial Proteins in Leydig Cells DISCUSSION

The most intriguing problem in the acute regulation of steroidogenesis remains the elucidation of the mechanism whereby the substratecholesterol is transferred from cellular stores and the outer mitochondrial membrane to the inner mitochondrial membrane where side-chain cleavage to yield pregnenolone occurs. Previous studies have shown that hormone stimulation of steroidogenic cells results in the rapid synthesis of several mitochondrial proteins with molecular masses of approximately 30 kDa (20-30). The appearance of theseproteins in atime frame that was consistent with increased steroid production made them candidates for the putative “rapidly synthesized, labile protein” thought to be instrumental inthe acute regulation of steroidogenesis ( 3 , l l 13). Theircandidacy is further strengthened by our observation that these mitochondrial proteins which are found only in hormone-stimulated MA-10mouseLeydig tumor cells, which have very low basal steroid production, are also found to be present in abundant levels in unstimulated K C rat Leydig tumor cells which produce high levels of steroid constitutively (29). In the present study we have shown in MA-10 cells that these mitochondrial proteins are synthesized in a dose-responsive manner with both the trophic hormone hCG and Bt2cAMP and that they are sensitive to CH inhibition. We have also shown that the 30-kDa proteins are synthesized in a time-dependent manner and continue to accumulate in the mitochondria. While the time required for these proteins to appear in MA-10 cells is significantly longer than thatshown for adrenal cells, it should be noted that the rate of acute hormone stimulatedsteroid production in adrenal cells is approximately 50-fold higher than found in MA-10 cells (20, 56). However, the failure of these proteins to disappear with kinetics which were similar to the decay of steroidogenesis observed in the presence of CH posed difficulties in explaining how they might be involved in steroidogenic regulation. Our finding that the 30-kDa mitochondrial proteins arise from larger precursor proteins may offer an explanation for this apparent discrepancy. We observed that the 32-kDa mitochondrial protein was synthesized prior to theappearance of large quantities of the 30-kDa proteins and that itwas maintained at a steady state level during the course of the stimulation. With time the 30-kDa proteins continued to accumulate in the mitochondria, presumably as a resultof the further and continuing processing of the 37- and 32-kDa precursor proteins and,as statedabove, did not display turnover kinetics consistent with the loss of steroidogenic capacity. Based on these observations, it is possible to form the hypothesis that the30-kDa proteins arise from two precursor proteins which are processed from larger to smaller molecular mass species during their import into the mitochondria. It is during this import process that contact sites may be formed between the outer and inner mitochondrial membranes and cholesterol transferred to the inner membrane and thusavailable for CSCC activity. The precursor proteins are transferred to theinner mitochondrial membrane concomitant with processing to the 30-kDa form. Once in the inner mitochondrial membrane contact sites are no longer present, and the 30kDa protein is no longer able to function in the transfer of cholesterol. Thus, the transferof additional cholesterol must await the continued de novo synthesis andprocessing of more precursor protein. It is for this reason that the30-kDa protein can continueto build up in theinner mitochondrial membrane and not disappear with a half-life similar to that of steroid production in the presence of CH. At this time we cannot

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speculate on the mechanism involved in cholesterol transfer. Perhaps these mitochondrial proteins have the capacity to bind and carry cholesterol to the inner mitochondrial membrane or perhaps cholesterol is transferred duringthe formation of contact sites between the membranes. In support of this hypothesis, it has been well documented that a number of cytosolic precursor mitochondrial proteins are imported at distinct areasof the mitochondrial membrane where contact sitesbetween the inner and outer mitochondrial membranes are found (45-48). Some proteins such as yeast cytochromes c1 and bp are synthesized as larger precursors and in the process of penetrating both mitochondrial membranes, are cleaved by both a matrix protease and a second protease located on theouter side of the inner mitochondrial membrane (57). During the insertion process, it appears that contact points are formed and subsequently lost as the processing from precursor to product occurs. We have shown that the 32-kDa protein canbe chased into the30-kDa form in the presence of unlabeled methionine and that this can be prevented by rn-CCCP. rn-CCCP causes disruption of the electrochemical potential across the inner mitochondrial membrane and, as such, blocks the transfer of a number of mitochondrial polypeptides into the matrix and inner membrane (58, 59). In this study we have also shown that rn-CCCP blocks the appearance of the 30-kDa proteins in the mitochondrial compartment using 2-D PAGE. Lastly, we have demonstrated that the 30-kDa proteins are located in the inner mitochondrial membrane as would be predicted with this model. Since the 37-, 32-, and 30-kDa proteins are all found associated with the mitochondria, it appears that binding and processing of the precursor proteins occurs very rapidly following de rwuo synthesis. In support of this, previous studies have indicated that cotranslational as well as post-translational import of precursor proteins into mitochondria can occur (60). The existence of mitochondrial precursor proteins which are cleaved during the processing and insertion mechanism have been demonstrated in the past (39,57,58). These precursors are usually present in small pools, have half-lives on the order of a few minutes and, as such, are difficult to detect. The proteins described in this studywould require two successive cleavages, and, in support of this mechanism, there are several mitochondrial proteins which have been shown to be processed in this manner (44,57,59,61,62). We observed that the 37-kDa protein accumulates in stimulated cells in the presence of OP, which blocks the proteolytic removal of the targeting sequences of precursor proteins, but not in the presence of rn-CCCP,whichblocks transfer of precursor proteins to the inner mitochondrial membrane. This would indicate that the 37-kDa protein cannot be processed to the 32- or 30-kDa forms without protease activity, but can be converted to the32-kDa protein in the absence of transfer to the inner mitochondrial membrane. There have been other protein candidates for the intramitochondrial transfer of cholesterol in steroidogenic cells (3338). The 28- and 30-kDa proteins which we and others have described have been shown to be highly regulated by trophic hormone treatment in adrenal, luteal, and Leydig cells (2030). The findings reported in this work indicate that the 30kDa mitochondrial proteins arise from precursor proteins in MA-10 Leydigtumor cells in response to hormone treatment. While it is possible that cholesterol transfer from the outer to inner mitochondrial membrane occurs during the processing, insertion, and concomitant formation of contact sites, further studies are required to determine if this relationship exists.

Mitochondrial in Proteins

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Acknowledgment-We would like to acknowledge the technical assistance of Deborah Alberts.

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