The Mitochondrial Environment Is Required for Activity of the Cholesterol Side-Chain Cleavage Enzyme' Cytochrome P450scc STEPHEN M

Proc. Natl. Acad. Sci. USA Vol. 91, pp. 7247-7251, July 1994 Cell Biology The mitochondrial environment is required for activity of the cholesterol side-chain cleavage enzyme' cytochrome P450scc STEPHEN M. BLACK, JENNIFER A. HARIKRISHNA, GRAZYNA D. SZKLARZ, AND WALTER L. MILLER* Department of Pediatrics and the Metabolic Research Unit, University of California, San Francisco, CA 94143-0978 Communicated by Seymour Lieberman, April 8, 1994 (receivedfor review April 15, 1993) ABSTRACT Sterodogen Is ni t by the conversion translocated to the inner membrane in a process that requires of colesterol to p egnel by mit il cytocome ATP but is not yet fully understood (10-12). The leader P4esocc [cholesterol, ruced-adrenal-ferredoxdnooxygen oxklo- peptides are removed by a specific peptidase found in the reductase (id ving); EC 1.14.15.6]. Several subsw mitochondrial matrix, and the mature P450scc, Adx, and quent steroidal coner occur In the endo c rc AdRed proteins then assume their normal location in the (ER), but the last step In the p in ofil and inner mitochondrial membrane (13). Mature mitochondrial mineralocorticolds again occurs In the nitochnr. Althop P450 proteins lack the highly hydrophobic membrane anchor cellua compa t i ofsteroldog enzynes appears sequences found in the N termini of the microsomal forms; to be a feature of all steroldogenic pathways, some reports thus it is not clear what mediates the correct association ofa hidIcate that cholesterol can be converted to preg e P450 with the inner mitochondrial membrane. outside the mit dr. To invesigte whether P4s can After pregnenolone is produced in mitochondria, conver- uction outside the mtohon, we cotrd vectors pro- sion to glucocorticoid and mineralocorticoid hormones reducing P45Oscc and various enzymes of P4Sbucc with quires both extramitochondrial and intramitochondrial en- electron-transport proteins and directed their expresion to zymes. Forexample, inthe synthesis ofcortisol, pregnenolone either the ER or the mtochondria. Whether tar to mito- must exit from the mitochondria to undergo conversions by chondri or to the ER, paid vectors e ing P450scc and 3p-hydroxysteroid dehydrogenase (a non-P450 microsomal fusion proteins ofP45Oscc with either rial or mcroso- enzyme) and by microsomal P450c17 and P450c21. The re- mal dectron-transport ins produced iunodee sulting product, 11-deoxycortisol, must then reenter the mi- protein. When expressed in cri, all ofthese construc- tochondria for conversion to cortisol by P450cll (for review, tin converted 22-hdroxy ol to penene, but see ref. 4). It is not clear how the steroidal intermediates are when exprsed i the ER noneofthem produced pR e. shuttled to the various cellular components, whether these These results show that P4&scc can fn only in the components are closely aggregated in space, or whether these mitochondrka. Furthermore, it appears to be the m honal specific subcellular locations are required for the functioning environment that is requred, rather than the spec mitocho- of the steroidogenic pathways (for review, see ref. 5). How- drial electron- tes. ever, the subcellular localization of enzymes has important consequences: the conversion ofcholesterol to pregnenolone by mitochondrial P450scc appears to be the rate-limiting The first and rate-limiting step in steroid hormone biosyn- reaction in steroidogenesis because transport of cholesterol thesis is the conversion ofcholesterol to pregnenolone (1-5). substrate into the mitochondria is slow, ratherthan because of This step involves three reactions: 20a-hydroxylation, 22- inherent inefficiency of P450scc (14-16). Experiments to hydroxylation, and scission of the C20-22 bond, all occur- transfer this three-component system to the ER to test the ring on the single active site of cytochrome P450scc [choles- requirements of subcellular localization have not yet been terol, reduced-adrenal-ferredoxin:oxygen oxidoreductase attempted because of the technical difficulty in ensuring that (side-chain-cleaving); EC 1.14.15.6] (6). This process re- all three components are each accurately expressed and tar- quires three pairs of electrons, one for each of the three geted to the ER in appropriate andreproducible quantities. We reactions, donated by NADPH. The electrons first pass to a have cloned the cDNAs for the three components of the flavoprotein (adrenodoxin reductase, AdRed), then to an human cholesterol side-chain cleavage system: P450scc (17), iron-sulfur protein (adrenodoxin, Adx), and finally to Adx (18), and AdRed (19), and we recently showed that these P450scc. P450scc is a typical mitochondrial P450 enzyme, all three components could be engineered into a single polypep- of which use the same electron-transfer proteins. However that a most cytochrome P450 enzymes, such as those involved in tide chain has enhanced enzymatic activity (20). Use of the metabolism ofxenobiotics, are found in the endoplasmic covalently linked, single-chain P450scc system facilitates reticulum (ER) (7). These enzymes receive electrons from studying its activity outside the mitochondrion. We have now NADPH via P450 oxidoreductase (OR), a flavoprotein that built a series of vectors that express P450scc fusion proteins differs from AdRed and that does not use an intermediate in the ER. By using soluble 22-hydroxycholesterol as a sub- iron-sulfur protein (7, 8). These microsomal forms of cy- strate, we can circumvent the mechanisms that transport tochrome P450 contain N-terminal sequences that encode an cholesterol to the mitochondria and thus test the requirements insertion/halt-transfer sequence (9) that targets the nascent for the electron donors to P450scc and the requirement for the polypeptide chain to the membranes of the ER and prevents mitochondrial environment for P45Oscc activity. its translocation into the lumen. By contrast, the mitochon- MATERIALS AND METHODS drial P450 enzymes have an amphipathic N-terminal leader sequence that allows the preprotein to bind to the mitochon- CostructionoERTargeting Psnids. The construction of drial surface at points where the inner and outer membranes the plasmids expressing Adx (21), P450scc (21), and AdRed are in close proximity (10). Mitochondrial proteins are then Abbreviations: AdRed, adrenodoxin reductase; Adx, adrenodoxin; OR, oxidoreductase; ER, endoplasmic reticulum. The publication costs ofthis article were defrayed in part by page charge *To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Pediatrics, Building MR-IV, Room 209, University of California, in accordance with 18 U.S.C. §1734 solely to indicate this fact. San Francisco, CA 94143-0978. 7247 Downloaded by guest on September 29, 2021 7248 Cell Biology: Black et al. Proc. Nadl. Acad. Sci. USA 91 (1994) (30) and ofthose encoding the fusion proteins F1-F3 (20) has supplemented with 5 x 10-6 M (22R)-hydroxycholesterol. been described. To construct fusion protein F4 (H2N- Twenty-four hours later, cells were harvested for luciferase P450scc-OR-COOH), the P450scc moiety was first prepared activity measurement, and pregnenolone in the medium was exactly as described for F1-F3 (20). The NADPH-dependent measured by immunoassay (27). P450 OR cDNA (8) was modified by PCR to remove the RNA and Protein Analysis. Forty-eight hours after trans- microsomal leader sequence, which consists of the first 56 fection, cells were washed twice in phosphate-buffered saline amino acids (22). A 418-bp segment from the 5' end ofthe OR and harvested with either 8 M guanidinium chloride for RNA cDNA was amplified by using primers 11 (5'-GACTAGTAT- preparation or into sucrose buffer (0.25 M sucrose/50 mM TCAGACATTGACCTCC-3') and 12 (5'-CAACCCCAGCT- ethanolamine/10 mM Tris-HCl, pH 7.4/1 mM EDTA) for CAAAGATGC-3'). Use of primer 11 removes the leader protein analysis. Northern analysis of RNA was done using sequence, adds an Spe I site for cloning, and encodes the Mops-formaldehyde denaturing gels and 32P-labeled human hinge sequence Thr-Asp-Gly-Thr-Ser to allow translation cDNAs for P450scc (17), Adx (18), AdRed (19), and OR (8) through both the P450scc and OR moieties to produce a as probe. fusion enzyme. The downstream primer 12 was chosen at a COS-1 cells were sonicated and fractionated into cytosol, naturally occurring Nar I site, allowing ligation to the re- mitochondria, and ER, as described (28). Total protein con- mainder of the OR cDNA. tent was determined after cell disruption with two 5-sec For the plasmids designated F4-F8, the mitochondrial bursts using a sonicator (Artek, Farmingdale, NY) atasetting targeting sequence of P450scc (amino acids 1-39) was re- of 20 and an equal volume of 2x loading buffer [50 mM placed by the ER insertion/halt-transfer sequence of rat Tris HCl, pH 6.8/2% SDS/5% 2-mercaptoetanol/1096 (vol/ P45011B1 (23). This was done using upstream oligonucleotide 13 (5'-GGGTACCATGGAGCCCAGTATCTTG-3') and vol) glycerol/0.005% bromophenol blue] was added. Samples downstream oligonucleotide 14 (S'-GACTAAGAGTAA- were boiled for 5 min and then separated by electrophoresis CAAGAAGCC-3') to prepare a 69-bp fragment encoding the on SDS/4-209o acrylamide gradient gels. The proteins were ER-targeting sequence (the first 23 residues) ofrat P450IIB1. then electrotransferred to nitrocellulose in Tris-HCl, pH Primer 13 adds a Kpn I site for cloning, and primer 14 8.4/193 mM glycine/20%o methanol for 1 hr at 4°C, and generates a blunt-ended site. A similar method was used to immunoblotting was done by using antisera specific to human remove the mitochondrial-targeting sequence from P450scc P450scc (27), Adx (27), AdRed (27), P450c17 (29), and OR to yield ablunt-ended fragment. Upstream oligonucleotide 15 (from C. R. Wolf, University of Edinburgh), as described (5'-ATCTCCACCCGCAGTCCTCGC-3') generated a blunt- (27). ended cDNA fragment beginning at the codon for amino acid 40 of P450scc (i.e., the first residue of the processed mature RESULTS intramitochondrial protein), and downstream oligonucleotide 16 (5'-TTGGGGCCCTCGGACTTAAAG-3') extended to Design and Construction of ER Targeting Psn. To test the Apa I site at codon 140. The two sequences were then the electron-transport requirements of P450scc and to test ligated together and subcloned into vector pUC-SF (20). A whether this enzyme requires the mitochondrial environ- Kpn I/EcoRV fragment was then isolated from this plasmid ment, we built a series of 18 expression vectors; their and used to replace the equivalent sequence in the F1-F4 encoded proteins are diagrammed in Fig. 1. The construction vectors. Similarly, the segment encoding the insertion/halt- of the plasmids encoding the fusion proteins designated F1 transfer sequence (amino acids 1-17) of human P450c17 (H2N-P450scc-AdRed-COOH), F2 (H2N-P450scc-AdRed- cDNA (24) was removed using PCR and replaced with the rat Adx-COOH), and F3 (H2N-P450scc-Adx-AdRed-COOH) P450IIB1 sequence. has been described (20). Protein F4, which is a fusion For the plasmids expressing F1AR+ and MARI, the between P450scc and NADPH-dependent P450 OR, was common, 18- form of AdRed cDNA was replaced with the constructed to examine the stringency of P450scc in accept- alternatively spliced 18+ form of AdRed cDNA (19, 25) by ing electrons from the mitochondrial electron-transfer sys- substitution into the Spe I/Nhe I site as described (20). To tem. The cDNA sequence that encodes the first 56 amino construct F2DM (double mutant), the F2AR+ construction was mutagenized by PCR using upstream oligonucleotide 17 P450scc DIII1iiZ Fl ER-P450scc (5'-TCTAGATATTGATGGCTTTGGTGCATATGAGG- E Adx lli11 Fl AR | I F5 GAACCCTGGCTTATTCAACCTAT-3') and downstream AR F22r oligonucleotide 10 (19). Oligonucleotide 17 creates the mu- | F6 tations C47W, C52W, and C55W in the Adx moiety of F2 by OR IIT F2ARft F7 changing three TGT (Cys) codons to TAT (Tyr), thus de- _ IZLE F2DM F8 stroying three of the four cysteines that coordinate the Fe2+ _II F3 ci 7wt ion in Adx (26). All PCR fragments and ligation junctions were sequenced to verify that no errors had occurred in the F4- 2B-c17 amplification or subcloning. FIG. 1. Constructions. Leader sequences at the N terminus (5' Trasection of COS-1 Ceils. COS-1 cells were transfected end, left) are the 39-amino acid mitochondrial leader sequence of by using either a calcium phosphate method or a DEAE- human P450scc (vertical lines) or the 23-amino acid microsomal (ER) dextran method. Plasmid DNA purified by cesium chloride leader sequence of rat P4S0IIB1 (checked boxes). Coding regions density gradients (>95% supercoiled) was used for each follow the leader sequences: black box, P450scc; stippled box, Adx; transfection. Each 10-cm dish (Falcon) received 2 pmol of white box, AdRed; wavy striped box, P450 OR. The vertical bar in vector plasmid and 5 jg of a Rous sarcoma virus-luciferase the F1AR+, F2AR+, and F2DM constructions indicates the presence (RSV-LUC) plasmid to control for transfection efficiency. of extra sequences in the 18+ form of AdRed or the 3 mutated After transfections were done on cultures at 60% confluency cysteine residues in Adx. The c17WT construction expresses the for 16 hr at 37°C in the medium was replaced with wild-type human P450c17 protein (diagonal lines), and 2B-c17 has the 5% CO2, same P450IIB1 microsomal leader sequence used in ER-P4S0scc and fresh Dulbecco's modified Eagle's medium-H21 (GIBCO) F5-8. Also shown are the constructions expressing wild-type human containing glucose (4.5 g/liter), 10% fetal calf serum, and Adx and AdRed, which use their own endogenous mitochondrial gentamicin (50 ,ug/ml). After 48 hr of transfection, the leader sequences (21), and the construction expressing human P450 medium was removed from the cells and replaced with a OR (29), which uses its own endogenous microsomal leader se- depleted medium containing only 0.5% fetal calf serum but quence. Downloaded by guest on September 29, 2021 Cell Biology: Black et al. Proc. Nati. Acad. Sci. USA 91 (1994) 7249 A lq B sequences predicted by their designs. The vector expressing 90<0 ER-P450scc, either when transfected alone or when cotrans- 0 fected with a vector expressing OR, expressed less mRNA than the corresponding normal P450scc vector with a mitochondrial leader sequence, either when it was transfected alone or triply transfected with vectors separately expressing AdRed and Adx. The reason for this is unclear. The abundances of the mRNAs produced by vectors F5-8 encoding microsomal proteins are very similarto the abundances ofthe D mRNAs produced by the corresponding vectors F1-4, which 5 0v( k~ b express mitochondrial proteins. Thus, the presence of the t GG sG99b leader sequence from rat P4501IB1 and thejunction between this leader and P450scc cannot be responsible for the poor expression (or poor mRNA stability) of the ER-P450scc construction. When the same Northern blot is reprobed with cDNAs for human Adx (Fig. 2B), AdRed (Fig. 2C), and OR (Fig. 2D), only the constructions predicted to encode these RNA segments are detected, and the sizes ofthe hybridizing bands on these different probings ofthe same gel correspond FIG. 2. Northern blot. RNA was prepared from COS-1 cells precisely. Although Adx (18) and AdRed (30) are expressed transfected with the various constructions indicated. ER-scc/OR in all tissues, the endogenous level of expression of these designates an RNA sample from cells doubly transfected with two mRNAs in COS-1 cells is below the level ofdetection on this vectors, one expressing ER-P450scc and the other expressing OR. Northern blot. By contrast, endogenous COS-1 cell OR TRIPLE designates COS-1 cells transfected with equimolar amounts mRNA is seen in all lanes (Fig. 2D). of three vectors separately expressing normal human P450scc, Expression AdRed, and Adx, and pECE is the expression vector with no cDNA of Fusion Proteins. To examine the translation insert. Samples of 20 pg of RNA were electrophoresed through a of the mRNAs encoded by the expression vectors shown in Mops-formaldehyde-1% agarose gel and transferred to Hybond-N Fig. 1, we isolated total protein from cells transfected with nylon membrane (Amersham). A single blot was sequentially probed each of the fusion constructions and analyzed it by immu- with 32P-labeled cDNAs forhuman P450scc (A), Adx (B), AdRed (C), noblotting with antibodies to human P450scc, Adx, AdRed, and OR (D). The blot was boiled in 10 mM Tris, pH 7.4/5 mM and OR (Fig. 3). The fusion proteins react with the expected EDTA/1% SDS and re-autoradiographed between probings to en- antisera: F1 and F5 react with antibodies to P450scc and sure removal ofall radioactivityfrom the previous probe. HindU-cut but not with antibodies to Adx or OR; F2 and F6 react bacteriophage PM-2, run in another lane, were used as markers and AdRed permitted alignment of the corresponding bands in the four autora- with antisera to P450scc, AdRed, and Adx, but not with diographs. antiserum to OR; and F4 and F8 react with antisera to P450scc and OR but not with antisera to AdRed or Adx. acids of OR, which are thought to be involved in the asso- Proteins encoded by the F3 and F7 constructions, which ciation ofOR with the ER (22), was deleted and replaced with should be the same size as the F2 and F6 proteins, could not a linker that encodes a unique Spe I site and also encodes the be detected with the P450scc or AdRed antibodies. However, hydrophilic hinge peptide Thr-Asp-Gly-Thr-Ser. Fusions a smaller (w100-kDa) band is detected with the Adxantibody, F1-F4 all possess the 39-residue N-terminal signal sequence suggesting lability due to a proteolytic cleavage. Withboth F3 of P450scc, which is responsible for targeting the protein to and F7, this same band can be detected with the P450scc mitochondria. In the proteins designated ER-P450scc and antibody, suggesting that there is a proteolytic cleavage that F5-F8, these 39 amino acids were replaced by the ER removes and degrades the AdRed moiety. The amount of insertion/halt-transfer sequence of rat P45011B1. protein produced by the constructions that target proteins to Transiription ofthe cDNA Expression Vectors. To examine the ER is generally lower than the amount ofthe correspond- the expression of the various cDNA expression construc- ing protein targeted to the mitochondria, even after normal- tions, we prepared RNA from transfected COS-1 cells and ization for differences in transfection efficiency. This result analyzed it by Northern blotting with probes for P450scc, may be from an inherent instability in the proteins caused by Adx, AdRed, and OR (Fig. 2). All of the vectors expressed their presence in a cellular compartment where they are not RNAs of the predicted sizes that contained hybridizing normally found.

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0.0Bs 46 CU ^ CM.+ *oo.V w . * w U0 T. + co in IL I* C N O0 ° Nt C- o.0o ,L QSSna uc CM IL ou C 0. IL w V V FIG. 5. Validation oftargeting to the ER with the P4501IB1 leader sequence. (A) Immunoblot of P450c17. Fifty-microgram samples of m protein from COS-1 cells transfected with vector (pECE) or from cells transfected with vectors expressing either P450c17 wild type (c17WT) or P450c17 with a P4501IB1 leader peptide (2B-c17) were displayed and analyzed with rabbit anti-human P450c17. (B) Enzy- FIG. 4. Biological activity of the fusion proteins. Conversion of matic activity of cells shown in A. Before cells were harvested, they 22-hydroxycholesterol to pregnenolone was measured by RIA and is were incubated with [14C]progesterone (PROG) for 2 hr, and the displayed as ng ofpregnenolone per ml ofculture medium, corrected production of 17a-hydroxy[l14C]progesterone (170HP) was assayed for transfection efficiency. COS-1 cells transfected with various by TLC of the culture medium. ORI, origin. (C) Immunoblot of expression vectors are designated as in Figs. 2 and 3. N.D., not cytosol, mitochondria (Mito.), and ER of COS-1 cells transfected detectable. with F2, F6, or pECE vector, probed with antiserum to Adx. Downloaded by guest on September 29, 2021 Cell Biology: Black et al. Proc. Natl. Acad. Sci. USA 91 (1994) 7251

F2 but showed no F2 protein in the cytosol or ER; similarly Institutes of Health Grants DK37927 and DK42154 and by March of the F6 protein was found only in the ER, and not in the Dimes Grant 6-0098, all to W.L.M. cytosol or mitochondria (Fig. 5C). Thus the mitochondrial leader from P450scc and the ER leader from P450IIB1 1. Simpson, E. R. (1979) Mol. Cell. Endocrinol. 13, 213-227. correctly target the fusion proteins to the predicted cellular 2. Kimura, T. (1981) Mol. Cell. Biochem. 36, 105-122. 3. Hall, P. F. (1985) Rec. Prog. Horm. Res. 41, 1-39. organelles. 4. Miller, W. L. (1988) Endocr. Rev. 9, 295-318. 5. Lieberman, S. & Prasad, V. V. K. (1990) Endocr. Rev. 11, DISCUSSION 469-493. 6. Lambeth, J. D. & Pember, S. 0. (1983) J. Biol. Chem. 258, The identities and activities of the three components of the 5596-5602. cholesterol side-chain cleavage system have been studied and 7. Gonzalez, F. J. (1989) Pharmacol. Rev. 40, 243-288. characterized in detail (for review, see refs. 1-5). P450scc is 8. Yamano, S., Aoyama, T., McBride, 0. W., Hardwick, J. P., the unique form of cytochrome P450 that catalyzes all three Gelboin, H. V. & Gonzalez, F. J. (1989) Mol. Pharmacol. 35, reactions needed to convert cholesterol to pregnenolone. 83-88. in mitochondria and its 9. Black, S. D. (1992) FASEB J. 6, 680-685. Although its activity is found pre- 10. Wickner, W. T. & Lodish, H. F. (1985) Science 230, 400-407. protein has a typical mitochondrial leader sequence (17, 34), 11. Hard, F.-U. & Newport, W. (1990) Science 247, 930-938. it has been suggested that cholesterol side-chain cleavage 12. Baker, K. P. & Schatz, G. (1991) Nature (London) 349, 205- activity can be found in other cellular compartments (for 208. review, see ref. 5). Because all cellular fractionation and 13. Hanukoglu, I., Suh, B. S., Himmelhoch, S. & Amsterdam, A. protein purification schemes are subject to cross contamina- (1990) J. Cell Biol. 111, 1373-1381. tion, a definitive test of this hypothesis has not previously 14. Lambeth, J. D., Xu, X. X. & Glover, M. (1987) J. Biol. Chem. been possible. While molecular biologic techniques permit 262, 9181-9188. targeting of P450scc to the ER or cytosol, the cotargeting of 15. Jefcoate, C. R., DiBartolomeis, M. J., Williams, C. A. & Mc- in a stoichiometric ratio is Namara, B. C. (1987) J. Steroid Biochem. 27, 721-729. its electron-transport proteins 16. lida, S., Papadopoulos, V. & Hall, P. F. (1989) Endocrinology needed to yield interpretable results. The engineering of 124, 2619-2624. variations of the entire cholesterol side-chain cleavage sys- 17. Chung, B., Matteson, K. J., Voutilainen, R., Mohandas, T. K. tem into a single polypeptide chain now permits such exper- & Miller, W. L. (1986) Proc. Natl. Acad. Sci. USA *3, 8962- iments. 8966. The ability of the four fusion proteins F1-F4 to convert 18. Picado-Leonard, J., Voutilainen, R., Kao, L., Chung, B., cholesterol to pregnenolone suggests that the P450scc moiety Strauss, J. F., III, & Miller, W. L. (1988) J. Biol. Chem. 263, may receive electrons from several different electron- 3240-3244, and correction (1988) 263, 11016. transfer proteins. An alternative interpretation is that the 19. Solish, S. B., Picado-Leonard, J., Morel, Y., Kuhn, R. W., Mohandas, T. K., Hanukoglu, I. & Miller, W. L. (1988) Proc. presence of any C-terminal extension on the P450scc moiety Natd. Acad. Sci. USA 85, 7104-7108. may facilitate its receipt of electrons from the low levels of 20. Harikrishna, J. A., Black, S. M., Szklarz, G. D. & Miller, endogenous COS-1 cell Adx. However, when the Adx moiety W. L. (1993) DNA Cell Biol. 12, 371-379. of F2 is mutated in the F2DM construction, all activity is lost, 21. Brentano, S. T. & Miller, W. L. (1992) Endocrinology 131, showing that the P450scc moiety of F2 is receiving electrons 3010-3018. from the covalently linked Adx moiety and not from the 22. Porter, T. D. & Kasper, C. B. (1985) Proc. Natl. Acad. Sci. COS-1 cell Adx. This result suggests that the F1 and F4 USA 82, 973-977. constructions may be catalytically active by receiving elec- 23. Monier, S., Van Luc, P., Kreibich, G., Sabatini, P. D. & trons from their covalently linked AdRed or OR moieties, Adesnik, M. (1988) J. Cell Biol. 107, 457-470. 24. Chung, B., Picado-Leonard, J., Haniu, M., Bienkowski, M., Adx. A rather broad of rather than from COS-1 cell range Hall, P. F., Shivley, J. E. & Miller, W. L. (1987) Proc. Nadl. acceptable electron donors for a P450 enzyme may not be Acad. Sci. USA 84, 407-411. wholly surprising, as microsomal P450c17 apparently can 25. Lin, D., Shi, Y. & Miller, W. L. (1990) Proc. Natl. Acad. Sci. receive electrons from either OR or cytochrome b, (35). USA 87, 8516-8520. Thus, the availability of the mitochondrial electron-transfer 26. Cupp, J. R. & Vickery, L. E. (1988) J. Biol. Chem. 263, system should not, in and of itself, prohibit P450scc from 17418-17421. being active if it, indeed, reached the ER or cytosol. 27. Black, S. M., Szklarz, G. D., Harikrishna, J. A., Lin, D., In these experiments, we used 22-hydroxycholesterol as Wolf, C. R. & Miller, W. L. (1993) Endocrinology 132, 539- substrate so that the activity of our P450scc constructions 545. 28. Black, S. M., Ellard, S., Meehan, R. R., Parry, J. M., would not depend on the mitochondrial cholesterol-transport Adesnik, M., Beggs, J. D. & Wolf, C. R. (1989) Carcinogenesis machinery and so that a metabolizable substrate would be 10, 2139-2143. available to enzymes in the ER. However, no form of 29. Lin, D., Black, S. M., Nagahama, Y. & Miller, W. L. (1993) P450scc, either the enzyme itself or the enzyme covalently Endocrinology 132, 2498-2506. linked to various demonstrably effective electron-transfer 30. Brentano, S. T., Black, S. M., Lin, D. & Miller, W. L. (1992) systems, is active when targeted to the ER. Sakaki et al. (36) Proc. Nati. Acad. Sci. USA 89, 4099-4103. have reported that mitochondrial P450c27 is active when 31. Brandt, M. E. & Vickery, L. E. (1992) Arch. Biochem. Bio- targeted to the ER of yeast that also express Adx and AdRed phys. 294, 735-740. as soluble, cytosolic forms. It is not known whether P450scc 32. Lin, D., Harikrishna, J. A., Moore, C. C. D., Jones, K. L. & would be active in the ER of mammalian cells if Miller, W. L. (1991) J. Biol. Chem. 266, 15992-15998. similarly 33. Clark, B. J. & Waterman, M. R. (1991) J. Biol. Chem. 266, soluble cytoplasmic Adx and AdRed were provided. How- 5898-5904. ever, the lack of activity of the F5-F8 constructs suggests 34. Morohashi, K., Fujii-Kuriyama, Y., Okada, Y., Sogawa, K., that the unique reducing environment ofthe mitochondrion is Hirose, T., Inayama, S. & Omura, T. (1984) Proc. Natl. Acad. essential for the P450scc activity. Sci. USA 81, 4647-4651. 35. Nakajin, S., Takahashi, M., Shinoda, M. & Hall, P. F. (1985) We thank Frank Gonzalez for the OR cDNA and C. R. Wolf for Biochem. Biophys. Res. Commun. 132, 708-713. the OR antibody and the rat P450IIB1 cDNA. This work was 36. Sakaki, T., Akiyoshi-Shibata, M., Yabusaki, Y. & Ohkawa, H. supported by a Glaxo Cardiovascular Discovery Grant, by National (1992) J. Biol. Chem. 267, 16497-16502. Downloaded by guest on September 29, 2021