Proc. Natl. Acad. Sci. USAVol. 91, pp. 7247-7251, July 1994Cell Biology

The mitochondrial environment is required for activity of thecholesterol side-chain cleavage enzyme' cytochrome P450sccSTEPHEN 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 (received for review April 15, 1993)

ABSTRACT Sterodogen Is ni t by the conversionof colesterol to p egnel by mit il cytocomeP4esocc [cholesterol, ruced-adrenal-ferredoxdnooxygen oxklo-reductase (id ving); EC 1.14.15.6]. Several subswquent steroidal coner occur In the endo c rc(ER), but the last step In thep in ofil andmineralocorticolds again occurs In the nitochnr. Althopcellua compa t i ofsteroldog enzynes appearsto be a feature of all steroldogenic pathways, some reportshidIcate that cholesterol can be converted to preg eoutside the mit dr. To invesigte whether P4s canuction outside the mtohon, wecotrd vectors pro-ducing P45Oscc and various enzymes of P4Sbucc withelectron-transport proteins and directed their expresion toeither the ER or the mtochondria. Whether tar to mito-chondri or to the ER, paid vectors e ing P450scc andfusion proteins ofP45Oscc with either rial or mcroso-mal dectron-transport ins produced iunodeeprotein. When expressed in cri, all of these construc-tin converted 22-hdroxy ol to penene, butwhen exprsed i the ER none ofthem produced pR e.These results show that P4&scc can fn only in themitochondrka. Furthermore, it appears to be them honalenvironment that is requred, rather than the spec mitocho-drial electron- tes.

The first and rate-limiting step in steroid hormone biosyn-thesis is the conversion of cholesterol to pregnenolone (1-5).This step involves three reactions: 20a-hydroxylation, 22-hydroxylation, and scission of the C20-22 bond, all occur-ring on the single active site of cytochrome P450scc [choles-terol, reduced-adrenal-ferredoxin:oxygen oxidoreductase(side-chain-cleaving); EC 1.14.15.6] (6). This process re-quires three pairs of electrons, one for each of the threereactions, donated by NADPH. The electrons first pass to aflavoprotein (adrenodoxin reductase, AdRed), then to aniron-sulfur protein (adrenodoxin, Adx), and finally toP450scc. P450scc is a typical mitochondrial P450 enzyme, allof which use the same electron-transfer proteins. Howevermost cytochrome P450 enzymes, such as those involved inthe metabolism of xenobiotics, are found in the endoplasmicreticulum (ER) (7). These enzymes receive electrons fromNADPH via P450 oxidoreductase (OR), a flavoprotein thatdiffers from AdRed and that does not use an intermediateiron-sulfur protein (7, 8). These microsomal forms of cy-tochrome P450 contain N-terminal sequences that encode aninsertion/halt-transfer sequence (9) that targets the nascentpolypeptide chain to the membranes of the ER and preventsits translocation into the lumen. By contrast, the mitochon-drial P450 enzymes have an amphipathic N-terminal leadersequence that allows the preprotein to bind to the mitochon-drial surface at points where the inner and outer membranesare in close proximity (10). Mitochondrial proteins are then

translocated to the inner membrane in a process that requiresATP but is not yet fully understood (10-12). The leaderpeptides are removed by a specific peptidase found in themitochondrial matrix, and the mature P450scc, Adx, andAdRed proteins then assume their normal location in theinner mitochondrial membrane (13). Mature mitochondrialP450 proteins lack the highly hydrophobic membrane anchorsequences found in the N termini of the microsomal forms;thus it is not clear what mediates the correct association of aP450 with the inner mitochondrial membrane.

After pregnenolone is produced in mitochondria, conver-sion to glucocorticoid and mineralocorticoid hormones re-quires both extramitochondrial and intramitochondrial en-zymes. Forexample, in the synthesis ofcortisol, pregnenolonemust exit from the mitochondria to undergo conversions by3p-hydroxysteroid dehydrogenase (a non-P450 microsomalenzyme) and by microsomal P450c17 and P450c21. The re-sulting product, 11-deoxycortisol, must then reenter the mi-tochondria for conversion to cortisol by P450cll (for review,see ref. 4). It is not clear how the steroidal intermediates areshuttled to the various cellular components, whether thesecomponents are closely aggregated in space, or whether thesespecific subcellular locations are required for the functioningof the steroidogenic pathways (for review, see ref. 5). How-ever, the subcellular localization of enzymes has importantconsequences: the conversion of cholesterol to pregnenoloneby mitochondrial P450scc appears to be the rate-limitingreaction in steroidogenesis because transport of cholesterolsubstrate into the mitochondria is slow, rather than because ofinherent inefficiency of P450scc (14-16). Experiments totransfer this three-component system to the ER to test therequirements of subcellular localization have not yet beenattempted because of the technical difficulty in ensuring thatall three components are each accurately expressed and tar-geted to the ER in appropriate and reproducible quantities. Wehave cloned the cDNAs for the three components of thehuman cholesterol side-chain cleavage system: P450scc (17),Adx (18), and AdRed (19), and we recently showed that thesethree components could be engineered into a single polypep-tide chain that has enhanced enzymatic activity (20). Use ofacovalently linked, single-chain P450scc system facilitatesstudying its activity outside the mitochondrion. We have nowbuilt a series of vectors that express P450scc fusion proteinsin the ER. By using soluble 22-hydroxycholesterol as a sub-strate, we can circumvent the mechanisms that transportcholesterol to the mitochondria and thus test the requirementsfor the electron donors to P450scc and the requirement for themitochondrial environment for P45Oscc activity.

MATERIALS AND METHODSCostructionoERTargeting Psnids. The construction of

the plasmids expressing Adx (21), P450scc (21), and AdRed

Abbreviations: AdRed, adrenodoxin reductase; Adx, adrenodoxin;OR, oxidoreductase; ER, endoplasmic reticulum.*To whom reprint requests should be addressed at: Department ofPediatrics, Building MR-IV, Room 209, University of California,San Francisco, CA 94143-0978.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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(30) and of those encoding the fusion proteins F1-F3 (20) hasbeen described. To construct fusion protein F4 (H2N-P450scc-OR-COOH), the P450scc moiety was first preparedexactly as described for F1-F3 (20). The NADPH-dependentP450 OR cDNA (8) was modified by PCR to remove themicrosomal leader sequence, which consists of the first 56amino acids (22). A 418-bp segment from the 5' end ofthe ORcDNA was amplified by using primers 11 (5'-GACTAGTAT-TCAGACATTGACCTCC-3') and 12 (5'-CAACCCCAGCT-CAAAGATGC-3'). Use of primer 11 removes the leadersequence, adds an Spe I site for cloning, and encodes thehinge sequence Thr-Asp-Gly-Thr-Ser to allow translationthrough both the P450scc and OR moieties to produce afusion enzyme. The downstream primer 12 was chosen at anaturally occurring Nar I site, allowing ligation to the re-mainder of the OR cDNA.For the plasmids designated F4-F8, the mitochondrial

targeting sequence of P450scc (amino acids 1-39) was re-placed by the ER insertion/halt-transfer sequence of ratP45011B1 (23). This was done using upstream oligonucleotide13 (5'-GGGTACCATGGAGCCCAGTATCTTG-3') anddownstream oligonucleotide 14 (S'-GACTAAGAGTAA-CAAGAAGCC-3') to prepare a 69-bp fragment encoding theER-targeting sequence (the first 23 residues) of rat P450IIB1.Primer 13 adds a Kpn I site for cloning, and primer 14generates a blunt-ended site. A similar method was used toremove the mitochondrial-targeting sequence from P450sccto yield a blunt-ended fragment. Upstream oligonucleotide 15(5'-ATCTCCACCCGCAGTCCTCGC-3') generated a blunt-ended cDNA fragment beginning at the codon for amino acid40 of P450scc (i.e., the first residue of the processed matureintramitochondrial protein), and downstream oligonucleotide16 (5'-TTGGGGCCCTCGGACTTAAAG-3') extended tothe Apa I site at codon 140. The two sequences were thenligated together and subcloned into vector pUC-SF (20). AKpn I/EcoRV fragment was then isolated from this plasmidand used to replace the equivalent sequence in the F1-F4vectors. Similarly, the segment encoding the insertion/halt-transfer sequence (amino acids 1-17) of human P450c17cDNA (24) was removed using PCR and replaced with the ratP450IIB1 sequence.For the plasmids expressing F1AR+ and MARI, the

common, 18- form of AdRed cDNA was replaced with thealternatively spliced 18+ form of AdRed cDNA (19, 25) bysubstitution into the Spe I/Nhe I site as described (20). Toconstruct F2DM (double mutant), the F2AR+ constructionwas mutagenized by PCR using upstream oligonucleotide 17(5'-TCTAGATATTGATGGCTTTGGTGCATATGAGG-GAACCCTGGCTTATTCAACCTAT-3') and downstreamoligonucleotide 10 (19). Oligonucleotide 17 creates the mu-tations C47W, C52W, and C55W in the Adx moiety of F2 bychanging three TGT (Cys) codons to TAT (Tyr), thus de-stroying three of the four cysteines that coordinate the Fe2+ion in Adx (26). All PCR fragments and ligation junctionswere sequenced to verify that no errors had occurred in theamplification or subcloning.Trasection of COS-1 Ceils. COS-1 cells were transfected

by using either a calcium phosphate method or a DEAE-dextran method. Plasmid DNA purified by cesium chloridedensity gradients (>95% supercoiled) was used for eachtransfection. Each 10-cm dish (Falcon) received 2 pmol ofvector plasmid and 5 jg of a Rous sarcoma virus-luciferase(RSV-LUC) plasmid to control for transfection efficiency.After transfections were done on cultures at 60% confluencyfor 16 hr at 37°C in 5% CO2, the medium was replaced withfresh Dulbecco's modified Eagle's medium-H21 (GIBCO)containing glucose (4.5 g/liter), 10% fetal calf serum, andgentamicin (50 ,ug/ml). After 48 hr of transfection, themedium was removed from the cells and replaced with adepleted medium containing only 0.5% fetal calf serum but

supplemented with 5 x 10-6 M (22R)-hydroxycholesterol.Twenty-four hours later, cells were harvested for luciferaseactivity measurement, and pregnenolone in the medium wasmeasured by immunoassay (27).RNA and Protein Analysis. Forty-eight hours after trans-

fection, cells were washed twice in phosphate-buffered salineand harvested with either 8 M guanidinium chloride forRNApreparation or into sucrose buffer (0.25 M sucrose/50 mMethanolamine/10 mM Tris-HCl, pH 7.4/1 mM EDTA) forprotein analysis. Northern analysis of RNA was done usingMops-formaldehyde denaturing gels and 32P-labeled humancDNAs for P450scc (17), Adx (18), AdRed (19), and OR (8)as probe.COS-1 cells were sonicated and fractionated into cytosol,

mitochondria, and ER, as described (28). Total protein con-tent was determined after cell disruption with two 5-secbursts using a sonicator (Artek, Farmingdale, NY) at a settingof 20 and an equal volume of 2x loading buffer [50 mMTris HCl, pH 6.8/2% SDS/5% 2-mercaptoetanol/1096 (vol/vol) glycerol/0.005% bromophenol blue] was added. Sampleswere boiled for 5 min and then separated by electrophoresison SDS/4-209o acrylamide gradient gels. The proteins werethen electrotransferred to nitrocellulose in Tris-HCl, pH8.4/193 mM glycine/20%o methanol for 1 hr at 4°C, andimmunoblotting was done by using antisera specific to humanP450scc (27), Adx (27), AdRed (27), P450c17 (29), and OR(from C. R. Wolf, University of Edinburgh), as described(27).

RESULTSDesign and Construction ofER TargetingPsn. To test

the electron-transport requirements of P450scc and to testwhether this enzyme requires the mitochondrial environ-ment, we built a series of 18 expression vectors; theirencoded proteins are diagrammed in Fig. 1. The constructionof the plasmids encoding the fusion proteins designated F1(H2N-P450scc-AdRed-COOH), F2 (H2N-P450scc-AdRed-Adx-COOH), and F3 (H2N-P450scc-Adx-AdRed-COOH)has been described (20). Protein F4, which is a fusionbetween P450scc and NADPH-dependent P450 OR, wasconstructed to examine the stringency of P450scc in accept-ing electrons from the mitochondrial electron-transfer sys-tem. The cDNA sequence that encodes the first 56 amino

P450scc DIII1iiZ Fl

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FIG. 1. Constructions. Leader sequences at the N terminus (5'end, left) are the 39-amino acid mitochondrial leader sequence ofhuman P450scc (vertical lines) or the 23-amino acid microsomal (ER)leader sequence of rat P4S0IIB1 (checked boxes). Coding regionsfollow the leader sequences: black box, P450scc; stippled box, Adx;white box, AdRed; wavy striped box, P450 OR. The vertical bar inthe F1AR+, F2AR+, and F2DM constructions indicates the presenceof extra sequences in the 18+ form of AdRed or the 3 mutatedcysteine residues in Adx. The c17WT construction expresses thewild-type human P450c17 protein (diagonal lines), and 2B-c17 has thesame P450IIB1 microsomal leader sequence used in ER-P4S0scc andF5-8. Also shown are the constructions expressing wild-type humanAdx and AdRed, which use their own endogenous mitochondrialleader sequences (21), and the construction expressing human P450OR (29), which uses its own endogenous microsomal leader se-quence.

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FIG. 2. Northern blot. RNA was prepared from COS-1 cellstransfected with the various constructions indicated. ER-scc/ORdesignates an RNA sample from cells doubly transfected with twovectors, one expressing ER-P450scc and the other expressing OR.TRIPLE designates COS-1 cells transfected with equimolar amountsof three vectors separately expressing normal human P450scc,AdRed, and Adx, and pECE is the expression vector with no cDNAinsert. Samples of 20 pg of RNA were electrophoresed through a

Mops-formaldehyde-1% agarose gel and transferred to Hybond-Nnylon membrane (Amersham). A single blot was sequentially probedwith 32P-labeled cDNAs forhuman P450scc (A), Adx (B), AdRed (C),and OR (D). The blot was boiled in 10 mM Tris, pH 7.4/5 mMEDTA/1% SDS and re-autoradiographed between probings to en-sure removal ofall radioactivityfrom the previous probe. HindU-cutbacteriophage PM-2, run in another lane, were used as markers andpermitted alignment of the corresponding bands in the four autora-diographs.

acids of OR, which are thought to be involved in the asso-ciation ofORwith the ER (22), was deleted and replaced witha linker that encodes a unique Spe I site and also encodes thehydrophilic hinge peptide Thr-Asp-Gly-Thr-Ser. FusionsF1-F4 all possess the 39-residue N-terminal signal sequenceof P450scc, which is responsible for targeting the protein tomitochondria. In the proteins designated ER-P450scc andF5-F8, these 39 amino acids were replaced by the ERinsertion/halt-transfer sequence of rat P45011B1.

Transiription of the cDNA Expression Vectors. To examinethe expression of the various cDNA expression construc-tions, we prepared RNA from transfected COS-1 cells andanalyzed it by Northern blotting with probes for P450scc,Adx, AdRed, and OR (Fig. 2). All of the vectors expressedRNAs of the predicted sizes that contained hybridizing

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sequences predicted by their designs. The vector expressingER-P450scc, either when transfected alone or when cotrans-fected with a vector expressing OR, expressed less mRNAthan the corresponding normal P450scc vector with a mito-chondrial leader sequence, either when it was transfectedalone or triply transfected with vectors separately expressingAdRed and Adx. The reason for this is unclear. The abun-dances of the mRNAs produced by vectors F5-8 encodingmicrosomal proteins are very similar to the abundances ofthemRNAs produced by the corresponding vectors F1-4, whichexpress mitochondrial proteins. Thus, the presence of theleader sequence from rat P4501IB1 and thejunction betweenthis leader and P450scc cannot be responsible for the poorexpression (or poor mRNA stability) of the ER-P450sccconstruction. When the same Northern blot is reprobed withcDNAs for human Adx (Fig. 2B), AdRed (Fig. 2C), and OR(Fig. 2D), only the constructions predicted to encode theseRNA segments are detected, and the sizes of the hybridizingbands on these different probings ofthe same gel correspondprecisely. Although Adx (18) and AdRed (30) are expressedin all tissues, the endogenous level of expression of thesemRNAs in COS-1 cells is below the level of detection on thisNorthern blot. By contrast, endogenous COS-1 cell ORmRNA is seen in all lanes (Fig. 2D).

Expression of Fusion Proteins. To examine the translationof the mRNAs encoded by the expression vectors shown inFig. 1, we isolated total protein from cells transfected witheach of the fusion constructions and analyzed it by immu-noblotting with antibodies to human P450scc, Adx, AdRed,and OR (Fig. 3). The fusion proteins react with the expectedantisera: F1 and F5 react with antibodies to P450scc andAdRed but not with antibodies to Adx or OR; F2 and F6 reactwith antisera to P450scc, AdRed, and Adx, but not withantiserum to OR; and F4 and F8 react with antisera toP450scc and OR but not with antisera to AdRed or Adx.Proteins encoded by the F3 and F7 constructions, whichshould be the same size as the F2 and F6 proteins, could notbe detected with the P450scc or AdRed antibodies. However,a smaller (w100-kDa) band is detected with the Adx antibody,suggesting lability due to a proteolytic cleavage. With both F3and F7, this same band can be detected with the P450sccantibody, suggesting that there is a proteolytic cleavage thatremoves and degrades the AdRed moiety. The amount ofprotein produced by the constructions that target proteins tothe ER is generally lower than the amount ofthe correspond-ing protein targeted to the mitochondria, even after normal-ization for differences in transfection efficiency. This resultmay be from an inherent instability in the proteins caused bytheir presence in a cellular compartment where they are notnormally found.

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*~9~-~ FIG. 3. Immunoblots. Variousamounts of -protein were loaded ineach lane as determined by normal-ization to a constant ratio ofprotein totransfection efficiency. Each gel ana-lyzed proteins from COS-1 cellstransfected with the vector alone(pECE), with vectors separately ex-pressing P450scc (scc), Adx, AdRed,or P450scc targeted to the ER (ER-scc) from cells doubly transfected

with vectors separately expressingER-P450scc and OR (ER-scc/OR) orfrom cells transfected with vectors:.sLb expressing fusion protein F1-F8.

Blots were probed with rabbit anti-human antibodies to P4S0scc (A),Adx (B), AdRed (C), and OR (D).

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Cell Biology: Black et al.

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Enzymatic Activities of Fusion Proteins. The enzymaticactivity of each fusion protein was measured by the abilitiesof the corresponding transfected cells to convert (22R)-hydroxycholesterol to pregnenolone (Fig. 4). (22R)-Hydroxy-cholesterol was chosen as a substrate because it is solubleand freely diffusible in the cell so that it is equally accessibleto enzymes in the ER and mitochondria. Only those proteinsexpressed in mitochondria exhibit detectable enzymatic ac-tivity, whereas those expressed in ER show no appreciableability to convert 22-hydroxycholesterol to pregnenolone.Thus, the mitochondrial environment is apparently requiredfor P450scc activity. The 4-fold increase in pregnenoloneproduced by F4 compared with P450scc alone shows thatP450scc can receive electrons from OR as well as fromAdRed. Thus, the ability of F1-F4 to convert cholesterol topregnenolone shows that P450scc can accept electrons froma variety of electron-transfer proteins. However, the loweractivity of F4 suggests some structural bias for the naturalelectron donor.To determine whether the P450scc moiety in F2 was

receiving its electrons from the covalently linked electron-transport proteins, we built F1AR+, F2AR+, and F2DM.When the active, 18- form ofAdRed in F1 was replaced withthe alternatively spliced, apparently inactive (25, 31) 18+form of AdRed, F1AR+ had only modestly reduced activity.Similarly, when the 18- form of AdRed in F2 was replacedwith the 18+ form, the activity ofF2AR+ was unchanged. The18+ form ofAdRed is inactive in assays in vitro (31). Our datacould be consistent with persistent activity of the 18+ form ofAdRed in vivo, or with F1AR+ and F2AR+ receiving elec-trons from the low levels of endogenous cellular Adx. Todetermine whether F2AR+ (or F2) can receive electrons fromendogenous cellular Adx, we constructed F2DM, in whichthree of the four cysteine residues that coordinate the Fe2+ion of Adx were mutated. This construction was completelyinactive, confirming that the P450scc moiety of F2 is cata-lytically active by receiving its electrons from the covalentlylinked Adx moiety and not from interaction with endogenouscellular Adx.

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Testing the Function of the Rat P450IIB1 Leader Sequence.Because all constructions containing the insertion/halt-transfer sequence of rat P450IIB1 failed to produce activeproteins, we asked whether this leader sequence might some-how be unsuitable for steroidogenic P450 enzymes. There-fore we tested the use of this leader to target P450c17, anothersteroidogenic P450 enzyme that is normally found in the ER(Fig. 5). P450c17 activity is easily assayed (29, 32), andremoval of its targeting sequence results in a cystolic form ofthe protein that is enzymatically inactive and rapidly de-graded (33). We built pECE vectors expressing P450c17 wildtype with its own leader sequence (c17WT) or P450c17 withthe leader sequence from P4501IB1 (2B-c17) and transientlytransfected these into COS-1 cells. The vectors expressingboth c17WT and 2B-c17 encode proteins that specificallycross-react with the P450c17 antiserum (Fig. 5A). The inten-sity ofeach is similar, indicating that each protein is producedin similar amounts after transfection and that both proteinsare stable. To determine whether P450c17 containing theP450IIB1 leader is enzymatically active, we measured theability of the 2B-c17 protein to catalyze the conversion ofprogesterone to 17-hydroxyprogesterone (Fig 5B). COS-1cells transfected with the pECE vector cannot convert pro-gesterone to 17-hydroxyprogesterone, whereas c17WT and2B-c17 exhibit comparable levels of 17a-hydroxylase activ-ity. Thus the rat P450IIB1 insertion/halt-transfer sequencecan localize steroidogenic cytochrome P450 enzymes to theER in a functional manner. To confirm the ability of ourconstructions to target the proteins to the desired subcellularcompartment, we prepared cytosol, mitochondria, and ERfrom cells transfected with F2, F6, and the empty pECEvector. Immunoblotting with antiserum to Adx showed theexpected band in the mitochondria of cells transfected with

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FIG. 4. Biological activity of the fusion proteins. Conversion of22-hydroxycholesterol to pregnenolone was measured by RIA and isdisplayed as ng ofpregnenolone per ml of culture medium, correctedfor transfection efficiency. COS-1 cells transfected with variousexpression vectors are designated as in Figs. 2 and 3. N.D., notdetectable.

46

FIG. 5. Validation oftargeting to the ER with the P4501IB1 leadersequence. (A) Immunoblot of P450c17. Fifty-microgram samples ofprotein from COS-1 cells transfected with vector (pECE) or fromcells transfected with vectors expressing either P450c17 wild type(c17WT) or P450c17 with a P4501IB1 leader peptide (2B-c17) weredisplayed and analyzed with rabbit anti-human P450c17. (B) Enzy-matic activity of cells shown in A. Before cells were harvested, theywere incubated with [14C]progesterone (PROG) for 2 hr, and theproduction of 17a-hydroxy[l14C]progesterone (170HP) was assayedby TLC of the culture medium. ORI, origin. (C) Immunoblot ofcytosol, mitochondria (Mito.), and ER of COS-1 cells transfectedwith F2, F6, or pECE vector, probed with antiserum to Adx.

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F2 but showed no F2 protein in the cytosol or ER; similarlythe F6 protein was found only in the ER, and not in thecytosol or mitochondria (Fig. 5C). Thus the mitochondrialleader from P450scc and the ER leader from P450IIB1correctly target the fusion proteins to the predicted cellularorganelles.

DISCUSSIONThe identities and activities of the three components of thecholesterol side-chain cleavage system have been studied andcharacterized in detail (for review, see refs. 1-5). P450scc isthe unique form of cytochrome P450 that catalyzes all threereactions needed to convert cholesterol to pregnenolone.Although its activity is found in mitochondria and its pre-protein has a typical mitochondrial leader sequence (17, 34),it has been suggested that cholesterol side-chain cleavageactivity can be found in other cellular compartments (forreview, see ref. 5). Because all cellular fractionation andprotein purification schemes are subject to cross contamina-tion, a definitive test of this hypothesis has not previouslybeen possible. While molecular biologic techniques permittargeting of P450scc to the ER or cytosol, the cotargeting ofits electron-transport proteins in a stoichiometric ratio isneeded to yield interpretable results. The engineering ofvariations of the entire cholesterol side-chain cleavage sys-tem into a single polypeptide chain now permits such exper-iments.The ability of the four fusion proteins F1-F4 to convert

cholesterol to pregnenolone suggests that the P450scc moietymay receive electrons from several different electron-transfer proteins. An alternative interpretation is that thepresence of any C-terminal extension on the P450scc moietymay facilitate its receipt of electrons from the low levels ofendogenous COS-1 cell Adx. However, when the Adx moietyof F2 is mutated in the F2DM construction, all activity is lost,showing that the P450scc moiety of F2 is receiving electronsfrom the covalently linked Adx moiety and not from theCOS-1 cell Adx. This result suggests that the F1 and F4constructions may be catalytically active by receiving elec-trons from their covalently linked AdRed or OR moieties,rather than from COS-1 cell Adx. A rather broad range of

acceptable electron donors for a P450 enzyme may not bewholly surprising, as microsomal P450c17 apparently canreceive electrons from either OR or cytochrome b, (35).Thus, the availability of the mitochondrial electron-transfersystem should not, in and of itself, prohibit P450scc frombeing active if it, indeed, reached the ER or cytosol.

In these experiments, we used 22-hydroxycholesterol assubstrate so that the activity of our P450scc constructionswould not depend on the mitochondrial cholesterol-transportmachinery and so that a metabolizable substrate would beavailable to enzymes in the ER. However, no form ofP450scc, either the enzyme itself or the enzyme covalentlylinked to various demonstrably effective electron-transfersystems, is active when targeted to the ER. Sakaki et al. (36)have reported that mitochondrial P450c27 is active whentargeted to the ER of yeast that also express Adx and AdRedas soluble, cytosolic forms. It is not known whether P450sccwould similarly be active in the ER of mammalian cells ifsoluble cytoplasmic Adx and AdRed were provided. How-ever, the lack of activity of the F5-F8 constructs suggeststhat the unique reducing environment ofthe mitochondrion isessential for the P450scc activity.

We thank Frank Gonzalez for the OR cDNA and C. R. Wolf forthe OR antibody and the rat P450IIB1 cDNA. This work was

supported by a Glaxo Cardiovascular Discovery Grant, by National

Institutes of Health Grants DK37927 and DK42154 and by March ofDimes Grant 6-0098, all to W.L.M.

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