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

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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 re- ducing 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.
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