Cell-free synthesis of precursor forms of mitochondrial steroid hydroxylase enzymes of the bovine adrenal cortex

ARCHIVESOF BIOCHEMISTRYAND BIOPHYSICS Vol. 215, No. 2, May, pp. 478-485, 1982

Cell-Free Synthesis of Precursor Forms of Mitochondrial Steroid Hydroxylase Enzymes of the Bovine Adrenal Cortex’ ROBERT E. KRAMER,*” RAYMOND N. DU BOIS,t EVAN R. SIMPSON,*,? CHRISTEN M. ANDERSON,? KIYOKO KASHIWAGI,? J. DAVID LAMBETH,* COLIN R. JEFCOATE,O AND MICHAEL R. WATERMAN? Departmentsof ~Biochemist~ and *Obstetrkx and Gyzecdogy, and the Cecil H. and Ida Green Center for Rep&dive Biology Sciacas, The University of Texas Health Science Center, Lkdlus, Texas :75235; #Departm.e& of Biochemistry, Emory University School of Me&&e, AUanta, Georgia $0332;and $Lkpa&nent of Phmmacobgy, University of Wiscansin Medical School, Ma&on, Wticqmin 55706 Received September 25, 1981, and in revised form December 15, 1981

Newly synthesized, [?S]methionine-labeled cholesterol side-chain cleavage cytochrome P-450, ll@hydroxylase cytochrome P-450, adrenodoxin, and adrenodoxin reductase were immunoisolated from radiolabeled bovine adrenocortical cells and from rabbit reticulocyte lysate translation systems programmed with bovine adrenocortical RNA. Cholesterol side-chain cleavage cytochrome P-450 immunoisolated from a reticulocyte lysate translation system had an apparent molecular weight of 54,500 whereas this cytochrome P-450 immunoisolated from radiolabeled bovine adrenocortical cells had an apparent molecular weight of 49,000, an apparent molecular weight identical to that of the purified protein. Similarly, newly synthesized, [?S]methionine-labeled llghydroxylase cytochrome P-450 immunoisolated from a reticulocyte lysate translation system had an apparent molecular weight 5500 daltons larger than that immunoisolated from radiolabeled adrenocortical cells (48,000) and the authentic cytochrome (48,000). The cell-free translation products of adrenodoxin and adrenodoxin reductase were also several thousand daltons larger than the corresponding mitochondrial proteins. The apparent molecular weight of adrenodoxin immunoisolated from a reticulocyte lysate translation system was 19,000, while that of the authentic protein was 12,000. Adrenodoxin reductase immunoisolated from a lysate translation system had an apparent molecular weight of 53,400; an apparent molecular weight 2300 daltons larger than that of adrenodoxin reductase immunoisolated from radiolabeled adrenocortical cells or purified by conventional techniques. These results demonstrate that all of the components of the mitochondrial steroid hydroxylase systems of the bovine adrenal cortex are synthesized as precursor molecules of higher molecular weight. Presumably, the precursor proteins are post-translationally converted to the mature enzymes upon insertion into the mitochondrion by a process which includes the proteolytic cleavage of the precursor segments. Steroidoeenesis in the adrenal cortex is catalyzed by enzymes associated with both the endoplasmic reticulum and the mitochondrion. Most of these enzymes are hemoproteins termed cytochromes P-450 (l4). Cytochromes P-450 are mixed-function oxidases, and each adrenal cytochrome catalyzes the hydroxylation of a steroid 0003-9861/82/060478-08$02.00/0 Copyrieht0 19%? by Academic Pman, he All rights of reproductions in my form retwved.

i This research was supported in part by U. S. Publie Health Service Grants GM2’7151, AM28350, AM18585, AM273’73, and HD13234 and Grant I-624 from The Robert A. Welch Foundation. R.E.K. is supported by the USPHS Service Training Grant T32HD07190; C.R.J. by Research Career Development Award CAOO25Ozand R.N.D. bv Research Service Award GM070621 2 To whom correspondence should be addressed.

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substrate at a specific carbon atom(s). Two of these cytochromes, cholesterol sidechain cleavage cytochrome P-450 (P-45O,)3 and llfi-hydroxylase cytochrome P-450 (P-450110),are integral components of the inner mitochondrial membrane (5-7). Cytochrome P-450,, catalyzes the conversion of cholesterol to pregnenolone (8, 9). This reaction is generally considered to be the rate-limiting step in corticosteroid biosynthesis and under acute hormonal control by ACTH (10). Cytochrome P-450118hydroxylates substrates such as ll-deoxycorticosterone and 11-deoxycortisol at the ll@-position to form corticosterone and cortisol, respectively (11, 12). Both cytochrome P-450,, and cytochrome P-4501,, require a flavoprotein (adrenodoxin reductase) and an iron-sulfur protein (adrenodoxin) for full hydroxylase activity (13-15). Adrenodoxin reductase and adrenodoxin are mitochondrial matrix proteins which appear to be associated with the inner aspect of the inner mitochondrial membrane (15-1’7) and function to transfer reducing equivalents from NADPH to cytochromes P-450,, and P-45011#9 (18, 19). The concentrations of the various hydroxylase enzymes within the mitochondrion directly affect its steroidogenic capacity and are dependent upon ACTH (2024). For example, mitochondrial cytochrome falls following hypophysectomy and is restored by administration of ACTH to hypophysectomized animals (20-22). Similarly, mitochondrial adrenodoxin and cytochrome P-450 levels were increased by ACTH in cultured rat adrenal tumor cells (23). ACTH has also been noted to increase cytochrome P-450 levels in cultured bovine adrenocortical cells (24). Cholesterol sidechain cleavage and lip-hydroxylase activities correlated with enzyme concentration. It has generally been assumed that the effect of ACTH to maintain mitochondrial concentrations of the various steroid hya Abbreviatiods used: ACTH, adrenocorticotropin: cytochrome P-450,, side-chain cleavage cytochrome P-450; cytochrome P-45011g, ll&hydroxylase cytochrome P-450; SDS, sodium dodecyl sulfate; IgG, serum immunoglobulin; TCA, trichloroacetic acid.

MITOCHONDRIAL

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droxylase components reflects an increase in specific protein synthesis. Nonetheless, relatively little attention has been directed to date toward the molecular mechanisms involved in the biosynthesis of these proteins or their incorporation into the mitochondrion. Studies recently carried out in this laboratory have demonstrated that the synthesis of cytochrome P-450,, by monolayer cultures of bovine adrenocortical cells is increased by ACTH (25). The increase in the rate of cytochrome P-450, synthesis was demonstrable both in radiolabeled adrenocortical cells and in a cell-free translation system programmed with RNA isolated from bovine adrenocortical cells. Additionally, it has been shown in this laboratory that cytochrome P-450,,, is synthesized as a precursor which requires post-translational conversion to the mature protein upon insertion into the mitochondrion (24). Nabi and co-workers (26, 27) have reported that cytochrome P-450118and adrenodoxin are also synthesized as precursor molecules. However, these authors failed to demonstrate a precursor form of cytochrome P-450,, or adrenodoxin reductase. In an attempt to clarify the mechanism of biosynthesis of the various components of the mitochondrial steroid hydroxylase systems, we have used specific immunoprecipitation techniques to isolate newly synthesized translation products of cytochromes P-450,, and P-450110, adrenodoxin, and adrenodoxin reductase from a cell-free rabbit reticulocyte lysate translation system programmed with bovine adrenocortical RNA. On the basis of the results of the present study we conclude that all of the components of the mitochondrial steroid hydroxylase systems of the bovine adrenal cortex are synthesized as higher molecular weight precursors. MATERIALS

AND

METHODS

Protein pur$v.xztion and antibody preparation. Cytochrome P-450, and cytochrome P-450118 were purified from bovine adrenocortical mitochondria using the methods described by Seybert et al. (28) and Takemori d al. (12), respectively. Adrenodoxin reductase and adrenodoxin were purified as previously de-

480

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scribed by Lambeth and Kamin (29). Antibodies to these purified bovine adrenocortical proteins were produced in rabbits after an initial injection into the popliteal lymph nodes and subsequent intradermal booster injections. The serum immunoglobulin (IgG) fraction was prepared by precipitation with 33% ammonium sulfate (30). Antigen-antibody interactions were shown to be specific by the double-immunodiffusion technique. RNA isolution and cell-free translation Bovine adrenal glands were obtained from a local abattoir. Adrenal glands were quickly removed and bisected. Cortical tissue was separated and immediately frozen in liquid nitrogen. Total adrenocortical RNA was isolated by extraction with guanidine hydrochloride as described by Deeley et al. (31). RNA was translated in a commercially available rabbit reticulocyte lysate system (New England Nuclear) in a total volume of 25 ~1 (32). Incubations were carried out at 37°C for 60 min. The incorporation of [35S]methionine into TCA-precipitable protein was measured to assay translational efficiency. Immurwprecipitation and electrophuresis. After translation, sample volumes were adjusted to contain the same amount of TCA-precipitable radioactivity when desired, diluted with l/5 vol of 50 mM methionine, vortexed, and then diluted with an equal volume of 10% SDS. Samples were next heated at 100°C for 2 min and diluted 1:lO with phosphate-buffered saline containing 1 mM EDTA and 0.5% NP-40. Specific newly synthesized proteins were isolated from total translational products by indirect immunoprecipitation (25) using Staphylowcczls awe-us cell membranes (Pansorbin; Calbiochem) as the source of protein A (33). Immunoprecipitates were suspended in 30 pl of Laemmli electrophoresis sample buffer (34) and heated to 100°C for 2 min to dissociate the immunoisolated protein from the immune IgG and the Pansorbin. Pansorbin was removed from suspension by centrifugation. Immunoisolates obtained with antibodies to adrenodoxin reductase and cytochromes P-450, and P-450,,, were subjected to SDS-7.5% to 12.5% polyacrylamide gradient gel electrophoresis. Adrenodoxin immunoisolates were subjected to electrophoresis on SDS-15% polyacrylamide gels. The gels were dried and subjected to autoradiography by standard techniques. Cell culture and protein labeling. Bovine adrenocortical cell dispersion and maintenance in monolayer culture were carried out as described previously (24, 25). Cell monolayers were incubated for 2 h in methionine-free Ham’s F-12 medium and then radiolabeled with [%S]methionine (60 &/ml) for an additional 2 h. Cells were harvested and lysed in phoshate-buffered saline containing 1% cholate and 0.1% SDS (25). Specific newly synthesized proteins were immunoisolated and subjected to electrophoresis as described above.

ET AL. RESULTS

Authentic cytochromes P-450118and P450,,, purified from bovine adrenocortical mitochondria had apparent molecular weights of 48,000 and 49,000, respectively (Fig. 1, Lanes 1 and 2). Newly synthesized cytochromes P-45011 and P-450,, immunoisolated from [AZSlmethionine-pulsed adrenocortical cells had apparent molecular weights identical to those of the purified proteins (Fig. 1; compare Lanes 1 and 3 and Lanes 2 and 4). In contrast, cytochrome P-450,,, synthesized in a cellfree translation system programmed with 5 pg of bovine adrenocortical RNA had an apparent molecular weight substantially greater than that of the authentic cytochrome (Fig. 1; compare Lanes 1 and 5). The apparent molecular weight of cytochrome P-450118synthesized in a cell-free translation system was 53,500, 5500 daltons larger than the mature mitochondrial protein. Cytochrome P-450,,, immunoisolated from a cell-free system programmed with bovine adrenocortical RNA also had an apparent molecular weight 5500 daltons greater than the authentic protein and that isolated from bovine adrenocortical cells previously maintained in monolayer culture (Fig. 1; compare Lanes 2, 4, and 6). Proteins immunoprecipitated when antibodies to adrenodoxin and adrenodoxin reductase were added to a cell-free translation system were also observed to have apparent molecular weights greater than purified adrenodoxin and adrenodoxin reductase. Authentic adrenodoxin had an apparent molecular weight of 12,000 (Fig. 2, Lane 1). Likewise, adrenodoxin immunoisolated from [35S]methionine-pulsed adrenocortical cells had an apparent molecular weight of 12,000 (Fig. 2, Lane 2). In contrast, adrenodoxin synthesized by translation of 5 pg of bovine adrenocortical RNA in a cell-free system had an apparent molecular weight of 19,000 (Fig. 2, Lane 3). Newly synthesized adrenodoxin reductase immunoisolated from cell-free translation system proirammedwith bovine adrenocortical RNA also had an apparent molecular weight greater than that immunoisolated from

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of the precursor form of adrenodoxin reductase was 53,400, 2300 daltons larger than the mature protein. The apparent molecular weights of the mature and precursor forms of the various mitochondrial hydroxylase components are summarized in Table I. The immunoisolation of newly synthesized, radiolabeled proteins with antibodies raised against cytochromes P-450118

1

2

3

4

5

6

7

8

9

FIG. 1. Electrophoretic comparison of mature and precursor forms of cytochromes P-4501i8 and P-450,. Cytochromes P-4501~~ (Lane 1) and P-450, (Lane 2) purified from bovine adrenocortical mitochondria were subjected to electrophoresis on an SDS-7.5% to 12.5% polyacrylamide gradient slab gel and stained with Coomassie blue. Newly synthesized cytochromes P-450,,, (Lane 3) and P-450, (Lane 4) immunoisolated from [?S]methionine-pulsed bovine adrenocortical cells previously maintained in monolayer culture were also subjected to electrophoresis and autoradiography. Autoradiograms of newly synthesized, [35S]methionine-labeled cytochromes P-450118 and P-460, immunoisolated from a cell-free translation system programmed with 5 kg of bovine adrenocortical RNA are shown in Lanes 5 and 6, respectively. Autoradiograms of cytochrome P-451118 immunoisolated from a cell-free translation system in the presence of 50 pg of authentic cytochrome P450,,# and 50 pg of authentic cytochrome P-450, are shown in Lanes 7 and 8, respectively. An autoradiogram of cytochrome P-450, immunoisolated from a cell-free translation system in the presence of 50 pg of authentic cytochrome P-450,, is shown in Lane 9.

adrenocortical cells or purified by conventional techniques (Fig. 3; compare Lanes 1,2, and 3). The apparent molecular weight

1

2

3

4

FIG. 2. Electrophoretic comparison of mature and precursor forms of adrenodoxin. Authentic, purified adrenodoxin was subjected to electrophoresis on an SDS-15% polyacrylamide gel and then stained with Coomassie blue (Lane 1). Newly synthesized, [?Zl]methionine-labeled adrenodoxin was immunoisolated from bovine adrenocortical cell monolayers (Lane 2) and from a cell-free translation system programmed with 5 pg of bovine adrenocortical RNA (Lane 3) and then subjected to electrophoresis and autoradiography. The result of immunoprecipitating newly synthesized, radiolabeled adrenodoxin from a cell-free translation system in the presence of 50 pg of authentic protein is shown in Lane 4.

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and adrenoand P-450,,, adrenodoxin, cloxin reductase was completely abolished by the addition of an excess of authentic

ET AL. TABLE APPARENT

MOLECULAR

I

WEICHT~

STEROID

HYDROX~LASE Apparent Mature protein

Cytochrome Cytochrome Adrenodoxin Adrenodoxin

1

2

3

4

FIG. 3. Electrophoretic comparison of mature and precursor forms of adrenodoxin reductase. Adrenodoxin reductase purified from bovine adrenocortical mitochondria was subjected to electrophoresis on an SDS-7.5% to 12.5% polyacrylamide gradient gel and then stained with Coomassie blue (Lane 1). Newly synthesized, pS]methionine-labeled adrenodoxin reductase was immunoisolated from monolayer cultures of bovine adrenocortical cells (Lane 2) and a cell-free translation system programmed with 5 pg of bovine adrenocortical RNA (Lane 3) and then subjected to electrophoresis and autoradiography. The result of immunoisolating newly synthesized, radiolabeled adrenodoxin reductase from a cell-free translation system in the presence of 50 pg of the authentic flavoprotein is shown in Lane 4.

OF THE

MATURE

AND PRECURSOR FORMS OF THE VARIOUS COMPONENTS OF THE ADREN~C~RTICAL MITOCHONDRIAL

P-450, P-45011, reductase

49,000 48,000 12.000 51,100

SYSTEMS molecular

weight Precursor

Precursor

segment

54,500

5500

53,500 19,000 53,400

5500 7ooo 2300

protein. For example, the addition of 50 pg of authentic cytochrome P-450,,, to the translation lysate prior to the addition of anti-cytochrome P-450118completely inhibited the immunoprecipitation of newly synthesized, [%]methionine -labeled cytochrome P-450118(Fig. 1, Lane 7). Similarly, the addition of 50 pg of purified cytochrome P-450 Seeto a cell-free translation system prevented the immunoisolation of newly synthesized, radiolabeled protein by anti-cytochrome P-450,, (Fig. 1, Lane 9), but not by anti-cytochrome P-450118(Fig. 1, Lane 8). The addition of 50 clg of adrenodoxin or aclrenodoxin reductase prior to the addition of anti-adrenodoxin or antiadrenodoxin reductase also abolished the immunoisolation of the corresponding specific [35S]methionine-labeled protein (Figs. 2 and 3, Lane 4). These results are indicative that the immunoisolates obtained in the absence of authentic nonradiolabeled proteins were, in fact, newly synthesized cytochromes P-450110and P45Oscc,adrenodoxin, and adrenodoxin reductase. DISCUSSION

Mitochondria of steroidogenic tissues contain not only the normal regimen of enzymes required for oxidative metabolism but also enzymes which function in the hydroxylation of steroid substrates. For example, mitochondria of the adrenal cortex contain adrenodoxin reductase, adrenodoxin, and cytochromes P-450,,, and P-450118(l-4). The amounts of these steroidogenic proteins within the mitochon-

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drion have been shown to affect the functional capacity of the adrenal cortex and to be directly dependent upon ACTH (2022). Nonetheless, the mechanisms involved in their synthesis and incorporation into the mitochondrion remain poorly understood. The mitochondrion has been shown to contain DNA and all of the enzymatic machinery required to carry out transcriptional and translational processes, but the majority of mitochondrial proteins, including those involved in steroidogenesis, appear to be coded for by nuclear genes, synthesized within the cytoplasm, and subsequently imported into the mitochondrion. The primary translation products of some (35-40), but not all (41, 42), mitochondrial proteins synthesized in the cytoplasm have apparent molecular weights several thousand daltons larger than the corresponding proteins contained within the mitochondrion. For example, the primary translation product of yeast mitochondrial cytochrome c peroxidase appears to be 6000 daltons larger than the mature protein (35). Various subunits of yeast mitochondrial F1-ATPase and cytochrome c oxidase also appear to be synthesized outside the mitochondrion as precursors 2000 to 6000 daltons larger than the mature subunits (36, 37). In addition, ornithine transcarbamoylase of liver mitochondria appears to be synthesized as a precursor of higher molecular weight (38). Similarly, in a recent report from this laboratory we showed that newly synthesized cytochrome P-450,, of bovine adrenocortical mitochondria was 5500 daltons larger than the mature protein (24). The results of the present investigation support the contention that cytochrome P450,, is synthesized as a precursor which must be post-translationally converted to the mature protein upon insertion into the mitochondrion. Newly synthesized cytochrome P-450118is also a precursor having an apparent molecular weight 5500 daltons larger than the authentic cytochrome. Thus, the mechanisms involved in the synthesis of mitochondrial cytochromes P-450 and their incorporation into the mitochondrial membrane appear to differ from those for cytochromes P-450

MITOCHONDRIAL

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483

destined for the endoplasmic reticulum. Larger molecular weight precursors have not been observed for hepatic (43-44) and adrenocortical (27) microsomal cytochromes P-450, in marked contrast to mitochondrial cytochromes P-450, and P4501i8. The other components of the mitochondrial steroid hydroxylase systems also are synthesized as precursor peptides. Adrenodoxin reductase synthesized in a reticulocyte lysate system programmed with bovine adrenocortical RNA had an apparent molecular weight 2300 daltons larger than the corresponding mitochondrial reductase. In addition, adrenodoxin synthesized in a cell-free translation system was 7000 daltons larger than the mature protein. These results are in partial agreement with those of Nabi and associates (26,27). These authors have also described higher molecular weight precursors of cytochrome P-450118 and adrenodoxin. The sizes of the precursor segments reported by Nabi and co-workers are in good agreement with those reported here. In contrast to the present study, however, they failed to observe a precursor for either adrenodoxin reductase or cytochrome P-450,. Although the reasons for this discrepancy are uncertain, it may reflect differences in the methodologies and cell-free translation systems employed in the two studies. Regardless, the results presented here and elsewhere (24, 25) clearly demonstrate that all of the components of the mitochondrial steroid hydroxylase systems of the bovine adrenal cortex are synthesized as higher molecular weight precursors. Results obtained in this laboratory (unpublished observations) and those of Omura (26,27) indicate that the synthesis of these precursor proteins occurs within the cytoplasm of the adrenocortical cell and is directed by both free and membrane-bound polysomes. Each precursor segment presumably has a specific peptide sequence(s) which serves to interact with a recognition site on the mitochondrion and perhaps also direct the protein to a specific mitochondrial locus. Yet, the precursor segments for adrenodoxin and adrenodoxin reductase, both of which are mitochondrial ma-

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trix proteins (E-17), are quite different in molecular weight. The precursors for adrenodoxin reductase and adrenodoxin are 2300 and 7000 daltons larger than the mature proteins, respectively. Interestingly, however, the precursor segments for cytochromes P-450,, and P-450118 both have molecular weights of 5500. Although the similarity in apparent molecular weights may be coincidental, it may reflect a homology in the precursor segments required for incorporation and proper orientation within the inner mitochondrial membrane so that each cytochrome can interact with adrenodoxin in a correct and similar fashion. Sequence analysis of each precursor segment will be required to substantiate this possibility. ACKNOWLEDGMENTS We gratefully assistance of Tuckey.

Ms.

acknowledge the expert technical Grace Rivera and Ms. Janette

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