Báo cáo khoa học: Effects of various N-terminal addressing signals on sorting and folding of mammalian CYP11A1 in yeast mitochondria doc

Effects of various N-terminal addressing signals on sorting and folding of mammalian CYP11A1 in yeast mitochondria Irina E. Kovaleva, Lyudmila A. Novikova, Pavel A. Nazarov, Sergei I. Grivennikov and Valentin N. Luzikov Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia Topogenesis of cytochrome P450 scc , a resident protein of the inner membrane of adrenocortical mitochondria, is still obscure. In particular, little is known about the cause of its tissue specificity. In an attempt to clarify this point, we examined the process in Saccharomyces cerevisiae cells synthesizing cytochrome P450 scc as its native precursor (pCYP11A1) or versions in which its N-terminal addressing presequence had been replaced with those of yeast mitoch- ondrial proteins: CoxIV(1–25) and Su9(1–112). We found the pCYP11A1 and CoxIV(1–25)-mCYP11A1 versions to be effectively imported into yeast mitochondria and subjec- ted to proteolytic processing. However, only minor portions of the imported proteins were incorporated into mito- chondrial membranes, whereas their bulk accumulated as aggregates insoluble in 1% Triton X-100. Along with pre- viously published data, this suggests that a distinguishing feature of the import of the CYP11A1 precursors into yeast mitochondria is their easy translocation into the matrix where the foreign proteins mainly undergo proteolysis or aggregation. The fraction of CYP11A1 that happens to be inserted into the inner mitochondrial membrane is effectively converted into the catalytically active holoenzyme. Experi- ments with the Su9(1–112)-mCYP11A1 construct bearing a re-export signal revealed that, after translocation of the fused protein into the matrix and its processing, the Su9(67–112) segment ensures association of the mCYP11A1 body with the inner membrane, but proper folding of the latter does not take place. Thus it can be said that the most specific stage of CYP11A1 topogenesis in adrenocortical mito- chondria is its confinement and folding in the inner mito- chondrial membrane. In yeast mitochondria, only an insignificant portion of the imported CYP11A1 follows this mechanism. Keywords: yeast mitochondria; import; sorting; folding; aggregation. Cytochrome P450 scc is a resident protein of adrenocortical mitochondria. In co-operation with adrenodoxin and adrenodoxin reductase it carries out conversion of choles- terol into pregnenolone. Cytochrome P450 scc is synthesized in the cytoplasm as a precursor [1] that is imported into mitochondria, where it becomes an integral protein of the inner membrane [2]. At least two peculiarities of its topogenesis are still unclear. First, this protein cannot be extracted from the membrane by carbonate treatment, although it does not contain any distinct transmembrane domains. Second, the import of the cytochrome P450 scc precursor into mitochondria is tissue-specific. In fact, pCYP11A1 is imported into adrenocortical and liver mito- chondria but not into heart mitochondria [3,4]. On the other hand, the import of various versions of pCYP11A1 has been demonstrated with mitochondria of COS-1 [5] and yeast [6] cells. Moreover, even plant mitochondria can import pCYP11A1 [7]. Mitochondria of transformed yeast cells exhibit side-chain cleavage activity in reconstituted systems, the activity being detected in the inner membrane fraction [6,8]. These data seem to suggest similar topogenesis of CYP11A1 in various organisms. However, no details of its import into heterologous mitochondria have been studied in the above publications. Efforts in this direction were undertaken in the experiments with isolated yeast mitochon- dria and in vitro synthesized bovine pCYP11A1 [9]. It turned out that upon import into mitochondria the protein accu- mulates mainly in the matrix in precursor and mature forms. Only a trace amount of the protein was found in the inner membrane fraction. This was in discord with the earlier data [10] according to which pCYP11A1 does not leave the inner membrane upon its import into isolated adrenocortical mitochondria and subsequent maturation. Thus, the crucial distinction of the import of pCYP11A1 into yeast mito- chondria is that, for unknown reasons, the protein is rapidly translocated into the mitochondrial matrix. The imported protein largely undergoes proteolysis by matrix protease Pim1p, which additionally testifies to such translocation [9]. Proteolysis competes with aggregation dominating in mito- chondria with mutant forms of Pim1p or mtHsp70. In contrast to pCYP11A1, the Su9(67–112)-CYP11A1(75–481) version was detected in a considerable amount in the inner membrane fraction where it was directed by the Ôre-exportÕ Correspondence to I. E. Kovaleva, Belozersky Institute of Physical and Chemical Biology, Moscow State University, 119899 Moscow, Russia. Fax: + 7 095 939 3181, E-mail: kovaleva@genebee.msu.su Abbreviations: pCYP11A1, precursor form of CYP11A1; mCYP11A1, mature form of CYP11A1; SMP, submito- chondrial particles. Enzymes: CYP11A1 (EC 1.14.15.6); adrenodoxin reductase (EC 1.14.15.4). (Received 26 March 2002, revised 6 November 2002, accepted 20 November 2002) Eur. J. Biochem. 270, 222–229 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03378.x mechanism [11] using the N-terminal transmembrane domain of the Su9 protein. The Su9(1–112) segment is known to ensure the initial stage of the import of Su9 into the matrix followed by two-stage processing and reinsertion of its N-terminus into the inner membrane in such a way that the transmembrane domain spans the membrane while the 14-mer extension becomes exposed into the intermembrane space. The Su9(1–112) sequence fused to N-termini of some foreign proteins is capable of carrying out the same transport function [12]. The membrane-bound Su9(67–112)- CYP11A1(75–493) form was degraded by the membrane Yta10p-12p proteolytic complex [9], which testifies to its improper folding. Unfortunately, the in vitro experiments could not provide information about conversion of CYP11A1 into a catalytically active holoenzyme. Using Saccharomyces cerevisiae yeast expressing CYP11A1 ver- sions (CYP11A1s) with different topogenic signals, in this work we planned to answer the following questions: (a) how do various topogenic signals influence the sorting of CYP11A1s in yeast mitochondria; (b) what is the relation between the contents of membrane-bound and aggregated forms of the imported CYP11A1 and (c) how effective is the conversion of membrane-bound CYP11A1 into the cataly- tically active form? Such information revealing specific features of CYP11A1 import into foreign mitochondria might be helpful both for understanding the topogenesis of this protein in nature and for assessing its possibilities in transgenic organisms. Materials and methods Media for cultivation of yeast and bacteria were from Difco Laboratories (USA). 22(R)-hydroxycholesterol and d-ami- nolevulinic acid were from Sigma (USA). Restriction endo- nucleases, Klenow fragment, and T4 DNA ligase were from Fermentas (Lithuania). Plasmids were maintained and am- plified in Escherichia coli JM-109 (Promega, USA). cDNAs encoding recombinant proteins were used for transformation of Saccharomyces cerevisiae 2805 (aMATpep4::HIS3 prb1-d can1GAL2His3ura3–5) deficient in vacuolar proteases. Rabbit polyclonal anticytochrome P450 scc IgG, adrenocor- tical mitochondria, purified bovine cytochrome P450 scc , adrenodoxin, and adrenodoxin reductase were generous gifts from V. M. Shkumatov (State University of Belarus, Minsk). DNAs and plasmids Yeast expressing the shuttle vector pYeDP1-8/2 (pYeDP) [13] was used for expression of cDNAs encoding recombin- ant proteins. This vector includes a galactose-inducible chimeric promoter GAL10-CYC1, a terminator of yeast phosphoglycerate kinase, and an auxotrophic marker URA3. The pYeDP/Cox(1–25)-mCYP11A1 with the pre- sequence of subunit IV of yeast cytochrome oxidase has been prepared earlier in this laboratory [6]. The pYeDP/ pCYP11A1 encoding CYP11A1 with its own N-terminal addressing presequence was constructed by inserting cDNA for human CYP11A1, a gift from W. L. Miller (University of California, San Francisco, USA), into pYeDP at the EcoRI and KpnI sites. cDNA for a fusion protein composed of mCYP11A1 preceded by the N-terminal fragment of subunit 9 (Su9) of yeast F 0 -ATPase, including its addressing presequence and the N-proximal transmembrane segment, was constructed on the basis of pGEM4/Su9(1–112)- CYP11A1(75–493) [9]. Full-size cDNA for mCYP11A1 was obtained by substituting the NcoI-BglII fragment of pTrc99A/mCYP11A1 [14] for the BamHI-BglIfragmentin pGEM4/Su9(1–112)-CYP11A1(75–493). Prior to ligation, the BamHI end of the plasmid and the NcoIendofthe excised cDNA were treated with Klenow fragment. Hybrid cDNA encoding the Su9(1–112)-mCYP11A1 fusion protein was excised from pGEM4/Su9(1–112)-mCYP11A1 at EcoRI and KpnI sites and inserted into pYeDP pretreated with BamHI and KpnI. The BamHI and EcoRI ends were treated with Klenow fragment prior to ligation. The resulting pYeDP/Su9(1–112)-mCYP11A1 plasmid was used to transform yeast cells. To achieve low-copy expression of CoxIV(1–25)-mCYP11A1, the corresponding cDNA was inserted into pINT2 (an integrative vector with phospho- glycerate kinase promoter and URA3 marker; kindly pro- vided by D. G. Kozlov, Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia) by excising this cDNA from pYeDP/CoxIV(1–25)-mCYP11A1 with EcoRI and XbaI and subcloning it sequentially in pUC19 and pUC18 to obtain appropriate ends for insertion into pINT2 at BamHI and EcoRI sites. Final ligation gave the pINT2/CoxIV(1–25)-mCYP11A1 construct. Transformation of yeast cells with pYeDP/pCYP11A1, pYeDP/Cox(1–25)-mCYP11A1, and pYeDP/Su9(1–112)- mCYP11A1 was carried out as described earlier [15]. The yeast cells were grown in SD medium (0.67% yeast nitrogen bases, 1% ammonium sulfate and 2% glucose) for 24 h, and then for 14 h in a selective medium with 2% galactose added for promoter induction. After this, the growth medium was also supplemented with 200 l M d-aminolevulinic acid. Isolation and fractionation of yeast mitochondria Yeast mitochondria were isolated as described in [16] and fractionated according to [4] with minor modifications. Mito- chondria were suspended in 5 m M Tris/HCl, pH 7.4, incu- bated for 20 min at 4 °C to disrupt the outer membranes, and subjected to sonic disintegration. The mitochondrial suspen- sion was then centrifuged for 15 min at 12 000 g.The resulting suspension was centrifuged for 1 h at 106 000 g to obtain the submitochondrial particles (SMP) fraction. Assessment of aggregation of recombinant proteins in yeast mitochondria Mitochondria (0.5 mg protein mL )1 ) were incubated in 20 m M Hepes/KOH, pH 7.4, with 150 m M NaCl, 1 m M phenylmethyl sulphonyl fluoride, and 1% Triton X-100 for 30 min on ice. The suspension was then centrifuged for 15 min at 25 000 g. The supernatant proteins were precipi- tated with 10% (v/v) trichloroacetic acid, and the pellets were dissolved in SDS/PAGE buffer. The content of CYP11A1 was estimated in Triton X-100 soluble and insoluble fractions by Western blot analysis. Western blot analysis To estimate the contents of recombinant CYP11A1 ver- sions in mitochondria and 12 000 g supernatants from Ó FEBS 2003 CYP11A1 topogenesis in yeast mitochondria (Eur. J. Biochem. 270) 223 mitochondrial homogenates, we used cytochrome P450 scc - calibrated immunoblotting. For this purpose, SDS/PAGE was carried out with fixed amounts of isolated cytochrome P450 scc in 2.5–20 or 25–200 ng ranges, with the contents of the protein in adjacent lanes differing by a factor of two. Then routine procedures were used for transferring the proteins to nitrocellulose membranes [17] and their detec- tion with rabbit anticytochrome P450 scc IgG and conjugate of anti-rabbit IgG with peroxidase. This approach allowed one to estimate the content of various CYP11A1 versions with an accuracy of ± 50%. Assays of cholesterol side-chain cleaving activity of various CYP11A1 versions Mitochondria isolated from yeast cells expressing CYP11A1 versions were subjected to hypotonic shock and sonication as indicated above. The assays were carried out with supernatants after centrifugation of mitochondrial homo- genates at 12 000 g, which removed large mitochondrial fragments and protein aggregates. The reaction mixture contained 200–500 lg of mitochondrial protein, purified bovine adrenodoxin (0.8 nmole), adrenodoxin reductase (0.2 nmole), and 25 nmoles of 22(R)-hydroxycholesterol in 0.5mLof30m M phosphate buffer, pH 7.5, supplemented with 0.05% (v/v) Tween 20. The samples were preincubated for 20 min at 25 °C. The reaction was started by addition of an NADPH-generating system including 0.1 m M NADPH, 5m M glucose-6-phosphate, and glucose-6-phosphate dehy- drogenase (1 UÆmL )1 ) (Serva, Germany). The reaction was stopped after 30 min by plunging the samples into boiling water for 15–20 s. Then the samples were mixed with 3 mL of 100 m M sodium phosphate buffer, pH 7.2, with 0.05% (v/v) Tween 20, and cholesterol oxidase (0.1 U per sample) (Serva, Germany) was added to convert pregnenolone formed from 22(R)-hydroxycholesterol into progesterone. After a 45-min incubation at 37 °C, the samples were treated with ethyl acetate to extract the steroids. The extracts were evaporated to dryness and the content of progesterone was determined with an ELISA test system based on antiprogesterone antibodies. The test kits were kindly provided by Dr A. G. Pryadko (Institute of Bioorganic Chemistry, Belarus). Results Import of recombinant forms of CYP11A1 into yeast mitochondria in vivo cDNAs encoding pCYP11A1, CoxIV(1-25)-mCYP11A1, and Su9(1–112)-mCYP11A1 have been inserted into the yeast expressing shuttle vector pYeDP. CoxIV(1–25) is the N-terminal addressing presequence of subunit IV of yeast mitochondrial cytochrome oxidase, and Su9(1–112) is the N-terminal part of the precursor of subunit 9 of yeast mitochondrial F o -ATPase. Yeast cells expressing the above recombinant proteins were used to isolate mitochondria in which various forms of CYP11A1 were detected by immunoblotting with anti-P450 scc IgG. Figure 1 shows that all the CYP11A1 forms were imported into mitochondria. As we failed to register CO difference spectra of yeast mitochondria containing the recombinant proteins, we have estimated their intramitochondrial contents by cytochrome P450 scc -calibrated immunoblotting. The values obtained for CoxIV(1–25)-mCYP11A1, Su9(1–112)-mCYP11A1, and pCYP11A1 were 0.3%, 0.5% and 0.1% of total mito- chondrial protein, respectively. Appreciable distinctions were found at the stage of processing of the imported proteins (Fig. 1). All of Su9(1)112)-mCYP11A1 is processed to a single product somewhat larger than mCYP11A1 because of the 45-mer addition at the N-terminus. CoxIV(1–25)-mCYP11A1 is also effectively processed, although immunoblotting reveals some precursor in yeast mitochondria. Similar results have been obtained upon expression of CYP11A1 with the presequence of subunit VI of yeast cytochrome oxidase [8]. A more complicated pattern was observed for pCY- P11A1 with its own N-terminal presequence. In this case one could see three forms of the protein, i.e. precursor, mature protein and semiprocessed precursor. The lower content of mCYP11A1 might have resulted from the poor match of the yeast mitochondrial processing peptidase to the indigenous processing site in pCYP11A1. Fig. 1. Western blots of mitochondria prepared from recombinant yeast strains producing pCYP11A1, CoxIV(1– – 25)-mCYP11A1, and Su9(1– – 112)-mCYP11A1. The samples of mitochondria isolated from recombinant yeast producing CoxIV-mCYP11A1, pCYP11A1, and Su9-mCYP11A1 were probed with anti-(cytochrome P450 scc )IgG. Mitochondria from these strains were loaded at 25, 25, and 10 lg protein per lane, respectively. Purified bovine cytochrome P450 scc was a marker at 20 and 500 ng protein per lane for the left and right panels, respectively. In the scheme, p, i, and m correspond to precursor, intermediate, and mature forms of CYP11A1. Light and dark shaded boxes are targeting signal (ts) and mature protein sequence, respect- ively; empty box is the transmembrane domain (tm) of the Su9 precursor. 224 I. E. Kovaleva et al.(Eur. J. Biochem. 270) Ó FEBS 2003 In as much as in the three above cases the mitochondrial fraction mainly contained mature or semiprocessed forms of CYP11A1, one may assume that this fraction was not significantly contaminated with the recombinant protein in a cytoplasmic aggregated form. Relationship between aggregated and membrane- bound forms of CYP11A1 in yeast mitochondria Obviously, early post-translocational changes of a protein imported into mitochondria can be revealed only in the short-term in vitro experiments. It has been shown [9] that upon import of the in vitro synthesized pCYP11A1 into isolated yeast mitochondria, only a minor portion of the protein remains in the inner membrane, whereas its bulk is transferred into the matrix where it undergoes proteolysis or aggregation. Therefore, expressing CYP11A1 with various addressing presequences in yeast cells, one would like to know the relationship between aggregated and membrane- bound forms of the protein imported into mitochondria, in particular, to estimate the efficiency of conversion of the imported protein into a catalytically active form in foreign surroundings. In our experiments, mitochondria isolated from the transformed yeast cells after 14-h induction of pCYP11A1, CoxIV(1–25)-mCYP11A1, and Su9(1–112)-mCYP11A1 were subjected to hypotonic shock followed by sonication and centrifugation at 12 000 g. Figure 2 shows the contents of CoxIV(1–25)-mCYP11A1 in whole mitochondria and the corresponding 12 000 g supernatant determined by SDS/PAGE and Western blotting of the samples containing equal amounts of total protein. According to these data, the content of the imported protein in the supernatant was 0.025% of total protein of the fraction, whereas that in mitochondria was approximately 0.3% of total protein. Obviously, the supernatant containing soluble mitochond- rial proteins and membrane vesicles had 10 times less mCYP11A1. In contrast to mCYP11A1, the specific content of phosphate carrier, a marker protein of the inner mitochondrial membrane, was almost the same in whole mitochondria and in the 12 000 g supernatant (Fig. 2). This suggests that CoxIV(1–25)-mCYP11A1 imported into yeast mitochondria does not accumulate in the inner membrane after its processing. Figure 2 shows also the data for the pCYP11A1 version expressed in yeast. In this case the difference in the combined contents of pCYP11A1 and iCYP11A1 in mitochondria and the 12 000 g supernatant was not so great as for the CoxIV(1–25)-CYP11A1 version (0.1% in mitochondria vs. 0.05% in the supernatant). It is remark- able that the 12 000 g supernatant does not contain a detectable amount of mCYP11A1. Perhaps, its content in whole mitochondria is too low to be detected in the supernatant. Unlike the above experiments with CoxIV(1–25)-mCY- P11A1 and pCYP11A1 constructs, fractionation of mito- chondria containing Su9(1–112)-mCYP11A1 yielded a 12 000 g supernatant with a considerable amount of the recombinant protein (Fig. 2) similar to that in whole mitochondria (0.5% of total protein in both cases). This suggests that the Su9(1–112) fragment ensures effective insertion or anchoring of mCYP11A1 in the inner mito- chondrial membrane. Recall that this amino acid sequence governs the import of fused proteins into the mitochondrial matrix and subsequent reinsertion into the inner membrane. The treatment of yeast mitochondria imported COX- IV - mCYP11A1 with 1% (v/v) Triton X-100 failed to wash out an appreciable amount of mCYP11A1 (Fig. 3), which was indicative of its predominant aggregation. Similarly, the pCYP11A1 and iCYP11A1 forms were insignificantly solubilized with 1% (v/v) Triton X-100 from yeast mito- chondria that had imported pCYP11A1 (Fig. 3), which testifies to their prevalent aggregation. Only the iCYP11A1 form was easily identified in the supernatant under these particular conditions; probably, pCYP11A1 and iCY- P11A1, which are the main forms of the imported foreign protein, differently associate with mitochondrial mem- branes. Figure 3 shows that Su9(67–112)-mCYP11A1 is also poorly solubilized from mitochondria with 1% (v/v) Triton X-100. Upon similar treatment of adrenocortical mitochondria, cytochrome P450 scc was essentially solubi- lized (Fig. 3). However, even in this case solubilization was incomplete; therefore, we could not use the above data to quantitatively estimate protein aggregation. Figure 4 demonstrates that when the 12 000 g super- natant of mitochondria that had imported CoxIV(1–25)- mCYP11A1 was centrifuged at 106 000 g, mCYP11A1 and a minor amount of the precursor were found in the pellet mainly containing the inner mitochondrial membrane fragments (sonic SMP). In the case of mitochondria from yeast cells expressing pCYP11A1, the analogous SMP fraction contained pCYP11A1 and iCYP11A1. In both experiments, we could not see any CYP11A1 form in the high-speed supernatant. Specific contents of mCYP11A1, pCYP11A1, and iCYP11A1 were much lower in the SMP fraction than in whole mitochondria, though the SMP fraction was enriched in phosphate carrier, the inner membrane marker (Fig. 4). In contrast, fractionation of adrenocortical mitochondria yielded SMP with a cyto- chrome P450 scc content higher than that in whole organelles [6]. One may conclude that in yeast mitochondria only minor amounts of the above CYP11A1 forms are associated Fig. 2. Comparative Western blots of mitochondria and their 12 000 g supernatant fractions from recombinant yeast producing CoxIV(1– – 25)- mCYP11A1, pCYP11A1 and Su9(1– – 112)-mCYP11A1. The samples of mitochondria (M) from yeast producing recombinant proteins and corresponding 12 000 g supernatant fraction (S) were probed with anti-(cytochrome P450 scc )IgG.Inallcases20lgofproteinwere loaded per lane. Markers: phosphate carrier (PC), an integral protein of the inner membrane; Mge1p, a soluble matrix protein. Ó FEBS 2003 CYP11A1 topogenesis in yeast mitochondria (Eur. J. Biochem. 270) 225 with the mitochondrial membrane(s) while the bulk thereof aggregates in the matrix. Clearly different results were obtained for the Su9(1– 112)-mCYP11A1 construct. As follows from Fig. 4, the specific contents of its processed form are close in whole yeast mitochondria and the SMP fraction, which is similar to the data for phosphate carrier. Thus, in contrast to pCYP11A1, iCYP11A1, and mCYP11A1, Su9(67–112)- mCYP11A1 is predominantly harboured by mitochondrial membranes. The impossibility of solubilizing this recom- binant protein by the Triton X-100 treatment of mitochon- dria (see above) suggests that it forms membrane-associated aggregates. However, these aggregates, unlike those of other CYP11A1 forms, are not sedimented at 12 000 g. This indirectly indicates that aggregates of pCYP11A1, iCYP11A1, and mCYP11A1 are not associated with mitochondrial membranes and accumulate in the matrix. It is known that in some cases the aggregation of proteins imported into mitochondria can be attenuated by lowering their concentration [18]. With this in mind, we have constructed a plasmid for expression of cDNA for CoxIV(1–25)-mCYP11A1 in an integrative vector under the control of the constitutive phosphoglycerate kinase promoter. In this case the content of recombinant protein in mitochondria was reduced by an order of magnitude. However, the bulk of CYP11A1 was again in the form of aggregates insoluble in 1% (v/v) Triton X-100 (data not shown). Assays of cholesterol side-chain cleavage activity of recombinant forms of CYP11A1 in yeast mitochondria Knowing that recombinant forms of CYP11A1 imported into yeast mitochondria can exert catalytic activity only in a nonaggregated state, we used for the assays the 12 000 g mitochondrial supernatants. As the latter obviously contain CYP11A1s as components of SMP, the results below should be taken as activities of the membrane-bound proteins. It follows from the Table 1 that the rates of conversion of 22(R)-hydroxycholesterol into pregnenolone related to the total protein content of the 12 000 g supernatant are similar for the cells expressing pCYP11A1 and CoxIV(1–25)-mCYP11A1. The activity of the 12 000 g supernatant containing Su9(67–112)-mCYP11A1) proved to be much lower. This became even more evident with the specific activities of CYP11A1s. Table 1 shows that for pCYP11A1 and CoxIV(1–25)-mCYP11A1 both the con- tents of the proteins and their specific activities are quite close. Within the experimental error, they match the values obtained for a system reconstituted of purified bovine cytochrome P450 scc , adrenodoxin, and adrenodoxin reduc- tase (Table 1) or for the solubilized membrane fraction of E. coli cells expressing mCYP11A1 supplemented with appropriate components [14]. Thus, once cotranslocation- ally inserted into the inner membrane of yeast mitochon- dria, CYP11A1 is efficiently converted into holocytochrome P450 scc . Especially remarkable is that in the experiments with pCYP11A1 the 12 000 g supernatant contains mainly pCYP11A1 and iCYP11A1, not mCYP11A1. As in this case the specific activity related to the total content of these Fig. 3. Treatment of mitochondria from recombinant yeast producing CoxIV(1– – 25)-mCYP11A1, pCYP11A1, or Su9-mCYP11A1, and adrenocortical mitochondria with Triton X-100. Mitochondria were treated with 1% (v/v) Triton X-100 and centrifuged at 25 000 g. The resulting pellet (P) and supernatant (S) were probed with anti-(cytochrome P450 scc ) IgG or anti-(cytochrome P450 scc ) and anti-Hsp78 IgGs. The presented P and S samples are from 50, 50, 60 and 20 lg of mitochondria including CoxIV-mCYP11A1, pCYP11A1, Su9-mCYP11A1, and cytochrome P450 scc , respectively. Probing with anti-Hsp78 IgG was made to demonstrate complete separation of soluble and aggregated/complexed proteins. Fig. 4. Comparative Western blots of mitochondria and SMP fractions from recombinant yeast strains producing CoxIV(1– – 25)-mCYP11A1, pCYP11A1, and Su9(1– – 112)-mCYP11A1. The samples of mitochon- dria (M) and corresponding SMP fractions (SMP) from yeast produ- cing recombinant proteins were probed with anti-(cytochrome P450 scc ) IgG. The M and SMP samples contained equal amounts of total protein, which were 18, 25 and 20 lg per lane in the experiments with mitochondria including CoxIV-mCYP11A1, pCYP11A1, and Su9- mCYP11A1, respectively. Markers: phosphate carrier (PC), an integral protein of the inner membrane; Mge1p, a soluble matrix protein. 226 I. E. Kovaleva et al.(Eur. J. Biochem. 270) Ó FEBS 2003 forms was close to the activity of cytochrome P450 scc in the reconstituted system, we suggest that pCYP11A1 and/or iCYP11A1 are catalytically active. The content of Su9(67–112)-mCYP11A1 was 10–20 times higher while its specific activity was several hundred times lower than the above values. Hence, the Su9(1–112) targeting signal governs effective binding of Su9(67–112)- mCYP11A1 to the inner mitochondrial membrane. How- ever, such binding does not result in proper folding of the polypeptide chain and formation of the active holoenzyme. Of course, cytochrome P450 scc -calibrated immunoblotting is only semiquantitative, but the observed effects exceed all possible experimental errors. Discussion In adrenocortical mitochondria, cytochrome P450 scc is known to be an integral protein of the inner membrane [2,19]. Two ways of achieving such localization have thus far been considered [reviewed in 20]: the Ôstop-transferÕ mech- anism, i.e. cotranslocational insertion of a polypeptide chain into the inner membrane using stretches of hydrophobic and nonpolar residues; and the Ôre-exportÕ mechanism, i.e. total or partial translocation of a polypeptide chain into the matrix followed by its processing and reinsertion into the membrane by means of a specific N-terminal hydrophobic stretch. Analysis of the amino acid sequence of mCYP11A1 [21] shows that this protein has no regions capable of realizing either of the above mechanisms. Thus, the mode of membrane insertion of CYP11A1 still remains puzzling. It has earlier been reported [10] that CYP11A1 perhaps remains membrane-bound during the entire period of its translocation into adrenocortical mitochondria and matur- ation. This might be accounted for by the presence of a specific mitochondrial partner of pCYP11A1 that retards its transmembrane translocation. Besides, the addressing pre- sequence of pCYP11A1 can be somehow involved in confining this protein in the inner mitochondrial membrane. This specific presequence has an N-terminal region enriched in hydrophobic amino acid residues, and negatively charged residues in its C-terminal region [21], which is not typical of the canonical presequences of cytoplasmically made mito- chondrial precursor proteins. In contrast to adrenocortical mitochondria, the in vitro synthesized pCYP11A1 is easily translocated into the matrix of isolated yeast mitochondria, as follows from the results of precise digitonin fractionation of mitochondria and the high sensitivity of either form of imported pCYP11A1 to the matrix protease Pim1p [9]. In this work we show that upon expression of pCYP11A1 and CoxIV(1–25)-mCYP11A1 in yeast the precursor, semiprocessed, and mature proteins (CYP11A1s) accumulate in mitochondria mainly as aggre- gates. Nevertheless, some amounts of CYP11A1s were found in the mitochondrial membranes. We suggest that in yeast mitochondria the CYP11A1 precursors meet no serious hindrances to their translocation and thus largely ÔslipÕ into the matrix, where the processed proteins cannot be properly folded and undergo proteolysis or aggregation. To a lesser degree this concerns pCYP11A1, which, owing to its noncanonical presequence, is better confined in the mem- brane of yeast mitochondria (Fig. 5). This can explain why the SMP fraction contains mainly the pCYP11A1 and iCYP11A1 forms, and why their insertion into the mito- chondrial membranes is more effective than that of Cox (1–25)-mCYP11A1. Table 1. The content and specific cholesterol side-chain cleavage activity of various forms of recombinant CYP11A1 imported into yeast mitochondria. The measurements were carried out with 12 000 g supernatants of mitochondria from yeast cells producing recombinant forms of CYP11A1. The activity is defined as the rate of conversion of 22(R)-hydroxycholesterol into pregnenolone at 37 °C. The latter was quantitatively oxidized to progesterone with cholesterol oxidase. Initial forms of recombinant proteins Content of CYP11A1 in assay sample, nmole (% total protein) Activity (10 4 nmole progesteroneÆmin )1 Æmg )1 total protein) Activity (10 3 nmole progesterone min )1 Ænmol )1 CYP11A1) pCYP11A1 0.003 (0.05%) 1052 13150 CoxIV(1–25)-mCYP11A1 0.0015 (0.025%) 530 13200 Su9(1–112)-mCYP11A1 0.048 (0.5%) 35 37 Purified bovine cytochrome P450 scc 0.003 13300 Fig. 5. Putative pathways of import of various CYP11A1 precursor versions into yeast mitochondria and their intramitochondrial sorting. (1) Su9(1–112)-mCYP11A1 is translocated into the matrix, processed, and reinserted into the membrane (yeast mitochondria); (2) CoxIV(1–25)- mCYP11A1 is rapidly processed and translocated into the matrix, with a smaller portion being inserted into the membrane (yeast mitochondria); (3) pCYP11A1 is rather slowly processed and trans- located into the matrix, with the more essential portion being cotranslocationally inserted into the membrane (yeast mitochondria); (4) pCYP11A1 is cotranslocationally processed and inserted into the inner membrane (adrenocortical mitochondria). Ó FEBS 2003 CYP11A1 topogenesis in yeast mitochondria (Eur. J. Biochem. 270) 227 It has earlier been found [8] that, upon expression of the pCoxVI-mCYP11A1 construct in S. cerevisiae, mCYP11A1 resides in the inner mitochondrial membrane and the intermembrane contact sites. However, these gradient centrifugation experiments with mitochondrial homogenates could not have quantitatively assessed the balance of the import. As follows from the present work, a considerable amount of the imported CYP11A1 could have been missed. The cytochrome P450 scc -calibrated immunoblotting allowed one to analyse the balance between the processes of membrane insertion of CYP11A1s and their aggregation, which takes place in the matrix, as follows from the in vitro experiments [9]. As the total protein contents in an aliquot of yeast mitochondria and the corresponding 12 000 g supernatant are close, one can compare the contents of CYP11A1s. Such a comparison clearly shows that for CoxIV(1–25)-mCYP11A1 the content of the imported and nonproteolysed protein is almost 10-fold higher in mito- chondria than in the 12 000 g supernatant, which suggests that only one-tenth of mCYP11A1 is inserted into the membrane, thus escaping aggregation in the matrix. It is evident that for pCYP11A1 the combined contents of precursor and intermediate forms of the recombinant protein in mitochondria and 12 000 g supernatant differ at least twofold. All the above membrane-bound CYP11A1s are effi- ciently converted into the catalytically active form. This follows from their high specific side-chain cleavage activities, which are of the same order of magnitude as that estimated for mCYP11A1 expressed in E. coli cells [14]. As to the Su9(67–112)-mCYP11A1 imported into mito- chondria, it almost completely accumulates in the mem- brane fraction: the mitochondria and the 12 000 g supernatant contain equal relative amounts of the recom- binant protein. The Su9(1–112) segment is known to ensure the initial stage of the import of Su9 into the matrix followed by two-stage processing and reinsertion of its N-terminus into the inner membrane [11]. Most plausibly, the Su9(67–112)-mCYP11A1 sequence imported into yeast mitochondria misfolds in the matrix and does not refold upon reinsertion into the inner membrane through the Su9(67–112) fragment. In fact, Su9(67–112)-mCYP11A1 detected mainly in the SMP fraction exhibits very low side-chain cleavage activity and is poorly solubilized with 1% (v/v) Triton X-100. Thus, different topogenic signals in the CYP11A1 versions predetermine their sorting and conversions in yeast mitochondria (Fig. 5). Catalytically active cyto- chrome P450 scc can be accumulated in yeast mitochondria in as much as the protein escapes aggregation in the matrix. However, some basic features of the import of its precursor into mitochondria inevitably result in a low yield of the active protein. Analysis of the above data definitely shows that the crucial moment in the topo- genesis of cytochrome P450 scc is not reception, trans- membrane translocation, or proteolytic processing of its precursor, but rather the confinement of the protein in the inner membrane upon its import into adrenocortical mitochondria. This still unknown mechanism cannot be adequately implemented in some foreign (e.g. yeast) mitochondria. A search for such a mechanism may extend the conventional notions on the topogenesis of mito- chondrial proteins. Acknowledgements The authors are indebted to S. A. Saveliev for the help in preparing the pYeDP/pCYP11A1 and to A. V. Galkin for editing the text. This work was supported by the Russian Foundation for Basic Research (grants 99-04-48003 and 00-15-97942 to VNL). References 1. Nabi, N., Kominami, S., Takemori, S. & Omura, T. (1983) Contributions of cytoplasmic free and membrane-bound ribo- somes to the synthesis of mitochondrial cytochrome P450 (SCC) and P450 (11b) and microsomal cytochrome P450 (C-21) in bovine adrenal cortex. J. Biochem. 94, 1517–1527. 2. Omura, T. & Ito, A. (1991) Biosynthesis and intracellular sorting of mitochondrial forms of cytochrome P450. Methods Enzymol. 206, 75–81. 3. Matocha, M.F. & Waterman, M.R. (1984) Discriminatory pro- cessing of the precursor forms of cytochrome P450scc and adre- nodoxin by adrenocortical and heart mitochondria. J. Biol. Chem. 259, 8672–8678. 4. Ogishima, T., Okada, T. & Omura, T. (1985) Import and pro- cessing of the precursor of cytochrome P450 (SCC) by bovine adrenal mitochondria. J. Biochem. 98, 781–791. 5.Zuber,M.X.,Mason,J.I.,Simpson,R.E.&Waterman,M.E. (1988) Simultaneous transfection of COS-1 cells with mitochon- drial and microsomal steroid hydroxylases: Incorporation of a steroidogenic pathway into nonsteroidogenic cells. Proc. Natl Acad. Sci. USA 85, 699–703. 6. Savel’ev, A.S., Novikova, L.A., Drutsa, V.L., Isaeva, L.V., Chernogolov,A.A.,Usanov,S.A.&Luzikov,V.N.(1997) Synthesis and some aspects of topogenesis of bovine cytochrome P450scc in yeast. Biochemistry (Moscow) 62, 779–786. 7. Luzikov, V.N., Novikova, L.A., Whelan, J., Hugosson, M. & Glaser, E. (1994) Import of mammalian cytochrome P450scc precursor into plant mitochondria. Biochem. Biophys. Res. Com- mun. 199, 33–36. 8. Cauet, G., Balbuena, D., Achstetter, T., Dumas, D. (2001) CYP11A1 stimulates the hydroxylase activity of CYP11B1 in mitochondria of recombinant yeast in vivo and in vitro. Eur. J. Biochem. 268, 4054–4062. 9. Savel’ev, A.S., Novikova, L.A., Kovaleva, I.E., Luzikov, V.N., Neupert, W. & Langer, T. (1998) ATP-dependent proteolysis in mitochondria: m-AAA protease and Pim1 protease exert over- lapping substrate specificity and cooperate with the mtHsp70 system. J. Biol. Chem. 273, 20596–20602. 10. Ou, W., Ito, A., Morohashi, K., Fujii-Kuriyama, Y. & Omura, T. (1986) Processing- independent in vitro translocation of cyto- chrome P450 (SCC) precursor across mitochondrial membranes. J. Biochem. 100, 1287–1296. 11. Rojo, E.E., Stuart, R.A. & Neupert, W. (1995) Conservative sorting of Fo-ATPase subunit 9: export from matrix requires pH across inner membrane and matrix ATP. EMBO J. 14, 3445– 3451. 12. Rojo, E.E., Guiard, B., Neupert, W. & Stuart, R.A. (1998) N-terminal tail export from the mitochondrial matrix: adherence to the procaryotic Ôpositive insideÕ rule of membrane protein topology. J. Biol. Chem. 274, 19617–19722. 13. Cullin, C. & Pompon, D. (1988) Synthesis of functional mouse P450 P1 and chimeric P450 P3–1 in yeast Saccharomyces cerevis- iae. Gene 65, 203–217. 14. Wada, A., Mathew, P.A., Barnes, H.J., Sanders, D., Estabrook, R.W. & Waterman, M.R. (1991) Expression of functional bovine 228 I. E. Kovaleva et al.(Eur. J. Biochem. 270) Ó FEBS 2003 cholesterol side chain cleavage cytochrome P450 (P450scc) in Escherichia coli. Arch. Biochem. Biophys. 290, 376–380. 15. Rech, E.L., Dobson, M.J. & Milligan, B.J. (1990) Introduction of a yeast artificial chromosome vector into Saccharomyces cerevisiae cells by electroporation. Nucl. Acids Res. 18, 1313–1319. 16. Akiyoshi-Shibata, M., Sakaki, T., Yabusaki, G., Murakami, H. & Ohkawa, H. (1991) Expression of bovine adrenodoxin and NADPH-adrenodoxin reductase cDNAs in Saccharomyces cere- visiae. DNA Cell Biol. 10, 613–621. 17. Sidhu, R.S. & Bollon, A.P. (1987) Analysis of a-factor secretion signals by fusing with acid phosphatase of yeast. Gene 54, 175–184. 18. Dibrov, E., Fu, S. & Lemire, B.D. (1998) The Saccharomyces cerevisiae TCM62 gene encodes a chaperone necessary for the assembly of the mitochondrial succinate dehydrogenase (Complex II). J. Biol. Chem. 48, 32042–32048. 19. Usanov, S.A., Chernogolov, A.A. & Chashchin, V.L. (1990) Is cytochrome P450scc a transmembrane protein? FEBS Lett. 275, 33–35. 20. Stuart, R.A. & Neupert, W. (1996) Topogenesis of inner membrane proteins of mitochondria. Trends Biochem. Sci. 21, 261–267. 21. Chung,B.C.,Matteson,K.J.,Voutilainen,R.,Mohandas,T.K.& Miller, W.L. (1986) Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning, assignment of the gene to chromosome 15, and expression in placenta. Proc.NatlAcad.Sci. USA 83, 8962–8966. Ó FEBS 2003 CYP11A1 topogenesis in yeast mitochondria (Eur. J. Biochem. 270) 229 . Effects of various N-terminal addressing signals on sorting and folding of mammalian CYP11A1 in yeast mitochondria Irina E. Kovaleva, Lyudmila A. Novikova,. confinement and folding in the inner mito- chondrial membrane. In yeast mitochondria, only an insignificant portion of the imported CYP11A1 follows this mechanism. Keywords: yeast mitochondria; import; sorting; . found in the pellet mainly containing the inner mitochondrial membrane fragments (sonic SMP). In the case of mitochondria from yeast cells expressing pCYP11A1, the analogous SMP fraction contained