Apocytochrome c requires the TOM complex for translocation across the mitochondrial outer membrane
2001; Springer Nature; Volume: 20; Issue: 20 Linguagem: Inglês
10.1093/emboj/20.20.5626
ISSN1460-2075
Autores Tópico(s)ATP Synthase and ATPases Research
ResumoArticle15 October 2001free access Apocytochrome c requires the TOM complex for translocation across the mitochondrial outer membrane Kerstin Diekert Kerstin Diekert Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Robert-Koch-Strasse 5, 35033 Marburg, Germany Search for more papers by this author Anton I.P.M. de Kroon Anton I.P.M. de Kroon Department Biochemistry of Membranes, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands Search for more papers by this author Uwe Ahting Uwe Ahting Adolf-Butenandt-Institut für Physiologische Chemie der Universität München, Butenandtstrasse 5, 81377 München, Germany Search for more papers by this author Brigitte Niggemeyer Brigitte Niggemeyer Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Robert-Koch-Strasse 5, 35033 Marburg, Germany Search for more papers by this author Walter Neupert Walter Neupert Adolf-Butenandt-Institut für Physiologische Chemie der Universität München, Butenandtstrasse 5, 81377 München, Germany Search for more papers by this author Ben de Kruijff Ben de Kruijff Department Biochemistry of Membranes, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands Search for more papers by this author Roland Lill Corresponding Author Roland Lill Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Robert-Koch-Strasse 5, 35033 Marburg, Germany Search for more papers by this author Kerstin Diekert Kerstin Diekert Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Robert-Koch-Strasse 5, 35033 Marburg, Germany Search for more papers by this author Anton I.P.M. de Kroon Anton I.P.M. de Kroon Department Biochemistry of Membranes, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands Search for more papers by this author Uwe Ahting Uwe Ahting Adolf-Butenandt-Institut für Physiologische Chemie der Universität München, Butenandtstrasse 5, 81377 München, Germany Search for more papers by this author Brigitte Niggemeyer Brigitte Niggemeyer Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Robert-Koch-Strasse 5, 35033 Marburg, Germany Search for more papers by this author Walter Neupert Walter Neupert Adolf-Butenandt-Institut für Physiologische Chemie der Universität München, Butenandtstrasse 5, 81377 München, Germany Search for more papers by this author Ben de Kruijff Ben de Kruijff Department Biochemistry of Membranes, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands Search for more papers by this author Roland Lill Corresponding Author Roland Lill Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Robert-Koch-Strasse 5, 35033 Marburg, Germany Search for more papers by this author Author Information Kerstin Diekert1, Anton I.P.M. de Kroon2, Uwe Ahting3, Brigitte Niggemeyer1, Walter Neupert3, Ben de Kruijff2 and Roland Lill 1 1Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Robert-Koch-Strasse 5, 35033 Marburg, Germany 2Department Biochemistry of Membranes, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands 3Adolf-Butenandt-Institut für Physiologische Chemie der Universität München, Butenandtstrasse 5, 81377 München, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:5626-5635https://doi.org/10.1093/emboj/20.20.5626 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The import of proteins into the mitochondrial intermembrane space differs in various aspects from the classical import pathway into the matrix. Apocytochrome c defines one of several pathways known to reach the intermembrane space, yet the components and pathways involved in outer membrane translocation are poorly defined. Here, we report the reconstitution of the apocytochrome c import reaction using proteoliposomes harbouring purified components. Import specifically requires the protease-resistant part of the TOM complex and is driven by interactions of the apoprotein with internal parts of the complex (involving Tom40) and the 'trans-side receptor' cytochrome c haem lyase. Despite the necessity of TOM complex function, the translocation pathway of apocytochrome c does not overlap with that of presequence-containing preproteins. We conclude that the TOM complex is a universal preprotein translocase that mediates membrane passage of apocytochrome c and other preproteins along distinct pathways. Apocytochrome c may provide a paradigm for the import of other small proteins into the intermembrane space such as factors used in apoptosis and protection from stress. Introduction Over the past two decades, it has become clear that the translocation of proteins into and across biological membranes requires the assistance of preprotein translocases (Schatz and Dobberstein, 1996). Generally, these translocases are organized as large complexes that contain surface-exposed preprotein receptors and membrane-embedded preprotein-conducting pores. Protein translocation into mitochondria is mediated by translocases in the outer (TOM complex) and inner (TIM complexes) membranes (Neupert, 1997; Koehler et al., 1999; Bauer et al., 2000; Pfanner and Geissler, 2001). The TOM complex is necessary and sufficient for insertion of outer membrane proteins and for translocation of constituents of the intermembrane space such as cytochrome haem lyases (Steiner et al., 1995; Lill and Neupert, 1996; Künkele et al., 1998a). During import of components of the inner membrane and the matrix, the TOM complex co-operates with the TIM complexes leading to simultaneous translocation of the polypeptide chain across both membranes. Translocation across the outer membrane occurs in at least two consecutive steps (Mayer et al., 1995c; Kanamori et al., 1999). First, the presequence interacts with the surface receptors Tom20/Tom22 (cis site). Then, the N-terminal part of the preprotein slides across the preprotein-conducting pore comprised by Tom40 to finally reach a binding site (trans site) on the internal face of the outer membrane (Bolliger et al., 1995; Mayer et al., 1995c; Moczko et al., 1997; Rapaport et al., 1997, 1998b; Kanamori et al., 1999). The import of apocytochrome c into the mitochondrial intermembrane space differs from that of typical preproteins in several aspects (Stuart and Neupert, 1990; Dumont, 1996; Kranz et al., 1998). For instance, apocytochrome c lacks an N-terminal presequence and does not undergo proteolytic cleavage after import (Dumont et al., 1988; Nicholson et al., 1988). Import does not require external energy sources such as ATP hydrolysis and rather seems to be driven by the interaction of apocytochrome c with cytochrome c haem lyase (CCHL) acting as specific binding partner in the intermembrane space (termed 'trans side receptor') (Dumont et al., 1991, Mayer et al., 1995d). Furthermore, translocation occurs independently of protease-sensitive outer membrane proteins, in particular of the surface receptors of the TOM complex (Stuart et al., 1990; Mayer et al., 1995d). Translocation of apocytochrome c is unaffected by blocking the TOM complex with import-competent porin (Pfaller et al., 1988). Together, these findings have generally been taken to suggest that the apoprotein does not use the TOM complex as a translocase. Apocytochrome c possesses a high tendency to insert into lipid bilayers containing negatively charged phospholipids (de Kruijff et al., 1992; Jordi et al., 1992). Therefore, the apoprotein has been suspected to spontaneously pass across the membrane without assistance of membrane proteins. In fact, apocytochrome c is slowly degraded when added to liposomes containing enclosed trypsin (Rietveld and de Kruijff, 1984; Miao et al., 2001). This has been regarded as evidence that lipids suffice to achieve membrane passage of apocytochrome c. Recently the requirement of a protease-resistant outer membrane component for transport of apocytochrome c into the intermembrane space has been demonstrated using purified outer membrane vesicles (OMV) (Mayer et al., 1995d). Until now, the component mediating transport has not been identified. The current investigation is aimed at (i) the identification of the translocase mediating apocytochrome c import and (ii) a better understanding of the translocation path way. We report the reconstitution of apocytochrome c translocation into proteoliposomes containing purified TOM complex and enclosed anti-apocytochrome c antibodies that served as high affinity trans-side receptors in the lumen of the proteoliposomes. Translocation of apocytochrome c was unaffected after blocking the default import pathway by addition of chemical amounts of preproteins. These results demonstrate that apocytoc uses the TOM complex for membrane passage. Yet, the protein follows a unique pathway that does not overlap with that used by other preproteins. Results Binding and import of apocytochrome c into outer membrane vesicles and into large unilamellar vesicles prepared by extrusion technology We first compared the ability of liposomes and purified OMV to bind and import apocytochrome c. To this end, large unilamellar vesicles comparable to OMV in both size and lipid composition (de Kroon et al., 1999) were prepared by an extrusion technique; these vesicles are the so-called large unilamellar vesicles prepared by extrusion technology (LUVET). In order to drive the translocation of the apoprotein across the membranes, IgGs specific for apocytochrome c were enclosed inside the LUVET and OMV by a freeze–thaw technique (Mayer et al., 1995a,d). IgG that was not entrapped in the lumen of the vesicles was removed by flotation centrifugation. Various amounts of the LUVET or OMV preparations were then used to analyse binding and import of [35S]methionine labelled apocytochrome c. To analyse the binding, material that was not associated with the vesicles was removed by membrane sedimentation. The resulting pellets were dissolved in buffer, proteins were precipitated by trichloroacetic acid (TCA) and the amount of bound apoprotein was analysed by SDS–PAGE and fluorography (Figure 1A and B, upper panels). Apocytochrome c bound efficiently to both LUVET and OMV. Quantification of binding by phosphorimager analysis shows that binding to LUVET was somewhat less efficient than that to OMV, but at higher lipid concentrations, a similar plateau of binding was reached with both vesicle types (Figure 1C). Binding was independent of the presence or absence of enclosed IgG (not shown). Figure 1.Apocytochrome c binds to both LUVET and OMV, but is only imported into OMV. Anti-apocytochrome c IgGs were enclosed into LUVET or purified OMV by a freeze–thaw procedure. After flotation centrifugation to remove non-enclosed IgG, the indicated amounts of LUVET or OMV (based on phospholipid-bound phosphate, PL-Pi) were incubated with radiolabelled apocytochrome c for 10 min at 25°C. Half the samples were centrifuged to re-isolate the membranes (Binding). The other half was incubated for 20 min on ice with proteinase K (50 μg/ml) to assay the import of apocytochrome c. Protease digestion was halted by addition of 2 mM PMSF followed by TCA precipitation. The proteins were separated by SDS–PAGE. Bound or imported apocytochrome c was visualized by autoradiography of the gel (A and B) and quantitated by phosphorimager analysis. (C) Binding or import of apocytochrome c in OMV containing 72 nmol of PL-Pi was set to 100. (D) Control experiments were performed to verify that apocytochrome c was not aggregated and degraded by proteinase K after addition of 0.1% Triton X-100 (Detergent). St., a standard containing 50% of added apocytochrome c; a.u., arbitrary units. Download figure Download PowerPoint Import of apocytochrome c was tested by treating the vesicles with proteinase K after the import reaction. No protease-resistant material was found associated with LUVET, not even at higher lipid concentrations (Figure 1A, lower panel). In contrast, about a third of the OMV-bound apoprotein was resistant to proteolytic attack indicating import into the OMV (Figure 1B, lower panel). Upon lysis of the OMV by detergent, apocytochrome c was completely degraded. This excludes the possibility that OMV-associated apocytochrome c was protease resistant due to aggregation (Figure 1D). These data demonstrate that apocytochrome c can efficiently bind to the surface of phospholipid membranes. For complete translocation across the lipid bilayer, however, a proteinaceous component of the mitochondrial outer membrane is necessary. The TOM complex is necessary for import of apocytochrome c Which component of the outer membrane might be able to facilitate membrane passage of apocytochrome c? We tested the possible involvement of the TOM complex in mediating translocation of apocytochrome c. Proteo liposomes were formed by diluting detergent solutions of purified TOM core complex [containing Tom40, Tom22, Tom7 and Tom6 but depleted in Tom20 and Tom70 (Ahting et al., 1999, 2001)] and phospholipids below the critical micellar concentration (CMC). The proteoliposomes were harvested by centrifugation. IgG directed against apocytochrome c was enclosed into the proteoliposomes by a freeze–thaw technique; import was followed after adding radiolabelled apocytochrome c as described above. About 30% of total added apoprotein became resistant to proteolytic attack indicating that the TOM complex could support efficient import into the proteoliposomes (Figure 2A). The imported apoprotein was completely degraded upon lysis of the liposomes by the addition of detergent. In contrast, no significant amounts of protease-protected apocytochrome c were detectable when liposomes were prepared by detergent dilution in the absence of TOM complex (Figure 2B). Hence, the TOM complex is able to support membrane translocation of apocytochrome c. Figure 2.The TOM complex is necessary for apocytochrome c translocation. (A) TOM complex-containing proteoliposomes. (B) Liposomes (each corresponding to 300 nmol phospholipid-bound phosphate) were prepared by detergent dilution; anti-apocytochrome c IgG was enclosed by a freeze–thaw technique. Radiolabelled apocytochrome c was added. After 10 min at 25°C, the samples were split and treated with proteinase K in the presence or absence of Triton X-100 (Detergent). Proteins were precipitated with TCA, separated by SDS–PAGE and blotted on to nitrocellulose. Quantitation was by phosphorimager analysis. Immunostaining was performed to visualize Tom40 (A) and the enclosed IgG. a.u., arbitrary units. Download figure Download PowerPoint Import of apocytochrome c was reported to occur independently of protease-sensitive components of the mitochondrial outer membrane (Nicholson et al., 1988; Stuart et al., 1990; Mayer et al., 1995d). We therefore tested whether the preprotein receptor Tom22 is necessary for membrane translocation of apocytochrome c. After generation of the TOM complex-containing proteoliposomes and inclusion of anti-apocytochrome c IgG, the vesicles were treated with trypsin. The protease removes the surface-exposed parts of Tom22, Tom7 and Tom6, but leaves Tom40 intact (Figure 3, bottom; compare with Ahting et al., 1999). Import of apocytochrome c into these vesicles was as efficient as that of import into untreated proteoliposomes (Figure 3, top panel). The surface receptors, in particular Tom22, are apparently not required for TOM complex-mediated membrane translocation of apocytochrome c. Hence, our results using TOM complex-reconstituted proteoliposomes closely resemble the situation obtained for apocytochrome c translocation into intact mitochondria or OMV (see above; Mayer et al., 1995d). Figure 3.Apocytochrome c translocation does not require protease-sensitive components of the TOM complex. After insertion of the TOM complex and enclosure of anti-apocytochrome c IgG, the proteoliposomes were incubated with 50 μg/ml trypsin for 15 min at 0°C. Soybean trypsin inhibitor (1 mg/ml) was added and the liposomes were re-isolated by flotation centrifugation. Import of apocytochrome c and further analysis was performed as in Figure 2. Anti-Tom22 antibodies were used for immunostaining. a.u., arbitrary units. Download figure Download PowerPoint Porin does not mediate membrane translocation of apocytochrome c The TOM complex contains a large preprotein-conducting pore that is formed by protease-resistant parts of the complex (Hill et al., 1998; Künkele et al., 1998a,b; Ahting et al., 2001). A pore of similar size is present in the mitochondrial outer membrane component porin (also termed VDAC) (Benz, 1994). We therefore asked whether a large pore such as porin might be sufficient for membrane translocation of apocytochrome c or whether the TOM complex is specifically required for import. To answer this question, purified porin was incorporated into proteoliposomes and anti-apocytochrome c IgG was enclosed. Import of apocytochrome c was not detectable in proteoliposomes containing porin (Figure 4A), and was only seen in vesicles harbouring the TOM complex. Figure 4.Membrane-embedded porin does not mediate translocation of apocytochrome c. (A) Liposomes were prepared by detergent dilution containing either no protein, purified porin or purified TOM complex, and anti-apocytochrome c antibodies were enclosed by a freeze–thaw technique. After an import reaction with radiolabelled apocytochrome c as in Figure 2, the samples were precipitated with TCA and subjected to SDS–PAGE. Proteins were blotted on to nitrocellulose and immunostained with antibodies against porin or Tom40. (B) LDH (50 μg/ml) was enclosed in the liposomes prepared as in (A) by a freeze–thaw technique. LDH activity was measured at 340 nm in SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM MOPS–KOH pH 7.0) by adding pyruvate (23 mM) and NADH (12 mM). To determine the total LDH activity per sample, the liposomes were lysed by adding 0.2% Triton X-100; this LDH activity was set to 100. Download figure Download PowerPoint To verify the functionality of the purified porin preparation, we performed two control experiments. First, porin incorporated into black lipid membranes was found to be electrophysiologically active and formed the characteristic voltage-dependent channels (Benz, 1994; data not shown). Secondly, the porin- or TOM complex-containing vesicles were used for enclosure of lactate dehydrogenase (LDH) by a freeze–thaw technique. Non-enclosed enzyme was removed by flotation centrifugation. The resulting vesicles were used to test the accessibility of the enclosed enzyme for the externally added substrates nicotinamide adenine dinucleotide (NADH) and pyruvate. In the absence of added membrane proteins, hardly any LDH activity was detectable, indicating that the liposomes were tightly sealed and did not allow access of the substrates to the enclosed enzyme (Figure 4B). LDH was active after opening the liposomes by the addition of detergent. When the liposomes contained porin, high LDH activity was measured (40% in comparison with the activity obtained after detergent lysis of the proteoliposomes; Figure 4B). This suggests strongly that porin was functionally reconstituted in the liposomes, i.e. mediating membrane passage of the low molecular mass substrates of LDH. A similar result was obtained with liposomes harbouring purified TOM complex, thus demonstrating that the preprotein-conducting pore of this complex is permeable for the enzyme substrates (Figure 4B). This result is in perfect agreement with a recent study demonstrating the importance of the TOM complex for metabolite trafficking in the absence of functional porin in intact mitochondria (Kmita and Budzinska, 2000). In conclusion, both the TOM complex and porin are capable of mediating membrane transport of small molecular mass compounds. However, only the TOM complex allows translocation of apocytochrome c across the lipid bilayer, demonstrating its specific function in membrane passage of apocytochrome c. Interaction of apocytochrome c with the TOM complex is sufficient for its membrane passage The function of the TOM complex in the translocation of apocytochrome c across the mitochondrial outer membrane suggests a direct interaction between these components. We therefore asked whether the TOM complex is able to mediate membrane passage of apocytochrome c in the absence of a trans-side receptor, i.e. IgG directed against apocytochrome c. To this end, liposomes formed with and without reconstituted TOM complex were loaded with anti-apocytochrome c IgG or left untreated and an import reaction was performed. Strikingly, even in the absence of enclosed IgG, a substantial amount of apocytochrome c became protease resistant (Figure 5A). The amount of protease-inaccessible material increased only slightly upon enclosure of IgG. Thus, the TOM complex appears to be sufficient for membrane translocation of apocytochrome c. These results support a direct interaction of apocytochrome c with the TOM complex preceding the association of the apoprotein with its trans-side receptor in the lumen of the vesicles (see below). Figure 5.The TOM complex is sufficient for membrane translocation of apocytochrome c. (A) Liposomes with or without reconstituted TOM complex and enclosed IgG were incubated with radiolabelled apocytochrome c; import was performed and analysed as described in Figure 2. (B) Purified OMV (15 μg/sample) with and without enclosed anti-apocytochrome c IgG were incubated with radiolabelled apocytochrome c and an import reaction was performed. Half the samples were lysed with Triton X-100 and import was analysed as in Figure 2. (C) Crosslinking of apocytochrome c with the TOM complex. Isolated TOM complex (6 μg) was incubated with or without apocytochrome c (10 μg) in the presence or absence of 0.25 mM DSS for 30 min on ice. The reaction was stopped by the addition of 5 mM Tris–HCl pH 7.0. The proteins were precipitated with TCA, separated by SDS–PAGE, blotted on to nitrocellulose and immunostained with anti-Tom40 antiserum. The crosslinking products are indicated (see Rapaport et al., 1998a). The asterisk indicates the crosslinking adduct between apocytochrome c and Tom40. The apparent molecular mass of this crosslinked product (∼52 kDa) fits well to an adduct between these two proteins. We tried to confirm the identity of apocytochrome c by immunostaining. However, the presence of a cross-reacting band (possibly a tetramer of apocytochrome c) in this molecular mass region (even in the absence of TOM complex) made this analysis impossible (data not shown). Download figure Download PowerPoint Dependence of apocytochrome c translocation upon the presence of enclosed anti-apocytochrome c IgG has previously been reported for an OMV import system (Mayer et al., 1995d). These findings are at apparent variance with the results obtained above. We therefore re-examined the import of apocytochrome c into OMV that were or were not loaded with anti-apocytochrome c IgG. We were able to reproduce the previous findings with apocytochrome c of Neurospora crassa, i.e. import was dependent upon the enclosure of IgG (not shown; compare with Mayer et al., 1995d). With the standard precursor of this study, the apoprotein of Saccharomyces cerevisiae, however, two major differences were observed. First, its import efficiency was considerably higher than that of the N.crassa precursor (30–40% versus 10%, respectively, relative to OMV-bound material; Figure 5B). Secondly, significant import of the yeast apoprotein was detected even in the absence of enclosed anti-apocytochrome c antibodies. Nevertheless, the presence of IgG increased the efficiency of import 2- to 3-fold, indicating that the apoprotein was pulled into the lumen of the vesicles by consecutive interactions with the TOM complex and with anti-apocytochrome c IgG. We examined the putative interaction between apocytochrome c and the TOM complex by a crosslinking approach. Purified TOM complex was incubated with or without chemical amounts of apocytochrome c in the presence of the homo-bifunctional crosslinker disuccinyldisuberate (DSS). After crosslinking, the samples were analysed by immunostaining for Tom40. In the absence of apocytochrome c, characteristic crosslinking products between Tom40 and Tom6 as well as between two Tom40 molecules were formed as described previously (Figure 5C; Rapaport et al., 1998a). Addition of apocytochrome c characteristically altered the crosslinking pattern, indicating an influence of added apocytochrome c on the interactions between the TOM complex components. A strikingly similar behaviour has been reported for the interaction of a presequence-containing preprotein or a presequence peptide with the TOM complex (Rapaport et al., 1998a). Thus, the TOM complex seems to undergo dynamic changes upon interaction with apocytochrome c. The vicinity of apocytochrome c and Tom40 is indicated by the presence of a Tom40 crosslinking band observed only upon addition of apocytochrome c (Figure 5C, *). Its molecular mass fits well with an adduct between Tom40 and apocytochrome c. We conclude from these data that apocytochrome c interacts with the TOM complex via Tom40. Protease resistance of the imported apoprotein indicates that the interaction takes place either within the outer membrane or on the intermembrane space side of the outer membrane. Consequently, mitochondrial import of apocytochrome c may be driven by consecutive interactions with (i) the TOM complex mediating membrane passage and (ii) anti-apocytochrome c antibodies serving as a trans-side receptor. The import pathways of apocytochrome c and of presequence-containing preproteins do not overlap Does apocytochrome c use a pathway similar to or distinct from that taken by presequence-containing preproteins? To distinguish between the two possibilities, we purified chemical amounts of a presequence-containing preprotein (pSu9-DHFR-His6) that undergoes a specific, high-affinity interaction with the TOM complex (Stan et al., 2000). The preprotein was used to block the import sites of either intact isolated mitochondria or of OMV (Mayer et al., 1995b; Rapaport et al., 1998b); the residual import efficiency of radiolabelled apocytochrome c was then tested. No significant reduction of the import efficiency of apocytochrome c was detectable in the presence of the preprotein (Figure 6). To verify the successful occupancy of the import sites by pSu9-DHFR-His6, various radiolabelled preproteins were imported in parallel experiments. Import of these preproteins into intact mitochondria (Figure 6A; see Künkele et al., 1998a; Rapaport and Neupert, 1999) and presequence translocation of pSu9-DHFR in OMV (Figure 6B; see Mayer et al., 1995c) was strongly inhibited by the addition of pSu9-DHFR-His6, demonstrating that the import sites were occupied to a large extent by this preprotein. Evidently, apocytochrome c follows an import pathway that does not detectably overlap with that of canonical mitochondrial preproteins, even though both types of preproteins depend on TOM complex function for membrane translocation. We conclude that the TOM complex offers more than one pathway to move preproteins across the mitochondrial outer membrane. Figure 6.Chemical amounts of pSu9-DHFR-His6 do not inhibit apocytochrome c translocation. (A) Isolated mitochondria (50 μg/sample) or (B) purified OMV (15 μg/sample) were incubated with radiolabelled apocytochrome c, the precursors (open circles in the autoradiographs) of matrix processing peptidase α-subunit (α-MPP), F1-ATPase β-subunit (pF1β) or Su9-DHFR as indicated, in the presence or absence of chemical amounts of pSu9-DHFR-His6 (4 μM final concentration; Künkele et al., 1998a). After 6 min at 25°C, mitochondria and OMV were re-isolated by centrifugation. Following proteinase K treatment (for apocytochrome c), the proteins were precipitated with TCA, analysed by SDS–PAGE, blotted on to nitrocellulose and subjected to phosphorimager analysis and autoradiography. Import into mitochondria (A) was estimated from the amounts of protease-resistant apocytochrome c and of the processed (mature) forms of the preproteins (closed circles). The signal in the absence of added pSu9-DHFR-His6 was set to 100. Translocation of the presequence of pSu9-DHFR in OMV (B) was taken from the amount of bound preprotein after pelleting the membranes in the presence of 100 mM NaCl (trans site binding; Mayer et al., 1995c). p, precursor; a.u., arbitrary units. Download figure Download PowerPoint Discussion In this investigation, we have reconstituted the import of apocytochrome c into the mitochondrial intermembrane space using proteoliposomes containing purified components. We show that apocytochrome c can interact efficiently with the lipid surface, but it is not translocated across the bilayer. For full membrane translocation, the protease-resistant part of the TOM complex is both necessary and sufficient. Entry into the lumen of the vesicles is driven by binding of the translocating apoprotein to the enclosed anti-apocytochrome c antibodies. In vivo, t
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