©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Translocation of Apocytochrome c across the Outer Membrane of Mitochondria (*)

Andreas Mayer , Walter Neupert , Roland Lill(§)

From the (1) Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie, der Universität München, Goethestrasse 33, 80336 München, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Apocytochrome c follows a unique pathway into mitochondria. Import does not require the general protein translocation machinery, protease-sensitive components of the outer membrane, or a membrane potential across the inner membrane. We investigated the membrane binding and translocation steps of the import reaction using purified outer membrane vesicles (OMV) from Neurospora crassa mitochondria. OMV specifically bound, but did not import apocytochrome c. If, however, specific antibodies were enclosed inside OMV, apocytochrome c was accumulated in soluble form in the lumen. Import was reversible, since apocytochrome c became accessible to external protease after release from the antibodies. Thus, OMV are competent of translocating apocytochrome c into their lumen, but lack a binding partner which traps the apoprotein. In intact mitochondria, cytochrome c heme lyase (CCHL), a peripheral protein of the inner membrane, serves such a function by stably associating with apocytochrome c in a complex which is detectable by co-immunoprecipitation. We suggest a model for the import mechanism of apocytochrome c in which the apoprotein specifically associates with and reversibly passes across the outer membrane. Translocation is rendered unidirectional by stable association with CCHL which serves as a ``trans side receptor.'' Finally, heme is attached by CCHL and the holoprotein folds into its native structure.


INTRODUCTION

Mitochondrial cytochrome c is a soluble protein of the intermembrane space which participates in the electron transfer between complex III and complex IV of the respiratory chain (for reviews, see Refs. 1, 2). As with most of the mitochondrial proteins, cytochrome c is encoded by a nuclear gene, synthesized on cytoplasmic ribosomes, and translocated into the organelle in a post-translational fashion (for reviews, see Refs. 3-5). In the intermembrane space the apoform of the protein is converted to holocytochrome c by the enzymatic action of cytochrome c heme lyase (CCHL)()(6, 7) , a peripheral protein of the mitochondrial inner membrane. In this reaction a reduced heme group becomes covalently linked via two thioether bonds to cysteines 14 and 17 of the apoprotein (8, 9) .

The mode of how apocytochrome c passes across the mitochondrial outer membrane defines a unique import pathway that differs in a number of criteria from that of other mitochondrial preproteins (for reviews, see Refs. 10, 11). Apocytochrome c does not carry a cleavable, N-terminal targeting sequence. Instead, two internal segments of the apoprotein have been reported to be important determinants for targeting (12, 13, 14) . Unlike for most other mitochondrial preproteins an electrochemical potential across the mitochondrial inner membrane is not needed for transport (15) . Protease-sensitive outer membrane proteins, in particular components of the mitochondrial receptor complex, do not appear to be required for import (6, 16) . Little is known, however, about the mechanism of passage across the outer membrane. Extensive studies using artificial liposomes demonstrated a unique membrane insertion activity of apocytochrome c which is dependent on the presence of negatively charged phospholipids (e.g. phosphatidylserine; for review, see Ref. 17). At least parts of the apoprotein are able to penetrate the lipid bilayer and thus become accessible to proteases added on the trans side of the membrane (18) . Based on these results the spontaneous membrane insertion activity was suggested to be an important feature of the membrane passage of apocytochrome c. However, it remained unclear from these studies, whether in mitochondria apocytochrome c passes across the lipid phase of the outer membrane, or whether yet unknown, protease-resistant membrane proteins assist in the transport reaction.

In addition to its enzymatic function during heme attachment, further involvements of CCHL in the biogenesis of apocytochrome c were suggested. For instance, it was proposed that CCHL serves as a high affinity binding site for apocytochrome c(6, 7) . Mutant mitochondria lacking CCHL showed only weak binding (7, 19) . In an in vivo study accumulation of apocytochrome c in mitochondria correlated well with different levels of CCHL expression (20) . However, a complex between CCHL and apocytochrome c has not been demonstrated directly. Thus, the function of CCHL as a ``trans side receptor'' for apocytochrome c remains to be proven. A possible third role of CCHL as a component assisting in the membrane passage of apocytochrome c remained elusive from previous work. In this function, CCHL was suggested to associate with apocytochrome c, while the latter is still penetrating the outer membrane (6) . This might trigger the entry of the apoprotein into the intermembrane space, e.g. by induction of a conformational change and/or by enzymatic conversion to the holoprotein (21) .

In this paper we have studied the import mechanism of apocytochrome c. For the mechanistic analysis of the membrane binding and translocation reactions, we took advantage of a protein translocation system with isolated outer membrane vesicles (OMV) from Neurospora crassa mitochondria (22, 23) . Since these OMV are devoid of CCHL, it was possible to investigate the outer membrane interaction of apocytochrome c without interference by this enzyme. We find that OMV specifically bound apocytochrome c, but did not import it, unless a high affinity binding partner was present on the trans side of the membrane. Since specific antibodies introduced into the lumen of the OMV were sufficient to support import, we conclude that CCHL is not required for outer membrane passage of apocytochrome c. Yet, the protein is needed for stable accumulation of apocytochrome c in the intermembrane space. Binding to CCHL may therefore provide the major driving force rendering the reversible translocation process unidirectional.


MATERIALS AND METHODS

Biochemical Procedures

The following published procedures were used: isolation of mitochondria from N. crassa strain 74A (24) , purification of OMV (22) , transcription and translation reactions using reticulocyte lysate (Promega) and [S]methionine (ICN Radiochemicals) as radioactive label (25), preparation of C-labeled apocytochrome c by reductive methylation (26) , and SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography or fluorography of the resulting gels (8) . Blotting of proteins onto nitrocellulose and immunostaining of blotted proteins using the ECL chemiluminescence detection system (Amersham) was according to the instructions of the supplier. The resulting films and those from fluorography of radioactive proteins were scanned on an Image Master densitometer (Pharmacia). Antisera were raised and immunoglobulin G (IgG) were purified as described (27) . IgG were concentrated by ultrafiltration in Centriprep tubes (Amicon). Protein concentrations were determined by the Coomassie dye binding assay (Bio-Rad).

Inclusion of Purified IgG into the Lumen of OMV

OMV (80 µg/ml) were diluted 3-fold with buffer EM (1 mM EDTA, 10 mM MOPS-KOH, pH 7.2) and pelleted for 30 min at 220,000 g in a TLA 100.3 rotor (Beckman). The OMV were resuspended in 1 ml of EM buffer and spun again for 15 min at 220,000 g to completely remove residual sucrose. After resuspending the pellet in inclusion buffer (IB; 5 mg/ml BSA, 10 mM MOPS-KOH, pH 6.5) at a protein concentration of 2-4 mg/ml, one volume of purified IgG in water was added (final concentration 5 mg/ml). The sample was immediately frozen in liquid nitrogen and then put into a metal block in an ice/water bath to permit slow thawing. This usually took 15-30 min. After addition of one-fifth volume of 100 mM MOPS-KOH, pH 7.5, the sample was incubated for 5 min at 25 °C and adjusted to a sucrose concentration of 45% by addition of 60% (w/v) sucrose in buffer EMK (EM plus 150 mM KCl). OMV were recovered from this mixture by flotation centrifugation (45 min, 140,000 g) through a gradient consisting of 45%, 40% (both in EMK), 32%, and 8% (both in EM) sucrose steps (500 µl/step) in a Beckman SW60 rotor. The OMV were harvested from the 32%/8% sucrose interphase by careful aspiration with a pipette tip and diluted to the desired protein concentration with SEM buffer (250 mM sucrose in EM buffer).

Import of Apocytochrome c into Mitochondria and OMV

S-Labeled apocytochrome c was synthesized in the reticulocyte lysate, precipitated with ammonium sulfate (66% saturation), and redissolved in the same volume of SEM buffer. This treatment removes most of the hemoglobin which interferes with the electrophoretic analysis of the supernatants of the translocation experiments. For import 5 µl of this solution were incubated with mitochondria (100 µg/sample) or OMV (7.5 µg/sample) for 5 min at 25 °C in SEM buffer in a total volume of 150 µl. Import reactions were chilled on ice, and proteinase K was added to a final concentration of 30 µg/ml. Protease digestion was stopped after 15 min at 0 °C by the addition of 2 mM PMSF and further incubation at 25 °C for 5 min. Mitochondria or OMV were reisolated (10 min at 15,000 g or 45 min at 125,000 g, respectively) and subjected to SDS-PAGE and fluorography or autoradiography. In binding experiments where treatment with proteinase K after the import incubation was omitted, all tubes were coated with fatty acid-free BSA (1 mg/ml, 15 min) before use in order to reduce unspecific interaction of apocytochrome c with tube walls. Pelleted OMV were spun again (5 min, 30,000 g) to remove residual liquid from the tube walls. Finally, the OMV were resuspended in 20 µl of SEM buffer and transferred to new tubes where they were dissolved in sample buffer.

Digitonin Fractionation and Sonication of OMV

After the import reactions, digitonin was added from a 1% (w/v) stock solution in SEM buffer, and the samples were incubated for 2 min at 0 °C, followed by 20-fold dilution with SEM buffer containing 100 mM KCl. OMV were reisolated by centrifugation (45 min, 125,000 g) in a Beckman TLA45 rotor. To the supernatant 50 µg of BSA or 50 µg of mitochondrial protein was added as a carrier to quantitatively recover apocytochrome c in the subsequent trichloroacetic acid precipitation. The trichloroacetic acid precipitates and the reisolated vesicle pellets were dissolved in sample buffer and analyzed by SDS-PAGE. For sonication experiments, OMV were reisolated after the import reactions and resuspended in 600 µl of SEM buffer containing 100 mM KCl. Samples were sonicated in a glass tube for 1 min at 0 °C (Branson Sonifier 250 with a microtip, intensity 4, 30% duty cycle). Membranes and associated material were reisolated and prepared for SDS-PAGE as described for the digitonin treatment.

Coimmunoprecipitation of Apocytochrome c with CCHL

IgG were prebound to protein A-Sepharose beads by shaking 25 µl of antiserum specific for CCHL with 10 mg of beads for 1 h in IP buffer (25 mM potassium phosphate pH 7.2, 100 mM KCl, and 2% (w/v) octyl glucoside). The Sepharose beads were washed once with IP buffer and then used for the precipitation. The samples to be analyzed were diluted 4-fold with IP buffer, incubated for 15 min at 0 °C, and spun for 15 min at 30,000 g. The supernatant was added to the Sepharose beads with prebound IgG and shaken gently for 30 min at 4 °C. The beads were washed twice with 1 ml of cold IP buffer and once with cold 25 mM potassium phosphate, pH 7.2, before resuspending in sample buffer.

Preparation of Liposomes

Lipids (from Sigma) were mixed in chloroform in the following molar ratios: 33% bovine brain phosphatidylcholine (P-6638), 30% Escherichia coli phosphatidylethanolamine (P-3511), 7% bovine liver phosphatidylinositol (P-2517), 4% bovine brain phosphatidylserine (P-6641), 1% bovine heart cardiolipin (C-1649), 25% ergosterol (E-6510). The solvent was removed in a rotary evaporator, and the lipid film was overlaid with EM buffer. Liposomes were formed by adding glass beads (2 mm diameter), shaking the tube vigorously on a vortex, and by subsequent sonication (Branson sonifier 250, stage 3, 20% duty cycle). The liposomes were pelleted by centrifugation (30 min, 125,000 g), resuspended in EM buffer at a lipid concentration of 2 mg/ml and stored at -20 °C. After thawing aliquots were sonicated briefly before use to disperse aggregates formed during the freeze-thaw cycle.


RESULTS

Mitochondria, But Not Isolated OMV, Can Accumulate Apocytochrome c in a Protease-protected Location

Purified OMV were incubated with S-labeled apocytochrome c. A considerable fraction of the added apoprotein (40%) was recovered with the OMV after reisolation by centrifugation, i.e. had bound to the OMV (Fig. 1A). All bound material was sensitive to externally added proteinase K and therefore had not become imported into the lumen of the OMV. The integrity of the outer membrane was assayed by the accessibility of MOM38 to added protease. This integral membrane protein is converted to a 26-kDa N-terminal fragment (MOM38*) by proteinase K digestion from outside, but becomes further degraded, when the outer membrane is opened (22).() In isolated OMV MOM38 was quantitatively converted to MOM38* demonstrating the sealed nature of the OMV, even after the import reaction (Fig. 1B). Apparently, purified OMV can bind a substantial fraction of added apocytochrome c, but they do not accumulate the apoprotein in a protease-protected form.


Figure 1: Apocytochrome c is imported into mitochondria but not into purified OMV. A, incubation of S-labeled apocytochrome c with mitochondria (Mit) or OMV was for 5 min at 25 °C. The samples were chilled to 0 °C, treated with the indicated amounts of proteinase K (15 min, 0 °C). Mitochondria or OMV were reisolated by centrifugation, and proteins were resolved by SDS-PAGE and blotted onto nitrocellulose. Imported apocytochrome c was visualized by autoradiography of the blot and quantitated by densitometry. B, to control for the sealed nature and for the right-side-out orientation of the OMV, the accessibility of various reporter proteins to proteinase K was tested. The blots from A were immunostained using antisera against porin, MOM38, and MOM19. Quantitation was by densitometry. MOM38* represents the N-terminal 26-kDa fragment of MOM38 generated by externally added proteinase K. The signals obtained in samples not treated with proteinase K were set to 100%. C, the intactness of the outer membrane of isolated mitochondria was analyzed as in B. In addition, the blots were immunodecorated with antibodies against CCHL. D, the more than 600-fold depletion of CCHL in purified OMV was estimated by subjecting mitochondria (10 µg) and OMV (120 µg) to SDS-PAGE and blotting of proteins onto nitrocellulose. CCHL was detected by immunostaining and quantitated by densitometry. The data contain a correction for the outer membrane content of mitochondria (6%, 22). a.u., arbitrary units.



Apocytochrome c became bound to isolated mitochondria even more efficiently (Fig. 1A). Two-thirds of bound apocytochrome c were resistant to externally added proteinase K, i.e. had become imported into mitochondria, even though the experiments were performed under non-reducing conditions which do not support the formation of the holoprotein (8, 9) . The integrity of the outer membrane was determined by using MOM38 and CCHL as marker proteins. 70% of the mitochondria contained an intact outer membrane (Fig. 1C). Since also about two-thirds of bound apocytochrome c were protected against proteolytic attack (Fig. 1A), we conclude that practically all of the mitochondria-associated apocytochrome c was bound to a site beyond the surface of the outer membrane. In an earlier investigation comparatively small amounts of protease-resistant apocytochrome c (10-15% of input) were observed (6) . The reason for this discrepancy most likely is that the outer membrane was opened in these experiments thus allowing added proteases access to the intermembrane space.

Based on previous models, a likely binding site for apocytochrome c was CCHL, which is located at the outer face of the inner membrane (data not shown, 20). We tried to directly demonstrate the interaction of apocytochrome c and CCHL in isolated mitochondria by co-immunoprecipitation experiments. Mitochondria-bound apocytochrome c was precipitated with antibodies specific for CCHL, but not by preimmune serum (Fig. 2). Precipitation was strongly decreased by including KCl in the binding reaction, a condition known to affect the binding of apocytochrome c to mitochondria (6, see below). Furthermore, much less material was co-immunoprecipitated if reduced heme was added to convert bound apocytochrome c to the holoprotein (6) . This results in the release of folded and soluble holocytochrome c into the intermembrane space. We conclude that apocytochrome c becomes efficiently imported into isolated mitochondria where it is found in a complex with CCHL. In contrast, OMV which are depleted in CCHL more than 600-fold (Fig. 1D) do not accumulate the apoprotein in their lumen, even though they are able to bind a substantial fraction.


Figure 2: Coimmunoprecipitation of apocytochrome c with CCHL. C-Labeled apocytochrome c was incubated with mitochondria (30 µg/sample) for 5 min at 25 °C in the presence or absence of 100 mM KCl. Mitochondria were reisolated and resuspended in SEM buffer. Aliquots of the sample received 1 µM heme and 1 mg/ml sodium dithionite (DT) or an equivalent amount of SEM buffer. After 3 min at 25 °C, the samples were chilled on ice and diluted 4-fold with IP buffer. Proteins were subjected to co-immunoprecipitation with antiserum against CCHL or with preimmune serum. Immunoprecipitated proteins were analyzed by SDS-PAGE and fluorography. The signal obtained with antiserum against CCHL in the absence of KCl and heme/DT was set to 100%. This represents about 15% of apocytochrome c bound to mitochondria.



OMV Containing IgG against Apocytochrome c Accumulate the Apoprotein in a Protease-resistant, Soluble Form

We addressed the question whether CCHL plays an active role in assisting outer membrane passage of apocytochrome c, or whether the enzyme only passively provides a binding site in the intermembrane space. In the latter case, it should be possible to replace CCHL by other binding partners for apocytochrome c in the lumen of the OMV. To this end, purified immunoglobulin G (IgG) which specifically recognized apocytochrome c (data not shown) was enclosed inside the OMV by a recently developed freeze-thaw procedure (28) . During the slow thawing period, the membranes are transiently opened allowing equilibration of the vesicle lumen with the external solution. The antibodies were recovered with the OMV only, when added before the freeze-thaw procedure, but not when this procedure was omitted, or when IgG was added after the freeze-thaw treatment (Fig. 3A). After the freeze-thaw step, the OMV maintained a right-side-out orientation as indicated by the protease sensitivity of MOM19. In addition, most of the OMV were sealed as judged from the conversion of MOM38 to its 26-kDa fragment, MOM38* (Fig. 3B, cf. Fig. 1, B and C).


Figure 3: Purified IgG can be entrapped in the lumen of OMV by a freeze-thaw treatment. A, OMV were incubated with purified IgG and subjected to a freeze-thaw treatment. IgG was added either before (B) or after (A) the freeze-thaw step. Samples which were not frozen remained on ice. After frozen samples had been thawed, all samples were incubated on ice for a further 20 min. OMV were reisolated by flotation centrifugation, and their IgG content was analyzed by SDS-PAGE, immunostaining, and quantitation by densitometry. B, the intactness of the membrane was analyzed with OMV which had been subjected to a freeze-thaw procedure or mock-treated on ice. OMV were reisolated and resuspended in SEM buffer. One aliquot was treated with proteinase K (40 µg/ml, 15 min, 0 °C), while another one was left on ice. Proteins in both samples were precipitated with trichloroacetic acid and subjected to SDS-PAGE and immunostaining using antibodies raised against MOM19 and an N-terminal peptide of MOM38. Generation of MOM38* (see Fig. 1B) was used as a marker for sealed nature of the OMV, and the protease accessibility of MOM19 for their correct orientation (22). Data are given as the fraction of the signals obtained with proteinase K-treated and untreated samples.



OMV preloaded with apocytochrome c-specific IgG were incubated with apocytochrome c. 15% of the added apoprotein became resistant to proteinase K, i.e. had completely become transported across the outer membrane (Fig. 4A). No protease-resistant material was observed when the freeze-thaw step had been omitted or when IgG had been added after the freeze-thaw step. Similarly, enclosure of IgG purified from preimmune serum did not result in protease resistance of apocytochrome c. In agreement with earlier import studies using intact mitochondria (6) , import into IgG-loaded OMV was unaffected, when surface components of the outer membrane were degraded by pretreatment with proteinase K (Fig. 4B). Taken together, our data show that for accumulation of significant amounts of apocytochrome c a high affinity binding site inside the lumen of the OMV is both necessary and sufficient. Apparently, neither protease-sensitive components on the surface of the outer membrane nor CCHL are essential for the transport of apocytochrome c across the outer membrane.


Figure 4: Specific antibodies introduced into the lumen of OMV can trap apocytochrome c in a protease-protected location. A, OMV were loaded with purified IgG specific for apocytochrome c (Apoc) or derived from preimmune serum (Pre-Imm.) as described in Fig. 3A. After reisolation the IgG-loaded OMV were used for import assays (5 min, 25 °C) using radiolabeled apocytochrome c synthesized in reticulocyte lysate. The samples were chilled to 0 °C and divided in half. One aliquot of each sample was treated with proteinase K (30 µg/ml, 15 min, 0 °C), the other one was left on ice. OMV were pelleted by centrifugation and subjected to SDS-PAGE and fluorography. For comparison a 15% input standard was included on the gel. B, OMV in EM buffer were treated with the indicated amounts of proteinase K for 15 min at 0 °C. After stopping proteolysis by addition of 1 mM PMSF, OMV were reisolated and resuspended in inclusion buffer. Apocytochrome c-specific IgG were enclosed and import was performed as described in A. a.u., arbitrary units.



Previous studies have shown that import of apocytochrome c into mitochondria and its interaction with CCHL is abolished at higher salt concentrations (6) . When tested in the OMV system, both the binding to OMV and the import into IgG-loaded OMV were salt-sensitive (Fig. 5A). The salt sensitivity of the latter reaction was identical to that reported for intact mitochondria (Fig. 5B, 6). Inhibition of import at higher salt concentrations was not a result of an inefficient interaction of apocytochrome c with the IgG enclosed inside the OMV, since apocytochrome c could be immunoprecipitated with the same efficiency under all conditions tested. Thus, in addition to the salt-sensitive interaction of apocytochrome c with CCHL (see above) binding to and transfer across the outer membrane appear to be affected at higher ionic strength. This makes it likely that apocytochrome c itself is the target of the salt sensitivity. The apoprotein may be rendered incompetent for both membrane insertion and CCHL interaction by undergoing a conformational change.


Figure 5: Binding and import of apocytochrome c are salt-sensitive. A, purified IgG specific for apocytochrome c were added to the OMV. After a freeze-thaw step or a corresponding mock treatment on ice, OMV were reisolated by flotation centrifugation. Import of apocytochrome c into the OMV was performed in the absence or presence of 300 mM KCl for 5 min at 25 °C. The samples were chilled on ice and divided in half. One aliquot was treated with proteinase K (30 µg/ml, 15 min, 0 °C) while the other one was left on ice. Then, the OMV were reisolated and subjected to SDS-PAGE and fluorography. B, to analyze the influence of KCl on the binding of apocytochrome c to IgG, purified IgG was prebound to protein A-Sepharose beads and incubated with radiolabeled apocytochrome c in SEM buffer (200 µl total volume) in the presence of increasing amounts of KCl (15 min, 25 °C). The Sepharose beads were recovered by centrifugation and washed three times with SEM buffer. Finally, the beads were treated with sample buffer, and solubilized apocytochrome c was visualized by SDS-PAGE and fluorography. Unspecific binding of apocytochrome c to protein A-Sepharose alone was negligible under these conditions (not shown). For direct comparison an import reaction into IgG-loaded OMV was performed under the same buffer conditions (for details see Fig. 4). a.u., arbitrary units.



We tested whether apocytochrome c translocated into IgG-loaded OMV was soluble in the lumen or was still associated with the outer membrane. After import the vesicles were opened by treatment with increasing amounts of digitonin (29) . Membranes were reisolated by centrifugation, and pellets and supernatants were analyzed for the content of apocytochrome c and IgG. Both proteins were released efficiently into the supernatant at digitonin concentrations which are known to selectively disrupt the outer membrane (Fig. 6A, cf. 22). Membrane proteins were not solubilized at these concentrations of digitonin, since the amount of porin in the pellet fractions remained unchanged. The soluble character of imported apocytochrome c was confirmed in sonication experiments. The import samples were briefly sonified and membranes were reisolated by centrifugation. The majority of imported apocytochrome c and of IgG was recovered in the supernatant, whereas porin as a control appeared in the pellet fraction (Fig. 6B). These independent approaches demonstrate that apocytochrome c became translocated into the lumen of the OMV, where it was in a soluble complex with IgG.


Figure 6: Apocytochrome c imported into IgG-containing OMV is soluble in the lumen. Enclosure of IgG directed against apocytochrome c (Apoc) and import of apocytochrome c was performed as in Fig. 4. A, identical aliquots of the import reaction were extracted with increasing concentrations of digitonin. After centrifugation the supernatants (sup) and pellets were analyzed by SDS-PAGE and blotted onto nitrocellulose. Apocytochrome c was visualized by autoradiography, whereas the amounts of porin and IgG were estimated by immunostaining of the same blot. The values obtained for the sample without digitonin treatment were set to 100%. B, another aliquot of the import reaction was sonicated, while a second one was left on ice. The two samples were centrifuged, and pellets (P) and supernatants (S) were analyzed as described in A. Data are given relative to the input measured by precipitating a third aliquot with trichloroacetic acid immediately after the import reaction.



Translocation of Apocytochrome c across the Outer Membrane Is Reversible

The results presented above show that apocytochrome c is accumulated on the intermembrane space side of the outer membrane, if a high affinity binding partner is available. This suggested that apocytochrome c can traverse the outer membrane in a reversible fashion. We sought to directly demonstrate the reversal of the translocation reaction by disrupting the interaction between apocytochrome c and IgG with 20 mM -mercaptoethanol and increasing amounts of urea. At 2 M urea, 60% of the imported apocytochrome c became accessible to added proteinase K (Fig. 7A). Release of imported apocytochrome c from the OMV was fast and occurred at 0 °C. OMV remained sealed as evident from the quantitative generation of the MOM38 fragment (Fig. 7B). The protease was still active at higher urea concentrations, since upon opening of the OMV by sonication or upon the addition of digitonin, MOM38 was completely degraded. Thus, in OMV imported apocytochrome c can regain access to the cytosolic face of the outer membrane by a retrograde translocation reaction.


Figure 7: Imported apocytochrome c becomes accessible to external protease after release from IgG. A, apocytochromec was imported into IgG-loaded OMV as described in Fig. 4. The sample was split, and aliquots were supplemented with 20 mM -mercaptoethanol and different concentrations of urea to disrupt the interaction between apocytochrome c and IgG. Each aliquot was again split into three portions. The first one (Intact) was treated with proteinase K (250 µg/ml, 15 min, 0 °C), the second one received the same treatment in the presence of 0.05% (w/v) digitonin (Dig.), and the third one was sonicated in the presence of the protease (30 s) and incubated for further 15 min at 0 °C. After adding 2 mM PMSF, all samples received 50 µg of BSA and were precipitated with trichloroacetic acid. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose. Protease-resistant apocytochrome c was determined by autoradiography of the blot. B, the intactness of the OMV at the various urea concentrations used was determined by immunodecoration of the blot from A using antiserum against the N-terminal segment of MOM38. MOM38*, 26-kDa fragment of MOM38.



Reversibility of apocytochrome c import has been reported for intact mitochondria (30) . This observation seemed to be in contradiction to other reports of a stable association of apocytochrome c with mitochondria (see above, 20). It was, however, obtained with a heterologous system which may explain the labile interaction between CCHL and apocytochrome c. We therefore examined in our homologous N. crassa system whether, at least in a longer time frame, dissociation from CCHL and consequently release from intact mitochondria would occur. Apocytochrome c was incubated with isolated mitochondria and, after reisolation of the mitochondria, proteinase K was added. Incubation was continued at 0 or 25 °C to test for the protease accessibility of imported apocytochrome c. In parallel, the integrity of the outer membrane was estimated by measuring the resistance against protease of endogenous CCHL. At 0 °C no significant decrease of protease-protected apocytochrome c was observed, even after prolonged times of incubation (Fig. 8A). Previous studies have shown that even at this low temperature import is fast and efficient (6) . In contrast, at 25 °C apocytochrome c became accessible to proteinase K with a half-time of 35 min (Fig. 8B). However, this decrease was paralleled by degradation of endogenous CCHL, and therefore was due to the opening of the outer membrane rather than the release of apocytochrome c from the intermembrane space. Thus, in intact mitochondria the stable interaction of apocytochrome c with CCHL precludes its dissociation from the intermembrane space and ensures the unidirectionality of the import reaction.


Figure 8: Imported apocytochrome c does not dissociate from mitochondria. Radiolabeled apocytochrome c (Apoc) was imported into isolated mitochondria for 5 min at 0 °C. Mitochondria were reisolated, resuspended in SEM buffer, and incubated in the presence of 20 µg/ml proteinase K at 0 °C (A) or in the presence of 7 µg/ml proteinase K at 0 °C (B). At the indicated time points, aliquots were withdrawn and supplemented with one volume of SEM buffer containing 2 mM PMSF. After reisolation the mitochondrial pellets were analyzed by SDS-PAGE and blotting onto nitrocellulose. Apocytochrome c was detected by autoradiography, porin and CCHL by immunostaining. The values obtained for the first aliquot withdrawn were set to 100%.



Binding of Apocytochrome c to the Mitochondrial Outer Membrane Is Specific

Finally, we asked whether the interaction of apocytochrome c with the outer membrane is specific or would also occur with other biological membranes or liposomes. Compared to OMV apocytochrome c bound very inefficiently to inverted vesicles derived from the inner membrane of E. coli or to liposomes composed of synthetic lipids similar to those present in the mitochondrial outer membrane (Fig. 9, 31). Similar amounts of apocytochrome c were found in the pellet fraction of a binding assay even in the absence of any added membranes. Therefore, we conclude that apocytochrome c exhibits a high preference for binding to the mitochondrial outer membrane. This preferential binding may be important to ensure correct targeting to mitochondria and may, in addition to the association with CCHL, contribute to the overall targeting specificity.


Figure 9: Binding of apocytochrome c to the mitochondrial outer membrane is specific. Apocytochrome c was incubated for 5 min at 25 °C with OMV (10 µg protein/sample), E. coli inverted inner membrane vesicles (20 µg protein/sample), liposomes (20 µg lipid/sample), or without any membranes. Binding was measured by centrifugation of the samples (45 min, 125,000 g), and the pelleted fractions were analyzed by SDS-PAGE and fluorography. The signal for apocytochrome c bound to OMV was set to 100%.




DISCUSSION

We have analyzed the membrane binding and translocation steps of apocytochrome c import into the mitochondrial intermembrane space. Together with previously obtained results our data lead to a model for the biogenesis of cytochrome c (Fig. 10). The precursor, apocytochrome c, is synthesized on cytosolic ribosomes without a cleavable mitochondrial targeting sequence at its N terminus. Targeting to mitochondria occurs in a post-translational fashion. Initial binding to the outer membrane surface may occur via electrostatic association with phospholipid headgroups and is followed by specific interaction with a protease-resistant component ( in Fig. 10 ). The apoprotein then becomes translocated across the outer membrane, a reaction which apparently can be reversed. Either during the release from the membrane or after diffusion across the intermembrane space as a soluble intermediate, apocytochrome c gains access to CCHL, a protein bound to the outer face of the inner membrane. CCHL serves as a high affinity binding partner on the trans side of the membrane thereby shifting the equilibrium of the transport reaction toward the intermembrane space. In addition to the specific binding to the outer membrane this complex formation with CCHL may be considered the major contribution to the overall targeting specificity. In a final step, heme is covalently attached to cysteines 14 and 17. This reaction is catalyzed by CCHL and requires heme in its reduced state. Folding to native holocytochrome c may then trigger the release from CCHL thus completing the process.


Figure 10: Model for the biogenesis of cytochrome c. denotes a putative component in the outer membrane (OM) facilitating specific binding and membrane passage of apocytochrome c. For further explanations see text. IMS, intermembrane space; IM, inner membrane; SH, thiol groups of cysteines 14 and 17.



Our investigations show that apocytochrome c can specifically bind to the mitochondrial outer membrane. Previous studies using intact mitochondria have failed to demonstrate this interaction, since interaction with the outer membrane is only transient, and due to its high affinity the apoprotein is further transported to CCHL (6) . At present it remains elusive what the chemical nature of the component contributing specificity of binding to the outer membrane might be. Based on extensive studies with artificial liposomes (for review, see Ref. 17) lipids may be expected to play an important role in this process. In particular, acidic phospholipids, e.g. phosphatidylserine, have been demonstrated to be required for an unusual membrane insertion activity of apocytochrome c. However, our study failed to demonstrate significant binding of apocytochrome c to artificial liposomes representing the lipid composition of the outer membrane, at least under the conditions used with OMV. Thus, if lipids alone are providing the basis for specific binding, then such a compound must be a special lipid of the outer membrane. Alternatively, an unknown proteinaceous component may be responsible for directing apocytochrome c to the mitochondrial surface. Such a component might not only assure specific binding of the apoprotein, but also function in passing it across the membrane. A recently identified component of cytochrome c biogenesis, Cyc2p, is an unlikely candidate, since the protein was reported to be localized to the mitochondrial inner membrane (5, 32) . Clearly, direct proof for the existence of such a component is required. Our studies using isolated OMV pave the way for approaching this goal by making it possible to reconstitute the binding and import processes with liposomes and detergent extracts of the outer membrane.

Despite being essential for in vivo and in vitro accumulation of apocytochrome c in the intermembrane space, CCHL does not appear to be required for the actual membrane translocation step. Transport across the mitochondrial outer membrane is observed if apocytochrome c-specific binding partners, like IgG, are present on the intermembrane space side. Without such a ``trap'' the apoprotein would leave the OMV in a retrograde translocation reaction. It seems unlikely to us that IgG functionally participates in the release of apocytochrome c from the outer membrane and thereby mimicks a possible function of CCHL in facilitating entry of the apoprotein into the intermembrane space. Thus, the outer membrane alone can accomplish full translocation of apocytochrome c, yet a binding site on the trans side of the membrane is required to shift the equilibrium of the import reaction. The question, when precisely CCHL starts to interact with translocating apocytochrome c, is difficult to answer. This might occur soon after the first appearance of segments of apocytochrome c in the intermembrane space. An early interaction of apocytochrome c with CCHL would fit well to a study in which holo conversion of membrane spanning apocytochrome c fusion proteins was observed (33) . However, the low efficiency of heme attachment in these experiments may indicate that this pathway is not the preferred one in the physiological situation. As an alternative, apocytochrome c might diffuse through the intermembrane space as a soluble intermediate before associating with CCHL. In this view, the function of CCHL is restricted to providing a binding site for apocytochrome c in the target compartment.

Despite the fundamental differences between the translocation of apocytochrome c and the general import routes used by other mitochondrial preproteins there are common principles underlying both processes. A reversible membrane passage is preceded and followed by steps introducing both specificity and unidirectionality to the reaction. Correct targeting is usually verified by the specific interaction of preproteins with components on the cis side of the membrane, e.g. targeting factors or surface receptors. In the case of apocytochrome c, the accuracy of targeting to the mitochondrial intermembrane space appears to be guaranteed by two sequential steps, namely by binding to the outer membrane and to CCHL. Reversibility of membrane translocation has been reported for a number of experimental systems, e.g. the bacterial inner membrane (34) , the membrane of the endoplasmic reticulum (35) , and the mitochondrial outer (23) and inner membranes (36). In comparison to these transport reactions, the system described here is remarkable in that the whole preprotein and not only segments thereof can undergo a retrograde translocation. This implies that apocytochrome c either passes through the lipid bilayer or uses a pore-like structure which can be entered and traversed from both sides.

Reversible membrane transit of preproteins allows for interaction with components on the trans side of the membrane. In most cases, this can be considered as the driving force for the whole reaction. For these interactions diverse mechanisms are used in the various membrane systems. For instance, repetitive cycles of binding to members of the Hsp70 family and ATP hydrolysis result in net movement across the endoplasmic reticulum and mitochondrial inner membranes (37, 38) . In case of the mitochondrial outer membrane, the N-terminal presequence, after passing across a putative translocation pore, associates with a specific binding site on the intermembrane space side of the membrane (23). For apocytochrome c, two reactions may be considered to serve as the driving force for import into mitochondria. First, the interaction with CCHL, and second, the conversion to the holoform, a reaction also catalyzed by CCHL. Heme attachment assures dissociation of holocytochrome c from CCHL. This presumably is triggered by folding into the native structure. In summary, despite taking a unique import pathway, apocytochrome c obeys general principles elucidated for other membrane translocation systems.


FOOTNOTES

*
This work was supported by Sonderforschungsbereich 184 Teilprojekt B19 of Deutsche Forschungsgemeinschaft and by a fellowship (to A. M.) by the Boehringer Ingelheim Fonds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Tel.: +49-89-5996-304; and Fax: +49-89-5996-270.

The abbreviations used are: CCHL, cytochrome c heme lyase; OMV, outer membrane vesicles; BSA, bovine serum albumin; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis.

M. Kiebler, unpublished results.


ACKNOWLEDGEMENTS

We thank Petra Heckmeyer and Marlies Braun for expert technical assistance and Dr. M. Müller for a gift of E. coli inner membranes. We are grateful to Dr. C. Hergersberg for his important role in starting these studies and for supplying antibodies specific for apocytochrome c.

Addendum-When large unilamellar vesicles prepared by the extruder technique (LUVETs) were used for lipid binding studies, the results of apocytochrome c binding were comparable to those presented (R. Baardman and A. Mayer, unpublished results). However, at higher concentrations of liposomes substantial amounts of apocytochrome c became bound to liposomes. These data support the conclusion drawn from the result of Fig. 9, and furthermore provide an explanation for the differences to previous lipid binding studies (as summarized by De Kruijff et al., 17).


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