The Import Route of ADP/ATP Carrier into Mitochondria Separates from the General Import Pathway of Cleavable Preproteins at thetrans Side of the Outer Membrane*

The ADP/ATP carrier (AAC) of the mitochondrial inner membrane is synthesized in the cytosol without a cleavable presequence. The preprotein preferentially binds to the mitochondrial surface receptor Tom70 and joins the import pathway of presequence-carrying preproteins at the cis side of the outer membrane. Little is known about the translocation of the AAC across the outer membrane and where its import route separates from that of cleavable preproteins. Here we have characterized a translocation intermediate of AAC during transfer across the outer membrane. The major portion of the preprotein is exposed to the intermembrane space, while a short segment is still accessible to externally added protease. This intermediate can be quantitatively chased to the fully imported form in the inner membrane. Its accumulation depends on Tom7, but not on the intermembrane space domain of Tom22 in contrast to cleavable preproteins. Moreover, opening of the intermembrane space inhibits the import of AAC, but not that of cleavable preproteins into mitoplasts. We conclude that the import route of AAC diverges from the general import pathway of cleavable preproteins already at the trans side of the outer membrane.

The ADP/ATP carrier (AAC) of the mitochondrial inner membrane is synthesized in the cytosol without a cleavable presequence. The preprotein preferentially binds to the mitochondrial surface receptor Tom70 and joins the import pathway of presequence-carrying preproteins at the cis side of the outer membrane. Little is known about the translocation of the AAC across the outer membrane and where its import route separates from that of cleavable preproteins. Here we have characterized a translocation intermediate of AAC during transfer across the outer membrane. The major portion of the preprotein is exposed to the intermembrane space, while a short segment is still accessible to externally added protease. This intermediate can be quantitatively chased to the fully imported form in the inner membrane. Its accumulation depends on Tom7, but not on the intermembrane space domain of Tom22 in contrast to cleavable preproteins. Moreover, opening of the intermembrane space inhibits the import of AAC, but not that of cleavable preproteins into mitoplasts. We conclude that the import route of AAC diverges from the general import pathway of cleavable preproteins already at the trans side of the outer membrane.
Most mitochondrial proteins are synthesized on cytosolic polysomes as preproteins with amino-terminal targeting sequences (presequences) that are cleaved off after import into the organelle (1)(2)(3)(4). The ADP/ATP carrier (AAC) 1 is the major representative of a large class of metabolite carriers of the mitochondrial inner membrane (5,6) that are synthesized without presequences and contain targeting information within the mature protein parts (7,8). Each carrier protein consists of about 300 amino acid residues, is predicted to contain six membrane-spanning segments, and is thought to have evolved by triplication of an ancient gene (5,6,9). The targeting signals in the carrier proteins are not exactly known. The available evidence indicates that the AAC contains at least two targeting signals distributed on distinct thirds of the preprotein (7,8).
In the past years, protein transport machineries have been identified in the mitochondrial outer and inner membranes, termed Tom and Tim, respectively. Thereby a detailed picture was obtained of how presequence-containing (cleavable) preproteins are translocated into mitochondria (2)(3)(4)10). The presequences are recognized by the receptors Tom20 and Tom22 on the cis side of the outer membrane, and with the help of the small protein Tom5, the preproteins are inserted into the import pore Tom40. On the trans side of the outer membrane, the presequences can interact with the intermembrane space domain of Tom22. Driven by the membrane potential ⌬, the preproteins are inserted into the translocase of the inner membrane, formed by Tim23 and Tim17, and are then translocated into the matrix with the help of a driving system containing Tim44 and the heat shock protein 70.
Based on import studies of AAC into mitochondria of the fungus Neurospora crassa, a number of import stages for inner membrane metabolite carriers were proposed (11)(12)(13). Stage I, the cytosolic transport form of AAC, probably in a complex with molecular chaperones; stage II, binding of AAC to the receptors on the cis side of the outer membrane (accumulation of AAC in the absence of ⌬ and ATP); stage III, accumulation of AAC in mitochondria in the absence of a ⌬, but presence of ATP, leading to a transport intermediate that is protected against a treatment of mitochondria with trypsin (or low concentrations of proteinase K); stage IV, ⌬-dependent insertion of AAC into the inner membrane; stage V, assembled AAC in the inner membrane. Only the stage II intermediate(s) has been characterized in detail, initially in N. crassa and then mainly in the yeast Saccharomyces cerevisiae. The carrier precursors are first recognized by the import receptor Tom70 (14 -17) and then join the import pathway of presequence-containing preproteins, probably at the level of Tom20/Tom22 (18,19). Tom5 is responsible for insertion of both cleavable and non-cleavable preproteins into the import pore of the outer membrane (20). Little is known about the requirements/components for the further steps of import of carrier proteins except of the strict ⌬ dependence for insertion into the inner membrane (stage IV) (11,21,22) and the recent identification of Tim22 (23). While Tim23-17 seems to be able to mediate a low efficient insertion of AAC (24), Sirrenberg et al. (23) reported that the main machinery for insertion of carriers into the inner membrane is distinct from the Tim23-17 complex and is formed by a complex containing Tim22. It is unknown if and where the import pathway of carrier proteins separates from that of presequence-containing preproteins.
For this report, we attempted to characterize the stage III intermediate of AAC to get insight into steps occurring between recognition by surface receptors of the outer membrane and insertion into the inner membrane. We used mitochondria from S. cerevisiae, the organism in which most mitochondrial import components have been identified and where mutant mitochondria are available. We describe a translocation intermediate of AAC during transfer across the outer membrane that can be quantitatively chased to the mature form in the inner membrane. The major portion of the translocation intermediate is exposed to the intermembrane space, while a small segment is still on the cytosolic side. Accumulation of the intermediate requires Tom7, but does not depend on the intermembrane space domain of Tom22. In contrast to cleavable preproteins, the productive accumulation of the translocation intermediate is insensitive to salt. Moreover, opening of the intermembrane space inhibits the import of AAC, but not that of cleavable preproteins. These results indicate that the import route of AAC diverges from the general import pathway already at the trans side of the outer membrane.

MATERIALS AND METHODS
The S. cerevisiae strains used are shown in Table I. Mitochondria were isolated as described by Daum et al. (25) and Hartl et al. (26). After in vitro transcription, preproteins were synthesized in rabbit reticulocyte lysate in the presence of [ 35 S]methionine and [ 35 S]cysteine (13). For the accumulation of the AAC at stage III, the mitochondria were incubated together with the radiolabeled preprotein in binding buffer (3% (w/v) BSA, 250 mM sucrose, 5 mM MgCl 2 , 5 mM sodium malate, 20 mM KP i , 1 M valinomycin, 10 mM MOPS/KOH, pH 7.2) at 25°C. (ATP was supplied from the reticulocyte lysate.) The mitochondria were either treated with proteinase K (150 -200 g/ml), reisolated, and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (13) or were reisolated and incubated in chase buffer (3% (w/v) BSA, 250 mM sucrose, 5 mM MgCl 2 , 5 mM sodium malate, 20 mM NaP i , 1 M valinomycin, 10 mM MOPS/NaOH, pH 7.2) at 25°C to allow the AAC to be imported to its final location in the mitochondrial inner membrane. Mitochondria were then treated with proteinase K (27). For swelling, the mitochondria were reisolated and resuspended in EM buffer (10 mM MOPS/KOH, pH 7.2, 1 mM EDTA) or 10 mM Tris/HCl, pH 7.4, for 15 min at 4°C (in case of a subsequent proteinase K treatment, 50 g/ml were used). In cases where preproteins were directly imported without an accumulation at the outer membrane, import was performed in BSA buffer (3% (w/v) BSA, 250 mM sucrose, 5 mM MgCl 2 , 80 mM KP i , 10 mM MOPS/ KOH, pH 7.2) containing 2 mM ATP and 2 mM NADH. To dissipate the membrane potential, a mixture of 1 M valinomycin, 20 M oligomycin, and 8 M antimycin A was added to the import reaction.
For the immunoprecipitation of the AAC, antibodies were added either directly to the mitochondria after the import reaction or to lysed mitochondria (28). Mitochondria were lysed with Triton X-100 buffer (1% Triton X-100, 10 mM Tris/HCl, pH 7.5, 300 mM NaCl). The lysed material was subjected to a clarifying spin, applied onto protein A-Sepharose, and incubated for 1 h. The bound material was eluted and analyzed by SDS-PAGE.
Alkaline extraction of mitochondria (100 mM Na 2 CO 3 ) and separation of pellet and supernatant were performed as published (27). For the salt extraction of accumulated AAC, mitochondria were incubated with radiolabeled AAC in binding buffer for 15 min at 25°C. Samples were split into two aliquots and were either reisolated or first treated with proteinase K and then reisolated. The pelleted mitochondria were resuspended in SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM MOPS/ KOH, pH 7.2) containing different concentrations of NaCl and incubated for 15 min at 4°C. For sonication, the mitochondria were resuspended in SEM buffer with 200 mM NaCl and sonified for 6 ϫ 6 s in a Branson sonifier 250 (50% duty cycle) (29), followed by separation into pellet and supernatant by centrifugation at 266,000 ϫ g for 90 min at 2°C and precipitation with 7.5% trichloroacetic acid in the presence of 0.0125% sodium deoxycholate.
Standard procedures were used for Western blotting onto nitrocellulose using enhanced chemiluminescence system (Amersham Pharmacia Biotech) and storage phosphorimaging technology (Molecular Dynamics).

Accumulation of AAC at the Intermembrane Space Side of the
Outer Membrane-The precursor of AAC was synthesized in rabbit reticulocyte lysates in the presence of [ 35 S]methionine/ cysteine and incubated with isolated yeast mitochondria in the presence of ATP, but the absence of a ⌬ across the inner membrane. The AAC accumulated at this stage III is protected against trypsin (or a low concentration of proteinase K) added to the mitochondria (13,30,31) but becomes accessible to the protease after opening of the intermembrane space by swelling of mitochondria and rupturing of the outer membrane (formation of mitoplasts) (30). This result suggested that the preprotein may be exposed to the intermembrane space. It could not be excluded, however, that the preprotein was enclosed by proteinaceous components of the import machinery and became accessible to the intermembrane space side only after proteolytic degradation of protecting components. We therefore applied a non-destructive assay to test for the localization of AAC, that is the accessibility to antibodies (we previously showed that AAC accumulated at the surface receptors (cis side of the outer membrane; no treatment with protease) was, as expected, accessible to antibodies (28)).
Deenergized mitochondria with AAC accumulated at stage III were treated with proteinase K at a low concentration (10 g/ml) to remove surface-bound preproteins. The mitochondria were reisolated, and the intermembrane space was opened. Then antibodies specifically directed against the AAC were added. After an incubation, antibodies that were not bound to the mitochondria were removed by reisolation of the mitochondria. Then the mitochondria were lysed in Triton X-100-containing buffer, and immunocomplexes were harvested by protein A-Sepharose. By this procedure we found that the accumulated AAC was efficiently recognized by the antibodies upon opening of the intermembrane space (Fig. 1A, upper panel, lane 2), while it was not accessible to the antibodies in intact mitochondria (Fig. 1A, upper panel, lane 1). The lack of immunoprecipitation from intact mitochondria excludes that antibodies added prior to lysis and reisolation of mitochondria became active in immunoprecipitation after the lysis. The total amount of immunoprecipitable AAC was then determined by addition of antibodies after the lysis of the mitochondria (Fig.  1A, middle panel, lanes [1][2][3][4]. AAC completely imported into the inner membrane in the presence of a ⌬ was not accessible to antibodies even after swelling (Fig. 1A, upper panel, lanes 3 and 4); it was precipitated only by antibodies added after lysis (Fig. 1A, middle panel, lanes 3 and 4). We conclude that AAC accumulated at stage III is exposed to the intermembrane his3-⌬200 leu2-⌬1 ura3-52 trp1-⌬63 tom22::HIS3 ϩ pRS414(TRP)-tom22- 2 39 space such that entire antibody molecules can efficiently bind to it.
To assess the localization of AAC accumulated in deenergized mitochondria, two assays were applied, treatment at alkaline pH and sonication. A treatment of the mitochondria at pH 11.5 (sodium carbonate) releases soluble proteins and peripheral membrane proteins (bound by protein-protein interactions) to the supernatant and leaves integral membrane proteins (protein-lipid interaction) in the membrane sheets (32,33). This treatment released the AAC (stage III) to the supernatant (Fig. 1B, column 2), while AAC completely imported into the inner membrane in the presence of a ⌬ (stage V) remained membrane-integrated (Fig. 1B, column 3) (11,27). To test if the AAC accumulated at stage III was soluble in the intermembrane space or still membrane-bound, the mitochondria were sonicated (29), and soluble proteins and membrane vesicles were separated. The AAC was found to be stably associated with the membrane vesicles (Fig. 1B, column 1). Together with the release at pH 11.5, this result suggests that the AAC at stage III is bound to the membranes by protein-protein interactions.
Is the AAC intermediate still in contact with the mitochondrial outer membrane? Deenergized mitochondria with accumulated AAC (Fig. 1C, upper panel, lane 1) were treated with proteinase K (150 g/ml), leading to the generation of fragments of AAC that were 2-4 kDa smaller than the full-length protein (Fig. 1C, lower panel, lane 1). Since we will show below that the AAC molecules giving rise to these fragments can be quantitatively chased to the fully imported form, they represent true translocation intermediates at stage III and are henceforth referred to as AAC III . The proteinase K treatment degraded surface-exposed components of the protein import machinery, such as Tom20 (Fig. 1C, upper panel, lane 6), but did not affect the outer membrane barrier, as evidenced by the full protection of the intermembrane space protein cytochrome b 2 (Fig. 1C, lower panel, lane 6) and of AAC transported into the inner membrane (Fig. 1C, lower panel, lane 2) (31). After opening of the intermembrane space by swelling, AAC III was no longer protected against the protease but completely degraded (Fig. 1C, lower panel, lane 3). We did not observe fragments that could be resolved by the SDS-PAGE suggesting that AAC at stage III was exposed to the intermembrane space with major portions.
In parallel reactions, we imported the precursor of AAC into energized mitochondria (stage V) (Fig. 1C, lane 2). A treatment with proteinase K did not lead to generation of AAC III (Fig. 1C, lower panel, lane 2). After swelling of the mitochondria, however, the proteinase K cleaved off a small piece (ϳ1 kDa) from the imported AAC (Fig. 1C, lower panel, lane 4). This fragmentation in swollen mitochondria is indicative of fully imported AAC (21,24,30) and is thus referred to as AAC V .
We conclude that AAC at stage III is mainly exposed to the intermembrane space but remains in stable contact with the outer membrane and exposes a small segment to the cytosolic side.
AAC Accumulated at Stage III Is Quantitatively Chased to the Mature Form-We asked if AAC giving rise to AAC III represented a true translocation intermediate, i.e. if the accumulated AAC could be chased to the fully imported form after regeneration of a ⌬. The 35 S-labeled precursor of AAC was accumulated in deenergized mitochondria (dissipation of ⌬ by valinomycin in the presence of potassium in the incubation buffer) with three different incubation times. In a first set, the accumulated AAC was directly analyzed (Fig. 2, lanes 1-6): a treatment of the mitochondria with proteinase K generated AAC III (Fig. 2, lanes 1-3), whereas after swelling proteinase K FIG. 1. The ADP/ATP carrier (AAC) accumulated in deenergized mitochondria (stage III) is associated with the outer membrane and exposed to the intermembrane space. A, accessibility of AAC to antibodies. Reticulocyte lysate with 35 S-labeled AAC was incubated with isolated S. cerevisiae mitochondria in the presence or absence of a membrane potential ⌬ for 10 min. The samples were treated with 10 g/ml proteinase K. The reaction was stopped by the addition of 1 mM phenylmethylsulfonyl fluoride. The samples were split. One aliquot was directly incubated with antiserum directed against AAC, in the other aliquot the mitochondria were first swollen and then incubated with antibodies. After reisolation, mitochondria were lysed in Triton X-100-containing buffer. After a clarifying spin, protein A-Sepharose was added, and antibody-AAC complexes were harvested and analyzed by SDS-PAGE and autoradiography (upper panel). The supernatant (after removal of the protein A-Sepharose by centrifugation) was added onto new protein A-Sepharose that had been preincubated with anti-AAC serum, representing the material where antibodies were added after lysis of mitochondria (middle panel). Total represents mitochondria that were lysed after the import reaction and then directly incubated with protein A-Sepharose carrying prebound anti-AAC antibodies (lower panel). B, AAC at stage III is membranebound, but extracted at alkaline pH. AAC was accumulated at the outer membrane in the absence of a ⌬ for 10 min at 25°C. The mitochondria were then subjected to sonication in the presence of 200 mM NaCl, followed by separation of membrane pellet and supernatant (sample 1). For alkaline extraction, mitochondria with AAC accumulated at stage III (Ϫ⌬; sample 2) or fully imported AAC (stage V; ϩ⌬; sample 3) were incubated with Na 2 CO 3 and separated into pellet and supernatant as described under "Materials and Methods." The fraction of AAC remaining in the pellet is shown. C, AAC at stage III exposes a short segment to the cytosolic side of mitochondria and is thus in contact with the outer membrane. 35 S-Labeled AAC was either accumulated in isolated mitochondria in binding buffer without a ⌬ or imported to its final location in the inner membrane in the presence of a ⌬ for 15 min at 25°C. Each sample was split into four aliquots. In one aliquot, the mitochondria were reisolated and analyzed by SDS-PAGE and digital autoradiography (sam- ples 1 and 2, upper panel). In the second aliquot, the mitochondria were treated with 150 g/ml proteinase K (samples 1 and 2, lower panel). In the third aliquot, the mitochondria were reisolated and swollen, and the resulting mitoplasts were analyzed (samples 3 and 4, upper panel). In the fourth aliquot, mitoplasts were generated, treated with 50 g/ml proteinase K, and then analyzed (samples 3 and 4, lower panel). As a control, mitochondria or mitoplasts that were treated as described above were blotted onto nitrocellulose and immunodecorated with antibodies against the marker proteins Tom20 or cytochrome b 2 (Cyt. b 2 ) (samples 5-7). AAC III , proteinase K-generated fragments of AAC accumulated at stage III (mitochondria); AAC V , proteinase K-generated fragment of AAC fully imported into the inner membrane (stage V; mitoplasts). completely degraded AAC III ; no fully imported AAC was detected (Fig. 2, lanes 4-6). In a second set (Fig. 2, lanes 7-12), the mitochondria with accumulated AAC were reisolated and were subjected to a second incubation in the presence of a ⌬ by generating a potassium diffusion potential (valinomycin in the absence of potassium in the buffer (27,34,35)). By this "chase incubation," the AAC III completely disappeared (non-swollen mitochondria; Fig. 2, lanes 7-9). After swelling of the mitochondria, proteinase K did not degrade the AAC, but led to removal of a small fragment in a considerable fraction of the AAC (Fig.  2, lanes 10-12), i.e. AAC V was generated. This demonstrates that AAC accumulated at stage III (AAC III ) was efficiently chased to the fully imported form.
Does the chase reaction involve the function of surface-exposed (cis) components of the protein import machinery of the outer membrane? We took advantage of the observation that AAC accumulated at stage III is resistant to a treatment of mitochondria with trypsin under conditions that lead to a removal of the cytosolic domains of the surface receptors Tom20, Tom22, and Tom70 (13,30,31). Therefore, in a third set of samples, the AAC was accumulated in deenergized mitochondria, followed by a trypsin treatment (Fig. 2, lanes 13-18). Then the mitochondria were reisolated and subjected to the chase incubation (generation of a ⌬). Lanes 13-18 of Fig. 2 demonstrate that the AAC accumulated at stage III was efficiently chased to the fully imported form (AAC V ), also after removal of the cytosolic domains of Tom20, Tom22, and Tom70.
Accumulation of AAC III Requires Tom7, but Not the Intermembrane Space Domain of Tom22-We asked if other components of the Tom machinery were involved in the accumulation of AAC III . Two small Tom proteins have been identified that modulate the dynamics of the outer membrane translocase. Studies with deletion mutants of yeast showed that Tom6 (36) promotes association of components of the Tom machinery (37) whereas Tom7 supports a dissociation of the Tom machinery (27). tom6⌬ mitochondria were able to generate AAC III (Fig.  3A, upper panel, lane 1) that was degraded by proteinase K after swelling of the mitochondria (Fig. 3A, lower panel, lane 1). In contrast, with tom7⌬ mitochondria no AAC III was observed (Fig. 3A, upper panel, lane 2). The tom7⌬ mitochondria were not unspecifically damaged since they were still able to import AAC in the presence of a ⌬, including formation of the fragment AAC V (Fig. 3A, lower panel, lane 6), although with a reduced efficiency (27). Moreover, proteinase K formed fragments of AAC in swollen mitochondria after import in the presence of a ⌬ that were 5-7 kDa smaller than full-length AAC (AAC IV ; Fig. 3A, lower panel, lane 6). These fragments, indicating incomplete insertion of AAC into the inner membrane, were found in only small amounts in wild-type and tom6⌬ mitochondria (Fig. 3A, lower panel, lane 5; Fig. 3B, columns 1 and 2), but were significantly more abundant in tom7⌬ mitochondria (Fig. 3A, lower panel, lane 6; Fig. 3B, column 3). This result supports an impairment of tom7⌬ mitochondria in import of AAC.
Tom6 and Tom7 function in an antagonistic manner in modulating the dynamics of the Tom machinery (27). If the inhibitory effect of a lack of Tom7 on the generation of AAC III was due to the role of Tom7 in dissociation of the Tom machinery, mitochondria from a double deletion strain tom6⌬ tom7⌬ (27) should be able to generate some AAC III intermediate. Lane 3 of Fig. 3A (upper panel) shows that this was indeed the case. We conclude that Tom7 is required for generation of AAC III in a manner antagonistic to the function of Tom6. Moreover, in the presence of a ⌬, the formation of the AAC IV fragments, indicative of incomplete insertion into the inner membrane, was reduced in tom6⌬ tom7⌬ mitochondria (Fig. 3A, lower panel, lane 7; Fig. 3B, column 4) compared with tom7⌬ mitochondria (Fig. 3, lower panel, lane 6; Fig. 3B, column 3).
The intermembrane space domain of Tom22 is critical for accumulation of presequence-containing preproteins at a trans site of the outer membrane, as shown with mutant mitochondria (tom22-2) selectively lacking this domain (38,39). We asked if the formation of AAC III required the same component at the intermembrane space side. tom22-2 mitochondria were not impaired in generation of AAC III (Fig. 3, upper panel, lane  4), suggesting that the trans sites for presequence-containing preproteins and AAC are different.
To obtain independent evidence for a difference in trans site accumulation, we assayed for a further characteristic of import of presequence-containing preproteins. The productive accumulation of a cleavable preprotein at the trans site of the outer membrane, determined by accumulation in the absence of a ⌬ and subsequent chase to the imported form (two-step import), is only partially resistant to a treatment of mitochondria with salt, i.e. is resistant to 100 mM NaCl, but inhibited at higher salt concentration; at 200 mM NaCl, the productive accumulation of a cleavable preprotein is blocked (39). We showed above that AAC accumulated at stage III remains membrane-associated during sonication at 200 mM NaCl, suggesting a difference FIG. 2. AAC III is chased to the fully imported form. Radiolabeled AAC was accumulated in isolated mitochondria in the absence of a ⌬ at 25°C for the times indicated as described under "Materials and Methods." In the upper panel, mitochondria were reisolated and either directly treated with proteinase K or first swollen (see "Materials and Methods") and then treated with proteinase K, followed by analysis by SDS-PAGE and digital autoradiography. In the middle panel, the reisolated mitochondria were subjected to a second incubation (inc.) for 20 min at 25°C in the presence of a ⌬ (Chase), followed by the same treatment and analysis as described for the upper panel. In the lower panel, AAC was first accumulated at the outer membrane by dissipation of the membrane potential (1st inc.). The mitochondria were then treated with 20 g/ml trypsin to remove the cytosolic domains of the receptors. After reisolation of the mitochondria, the AAC was chased to the fully imported form and treated as described for the middle panel (2nd inc.).
to cleavable preproteins. To directly determine if the salt-resistant AAC represented a productive intermediate, we assayed for the two-step import of AAC at distinct salt concentrations. Mitochondria with AAC accumulated in the absence of a ⌬ were reisolated and treated with NaCl up to concentrations of 400 mM. A first set of samples was treated with proteinase K to demonstrate the formation of AAC III (Fig. 4, lanes  1-4) that was accessible to the protease in swollen mitochondria (Fig. 4, lanes 5-8). A second set of samples was subjected to the chase reaction (generation of ⌬). Thereby, the accumulated AAC was fully imported, evidenced by the formation of AAC V , independently of the concentration of NaCl during the salt treatment (Fig. 4, lanes 13-16). Therefore the productive accumulation of AAC is clearly different from that of presequencecontaining preproteins.
Opening of the Intermembrane Space Inhibits the Import of AAC, but Not of a Cleavable Preprotein-After swelling of mitochondria, cleavable preproteins have been shown to be directly translocated across the inner membrane without a requirement for the Tom machinery of the outer membrane (40,41). We compared the import of the precursor of F 1 -ATPase subunit ␤ (F 1 ␤) to that of AAC into mitoplasts. To prevent the use of the Tom machinery, mitochondria were pretreated with trypsin to remove the surface receptors. Thereby the import of both F 1 ␤ and AAC into non-swollen mitochondria was strongly inhibited (Fig. 5, lower panel, lanes 2 and 8; compare with lanes  1 and 7 (no trypsin treatment)). After swelling of the trypsintreated mitochondria, the import of F 1 ␤ was restored to a large extent, demonstrating direct translocation across the inner membrane in a ⌬-dependent manner (Fig. 5, lanes 3). In contrast, the import of AAC into the mitochondria was still strongly inhibited (Fig. 5, lower panel, lane 9).
Two possibilities are conceivable to explain this import inhibition of AAC, the lack of interaction with the outer membrane machinery or a loss of intermembrane space function required for import of AAC. To distinguish between the possibilities, non-trypsinized mitochondria were subjected to swelling and subsequently incubated with preproteins. The import of F 1 ␤ was very efficient (Fig. 5, lanes 5), whereas the import of AAC was still inhibited (Fig. 5, lower panel, lane 11). Under these conditions, the AAC can use the intact Tom machinery, but is still unable to translocate into the inner membrane. We con- FIG. 3. The generation of AAC III requires Tom7, but not the intermembrane space domain of Tom22. A, mitochondria were isolated from S. cerevisiae strains lacking Tom6 (tom6⌬), Tom7 (tom7⌬), Tom6 and Tom7 (tom6⌬ tom7⌬), or the intermembrane space domain of Tom22 (tom22-2), respectively. 35 S-Labeled AAC was either accumulated in the absence of a ⌬ or was imported in the presence of a ⌬ for 15 min at 25°C. After the binding or import reaction, the mitochondria were either directly treated with proteinase K or were swollen and then treated with proteinase K. The mitochondria/mitoplasts were reisolated and analyzed by SDS-PAGE and digital autoradiography. AAC IV , fragments of not fully imported AAC (ϩ⌬) generated by a proteinase K treatment of mitoplasts. B, the experiment was performed as described for A (ϩ⌬, ϩSwelling) with the following modifications. Wild-type mitochondria were used instead of tom22-2 mitochondria. The amounts of AAC IV and AAC V were quantified by digital autoradiography, and the ratio is shown (averages of at least three independent experiments).

FIG. 4. The productive accumulation of AAC is not inhibited by salt.
In a first incubation, AAC was accumulated in isolated yeast wild-type mitochondria in the absence of a ⌬ for 15 min at 25°C. After reisolation of the mitochondria, the samples were incubated in binding buffer described under "Materials and Methods" supplemented with different concentrations of sodium chloride as indicated for 15 min at 4°C. Each sample was split into three aliquots, respectively, of which the third contained double the volume. One aliquot was directly treated with proteinase K, and the mitochondria were reisolated and analyzed by SDS-PAGE and digital autoradiography. In the second aliquot, the mitochondria were swollen (see "Materials and Methods"), and the resulting mitoplasts were treated with proteinase K. The third aliquot was incubated in chase buffer (presence of a ⌬) for 20 min at 25°C and was then split into two halves. One half was directly treated with proteinase K; in the other half, the mitochondria were first swollen and then treated with the protease. clude that opening of the outer membrane by swelling leads to a loss of an intermembrane space function that is essential for import of AAC. DISCUSSION We have identified a translocation intermediate of the AAC during transfer of the preprotein across the yeast mitochondrial outer membrane and passage into the intermembrane space. Thereby the first detailed characterization of the import stage III of a mitochondrial metabolite carrier, i.e. the transport pathway between the recognition of the preprotein at the outer membrane surface (stage II) and the insertion into the inner membrane (stage IV), has been possible (Fig. 6). Previous work has indicated that stage III intermediates, accumulated in the absence of a ⌬, are protected against trypsin added to the isolated mitochondria (11)(12)(13)31) but become accessible to the protease after opening of the intermembrane space (30). However, most characteristics of the stage III intermediates have been unknown, such as the topology, the dependence on Tom components, and the relation to the import pathway of presequence-containing preproteins.
The stage III intermediate of AAC is stably membranebound, although not as integral membrane protein, but apparently by protein-protein interactions. The preprotein exposes major parts to the intermembrane space such that entire antibody molecules have access to the preprotein after swelling of mitochondria. A striking observation is the cleavage of the stage III intermediate by proteinase K (150 g/ml added to non-swollen mitochondria) to a few fragments of closely related length, termed AAC III (2-4 kDa smaller than full-length AAC). This demonstrates that the stage III intermediate spans the outer membrane and exposes a short segment to the cytosolic side. In a chase reaction after establishing a ⌬, all AAC molecules that can give rise to the formation of AAC III are quantitatively chased to the fully imported form. This remarkably high chase efficiency demonstrates that AAC III is indicative of a true translocation intermediate. For the accumulation of the stage III intermediate, the ⌬ across the inner membrane is selectively dissipated while the other known requirements for import, in particular the presence of ATP, are not impaired. The formation of the AAC III fragments shows that the stage III intermediate is still spanning the outer membrane, demonstrating that the prevention of AAC insertion into the inner membrane inhibits the complete translocation across the outer membrane. This suggests a coupling between the completion of outer membrane translocation and the ⌬-dependent insertion into the inner membrane.
Tom7, a small subunit of the outer membrane translocase, has been shown to support a dissociation of the Tom machinery (27). We find here that Tom7 is important for the generation of AAC III . Tom6 functions in an antagonistic manner to Tom7, in that Tom6 promotes an association of components of the Tom  5. Swelling of mitochondria inhibits the import of AAC, but not of a cleavable preprotein. Where indicated, isolated yeast wild-type mitochondria were pre-treated with trypsin to remove the import receptors at the outer membrane surface and/or were subjected to osmotic shock to disrupt the outer membrane and form mitoplasts (30,31). Reticulocyte lysates containing the 35 S-labeled precursors of F 1 ␤ and AAC were incubated with the mitochondria/mitoplasts in the presence or absence of a ⌬. The import reactions were carried out in isotonic import buffer at 25°C for 10 min. The samples were subsequently cooled on ice and split into halves, one of each was treated with proteinase K. After addition of phenylmethylsulfonyl fluoride, the mitochondria were reisolated by centrifugation and analyzed by SDS-PAGE. The amount of protease-protected mature-sized F 1 ␤ or AAC was quantified. The import efficiency into non-trypsinized and non-swollen mitochondria in the presence of a ⌬ was set to 100% (control), respectively. p, precursor protein; m, mature protein after cleavage of the presequence. machinery (27,37). Indeed, a lack of Tom6 suppresses the inhibitory effect of a lack of Tom7 on the formation of AAC III . These findings suggest that the formation of AAC III requires a dynamic behavior (dissociation) of the Tom machinery.
Interestingly, a lack of Tom7 only partially inhibits the transport of AAC into the inner membrane when a ⌬ is present during the whole import reaction (this retardation of translocation can be monitored by fragments, 5-7 kDa smaller than full-length AAC, generated by proteinase K in mitoplasts and termed AAC IV (see Figs. 3 and 6)), while in the absence of a ⌬ the formation of AAC III is almost completely inhibited. The pulling force provided by ⌬ on the preprotein (35) may promote a continuation of the import process despite a slow-down of the translocation reaction due to a higher rigidity of the outer membrane translocase. In the absence of a ⌬, the translocation across the outer membrane cannot be completed and thus the dynamic behavior of the Tom machinery (dissociation by Tom7) seems to be crucial for the accumulation of sufficient quantities of AAC preproteins.
A major question is if and where the import route of metabolite carriers diverges from that of cleavable preproteins. Both import pathways join for preprotein insertion into the general import pore (Tom20/22, Tom5 (18 -20, 42)). The trans site at the outer membrane for cleavable preproteins is marked by the intermembrane space domain of Tom22 that is required for productive accumulation of preproteins (38,39). AAC III has been translocated to the trans side of the outer membrane with major portions; however, its generation is not impaired in mutant mitochondria lacking the intermembrane space domain of Tom22 (tom22-2). To obtain independent evidence for the existence of distinct trans sites for cleavable preproteins and AAC, the salt resistance of accumulated preproteins was analyzed. Cleavable preproteins accumulated at the trans site are released by 200 mM salt (38,39), whereas AAC accumulated at stage III was resistant to salt concentrations up to 400 mM and could subsequently be chased to the fully imported form. Together with the efficient import of AAC into tom22-2 mitochondria (39) it is evident that the accumulation of AAC at the trans side of the outer membrane occurs by a mechanism distinct from that of cleavable preproteins. We conclude that the import route of AAC separates early from the import pathway of cleavable preproteins, i.e. already at the intermembrane space side of the outer membrane.
Are the import pathways also distinct with regard to functions of the intermembrane space? We used the observation by Schatz and colleagues (40,41) that cleavable preproteins can be imported into swollen mitochondria that have lost their intermembrane space content. Thereby the preproteins can circumvent the import machinery of the outer membrane and are efficiently imported even after removal of the Tom receptors by protease treatment. We find that the AAC shows a different behavior. The import of AAC into swollen mitochondria is strongly inhibited. Even when the translocation across the outer membrane is not impaired (by omitting the proteolytic removal of Tom receptors), the import of AAC into mitochondria with opened intermembrane space is strongly decreased compared with that into non-swollen mitochondria. These results thus show a fundamental difference between AAC and cleavable preproteins with regard to a need for intermembrane space function/components. While numerous components of the mitochondrial protein import machinery have been identified in the outer membrane and inner membrane, little has been known about import components in the intermembrane space. Schweyen and colleagues (43,44) identified two essential intermembrane space proteins, Mrs5p and Mrs11p (now termed Tim12 and Tim10), implicated to function in mitochondrial biogenesis. It was found very recently that both proteins are required for import of carrier proteins, but not of cleavable preproteins (45,46). A loss of these intermembrane space proteins, in particular Tim10, by swelling of mitochondria 2 can explain the striking difference of import of cleavable preproteins and AAC into mitoplasts.
The implication derived from characterization of AAC III that the import pathways of cleavable preproteins and carrier proteins diverge early is thus supported by the differential requirement for intermembrane space components. We conclude that AAC and cleavable preproteins use common components for insertion into the general import pore of the outer membrane, but that already at the exit of the general import pore the pathways separate and probably remain distinct for transfer through the intermembrane space and insertion into the inner membrane.