Functional Cooperation and Stoichiometry of Protein Translocases of the Outer and Inner Membranes of Mitochondria*

The qualitative relationship between preprotein translocases in the mitochondrial outer and inner membranes was determined by both a functional analysis and a determination of characteristic components of the translocases. Translocation contact sites of isolated mitochondria were saturated with intermediates of a matrix-targeted precursor of the β-subunit of the F1-ATPase (pF1β), and import of preproteins into the different mitochondrial subcompartments was monitored. A strong inhibition (75–95%) was observed for preproteins with an N-terminal matrix targeting signal, indicating that a significant portion of the contact sites was blocked by accumulated F1β. Insertion of preproteins into the outer membrane and import into the intermembrane space of preproteins without matrix targeting signals was inhibited by about 45%, indicating that functional outer membrane translocases were available despite saturation of contact sites. Similarly, import of members of the mitochondrial carrier family into the inner membrane was only partly inhibited (40–50%), demonstrating that functional Tim22 translocases were available to cooperate with the Tom machinery in the import of carrier proteins. The stoichiometry of Tom40, Tim23, and Tim22 in mitochondria was determined to be 5:1:0.22. We conclude that translocases of the outer membrane are present in excess over translocases of the inner membrane.

Import of nuclear encoded mitochondrial preproteins into the mitochondria is a multistep process (1). To reach the mitochondrial matrix, precursor proteins are translocated across the mitochondrial outer and inner membranes. Biochemical and morphological studies revealed that protein import into the matrix proceeds through proteinaceous channels at so-called translocation contact sites (2)(3)(4)(5)(6)(7). In the electron microscope, regions of close proximity of the mitochondrial outer membrane to the inner membrane have been observed (5). These areas are particularly conspicuous in mitochondria in which the matrix space has been shrunk and the inner membrane is pulled away from the outer membrane. They are often referred to as morphological contact sites. Contact sites may be the preferred location where translocation contacts of the Tom and Tim com-plexes are formed, but this issue has not been finally settled. With biochemical means, contact sites between Tom and Tim complexes were only detected in the presence of a translocating polypeptide chain (6,7). Components of the Tom machinery and Tim23, Tim17, as well as mt-Hsp70 and Tim44 were found in such super-complexes.
For import of members of the mitochondrial carrier family, such as the ADP/ATP carrier, the Tom machinery cooperates with Tim22 (8). This pathway does not require functional Tim23 and mt-Hsp70 (8). The translocation machinery of the outer membrane can also act independent of the translocases of the inner membrane (9). Similarly, in mitoplast preparations, preproteins are imported directly via Tim23 into the matrix (10). This raises the question of how the Tom machinery interacts with the inner membrane translocases Tim23⅐Tim17 and Tim22 and how the translocases are related stoichiometrically.
In this study, chemical amounts of a matrix-targeted precursor protein were incubated with isolated mitochondria under conditions optimal for import. The effect of accumulation of this precursor in a membrane-spanning fashion on the subsequent import of preproteins into the different mitochondrial subcompartments was analyzed. In this situation, import of preproteins into the matrix is blocked. The results demonstrate that translocation contact sites for import of preproteins into the matrix can be saturated. However, functional Tom machinery is still available for import of proteins into the outer membrane and the intermembrane space, suggesting that Tom complexes are in excess over Tim23⅐Tim17 complexes. Tom machinery is also available for cooperation with Tim22 to facilitate import of members of the mitochondrial carrier family into the inner membrane. A quantification of the mitochondrial content of Tom40, Tim23, and Tim22 supports the notion that translocases of the mitochondrial outer membrane are present in excess over inner membrane import machinery. The data suggest that a preprotein associates with the Tom complex and then recruits the appropriate machinery required for its sorting to mitochondrial subcompartments.

EXPERIMENTAL PROCEDURES
Preparation of Radiolabeled pF 1 ␤-The c-DNA encoding the precursor of the ␤-subunit of the F 1 F 0 ATPase (pF 1 ␤) from Neurospora crassa (11) was modified as follows. An NdeI restriction site was introduced in front of the start codon by oligonucleotide-directed site-specific mutagenesis using the polymerase chain reaction. The resulting DNA fragment was ligated into pJLA503 (12), which was cleaved with NdeI and EcoRI. The plasmid was used for transformation of Escherichia coli DH1.
A single colony of the transformed E. coli strain was grown overnight at 28°C in M9 minimal medium (0.4% (w/v) glucose, 0.5% NaCl, 6% Na 2 HPO 4 , 3% KH 2 PO 4 , 1% NH 4 Cl, 1 mM Mg 2 Cl, 1 mM CaCl 2 , 1 mg/liter thiamin) containing 0.4% (w/v) casamino acids and 100 mg/liter ampicillin. This culture was diluted to an A 578 of 0.1 into M9 medium containing 40 mg/liter of each amino acid (except methionine and cysteine), 25 M MgSO 4 , and 50 mg/liter ampicillin. At A 578 ϭ 1.0, the cells were reisolated in the same volume M9 medium (containing 40 mg/liter of each amino acid except methionine and cysteine), prewarmed to 42°C, and supplied with 50 Ci of [ 35 S]sulfate at a final concentration of 10 M. Incubation at 42°C was continued for 2 h.
Cells were reisolated in 2% of the original volume in buffer A (25% (w/v) sucrose, 50 mM Tris-HCl, pH 8.0), 1 mg/ml lysozyme and incubated for 30 min at room temperature. Then, the volume was adjusted to 20% of the original culture volume with buffer A supplied with 25 mM EDTA, 1% (w/v) Triton X-100, 10 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. The cells were sonicated in a Branson sonifier (3 ϫ 10 pulses, 40% duty, maximal setting), and inclusion bodies of pF 1 ␤ were pelleted by centrifugation for 30 min at 40,000 ϫ g. The pellet was resuspended by sonication in buffer B (20 mM Tris, pH 7.5, 1 mM EDTA, 10 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.1% Triton X-100) and centrifuged as above. Finally, the pellet was washed twice as above in buffer B lacking Triton X-100. The final pellet was solubilized in buffer C (7 M urea, 10 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 30 mM histidine, pH 6.0). The pF 1 ␤ protein was purified from these inclusion bodies by chromatography on DEAE-Sepharose equilibrated with buffer C. The column was developed with a gradient of buffer C from 0 mM to 200 mM NaCl. The protein eluted at 50 mM NaCl.
In Vitro Synthesis of Precursor Proteins and Import into Mitochondria-Precursor proteins were synthesized by coupled transcription/ translation in reticulocyte lysate in the presence of [ 35 S]methionine (Amersham) as described (13). Wild-type Saccharomyces cerevisiae strain MC3:YIPGALOTC (14) was grown overnight in lactate medium, cells were harvested at A 578 ϭ 1-1.5, and mitochondria were isolated (15).
Import reactions were carried out at 25°C in 200 l of import buffer (600 mM sorbitol, 1 mg/ml bovine serum albumin, 50 mM KCl, 10 mM MgCl 2 , 2.5 mM EDTA, 2 mM KH 2 PO 4 , 2 mM ATP, 5 mM NADH, 50 mM HEPES, pH 7.2) containing 20 g of mitochondria and 5-10% reticulocyte lysate with radiolabeled precursor. Where indicated, the membrane potential was dissipated with 50 M carbonyl cyanide p-chlorophenylhydrazone. Import was assayed by treating the mitochondria with 100 g/ml proteinase K for 15 min on ice, and samples were analyzed by SDS-PAGE 1 and fluorography.
Immunoprecipitation-Yeast cells were grown overnight at 30°C in the presence of [ 35 S]sulfate (300 mCi/mol). Mitochondria were prepared, and aliquots corresponding to 50 g of protein were solubilized in TBS containing 0.05% SDS and 0.5% Triton X-100. After a clarifying spin, the supernatant was subjected to immunoprecipitation with anti-Tom40 IgG (10 g), with affinity-purified anti-Tim23 IgG (0.5 g) and affinity-purified anti-Tim17 IgG (0.5 g), respectively. Immunoprecipitates were analyzed by SDS-PAGE and quantified with a phosphorimaging system.

Accumulation of Translocation Intermediates in a Membrane-spanning Fashion-
The precursor of the ␤-subunit of the mitochondrial ATP synthase (pF 1 ␤) (11) (0.1 g) was diluted out of urea into an import assay containing S. cerevisiae mitochondria (30 g). When the import reaction was carried out for 30 min at 10°C, the preprotein was efficiently processed to the mature form; however, only about one-third of the processed species was resistant to externally added proteinase K (PK) (Fig. 1). This indicates that the kinetics of translocation of the polypeptide chain were slow at low temperature, and a significant fraction of the protein was present in a membrane-spanning configuration, with the N terminus processed in the matrix and the C terminus on the outside and accessible to externally added PK. To saturate the mitochondrial import sites with membrane-spanning translocation intermediates, import reactions with pF 1 ␤ were performed at 10°C, and antibodies directed against the mature F 1 ␤ (mF 1 ␤) were added. The amount of precursor recovered with the mitochondrial pellet increased with increasing amounts of anti-F 1 ␤ IgG. This may reflect cross-linking of the preprotein by the antibody. In the presence of 10 -100 g of anti-F 1 ␤ IgG, about 30 ng of F 1 ␤ were processed. This was slightly more than without IgG, sug-gesting that the antibodies might help to maintain the preprotein in a translocation-competent form after dilution out of denaturant. However, the majority of the processed form was degraded by added PK, indicating that the protein was arrested in a membrane-spanning fashion. In the presence of 100 g of anti-F 1 ␤ IgG, more than 95% of the processed form were present as a membrane-spanning translocation intermediate. About 0.5 g of F 1 ␤ per mg of mitochondrial protein was arrested in a membrane-spanning fashion. Assuming all mitochondria are active in translocation, this corresponds to approximately 9 pmol of import sites per mg of mitochondrial protein. The arrested translocation intermediate remained sensitive to PK treatment for at least 2 h, indicating that the membrane-spanning F 1 ␤ was arrested in a stable manner (not shown).
Second Stage Import of Preproteins into Mitochondria with Arrested Translocation Intermediates-We investigated whether the accumulation of F 1 ␤ across the membranes blocked subsequent import of mitochondrial precursor proteins into the different mitochondrial subcompartments. In a first stage import reaction, mitochondria were saturated with the F 1 ␤ translocation intermediates in the presence of anti-F 1 ␤ IgG. Control reactions received pF 1 ␤ together with preimmune IgG. Subsequently, mitochondria were reisolated and used in a second stage import reaction at 25°C.
Second stage import of radiochemical amounts of the matrixtargeted chimeric preprotein pb 2 ⌬19(167)DHFR (16) into mitochondria with accumulated F 1 ␤ protein was inhibited by more than 90% (Fig. 2), whereas import into mock-treated mitochondria was as efficient as into untreated mitochondria. Similarly, second stage import of the matrix-targeted fusion protein pSu9(1-69)DHFR (17) was efficiently inhibited. This indicates that accumulation of F 1 ␤ in a membrane-spanning fashion blocks the sites for protein import into the mitochondrial matrix.
The precursor of cytochrome b 2 and the precursor of cytochrome c 1 , which are sorted to the intermembrane space, are synthesized with a bipartite presequence. The N-terminal matrix-targeting signal is cleaved after import into the matrix by the matrix-processing peptidase before sorting of these proteins to the intermembrane space (18). Import of pb 2 (167)-DHFR, a chimeric preprotein that contains the first 167 amino acid residues of cytochrome b 2 was strongly inhibited by accumulated F 1 ␤ translocation intermediates (Fig. 2). Also, import of cytochrome c 1 was inhibited by more than 95%. The import of the precursor of the Rieske iron-sulfur protein (pFe/S), which becomes imported into the matrix, processed, and subsequently sorted to the intermembrane space (19), was inhibited by 85% after accumulation of F 1 ␤. Second stage import of the outer membrane protein porin and of cytochrome c heme lyase into the intermembrane space was inhibited by about 40% (Fig. 2). Both proteins are synthesized without cleavable presequence and imported via the Tom complex of the outer membrane (10,20). These data suggest that despite saturation of translocation contact sites for protein import into the matrix, functional Tom machinery is available for import of proteins into the outer membrane and the intermembrane space.
The integral inner membrane proteins ADP/ATP carrier (AAC) and inorganic phosphate carrier (PiC) are synthesized without a cleavable presequence. Their import into the inner membrane requires the membrane potential ⌬⌿ and is facilitated by Tim22 in a reaction that does not need functional Tim23 and mt-Hsp70 (8). When F 1 ␤ was accumulated in a membrane-spanning fashion, the subsequent import of AAC and PiC was inhibited by 40 and 50%, respectively.
These observations demonstrate that accumulation of F 1 ␤ in a membrane-spanning fashion efficiently blocks the import of other mitochondrial preproteins with an N-terminal matrixtargeting signal. Import of proteins, however, without matrixtargeting signals into the inner membrane, the intermembrane space, and into the outer membrane is only partly affected. Thus, functional outer membrane import sites seem to be present in excess over components required for the formation of translocation contact sites for protein import into the matrix. Outer membrane import sites that are not engaged in contact site formation are available for import of members of the mitochondrial carrier family via Tim22.
Quantification of Components of the Mitochondrial Protein Import Apparatus-To estimate the stoichiometry of mitochondrial protein translocases in the outer and inner membrane, Tom40 (21), a component of outer membrane import machinery, Tim23 (22), a component of the inner membrane translocase for matrix-targeted proteins, and Tim22 (8), a component for import of members of the mitochondrial carrier family, were quantified from immunoprecipitates. Yeast cells were metabolically labeled with [ 35 S]sulfate. Mitochondria were prepared and lysed with Triton X-100 and SDS, and the antigens were depleted from detergent extracts by immunoprecipitation. The immunoprecipitated antigens were then analyzed by SDS-PAGE and quantified with a phosphorimaging system (Fig. 3). , and mitochondria were prepared. They were lysed with 0.05% SDS and 0.5% Triton X-100. Detergent extracts corresponding to 12.5, 25, and 50 g of mitochondrial protein were used for immunoprecipitation with 0.5 g of affinity-purified anti-Tim23 IgG. A, immunoprecipitates were analyzed by SDS-PAGE and autoradiography. B, immunoprecipitated Tim23 was quantified with a phosphorimaging system. The phosphorimager signal obtained from 12.5 g of total mitochondrial protein was 1.1 ϫ 10 5 . C, molar ratio of Tom40:Tim23:Tim22 in mitochondria. Immunoprecipitation with anti-Tom40 IgG, affinity-purified anti-Tim23 IgG, and affinity-purified anti-Tim22 IgG were performed and quantified as described above. To obtain molar ratios of proteins, the phosphorimager signals were corrected for differences in radioactivity incorporated into Tom40 (10 35 S), Tim23 (9 35 S), and Tim22 (13 35 S). The amount of Tim23 was set equal to 1. spanning fashion specifically blocked subsequent import of preprotein with a matrix-targeting signal, whereas import of precursors without such a signal into the outer membrane, the intermembrane space, and into the inner membrane was only partly blocked. This suggests that Tim23⅐Tim17 complexes, which mediate translocation of matrix-targeted preproteins across the inner membrane, are less abundant than Tom complexes, which constitute the general insertion pore for preproteins in the outer membrane (23). We have determined that mitochondria contain about 85 pmol of Tom40, 17 pmol of Tim23, and 3.7 pmol of Tim22 per mg of mitochondrial protein.
The molar ratio of these components seems to be independent of the functional state of the mitochondria. Although the stoichiometry of the quantified components within functional Tom and Tim translocases is not known, it is likely that Tom complexes are in excess over Tim23⅐Tim17 complexes. The native molecular mass of the Tom complex is about 400 kDa (24,25). Thus, the Tom complex could contain about five copies of Tom40 if the other Tom components (26) are present in only single copies. Accordingly, mitochondria should contain at least 17 pmol of Tom complexes per mg of protein. The Tim23⅐Tim17 complex functions as a dimeric or higher oligomeric species (27), corresponding to 8.5 pmol/mg. Tom complexes should therefore be present in at least 2-fold molar excess over Tim17⅐23 complexes. In agreement with this estimation, saturation of contact sites between Tom complexes and Tim17⅐23 complexes by matrix-targeted pF 1 ␤ almost completely blocked subsequent protein import into the matrix. The remaining Tom complexes that are not engaged in formation of contact sites are obviously available for import pathways that are independent of Tim23⅐Tim17 complexes. These are pathways for import of proteins into the outer membrane and the intermembrane space as well as the pathway for import of proteins into the inner membrane via the Tim22 complex. Protein import via these pathways was reduced by about 40% when translocation contact sites were saturated by pF 1 ␤. This reduction could reflect an accumulation of a fraction of the precursors at receptors of Tom complexes that are blocked by membrane-spanning F 1 ␤.
In conclusion, the interaction of the Tom machinery with translocases in the inner membrane is highly dynamic: a preprotein initially binds to a Tom complex and then recruits the appropriate machinery required for its further import and submitochondrial sorting.