Article |
Address correspondence to Carla M. Koehler, Dept. of Chemistry and Biochemistry, UCLA, Box 951569, 607 Charles Young Dr. East, Los Angeles, CA 90095-1569. Tel.: (310) 794-4834. Fax: (310) 206-4038. E-mail: koehler{at}chem.ucla.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: protein import; TIM complex; Saccharomyces cerevisiae; mitochondria; protein translocation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Proteins targeted to the mitochondrial inner membrane require an adapted second pathway (Káldi and Neupert, 1998; Pfanner, 1998; Koehler et al., 1999b). Substrates of this pathway are the mitochondrial carrier family including the ADP/ATP carrier (AAC) (Palmieri et al., 1996), Tim22p, and Tim23p (Koehler et al., 1998a; Sirrenberg et al., 1998; Leuenberger et al., 1999); these precursors are synthesized without a cleavable NH2-terminal presequence. Instead, the targeting information resides within the mature protein (Pfanner et al., 1987; Davis et al., 1998; Endres et al., 1999). The carrier protein sequence is a tripartite repeat, in which each repeat consists of a pair of membrane-spanning domains connected by a matrix-sided loop. Each module contains targeting information that cooperates in binding to the receptor Tom70 such that three Tom70 dimers are recruited per precursor (Ryan et al., 1999; Wiedemann et al., 2001). The carrier then passes through the TOM pore in a loop formation. After passing through the TOM pore, the hydrophobic precursors are escorted through the intermembrane space by two different soluble 70-kD protein complexes consisting of either Tim9pTim10p or Tim8pTim13p (Adam et al., 1999; Koehler et al., 1998b, 1999a; Leuenberger et al., 1999; Davis et al., 2000; Murphy et al., 2001). By binding to the hydrophobic transmembrane segments of AAC, the Tim9pTim10p complex potentially prevents the precursor from aggregating while passing through the aqueous intermembrane space (Curran et al., 2002). Insertion into the inner membrane is mediated by the 300-kD TIM22 complex, consisting of Tim12p, Tim18p, Tim22p, Tim54p, and a small fraction of Tim9p and Tim10p (Kerscher et al., 1997, 2000; Koehler et al., 1998a, 1998b, 2000; Sirrenberg et al., 1998; Adam et al., 1999).
The members of the family of small Tim proteins (Tim8p, Tim9p, Tim10p, Tim12p, and Tim13p) share 25% identity and 50% similarity and contain a conserved twin CX3C motif (Koehler et al., 1999a, 1999b). Previous studies suggested that the twin CX3C motif might coordinate zinc and form a zinc finger-like structure to mediate substrate binding (Sirrenberg et al., 1998; Adam et al., 1999; Paschen et al., 2000). Recently, we have shown that the cysteine residues in the Tim9pTim10p complex seemingly form disulfide bonds and do not coordinate zinc (Curran et al., 2002). Tim8p assembles with Tim13p and Tim9p assembles with Tim10p to form distinct 70-kD complexes with different substrate specificity: Tim8pTim13p binds to Tim23p, whereas Tim9pTim10p binds to members of the carrier family, Tim17p and Tim22p (Endres et al., 1999; Leuenberger et al., 1999; Davis et al., 2000; Paschen et al., 2000). The function of the 70-kD complexes is not essential for viability but facilitates import across the intermembrane space (Murphy et al., 2001).
To determine if the translocation mechanism via a loop formation for the carrier family is general for polytopic inner membrane proteins, we have extended our studies to characterize the Tim8pTim13p complex and its role in mediating the import of Tim23p. Tim23p does not consist of the tripartite repeat like the carrier proteins. Instead, the NH2-terminal half of Tim23p forms a soluble domain in the intermembrane space and potentially inserts in the outer membrane (Donzeau et al., 2000), and the COOH-terminal half consists of four membrane spanning domains, which contains the mitochondrial targeting and membrane insertion signals (Davis et al., 1998, 2000; Káldi et al., 1998). We report that Tim23p follows an import pathway similar to that of the carrier proteins and propose that this is a general translocation mechanism for polytopic inner membrane proteins, thus enabling the small Tim proteins to bind to the hydrophobic membrane domains in the aqueous intermembrane space to prevent subsequent aggregation.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Recombinant and native Tim8pTim13p complex have similar properties
To study the mechanism by which the Tim8pTim13p complex mediates the import of Tim23p, we developed a method to purify the recombinant Tim8pTim13p complex from an Escherichia coli strain. Specifically, TIM8 and TIM13 with their own E. coli ribosomal binding site were cloned in tandem in an expression plasmid and transformed into the E. coli host BL21(DE3). Affinity tags were not utilized because Tim8p and Tim13p have a molecular mass near 10 kD; the addition of tags to the monomers would significantly increase their molecular mass and could interfere with assembly of the complex. The Tim8pTim13p complex was purified to homogeneity from an E. coli lysate by subsequent chromatography steps using an anion exchange column, followed by a cation exchange column and a gel filtration column. The Tim8pTim13p complex eluted at 6080 mM NaCl on ion exchange columns and eluted in the 6070 kD mass range from the gel filtration column; the complex was purified to 95% homogeneity as shown by SDS-PAGE and Coomassie staining (Fig. 2 A). The Tim8pTim13p complex seemingly assembled efficiently in the E. coli cytoplasm, because Tim8p and Tim13p were only detectable in a 70-kD complex when separated by blue native gel electrophoresis (Fig. 2 B). In addition, the recombinant complex and the Tim8pTim13p complex from an intermembrane space fraction migrated identically in a native gel, indicating that the two complexes were identical with regard to shape and charge (Fig. 2 C).
|
|
Because the native and recombinant complexes were seemingly identical, the physical properties of the recombinant Tim8pTim13p complex were examined in further detail. The secondary structure of the recombinant Tim8pTim13p complex was analyzed by circular dichroism spectroscopy (Fig. 4, top). Computational analysis of the spectra (curves a and c) between 195 and 250 nm showed that the Tim8pTim13p complex was composed of 70.7% alpha helix, 5.2% beta sheet, 10.7% turn, and 13.4% undetermined (Fig. 4, bottom) (Perczel et al., 1992; Sreerama and Woody, 1993). Under thermal denaturation conditions of 95°C (curve b), the proteins adapted a random coil confirmation as expected. When cooled back to 4°C the subunits refolded and reassociated into the native complex with similar secondary structure to the undenatured sample (Fig. 4, curve c). In addition, analysis of the refolded complex on blue native gels showed that Tim8p and Tim13p were only detectable in a 70-kD complex (unpublished data) and thus confirmed that refolding and reassembly were efficient.
|
The Tim8pTim13p complex does not coordinate zinc
The small Tim proteins contain the conserved twin CX3C motif, in which two cysteine residues are separated by three amino acids and the spacing between each CX3C is 1116 amino acids. (Koehler et al., 1999b) Previous studies have yielded conflicting results: the small Tim proteins as monomers coordinated zinc in a 1:1 molar ratio (Sirrenberg et al., 1998; Paschen et al., 2000; Rothbauer et al., 2001), but the assembled Tim9pTim10p complex did not contain zinc (Curran et al., 2002). We investigated the state of the cysteine residues using several techniques, including thiol-trapping assays, reductant and metal chelator sensitivity assays, inductively coupled plasmonatomic emission spectrometry (ICP-AE), and protein import assays.
Previously, we employed a thiol-trapping method that allowed the separation and visualization of oxidized and reduced species on Tricine gels to show that the cysteines residues in the Tim9pTim10p were occupied (Fig 5 A, schematic) (Jakob et al., 1999; Curran et al., 2002). The modification of Tim8p was followed by immunoblot analysis (Fig. 5 A). First, all accessible thiol groups in the intermembrane space fraction were alkylated with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS). AMS is a thiol-reactive reagent that alkylates cysteines residues, thereby adding 0.5 kD to the molecular mass of each thiol group. As a control, completely reduced Tim8p (pretreated with DTT; Fig. 5 A, lane 2) showed a slower mobility on tricine gels due to the addition of four AMS molecules to the four cysteine residues in Tim8p; this translated into a 2-kD change in molecular mass that was easily observed in the 8-kD protein. In contrast, completely oxidized Tim8p (pretreated with hydrogen peroxide; Fig. 5 A, lane 3) migrated at the same molecular weight as the untreated complex (Fig. 5 A, lane 4) because the sulfhydryl residues were oxidized and unreactive to AMS. When the sample was alkylated with AMS, Tim8p migrated identically to the oxidized sample; AMS was not bound toTim8p. To further confirm that sulfhydryls might form disulfide linkages, the experiment was modified as follows. All accessible thiol groups were first blocked irreversibly with iodoacetamide (IAA). After removal of the excess IAA, disulfide bonds present in the protein were reduced with DTT, followed by AMS alkyation to trap any free sulfhydryl groups (Fig. 5 A, schematic). With this modification, Tim8p in the sample that was first pretreated with DTT was not modified by AMS (Fig. 5 A, lane 5), because the cysteines residues were already reduced by DTT and subsequently blocked by IAA. But Tim8p in the sample that was first oxidized with hydrogen peroxide was modified by AMS (Fig. 5 A, lane 6), because IAA was not able to block the thiols in the first step. As expected, when the sample was alkylated with IAA (avoiding both reduction and oxidation), Tim8p migrated at a molecular weight identical to that of the oxidized Tim8p (Fig. 5 A, lane 7); four AMS molecules covalently bound to Tim8p. This observation thus indicates that the cysteine residues are not reduced and accessible to IAA, but instead are occupied, potentially in disulfide bonds, because the cysteines residues were modified after reduction with DTT. Analysis of Tim13p by thiol-trapping yielded the same results; the cysteines residues in Tim13p are occupied (unpublished data).
|
In addition, the Tim8pTim13p complex was separated on a denaturing nonreducing gel to determine if the thiol modifications were intra- or intermolecular. Tim8p and Tim13p migrated at a molecular mass near 10 kD that was almost indistinguishable from the separation profile on a reducing denaturing gel (unpublished data). This observation further suggests that the cysteine modifications are intramolecular.
Although Zn2+ is not essential for formation of the 70-kD complex, it is possible that the Tim8pTim13p complex coordinates zinc at sites other than the cysteine residues. To determine the total metal content of the Tim8pTim13p complex, we utilized ICP-AE spectrometry (Table I). Fractions containing the Tim8pTim13p complex from the final purification step on the size-exclusion column from three independent purifications were analyzed. As controls, fractions eluting before and after the Tim8pTim13p complex were included. The amount of protein in each sample was determined by quantitative amino acid analysis and added to the assay to provide a minimum detection of 30 ppb zinc, which is within the sensitivity of the instrument. This calculation was based upon the assumption that the Tim8pTim13p complex coordinates one Zn2+. It is possible that each Tim8p and Tim13p could bind a single zinc ion, which would result in detection of 180 ppb zinc. ICP-AE analysis of the recombinant Tim8pTim13p complex failed to detect significant levels of zinc above background (Table I). Manganese, iron, copper, and cobalt were also not detected at levels higher than that of the buffer blank (unpublished data). Because it is plausible that the availability of zinc was limiting during induction, the Tim8pTim13p complex was purified from cells that were induced and lysed in the presence of 1 mM zinc acetate. However, even under conditions of zinc loading, Zn2+ was not bound to the Tim8pTim13p complex (Table I).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tim23p crossed the TOM complex as a loop and was inserted into the mitochondrial inner membrane. Fusion constructs containing DHFR on both the NH2- and COOH-termini were imported and inserted into the inner membrane. Specifically, the DHFRTim23pDHFR precursor complexed with methotrextate was arrested at the outer membrane and was crosslinked to both Tim8p and Tim13p. When the DHFRTim23pDHFR construct was synthesized in the absence of methotrexate (import competent because DHFR is no longer tightly folded), it was inserted into the inner membrane as shown by manipulations involving protease treatment, osmotic lysis, and resistance to carbonate extraction. Thus, these experiments confirm that the NH2 and COOH terminus are not required for import of Tim23p. Previous studies have shown that the polytopic carrier proteins, which also lack an NH2-terminal targeting sequence, cross the outer membrane as a loop. It has been demonstrated that AAC, when flanked by DHFR on both the NH2 and COOH termini, can span the TOM complex as a loop and be crosslinked to Tim9p and Tim10p in the intermembrane space (Ryan et al., 1999; Wiedemann et al., 2001); however, the ability of these AACDHFR fusion constructs to insert into the inner membrane was not tested. In addition, the central matrix loop of the carrier uncoupling protein-1 drives protein translocation across the outer membrane (Schleiff and McBride, 2000). Taken together, we propose that polytopic inner membrane proteins follow a conserved translocation mechanism to reach the inner membrane. The precursor crosses the TOM complex as a loop and then the small Tim proteins bind to the hydrophobic membrane spanning domains as they reach the aqueous intermembrane space. Thus, the small Tim proteins act as putative chaperones to potentially prevent aggregation and maintain the unfolded precursor in an import-competent state.
The Tim8pTim13p complex does not coordinate zinc
In addition to characterizing the interaction between the Tim8pTim13p complex and Tim23p, we characterized the biochemical and biophysical properties of the Tim8pTim13p complex. The Tim8pTim13p complex is a hexamer. Tim8p may form the core of the complex because Tim8p was more protease-resistant than Tim13p. From circular dichroism studies, the complex was mostly alpha-helical and could refold efficiently after thermal denaturation. The small Tim proteins contain the twin CX3C motif (Koehler et al., 1999b). This motif has been suggested to form a zinc finger-like structure despite its divergence from the canonical zinc finger in which two amino acids separate the cysteines (Mackay and Crossley, 1998; Laity et al., 2001). Mutations in the human homologue of Tim8p, DDP1/TIMM8a (deafness dystonia protein) result in Mohr-Tranebjaerg syndrome (Jin et al., 1996). The majority of patients with Mohr-Tranebjaerg syndrome have frameshift/nonsense mutations or deletions at the DDP1/TIMM8a locus resulting in a truncated or absent protein. Recently, a de novo mutation in DDP1/TIMM8a was identified in which the fourth cysteine in the twin CX3C motif was changed from cysteine-66 to tryptophan-66, C66W (Tranebjaerg et al., 2000). We made a similar mutation in yeast, designated Tim8pC68W, and showed that the mutant Tim8pC68W protein imported into mitochondria but failed to assemble into stable 70kDa complex (Roesch et al., 2001). These results further confirm that the cysteine residues are important for assembly of the Tim8pTim13p complex.
Conflicting results have been reported about whether the Tim9pTim10p and Tim8pTim13p complexes coordinate zinc. Monomeric Tim8p and Tim13p have been shown to bind zinc in a 1:1 molar ratio and binding of the Tim8pTim13p complex to Tim23p is abolished in mitochondria incubated with metal chelators (Paschen et al., 2000; Rothbauer et al., 2001). Recently, Millar and colleagues have developed an elegant assay in which they can import the carrier proteins into plant mitoplasts (mitochondria that lack the outer membrane); they show that the addition of Zn2+ or Cd2+ stimulates import of the carriers and treatment with metal chelators inhibits import (Lister et al., 2002). Because the mechanism by which the 70-kD complexes assemble in the intermembrane space is not known, a divalent metal ion may play a critical role in assembly of the complex; such a mechanism could explain why the monomers bind zinc. From our studies, the assembled Tim8pTim13p complex did not coordinate zinc or any other metal ion, even when expression of the Tim8pTim13p complex was induced in the presence of Zn2+. Furthermore, the chelators EDTA and o-phe did not affect the binding of the Tim8pTim13p complex to Tim23p in intact mitochondria. In similar studies, the Tim9pTim10p complex did not coordinate metal ions (Curran et al., 2002). While it is plausible that the metal ions were lost during purification, the Tim8pTim13p complex folded efficiently in the presence of the chelator EDTA. The cysteine residues most likely form disulfide bridges because the complex was unable to refold in the presence of DTT. In contrast, Harrison and colleagues have shown that Lck (a lymphoid-specific, Src family protein-tyrosine kinase) interacts with the T-cell co-receptor CD4 through a cocoordinated Zn2+ ion with two cysteines in Lck and two cysteines in CD4 (Huse et al., 1998). This interaction is disrupted with low concentrations of EDTA but DTT and ß-mercaptoethanol have no effect. Taken together, these findings suggest that the cysteine residues in the twin CX3C do not coordinate zinc but instead may form disulfide bonds for structural stability. The elucidation of a high resolution structure of the Tim8pTim13p complex should provide helpful clues to the role of the cysteine residues.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Purification of the Tim8pTim13p complex from E. coli
Frozen E. coli cell pellets containing the recombinant Tim8pTim13p complex were thawed and lysed by sonication on ice. Cell debris was removed by centrifugation (30 min, 100,000 g, 4°C) and proteins were precipitated by addition of polyethylene glycol (ave. MW 3350; J.T. Baker, Inc.) to 25% and NaCl to 250 mM. After centrifugation, the pellet was solubilized in Buffer A (20 mM Hepes-KOH, pH 7.4) and loaded onto a Q-Sepharose Fast Flow column (5 x 30 cm; Amersham Biosciences). The column was washed with Buffer A at a flow rate of 5 ml/min, and bound proteins were eluted with 500 ml of a linear gradient (0200 mM NaCl) in Buffer A. 15-ml fractions were collected and analyzed by SDS-PAGE followed by immunoblotting, Coomassie staining, and blue native gel electrophoresis. After desalting and concentration in buffer A, the fractions containing the Tim8pTim13p complex were loaded onto a Source S column (1.6 x 10 cm; Amersham Biosciences). The column was washed with Buffer A at a flow rate of 5 ml/min and bound proteins were eluted with 50 ml of a linear gradient (0100 mM NaCl) in buffer A. 1-ml fractions were collected and analyzed as described above. These fractions were concentrated to 200 µl in buffer A containing 100 mM NaCl and loaded onto a Superose-12 gel filtration column (1.0 x 30 cm; Amersham Biosciences). The column was developed with buffer A containing 100 mM NaCl at a flow rate of 0.2 ml/min. 1-ml fractions were collected and analyzed as described above. The molecular mass standards for the Superose 12 column were ribonuclease A (13.7 kD); chymotrypsinogen A (25 kD); ovalbumin (43 kD); bovine serum albumin (67 kD); and aldolase (158 kD).
Import of radiolabeled proteins into isolated mitochondria
Mitochondria were purified from lactate-grown yeast cells (Glick and Pon, 1995) and assayed for in vitro protein import as described (Rospert and Schatz, 1998). Proteins were synthesized in a rabbit reticulocyte lysate in the presence of [35S]-methionine after in vitro transcription of the corresponding gene by SP6 polymerase. The reticulocyte lysate containing the radiolabeled precursor was incubated with isolated mitochondria at the indicated temperatures in import buffer (1 mg/ml bovine serum albumin, 0.6 M sorbitol, 150 mM KCl, 10 mM MgCl2, 2.5 mM EDTA, 2 mM ATP, 2 mM NADH, 20 mM K+-Hepes, pH 7.4). Where indicated, the potential across the mitochondrial inner membrane was dissipated with 1 µM valinomycin. Nonimported radiolabeled proteins were removed by treatment with 100 µg/ml trypsin or 50 µg/ml proteinase K for 1530 min on ice; trypsin was inhibited with 200 µg/ml soybean trypsin inhibitor and proteinase K with 1 mM PMSF, respectively. Cross-linking studies were performed as previously described (Leuenberger et al., 1999).
ICP-AE studies
Protein samples were diluted to 7 ml in metal-free water, and these samples were analyzed using a Thermo-Jarrel Ash Iris 1000 ICP-AE spectrometer.
Circular dichroism analysis
Circular dichroism (CD) analysis was performed on a JASCO J-600 spectropolarimeter by using a scan speed of 10 nm/min, a time constant of 8 s, and a bandwidth of 0.5 nm. Eight scans were averaged for each spectrum. The baseline correction option was used to subtract a buffer baseline. Spectra were recorded from 260 to 190 nm in 1-mm pathlength cells with protein concentrations of 0.10.2 mg/ml. Spectra were analyzed for secondary structure by using a self consistent method and/or the convex constraint algorithm for secondary structure prediction (Perczel et al., 1992; Sreerama and Woody, 1993).
Analytical ultracentrifugation
Sedimentation equilibrium measurements were performed at 20°C in an analytical ultracentrifuge (Beckman XLA) equipped with an optical absorption system. Sedimentation equilibrium was measured with 100 µl samples at 19,000 rpm; absorbance was recorded at 280 nm. The molecular mass was determined using a linear regression computer program that adjusts the baseline absorption to obtain the best linear fit of lnA versus r2 (A = absorption, r2 = radial distance from the rotor center). Measurements were in the presence of 20 mM potassium phospahte-KOH pH 7.4, 100 mM NaCl.
Screening of peptide scans with the Tim8pTim13p complex
The cellulose-bound peptide scans were prepared by automated spot synthesis (Jerini). Multiple 13-mer peptides with a 10 amino acid overlap were synthesized according to the sequence of Tim23p. The membranes were incubated with 200 nM of recombinant Tim8pTim13p complex or Tim9pTim10p complex in binding buffer (100 mM KCl, 5% sucrose, 1% bovine serum albumin, 30 mM Tris-HCl, pH 7.4) at 25°C for 2 h as described by Brix et al. (1999) After extensive washing, the bound Tim8pTim13p complex was transferred to a polyvinylidene difluoride membrane, followed by detection with antibodies against Tim8p or Tim13p and [125I]-protein A. Binding data was acquired by scanning laser densitometry (Personal Densitometer SI; Molecular Dynamics) and quantitated utilizing ImageQuaNT (version 4.2a; Molecular Dynamics). The mean of at least three independent experiments for each peptide spot was used and the local background of each peptide spot was subtracted. Binding data were identical for antibodies against Tim8p and Tim13p.
Blue native gel electrophoresis
Mitochondria (2.5 mg/ml) were solubilized in 20 mM K+-Hepes, pH 7.4, 50 mM NaCl, 10% glycerol, 2.5 mM MgCl2. 1 mM EDTA, 0.16% n-dodecylmaltoside (Boehringer Mannheim) for 30 min on ice. Insoluble material was removed by centrifugation at 100,000 g for 10 min, and the solubilized proteins were analyzed by blue native gel electrophoresis on a 616% linear polyacrylamide gradient (Schägger et al., 1994).
Miscellaneous
Mitochondrial proteins were analyzed by SDS-PAGE using a 10 or 16% polyacrylamide gel and a Tricine-based running buffer (Schägger and von Jagow, 1987). Proteins were detected by immunoblotting using nitrocellulose- or PVDF membranes and visualization of immune complexes with [125I]-protein A. Protein concentration was assayed by the bicinchoninic acid method (Pierce Chemical Co.) using bovine serum albumin as the standard. Thiol-trapping studies were previously described (Curran et al., 2002).
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
C.M. Koehler is a Damon Runyon-Walter Winchell Scholar. This work was supported by the Damon Runyon-Walter Winchell Cancer Research Foundation (DRS18), the American Heart Association (0030147N), Burroughs Wellcome Fund New Investigator Award in the Toxicological Sciences (1001120), Research Corporation (RI0459), and the National Institutes of Health (1R01GM61721-01). S.P. Curran is funded by the United States Public Health Service National Research Service Award (GM07185). E. Schmidt is funded by the Dystonia Research Foundation.
Submitted: 29 May 2002
Revised: 30 July 2002
Accepted: 30 July 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adam, A., M. Endres, C. Sirrenberg, F. Lottspeich, W. Neupert, and M. Brunner. 1999. Tim9, a new component of the TIM22.54 translocase in mitochondria. EMBO J. 18:313319.
Bohni, P.C., G. Daum, and G. Schatz. 1983. Import of proteins into mitochondria. Partial purification of a matrix-located protease involved in cleavage of mitochondrial precursor polypeptides. J. Biol. Chem. 258:49374943.
Brix, J., S. Rudiger, B. Bukau, J. Schneider-Mergener, and N. Pfanner. 1999. Distribution of binding sequences for the mitochondrial import receptors Tom20, Tom22, and Tom70 in a presequence-carrying preprotein and a non-cleavable preprotein. J. Biol. Chem. 274:1652216530.
Curran, S.P., D. Leuenberger, W. Oppliger, and C.M. Koehler. 2002. The Tim9p-Tim10p complex binds to the transmembrane domains of the ADP/ATP carrier. EMBO J. 21:942953.
Davis, A.J., K.R. Ryan, and R.E. Jensen. 1998. Tim23p contains separate and distinct signals for targeting to mitochondria and insertion into the inner membrane. Mol. Biol. Cell. 9:25772593.
Davis, A.J., N.B. Sepuri, J. Holder, A.E. Johnson, and R.E. Jensen. 2000. Two intermembrane space TIM complexes interact with different domains of Tim23p during its import into mitochondria. J. Cell Biol. 150:12711282.
Endres, M., W. Neupert, and M. Brunner. 1999. Transport of the ADP/ATP carrier of mitochondria from the TOM complex to the TIM22.54 complex. EMBO J. 18:32143221.
Guthrie, C., and G.R. Fink. 1991. Guide to Yeast Genetics and Molecular Biology. Academic Press, San Diego, CA. 933 pp.
Huse, M., M.J. Eck, and S.C. Harrison. 1998. A Zn2+ ion links the cytoplasmic tail of CD4 and the N-terminal region of Lck. J. Biol. Chem. 273:1872918733.
Jin, H., M. May, L. Tranebjaerg, E. Kendall, G. Fontan, J. Jackson, S.H. Subramony, F. Arena, H. Lubs, S. Smith, et al. 1996. A novel X-linked gene, DDP, shows mutations in families with deafness (DFN-1), dystonia, mental deficiency and blindness. Nat. Genet. 14:177180.[Medline]
Káldi, K., M.F. Bauer, C. Sirrenberg, W. Neupert, and M. Brunner. 1998. Biogenesis of Tim23 and Tim17, integral components of the TIM machinery for matrix-targeted preproteins. EMBO J. 17:15691576.
Kerscher, O., J. Holder, M. Srinivasan, R.S. Leung, and R.E. Jensen. 1997. The Tim54p-Tim22p complex mediates insertion of proteins into the mitochondrial inner membrane. J. Cell Biol. 139:16631675.
Kerscher, O., N.B. Sepuri, and R.E. Jensen. 2000. Tim18p is a new component of the Tim54p-Tim22p translocon in the mitochondrial inner membrane. Mol. Biol. Cell. 11:103116.
Koehler, C.M., E. Jarosch, K. Tokatlidis, K. Schmid, R.J. Schweyen, and G. Schatz. 1998a. Import of mitochondrial carriers mediated by essential proteins of the intermembrane space. Science. 279:369373.
Koehler, C.M., S. Merchant, W. Oppliger, K. Schmid, E. Jarosch, L. Dolfini, T. Junne, G. Schatz, and K. Tokatlidis. 1998b. Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial carrier proteins. EMBO J. 17:64776486.
Koehler, C.M., D. Leuenberger, S. Merchant, A. Renold, T. Junne, and G. Schatz. 1999a. Human deafness dystonia syndrome is a mitochondrial disease. Proc. Natl. Acad. Sci. USA. 96:21412146.
Koehler, C.M., M.P. Murphy, N. Bally, D. Leuenberger, W. Oppliger, L. Dolfini, T. Junne, G. Schatz, and E. Or. 2000. Tim18p, a novel subunit of the inner membrane complex that mediates protein import into the yeast mitochondrial inner membrane. Mol. Cell. Biol. 20:11871193.
Leuenberger, D., N.A. Bally, G. Schatz, and C.M. Koehler. 1999. Different import pathways through the mitochondrial intermembrane space for inner membrane proteins. EMBO J. 17:48164822.[CrossRef]
Mackay, J.P., and M. Crossley. 1998. Zinc fingers are sticking together. Trends Biochem. Sci. 23:14.[CrossRef][Medline]
Murphy, M.P., D. Leuenberger, S.P. Curran, W. Oppliger, and C.M. Koehler. 2001. The essential function of the small Tim proteins in the TIM22 import pathway does not depend on formation of the soluble 70-kilodalton complex. Mol. Cell. Biol. 21:61326138.
Paschen, S.A., and W. Neupert. 2001. Protein import into mitochondria. IUBMB Life. 52:101112.[Medline]
Paschen, S.A., U. Rothbauer, K. Káldi, M.F. Bauer, W. Neupert, and M. Brunner. 2000. The role of the TIM8-13 complex in the import of Tim23 into mitochondria. EMBO J. 19:63926400.
Pfanner, N. 1998. Mitochondrial import: crossing the aqueous intermembrane space. Curr. Biol. 8:R262R265.[Medline]
Pfanner, N., P. Hoeben, M. Tropschug, and W. Neupert. 1987. The carboxyl-terminal two-thirds of the ADP/ATP carrier polypeptide contains sufficient information to direct translocation into mitochondria. J. Biol. Chem. 262:1485114854.
Roesch, K., S.P. Curran, L. Tranebjaerg, and C.M. Koehler. 2001. Human deafness dystonia syndrome is caused by a defect in assembly of the DDP1/TIMM8a-TIMM13 complex. Hum. Mol. Genet. 11:477486.[CrossRef][Medline]
Rospert, S., and G. Schatz. 1998. Protein translocation into mitochondria. In Cell Biology: A Laboratory Handbook. Vol. 2. J.E. Celis, editor. Academic Press, San Diego. 277285.
Rothbauer, U., S. Hofmann, N. Muhlenbein, S.A. Paschen, K.D. Gerbitz, W. Neupert, M. Brunner, and M.F. Bauer. 2001. Role of the deafness dystonia peptide 1 (DDP1) in import of human Tim23 into the inner membrane of mitochondria. J. Biol. Chem. 276:3732737334.
Ryan, M.T., H. Muller, and N. Pfanner. 1999. Functional staging of ADP/ATP carrier translocation across the outer mitochondrial membrane. J. Biol. Chem. 274:2061920627.
Schägger, H., W.A. Cramer, and G. von Jagow. 1994. Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal. Biochem. 217:220230.[CrossRef][Medline]
Schatz, G., and B. Dobberstein. 1996. Common principles of protein translocation across membranes. Science. 271:15191526.[Abstract]
Schleiff, E., and H. McBride. 2000. The central matrix loop drives import of uncoupling protein 1 into mitochondria. J. Cell Sci. 113:22672272.
Sikorski, R.S., and P. Hieter. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 122:1927.
Sreerama, N., and R.W. Woody. 1993. A self-consistent method for the analysis of protein secondary structure from circular dichroism. Anal. Biochem. 209:3244.[CrossRef][Medline]
van Dijl, J.M., E. Kutejova, K. Suda, D. Perecko, G. Schatz, and C.K. Suzuki. 1998. The ATPase and protease domains of yeast mitochondrial Lon: roles in proteolysis and respiration-dependent growth. Proc. Natl. Acad. Sci. USA. 95:1058410589.
Wiedemann, N., N. Pfanner, and M.T. Ryan. 2001. The three modules of ADP/ATP carrier cooperate in receptor recruitment and translocation into mitochondria. EMBO J. 20:951960.