Correspondence to: Walter Neupert, Institut für Physiologische Chemie der Universität München, Goethestraße 33, D-80336 München, Germany. Tel:49 (89) 5996 312
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Abstract |
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Translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane TOM complex. We have isolated the TOM core complex of Neurospora crassa by removing the receptors Tom70 and Tom20 from the isolated TOM holo complex by treatment with the detergent dodecyl maltoside. It consists of Tom40, Tom22, and the small Tom components, Tom6 and Tom7. This core complex was also purified directly from mitochondria after solubilization with dodecyl maltoside. The TOM core complex has the characteristics of the general insertion pore; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner. It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles. Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of 2.1 nm and a height of
7 nm. Tom40 is the key structural element of the TOM core complex.
Key Words: TOM complex, mitochondria, protein translocation channel, electron tomography, protein targeting
TRANSPORT of nuclear-encoded proteins into mitochondria is mediated by distinct translocation machineries located in the outer and inner membranes of mitochondria. Components in the outer membrane, which facilitate the recognition of preproteins, their transfer through the outer membrane, and the insertion of resident outer membrane proteins, are organized in the TOM complex (for review see
Three import receptors, Tom20 (
The recent isolation and purification of the TOM holo complex of Neurospora has provided information about its composition, structure, and channel function (
A deep understanding of protein translocation across the outer membrane of mitochondria requires structural information of the TOM protein-conducting channel itself. Here, we report on the isolation and the structure of the TOM core complex comprising the general import pore. Its constituents are Tom40, the major pore forming protein, Tom22, and the small Tom components, Tom7 and Tom6. An as yet unidentified band of the size of yeast Tom5 is also present in the core complex. The isolated core complex forms a high conductance channel in lipid membranes with properties similar to those of the holo complex. Analysis of the binding activity of the core complex to a translocation substrate in detergent solution indicates that it fulfills protein import functions. Single particle EM reveals particles, the majority of which contain two centers of stain accumulation and a less abundant complex with one center. These likely represent protein-conducting channels. Electron tomography and three-dimensional (3D)1 image reconstruction yielded a map of the TOM core complex with two channels crossing the outer membrane.
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Materials and Methods |
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Isolation and Purification of the TOM Core Complex
TOM holo complex and TOM core complex were isolated and purified from mitochondrial membranes of a Neurospora strain (GR-107) in which the wild-type Tom22 is replaced with a version of Tom22 encoding a protein with a hexahistidinyl tag at its COOH terminus. Growth of the Neurospora cells and preparation of mitochondria were performed as described previously (510 mg ml-1 at 4°C. The identity of the individual Tom proteins was verified by immunodetection with antibodies specific for the Tom components. An average preparation of the TOM core complex started with
1.5 kg of Neurospora cells (wet wt) which yielded
7 g of mitochondrial protein. The final preparation contained
1015 mg pure TOM core complex.
For determination of the stoichiometry of Tom components, core complex was isolated from strain GR-107 grown in the presence of 35S-sulfate. The purified radio-labeled TOM core complex was subjected to SDS-PAGE. For the detection and quantification of radio-labeled proteins, dried gels were analyzed by phosphorimaging analysis.
Holo complex containing all established Tom components was purified from isolated Neurospora mitochondrial outer membrane vesicles in 0.5% (wt/vol) digitonin as previously described (
For preparing of TOM core complex that lacks the hydrophilic receptor domains, core complex (900 µg) was incubated with 100 µg ml-1 trypsin in 100 µl 50 mM potassium acetate, 10 mM MOPS, pH 7.0, 20% glycerol, and 0.1% DDM at 0°C for 30 min. Proteolysis was stopped with trypsin inhibitor (0.5 mg ml-1) and proteolytic cleavage of TOM complex was assessed by SDS-PAGE.
Preparation of Chemical Amounts of pSu9-DHFR
A COOH terminally His-tagged fusion protein consisting of the presequence of subunit 9 of the F0-ATPase (residues 169) and dihydrofolate reductase (pSu9-DHFR) was expressed in Escherichia coli and purified by Ni-NTA chromatography. In brief, bacteria were grown overnight in 250 ml LB medium at 37°C. The overnight culture was diluted and grown to an OD600 of 0.80.9. Expression of pSu9-DHFR was induced by adding isopropyl thiogalactoside to a final concentration of 2 mM. Cells were grown for 1 h and harvested by centrifugation. The bacterial pellet was resuspended in buffer containing 50 mM NaHPO4, pH 8.0, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 1 mM PMSF, 10 µg ml-1 -macroglobulin, 10 µg ml-1 leupeptin, and 10 µg ml-1 pepstatin, and then sonicated using a Branson 250 sonifier. Unbroken cells were removed by centrifugation and supernatants were loaded onto a Ni-NTA affinity column. The column was washed with 10 column vol phosphate buffer, and bound material was eluted by a linear 10300 mM imidazole gradient in the same buffer. The peak fractions containing 34 mg ml-1 of purified pSu9-DHFR were stored at -80°C.
Size Exclusion Chromatography
Purified TOM complex (100 µg) was loaded onto a TosoHaas TSK G4000 PWXL size-exclusion column equilibrated with 50 mM potassium acetate, 10 mM MOPS, pH 7.0, 10% glycerol, and 0.5% digitonin at room temperature using an Äkta chromatography system (Pharmacia Biotech, Inc.). Protein was eluted at a flow rate of 0.45 ml min-1. The absorbance of the eluant was monitored at 280 nm. The molecular weights of TOM complexes were calculated using thyroglobulin (669 kD), apoferritin (443 kD), alcohol dehydrogenase (155 kD), and carboanhydrase (29 kD) as protein standards.
TOM core complex used for EM was passed over a Superose 6 gel filtration column (Pharmacia Biotech), equilibrated with 50 mM potassium acetate, 10 mM MOPS, pH 7.0, 20% glycerol, and 0.1% DDM.
Gel Electrophoresis
Native PAGE was performed using a 415% acrylamide gradient (PhastGel; Pharmacia Biotech, Inc.). For blue native polyacrylamide electrophoresis (
SDS-PAGE was performed according to the procedure described by
Conductance Measurements
Conductance measurements of TOM complex in planar black lipid membranes were carried out as previously described (0.1 mm2) in the wall of a Teflon cell separating two aqueous compartments of 5 ml each. The aqueous solutions contained 1 M KCl, 5 mM Hepes-KOH, pH 7.0 (
0 = 96.7 mS cm-1).
The channel size of native TOM complex and TOM subcomplexes was determined by analyzing the partitioning of differently sized PEGs into the TOM complex channel. The electrolyte contained 1 M KCl, 5 mM Hepes-KOH, pH 7.0, and 20% (wt/vol) polyethylene glycol (PEG) of various molecular weight (Fluka; Sigma Chemical Co.). The bulk electrolyte conductances, PEG, were the same for all PEG solutions (
PEG = 58.1 ± 0.4 mS cm-1, mean ± SEM). Membrane currents were measured at a membrane potential of +20 mV with a pair of Ag/AgCl electrodes (Metrohm) using a Keithley 428 current amplifier. Amplified signals were monitored with an analogue/digital storage oscilloscope (Hameg HM 407) and recorded with a strip chart recorder (Philips PM8100). The conductance of each buffer solution,
0 and
PEG, was measured using a Greisinger GLM 200A conductance meter.
Electron Microscopy Analysis
Purified TOM complex preparations (0.1 mg protein ml-1) were adsorbed to glow-discharged carbon-coated specimen support grids (Cu, 600 mesh or 400 x 100 mesh) for 30 s. The grids were washed twice with deionized water, blotted with filter paper, and stained with 2% (wt/vol) aqueous uranyl acetate for 60 s.
Projection images of isolated TOM complex were recorded at 0° tilt in a Philips CM12 transmission electron microscope operating at 120 kV at 43,800x and an underfocus of between 1 and 2.5 µm. Single particle images were processed as described (
Electron tomography of TOM core complex stained with 2% (wt/vol) uranyl acetate was carried out using a Philips 200 FEG electron transmission microscope equipped with a VIPS-1000 computer (TVIPS) and a large-area CCD camera (Photometrics; 1,600 e- nm-2. The direction of the tilt axis was determined from an independent tilt series of a specimen with 10-nm gold clusters.
Image processing of the tomographic data included the alignment of the projections of each tilt series to a common origin, the selection of single particles at 0° tilt, and the 3D reconstruction of individual particles by means of weighted backprojection.
From the 3D maps of 321 particles, projection images were calculated, aligned, and subjected to MSA. Particles that did not show two pores were excluded from the data set. For calculation of the final 3D model, the largest homogenous class of particles (n = 116) was subjected to refined 3D alignment with respect to all six alignment parameters (i.e., three Cartesian coordinates and three angles) using the 3D map of the previous cycle as a reference. To avoid biased 3D alignment and merging molecules with different up and down orientation, each individual particle was allowed to rotate by all three Euler angles in each refinement cycle.
For the visualization of the 3D model of the TOM core complex, the AVS/Express 4.0 software package (Advanced Visual Systems Inc.) was used. The surface models were, based on a protein density of 1.3 g cm-3, thresholded to a volume with an expected mass of 410 kD.
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Results |
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Isolation of TOM Core Complex
The TOM core complex was prepared in two different ways: either by treatment of purified TOM holo complex with high concentrations of nonionic detergent and size-exclusion chromatography or by direct isolation from detergent-solubilized mitochondria in DDM.
The TOM holo complex containing all the established Tom components was purified from isolated mitochondrial outer membrane vesicles prepared from a Neurospora strain bearing a hexahistidinyl-tagged form of Tom22 (Fig 1 A, left; 490 kD, in agreement with earlier studies (
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Incubation of the holo complex with DDM at a concentration of 0.33% (wt/vol) at 37°C for 1 h led to dissociation of the import receptors, Tom70 and Tom20, and formation of a defined subcomplex. Size-exclusion chromatography of this material gave a profile similar to that of the holo complex, which was slightly shifted towards the low molecular weight range (Fig 1 C). The main fraction contained nearly all of Tom40, Tom22, and the smaller Tom components, Tom7 and Tom6. Tom40 and Tom22 were present at roughly the same proportion as in the holo complex, as indicated by quantification of Coomassie staining (Fig 1 A, right). Most of Tom70 and Tom20 eluted in fractions corresponding to lower molecular weight. Similar results were obtained when intact TOM complex was treated with the nonionic detergent Triton X-100 at concentrations above 0.33% (data not shown). Treatment of the TOM holo complex with SDS, in contrast, completely dissociated the complex into its individual components (data not shown). Thus, Tom40, Tom22, Tom7, and Tom6 form a defined, and rather stable subcomplex that we designated as the TOM core complex. According to the elution from the sizing column, the apparent molecular mass of this complex was estimated to be 410 kD.
To isolate the TOM core complex directly from mitochondria in high amounts, mitochondria from a strain with a hexahistidinyl-tag on Tom22 were solubilized in DDM at a concentration of 1% (wt/vol). The extract was loaded onto a Ni-NTA affinity column and, after extensive washing, bound material was eluted with an imidazole gradient. Tom40, Tom22, and the smaller Tom components all coeluted within five major fractions that accounted for 0.2% of protein loaded onto the column (Fig 2 A, lane 2). Further anion-exchange chromatography resulted in a virtually pure TOM core complex (Fig 2 A, lane 3). The yield of purified core complex was
2 mg complex per 1 g isolated mitochondrial protein.
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Upon size exclusion chromatography, the isolated TOM core complex was recovered in a single peak (Fig 2 B) that contained only Tom40, Tom22, and the smaller Tom components (Fig 2 C). Tom70 and Tom20 were not detected in this complex. Low amounts of Tom20 were present in the preparation eluted from the Ni-NTA column but, as determined by immunoblotting, these were completely removed after the gel filtration step (Fig 2 D).
TOM core complex was purified from Neurospora cells that were grown in the presence of 35S-sulfate. In the purified complex, Tom40, Tom22, Tom7, and Tom6 were present in molar ratios of 8:4:2:2 (n = 2).
Characterization of the Isolated Core Complex
Isolated core complex was incubated with low amounts of trypsin (Fig 3) and analyzed by size-exclusion chromatography. Proteolytic cleavage left Tom40 intact and removed the hydrophilic domains of Tom22 and the small Tom components, yielding fragments <35 kD. Immunoblotting using specific antisera against the COOH and NH2 terminus of Tom22 did not recognize a fragment of the protein. With antiserum against Tom6, no protein could be detected (data not shown).
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The trypsinized complex eluted in a defined peak corresponding to a high molecular mass complex of 410 kD. A similar observation was made when the TOM holo complex, isolated in digitonin, was treated with protease (data not shown). This result indicated that the hydrophilic domains of Tom22 and of the small Tom components are not important for the structural integrity of the core complex.
To further confirm the tight association of Tom40, Tom22, and the smaller Tom proteins in a defined subcomplex, purified TOM holo complex and TOM core complex were examined by native PAGE. Single high molecular weight bands were observed upon staining with Coomassie brilliant blue (Fig 4 A). The different migration behavior of the complexes is due to the different detergents. The holo complex was solubilized in digitonin whereas the core complex has been purified in DDM.
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Immunoblotting of the holo complex with monospecific antisera confirmed the presence of Tom70, Tom40, Tom22, Tom20, Tom7, and Tom6 (data not shown). The band representing the core complex yielded a positive signal using antibodies against Tom40, Tom22, and Tom6.
When the holo complex was treated with DDM (0.33%) or Triton X-100 (0.33%), the resulting core complex had the same electrophoretic mobility as the core complex isolated from mitochondria, and Tom70 migrated close to the running front (data not shown). Only Tom40, Tom22, and the smaller Tom components were detected in the band corresponding to the core complex.
Examination of the holo and core complexes by blue native gel electrophoresis, a method in which the binding of Coomassie brilliant blue adds negative charges to the protein complexes, gave results similar to those obtained without inclusion of a dye (Fig 4 B). However, these conditions resulted in partial dissociation of the TOM holo complex since Western blotting and decoration of proteins with specific antibodies revealed that Tom70, and most of Tom20, no longer comigrated with Tom40 (data not shown). This partial disintegration of the TOM holo complex can be attributed to a destabilizing effect of the negatively charged dye used on the complex (
Channel Activity of the Isolated Core Complex
To test whether the isolated core complex contains pores, we analyzed its channel forming activity after reconstitution into lipid membranes. Purified core complex was added to both sides of a black lipid membrane bilayer. Current recordings showed characteristic steps of conductance increase that reflect insertion of the core complex into the lipid bilayer. An average conductance of 2.3 nS in the presence of 1 M KCl was observed (Fig 5 A). The trypsin-treated TOM holo complex had an average conductance of 2.7 nS (data not shown). These average conductances were similar to that of the holo complex (2.3 nS in 1 M KCl;
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To probe the channel size of the isolated core complex, we studied its conductance in the presence of differently sized nonelectrolyte polymers. Low molecular weight polyethylene glycol, PEG1000, led to decreased channel conductances (Fig 5 B). High molecular weight polyethylene glycols, such as PEG8000, affected the channel conductance to a lesser degree (Fig 5 C). The results indicate that the TOM core complex channel can be blocked by molecules of up to 6 kD (Fig 5 D).
Binding of Preprotein by the TOM Core Complex
Does the TOM core complex retain its ability to bind a chemically pure preprotein in the presence of detergent? To address this question, we incubated chemical amounts of pure preprotein (pSu9-DHFR) with mitochondrial outer membrane vesicles. Membranes were solubilized with DDM at a concentration identical to that used for the isolation of the core complex directly from mitochondria, and the lysate was subjected to size exclusion chromatography. All column fractions were analyzed by SDS-PAGE and immunoblotting. A large fraction of pSu9-DHFR coeluted with Tom22 (Fig 6) and Tom40 (not shown) in a high molecular weight complex. Only background binding was observed when DHFR lacking a mitochondrial presequence was analyzed, excluding the possibility that formation of the TOM-pSu9-DHFR complex was the result of unspecific binding. Thus, pSu9-DHFR remained firmly bound to the TOM core complex in a signal-sequence dependent manner, even at high levels of nonionic detergent.
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Structure of the Isolated TOM Core Complex
Electron micrographs of negatively stained TOM core complex particles displayed predominantly two stain filled openings or pores, but particles representing a single ring were also present (Fig 7 A). The length of the two pore particles was 12 nm, with a width of
7 nm. For further image processing, a total of 1,598 particle images were extracted and aligned with respect to translation and rotation via cross-correlation (
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Projection maps of the TOM core complex treated with trypsin (Fig 7 E) were calculated. The class averages of the trypsinized complex predominantly showed particles with two pores (n = 326) and one pore (n = 254; Fig 7 F). Frequently, one of the channels appeared less distinct. This may be due to stain fluctuations, as can be seen in the original micrograph (Fig 7A and Fig E). The overall structure of the trypsinized core complex was similar to that of the intact core complex.
Do the stain filled openings of the TOM core complex, as seen in the 2D projections, span the entire complex? A 3D map of the TOM core complex was constructed by means of electron tomography. A total of 321 core complex particles were individually reconstructed in three dimensions from 6,741 projections, and subjected to 3D alignment and classification. As mentioned in Materials and Methods, the orientation of each particle was checked before averaging. As a result, 19% of the particles had to be flipped from up to down orientation. We cannot exclude, however, that the final 3D average is slightly distorted, due to possible flattening of the molecules that would mimic a common orientation of actually differently oriented particles.
An average of 116 reconstructions corresponding to the most prominent class of particles is shown in Fig 8. The resolution of this average was 1/2.4 nm-1, based on the Fourier shell correlation function and 1/3.4 nm-1 following the stronger phase residual criterion (
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The top view of the 3D model shows a two-ring structure. The density of the contacts between the two rings is as strong as the density of the walls of the rings, thus excluding the possibility that the two-ring structure was due to association of two independent translocation pores. The diameters of the two channels measure 2.1 nm. Based on the final reconstruction, and taking into account possible flattening and incomplete staining effects occurring during specimen preparation, the height of the TOM complex is
7 nm.
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Discussion |
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We have isolated and analyzed the TOM core complex, which lacks the receptors Tom70 and Tom20, but retains several of the essential properties of the TOM holo complex. Tom40 is present in the holo and the core complex in the same number and constitutes the main component of the protein conducting channel. Tom22, which appears to have both a receptor function and a role in translocation, is firmly associated with Tom40. Likewise, the small Tom components, Tom6 and Tom7, which are believed to be required for the stability of the TOM complex (
Our data also allows us to define the minimal structural requirement of the translocation pore to Tom40, the transmembrane segments of Tom22, and the small Tom components. Whether the transmembrane segments are essential for the formation of the double pore structure remains an open question.
Although the sequence homology between Tom40, mitochondrial and bacterial porins is limited, circular dichroism data suggest a common ß-barrel-like structure (
EM and image analysis of the isolated TOM core complex revealed mainly double ring structures, but a significant fraction of single ring particles were also observed (19%). This percentage of single ring particles is higher than observed with the holo complex (
2%;
410 kD, which correspond to the double ring structures. Therefore, the TOM core complex may display a somewhat increased instability, or the placement on grids and negative staining of samples may promote disassembly.
The height of the TOM core complex of 7 nm is
2 nm larger than the thickness of a lipid bilayer. In fact, the extra membrane loops of Tom40 and receptors, or intermembrane space domains of Tom22, and the small Tom components would not be able to form large masses on either side of the complex. When edge-on views of the core complex become available, it should be possible to resolve the cytosolic and intermembrane space domains of the TOM core complex in more detail.
The 3D reconstruction of the TOM core complex shows several globular elements. Given that the TOM core complex is composed of about eight Tom40 molecules, these elements could represent dimers of Tom40.
At present, the cytosolic and intermembrane space sides of the TOM core complex cannot be distinguished. Probing both surfaces of the molecule with tags or antibodies should help resolve this issue. As higher resolution images become available, it will also be possible to better resolve the surface boundaries and internal surfaces of the putative translocation channels.
The size of the pores of the TOM core complex, as derived from single particle analysis, can be compared with that calculated from conductance measurements of the TOM core complex in the presence of differently sized nonelectrolyte polymers. Low molecular weight polyethylene glycols that were able to partition into the pores of the core complex reduced the mean conductance of the complex. Intermediate and large size polyethylene glycols with molecular weights of >6,000 reduced the currents mediated by the TOM complex to a lesser extent. Apparently, molecules with hydrodynamic radii larger than that of PEG6000 were not able to penetrate the core complex channel. Given a radius of 2.5 nm of PEG6000 (
2.1 nm). High molecular weight PEG molecules might block the entrances of the import channels of the TOM complex.
Conceivably, the two-ring structure is a dynamic assembly. A structural flexibility of the TOM complex in terms of alterations of subunit interactions during import of matrix-targeted preproteins has indeed been observed (
The TOM core complex shares a number of interesting characteristics with the Sec61p complex, which facilitates protein translocation in the ER (, respectively, and contain additional small components that span the membrane only once. Further, both complexes form ring-like structures with the characteristics of hydrophilic pores (
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Footnotes |
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1 Abbreviations used in this paper: 3D, three-dimensional; DHFR, dihydrofolate reductase; DDM, n-dodecyl ß-D-maltoside; MSA, multivariate statistical analysis; Ni-NTA, nickel-nitrilotriacetic acid; pSu9, presequence of subunit 9 of the F0-ATPase.
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Acknowledgements |
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We thank W. Baumeister for continuous support and R. Benz for his support in the electrophysiology measurements. We also thank D. Rapaport for critically reading the manuscript, and M. Braun and U. Staudinger for technical assistance.
This research was supported by grants of the Deutsche Forschungsgemeinschaft (S. Nussberger and W. Neupert), the Münchener Medizinische Wochenschrift (S. Nussberger), and the Medical Research Council of Canada (F.E. Nargang).
Submitted: 2 September 1999
Revised: 13 October 1999
Accepted: 19 October 1999
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References |
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