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© The Rockefeller University Press,
0021-9525/2001//1151 $5.00
The Journal of Cell Biology, Volume 153, Number 6,
, 2001 1151-1160
Original Article |
Tom40, the Pore-Forming Component of the Protein-Conducting Tom Channel in the Outer Membrane of Mitochondria
neupert{at}bio.med.uni-muenchen.de
Tom40 is the main component of the preprotein translocase of the outer membrane of mitochondria (TOM complex). We have isolated Tom40 of Neurospora crassa by removing the receptor Tom22 and the small Tom components Tom6 and Tom7 from the purified TOM core complex. Tom40 is organized in a high molecular mass complex of
350 kD. It forms a high conductance channel. Mitochondrial presequence peptides interact specifically with Tom40 reconstituted into planar lipid membranes and decrease the ion flow through the pores in a voltage-dependent manner. The secondary structure of Tom40 comprises 31% β-sheet, 22% 
-helix, and 47% remaining structure as determined by circular dichroism measurements and Fourier transform infrared spectroscopy. Electron microscopy of purified Tom40 revealed particles primarily with one center of stain accumulation. They presumably represent an open pore with a diameter of 2.5 nm, similar to the pores found in the TOM complex. Thus, Tom40 is the core element of the TOM translocase; it forms the protein-conducting channel in an oligomeric assembly.
Key Words: TOM complex • Tom40 • mitochondria • protein translocation channel • protein targeting
© 2001 The Rockefeller University Press
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Introduction |
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Biochemical and biophysical characterization of the TOM complex of Neurospora crassa and Saccharomyces cerevisiae revealed Tom40 as the key structural component of the protein-conducting channel in the mitochondrial outer membrane (Hill et al. 1998; Künkele et al. 1998a,Künkele et al. 1998b). Tom40 is an integral membrane protein that is essential for viability in yeast and Neurospora (Vestweber et al. 1989; Kiebler et al. 1990). Multiple copies of this protein are organized in the TOM core complex together with up to three small membrane-embedded subunits, Tom5 (Dietmeier et al. 1997), Tom6 (Kassenbrock et al. 1993; Alconada et al. 1995; Cao and Douglas 1995), and Tom7 (Hönlinger et al. 1996) and Tom22 (Kiebler et al. 1993; Lithgow et al. 1994; Hönlinger et al. 1995; Nakai and Endo 1995), a subunit with hydrophilic domains exposed to both sides of the outer membrane. The TOM holo complex in addition comprises the receptors Tom20 (Söllner et al. 1989; Ramage et al. 1993) and Tom70 (Hines et al. 1990; Söllner et al. 1990), single-spanning membrane proteins with hydrophilic domains exposed to the cytosol. They are only loosely attached to the TOM core complex (Dekker et al. 1998; Ahting et al. 1999).
The recent isolation and purification of the TOM holo complex of N. crassa has provided information about its composition, structure, and function as a protein-conducting channel. EM revealed particles, the majority of which contained two and three centers of stain accumulation (Künkele et al. 1998a). Electron tomography and three-dimensional image reconstruction of the TOM core complex yielded a map with two channels traversing the complex. Removal by proteolysis of the cytosolic or the intermembrane space domains of Tom22 did not interfere with the structural integrity of the complex of Neurospora (Ahting et al. 1999). On the other hand, in S. cerevisiae Tom22 was proposed to be crucial for the high level organization of the complex (van Wilpe et al. 1999).
Here, we report on the isolation and biochemical and biophysical characterization of a TOM subcomplex, consisting exclusively of Tom40 of N. crassa. Using circular dichroism (CD) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), we analyze the secondary structure of the protein and compare it with those of purified TOM core complex and the voltage-dependent anion channel (VDAC), the mitochondrial porin. The results provide insights into the secondary structure of Tom40, revealing both β-sheet and -helical elements. Electrophysiological measurements suggest Tom40 to be the essential element in the formation of the pore. Analysis of purified Tom40 by EM and image analysis reveals particles, most of which contain one center of stain accumulation. The results indicate that Tom6, Tom7, and Tom22 are important for the stability of the TOM complex but play only a relatively minor structural role in the formation of the protein translocation channel.
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50 mg/ml and stored in aliquots at –20°C.
Isolation and Purification of TOM Core Complex
TOM core complex was purified as described previously (Ahting et al. 1999) with minor modifications. In brief, isolated mitochondria were solubilized in 50 mM potassium acetate, 10 mM MOPS, pH 7.0, 20% glycerol, and 1% (wt/vol) n-dodecyl β-D-maltoside (DDM; Anatrace, Inc.) in the presence of 1 mM PMSF and a cocktail of protease inhibitors at a protein concentration of 10 mg/ml for 30 min at 4°C. Insoluble material was removed by centrifugation, and the clarified extract was loaded onto a nickel nitrilotriacetic acid agarose column (Ni-NTA; QIAGEN) using 4 ml resin per 1 g of total mitochondrial protein. The column was washed with 20 column volumes of a buffer containing 50 mM potassium acetate, 10 mM MOPS, pH 7.0, 20% glycerol, 0.1% DDM, and 40 mM imidazole. Specifically bound material was eluted with 300 mM imidazole in the same buffer. Fractions containing TOM core complex were pooled and loaded onto a Resource Q anion exchange column (Amersham Pharmacia Biotech) equilibrated with 50 mM potassium acetate, 10 mM MOPS, pH 7.0, 20% glycerol, and 0.1% DDM. The complex was eluted by a linear 0–500 mM KCl gradient in the same buffer. Stock solutions of purified TOM core complex were stored at a protein concentration of 5 mg/ml at 4°C.
Preparation of Tom40
For the isolation of Tom40, purified TOM core complex (1–5 mg) was reloaded onto an Ni-NTA affinity column (1 ml resin) equilibrated with 50 mM potassium acetate, 10 mM MOPS, pH 7.0, 10% glycerol, and 0.1% (wt/vol) DDM at 4°C. The column was washed with two column volumes of equilibration buffer. Tom40 and Tom7 were eluted with 3% (wt/vol) n-octyl β-D-glucopyranoside (OG; Fluka) in the same buffer using a column flow rate of 0.05 ml/min. 10 column fractions of a volume of 1 ml were collected. Specifically bound material was eluted with 300 mM imidazole in the same buffer containing 1% (wt/vol) OG. To prevent Tom40 from aggregation, the fractions containing Tom40 were supplemented with 0.5% (wt/vol) DDM (final concentration) and subsequently loaded onto a sucrose gradient (7–35%) containing 50 mM potassium acetate, 10 mM MOPS, pH 7.0, and 0.5% (wt/vol) DDM and centrifuged overnight to replace OG with DDM. Alternatively, detergent exchange was performed by anion exchange chromatography using a Resource Q column (Amersham Pharmacia Biotech). Isolated Tom40 was stored in 0.5% DDM at a protein concentration of 0.8–1 mg/ml at 4°C. The purity of protein was assessed by denaturing gel electrophoresis (Laemmli 1970).
Purification of Mitochondrial Porin
Mitochondrial porin (VDAC) was isolated from N. crassa according to Freitag et al. 1982. In brief, mitochondrial outer membrane vesicles (1 mg protein per ml) were prepared as described by Künkele et al. 1998a and solubilized by incubation in 1% DDM, 50 mM potassium acetate, pH 7.0, 10 mM MOPS, 20% glycerol, and 1 mM PMSF at 4°C for 30 min. Insoluble material was removed by centrifugation at 226,200 g, and the supernatant was passed over a Resource Q anion exchange column (1 ml resin; Amersham Pharmacia Biotech). Mitochondrial porin was recovered from the flow through fraction and stored at 4°C at a protein concentration of 3 mg/ml.
Size Exclusion Chromatography
For determination of the molecular mass of Tom40, 100 µg of protein was loaded onto a Superose 6 size exclusion column (Amersham Pharmacia Biotech) equilibrated with 50 mM potassium acetate, 10 mM MOPS, pH 7.0, 10% glycerol, and 0.1% DDM at 4°C. Protein was eluted at a flow rate of 0.5 ml/min. Control experiments were carried out using purified TOM core complex. The molecular masses of Tom40 complexes were estimated using thyroglobulin (669 kD), apoferritin (443 kD), alcohol dehydrogenase (155 kD), and carboanhydrase (29 kD) as protein standards.
CD Spectroscopy
CD measurements were performed using a Jasco J-715 spectrometer in quartz cuvettes of 0.1-cm path length. Spectra were recorded at 4°C from 198 to 250 nm with a resolution of 0.1 nm and an acquisition time of 50 nm/min. Final CD spectra were obtained by averaging 10 consecutive scans and corrected for background by subtraction of spectra of protein-free samples recorded under the same conditions. Mean ellipticities per residue 
R were calculated based on the molar protein concentration and the amino acid composition (Tom40 and mitochondrial porin) or based on the absolute protein concentration and a mean residue molecular weight of 113 (TOM core complex). The concentrations of Tom40 and mitochondrial porin were determined by ultraviolet absorbance spectroscopy after unfolding of protein in 7.2 M urea and using extinction coefficients 
W,280nm 5,600 M–1cm–1 for tryptophan and Y,280nm 1,200 M–1cm–1 for tyrosine (Pace et al. 1995). Secondary structure predictions were based on algorithms by Sreerama and Woody 1993 using the Dicroprot version 2.5 software package by G. Deleage (CNRS).
ATR-FTIR
ATR-FTIR was performed using a Nicolet 740 FT spectrometer. The spectrometer was purged continuously with nitrogen gas to remove water vapor. The internal reflection element was a germanium crystal (ATR plate) with an aperture angle of 45°. Purified TOM core complex, Tom40, or mitochondrial porin was dialyzed against 5 mM phosphate buffer at 4°C. 50–100 µg of protein was applied onto one side of the glow-discharged ATR plate and taken to dryness under a stream of nitrogen. The ATR plate was sealed in a home built sample holder. Infrared (IR) spectra were recorded from 600 to 4,000 cm–1 at a resolution of better than 2 cm–1. The data are means of 1,024 scans. To differentiate -helical components from random coil, the sample compartment was flushed with D2O-saturated nitrogen for 120 min at room temperature. This shifted the absorption peak corresponding to the random coil structure elements from 1,655 to 1,642 cm–1. The exchange of hydrogen with deuterium was monitored by repeated measurements and judged by decrease of the amide band II centered around 1,530 cm–1. The content of β-sheet, random coil, and -helical secondary structure elements was estimated by analyzing the amide I region between 1,600 and 1,700 cm–1 using Fourier self deconvolution according to Kauppinen et al. 1981 for determining the position of absorption bands, and constrained band fitting to original spectra essentially by following the approach described by Byler and Susi 1986. Spectra of mitochondrial porin revealed a significant contribution of residual lipid, indicated by carbonyl ester vibrations 1,730 cm–1. These absorptions were fitted and subtracted from porin spectra before further quantitative analysis.
Electrophysiological Procedures
Conductance measurements of Tom40 in planar black lipid membranes were carried out as described previously (Benz et al. 1978; Künkele et al. 1998a). Membranes were formed from a 1% (wt/vol) solution of diphytanoyl phosphatidyl choline (Avanti Polar Lipids) in n-decane/butanol (9:1 vol/vol) across circular holes (surface area 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). To improve the insertion of protein into the lipid membranes, purified TOM core complex and Tom40 were mixed with an aqueous suspension of ergosterol (Fluka) before addition to the aqueous phase bathing the black lipid membrane. Membrane currents were measured at a membrane potential of +20 mV with a pair of Ag/AgCl electrodes (Metrohm) using a 428-current amplifier (Keithley Instruments, Inc.). Amplified signals were monitored with an analogue/digital storage oscilloscope (HM 407; Hameg) and recorded with a strip chart recorder. Single channel analysis was carried out according to previously described methods (Künkele et al. 1998b). Voltages are given as Vcis–Vtrans.
EM
Purified Tom40 (0.1 mg protein/ml) was adsorbed to glow-discharged carbon-coated grids (Cu, 600 mesh) for 30 s. The grids were washed twice with deionized water, blotted with filter paper, and stained with 2% (wt/vol) uranyl acetate for 30–60 s. EM images of Tom40 were recorded using a Philips CM 12 electron transmission microscope equipped with a VIPS computer (TVIPS) and a large area CCD camera (Photometrics). The microscope was operated at 120 kV. Images were taken at an underfocus of 1.5 µm, a nominal EM magnification of 35.000x and a postmagnification factor of 1.934 on the CCD camera. This corresponded to a pixel size of 0.355 nm at the specimen.
Single particle image processing was carried out on a Silicon graphics workstation using the EM software (Hegerl 1996). Images were low-pass filtered to the first zero of the electron microscope transfer function corresponding to a cutoff frequency of 2.3 nm–1. A total of 1,550 particles were manually marked in the digitized images. After extraction of frames with 64 x 64 pixels, images were subjected to multireference alignment using synthetic model images with one, two, three, and four pores as first references (Frank et al. 1981). Particle images of the two most prominent classes were resubjected to separate alignment, multivariate statistical analysis (Frank and van Heel 1982), and averaging. 20 eigenimages of each class were calculated. Each data set was subsequently split into 20 groups using the 6 most significant eigenimages.
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350 kD (Fig. 1 D), indicating that Tom40 is organized in a high molecular mass complex similar to the TOM core complex.
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7%) than one pore particles (36%). This suggests that the components Tom22, Tom7, and Tom6 are not necessary for the formation of the two pore form of the TOM complex but help to stabilize this form. The pore size of the Tom40 particles ranged between 2 and 3 nm and was comparable with that of the TOM core complex and the TOM holo complex (Ahting et al. 1999).
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min), the wavelength at which the ellipticity equals zero (crossover), and the spectral deconvolution results. Tom40 revealed a spectrum with a crossover of the baseline at 202 nm and a minimum at 216.5 nm (Fig. 3 A). At wavelengths >245 nm, the CD spectrum approached ellipticity values close to zero, indicating that the Tom40 preparation was virtually free of higher order aggregates, which would cause light scattering effects and interfere with the interpretation of the data. CD measurements of the TOM core complex yielded a spectrum with additional local minima at 208 and 222 nm, which are characteristic of -helical structure (Fig. 3 B). Both spectra were markedly different from that of mitochondrial porin (Fig. 1 C), which yielded a spectrum with a large positive ellipticity <206 nm and a less intense minimum centered at 216 nm (Fig. 3 C). The content of -helix of Tom40 was higher than proposed previously on the basis of structure predictions (Court et al. 1995; Mannella et al. 1996).
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-helical, random coil, and β-sheet structure elements (Fig. 4, A–D). Evaluation of this frequency region by Fourier self deconvolution and curve fitting revealed for Tom40 and the TOM core complex a content of β-sheet structure of 31 and 30%, respectively (Table ). This was significantly less than that of mitochondrial porin of Neurospora (Fig. 4 E). The shape of the amide I band of porin was typical of proteins with a high content of antiparallel β-sheet. Also for Tom40, the spectral component at 1,695 cm–1 indicated the existence of antiparallel β-strands. Analysis of the spectra of deuterated proteins (Fig. 4B and Fig. D) resulted in estimates of 22 and 33% -helical structure for Tom40 and the TOM core complex. In agreement with the CD measurements, the content of -helical structure of Tom40 was again larger than that of mitochondrial porin (Fig. 4 F). The amount of β-sheet structure of Tom40 based on CD was less than that calculated from IR spectroscopy. It should be noted that the prediction of β-strand structures by CD measurements tends generally to underestimate β-sheet relative to -helical structures.
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2.8 nS (Fig. 5 B). The mean conductance of isolated Tom40 insertions was comparable to those of the TOM holo complex (Künkele et al. 1998a), the TOM core complex, and protease-treated core complex lacking the hydrophilic domains of the receptor component Tom22 (Ahting et al. 1999).
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0 mV, they were fully open, and they closed with slow kinetics at potentials of either polarity. In addition, a fast flicker between the three main conductance levels occurred only at voltages of one polarity around 70 mV (Fig. 6 A). These characteristics are similar to those of the dimeric peptide-sensitive channel (PSC) identified in outer membrane and holo complex fractions of Neurospora (Künkele et al. 1998b). The channels most often found in the Tom40 fraction had similar selectivity and voltage-dependence properties, but their maximum conductance (550 pS in 150 mM KCl) was half that of the channels described above. They exhibited only two main conductance levels separated by jumps of 440 pS and a fast flicker at voltages of one polarity (Fig. 6 B). They are thus similar to the monomeric form of the Neurospora PSC described previously (Künkele et al. 1998b). This form was also found in the core complex fraction but with a lower probability than the dimeric form. These results indicate that Tom40 can form the protein translocation channel of the mitochondrial outer membrane.
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When Tom40-containing proteoliposomes were fused to bilayers, not only monomeric PSC-type channels were observed but, with a frequency of <25% of all channels recorded, two other types of channels were observed (Fig. 6 D), which were not seen using holo or core complex proteoliposomes. Like PSCs, both types had cationic selectivity, but their maximum conductance and voltage dependence were different. The first type was characterized by multiple conductance levels and rectification. The second type was a pore devoid of voltage dependence. Differing from the PSCs, the two types of channels were not blocked by pF1β at the concentration of 1 µM (data not shown). A likely explanation for the occurrence of these unusual channels is the relative instability of Tom40 compared with the very stable TOM core complex; this could lead to the formation of nonnative Tom40 channels and nontypical Tom40 particles in the electron microscopic pictures in the purified Tom40 preparations (see Fig. 2).
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Analysis of Tom40 preparations was reported previously for yeast Tom40 obtained by expression in Escherichia coli and refolding from urea-solubilized inclusion bodies (Hill et al. 1998). These preparations are different in several aspects from the Tom40 purified from TOM complex under nondenaturing conditions described here.
The maximum conductance level of recombinant Tom40 was reported to be 360 pS in 250 mM KCl (Hill et al. 1998). This is a rather low conductance that does not fit to previous measurements of TOM complex channels in planar lipid membranes. The conductance of single channels (the major form found in the present study, which corresponds to one pore particle) is 550 pS in 150 mM KCl, corresponding to 900 pS in 250 mM KCl both in TOM40 (this study) and holo complex fractions (Künkele et al. 1998a,Künkele et al. 1998b). Similar values were observed previously using different techniques for S. cerevisiae and adrenal cortex channels (Thieffry et al. 1992; Bathori et al. 1996).
Also, the spectral properties of purified Neurospora Tom40 were different from those of recombinant Tom40. The CD spectra of recombinant yeast Tom40 showed crossovers of the ellipticity to the baseline at 217 nm and a broad minimum >230 nm (Hill et al. 1998). Again, this may suggest that the folding of refolded recombinant membrane protein Tom40 differs considerably from that of Tom40 isolated from mitochondria. We presume that gentle isolation of Tom40 from the native complex conserves the basic structure and function of the channel, whereas expression in E. coli as inclusion bodies and renaturation from 8 M urea may not produce the correct higher order structure.
Tom40 was predicted to traverse the mitochondrial outer membrane as a series of antiparallel β-strands that form a β-barrel (Court et al. 1995; Mannella et al. 1996). A novel multiple alignment algorithm, called the Gibbs sampling algorithm, was used previously to detect motif-encoding regions in sequences of bacterial outer membrane proteins that correspond to transmembrane β-strands in bacterial porins (Neuwald et al. 1995). This bacterial motif has been used to screen sets of mitochondrial membrane protein sequences. Matches occurred in two proteins: mitochondrial porin and the outer membrane protein import pore Tom40. This suggested a structural relatedness between Tom40 and the bacterial and mitochondrial pore proteins (Mannella et al. 1996). CD measurements of bacterially expressed Tom40 of S. cerevisiae were suggested to indicate a predominance of >60% β-sheet (Hill et al. 1998) and high structural similarity to members of the porin membrane protein family.
We have performed CD and FTIR spectroscopy measurements with purified Neurospora Tom40. A maximum of 31% of Neurospora Tom40 was found to adopt β-sheet topology, whereas the calculated helix content is 
22%. Surprisingly, the content of β-sheet is markedly less than that of mitochondrial porin, which in agreement with previous studies (Shao et al. 1996; Koppel et al. 1998) revealed predominantly β-sheet structure (48% β-sheet, 15% -helix).
An important question is whether a single Tom40 protomer can form a protein translocation pore. Our data predict that 108 amino acid residues of Tom40 may be organized in β secondary structure elements. The mean radius of a regular β-barrel can be computed according to the number of β-strands and the shear number (Murzin et al. 1994). Given an inner diameter of 2.5 nm of the barrel, implying a larger diameter of the barrel backbone, and applying common values for the shear number (Murzin et al. 1994), >14 β-strands are necessary to fulfill the structural requirements. This denotes that the β-strands of Tom40 consist of less than seven to eight amino acid residues on average and cannot be expected to span the hydrophobic region of the membrane, if the shear number is close to or greater than the number of β-strands. In fact, the average of β-strands in bacterial outer membrane proteins are made up by 12 amino acid residues (Buchanan 1999; Koebnik et al. 2000; Schulz 2000). If this would also apply to Tom40, our data suggest the existence of only 8–10 β-strands in Tom40. This number of strands can hardly be expected to form stain-filled and open channels of the observed size.
On the basis of these considerations, we speculate that a β-strand solvent-accessible pore with a diameter of 2.5 nm could only be formed by β-barrel structures contributed by more than one Tom40 protomer. These protomers could be imagined to assemble in a way similar to staphylococcal -hemolysin (Song et al. 1996) or the bacterial multidrug efflux and protein export channel TolC (Koronakis et al. 2000). However, to fully understand the structural basis of pore formation, further studies addressing the oligomeric state of Tom40 will be required. It seems possible that different oligomeric structures of Tom40 exist, similar to the bacterial protein translocase SecYEG, which forms primarily dimeric but also tetrameric structures. Formation of the tetramer is induced by SecA (Manting et al. 2000).
EM and image analysis of Tom40 revealed mainly single ring structures, whereas the TOM core complex consists predominantly of double rings. The core complex is composed of about eight Tom40 molecules (Ahting et al. 1999). From the size of the Tom40 complex as indicated by gel filtration analysis, the stoichiometry cannot be determined with certainty. Higher resolution images of the Tom40 complex are required to resolve structural symmetries.
It seems clear from the findings reported here that oligomeric Tom40 forms the basic structure of the TOM complex. At the same time, our results demonstrate that the other constituents of the core complex Tom22, Tom7, and Tom6 have important functions in generating a stable two pore channel. This agrees well with genetic experiments in which the genes for these components were deleted (Hönlinger et al. 1996; van Wilpe et al. 1999).
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Acknowledgments |
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This research was supported by the Sonderforschungsbereich 184 of the Deutsche Forschungsgemeinschaft (to S. Nussberger and W. Neupert) and the Münchner Medizinische Wochenschrift (to S. Nussberger).
Submitted: 7 March 2001
Revised: 26 April 2001
Accepted: 30 April 2001
Abbreviations used in this paper: ATR, attenuated total reflection; CD, circular dichroism; DDM, n-dodecyl β-D-maltoside; FTIR, Fourier transform IR spectroscopy; IR, infrared; Ni-NTA, nickel nitrilotriacetic acid agarose; OG, n-octyl β,D-glucopyranoside; PSC, peptide-sensitive channel; TOM, translocase of the outer mitochondrial membrane; VDAC, voltage-dependent anion channel.
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