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Protein translocase of the outer mitochondrial membrane: role of import receptors in the structural organization of the TOM complex1
doi:10.1006/jmbi.2001.5365 available online at http://www.idealibrary.com on
J. Mol. Biol. (2002) 316, 657±666
Protein Translocase of the Outer Mitochondrial Membrane: Role of Import Receptors in the Structural Organization of the TOM Complex Kirstin Model1, Thorsten Prinz2, Teresa Ruiz1, Michael Radermacher1 Thomas Krimmer2, Werner KuÈhlbrandt1, Nikolaus Pfanner2* and Chris Meisinger2 1
Department of Structural Biology, Max-Planck-Institut fuÈr Biophysik, D-60528 Frankfurt am Main, Germany 2
Institut fuÈr Biochemie und Molekularbiologie, UniversitaÈt Freiburg, Hermann-HerderStraûe 7, D-79104, Freiburg Germany
The mitochondrial outer membrane contains a multi-subunit machinery responsible for the speci®c recognition and translocation of precursor proteins. This translocase of the outer membrane (TOM) consists of three receptor proteins, Tom20, Tom22 and Tom70, the channel protein Tom40, and several small Tom proteins. Single-particle electron microscopy analysis of the Neurospora TOM complex has led to different views with two or three stain-®lled centers resembling channels. Based on biochemical and electron microscopy studies of the TOM complex isolated from yeast mitochondria, we have discovered the molecular reason for the different number of channel-like structures. The TOM complex from wild-type yeast contains up to three stain-®lled centers, while from a mutant yeast selectively lacking Tom20, the TOM complex particles contain only two channel-like structures. From mutant mitochondria lacking Tom22, native electrophoresis separates an 80 kDa subcomplex that consists of Tom40 only and is functional for accumulation of a precursor protein. We conclude that while Tom40 forms the import channels, the two receptors Tom22 and Tom20 are required for the organization of Tom40 dimers into larger TOM structures. # 2002 Elsevier Science Ltd.
*Corresponding author
Keywords: electron microscopy; image analysis; mitochondria; protein sorting; Saccharomyces cerevisiae
Introduction The vast majority of mitochondrial proteins are synthesized as precursor proteins on cytosolic polysomes. The precursor proteins are recognized by receptors on the mitochondrial surface and are translocated through a general import pore (GIP) across the outer mitochondrial membrane.1 ± 4 The receptors and GIP are assembled into a large oligomeric complex, referred to as the translocase of the Abbreviations used: BN-PAGE, blue native polyacrylamide gel electrophoresis; GIP, general import pore; FRC, Fourier ring correlation; IEF, isoelectric focussing; MSA, multivariate statistical analysis; MTX, methotrexate; SOM, self-organizing maps; TOM, translocase of the outer mitochondrial membrane; TomX, subunit of the translocase of the outer membrane of X kDa. E-mail address of the corresponding author: [email protected] 0022-2836/02/030657±10 $35.00/0
outer membrane (TOM). Two main classes of precursor proteins can be distinguished. Preproteins with a cleavable, N-terminal targeting signal (presequence) are recognized initially by the receptor Tom20 and are then transferred to the central receptor, Tom22. Proteins with internal targeting signals are recognized preferentially by Tom70 and are transferred subsequently to Tom22. The channel across the outer membrane is formed by Tom40, a major component of the TOM complex that is essential for cell viability under all growth conditions.5 ± 8 The receptor Tom22 as well as three small Tom proteins, Tom5, Tom6 and Tom7, are associated tightly with the Tom40 channel, forming the so-called GIP complex or TOM core complex, while Tom20 and Tom70 are associated with this core complex more loosely.8 ± 14 The structural organization of the TOM machinery is still a subject of debate, in particular concerning the number of channel-like structures per TOM complex. Structural studies have been carried out # 2002 Elsevier Science Ltd.
658 on the TOM complex isolated from mitochondria of the fungus Neurospora crassa. In the presence of the detergent digitonin, both Tom20 and Tom70 were associated with the TOM complex and singleparticle analysis revealed the presence of three stain-®lled centers.12 Using the detergent dodecylmaltoside, a TOM core complex lacking both Tom20 and Tom70 was isolated. This complex contained only two stain-®lled channels.13 It is not certain whether the use of a different detergent or the presence of Tom20 and Tom70, or both, was responsible for the different number of putative pores.13,15 A biochemical analysis of the TOM complex from the yeast Saccharomyces cerevisiae suggested the presence of up to three functional Tom40 channel units.7,11 An advantage of the yeast system is that individual Tom proteins can be deleted easily. However, the puri®cation of the yeast TOM complex in a suitable form for structural studies has not been reported up to now. For the work presented here, we puri®ed the yeast mitochondrial TOM complex via a Histagged Tom22 and investigated its structure by single-particle electron microscopy and image analysis. We show that the presence of Tom20 is selectively responsible for the difference between TOM complexes containing two or three stain-®lled pits. Moreover, biochemical analysis suggests that the deletion of Tom22 results in small functional Tom40 units that are single channels. These ®ndings indicate that the receptors Tom22 and Tom20 have well-de®ned, different roles in the organization of Tom40 channel units into larger complexes.
Results and Discussion Wild-type yeast TOM complex has up to three pore-like structures We used a yeast strain where the chromosomal copy of Tom22 was tagged with an extension coding for ten C-terminal histidine residues.14 The resulting strain grew like wild-type yeast. Since defects in the TOM machinery cause an impairment of yeast growth,5,7,16 ± 19 the tagging of Tom22 did not affect critical functions of the TOM machinery and thus the strain is referred to as wild-type here. Mitochondria were isolated and separated into membranes and soluble fraction. Upon lysis of the membranes with digitonin, the TOM complex was isolated via Ni-NTA af®nity chromatography (Figure 1(a)). The eluted complex showed a uniform running behavior on blue native polyacrylamide gel electrophoresis (BN-PAGE), migrating in the 400-500 kDa range (Figure 1(b), lane 1). Silver staining did not reveal the presence of complexes with different mobility. Immunodecoration with antibodies directed against Tom40 con®rmed that the TOM complex had been isolated (Figure 1(b), lane 2). The complex contained Tom40, Tom22, Tom20 and small Tom proteins (Figure 1(c), lane 2). While Tom20 was present in the complex under
Translocase of Outer Mitochondrial Membrane
Figure 1. Puri®cation of the TOM complex of yeast wild-type mitochondria. (a) Puri®cation scheme. (b) BNPAGE of the puri®ed TOM complex. Lane 1, silver stain. Lane 2, immunodecoration with antibodies directed against Tom40. (c) SDS-PAGE of the isolated TOM complex, followed by immunodecoration with antibodies directed against Tom70, Tom40, Tom22, Tom20 or Tom5 (lane 2; 10 % of eluted material is shown). For comparison, lane 1 shows 0.15 % of the total material before puri®cation. (d) Two-dimensional electrophoresis of the puri®ed TOM complex with IEF (pH 3-9) in the ®rst dimension and SDS-PAGE in the second dimension. The gel was stained with Coomassie brilliant blue. Proteins were identi®ed by immunodecoration with speci®c antibodies. Tom40 is found in one major spot and additional spots of low abundance (with different isoelectric point, but the same mobility on SDSPAGE).
these mild solubilization conditions (low concentration of digitonin), Tom70 was not found in the puri®ed complex (Figure 1(c), lane 2).14 The purity of the complex was also analyzed by two dimensional gel electrophoresis, isoelectric focussing followed by SDS-PAGE and staining with Coomassie blue (separating the proteins above 14 kDa), demonstrating the presence of Tom40, Tom22 and Tom20 (Figure 1(d)). The quaternary structure of the puri®ed TOM complex was analyzed by transmission electron microscopy, using uranyl acetate as the negative
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Translocase of Outer Mitochondrial Membrane
position of aligned images from the wild-type TOM complex revealed an average projection map with three centers of stain accumulation (Figure 2(c)). The resolution of the overall average Ê as determined by Fourier ring image was 25-27 A correlation (Figure 2(d)). The largest dimension of Ê . The stain-®lled the particles varied around 125 A centers differed slightly in contrast and de®nition against the surrounding protein mass. The diamÊ was consistent with eter of each center of 20-25 A the electrophysiological characterization of channels formed by puri®ed yeast Tom40 and the determination of their size with the polymer exclusion method,6 suggesting that these centers represented pores that may extend through the complex. Thus, the images represent top or bottom view projections of the complex (views perpendicular to the mitochondrial membrane plane) when stained with uranyl acetate and imaged at 0 tilt. The overall view of the yeast TOM complex, including size and number of stain-®lled centers, is therefore comparable to that of the N. crassa holo TOM complex.12 However, there is an important difference in molecular composition: Tom70 is present in the puri®ed Neurospora complex, but is absent in the yeast complex, demonstrating that Tom70 is not critical for the formation of the channel-like structures.
Figure 2. Electron microscopy of the isolated TOM complex from wild-type yeast. (a) Electron micrograph of negatively stained TOM complex isolated from wildtype yeast mitochondria. Tobacco mosaic virus (TMV) was included in the sample as a standard for the purpose of calibrating the magni®cation. Particles were stained with 2 % uranyl acetate. The scale bar represents 200 nm. (b) Initial reference generated by reference-free alignment (see Materials and Methods). (c) Total average calculated from 1010 particles. The scale bar represents 10 nm. (d) Fourier ringpcorrelation and noise p correlation reference curves (5/ n for FRC5 and 3/ n for FRC3). The resolution of the overall average image Ê based on the Fourier ring was determined to be 27 A Ê in the case of FRC3).34 correlation criterion FRC5 (25 A
stain. A total of 1010 particles were selected from electron micrographs (Figure 2(a)). The particles seemed to attach to the carbon ®lm in a preferred orientation. In order to generate an initial bias-free reference, we applied a reference-free alignment algorithm (see Materials and Methods) to the data set of centered particles. The resulting initial reference particle had an oval outline with three darkly staining regions (Figure 2(b)). After iterative rounds of re®nement in translational and rotational alignment, the projections of all selected molecules were averaged, thereby improving the signal to noise ratio. The superim-
The TOM complex of yeast mitochondria lacking Tom20 has two channels There were two possible explanations for the difference between the three stain-accumulating centers observed in the TOM holo complex12 and the two pores found in the TOM core complex13 of Neurospora: it could be due either to the presence or absence of Tom20 or to the use of different detergents. To distinguish between these two possibilities, we generated a yeast strain that carried His-tagged Tom22, but lacked Tom20 (tom20). Mitochondria that were isolated from the tom20 strain selectively lacked Tom20, while all other Tom proteins as well as marker proteins such as the most abundant outer membrane protein, porin, were present in wild-type amounts (Figure 3(a)). Upon lysis of the mitochondrial membranes with digitonin and separation by BN-PAGE, the mobility of the TOM complex of wild-type and tom20 mitochondria was analyzed by immunodecoration with anti-Tom40. The TOM complex from tom20 mitochondria indeed migrated faster than the wild-type complex (Figure 3(b)). We then puri®ed the tom20 TOM complex under conditions identical with those used for isolating the wild-type complex (Figure 1(a)), including the same detergent, such that the only difference between the puri®ed TOM complexes was the presence or absence of Tom20. The mobility on BN-PAGE of the puri®ed tom20 TOM complex was again faster than that of the wild-type TOM complex (Figure 3(c)).
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Translocase of Outer Mitochondrial Membrane
Figure 3. Puri®cation of the TOM complex from tom20 yeast mitochondria. (a) SDS-PAGE of total mitochondrial proteins, followed by immunodecoration with antibodies against the proteins indicated. WT, wild-type mitochondria; tom20, mitochondria lacking Tom20. (b) BN-PAGE of digitoninsolubilized mitochondria, followed by immunodecoration with antibodies directed against Tom40. (c) BN-PAGE of puri®ed TOM complexes and immunodecoration with anti-Tom40. The lengths of the blue native gels in (b) and (c) were adjusted such that the position of the marker proteins was comparable.
We performed an analogous electron microscopy study using a set of 1281 TOM particles (Figure 4(a)). The particles were found to attach to the carbon layer in orientations similar to that of the wild-type complex, providing a point of reference for comparison. The ®rst reference of the mutant TOM complex generated by reference-free alignment appeared as a particle with only two centers of stain accumulation (Figure 4(b)). This result revealed a signi®cant difference in overall appearance to the wild-type complex already at this early stage of image processing. This difference was con®rmed by the average image of the aligned particles from the mutant TOM complex, which revealed only two stain-®lled pits at a resolution similar to that of the wild-type average image (Figure 4(c) and (d)). These results suggest that the lack of Tom20 is responsible for the lack of a third pore-like structure. The size and relative position of these stain-®lled pits in the tom20 complex are similar to those found in the Neurospora TOM core complex isolated from wild-type mitochondria in the presence of dodecyl-maltoside.13 The Neurospora pits have been shown to be stain-®lled channels by tomography. We are therefore con®dent that the two stain-®lled centers seen in the tom20 complex and at least two of the centers seen in the wild-type complex are projection views of stain-®lled channels extending through the whole depth of the complex. Comparison of wild-type and tom20 TOM images We used two independent statistical approaches to analyze the differences between wild-type and tom20 TOM complexes. In the ®rst approach, correspondence analysis.20-22 with eight factors was
applied to analyze the variability of the data set. In two-dimensional projection maps of the eightdimensional space, the in¯uence of two out of eight factors can be illustrated (Figure 5). Out of 16 eigen-images we chose those that differentiated between two and three stain-®lled pore-like structures and calculated the corresponding factor maps (Figure 5(a) and (d)). Classi®cation of the data sets into separate groups was carried out by visual inspection of the factor maps according to the number of stain-®lled centers. The wild-type complex projections revealed that the particles could be divided roughly into three clusters (Figure 5(b)). The majority of particles (up to 70 %) belonged to the class with two fully visible channels and one less distinct staining center, i.e. an area in the upper section of the image that showed two spots of higher protein density separated by a low-density area. Particles with three fully visible centers of stain accumulation represented 15-20 % of the data set. About 15 % of the particles showed one fully visible channel and two less distinct pits. Superimposed density contours con®rmed the presence of three round low-density regions (Figure 5(c)). In contrast, with the TOM complex from tom20 mitochondria, particles with three staining centers could not be detected at all (Figure 5(d)). By far the most particles (> 95 %) had a de®ned double ring structure (Figure 5(e)), as illustrated by the contoured class image (Figure 5(f)). The lower pore exhibited sizes of up Ê in diameter. A minor fraction (4 % of to 30 A images) showed a reduced protein density at one end of the double ring (Figure 5(e)). In our second approach to analyzing the observed structural differences, we used neural networks to classify images based on an unsupervised intrinsically parallel method, by which
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Translocase of Outer Mitochondrial Membrane
Figure 4. Image analysis of the isolated TOM complex from tom20 yeast mitochondria. (a) Electron micrograph of negatively stained TOM complex isolated from the mutant yeast tom20. Particles were stained with 2 % uranyl acetate. The scale bar represents 200 nm. (b) Initial reference generated by reference-free alignment. (c) Total average calculated from 1281 particles. The scale bar represents 10 nm. (d) Fourier ring correlation and noise correlation reference curves. The resolution of the overall average image was determined Ê based on the Fourier ring correlation criterion to be 26 A Ê in case of FRC3). FRC5 (23 A
images may belong to several groups and can in¯uence their neighbors.23 Thus adjacent ``nodes'' are more similar than the more distant ones. The calculated self-organizing maps (SOM) shown in Figure 6 indicate some variations. In the left panel for the wild-type complex (Figure 6(a)), the node in the upper right corner resembles the total average (Figure 2(c)). Following the direction down to the bottom right corner of the panel or to the upper left corner, we observed a continuous transition from triple to double pore structures, while the different channels in the multi-channel structures seem to be more or less distinct. This may be due to differences in stain distribution or to small differences in the particle orientation on the carbon support ®lm. The channels of slightly tilted particles in projection will appear to have different
sizes and contrast depending on the tilt angle and direction of the tilt. The neural network analysis does not indicate any side views, which would be expected if the complexes were oriented randomly with respect to the carbon ®lm. It is therefore likely that the TOM complex attaches to the carbon support ®lm in one preferential orientation, although we cannot exclude the possibility that the particles are oriented up or down, as highly asymmetrical class averages in Figure 5(a) and (d) are absent. Hence the total averages shown in Figures 2 and 4 could be a combination of both views. However, as these two views would be similar at the current resolution, this would not in¯uence the interpretation of our results. In particular, the number of pores observed remains the same. Stain-excluding densities surrounding the pores were not symmetrical (Figures 5 and 6). Thus no symmetry was imposed at any stage of the analysis to avoid losing structural details. The three porelike structures of the wild-type complex are not distributed equally, since two of them are closer Ê between their together, with a distance of 34 A midpoints, while the center-to-center distance to Ê . In the case of the third pore measures up to 48 A the mutant complex lacking Tom20, a de®ned double ring structure is the predominant feature. It shows only minor variations in the protein densities forming the channels (Figure 6(b)). The most intriguing changes are found in the pore diameter (especially in the lower of the two rings in the Ê. Figure), which ranges from 20-30 A In summary, the main source of variability between images of wild-type and mutant complex is the presence or absence of a third annular feature due to the presence or absence of the receptor Tom20. A functional import unit formed by Tom40 alone The results shown so far indicate a strikingly different role of the receptors Tom20 and Tom70 in the structural organization of the TOM complex. While Tom70 is not required to obtain TOM particles with three stain-®lled centers, the presence or absence of Tom20 correlates with the observation of three versus two stain-®lled centers. This raises the question of the function of the third receptor, Tom22, in the complex. Biochemical evidence has shown that the lack of Tom22 in yeast results in TOM subcomplexes of 100 kDa consisting of a dimer of Tom40 and the small Tom proteins (Figure 7(a), lane 2).7 Use of the detergent Triton X-100 causes the dissociation of small Tom proteins as well as Tom20 from Tom40 under both wildtype and tom22 conditions.7,11,14 A large wildtype TOM complex of 300 kDa, the Tom40Tom22 oligomer, is stable in the presence of Triton X-100 (Figure 7(a), lane 3) and functional for accumulation of precursor proteins.11,14 The TOM subcomplex isolated from tom22 mitochondria
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Translocase of Outer Mitochondrial Membrane
Figure 5. Multivariate statistical analysis (MSA) and subsequent classi®cation. (a)-(c) Wild-type TOM complex. (d)(f) tom20 TOM complex. (a) and (d) Factor maps showing local averages of images calculated over a regular grid pattern covering the chosen factors: factor 3 versus factor 5 in the case of the wild-type TOM complex and factor 5 versus factor 7 in the case of the TOM complex lacking Tom20. (b) and (e) Average images of the classes obtained upon visual inspection of the factor maps. Either one, two or three fully visible stain-®lled centers are observed. (c) and (f) Selected class averages from (b) and (e) with superimposed density contours. The scale bars represent 10 nm.
that were lysed with Triton X-100 migrates at 80 kDa (Figure 7(a), lane 4). This subcomplex most likely consists of a single Tom40 dimer.7 We used two-dimensional gels, BN-PAGE followed by SDS-PAGE, to characterize this small subcomplex. The right half of Figure 7(b) shows tom22 mitochondria treated with Triton X-100. Tom40 migrated at 80 kDa, while the small Tom proteins
dissociated from the complex and migrated close to the front of the gel (Tom6 and Tom7 behaved like Tom5).7,11,14 Moreover, both electrophysiological data on recombinant yeast Tom40 and image analysis of puri®ed Neurospora Tom40 suggested that Tom40 alone was able to form a pore.6,8 Taken all these results together, the following hypothesis is appealing: Tom40 alone forms small channel
Figure 6. Neural network comparison of TOM complexes. Self-organizing maps of (a) wild-type and (b) tom20 TOM complexes are shown. Nine node images were selected as indicated and used as references for multireference alignment.
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Translocase of Outer Mitochondrial Membrane
Figure 7. Accumulation of a precursor protein in Tom40 units of tom22 mitochondria. (a) BNPAGE of wild-type (WT) and tom22 mitochondria solubilized either in 1 % digitonin (lanes 1 and 2) or 0.5 % Triton X-100 (lanes 3 and 4). The gel was blotted onto PVDF-membranes and immunodecorated with antibodies directed against Tom40. TOM, large complex of wild-type mitochondria solubilized in digitonin; TOM0 , large TOM complex of wild-type mitochondria solubilized in Triton X-100; 100 K, 80 K, small complexes of tom22 mitochondria solubilized in digitonin or Triton X100, respectively. (b) Two-dimensional gel ectrophoresis (BN-PAGE followed by SDS-PAGE) of wildtype mitochondria solubilized in 1 % digitonin and tom22 mitochondria solubilized in 0.5 % Triton X-100. The gels were blotted onto PVDF membranes and immunodecorated with antibodies directed against Tom40, Tom22 and Tom5. The asterisk (*) marks the running front of the blue native gel. (c) Radiolabelled AAC-DHFR was imported into mitochondria of WT and tom22 strains in the presence (lanes 3 and 5) or absence (lanes 2 and 4) of methotrexate (MTX). The mitochondria were reisolated. After solubilization in 1 % digitonin (WT) or 0.5 % Triton X-100 (tom22), the samples were separated by BNPAGE followed by transfer onto PVDF membranes. The endogenous Tom40 was shown by immunodecoration (lanes 1 and 6). The arrested precursor protein was detected by autoradiography.
units. The receptors Tom22 and Tom20 are required to organize several Tom40 dimers into larger TOM structures, leading to two-pore or three-pore complexes. However, one crucial piece of evidence to support this hypothesis has been missing. It has never been shown that Tom40 alone is indeed able to accumulate a precursor protein in transit. To provide this evidence, we used the ADP/ATP carrier fused to dihydrofolate reductase as a model precursor protein. A radiolabelled form of this model precursor, AAC-DHFR, was synthesized in rabbit reticulocyte lysate. When the DHFR domain was stabilized by the speci®c ligand methotrexate, the mitochondrial import machinery was not able to unfold it, and therefore AAC-DHFR accumulated in the large GIP (TOM) complex.7,14,24 Figure 7(c) demonstrates that the fusion protein (55 kDa) migrated at the expected position, i.e. 50 kDa above the band observed for the GIP complex (Figure 7(c), lanes 3 versus 1). Since only very small
amounts of the radiochemically labelled precursor protein were present, the mobility of the bulk of GIP complexes as analyzed by Western blotting did not change, and only the fraction binding the radiolabelled precursor was shifted to the corresponding higher molecular mass.24 We then performed the equivalent experiment with tom22 mitochondria, which were then lysed with Triton X-100. In the presence of methotrexate, AACDHFR indeed migrated at a position of 130 kDa (Figure 7(c), lane 5), corresponding to the size of the precursor protein plus the 80 kDa Tom40 dimer. We conclude that Tom40 alone can indeed form a functional unit for accumulation of precursor proteins.
Conclusions We report that the receptor Tom20 is selectively responsible for the presence of a third pore-like structure in images of the mitochondrial TOM
664 complex. The possibility that Tom70 with its seven tetratrico peptide repeat (TPR) motifs15 is critical for formation of a third pore can be excluded, since the puri®ed yeast TOM complex that shows three pore-like structures in our analysis does not contain Tom70. Moreover, the use of different detergents as a possible explanation for the appearance of three versus two pore-like structures in the Neurospora complex12,13 can be excluded, since the yeast TOM complexes from wild-type and tom20 mitochondria were isolated under identical conditions. There is no reason to assume that Tom20, which contains only one membrane-spanning segment and a single TPR motif,25,26 forms a pore by itself. Taken together, this and previous work6 ± 8 now demonstrate unambiguously that Tom40 alone is the pore-forming unit for precursor proteins in transit. We show that the receptors Tom22 and Tom20 are each crucial for a stable organization of Tom40 channel units into larger assemblies consisting of two or three pore-like structures, respectively. The re®ned statistical analysis indicates a dynamic behavior of TOM complexes. While some aspects, such as the images of partial pits, may be related to stain variation or slightly different orientations of TOM complexes on the grid, the variation in pore size of the fully visible pits points to dynamic changes of the pores. In particular, one of the pores of the double-pore particle from tom20 mitochondria reveals pore diameters between 20 Ê . In the wild-type TOM complex, the and 30 A three stain-accumulation centers are not distributed equally, but two are closer together, raising the possibility that upon removal of Tom20 these two pores may fuse to form a larger pore-like structure. The comparatively small difference in the mobility on BN-PAGE of the three pore-particle (wild-type) versus the two-pore particle (tom20) indicates that the loss of the third pore-like structure does not correlate with the loss of one-third of the mass of the TOM complex, but supports the view of a structural rearrangement of Tom40 molecules and other Tom proteins upon removal of Tom20. In summary, we conclude that the two receptors, Tom22 and Tom20, are critical for the assembly of Tom40 channel units and have distinct roles as organizers of Tom40 dimers into larger TOM structures.
Materials and Methods Isolation of the TOM complex from mitochondrial membranes via tagged Tom22 We used the S. cerevisiae strains MR103 (MATa his3200 leu2-1 ade2-101 trp1-63 ura3-52 lys2-801 tom22His10-HIS3; referred to as wild-type)14 and TK12 (MATa his3-200 leu2-1 ade2-101 trp1-63 ura3-52 lys2-801 tom20::URA3 tom22-His10-HIS3; referred to as tom20). TK12 was constructed from the original tom20 mutant strain17 MM112 by extending the open reading frame of TOM22 with a region coding for ten histidine residues.
Translocase of Outer Mitochondrial Membrane After growth on YPG medium (1 % (w/v) yeast extract, 2 % (w/v) Bacto peptone, 3 % (v/v) glycerol), mitochondria were isolated. The mitochondria (100 mg of protein) were resuspended in swelling buffer (10 mM Mops (pH 7.2), 1 mM EDTA), incubated for 30 minutes at 0 C and subjected to sonication at 0 C (3 10 pulses, settings: duty 60 % and level 6). The membrane fraction was collected by centrifugation at 100,000 g and 4 C for 15 minutes. After solubilization with 0.5 % (w/v) digitonin, 20 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 10 % (v/ v) glycerol, 50 mM NaCl and a clarifying spin, the supernatant was mixed with 1 ml of Ni-NTA resin (Qiagen) in loading buffer (20 mM Tris, pH 7.4, 0.5 mM EDTA, 10 % (v/v) glycerol, 0.2 % (w/v) digitonin, 250 mM NaCl, 30 mM imidazole). Washing was performed essentially with the same buffer containing 50 or 80 mM imidazole. The TOM complex was eluted with 200-400 mM imidazole and concentrated via a Microcon YM-100 (Millipore) to 5 % of its elution volume. During this procedure the elution buffer was exchanged against a concentration buffer (20 mM Tris-HCl (pH 7.2), 10 % (v/v) glycerol, 0.2 % (w/v) digitonin, 50 mM NaCl). All buffers contained 1 mM phenylmethylsulfonyl ¯uoride (PMSF). The integrity of the isolated TOM complex was con®rmed via BN-PAGE and its individual components were identi®ed by SDS-PAGE. BN-PAGE and two-dimensional gel electrophoresis Digitonin-solubilized mitochondria or isolated TOM complex in 0.2 % (w/v) digitonin, 20 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 10 % (v/v) glycerol, 50 mM NaCl were mixed with loading dye (100 mM bis-Tris (pH 7.0), 500 mM 6-aminocaproic acid, 5 % (w/v) Coomassie brilliant blue G250) and subjected to a 6 % to 16.5 % (w/v) polyacrylamide gradient gel.27 Two-dimensional gel electrophoresis (BN/SDS) To analyze co-migration of individual Tom proteins on BN-PAGE, single lanes from the ®rst-dimension BNPAGE were polymerized on top of a second-dimension SDS/polyacrylamide gel. After electrophoresis, gels were transferred onto PVDF membranes and Tom proteins were identi®ed by immunodecoration. Two-dimensional gel electrophoresis (IEF/SDS) Isolated TOM complex in a rehydration solution (2 M urea, 7 M thiourea, 0.2 % (w/v) digitonin, 2 % (w/v) Chaps, 0.4 % (w/v) DTT, and 1.25 % (v/v) ampholytes pH 3.5-10) was applied to an immobilized pH gradient gel (13 cm, pH 3-10L, Pharmacia). Upon rehydration for ten hours at 50 V and 17 C, iso-electric focussing (IEF) of the ®rst dimension was performed for 56,000 Vh at 17 C with the following steps: one hour at 300 V, one hour at 1000 V, one hour at 2000 V, and 15 hours at 3500 V. For the second dimension, 4 % to 16 % gradient Tricine-SDS-PAGE28 was used. The gel was stained with Coomassie brilliant blue. Accumulation of a precursor protein in the TOM complex Mitochondria were isolated from the TOM22-de®cient yeast strain OL201 (his3-200 leu2-1 ura3-52 trp1-63 tom22::HIS3 rho0 ; referred to as tom22) and the corresponding wild-type strain OL223 (his3-200 leu2-1
665
Translocase of Outer Mitochondrial Membrane ura3-52 trp1-63 rho0).7 The 35S-labeled fusion protein AAC-DHFR was generated by in vitro transcription and translation in rabbit reticulocyte lysate (Amersham) in the presence of [35S]methionine/cysteine (Amersham). Mitochondria (50 mg of protein) were incubated with the fusion protein in import buffer (3 % (w/v) bovine serum albumin, 250 mM sucrose, 5 mM MgCl2, 80 mM KCl, 10 mM Mops-KOH, pH 7.2) containing 2 mM NADH and 2 mM ATP for 25 minutes at 25 C. Arrest of the precursor protein was achieved by addition of 20 mM methotrexate (MTX) prior to import. The reisolated mitochondria were lysed in 50 ml of solubilization buffer containing 20 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 50 mM NaCl, 10 % (v/v) glycerol, 1 mM PMSF and either 1 % (w/v) digitonin or 0.5 % (w/v) Triton X-100. After a clarifying spin, the supernatant was loaded onto a 6 %-13 % blue native gel and accumulated precursor proteins were analyzed by autoradiography using the PhosphorImage technology (Molecular Dynamics).
Electron microscopy and image analysis For electron microscopy analysis, the isolated TOM complex (40 mg/ml in 0.2 % (w/v) digitonin, 20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 10 % (w/v) glycerol) was applied to freshly glow-discharged carbon-coated copper grids (400 mesh) and stained with 2 % (w/v) uranyl acetate following the deep staining technique.29 Micrographs were recorded in a Philips CM 120 electron microscope at an accelerating voltage of 100 kV with a calibrated magni®cation of 57.900 under lowdose conditions at an underfocus of about 1.5 mm. The quality of the micrographs was assessed by optical diffraction. Micrographs were scanned on a ¯at-bed SCAI (Zeiss) microdensitometer with a pixel size of 7 mm. Groups of 3 3 pixels were averaged, resulting in a ®nal Ê on the specimen scale. Image analysis pixel size of 3.6 A was performed using the SPIDER/WEB software30 with extensions and the XMIPP package.31 A minimum of 1000 particle images of each TOM complex preparation were extracted and normalized in contrast. The images were aligned by cross-correlation methods. A ®rst reference was created by a referencefree alignment algorithm,32 followed by iterations of rotational and translational alignments with updated references. Multivariate statistical analysis using eight factors20,33 was applied to check homogeneity and discard poorly de®ned images (less than 10 % in each preparation). Out of 16 eigen-images calculated at both ends of each factor, those were chosen that contributed to the dissection between two and three pore-like structures. Taking the corresponding two factors (factor 3 versus factor 5 for the wild-type complex, factor 5 versus factor 7 for the mutant complex), the factor maps were calculated. Classi®cation was based on visual inspection of the factor maps generated by correspondence analysis. The class averages showed either one, two or three fully visible stain-®lled centers. To assess the consistency of the data set and perform unsupervised classi®cation, the algorithm of self-organizing maps was applied.23 Based on nine selected nodes of the self-organizing maps, a multireference alignment was performed. A subsequent neural network analysis con®rmed the result of the initial neural network analysis both for wild-type and tom20. The resolution was calculated using the Fourier ring correlation criterion FRC5 (®ve times over noise correlation).34,35
Acknowledgements We thank Birgit SchoÈn®sch for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, the Sonderforschungsbereich 388, the Fonds der Chemischen Industrie/BMBF (N.P.), and the NSF grant DBI-9515518 (M.R).
References 1. Schatz, G. & Dobberstein, B. (1996). Common principles of protein translocation across membranes. Science, 271, 1519-1526. 2. Neupert, W. (1997). Protein import into mitochondria. Annu. Rev. Biochem. 66, 863-917. 3. Pfanner, N. & Geissler, A. (2001). Versatility of the mitochondrial protein import machinery. Nature Rev. Mol. Cell. Biol. 2, 339-349. 4. Truscott, K. N., Pfanner, N. & Voos, W. (2001). Transport of proteins into mitochondria. Rev. Physiol. Biochem. Pharmacol. 143, 81-136. 5. Baker, K. P., Schaniel, A., Vestweber, D. & Schatz, G. (1990). A yeast mitochondrial outer membrane protein essential for protein import and cell viability. Nature, 348, 605-609. 6. Hill, K., Model, K., Ryan, M. T., Dietmeier, K., Martin, F., Wagner, R. & Pfanner, N. (1998). Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins. Nature, 395, 516-521. 7. van Wilpe, S., Ryan, M. T., Hill, K., Maarse, A. C., Meisinger, C., Brix, J. et al. (1999). Tom22 is a multifunctional organizer of the mitochondrial preprotein translocase. Nature, 401, 485-489. 8. Ahting, U., Thieffry, M., Engelhardt, H., Hegerl, R., Neupert, W. & Nussberger, S. (2001). Tom40, the pore-forming component of the protein-conducting TOM channel in the outer membrane of mitochondria. J. Cell Biol. 153, 1151-1160. 9. HoÈnlinger, A., BoÈmer, U., Alconada, A., Eckerskorn, C., Lottspeich, F., Dietmeier, K. & Pfanner, N. (1996). Tom7 modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related role in protein import. EMBO J. 15, 2125-2137. 10. Dietmeier, K., HoÈnlinger, A., BoÈmer, U., Dekker, P. J. T., Eckerskorn, C., Lottspeich, F. et al. (1997). Tom5 functionally links mitochondrial preprotein receptors to the general import pore. Nature, 388, 195-200. 11. Dekker, P. J. T., Ryan, M. T., Brix, J., MuÈller, H., HoÈnlinger, A. & Pfanner, N. (1998). Preprotein translocase of the outer mitochondrial membrane: molecular dissection and assembly of the general import pore complex. Mol. Cell. Biol. 18, 6515-6524. 12. KuÈnkele, K. P., Heins, S., Dembowski, M., Nargang, F. E., Benz, R., Thieffry, M. et al. (1998). The preprotein translocation channel of the outer membrane of mitochondria. Cell, 93, 1009-1019. 13. Ahting, U., Thun, C., Hegerl, R., Typke, D., Nargang, F. E., Neupert, W. & Nussberger, S. (1999). The TOM core complex: the general protein import pore of the outer membrane of mitochondria. J. Cell Biol. 147, 959-968. 14. Meisinger, C., Ryan, M. T., Hill, K., Model, K., Lim, J. H., Sickmann, A. et al. (2001). Protein import channel of the outer mitochondrial membrane: a highly
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stable Tom40-Tom22 core structure differentially interacts with preproteins, small Tom proteins, and import receptors. Mol. Cell. Biol. 21, 2337-2348. Verschoor, A. & Lithgow, T. (1999). A ®rst glimpse at the structure of the TOM translocase from the mitochondrial outer membrane. J. Cell Biol. 147, 905907. Ramage, L., Junne, T., Hahne, K., Lithgow, T. & Schatz, G. (1993). Functional cooperation of mitochondrial protein import receptors in yeast. EMBO J. 12, 4115-4123. Moczko, M., Ehmann, B., GaÈrtner, F., HoÈnlinger, A., SchaÈfer, E. & Pfanner, N. (1994). Deletion of the receptor MOM19 strongly impairs import of cleavable preproteins into Saccharomyces cerevisiae mitochondria. J. Biol. Chem, 269, 9045-9051. Lithgow, T., Junne, T., Suda, K., Gratzer, S. & Schatz, G. (1994). The mitochondrial outer membrane protein Mas22p is essential for protein import and viability of yeast. Proc. Natl Acad. Sci. USA, 91, 11973-11977. HoÈnlinger, A., KuÈbrich, M., Moczko, M., GaÈrtner, F., Mallet, L., Bussereau, F. et al. (1995). The mitochondrial receptor complex: Mom22 is essential for cell viability and directly interacts with preproteins. Mol. Cell. Biol. 15, 3382-3389. van Heel, M. & Frank, J. (1981). Use of multivariate statistics in analysing the images of biological macromolecules. Ultramicroscopy, 6, 187-194. Frank, J. (1990). Classi®cation of macromolecular assemblies studied as ``single particles''. Quart. Rev. Biophys. 23, 281-329. Frank, J. & Radermacher, M. (1992). Three-dimensional reconstruction of single particles negatively stained or in vitreous ice. Ultramicroscopy, 46, 241262. Marabini, R. & Carazo, J. M. (1994). Pattern recognition and classi®cation of images of biological macromolecules using arti®cial neural networks. Biophys. J. 66, 1804-1814. Ryan, M. T., MuÈller, H. & Pfanner, N. (1999). Functional staging of ADP/ATP carrier translocation across the outer mitochondrial membrane. J. Biol. Chem. 27, 20619-20627.
25. Schneider, H., SoÈllner, T., Dietmeier, K., Eckerskorn, C., Lottspeich, F., TruÈlzsch, B. et al. (1991). Targeting of the master receptor MOM19 to mitochondria. Science, 254, 1659-1662. 26. Abe, Y., Shodai, T., Muto, T., Mihara, K., Torii, H., Nishikawa, S. et al. (2000). Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20. Cell, 100, 551-560. 27. SchaÈgger, H. & von Jagow, G. (1991). Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 199, 223-231. 28. SchaÈgger, H. & von Jagow, G. (1987). Tricinesodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368-379. 29. Stoops, J. K., Baker, T. S., Schroeter, J. P., Kolodziej, S. J., Niu, X. D. & Reed, L. J. (1992). Three-dimensional structure of the truncated core of the Saccharomyces cerevisiae pyruvate dehydrogenase complex determined from negative stain and cryoelectron microscopy images. J. Biol. Chem. 267, 24769-24775. 30. Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M. & Leith, A. (1996). SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related ®elds. J. Struct. Biol. 116, 190-199. 31. Marabini, R., Masegosa, I. M., San Martin, M. C., Marco, S., Fernandez, J. J., de la Fraga, L. G. et al. (1996). XMIPP: an image processing package for electron microscopy. J. Struct. Biol. 116, 237-240. 32. Marco, S., Chagoyen, M., Delafraga, L. G., Carazo, J. M. & Carrascosa, J. L. (1996). A variant to the random approximation of the reference-free alignment algorithm. Ultramicroscopy, 66, 5-10. 33. Frank, J. & van Heel, M. (1982). Correspondence analysis of aligned images of biological particles. J. Mol. Biol. 161, 134-137. 34. Saxton, W. O. & Baumeister, W. (1982). The correlation averaging of a regularly arranged bacterial cell envelope protein. J. Microsc. 127, 127-138. 35. Radermacher, M. (1988). Three-dimensional reconstruction of single particles from random and nonrandom tilt series. J. Electr. Microsc. Tech. 9, 359-394.
Edited by M. Yaniv (Received 23 August 2001; received in revised form 5 December 2001; accepted 10 December 2001)
J. Mol. Biol. (2002) 316, 657±666
Protein Translocase of the Outer Mitochondrial Membrane: Role of Import Receptors in the Structural Organization of the TOM Complex Kirstin Model1, Thorsten Prinz2, Teresa Ruiz1, Michael Radermacher1 Thomas Krimmer2, Werner KuÈhlbrandt1, Nikolaus Pfanner2* and Chris Meisinger2 1
Department of Structural Biology, Max-Planck-Institut fuÈr Biophysik, D-60528 Frankfurt am Main, Germany 2
Institut fuÈr Biochemie und Molekularbiologie, UniversitaÈt Freiburg, Hermann-HerderStraûe 7, D-79104, Freiburg Germany
The mitochondrial outer membrane contains a multi-subunit machinery responsible for the speci®c recognition and translocation of precursor proteins. This translocase of the outer membrane (TOM) consists of three receptor proteins, Tom20, Tom22 and Tom70, the channel protein Tom40, and several small Tom proteins. Single-particle electron microscopy analysis of the Neurospora TOM complex has led to different views with two or three stain-®lled centers resembling channels. Based on biochemical and electron microscopy studies of the TOM complex isolated from yeast mitochondria, we have discovered the molecular reason for the different number of channel-like structures. The TOM complex from wild-type yeast contains up to three stain-®lled centers, while from a mutant yeast selectively lacking Tom20, the TOM complex particles contain only two channel-like structures. From mutant mitochondria lacking Tom22, native electrophoresis separates an 80 kDa subcomplex that consists of Tom40 only and is functional for accumulation of a precursor protein. We conclude that while Tom40 forms the import channels, the two receptors Tom22 and Tom20 are required for the organization of Tom40 dimers into larger TOM structures. # 2002 Elsevier Science Ltd.
*Corresponding author
Keywords: electron microscopy; image analysis; mitochondria; protein sorting; Saccharomyces cerevisiae
Introduction The vast majority of mitochondrial proteins are synthesized as precursor proteins on cytosolic polysomes. The precursor proteins are recognized by receptors on the mitochondrial surface and are translocated through a general import pore (GIP) across the outer mitochondrial membrane.1 ± 4 The receptors and GIP are assembled into a large oligomeric complex, referred to as the translocase of the Abbreviations used: BN-PAGE, blue native polyacrylamide gel electrophoresis; GIP, general import pore; FRC, Fourier ring correlation; IEF, isoelectric focussing; MSA, multivariate statistical analysis; MTX, methotrexate; SOM, self-organizing maps; TOM, translocase of the outer mitochondrial membrane; TomX, subunit of the translocase of the outer membrane of X kDa. E-mail address of the corresponding author: [email protected] 0022-2836/02/030657±10 $35.00/0
outer membrane (TOM). Two main classes of precursor proteins can be distinguished. Preproteins with a cleavable, N-terminal targeting signal (presequence) are recognized initially by the receptor Tom20 and are then transferred to the central receptor, Tom22. Proteins with internal targeting signals are recognized preferentially by Tom70 and are transferred subsequently to Tom22. The channel across the outer membrane is formed by Tom40, a major component of the TOM complex that is essential for cell viability under all growth conditions.5 ± 8 The receptor Tom22 as well as three small Tom proteins, Tom5, Tom6 and Tom7, are associated tightly with the Tom40 channel, forming the so-called GIP complex or TOM core complex, while Tom20 and Tom70 are associated with this core complex more loosely.8 ± 14 The structural organization of the TOM machinery is still a subject of debate, in particular concerning the number of channel-like structures per TOM complex. Structural studies have been carried out # 2002 Elsevier Science Ltd.
658 on the TOM complex isolated from mitochondria of the fungus Neurospora crassa. In the presence of the detergent digitonin, both Tom20 and Tom70 were associated with the TOM complex and singleparticle analysis revealed the presence of three stain-®lled centers.12 Using the detergent dodecylmaltoside, a TOM core complex lacking both Tom20 and Tom70 was isolated. This complex contained only two stain-®lled channels.13 It is not certain whether the use of a different detergent or the presence of Tom20 and Tom70, or both, was responsible for the different number of putative pores.13,15 A biochemical analysis of the TOM complex from the yeast Saccharomyces cerevisiae suggested the presence of up to three functional Tom40 channel units.7,11 An advantage of the yeast system is that individual Tom proteins can be deleted easily. However, the puri®cation of the yeast TOM complex in a suitable form for structural studies has not been reported up to now. For the work presented here, we puri®ed the yeast mitochondrial TOM complex via a Histagged Tom22 and investigated its structure by single-particle electron microscopy and image analysis. We show that the presence of Tom20 is selectively responsible for the difference between TOM complexes containing two or three stain-®lled pits. Moreover, biochemical analysis suggests that the deletion of Tom22 results in small functional Tom40 units that are single channels. These ®ndings indicate that the receptors Tom22 and Tom20 have well-de®ned, different roles in the organization of Tom40 channel units into larger complexes.
Results and Discussion Wild-type yeast TOM complex has up to three pore-like structures We used a yeast strain where the chromosomal copy of Tom22 was tagged with an extension coding for ten C-terminal histidine residues.14 The resulting strain grew like wild-type yeast. Since defects in the TOM machinery cause an impairment of yeast growth,5,7,16 ± 19 the tagging of Tom22 did not affect critical functions of the TOM machinery and thus the strain is referred to as wild-type here. Mitochondria were isolated and separated into membranes and soluble fraction. Upon lysis of the membranes with digitonin, the TOM complex was isolated via Ni-NTA af®nity chromatography (Figure 1(a)). The eluted complex showed a uniform running behavior on blue native polyacrylamide gel electrophoresis (BN-PAGE), migrating in the 400-500 kDa range (Figure 1(b), lane 1). Silver staining did not reveal the presence of complexes with different mobility. Immunodecoration with antibodies directed against Tom40 con®rmed that the TOM complex had been isolated (Figure 1(b), lane 2). The complex contained Tom40, Tom22, Tom20 and small Tom proteins (Figure 1(c), lane 2). While Tom20 was present in the complex under
Translocase of Outer Mitochondrial Membrane
Figure 1. Puri®cation of the TOM complex of yeast wild-type mitochondria. (a) Puri®cation scheme. (b) BNPAGE of the puri®ed TOM complex. Lane 1, silver stain. Lane 2, immunodecoration with antibodies directed against Tom40. (c) SDS-PAGE of the isolated TOM complex, followed by immunodecoration with antibodies directed against Tom70, Tom40, Tom22, Tom20 or Tom5 (lane 2; 10 % of eluted material is shown). For comparison, lane 1 shows 0.15 % of the total material before puri®cation. (d) Two-dimensional electrophoresis of the puri®ed TOM complex with IEF (pH 3-9) in the ®rst dimension and SDS-PAGE in the second dimension. The gel was stained with Coomassie brilliant blue. Proteins were identi®ed by immunodecoration with speci®c antibodies. Tom40 is found in one major spot and additional spots of low abundance (with different isoelectric point, but the same mobility on SDSPAGE).
these mild solubilization conditions (low concentration of digitonin), Tom70 was not found in the puri®ed complex (Figure 1(c), lane 2).14 The purity of the complex was also analyzed by two dimensional gel electrophoresis, isoelectric focussing followed by SDS-PAGE and staining with Coomassie blue (separating the proteins above 14 kDa), demonstrating the presence of Tom40, Tom22 and Tom20 (Figure 1(d)). The quaternary structure of the puri®ed TOM complex was analyzed by transmission electron microscopy, using uranyl acetate as the negative
659
Translocase of Outer Mitochondrial Membrane
position of aligned images from the wild-type TOM complex revealed an average projection map with three centers of stain accumulation (Figure 2(c)). The resolution of the overall average Ê as determined by Fourier ring image was 25-27 A correlation (Figure 2(d)). The largest dimension of Ê . The stain-®lled the particles varied around 125 A centers differed slightly in contrast and de®nition against the surrounding protein mass. The diamÊ was consistent with eter of each center of 20-25 A the electrophysiological characterization of channels formed by puri®ed yeast Tom40 and the determination of their size with the polymer exclusion method,6 suggesting that these centers represented pores that may extend through the complex. Thus, the images represent top or bottom view projections of the complex (views perpendicular to the mitochondrial membrane plane) when stained with uranyl acetate and imaged at 0 tilt. The overall view of the yeast TOM complex, including size and number of stain-®lled centers, is therefore comparable to that of the N. crassa holo TOM complex.12 However, there is an important difference in molecular composition: Tom70 is present in the puri®ed Neurospora complex, but is absent in the yeast complex, demonstrating that Tom70 is not critical for the formation of the channel-like structures.
Figure 2. Electron microscopy of the isolated TOM complex from wild-type yeast. (a) Electron micrograph of negatively stained TOM complex isolated from wildtype yeast mitochondria. Tobacco mosaic virus (TMV) was included in the sample as a standard for the purpose of calibrating the magni®cation. Particles were stained with 2 % uranyl acetate. The scale bar represents 200 nm. (b) Initial reference generated by reference-free alignment (see Materials and Methods). (c) Total average calculated from 1010 particles. The scale bar represents 10 nm. (d) Fourier ringpcorrelation and noise p correlation reference curves (5/ n for FRC5 and 3/ n for FRC3). The resolution of the overall average image Ê based on the Fourier ring was determined to be 27 A Ê in the case of FRC3).34 correlation criterion FRC5 (25 A
stain. A total of 1010 particles were selected from electron micrographs (Figure 2(a)). The particles seemed to attach to the carbon ®lm in a preferred orientation. In order to generate an initial bias-free reference, we applied a reference-free alignment algorithm (see Materials and Methods) to the data set of centered particles. The resulting initial reference particle had an oval outline with three darkly staining regions (Figure 2(b)). After iterative rounds of re®nement in translational and rotational alignment, the projections of all selected molecules were averaged, thereby improving the signal to noise ratio. The superim-
The TOM complex of yeast mitochondria lacking Tom20 has two channels There were two possible explanations for the difference between the three stain-accumulating centers observed in the TOM holo complex12 and the two pores found in the TOM core complex13 of Neurospora: it could be due either to the presence or absence of Tom20 or to the use of different detergents. To distinguish between these two possibilities, we generated a yeast strain that carried His-tagged Tom22, but lacked Tom20 (tom20). Mitochondria that were isolated from the tom20 strain selectively lacked Tom20, while all other Tom proteins as well as marker proteins such as the most abundant outer membrane protein, porin, were present in wild-type amounts (Figure 3(a)). Upon lysis of the mitochondrial membranes with digitonin and separation by BN-PAGE, the mobility of the TOM complex of wild-type and tom20 mitochondria was analyzed by immunodecoration with anti-Tom40. The TOM complex from tom20 mitochondria indeed migrated faster than the wild-type complex (Figure 3(b)). We then puri®ed the tom20 TOM complex under conditions identical with those used for isolating the wild-type complex (Figure 1(a)), including the same detergent, such that the only difference between the puri®ed TOM complexes was the presence or absence of Tom20. The mobility on BN-PAGE of the puri®ed tom20 TOM complex was again faster than that of the wild-type TOM complex (Figure 3(c)).
660
Translocase of Outer Mitochondrial Membrane
Figure 3. Puri®cation of the TOM complex from tom20 yeast mitochondria. (a) SDS-PAGE of total mitochondrial proteins, followed by immunodecoration with antibodies against the proteins indicated. WT, wild-type mitochondria; tom20, mitochondria lacking Tom20. (b) BN-PAGE of digitoninsolubilized mitochondria, followed by immunodecoration with antibodies directed against Tom40. (c) BN-PAGE of puri®ed TOM complexes and immunodecoration with anti-Tom40. The lengths of the blue native gels in (b) and (c) were adjusted such that the position of the marker proteins was comparable.
We performed an analogous electron microscopy study using a set of 1281 TOM particles (Figure 4(a)). The particles were found to attach to the carbon layer in orientations similar to that of the wild-type complex, providing a point of reference for comparison. The ®rst reference of the mutant TOM complex generated by reference-free alignment appeared as a particle with only two centers of stain accumulation (Figure 4(b)). This result revealed a signi®cant difference in overall appearance to the wild-type complex already at this early stage of image processing. This difference was con®rmed by the average image of the aligned particles from the mutant TOM complex, which revealed only two stain-®lled pits at a resolution similar to that of the wild-type average image (Figure 4(c) and (d)). These results suggest that the lack of Tom20 is responsible for the lack of a third pore-like structure. The size and relative position of these stain-®lled pits in the tom20 complex are similar to those found in the Neurospora TOM core complex isolated from wild-type mitochondria in the presence of dodecyl-maltoside.13 The Neurospora pits have been shown to be stain-®lled channels by tomography. We are therefore con®dent that the two stain-®lled centers seen in the tom20 complex and at least two of the centers seen in the wild-type complex are projection views of stain-®lled channels extending through the whole depth of the complex. Comparison of wild-type and tom20 TOM images We used two independent statistical approaches to analyze the differences between wild-type and tom20 TOM complexes. In the ®rst approach, correspondence analysis.20-22 with eight factors was
applied to analyze the variability of the data set. In two-dimensional projection maps of the eightdimensional space, the in¯uence of two out of eight factors can be illustrated (Figure 5). Out of 16 eigen-images we chose those that differentiated between two and three stain-®lled pore-like structures and calculated the corresponding factor maps (Figure 5(a) and (d)). Classi®cation of the data sets into separate groups was carried out by visual inspection of the factor maps according to the number of stain-®lled centers. The wild-type complex projections revealed that the particles could be divided roughly into three clusters (Figure 5(b)). The majority of particles (up to 70 %) belonged to the class with two fully visible channels and one less distinct staining center, i.e. an area in the upper section of the image that showed two spots of higher protein density separated by a low-density area. Particles with three fully visible centers of stain accumulation represented 15-20 % of the data set. About 15 % of the particles showed one fully visible channel and two less distinct pits. Superimposed density contours con®rmed the presence of three round low-density regions (Figure 5(c)). In contrast, with the TOM complex from tom20 mitochondria, particles with three staining centers could not be detected at all (Figure 5(d)). By far the most particles (> 95 %) had a de®ned double ring structure (Figure 5(e)), as illustrated by the contoured class image (Figure 5(f)). The lower pore exhibited sizes of up Ê in diameter. A minor fraction (4 % of to 30 A images) showed a reduced protein density at one end of the double ring (Figure 5(e)). In our second approach to analyzing the observed structural differences, we used neural networks to classify images based on an unsupervised intrinsically parallel method, by which
661
Translocase of Outer Mitochondrial Membrane
Figure 4. Image analysis of the isolated TOM complex from tom20 yeast mitochondria. (a) Electron micrograph of negatively stained TOM complex isolated from the mutant yeast tom20. Particles were stained with 2 % uranyl acetate. The scale bar represents 200 nm. (b) Initial reference generated by reference-free alignment. (c) Total average calculated from 1281 particles. The scale bar represents 10 nm. (d) Fourier ring correlation and noise correlation reference curves. The resolution of the overall average image was determined Ê based on the Fourier ring correlation criterion to be 26 A Ê in case of FRC3). FRC5 (23 A
images may belong to several groups and can in¯uence their neighbors.23 Thus adjacent ``nodes'' are more similar than the more distant ones. The calculated self-organizing maps (SOM) shown in Figure 6 indicate some variations. In the left panel for the wild-type complex (Figure 6(a)), the node in the upper right corner resembles the total average (Figure 2(c)). Following the direction down to the bottom right corner of the panel or to the upper left corner, we observed a continuous transition from triple to double pore structures, while the different channels in the multi-channel structures seem to be more or less distinct. This may be due to differences in stain distribution or to small differences in the particle orientation on the carbon support ®lm. The channels of slightly tilted particles in projection will appear to have different
sizes and contrast depending on the tilt angle and direction of the tilt. The neural network analysis does not indicate any side views, which would be expected if the complexes were oriented randomly with respect to the carbon ®lm. It is therefore likely that the TOM complex attaches to the carbon support ®lm in one preferential orientation, although we cannot exclude the possibility that the particles are oriented up or down, as highly asymmetrical class averages in Figure 5(a) and (d) are absent. Hence the total averages shown in Figures 2 and 4 could be a combination of both views. However, as these two views would be similar at the current resolution, this would not in¯uence the interpretation of our results. In particular, the number of pores observed remains the same. Stain-excluding densities surrounding the pores were not symmetrical (Figures 5 and 6). Thus no symmetry was imposed at any stage of the analysis to avoid losing structural details. The three porelike structures of the wild-type complex are not distributed equally, since two of them are closer Ê between their together, with a distance of 34 A midpoints, while the center-to-center distance to Ê . In the case of the third pore measures up to 48 A the mutant complex lacking Tom20, a de®ned double ring structure is the predominant feature. It shows only minor variations in the protein densities forming the channels (Figure 6(b)). The most intriguing changes are found in the pore diameter (especially in the lower of the two rings in the Ê. Figure), which ranges from 20-30 A In summary, the main source of variability between images of wild-type and mutant complex is the presence or absence of a third annular feature due to the presence or absence of the receptor Tom20. A functional import unit formed by Tom40 alone The results shown so far indicate a strikingly different role of the receptors Tom20 and Tom70 in the structural organization of the TOM complex. While Tom70 is not required to obtain TOM particles with three stain-®lled centers, the presence or absence of Tom20 correlates with the observation of three versus two stain-®lled centers. This raises the question of the function of the third receptor, Tom22, in the complex. Biochemical evidence has shown that the lack of Tom22 in yeast results in TOM subcomplexes of 100 kDa consisting of a dimer of Tom40 and the small Tom proteins (Figure 7(a), lane 2).7 Use of the detergent Triton X-100 causes the dissociation of small Tom proteins as well as Tom20 from Tom40 under both wildtype and tom22 conditions.7,11,14 A large wildtype TOM complex of 300 kDa, the Tom40Tom22 oligomer, is stable in the presence of Triton X-100 (Figure 7(a), lane 3) and functional for accumulation of precursor proteins.11,14 The TOM subcomplex isolated from tom22 mitochondria
662
Translocase of Outer Mitochondrial Membrane
Figure 5. Multivariate statistical analysis (MSA) and subsequent classi®cation. (a)-(c) Wild-type TOM complex. (d)(f) tom20 TOM complex. (a) and (d) Factor maps showing local averages of images calculated over a regular grid pattern covering the chosen factors: factor 3 versus factor 5 in the case of the wild-type TOM complex and factor 5 versus factor 7 in the case of the TOM complex lacking Tom20. (b) and (e) Average images of the classes obtained upon visual inspection of the factor maps. Either one, two or three fully visible stain-®lled centers are observed. (c) and (f) Selected class averages from (b) and (e) with superimposed density contours. The scale bars represent 10 nm.
that were lysed with Triton X-100 migrates at 80 kDa (Figure 7(a), lane 4). This subcomplex most likely consists of a single Tom40 dimer.7 We used two-dimensional gels, BN-PAGE followed by SDS-PAGE, to characterize this small subcomplex. The right half of Figure 7(b) shows tom22 mitochondria treated with Triton X-100. Tom40 migrated at 80 kDa, while the small Tom proteins
dissociated from the complex and migrated close to the front of the gel (Tom6 and Tom7 behaved like Tom5).7,11,14 Moreover, both electrophysiological data on recombinant yeast Tom40 and image analysis of puri®ed Neurospora Tom40 suggested that Tom40 alone was able to form a pore.6,8 Taken all these results together, the following hypothesis is appealing: Tom40 alone forms small channel
Figure 6. Neural network comparison of TOM complexes. Self-organizing maps of (a) wild-type and (b) tom20 TOM complexes are shown. Nine node images were selected as indicated and used as references for multireference alignment.
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Translocase of Outer Mitochondrial Membrane
Figure 7. Accumulation of a precursor protein in Tom40 units of tom22 mitochondria. (a) BNPAGE of wild-type (WT) and tom22 mitochondria solubilized either in 1 % digitonin (lanes 1 and 2) or 0.5 % Triton X-100 (lanes 3 and 4). The gel was blotted onto PVDF-membranes and immunodecorated with antibodies directed against Tom40. TOM, large complex of wild-type mitochondria solubilized in digitonin; TOM0 , large TOM complex of wild-type mitochondria solubilized in Triton X-100; 100 K, 80 K, small complexes of tom22 mitochondria solubilized in digitonin or Triton X100, respectively. (b) Two-dimensional gel ectrophoresis (BN-PAGE followed by SDS-PAGE) of wildtype mitochondria solubilized in 1 % digitonin and tom22 mitochondria solubilized in 0.5 % Triton X-100. The gels were blotted onto PVDF membranes and immunodecorated with antibodies directed against Tom40, Tom22 and Tom5. The asterisk (*) marks the running front of the blue native gel. (c) Radiolabelled AAC-DHFR was imported into mitochondria of WT and tom22 strains in the presence (lanes 3 and 5) or absence (lanes 2 and 4) of methotrexate (MTX). The mitochondria were reisolated. After solubilization in 1 % digitonin (WT) or 0.5 % Triton X-100 (tom22), the samples were separated by BNPAGE followed by transfer onto PVDF membranes. The endogenous Tom40 was shown by immunodecoration (lanes 1 and 6). The arrested precursor protein was detected by autoradiography.
units. The receptors Tom22 and Tom20 are required to organize several Tom40 dimers into larger TOM structures, leading to two-pore or three-pore complexes. However, one crucial piece of evidence to support this hypothesis has been missing. It has never been shown that Tom40 alone is indeed able to accumulate a precursor protein in transit. To provide this evidence, we used the ADP/ATP carrier fused to dihydrofolate reductase as a model precursor protein. A radiolabelled form of this model precursor, AAC-DHFR, was synthesized in rabbit reticulocyte lysate. When the DHFR domain was stabilized by the speci®c ligand methotrexate, the mitochondrial import machinery was not able to unfold it, and therefore AAC-DHFR accumulated in the large GIP (TOM) complex.7,14,24 Figure 7(c) demonstrates that the fusion protein (55 kDa) migrated at the expected position, i.e. 50 kDa above the band observed for the GIP complex (Figure 7(c), lanes 3 versus 1). Since only very small
amounts of the radiochemically labelled precursor protein were present, the mobility of the bulk of GIP complexes as analyzed by Western blotting did not change, and only the fraction binding the radiolabelled precursor was shifted to the corresponding higher molecular mass.24 We then performed the equivalent experiment with tom22 mitochondria, which were then lysed with Triton X-100. In the presence of methotrexate, AACDHFR indeed migrated at a position of 130 kDa (Figure 7(c), lane 5), corresponding to the size of the precursor protein plus the 80 kDa Tom40 dimer. We conclude that Tom40 alone can indeed form a functional unit for accumulation of precursor proteins.
Conclusions We report that the receptor Tom20 is selectively responsible for the presence of a third pore-like structure in images of the mitochondrial TOM
664 complex. The possibility that Tom70 with its seven tetratrico peptide repeat (TPR) motifs15 is critical for formation of a third pore can be excluded, since the puri®ed yeast TOM complex that shows three pore-like structures in our analysis does not contain Tom70. Moreover, the use of different detergents as a possible explanation for the appearance of three versus two pore-like structures in the Neurospora complex12,13 can be excluded, since the yeast TOM complexes from wild-type and tom20 mitochondria were isolated under identical conditions. There is no reason to assume that Tom20, which contains only one membrane-spanning segment and a single TPR motif,25,26 forms a pore by itself. Taken together, this and previous work6 ± 8 now demonstrate unambiguously that Tom40 alone is the pore-forming unit for precursor proteins in transit. We show that the receptors Tom22 and Tom20 are each crucial for a stable organization of Tom40 channel units into larger assemblies consisting of two or three pore-like structures, respectively. The re®ned statistical analysis indicates a dynamic behavior of TOM complexes. While some aspects, such as the images of partial pits, may be related to stain variation or slightly different orientations of TOM complexes on the grid, the variation in pore size of the fully visible pits points to dynamic changes of the pores. In particular, one of the pores of the double-pore particle from tom20 mitochondria reveals pore diameters between 20 Ê . In the wild-type TOM complex, the and 30 A three stain-accumulation centers are not distributed equally, but two are closer together, raising the possibility that upon removal of Tom20 these two pores may fuse to form a larger pore-like structure. The comparatively small difference in the mobility on BN-PAGE of the three pore-particle (wild-type) versus the two-pore particle (tom20) indicates that the loss of the third pore-like structure does not correlate with the loss of one-third of the mass of the TOM complex, but supports the view of a structural rearrangement of Tom40 molecules and other Tom proteins upon removal of Tom20. In summary, we conclude that the two receptors, Tom22 and Tom20, are critical for the assembly of Tom40 channel units and have distinct roles as organizers of Tom40 dimers into larger TOM structures.
Materials and Methods Isolation of the TOM complex from mitochondrial membranes via tagged Tom22 We used the S. cerevisiae strains MR103 (MATa his3200 leu2-1 ade2-101 trp1-63 ura3-52 lys2-801 tom22His10-HIS3; referred to as wild-type)14 and TK12 (MATa his3-200 leu2-1 ade2-101 trp1-63 ura3-52 lys2-801 tom20::URA3 tom22-His10-HIS3; referred to as tom20). TK12 was constructed from the original tom20 mutant strain17 MM112 by extending the open reading frame of TOM22 with a region coding for ten histidine residues.
Translocase of Outer Mitochondrial Membrane After growth on YPG medium (1 % (w/v) yeast extract, 2 % (w/v) Bacto peptone, 3 % (v/v) glycerol), mitochondria were isolated. The mitochondria (100 mg of protein) were resuspended in swelling buffer (10 mM Mops (pH 7.2), 1 mM EDTA), incubated for 30 minutes at 0 C and subjected to sonication at 0 C (3 10 pulses, settings: duty 60 % and level 6). The membrane fraction was collected by centrifugation at 100,000 g and 4 C for 15 minutes. After solubilization with 0.5 % (w/v) digitonin, 20 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 10 % (v/ v) glycerol, 50 mM NaCl and a clarifying spin, the supernatant was mixed with 1 ml of Ni-NTA resin (Qiagen) in loading buffer (20 mM Tris, pH 7.4, 0.5 mM EDTA, 10 % (v/v) glycerol, 0.2 % (w/v) digitonin, 250 mM NaCl, 30 mM imidazole). Washing was performed essentially with the same buffer containing 50 or 80 mM imidazole. The TOM complex was eluted with 200-400 mM imidazole and concentrated via a Microcon YM-100 (Millipore) to 5 % of its elution volume. During this procedure the elution buffer was exchanged against a concentration buffer (20 mM Tris-HCl (pH 7.2), 10 % (v/v) glycerol, 0.2 % (w/v) digitonin, 50 mM NaCl). All buffers contained 1 mM phenylmethylsulfonyl ¯uoride (PMSF). The integrity of the isolated TOM complex was con®rmed via BN-PAGE and its individual components were identi®ed by SDS-PAGE. BN-PAGE and two-dimensional gel electrophoresis Digitonin-solubilized mitochondria or isolated TOM complex in 0.2 % (w/v) digitonin, 20 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 10 % (v/v) glycerol, 50 mM NaCl were mixed with loading dye (100 mM bis-Tris (pH 7.0), 500 mM 6-aminocaproic acid, 5 % (w/v) Coomassie brilliant blue G250) and subjected to a 6 % to 16.5 % (w/v) polyacrylamide gradient gel.27 Two-dimensional gel electrophoresis (BN/SDS) To analyze co-migration of individual Tom proteins on BN-PAGE, single lanes from the ®rst-dimension BNPAGE were polymerized on top of a second-dimension SDS/polyacrylamide gel. After electrophoresis, gels were transferred onto PVDF membranes and Tom proteins were identi®ed by immunodecoration. Two-dimensional gel electrophoresis (IEF/SDS) Isolated TOM complex in a rehydration solution (2 M urea, 7 M thiourea, 0.2 % (w/v) digitonin, 2 % (w/v) Chaps, 0.4 % (w/v) DTT, and 1.25 % (v/v) ampholytes pH 3.5-10) was applied to an immobilized pH gradient gel (13 cm, pH 3-10L, Pharmacia). Upon rehydration for ten hours at 50 V and 17 C, iso-electric focussing (IEF) of the ®rst dimension was performed for 56,000 Vh at 17 C with the following steps: one hour at 300 V, one hour at 1000 V, one hour at 2000 V, and 15 hours at 3500 V. For the second dimension, 4 % to 16 % gradient Tricine-SDS-PAGE28 was used. The gel was stained with Coomassie brilliant blue. Accumulation of a precursor protein in the TOM complex Mitochondria were isolated from the TOM22-de®cient yeast strain OL201 (his3-200 leu2-1 ura3-52 trp1-63 tom22::HIS3 rho0 ; referred to as tom22) and the corresponding wild-type strain OL223 (his3-200 leu2-1
665
Translocase of Outer Mitochondrial Membrane ura3-52 trp1-63 rho0).7 The 35S-labeled fusion protein AAC-DHFR was generated by in vitro transcription and translation in rabbit reticulocyte lysate (Amersham) in the presence of [35S]methionine/cysteine (Amersham). Mitochondria (50 mg of protein) were incubated with the fusion protein in import buffer (3 % (w/v) bovine serum albumin, 250 mM sucrose, 5 mM MgCl2, 80 mM KCl, 10 mM Mops-KOH, pH 7.2) containing 2 mM NADH and 2 mM ATP for 25 minutes at 25 C. Arrest of the precursor protein was achieved by addition of 20 mM methotrexate (MTX) prior to import. The reisolated mitochondria were lysed in 50 ml of solubilization buffer containing 20 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 50 mM NaCl, 10 % (v/v) glycerol, 1 mM PMSF and either 1 % (w/v) digitonin or 0.5 % (w/v) Triton X-100. After a clarifying spin, the supernatant was loaded onto a 6 %-13 % blue native gel and accumulated precursor proteins were analyzed by autoradiography using the PhosphorImage technology (Molecular Dynamics).
Electron microscopy and image analysis For electron microscopy analysis, the isolated TOM complex (40 mg/ml in 0.2 % (w/v) digitonin, 20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 10 % (w/v) glycerol) was applied to freshly glow-discharged carbon-coated copper grids (400 mesh) and stained with 2 % (w/v) uranyl acetate following the deep staining technique.29 Micrographs were recorded in a Philips CM 120 electron microscope at an accelerating voltage of 100 kV with a calibrated magni®cation of 57.900 under lowdose conditions at an underfocus of about 1.5 mm. The quality of the micrographs was assessed by optical diffraction. Micrographs were scanned on a ¯at-bed SCAI (Zeiss) microdensitometer with a pixel size of 7 mm. Groups of 3 3 pixels were averaged, resulting in a ®nal Ê on the specimen scale. Image analysis pixel size of 3.6 A was performed using the SPIDER/WEB software30 with extensions and the XMIPP package.31 A minimum of 1000 particle images of each TOM complex preparation were extracted and normalized in contrast. The images were aligned by cross-correlation methods. A ®rst reference was created by a referencefree alignment algorithm,32 followed by iterations of rotational and translational alignments with updated references. Multivariate statistical analysis using eight factors20,33 was applied to check homogeneity and discard poorly de®ned images (less than 10 % in each preparation). Out of 16 eigen-images calculated at both ends of each factor, those were chosen that contributed to the dissection between two and three pore-like structures. Taking the corresponding two factors (factor 3 versus factor 5 for the wild-type complex, factor 5 versus factor 7 for the mutant complex), the factor maps were calculated. Classi®cation was based on visual inspection of the factor maps generated by correspondence analysis. The class averages showed either one, two or three fully visible stain-®lled centers. To assess the consistency of the data set and perform unsupervised classi®cation, the algorithm of self-organizing maps was applied.23 Based on nine selected nodes of the self-organizing maps, a multireference alignment was performed. A subsequent neural network analysis con®rmed the result of the initial neural network analysis both for wild-type and tom20. The resolution was calculated using the Fourier ring correlation criterion FRC5 (®ve times over noise correlation).34,35
Acknowledgements We thank Birgit SchoÈn®sch for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, the Sonderforschungsbereich 388, the Fonds der Chemischen Industrie/BMBF (N.P.), and the NSF grant DBI-9515518 (M.R).
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Edited by M. Yaniv (Received 23 August 2001; received in revised form 5 December 2001; accepted 10 December 2001)