The Isolated Complex of the Translocase of the Outer Membrane of Mitochondria

The complex of the translocase mitochondrial outer membrane (TOM), mediates recognition, unfolding, and translocation of preproteins. We have used a combination of biochemical and electrophysiological methods to study the properties of the preprotein-conducting pore of the purified TOM complex. The pore is cation-selective and voltage-gated. It shows three main conductance levels with characteristic slow and fast kinetics transitions to states of lower conductance following application of transmembrane voltages. These electrical properties distinguish it from the mitochondrial voltage-dependent anion channel (porin) and are identical to those of the previously described peptide-sensitive channel. Binding of antibodies to the C terminus of Tom40 on the intermembrane space side of the outer membrane modifies the channel properties and allows determination of the orientation of the channel within the lipid bilayer. Mitochondrial presequence peptides specifically interact with the pore and decrease the ion flow through the channel in a voltage-dependent manner. We propose that the presequence-induced closures of the pore are related to structural alterations of the TOM complex observed during the various stages of preprotein movement across the mitochondrial outer membrane.

The complex of the translocase mitochondrial outer membrane (TOM), mediates recognition, unfolding, and translocation of preproteins. We have used a combination of biochemical and electrophysiological methods to study the properties of the preprotein-conducting pore of the purified TOM complex. The pore is cation-selective and voltage-gated. It shows three main conductance levels with characteristic slow and fast kinetics transitions to states of lower conductance following application of transmembrane voltages. These electrical properties distinguish it from the mitochondrial voltagedependent anion channel (porin) and are identical to those of the previously described peptide-sensitive channel. Binding of antibodies to the C terminus of Tom40 on the intermembrane space side of the outer membrane modifies the channel properties and allows determination of the orientation of the channel within the lipid bilayer. Mitochondrial presequence peptides specifically interact with the pore and decrease the ion flow through the channel in a voltage-dependent manner. We propose that the presequence-induced closures of the pore are related to structural alterations of the TOM complex observed during the various stages of preprotein movement across the mitochondrial outer membrane.
Translocation of preproteins across biological membranes involves proteinaceous machineries consisting of multiple components (for reviews, see Refs. [1][2][3][4][5][6][7]. Some of these components are exposed to the cytosol and act as receptors by specifically deciphering the targeting information contained in the transported preproteins. Other proteins that are more embedded in the membrane also provide specific recognition sites for these targeting sequences and are responsible for the movement of the preproteins across the membrane, presumably by forming a preprotein-conducting hydrophilic pore. Only a few candidates for such aqueous channels have been described so far by electrophysiological methods (8 -11). Because entire membranes were used in those early studies, the connection between the observed channel activity and the preprotein translocation machinery could not be established with certainty. Recently, a purified component of the translocase of the chloroplast envelope membrane, termed Toc75, has been reported to form a voltage-gated ion-conducting pore, which appears to be involved in preprotein translocation (12).
In mitochondria, translocation of preproteins is mediated by the translocases of the outer (TOM 1 complex) and inner (translocase of the inner membrane at mitochondria complexes) membranes. The TOM complex consists of protease-sensitive receptors that interact with preproteins during specific recognition of the cognate preproteins at the mitochondrial surface (e.g. see Refs. [13][14][15][16]. Another set of TOM complex components is embedded in the outer membrane and is involved in the passage of the polypeptide chain into and across the membrane (17)(18)(19). These proteins provide a second presequence recognition site on the intermembrane space side of the membrane and were proposed to form the translocation pore. Much has recently been learned about the recognition of preproteins at the mitochondrial surface and about the various steps leading to movement of the polypeptide chain across the mitochondrial outer membrane (reviewed in Ref. 20). The existence of an ion-conducting pore in the isolated TOM complex has been reported recently (21), but a detailed characterization of this pore has not been performed. It was noted that the TOM complex channel resembles some properties of the cation-selective channel of large conductance termed the peptide-sensitive channel (PSC) 1 (22). This channel has been characterized as a constituent of the outer membrane (23) and suggested to be related to the preprotein translocation machinery (24). Definite proof for the identity of both channels requires an analysis of the ion-conducting properties of the purified TOM complex channel. Here, we report a detailed characterization of the properties of the purified TOM complex from the outer mem-brane of Neurospora mitochondria by employing a combination of biochemical and electrophysiological measurements.

EXPERIMENTAL PROCEDURES
Construction of a Neurospora Strain Expressing a Hexahistidinyltagged TOM22 Protein-To facilitate purification of the Neurospora crassa TOM complex, a hexahistidinyl tag was attached to the C terminus of Tom22 by adding six histidine codons to the wild-type tom22 gene. A PCR product containing about 700 base pairs of upstream sequence and the wild-type tom22 coding sequence with an additional six histidine codons at the C terminus was generated from a genomic tom22 clone. The resulting tom22 his6 gene was inserted into pGEM-T (Promega), and a clone containing the correct sequence (pHT22-11) was co-transformed with bleomycin resistance plasmid pAB520 (27) into spheroplasts of the sheltered heterokaryon ND-113-1. One of the nuclei in ND-113-1 carries a requirement for histidine, resistance to p-fluorophenylalanine, and a disrupted version of the essential tom22 gene (28). Strains bearing this nucleus are inviable as homokaryons. To select for transformation of this nucleus, transformants were plated on medium containing bleomycin, p-fluorophenylalanine, and histidine. Colonies were picked to slants containing histidine and p-fluorophenylalanine, allowed to conidiate, and purified once by streaking for single colony isolates on the same medium. Isolated strains requiring histidine should be homokaryotic for the nucleus containing that marker. Restoration of viability to this nucleus in a homokaryon indicates that the tom22 his6 gene has restored Tom22 function. Mitochondria isolated from histidine-requiring strains contained Tom22 his6 with slightly higher molecular mass than the normal protein in amounts indistinguishable from that in wild-type cells. Strain GR107 was selected for further studies. The growth behavior of the obtained strain (GR107) was identical to wild-type cells (not shown). Mitochondria isolated from strain GR107 imported preproteins at the same efficiency as wild-type organelles.
Growth of Cells and Biochemical Procedures-The following published procedures were used: growth of N. crassa strain GR107 (requiring addition of 1 mM histidine to the culture medium), preparation of mitochondria and outer membrane vesicles (OMVs) (29,30), raising antisera and purification of IgG (31), SDS-PAGE, blotting of proteins onto nitrocellulose, immunostaining of blotted proteins using the ECL detection system (Amersham Pharmacia Biotech), and quantitation of the resulting films on an Image Master densitometer (Amersham Pharmacia Biotech) (30). Protein concentrations were determined by the Coomassie dye binding assay (Bio-Rad) or by the BCA reagent (Pierce) with bovine serum albumin as a standard.
Purification of the TOM Complex-Purification of the TOM complex was performed as described (21), with minor modifications: outer membrane vesicles with a protein concentration of 1 mg/ml were solubilized for 30 min in Buffer A (50 mM KCl, 10 mM MOPS, pH 7.0, and 1% digitonin) in the presence of 1 mM phenylmethylsulfonyl fluoride. The clarified extract (226, 200 ϫ g for 15 min) was bound for 1 h to a nickel-nitrilotriacetic acid affinity resin (Qiagen; 1 ml of nickel-nitrilotriacetic acid per 10 mg of OMV protein). The column was washed with 50 volumes of Buffer B (50 mM KCl, 10 mM MOPS, pH 7.0, and 0.5% digitonin) containing a gradient of 0 -60 mM imidazole. Bound material was eluted with 300 mM imidazole in Buffer B. The typical yield was 3% of total OMV protein content or about 50% of TOM complex content.
Electrophysiological Procedures-Phospholipids were from Avanti Polar Lipids (Alabaster, AL). Liposomes were prepared by extrusion through 100-nm-pore filters (Avestin Inc., Ottawa, Ontario, Canada) of a 7:3 mixture of bovine brain phosphatidylethanolamine (BBPE) and bovine brain phosphatidylserine (BBPS) in 20 mM Hepes/KOH, pH 7.5 (10 mg of phospholipid/ml) (32). Formation of proteoliposomes was achieved by adding 25 l (for "tip-dip" experiments) or 7 l (for planar bilayer experiments) of the protein suspension to 75 l of the liposome solution. The mixture was frozen in liquid nitrogen and stored at -80°C. Before use, it was thawed at room temperature and submitted to two additional freeze-thaw cycles. In tip-dip experiments, the bilayer was formed at the tip of microelectrodes by apposition of two monolayers derived from the proteoliposomes as described previously (25). In planar bilayer experiments, the bilayer was formed by the Mueller-Rudin method (33) using synthetic phospholipids dissolved in n-decane at 20 mg/ml. Channels were incorporated into the bilayer by fusion with the proteoliposomes in the presence of an osmotic gradient as previously reported (34). Voltages are given as V pipette Ϫ V bath in tip-dip records and V cis Ϫ V trans in planar bilayer records. Some experiments were performed using the porin-deficient yeast strain B5 (35). (30) were isolated from N. crassa strain GR107 containing a hexahistidinyl-tagged version of Tom22. The TOM complex was purified by affinity chromatography on nickel-nitrilotriacetic acid (21). For our electrophysiological measurements the depletion of other proteins, especially of the major outer membrane protein porin, the voltage-dependent anion channel (VDAC) was critical (36,37). Therefore, the purity of the isolated TOM complex was estimated by immunostaining analysis using specific antisera directed against Tom70, Tom40, Tom22, Tom20, and porin. The recovery of the purified TOM complex proteins was about 50% of total TOM complex present in the starting OMV preparation. Porin was not detectable in the isolated TOM complex by immunostaining (Fig. 1). Based on the minimum amount possible to detect by this method, the enrichment of TOM complex over porin can be calculated to be more than 1250-fold. The TOM complex in the outer membrane makes up about 6% of total outer membrane protein. We conclude that the purified TOM complex is virtually free of other outer membrane proteins, especially of porin.

The Isolated TOM Complex Is Free of Porin-OMVs
The Purified TOM Complex Contains a Cation-selective Pore That Is Identical in Its Properties to PSC-The ion-conducting pore of the purified TOM complex was analyzed by two independent electrophysiological methods, the planar bilayer technique (33) and the tip-dip method (38). For planar bilayer experiments, the TOM complex was inserted into liposomes by a freeze-thaw technique (25). A planar bilayer was formed from phospholipids in the presence of an ionic gradient between the cis and the trans compartments. The proteoliposomes were added to the cis compartment and allowed to fuse spontaneously with the bilayer at 0 mV (39). Fusion events leading to the insertion of a single channel were studied further. Channel incorporation into the bilayer always resulted in a positive current flowing from the cis to the trans compartment ( Fig. 2A). No such currents were detected using liposomes lacking the TOM complex (not shown). This suggested that the TOM complex channel has cationic selectivity.
To investigate the properties of the channel in detail, the salt concentrations of the cis and trans compartments were made The TOM complex (0.47 mg) was purified from OMVs (15 mg) isolated from Neurospora strain GR107 as described under "Experimental Procedures." Fractions of OMVs or purified TOM complex were analyzed by immunostaining for the indicated TOM complex proteins and for porin. The relative amounts of these proteins in the isolated TOM complex and in OMVs were determined by densitometry. To ensure evaluation of the immunostaining data in the linear range, various amounts were loaded on the gel. For optimal estimation of the depletion of porin in the purified TOM complex, a 10-fold higher amount of total isolated TOM complex as compared with the amount of OMVs was analyzed. Enrichment is defined as the amount of the respective component per g of purified TOM complex over the amount of the same component per g of total OMV protein.
identical, and currents were recorded at different potentials. The channel exhibited several conductance levels and a complex voltage dependence (Fig. 2B). Between ϩ30 and Ϫ30 mV, the channel was mostly in a state (termed level 3) corresponding to a conductance of 550 pS in 40 mM KCl. At higher voltages, it closed to conductance levels termed 2 and 1, which were separated from level 3 by 230 and 460 pS, respectively, in 40 mM KCl (Fig. 2B). No direct jumps between levels 1 and 3 and vice versa were detected. Two types of closures were observed. One type corresponded to a fast flicker between the three levels ( Fig. 2B, trace recorded at Ϫ70 mV). It was characterized by a voltage dependence in which the most probable state changed over a range of 20 mV from levels 3 to 2 to 1 at around Ϫ60 mV (Fig. 2C). For a given channel, these fast fluctuations occurred only at potentials of one polarity. Analysis of many channels revealed a similar frequency of the fast flicker at either positive or negative voltages, indicating a random orientation of the TOM complex channel in the planar bilayer.
A second type of closure was characterized by slow kinetics transitions to lower conductance levels. These closures were observed at both negative and positive voltages (see, e.g. traces at Ϫ40 and ϩ70 mV in Fig. 2B), distinguishing this channel property from the fast flicker. They were best illustrated in records in which a sawtooth voltage of low frequency was applied. (Fig. 3). Such records showed that the probability for the channel being in the lowest conductance levels increased with the voltage magnitude. A complete closure of the channel was observed neither when steady state voltages of high magnitude (Ͼ100 mV) of either polarity were applied for long duration (Ͼ1 min) nor during application of sawtooth voltages (Fig. 3).
Occasionally, cationic channels of lower conductance (275 pS in 40 mM KCl) were detected (not shown). They closed in one step with slow kinetics during voltage pulses of long duration of either polarity to a state of about 50 pS. This conductance jump represents half of the difference between levels 3 and 1 of the channel exhibiting three conductance levels. Even during long applications of high voltages, no complete closure occurred. These channels also exhibited fast fluctuations between the open and "closed" states at voltages around 60 mV of one polarity only. The similarity of the electrical properties of the two types of channels suggests that the channel exhibiting three main conductance levels represents a dimeric form composed of two identical and independent subunits.
The qualitative properties of the TOM complex channel did not depend on the ionic composition of the buffer solutions used in the bilayer experiment (see e.g. Fig. 2D). The channel exhibited a preference for K ϩ over Na ϩ . The conductance of level 3 was 850 pS in 150 mM NaCl and 1.25 nS in 150 mM KCl. The latter value is lower than expected from the value in 40 mM KCl (550 pS), which indicates a nonlinear dependence of the conductance on salt concentration. In conclusion, the isolated TOM complex encompasses an ion-conducting pore that is voltagegated. Our detailed electrophysiological characterization of this cation-selective channel indicates that it is identical to the previously described PSC (22).
The cationic channel was the only type of pore-forming activity detected in the purified TOM complex fraction. When proteoliposomes were prepared from OMVs and allowed to fuse with the planar bilayers, two types of channels were found. One exhibited the same electrical properties as that detected in the purified TOM complex (Fig. 4A). The ion selectivity was identical with a reversal potential of Ϫ54.4 Ϯ 1.6 mV (n ϭ 26) and Ϫ54.3 Ϯ 1.2 mV (n ϭ 27) for the channel in the TOM complex and the OMVs, respectively, recorded in 40 mM KCl, 5 mM MOPS/KOH, pH 7 (cis), and 5 mM MOPS/ KOH, pH 7 (trans), using a diphytanoyl phosphatidylcholine (DPPC) bilayer. The other frequently detected type of channel showed anionic selectivity. The electrophysiological properties of this latter channel were characteristic for the VDAC, the mitochondrial porin (36,37). At 0 mV and in the presence of an ionic gradient, this channel switched with low kinetics between one anionic and several cationic or nonselective states (Fig. 4B). This is in agreement with earlier observations demonstrating anionic selectivity of the open state of the VDAC and cationic selectivity for its different closed states. The VDAC was never observed in more than 70 experiments using proteoliposomes prepared from the isolated TOM complex. This is consistent with the absence of porin in the purified TOM complex (see Fig. 1).
As an independent method to study the electrophysiological properties of the isolated TOM complex, we used the tip-dip technique, in which the lipid bilayer is formed at the tip of a microelectrode. This method allowed the estimation of the relative PSC content of various membrane fractions (23). VDAC cannot be detected by the tip-dip method (40). The cation-selective channel of the purified TOM complex identified by this method exhibited the same properties as the channel found in the bilayer experiments (not shown). It was observed frequently in samples derived from outer membranes or the purified TOM complex (Table I). The frequency of detection of the channel correlated with the concentration of TOM complex present in the analyzed fraction. The channel could not be detected in those fractions of the TOM complex purification that did not contain the TOM complex. These data support the idea that the observed channel is related to the TOM complex.
In conclusion, we characterized the ion-conducting pore in the purified TOM complex by two independent techniques. All of the characteristics of this channel are identical to those of the previously described PSC (22).
Antibodies against the C Terminus of Tom40 Modify the Properties of the TOM Complex Channel and Allow Determina-  The mixture was subjected to three cycles of freezing and thawing. 10 l of this proteoliposome solution were added to 200 l of the recording solution, leading to the formation at the surface of the bath of a monolayer having the same composition in phospholipids and proteins as the proteoliposomes (52). Bilayers were formed by apposition at the tip of microelectrodes using the tip-dip technique (25). For each fraction, the frequency of detection of the cationic channel is expressed as the number of channels detected relative to the total number of bilayers formed. The control fractions contained buffer or samples from the purification procedure that did not contain any detectable TOM complex.  3. Current flow through the TOM complex channel in response to a sawtooth voltage. The purified Neurospora TOM complex was incorporated into a DPPC planar lipid bilayer as described in Fig. 2. A 5-mHz sawtooth voltage varying between 105 and -100 mV was applied, and the current was recorded in 150 mM KCl, 10 mM MOPS, pH 7.0 in both compartments. Data were filtered at 25 Hz and sampled at 50 Hz. Due to filtering and time scale, the flicker, which occurred at positive potentials, appears as unresolved fluctuations, whereas individual slow kinetics closures can be identified. The dashed line indicates the 0 pA level.

FIG. 4. Electrical properties of the two types of channels found in the OMV fraction.
A, a channel from the OMV fraction exhibiting cationic selectivity was fused to a DPPC bilayer, and currents were recorded under the experimental conditions used in the experiment shown in Fig. 2B. B, VDAC was fused with a DPPC bilayer, and the current was recorded at 0 mV immediately after fusion. The buffer solution of the cis compartment was 40 mM KCl, 5 mM MOPS/ KOH, pH 7.0, and that of the trans compartment 5 mM MOPS/KOH, pH 7.0. Under these conditions, the parts of the trace below and above the dashed line indicate states of anionic and cationic selectivity, respectively. tion of the Channel Orientation in the Lipid Bilayer-For further characterization of the TOM complex, we studied the effect of various antibodies on the electrical properties of the pore. Either the purified TOM complex or proteins solubilized from OMVs were incorporated into planar bilayers, and after successful detection of the cation-selective channel, various antibodies were added to the cis or trans compartments. An antibody directed against the C-terminal 12 residues of N. crassa Tom40 (␣-nTom40C IgG) was found to decrease the conductance of levels 2 and 3 by 25% at all potentials applied (Fig. 5A). No significant effect of this antibody was seen on the lowest conductance, level 1. The ␣-nTom40C IgG also decreased the steepness of the voltage dependence. The voltage at which the flicker was observed was increased by about 20 mV (not shown). These two alterations of the PSC properties occurred simultaneously. Both effects were only seen when the antibodies were applied from one side of the bilayer (Fig. 5A, middle  and right panels). Addition of the antibodies to the opposite compartment had no detectable influence on any of the channel properties and did not interfere with the effect of ␣-nTom40C IgG, which was subsequently added to the other side. The sensitive side depended on the orientation of the channel in the bilayer (see below and Fig. 6). Channels exhibiting the characteristic flicker at negative voltages were affected by addition of the ␣-nTom40C antibodies to the cis side and were insensitive to addition to the trans side. Channels with the reverse orientation showed the opposite behavior. The effect was observed within a few minutes in all of the 18 experiments where ␣-nTom40C IgG was added to the sensitive side.
To demonstrate the specificity of the effects of the ␣-nTom40C IgG on the channel properties, two types of experiments were performed. First, the effects of antibodies derived from preimmune serum or antibodies directed against other proteins of the N. crassa import apparatus (e.g. Tom20, Tom22, and Tom70) were tested. None of these antibodies elicited any specific alterations of the channel properties when added to either compartment (Fig. 5B), even if 5-fold higher concentrations of IgG were used in these experiments (not shown). Subsequent addition of ␣-nTom40C IgG to the sensitive side still induced the characteristic effects of this latter antibody on the FIG. 5. The cation-selective channel contained in the TOM complex is specifically modified by antibodies against the C terminus of Tom40. A, the purified Neurospora TOM complex was inserted into proteoliposomes and allowed to fuse with DPPC lipid bilayers as in Fig. 2A. Currents were recorded in 40 mM KCl, 5 mM MOPS, pH 7, at different potentials before (left) or after addition of 35 g/ml IgG specific for the C terminus of Neurospora Tom40 (anti-nTom40C) to the trans (middle) and then also to the cis compartment (right). B, the purified Neurospora TOM complex channel was inserted into a BBPE/BBPS (7:3) bilayer as in Fig. 2A. Currents were recorded in 40 mM KCl, 5 mM MOPS, pH 7.0, before addition (left) or 30 min after addition of 35 g/ml IgG derived from preimmune serum (middle) to the cis compartment. In the right panel, 35 g/ml anti-nTom40C antibodies were added to the same compartment after the preimmune IgG incubation, and the currents were recorded as above. All data were filtered at 0.2 kHz and sampled at 0.4 kHz. The dashed line indicates the 0 pA level.
FIG. 6. The N and C termini of Neurospora Tom40 are exposed to the intermembrane space. A, isolated mitochondria (100 g of protein per sample) were treated with or without 50 g/ml proteinase K (PK) in SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM MOPS, pH 7.2) for 20 min at 0°C. An alkaline extraction with 100 mM sodium carbonate was performed (50), and membrane-associated material (Pellet) was separated from soluble proteins (Sup.) by centrifugation. Proteins were separated by SDS-PAGE followed by blotting onto nitrocellulose membranes. Samples were immunostained with antibodies directed against the C-and N-terminal 12 residues of Neurospora Tom40 (anti-nTom40C and anti-nTom40N, respectively). B, mitoplasts (MP) were generated by treatment of isolated mitochondria (M) with 0.15% digitonin for 3 min at 0°C. The opening of the outer membrane was monitored by protease accessibility of the intermembrane space enzyme cytochrome c heme lyase (not shown) (42). Mitoplasts and intact mitochondria were incubated with or without 5 g/ml proteinase K or 50 g/ml elastase (Ela) in SEM buffer for 20 min at 0°C. After precipitation with trichloroacetic acid, proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes. All samples were analyzed by immunostaining using anti-nTom40C and anti-nTom40N antibodies as in A or a polyclonal antibody raised against the entire Tom40 protein (anti-nTom40P). The latter antibody recognizes internal epitopes of Tom40. Molecular masses of marker proteins are given on the left. channel activity (Fig. 5B, right panel). Second, ␣-nTom40C IgG was preincubated with the peptide used for antigen production (nTom40C). Under these conditions, ␣-nTom40C IgG had no effect on the channel properties (not shown). Reversal of the effects of the ␣-nTom40C IgG by subsequent addition of the nTom40C peptide was not observed, indicating that the antibody was bound to the C terminus of Tom40 in a rather stable manner. This result is in keeping with earlier studies employing this antibody for immunoprecipitation of the entire TOM complex (see e.g. Ref. 41).
A different result was obtained with an antibody directed against the N-terminal 12 residues of N. crassa Tom40 (␣-nTom40N). Although this antibody per se did not alter the properties of the channel, it prevented the effects of subsequently added ␣-nTom40C IgG (not shown). This effect was only detected when ␣-nTom40N antibody was added to that side of the TOM complex channel sensitive to ␣-nTom40C antibodies. Most likely, the interference of ␣-nTom40N antibody with the binding of the C-terminal IgG is caused by steric hindrance of IgG attachment to the C-terminal epitope. These results suggested that both termini of Tom40 are located on the same side of the outer membrane.
To investigate the localization of the N and C termini of Tom40 in the outer membrane, the protease accessibility of Tom40 was tested. Upon treatment of intact mitochondria with proteinase K, Tom40 was cleaved into two fragments of 26 and 14 kDa, corresponding to the N-and C-terminal portions of Tom40 (Fig. 6A). Both pieces were tightly associated with the outer membrane and could not be released from the membrane by extraction with alkaline buffers. In mitoplasts, i.e. mitochondria with an opened outer membrane (42), treatment with proteinase K generated a Tom40 fragment of 25 kDa. It corresponds to an internal piece of Tom40, because it is not detected with antibodies against the N and C termini. Tom40 was not accessible to proteolytic attack by elastase in intact mitochondria, whereas in mitoplasts, a 36-kDa fragment was generated that could be detected with ␣-nTom40N antibodies (Fig. 6B). These data suggest that a small N-terminal piece of 1 kDa and a C-terminal piece of 2 kDa became accessible for proteases only after opening of the outer membrane. We conclude from these results that both the N and C termini of Neurospora Tom40 are located toward the intermembrane space side of the outer membrane. The orientation of Tom40 in the membrane is consistent with the differential effects of the antibodies against these regions observed in the bilayer experiments (see above). In combination, the results using the antibodies and the char-acteristic flicker observed at negative voltages allow us to define the orientation of the channel in the bilayer.
A channel with the characteristics of PSC has been previously found in isolated mitochondria of Saccharomyces cerevisiae (43,26,44). We investigated the influence of antibodies directed against the C-terminal 15 residues of yeast Tom40 (␣-yTom40C IgG) on the electrical properties of this channel. To avoid any interference of VDAC in these experiments, a porin-deficient yeast mutant was utilized (35). Mitochondrial membranes isolated from this strain were fused with liposomes and those subsequently fused with the planar bilayer. Insertion of PSC was detected through its characteristic electrical properties (26). Upon addition of ␣-yTom40C IgG, a decrease in conductance of levels 2 and 3 by about 20% was measured at all potentials (Fig. 7), similar to what was observed for the corresponding Neurospora antibody and the channel present in the purified Neurospora TOM complex (see Fig. 5A). No specific alterations of the properties of the yeast channel were detectable after addition of Neurospora-specific ␣-nTom40C IgG to either side of the bilayer (not shown). Likewise, ␣-yTom40C antibodies did not affect the channel associated with the Neurospora TOM complex. Together, these data demonstrate the specificity of the effects elicited by antibodies against the C terminus of Tom40 and provide strong evidence for PSC being associated with the TOM complex. Presumably, the antibody bound at the C terminus of Tom40 is localized close to the exit of the ion-conducting pore and modifies the energy profile of the ion pathway and the sensitivity to applied voltage possibly by inducing a structural change in Tom40.
A Mitochondrial Presequence Peptide Modulates the Properties of the TOM Complex Channel-We finally asked whether the channel contained within the TOM complex can be influenced in its properties by mitochondrial presequences. Mitochondrial presequence peptides have recently been shown to alter the structure of Tom40 within the TOM complex (45) and thus may be expected to have a profound influence on the channel properties. Furthermore, interaction with presequence peptides is a characteristic of PSC of yeast and mammalian mitochondria (22). The purified Neurospora TOM complex was inserted into the lipid bilayer and a peptide corresponding to the presequence of the precursor of the ␤-subunit of yeast F 1 -ATPase (pF 1 ␤) was added. Peptide pF 1 ␤ reduced the current flowing through the channel in a voltage-dependent manner (Fig. 8). Control peptides unrelated to presequences (Fig. 8, ϩ CH4 peptide) had no influence on the channel. When the presequence peptide was added to the cytosolic side, the effect appeared as brief closures (Ͻ1 ms) occurring at low positive voltages. At further increased voltages, the number of closures strongly increased (Fig. 8, top; trace recorded at 20 mV). Around 50 mV, the current was even further reduced, the channel remaining mainly at conductance level 1 and even below. No effect was detectable at negative voltages. Similar effects were observed when pF 1 ␤ was added to the intermembrane space side. In this case, the voltage dependence of the effect was reversed, i.e. the current block increased when the voltage was decreased. Although the effects are similar, they are not exactly identical on both sides, as shown by the smaller size of the residual current observed when the peptide was applied to the cytosolic side. In summary, the conductance of the TOM complex channel is influenced in a specific fashion by mitochondrial presequence peptides. These changes are likely related to the structural alterations of the TOM complex observed during interaction of the presequence part of preproteins with specific sites of the translocase (45). DISCUSSION The isolated N. crassa preprotein translocase of the mitochondrial outer membrane, the TOM complex, represents a stable entity and can be inserted into lipid bilayers in a func-tional way (21). This facilitated a thorough analysis of the characteristics of the preprotein-conducting channel of the purified TOM complex by a combination of biochemical and electrophysiological methods. The electrophysiological properties of the channel indicate a homodimeric structure of the pore and show a cation selectivity and a large conductance with three main levels of 90, 320, and 550 pS at 40 mM KCl. Following application of potentials, the channel undergoes characteristic slow and fast transitions to the low conductance levels, but a complete closure is not observed. In addition, the properties of the channel are modified by presequence peptides added from both sides of the channel. All of these characteristics are identical to those of the previously described PSC of mammals (25) and yeast (26). We therefore conclude that the TOM complex contains PSC.
Several observations presented here support the notion that the channel observed in the isolated TOM complex is not related to other proteins, in particular not to porin. First, porin was not detectable in our TOM complex preparations, even when higher amounts of protein were analyzed. Second, channels corresponding to VDAC were never detected in the purified TOM complex fractions, whereas VDAC was frequently observed using OMVs or flow-through fractions of the TOM complex purification. Third, PSC is present in porin-deficient yeast mitochondria (26). Fourth, the TOM complex channel is distinguished from mitochondrial porin by several characteristic properties, such as the ion selectivity, the sensitivity to presequence peptides, and the conductance of the pores. Finally, by using the tip-dip technique, the cationic PSC was detected in fractions of the TOM complex purification at a frequency that correlated with the concentration of TOM complex (Table I).
Antibodies against the C terminus of Tom40 alter the electrical properties of the TOM complex channel. A similar decrease in conductance of levels 2 and 3 was found for the cationic channels of both Neurospora and yeast. In contrast, antibodies against the N terminus did not elicit any detectable alterations of the channel properties per se. Instead, these antibodies prevented the effect of the C-terminal antibody, when added to the same side of the membrane. Presumably, attachment of the N-terminal antibody to Tom40 inhibits the subsequent association of the C-terminal antibody, most likely by steric hindrance. This interpretation is in keeping with the localization of the N and C termini of Neurospora Tom40 in the outer membrane. They are both facing the intermembrane space and can be cleaved by proteases after opening the outer membrane. The effects of the C-terminal antibodies on the TOM complex channel fit nicely to the former observation that antibodies directed against the N and C termini of yeast Tom40 could immunoprecipitate PSC activity (24). On the basis of these results, it is now possible to deduce the orientation of the TOM complex within the bilayer from the polarity of the voltages at which a characteristic flicker of the channel is observed. Channels flickering at negative voltages have their antibody-sensitive side (corresponding to the intermembrane space side) oriented toward the cis compartment. This information will aid future studies addressing mechanistic questions of preprotein transfer across the outer membrane by electrophysiological means.
Even though the precise molecular composition of the translocation channel remains to be determined, Tom40 appears to represent a crucial component. First, Tom40 has been suggested to adopt a ␤-barrel structure (46,47) and is one of the few proteins of the TOM complex that is highly resistant to proteases such as trypsin; thus, it may be directly involved in forming the pore. Second, our experiments using anti-Tom40 antibodies suggest that the exit of the pore toward the intermembrane space is located in the vicinity of Tom40. Finally, FIG. 8. Effects of a mitochondrial presequence peptide on the current flowing through the TOM complex channel. The purified Neurospora TOM complex was incorporated into the DPPC planar lipid bilayer as described in Fig. 2. The orientation in the bilayer was determined from the characteristic flicker at positive voltages (see explanations to Fig. 5). A peptide corresponding to the first 25 amino acid residues of Neurospora cytochrome c heme lyase (CH4) was added to either the cytosolic (cis compartment) or the intermembrane space side (trans compartment) of the TOM complex at a final concentration of 2 M. This peptide unrelated to mitochondrial presequences (see Ref. 51) had no effect on the channel. A peptide corresponding to the presequence of the precursor of the ␤-subunit of yeast F 1 -ATPase (pF1␤) was further added to the same compartment at a final concentration of 400 nM (cytosolic side) or 1 M (intermembrane side). Currents were recorded in 150 mM KCl, 10 mM MOPS/KOH, pH 7.0, before (Control) or 2 min after addition of the peptides. Data were filtered at 0.2 kHz and sampled at 0.4 kHz. The dashed line indicates the 0 pA level. Tom40 appears to be the major component of the TOM complex, as is evident from a recent determination of the stoichiometry of the TOM complex components (21). Further structural studies are needed to determine whether other components of the TOM complex participate in forming the pore.
A role of PSC in preprotein translocation across the mitochondrial outer membrane was proposed soon after its discovery. The proposal was based on its large conductance and its sensitivity to presequences and basic peptides (8,34). Presequences affected the PSC properties from both sides. In addition, basic peptides unrelated to mitochondrial physiology, but blocking PSC in the same way, were shown to function as presequences that target passenger proteins to mitochondria (24). Like presequences (48,41) these basic peptides could be cross-linked to Tom40 (24). Import directed by these peptides occurred on the same pathways as with natural presequences (24). By clarifying the molecular identity of PSC, we now provide firm experimental evidence for the function of PSC in protein translocation across the mitochondrial outer membrane.
We demonstrate that the presequence peptide of F 1 ␤ alters the ion flow through the channel in a voltage-dependent manner, when added from either side of the membrane. Similar observations have been made previously for mammalian and yeast PSC exposed to presequences or basic peptides (34). A precise molecular model explaining the influence of the presequence peptide on the TOM complex channel cannot be derived at present. However, insertion of the presequence peptide into the pore would be expected to reduce the ion flow and could provide a possible explanation for the effects described here. The voltage dependence of the blocking intensity agrees well with the action of the electrical field within the channel on a positively charged molecule. Potentials attracting the peptide into the channel stabilize its insertion leading to plugging of the pore. Opposite potentials weaken the interaction between the presequence peptide and the channel and, at higher potentials, overcome the tendency of the peptide to insert into the pore.
The effect of presequence peptides on either side of the membrane can be understood on the basis of the reversible character of translocation across the mitochondrial outer membrane (49). By forward and/or reverse translocation, a presequence peptide added to one side of the TOM complex could induce the observed alterations of PSC. However, the complex electrical patterns observed in the presence of the peptide may reflect not only insertion of the peptide into the channel but also conformational changes of the pore triggered by the binding of the presequence. We have recently demonstrated that the TOM complex undergoes structural rearrangements during preprotein transfer across the outer membrane (45). These structural alterations within the TOM complex may be the molecular manifestation of the effects of presequence peptides on PSC. Under physiological conditions, the outer membrane is subject to little or no transmembrane voltage, so the TOM complex channel is likely to be in the open state. In this view, the voltage gating of the TOM complex channel would not be relevant for its physiological role. Rather, the channel would act as a presequence-gated pore that facilitates the movement of the translocating polypeptide chain by presequence binding to various sites and associated conformational changes (45). On the basis of our results, elucidation of the precise mechanism of the interaction of peptides with the TOM complex channel will be possible in future studies.