The Mitochondrial Oxidase Assembly Protein1 (Oxa1) Insertase Forms a Membrane Pore in Lipid Bilayers*

Background: Oxa1 mediates the insertion of mitochondrion-encoded precursors into the inner mitochondrial membrane. Results: Oxa1 forms a voltage- and substrate-dependent membrane pore. Conclusion: The channel properties of the Oxa1 pore are compatible with the membrane-potential regulated protein insertase. Significance: This is the first report on the pore-forming capacity of Oxa1, providing mechanistic insight into the insertase mechanism of Oxa1. The inner membrane of mitochondria is especially protein-rich. To direct proteins into the inner membrane, translocases mediate transport and membrane insertion of precursor proteins. Although the majority of mitochondrial proteins are imported from the cytoplasm, core subunits of respiratory chain complexes are inserted into the inner membrane from the matrix. Oxa1, a conserved membrane protein, mediates the insertion of mitochondrion-encoded precursors into the inner mitochondrial membrane. The molecular mechanism by which Oxa1 mediates insertion of membrane spans, entailing the translocation of hydrophilic domains across the inner membrane, is still unknown. We investigated if Oxa1 could act as a protein-conducting channel for precursor transport. Using a biophysical approach, we show that Oxa1 can form a pore capable of accommodating a translocating protein segment. After purification and reconstitution, Oxa1 acts as a cation-selective channel that specifically responds to mitochondrial export signals. The aqueous pore formed by Oxa1 displays highly dynamic characteristics with a restriction zone diameter between 0.6 and 2 nm, which would suffice for polypeptide translocation across the membrane. Single channel analyses revealed four discrete channels per active unit, suggesting that the Oxa1 complex forms several cooperative hydrophilic pores in the inner membrane. Hence, Oxa1 behaves as a pore-forming translocase that is regulated in a membrane potential and substrate-dependent manner.

The inner membrane of mitochondria is especially proteinrich. To direct proteins into the inner membrane, translocases mediate transport and membrane insertion of precursor proteins. Although the majority of mitochondrial proteins are imported from the cytoplasm, core subunits of respiratory chain complexes are inserted into the inner membrane from the matrix. Oxa1, a conserved membrane protein, mediates the insertion of mitochondrion-encoded precursors into the inner mitochondrial membrane. The molecular mechanism by which Oxa1 mediates insertion of membrane spans, entailing the translocation of hydrophilic domains across the inner membrane, is still unknown. We investigated if Oxa1 could act as a protein-conducting channel for precursor transport. Using a biophysical approach, we show that Oxa1 can form a pore capable of accommodating a translocating protein segment. After purification and reconstitution, Oxa1 acts as a cation-selective channel that specifically responds to mitochondrial export signals. The aqueous pore formed by Oxa1 displays highly dynamic characteristics with a restriction zone diameter between 0.6 and 2 nm, which would suffice for polypeptide translocation across the membrane. Single channel analyses revealed four discrete channels per active unit, suggesting that the Oxa1 complex forms several cooperative hydrophilic pores in the inner membrane. Hence, Oxa1 behaves as a pore-forming translocase that is regulated in a membrane potential and substrate-dependent manner.
Membrane protein biogenesis demands the translation on ribosomes, insertion into a target membrane in the topologically correct orientation, and proper three-dimensional folding into a functional state. In particular, the latter two processes, membrane insertion and folding of membrane proteins, are only poorly understood. The bacterial inner membrane and the endoplasmic reticulum of eukaryotic cells contain structurally related translocation machineries referred to as SecY or Sec61 complexes, respectively (1,2). These complexes represent pore-forming structures that not only allow translocation of substrate proteins across the lipid bilayer but also their lateral integration into the membrane. In bacteria, membrane protein insertion is assisted by the conserved protein YidC (3,4). YidC is present in the inner membrane at much higher concentrations than SecY (5). YidC facilitates SecY-independent membrane insertion of membrane proteins that do not expose large hydrophilic domains into the periplasmic space (6,7). Moreover, YidC increases the folding rate of membrane proteins, suggesting that it enables membrane proteins to fold into their native structure (8,9).
The inner membrane of mitochondria lacks a SecY-like translocation complex (10). Instead, the membrane protein Oxa1, which is closely related to YidC, facilitates protein insertion in mitochondria (11,12). Oxa1 and YidC share a conserved domain consisting of five transmembrane spans, which catalyze protein insertion reactions. In contrast to YidC, Oxa1 contains a positively charged C-terminal domain of helical structure (13,14). This matrix-exposed region binds mitochondrial ribosomes to facilitate co-translational protein insertion (13)(14)(15). Subunit 2 of cytochrome c oxidase (Cox2) of yeast is the best studied substrate of Oxa1. This mitochondrion-encoded protein directly interacts with Oxa1 early during its biosynthesis and is inserted in a strictly Oxa1-dependent manner. Cox2 contains an N-terminal leader peptide that is removed after translocation by the intermembrane space peptidase Imp1 (16,17).
Although the relevance of Oxa1 in regard to the biogenesis of membrane proteins is well documented by many studies, the molecular function of Oxa1 in the process of substrate integration into the lipid bilayer is not understood. It was speculated that Oxa1 might form a pore to allow for the translocation of hydrophilic domains of its substrates (14). To test directly the pore-forming ability of Oxa1, we purified Oxa1 and reconstituted it into planar lipid bilayers for electrophysiological analyses. Our experiments show that Oxa1 forms a membrane channel in bilayers resembling the lipid composition of the inner mitochondrial membrane. As expected of an inner membrane channel, it was shown to maintain an electrochemical gradient, as seen in the inner membrane of mitochondria. The Oxa1 channel closes at the physiological membrane potential; however, Oxa1 responds to a substrate-peptide indicating its substrate-induced activation. Electrophysiological single channel analyses show that the active unit of the native Oxa1 complex contains four distinct pores, two of which display cooperativity in their gating behavior. Each pore is large enough to accommodate polypeptides in an unfolded or secondary structured form. Thus, our analyses provide the first experimental evidence that the Oxa1 insertase can act as a signal-regulated proteinconducting channel for the export of mitochondrion-encoded precursor proteins.
For proteins purified from Saccharomyces cerevisiae, liposomes were resuspended in DDM buffer (100 mM KCl 0.4% DDM, 20 mM Mops/Tris, pH 7.0) and mixed with solubilized protein at a phospholipid concentration of 5 mg/ml. The mixture was incubated at 4°C for 30 min and subsequently added to pre-washed and equilibrated Biobeads (Bio-Rad). Samples were shaken for 30 min at room temperature, following which the Biobeads were replaced and shaken overnight at 4°C.
Liposome Flotation Assay-Proteoliposomes were floated in a discontinuous Nycodenz density gradient. The buffer contained 100 mM KCl, 10 mM Mops/Tris, pH 7.0. Nycodenz and proteoliposomes were solubilized in the same buffer and mixed to a Nycodenz concentration of 40%. Subsequent layers of decreasing Nycodenz concentration were added (20, 10, 5, and 2%). Percentages indicated in the figures are the final concentrations of Nycodenz. The gradient was centrifuged at 4°C for 60 min at 100,000 ϫ g. Subsequently, the gradient was fractionated by taking equal volumes from the top. Fractions were analyzed using SDS-PAGE and Western blotting.
CD Spectroscopy-Purified proteins were reconstituted as described above. Small liposomes were formed as described previously and lysed in a buffer containing 80 mM Mega-9, 10 mM Mops/Tris, pH 7.0. Purified proteins were diluted 10-fold into the mixture. Proteoliposomes were formed by dialysis in a buffer containing 10 mM KCl, 10 mM Mops/Tris, pH 7.0. Samples were adjusted to a protein concentration of 250 g/ml. CD spectra were recorded with a Jasco J-810 spectropolarimeter after calibration with (ϩ)-10-camphorsulfonic acid. The measurements were performed at room temperature in a quartz cell (0.01-cm optical path length). Scans were performed at a rate of 50 nm/min with a sampling interval of 1 nm and averaged (n ϭ 32) to improve the signal/noise ratio. Spectra were corrected with the corresponding buffer spectra collected under identical conditions. Data sets were converted to mean residue ellipticity and deconvoluted by a neural network approach (18) and the CDPro package (19 -21).
Electrophysiological Setup and Measurements-Using the painting technique, we produced planar lipid bilayers. Proteoliposomes were dropped directly below the bilayer in the cis chamber. Buffer conditions were asymmetrical (250 mM KCl, 10 mM Mops/Tris, pH 7.0, in the cis compartment and 20 mM KCl, 10 mM Mops/Tris, pH 7.0, in the trans compartment). This led to osmotically induced fusion of proteoliposomes with the bilayer. Ag/AgCl electrodes were connected by 2 M KCl-agar bridges. The electrode in the trans chamber was connected to the head stage (CV-5-1GU) of a GeneClamp 500 current amplifier (Axon Instruments) and thus was the reference for reported membrane potentials. Current recordings were carried out using a Digidata 1200 A/D converter. Data analysis was performed by a self-written Windows-based single-channel investigation program (SCIP) in combination with Origin 7.0 (Microcal Software). After incorpo-FIGURE 2. A, current recordings of a bilayer after fusion of an Oxa1-containing proteoliposome (membrane potential as indicated). Zoom plots show single gating events in main and subconductance states. B, conductance state histograms of Oxa1 at positive and negative applied holding potentials. C, current recordings of a bilayer after fusion of an Oxa1 rec -containing proteoliposome (left) and from a bilayer fused with mock-treated sample liposomes obtained from cells expressing nontagged Oxa1 rec that were added to the cis compartment (membrane potential of the voltage gate as indicated). D, current recordings of a bilayer after fusion of an Oxa1 rec -containing proteoliposome in the absence (left) and presence of Pam17 antiserum (membrane potential of the voltage gate as indicated).
ration of Oxa1 into the bilayer, currents in response to varying applied voltages were recorded. Subsequently, antibodies or Cox2 peptides were added either to proteoliposomes before osmotically induced fusion or directly into the recording chamber after Oxa1 incorporation.

Oxa1
Purified from Yeast Mitochondria Displays Ion Channel Activity-Oxa1 mediates the export of mitochondrion-encoded proteins across the inner mitochondrial membrane and represents the bottom of the gradient after centrifugation where aggregated or not incorporated protein is found. The majority of proteoliposomes floats into the 5% fraction of the gradient. C, CD spectrum of Oxa1 rec in DDM. D, current recordings of a bilayer after the fusion of Oxa1 rec (membrane potential as indicated) under symmetrical buffer conditions (see Fig. 1B). Diagram to the right shows that full channel closure occurs in four main conductance state gating events. E, mean variance analysis of D, with upper current trace showing that four gating events with the main conductance state led to complete channel closure. F, current voltage relationship calculated from over 5,000 single gating events. Conductance states are indicated. Buffer conditions are as in Fig. 1B. G, current-voltage ramp recorded under asymmetrical buffer conditions (as in Fig. 1D). Reversal potential as indicated. H, voltage-dependent open probability of Oxa1 rec . Quantification was performed by comparing the mean current determined over a range of 1 min with the maximum current at a constant holding potential. I, current recordings of an Oxa1 reccontaining bilayer before (black) and after the addition of anti-Oxa1 antibodies (upper gray). the membrane insertion of protein domains following their matrix import via the TIM23 translocase in a process referred to as conservative sorting (22)(23)(24). It is controversially debated how Oxa1 or its homologs mediate the transfer of polypeptide chains across a lipid bilayer. Thus, it has been proposed that Oxa1 may act as a protein-conducting channel in the inner membrane of mitochondria. To address the possible channel activity of Oxa1 directly, we purified Oxa1 from S. cerevisiae mitochondria, in which Oxa1 was expressed with a C-terminal double affinity ZZ/His 10 tag (Oxa1 ZZ/His ) from the chromosomal locus (Fig. 1A) (25). Purified mitochondria were lysed under denaturing conditions. After dilution, Oxa1 ZZ/His was purified in Triton X-100-containing buffer and subjected to sequential affinity chromatographic steps utilizing first Ni-NTA-agarose and then IgG-Sepharose. Oxa1 was released from IgG-Sepharose by TEV-protease cleavage. Because of the low amount of purified protein, purity was monitored by Western blotting (Fig. 1A). Next, purified Oxa1 was refolded into liposomes by a detergent-mediated reconstitution. It is important to note that successful reconstitution of the Oxa1 channel required the lipid composition of pre-formed proteoliposomes resembling the composition of the inner mitochondrial membrane (26).
When Oxa1 containing proteoliposomes were fused to a planar lipid bilayer by osmotic fusion (27,28), ion channel activity could readily be detected (Figs. 1B and 2A). As a control, mock samples from yeast cells expressing untagged Oxa1, which had been subjected to the same purification and reconstitution routine, showed no ion channel activity (Fig. 2C).
Oxa1 channels displayed distinct gating events with a mean maximal conductance of G max Х500 pS and with a minimal subconductance of G min Х75 pS at positive membrane poten-  tials (V m ) and 250 mM KCl on both sides of the membrane. Conductance states varied slightly between negative and positive membrane potentials, and several subconductance states could be detected. This resulted in complex gating behavior of the ion channel (Figs. 1, B and C, and 2, A and B). Under asymmetrical buffer conditions (250/20 mM KCl, cis/trans) a reversal potential of U rev ϭ 44 mV could be detected. Using the Goldman-Hodgkin-Katz approach, a cation-selective pore with a ratio for P K ϩ/P Cl Ϫ of 10:1 (Fig. 1D) could be calculated.
To assess the specificity of the aforementioned channel activity, we utilized antiserum directed against the intermembrane space domain of Oxa1. After incubation of Oxa1-containing proteoliposomes with the antiserum prior to liposome osmotic fusion with the planar lipid bilayer, ion channel incorporation into the membrane was drastically decreased. In addition, the remaining channel activity was limited to a very low conductance state, comprising only 10% of the main conductance in the absence of antisera (Fig. 1E). As a control, incubation with an antiserum directed against an unrelated inner mitochondrial membrane protein, Pam17, did not lead to any changes in either the fusion rate or ion channel characteristics (Fig. 2D). Thus, we concluded that the channel activity of the purified protein was specific for the reconstituted yeast Oxa1 channel.
Oxa1 Forms a Voltage-gated Ion Channel-Despite the fact that we isolated Oxa1 from mitochondria under stringent and denaturing conditions, we wanted to rule out that a contaminating yeast protein was responsible for the pore forming activity. Therefore, we expressed S. cerevisiae Oxa1 as a His 10 -tagged version in E. coli. Oxa1 (Oxa1 rec ) was purified by affinity chromatography from E. coli membranes (Fig. 3A) and reconstituted in the presence of detergent into liposomes (29,30). Reconstitution efficiency was assessed by flotation of liposomes, which revealed that Oxa1 rec was efficiently incorporated into liposomes (Fig. 3B). In the absence of liposomes, Oxa1 rec did not float in a density gradient and could be found at the bottom of the gradient after centrifugation (Fig. 4C). In addition, we followed refolding by assessing the secondary structure of Oxa1 rec in dodecylmaltoside micelles using circular dichroism (CD) spectroscopy (Fig. 3C). The Oxa1 CD spectrum revealed a shape characteristic of a predominantly ␣-helical protein (Table 1).
When Oxa1 rec proteoliposomes were fused with planar lipid bilayers, ion channel activity could be detected (Figs. 3D and 5A). The basic characteristics resembled those of Oxa1 channels purified from yeast with a mean maximal conductance state for Oxa1 rec of G max ϭ 530 pS at positive V m and 250 mM KCl (cis/trans), channel asymmetry in conductance states between negative and positive membrane potentials, and multiple subconductance states (Figs. 3F and 5B). Based on a conductance state of about 500 pS, we calculated a pore diameter of ϳ1.9 nm (28,31,32). The calculated pore size would accommodate polypeptide chains with secondary or unfolded structure, as assessed by a typical ␣-helix diameter being roughly 0.5 nm (33,34).
Remarkably, the minimal unit incorporated into the bilayer by a single fusion event always displayed a maximal current   SEPTEMBER 28, 2012 • VOLUME 287 • NUMBER 40

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corresponding to four single channel (G max ) currents (Fig. 3E). As observed in Oxa1 purified from yeast, the Oxa1 rec channel was cation-selective with a reversal potential of U rev ϭ 44 mV under asymmetrical salt conditions (250/20 mM KCl, cis/trans) corresponding to a permeability ratio of P K ϩ/P Cl Ϫ ϭ 10:1 (Fig.  3G).
The observed voltage-dependent open probability of a fourpore-containing Oxa1 channel unit showed clear asymmetric behavior, indicating that the channel incorporated unidirectionally into the membrane (Fig. 3H). Considering the polarization of the membrane potential across the inner mitochondrial membrane, it is tempting to speculate that the cis side of the channel in vitro corresponds to the intermembrane space side in vivo, implying that at a negative V m of Ͼ120 mV the pore will close. Thus, similar to what has been observed for the Tim23 channel of the presequence translocase and the Tim22 channel of the carrier translocase, at physiological inner membrane potentials of Ͼ150 mV, the Oxa1 channel will reside in the closed state thereby preventing deleterious ion leakage across this energy-coupling membrane.
As seen with Oxa1 purified from yeast, incubation of Oxa1 rec proteoliposomes with anti-Oxa1 antibodies led to a reduction of proteoliposome fusion rates as well as to an almost complete block of channel conductance (Fig. 3I). Accordingly, these results, together with the ones obtained for Oxa1 purified from yeast, show that Oxa1 forms an ion channel in a lipid membrane.
Oxa1 Channels Are Activated by Substrate Peptides-Oxa1 mediates export of mitochondrion-encoded proteins across the inner membrane. A well known Oxa1 substrate is the precursor of Cox2 (pCox2) (22,24). After processing by the IMP protease, mature Cox2 assembles into the core of the cytochrome c oxidase. To directly test if Oxa1 specifically recognizes transport substrates, we generated a synthetic peptide corresponding to the targeting signal of Cox2, comprising of the 19 N-terminal amino acids (MLDLLRLQLTTFIMNDVPT). Incubation of Oxa1 rec proteoliposomes with Cox2 peptides prior to osmotic fusion led to a drastic increase in the gating frequency (flickering) of the ion channel (Fig. 6A). For example, the analysis of a 60-s current sample at a holding potential of V-h ϭ ϩ100 mV (Fig. 6A) yielded an average value f gating of 0.5 Hz, whereas Cox2 incubated channels showed a gating frequency of roughly 60 Hz. This increase in gating frequency was accompanied by changes in the conductance state distribution. Although the main conductance state could still be detected (Fig. 6B), channel flickering occurred not between the closed and the fully open state, but rather between the varieties of subconductance states (compare Fig. 5C with A and B, which are without Cox2)). Although no change in the reversal potential was observed (Fig.  6C), the peptide-induced activation of gating leads to a decreased voltage sensitivity of (P o ) (Fig. 6D). One might speculate that the interaction of the channel with the peptide shifts the channel from an idle nonactive state into an active state with high gating frequency, representing a highly flexible pore state. Control measurements in which a presequence peptide of Cox4, a substrate of the Tim23 channel, was used instead of Cox2 showed no significant changes of the Oxa1 channel activity (Fig. 5D).
Oxa1 Complex Displays Two Distinct Activity States-So far our analyses assessed the channel activity of Oxa1 after purification under denaturing conditions, thereby excluding unspecific channel activities. We reasoned that in the inner membrane of mitochondria, Oxa1 likely exists in a different oligomeric state then after reconstitution of an unfolded polypeptide. In previous analyses, we successfully isolated protein complexes from solubilized membranes and reconstituted these for electrophysiological studies (35,36). Therefore, we purified Oxa1 under native conditions from mitochondria after solubilization in digitonin-containing buffer. Oxa1 containing a C-terminal ZZ-tag has been previously shown to be defective in association with mitochondrial ribosomes (25). After purification of Oxa1 complexes by IgG chromatography, native complexes were released from the resin by TEV protease cleavage of the tag. We confirmed efficient isolation of Oxa1 and purity of the sample by SDS-PAGE (Fig. 7A). When purified Oxa1 complexes were analyzed by blue native-PAGE, two major complexes were detected, one that migrated at ϳ70 kDa and a larger one that migrated at 180 kDa (Fig. 7B). Although the smaller complex could represent a dimeric form of Oxa1, the larger complex was reminiscent of the tetrameric form of the protein that has been previously suggested for Oxa1 from S. cerevisiae and Neurospora crassa (37,38). The ratio of dimeric to tetrameric Oxa1 that is visible on the blue native-PAGE does not necessarily reflect the ratio of complexes in the lipid membrane due to the presence of detergent and Coomassie. To assess the channel properties of native Oxa1, we purified Oxa1 complexes in preparative scale from mitochondria. Oxa1 complexes (Oxa1 complex ) were reconstituted into liposomes using mixed detergents with subsequent dialysis. Incorporation success was monitored by flotation of Oxa1containing proteoliposomes (Fig. 7C). After fusion of these proteoliposomes with planar lipid bilayers, ion channel activity could be observed. The basic characteristics resembled those of the reconstituted Oxa1 yeast and Oxa1 rec channel properties. Strikingly, the OXA1 complex channel appeared in two distinguishable forms, a low frequency and a high frequency gating state (Fig. 7, D and E). Both states showed a mean maximal conductance of G max Х500 pS at positive V m as well as multiple subconductance states (Figs.   F and G, and 9A). An asymmetric conductance state distribution, as observed with both channels described above, could also be seen (Figs. 7, F and G, and 9A). The reversal potential (250/20 mM KCl, cis/trans) was determined to be U rev ϭ 45 mV in either activity state corresponding to a cation-selective channel with a permeability ratio of P K ϩ/ P Cl Ϫ ϭ 10:1 (Fig. 7, H and J). Interestingly, the voltage-dependent open probability showed remarkable differences for the two states. The low frequency gating activity showed steep voltage-dependent channel closure above threshold potentials of V m Ͼ Ϫ100 mV. This is in line with the voltage dependence observed for Oxa1 rec and indicates that the channel is closed under physiological membrane potentials (Fig. 7, I and K). Interestingly, the channel showed a rectifying current voltage relationship with reduced conductance at negative membrane potentials (Fig. 4B). Thus, if the cis compartment was made equivalent to the intermembrane space side of the channel, this would counteract ion leakage in vivo under conditions where the pore is in the open state.
The Oxa1 complex channel in the high frequency gating state was less sensitive to high voltage-induced channel closure and therefore resembles Oxa1 rec in the presence of the Cox2 substrate. However, Oxa1 complex in the low frequency state and Oxa1 rec displayed a similar gating frequency. Furthermore, when we analyzed the relative occupancies of the different conductance states, we found remarkable similarities between the Oxa1 complex in its high frequency gating state and the recombinant Oxa1 channel in the presence of the Cox2 peptide (Fig.  8). A comparison of all the conductance occupancies of the two channels in their different activation states reveals that the increase in gating frequency mainly effects conductance state 2 (Fig. 8, D and H).
In addition, we found that a single fusion event of an Oxa1 complex proteoliposome yielded a maximal bilayer current in a multiple of four with respect to the mean maximal conductance state (G max ) of the single Oxa1 rec channel (Fig. 9A). At a holding potential of V-h ϭ ϩ120 mV, the maximal observed current was I max Х250 pA, which at a bilayer resistance of R bilayer ϭ 10 gigaohms corresponds to four times G max Х480 pS, the main single channel conductance. As this behavior was also observed with the Oxa1 rec channel activity, we analyzed the time course of gating events of Oxa1 complex in the high frequency state. Remarkably, the analysis showed statistically significant numbers of direct gating transitions between either two of the four conductance levels of the pore (for example, see Equation 1) (Fig. 9, B and C).
G max 4 (open) 7 G max 3 (closed) REACTION 1 We therefore conclude the pores could be coupled pairwise in their gating activity, presumably forming a common pore unit implying the Oxa1 channel assembles into a tetramer of two functionally coupled dimers (a dimer of dimers).

DISCUSSION
The above-described experiments show that Oxa1 reconstituted into a lipid environment resembling the lipid composition of the inner mitochondrial membrane is able to form a hydrophilic pore. The channel shows selectivity for cations and can be activated by peptides corresponding to a physiological substrate. The dimensions of the pore and its overall properties are similar to that of other protein translocation pores in the inner mitochondrial membrane, namely Tim23 and Tim22. The Oxa1 channel displays sufficient diameter to accommodate secondary structured polypeptides. Importantly, the channel closes in a voltage-dependent manner suggesting that the membrane potential across the inner membrane is maintained with the channel in a closed state in the absence of substrates. In particular, the substrate and voltage dependence of the Oxa1 channel properties resemble those of the previously extensively characterized Tim22 channel, implying similar inner mitochondrial membrane protein insertion mechanisms might be at work in both pathways (39,40). Moreover, it has been shown for Tom40, Tim22, and Tim23 that the specific interaction of these protein translocation pores with signal peptides represent initial translocation steps leading to fast channel gating (flickering) (37, 39 -41). Thus, the high frequency gating state of Oxa1 may be envisaged as a transport initiation state. Because Oxa1 directly binds to ribosomes mediating co-translational precursor protein transport across the inner membrane, it is tempting to speculate that the membrane potential-dependent closure observed here for the ribosome-free Oxa1 reflects its ability to prevent ion leakage in the absence of translocating substrates. FIGURE 9. Oxa1 complex gating shows four pores of which two are coupled in each case. A, current recording of an Oxa1 complex containing bilayer at ϩ120 mV. B, mean variance analysis of the current trace depicted in A (zoom plot). C, schematic representation of the gating transitions shown in B. B and C mainly show gating events of the main conductance state (4 to 3, 3 to 2, etc, but also gating events over two main conductance states (i.e. 4 to 2, 3 to 1, and 2 to fully closed) indicating two of the four pores are coupled in each case.