Interactions of Mitochondrial Presequence Peptides with the Mitochondrial Outer Membrane Preprotein Translocase TOM

Abstract

TOM protein-conducting channels serve as the main entry sites into mitochondria for virtually all mitochondrial proteins. When incorporated into lipid bilayers, they form large, relatively nonspecific ion channels that are blocked by peptides derived from mitochondrial precursor proteins. Using single-channel electrical recordings, we analyzed the interactions of mitochondrial presequence peptides with single TOM pores. The largest conductance state of the translocon represents the likely protein-conducting conformation of the channel. The frequency (but not the duration) of the polypeptide-induced blockage is strongly modulated by the substrate concentration. Structural differences between substrates are reflected in characteristic blockage frequencies and duration of blockage. To our knowledge, this study provides first quantitative data regarding the kinetics of polypeptide interaction with the mitochondrial TOM machinery.

Introduction

The channeling of proteins across biological membranes through nanometer-scale pores is a process common to all living organisms. Examples include the protein secretory pathways of bacteria (1), the protein import pathways in the endoplasmic reticulum (2), mitochondria (3–6), chloroplasts (7), and peroxisomes (8). The transfer of proteins across a lipid bilayer requires an assembly of proteins (e.g., SecY, TOM, SAM, TIM23, TIM22, TOC, TIC, and PEX complexes, respectively) that recognize the polypeptide to be translocated, mediate its insertion into the pore, facilitate the transfer through the pore, and drive the movement so that it becomes vectorial.

In mitochondrial outer membranes, the TOM complex (Fig. 1) represents the primary selection filter for the import of virtually all mitochondrial preproteins. Various receptor proteins selectively recognize different substrates. Previous studies assigned the central subunit organizing the translocation pore to Tom40 (9–11). Low-resolution electron microscopy (9,10,12–15), electrophysiology (9,10,13,16), and biochemical studies measuring the effect of rigid gold labels introduced into precursor proteins on import (17) indicated pore diameters of ∼20 Å, which is sufficient to accommodate unfolded or partially folded proteins.

Figure 1.

Electron microscopy-based model and purification of the TOM core complex channel from N. crassa. (A) Representation of the N. crassa TOM core complex (10) inserted into a planar lipid bilayer with a polypeptide traversing the channel. The inner diameter of the pore is ∼20 Å. (B) Mitochondria from an N. crassa strain carrying a hexahistidinyl tag on Tom22 were solubilized in 0.1% dodecyl-β-D-maltoside and applied to Ni-NTA affinity and anion exchange column chromatography. A Coomassie Blue-stained sodium dodecyl sulfate-polyacrylamide gel of purified TOM core complex is shown. The complex contains all known subunits (Tom40, Tom22, Tom7, Tom6, and Tom5).

Import studies with radio- and fluorescence-labeled synthetic preprotein peptides have demonstrated that peptides are capable of serving as a TOM substrate and are imported into isolated mitochondria (18–20). More-detailed studies addressing the interaction of preproteins with purified TOM complex at high temporal resolution, however, have been hampered by the considerable complexity of the translocation machinery and the complications caused by the intrinsic gating of the channel between different conformational substates.

In this work we explored the interaction of a natural and a synthetic model peptide with TOM pores (Fig. 1) by obtaining single-channel electrical recordings and probing the frequency and duration of transient polypeptide-induced current blockage at high temporal resolution. Using mitochondrial presequence peptides (Fig. 2), we observed concentration-dependent channel blockage at the single-molecule level at voltages where the channel did not show endogenous gating. The frequency of channel blockage progressively increased with peptide concentration and was dependent on the membrane voltage, indicating an open blocker mechanism. Our results provide the rate constants of substrate association and dissociation, and the first glimpse (to our knowledge) into the kinetics of protein translocation through the mitochondrial TOM machinery.

Figure 2.

Structure of the model substrate peptides used in this study. pF1β is a leader peptide corresponding to the first 31 residues of the precursor of the β-subunit of mitochondrial F1-ATPase. pAK₅ is a synthetic α-helical peptide that has previously been used in protein translocation experiments with α-hemolysin as a model pore (22,23). (A) Helical wheel plots of peptides. (B) Helical net plots.

Materials and Methods

Isolation of the TOM core complex

The TOM core complex was purified from Neurospora crassa strain GR-107 (12), which contains a hexahistidinyl-tagged form of Tom22 (Fig. 1 B). Protein was isolated from detergent-solubilized mitochondria according to established protocols (10) by nickel-nitrilotriacetic acid affinity and anion exchange chromatography with minor modifications regarding the buffer (0.1% (w/v) n-dodecyl-β-D-maltoside, 20 mM Tris, pH 8.5) for the first isolation steps. The purity of the isolated protein complex was assessed by means of sodium dodecylsulfate gel electrophoresis and Coomassie Brilliant Blue staining. Protein concentrations were determined with the method described by Bradford (21), and purified protein was stored at a final concentration of 1 mg/mL at 4°C.

Design and synthesis of blocking peptides

A polypeptide corresponding to the first 31 residues of the precursor of the Saccharomyces cerevisiae F1-ATPase β-subunit (Ac-MVLPRLYTATSRAAFKAAKQSAPLLSTSWKR-NH₂, pF1β, MW = 3451.0, pI = 11.7 (9)) or an alanine-based model polypeptide (Ac-[AAKAA]₅Y-NH₂, pAK₅, MW = 2243.6, pI = 10.3 (22,23)) were used as substrates for the TOM core complex. Helical wheel plots for both peptides are shown in Fig. 2. The physical properties of the polypeptides are summarized in Table 1. Both polypeptides were custom-synthesized and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and had a high-performance liquid chromatography purity of at least 95% (Biosyntan, Berlin, Germany). Fresh stock solutions of polypeptides were prepared for each experiment with peptide concentrations of 0.1 mg/mL in water and kept at 4°C for a maximum of 1 day. For channel blockage experiments, the peptide concentrations were in the micromolar range. Nonspecific absorption of peptides to the bilayer cuvette below micromolar concentrations made it difficult to obtain measurements.

Table 1.

Physical properties of the substrate polypeptides

Polypeptide	Length (residues)	Charge	Kyte-Doolittlehydropathy index
pAK5	26	+5	+0.585
pF1β	31	+6	-0.145

Electrophysiology

The purified TOM core complex was reconstituted into planar lipid bilayers using standard protocols (24). Planar lipid bilayers were formed with the monolayer opposition technique across a 40–60 μm diameter circular aperture in a 25 μm thick polytetrafluoroethylene film (Goodfellow, Cambridge, UK) tightly glued between two Delrin chambers. Each chamber contained 2 mL of an aqueous bathing solution (1 M KCl, 20 mM HK₂PO₄, pH 7.5). Lipid bilayers were formed by the layering of 2 μL of a 5 mg/mL solution of 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC; Avanti Polar Lipids, Alabaster, AL) dissolved in pentane on the buffer surface on each side. The purified TOM core complex was added to the cis side of the membrane (amplifier ground) at a final concentration of 5 × 10⁻⁴ mg/mL, and protein insertion was facilitated by applying a transmembrane potential of 200 mV and mixing the contents of the chamber.

Electrical recordings were made with a pair of Ag/AgCl electrodes (World Precision Instruments, Sarasota, FL) attached to an Axon Instruments 200B amplifier with a capacitive headstage. Current signals were filtered by an analog low-pass, four-pole Bessel filter at 10 kHz and digitally sampled at 50 kHz with an Axon Digidata 1322A digitizer controlled by the Clampex 10.0 software (all from Molecular Devices, Sunnyvale, CA). Data analysis was carried out using the Clampfit 10.0 software (Molecular Devices).

Before adding the peptide, we always characterized the endogenous channel gating by recording the current traces at constant voltage between −100 and +100 mV. It should be noted that the degree of endogenous gating depends on the protein batch. In agreement with results for other porins, we could not correlate this to the particular boundary conditions of the experimental setting. Possible explanations for this phenomenon are the oligomeric heterogeneity of the purified TOM core complex (10) or the loss of individual subunits with time. Peptide interactions were thus analyzed only in the absence of intrinsic gating of the TOM core complex at low voltage. Peptides were added to the side of the lipid bilayer yielding the lowest intrinsic channel gating as indicated for each measurement. Antibodies against the C-terminus of Tom40 oriented toward the mitochondrial intermembrane space have been reported to modify the asymmetric gating properties of the TOM channel and allow determination of the channel orientation in the lipid bilayer (25). Accordingly, we assumed that peptides were added to the cytosolic side of the TOM complex.

Polypeptide-induced channel closures were analyzed at voltages between −40 and +40 mV. For noise analysis and determination of blockage rates, we used standard routines available in Clampfit 10.0, which yielded the number of blockage events and the mean blockage time (also called the mean residence time).

Results

Single-channel activity of the TOM core complex in the absence of polypeptide substrates

A single TOM core complex channel was reconstituted into the planar lipid bilayer and the single-channel properties were characterized in the absence of peptide at voltage differences between −100 and +100 mV. For example, Fig. 3 shows representative single-channel recordings of a TOM core complex channel reconstituted in a planar lipid bilayer at transmembrane voltages between −20 mV and −100 mV. The channel shows the typical switching between open and closed states (Fig. 3 A) with a prevalence of closed-state conductance at higher voltages pertinent to the voltage-dependent open probability distribution of the channel (13). The corresponding amplitude histograms (Fig. 3 B) show that the majority of transitions occur at higher voltage differences (>40 mV) between the fully open state and a defined low-conductance state (13), hereafter called the closed state. At low voltages (<40 mV) the gating frequency is substantially reduced and the channel is widely found in the fully open state. The mean closure time τ increases from ∼0.1 to 5 ms from low to high transmembrane voltage differences. The open single-channel conductance was characterized as 2.9 ± 0.2 nS in 25 independent experiments, and was in agreement with previous studies (10,13). By following the asymmetry of voltage-dependent gating (data not shown), we defined a virtual orientation of the TOM core complex in the lipid membrane. In contrast to other porins, such as OmpF and LamB (26,27), this virtual orientation was random but consistent throughout one measurement.

Figure 3.

Kinetic analysis of single TOM core complex channels in the absence of substrate. (A) Membrane currents of TOM core complex reconstituted into planar lipid bilayers were recorded at constant voltage in symmetrical 1 M KCl, 20 mM K₂HPO₄, pH 7.5 solutions. The channels are basically open at voltages below −40 mV and start to gate at −60 mV. At higher voltages, the TOM-mediated currents are voltage-sensitive and switch between various conductance states, predominantly between two states of the channel. The dwell times of the low-conductance state as a function of applied voltage are indicated. (B) Amplitude histograms. Amplitude histograms and dwell times were determined from ∼3 min of channel activity. Bin width: 2 pA.

Single-channel activity of the TOM core complex in the presence of polypeptide substrates

Since some of the behavior originating from voltage gating has characteristics similar to what would be expected for polypeptide interaction, we decided to test the influence of substrate peptides on channel gating only at voltages where intrinsic channel gating was virtually absent. Current fluctuations of the TOM core complex channels were first analyzed in the absence of peptides at voltages between −100 and + 100 mV. The effect of polypeptide interaction was then tested only for channels that showed no intrinsic gating at voltages between −40 and +40 mV.

For the substrates we chose a mitochondrial presequence peptide corresponding to the first 31 residues of the precursor of the S. cerevisiae F1-ATPase β-subunit pF1β (9) and a synthetic alanine-lysine-based model polypeptide, pAK₅ (Fig. 2). Both polypeptides form α-helical structures (22,28,29). They are similar in length and have a net positive charge of +5 or +6 at physiological pH. The polypeptide pAK₅ was recently used to study the kinetics of polypeptide interaction with model pores formed by Staphylococcal aureus α-hemolysin (22,30).

As shown in Fig. 4, the addition of 10 μM pAK₅ into the lipid bilayer chamber induced hardly any ion current blockage events. With an increase in concentration of the peptides to 20 μM, very short and less frequent blockage events were visible. A single-channel analysis was performed to study the kinetic parameters of the polypeptide binding to the channel. The residence time was calculated from current recordings of at least 1 min to be <100 μs, which is the resolution limit of the instrument. In this case, quantification of peptide translocation is not possible. We hypothesize that either the pAK₅ polypeptide does not translocate through the channel and binds to the channel only briefly, or it translocates too fast to resolve the binding kinetics.

Figure 4.

Representative single-channel electrical recordings with a TOM core complex channel in the presence of pAK₅ polypeptide. (A) Membrane currents of the TOM core complex reconstituted on planar lipid bilayers were recorded at constant voltage of +40 mV in the presence of 0, 10, and 20 μM pAK₅ in symmetrical 1 M KCl, 20 mM K₂HPO₄, pH 7.5 buffers. pAK₅ was added to the trans side of the lipid bilayer. (B) All point histograms were generated from ∼1 min of channel activity and fitted with simple (control) or double Gaussian peak functions. Bin width: 2 pA.

In contrast to pAK₅, the addition of the mitochondrial presequence peptide pF1β to the bilayer chamber resulted in concentration- and voltage-dependent ion current blockades of the TOM complex channel (Fig. 5, Fig. S1 in the Supporting Material). At voltage differences < 40 mV, pF1β polypeptide-induced current blockade events could easily be witnessed. The number of blockades increased with the polypeptide concentration from ∼500 events/s at 10 μM pF1β (440 ±140 events/s, n = 6) to 900 events/s at 20 μM pF1β (960 ± 80 events/s, n = 2), applying a membrane potential of +40 mV. Peptide-binding events were only observed when the external voltage could facilitate translocation. Dilution of peptides resulted in decreased blockage frequency, which indicates that the current blockage is reversible.

Figure 5.

Interaction of a mitochondrial presequence peptide with TOM core complex channels reconstituted in planar lipid bilayers. (A) Membrane currents of TOM core complex channels were measured at a holding potential of +40 mV in the absence of peptide. Then pF1β was added to the trans side of the lipid bilayer. The frequency of polypeptide-induced current blockages was strongly dependent on the polypeptide concentration. The mean current blockage time (B) remained virtually unaltered with increasing polypeptide concentration. (C) Analysis of corresponding ion current traces shown in A. All point histograms were generated from at least 1 min of channel activity and fitted with simple (control) or double (10 μM and 20 μM) Gaussian peak functions. Bin width: 2 pA. Buffer solution: 1 M KCl, 20 mM K₂HPO₄, pH 7.5.

Effect of transmembrane voltage polarity

To elucidate the channel properties with respect to the applied voltage polarity, we repeated the measurements as shown in Fig. 5 at −40 and +40 mV. Fig. 6 A shows the typical difference between the intrinsic gating of the TOM core complex at different polarities. In contrast to Fig. 3 A, channel gating is almost negligible and can easily be used to demonstrate the effect of peptides. The addition of 10 μM pF1β to the cis side of the lipid bilayer caused substantial blocking events at a transmembrane potential of −40 mV (Fig. 6 B), corresponding to the trans addition and application of +40 mV shown in Fig. 5. There was no substantial peptide-induced gating at +40 mV. As expected, symmetric peptide addition showed peptide-induced blockage at both voltages (Fig. 6 C).

Figure 6.

Interaction of a mitochondrial presequence peptide with the TOM core complex after asymmetrical and symmetrical addition of the peptide. Current traces were recorded at a holding potential of −40 mV and +40 mV. (A) TOM core complex current traces in the absence of mitochondrial presequence peptide. (B) TOM core complex current traces in the presence of 10 μM pF1β added to the cis side of the lipid bilayer chamber. (C) TOM core complex current traces in the presence of 10 μM pF1β added to both the cis and trans sides of the chamber. Enlarged views of the each current trace are included (3 ms). Buffer solution as in Fig. 5.

Binding kinetics

Single-channel (Fig. 7) and noise (Fig. 8) analyses were used to determine the peptide-binding kinetics. The average blockage time τ_c was calculated from three independent experiments to be 150 ± 30 μs at 10 μM pF1β. The blockade time was virtually independent of polypeptide concentration and was the same irrespective of the side of peptide addition and change of transmembrane potential polarity (Table 2).

Figure 7.

Dwell time histogram of current blockades of TOM core complex pores in the presence of 10 μM pF1β. (A) Current trace recorded as in Fig. 5 at a transmembrane potential of +40 mV, representing the blockage time τ_c for several events. (B) Blockage time histogram of polypeptide-induced current blockades determined from ∼3 min of channel activity. The histogram was fitted to a single exponential function with a mean blockage time of 150 ± 30 μs.

Figure 8.

Examples of power spectra of ion current traces shown in Figs. 3–5. The power density spectra increased with applied voltage. In the absence of peptides, the background noise (lines 1 (20 mV) and 2 (40 mV)) increased by almost an order of magnitude from 20 to 40 mV. The addition of 20 μM pF1β (lines 3 (20 mV) and 4 (40 mV)) increased the noise by almost two orders of magnitudes. Note that in the absence of peptides the noise decreases rather linearly with frequency, whereas in the presence of peptides the spectra show the $1/f^2$ dependence typically found for channels with a binding site for substrates.

Table 2.

Rate constants of pF1β association k_on and dissociation k_off of the interaction between pF1β and the TOM core complex channel

Peptide concentration(μM)	$k_{on}^{cis}\left(20mV\right)$(s-1M−1)	$k_{on}^{cis}\left(40mV\right)$(s−1M−1)	$k_{off}\left(20mV\right)$(s−1)	$k_{off}\left(40mV\right)$(s−1)
10	no data	50 × 106	no data	6000
20	50 × 106	45 × 106	5000	6000

The association rate constants k_on were calculated from the frequency of blockades (number of events, counts) as a function of substrate concentration (k_on = number of events s⁻¹/[concentration of the peptide]). The dissociation constants k_off were calculated from the residence (blockage) time τ_c (k_off =1/τ_c) (24). Noise analysis revealed the power spectra of ion currents through single TOM core complex channels in the absence and presence of peptides to further illustrate peptide binding (Fig. 8). The power spectrum is voltage-dependent. Raising the transmembrane voltage from 20 to 40 mV in the absence of peptide enhances the background noise by almost an order of magnitude, in agreement with Fig. 3. The presence of peptides (20 μM pF1β) resulted in additional current fluctuations. The spectra obtained show two orders of magnitude of excess noise compared to one in the absence of peptide. Inspection of the on- and off-rates of peptide interaction obtained from the single-channel analysis above indicates that the power spectra are independent of the peptide concentration in the micromolar range. In agreement with this, the excess noise was not influenced by a variation of the peptide concentration in the micromolar range (data not shown).

Discussion

The transfer of proteins across a lipid bilayer requires an assembly of proteins that recognize the polypeptide to be translocated, mediate its insertion into a protein pore, facilitate the transfer through the pore, and drive the movement so that it becomes vectorial.

The aim of this study was to shed light on the interaction of mitochondrial preproteins through the protein-conducting channel of the TOM complex at high temporal resolution.

Using a purified TOM core complex consisting of five subunits (Tom40, Tom22, Tom7, Tom6, and Tom5 (10)), we established a reduced system containing only the channel part. The driving force for polypeptide translocation across the outer membrane of mitochondria in vivo remains to be determined (31). One hypothesis is based on biased diffusion and a Brownian ratchet mechanism that binds receptor proteins at the trans site of the membrane. The power stroke model proposes that mitochondrial chaperones, such as mtHsp70, bind to an incoming precursor chain, and then undergo a conformational change and actively pull the precursor through the pore (32,33). In our system, the main driving force for peptide binding and translocation is clearly the transmembrane potential.

Experimental insights into the translocation of polymers or small organic solutes through aqueous nanopores have been obtained with the use of high-resolution electrophysiological techniques and model pores such as α-hemolysin and macromolecules (e.g., oligonucleotides and peptides (22,23)) or mitochondrial porin and ATP (34). Substrate-pore interactions and partitioning of the substrate into individual pores were explored by probing the frequency and duration of transient substrate-induced current blockages. Current blockages were found to be sensitive to the properties of the biopolymers and provided a rich source of information to elucidate the mechanism of polymer capture and transport on physical grounds.

The first quantitative data addressing peptide translocation through mitochondrial import channels (TIM and TOM) were based on patch-clamp experiments using isolated mitochondrial membrane fractions (25). We focused on the reconstitution of purified channels to separate the individual contributions of different parts of the complex in peptide interactions (9,10) to identify the pore-forming subunit of the translocation machinery.

In this study, we attempted to separate the different contributions of intrinsic, voltage-dependent, and polypeptide-induced channel gating. The results of our single-channel measurements show a large variance of intrinsic gating in the absence of peptide and independence of the voltage. This may have been caused by some instability of the purified 400 kDa TOM core complex, as also suggested by the observation that older protein batches showed more intrinsic gating. By focusing on stable channels with no intrinsic gating, we were able to enhance the resolution of the peptide-channel interaction. Using this approach, the number of subconductance states was reduced to the previously characterized fully open state (S5) and one low-conductance state (S3 (13)).

We find substantial differences in the interaction of the TOM core complex with mitochondrial and unrelated substrates. As expected, the mitochondrial pF1β peptide was significantly more potent in blocking the pore than the synthetic nonmitochondrial model peptide pAK₅, which was similar in length and charge. Therefore, the pF1β peptide is an ideal substrate for studying the mechanism of peptide interactions with TOM complex channels at the single-molecule level. Micromolar concentrations of pF1β appear to be sufficient to induce TOM channel blockage. They are also in the relevant range to inhibit protein import into mitochondria (35).

A quantitative analysis of ion current traces in the presence of pF1β peptides revealed the number of binding events and peptide-induced blockage time. To date, the connection between substrate blockage and translocation of single peptides through TOM from one chamber to the other has not been experimentally verified. Direct evidence for translocation may be obtained in future work by combining nanopore analysis with optical single-molecule approaches that allow simultaneous monitoring of single ion-channel currents and detection of molecules residing inside a pore (36).

According to our results, the addition of 20 μM peptides and application of +40 mV (Fig. 5) revealed ∼1000 blockage events per second. This may result in ∼500 translocation events per second with an average blockage time of ∼150 μs. These values are in the expected range for similar peptides translocating through other channels, such as COX-IV moving through α-hemolysin pores (22,23,30).

Assuming a two-state channel (open and closed corresponding to unbound and bound peptides), fluctuation theory predicts that the corner frequencies ${\omega }_0$ at which power spectra amplitudes decay to 1/2 S₀ are related to the on- and off-rates of substrate interaction according to ${\omega }_0=2\pi f_c=k_{on}\left[M\right]+k_{off}$, where $\left[M\right]$ is the peptide concentration (37). According to this model, our data obtained from the single-channel analysis (Table 2) predict a corner frequency $f_c$ between 3 and 3.5 kHz. This is in agreement with the fluctuation analysis of the ion current traces shown in Figs. 3–5. Note that the excess noise in the power spectra caused by a transmembrane voltage has a different decay than the excess noise induced by peptide addition. Fig. 8 clearly indicates that peptides interact with the channel, and not by causing a Donnan potential.

The single-molecule experiments presented here may open exciting opportunities to further explore the mechanism by which mitochondrial preproteins interact with the TOM machinery. They can complement existing and future biochemical studies that focus on the molecular properties of the translocation machinery to elucidate its cellular role.