Polypeptide Translocation Through the Mitochondrial TOM Channel

The TOM protein complex facilitates the transfer of nearly all mitochondrial preproteins across outer mitochondrial membranes. Here we characterized t...
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Letter pubs.acs.org/JPCL

Polypeptide Translocation Through the Mitochondrial TOM Channel: Temperature-Dependent Rates at the Single-Molecule Level Kozhinjampara R. Mahendran,† Usha Lamichhane,† Mercedes Romero-Ruiz,‡ Stephan Nussberger,‡ and Mathias Winterhalter*,† †

School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, D-28759 Bremen, Germany Biophysics Department, Institute of Biology, University of Stuttgart, D-70550 Stuttgart, Germany

‡

S Supporting Information *

ABSTRACT: The TOM protein complex facilitates the transfer of nearly all mitochondrial preproteins across outer mitochondrial membranes. Here we characterized the effect of temperature on facilitated translocation of a mitochondrial presequence peptide pF1β. Ion current fluctuations analysis through single TOM channels revealed thermodynamic and kinetic parameters of substrate binding and allowed determining the energy profile of peptide translocation. The activation energy for the on-rate and off-rate of the presequence peptide into the TOM complex was symmetric with respect to the electric field and estimated to be about 15 and 22 kT per peptide. These values are above that expected for free diffusion of ions in water (6 kT) and reflect the stronger interaction in the channel. Both values are in the range for typical enzyme kinetics and suggest one process without involving large conformational changes within the channel protein. SECTION: Biomaterials, Surfactants, and Membranes polypeptide corresponding to the first 31 residues of the mitochondrial precursor of the S. cerevisiae F1-ATPase β-subunit pF1β.10,11 We observed temperature and voltage-dependent channel blockages in the presence of the mitochondrial presequence peptide pF1β at a single molecule level. An analysis of the temperature dependent rates gives information on the energy barrier for translocation. Isolated TOM core complex reconstituted in stable DPhPC lipid bilayers showed characteristic single channel conductance of ∼3.0 nS in 1 M KCl, 20 mM phosphate and pH 7.4 at room temperature. Increasing temperature cause the bulk conductance to increase. As expected, increasing the temperature from 2 to 30 °C increases the open channel conductance. Moreover, TOM channels show voltage and temperature-dependent channel closure. The higher the voltage and temperature, the more gating occurs (data not shown). TOM core complex showed an asymmetric channel closure and conductance with respect to the polarity of the applied voltage as shown previously.10,11 At 2 °C (Figure 1A), the channel is open without any gating perturbations with stable ion current. Addition of peptide pF1β to the trans side produces few ion current blockages with typical blocking times of 1 ms at 2 °C (Figure 1A). Increasing the temperature to 10 °C results in an increase of the number of blockage events and a decrease of the residence time of peptide inside the channel (Figure 1B). At higher temperatures

T

he transfer of proteins across lipid membranes is a fundamental process in biology. Understanding the mechanism of this process has been one of the most challenging tasks of the past decade. Extensive studies of protein import into mitochondria in fungi, plants, and mammalian cells revealed several protein translocation machineries in inner and outer mitochondrial membranes.1 In particular, the outer membrane preprotein translocase TOM acts as the main entry gate for nearly all proteins of mitochondria. Most components of the mitochondrial outer membrane import machinery are known and form multimeric protein complexes with a molecular mass between 350 and 500 kDa.1−5 Electron microscopy studies on TOM purified from Neurospora crassa and yeast revealed the overall shape of the holo complex and the core complex, which consists of the components Tom40, 22, 7, 6, and 5.2,5,6 Electrophysiological studies suggested that the general import pore Tom40 itself forms one pore.6−8 Single-channel recordings of TOM core complex from Saccharomyces cerevisiae and N. crassa as well as isolated Tom40 reconstituted into planar lipid bilayers revealed a voltage-dependent increase of channel closures in the presence of mitochondrial targeting peptides.7−12 However, since TOM channels show spontaneous voltage-dependent gating behavior in the absence of substrate, interactions between peptides and TOM were analyzed at low voltages only where endogenous channel gating was significantly reduced. 10,11 Here, we investigated the effect of temperature on peptide translocation through TOM core complex isolated and purified from N. crassa mitochondria (Supporting Information A). We selected a © XXXX American Chemical Society

Received: November 4, 2012 Accepted: December 11, 2012

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Figure 1. Ion current recordings through a single TOM core complex channel in the absence and presence of 10 μM pF1β peptide (trans side) at +40 mV and at (A) 2 °C, (B) 10 °C and (C) at 20 °C. The buffer contained 1 M KCl, 20 mM phosphate, pH 7.4.

(≥20 °C), the blocking events are shorter and become more frequent (Figure 1C). It should be noted that addition of the cationic pF1β peptide to the trans side of the membrane required positive potentials on the opposite side (cis) to promote the interaction between peptide and TOM complex, whereas reversing the voltage inhibits the entry of peptides, and no ion current blockages were observed. Statistical analysis of the ion trace from 5 to 30 °C revealed the average residence time of peptide and number of blockage events. Figure 2A shows that the average residence time of peptide decreases with increasing temperature from 5 to 30 °C. Moreover, the average residence time of peptide decreased with the increase in the applied transmembrane voltage. Furthermore, the number of blocking events increases exponentially with the temperature (Figure 2B) about 5-fold from 5 to 30 °C. In addition, the number of peptide blockage events is strongly voltage dependent and with increase in voltage from 20 mV to 40 mV, the number of events increased, which indicates that the electric field pulled the peptides from the bath solution into an affinity site inside the channel. To elucidate possible asymmetries in translocation, we added pF1β peptide to the cis side of the TOM core complex and investigated translocation from the opposite side. In Figure 3, we show ionic current fluctuations through TOM core complex in the absence and presence of 10 μM pF1β added to the cis side of the membrane recorded at temperatures of 5 and 25 °C. Application of negative potentials induced ion current blockages through the TOM complex in the presence of the peptide. At

Figure 2. (A) The peptide residence time in the channel and (B) the number of pF1β peptide binding events as a function of temperature from 5 to 30 °C at 20 and 40 mV. The buffer contained 1 M KCl, 20 mM phosphate, pH 7.4, and 10 μM pF1β peptide added to the trans side of the lipid membrane.

25 °C, the peptide caused significant ion current fluctuations, whereas few blocking events were visible at 5 °C. 79

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Figure 3. Ion current recordings through a single TOM core complex channel in the absence and presence of 10 μM pF1β peptide (same setup as in Figure 2 but with peptide addition on the cis side) at −40 mV: (A) T = 5 °C; (B) T = 25 °C. The buffer contained 1 M KCl, 20 mM phosphate, and pH 7.4.

Similar to trans side addition of peptide, the average residence time of the peptide in the cis side decreases with the increase in the temperature (Figure 4A). In both cases, the number of peptide blocking events increased with the increase in the temperature and applied voltage (Figure 4B). It should be noted, that the number of events and residence times show a broad static distribution between individual channels requiring measurements of a complete set on one channel prior averaging kinetic properties.13,14 Within the experimental error the number of blockage events and residence times were symmetric with respect to applied voltage and side of addition. In this context we should mention that the on-rate is calculated from the number of blockage events, and the off-rate is calculated from the average residence time of the peptide blockage.15 Regarding TOM complex as an enzyme catalyzing the translocation, we measure peptide entry and exit from the channel for a broad temperature range. The variation of the onand off-rates with temperature can be transferred into an Arrhenius plot giving an activation barrier Ea. For further analysis, we may write for the forward rate kon = Aon exp(−Eaon /kT) and a similar one for the off-rate. In Figure 5 we present on- and off-rates in an Arrhenius plot for trans side addition of the peptide at 40 mV. The best fit for the on- and off-rates revealed an activation barrier of Ea = 15 and 22 kT, respectively. Similarly, we calculated the activation barrier for the cis side addition of the peptide (Supporting Information Figure 1). Inspection of the rates reveal that translocation is not simply diffusion limited.16,17 The energy barrier contains several contributions, the most obvious one originating from the

Figure 4. (A) The residence time and (B) the number of peptide binding events as a function of temperature from 5 to 30 °C at −20 and −40 mV. The buffer contained 1 M KCl, 20 mM phosphate, pH 7.4, and 10 μM pF1β peptide added to the cis side of the lipid membrane.

viscosity of the solvent itself. For example, KCl diffusion in bulk water has an activation barrier of about 6 kT, whereas in 80

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regulation (Dagan). Purified TOM core complex was added to the cis side of the membrane, and protein insertion was facilitated by mixing the contents of the chamber. Electrical recordings were made through a pair of Ag/AgCl electrodes (World Precision Instruments, Sarasota, FL, USA). One electrode was used as the ground (cis), and the other (trans) was connected to an Axon Instruments 200B amplifier with a capacitive headstage, digitized by an Axon Digidata 1440A digitizer and computer controlled by Clampex 10.0 software (Axon Instruments, Foster City, CA, USA). The data was filtered by an analog low-pass 4-pole Bessel filter at 10 kHz, digitally sampled at 50 kHz, and analyzed using the pClamp software. The mitochondrial signal sequence peptide pF1β (Ac-MVLPRLYTATSRA-AFKAAKQSAPLLSTSWKRNH2, charge +6) used for the measurements were customsynthesized, confirmed by MALDI-TOF mass spectrometry, and had an HPLC purity of at least 95% (Biosyntan, Berlin, Germany).

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ASSOCIATED CONTENT

S Supporting Information *

(A) Isolation of TOM core complex; (B) Arrhenius plot of the kon and koff as a function of temperature for cis side peptide addition; (C) Arrhenius plot for the conductance. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 5. Arrhenius plot of (A) koff and (B) kon as a function of temperature from 5 to 30 °C resulting in a linear slope with an energy barrier of about 22 and 15 kT. The buffer contained 1 M KCl, 20 mM phosphate, pH 7.4, and 10 μM pF1β peptide added to the trans side of the lipid membrane at applied voltage of 40 mV.

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AUTHOR INFORMATION

Corresponding Author

TOM channels, a slightly higher activation of 6.4 kT is obtained using our temperature-dependent channel conductance (Supporting Information Figure 2). The additional 8.6 kT or 14.6 kT reflects the additional interaction of the peptide with the channel. The activation barrier is a characteristic of the channel. For example, the modulation of the activation energy can be compared with OmpF. Here the interaction of KCl ions with the channel surface results in an enhanced barrier of 8.5 kT and has been reproduced by all-atom modeling.18,19 In this context, we should mention a previous study using α-hemolysin as a model system to study the interaction between artificial cationic polypeptides and the β-barrel protein pore.20−22 In the absence of peptide channel interactions, the residence times of peptides inside the channel would rather be in the nanosecond range.23 We conclude that the transport of pF1β peptide through the TOM complex is effectively not diffusion limited. We have shown that temperature plays a very important role in the strength of solute interaction and the energy profile of the translocation can be demonstrated. Single-molecule experiment opens many exciting opportunities for exploring the mechanisms by which the proteins interact with receptors or protein translocase. Further experimentation combined with structural analysis and computational modeling24 will be used in the future studies to understand the molecular mechanism involved in the protein translocation machinery.

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank Beate Nitschke for expert technical assistance. The work was supported by a Competence Network on Functional Nanostructures grant of the Baden-Württemberg Stiftung (TP-A03) to S.N. and a grant of the Deutsche Forschungsgemeinschaft (Wi2278/18-1) to M.W.

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REFERENCES

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EXPERIMENTAL METHOD Single-Channel Recordings. Virtually solvent-free planar lipid membranes were formed using 1,2-diphytanoyl-sn-glycero-3phosphatidylcholine (DPhPC) (Avanti Polar Lipids, Alabaster, AL, USA) according to the Montal−Mueller technique with slight modifications.10−12,25 Most of the experiments were carried out with a Teflon septum of 40 μm aperture that yielded stable bilayers prepainted with 1% squalene. The measurements were carried out with buffer containing 1 M KCl, 20 mM Phosphate, pH 7.4. Peltier element was included for temperature 81

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