Protein translocation through Tom40: kinetics of peptide release

Abstract

Mitochondrial proteins are almost exclusively imported into mitochondria from the cytosol in an unfolded or partially folded conformation. Regardless of whether they are destined for the outer or inner membrane, the intermembrane space, or the matrix, proteins begin the importation process by crossing the mitochondrial outer membrane via a specialized protein import machinery whose main component is the Tom40 channel. High-resolution ion conductance measurements through the Tom40 channel in the presence of the mitochondrial presequence peptide pF(1)β revealed the kinetics of peptide binding. Here we show that the rates for association k(on) and dissociation k(off) strongly depend on the applied transmembrane voltage. Both kinetic constants increase with an increase in the applied voltage. The increase of k(off) with voltage provides strong evidence of peptide translocation. This allows us to distinguish quantitatively between substrate blocking and permeation.

Figure 1

Purification and single-channel recordings of N. crassa Tom40. (A) Coomassie Blue-stained SDS-polyacrylamide gel of purified Tom40. Tom40 was purified from the TOM core complex of N. crassa mitochondria as previously described (15, 29). (B and C) Ionic currents through a single Tom40 channel at +50 and −50 mV. At 50 mV, the channel exits in one open conductance state, whereas at −50 mV the channel fluctuates between an open and a closed conductance state. The corresponding amplitude histogram is shown on the right side. Experimental conditions are 1 M KCl, 20 mM MES, pH 6, at room temperature.

Figure 2

Typical ion current recordings through a single Tom40 channel in the presence of (A) 0 μM, (B) 5 μM, and (C) 10 μM pF₁β peptide added to the trans side of the lipid membrane with applied voltage of +50 mV. Experimental conditions are 1 M KCl, 20 mM MES, pH 6, at room temperature.

Figure 3

(A) Number of binding events increases with an increase in the concentration of the peptide from 2 μM to 20 μM measured at 20 mV and 50 mV. (B) The number of binding events increases with an increase in the applied transmembrane voltage from 20 mV to 75 mV at 10 μM peptide. Experimental conditions are 1 M KCl, 20 mM MES, pH 6, and peptide added to the trans side at room temperature (n = 5, mean ± SE).

Figure 4

(A) Single exponential fitting of a dwell-time histogram for peptide blockage. (B) The residence time decreases with an increase in the applied transmembrane voltage from 20 mV to 75 mV at 10 μM peptide. (C) The residence time does not depend on the concentration of peptide and remains the same irrespective of an increase in peptide concentration from 2 μM to 20 μM measured at 20 mV and 50 mV. Experimental conditions are 1 M KCl, 20 mM MES, pH 6. Peptide was added to the trans side of the lipid bilayer at room temperature (n = 5, mean ± SE).

Figure 5

Typical ion current recordings through a single Tom40 channel in the presence of (A) 0 μM and (B) 5 μM pF₁β peptide added to the trans side of the lipid membrane with applied voltage of +50 mV. Experimental conditions are 150 mM KCl, 10 mM MES, pH 6, at room temperature.

Figure 6

(A) Power spectral densities of the fluctuations in the ion current caused by peptide blocking a single Tom40 channel at different peptide concentrations ranging from 5 μM to 20 μM at 50 mV. (B) Power spectral densities of the fluctuations in the ion current caused by peptide blocking a single Tom40 channel at different applied voltages ranging from 20 to 50 mV at 10 μM pF₁β peptide. Experimental conditions are 1 M KCl, 20 mM MES, pH 6. Peptides were added to the trans side of the channels at room temperature.

Figure 7

Typical ion current recordings through single Tom40 channels (A) in the absence of peptide at −50 mV and 50 mV, (B) with 5 μM peptide added to the cis side of lipid membrane with applied voltage of −50 mV and + 50 mV, and (C) with 5 μM peptide added to the trans side of lipid membrane with applied voltage of −50 mV and + 50 mV. Experimental conditions are 1 M KCl, 20 mM MES, pH 6, at room temperature. The schematic representations showing peptide translocation through N. crassa Tom40 driven by applied transmembrane potential are based on the work of Gessmann et al. (44).

Figure 8

(A) Number of binding events increases with a decrease in the applied transmembrane voltage from −20 mV to −75 mV at 10 μM peptide. (B) The residence time decreases with a decrease in the applied transmembrane voltage from −20 mV to −75 mV at 10 μM peptide. Experimental conditions are 1 M KCl, 20 mM MES, pH 6, and peptide was added to the cis side of the channels at room temperature (n = 5, mean ± SE).