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.