期刊
BIOPHYSICAL JOURNAL
卷 95, 期 3, 页码 1511-1522出版社
CELL PRESS
DOI: 10.1529/biophysj.108.131003
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The protein translocase of the outer mitochondrial membrane (TOM) serves as the main entry site for virtually all mitochondrial proteins. Like many other protein translocases; it also has an ion channel activity that can be used to study the dynamical properties of this supramolecular complex. We have purified TOM core complex and Tom40, the main pore forming subunit, from mitochondria of the filamentous fungus Neurospora crassa and incorporated them into planar lipid bilayers. We then examined their single channel properties to provide a detailed description of the conformational dynamics of this channel in the absence of its protein substrate. For isolated TOM core complex we have found at least six conductance states. Transitions between these states were voltage-dependent with a bell-shaped open probability distribution and distinct kinetics depending on the polarity of the applied voltage. The states with the largest conductance followed an Ohmic IN characteristic consistent with a large cylindrical pore with very little interaction with the permeating ions. For the lower conductance states, however, we have observed inverted S-shaped nonlinear current-voltage curves reminiscent to those of much narrower pores where the permeating ions have to surmount an electrostatic energy barrier. At low voltages (<+/- 70 mV), purified Tom40 protein did not show any transitions between its conductance states. Prolonged exposure to higher voltages induced similar gating behavior to what we observed for TOM core complex. This effect was time-dependent and reversible, indicating that Tom40 forms not only the pore but also contains the gating machinery of the complex. However, for proper functioning, additional proteins (Tom22, Tom7, Tom6, and Tom5) are required that act as a modulator of the pore dynamics by significantly reducing the energy barrier between different conformational states.
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