Investigating Tom40 Structure and Function Relationship using Single Channel Analysis

Abstract

Nearly all mitochondrial proteins are coded by nuclear genome and must be transported across mitochondrial membranes to their final functional locations. This translocation of partially folded proteins is accomplished by several multi-protein complexes in the mitochondrial outer and inner membranes. Tom40 is the central pore-forming subunit of the multi-protein translocase complex in the mitochondrial outer membrane (TOM). Although much is known regarding the general organization of the TOM complex, the molecular basis of Tom40's ability to translocate both acidic pre-sequences of matrix proteins and hydrophobic membrane proteins remains unclear. Here we have functionally investigated the role of structural domains of Tom40. Fungal Tom40 is predicted to be comprised of a membrane-spanning beta-barrel domain with strongly conserved alpha-helical domains at N- and C-termini. We have refolded and purified a full-length (FL) fungal Tom40 and N-terminal truncation mutant (Tom40-319), and characterized their electrophysiological behavior in planar lipid membranes. Our results demonstrate that recombinant Tom40 forms cation-selective channels with four well-defined conductive states, thus reconciling conflicting reports in literature regarding conductance of the channel. High cation selectivity of all conducting sublevels of Tom40 suggests the presence of a constriction zone within the channel lumen. Tom40-319 has a lower selectivity for cations, suggesting that the N-terminal alpha-helix is located in the channel lumen. Both Tom40-FL and the 319 mutant interact with specific polycationic precursor peptide of the F1β subunit of ATP synthase in a concentration- and voltage-dependent manner. However, Tom40-319 has a lower apparent affinity for pF1β than FL, which also suggests that the N-terminal alpha-helix is located inside the channel lumen. Furthermore, both Tom40 FL and the 319 mutant do not show interactions with SynB2, a synthetic peptide of similar length and charge as pF1β, thus confirming Tom40 specificity for the presequence peptide.