Evidence of Distinct Channel Conformations for the Mitochondrial Outer Membrane Translocase Tom40

Abstract

Nearly all mitochondrial proteins must be transported across mitochondrial membranes to their final functional locations by the translocase of the outer membrane (TOM) complex. Tom40 is the central pore-forming subunit of the TOM complex. While extensive studies have elucidated the substrate processing of the TOM complex, the structural basis of Tom40's function remains unclear. Fungal Tom40 is predicted to comprise a membrane-spanning β-barrel domain with conserved α-helical domains at the N- and C-termini. Using recombinant Tom40 from the yeast C. glabrata, as well as truncated forms lacking the N- and C-termini, we characterized the channels they formed upon reconstitution into planar lipid membranes. Our results demonstrate that all forms of Tom40 function as cation-selective channels with up to four conducting levels, indicating that the N- and C-termini are not essential structural elements of the channel. Cation selectivity ratios are markedly high for all four conductive sub-levels, suggesting the presence of a constriction zone within the channel lumen. Full length and truncated Tom40 constructs interact with a presequence peptide from the F1β subunit of mitochondrial ATP synthase in a concentration- and voltage-dependent manner. Unexpectedly, substrate binding affinity was dependent upon the channel conductive state. These results provide the first evidence for structurally distinct Tom40 conformations that may play a role in substrate recognition and translocation.