Spatiotemporal stop-and-go dynamics of the mitochondrial TOM core complex correlates with channel activity

Abstract

The TOM complex of the outer membrane of mitochondria is the main entry gate for nuclearencoded proteins from the cytosol into mitochondria. Single-molecule studies can reveal phenomena that remain hidden in ensemble measurements. In this thesis, total internal reflection fluorescence (TIRF) microscopy has been used to study the correlation between lateral protein diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC) in membranes resting on ultrathin hydrogel films. Using electrode-free optical recordings of ion flux, it is shown that TOM-CC switches reversibly between three states of ion permeability associated with protein diffusion. While freely diffusing TOM-CC molecules are predominantly in a high permeability state, non-mobile molecules are mostly in an intermediate or low permeability state. This behavior can be explained by the mechanical binding of the two protruding Tom22 subunits to the hydrogel and a concomitant combinatorial opening and closing of the two β-barrel pores of TOM-CC. TOM-CC is thus the first β-barrel membrane protein complex to exhibit membrane state-dependent mechanosensitive properties.