Nussberger, S. | Search bioRxiv

bioRxiv2023

Two conformations of the Tom20 preprotein receptor in the TOM holo complex

The TOM complex is the main entry point for precursor proteins into mitochondria. Precursor proteins containing targeting sequences are recognized by the TOM complex and imported into the mitochondria. We have determined the structure of the TOM core complex from Neurospora crassa by single-particle cryoEM at 3.3 [A] resolution, showing its interaction with a bound presequence at 4 [A] resolution, and of the TOM holo complex including the Tom20 receptor at 6-7 [A] resolution. TOM is a transmembrane complex consisting of two {beta}-barrels, three receptor subunits and three short transmembrane subunits. Tom20 has a transmembrane helix and a receptor domain on the cytoplasmic side. We propose that Tom20 acts as a dynamic gatekeeper, guiding precursor proteins into the pores of the TOM complex. We analyze the interactions of Tom20 with other TOM subunits, present insights into the structure of the TOM holo complex, and suggest a translocation mechanism.

biophysics↗

bioRxiv2021

Correlation of mitochondrial TOM core complex stop-and-go and open-closed channel dynamics

The role of lateral diffusion of proteins in the membrane in the context of function has not been examined extensively. Here, we explore the relationship between protein lateral diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC). Optical ion flux sensing through single TOM-CC molecules shows that TOM-CC can occupy three ion permeability states. Whereas freely diffusing TOM-CC molecules are preferentially found in a high permeability state, physical tethering to an agarose support causes the channels to transition to intermediate and low permeability states. This data shows that combinatorial opening and closing of the two pores of TOM-CC correlates with lateral protein diffusion in the membrane plane, and that the complex has mechanosensitive-like properties. This is the first demonstration of {beta}-barrel protein mechanosensitivity, and has direct conceptual consequences for the understanding of the process of mitochondrial protein import. Our approach provides a novel tool to simultaneously study the interplay of membrane protein diffusion and channel dynamics.

biophysics↗