Mapping protein interactions in the active TOM-TIM23 supercomplex

Ridhima Gomkale,Peter Rehling,Blanche Schwappach,Ralf Ficner,Olexandr Dybkov,Christian Schulz,Stefan Stoldt,Luis Daniel Cruz-Zaragoza,Piotr Neumann,Stefan Jakobs,Henning Urlaub,Alexander Benjamin Schendzielorz,Andreas Linden,Markus Kilisch

doi:10.1038/s41467-021-26016-1

Abstract

Nuclear-encoded mitochondrial proteins destined for the matrix have to be transported across two membranes. The TOM and TIM23 complexes facilitate the transport of precursor proteins with N-terminal targeting signals into the matrix. During transport, precursors are recognized by the TIM23 complex in the inner membrane for handover from the TOM complex. However, we have little knowledge on the organization of the TOM-TIM23 transition zone and on how precursor transfer between the translocases occurs. Here, we have designed a precursor protein that is stalled during matrix transport in a TOM-TIM23-spanning manner and enables purification of the translocation intermediate. Combining chemical cross-linking with mass spectrometric analyses and structural modeling allows us to map the molecular environment of the intermembrane space interface of TOM and TIM23 as well as the import motor interactions with amino acid resolution. Our analyses provide a framework for understanding presequence handover and translocation during matrix protein transport.

Highlights

Nuclear-encoded mitochondrial proteins destined for the matrix have to be transported across two membranes
Based on molecular modeling approaches of protein interactions, we propose a model of the TOM-TIM23 transition zone and provide insight into precursor transport from the outer membrane TOM to the inner membrane TIM23 complex and on interactions in the mitochondrial import motor
Upon inner membrane translocation of the precursor a translocation intermediate spanning both TOM and TIM23 complexes is established by the precursor[39]

Summary

Introduction

Nuclear-encoded mitochondrial proteins destined for the matrix have to be transported across two membranes. The cross-linking data allowed us to define interaction sites between TOM, TIM23, and PAM (presequence translocase-associated import motor) complex constituents (Fig. 3a, b). The cross-linking analyses identified multiple interaction sites between the import motor and the membrane module constituents of the TIM23 complex (Fig. 3b).

Results

Conclusion