Cation selectivity of the presequence translocase channel Tim23 is crucial for efficient protein import.

Niels Denkert,Michael Meinecke,Lennart Versemann,Peter Rehling,Alexander Benjamin Schendzielorz,Frank Richter,Mariam Barbot

doi:10.7554/elife.28324

Abstract

Virtually all mitochondrial matrix proteins and a considerable number of inner membrane proteins carry a positively charged, N-terminal presequence and are imported by the TIM23 complex (presequence translocase) located in the inner mitochondrial membrane. The voltage-regulated Tim23 channel constitutes the actual protein-import pore wide enough to allow the passage of polypeptides with a secondary structure. In this study, we identify amino acids important for the cation selectivity of Tim23. Structure based mutants show that selectivity is provided by highly conserved, pore-lining amino acids. Mutations of these amino acid residues lead to reduced selectivity properties, reduced protein import capacity and they render the Tim23 channel insensitive to substrates. We thus show that the cation selectivity of the Tim23 channel is a key feature for substrate recognition and efficient protein import.

Highlights

Double membrane bounded mitochondria import over 1000 different proteins synthesized on cytosolic ribosomes (Endo and Yamano, 2009; Neupert and Herrmann, 2007; Schmidt et al, 2010)
The ability of TIM23 mutants to complement Tim23 function was monitored by plasmid shuffle on 5-fluoroorotic acid (5FOA)-containing medium (Figure 1B)
Due to lack of structural information the current understanding is little more than that at the heart of the TIM23 complex a water-filled pore facilitates the passage of preproteins across the inner membrane

Summary

Introduction

Double membrane bounded mitochondria import over 1000 different proteins synthesized on cytosolic ribosomes (Endo and Yamano, 2009; Neupert and Herrmann, 2007; Schmidt et al, 2010). The TIM23 complex transports precursor proteins across the inner membrane, or, if they contain additional sorting signals, inserts them into the IM (Neupert and Herrmann, 2007; van der Laan et al, 2007). DÉ is necessary and sufficient for membrane insertion of IM proteins (van der Laan et al, 2007), whereas full translocation of proteins into the mitochondrial matrix depends on additional energy provided by the ATP consuming presequence translocase-associated import motor PAM (Neupert and Brunner, 2002; Schendzielorz et al, 2017). The receptor protein Tim as well as Mgr are constitutive subunits of the presequence translocase, whereas Tim is specific to the TIM23 complex in the absence of the PAM motor

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