Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes

Holly C Ford, William J Allen, Gonçalo C Pereira, Xia Liu, Mark Simon Dillingham, Ian Collinson*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

14 Citations (Scopus)
75 Downloads (Pure)

Abstract

Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-sequence translocase of the inner membrane (TIM23-complex) imports proteins with cleavable pre-sequences. Progress in understanding these transport mechanisms has been hampered by the poor sensitivity and time resolution of import assays. However, with the development of an assay based on split NanoLuc luciferase, we can now explore this process in greater detail. Here, we apply this new methodology to understand how ∆ψ and ATP hydrolysis, the two main driving forces for import into the matrix, contribute to the transport of pre-sequence-containing precursors (PCPs) with varying properties. Notably, we found that two major rate-limiting steps define PCP import time: passage of PCP across the outer membrane and initiation of inner membrane transport by the pre-sequence - the rates of which are influenced by PCP size and net charge. The apparent distinction between transport through the two membranes (passage through TOM is substantially complete before PCP-TIM engagement) is in contrast with the current view that import occurs through TOM and TIM in a single continuous step. Our results also indicate that PCPs spend very little time in the TIM23 channel - presumably rapid success or failure of import is critical for maintenance of mitochondrial fitness.

Original languageEnglish
Article numbere75426
JournaleLife
Volume11
DOIs
Publication statusPublished - 8 Jun 2022

Bibliographical note

Funding Information:
We would like to thank Prof. Andrew Halestrap for his insight and enthusiastic discussions on the mysteries of mitochondrial bioenergetics. We also thank past and present members of the Collinson lab who helped to get this project off the ground, particularly Drs Andrew Richardson and Dylan Noone. Funding: This research was funded by the Wellcome Trust: Investigator Award to IC (104632/Z/14/Z).

Publisher Copyright:
‍@ Ford et al.

Keywords

  • Carrier Proteins/metabolism
  • Luciferases
  • Mitochondrial Precursor Protein Import Complex Proteins/metabolism
  • Mitochondrial Proteins/metabolism
  • Protein Transport
  • Saccharomyces cerevisiae/metabolism
  • Saccharomyces cerevisiae Proteins/metabolism

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