Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes
- University of Bristol, United Kingdom
- University of Cambridge, United Kingdom
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 properties such as 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 maintaining mitochondrial fitness.
Data availability
We present secondary analysis of raw optical readout data. All raw data is included in the manuscript, supplementary information and source data.
Article and author information
Author details
Mark Simon Dillingham
Funding
Wellcome Trust (104632)
- Holly C Ford
Wellcome Trust (104632)
- William J Allen
Wellcome Trust (104632)
- Gonçalo C Pereira
Wellcome Trust (104632)
- Xia Liu
Wellcome Trust (104632)
- Ian Collinson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Ford et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes
eLife 11:e75426.
https://doi.org/10.7554/eLife.75426
