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Structural changes in the mitochondrial Tim23 channel are coupled to the proton-motive force

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Abstract

Tim23, the central subunit of the TIM23 protein-translocation complex, forms a voltage-gated channel in the mitochondrial inner membrane (MIM), an energy-conserving membrane that generates a proton-motive force to drive vital processes. Using high-resolution fluorescence map** of a channel-facing transmembrane segment (TMS2) of Tim23 from Saccharomyces cerevisiae, we demonstrate that changes in the energized state of the MIM cause marked structural alterations in the channel region. In an energized membrane, TMS2 forms a continuous α-helix that is inaccessible to the aqueous intermembrane space (IMS). Upon depolarization, the helical periodicity of TMS2 is disrupted, and the channel becomes exposed to the IMS. Kinetic measurements confirm that changes in TMS2 conformation coincide with depolarization. These results reveal how the energized state of the membrane drives functionally relevant structural dynamics in membrane proteins coupled to processes such as channel gating.

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Figure 1: CCCP-coupled changes in Δψm and TIM23-complex transport activity.
Figure 2: Fluorescence properties of probes along the Tim23 TMS2 helical axis.
Figure 3: Analysis of NBD-detected structural changes with increasing depolarization.
Figure 4: IASD labeling tests for the exposure of key Tim23 sites to the IMS.
Figure 5: Time-course measurements of NBD-detected structural changes and membrane depolarization.
Figure 6: Summary of Δψm-coupled structural changes in Tim23 TMS2 and working model.

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Acknowledgements

This work was supported by grants from the US National Science Foundation (MCB-1024908 to N.N.A.), the US National Institutes of Health (GM26494 to A.E.J.) and the Robert A. Welch Foundation (Chair Grant BE-0017 to A.E.J.).

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Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA

    Ketan Malhotra, Murugappan Sathappa, Judith S Landin & Nathan N Alder

  2. Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas, USA

    Arthur E Johnson

  3. Department of Chemistry, Texas A&M University, College Station, Texas, USA

    Arthur E Johnson

  4. Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA

    Arthur E Johnson

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  1. Ketan Malhotra
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  2. Murugappan Sathappa
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  3. Judith S Landin
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  5. Nathan N Alder
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Contributions

All authors conceived of and designed the research; K.M., M.S. and N.N.A. conducted experiments; all authors analyzed data; N.N.A. supervised the project and prepared the manuscript and supplementary material.

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Correspondence to Nathan N Alder.

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Malhotra, K., Sathappa, M., Landin, J. et al. Structural changes in the mitochondrial Tim23 channel are coupled to the proton-motive force. Nat Struct Mol Biol 20, 965–972 (2013). https://doi.org/10.1038/nsmb.2613

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