Peptide Library Approach with a Disulfide Tether to Refine the Tom20 Recognition Motif in Mitochondrial Presequences

Takayuki Obita, Takanori Muto, Toshiya Endo, Daisuke Kohda

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    48 Citations (Scopus)


    Many mitochondrial matrix and inner-membrane proteins are synthesized in the cytosol as precursor proteins with an N-terminal presequence, and are imported into the mitochondria. Although no distinct sequence homology has been found among mitochondrial presequences, Tom20, a general import receptor in the outer mitohcondrial membrane, binds to presequences, and distinguishes mitochondrial proteins from non-mitochonrial proteins. The recently determined structure of the cytosolic domain of Tom20 (ΔTom20) in a complex with the presequence of rat aldehyde dehydrogenase (ALDH) showed that a short stretch of the presequence forms an amphiphilic helix, and its hydrophobic surface interacts with the hydrophobic-binding groove of Tom20. The following NMR analyses revealed a common five-residue pattern for Tom20 binding in five different presequences. To refine the common amino acid motif for the recognition by Tom20, we introduced a new peptide library approach in this study: we prepared a mixture of ALDH presequence variants, tethered these peptides to ΔTom20 in a competitive manner by an intermolecular disulfide bond, and determined the relative affinities by MALDI-TOF mass spectrometry. We successfully deduced a refined, common motif for the recognition by Tom20, and found that the segment consisting of residues 14-20 of the ALDH presequence was locally optimized in the sequence space, with respect to Tom20 binding.

    Original languageEnglish
    Pages (from-to)495-504
    Number of pages10
    JournalJournal of Molecular Biology
    Issue number2
    Publication statusPublished - Apr 25 2003

    All Science Journal Classification (ASJC) codes

    • Structural Biology
    • Molecular Biology


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