Peptide library approach with a disulfide tether to refine the Tom20 recognition motif in mitochondrial presequences.
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 (DeltaTom20) 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 DeltaTom20 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.
Obita
Takayuki
T
Department of Structural Biology, Biomolecular Engineering Research Institute, Suita, Osaka 565-0874, Japan.
Muto
Takanori
T
Endo
Toshiya
T
Kohda
Daisuke
D
D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D051381:Rats; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004220:Disulfides; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D019151:Peptide Library; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C489998:Tomm20 protein, rat; D000444:Aldehyde Dehydrogenase; D003545:Cysteine