title: Chloroplast Preproteins Bind to the Dimer Interface of the Toc159 Receptor during Import.
abstract: Most chloroplast proteins are synthesized in the cytosol as higher molecular weight preproteins and imported via the translocons in the outer (TOC) and inner (TIC) envelope membranes of chloroplasts. Toc159 functions as a primary receptor and directly binds preproteins through its dimeric GTPase domain. As a first step toward a molecular understanding of how Toc159 mediates preprotein import, we mapped the preprotein-binding regions on the Toc159 GTPase domain (Toc159G) of pea (Pisum sativum) using cleavage by bound preproteins conjugated with the artificial protease FeBABE and cysteine-cysteine cross-linking. Our results show that residues at the dimer interface and the switch II region of Toc159G are in close proximity to preproteins. The mature portion of preproteins was observed preferentially at the dimer interface, whereas the transit peptide was found at both regions equally. Chloroplasts from transgenic plants expressing engineered Toc159 with a cysteine placed at the dimer interface showed increased cross-linking to bound preproteins. Our data suggest that, during preprotein import, the Toc159G dimer disengages and the dimer interface contacts translocating preproteins, which is consistent with a model in which conformational changes induced by dimer-monomer conversion in Toc159 play a direct role in facilitating preprotein import.
authors.affiliation: Molecular and Cell Biology, Taiwan International Graduate Program, Academia Sinica and National Defense Medical Center, Taipei 11529, Taiwan (J.-S.C., H.-m.L.); and.|Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan (J.-S.C., L.-J.C., Y.-H.Y., C.-D.H., H.-m.L.).
authors.forename: Jun-Shian
authors.identifier: 0000-0001-8199-5983
authors.initials: JS
authors.lastname: Chang
authors.affiliation: Molecular and Cell Biology, Taiwan International Graduate Program, Academia Sinica and National Defense Medical Center, Taipei 11529, Taiwan (J.-S.C., H.-m.L.); and.|Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan (J.-S.C., L.-J.C., Y.-H.Y., C.-D.H., H.-m.L.).
authors.forename: Lih-Jen
authors.initials: LJ
authors.lastname: Chen
authors.affiliation: Molecular and Cell Biology, Taiwan International Graduate Program, Academia Sinica and National Defense Medical Center, Taipei 11529, Taiwan (J.-S.C., H.-m.L.); and.|Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan (J.-S.C., L.-J.C., Y.-H.Y., C.-D.H., H.-m.L.).
authors.forename: Yi-Hung
authors.initials: YH
authors.lastname: Yeh
authors.affiliation: Molecular and Cell Biology, Taiwan International Graduate Program, Academia Sinica and National Defense Medical Center, Taipei 11529, Taiwan (J.-S.C., H.-m.L.); and.|Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan (J.-S.C., L.-J.C., Y.-H.Y., C.-D.H., H.-m.L.).
authors.forename: Chwan-Deng
authors.initials: CD
authors.lastname: Hsiao
authors.affiliation: Molecular and Cell Biology, Taiwan International Graduate Program, Academia Sinica and National Defense Medical Center, Taipei 11529, Taiwan (J.-S.C., H.-m.L.); and mbhmli@gate.sinica.edu.tw.|Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan (J.-S.C., L.-J.C., Y.-H.Y., C.-D.H., H.-m.L.) mbhmli@gate.sinica.edu.tw.
authors.forename: Hsou-Min
authors.identifier: 0000-0002-0211-7339
authors.initials: HM
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D060365:Chloroplast Proteins / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D018532:Pisum sativum / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011485:Protein Binding; D055503:Protein Multimerization; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid
chemical_list: D029681:Arabidopsis Proteins; D060365:Chloroplast Proteins; D008565:Membrane Proteins; D010940:Plant Proteins; D011498:Protein Precursors; C403444:TOC159 protein, Arabidopsis; D020558:GTP Phosphohydrolases