title: Structural insights into cargo recognition by the yeast PTS1 receptor.
abstract: The peroxisomal matrix protein import is facilitated by cycling import receptors that shuttle between the cytosol and the peroxisomal membrane. The import receptor Pex5p mediates the import of proteins harboring a peroxisomal targeting signal of type I (PTS1). Purified recombinant Pex5p forms a dimeric complex with the PTS1-protein Pcs60p in vitro with a KD of 0.19 μm. To analyze the structural basis for receptor-cargo recognition, the PTS1 and adjacent amino acids of Pcs60p were systematically scanned for Pex5p binding by an in vitro site-directed photo-cross-linking approach. The cross-linked binding regions of the receptor were subsequently identified by high resolution mass spectrometry. Most cross-links were found with TPR6, TPR7, as well as the 7C-loop of Pex5p. Surface plasmon resonance analysis revealed a bivalent interaction mode for Pex5p and Pcs60p. Interestingly, Pcs60p lacking its C-terminal tripeptide sequence was efficiently cross-linked to the same regions of Pex5p. The KD value of the interaction of truncated Pcs60p and Pex5p was in the range of 7.7 μm. Isothermal titration calorimetry and surface plasmon resonance measurements revealed a monovalent binding mode for the interaction of Pex5p and Pcs60p lacking the PTS1. Our data indicate that Pcs60p contains a second contact site for its receptor Pex5p, beyond the C-terminal tripeptide. The physiological relevance of the ancillary binding region was supported by in vivo import studies. The bivalent binding mode might be explained by a two-step concept as follows: first, cargo recognition and initial tethering by the PTS1-receptor Pex5p; second, lock-in of receptor and cargo.
authors.affiliation: From the Institute of Biochemistry and Pathobiochemistry, Faculty of Medicine, System Biochemistry, Ruhr-University Bochum, D-44780 Bochum, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Hagen
authors.affiliation: the Department of Biochemistry and Functional Proteomics, Faculty of Biology and BIOSS Centre for Biological Signaling Studies, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Friedel
authors.initials: F
authors.lastname: Drepper
authors.affiliation: the Department of Biochemistry and Functional Proteomics, Faculty of Biology and BIOSS Centre for Biological Signaling Studies, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Fischer
authors.affiliation: the Department of Biochemistry, Eötvös Loránd University, H-1117 Budapest, Hungary.
authors.forename: Krisztian
authors.initials: K
authors.lastname: Fodor
authors.affiliation: the European Molecular Biology Laboratory at Hamburg, D-22607 Hamburg, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Passon
authors.affiliation: the Institute of Biochemistry and Pathobiochemistry, Faculty of Medicine, Biochemistry of Intracellular Transport Mechanism, Ruhr-University Bochum, D-44781 Bochum, Germany, and.
authors.forename: Harald W
authors.initials: HW
authors.lastname: Platta
authors.affiliation: the Biaffin GmbH and Co., KG, D-34132 Kassel, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Zenn
authors.affiliation: From the Institute of Biochemistry and Pathobiochemistry, Faculty of Medicine, System Biochemistry, Ruhr-University Bochum, D-44780 Bochum, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schliebs
authors.affiliation: From the Institute of Biochemistry and Pathobiochemistry, Faculty of Medicine, System Biochemistry, Ruhr-University Bochum, D-44780 Bochum, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Girzalsky
authors.affiliation: the European Molecular Biology Laboratory at Hamburg, D-22607 Hamburg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Wilmanns
authors.affiliation: the Department of Biochemistry and Functional Proteomics, Faculty of Biology and BIOSS Centre for Biological Signaling Studies, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Warscheid
authors.affiliation: From the Institute of Biochemistry and Pathobiochemistry, Faculty of Medicine, System Biochemistry, Ruhr-University Bochum, D-44780 Bochum, Germany, ralf.erdmann@rub.de.
authors.forename: Ralf
authors.initials: R
authors.lastname: Erdmann
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015966:Gene Expression Regulation, Fungal; D007700:Kinetics; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013816:Thermodynamics; D014162:Transfection
chemical_list: D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C499509:PEX5 protein, S cerevisiae; D000074435:Peroxisome-Targeting Signal 1 Receptor; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D008025:Ligases; C101849:PCS60 protein, S cerevisiae
keywords: mass spectrometry (MS); peroxisome; protein cross-linking; protein targeting; protein translocation; protein-protein interaction; yeast