title: Direct interaction of mitochondrial targeting presequences with purified components of the TIM23 protein complex.
abstract: Precursor proteins that are imported from the cytosol into the matrix of mitochondria carry positively charged amphipathic presequences and cross the inner membrane with the help of vital components of the TIM23 complex. It is currently unclear which subunits of the TIM23 complex recognize and directly bind to presequences. Here we analyzed the binding of presequence peptides to purified components of the TIM23 complex. The interaction of three different presequences with purified soluble domains of yeast Tim50 (Tim50IMS), Tim23 (Tim23IMS), and full-length Tim44 was examined. Using chemical cross-linking and surface plasmon resonance we demonstrate, for the first time, the ability of purified Tim50IMS and Tim44 to interact directly with the yeast Hsp60 presequence. We also analyzed their interaction with presequences derived from precursors of yeast mitochondrial 70-kDa heat shock protein (mHsp70) and of bovine cytochrome P450SCC. Moreover, we characterized the nature of the interactions and determined their KDs. On the basis of our results, we suggest a mechanism of translocation where stronger interactions of the presequences on the trans side of the channel support the import of precursor proteins through TIM23 into the matrix.
authors.affiliation: Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Milit
authors.initials: M
authors.lastname: Marom
authors.affiliation: Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Dana
authors.initials: D
authors.lastname: Dayan
authors.affiliation: Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Keren
authors.initials: K
authors.lastname: Demishtein-Zohary
authors.affiliation: Institute for Physiological Chemistry, University of Munich, Munich 81377, Germany.
authors.forename: Dejana
authors.initials: D
authors.lastname: Mokranjac
authors.affiliation: Max Planck Institute of Biochemistry, Martinsried 82152, Germany.
authors.forename: Walter
authors.initials: W
authors.lastname: Neupert
authors.affiliation: Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Electronic address: azema@tauex.tau.ac.il.
authors.forename: Abdussalam
authors.initials: A
authors.lastname: Azem
mesh_terms: D000818:Animals; D001665:Binding Sites; D001703:Biophysics / Q000379:methods; D001710:Biotin / Q000737:chemistry; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008928:Mitochondria / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D020349:Surface Plasmon Resonance
chemical_list: D003432:Cross-Linking Reagents; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; C083081:TIM23 protein, S cerevisiae; D001710:Biotin; D002786:Cholesterol Side-Chain Cleavage Enzyme