title: Binding of mitochondrial precursor proteins to the cytoplasmic domains of the import receptors Tom70 and Tom20 is determined by cytoplasmic chaperones.
abstract: We have reconstituted the early steps of precursor targeting to mitochondria in a defined and soluble system consisting of the cytosolic domains of the yeast mitochondrial import receptors Tom20 and Tom70, precursor to bovine adrenal adrenodoxin (which has a cleavable targeting signal) and rat liver cytosolic chaperones hsp70 and mitochondrial import-stimulating factor (MSF). The Tom70 domain only bound the precursor in the presence of MSF, yielding a precursor-MSF-Tom70 complex; ATP hydrolysis by MSF released MSF and generated a precursor-Tom70 complex whose formation was inhibited by an excess of a functional presequence peptide, but not by 150 mM NaCl. In the presence of the Tom20 domain, ATP caused transfer of the precursor from the precursor-MSF-Tom70 complex to Tom20. The Tom20 domain alone only bound the precursor in the presence of hsp70; hsp70 itself was not incorporated into the resulting complex. Formation of the Tom20-precursor complex was inhibited by excess presequence peptide or by 150 mM NaCl. Similar results were obtained with the ADP/ATP carrier and porin precursors, which both lack a cleaved targeting signal. Correct targeting of a precursor to mitochondrial import receptors thus requires cytosolic chaperones, irrespective of the presence or absence of a cleavable presequence.
authors.affiliation: Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Komiya
authors.forename: S
authors.initials: S
authors.lastname: Rospert
authors.forename: G
authors.initials: G
authors.lastname: Schatz
authors.forename: K
authors.initials: K
authors.lastname: Mihara
mesh_terms: D048948:14-3-3 Proteins; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000325:Adrenodoxin / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D018272:Porins; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface; D012965:Sodium Chloride / Q000494:pharmacology; D050994:Voltage-Dependent Anion Channels; D000099221:Protein Kinase C zeta
chemical_list: D048948:14-3-3 Proteins; D000255:Adenosine Triphosphate; D000325:Adrenodoxin; D003577:Cytochrome P-450 Enzyme System; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000226:Mitochondrial ADP, ATP Translocases; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D018272:Porins; D011498:Protein Precursors; D011956:Receptors, Cell Surface; D012965:Sodium Chloride; D050994:Voltage-Dependent Anion Channels; C490392:Prkcz protein, rat; D000099221:Protein Kinase C zeta; C489998:Tomm20 protein, rat; C490391:Ywhaz protein, rat; C090759:mitochondrial import stimulation factor, rat