Binding of mitochondrial precursor proteins to the cytoplasmic domains of the import receptors Tom70 and Tom20 is determined by cytoplasmic chaperones.
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.
Komiya
T
T
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
Rospert
S
S
Schatz
G
G
Mihara
K
K
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
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