Characterization of the mitochondrial binding and import properties of purified yeast F1-ATPase beta subunit precursor. Import requires external ATP.
To better understand the early events of the mitochondrial protein import process, we purified the precursor of the F1-ATPase beta subunit (pre-F1 beta) and examined its import into isolated mitochondria. Import of purified urea-denatured pre-F1 beta did not require cytosolic factors. However, the period of productive import was prolonged by the addition of reticulocyte lysate, suggesting that cytosolic factors such as molecular chaperones were acting to extend the period of import competence of pre-F1 beta. Purified pre-F1 beta bound extensively to both cardiolipin-containing liposomes and to intact mitochondria, indicating that a direct interaction between mitochondrial precursors and the mitochondrial outer membrane surface can occur. The ability to chase this surface-bound pre-F1 beta into mitochondria suggests that precursors bound to the mitochondrial surface can be maintained in an import competent conformation. Finally, our defined mitochondrial import system was used to characterize the ATP requirements of pre-F1 beta import in the absence of cytosol. We found a strong requirement for ATP on both sides of the mitochondrial inner membrane, suggesting that one or more previously undetected mitochondrial proteins outside the inner membrane utilize ATP to promote efficient pre-F1 beta import.
Hajek
P
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Department of Microbiology, University of Alabama at Birmingham 35294.
Bedwell
D M
DM
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D002308:Cardiolipins / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008081:Liposomes; D008565:Membrane Proteins / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012156:Reticulocytes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism
D002308:Cardiolipins; D004792:Enzyme Precursors; D008081:Liposomes; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D000255:Adenosine Triphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases