A cooperative action of the ATP-dependent import motor complex and the inner membrane potential drives mitochondrial preprotein import.
The import of mitochondrial preproteins requires an electric potential across the inner membrane and the hydrolysis of ATP in the matrix. We assessed the contributions of the two energy sources to the translocation driving force responsible for movement of the polypeptide chain through the translocation channel and the unfolding of preprotein domains. The import-driving activity was directly analyzed by the determination of the protease resistances of saturating amounts of membrane-spanning translocation intermediates. The ability to generate a strong translocation-driving force was solely dependent on the activity of the ATP-dependent import motor complex in the matrix. For a sustained import-driving activity on the preprotein in transit, an unstructured N-terminal segment of more than 70 to 80 amino acid residues was required. The electric potential of the inner membrane was required to maintain the import-driving activity at a high level. The electrophoretic force of the potential exhibited only a limited capacity to unfold preprotein domains. We conclude that the membrane potential increases the probability of a dynamic interaction of the preprotein with the import motor. Polypeptide translocation and unfolding are mainly driven by the inward-directed translocation activity based on the functional cooperation of the import motor components.
Krayl
Martin
M
Institut für Biochemie und Molekularbiologie, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany.
Lim
Joo Hyun
JH
Martin
Falk
F
Guiard
Bernard
B
Voos
Wolfgang
W
D000255:Adenosine Triphosphate / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D042844:L-Lactate Dehydrogenase (Cytochrome) / Q000235:genetics / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000502:physiology*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D020409:Molecular Motor Proteins / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013367:Submitochondrial Particles / Q000235:genetics / Q000502:physiology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D020409:Molecular Motor Proteins; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C083081:TIM23 protein, S cerevisiae; D000255:Adenosine Triphosphate; D042844:L-Lactate Dehydrogenase (Cytochrome); D013762:Tetrahydrofolate Dehydrogenase