The mitochondrial protein import motor: dissociation of mitochondrial hsp70 from its membrane anchor requires ATP binding rather than ATP hydrolysis.
During protein import into mitochondria, matrix-localized mitochondrial hsp70 (mhsp70) interacts with the inner membrane protein Tim44 to pull a precursor across the inner membrane. We have proposed that the Tim44-mhsp70 complex functions as an ATP-dependent "translocation motor" that exerts an inward force on the precursor chain. To clarify the role of ATP in mhsp70-driven translocation, we tested the effect of the purified ATP analogues AMP-PNP and ATP gamma S on the Tim44-mhsp70 interaction. Both analogues mimicked ATP by causing dissociation of mhsp70 from Tim44. ADP did not disrupt the Tim44-mhsp70 complex, but did block the ATP-induced dissociation of this complex. In the presence of ADP, mhsp70 can bind simultaneously to Tim44 and to a peptide substrate. These data are consistent with a model in which mhsp70 first hydrolyzes ATP, then associates tightly with Tim44 and a precursor protein, and finally undergoes a conformational change to drive translocation.
Horst
M
M
Biozentrum, Basel, Switzerland. horst@ubaclu.unibas.ch
Oppliger
W
W
Feifel
B
B
Schatz
G
G
Glick
B S
BS
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011485:Protein Binding; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins
D002352:Carrier Proteins; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D029701:Saccharomyces cerevisiae Proteins; C077016:TIM44 protein, S cerevisiae; D000255:Adenosine Triphosphate