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 mitochondria l hsp70 (mhsp70) interacts with the in ner membrane protein Tim44 to p ull a precursor across the inner membrane. We have proposed that the T im44-mhsp70 complex functions as an ATP-dependent ''translocation moto r'' that exerts an inward force on the precursor chain. To clarify the role of ATP in mhsp70-driven translocation, we tested the effect of t he purified ATP analogues AMP-PNP and ATP gamma S on the Tim44-mhsp70 interaction. Both analogues mimicked ATP by causing dissociation of mh sp70 from Tim44. ADP did not disrupt the Tim44-mhsp70 complex, but did block the ATP-induced dissociation of this complex. In the presence o f ADP, mhsp70 can bind simultaneously to Tim44 and to a peptide substr ate. These data are consistent with a model in which mhsp70 first hydr olyzes ATP, then associates tightly with Tim44 and a precursor protein , and finally undergoes a conformational change to drive translocation .