THE MITOCHONDRIAL PROTEIN IMPORT MACHINERY - ROLE OF ATP IN DISSOCIATION OF THE HSP70-CENTER-DOT-MIM44 COMPLEX

Interaction of preproteins with the heat shock protein Hsp70 in the mi tochondrial matrix is required for driving protein transport across th e mitochondrial inner membrane. Binding of mt-Hsp70 to the protein Mim 44 of the inner membrane import site seems to be an essential part of an ATP-dependent reaction cycle. However, the available results on the role played by ATP are controversial. Here we demonstrate that the mt -Hsp70 . Mim44 complex contains ADP and that a nonhydrolyzable analog of ATP dissociates the mt-Hsp70 . Mim44 complex in the presence of pot assium ions. The previously reported requirement of ATP hydrolysis for complex dissociation was due to the use of a nonphysiological concent ration of sodium ions. In the presence of potassium ions, mt-Hsp70 und ergoes a conformational change that is not observed with a mutant Hsp7 0 defective in binding to Mim44. The mutant Hsp70 is able to bind subs trate proteins, differentiating binding to Mim44 from binding to subst rate proteins. We conclude that binding of ATP, not hydrolysis, is req uired to dissociate the mt-Hsp70 . Mim44 complex and that the reaction cycle includes an ATP-induced conformational change of mt-Hsp70.