Mitochondrial protein import motor: the ATPase domain of matrix Hsp70 is crucial for binding to Tim44, while the peptide binding domain and the carboxy-terminal segment play a stimulatory role

The import motor for preproteins that are targeted into the mitochondrial m atrix consists of the matrix heat shock protein Hsp70 (mtHsp70) and the tra nslocase subunit Tim44 of the inner membrane. mtHsp70 interacts with Tim44 in an ATP-dependent reaction cycle, binds to preproteins in transit, and dr ives their translocation into the matrix, While different functional mechan isms are discussed for the mtHsp70-Tim44 machinery, little is known about t he actual mode of interaction of both proteins, Here, we have addressed whi ch of the three Hsp70 regions, the ATPase domain, the peptide binding domai n, or the carboxy-terminal segment, are required for the interaction with T im44, By two independent means, a two-hybrid system and coprecipitation of mtHsp70 constructs imported into mitochondria, we show that the ATPase doma in interacts with Tim44, although with a reduced efficiency compared to the full-length mtHsp70, The interaction of the ATPase domain with Tim44 is AT P sensitive, The peptide binding domain and carboxy-terminal segment are un able to bind to Tim44 in the absence of the ATPase domain, but both regions enhance the interaction with Tim44 in the presence of the ATPase domain. W e conclude that the ATPase domain of mtHsp70 is essential for and directly interacts with Tim44, clearly separating the mtHsp70-Tim44 interaction from the mtHsp70-substrate interaction.