The J-related segment of Tim44 is essential for cell viability: A mutant Tim44 remains in the mitochondrial import site, but inefficiently recruits mtHsp70 and impairs protein translocation

Tim44 is a protein of the mitochondrial inner membrane and serves as an ada ptor protein for mtHsp70 that drives the import of preproteins in an ATP-de pendent manner. In this study we have modified the interaction of Tim44 wit h mtHsp70 and characterized the consequences for protein translocation, By deletion of an 18-residue segment of Tim44 with limited similarity to J-pro teins, the binding of Tim44 to mtHsp70 was weakened. We found that in the y east Saccharomyces cerevisiae the deletion of this segment is lethal. To in vestigate the role of the 18-residue segment, we expressed Tim44(Delta 18) in addition to the endogenous wild-type Tim44. Tim44(Delta 18) is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44(Delta 18) together with wildtype Tim44, however, does not stimulate protein import, but reduces its efficiency. In particula r; the promotion of unfolding of preproteins during translocation is inhibi ted. mtHsp70 is still able to bind to Tim44(Delta 18) in an ATP-regulated m anner, but the efficiency of interaction is reduced. These results suggest that the J-related seg ment of Tim44 is needed for productive interaction w ith mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates th e translocation of loosely folded preproteins and plays a crucial role in t he impart of preproteins which contain a tightly folded domain.