Residues of Tim44 Involved in Both Association with the Translocon of the Inner Mitochondrial Membrane and Regulation of mtHsp70 Tethering

Department of Biochemistry, University of Wisconsin – Madison, Madison WI 53706

^* To whom correspondence should be addressed. Email: ecraig{at}wisc.edu.

	Abstract

Translocation of proteins from the cytosol across the mitochondrial inner membrane is driven by the action of the import motor, which is associated with the translocon on the matrix side of the membrane. It is well-established that an essential peripheral membrane protein Tim44 tethers mitochondrial Hsp70 (mtHsp70), the core of the import motor, to the translocon. This Tim44:mtHsp70 interaction, which can be recapitulated in vitro, is destabilized by binding of mtHsp70 to a substrate polypeptide. Here we report that the N-terminal 167 amino acid segment of mature Tim44 is sufficient for both interaction with mtHsp70 and destabilization of a Tim44:mtHsp70 complex caused by client protein binding. Amino acid alterations within a 30 amino acid segment affected both the release of mtHsp70 upon peptide binding and the interaction of Tim44 with the translocon. Our results support the idea that Tim44 plays multiple roles in mitochondrial protein import by recruiting Ssc1 and its J-protein co-chaperone to the translocon and coordinating their interactions to promote efficient protein translocation in vivo.