Direct interaction of mitochondrial targeting presequences with purified components of the TIM23 protein complex

Precursor proteins that are imported from the cytosol into the matrix of mitochondria carry positively charged amphipathic presequences and cross the inner membrane with the help of vital components of the TIM23 complex. It is currently unclear which subunits of the TIM23 complex recognize and directly bind to presequences. Here we analyzed the binding of presequence peptides to purified components of the TIM23 complex. The interaction of three different presequences with purified soluble domains of yeast Tim50 (Tim50 _IMS), Tim23 (Tim23 _IMS), and full-length Tim44 was examined. Using chemical crosslinking and surface plasmon resonance we demonstrate, for the first time, the ability of purified Tim50 _IMS and Tim44 to interact directly with the yeast Hsp60 presequence. We also analyzed their interaction with presequences derived from precursors of yeast mitochondrial 70-kDa heat shock protein (mHsp70) and of bovine cytochrome P450 _SCC. Moreover, we characterized the nature of the interactions and determined their K _Ds. On the basis of our results, we suggest a mechanism of translocation where stronger interactions of the presequences on the trans side of the channel support the import of precursor proteins through TIM23 into the matrix.