Abstract
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 (Tim50IMS), Tim23 (Tim23IMS), and full-length Tim44 was examined. Using chemical cross-linking and surface plasmon resonance we demonstrate, for the first time, the ability of purified Tim50IMS 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 P450SCC. Moreover, we characterized the nature of the interactions and determined their KDs. 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.
Highlights
The vital interaction between components of the TIM23 import complex and presequences is poorly characterized
In the first method, biotinylated presequence of yeast Hsp60 (pHsp60) was incubated with Tim50IMS to allow complex formation, followed by mild crosslinking with a low concentration of disuccinimidyl suberate (DSS), resolution of the crosslinking products by SDS-PAGE, and detection with streptavidin conjugated to horseradish peroxidase
We examined binding at increasing concentrations of pHsp60 (Fig. 1A)
Summary
The vital interaction between components of the TIM23 import complex and presequences is poorly characterized. 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. Tim could be cross-linked to various preproteins transiting the import channel, and the function of a receptor was attributed to this subunit (7, 9 –11) Another component on the cis side, the soluble domain of Tim, was suggested to receive precursor proteins from Tim and to transfer them further into the channel [7, 22,23,24]. The significance of our results is discussed in light of what is known about the mechanism of protein translocation across the mitochondrial inner membrane
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
