Protein import into mitochondria : a study on hTom20

Tom20 is a major receptor for protein translocation located on the outer mitochondrial membrane. This protein is a member of a multicomponent complex and is found to stimulate import of all classes of preproteins. In here, the biochemical and biophysical characterization of the human homologue will be presented. A new binding method was developed in order to study the properties of the protein. The receptor contains two distinct domains for recognition of different classes of preproteins and both domains show different binding characteristics. The interaction with terminal targeting sequences has an electrostatic character and is initiated by formation of the amphiphilic alpha helix of the signal. The signal is recognized by the protein in a membrane bound state. The dissociation constant found for this process is in the muM range and is increased upon formation of the amphiphilic alpha-helix. Binding to internal targeting signals is mediated by hydrophobic interaction. Neither interaction is mediated by chaperones and they are not ATP or DTT sensitive. In here, it will be demonstrated how these properties can be used to identify targeting signals of preproteins, for example, in the uncoupling protein (UCP). In addition, a low-resolution model is proposed based on biochemical and biophysical characterization and protein modeling. Using these techniques a 4-helix bundle structure is predicted. The protein contains a "Glutamine face" involved in interaction with internal targeting signals and predicted to face the surface of the protein. Furthermore, it was determined that Tom20 can stimulate insertion of outer mitochondrial membrane proteins like VDAC. The insertion process is strongly dependent on Tom20 and the inserted protein is active. This suggest that Tom20 acts as receptor and translocator, a double function not found yet for any other receptor involved in recognition of preproteins to be imported into mitochondria