| Abstract |
Erv1 and Mia40 constitute the two important components of the disulfide relay system that mediates oxidative protein folding of the small Tim proteins and other cysteine rich substrates in the mitochondrial intermembrane space. The small Tims chaperone hydrophobic precursors across the mitochondrial intermembrane space. Tim9 and Tim10 form the soluble TIM10 complex that binds precursors exiting from the outer membrane. Tim12 functions downstream, as the only small Tim peripherally attached on the inner membrane. The aim of the first part of this study was to investigate the funvtion that renders Tim12 essential for the cell, and the domain via which it is mediated. It is shown that Tim12 has an intrinsic affinity for the lipids of the inner mitochondrial membrane. The C-terminal end of Tim12 is essential in vivo and it determines the interaction with the membrane and the assembly of the protein in complexes with the other Tims. The N-terminal end, is dispensable and it both contains targeting information and mediates direct binding of Tim12 to the substrates. These results provide a molecular basis for the concept that the essential role of Tim12 relies on its unique properties that allow it to act as a bridge for the soluble and membrane-embedded translocases in the carrier import pathway.
Mia40 is the import receptor that recognizes the substrates that target the intermembrane space of mitochondria introducing disulfide bonds to promote their folding. A key function of Erv1 is to recycle Mia40 to its active oxidative state. The aims in the second part of this study, were to dissect the domain of Erv1 that mediates the interaction with Mia40 and to investigate the intamolecular communication of the two Erv1 domains. It is established that the N’ terminal flexible domain of Erv1 is necessary and sufficient for both the covalent and the non-covalent interaction with Mia40 to occur. Furthermore, we provide evidence for the intramolecular electron transfer from the shuttle cysteine pair of Erv1 to the catalytic core of the protein.
The final part of this study focuses on the mitochondrial targeting of the bacterial toxin, EspF. EspF is injected from the type II secretion system of Enteropathogenic E. coli (EPEC) directly into the gut epithelial cell cytoplasm where it exerts its pathogenic role, which includes mitochondrial membrane potential disruption and cytochrome c release. It is shown that EspF is targeted specifically to yeast mitochondrial matrix and causes limited cystochrome c release, very early on. In the
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mitochondrial matrix EspF interacts with the inner membrane. Co-purification studies showed that EspF interacts with 3 proteins of the matrix targeted import pathway. The most abundant protein partner identified is the mtHsp70. The model that is proposed for the pathogenic function of EspF, is that by blocking TIM23 import pathway during its import into the mitochondrial matrix, it causes depolarization of the inner membrane, and cytochrome c release.
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Μελέτη των μηχανισμών εισόδου πρωτεϊνών στα μιτοχόνδρια
