| Abstract |
The place between the inner and outer membrane of mitochondria, which is called intermembrane space (IMS), possess a number of many and different proteins that have disulfide bonds in their structure. Recently, a disulfide oxidative system in the intermembrane space has been identified in which two proteins, sulfhydryl oxidase Erv1 and the import receptor of proteins-substrates that is redox regulated, Mia40/Tim40 have a crucial role. This redox system, which is called Mia40-Erv1 pathway, drives the import of proteins that are rich in cysteines into IMS of mitochondria through a mechanism of oxidative folding.
In the present study, the aim was the finding of possibly new substrates of sulfhydryl oxidase Erv1, which is located in the intermembrane space of mitochondria of yeast Saccharomyces cerevisiae. By biochemical experiments, like gel filtration and binding of this protein as histidin-tagged at its carboxyl terminal end, on nickel beads, we show that the complex that is appeared to be isolated from the mitochondria of Erv1His strain of yeast has approximately 70kDa molecular weight. This molecular weight may refers to the Erv1 dimer or a complex between Erv1 and a similar molecular weight protein with, as from the experimental data it does not seem to conclude the proteins Mia40 and cytochrome c (cytc), two proteins that interact with Erv1 protein. In parallel, an effort to quantify the proteins Erv1, Mia40 and cytochrome c in wild type mitochondria took place under steady state conditions. The result showed that while cytochrome c seems to appear in a bigger quantity per mg of mitochondria compared to the other two proteins, the protein Mia40 seems to exist in substoichiometric amounts of Erv1, which is known to oxidize Mia40 and therefore recycling it. Moreover, we studied the interaction of the Erv1 and Mia40 in two steps: during the import of Erv1 protein as a substrate of Mia40 protein and in a second way the interaction between them when Erv1 has been folded into the intermembrane space of yeast mitochondria and has been functional. It seems like some hydrophobic residues that are downstream of the cysteine motif CRSC of wild type Erv1 (yErv1) are very important for the formation of the “Μia40-Εrv1” mixed disulfide bonded intermediate while they are not important for the import of this protein as a substrate and therefore for its recognition by Mia40 protein. In the end, we tried to work on human ortholog of Erv1, the protein hALR, that has two isoforms: the long form (lf) and short form (sf). Many of the tools for the study of these specific proteins in yeast mitochondria are ready to answer many and important questions in this heterologous system.
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Μελέτη της σουλφυδριλοξείδασης Erv1 στο μιτοχόνδριο του σακχαρομύκητα S. cerevisiae
