The N-terminal shuttle domain of Erv1 determines the affinity for Mia40 and mediates electron transfer to the catalytic Erv1 core in yeast mitochondria.
Erv1 and Mia40 constitute the two important components of the disulfide relay system that mediates oxidative protein folding in the mitochondrial intermembrane space. Mia40 is the import receptor that recognizes the substrates introducing disulfide bonds while it is reduced. A key function of Erv1 is to recycle Mia40 to its active oxidative state. Our aims here were to dissect the domain of Erv1 that mediates the protein-protein interaction with Mia40 and to investigate the interactions between the shuttle domain of Erv1 and its catalytic core and their relevance for the interaction with Mia40. We purified these domains separately as well as cysteine mutants in the shuttle and the active core domains. The noncovalent interaction of Mia40 with Erv1 was measured by isothermal titration calorimetry, whereas their covalent mixed disulfide intermediate was analyzed in reconstitution experiments in vitro and in organello. We established that the N-terminal shuttle domain of Erv1 is necessary and sufficient for interaction to occur. Furthermore, we provide direct evidence for the intramolecular electron transfer from the shuttle cysteine pair of Erv1 to the core domain. Finally, we reconstituted the system by adding in trans the N- and C- terminal domains of Erv1 together with its substrate Mia40.
Lionaki
Eirini
E
Institute of Molecular Biology and Biotechnology, Crete, Greece.
Aivaliotis
Michalis
M
Pozidis
Charalambos
C
Tokatlidis
Kostas
K
D002352:Carrier Proteins / Q000378:metabolism; D020134:Catalytic Domain; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004579:Electron Transport; D008928:Mitochondria / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D015003:Yeasts / Q000378:metabolism
D002352:Carrier Proteins; D004220:Disulfides; C495320:MIA40 protein, S cerevisiae; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C073790:ERV1 protein, S cerevisiae; D003545:Cysteine