Functional characterization of Mia40p, the central component of the disulfide relay system of the mitochondrial intermembrane space.
Mia40p and Erv1p are components of a translocation pathway for the import of cysteine-rich proteins into the intermembrane space of mitochondria. We have characterized the redox behavior of Mia40p and reconstituted the disulfide transfer system of Mia40p by using recombinant functional C-terminal fragment of Mia40p, Mia40C, and Erv1p. Oxidized Mia40p contains three intramolecular disulfide bonds. One disulfide bond connects the first two cysteine residues in the CPC motif. The second and the third bonds belong to the twin CX(9)C motif and bridge the cysteine residues of two CX(9)C segments. In contrast to the stabilizing disulfide bonds of the twin CX(9)C motif, the first disulfide bond was easily accessible to reducing agents. Partially reduced Mia40C generated by opening of this bond as well as fully reduced Mia40C were oxidized by Erv1p in vitro. In the course of this reaction, mixed disulfides of Mia40C and Erv1p were formed. Reoxidation of fully reduced Mia40C required the presence of the first two cysteine residues in Mia40C. However, efficient reoxidation of a Mia40C variant containing only the cysteine residues of the twin CX(9)C motif was observed when in addition to Erv1p low amounts of wild type Mia40C were present. In the reconstituted system the thiol oxidase Erv1p was sufficient to transfer disulfide bonds to Mia40C, which then could oxidize the variant of Mia40C. In summary, we reconstituted a disulfide relay system consisting of Mia40C and Erv1p.
Grumbt
Barbara
B
Adolf-Butenandt-Institut für Physiologische Chemie, Ludwig-Maximilians-Universität München, Butenandtstrasse 5, 81377, München, Germany.
Stroobant
Vincent
V
Terziyska
Nadia
N
Israel
Lars
L
Hell
Kai
K
D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D015966:Gene Expression Regulation, Fungal; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000737:chemistry; D008954:Models, Biological; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry; D050862:Oxidoreductases Acting on Sulfur Group Donors; D010100:Oxygen / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013696:Temperature
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; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; C073790:ERV1 protein, S cerevisiae; D003545:Cysteine; D010100:Oxygen