Abstract
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.
Highlights
The pathways along which these proteins are sorted in the intermembrane space (IMS) are diverse [1,2,3,4,5]
This segment of Mia40p was sufficient to confer viability to yeast cells when targeted to the intermembrane space of mitochondria lacking authentic Mia40p
It contains that part of the protein which is conserved throughout the eukaryotic kingdom [12,13,14]
Summary
Plasmids and Yeast Strains—To generate pGEX-6P-1Mia40C, amino acid residues 284 – 403 of Mia40p were amplified by polymerase chain reaction using the primers Mia40For850BamH1 (5Ј-CTCGGATCCGCTTATAACCCAGACACTGG-3Ј) and Mia40Sc_StBglXho (5Ј-CTCCTCGAGAGATCTTTAAGGTTTGGATTCCTCATTC-3Ј) and the plasmid pGEM4-Mia as template. The beads were resuspended with 30 ml of 20 mM Tris-HCl, pH 7.0, 200 mM NaCl. Prescission protease was added to a final concentration of 0.05 mg/ml. Redox Reactions with Recombinant Proteins—In the reconstitution experiments of the Mia40p-Erv1p disulfide transfer system, various redox forms of Mia40C were used. To generate the partially reduced Mia40p containing two free thiol groups, the protein was incubated in the presence of 5 mM DTT for 10 min at 25 °C. Reduced Mia40p was obtained by heat denaturation of the protein for 10 min at 95 °C in the presence of 5 mM DTT. Final CD spectra were obtained by averaging four consecutive scans, and the secondary structure content was estimated
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