Redox-regulated dynamic interplay between Cox19 and the copper-binding protein Cox11 in the intermembrane space of mitochondria facilitates biogenesis of cytochrome c oxidase.

Members of the twin Cx9C protein family constitute the largest group of proteins in the intermembrane space (IMS) of mitochondria. Despite their conserved nature and their essential role in the biogenesis of the respiratory chain, the molecular function of twin Cx9C proteins is largely unknown. We performed a SILAC-based quantitative proteomic analysis to identify interaction partners of the conserved twin Cx9C protein Cox19. We found that Cox19 interacts in a dynamic manner with Cox11, a copper transfer protein that facilitates metalation of the Cu(B) center of subunit 1 of cytochrome c oxidase. The interaction with Cox11 is critical for the stable accumulation of Cox19 in mitochondria. Cox19 consists of a helical hairpin structure that forms a hydrophobic surface characterized by two highly conserved tyrosine-leucine dipeptides. These residues are essential for Cox19 function and its specific binding to a cysteine-containing sequence in Cox11. Our observations suggest that an oxidative modification of this cysteine residue of Cox11 stimulates Cox19 binding, pointing to a redox-regulated interplay of Cox19 and Cox11 that is critical for copper transfer in the IMS and thus for biogenesis of cytochrome c oxidase.

The role of the Tra1p transcription factor of Magnaporthe oryzae in spore adhesion and pathogenic development.

Transcription factors play a critical regulatory role in development by binding DNA and initiating alterations in gene transcription. The transcript of the putative Magnaporthe oryzae transcription factor-encoding gene TRA1 accumulates during germination and this accumulation was previously found to depend on the transcription factor Con7p. In the current work tra1⁻ mutants were generated and these strains were found to exhibit a reduced attachment, germination, appressorium formation and virulence. Adhesion to artificial and plant surfaces was affected, and FITC-labelled concanavalin A, a lectin which inhibits attachment of Magnaporthe spores, showed a reduced affinity for mutant spore tip where it normally preferentially binds. We used microarray analysis to identify Tra1p-dependent genes from two different sources: aerial structures and conidia. Mutation of 11 Tra1p-dependent genes showed that the predicted transcription factor encoding gene TDG2 is required for normal adhesion and virulence, that the genes TDG7 and TDG4 are required for normal sporulation and that TDG6 is required for wild-type levels of spore adhesion.

Inaccurately assembled cytochrome c oxidase can lead to oxidative stress-induced growth arrest.

AIMS: To identify yeast mutants that show a strong redox dependence of the ability to respire, we systematically screened a yeast deletion library for mutants that require the presence of reductants for growth on nonfermentable carbon sources. RESULTS: Respirative growth of 44 yeast mutants was significantly improved by the addition of dithiothreitol or glutathione. Two mutants that were strongly stimulated by reductants lacked the proteins Cmc1 and Coa4. Both proteins belong to the family of "twin Cx(9)C" proteins present in the intermembrane space of mitochondria. Deletion of CMC1 or COA4 leads to assembly defects of cytochrome c oxidase, in particular to the lack of Cox1 and rapid degradation of Cox2 and Cox3. Interestingly, the presence of the reductants does not suppress these assembly defects and the levels of cytochrome c oxidase remain reduced. Reductants and antioxidants such as ascorbic acid rather counteract the effects of hydrogen peroxide that is produced from partially assembled cytochrome c oxidase intermediates. INNOVATION: Here we show that oxidative stress generated by the accumulation of partially assembled respiratory chain complexes prevents growth on carbon sources that force cells to respire. CONCLUSION: Defects in the assembly of cytochrome c oxidase can lead to increased production of hydrogen peroxide, which is sensed in cells and blocks their proliferation. We propose that this redox-regulated feedback regulation specifically slows down the propagation of cells carrying respiratory chain mutations in order to select for cells of high mitochondrial fitness.