The presence of disulfide bonds reveals an evolutionarily conserved mechanism involved in mitochondrial protein translocase assembly

Disulfide bonds are covalent links between two cysteine residues of proteins, typically introduced posttranslationally through thiol-disulfide exchange reactions. Disulfide bond formation can be crucial for the correct folding of a protein, maintenance of protein structure, and regulation of its redox-dependent functions. Most proteins that contain intramolecular disulfide bonds perform their function as secreted proteins in the extracellular milieu. However, a substantial group of proteins that require disulfide bonds is present in the intermembrane space (IMS) compartment of mitochondria. The majority of the IMS proteome are proteins that contain characteristic twin cysteine motifs, arranged as CX3C or CX9C, in their amino acid sequence1,2,3,4. The import and oxidative folding of such precursor proteins are mediated by the mitochondrial IMS assembly (MIA) pathway5,6. Representatives of classic substrates of the MIA pathway include small Tim chaperones, such as Tim9 or Tim10, and proteins that are involved in the proper function and assembly of respiratory chain complexes, including Cox12, Cox17, and Cox19. The activity of this pathway depends on two essential components, oxidoreductase Mia40 and sulfhydryl oxidase Erv12,3,7. The mechanisms that are utilized by the MIA pathway are unique as the oxidation of incoming precursor proteins provides a means to trap them in the IMS and prevent their escape8,9. Redox reactions are also important during the biogenesis of atypical IMS proteins. One example is the IMS protease Atp23, which is a heavily oxidized protein that is peripherally associated with the inner mitochondrial membrane. The import and maturation of Atp23 were found to be assisted by Mia4010. In addition, Mia40 catalyzes the formation of disulfide bonds in anamorsin, a protein that has been implicated in Fe/S cluster assembly11. Beside the crucial role in mitochondrial protein structure, cysteine residues oxidation is important in the regulation of protein function within mitochondria. The conserved cysteine residues of the mitochondrial ADP/ATP carrier were shown to be important for its maturation12. Recently, Mia40-mediated formation of an intramolecular disulfide bond between the IMS proteins MICU1 and MICU2 was shown to be critical for the control of mitochondrial Ca2+ uniporter function in human cells13. The formation of the disulfide bond can also be part of a protein quality control system. The presence of the disulfide bond within the structure of the mitochondrial ribosomal protein Mrp10 prevents its proteolytic degradation in the IMS prior to mitochondrial matrix translocation14. Thus, the importance of disulfide bond formation in soluble proteins is well defined2,3,7. Recent work revealed that membrane protein Tim22, the core component of the TIM22 protein translocase complex responsible for the import of multispanning membrane proteins in the yeast Saccharomyces cerevisiae15, was found in the oxidized state16,17. The oxidation of Tim22 during import supports its proper membrane integration and assembly of the mature TIM22 complex16,17. Furthermore, this oxidation is facilitated by a direct interaction between Mia40 and Tim22 during its biogenesis16. The role of cysteine oxidation in the biogenesis of the yeast multi-spanning membrane protein Tim22 led us to systematically determine the presence of this modification in the Tim17/Tim22/Tim23 family. Moreover, we were interested in disulfide bond conservation within eukaryotes and the presence of this modification relative to membrane topology. We demonstrated a conserved pattern of cysteine residues in Tim17 and Tim22 but not Tim23. Our analysis revealed the formation of disulfide bonds in Tim17 and Tim22 proteins in S. cerevisiae, C. albicans, and H. sapiens. Moreover, we provided membrane topology models for Tim17 and Tim22, including the positioning of disulfide bond(s). Thus, oxidation of Tim17 and Tim22, which promotes translocase complex biogenesis, is a highly conserved feature across eukaryotes.