Mitochondrial translocation of APE1 relies on the MIA pathway

APE1 is a multifunctional protein with a fundamental role in repairing nuclear and mitochondrial DNA lesions caused by oxidative and alkylating agents. Unfortunately, comprehensions of the mechanisms regulating APE1 intracellular trafficking are still fragmentary and contrasting. Recent data demonstrate that APE1 interacts with the mitochondrial import and assembly protein Mia40 suggesting the involvement of a redox-assisted mechanism, dependent on the disulfide transfer system, to be responsible of APE1 trafficking into the mitochondria. The MIA pathway is an import machinery that uses a redox system for cysteine enriched proteins to drive them in this compartment. It is composed by two main proteins: Mia40 is the oxidoreductase that catalyzes the formation of the disulfide bonds in the substrate, while ALR reoxidizes Mia40 after the import. In this study, we demonstrated that: (i) APE1 and Mia40 interact through disulfide bond formation; and (ii) Mia40 expression levels directly affect APE1's mitochondrial translocation and, consequently, play a role in the maintenance of mitochondrial DNA integrity. In summary, our data strongly support the hypothesis of a redox-assisted mechanism, dependent on Mia40, in controlling APE1 translocation into the mitochondrial inner membrane space and thus highlight the role of this protein transport pathway in the maintenance of mitochondrial DNA stability and cell survival

COA6 facilitates cytochrome c oxidase biogenesis as thiol-reductase for copper metallochaperones in mitochondria.

The mitochondrial cytochrome c oxidase, the terminal enzyme of the respiratory chain, contains heme and copper centers for electron transfer. The conserved COX2 subunit contains the CuA site, a binuclear copper center. The copper chaperones SCO1, SCO2, and COA6 are required for CuA center formation. Loss of function of these chaperones and the concomitant cytochrome c oxidase deficiency cause severe human disorders. Here we analyzed the molecular function of COA6 and the consequences of COA6 deficiency for mitochondria. Our analyses show that loss of COA6 causes combined complex I and complex IV deficiency and impacts membrane potential driven protein transport across the inner membrane. We demonstrate that COA6 acts as a thiol-reductase to reduce disulphide bridges of critical cysteine residues in SCO1 and SCO2. Cysteines within the CX3CXNH domain of SCO2 mediate its interaction with COA6 but are dispensable for SCO2-SCO1 interaction. Our analyses define COA6 as thiol-reductase, which is essential for CuA biogenesis

Determinants of the cytosolic turnover of mitochondrial intermembrane space proteins

Background; The proteome of mitochondria comprises mostly proteins that originate as precursors in the cytosol. Before import into the organelle, such proteins are exposed to cytosolic quality control mechanisms. Multiple lines of evidence indicate a significant contribution of the major cytosolic protein degradation machinery, the ubiquitin-proteasome system, to the quality control of mitochondrial proteins. Proteins that are directed to the mitochondrial intermembrane space (IMS) exemplify an entire class of mitochondrial proteins regulated by proteasomal degradation. However, little is known about how these proteins are selected for degradation. Results: The present study revealed the heterogeneous cytosolic stability of IMS proteins. Using a screening approach, we found that different cytosolic factors are responsible for the degradation of specific IMS proteins, with no single common factor involved in the degradation of all IMS proteins. We found that the Cox12 protein is rapidly degraded when localized to the cytosol, thus providing a sensitive experimental model. Using Cox12, we found that lysine residues but not conserved cysteine residues are among the degron features important for protein ubiquitination. We observed the redundancy of ubiquitination components, with significant roles of Ubc4 E2 ubiquitin-conjugating enzyme and Rsp5 E3 ubiquitin ligase. The amount of ubiquitinated Cox12 was inversely related to mitochondrial import efficiency. Importantly, we found that precursor protein ubiquitination blocks its import into mitochondria. Conclusions: The present study confirms the involvement of ubiquitin-proteasome system in the quality control of mitochondrial IMS proteins in the cytosol. Notably, ubiquitination of IMS proteins prohibits their import into mitochondria. Therefore, ubiquitination directly affects the availability of precursor proteins for organelle biogenesis. Importantly, despite their structural similarities, IMS proteins are not selected for degradation in a uniform way. Instead, specific IMS proteins rely on discrete components of the ubiquitination machinery to mediate their clearance by the proteasome

The oxidation status of Mic19 regulates MICOS assembly.

The function of mitochondria depends on the proper organization of mitochondrial membranes. The morphology of the inner membrane is regulated by the recently identified mitochondrial contact site and cristae organizing system (MICOS) complex. MICOS mutants exhibit alterations in crista formation, leading to mitochondrial dysfunction. However, the mechanisms that underlie MICOS regulation remain poorly understood. MIC19, a peripheral protein of the inner membrane and component of the MICOS complex, was previously reported to be required for the proper function of MICOS in maintaining the architecture of the inner membrane. Here, we show that human and yeast MIC19 proteins undergo oxidation in mitochondria and require the mitochondrial intermembrane space assembly (MIA) pathway, which couples the oxidation and import of mitochondrial intermembrane space proteins for mitochondrial localization. Detailed analyses identified yeast Mic19 in two different redox forms. The form that contains an intramolecular disulfide bond is bound to Mic60 of the MICOS complex. Mic19 oxidation is not essential for its integration into the MICOS complex but plays a role in MICOS assembly and the maintenance of proper inner membrane morphology. These findings suggest that Mic19 is a redox-dependent regulator of MICOS function

Tim50’s presequence receptor domain is essential for signal driven transport across the TIM23 complex

The Tim50 subunit of the mitochondrial TIM23 complex contains a presequence-binding domain that is essential for viability and precursor transport across the inner membrane

Mitochondrial stress-dependent regulation of cellular protein synthesis

The production of newly synthesized proteins is vital for all cellular functions and is a determinant of cell growth and proliferation. The synthesis of polypeptide chains from mRNA molecules requires sophisticated machineries and mechanisms that need to be tightly regulated, and adjustable to current needs of the cell. Failures in the regulation of translation contribute to the loss of protein homeostasis, which can have deleterious effects on cellular function and organismal health. Unsurprisingly, the regulation of translation appears to be a crucial element in stress response mechanisms. This review provides an overview of mechanisms that modulate cytosolic protein synthesis upon cellular stress, with a focus on the attenuation of translation in response to mitochondrial stress. We then highlight links between mitochondrion-derived reactive oxygen species and the attenuation of reversible cytosolic translation through the oxidation of ribosomal proteins at their cysteine residues. We also discuss emerging concepts of how cellular mechanisms to stress are adapted, including the existence of alternative ribosomes and stress granules, and the regulation of co-translational import upon organelle stress

Mitochondrial Biogenesis: Cell-Cycle-Dependent Investment in Making Mitochondria

SummaryMitochondria cannot be made de novo, so pre-existing mitochondria must be inherited at each cell division. A new study demonstrates cell-cycle-dependent regulation of the activity of the TOM translocase complex to induce mitochondrial biogenesis during the M phase of the cell cycle

Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase

The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins. The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum. A disulfide-generating enzyme/sulfhydryl oxidase oxidizes a disulfide carrier protein, which in turn transfers a disulfide to the substrate protein. Current views suggest that the disulfide carrier alternates between binding to the oxidase and the substrate. We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components. The ternary complex represents a transient and intermediate step in the oxidation of intermembrane space precursors, where the oxidase Erv1 promotes disulfide transfer to the precursor while both oxidase and precursor are associated with the disulfide carrier Mia40