Vectorial import via a metastable disulfide-linked complex allows for a quality control step and import by the mitochondrial disulfide relay

Disulfide formation in the mitochondrial intermembrane space (IMS) is an essential process. It is catalyzed by the disulfide relay machinery, which couples substrate import and oxidation. The machinery relies on the oxidoreductase and chaperone CHCHD4-Mia40. Here, we report on the driving force for IMS import and on a redox quality control mechanism. We demonstrate that unfolded reduced proteins, upon translocation into the IMS, initiate formation of a metastable disulfide-linked complex with CHCHD4. If this interaction does not result in productive oxidation, then substrates are released to the cytosol and degraded by the proteasome. Based on these data, we propose a redox quality control step at the level of the disulfide-linked intermediate that relies on the vectorial nature of IMS import. Our findings also provide the mechanistic framework to explain failures in import of numerous human disease mutants in CHCHD4 substrates

The C-terminal region of the oxidoreductase MIA40 stabilizes its cytosolic precursor during mitochondrial import

Background The mitochondrial intermembrane space (IMS) is home to proteins fulfilling numerous essential cellular processes, particularly in metabolism and mitochondrial function. All IMS proteins are nuclear encoded and synthesized in the cytosol and must therefore be correctly targeted and transported to the IMS, either through mitochondrial targeting sequences or conserved cysteines and the mitochondrial disulfide relay system. The mitochondrial oxidoreductase MIA40, which catalyzes disulfide formation in the IMS, is imported by the combined action of the protein AIFM1 and MIA40 itself. Here, we characterized the function of the conserved highly negatively charged C-terminal region of human MIA40. Results We demonstrate that the C-terminal region is critical during posttranslational mitochondrial import of MIA40, but is dispensable for MIA40 redox function in vitro and in intact cells. The C-terminal negatively charged region of MIA40 slowed import into mitochondria, which occurred with a half-time as slow as 90 min. During this time, the MIA40 precursor persisted in the cytosol in an unfolded state, and the C-terminal negatively charged region served in protecting MIA40 from proteasomal degradation. This stabilizing property of the MIA40 C-terminal region could also be conferred to a different mitochondrial precursor protein, COX19. Conclusions Our data suggest that the MIA40 precursor contains the stabilizing information to allow for postranslational import of sufficient amounts of MIA40 for full functionality of the essential disulfide relay. We thereby provide for the first time mechanistic insights into the determinants controlling cytosolic surveillance of IMS precursor proteins

Spatial and temporal control of mitochondrial H2O2 release in intact human cells

Hydrogen peroxide (H2O2) has key signaling roles at physiological levels, while causing molecular damage at elevated concentrations. H2O2 production by mitochondria is implicated in regulating processes inside and outside these organelles. However, it remains unclear whether and how mitochondria in intact cells release H2O2. Here, we employed a genetically encoded high-affinity H2O2 sensor, HyPer7, in mammalian tissue culture cells to investigate different modes of mitochondrial H2O2 release. We found substantial heterogeneity of HyPer7 dynamics between individual cells. We further observed mitochondria-released H2O2 directly at the surface of the organelle and in the bulk cytosol, but not in the nucleus or at the plasma membrane, pointing to steep gradients emanating from mitochondria. Gradient formation is controlled by cytosolic peroxiredoxins, which act redundantly and with a substantial reserve capacity. Dynamic adaptation of cytosolic thioredoxin reductase levels during metabolic changes results in improved H2O2 handling and explains previously observed differences between cell types. Our data suggest that H2O2-mediated signaling is initiated only in close proximity to mitochondria and under specific metabolic conditions