Spatial and temporal control of mitochondrial H2 O2 release in intact human cells.

Hydrogen peroxide (H2 O2 ) has key signaling roles at physiological levels, while causing molecular damage at elevated concentrations. H2 O2 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 H2 O2 . Here, we employed a genetically encoded high-affinity H2 O2 sensor, HyPer7, in mammalian tissue culture cells to investigate different modes of mitochondrial H2 O2 release. We found substantial heterogeneity of HyPer7 dynamics between individual cells. We further observed mitochondria-released H2 O2 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 H2 O2 handling and explains previously observed differences between cell types. Our data suggest that H2 O2 -mediated signaling is initiated only in close proximity to mitochondria and under specific metabolic conditions.

Proteasomal degradation induced by DPP9-mediated processing competes with mitochondrial protein import.

Plasticity of the proteome is critical to adapt to varying conditions. Control of mitochondrial protein import contributes to this plasticity. Here, we identified a pathway that regulates mitochondrial protein import by regulated N-terminal processing. We demonstrate that dipeptidyl peptidases 8/9 (DPP8/9) mediate the N-terminal processing of adenylate kinase 2 (AK2) en route to mitochondria. We show that AK2 is a substrate of the mitochondrial disulfide relay, thus lacking an N-terminal mitochondrial targeting sequence and undergoing comparatively slow import. DPP9-mediated processing of AK2 induces its rapid proteasomal degradation and prevents cytosolic accumulation of enzymatically active AK2. Besides AK2, we identify more than 100 mitochondrial proteins with putative DPP8/9 recognition sites and demonstrate that DPP8/9 influence the cellular levels of a number of these proteins. Collectively, we provide in this study a conceptual framework on how regulated cytosolic processing controls levels of mitochondrial proteins as well as their dual localization to mitochondria and other compartments.