PROJECT SUMMARY Mitochondria are essential organelles that supply cells with ATP and metabolic building blocks, but also play key roles as signaling hubs that orchestrate the immune response or cell survival. Mutations in mitochondrial proteins impede development and cause diseases, such as neurodegeneration, and a decline in mitochondrial activity is considered a hallmark of aging. To prevent such consequences, cells employ conserved signaling pathways that detect and alleviate mitochondrial dysregulation. It is a major goal of this proposal to dissect the regulation of a mitochondrial signaling pathway, the reductive stress response, which safeguards activation of the electron transport chain (ETC) through sensing an invariant ETC byproduct, reactive oxygen species (ROS). The reductive stress response is built on a Cys redox switch: in healthy cells, Cys residues in FNIP1 are oxidized, which stabilizes this protein and allows it to downregulate ETC activity. When cells run out of ROS, however, the FNIP1 Cys residues become reduced and FNIP1 is recognized by the E3 ligase CUL2FEM1B. The subsequent ubiquitylation and proteasomal degradation of FNIP1 removes this mitochondrial gatekeeper to re- activate the ETC and re-supply cells with ROS. FNIP1 and CUL2FEM1B are therefore the sensory module of the reductive stress response. Importantly, mutations in FEM1B that hyperactivate this E3 ligase cause syndromic developmental delay, showing that tissue formation and homeostasis require tight regulation of the reductive stress response. This proposal will dissect three crucial modes of regulation that ensure accurate reductive stress signaling. We will first investigate spatial control of reductive stress signaling. As with all ubiquitylation reactions, FNIP1 modification by CUL2FEM1B takes places in the cytoplasm, yet how cells can modulate the oxidation state of the critical FNIP1 Cys residues in this already reducing environment is unclear. We found that substrate and enzyme of the reductive stress response are anchored on the outer mitochondrial membrane via the TOM complex, a channel that connects the oxidative mitochondrial intermembrane space with the reducing cytoplasm. In our first aim, we will dissect how this localization impacts reductive stress signaling. We expect that this work will reveal a novel function of a membrane channel as an E3 ligase co-adaptor. Moreover, it will likely allow us to pinpoint the source of ROS that mediate reductive stress signaling, thereby revealing a sought-after physiological role for ROS in signaling. We will next focus on the regulation of reductive stress signaling by the cell cycle. ROS have long been suggested to control cell division, and we had indeed found that hyperactivation of CUL2FEM1B inhibits proliferation. This result implied that ROS control the cell cycle via the reductive stress response. In line with this notion, we identified the cell cycle regulator RNF187, which promotes cell cycle prog...