Mitochondrial fidelity is tightly linked to overall cellular homeostasis and is 
compromised in ageing and various pathologies1-3. Mitochondrial malfunction 
needs to be relayed to the cytosol, where an integrated stress response is 
triggered by the phosphorylation of eukaryotic translation initiation factor 2α 
(eIF2α) in mammalian cells4,5. eIF2α phosphorylation is mediated by the four 
eIF2α kinases GCN2, HRI, PERK and PKR, which are activated by diverse types of 
cellular stress6. However, the machinery that communicates mitochondrial 
perturbation to the cytosol to trigger the integrated stress response 
remains unknown1,2,7. Here we combine genome engineering and haploid genetics to 
unbiasedly identify genes that affect the induction of C/EBP homologous protein 
(CHOP), a key factor in the integrated stress response. We show that the 
mitochondrial protease OMA1 and the poorly characterized protein DELE1, together 
with HRI, constitute the missing pathway that is triggered by mitochondrial 
stress. Mechanistically, stress-induced activation of OMA1 causes DELE1 to be 
cleaved into a short form that accumulates in the cytosol, where it binds to and 
activates HRI via its C-terminal portion. Obstruction of this pathway can be 
beneficial or adverse depending on the type of mitochondrial perturbation. In 
addition to the core pathway components, our comparative genetic screening 
strategy identifies a suite of additional regulators. Together, these findings 
could be used to inform future strategies to modulate the cellular response to 
mitochondrial dysfunction in the context of human disease.