Cytosolic retention of the mitochondrial protease HtrA2 during mitochondrial protein import stress (MPIS) triggers the DELE1-HRI pathway

Mitochondrial stress inducers, such as the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and the ATPase pump inhibitor oligomycin, trigger the DELE1-HRI branch of the integrated stress response (ISR) pathway. Previous studies performed using epitope-tagged forms of DELE1 showed that these stresses induced the accumulation of a cleaved form of DELE1, DELE1-S, which stimulates HRI. Here, we report that mitochondrial protein import stress (MPIS) is an overarching stress that triggers the DELE1-HRI pathway, and that endogenous DELE1 could be cleaved into two forms, DELE1-S and DELE1-VS, the latter accumulating only upon non- depolarizing MPIS. We further showed that DELE1 specifically senses MPIS triggered by the inhibition of the TIM23 complex at the inner mitochondrial membrane (IMM). While MPIS can also cause mitophagy induction through engagement of the NLRX1-RRBP1 pathway, we observed that DELE1-HRI and NLRX1-RRBP1 signaling were engaged independently upon MPIS. Surprisingly, our results suggest that in our cellular model the mitochondrial protease OMA1 was dispensable for DELE1 cleavage upon MPIS. Instead, we identified a key role for another mitochondrial protease, HtrA2, in mediating the cleavage of DELE1 into DELE1-S and DELE1- VS. Our data further suggest that DELE1 is likely cleaved into DELE1-S by HtrA2 in the cytosol, while the DELE1-VS form might be generated during halted translocation of the protein into mitochondria. Together, this study identifies MPIS as the overarching stress detected by DELE1 and identifies HtrA2 as a critical protease involved in DELE1 processing.