Abstract
During apoptosis, the permeabilization of the mitochondrial inner membrane (MIM) through still-unclear mechanisms releases mtDNA into the cytosol, triggering the inflammatory cGAS/STING pathway under low caspase activity. Here, we report that, in apoptosis, the active form of the pore-forming protein Gasdermin E (GSDME-N) damages mitochondria before plasma membrane disruption. We visualize GSDME-N pore-like nano-assemblies in the MIM of apoptotic cells and of isolated mitochondria, which we bridge with the cryo-EM structure of the GSDME-N pore in mitochondria-like membranes. Deep membrane insertion of the anchor domain, which acts as a determinant of GSDME-N cardiolipin binding preferences, supports a role in pore formation. Notably, GSDME depletion results in a reduction in cristae swelling, MIM extrusion and mtDNA release during apoptosis. Subsequently, this decreases STING activation and inflammatory responses under caspase inhibition. Our findings reveal that GSDME mediates MIM permeabilization and mtDNA release during apoptosis and define the mechanism of GSDME-mediated membrane damage.