ESCRT-III-mediated membrane fission is an ancestral activity predating eukaryogenesis, executed through a conserved N-terminal amphipathic helix (Hofund) that drives fission via deep membrane insertion and lipid destabilization in Asgard archaea, with this mechanism preserved but fragmented across multiple eukaryotic ESCRT-III paralogs that cooperatively reconstitute the ancestral fission trigger during intraluminal vesicle formation.

The emergence of cell compartmentalization depends on membrane fission to create the endomembrane compartments. In eukaryotes, membrane fission is commonly executed by ESCRT-III, a protein complex conserved in all domains of life. However, whether membrane fission was an ancestral ESCRT-III activity predating eukaryogenesis remains unknown. Here we show that ESCRT-IIIA from Asgard Heimdallarchaeota, the closest archaeal relatives of eukaryotes, performs membrane fission through an N-terminal amphipathic helix, which we term Hofund. In eukaryotes, Hofund is fragmented across ESCRT-IIIA paralogs, and disrupting these regions causes severe fission defect in yeast. Remarkably, Heimdallarchaeota Hofund restores fission when fused to defective eukaryotic paralogs. These findings suggest that ESCRT-III-mediated fission arose before eukaryogenesis and later diversified to support the regulatory complexity of eukaryotic compartmentalization.