Mechanisms of RCD-1 pore formation and membrane bending.

Regulator of cell death-1 (RCD-1) governs the heteroallelic expression of RCD-1-1 and RCD-1-2, a pair of fungal gasdermin (GSDM)-like proteins, which prevent cytoplasmic mixing during allorecognition and safeguard against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (e.g., senescence plasmids) by effecting a form of cytolytic cell death. However, the underlying mechanisms by which RCD-1 acts on the cell membrane remain elusive. Here, we demonstrate that RCD-1 binds acidic lipid membranes, forms pores, and induces membrane bending. Using atomic force microscopy (AFM) and AlphaFold, we show that RCD-1-1 and RCD-1-2 form heterodimers that further self-assemble into ~14.5 nm-wide transmembrane pores (~10 heterodimers). Moreover, through AFM force spectroscopy and micropipette aspiration, we reveal that RCD-1 proteins bend membranes with low bending moduli. This combined action of pore formation and membrane deformation may constitute a conserved mechanism within the broader GSDM family.