Xkr regulates phosphatidylserine transport via enhanced ER-PM connectivity to promote apoptotic cell clearance

Exposure of the apoptotic “eat me” signal factor, phosphatidylserine, is important for cell corpse clearance (efferocytosis) through macrophage recognition, which is induced by scramblases, such as Xkr8, in a caspase-dependent manner. Drosophila Xkr, the Xkr8 homolog, lacks caspase recognition sites, implying an alternative efferocytosis pathway. Here, we show that Xkr exhibits scramblase activity to mediate PS exposure in apoptosis through direct interaction with the transmembrane protein TM9SF4 and the endoplasmic reticulum (ER) phospholipase Sac1. Additionally, Xkr specifically accumulates at ER-plasma membrane (PM) contact sites by directly interacting with the pleckstrin homology domain of Drosophila OSBP (dORP9). The formation of this Xkr-dORP9-TM9SF4-Sac1 complex structurally stabilizes ER-PM junctions to enable non-vesicular PS translocation to the PM. Moreover, the suppression of Xkr or other complex components disrupts non-vesicular PS transport, consequently impairing efferocytosis in vitro and in vivo. Similarly, mammalian ORP8 mediates apoptotic PS exposure through its interaction with Xkr8. These results reveal a previously unrecognized function of the lipid scramblase, Xkr, as a structural tethering factor that helps maintain ER-PM contact sites to mediate PS transport and exposure in efferocytosis.