A classic feature of apoptotic cells is the cell-surface exposure of phosphatidylserine (PtdSer) as an "eat me" signal for engulfment. We show that the Xk-family protein Xkr8 mediates PtdSer exposure in response to apoptotic stimuli. Mouse Xkr8(-/-) cells or human cancer cells in which Xkr8 expression was repressed by hypermethylation failed to expose PtdSer during apoptosis and were inefficiently engulfed by phagocytes. Xkr8 was activated directly by caspases and required a caspase-3 cleavage site for its function. CED-8, the only Caenorhabditis elegans Xk-family homolog, also promoted apoptotic PtdSer exposure and cell-corpse engulfment. Thus, Xk-family proteins have evolutionarily conserved roles in promoting the phagocytosis of dying cells by altering the phospholipid distribution in the plasma membrane.
Background: Xkr8 is essential to scramble phospholipids during apoptosis. The function of other Xkr family members is unknown. Results: Xkr4, Xkr8, and Xkr9 rescued apoptotic lipid scrambling in Xkr8 Ϫ/Ϫ cells.
Xk-related protein (Xkr) 8, a protein carrying 10 transmembrane regions, is essential for scrambling phospholipids during apoptosis. Here, we found Xkr8 as a complex with basigin (BSG) or neuroplastin (NPTN), type I membrane proteins in the Ig superfamily. In BSG −/− NPTN −/− cells, Xkr8 localized intracellularly, and the apoptosis stimuli failed to expose phosphatidylserine, indicating that BSG and NPTN chaperone Xkr8 to the plasma membrane to execute its scrambling activity. Mutational analyses of BSG showed that the atypical glutamic acid in the transmembrane region is required for BSG's association with Xkr8. In cells exposed to apoptotic signals, Xkr8 was cleaved at the C terminus and the Xkr8/BSG complex formed a higher-order complex, likely to be a heterotetramer consisting of two molecules of Xkr8 and two molecules of BSG or NPTN, suggesting that this cleavage causes the formation of a larger complex of Xkr8-BSG/NPTN for phospholipid scrambling.phospholipid scramblase | Xkr8 | chaperone | basigin | neuroplastin P hospholipids are asymmetrically distributed in plasma membranes by flippases that actively translocate phosphatidylserine (PtdSer) and phosphatidylethanolamine from the outer to inner leaflets of the membrane (1, 2). This asymmetrical distribution is disrupted in the activated platelets and apoptotic cells (3), in which the PtdSer exposed on the cell surface serves as a scaffold for blood clotting factors and as an "eat me" signal, respectively (4, 5). ATP11A and ATP11C, members of the P4-type ATPase family, act as flippases at the plasma membrane in most cells (6, 7). Two processes, flippase inactivation and scramblase activation, must occur to disrupt the asymmetrical phospholipid distribution and expose PtdSer on the cell surface (8).Scramblases are membrane proteins that nonspecifically and bidirectionally transport phospholipids between the two plasma membrane leaflets (9). Ca 2+ -activated phospholipid scrambling is mediated by membrane proteins that belong to the transmembrane protein (TMEM)16 (also called ANO) family (8). Of 10 human TMEM16-family members, 5 are Ca 2+ -activated phospholipid scramblases at plasma membranes. TMEM16F exposes PtdSer in activated platelets and osteoblasts (10-12). The tertiary structure of fungal TMEM16 and the biochemical characterization of mouse TMEM16 family members indicate that TMEM16 forms a homodimer that directly binds Ca 2+ (13).Phospholipid scrambling and PtdSer exposure in apoptotic cells is mediated by another family of membrane proteins, the XK-related (Xkr) proteins (8). Of 10 human Xkr family members, Xkr8 (ubiquitously expressed) and Xkr4 and Xkr9 (expressed in specific tissues) are cleaved by caspase during apoptosis to expose PtdSer (14, 15), but how the cleavage activates these Xkrs to scramble phospholipids is unknown. XK, the founding member of the Xkr family, associates with Kell, a type II membrane protein (16). Whether Xkr8 and other Xkr-family members associate with other proteins has not been addressed.In this report, we found that...
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