Identification and functional analyses of disease-associated P4-ATPase phospholipid flippase variants in red blood cells

Abstract

Edited by George M. Carman ATP-dependent phospholipid flippase activity crucial for generating lipid asymmetry was first detected in red blood cell (RBC) membranes, but the P4-ATPases responsible have not been directly determined. Using affinity-based MS, we show that ATP11C is the only abundant P4-ATPase phospholipid flippase in human RBCs, whereas ATP11C and ATP8A1 are the major P4-ATPases in mouse RBCs. We also found that ATP11A and ATP11B are present at low levels. Mutations in the gene encoding ATP11C are responsible for blood and liver disorders, but the disease mechanisms are not known. Using heterologous expression, we show that the T415N substitution in the phosphorylation motif of ATP11C, responsible for congenital hemolytic anemia, reduces ATP11C expression, increases retention in the endoplasmic reticulum, and decreases ATPase activity by 61% relative to WT ATP11C. The I355K substitution in the transmembrane domain associated with cholestasis and anemia in mice was expressed at WT levels and trafficked to the plasma membrane but was devoid of activity. We conclude that the T415N variant causes significant protein misfolding, resulting in low protein expression, cellular mislocalization, and reduced functional activity. In contrast, the I355K variant folds and traffics normally but lacks key contacts required for activity. We propose that the loss in ATP11C phospholipid flippase activity coupled with phospholipid scramblase activity results in the exposure of phosphatidylserine on the surface of RBCs, decreasing RBC survival and resulting in anemia. The asymmetrical distribution of lipids across membranes of eukaryotic cells plays a crucial role in many cellular processes, including blood coagulation, apoptosis, phagocytosis, vesicle trafficking, axon elongation, sensory functions, and metabolic

Expression and functional characterization of missense mutations in ATP8A2 linked to severe neurological disorders

“…One possibility is that other P4‐ATPases with similar substrate specificities as ATP8A2 compensate for the loss in function of ATP8A2 in certain tissues. ATP8A1, ATP11A, ATP11B and ATP11C like ATP8A2 actively flip PS and PE across cell membranes (Liou, Molday, Wang, Andersen, & Molday, ; Takatsu et al, ; Wang et al, ). Of these P4‐ATPases, ATP8A1 is most similar to ATP8A2 sharing 67% identity in sequence to ATP8A2 and it is widely expressed in various tissues.…”

Flagging fusion: Phosphatidylserine signaling in cell–cell fusion

“…Estimates from model membranes suggest that, once oriented, PS should not spontaneously return to the exofacial/luminal leaflets on biologically relevant timescales (days) ( 63 , 64 ). Loss of P4-ATPase function ablates PS asymmetry in eukaryotic membranes and leads to human disease ( 65 , 66 , 67 , 68 ).…”

Phosphatidylserine exposure in living cells

“…Mammalian P4-ATPases exhibit strict substrate specificities (Table 1), and PSspecific P4-ATPases are responsible for the removal of cell surface PS (Figure 1). Among the P4-ATPases summarized in Table 1, ATP11A and ATP11C are major plasma membrane PS flippases in many cell types (Liou et al, 2019;Segawa et al, 2014;Siggs et al, 2011a;Takada et al, 2015;Tsuchiya et al, 2018;Wang et al, 2018;Yabas et al, 2011) and ATP8A1 and ATP8A2 also flip PS (Coleman et al, 2009;Paterson et al, 2006). ATP8B1, ATP8B2, and ATP10A preferentially flip PC at the plasma membrane (Naito et al, 2015;Takada et al, 2015;Takatsu et al, 2014), and ATP10D translocates a glycosphingolipid, glucosylceramide, but not any other phospholipids (Roland et al, 2019).…”