Identification of P4-ATPase Phospholipid Flippases in Red Blood Cells and Functional Analysis of Disease-associated Mutants

ATP-dependent phospholipid flippase activity crucial for generating lipid asymmetry in membranes was first detected in red blood cells (RBCs), but the P4-ATPases responsible have not been directly determined. Using affinity-based mass spectrometry, we show that ATP11C is the only abundant P4-ATPase in human RBCs, while ATP11C and ATP8A1 are the major P4-ATPases in mouse RBCs. ATP11A and ATP11B are detectable at low levels. Mutations in ATP11C are responsible for blood and liver disorders, but the mechanisms are not known. Using heterologous expression, we show that the T418N mutation localized in the conserved phosphorylation motif of ATP11C and responsible for human congenial hemolytic anemia expresses at low levels, is retained in the endoplasmic reticulum, and has 33% of wild-type ATP11C activity. The I355K mutant in the transmembrane domain associated with cholestasis and anemia in mice expresses at wild-type levels and traffics to the plasma membrane, but is devoid of activity. We conclude that the T418N mutation of ATP11C causes significant protein misfolding, whereas the I355K mutant folds normally, but lacks key contacts required for functional activity.

介导膜脂质不对称性生成的ATP依赖型磷脂翻转酶（ATP-dependent phospholipid flippase）活性最初于红细胞（red blood cells, RBCs）中被检测到，但负责该催化活性的P4-ATP酶（P4-ATPases）尚未被直接鉴定。本研究通过亲和质谱法（affinity-based mass spectrometry）证实，人类红细胞中仅ATP11C为丰度最高的P4-ATP酶，而小鼠红细胞中的主要P4-ATP酶则为ATP11C与ATP8A1；ATP11A与ATP11B仅能以低水平被检出。ATP11C的突变可引发血液与肝脏疾病，但其具体分子机制尚未阐明。通过异源表达（heterologous expression）实验，我们发现定位于ATP11C保守磷酸化基序（phosphorylation motif）、且与人类先天性溶血性贫血相关的T418N突变体，其蛋白表达水平较低，会在内质网（endoplasmic reticulum）中发生滞留，且仅保留野生型ATP11C 33%的酶活性。而位于跨膜结构域（transmembrane domain）、与小鼠胆汁淤积（cholestasis）及贫血症状相关的I355K突变体，其表达水平与野生型一致，可正常转运至质膜（plasma membrane），但完全丧失酶活性。综上，ATP11C的T418N突变会引发显著的蛋白质错误折叠，而I355K突变体则可正常折叠，但缺失维持功能活性所必需的关键相互作用位点。