Phospholipid Flippase Activities and Substrate Specificities of Human Type IV P-type ATPases Localized to the Plasma Membrane
Phospholipid Flippase Activities and Substrate Specificities of Human Type IV P-type ATPases Localized to the Plasma Membrane
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DOI：
10.1074/jbc.m114.593012
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发表时间：
2014-11-28
影响因子：
4.8
通讯作者：
Shin, Hye-Won
中科院分区：
文献类型：
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作者：
Takatsu, Hiroyuki;Tanaka, Gaku;Shin, Hye-Won
IV型P-型ATP酶（P4-ATP酶）被认为将氨基磷脂从细胞膜的细胞外质转移到细胞质小叶。酵母P4-ATP酶Drs 2 p和Dnf 1 p/Dnf 2 p分别翻转高尔基复合体上的硝基苯并恶二唑标记的磷脂酰丝氨酸和质膜上的硝基苯并恶二唑标记的磷脂酰胆碱（PC）。然而，哺乳动物P4-ATP酶的翻转酶活性和底物特异性仍然不完全表征。在这项研究中，我们建立了一个测定磷脂翻转酶活性的质膜定位的P4-ATP酶使用人类细胞株稳定表达ATP 8B 1，ATP 8B 2，ATP 11 A，和ATP 11 C。我们发现ATP 11 A和ATP 11 C对磷脂酰丝氨酸和磷脂酰乙醇胺有翻转酶活性，但对PC和鞘磷脂没有翻转酶活性。相比之下，ATP酶缺陷突变体的ATP 11 A和ATP 11 C没有表现出任何翻转酶活性，表明这些酶催化翻转的ATP酶依赖性的方式。此外，ATP 8B 1和ATP 8B 2表现出对PC的优先翻转酶活性。在进行性家族性肝内胆汁淤积症1型（PFIC 1）患者中发现的一些ATP 8B 1突变体，尽管它们被递送到质膜，但未能易位PC，PFIC 1是一种由胆汁流动受损引起的严重肝病。此外，由ATP 8B 1介导的PC掺入可以通过同时表达ABCB 4（PFIC 3患者中突变的PC翻转酶）来逆转。我们的研究结果阐明了翻转酶的活动和质膜定位的人P4-ATP酶的底物特异性，并表明，一些PFIC 1患者的表型导致ATP 8B 1的PC翻转酶活性受损。
Type IV P-type ATPases (P4-ATPases) are believed to translocate aminophospholipids from the exoplasmic to the cytoplasmic leaflets of cellular membranes. The yeast P4-ATPases, Drs2p and Dnf1p/Dnf2p, flip nitrobenzoxadiazole-labeled phosphatidylserine at the Golgi complex and nitrobenzoxadiazole-labeled phosphatidylcholine (PC) at the plasma membrane, respectively. However, the flippase activities and substrate specificities of mammalian P4-ATPases remain incompletely characterized. In this study, we established an assay for phospholipid flippase activities of plasma membrane-localized P4-ATPases using human cell lines stably expressing ATP8B1, ATP8B2, ATP11A, and ATP11C. We found that ATP11A and ATP11C have flippase activities toward phosphatidylserine and phosphatidylethanolamine but not PC or sphingomyelin. By contrast, ATPase-deficient mutants of ATP11A and ATP11C did not exhibit any flippase activity, indicating that these enzymes catalyze flipping in an ATPase-dependent manner. Furthermore, ATP8B1 and ATP8B2 exhibited preferential flippase activities toward PC. Some ATP8B1 mutants found in patients of progressive familial intrahepatic cholestasis type 1 (PFIC1), a severe liver disease caused by impaired bile flow, failed to translocate PC despite their delivery to the plasma membrane. Moreover, incorporation of PC mediated by ATP8B1 can be reversed by simultaneous expression of ABCB4, a PC floppase mutated in PFIC3 patients. Our findings elucidate the flippase activities and substrate specificities of plasma membrane-localized human P4-ATPases and suggest that phenotypes of some PFIC1 patients result from impairment of the PC flippase activity of ATP8B1.