Phospholipid Flippase Activities and Substrate Specificities of Human Type IV P-type ATPases Localized to the Plasma Membrane

• Published in: The Journal of biological chemistry Vol. 289; no. 48; pp. 33543 - 33556
• Main Authors: Takatsu, Hiroyuki, Tanaka, Gaku, Segawa, Katsumori, Suzuki, Jun, Nagata, Shigekazu, Nakayama, Kazuhisa, Shin, Hye-Won
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.11.2014; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Animals; ATPase; Biological Transport, Active - genetics; Cell Biology; Cell Membrane - genetics; Cell Membrane - metabolism; Cell Membrane - pathology; CHO Cells; Cholestasis, Intrahepatic - genetics; Cholestasis, Intrahepatic - metabolism; Cholestasis, Intrahepatic - pathology; Cricetinae; Cricetulus; HEK293 Cells; HeLa Cells; Humans; Lipid Bilayer; Lipid Metabolism, Inborn Errors - genetics; Lipid Metabolism, Inborn Errors - metabolism; Lipid Metabolism, Inborn Errors - pathology; Lipid Transport; Membrane Proteins - genetics; Membrane Proteins - metabolism; Phospholipid; Phospholipids - genetics; Phospholipids - metabolism; Plasma Membrane; Substrate Specificity - genetics; Plasma Membrane; Phospholipid; Lipid Bilayer; Lipid Transport; ATPase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.593012

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. Background: The enzymatic activities of mammalian P4-ATPases are incompletely characterized. Results: ATP11A and ATP11C catalyze flipping of NBD-PS and NBD-PE, whereas ATP8B1 preferentially catalyzes flipping of NBD-PC. Furthermore, some PFIC1 mutants of ATP8B1 failed to flip PC. Conclusion: ATP11A/ATP11C and ATP8B1/ATP8B2 preferentially translocate aminophospholipids and PC, respectively. Significance: This is the first evidence showing that the PC-flipping activity of ATP8B1 is associated with the episode of PFIC1.