title: Phospholipid flippase activities and substrate specificities of human type IV P-type ATPases localized to the plasma membrane.
abstract: 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.
authors.affiliation: From the Career-path Promotion Unit for Young Life Scientists and Graduate Schools of Pharmaceutical Sciences and.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takatsu
authors.affiliation: Graduate Schools of Pharmaceutical Sciences and.
authors.forename: Gaku
authors.initials: G
authors.lastname: Tanaka
authors.affiliation: Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Katsumori
authors.initials: K
authors.lastname: Segawa
authors.affiliation: Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Suzuki
authors.affiliation: Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Shigekazu
authors.initials: S
authors.lastname: Nagata
authors.affiliation: Graduate Schools of Pharmaceutical Sciences and.
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Nakayama
authors.affiliation: From the Career-path Promotion Unit for Young Life Scientists and Graduate Schools of Pharmaceutical Sciences and shin@pharm.kyoto-u.ac.jp.
authors.forename: Hye-Won
authors.initials: HW
authors.lastname: Shin
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active / Q000235:genetics; D016466:CHO Cells; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002780:Cholestasis, Intrahepatic / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006224:Cricetinae; D003412:Cricetulus; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008052:Lipid Metabolism, Inborn Errors / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D008565:Membrane Proteins; D010743:Phospholipids; D000251:Adenosine Triphosphatases
keywords: ATPase; Lipid Bilayer; Lipid Transport; Phospholipid; Plasma Membrane