RRC ID 47459
Author Takatsu H, Tanaka G, Segawa K, Suzuki J, Nagata S, Nakayama K, Shin HW.
Title Phospholipid flippase activities and substrate specificities of human type IV P-type ATPases localized to the plasma membrane.
Journal J Biol Chem
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.
Volume 289(48)
Pages 33543-56
Published 2014-11-28
DOI 10.1074/jbc.M114.593012
PII S0021-9258(20)47429-0
PMID 25315773
PMC PMC4246107
MeSH Adenosine Triphosphatases / genetics Adenosine Triphosphatases / metabolism* Animals Biological Transport, Active / genetics CHO Cells Cell Membrane / genetics Cell Membrane / metabolism* Cell Membrane / pathology Cholestasis, Intrahepatic / genetics Cholestasis, Intrahepatic / metabolism Cholestasis, Intrahepatic / pathology Cricetinae Cricetulus HEK293 Cells HeLa Cells Humans Lipid Metabolism, Inborn Errors / genetics Lipid Metabolism, Inborn Errors / metabolism Lipid Metabolism, Inborn Errors / pathology Membrane Proteins / genetics Membrane Proteins / metabolism* Phospholipids / genetics Phospholipids / metabolism* Substrate Specificity / genetics
IF 4.238
Times Cited 50
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
DNA material pCMV-VSV-G-RSV-Rev (RDB04393) pMx/ATP8A1-HA (RDB20147) pMx/ATP8B1-HA (RDB20148) pMx/ATP8B3-HA (RDB20150) pMx/ATP8B4-HA (RDB20151) pMx/ATP11A-HA (RDB20157) pMx/ATP11B-HA (RDB20158) pMx/ATP11C-HA (RDB20159) pCAG/ATP8A1(E191Q)-HA (RDB20162) pCAG/ATP8B1(E234Q)-HA (RDB20163) pCAG/ATP8B2(E171Q)-HA (RDB20164) pCAG/ATP8B4(E170Q)-HA (RDB20165) pCAG/ATP11A(E186Q)-HA (RDB20171) pCAG/ATP11C(E184Q)-HA (RDB20173) pMx/ATP8A1(E191Q)-HA (RDB20174) pMx/ATP8B1(E234Q)-HA (RDB20175) pMx/ATP8B4(E170Q)-HA (RDB20177) pMx/ATP11A(E186Q)-HA (RDB20183) pMx/ATP11C(E184Q)-HA (RDB20185)