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.
Highlights
The enzymatic activities of mammalian P4-ATPases are incompletely characterized
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
ATP11A and ATP11C Flip Aminophospholipids—Mutant P4-ATPase proteins with alterations in the catalytically critical aspartate residue, which undergoes phosphorylation and dephosphorylation in the ATPase cycle, are commonly used as ATPase-deficient mutants in yeast [32]. We found that such aspartate mutants of ATP8B1, ATP11A, and ATP11C failed to localize to the plasma membrane and were instead retained in the endoplasmic reticulum (ER) even when coexpressed with CDC50A (Fig. 2, A–D) in HeLa cells
Summary
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. 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. ATPase-deficient mutants of ATP11A and ATP11C did not exhibit any flippase activity, indicating that these enzymes catalyze flipping in an ATPase-dependent manner. 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. We established an assay for plasma membranelocalized phospholipid flippases by modifying previously described methods [8, 24] Using this method, we showed that ATP11A and ATP11C catalyze flipping of NBD-PS and NBD-PE but not NBD-PC or NBD-SM, whereas ATP8B1 preferentially catalyzes flipping of NBD-PC. We found that some PFIC1-type mutants of ATP8B1 failed to flip PC and that exogenous expression of ABCB4 diminished PC translocation mediated by ATP8B1
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