Two types of type IV P-type ATPases independently re-establish the asymmetrical distribution of phosphatidylserine in plasma membranes

Abstract

Phospholipids are asymmetrically distributed between the lipid bilayer of plasma membranes in which phosphatidylserine (PtdSer) is confined to the inner leaflet. ATP11A and ATP11C, type IV P-Type ATPases in plasma membranes, flip PtdSer from the outer to the inner leaflet, but involvement of other P4-ATPases is unclear. We herein demonstrated that once PtdSer was exposed on the cell surface of ATP11A−/−ATP11C−/− mouse T cell line (W3), its internalization to the inner leaflet of plasma membranes was negligible at 15 °C. However, ATP11A−/−ATP11C−/− cells internalized the exposed PtdSer at 37 °C, a temperature at which trafficking of intracellular membranes was active. In addition to ATP11A and 11C, W3 cells expressed ATP8A1, 8B2, 8B4, 9A, 9B, and 11B, with ATP8A1 and ATP11B being present at recycling endosomes. Cells deficient in four P4-ATPases (ATP8A1, 11A, 11B, and 11C) (QKO) did not constitutively expose PtdSer on the cell surface but lost the ability to re-establish PtdSer asymmetry within 1 hour, even at 37 °C. The expression of ATP11A or ATP11C conferred QKO cells with the ability to rapidly re-establish PtdSer asymmetry at 15 °C and 37 °C, while cells expressing ATP8A1 or ATP11B required a temperature of 37 °C to achieve this function, and a dynamin inhibitor blocked this process. These results revealed that mammalian cells are equipped with two independent mechanisms to re-establish its asymmetry: the first is a rapid process involving plasma membrane flippases, ATP11A and ATP11C, while the other is mediated by ATP8A1 and ATP11B, which require an endocytosis process. Phospholipids are asymmetrically distributed between the lipid bilayer of plasma membranes in which phosphatidylserine (PtdSer) is confined to the inner leaflet. ATP11A and ATP11C, type IV P-Type ATPases in plasma membranes, flip PtdSer from the outer to the inner leaflet, but involvement of other P4-ATPases is unclear. We herein demonstrated that once PtdSer was exposed on the cell surface of ATP11A−/−ATP11C−/− mouse T cell line (W3), its internalization to the inner leaflet of plasma membranes was negligible at 15 °C. However, ATP11A−/−ATP11C−/− cells internalized the exposed PtdSer at 37 °C, a temperature at which trafficking of intracellular membranes was active. In addition to ATP11A and 11C, W3 cells expressed ATP8A1, 8B2, 8B4, 9A, 9B, and 11B, with ATP8A1 and ATP11B being present at recycling endosomes. Cells deficient in four P4-ATPases (ATP8A1, 11A, 11B, and 11C) (QKO) did not constitutively expose PtdSer on the cell surface but lost the ability to re-establish PtdSer asymmetry within 1 hour, even at 37 °C. The expression of ATP11A or ATP11C conferred QKO cells with the ability to rapidly re-establish PtdSer asymmetry at 15 °C and 37 °C, while cells expressing ATP8A1 or ATP11B required a temperature of 37 °C to achieve this function, and a dynamin inhibitor blocked this process. These results revealed that mammalian cells are equipped with two independent mechanisms to re-establish its asymmetry: the first is a rapid process involving plasma membrane flippases, ATP11A and ATP11C, while the other is mediated by ATP8A1 and ATP11B, which require an endocytosis process. Major glycerophospholipids in the plasma membranes of mammalian cells are phosphatidylcholine, phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns), and phosphatidic acid (1Harayama T. Riezman H. Understanding the diversity of membrane lipid composition.Nat. Rev. Mol. Cell Biol. 2018; 19: 281-296Google Scholar, 2Yang Y. Lee M. Fairn G.D. Phospholipid subcellular localization and dynamics.J. Biol. Chem. 2018; 293: 6230-6240Google Scholar). In addition, sphingomyelin (SM) and cholesterol are abundantly present in the plasma membrane. These lipids are asymmetrically distributed between the inner and outer leaflets of the plasma membranes. PtdSer, PtdEtn, and phosphatidylinositol are confined to the inner leaflet, whereas PtdCho and SM are enriched in the outer leaflet (3van Meer G. Voelker D.R. Feigenson G.W. Membrane lipids: where they are and how they behave.Nat. Rev. Mol. Cell Biol. 2008; 9: 112-124Google Scholar, 4Murate M. Abe M. Kasahara K. Iwabuchi K. Umeda M. Kobayashi T. Transbilayer distribution of lipids at nano scale.J. Cell Sci. 2015; 128: 1627-1638Google Scholar). PtdSer at the inner leaflet (or cytoplasmic side) of the plasma membrane plays an important role in the recruitment of proteins to the plasma membrane for their activation (5Zhou Y. Hancock J.F. Lipid profiles of RAS nanoclusters regulate RAS function.Biomolecules. 2021; 11: 1439Google Scholar, 6Sartorel E. Unlu C. Jose M. Massoni-Laporte A. Meca J. Sibarita J.B. et al.Phosphatidylserine and GTPase activation control Cdc42 nanoclustering to counter dissipative diffusion.Mol. Biol. Cell. 2018; 29: 1299-1310Google Scholar, 7Lenoir G. D'Ambrosio J.M. Dieudonne T. Copic A. Transport pathways that contribute to the cellular distribution of phosphatidylserine.Front Cell Dev Biol. 2021; 9737907Google Scholar). In various biological processes, the asymmetrical distribution of PtdSer is broken, which exposes it on the cell surface (8Bevers E.M. Williamson P.L. Getting to the outer leaflet: physiology of phosphatidylserine exposure at the plasma membrane.Physiol. Rev. 2016; 96: 605-645Google Scholar, 9Nagata S. Sakuragi T. Segawa K. Flippase and scramblase for phosphatidylserine exposure.Curr. Opin. Immunol. 2020; 62: 31-38Google Scholar). Apoptotic cells irreversibly expose PtdSer as an “eat me” signal for macrophages (10Segawa K. Nagata S. An apoptotic 'eat Me' signal: phosphatidylserine exposure.Trends Cell Biol. 2015; 25: 639-650Google Scholar, 11Nagata S. Segawa K. Sensing and clearance of apoptotic cells.Curr. Opin. Immunol. 2021; 68: 1-8Google Scholar). In contrast, PtdSer is transiently and reversibly exposed by activated lymphocytes and platelets (12Ryoden Y. Fujii T. Segawa K. Nagata S. Functional expression of the P2X7 ATP receptor requires Eros.J. Immunol. 2020; 204: 559-568Google Scholar, 13Bevers E.M. Tilly R.H. Senden J.M. Comfurius P. Zwaal R.F. Exposure of endogenous phosphatidylserine at the outer surface of stimulated platelets is reversed by restoration of aminophospholipid translocase activity.Biochemistry. 1989; 28: 2382-2387Google Scholar), myoblasts and trophoblasts undergoing cell fusion (14van den Eijnde S.M. van den Hoff M.J. Reutelingsperger C.P. van Heerde W.L. Henfling M.E. Vermeij-Keers C. et al.Transient expression of phosphatidylserine at cell-cell contact areas is required for myotube formation.J. Cell Sci. 2001; 114: 3631-3642Google Scholar, 15Das M. Xu B. Lin L. Chakrabarti S. Shivaswamy V. Rote N.S. Phosphatidylserine efflux and intercellular fusion in a BeWo model of human villous cytotrophoblast.Placenta. 2004; 25: 396-407Google Scholar), and capacitated sperm (16Gadella B.M. Harrison R.A. Capacitation induces cyclic adenosine 3',5'-monophosphate-dependent, but apoptosis-unrelated, exposure of aminophospholipids at the apical head plasma membrane of boar sperm cells.Biol. Reprod. 2002; 67: 340-350Google Scholar), indicating the ability of mammalian cells to break and re-establish PtdSer asymmetry in the plasma membrane. The distribution of phospholipids in the lipid bilayer is regulated by flippases and scramblases (9Nagata S. Sakuragi T. Segawa K. Flippase and scramblase for phosphatidylserine exposure.Curr. Opin. Immunol. 2020; 62: 31-38Google Scholar, 17Montigny C. Lyons J. Champeil P. Nissen P. Lenoir G. On the molecular mechanism of flippase- and scramblase-mediated phospholipid transport.Biochim. Biophys. Acta. 2016; 1861: 767-783Google Scholar). Flippases translocate phospholipids from the outer to the cytoplasmic side of the lipid bilayer in an ATP-dependent while scramblases phospholipids the membrane in an of the and as and J. Umeda M. Nagata S. phospholipid by Scholar, J. E. Nagata S. and phosphatidylserine exposure in apoptotic Scholar). IV ATPases to as flippases M. P4-ATPases as phospholipid function, and 2016; Scholar, K. K. T. of phospholipid Scholar). and a cytoplasmic are and 15 for human and mouse that at the plasma and of require as an for their and from the human P4-ATPases (ATP8A1, 11A, 11B, and 11C) to with proteins or a S. Y. J. T. T. T. et recycling requires recruitment by a phosphatidylserine J. 2015; Scholar, J. T. T. et and of phospholipid flippases from 2018; Scholar, Y. Y. et the activation of human lipid Sci. A. Scholar, T. M.J. M. C. K. et and by of a human lipid with 11: Scholar). We and demonstrated that 11A, and at the plasma membrane and the ability to flip PtdSer K. S. Nagata S. type IV ATPases that as plasma membrane phospholipid flippases and their by and Biol. Chem. 2016; Scholar, H. G. Segawa K. J. Nagata S. K. et and of human type IV ATPases to the plasma Biol. Chem. Scholar). On the other ATP8A1 and ATP11B at as and the S. Y. J. T. T. T. et recycling requires recruitment by a phosphatidylserine J. 2015; Scholar, H. K. T. H. Umeda M. et a aminophospholipid to the in a Biol. Chem. Scholar, S. T. Y. K. H. et or cytoplasmic of P4-ATPases their cellular Biol. Cell. 2020; Scholar), it intracellular P4-ATPases contribute to the asymmetrical distribution of PtdSer at the plasma membrane. ATP11A and ATP11C are expressed in various cell while the expression of is to the or J. T. T. et and of phospholipid flippases from 2018; Scholar, K. S. Nagata S. type IV ATPases that as plasma membrane phospholipid flippases and their by and Biol. Chem. 2016; Scholar). We that at the plasma membrane of the ATP11A−/−ATP11C−/− mouse W3 cell line was negligible K. Y. K. C. Y. Nagata S. Phospholipid flippases cells to by Sci. A. 2018; Scholar). In the present demonstrated that the exposure of PtdSer, cells the asymmetrical distribution of PtdSer at the plasma membrane at 37 °C, but not at 15 °C. at 37 °C was by a dynamin inhibitor that blocked a endocytosis and membrane trafficking A. G. A. et a the dynamin and Scholar). the ATP8A1 and ATP11B deficient (QKO) cells lost the ability to re-establish PtdSer asymmetry at the plasma membrane. PtdSer asymmetry was rapidly in QKO cells expressing ATP11A or ATP11C, but and in a in QKO cells expressing ATP8A1 or ATP11B, and that was blocked by a dynamin QKO cells or within plasma membrane PtdSer asymmetry at 37 °C its that the cell the PtdSer asymmetry by an process that is independent of the four QKO cells the PtdSer asymmetry in the plasma membrane. on that mammalian cells are equipped with two of PtdSer flippases at the plasma membrane or that PtdSer asymmetry in the plasma membrane its We that W3 a mouse cell expressed P4-ATPases (ATP8A1, 8B2, 8B4, 9A, 9B, 11A, 11B, and 11C) K. S. Y. Nagata S. of phospholipid for apoptotic phosphatidylserine Scholar), while cells lost PtdSer at the plasma membrane K. S. Nagata S. type IV ATPases that as plasma membrane phospholipid flippases and their by and Biol. Chem. 2016; Scholar, K. Y. K. C. Y. Nagata S. Phospholipid flippases cells to by Sci. A. 2018; Scholar). membrane flippases the PtdSer We the and of temperature on in cells the inner leaflet at 15 °C, and its at for at 37 °C, a by In contrast, a in was not in cells at 15 °C or 37 °C, indicating that PtdSer on the cell surface of cells was We endogenous PtdSer distribution on the cell surface with a and that they did not expose PtdSer the of PtdSer contrast, W3 and cells with a they exposed PtdSer on the cell surface within the activation of the J. Umeda M. Nagata S. phospholipid by Scholar). that the asymmetrical distribution of PtdSer on the cell surface was plasma membrane flippases or was mediated by intracellular We the process to re-establish the asymmetrical distribution of endogenous PtdSer the exposure of PtdSer by the was from the W3 cells rapidly exposed PtdSer the inner leaflet of the plasma membrane at 15 °C and 37 °C and of the internalized In contrast, cells lost the ability to flip exposed PtdSer at 15 °C, it on the cell surface that ATP11A and ATP11C are for PtdSer asymmetry at 15 °C. However, an in temperature to 37 °C, cells internalized PtdSer from the surface 15 and PtdSer on cells within PtdSer asymmetry These results that cells a to re-establish the asymmetrical distribution of and intracellular membrane trafficking are at 37 °C and blocked at 15 °C B. of and on Cell Scholar, E. J. as to membrane Cell Biol. 1989; Scholar). W3 cells with at 37 °C, it was cells and intracellular and process was blocked at 15 °C or by a dynamin that the endocytosis or membrane trafficking A. G. A. et a the dynamin and and that W3 cells endocytosis and membrane trafficking We that intracellular P4-ATPases the of the asymmetrical distribution of PtdSer at the plasma membrane membrane trafficking to re-establish PtdSer asymmetry at 37 °C in a and and mammalian P4-ATPases to PtdSer S. Y. J. T. T. T. et recycling requires recruitment by a phosphatidylserine J. 2015; Scholar, J. T. T. et and of phospholipid flippases from 2018; Scholar, Y. Y. et the activation of human lipid Sci. A. Scholar, T. M.J. M. C. K. et and by of a human lipid with 11: Scholar, K. S. Nagata S. type IV ATPases that as plasma membrane phospholipid flippases and their by and Biol. Chem. 2016; Scholar, H. G. Segawa K. J. Nagata S. K. et and of human type IV ATPases to the plasma Biol. Chem. Scholar), and ATP8A1 and ATP11B at or the the of ATP8A1 and ATP11B on the of the asymmetrical distribution of PtdSer in The ATP8A1 ATP11B on cells and and QKO QKO cells as as cells and did not in PtdSer at the plasma membrane did not between and QKO cells at 15 °C or 37 °C, indicating that ATP8A1 and ATP11B did not contribute to this process. In contrast, the of PtdSer asymmetry by cells at 37 °C the exposure of PtdSer was in the required for this process from with cells to and with and and the of cells was in QKO of ATP11B on the ATP8A1, which with of ATP11B in W3 cells K. S. Y. Nagata S. of phospholipid for apoptotic phosphatidylserine Scholar). The exposure of PtdSer on QKO cells was at 37 °C for These results that the four P4-ATPases (ATP8A1, ATP11B, and in PtdSer asymmetry in W3 how to the they with and expressed in QKO cells and with or by with in and and ATP11C with that the plasma membrane M. T. M. Y. for and of plasma 2020; Scholar). On the other with S. Y. J. T. T. T. et recycling requires recruitment by a phosphatidylserine J. 2015; Scholar, H. K. T. H. Umeda M. et a aminophospholipid to the in a Biol. Chem. Scholar), of the of ATP8A1 and ATP11B with in recycling of and cells Cell Sci. and with The expression of ATP11A and ATP11C conferred rapid to re-establish PtdSer asymmetry in QKO cells the exposure of PtdSer, which was not by temperature On the other ATP8A1 and ATP11B internalized PtdSer as as ATP11A or ATP11C at 37 °C, but at 15 °C. of QKO expressing ATP8A1 cells internalized PtdSer at 15 °C, which with the localization of ATP8A1 at plasma membranes did not the of PtdSer asymmetry by ATP11A or ATP11C at 37 °C it in the or on that in to ATP11A and ATP11C, which at the plasma the ability of ATP8A1 and ATP11B to re-establish PtdSer asymmetry was on a endocytosis or recycling between and plasma membranes. We that PtdSer the of ATP11A and ATP11C with activation and of and and and K. S. Nagata S. type IV ATPases that as plasma membrane phospholipid flippases and their by and Biol. Chem. 2016; Scholar). On the other et J. T. T. et and of phospholipid flippases from 2018; that the and of human ATP11B for PtdSer and the of the and and it with that of a plasma membrane ATP8A1, ATP11B, and ATP11C with and expressed in cells with their human with Cells with and the was with an as K. S. Nagata S. The is required for a with and phospholipid flippases to the plasma Biol. Chem. 2018; 293: Scholar). An by by that ATP8A1, ATP11B, and ATP11C in the the of was in at of to which to of An by by or with and a at to and the of in a The to but not to or SM for the of ATP11B and ATP11C with of while it activated the of ATP8A1 with of that ATP11B and ATP11C to ATP8A1 that for was the P4-ATPases (ATP8A1, ATP11B, and of to whereas of ATP8A1 for was of ATP11B and ATP11C and These results with the for the human ATP11A and ATP11C K. S. Nagata S. type IV ATPases that as plasma membrane phospholipid flippases and their by and Biol. Chem. 2016; Scholar), ATP8A1 and ATP11B at as flippases for On the other to by et J. T. T. et and of phospholipid flippases from 2018; Scholar). not a for this it to the or of a of in the in J. T. T. et and of phospholipid flippases from 2018; or in this We and that the of the exposure of PtdSer in W3 and various of cells K. S. Y. Nagata S. of phospholipid for apoptotic phosphatidylserine Scholar, M. Y. M. K. A. et surface of phosphatidylserine is for myotube 2018; 9: Scholar, J. Y. E. Lee J. 2021; Scholar, M. J.F. S. A. et is required for aminophospholipid and cell fusion in mouse Cell Sci. Scholar). W3 cells W3 cells expressed of P4-ATPases (ATP8A1, 8B2, 8B4, 9A, 9B, 11A, 11B, and and are present in the plasma they not for PtdSer or other in a phospholipid K. S. Nagata S. type IV ATPases that as plasma membrane phospholipid flippases and their by and Biol. Chem. 2016; Scholar). In addition, and not with and not require for their cellular localization H. K. T. H. Umeda M. et a aminophospholipid to the in a Biol. Chem. Scholar). QKO cells ATP8A1, 11A, 11B, and to to cells and constitutively expose However, the of PtdSer exposed on the surface of QKO cells was at to of that in cells These results the of that constitutively the asymmetrical distribution of this the of PtdSer asymmetry for a in QKO cells with a QKO cells to expose PtdSer at 37 °C for the of the However, cells with asymmetrically distributed PtdSer to a of cells lost the ability to QKO to with a of that the of PtdSer from the cell surface in QKO cells with cell The plasma membranes of cells a lipid and PtdSer at the inner leaflet. In the present a mouse T cell and two independent the asymmetrical distribution of The first is mediated by two ATP11A and ATP11C, which translocate PtdSer from the outer leaflet to the inner leaflet in the plasma membrane. is rapid and to with involvement of intracellular requires ATP8A1 or ATP11B, other P4-ATPases that in endosomes. is the first and requires a temperature of 37 °C. a dynamin inhibitor blocked the ATP8A1 and ATP11B required a process as endocytosis and recycling of to PtdSer asymmetry in the plasma membrane. cells and the of with and the human and K. K. T. of phospholipid Scholar, Xu P. Phospholipid membranes and Biophys. Acta. Scholar). In the present that human at in the plasma membrane or in the asymmetrical distribution of PtdSer in the plasma membrane. These four P4-ATPases (ATP8A1, ATP11B, and are expressed in and J. T. T. et and of phospholipid flippases from 2018; Scholar, K. S. Nagata S. type IV ATPases that as plasma membrane phospholipid flippases and their by and Biol. Chem. 2016; human PtdSer asymmetry at the plasma membrane in cell to for P4-ATPases to the asymmetrical distribution of phospholipids at the plasma membrane in L. The aminophospholipid and are not required for flip the plasma membrane of Biol. Chem. 2008; Scholar, L. T. of ATPases and aminophospholipid and asymmetry in Biol. Cell. Scholar). On the other a in ATP11A and ATP11C, but not in ATP8A1 or ATP11B, in and to their expression in cell C. E. Y. Lin P. et ATP11C is for in Immunol. Scholar, B. B. B. in mouse to of the Sci. A. Scholar, Y. C. Segawa K. Y. Nagata S. of in the mouse Sci. A. Scholar, K. A. T. Y. K. C. et ATP11A with in plasma 2021; Scholar, K. M. Y. ATP11C a to membrane Biophys. These results that ATP11A and ATP11C a role in the asymmetrical distribution of PtdSer at the plasma and ATP8A1 and ATP11B as are to mouse the ATP8A1 and in to the role of flippases at and their for PtdSer asymmetry in plasma membranes. ATP8A1 and ATP11C a and are to translocate PtdSer in the lipid bilayer with a mechanism M. K. T. the of the Scholar, H. K. Segawa K. K. Y. Nagata S. et of a human plasma membrane phospholipid Biol. Chem. 2020; Scholar). The mechanisms by which ATP8A1 and ATP11B in regulate the distribution of PtdSer at the plasma membrane unclear. We two In the first ATP8A1 and ATP11B are between the and plasma membranes as in the of or M. J. Getting in Rev. Mol. Cell Biol. Scholar). These flippases transiently to the plasma membrane to flip PtdSer and to the endosomes. The other mechanism ATP8A1 and ATP11B as flippases at with recycling or lipid as and PtdSer from to the plasma membrane J. K. L. H. et at and membrane 2015; Scholar). to ATP8A1 and ATP11B between the plasma membrane and the PtdSer We and that the in various cell K. S. Y. Nagata S. of phospholipid for apoptotic phosphatidylserine Scholar, M. Y. M. K. A. et surface of phosphatidylserine is for myotube 2018; 9: Scholar, J. Y. E. Lee J. 2021; Scholar, M. J.F. S. A. et is required for aminophospholipid and cell fusion in mouse Cell Sci. Scholar). is an for (ATP8A1, and for and K. S. Nagata S. type IV ATPases that as plasma membrane phospholipid flippases and their by and Biol. Chem. 2016; Scholar, H. K. T. H. Umeda M. et a aminophospholipid to the in a Biol. Chem. Scholar). QKO cells the PtdSer flippases expressed on the plasma they did not expose PtdSer on the cell In addition to the four P4-ATPases (ATP8A1, 11A, 11B, and W3 cells and and P4-ATPases contribute to the asymmetrical distribution of PtdSer in QKO for this is that and other that not to the but is in PtdSer asymmetry in the plasma membrane. is important to that et with in mouse and demonstrated that proteins other P4-ATPases a with J. T. T. et and of phospholipid flippases from 2018; Scholar). of proteins contribute to the of PtdSer asymmetry in the plasma membrane in a We that QKO cells plasma membranes in which PtdSer was asymmetrically distributed between the two PtdSer exposed on the surface of QKO cells by with the from the surface with a of The required for the of PtdSer was to the of QKO The of the plasma membrane is for cell The scramblase activated by the is to a distribution of PtdSer between the outer and inner leaflets of the plasma membrane. The mechanisms by which QKO cells the PtdSer asymmetry cells unclear. the P4-ATPases and or other contribute to this process. it PtdSer in the cytoplasmic leaflet of to the inner leaflet of the plasma membrane a or lipid G. D'Ambrosio J.M. Dieudonne T. Copic A. Transport pathways that contribute to the cellular distribution of phosphatidylserine.Front Cell Dev Biol. 2021; 9737907Google Scholar, Phospholipid and in mammalian 2015; Scholar). The molecular mechanisms by which the plasma membrane is cell not in D.R. membrane the cell and Opin. Cell Biol. Scholar, E. J. S. A. et cells regulate their lipid and Scholar). QKO in which cell to an important role in asymmetrical PtdSer for this process. cells in W3 is a mouse T cell expressing mouse J. M. A. T. Y. et of the in Scholar), and was in with The ATP11A−/−ATP11C−/− and W3 K. Y. K. C. Y. Nagata S. Phospholipid flippases cells to by Sci. A. 2018; Scholar, K. S. Nagata S. The is required for a with and phospholipid flippases to the plasma Biol. Chem. 2018; 293: Scholar, Y. C. Segawa K. Y. Nagata S. of in the mouse Sci. A. Scholar). and QKO W3 cell from cells by a the L. Lin S. et Scholar). In cells with the the to for ATP8A1 and ATP8A1, and ATP11B, are at was by The and T. Y. T. H. T. et and expression in from T. of of The was from H. was from for ATP8A1 ATP11A ATP11B and ATP11C K. S. Nagata S. type IV ATPases that as plasma membrane phospholipid flippases and their by and Biol. Chem. 2016; Scholar). with or at the or S. Nagata S. a mammalian expression Scholar), and to W3 or proteins in was from An and from and and from and and (SM) from was from and from The of W3 cells was as K. S. Nagata S. type IV ATPases that as plasma membrane phospholipid flippases and their by and Biol. Chem. 2016; Scholar). the for human ATP8A1, ATP11B, and ATP11C was cells by with and The by or the and to W3 Cells in the of 1 and cells to with for the expression of Flippase was as K. S. Nagata S. type IV ATPases that as plasma membrane phospholipid flippases and their by and Biol. Chem. 2016; Scholar, K. S. Y. Nagata S. of phospholipid for apoptotic phosphatidylserine Scholar). In cells with in of 1 and An was with and PtdSer exposure and its to the intracellular side as K. Y. K. C. Y. Nagata S. Phospholipid flippases cells to by Sci. A. 2018; Scholar). In 1 cells with and in 1 of and a at °C for to cells with at °C for and to at of a and and by the with to of and in the or of cells by and at 15 °C or 37 °C for the in 1 of in the or of PtdSer on the surface was by with as The internalization of the was as cells on for with 1 and and in 1 of of the and of by an at 37 °C for 1 cells and in of of and Cells to a and an The and of the as K. S. Nagata S. type IV ATPases that as plasma membrane phospholipid flippases and their by and Biol. Chem. 2016; Scholar, K. S. Nagata S. The is required for a with and phospholipid flippases to the plasma Biol. Chem. 2018; 293: Scholar). In C. S. P. G. et process for and Scholar), the for or was cells with cells with and a of Cell to and proteins with and The of was as K. S. Nagata S. type IV ATPases that as plasma membrane phospholipid flippases and their by and Biol. Chem. 2016; Scholar). In of and of the was on at temperature for to the and at 37 °C for was with and in the of and the was at a the was at temperature for in and and by on a or as molecular was to a J. E. Nagata S. phospholipid in apoptotic phosphatidylserine Sci. A. 2016; Scholar). the in was with of and to at at °C for the of in the was from to and the was at for of an and and inhibitor as molecular In proteins to membranes at temperature for 15 in and and to membranes. with or by with or In proteins on membranes with S. are in this K. Y. is on a of from We M. and for and K. S. and S. K. Y. and K. Y. K. S. and Y. M. K. S. K. Y. and S. and K. S. and S. K. S. and S. was in by from the for the of to K. and to S. Kobayashi of for and to K. S. and S.

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