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.

Keywords: ATPase; Lipid Bilayer; Lipid Transport; Phospholipid; Plasma Membrane.

FIGURE 1.

Flippase activities of ATP11C across the leaflets of the plasma membrane in Ba/F3 cells. Parental Ba/F3 cells (−) or Ba/F3 cells infected with empty retrovirus vector (Vector) or recombinant virus vector encoding HA-tagged ATP11C(WT) or ATP11C(E184Q) were either processed for immunoblot analysis with antibodies against HA and TfnR (as an internal control) (A) or stained with Alexa Fluor 555-conjugated wheat germ agglutinin (WGA) to visualize the plasma membrane followed by immunostaining for HA and giantin (a Golgi marker) (B). Scale bar, 10 μm. C, Ba/F3 cells were incubated with the indicated NBD-lipids at 15 °C for the indicated times (x axis). After extraction with fatty acid-free BSA, the residual fluorescence intensity associated with the cells was determined by flow cytometry. Graphs are representative of two independent experiments, and results display averages from triplicates ±S.D. D, -fold increase of NBD-lipid uptake compared with parental cells (−) is shown at the 10- (NBD-PS) or 15-min (NBD-PE, -PC, and -SM) time point from C. Graphs are representatives of three independent experiments, and results display averages from triplicates ±S.D. (*, p < 0.0001; **, p < 0.0005). Error bars represent S.D.

FIGURE 2.

Plasma membrane localization of P4-ATPases and their ATPase-deficient mutants in HeLa cells. HeLa cells were transiently co-transfected with expression vectors for FLAG-tagged CDC50A and HA-tagged ATP8B1(WT), ATP8B1(E234Q), or ATP8B1(D454G) (a BRIC mutant, see Fig. 7) (A); ATP8B2(WT) or ATP8B2(E204Q) (B); ATP11A(WT), ATP11A(E186Q), or ATP11A(D414N) (C); or ATP11C(WT), ATP11C(E184Q), or ATP11C(D412N) (D). Before fixation, cells were incubated with Alexa Fluor 488-conjugated anti-CD147 antibody for 5 min at room temperature to label the plasma membrane. The fixed cells were then incubated with anti-HA and anti-FLAG (M2) antibodies followed by Cy3-conjugated anti-rat and DyLight649-conjugated anti-mouse secondary antibodies. For protein-disulfide isomerase (PDI; an endoplasmic reticulum marker) staining, fixed cells were incubated with anti-HA, anti-FLAG, and anti-protein-disulfide isomerase antibodies followed by Cy3-conjugated anti-rat, Alexa Fluor 647-conjugated anti-rabbit, and Alexa Fluor 488-conjugated anti-mouse secondary antibodies. Insets indicate FLAG-CDC50A-expressing cells. Scale bars, 10 μm.

FIGURE 3.

Cell surface expression of P4-ATPases in HeLa stable cell lines. HeLa cell lines stably expressing HA-tagged P4-ATPase were established by plasmid transfection followed by selection in the presence of antibiotics. Two or three independent clones of cells expressing ATP8B1(WT), ATP8B2(WT), ATP11A(WT), or ATP11C(WT) or one clone expressing ATP8B1(E234Q), ATP11A(E186Q), or ATP11C(E184Q) was examined. A and C, the total expression level of the P4-ATPase in each cell line was analyzed by immunoblotting with anti-HA and anti-TfnR antibodies (as an internal control). 4% of the input of the biotinylation reaction was loaded in each lane. Arrow, ATP8B2; open arrow, ATP8B1. B and D, the cell surface level of the P4-ATPase in each cell line was analyzed after surface biotinylation. Proteins precipitated with streptavidin-agarose beads were subjected to immunoblot analysis for HA and TfnR (as an internal control). The numbers show the relative expression level of proteins, which were normalized with the level of the internal control, TfnR, and were used for normalizing the enzymatic activities shown in Fig. 4 (J and K). In A and B, open and solid arrows indicate positions of ATP8B1 and ATP8B2, respectively. In A–D, middle panels show overexposures. WT(vi), cell lines established by infection of recombinant retrovirus for each P4-ATPase.

FIGURE 4.

Flippase activities of P4-ATPases across the leaflets of the plasma membrane in HeLa cells. A–D, parental HeLa cells (−; open squares) and a cell line stably expressing ATP8B1 (clone 12; blue), ATP11A (clone 14; green), or ATP11C (clone 38; orange) were incubated with the indicated NBD-lipids at 15 °C for the indicated times (x axis). After extraction with fatty acid-free BSA, the residual fluorescence intensity associated with the cells was determined by flow cytometry. Graphs are representatives of two independent experiments, and results display averages from triplicates ±S.D. E–H, each of the clonal cell lines shown in Fig. 2 was incubated with the indicated NBD-lipids at 15 °C for 15 min (or 10 min for NBD-PS). After extraction with fatty acid-free BSA, the residual fluorescence intensity associated with the cells was determined by flow cytometry. -Fold increase of NBD-lipid uptake compared with parental HeLa cells (−) is shown. Graphs are representative of at least two independent experiments, and results display averages from triplicates ±S.D. (*, p < 0.001; **, p < 0.005). Error bars represent S.D. I, a histogram of a representative experiment displaying the differences in the fluorescence intensity among parental HeLa cells in the absence (w/o NBD-lipids; dashed line) or presence (gray area) of NBD-lipids or a cell line stably expressing ATP8B1 (clone 12; blue), ATP11A (clone 14; green), or ATP11C (clone 38; orange) in the presence of indicated NBD-lipids at 15 °C for the indicated times. J and K, the enzymatic activities of WTs and glutamate mutants were normalized with the level of biotinylated P4-ATPases shown in Fig. 3D.

FIGURE 5.

Cell surface expression and flippase activities of P4-ATPases in retrovirus-infected HeLa stable cells. HeLa cells stably expressing the indicated HA-tagged P4-ATPases were established by infection with recombinant retrovirus without clonal selection and processed for analysis of the total (A) or cell surface (B) expression level of the P4-ATPase proteins as described in the legend for Fig. 3. The middle panels of A and B show overexposures of the blots shown in the top panels. Cells expressing ATP8B1, ATP8B2, and ATP11A were incubated with NBD-PS (C), NBD-PE (D), NBD-PC (E), or NBD-SM (F) at 15 °C for 15 min (or 10 min for NBD-PS). After extraction with fatty acid-free BSA, the residual fluorescence intensity associated with the cells was determined by flow cytometry. -Fold increase of NBD-lipid uptake relative to parental HeLa cells (−) is shown. The graphs are representative of two independent experiments, and results display averages from triplicates ±S.D. (*, p < 0.001). Error bars represent S.D. G–I, the enzymatic activities were normalized with the level of biotinylated P4-ATPases shown in B.

FIGURE 6.

Flippase activities of P4-ATPases at 4 °C. HeLa cell lines stably expressing the HA-tagged ATP11A(clone 14) and ATP8B1(clone 12) were incubated with NBD-PS (A), NBD-PE (B), or NBD-PC (C) at 4 °C for the indicated times. The graph displays averages from two independent experiments ±S.D. Error bars represent S.D.

FIGURE 7.

Flippase activity of ATP8B1 across the leaflets of the plasma membrane in CHO-K1 cells. CHO-K1 cell lines stably expressing HA-tagged ATP8B1(WT) or ATP8B1(E234Q) were established by infection with recombinant retrovirus followed by selection in the presence of antibiotics. A, the total expression level of ATP8B1 was analyzed by immunoblotting with antibodies against HA and TfnR (as an internal control). 4% of the input of the biotinylation reaction was loaded in each lane. B, the cell surface expression level of ATP8B1 was determined by surface biotinylation analysis. The middle panels show overexposures. C, cells were incubated with NBD-lipids and processed for flippase assays as described in the legend for Fig. 4. -Fold increase of NBD-lipid uptake relative to parental CHO-K1 cells (−) is shown. The results display averages from triplicates ±S.D. (*, p < 0.001). Error bars represent S.D.

FIGURE 8.

Localization and flippase activity of ATP8B1 mutants associated with cholestasis. HeLa cell lines stably expressing the HA-tagged ATP8B1 mutants indicated were established by infection of recombinant retroviral vectors. A and C, the total expression level of each ATP8B1 protein was analyzed by immunoblotting with anti-HA and anti-TfnR antibodies. 4% of the input of the biotinylation reaction was loaded in each lane. B and D, cell surface expression level of each ATP8B1 protein was analyzed after surface biotinylation. The numbers in B show the relative expression level of proteins, which were normalized with the level of the internal control, TfnR. C and D, the cells were treated with 10 μm MG-132 for 12 h. E, cells expressing the indicated ATP8B1 protein were processed for immunostaining. Before fixation, cells were incubated with Alexa Fluor 488-conjugated anti-CD147 antibody for 5 min at room temperature to label the plasma membrane. The fixed cells were then incubated with anti-HA antibody followed by Cy3-conjugated anti-rat secondary antibody. Cells treated with MG-132 are indicated. Scale bar, 10 μm. Each cell line was incubated with NBD-PC (F) or NBD-PS (H) and then processed for the flippase assay as described in the legend for Fig. 4. -Fold increase of NBD-lipid uptake compared with parental HeLa cells (−) is shown. The graph is representative of two independent experiments, and results display averages from triplicates ±S.D. (*, p < 0.001). Error bars represent S.D. G, the PC flippase activities were normalized with the level of biotinylated P4-ATPases shown in B.

FIGURE 9.

Exogenous expression of ABCB4 antagonizes PC flipping mediated by ATP8B1. HeLa cell lines stably expressing HA-tagged ATP8B1, FLAG-tagged ABCB4(WT), FLAG-tagged ABCB4(K435M), ATP8B1-HA + FLAG-ABCB4(WT), or ATP8B1-HA + FLAG-ABCB4(K435M) were established by infection with recombinant retroviral vectors. A, the total expression level of each protein was analyzed by immunoblotting with anti-HA, anti-DYKDDDDK (which recognizes the FLAG epitope), and anti-β-tubulin antibodies. B, cells expressing the indicated proteins singly or doubly were processed for immunostaining with anti-HA and anti-DYKDDDDK antibodies followed by Cy3-conjugated anti-rat and DyLight649-conjugated anti-mouse secondary antibodies. Scale bar, 20 μm. C, cells were incubated with NBD-PC at 37 °C for the indicated times (x axis). After extraction with fatty acid-free BSA, the residual fluorescence intensity associated with the cells was determined by flow cytometry. Graphs display averages from three independent experiments ±S.D. (*, p < 0.005). D, -fold increase of NBD-PC uptake compared with parental HeLa cells (−) at the 5-min time point of C is shown. Graphs display averages from three independent experiments ±S.D. (*, p < 0.001 versus −; #, p < 0.001 versus ATP8B1). Error bars represent S.D.