ABCMdb

ABCB4 p.Lys435Met

Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (91%), I: D (95%), L: D (95%), M: D (91%), N: D (95%), P: D (95%), Q: D (91%), R: D (91%), S: D (91%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Complementary functions of the flippase ATP8B1 and... Gastroenterology. 2011 Nov;141(5):1927-37.e1-4. Epub 2011 Aug 4. Groen A, Romero MR, Kunne C, Hoosdally SJ, Dixon PH, Wooding C, Williamson C, Seppen J, Van den Oever K, Mok KS, Paulusma CC, Linton KJ, Oude Elferink RP
Complementary functions of the flippase ATP8B1 and the floppase ABCB4 in maintaining canalicular membrane integrity.
Gastroenterology. 2011 Nov;141(5):1927-37.e1-4. Epub 2011 Aug 4., [PMID:21820390]

Abstract [show]
BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis can be caused by mutations in ABCB4 or ATP8B1; each encodes a protein that translocates phospholipids, but in opposite directions. ABCB4 flops phosphatidylcholine from the inner to the outer leaflet, where it is extracted by bile salts. ATP8B1, in complex with the accessory protein CDC50A, flips phosphatidylserine in the reverse direction. Abcb4(-/-) mice lack biliary secretion of phosphatidylcholine, whereas Atp8b1-deficient mice have increased excretion of phosphatidylserine into bile. Each system is thought to have a role protecting the canalicular membrane from bile salts. METHODS: To investigate the relationship between the mechanisms of ABCB4 and ATP8B1, we expressed the transporters separately and together in cultured cells and studied viability and phospholipid transport. We also created mice with disruptions in ABCB4 and ATP8B1 (double knockouts) and studied bile formation and hepatic damage in mice fed bile salts. RESULTS: Overexpression of ABCB4 was toxic to HEK293T cells; the toxicity was counteracted by coexpression of the ATP8B1-CDC50A complex. In Atp8b1-deficient mice, bile salts induced extraction of phosphatidylserine and ectoenzymes from the canalicular membrane; this process was not observed in the double-knockout mice. CONCLUSIONS: ATP8B1 is required for hepatocyte function, particularly in the presence of ABCB4. This is most likely because the phosphatidylserine flippase complex of ATP8B1-CDC50A counteracts the destabilization of the membrane that occurs when ABCB4 flops phosphatidylcholine. Lipid asymmetry is therefore important for the integrity of the canalicular membrane; ABCB4 and ATP8B1 cooperate to protect hepatocytes from bile salts.

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23 Materials and Methods Plasmids Generation of the pcDNA3.1ϩ vector (Invitrogen, Carlsbad, CA) expressing human ABCB4 and its K435M derivative (replacing the conserved lysine within the Walker A motif of the first nucleotide binding domain to inhibit adenosine triphosphate binding) was described previously.11 The E558Q derivative (replacing a conserved glutamate in the Walker B motif with a glutamine to prevent adenosine triphosphate hydrolysis) was derived from the wild-type ABCB4 by site-directed mutagenesis using the mutagenic oligonucleotide 5=- GATCCTTCTGCTG- GATCAGGCGACGTCAGCATTGGAC-3= and its reverse complement to prime synthesis of the mutant vector using Quikchange II (Agilent Technologies, Santa Clara, CA). Login to comment
65 We also noted that inactive derivatives of ABCB4 carrying a point mutation in either the Walker A (K435M) or Walker B (E558Q) motifs of the first nucleotide binding domain, expressed to much higher levels than wild-type protein, suggesting a negative selective pressure against ABCB4 function (Figure 1A). Login to comment
67 In contrast, the nonfunctional K435M mutant was easily detectable on the plasma membrane and there was less evidence of nuclear breakdown (Supplementary Figure 3). Login to comment
68 Transient expression of wild-type ABCB4, but not the K435M mutant, was also associated with high levels of LDH activity in the culture media indicative of leakage of the cytoplasmic LDH from damaged cells (Figure 1D). Login to comment
88 (A) Western analysis of mutant and wild-type ABCB4 in HEK293T whole cell lysates. Lane 1, mock-transfected cells; lane 2, wild-type ABCB4 alone; lane 3, ABCB4 mutant K435M alone; lane 4, ABCB4 mutant E558Q alone. Login to comment
66 We also noted that inactive derivatives of ABCB4 carrying a point mutation in either the Walker A (K435M) or Walker B (E558Q) motifs of the first nucleotide binding domain, expressed to much higher levels than wild-type protein, suggesting a negative selective pressure against ABCB4 function (Figure 1A). Login to comment
69 Transient expression of wild-type ABCB4, but not the K435M mutant, was also associated with high levels of LDH activity in the culture media indicative of leakage of the cytoplasmic LDH from damaged cells (Figure 1D). Login to comment
89 (A) Western analysis of mutant and wild-type ABCB4 in HEK293T whole cell lysates. Lane 1, mock-transfected cells; lane 2, wild-type ABCB4 alone; lane 3, ABCB4 mutant K435M alone; lane 4, ABCB4 mutant E558Q alone. Login to comment

[hide] Bile salt-dependent efflux of cellular phospholipi... Hepatology. 2007 Jul;46(1):188-99. Morita SY, Kobayashi A, Takanezawa Y, Kioka N, Handa T, Arai H, Matsuo M, Ueda K
Bile salt-dependent efflux of cellular phospholipids mediated by ATP binding cassette protein B4.
Hepatology. 2007 Jul;46(1):188-99., [PMID:17523162]

Abstract [show]
Human ABCB4 (multidrug resistance [MDR]3 P-glycoprotein) is expressed in the canalicular membrane of the hepatocyte. ABCB4 has been shown to be required for phosphatidylcholine (PC) secretion into the bile and to translocate PC across the plasma membrane. To further investigate the function of ABCB4, we established a cell line stably expressing ABCB4 (human embryonic kidney [HEK]/ABCB4). The efflux of phospholipids from HEK/ABCB4 cells was remarkably increased by the addition of taurocholate. In addition, the cholesterol efflux from HEK/ABCB4 cells was also enhanced in the presence of taurocholate. Light scattering measurements suggested that the taurocholate monomer plays an important role in ABCB4-mediated lipid secretion. On the other hand, the efflux of phospholipids and cholesterol was not mediated by ABCB1 (MDR1) even in the presence of taurocholate. Taurocholate promoted the efflux of phospholipids and cholesterol from HEK/ABCB4 cells more efficiently than glycocholate and cholate. ABCB4-K435M and ABCB4-K1075M, Walker A lysine mutants, did not mediate the phospholipid and cholesterol efflux in the presence of taurocholate, suggesting that ATP hydrolysis is essential for the efflux. Verapamil completely inhibited the taurocholate-dependent efflux of phospholipids and cholesterol from HEK/ABCB4 cells. Mass spectrometry revealed that, in the presence of taurocholate, HEK/ABCB4 cells preferentially secreted PC compared to sphingomyelin. PC vesicles induced cholesterol diffusion from cell membrane, but did not accept cholesterol from ABCB4. CONCLUSION: ABCB4 mediates the efflux of phospholipids into the canalicular lumen in the presence of bile salts, and plays a crucial role in bile formation and lipid homeostasis.

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8 ABCB4-K435M and ABCB4-K1075M, Walker A lysine mutants, did not mediate the phospholipid and cholesterol efflux in the presence of taurocholate, suggesting that ATP hydrolysis is essential for the efflux. Login to comment
42 ABCB4 WalkerA lysine mutants (ABCB4-K435M and -K1075M) were prepared with the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) as described by the manufacturer. Login to comment
43 The human ABCB4 gene and its mutant genes were inserted into the HindIII-XbaI site of pcDNA3.1/Hygro(ϩ) (Invitrogen, Carlsbad, CA) to make an expression vector for pcDNA3.1/Hygro(ϩ)/ABCB4, pcDNA3.1/Hygro(ϩ)/ABCB4-K435M and pcDNA3.1/ Hygro(ϩ)/ABCB4-K1075M.ThehumanABCB1genewas fused to His tag in the pCAGGSP vector (pCAGGSP/ MDR1-His). Login to comment
47 HEK293 cells were transfected with pcDNA3.1/ Hygro(ϩ)/ABCB4, pcDNA3.1/Hygro(ϩ)/ABCB4-K435M, or pcDNA3.1/Hygro(ϩ)/ABCB4-K1075M using LipofectAMINE (Invitrogen) according to the manufacturer`s instructions. Login to comment
188 To study the involvement of ATP hydrolysis in ABCB4-mediated secretion of phospholipids and cholesterol in the presence of NaTC, we established HEK293 cells stably expressing ABCB4-K435M (HEK/ABCB4-K435M) and ABCB4-K1075M (HEK/ABCB4-K1075M), in which the Walker A lysine in either nucleotide binding domain was substituted by methionine. Login to comment
189 NaTC-dependent efflux of phospholipids and cholesterol was not observed with HEK/ABCB4-K435M or HEK/ABCB4-K1075M cells (Fig. 6F,G), although the expression levels, glycosylation, and the surface expression of mutant ABCB4 were comparable to those of wild-type ABCB4 (Fig. 6A-E). Login to comment
192 (A) Cell lysates (32.0 ␮g of proteins) from HEK/ABCB4-WT cells (lane 1), HEK/ABCB4-K435M cells (lane 2) and HEK/ ABCB4-K1075M cells (lane 3) were separated by 7% polyacrylamide gel electrophoresis. Login to comment
193 ABCB4-WT, -K435M and -K1075M were detected with mouse monoclonal antibody C219. Login to comment
194 (B-E) HEK293 cells (B), HEK/ABCB4-WT cells (C), HEK/ABCB4-K435M cells (D), and HEK/ ABCB4-K1075M cells (E) were fixed in 70% ethanol and reacted with monoclonal antibody C219 and Alexa488-conjugated anti-mouse IgG. Login to comment
196 (F-G) HEK/ ABCB4 cells, HEK/ABCB4-K435M cells and HEK/ABCB4-K1075M cells were incubated for 24 hours at 37°C with 0.02% BSA in the absence (control, open bars) or presence (filled bars) of 0.5 mM NaTC. Bars represent the mean Ϯ SE of 3 measurements. Login to comment
217 Because the excretion of phospholipids and cholesterol was not observed with ABCB4-K435M or ABCB4-K1075M, in which the Walker A lysine in either nucleotide binding domain was substituted by methionine, ABCB4 mediates lipid efflux in an ATP-dependent manner, like other ABC transporters. Login to comment

[hide] Phospholipid flippase activities and substrate spe... J Biol Chem. 2014 Nov 28;289(48):33543-56. doi: 10.1074/jbc.M114.593012. Epub 2014 Oct 14. Takatsu H, Tanaka G, Segawa K, Suzuki J, Nagata S, Nakayama K, Shin HW
Phospholipid flippase activities and substrate specificities of human type IV P-type ATPases localized to the plasma membrane.
J Biol Chem. 2014 Nov 28;289(48):33543-56. doi: 10.1074/jbc.M114.593012. Epub 2014 Oct 14., [PMID:25315773]

Abstract [show]
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.

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207 To address this hypothesis, we established cell lines stably expressing FLAG-tagged ABCB4(WT) or ABCB4(K435M), which is nonfunctional due to a mutation in the Walker A motif in the nucleotide-binding domain (41), with or without ATP8B1-HA. Login to comment
208 In both the singly and doubly expressing cell lines, comparable expression levels of ABCB4(WT) and ABCB4(K435M) were confirmed by immunoblotting, and the plasma membrane localization of all proteins was visualized by immunostaining with anti-FLAG antibody (Fig. 9, A and B). Login to comment
210 ABCB4(WT) and ABCB4- (K435M) colocalized with ATP8B1 at the plasma membrane (Fig. 8B). Login to comment
211 Importantly, coexpression of ATP8B1 with ABCB4- (WT), but not ABCB4(K435M), significantly suppressed PC incorporation at all time points examined relative to that in cells expressing only ATP8B1 (Fig. 9, C and D). Login to comment
214 Although we did not detect a decrease in PC incorporation in cells expressing ABCB4 alone at 5 min, we did detect a small but significant decrease at 30 min relative to parental cells or cells expressing ABCB4(K435M) (Fig. 9C). Login to comment
219 HeLa cell lines stably expressing HA-tagged ATP8B1, FLAG-tagged ABCB4(WT), FLAG-tagged ABCB4(K435M), ATP8B1-HA af9; FLAG-ABCB4(WT), or ATP8B1-HA af9; FLAG-ABCB4(K435M) were established by infection with recombinant retroviral vectors. Login to comment