Fig 1: ATPase activity and phospholipid flippase activity of ATP11-CDC50A complexes. (A) ATPase activity of ATP11A, ATP11B and ATP11C in the presence of 90% DOPC and either 10% DOPS (PS) or 10% NBD-PS. Activity is normalized to 100% ATPase activity in 10% DOPS. (B) NBD-phospholipid flippase activity. ATP11A, ATP11B and ATP11C associated with CDC50A were purified and reconstituted into DOPC liposomes containing 2.5% NBD-PS, 2.5% NBD-PE or 2.5% NBD-PS plus 30% DOPS. The activity was normalized to samples containing NBD-PS. Addition of 30% unlabeled DOPS effectively competed with NBD-PS to reduce the NBD-PS flipping. Data is the average of 3 experiments ± SD.
Fig 2: Purification and ATPase activity of WT ATP11 and mutants with the E?Q mutation in the activator domain. ATP11A, ATP11B, and ATP11C containing a 1D4 tag were co-expressed with CDC50A in HEK293 cells and purified by immunoaffinity chromatography on a Rho1D4-Sepharose matrix. SDS gels and Western blots of the HEK293 cell extracts (Input) and 1D4 peptide eluted WT or E?Q mutants for ATP11A (A), ATP11B (B) and ATP11C (C). SDS gels were stained with Coomassie Blue (CB) and Western blots were labeled with the Rho 1D4 antibody (ATP11) or Cdc50-7F4 antibody (CDC50A). ATPase activity for WT and E?Q mutants in the presence of brain polar lipid is shown for ATP11A (D), ATP11B (E), and ATP11C (F). Data shown as the mean ± SD for n = 3.
Fig 3: The effect of PS, PE and ATP concentration on the ATPase activity of purified ATP11A-CDC50A, ATP11B-CDC50A, and ATP11C- CDC50A complexes isolated from transfected HEK293 cells. The purified proteins were reconstituted with DOPC as the base phospholipid and varying concentrations of DOPS (A) or DOPE (B) and the ATPase assays were carried out with 5 mM ATP. (C) The effect of ATP concentration on the ATPase activity for ATP11A, ATP11B and ATP11C complexes reconstituted with 70% DOPC and 30% DOPS. Measurements were performed in triplicate and results were averaged. Error bars represent ± SD. Curves were fitted with a Michaelis-Menten equation using the parameters summarized in Table 2.
Fig 4: Effect of specific phospholipids, nucleotides and inhibitors on the ATPase activity of ATP11 proteins. (A) ATP11A, ATP11B and ATP11C were co-expressed with CDC50A in HEK293 cells, purified by immunoaffinity chromatography, and reconstituted with 100% DOPC (PC) or 90% DOPC and 10% DOPS (PS), DOPE (PE), DOPG (PG), DOPI (PI), sphingomyelin (SM), DOPA (PA) or cholesterol (Chol) for determination of their ATPase activity. (B) The ATPase activities were determined for 0.5 mM ATP or 0.5 mM non-hydrolyzable ATP analogue AMP-PNP. For inhibition studies, the proteoliposomes were pre-incubated with 100 µM NaF, 100 µM vanadate, 5 mM N-ethylmaleimide (NEM), or 1 mM ouabain prior to the addition of 0.5 mM ATP for ATPase measurements. The ATPase activity was normalized to the activity of proteoliposomes in the presence of ATP, but in the absence of inhibitors. Data shown as the mean ± SD for n = 3.
Fig 5: SDS gels and Western blots of P4-ATPase complexes from brain, kidney, testes, and liver. (A) Proteins from membrane fractions of mouse brain (B), kidney (K), testes (T), and liver (L) were resolved on a SDS gel and either stained with Coomassie Blue (CB) or transferred to Immobilon membranes and labeled for CDC50A with the Cdc50-7F4 monoclonal antibody. Approximately, 30 µg of protein was applied to each lane. (B) Western blots of P4-ATPases in mouse tissues isolated by immunoaffinity chromatography. Membranes from brain (B), kidney (K), testes (T), and liver (L) were solubilized in CHAPS detergent and P4-ATPase complexes were isolated by immunoaffinity chromatography on a Cdc50-7F4 immunoaffinity matrix. Western blots were labeled with antibodies to ATP8A1, ATP11C, ATP11A and CDC50A.
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