ATP2, The essential P4-ATPase of malaria parasites, catalyzes lipid-stimulated ATP hydrolysis in complex with a Cdc50 β-subunit

Figures & data

Figure 1. Analysis of PcATP2 expression in S. cerevisiae membranes, alone or co-expressed with each of the three putative PcCdc50 subunits. 5 µg of total protein were loaded on each lane. Top panels, western blots revealed with the probe against the BAD tag. Bottom panels, western blots revealed with the HisProbe^TM to detect the 10xHis tag. Gel bands corresponding to PcATP2 and the P. chabaudi Cdc50 subunits are indicated by arrows. Lane 1: single expression of PcATP2-BAD. Lane 2: single expression of BAD-PcATP2. Lane 3: co-expression of PcATP2-BAD and PcCdc50B-His. Lane 4: co-expression of BAD-PcATP2 and PcCdc50B-His. Lane 5: co-expression of PcATP2-BAD and PcCdc50C-His. Lane 6: co-expression of BAD-PcATP2 and PcCdc50C-His. Lane 7: co-expression of PcATP2-BAD and PcCdc50A-His. Lane 8: co-expression of BAD-PcATP2 and PcCdc50A-His. Lane 9: empty Vector, membranes expressing no Plasmodium proteins (negative control). The theoretical molecular weight mass of PcATP2-BAD or BAD-PcATP2 is 180 kDa. The theoretical molecular weight masses of PcCdc50B-His, PcCdc50C-His and PcCdc50A-His are, respectively, 45, 61 and 51 kDa. Lanes 1–6 were run in a 8% acrylamide gel. Lanes 7–9 were run in a gel containing a gradient of acrylamide between 4–15%. Naturally occurring biotinylated S. cerevisiae proteins are indicated in lane 9: Acc1p, acetyl-CoA carboxylase; Dur 1/2p, urea carboxylase; Pyc 1/2p, pyruvate carboxylase isoforms 1 and 2, and Arc 1p, complex acyl-RNA[40].

Figure 2. Analysis of membrane fractions of S. cerevisiae co-expressing PcATP2 with either PcCdc50B or PcCdc50A. Top panels, western blots revealed with the probe against the BAD. Bottom panels, western blots revealed with the HisProbe^TM to detect the 10xHis tag. (A) membrane fraction co-expressing BAD-PcATP2 and PcCdc50B-His. (B) membrane fraction co-expressing BAD-PcATP2 and PcCdc50A-His. The bands corresponding to BAD-PcATP2, PcCdc50B-His and PcCdc50A-His are indicated. P2 and S2 are, respectively, the membrane pellet and the supernatant obtained after centrifugation at 20,000 × g. P3 and S3 are, respectively, the membrane pellet and the supernatant obtained after centrifugation at 125,000 × g. Each lane was loaded with the corresponding membrane fraction or supernatant containing 1 µg of total protein. Biotinylated S. cerevisiae proteins are also indicated: Acc1p: acetyl-CoA carboxylase, Dur1/2p: urea carboxylase, and Pyc1/2p: pyruvate carboxylase isoforms 1 and 2[40].

Figure 3. Deglycosylation assays of PcCdc50B and PcCdc50A co-expressed with PcATP2. Membranes co-expressing PcATP2/PcCdc50B, PcATP2/PcCdc50A or Drs2p/Cdc50p were enzymatically deglycosylated with PNGase F or EndoH, as indicated. 10 µg total protein were loaded in the experiments with PcATP2/PcCdc50B and PcATP2/PcCdc50A, whereas 5 µg of total protein were loaded in the control experiment with Drs2p/Cdc50p. The western blots were revealed with the HisProbe^TM to detect the 10xHis tag.

Figure 4. Co-immunoprecipitation of PcATP2-GFP-BAD with PcCdc50B-His or PcCdc50A-His. Lanes 1–4 correspond to the different steps of the assay with membranes co-expressing PcATP2-GFP-BAD and PcCdc50B-His (A) or PcCdc50A-His (B): (1) input, detergent-solubilized membranes, (2) flow-through or non-bound protein fraction, (3) washings, and (4) elution. Lanes 5–8 are equivalent to lanes 1–4 but with membranes co-expressing PcATP2-BAD and either, PcCdc50B-His (A) or PcCdc50A-His (B). The blots on the top panel were revealed with an antibody against the GFP to detect PcATP2-GFP-BAD, and the bottom one with the HisProbe^TM to detect PcCdc50B-His and PcCdc50A-His (labelled with an asterisk).

Figure 5. Fluorescence-detection Size Exclusion Chromatography of detergent solubilized PcATP2 and PcCdc50B. Membranes containing 5 mg/ml of total protein concentration were solubilized with 1% (w/v) DDM, 0.2% (w/v) CHS, and the supernatant after ultracentrifugation was loaded into a Superose 6 10/300 GL gel-filtration column equilibrated with 20 mM Tris-HCl pH 7.8, 150 mM NaCl, 10% (v/v) Glycerol, 0.1 mg/mL DDM, 0.02 mg/mL CHS, and connected to a fluorescence detector. Normalized FSEC profiles of single-expression of PcATP2-GFP-BAD (dotted lines) and co-expression of PcATP2-GFP-BAD with PcCdc50B-His (solid line). A profile of membranes expressing no protein is shown (in grey shadow). Normalized FSEC profile of single-expression PcCdc50B-GFP-His (dashed line). The presence of free eGFP is indicated. On top of the figure, analysis of PcCdc50B-His co-elution with PcATP2-GFP-BAD (solid line chromatogram). Collected fractions of 200 µl between 10.4–12 ml of the PcATP2-GFP-BAD elution were analyzed by western blot using the HisProbe^TM to detect the presence of PcCdc50B-His.

Figure 6. Lipid-stimulated ATPase activity of PcATP2/PcCdc50B complex. The apparent ATPase activity of the PcATP2/PcCdc50B complex was measured by spectrophotometric quantification of the released inorganic phosphate in the absence (no lipid) and in the presence of the indicated lipids. POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine, and POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine. As indicated, ATPase assays were also performed in the presence (+) or in the absence (-) of phosphatidylinositol-4-phosphate (PI4P). Gray bars and white bars represent, respectively, the activity of the wild-type (WT) and the functionally-impaired D596N mutant (DN) at each experimental condition. The reaction was performed at 37°C during 25 min, and initiated by the addition of 1 mM ATP-Mg²⁺. The bars display the average value of n = 3 or n = 4 experimental datapoints, and the indicated error is the standard deviation. The upper panel displays the SDS-PAGE of the acid-eluted content of the immobilized wild-type (WT) or D596N mutant (DN) agarose beads used for this experiments. (A) Coomassie-stained, (B) western blot using the antibody against the GFP to detect PcATP2-GFP, and (C) western blot using the HisProbe^TM to detect the 10xHis tag fused to PcCdc50B .* P < 0.05 (paired t-test). Note that the DN mutant of PcATP2 runs slightly faster because it lacks the BAD domain.

Figure 7. Alignment of the cryo-EM structure of Drs2p and a 3D structural model of PcATP2. The 3D structural model of PcATP2 (cartoon representation) was aligned with the cryo-EM structure of the autoinhibited conformation of Drs2p (PDB ID 6roh, mesh representation [23]). Protein domains of PcATP2 in the cytoplasmic region between TMs 2 and 3 that are absent in Drs2p are depicted in blue. Protein domains of PcATP2 in the cytoplasmic region between TMs 4 and 5 that are absent in Drs2p are depicted in magenta. (A) Lateral perspective of the molecule. (B) Cytoplasmic view after 90° rotation of A through the x axis. The grey shadow square in A represents the putative position of the surrounding membrane.

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