Table 1.
Dissociation constants for P. falciparum aldolase binding to cytoplasmic domains.
Table 2.
CTD peptide phosphorylation by CDPK1 or PKA in vitro.
Fig 1.
A) CDPK1-mediated phosphorylation of recombinant CTDs fused to GST in vitro. MTIP and PTRAMP were used as positive and negative control substrates, respectively. The upper panel shows the proteins stained with Coomassie Blue staining and the lower panel shows 32P labelling detected by autoradiography resulting from protein phosphorylation. B) Analysis of the phosphorylated MTRAP-GST fusion protein by electrospray mass spectrometry, with the peaks corresponding to the protein and its phosphorylated forms identified. The unlabelled peaks at 32584.4 and 32813.6 correspond to the dual phosphorylated product without the terminal methionine and with an additional H2SO4 (a known electrospray artefact), respectively. C) Circular dichroism spectrum of MTRAP CTD (black), and the forms phosphorylated at either Thr-459 (blue), Thr-467 (green) residues or both residues (red). The CD absorption coefficient calculated on a mean residue weight basis (Δεmrw) is plotted against wavelength.
Fig 2.
Binding of protein CTDs to aldolase measured by biolayer interferometry.
A) Binding assays of aldolase to MTRAP CTD, either non-phosphorylated (blue circles) or phosphorylated at Thr-459 (yellow diamonds), Thr-467 (red triangles), and both Thr residues (green squares); B), C) and D) Binding of aldolase to either non-phosphorylated (blue circles) or phosphorylated (yellow diamonds) EBA181, EBA175 and EBA140, respectively. Data were obtained using the Octet Red system (ForteBio).
Table 3.
P. falciparum aldolase binding to phosphorylated cytoplasmic domains.
Fig 3.
MTRAP competes with AMA1 and RH4 for binding to aldolase.
(A) AMA1 was loaded onto the Octet Red sensor and incubated with 70 μM aldolase in the presence of increasing concentrations of MTRAP CTD peptide. (B) RH4 was loaded on the sensor and incubated with 20 μM aldolase in the presence of increasing concentrations of MTRAP CTD. The experiments were performed in duplicate and the mean values +/- S.D. are plotted on the graph as a percentage reduction in aldolase binding to AMA1 or RH4 at increasing concentrations of MTRAP CTD.
Fig 4.
Expression of GFP-tagged MTRAP in P. falciparum asexual blood stage parasites.
A. A schematic representation of the GFP-tagging of MTRAP by single crossover homologous recombination into the mtrap locus. The modified mtrap containing sequence coding for GFP inserted between the regions coding for the transmembrane (TM) and cytoplasmic domain (CTD) was cloned as the pHH4-MTRAP-GFP plasmid and transfected into parasites. The locations of PCR primers diagnostic of integration are indicated by arrows. B. PCR analysis of genomic DNA from wild type (WT) parasites and two clones with integrated sequence, using primers F1 and R1 (lane 1), F1 and R2 (lane 2) and F1 and R3 (lane 3); the expected size of the product is 1857, 2284 and 2510 bp, respectively. C. Western blot analysis with anti-GFP antibodies of lysates of schizonts prepared from WT or clone 2. Hypotonic buffer lysate was divided into soluble (S1) or insoluble (P1) fractions and a similar fractionation was performed on lysate prepared with a buffer containing NP40 detergent (S2 and P2, respectively). D. Fluorescent images of live parasites expressing GFP-tagged MTRAP (green); nuclei (blue) were stained with Hoechst dye prior to microscopy. Merged and bright-field images are also shown.