PbCP1-GFP localises to the parasite-induced structures.

<p>(A) IEM was performed on the PbCP1-GFP expressing cell line to localise the protein at the ultrastructural level. Anti-GFP conjugated gold particles decorate the extra-parasitic structures (white arrow heads), indicating that PbCP1-GFP associates with these parasite-induced membranous compartments. (B) In contrast, no labeling of the erythrocytic endoplasmatic reticulum (ER) could be detected. Enlargement of the regions boxed in the panels A and B are shown to their right. The parasite (P), parasite plasma membrane (PM), the parasitophorous vacuole membrane (PVM and the red blood cell membrane (RBCM) are indicated.</p

The PbCP1 2TMD region mediates trafficking to the discrete punctuate structures in the RBC cytosol.

<p>(A) Alignment of PbCP1 and its paralogue Pb400 shows 77% identity. The signal peptide is highlighted in yellow, the PEXEL motif in purple and the predicted 2TMD region in red (Pb400) and blue (PbCP1), respectively. (B) Wild-type Pb400 is episomally expressed as a GFP fusion protein and localises to the RBC cytosol in live PbANKA parasites. Replacement of the predicted Pb400 2TMD and downstream region with that of PbCP1 led to the localisation of the resulting Pb400_PbCP1TMDGFP chimera to the discrete extra-parasitic structures in the RBC cytosol. In contrast, replacement of the putative PbCP1 2TMD and downstream region with that of Pb400 abolished trafficking of PbCP1_Pb400TMDGFP to the punctuate structures.</p

RxLxD containing proteins Pb021540-GFP and Pb000080-GFP are exported into the RBC cytosol.

<p>Pb021580-GFP was not expressed in two independent transgenic parasite lines. GFP fluorescence is indicated by GFP (green), parasite nuclei are stained with DAPI (blue) and merged images include the bright field. A schematic structure of all GFP chimera is depicted beside the panel: signal peptide/hydrophobic stretch (yellow), PEXEL (orange), predicted TMD region (black) and GFP (green).</p

The TMD region of IBIS1 is not sufficient to mediate trafficking to the extra-parasitic structures.

<p>The predicted PbCP1 and Pb400 2TMD and downstream region, respectively, were replaced with the single TMD (highlighted in brown in the schematic structure beside the panel) and downstream region of IBIS1 (17). The resulting fusion proteins PbCP1_IBIS1TMDGFP and Pb400_IBIS1TMDGFP, however, did not display the punctuate localisation pattern as seen in wild-type IBIS1-GFP and were only exported into the RBC cytosol.</p

All RxLxY containing proteins are exported into the RBC.

<p>Lower expression levels in the RBC cytosol and accumulation of GFP chimera at the ER and/or PV(M) could also be observed for Pb124710-GFP and Pb031630-GFP. Pb124660-GFP (PbCP1) was also found to localise to extra-parasitic structures within the RBC cytosol (white arrows). GFP fluorescence is indicated by GFP (green), parasite nuclei are stained with DAPI (blue) and merged images include the bright field. A schematic structure of all GFP chimera is depicted beside the panel: signal peptide/hydrophobic stretch (yellow), PEXEL (purple), predicted TMD region (blue) and GFP (green).</p

Investigation of the RxLxY PEXEL motif in PbCP1.

<p>Substitution of the first PEXEL residue (R) abolished export of PbCP1_R>AGFP and led to the accumulation of the respective GFP chimera within the parasite and the PV(M). Different phenotypes could be observed for the PbCP1_L>AGFP mutant. Mutation of the last PEXEL residue (Y) did not influence export of the resulting PbCP1_Y>AGFP fusion protein to the RBC cytosol and the extra-parasitic structures.</p

<i>P. berghei</i> induces membranous vesico-tubular structures in the RBC cytsosol.

<p>TEM reveals different membranous structures in reticulocytes: The endoplasmatic reticulum, which is characteristic for premature RBCs is indicated by white arrows in uninfected reticulocytes (A+B) and in infected reticulocytes (C+D). The parasite is indicated by ‘P’. Enlargement of the regions boxed in panels A, C and E are shown in panels B, D and F, respectively. Infected RBCs also contain condensed membranous structures within the RBC cytosol (E–I, white arrowheads). Electron tomography performed on an entire infected reticulocyte reveals the 3D architecture of the extra-parasitic membranous structure: G, H and I represent the orthoslices (xy, zy and xz, respectively) of the parasite-induced structure rendered in J. Scale bars: A, C, E 1 mm; B, D, F 200 nm and G–J 100 nm.</p

PbCP1-GFP is a membrane-bound protein.

<p>(A) PbCP1-GFP mainly associates with the Triton X-100 soluble (TX-100 SN) and insoluble pellet fractions (TX-100 P) as indicated by an ∼52 kDa protein band (black *) in Western blot analysis. A small fraction is also detected in the supernatant (SN) after Na₂CO₃ extraction (red *). (B) The PbCP1Δ2-GFP mutant lacking the 2TMD is soluble. Cross-reactive PfADF1 antibodies detected the soluble protein exclusively in the supernatant after hypotonic lysis at the predicted MW of ∼13 kDa. The ∼27 kDa protein band is indicative of cleaved GFP, which no longer associates with the Triton X-100 soluble or insoluble fractions.</p

The localization of DPAP3 is distinct from proteins that localize to the apical organelles.

<p><b>A.</b> IFA on RBCs infected with PfDPAP3-HAglmS parasites and fixed with acetone/methanol. DPAP3 is labeled with the anti-HA antibody. The scale bars represent 5 μm. <b>B.</b> Immuno-electron microscopy of PfDPAP3-HAglmS parasites with anti-HA antibody shows labelling for DPAP3 (as indicated by the arrowheads) in a mature schizont at the periphery of the rhoptry bulb (RB), rhoptry neck (RN) as well as at the PV/parasitophorous vacuole membrane (PVM).</p

DPAP3 expression in <i>P</i>. <i>falciparum</i> can be conditionally regulated.

<p><b>A.</b> Western blot analysis showing dose-dependent PfDPAP3-HA protein expression in two independent clones after treatment with glucosamine (GlcN) for one (upper panel) or two (lower panel) cell cycles (Cyc1 and Cyc2, respectively). EXP2 serves as the loading control. <b>B.</b> Densitometry of PfDPAP3 expression levels in PfDPAP3-HAglmS Clone 1 parasites grown in the presence or absence of glucosamine for one cycle revealed DPAP3 expression levels were knocked down by 91%. Error bars represent ± SEM from three independent experiments. <b>C.</b> IFA showing DPAP3-HA expression is significantly reduced within 24 hrs after addition of 2.5 mM GlcN when the parasites are at schizont stage. Scale bars represent 5 μm.</p