Fig. 3

Transport of PfRab5b^Q94L-YFP-DD to the cytoplasmic face of infected red blood cells on the TVN. a The selective permeabilization scheme. Saponin permeabilizes RBC plasma membrane (RPM) and PVM, which allows the detection of proteins localized to the outside of the parasite plasma membrane (PPM) or both sides of the PVM. Triton X-100 permeabilizes RPM, PVM, and PPM, allowing staining of the iRBCs, PV, and parasite cytosol with antibody. b Cells expressing PfRab5b^Q94L-YFP-DD (green) were subjected to an antibody accessibility assay with anti-GFP antibody (red) prior to permeabilization with saponin (upper panel, SAP) or Triton X-100 (lower panel, Triton). After the saponin treatment, anti-GFP antibody labelled PfRab5b^Q94L-YFP-DD secreted to the TVN (arrowhead), whereas PfRab5b^Q94L-YFP-DD in the parasite cytosol was not labelled. Permeabilization with Triton X-100 allowed labelling of both the parasite cytosolic and TVN-localized PfRab5b^Q94L-YFP-DD with anti-GFP antibody. c Schematic of the Shld1 washout assay and the protease accessibility assay. After removal of Shld1, cytosolic DD-tagged proteins are degraded (red cross and blue circle) by the parasite proteasome (yellow cylinder). DD-tagged proteins, inside of intracellular organelles or transported to the outside of the parasite (blue circular), are resistant to the proteasomal degradation. Since streptolysin O (SLO) permeabilizes RPM but not PVM, DD-tagged proteins in the RBC cytoplasm were selectively degraded by extracellular proteinase K (ProK) after permeabilization with SLO (black cross and blue circle). d Sub-cellular localization of PfRab5b^Q94L-YFP-DD (green) after Shld1 stabilization for 24 h (upper panel, Shld1) and 2 h after Shld1 washout (lower panel, washout). After the removal of Shld1, the punctate signal of PfRab5b^Q94L-YFP-DD localized to the TR-ceramide (red)-labelled parasite periphery. Bars 5 µm. e Parasites expressing PfRab5b^Q94L-YFP-DD were subjected to Shld1 washout after stabilization (Fig. 3d), and then permeabilized with SLO before treatment with proteinase K. Anti-PfEXP2 and anti-TPx-1 antibodies were used as a control which is not processed with proteinase K, and a loading control for immunoblot analysis, respectively. Representative image from three independent experiments is shown. f The intensity of each band in Fig. 3e were quantitated and the intensity of the band with ProK (+) was divided by that of ProK(−) band. Significance was evaluated by the Student’s t test. Bars standard deviation (n = 3)