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. 2003 Apr;14(4):1529–1544. doi: 10.1091/mbc.E02-04-0240

Figure 5.

Figure 5

ETRAMPs are located in the PVM with their C termini facing the RBC cytosol. (A–C) Distribution of ETRAMP4 around the parasite periphery in fixed cells. Top two panels of A depict ETRAMP4 staining (FITC) and nuclear staining (DAPI) of a schizont stage parasite; spots of more intense staining are indicated by small arrows. Bottom two panels of A show ETRAMP4 (FITC) and nuclear staining (DAPI) of a late trophozoite stage parasite. The single patch of strong staining is indicated by a large arrow. (B) Extensions from the parasite body are labeled by anti-ETRAMP4 serum, indicating localization in blebbing vesicles or the TVM. The top panel shows ETRAMP4 (FITC) detected in a young trophozoite; below the nucleus is shown (DAPI). The extension is indicated by an arrow. (C) Costaining with a rabbit anti-aldolase serum verifies that the periphery of the parasite is decorated by ETRAMP4 antiserum; the four panels represent the same section depicting a young trophozoite. From top to bottom, ETRAMP4 (FITC), aldolase (Cy3), nucleus (DAPI), and aldolase and ETRAMP4 merged. Bars, 5 μm. (D) Representation of the selective permeabilization scheme: 1) an untreated IRBC (P, parasite; EPM, erythrocyte plasma membrane; PPM, parasite plasma membrane); 2) a cell permeabilized with SLO, enabling access of antibodies through the perforated RBC membrane, resulting in a fluorescence signal if the corresponding antigen is located in the PVM facing the RBC cytosol; 3) a cell permeabilized with saponin, which allows to detect antigens facing the outer side of the PPM or both sides of the PVM. (E) Formaldehyde (1%)-fixed and saponin-permeabilized ring stage parasites showed a signal for ETRAMP10.1 (green, FITC, top), but not for aldolase (red, Cy3, bottom). Both panels show the same section. (F) Aldolase is strongly detected under similar fixation conditions and treatment with Triton X-100, indicating that aldolase is well detectable in ring stages. (G) Western analysis with extracts of saponin-released parasites that were either treated with (+) or without (−) trypsin before protein extraction, shows that the ETRAMP4 (left) and ETRAMP10.1 (right) C termini were digested, but not the cytoplasmic parasite enzyme GAPDH. Molecular mass (in kilodaltons) is indicated on the right.