PfEMP1 cannot be loaded onto Maurer clefts in CS2ΔSBP1 transgenic cells. (A) Immunofluorescence study to assess trafficking of PfEMP1 in erythrocytes infected with parental CS2 and CS2ΔSBP1. Parental cells probed with anti-ATS and anti-SBP1 antibodies showed typical PfEMP1 localization in Maurer clefts in the erythrocyte (first row of panel), whereas PfEMP1 was accumulated in the parasite surrounding parasitophorous vacuole in CS2ΔSBP1-infected cells (second row of panel). Arrowheads show colocalization. DAPI was used to stain the nuclear DNA in all panels. (B) Colocalization with anti-ATS and anti-MAHRP antibodies to Maurer clefts is shown in parental CS2-infected erythrocytes (first row of panel, arrowheads), whereas in the CS2ΔSBP1-infected cells there is no overlap of the 2 proteins in Maurer clefts. (C) Schematic representation of the plasmid to express a truncated PfEMP1 fused to YFP. The PfEMP1-YFP gene is flanked by an hsp86 promoter and a PbDT 3′ terminator. Blasticidin-S deaminase is used as a selection cassette. (D) Expression of PfEMP1-YFP in CS2 wild-type– and CS2ΔSBP1-infected erythrocytes are shown by anti-GFP labeling. In erythrocytes infected with parental CS2 (first row, top panel), the PfEMP1-YFP fusion is localized in the parasite, but the majority of the protein is transported into the erythrocyte cytosol and shows a characteristic Maurer clefts staining. In the CS2ΔSBP1-infected erythrocytes (first row, bottom panel), however, the fusion protein can only be detected within the confines of the parasitophorous vacuole. Both cell lines were also probed with anti-ATS antibody to check for possible trafficking defects caused by the YFP chimera (bottom panels).