HT-dependent protein sorting into clefts was not influenced by deletion of putative substrate binding domain in PFE0055c. (A) Deduced amino acid sequence of PFE0055c with an N-terminal ER-type signal sequence (brown), HT motif (bold), followed by sequences containing DnaJ region (orange) with the characteristic HPD (green) motif. Further downstream region include a glycine/phenylalanine-rich stretch and C-terminal substrate-binding domain (underlined). Sequences in blue indicate region deleted in parasite line generated by single crossover recombination as shown in panels D-F. (B) Western blot, using anti-PFE0055c antibodies, detecting the presence of a 42-kDa protein in infected erythrocyte (arrowhead, lane 2) but not uninfected erythrocyte (lane 1). (C) Single optical section of a trophozoite-infected erythrocyte fixed and probed with peptide antibodies to PFE0055c (green) and the cleft protein SBP1 (red). Arrow in merge image shows proximal location of PFE0055c to clefts. (D) Strategy for deletion in the C-terminal substrate-binding region of PFE0055c by single crossover recombination with the chromosomal copy of pfe0055c. P falciparum parasites were transfected with plasmids containing an in-frame fusion of the neomycin resistance gene (npt, green) to an internal fragment of pfe0055c (orange) without sequences encoding for C-terminal substrate-binding domain. Only chromosomal integration of the vector by single crossover with the native pfe0055c (pink) drives npt expression under the control of pfe0055c promoter (Ppfe0055c), thus conferring resistance of antibiotic G418. (E) PCR-based detection for the loss of chromosomal copy of pfe0055c. Positions for primer pairs used for amplification analyses of single crossover recombination are highlighted in panel D. (F) Western blot analysis showing the detection of PFE0055c-NPT fusion protein of 45-kDa in transfected line (arrowhead, lane 1) but in not parental line (lane 2) using antibodies to NPT (top). Parasite protein PfFKBP serves as a loading control (bottom). (G) 0° projection of live infected erythrocyte expressing HT-GFP^membmyc in 3D7 strain with chromosomal deletion of pfe0055c viewed under GFP optics and merged with bright field. Arrow indicates that the export of HT-GFP^membmyc to cleft is not altered by truncation in PFE0055c. Parasite nucleus (p) in all cases is stained with Hoechst 33342 (blue). Bar represents 2 μm.