Fig. 1.

Conditional ER retrieval of a secreted reporter does not impact parasite fitness. (A) Schematic of reporter expression cassette installed at the attB site of chromosome 6. An mNeonGreen reporter is fused to the signal peptide of EXP2 and expressed under control of the exp2 promoter. A 3xFLAG tag and stop codon are flanked by loxP-containing introns (triangles) followed by a 3xHA-KDEL, stop codon, and 10× aptamer array (10×-Apt). Aptamer interaction with TetR-DOZI (expressed from another cassette in the plasmid) prevents translation of the reporter, which is relieved in the presence of anhydrotetracycline (aTc) to enable expression. Activation of DiCre by rapamycin treatment excises the FLAG tag, bringing into frame the HA-KDEL tag. (B) Time course of excision following rapamycin treatment detected by PCR using primers P7/32. (C) Western blot of mNG reporter 24 h posttreatment with DMSO or rapamycin. Molecular weights after signal peptide cleavage are predicted to be 30.8 kDa for mNG-3xFLAG and 30.9 kDa for mNG-3xHA-KDEL. (D) Live microscopy of DMSO- or rapamycin-treated parasites 24 h posttreatment. (E) Quantification of percent internal mNG fluorescence 24 h and 168 h posttreatment with DMSO or rapamycin. Data are pooled from two independent experiments and bar indicates mean (****P < 0.0001; unpaired t test). (F) Representative growth of asynchronous parasites (n = 2 biological replicates) treated with DMSO or rapamycin. Data are presented as means ± SD from one biological replicate (n = 3 technical replicates). All cultures were maintained in media supplemented with 500 nM aTc. (Scale bar, 5 μm.)