Fig. 4. The BFA-sensitive step in apicoplast protein import occurs before transit peptide cleavage. Intracellular T. gondii expressing ACP-GFP were labeled with 35S amino acids in the presence of 1 µg/ml BFA for 1 hour (pulse) then chased in control medium for various times (-BFA, hours). Alternatively (three right lanes), the samples were chased without BFA for the indicated times (-BFA, hours) and then BFA was added to 1 µg/ml and the incubation continued for an additional period (+BFA, hours). In these BFA add-back experiments, the total incubation time was 4 hours. GFP was immunoprecipitated and resolved by SDS-PAGE; t-GFP marks the species containing the transit peptide, m-GFP marks the mature form in which lacks the transit peptide. The amount of precursor and mature protein was quantified by phosphorimaging and the percentage of protein in the mature form is indicated below each lane.