Figure 2. C1 and C2 do not affect expression, trafficking or maturation of PfSUB1.

(A) IFA of mature wt 3D7 parasites following treatment for 6 h with DMSO only (1% v/v) or 2.5 µM C1, probed with the anti-PfSUB1 mAb NIMP.M7 or a rabbit anti-PfAMA1 serum. The PfPKG inhibitor had no effect on localisation or expression of PfSUB1 or PfAMA1. For clarity, the anti-PfSUB1 and anti-PfAMA1 signals are shown merged in addition to a final merge with the DAPI signal. Similar results were obtained following treatment with 1.5 µM C2 (not shown). Note that treatment with the PKG inhibitors also had no effect on schizont replication as determined by counts of the average number of nuclei per schizont (data not shown, but compare DAPI staining of untreated and treated schizonts). (B) Progress curves showing cleavage in vitro of fluorogenic PfSUB1 substrate SERA5st1F-6R by extracts of wt 3D7 schizonts produced following culture for 6 h in the presence of C1 or C2, or DMSO only (control). Identities of the extracts used (tested in triplicate) are colour coded. (C) Maturation of PfSUB1 is unaffected by C1. Schizonts biosynthetically pulse-radiolabeled with [³⁵S]-methionine/cysteine were further cultured for 1 h in medium containing DMSO only (control), C1 (2.5 µM), or brefeldin A (BFA; 10 µg/ml). Schizonts were then harvested and PfSUB1 immunoprecipitated with a rabbit anti-PfSUB1 antibody and visualised by SDS PAGE and fluorography. Positions of the full-length (∼82 kDa) PfSUB1 primary translation product, as well as the p54 and p47 maturation products, are arrowed. As expected, BFA inhibited conversion of p54 to p47 as this occurs in a parasite Golgi or post-Golgi compartment [40]. C1 had no effect on PfSUB1 maturation. The intense band at ∼45 kDa in all tracks is not derived from PfSUB1, but is non-specifically bound by the antibodies [40]. (D) Processing of endogenous PfSUB1 substrates following release of a C1-mediated egress block. Schizonts cultured for 7 h in the presence of C1 were washed and incubated for a further 30 min in medium containing C1 or E64 only, then examined by Western blot. An antibody to RBC spectrin was used as a loading control. See also Figure S2 in Text S1, and Movies S1 and S2.