Proteolytic processing of AMA1 requires PfERC. In these parasite lines, the endogenous locus of PfERC was tagged with the inducible version of the ribozyme gene, glmS, or with the inactive version of the ribozyme, M9 (termed PfERC-glmS and PfERC-M9, respectively). (A) Parasites were grown in the presence of GlcN for 48 h and then incubated with compound 1 for 4 h. Compound 1 was then removed, and parasites were incubated further with E-64 for 6 h and stained with anti-AMA1 as well as the nuclear stain DAPI. Representative SIM images of these PfERC-glmS and PfERC-M9 schizonts are shown. From top to bottom, the images are anti-AMA1 (green), DAPI (blue), and fluorescence merge. Bar, 2 mm. Synchronized PfERC-M9 or PfERC-glmS schizonts for which knockdown had been initiated during the previous cycle were incubated with C1 to achieve tight synchronization. Then, C1 was washed off and the parasites were incubated with E-64 to trap merozoites that had initiated egress within the RBC membrane (12). Using SIM, we observed AMA1 localization either in micronemes or on the surface of merozoites.

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