Figure 6. Proteolytic processing of RhopH3 compromises post-translational knockdown and accounts for the preserved PSAC activity in R3-DDD parasites.
(A) Ribbon schematic showing RhopH3 (blue), the engineered C-terminal 3HA-DDD tag (orange), and the sites recognized by the indicated antibodies. The ribbon and the red bar indicating the epitope used to produce anti-RhopH3 antibody are drawn to scale. The data in this figure implicate proteolytic processing at a site marked with a black arrow. (B) Immunoblot of schizont-, ring-, and trophozoite-infected cell lysates from R3-DDD and of spent culture medium collected after invasion of synchronous cultures, probed with anti-HA. A red arrow points to the soluble cleavage product detected in schizonts. CM, culture medium not exposed to parasites. S and P, soluble and ultracentrifugation pellet fractions. (C) Immunoblot of a schizont-infected R3-DDD membrane fraction, probed with anti-RhopH3. A primary 100 kDa processed band and a less intense 130 kDa unprocessed band are apparent. (D) IFA of R3-DDD probed with the indicated antibodies at each parasite stage. Note that both antibodies detect protein in schizont rhoptries, but that only the anti-RhopH antibody detects export to the host membrane in trophozoites. Scale bars, 5 µm. (E) Trophozoite-stage IFAs of indicated parasites cultivated with and without TMP for 48 hr, showing that knockdown abolishes each member of the complex in R2-DDD but not in R3-DDD. Co-localization is shown with the exported parasite proteins KAHRP or SBP1 (red or green in merge panels, respectively). Scale bars, 5 µm. (F) Trophozoite-stage immunoblots using the indicated antibodies. R3-DDD was cultivated with or without TMP for 48 hr (the ‘max’ knockdown culture in Figure 5B).