Figure 6. ASP5 processes the novel dense granule protein MYR1 near the C-terminus.
(A) Antibodies to the N-terminus of MYR1 show that it is processed in wild-type parasites (migrating at ~80 kDa) and a loss of processing in Δasp5 parasites resulting in the appearance of a larger molecular weight species (migrating at ~105 kDa). αSAG1 serves as the parasite loading control. For increased resolution, the samples from the left panel were separated on a 3–8% Tris-acetate gel (right panel), which revealed that the ~80 kDa band migrates at ~70 kDa on this gel and comprises a doublet, with the upper band absent in Δasp5 parasites, confirming lack of cleavage. * = suspected cross-reactive species. As a consequence of increased running time, SAG1 migrated off the 3–8% gel and was not transferred. (B) ASP5WT-HA3 cleavage of DABCYL/EDANS peptides containing the TEXEL of MYR1 and associated mutations (red residues). Peptides containing RRLSE from MYR1 and RRLAE from GRA16 are cleaved, but peptides with point mutations in P1, P2 or P3 are not cleaved. The serine at P1’ (compared to Ala in GRA16) does not interfere with cleavage by ASP5. (C) Schematic of MYR1 with an N-terminal SP, the RRLSE TEXEL at AA 557–581 and a C-terminal HA tag. (D) Immunoblot using αHA antibodies against Δmyr1:MYR1-HA parasites where Δmyr1:MYR1WT-HA runs at ~32 kDa, whereas Δmyr1:MYR1ARLSE-HA and Δmyr1:MYR1ARASA-HA mutants run at ~105 kDa. αSAG1 serves as a loading control. ASP5, Aspartyl Protease 5; HA3, triple-hemagglutinin; TEXEL, Toxoplasma export element.