FIG 5.

GRA44 and GRA45, but not MYR4, show evidence for processing at RRL sites. (A) Schematic of GRA44, GRA45, and MYR4 protein sequences, showing the locations of predicted signal peptides (SP), RRL tripeptide sequences, previously identified phosphorylated serine residues (S*) (52), and conserved domains, numbered in amino acid residues relative to the predicted N terminus of the primary translation product. The approximate molecular weights (in kilodaltons) of the indicated portions are indicated. The amino acid sequence of MYR4 was determined using the fourth in-frame methionine relative to the protein predicted in ToxoDB (v45). Transmembrane domain prediction was based on Phobius (51). (B) Western blot of protein processing. (Left) The indicated parasite lines were transiently transfected with plasmids expressing C-terminally V5-tagged versions of either the indicated wild-type protein or a mutant version with the indicated RRL mutated to ARL (the numbers at the top indicate the amino acid position of the mutated arginine). These were then used to infect HFFs for 18 h. Lysates were analyzed by Western blotting using mouse anti-V5 antibodies to detect the C-terminally V5-tagged portions of each protein. The approximate migration of a ladder of size standards (indicated in kilodaltons on the left) is indicated. (Right) A longer exposure of the right-most two lanes of the left panel. (C) Western blot of MYR4 processing. RHΔhpt (RH) parasites were transiently transfected with either the wild type or an RRL → AAA-mutated version of C-terminally V5-tagged MYR4 and allowed to infect HFFs for 24 h before lysates were generated for immunoblotting. Lysates were analyzed by Western blotting using mouse anti-V5 antibodies to detect MYR4. The approximate migration of a ladder of size standards (indicated in kilodaltons on the left) is indicated.