<i>Toxoplasma</i> PKAc1 localises to the tachyzoite periphery likely by dual acylation of its regulatory subunit PKAr1.

(A) Localisation of HA-tagged PKAc1 by IFA, co-staining with the peripheral marker IMC1 (i) and the apical IMC marker ISP1 [30] (ii). (B) Identification of PKAc1-interacting proteins by anti-HA immunoprecipitation, whole eluate tryptic digestion, mass spectrometry, and label-free quantification. Red and green lines signify 2-fold and 4-fold enrichment, as compared to the parental line (see S1 Table for full list of identified proteins). (C) Identification of PKAc1-interacting proteins by anti-HA immunoprecipitation, SYPRO Ruby staining of eluates followed by in-gel digest and mass spectrometry. Table denotes proteins and coverage identified in each band. (D) Western blot of Ty epitope-tagged PKAr1 (TGME49_242070). (E) Localisation of Ty epitope tagged PKAr1 by IFA, co-stained with either (i) IMC1 antibodies or (ii) the antibodies to the apical IMC protein ISP1, outlining localisation to the developing daughter cells. (F) Mutational analysis of putative myristoylation and palmitoylation sites at the N-terminus of PKAr1. (i) The first 15 amino acids of PKAr1 (PKAr1(1–15)WT) were fused to GFP and colocalised with GAP45 and (ii) ISP1, showing localisation to the periphery and to the developing daughter cells, respectively. (iii) Putative myristoylation site—glycine at position 2—was mutated to alanine (PKAr1(1–15)G2A), (iv) putative palmitoylated cysteine residues at positions 5 and 7 were mutated to alanine (PKAr1(1–15)C5AC7A). (v) Both putative myristoylation and palmitoylation sites were mutated (PKAr1(1–15)G2AC5AC7A). In each case, localisation of GFP fusions were compared to the peripheral marker GAP45 and counterstained with DAPI. (G) Western blot on PKAr1(1–15)WT-GFP and point mutants. C5AC7A, cysteine-to-alanine-mutations at amino acid 5 and 7; GAP45, glideosome-associated protein 45; GFP, green fluorescent protein; G2A, glycine to alanine mutation at amino acid 2; HA, haemagglutinin; IFA, immunofluorescence assay; IMC, inner membrane complex; IP, immunoprecipitation; ISP1, IMC subcompartment protein 1; PKAc1, protein kinase A catalytic subunit 1; PKAr1, protein kinase A regulatory subunit 1; WT, wild type.