Identification of an IMC-associated protein recruiting the MyoC-glideosome to the basal polar ring.

<p>A. Parasites stably expressing MycGFPCtGAP45 and MycGFPCtGAP80 as a second copy were subjected to co-IP with anti-Myc antibodies after metabolic labeling. Eluted proteins were boiled (left panel) or not (right panel) before migration on a SDS-page gel and then visualized by autoradiography. Stars indicate the Myc-tagged proteins. Proteins 1 and 2 are the candidates analyzed by mass spectrometry. B. Total lysates from parasites expressing an endogenous Ty-tagged full-length version of IAP1 (KI-IAP1-3Ty) in a Ku80-KO background or an endogenous Myc-tagged truncated version of IAP1 (KI-Nt-IAP1-3Myc) in the KI-GAP80Ty were analyzed by western blot using respectively anti-Ty and anti-Myc antibodies and the catalase as loading control. C. Localization of KI-IAP1-3Ty at the posterior polar ring and in the last third of the IMC assessed in intracellular parasites using anti-Ty, anti-GAP45 and anti-IMC1 antibodies. Scale bar: 2 µm. D. Schematic representation of the IAP1 constructs used in this study and highlighting the position of the cysteine residues. In red are the cysteines predicted to be palmitoylated by CSS-Palm 3.0 with the highest threshold <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004504#ppat.1004504-Ren1" target="_blank">[26]</a> and in blue are the ones mutated to alanines. E. Co-IP performed with anti-Ty antibodies on parasites expressing KI-IAP1-3Ty and KI-ILP1-3Ty (as control) and revealed by western blot using anti-MLC1 and anti-GAP40 antibodies. Arrows show MLC1 and GAP40, respectively. The asterisks indicate the Ig heavy and light chains that cross-react with the antibodies. F. The truncated version of IAP1 (KI-Nt-IAP1-3Myc) was generated in wild type and KI-GAP80Ty backgrounds. It localizes in the last third of the IMC and led to the relocalization of GAP80Ty in the same area of the IMC. Scale bars: 2 µm. G. A wild type second copy of IAP1 (IAP1-Ty) stably expressed in RH strain localized to the posterior part of the IMC while a second copy of IAP1 mutated on the three first cysteines (AAA-IAP1-Ty) was found in the cytoplasm. Scale bars: 2 µm. H. The solubility of strains expressing IAP1 (KI-IAP1-3Ty, IAP1-Ty and AAA-IAP1-Ty) was assessed by fractionation after extraction in PBS, PBS/NaCl, PBS/Na₂CO₃ or PBS/Triton X-100. Their distribution in different fractions was assessed by western blot using anti-Ty antibodies and the soluble catalase (CAT) was used as control for the correct fractionation.