Figure 1. Expression and localization of PI3P in T. gondii.

(A) Extracellular tachyzoites were labelled with [³²P]orthophosphate, and phosphatidylinositol-monophosphates were analyzed by a combination of thin layer chromatography and HPLC. Retention times of 36 and 45.5 min identified the peaks as PI3P and PI4P, respectively. (B) Schematic representation of constructs expressed in T. gondii. PI3P-binding capacity of the constructs, the ability to generate stable parasite clones, and the observed localizations are indicated. (C) Fluorescence microscopy analysis of parasites transiently transfected with GFP-2xFYVE. Arrowheads indicate the PI3P-enriched compartment at the sub-apical pole of the parasite and arrows point to the accumulation of the FYVE marker in the residual body. Scale bar  = 2 µm. (D) Western blot analysis of ddFYVE and ddFYVEm proteins. Intracellular parasites grown with or without Shield-1 (1 µM) were lysed from host cells by passage through a 26-gauge needle and boiled in reducing sample buffer and then separated by SDS PAGE, before probing with anti-GFP. Equal numbers of parasites were loaded in each lane. Time of Shield-1 incubation is indicated. The difference of mobility between ddFYVE and ddFYVEm has also been observed for GST-FYVE and GST-FYVEm recombinant proteins produced in E. coli (our unpublished data), and thus appears to be a consequence of these mutations. Anti-SAG1 antibody was used as a loading control. (E) ddFYVE expressing parasites were transiently transfected with Rab51-HA and processed for IFA in absence of Shield-1 using the anti-HA antibodies. ddFYVE is enriched in a compartment that is distinct from the one labelled with the endosomal marker Rab51-HA. (F) Fluorescence analysis of a parasite clone expressing both ddFYVE and FNR-RFP shows co-localization of both markers in the absence of Shield-1. (G) Stable transfected parasites with the mutated construct ddFYVEm show diffuse cytosolic staining after 20 min of Shield-1 induction (fluorescence of ddFYVEm was not observed in the absence of Shield-1). Scale bar  = 2 µm.