FIG. 7.

GRA14 is inserted into the PVM as an integral membrane protein with its C terminus exposed to the host cell cytoplasm. (A) Differential permeabilization conditions with 0.001% saponin were used in IFAs of cells infected with GRA14-HA parasites. All cells were stained with SAG1 (to control for permeabilization of the PVM) and stained with either anti-HA or anti-GRA14 antiserum or anti-ROP2. PVs stained with the HA antibody (showing a rim-like pattern) were SAG1 negative, indicating that the GRA14 C terminus is facing the host cell cytosol. GRA14 antiserum (N-terminal) staining was never observed in cells that were also SAG1 negative. ROP2 staining, which showed immunoreactivity on the outside of the PVM (in SAG1-negative vacuoles), was used as a positive control. (B) Infected cells were fully permeabilized in 0.15% saponin and stained with the same antibodies as in panel A. All PVs were SAG1 positive and either HA, GRA14N, or ROP2 positive. The arrow indicates internal staining within the PV. (C) Cartoon showing the topology of the characterized transmembrane-containing GRA proteins and PVM-associating ROP2 family proteins. The question marks represent the uncertainty of the topology of ROP2 family members at the PVM.