Different fractional domains in the parasitophorous vacuole membrane of Toxoplasma gondii

The obligate intracellular parasite Toxoplasma gondii resides inside the host cells in a specialized vacuole known as the parasitophorous vacuole (PV) which is delimited by the parasitophorous vacuole membrane (PVM). The PV and the PVM are modified by the secretion of dense granule (DO) proteins of which GRA3 is specifically targeted to the PVM. Using a chimeric construct with the GRA3 gene fused at the C-terminus to E. coli alkaline phosphatase (BAP) we analyzed targeting of GRA3 to the PVM. In immunoblots of GRA3-BAP transgenic parasites, me fusion protein was recognized by the rabbit polyclonal antibody to GRA3 and BAP but not by the mAb to GRA3 suggesting that GRA3 within GRA3-BAP is in a different conformation than native GRA3. By double IF, the fusion protein was targeted to the PVM but co-localized only imperfectly with the endogenous GRA3. The GRA3-BAP fusion had altered PVM association as assessed by subcellular-fractionauon, and altered TX-114 partitioning for it partitioned as a completely aqueous protein, unlike GRA 3. By immuno EM, unlike endogenous GRA3, the fusion protein was excluded from specific sites in the PVM where host mitochondria associate tightly. These experiments suggest that 1) the C-terminus of GRA3 may participate in PVM association, 2) there are different domains in the PVM, 3) the mitochondrial association sites in the PVM may have a functional significance.