GRA12, a Toxoplasma dense granule protein associated with the intravacuolar membranous nanotubular network

The intracellular protozoan parasite Toxoplasma gondii develops within the parasitophorous vacuole (PV), an intracellular niche in which it secretes proteins from secretory organelles named dense granules and rhoptries. Here, we describe a new dense granule protein that should now be referred to as GRA12, and that displays no homology with other proteins. Immunofluorescence and immuno-electron microscopy showed that GRA12 behaves similarly to both GRA2 and GRA6. It is secreted into the PV from the anterior pole of the parasite soon after the beginning of invasion, transits to the posterior invaginated pocket of the parasite where a membranous tubulovesicular network is first assembled, and finally resides throughout the vacuolar space, associated with the mature membranous nanotubular network. GRA12 fails to localise at the parasite posterior end in the absence of GRA2. Within the vacuolar space, like the other GRA proteins, GRA12 exists in both a soluble and a membrane-associated form. Using affinity chromatography experiments, we showed that in both the parasite and the PV soluble fractions, GRA12 is purified with the complex of GRA proteins associated with a tagged version of GRA2 and that this association is lost in the PV membranous fraction.