Purification of Toxoplasma dense granule proteins reveals that they are in complexes throughout the secretory pathway.

Dense granules are Apicomplexa specific secretory organelles. In Toxoplasma gondii, the dense granules proteins, named GRA proteins, are massively secreted into the parasitophorous vacuole (PV) shortly after invasion. Despite the presence of hydrophobic membrane segments, they are stored as both soluble and aggregated forms within the dense granules and are secreted as soluble forms into the vacuolar space where they further stably associate with PV membranes. In this study, we explored the unusual biochemical behavior of GRA proteins during their trafficking. Conventional chromatography indicated that the GRA proteins form high globular weight complexes within the parasite. To confirm these results, DeltaGRA knocked-out parasites were stably complemented with their respective HA-FLAG tagged GRA2 or GRA5. Purification of the tagged proteins by affinity chromatography showed that within the parasite and the PV soluble fraction, both the soluble GRA2-HA-FLAG and GRA5-HA-FLAG associate with several GRA proteins, the major ones being GRA3, GRA6 and GRA7. Following their insertion into the PV membranes, GRA2-HA-FLAG associated with GRA5 and GRA7 while GRA5-HA-FLAG associated with GRA7 only. Taken together, these data suggest that the GRA proteins form oligomeric complexes that may explain their solubility within the dense granules and the vacuolar matrix by sequestering their hydrophobic domains within the interior of the complex. Insertion into the PV membranes correlates with the decrease of the GRA partners number.