The fate of Toxoplasma gondii dense-granule (GRA2, GRA3), rhoptry (ROP1), and surface (SAG1) proteins was followed by immunofluorescence assay (IFA) and immunoelectron microscopy at different stages after infection. Dense-granule exocytosis occurred in the apical area of the tachyzoite within minutes of invasion. Several exocytic events were found simultaneously in the same organism, both by serial sectioning and by freeze-fracture studies. Dense-granule contents were first found as a dense material trapped between parasite and vacuole membranes before either the vacuolar network or the vacuole membrane could be immunolabeled with specific antibodies. The vacuolar network was strongly labeled with dense-granule antibodies but not with the SAG1-specific probe, which suggests that the network is not enriched in membrane proteins. In addition to strongly labeling the vacuole membrane, GRA3 antibodies also labeled strands extending from the parasitophorous vacuoles into the host-cell cytoplasm.

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The biosynthesis and fate of 4 different dense granule proteins of Toxoplasma gondii were studied with 3 monoclonal antibodies raised against tachyzoites and 1 polyclonal antibody raised against a recombinant protein. These proteins have the following molecular weights: 27 kDa (GRA 1), 28 kDa (GRA 2), 30 kDa (GRA 3) and 40 kDa (GRA 4). All four proteins were found in dense granules by immunoelectron microscopy; in T. gondii-infected cells, they were found in the vacuolar network but, in addition, GRA 3 was also detected on the parasitophorous vacuole membrane. Therefore, dense granule contents undergo differential targeting when exocytosed in the parasitophorous vacuole. Metabolic labelling and immunoprecipitation showed that GRA 2 and GRA 3 were processed from lower molecular weight precursors, and that GRA 2 and GRA 4 incorporated [3H] glucosamine and are thus likely to be glycosylated.