THE PROTOZOAN PARASITE TOXOPLASMA-GONDII TARGETS PROTEINS TO DENSE GRANULES AND THE VACUOLAR SPACE USING BOTH CONSERVED AND UNUSUAL MECHANISMS

All known proteins that accumulate in the vacuolar space surrounding t he obligate intracellular protozoan parasite Toxoplasma gondii are der ived from parasite dense granules. To determine if constitu tive secre tory vesicles could also mediate delivery to the vacuolar space, T. go ndii was stably transfected with soluble Escherichia coli alkaline pho sphatase and E. coli beta-lactamase, Surprisingly, both foreign secret ory reporters were delivered quantitatively into parasite dense granul es and efficiently secreted into the vacuolar space. Addition of a gly cosylphosphatidylinositol membrane anchor rerouted alkaline phosphatas e to the parasite surface. Alkaline phosphatase fused to the transmemb rane domain and cytoplasmic tail from the endogenous dense granule pro tein GRA4 localized to dense granules, The protein was secreted into a tuboreticular network in the vacuolar space, in a fashion dependent u pon the cytoplasmic tail, but not upon a tyrosine-based motif within t he tail. Alkaline phosphatase fused to the vesicular stomatitis virus G protein transmembrane domain and cytoplasmic tail localized primaril y to the Golgi, although staining of dense granules and the intravacuo lar network was also detected; truncating the cytoplasmic tail decreas ed Golgi staining and increased delivery to dense granules but blocked delivery to the intravacuolar network. Targeting of secreted proteins to T. gondii dense granules and the plasma membrane uses general mech anisms identified in higher eukaryotic cells but is simplified and exa ggerated in scope, while targeting of secreted proteins beyond the bou ndaries of the parasite involves unusual sorting events.