Targeting of proteins in the parasitophorous vacuolar space of Toxoplasma gondii

One of the hallmarks of the vacuolar space (PV) surrounding Toxoplasma gondii inside cells is the intravacuolar network of tubuloreticular elements that is elaborated by the parasite, which is modified by secretion of proteins from its dense granule (DG) organdies. All DG proteins associate with the network by immuno EM despite the absence of common motifs which might mediate network binding. In order to explore this observation, we analyzed network targeting of soluble, foreign secretory reporters E. coli β lactamase (BLA) or alkaline phosphatase (BAP) expressed stably in T. gondii. These foreign reporters should lack specific network targeting signals. By immunofluorescence and immuno EM, the reporters were localized to the DG within the parasites and like the endogenous proteins GRA3 and NTPase were associated with the network. However, in subcellular fractionation experiments of low speed supernatant enriched for network material, more than 80% of GRA3 in the PV pelleted at 10, 000 X g, whereas greater man 75% of NTPase, BLA, and BAP remained in the 100,000 X g supernatant. In sucrose flotation experiments using the differentially pelleted material, the little NTPase, BLA and BAP that pelleted did float up to lower density interfaces, co-fractionating with membrane associated GRA3, suggesting that this fraction is either membrane associated or in common vesicles. These results indicate that although NTPase, BLA and BAP associate with the network by EM, they do so minimally by subcellular fractionation, and that soluble proteins in the PV associate with the network by default. The fact that foreign reporter proteins, presumably lacking in T. gondii network targeting signals, also associate with the network suggests that such an association may not have any functional significance.