Differential membrane targeting of the secretory proteins GRA4 and GRA6 within the parasitophorous vacuole formed by Toxoplasma gondii

Functional Analysis of Toxoplasma gondii Protease Inhibitor 1

“…Vacuole Fractionations-Membranous fractions were isolated from vacuoles as described previously (25). Briefly, tachyzoites were inoculated onto HFF monolayers and incubated 16 h. Monolayers were washed twice with PBS to remove extracellular tachyzoites, and vacuoles were lysed by passage through 23-and 25-gauge syringes.…”

“…TgPI-1 Remains Soluble after Secretion into the PV-After secretion into the PV, DG proteins can be targeted to the PV membrane (29,30) or the intravacuolar network of tubular membranes within the PV (25) or remain soluble (31). The solubility characteristics of a DG protein are important for predicting whether the protein will remain confined to the host cell after parasite egress or whether it will be released to…”

GRA Proteins of Toxoplasma gondii: Maintenance of Host-Parasite Interactions across the Parasitophorous Vacuolar Membrane

“…The molecular basis of targeting to a network of membranous tubules that connect with the vacuolar membrane is dependent on the 2 amphipathic α -helices [29]. Cross-linking studies established that GRA4 and GRA6 specifically interact with GRA2 to form a multimeric complex that is stably associated with the intravacuolar network [20], which is based on both protein-protein and hydrophobic interactions, may participate in nutrient or protein transport within the vacuole.…”

“…Hence, a fraction of the GRA1, GR-A3, GRA7, and NTPases pools, as well as GRA2, GRA4, GRA6, GRA9, GRA12, and GRA14 are detected more specifically associated with the vacuolar network membranes. In these membranes, GRA2, GRA4 and GRA6 participate to the formation of a multimeric protein complex [20]. In contrast, GRA3, GRA5, GRA7, GRA8 and GRA10 are preferentially detected as PVMassociated proteins [10].…”

The acidocalcisome Ca2+‐ATPase (TgA1) of Toxoplasma gondii is required for polyphosphate storage, intracellular calcium homeostasis and virulence

“…Monoclonal antibody 6D10 (anti-MIC2) (Wan et al, 1997) was diluted at 1:5000, and the samples were processed for chemiluminiscence detection as per the manufacturer's instructions. The rabbit polyclonal antibody against GRA4 was used at 1:5000 dilution and it was a gift from Dr L. David Sibley (Labruyere et al, 1999). Densitometric analysis of Western blots was performed with the IS1-1000 digital imaging system (Alpha Inotech).…”