Differential membrane targeting of the secretory proteins GRA4 and GRA6 within the parasitophorous vacuole formed by Toxoplasma gondii.
Following secretion into the parasitophorous vacuole, dense granule proteins, referred to as GRA proteins, are targeted to different locations including a complex of tubular membranes that are connected with the vacuolar membrane. To further define the formation of this intravacuolar network, we have investigated the secretion, trafficking and membrane association of GRA4 and GRA6 within the parasitophorous vacuole. In extracellular parasites, GRA4 and GRA6 were found exclusively in dense secretory granules where they were packaged primarily as soluble proteins. Following release into the vacuole, GRA6 was rapidly translocated to the posterior end of the parasite where, like previously reported for GRA2, it bound to a cluster of multi-lamellar vesicles that give rise to the network. In contrast, GRA4 was distributed throughout the lumen of the vacuole and only later became associated with the mature network that is found dispersed throughout the vacuole. Cell fractionation and treatment with denaturing agents established that the association of GRA4 with the network membranes was mediated by strong protein-protein interactions. In contrast, GRA6 was predominantly influenced by hydrophobic interactions, and a phosphorylated form of this protein present within the vacuole showed increased association with the network membranes. 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. Formation of this protein complex, which is based on both protein-protein and hydrophobic interactions, may participate in nutrient or protein transport within the vacuole.
Labruyere
E
E
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Lingnau
M
M
Mercier
C
C
Sibley
L D
LD
D000818:Animals; D000953:Antigens, Protozoan; D015153:Blotting, Western; D002458:Cell Fractionation; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D006801:Humans; D008566:Membranes / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D011233:Precipitin Tests; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014122:Toxoplasma / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism / Q000469:parasitology*
D000953:Antigens, Protozoan; C079248:GRA4 protein, Toxoplasma gondii; C095047:GRA6 protein, Toxoplasma gondii; D015800:Protozoan Proteins; D011994:Recombinant Proteins