Differential targeting of dense granule proteins in the parasitophorous vacuole ofToxoplasma gondii

Abstract

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.

Toxoplasma gondii Rab6 Mediates a Retrograde Pathway for Sorting of Constitutively Secreted Proteins to the Golgi Complex

“…To address the possibility that protein modification in the parasite Golgi could be altered in the presence of the Rab6 mutants due to retrograde imbalance of glycosylation enzymes, the O-linked glycosylation of GRA2 was examined by metabolic pulse-chase. Within minutes of synthesis, GRA2 is modified by O-linked glycosylation from an ϳ26-kDa precursor to an ϳ28-kDa mature protein (27,32). At 15 min of chase, a kinetic lag was observed in the mobility of GRA2 in parasites stably expressing HARab6(T25N), whereas GRA2 mobility in parasites expressing HA-Rab6(Q70L) was unaltered compared with wild-type parasites (Fig.…”

“…Coverslips were prepared for immunofluorescence assay as previously described (12). Primary antibodies included mouse anti-HA-11 (Covance), rabbit anti-BAP (5 Prime 3 3 Prime, Inc.), anti-IMC1 monoclonal antibody (mAb) 45.5 (gift of Gary Ward, University of Vermont, Burlington, VT), and anti-GRA1 mAb T5-2B4 and anti-GRA2 mAb T4-1F5 (27). Secondary antibodies included fluorescein isothiocyanate-conjugated sheep anti-mouse IgG (Calbiochem), rhodamine-conjugated goat anti-rabbit IgG (Calbiochem), and Alexa-conjugated equivalents (Molecular Probes, Inc.).…”

Transmembrane Insertion of theToxoplasma gondiiGRA5 Protein Occurs after Soluble Secretion into the Host Cell

“…After their release within the vacuole, the GRA proteins are selectively targeted to specific locations of the PV where they are found either, like GRA1, as a soluble protein in the lumen of the vacuole or, like GRA2, as both soluble and membrane forms, the latter being closely associated with a reticulum of membranous tubules called the intravacuolar network (CesbronDelauw, 1994;Sibley et al, 1995). Others, like GRA3 and GRA5 are specifically targeted to the PVM (Achbarou et al, 1991;Lecordier et al, 1993). The mechanisms underlying the secretion of the GRA proteins and the basis of their targeting to specific locations in the PV are not known.…”

“…Others, like GRA3 and GRA5 are specifically targeted to the PVM (Achbarou et al, 1991;Lecordier et al, 1993). The mechanisms underlying the secretion of the GRA proteins and the basis of their targeting to specific locations in the PV are not known.…”

CD40, autophagy and Toxoplasma gondii

“…A critical question was whether parasitophorous vacuoles, which by definition have been considered non-fusogeneic, fused with late endosomeslysosomes. The formation of parasitophorous vacuoles involves secretion of contents of parasite organelles located in the apex of the organism (Archbarou et al 1991, Cesbron-Delauw 1994, Carruthers & Sibley 1997. The use of transgenic parasites that express a fluorescent protein targeted to the dense granules allowed to demonstrate that vacuoles where contents of the dense granules are secreted into the vacuolar lumen fuse with late endosomes-lysosomes (Andrade et al 2006).…”