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

Abstract

Toxoplasma gondii relies on protein secretion from specialized organelles for invasion of host cells and establishment of a parasitophorous vacuole. We identify T. gondii Rab6 as a regulator of protein transport between post-Golgi dense granule organelles and the Golgi. Toxoplasma Rab6 was localized to cisternal rims of the late Golgi and trans-Golgi network, associated transport vesicles, and microdomains of dense granule and endosomal membranes. Overexpression of wildtype Rab6 or GTP-activated Rab6(Q70L) rerouted soluble dense granule secretory proteins to the Golgi and endoplasmic reticulum and augmented the effect of brefeldin A on Golgi resorption to the endoplasmic reticulum. Parasites expressing a nucleotide-free (Rab6(N124I)) or a GDP-bound (Rab6(T25N)) mutant accumulated dense granule proteins in the Golgi and associated transport vesicles and displayed reduced secretion of GRA4 and a delay in glycosylation of GRA2. Activated Rab6 on Golgi membranes colocalized with centrin during mitosis, and parasite clones expressing Rab6 mutants displayed a partial shift in cytokinesis from endodyogeny (formation of two daughter cells) to endopolygeny (multiple daughter cells). We propose that Toxoplasma Rab6 regulates retrograde transport from post-Golgi secretory granules to the parasite Golgi.

Dissection of brefeldin A-sensitive and -insensitive steps in apicoplast protein targeting

“…BFA is effective at blocking transport in mammalian cells at concentrations of 50-100 ng/ml, and has generally been used at concentrations of 5-10 µg/ml for studies in T. gondii (Brydges et al, 2000;Soldati et al, 1998;Stedman et al, 2003), although 1 µg/ml has been shown to prevent secretion of GRA1 (Coppens et al, 1999). Preliminary studies showed that T. gondii were able to proliferate following treatment with 1 µg/ml BFA for 4 hours, but not if the concentration was raised to 10 µg/ml (not shown).…”

Golgi and centrosome cycles in Toxoplasma gondii

“…But some of these observations have never been followed-up or published and others have not been quantified and moreover have been made in fixed images, so that nothing could be told about the dynamics of this relationship. [69] Interestingly, just recently a study found that another Toxoplasma gondii organelle, the apicoplast, is divided by association with the centrosomes. The ends of the dividing apicoplast are closely linked to the centrosome.…”

“…A characteristic spatial relation of centrioles and Golgi has been noticed, but not further studied ( [69] and D.S. Roos, University of Pennsylvania, personal communication).…”

Re-defining the Golgi complex inPlasmodium falciparumusing the novel Golgi markerPfGRASP

“…Another explanation for the loose association of Rab6 and the Golgi might be the multifunctional role of Rab6 in vesicular transport. In the mammalian system it could be shown that Rab6 (with its two isoforms) does not only function in intra-Golgi transport (Martinez et al, 1994) but is also required for protein recycling between endosomes and the trans-Golgi network (Mallard et al, 2002;Stedman et al, 2003) and even to the ER (White et al, 1999). In yeast, the Rab6 homologue Ypt6p has also been implicated in multiple roles, for example the retrieval of proteins from endosomes to the trans-Golgi network, intra-Golgi retrograde and Golgi to ER transport (Siniossoglou et al, 2001;Bensen et al, 2001;Luo and Gallwitz, 2003).…”