Targeting of soluble proteins to the rhoptries and micronemes in Toxoplasma gondii

Rhoptry and microneme organelles of the protozoan parasite Toxoplasma gondi i are closely associated with host cell adhesion/invasion and establishment of the intracellular parasitophorous vacuole. In order to study the target ing of proteins to these specialized secretory organelles, we have engineer ed green fluorescent protein (GFP) fusions to the rhoptry protein ROP1 and the microneme protein MIC3. Both chimeras are correctly targeted to the app ropriate organelles, permitting deletion analysis to map protein subdomains critical for targeting. The propeptide and a central 146 amino acid region of ROP1 are sufficient to target GFP to the rhoptries. More extensive dele tions result in a loss of rhoptry targeting; the GFP reporter is diverted i nto the parasitophorous vacuole via dense granules. Certain MIC3 deletion m utants were also secreted into the parasitophorous vacuole via dense granul es, supporting the view that this route constitutes: the default pathway in T. gondii. and that specific signals are required for sorting to rhoptries and micronemes. Deletions within the cysteine-rich central region of MIC3 cause this protein to be arrested at various locations within the secretory pathway, presumably due to improper folding. Although correctly targeted t o the appropriate organelles in living parasites, ROP1-GFP and MIC3-GFP fus ion proteins were not secreted during invasion. GFP fusion proteins were re adily secreted from dense granules, however, suggesting that protein secret ion from rhoptries and micronemes might involve more than a simple release of organellar contents. (C) 2001 Elsevier Science B.V. All rights reserved.