Interactions between GRA proteins of dense granules in Toxoplasma gondii and host cell proteins were analyzed by yeast two-hybrid technique. The cMyc-GRA fusion proteins expressed from pGBKT7 plasmid in Y187 yeast were bound to host cell proteins from pGADT7-Rec-HeLa cDNA library transformed to AH109 yeast by mating method. By the selection procedures, a total of 939 colonies of the SD/-AHLT culture, 348 colonies of the X-alpha-gal positive and PCR, 157 colonies of the X-beta-gal assay were chosen for sequencing the cDNA and finally 90 colonies containing ORF were selected to analyze the interactions. GRA proteins interacted with a variety of host cell proteins such as enzymes, structural and functional proteins of organellar proteins of broad spectrum. Several specific bindings of each GRA protein to host proteins were discussed presumptively the role of GRA proteins after secreting into the parasitophorous vacuoles (PV) and the PV membrane in the parasitism of this parasite.
Vacuoles/*metabolism ; Two-Hybrid System Techniques ; Toxoplasma/metabolism/*pathogenicity ; Protozoan Proteins/*metabolism/secretion ; Proteins/*metabolism ; Organelles/metabolism ; Intracellular Membranes/*metabolism ; Humans ; Hela Cells ; Gene Library ; Cytoplasmic Granules ; Animals

Monoclonal antibody (mAb) Tg786 against Toxoplasma gondii has been found to detect a 42-kDa rhoptry protein (ROP6) which showed protease activity and host cell binding characteristics after secretion. Using the mAb, a colony containing a 3'-UTR was probed in a T. gondii cDNA expression library. A full length cDNA sequence of the rhoptry protein was completed after 5'-RACE, which consisted of 1,908 bp with a 1,443 bp ORF. The deduced amino acid sequence of ROP6 consisted of a polypeptide of 480 amino acids without significant homology to any other known proteins. This sequence contains an amino terminal stop transfer sequence downstream of a short neutral sequence, hydrophilic middle sequence, and hydrophobic carboxy terminus. It is suggested that the ROP6 is inserted into the rhoptry membrane with both N- and C-termini.
Toxoplasma/enzymology/*genetics ; Protozoan Proteins/*genetics/metabolism ; Peptide Hydrolases/genetics/metabolism ; Molecular Sequence Data ; Gene Library ; Cloning, Molecular ; Animals ; Amino Acid Sequence ; 3' Untranslated Regions

Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.
Alpha-Amanitin/pharmacology ; Animals ; Antibodies, Monoclonal/analysis/metabolism ; Antibodies, Protozoan/analysis/metabolism ; Dactinomycin/pharmacology ; Fluorescent Antibody Technique, Direct ; Gene Expression/*physiology ; Green Fluorescent Proteins/genetics ; Hela Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Nucleic Acid Synthesis Inhibitors/pharmacology ; Nucleolus Organizer Region/drug effects/*metabolism ; Pol1 Transcription Initiation Complex Proteins/metabolism ; Protein Sorting Signals/physiology ; Protozoan Proteins/*biosynthesis/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; Toxoplasma/*physiology ; Transfection