Fig. 2: Tachyzoites express a functional PtdIns synthase in the Golgi complex.
a The primary structures of TgPIS and selected orthologs from different organisms. CDP-alcohol phosphotransferase domains and transmembrane regions were predicted by Simple Modular Architecture Research Tool (SMART) and transmembrane Hidden Markov Model (HMM). Numerals indicate the size of the N-termini and entire open reading frames in amino acids. Other relevant sequence information such as, GenBank IDs are shown in Supplementary Table 1. Ef, Eimeria falciformis; Pf, Plasmodium falciparum; Tb, Trypanosoma brucei; Gs, Galdieria sulphuraria; Ap, Auxenochlorella protothecoides; At, Arabidopsis thaliana; Sc, Saccharomyces cerevisiae; Mm, Mus musculus. b Immunoblot of E. coli M15/pREP4 strains harboring pQE60 (N.C., negative control), pQE60-TgPIS-6xHis or pQE60-TgPIS49–258-6xHis plasmids. Induction was performed by IPTG as reported in “Methods”. The total bacterial extract was resolved by 12% SDS-PAGE, blotted and probed with the mouse anti-His antibody (1:3000). c TLC of lipids isolated from E. coli samples as shown in b. Expression was induced by 1 mM IPTG, and cultures were incubated with specified amounts of myo-inositol. Lipids were visualized under the UV light after spraying 8-anilino-1-naphthalenesulfonic acid. The unlabeled bands in samples on the TLC plate represent unknown lipids, some of which appear upon expression of PIS isoforms regardless of myo-inositol substrate in medium. d Lipid-phosphorous quantification of bands corresponding to PtdIns in c. Silica scraping containing the individual lipid bands were subjected to lipid-phosphorus measurements along with defined standards. Graph shows the mean values with S.E. from 3 different assays. e, f Immunofluorescence images of the transgenic tachyzoites expressing either TgPIS-HA or TgPIS49–258-HA. The strain expressing TgPIS-HA under the control of its native promoter and TgDHFR-TS-3′UTR was constructed as described in Fig. 3a. The truncated isoform lacking the extended N-terminal sequence (1–48 residues, a) was regulated by pTETO7SAG1 promoter and TgNTP3–3′UTR. The TgPIS49–258-HA expression cassette was inserted at the UPRT locus by negative selection using 5-fluorodeoxyuridine. Transgenic parasites expressing the either variants were transfected with a vector expressing TgERD2-Ty1 (regulated by TgGRA1 elements) for co-localization study. Immunostaining was performed 24 h post-infection using α-HA/Alexa594 (red) and α-Ty1/Alexa488 (green) antibodies. Nuclei were stained with DAPI (blue). Scale bars, 2 μ; DIC, differential interference contrast.
