A single Na+-Pi cotransporter in Toxoplasma plays key roles in phosphate import and control of parasite osmoregulation
Fig 2
Motif features and expression of TgPiT in Toxoplasma.
A. Phylogenetic analysis of the PHS and PiT family proteins. Amino acid sequences from different unicellular eukaryotic species were aligned and phylogenetic analysis was performed using MEGA 5.2.2 software. The PiT family members include PHO-4 from Neurospora crassa (GenBank: AAA33607.1), ScPHO89 from Saccharomyces cerevisiae (GenBank: NP_009855.1), PfPiT from Plasmodium falciparum (GenBank: CAE30463.1), LiPHO89 p from Leishmania infantum (GenBank: XP_001466587.1) and TcPHO89 p from Trypanosoma cruzi (GenBank: XP_813912.1), The PHS family members include TcPHO84 p from T. cruzi (GenBank:XM_809326.1), LiPHO84 p from L. infantum (GenBank:AFJ96967.1), NcPHO-5 from N. crassa (GenBank: AAA74899.1), CgPHO84 from Candida glabrata (GenBank: XM_445078.1) and ScPHO84 from S. cerevisiae (GenBank: CAA89157.1). p = putative sequence based on functional motif identifications. Outgroup: HsGAPDH from Homo sapiens (GenBank: NP_002037.2). B. Conserved domains in TgPiT. TgPiT has two conserved PHO4 domains (I and II) containing GLU residues required for Pi translocation (turquoise boxes), present in the PiT of yeast (ScPHO89) and P. falciparum (PfPiT; MAL13P1.206). TgPiT contains 12 putative TMD (black boxes) and a large intracellular loop at between TMD 7 and 8. C. Expression of TgPiT. Immunoblots of Toxoplasma lysates (10E7 parasites per lane) were incubated with anti-TgPiT antibodies showing a band at ~95 kD. D. Biochemical analysis of TgPiT in isolated parasites. Solubilization of TgPiT: after washing, parasites isolated from cells were lysed in buffer containing 1% TritonX-100 (Tx-100) for 15 min before centrifugation of the lysate and collection of the supernatant (S; detergent-solubilized fraction) and pellet (P; membrane fraction) for SDS-PAGE and Western blotting using antibodies against TgPiT or the surface protein SAG1 as positive control (panel a). Surface-exposure of TgPiT: after washing, extracellular Toxoplasma were incubated 30 min in the presence of 0.1 mg/ml of proteinase K (Prot K) or reaction buffer alone at 22°C before adding PMSF to inactivate proteinase K, centrifugation to collect the supernatant (S; Proteinase K-sensitive fraction) and the pellet (P; Proteinase K-resistant fraction) for SDS-PAGE and Western blotting using antibodies against TgPiT, SAG1 (plasma membrane) and Hsp70 (cytosol) antibodies as controls for surface-proteolysis (panel a). Panel b shows the quantification of the ECL signal on immunoblots from 3 independent assays (means ± SD) and expressed in percent of ratios of S fractions to P fractions for TritonX-100 or proteinase K assays.