We have identified a phosphate transporter (TcPho91) localized to the bladder of the contractile vacuole complex (CVC) of oocytes followed by two-electrode voltage clamp showed that TcPho91 is a low affinity transporter with a for Pi in the millimolar range and sodium-dependency. polyP levels. Our results indicate that TcPho91 is usually a phosphate sodium symporter involved in Pi homeostasis in that localizes to the vacuolar membrane and was postulated to export Pi from your vacuolar lumen to the cytosol (Hurlimann et al. 2007 In we recognized a Pi transporter (previously annotated as a sulfate/sodium symporter) with similarity to Pho91p localized it to the CVC by expressing the green fluorescent protein (GFP)-tagged protein and suggested that this transporter TcPho1 could be involved in returning Pi to the cytosol once water is discharged by the CVC Tenovin-1 upon hyposmotic stress (Ulrich et al. 2011 In this study we have characterized this Pi transporter (now renamed as TcPho91) at the molecular and electrophysiological levels and statement its relevance in Pi and polyP homeostasis and growth in trypanosomes. Results Characteristics and localization of TcPho91 One gene (TcCLB.508831.60) annotated as sulfate/sodium symporter and encoding for any putative orthologue was found in the genome (http://tritrypdb.org/tritrypdb/) and named was cloned by PCR amplification from your CL strain of confirmed by sequencing and shown to be identical to the gene in the database. The orthologs recognized in (Tb927.11.11160) (Huang et al. 2014 and (LmjF.28.2930) shared 65% and 59% amino Rabbit Polyclonal to ENDOGL1. acid identity respectively to TcPho91 (Fig. S1A). The ORF predicts a 727 amino acid protein with an apparent molecular excess weight of 80 kDa 12 transmembrane domains (Fig. S1B) an N-terminal regulatory SPX domain and an anion-permease domain also present in other anion transporters (Fig. S1C). Interestingly only one of the alleles (30s) encodes for the Tenovin-1 complete protein while the other one (30p) seems to be truncated and only contains the sequence corresponding to the SPX domain name (Fig. S1C). In previous work we reported the with the green fluorescent protein (and also to other intracellular membranes (Ulrich et al. 2011 Fig. 1A shows that TcPho91-GFP localizes mainly to the bladder of the CVC of epimastigotes trypomastigotes and amastigotes as revealed by its circular staining pattern (Fig. 1A cRNA or the same volume of water (control = ?19.8 ± 3.5 mV n = 20; = ?22.7 ± 2.7 mV n = 28) indicating that expression of the gene was not toxic to the cells (Fig. 2B). Fig. 2C show representative currents recorded in response to the voltage step protocol indicated in oocytes expressing TcPho91 when perfused in ND96 solution in the absence ((Fig. 2D) shows a significant increase in the current at positive and negative potential when comparing the control conditions in ND96 without Pi (< 0.05 n = 25). Fig. 2E shows the currents elicited at holding potential (?60 mV) by increasing concentrations of Pi in oocytes expressing with bath solution ND96. In the presence of Na+ 15 mM Pi induces an inward current of ?237 ± 12 nA at a holding potential of ?60 mV (n = 6). Water injected oocytes showed no significant changes in current (data not shown). Fig. 2F shows steady state kinetic parameters obtained by fitting the peak currents recorded in the presence of increasing concentrations of Pi according with the Hill equation (IPi= Ipimax * SnH/(SnH + KmnH) where IPi is the peak current in presence of Pi IPimax is the maximum current elicited by Pi S in the concentration of Pi nH represents the Hill coefficient and the apparent affinity constant. The calculated for Pi is 7.43 ± 0.28 mM and demonstrates that TcPho91 is a low affinity transporter. Values are mean ± Tenovin-1 SD of six independent experiments. Fig. 2 Electrophysiological characterization of TcPho91. A. Western blot analysis of oocytes lysates Tenovin-1 control (C) or transfected with cRNA using anti-GFP antibody. Markers are shown on the (in kDa). B. Resting membrane potential (< 0.05 n = 5). No significant increase in current was observed when Na+ was replaced by K+ (data not shown). Anion selectivity was demonstrated by applying voltage pulses as described before (Fig. 3A) in the presence of either 15 mM Pi (Fig. 3C < Tenovin-1 0.01) (Fig. 3C). Fig. 3 A. Sodium-dependency of TcPho91 activity. Currents elicited by Pi in the presence or absence of Na+ when a voltage pulse protocol was applied. When Na+ is replaced by N-methyl-D-glucamine (NMDG) the steady-state and inactivation currents are smaller. ... Voltage dependency.