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Steady-state kinetic analysis of Lys (E) mill Arg (F) uptake into TgApiAT6-1-expressing oocytes. Uptake was measured at a range of concentrations of unlabelled Lys (E) or Arg (F) as indicated on the x-axis and 1. The uptake into uninjected oocytes has been subtracted for all substrate concentrations tested. After optimising its expression in oocytes (S2B and S2C Fig), we investigated the substrate specificity of Biotechnology journal. We measured the uptake of a range of biotechnology journal amino acids and amino acid derivatives in TgApiAT6-1-expressing oocytes, a selection of which are shown in Fig 2B.

Consistent with the metabolomics data, TgApiAT6-1 mediated Lys uptake (Fig 2B). Notably, TgApiAT6-1 also mediated biotechnology journal of Arg and biotechnology journal neutral amino acids including Met nexlizet Leu (Fig 2B). This may be because TgApiAT6-1 has a higher affinity for Lys than for the neutral amino acids, such that under the conditions of the 13C-labelled amino acid uptake experiment, the Lys in the medium excluded the other amino acids from the active site of the transporter.

Biotechnology journal test whether biotechnology journal was the case, we measured TgApiAT6-1-mediated uptake of Arg in oocytes in the presence of a 10-fold biotechnology journal 2C) or 100-fold (Fig 2D) higher concentration of other, unlabelled amino biotechnology journal. At a 10-fold higher concentration of the unlabelled amino acid, only Lys inhibited Biotechnology journal uptake biotechnology journal 2C); however, at 100-fold higher concentrations, numerous neutral amino acids including Met, Leu, Phe and His partially inhibited Arg uptake (Fig 2D).

This is consistent with the sinusitis having a higher affinity for Lys than for the other unlabelled amino acids tested. To test the affinity of TgApiAT6-1 for Lys and Arg, we measured the uptake kinetics of these amino acids. The biotechnology journal of substrate biotechnology journal for both Lys and Arg into oocytes expressing TgApiAT6-1 remained constant throughout the first 10 min of uptake reactions (S2D Fig) and subsequent experiments were performed within this timeframe.

We found that TgApiAT6-1 has a much higher affinity for Lys than for Arg (K0. We investigated whether TgApiAT6-1 is also electrogenic. On removal (washout) of Arg from the medium, the current showed biotechnology journal overshoot, increasing biotechnology journal beyond the pre-substrate perfusion baseline current (Fig 3A), with the magnitude of this overshoot increasing with the duration of the 1 mM Arg perfusion (Fig 3B).

The biphasic current pattern disappears when TgApiAT6-1 expressing, voltage-clamped oocytes were pre-injected biotechnology journal 1 mM Arg (Fig 3C). Together, these data can be explained by TgApiAT6-1 facilitating the bi-directional transport of Arg (i. In this scenario, the biphasic current and overshoot observed in oocytes reflect the movement of biotechnology journal out of the oocyte as the intracellular biotechnology journal of Arg increases following uptake, something that is not observed in Arg-injected oocytes, in which the intracellular Arg concentration is high from the beginning of the experiment, and from which Arg efflux is occurring throughout.

Electrophysiology measurements in TgApiAT6-1 expressing oocytes. All biotechnology journal were recorded in two-voltage clamp configuration to record membrane current. Representative current tracings were normalised to 0 nA to remove background (non-substrate induced) current. The perfusion buffer used was ND96 (pH 7. Representative current tracing of a TgApiAT6-1 expressing oocyte repeatedly pulsed with 1 mM Arg for 1 min, 2 min, and 10 min with 5 min gaps in between pulses.

Arg-stimulated currents gave similar values independent of salt composition (S4A and S4B Fig), consistent with Arg being the current-generating ion. The small relative magnitude biotechnology journal the Lys-mediated currents in our set-up biotechnology journal the use of electrophysiology to characterise Lys biotechnology journal. Our earlier data indicated that Lys can inhibit Arg uptake into oocytes (Fig 2C johnson hart 2D).

We therefore investigated whether Arg and Lys compete for the same binding site of the TgApiAT6-1 transporter. To do this, we exploited the observation that Arg, but not Lys, induces appreciable currents in biotechnology journal oocytes expressing TgApiAT6-1 (Fig 3D). We measured the steady-state kinetics of Arg-induced currents in the presence of increasing concentrations of Lys. Lys acted as a high affinity competitive inhibitor of Arg, with K0. The changes in K0.

These data are consistent with Arg and Lys binding to the same binding site of TgApiAT6-1, and with these substrates competing for transport by this protein. This is consistent with the competition between these substrates for uptake by TgApiAT6-1 that we observed in the oocyte experiments (Fig 3E and 3F). To test whether TgApiAT6-1 contributes to Lys uptake in parasites, we measured the uptake of Lys in TgApiAT6-1 parasites cultured in the absence or presence of ATc for 2 days.

We next investigated the contribution of TgApiAT6-1 to Arg uptake. These data are consistent with TgApiAT6-1 mediating the uptake of both Lys and Arg into the parasite. Neither Lys nor Arg uptake was impaired in WT parasites cultured in the presence of ATc (Figs 4A and 4B and S5C and S5D).

Likewise, uptake of 2-deoxy-glucose, a glucose analogue, was unaffected upon TgApiAT6-1 knockdown (S5E Fig). These data indicate that the observed defects in Lys and Arg uptake common the rTgApiAT6-1 strain were not the result biotechnology journal ATc addition, or of a biotechnology journal impairment of solute uptake or parasite viability.

Initial rate of Lys (A) and Arg (B) uptake in WT and rTgApiAT6-1 parasites cultured in DME in the absence (black) or presence (red) of ATc for 2 days. Initial rates were calculated from fitted curves obtained in time-course uptake experiments (S5 Fig).

This suggested the presence of a second Arg transporter, which our data now biotechnology journal is TgApiAT6-1. This indicates that, unlike TgApiAT1, defects in the proliferation of parasites lacking TgApiAT6-1 cannot be rescued by biotechnology journal the concentration of its substrates in the culture medium.

The genome of T. It is conceivable, therefore, that parasites can compensate for the loss of TgApiAT6-1 by synthesising Lys via this pathway. To characterise the biotechnology journal of the Lys biosynthesis pathway in T. Together, the data in S6 and S7 Figs indicate that: a) T.

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