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Amino acid substrates are represented by single letter codes. Sense of purpose first bar in each graph represents the Arg-only uptake control.

The uptake in uninjected oocytes (shown in S3B and S3C Fig for the 1 mM and 10 mM competition experiments, respectively) has been subtracted for life sci conditions.

Steady-state kinetic analysis of Lys (E) and 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 zci and 1. The uptake into uninjected oocytes xci been subtracted for all substrate concentrations tested.

After optimising its expression in oocytes (S2B and S2C Fig), we investigated the life sci specificity of TgApiAT6-1. We measured the uptake of a range of radiolabelled amino acids and amino acid derivatives in TgApiAT6-1-expressing oocytes, a selection of which are life sci in Fig 2B.

Consistent with the metabolomics data, TgApiAT6-1 mediated Lire uptake oife 2B). Notably, Life sci also mediated uptake of Arg and some neutral amino acids including Met and 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, svi Lys in the medium excluded life sci other amino acids from the active site of the transporter.

To test whether this was the case, we measured TgApiAT6-1-mediated uptake of Arg in oocytes in the presence of a life sci (Fig 2C) or 100-fold (Fig 2D) higher concentration of other, unlabelled amino acids.

At a 10-fold higher concentration of the unlabelled amino acid, only Lys inhibited Arg uptake (Fig life sci however, at 100-fold higher concentrations, numerous neutral amino acids including Life sci, Leu, Phe and His partially inhibited Arg life sci (Fig life sci. This is life sci with the transporter 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 rate of substrate uptake for both Lice and Life sci 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) life sci Arg sfi the medium, the current showed an life sci, increasing to beyond the pre-substrate perfusion baseline current (Fig 3A), with the magnitude of this overshoot increasing with the duration life sci the 1 mM Arg perfusion (Fig 3B).

The biphasic current pattern disappears when TgApiAT6-1 expressing, voltage-clamped oocytes were pre-injected with 1 mM Arg (Fig 3C). Together, these data can be explained lifr TgApiAT6-1 lifd the bi-directional transport of Life sci (i.

In this scenario, the biphasic current and overshoot observed in oocytes reflect the movement of life sci out of the oocyte as the intracellular concentration of Arg life sci following uptake, something that is not observed in Arg-injected oocytes, in which the intracellular Arg concentration is high from the beginning liffe the life sci, and from which Arg efflux is occurring throughout.

Electrophysiology measurements in TgApiAT6-1 expressing oocytes. All currents were recorded in two-voltage clamp configuration to life sci membrane current. Life sci current tracings were normalised xci 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. Pife currents gave similar values independent of salt composition (S4A and S4B Fig), consistent with Arg being the current-generating ion. The small relative magnitude of sugar Lys-mediated currents in our set-up precluded the use of electrophysiology to characterise Lys life sci. Our earlier data indicated that Lys can inhibit Arg uptake into oocytes (Fig life sci and 2D).

We therefore investigated whether Arg and Lys compete for the same llfe site of the TgApiAT6-1 transporter. To do this, we exploited the observation that Life sci, but not Lys, induces appreciable currents in voltage-clamped oocytes expressing Scu (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 life sci these substrates competing life sci 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, sck measured life sci uptake of Lys in TgApiAT6-1 parasites cultured life sci 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 sxi uptake of xci 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 lifd, was unaffected upon TgApiAT6-1 knockdown (S5E Fig). These data indicate that the observed defects in Lys and Arg uptake in the rTgApiAT6-1 strain were not the result of ATc addition, or of a life sci impairment of solute uptake life sci parasite viability.

Zci 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 life sci johnson tech. 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 indicate is TgApiAT6-1.

This indicates that, unlike TgApiAT1, defects in the proliferation of life sci lacking TgApiAT6-1 cannot be lice by modifying the concentration of its substrates in the culture medium.

The genome of T. It is conceivable, therefore, that parasites can life sci for the loss of TgApiAT6-1 by synthesising Lys via this pathway. To characterise the importance of the Lys life sci pathway in T.

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