Unlike all other organisms, parasitic protozoa of the family Trypanosomatidae maintain a large cellular pool of proline that, together with the alanine pool, serve as alternative carbon sources as well as reservoirs of organic osmolytes. These reflect adaptation to their insect vectors whose haemolymphs are exceptionally rich in the two amino acids. In the present study we identify and characterize a new neutral amino acid transporter, LdAAP24, that translocates proline and alanine across the Leishmania donovani plasma membrane. This transporter fulfils multiple functions: it is the sole supplier for the intracellular pool of proline and contributes to the alanine pool; it is essential for cell volume regulation after osmotic stress; and it regulates the transport and homoeostasis of glutamate and arginine, none of which are its substrates. Notably, we provide evidence that proline and alanine exhibit different roles in the parasitic response to hypotonic shock; alanine affects swelling, whereas proline influences the rate of volume recovery. On the basis of our data we suggest that LdAAP24 plays a key role in parasite adaptation to its varying environments in host and vector, a phenomenon essential for successful parasitism.

Long N-terminal tails of amino acid transporters are known to act as sensors of the internal pool of amino acids and as positive regulators of substrate flux rate. In this study we establish that N-termini of amino acid transporters can also determine substrate specificity. We show that due to alternative trans splicing, the human pathogen Leishmania naturally expresses two variants of the proline/alanine transporter, one 18 amino acid shorter than the other. We demonstrate that the longer variant (LdAAP24) translocates both proline and alanine, whereas the shorter variant (∆18LdAAP24) translocates just proline. Remarkably, co-expressing the hydrophilic N-terminal peptide of the long variant with ∆18LdAAP24 was found to recover alanine transport. This restoration of alanine transport could be mediated by a truncated N-terminal tail, though truncations exceeding half of the tail length were no longer functional. Taken together, the data indicate that the first 18 amino acids of the negatively charged N-terminal LdAAP24 tail are required for alanine transport and may facilitate the electrostatic interactions of the entire negatively charged N-terminal tail with the positively charged internal loops in the transmembrane domain, as this mechanism has been shown to underlie regulation of substrate flux rate for other transporters.

Leishmania are obligatory intracellular parasites that cycle between the sand fly midgut (extracellular promastigotes) and mammalian macrophage phagolysosomes (intracellular amastigotes). They have developed mechanisms of adaptation to the distinct environments of host and vector that favor utilization of both proline and alanine. LdAAP24 is the L. donovani proline-alanine transporter. It is a member of Leishmania system A that translocates neutral amino acids. Since system A is promastigote-specific, we aimed to assess whether LdAAP24 is also expressed exclusively in promastigotes. Herein, we established that upon exposing L. donovani promastigotes to amastigote differentiation signal (pH 5.5 and 37 °C), parasites rapidly and completely degrade LdAAP24 protein in both axenic and in spleen-derived amastigotes. In contrast, LdAAP24 mRNA remained unchanged throughout differentiation. Addition of either MG132 or Bafilomycin A1 partially inhibited LdAAP24 protein degradation, indicating a role for both lysosome- and proteasome-mediated degradation. This work provides the first evidence for post-translational regulation of stage-specific expression of LdAAP24.