טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentSchlisselberg Doreen
SubjectAssessment of the Regulatory Role of LdAAP24 N-terminus in
Proline and Alanine Transport
DepartmentDepartment of Biology
Supervisor Professor Emeritus Dan Zilberstein
Full Thesis textFull thesis text - English Version


Abstract

________ Parasitic protozoa of the genus Leishmania are the causative agents of a wide range of cutaneous, mucocutaneous and visceral diseases in humans. During their life, parasites encounter changes in the environment from relatively alkaline, sugar- and amino acid-rich, to acidic, fatty acid- and amino acid-rich environments. Leishmania cells have developed mechanisms of adaptation that favor utilization of amino acids. Proline and alanine are highly abundant in the hemolymph of flying insects, and are used as metabolites for flight-muscles. Leishmania promastigotes have adapted to this environment and use proline and alanine as carbon sources as well as enriching the intracellular amino-acid pool with proline and alanine in order to maintain osmoregulation. 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 work 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 donovani naturally expresses two variants of LdAAP24, the proline/alanine transporter- one variant is 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. We also show that further truncating the N-terminus of LdAAP24 did not affect proline transport. Remarkably, co-expressing the hydrophilic N-terminal peptide of the long variant with ∆18LdAAP24 or with ∆44LdAAP24 was found to fully recover alanine transport. Truncations exceeding the first 44 amino-acids of the N-terminal tail were no longer functional, and could not translocate neither proline nor alanine, albeit intact localization to the parasite’s plasma membrane. Co-expression of the full-length soluble N-terminus could not recover proline or alanine transport in these parasites. Deleting the element between amino-acids 44 and 54, ∆(44-54)LdAAP24 could not translocate alanine, and recovery in proline transport was partial (half of the recovery in (∆18LdAAP24 and (∆44LdAAP24), suggesting that this region serves as a regulatory element for proline specificity in LdAAP24. 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. We also indicate that the N-terminus interacts with cytosolic proteins in Leishmania donovani, suggesting a coordinated mechanism of alanine and proline transport regulation.  We hypothesize that the two natural variants of LdAAP24 balance alanine uptake in the alanine-rich environment in the insect vector. This work provides the first evidence that an intracellular N-terminal tail regulate specificity of a transporter to its substrates._