טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLiat Iancovici
SubjectThe Hydrophilic N-Terminus of the Proline-Alanine
Transporter in the Human Pathogen Leishmania
Regulates Substrate Specificity
DepartmentDepartment of Biology
Supervisor Professor Emeritus Zilberstein Dan
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.  Leishmania are obligatory intracellular parasites that cycle between the sand fly midgut (extracellular promastigotes) and mammalian macrophage phagolysosomes (intracellular amastigotes). During their life cycle, 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 insects and are utilized as a source of muscular metabolic energy for flight.  Leishmania promastigotes have adapted to this environment and use these amino acids as alternative sources of carbon and as osmolytes. LdAAP24 is the L. donovani proline-alanine transporter. Our laboratory had previously established that N termini of amino acid transporters can determine substrate specificity. The first 18 amino acids of the N-terminal LdAAP24 tip were previously proven to be required for alanine transport. LdAAP24 was shown to have two isoforms which are functionally different. The full-length LdAAP24 translocates both proline and alanine while the 18 amino acid shorter LdAAP24 transports only proline. Moreover, unpublished data have shown that the sequence at the tip of the N terminus is important for LdAAP24 substrate specificity and not only its length. In this work, we further investigated the mechanism by which LdAAP24 N terminus control alanine and proline transport. We showed that three phosphorylation sites located within the first 18 amino acids of the LdAAP24 N terminus - Serine 6, threonine 7 and Serine 9, have a role in regulating transport activity. We demonstrate that phosphorylation of Threonine 7 is essential for alanine transport, while Serine 6 is essential for both proline and alanine transport activity by LdAAP24.  Interestingly, a bioinformatics analysis indicated that the full length N terminus is a feature of the old world while the new world have lost this trait. We also show that LdAAP24 forms a complex of four subunits; two of LdAAP24 and two of proline-dehydrogenases. Additionally, we provide evidence that the length of the N terminus affects complex size. Our findings bring to light an additional level of regulation, phosphorylation sites located within the N termini that are responsible for regulating transport activity. Furthermore, uncovering LdAAP24 is found in the plasma membrane as a complex, could be the first step to unraveling the mechanism of transport activity regulation by the N terminus.