Development
of a model system to study the topogenesis of the nucleobase-ascorbate
transporter family.
Areti Pantazopoulou,
Marina Koukaki, Anna Vlanti, George Diallinas
Faculty of Biology, University of Athens, Panepistimioupolis, Athens, 15781,
Greece (diallina@biol.uoa.gr)
Transporter trafficking and translocation in the plasma membrane is assisted by general secretory pathway chaperones and family-specific factors. We describe an approach to identify the protagonists involved in the topogenesis of UapA, the Aspergillus nidulans uric acid-xanthine transporter, which belongs to the ubiquitous Nucleobase-Ascorbate Transporter family. We constructed a strain lacking all genes encoding endogenous purine transporters while expressing functionally only a UapA-GFP chimeric protein. Using this strain, we investigated the possible role of a motif, in the first transmembrane section (TMS1) of all NAT proteins, as a cis-acting element involved in interactions with chaperones. We showed that substitutions of Q126 within this motif lead to problematic UapA translocation in the plasma membrane. We also obtained evidence that overexpression of TMS1 leads to a dominant negative phenotype compatible with interactions with factors involved in UapA topogenesis. Finally, using an appropriate genetic screen we selected uapB, a novel mutation affecting UapA function. We will discuss current studies investigating the role of TMS1 and UapB in UapA topogenesis.