eng URI http://hdl.handle.net/10795/3236 βιολογία βιοχημεία κυτταρολογία υγεία Earlier, we identified mutations in the first transmembrane segment (TMS1) of UapA, a uric acid-xanthine transporter in Aspergillus nidulans, that affect its turnover and subcellular localization. Here, we use one of these mutations (H86D) and a novel mutation (I74D) as well as genetic suppressors of them, to show that TMS1 is a key domain for proper folding, trafficking and turnover. Kinetic analysis of mutants further revealed that partial misfolding and deficient trafficking of UapA does not affect its affinity for xanthine transport, but reduces that of uric acid and confers a degree of promiscuity towards the binding of other purines. This result strengthens the idea that subtle interactions among domains not directly involved in substrate binding refine the selectivity of UapA. Characterization of second-site suppressors of H86D revealed a genetic interaction of TMS1 with TMS3, the latter segment shown for the first time to be important for UapA function. Systematic mutational analysis of polar and conserved residues in TMS3 showed that Ser154 is crucial for UapA transport activity. Our results are in agreement with a topological model of UapA built on the recently published structure of UraA, a bacterial homolog of UapA. 14 pp. LOMv1.0 creator LOMv1.0 publisher LOMv1.0 Scientific Coordinator LOMv1.0 Project Executing Organisation 2011-06-29 Digital Library of the Operational Programme "Education and Lifelong Learning" abstract types Text Aspergillus nidulans Nucleobase ascorbate Transporter (NAT) family Suppressors Uric acid Xanthine Topological modeling Crystal structure Proteins UapA Μικροβιολογία Κρυσταλλογραφική δομή Πρωτεΐνες Ουρικό οξύ http://www.sciencedirect.com/science/article/pii/S002228361100670X 1529358 application/pdf http://repository.edulll.gr/edulll/bitstream/10795/3236/2/3236_1.64_%ce%94%ce%97%ce%9c_29_6_11.pdf URI http://hdl.handle.net/10795/3236 LOMv1.0creator 2016-05-19T07:59:14Z LOMv1.0validator 2016-05-19T07:59:14Z LOMv1.0 gre no no Copyright EYD-EPEDBM (Operational Programme "Education and Lifelong Learning")