The first transmembrane segment (TMS1) of UapA contains determinants necessary for expression in the plasma membrane and purine transport.
UapA, a member of the NAT/NCS2 family, is a high affinity, high capacity, uric acid-xanthine/H+ symporter in Aspergillus nidulans. Determinants critical for substrate binding and transport lie in a highly conserved signature motif downstream from TMS8 and within TMS12. Here we examine the role of TMS1 in UapA biogenesis and function. First, using a mutational analysis, we studied the role of a short motif (Q85H86), conserved in all NATs. Q85 mutants were cryosensitive, decreasing (Q85L, Q85N, Q85E) or abolishing (Q85T) the capacity for purine transport, without affecting physiological substrate binding or expression in the plasma membrane. All H86 mutants showed nearly normal substrate binding affinities but most (H86A, H86K, H86D) were cryosensitive, a phenotype associated with partial ER retention and/or targeting of UapA in small vacuoles. Only mutant H86N showed nearly wild-type function, suggesting that His or Asn residues might act as H donors in interactions affecting UapA topology. Thus, residues Q85 and H86 seem to affect the flexibility of UapA, in a way that affects either transport catalysis per se (Q85), or expression in the plasma membrane (H86). We then examined the role of a transmembrane Leu Repeat (LR) motif present in TMS1 of UapA, but not in other NATs. Mutations replacing Leu with Ala residues altered differentially the binding affinities of xanthine and uric acid, in a temperature-sensitive manner. This result strongly suggested that the presence of L77, L84 and L91 affects the flexibility of UapA substrate binding site, in a way that is necessary for high affinity uric acid transport. A possible role of the LR motif in intramolecular interactions or in UapA dimerization is discussed.
Pantazopoulou
Areti
A
Faculty of Biology, Department of Botany, University of Athens, Panepistimioupolis, Athens, Greece.
Diallinas
George
G
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D004252:DNA Mutational Analysis; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011687:Purines / Q000378:metabolism*
D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D011687:Purines; C083852:UAPA protein, Aspergillus nidulans