The nucleobase-ascorbate transporter (NAT) signature motif in UapA defines the function of the purine translocation pathway.
UapA, a member of the NAT/NCS2 family, is a high affinity, high capacity, uric acid-xanthine/H+ symporter of Aspergillus nidulans. We have previously presented evidence showing that a highly conserved signature motif ([Q/E/P]408-N-X-G-X-X-X-X-T-[R/K/G])417 is involved in UapA function. Here, we present a systematic mutational analysis of conserved residues in or close to the signature motif of UapA. We show that even the most conservative substitutions of residues Q408, N409 and G411 modify the kinetics and specificity of UapA, without affecting targeting in the plasma membrane. Q408 substitutions show that this residue determines both substrate binding and transport catalysis, possibly via interactions with position N9 of the imidazole ring of purines. Residue N409 is an irreplaceable residue necessary for transport catalysis, but is not involved in substrate binding. Residue G411 determines, indirectly, both the kinetics (K(m), V) and specificity of UapA, probably due to its particular property to confer local flexibility in the binding site of UapA. In silico predictions and a search in structural databases strongly suggest that the first part of the NAT signature motif of UapA (Q(408)NNG(411)) should form a loop, the structure of which is mostly affected by mutations in G411. Finally, substitutions of residues T416 and R417, despite being much better tolerated, can also affect the kinetics or the specificity of UapA. Our results show that the NAT signature motif defines the function of the UapA purine translocation pathway and strongly suggest that this might occur by determining the interactions of UapA with the imidazole part of purines.
Koukaki
Marina
M
Faculty of Biology, Department of Botany, University of Athens, Panepistimioupolis, Athens 15781, Greece.
Vlanti
Anna
A
Goudela
Sophia
S
Pantazopoulou
Areti
A
Gioule
Harris
H
Tournaviti
Stella
S
Diallinas
George
G
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001692:Biological Transport; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D030562:Databases, Protein; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry; D020128:Inhibitory Concentration 50; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000502:physiology*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011687:Purines / Q000737:chemistry / Q000378:metabolism; D012984:Software; D013379:Substrate Specificity; D019820:Xanthine / Q000737:chemistry
D017931:DNA Primers; D005656:Fungal Proteins; D007093:Imidazoles; D026901:Membrane Transport Proteins; D011687:Purines; C083852:UAPA protein, Aspergillus nidulans; D049452:Green Fluorescent Proteins; D019820:Xanthine; C029899:imidazole