A novel-type substrate-selectivity filter and ER-exit determinants in the UapA purine transporter.
We present a functional analysis of the last alpha-helical transmembrane segment (TMS12) of UapA, a uric acid-xanthine/H+ symporter in Aspergillus nidulans and member of the nucleobase-ascorbate transporter (NAT) family. First, we performed a systematic mutational analysis of residue F528, located in the middle of TMS12, which was known to be critical for UapA specificity. Substitution of F528 with non-aromatic amino acid residues (Ala, Thr, Ser, Gln, Asn) did not affect significantly the kinetics of UapA for its physiological substrates, but allowed high-capacity transport of several novel purines and pyrimidines. Allele-specific combinations of F528 substitutions with mutations in Q408, a residue involved in purine binding, led to an array of UapA molecules with different kinetic and specificity profiles. We propose that F528 plays the role of a novel-type selectivity filter, which, in conjunction with a distinct purine-binding site, control UapA-mediated substrate translocation. We further studied the role of TMS12 by analysing the effect of its precise deletion and chimeric molecules in which TMS12 was substituted with analogous domains from other NATs. The presence of any of the TMS12 tested was necessary for ER-exit while their specific amino acid composition affected the kinetics of chimeras.
Vlanti
Anna
A
Faculty of Biology, Department of Botany, University of Athens, Panepistimioupolis, Athens 15781, Greece.
Amillis
Sotiris
S
Koukaki
Marina
M
Diallinas
George
G
D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001233:Aspergillus nidulans / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C083852:UAPA protein, Aspergillus nidulans; D005973:Glutamine; D010649:Phenylalanine; D001216:Asparagine