Cysteine-scanning analysis of putative helix XII in the YgfO xanthine permease: ILE-432 and ASN-430 are important.
Transmembrane helix XII of UapA, the major fungal homolog of the nucleobase-ascorbate transporter (NAT/NCS2) family, has been proposed to contain an aromatic residue acting as a purine-selectivity filter, distinct from the binding site. To analyze the role of helix XII more systematically, we employed Cys-scanning mutagenesis of the Escherichia coli xanthine-specific homolog YgfO. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence 419ILPASIYVLVENPICAGGLTAILLNIILPGGY450 (the putative helix XII is underlined) was replaced individually with Cys. Of the 32 single-Cys mutants, 25 accumulate xanthine to 80-130% of the steady state observed with C-less YgfO, six (P421C, S423C, I424C, Y425C, L427C, G436C) accumulate to low levels (15-40%), and I432C is inactive. Immunoblot analysis shows that P421C and I432C display low expression in the membrane. Extensive mutagenesis reveals that replacement of Ile-432 with equally or more bulky side chains abolishes active transport without affecting expression, whereas replacement with smaller side chains allows activity but impairs affinity for the analogues 1-methyl and 6-thioxanthine. Only three of the single-Cys mutants of helix XII (V426C, N430C, and N443C) are sensitive to inactivation by N-ethylmaleimide. N430C is highly sensitive, with an IC50 of 10 microm, and is completely protected against inactivation in the presence of 2-thioxanthine, a high affinity substrate analogue. Other xanthine analogues are poorly bound by N430C, whereas replacement of Asn-430 with Thr inactivates the permease. The findings suggest that Ile-432 and Asn-430 of helix XII are crucial for purine uptake and affinity, and Asn-430 is probably at the vicinity of the binding site.
Papakostas
Konstantinos
K
Laboratory of Biological Chemistry, University of Ioannina Medical School, 45110 Ioannina, Greece.
Georgopoulou
Ekaterini
E
Frillingos
Stathis
S
D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial; D020128:Inhibitory Concentration 50; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics*; D008954:Models, Biological; D008969:Molecular Sequence Data; D033702:Nucleobase Transport Proteins / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D033702:Nucleobase Transport Proteins; C529797:YgfO protein, E coli; D007532:Isoleucine; D001216:Asparagine; D005033:Ethylmaleimide