PMID:18359771

Citation	Papakostas, K, Georgopoulou, E and Frillingos, S (2008) Cysteine-scanning analysis of putative helix XII in the YgfO xanthine permease: ILE-432 and ASN-430 are important. J. Biol. Chem. 283:13666-78
Abstract	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.
Links	PubMed Online version:10.1074/jbc.M800261200
Keywords	Amino Acid Sequence; Asparagine/chemistry; Binding Sites; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Ethylmaleimide/pharmacology; Gene Expression Regulation, Bacterial; Inhibitory Concentration 50; Isoleucine/chemistry; Kinetics; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/genetics; Models, Biological; Molecular Sequence Data; Nucleobase Transport Proteins/chemistry; Nucleobase Transport Proteins/genetics; Protein Conformation; Protein Structure, Secondary; Sequence Homology, Amino Acid
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