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J. Biol. Chem., Vol. 283, Issue 20, 13666-13678, May 16, 2008

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Cysteine-scanning Analysis of Putative Helix XII in the YgfO Xanthine Permease

ILE-432 AND ASN-430 ARE IMPORTANT^*

From the Laboratory of Biological Chemistry, University of Ioannina Medical School, 45110 Ioannina, Greece

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 µM, 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.

Received for publication, January 10, 2008

^* This paper is part of the 05NONEU547 research project, implemented within the framework of "Collaborations with Research and Technology Organizations outside Europe" and co-financed by National and Community Funds (25% from the Greek Ministry of Development, Secretariat for Research and Technology, and 75% from the European Union European Social Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.

¹ To whom correspondence should be addressed: University of Ioannina Medical School, Laboratory of Biological Chemistry, 45110 Ioannina, Greece. Tel.: 30-26510-97814; Fax: 30-26510-97868; E-mail: efriligo{at}cc.uoi.gr.