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J. Biol. Chem., Vol. 281, Issue 32, 22647-22655, August 11, 2006

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Comprehensive Examination of Charged Intramembrane Residues in a Nucleoside Transporter^*

Raquel Valdés, Wei Liu, Buddy Ullman, and Scott M. Landfear¹

From the Departments of Molecular Microbiology and Immunology and Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239

Permeases of the equilibrative nucleoside transporter family mediate the uptake of nucleosides and/or nucleobases in a diverse array of eukaryotes and transport a host of drugs used for treatment of cancer, heart disease, AIDS, and parasitic infections. To identify residues that play central roles in transport function, we have systematically substituted by site-directed mutagenesis all the charged residues located within predicted transmembrane domains of the Leishmania donovani nucleoside transporter 1.1, LdNT1.1, which transports adenosine and the pyrimidine nucleosides. Substitution of three of these ten residues by uncharged amino acids resulted in loss of >95% transport activity, and we hence designated them "key" residues. These amino acids were Glu⁹⁴, Lys¹⁵³, and Arg⁴⁰⁴ located in transmembrane domains 2, 4, and 9, respectively. In addition, previous studies on the related LdNT2 inosine/guanosine transporter identified the highly conserved Asp³⁸⁹ and Arg³⁹³ (equivalent to Asp³⁷⁴ and Arg³⁷⁸ in LdNT1.1) in transmembrane domain 8 as key residues. Among these residues, the mutants in Arg³⁹³ (LdNT2) and Arg⁴⁰⁴ were strongly impaired in trafficking to the plasma membrane, but the other mutants were expressed with high to moderate efficiency at the cell surface, indicating that their mutation impaired transport activity per se. A conservative K153R substitution exhibited a change in substrate specificity, acquiring the ability to transport inosine, a nucleoside that is not a substrate for the wild-type LdNT1.1 permease. These results imply that the Glu⁹⁴, Lys¹⁵³, and Asp³⁷⁴ residues may play central roles in the mechanism of substrate translocation in LdNT1.1.

Received for publication, March 13, 2006 , and in revised form, May 5, 2006.

^* This work was supported by Grants AI 25920 and AI 44138 (to S. M. L.) and AI 23682 (to B. U.) from the National Institutes of Health and by a postdoctoral fellowship 0325453Z (to R. V.) from the American Heart Association, Pacific Mountain Chapter. 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.

¹ To whom correspondence should be addressed: Dept. of Molecular Microbiology and Immunology, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97239. Tel.: 503-494-2426; Fax: 503-494-6862; E-mail: landfear{at}ohsu.edu.

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