title: Comprehensive examination of charged intramembrane residues in a nucleoside transporter.
abstract: 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.
authors.affiliation: Department of Molecular Microbiology and Immunology and Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, USA.
authors.forename: Raquel
authors.initials: R
authors.lastname: Valdés
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: Buddy
authors.initials: B
authors.lastname: Ullman
authors.forename: Scott M
authors.initials: SM
authors.lastname: Landfear
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001692:Biological Transport; D002462:Cell Membrane / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D007893:Leishmania donovani / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D049990:Membrane Transport Modulators / Q000378:metabolism; D033703:Nucleoside Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D013379:Substrate Specificity
chemical_list: C114230:LdNT1 protein, Leishmania donovani; D049990:Membrane Transport Modulators; D033703:Nucleoside Transport Proteins; D015800:Protozoan Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D008239:Lysine