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J Biol Chem, Vol. 275, Issue 8, 5687-5693, February 25, 2000

Four Conserved Cytoplasmic Sequence Motifs Are Important for Transport Function of the Leishmania Inositol/H⁺ Symporter^*

Andreas Seyfang and Scott M. Landfear^§

From the Department of Molecular Microbiology and Immunology, School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201

The protozoan Leishmania donovani has a myo-inositol/proton symporter (MIT) that is a member of a large sugar transporter superfamily. Active transport by MIT is driven by the proton electrochemical gradient across the parasite membrane, and MIT is a prototype for understanding the function of an active transporter in lower eukaryotes. MIT contains two duplicated 6- or 7-amino acid motifs within cytoplasmic loops, which are highly conserved among 50 members of the sugar transporter superfamily and are designated A₁, A₂ ((V)(D/E)(R/K)GR(R/K)), and B₁ (PESPRL), B₂ (VPETKG). In particular, the three acidic residues within these motifs, Glu¹⁸⁷(B₁), Asp³⁰⁰(A₂), and Glu⁴²⁹(B₂) in MIT, are highly conserved with 96, 78, and 96% amino acid identity within the analyzed members of this transporter superfamily ranging from bacteria, archaea, and fungi to plants and the animal kingdom. We have used site-directed mutagenesis in combination with functional expression of transporter mutants in Xenopus oocytes and overexpression in Leishmania transfectants to investigate the significance of these three acidic residues in the B₁, A₂, and B₂ motifs. Alteration to the uncharged amides greatly reduced MIT transport function to 23% (E187Q), 1.4% (D300N), and 3% (E429Q) of wild-type activity, respectively, by affecting V_max but not substrate affinity. Conservative mutations that retained the charge revealed a less pronounced effect on inositol transport with 39% (E187D), 16% (D300E) and 20% (E429D) remaining transport activity. Immunofluorescence microscopy of oocyte cryosections confirmed that MIT mutants were expressed on the oocyte surface in similar quantity to MIT wild type. The proton uncouplers carbonylcyanide-4-(trifluoromethoxy) phenylhydrazone and dinitrophenol inhibited inositol transport by 50-70% in the wild type as well as in E187Q, D300N, and E429Q, despite their reduced transport activities, suggesting that transport in these mutants is still proton-coupled. Furthermore, temperature-dependent uptake studies showed an increased Arrhenius activation energy for the B₁-E187Q and the B₂-E429Q mutants, which supports the idea of an impaired transporter cycle in these mutants. We conclude that the conserved acidic residues Glu¹⁸⁷, Asp³⁰⁰, and Glu⁴²⁹ are critical for transport function of MIT.

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