Four conserved cytoplasmic sequence motifs are important for transport function of the Leishmania inositol/H+ symporter

Andreas Seyfang, Scott M. Landfear

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20 Scopus citations


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)ΦGR(R/K)), and B1 (PESPRΦL), 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 preton-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.

Original languageEnglish (US)
Pages (from-to)5687-5693
Number of pages7
JournalJournal of Biological Chemistry
Issue number8
StatePublished - Feb 25 2000

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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