Aspartate 19 and Glutamate 121 Are Critical for Transport Function of the myo-Inositol/H+ Symporter fromLeishmania donovani

Andreas Seyfang,Michael P Kavanaugh,Scott M Landfear

doi:10.1074/jbc.272.39.24210

Andreas Seyfang, Michael P Kavanaugh + Show 1 more

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https://doi.org/10.1074/jbc.272.39.24210

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Abstract

The protozoan flagellate Leishmania donovani has an active myo-inositol/proton symporter (MIT), which is driven by a proton gradient across the parasite membrane. 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 acidic transmembrane residues for proton relay and inositol transport. MIT has only three charged amino acids within predicted transmembrane domains. Two of these residues, Asp19 (TM1) and Glu121 (TM4), appeared to be critical for transport function of MIT, with a reduction of inositol transport to about 2% of wild-type activity when mutated to the uncharged amides D19N or E121Q and 20% (D19E) or 4% (E121D) of wild-type activity for the conservative mutations that retained the charge. Immunofluorescence microscopy of oocyte cryosections showed that MIT mutants were expressed on the oocyte surface at a similar level as MIT wild type, confirming that these mutations affect transport function and do not prevent trafficking of the transporter to the plasma membrane. The proton uncouplers carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone and dinitrophenol inhibited inositol transport by 50-70% in the wild-type as well as in E121Q, despite its reduced transport activity. The mutant D19N, however, was stimulated about 4-fold by either protonophore and 2-fold by cyanide or increase of pH 7.5 to 8.5 but inhibited at pH 6.5. The conservative mutant D19E, in contrast, showed an inhibition profile similar to MIT wild type. We conclude that Asp19 and Glu121 are critical for myo-inositol transport, while the negatively charged carboxylate at Asp19 may be important for proton coupling of MIT.

Highlights

In kinetoplastid protozoa such as Leishmania myo-inositol plays an especially important role as the precursor for various inositol phospholipids found in the great majority of surface molecules in these parasites
Functional expression of myo-inositol transporter (MIT) in Xenopus laevis oocytes has revealed that it is an active myo-inositol/proton symporter driven by a proton gradient across the cell membrane [12]
Inositol Uptake in MIT Mutants—As shown in Fig. 2, alteration of either Asp19 (TM1) or Glu121 (TM4) from the charged carboxylate to the uncharged amide reduced transport activity to about 2% of wild-type activity when the mutants were expressed in Xenopus oocytes

Summary

Introduction

In kinetoplastid protozoa such as Leishmania myo-inositol plays an especially important role as the precursor for various inositol phospholipids found in the great majority of surface molecules in these parasites These include glycosyl-phosphatidylinositol-anchored surface proteins such as the major surface glycoprotein, gp63 [1, 2], or abundant inositol-containing glycolipids such as lipophosphoglycan [3] and glycoinositolphos-. Functional expression of MIT in Xenopus laevis oocytes has revealed that it is an active myo-inositol/proton symporter driven by a proton gradient across the cell membrane [12]. Extensive mutagenesis of lac permease has revealed only four amino acids essential for transport, and all are thought to be charged transmembrane residues [18]. We have used site-directed mutagenesis, in combination with functional expression of MIT mutants in X. laevis oocytes and overexpression in L. donovani, to investigate the significance of these acidic transmembrane residues in MIT for inositol uptake

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