Four Conserved Cytoplasmic Sequence Motifs Are Important for Transport Function of the LeishmaniaInositol/H+ Symporter

Andreas Seyfang,Scott M Landfear

doi:10.1074/jbc.275.8.5687

Andreas Seyfang, Scott M Landfear

Open Access

https://doi.org/10.1074/jbc.275.8.5687

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Abstract

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 A(1), A(2) ((V)(D/E)(R/K)PhiGR(R/K)), and B(1) (PESPRPhiL), B(2) (VPETKG). In particular, the three acidic residues within these motifs, Glu(187)(B(1)), Asp(300)(A(2)), and Glu(429)(B(2)) 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 B(1), A(2), and B(2) 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 B(1)-E187Q and the B(2)-E429Q mutants, which supports the idea of an impaired transporter cycle in these mutants. We conclude that the conserved acidic residues Glu(187), Asp(300), and Glu(429) are critical for transport function of MIT.

Highlights

Parasitic protozoa of the genus Leishmania are important human pathogens [1] with more than 200 million people exposed to infection worldwide and an incidence of over 500,000 new cases annually of fatal visceral leishmaniasis [2]
Four Conserved Cytoplasmic Sequence Motifs—In order to identify functionally important sequence motifs in myo-inositol/proton symporter (MIT), we have compared the MIT amino acid sequence with 48 representative members of the sugar transporter superfamily that represent each kingdom of living organisms, including bacteria, archaea, fungi, plants and animals (Fig. 2)
MIT contains two duplicated 6- or 7-amino acid motifs within cytoplasmic loops that are highly conserved between the 50 transporter proteins analyzed, designated A1 (AAFGRR), B1 (PE187SPRWL), A2 (VD300RFGRR), and B2 (AVE429TKG) in MIT, respectively (Fig. 1 and Table II)

Summary

Introduction

Parasitic protozoa of the genus Leishmania are important human pathogens [1] with more than 200 million people exposed to infection worldwide and an incidence of over 500,000 new cases annually of fatal visceral leishmaniasis [2]. For myo-inositol uptake in mammals, a Naϩ/myo-inositol transporter SMIT has been identified [14] that is unrelated to the above sugar transporter superfamily but closely related to the intestinal Naϩ/glucose transporter SGLT [15] and related sodium cotransporters like the vitamin transporter SMVT [16], which are members of the sodium/solute symporter superfamily [17] This difference between the mammalian and parasite myoinositol transporters, together with the high abundance of inositol in flagellate surface molecules, suggests that MIT would make an attractive target for parasite-specific drug design. Nucleotide position (bold italics) is numbered in sense orientation according to Langford et al [7]

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