Aspartate 19 and glutamate 121 are critical for transport function of the myo-inositol/H+ symporter from Leishmania donovani.

摘要: 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 Xenopus oocytes and overexpression transfectants to investigate significance acidic transmembrane residues for relay inositol transport. MIT only three charged amino acids within predicted domains. Two these residues, Asp19 (TM1) Glu121 (TM4), appeared be critical transport function MIT, reduction about 2% wild-type activity when mutated uncharged amides D19N or E121Q 20% (D19E) 4% (E121D) conservative mutations that retained charge. Immunofluorescence microscopy oocyte cryosections showed were expressed on surface at similar level as wild type, confirming affect do not prevent trafficking plasma uncouplers carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone dinitrophenol inhibited 50-70% well E121Q, despite its reduced activity. mutant D19N, however, was stimulated 4-fold either protonophore 2-fold cyanide increase pH 7.5 8.5 but 6.5. D19E, contrast, inhibition profile type. conclude are myo-inositol transport, while negatively carboxylate may important coupling MIT.