ASPARTATE-19 AND GLUTAMATE-121 ARE CRITICAL FOR TRANSPORT FUNCTION OFTHE MYO-INOSITOL H+ SYMPORTER FROM LEISHMANIA-DONOVANI/

The protozoan flagellate Leishmania donovani has an active myo-inosito l/proton symporter (MIT), which is driven by a proton gradient across the parasite membrane. We have used site-directed mutagenesis in combi nation with functional expression of transporter mutants in Xenopus oo cytes and overexpression in Leishmania transfectants to investigate th e significance of acidic transmembrane residues for proton relay and i nositol transport, MIT has only three charged amino acids within predi cted transmembrane domains. Two of these residues, Asp(19) (TM1) and G lu(121) (TM4), appeared to be critical for transport function of MIT, with a reduction of inositol transport to about 2% of wild-type activi ty when mutated to the uncharged amides D19N or E121Q and 20% (D19E) o r 4% (E121D) of wild-type activity for the conservative mutations that retained tare charge. Immunofluorescence microscopy oocyte cryosectio ns showed that MIT mutants were expressed on the oocyte surface at a s imilar level as MIT wild type, confirming that these mutations that tr ansport function and do not prevent trafficking of the transporter to the plasma membrane, The proton uncouplers carbonylcyanide-4-(trifluor omethoxy)phenylhydrazone and dinitrophenol inhibited inositol transpo rt 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 bg cyanide or increase of pH 7. 5 to 8.5 but inhibited at pH 6.5. The conservative mutant D19E, ire co ntrast, showed an inhibition profile similar to MIT wild type. We conc lude that Asp(19) and Glu(121) are critical for myo-inositol transport , while the negatively charged carboxylate at Asp(19) may be important for proton coupling of MIT.