A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and catalytic activity is essential in parasitic Leishmania major

Glycoconjugates play major roles in the infectious cycle of the trypanosomatid parasite Leishmania. Recently we showed that GDP-Fucose synthesis is essential, although fucosylated glycoconjugates have not been reported in Leishmania major. The Leishmania genome predicts at least five candidate fucosyltransferases, four of which appear targeted to the secretory pathway; SCA1 and SCA2 play a role in side-chain modifications of the abundant surface glycoconjugate lipophosphoglycan, while gene deletion studies here showed that FUT2 and SCAL were not essential. Unlike most eukaryotic glycosyltransferases, the predicted α 1-2 fucosyltransferase encoded by FUT1 localized to the mitochondrion. Enzymatic assays of tagged proteins expressed in vivo or of purified recombinant FUT1 showed it to have fucosyltransferase activity, with a relative broad substrate specificity including glycans and peptide substrates. A quantitative plasmid segregation assay, expressing FUT1 from the multicopy episomal pXNG vector in a chromosomal null Δfut1- background established that FUT1 is essential. We used plasmid shuffling to confirm that both enzymatic activity and mitochondrial localization were essential for viability, comparing import-blocked or catalytically inactive enzymes respectively. Unexpectedly a single rare Δfut1s mutant was obtained, which showed severe growth defects accompanied by mitochondrial dysfunction and loss, all of which were restored upon FUT1 re-expression. Thus, FUT1 along with the similar Trypanosoma brucei enzyme TbFUT1 (1) joins the eukaryotic O-GlcNAc transferases as one of the few glycosyltransferases acting within the mitochondrion. Current work is now oriented towards identifying the Leishmania fucosylated targets therein.