GOLGI GDP-MANNOSE UPTAKE REQUIRES LEISHMANIA LPG2 - A MEMBER OF A EUKARYOTIC FAMILY OF PUTATIVE NUCLEOTIDE-SUGAR TRANSPORTERS

The synthesis of glycoconjugates within the secretory pathway of eukar yotes requires the provision of lumenal nucleotide-sugar substrates. T his is particularly important for eukaryotic microbes such as Leishman ia because they must synthesize considerable amounts of extracellular and cell surface glycoconjugates that play significant roles in the in fectious cycle. Here we used properly oriented sealed microsomes to ch aracterize lumenal uptake of GDP-Man in Leishmania donovani. In this s ystem, GDP-Man uptake was saturable with an apparent K-m for GDP-Man o f 0.3 mu M and facilitated its use as a donor substrate for lipophosph oglycan (LPG) synthesis, A lpg2(-) deletion mutant showed loss of GDP- Man but not UDP-Gal uptake, which was restored by introduction of the gene LPG2, Immunoelectron microscopy localized an active, epitope-tagg ed LPG2 protein to the Golgi apparatus. Thus, LPG2 is required for nuc leotide-sugar transport activity and probably encodes this Golgi trans porter. LPG2 belongs to a large family of eukaryotic genes that potent ially encode transporters with different substrate specificities and/o r cellular locations. In the future, the amenability of the Leishmania system to biochemical and genetic manipulation will assist in functio nal characterization of nucleotide-sugar transports from this and othe r eukaryotes. Furthermore, since LPG2 plays an important role in the L eishmania infectious cycle and mammalian cells lack a Golgi GDP-Man tr ansporter, this activity may offer a new target for chemotherapy.