The synthesis oi'glytoconjugatcs within the secretory pathway of eukaryotes requires the provision of lumenal nucleotide-sugar substrates. This is particularly important for l.i'ishmunia because their cell surface giycoconjugates play significant roles in the infectious cycle. We used properly oriented sealed microsomes to characterize lumenal uptake of'GDP-Man in J.. dimovani. In this system. GDP-Man uptake was saturable with un apparent Krll of 0.3 uM and facilitated its use as a donor substrate for lipophosphoglycan (LPG) synthesis. A lps>2deletion mutant showed loss of GDP-Man but not UDPGal uptake, which was restored by introduction o( I.l'(i2 gene. An active I.PG2 protein was localized to Golgi. Thus, I.FH2 is required for nucleotidesugar (NS) transport and probably encodes this Golgi transporter. The overexpressed EPG2 protein in IIEK cells was found to be active. LPG2 belongs to a large family of eukaryotic genes that potentially encode different NS transporters. The amenability of the Leishmania system to biochemical and genetic manipulation will assist in functional characterization of NS transporters from this and other eukaryotes. Since 1.PC2 plays an important role in the Lcithmania infectious cycle and mammalian cells lack a Golgi GDP-Man transporter, this activity may offer a new target for chemotherapy. Ibis work uas supported by National Institute of Health, USA.
|State||Published - 1997|