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 eukaryotes requires the provision of lumenal nucleotide-sugar substrates. This is particularly important for eukaryotic microbes such as Leishmania because they must synthesize considerable amounts of extracellular and cell surface glycoconjugates that play significant roles in the infectious cycle. Here we used properly oriented sealed microsomes to characterize lumenal uptake of GDP-Man in Leishmania donovani. In this system, GDP-Man uptake was saturable with an apparent Km for GDP-Man of 0.3 microM and facilitated its use as a donor substrate for lipophosphoglycan (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-tagged LPG2 protein to the Golgi apparatus. Thus, LPG2 is required for nucleotide-sugar transport activity and probably encodes this Golgi transporter. LPG2 belongs to a large family of eukaryotic genes that potentially encode transporters with different substrate specificities and/or cellular locations. In the future, the amenability of the Leishmania system to biochemical and genetic manipulation will assist in functional characterization of nucleotide-sugar transports from this and other eukaryotes. Furthermore, since LPG2 plays an important role in the Leishmania infectious cycle and mammalian cells lack a Golgi GDP-Man transporter, this activity may offer a new target for chemotherapy.
Ma
D
D
Department of Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky 40536, USA.
Russell
D G
DG
Beverley
S M
SM
Turco
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D002855:Chromatography, Thin Layer; D006028:Glycosphingolipids / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism*; D007893:Leishmania donovani; D008565:Membrane Proteins / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D014162:Transfection
D006028:Glycosphingolipids; C095909:LPG2 protein, Leishmania; D008565:Membrane Proteins; D015800:Protozoan Proteins; D006155:Guanosine Diphosphate Mannose