The Leishmania GDP-mannose transporter is an autonomous, multi-specific, hexameric complex of LPG2 subunits

LPG2 (a gene involved in lipophosphoglycan assembly) encodes the Golgi GDP- Man transporter of the protozoan parasite Leishmania and is a defining memb er of a new family of eukaryotic nucleotide-sugar transporters (NSTs). Alth ough NST activities are widespread, mammalian cells lack a GDP-Man NST, the reby providing an ideal heterologous system for probing the LPG2 structure and activity. LPG2 expression constructs introduced into either mammalian c ells or a Leishmania lpg2(-) mutant conferred GDP-Man, GDP-Ara, and GDP-Fuc (in Leishmania only) uptake in isolated microsomes. LPG2 is the first NST to be associated with multiple substrate specificities. Uptake activity sho wed latency, exhibited an antiport mechanism of transport with GMP, and was susceptible to the anion transport inhibitor DIDS. The apparent K-m for GD P Man uptake was similar in transfected mammalian cells (12.2 mu M) or Leis hmania (6.9 mu M). Given the evolutionary distance between protozoans and v ertebrates, these data suggest that LPG2 functions autonomously to provide transporter activity. Using epitope-tagged LPG2 proteins, we showed the exi stence of hexameric LPG2 complexes by immunoprecipitation experiments, glyc erol gradient centrifugation, pore-limited native gel electrophoresis, and cross-linking experiments. This provides strong biochemical evidence for a multimeric complex of NSTs, a finding with important implications to the st ructure and specificity of NSTs in both Leishmania and other organisms. Inh ibition of essential GDP-Man uptake in fungal and protozoan systems offers an attractive target for potential chemotherapy.