The Leishmania GDP-Mannose Transporter Is an Autonomous, Multi-specific, Hexameric Complex of LPG2 Subunits
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- 作者：Kyoungja Hong ; Deqin Ma ; Stephen M. Beverley ; and Salvatore J. Turco
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：February 29, 2000
- 年：2000
- 卷：39
- 期：8
- 页码：2013 - 2022
- 全文大小：317K
- 年卷期：v.39,no.8(February 29, 2000)
- ISSN：1520-4995
文摘
LPG2 (a gene involved in lipophosphoglycan assembly) encodes the Golgi GDP-Man transporterof the protozoan parasite Leishmania and is a defining member of a new family of eukaryotic nucleotide-sugar transporters (NSTs). Although NST activities are widespread, mammalian cells lack a GDP-ManNST, thereby providing an ideal heterologous system for probing the LPG2 structure and activity. LPG2expression constructs introduced into either mammalian cells or a Leishmania lpg2- mutant conferredGDP-Man, GDP-Ara, and GDP-Fuc (in Leishmania only) uptake in isolated microsomes. LPG2 is thefirst NST to be associated with multiple substrate specificities. Uptake activity showed latency, exhibitedan antiport mechanism of transport with GMP, and was susceptible to the anion transport inhibitor DIDS.The apparent Km for GDP-Man uptake was similar in transfected mammalian cells (12.2 
M) or Leishmania(6.9 M). Given the evolutionary distance between protozoans and vertebrates, these data suggest thatLPG2 functions autonomously to provide transporter activity. Using epitope-tagged LPG2 proteins, weshowed the existence of hexameric LPG2 complexes by immunoprecipitation experiments, glycerol gradientcentrifugation, pore-limited native gel electrophoresis, and cross-linking experiments. This provides strongbiochemical evidence for a multimeric complex of NSTs, a finding with important implications to thestructure and specificity of NSTs in both Leishmania and other organisms. Inhibition of essentialGDP-Man uptake in fungal and protozoan systems offers an attractive target for potential chemotherapy.
M) or Leishmania(6.9 M). Given the evolutionary distance between protozoans and vertebrates, these data suggest thatLPG2 functions autonomously to provide transporter activity. Using epitope-tagged LPG2 proteins, weshowed the existence of hexameric LPG2 complexes by immunoprecipitation experiments, glycerol gradientcentrifugation, pore-limited native gel electrophoresis, and cross-linking experiments. This provides strongbiochemical evidence for a multimeric complex of NSTs, a finding with important implications to thestructure and specificity of NSTs in both Leishmania and other organisms. Inhibition of essentialGDP-Man uptake in fungal and protozoan systems offers an attractive target for potential chemotherapy.