Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Oct;15(10):5508–5515. doi: 10.1128/mcb.15.10.5508

Functional expression of a myo-inositol/H+ symporter from Leishmania donovani.

M E Drew 1, C K Langford 1, E M Klamo 1, D G Russell 1, M P Kavanaugh 1, S M Landfear 1
PMCID: PMC230801  PMID: 7565702

Abstract

The vast majority of surface molecules in such kinetoplastid protozoa as members of the genus Leishmania contain inositol and are either glycosyl inositol phospholipids or glycoproteins that are tethered to the external surface of the plasma membrane by glycosylphosphatidylinositol anchors. We have shown that the biosynthetic precursor for these abundant glycolipids, myo-inositol, is translocated across the parasite plasma membrane by a specific transporter that is structurally related to mammalian facilitative glucose transporters. This myo-inositol transporter has been expressed and characterized in Xenopus laevis oocytes. Two-electrode voltage clamp experiments demonstrate that this protein is a sodium-independent electrogenic symporter that appears to utilize a proton gradient to concentrate myo-inositol within the cell. Immunolocalization experiments with a transporter-specific polyclonal antibody reveal the presence of this protein in the parasite plasma membrane.

Full Text

The Full Text of this article is available as a PDF (616.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Arriza J. L., Kavanaugh M. P., Fairman W. A., Wu Y. N., Murdoch G. H., North R. A., Amara S. G. Cloning and expression of a human neutral amino acid transporter with structural similarity to the glutamate transporter gene family. J Biol Chem. 1993 Jul 25;268(21):15329–15332. [PubMed] [Google Scholar]
  3. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  4. Bordier C., Etges R. J., Ward J., Turner M. J., Cardoso de Almeida M. L. Leishmania and Trypanosoma surface glycoproteins have a common glycophospholipid membrane anchor. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5988–5991. doi: 10.1073/pnas.83.16.5988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Caspary W. F., Crane R. K. Active transport of myo-inositol and its relation to the sugar transport system in hamster small intestine. Biochim Biophys Acta. 1970 Apr 21;203(2):308–316. doi: 10.1016/0005-2736(70)90145-8. [DOI] [PubMed] [Google Scholar]
  6. Chang C. S., Chang K. P. Monoclonal antibody affinity purification of a Leishmania membrane glycoprotein and its inhibition of leishmania-macrophage binding. Proc Natl Acad Sci U S A. 1986 Jan;83(1):100–104. doi: 10.1073/pnas.83.1.100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cruz A., Coburn C. M., Beverley S. M. Double targeted gene replacement for creating null mutants. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7170–7174. doi: 10.1073/pnas.88.16.7170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Culbertson M. R., Donahue T. F., Henry S. A. Control of inositol biosynthesis in Saccharomyces cerevisiae: properties of a repressible enzyme system in extracts of wild-type (Ino+) cells. J Bacteriol. 1976 Apr;126(1):232–242. doi: 10.1128/jb.126.1.232-242.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deshusses J., Reber G. Myo-inositol transport in Aerobacter aerogenes. Biochim Biophys Acta. 1972 Aug 9;274(2):598–605. doi: 10.1016/0005-2736(72)90206-4. [DOI] [PubMed] [Google Scholar]
  10. Gould G. W., Bell G. I. Facilitative glucose transporters: an expanding family. Trends Biochem Sci. 1990 Jan;15(1):18–23. doi: 10.1016/0968-0004(90)90125-u. [DOI] [PubMed] [Google Scholar]
  11. Hauser G. Myo-inositol transport in slices of rat kidney cortex. I. Effect of incubation conditions and inhibitors. Biochim Biophys Acta. 1969 Mar 11;173(2):257–266. doi: 10.1016/0005-2736(69)90109-6. [DOI] [PubMed] [Google Scholar]
  12. Iovannisci D. M., Ullman B. High efficiency plating method for Leishmania promastigotes in semidefined or completely-defined medium. J Parasitol. 1983 Aug;69(4):633–636. [PubMed] [Google Scholar]
  13. Johnstone R. M., Sung C. P. Transport of myo-inositol in Ehrlich ascites cells. Biochim Biophys Acta. 1967;135(5):1052–1055. doi: 10.1016/0005-2736(67)90074-0. [DOI] [PubMed] [Google Scholar]
  14. Kavanaugh M. P., Arriza J. L., North R. A., Amara S. G. Electrogenic uptake of gamma-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes. J Biol Chem. 1992 Nov 5;267(31):22007–22009. [PubMed] [Google Scholar]
  15. Kwon H. M., Yamauchi A., Uchida S., Preston A. S., Garcia-Perez A., Burg M. B., Handler J. S. Cloning of the cDNa for a Na+/myo-inositol cotransporter, a hypertonicity stress protein. J Biol Chem. 1992 Mar 25;267(9):6297–6301. [PubMed] [Google Scholar]
  16. Langford C. K., Ewbank S. A., Hanson S. S., Ullman B., Landfear S. M. Molecular characterization of two genes encoding members of the glucose transporter superfamily in the parasitic protozoan Leishmania donovani. Mol Biochem Parasitol. 1992 Oct;55(1-2):51–64. doi: 10.1016/0166-6851(92)90126-5. [DOI] [PubMed] [Google Scholar]
  17. Langford C. K., Little B. M., Kavanaugh M. P., Landfear S. M. Functional expression of two glucose transporter isoforms from the parasitic protozoan Leishmania enriettii. J Biol Chem. 1994 Jul 8;269(27):17939–17943. [PubMed] [Google Scholar]
  18. LeBowitz J. H., Coburn C. M., McMahon-Pratt D., Beverley S. M. Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9736–9740. doi: 10.1073/pnas.87.24.9736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Maiden M. C., Davis E. O., Baldwin S. A., Moore D. C., Henderson P. J. Mammalian and bacterial sugar transport proteins are homologous. Nature. 1987 Feb 12;325(6105):641–643. doi: 10.1038/325641a0. [DOI] [PubMed] [Google Scholar]
  20. McConville M. J., Ferguson M. A. The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes. Biochem J. 1993 Sep 1;294(Pt 2):305–324. doi: 10.1042/bj2940305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nikawa J., Nagumo T., Yamashita S. Myo-inositol transport in Saccharomyces cerevisiae. J Bacteriol. 1982 May;150(2):441–446. doi: 10.1128/jb.150.2.441-446.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nikawa J., Tsukagoshi Y., Yamashita S. Isolation and characterization of two distinct myo-inositol transporter genes of Saccharomyces cerevisiae. J Biol Chem. 1991 Jun 15;266(17):11184–11191. [PubMed] [Google Scholar]
  23. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Piper R. C., Xu X., Russell D. G., Little B. M., Landfear S. M. Differential targeting of two glucose transporters from Leishmania enriettii is mediated by an NH2-terminal domain. J Cell Biol. 1995 Feb;128(4):499–508. doi: 10.1083/jcb.128.4.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Russell D. G., Wilhelm H. The involvement of the major surface glycoprotein (gp63) of Leishmania promastigotes in attachment to macrophages. J Immunol. 1986 Apr 1;136(7):2613–2620. [PubMed] [Google Scholar]
  26. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  27. Sauer N., Caspari T., Klebl F., Tanner W. Functional expression of the Chlorella hexose transporter in Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7949–7952. doi: 10.1073/pnas.87.20.7949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sauer N., Friedländer K., Gräml-Wicke U. Primary structure, genomic organization and heterologous expression of a glucose transporter from Arabidopsis thaliana. EMBO J. 1990 Oct;9(10):3045–3050. doi: 10.1002/j.1460-2075.1990.tb07500.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  30. Ter Kuile B. H., Opperdoes F. R. A chemostat study on proline uptake and metabolism of Leishmania donovani. J Protozool. 1992 Sep-Oct;39(5):555–558. doi: 10.1111/j.1550-7408.1992.tb04850.x. [DOI] [PubMed] [Google Scholar]
  31. Ter Kuile B. H., Opperdoes F. R. Uptake and turnover of glucose in Leishmania donovani. Mol Biochem Parasitol. 1993 Aug;60(2):313–321. doi: 10.1016/0166-6851(93)90142-k. [DOI] [PubMed] [Google Scholar]
  32. Turco S. J., Descoteaux A. The lipophosphoglycan of Leishmania parasites. Annu Rev Microbiol. 1992;46:65–94. doi: 10.1146/annurev.mi.46.100192.000433. [DOI] [PubMed] [Google Scholar]
  33. Wright E. M. The intestinal Na+/glucose cotransporter. Annu Rev Physiol. 1993;55:575–589. doi: 10.1146/annurev.ph.55.030193.003043. [DOI] [PubMed] [Google Scholar]
  34. Zilberstein D., Dwyer D. M. Identification of a surface membrane proton-translocating ATPase in promastigotes of the parasitic protozoan Leishmania donovani. Biochem J. 1988 Nov 15;256(1):13–21. doi: 10.1042/bj2560013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zilberstein D., Dwyer D. M. Protonmotive force-driven active transport of D-glucose and L-proline in the protozoan parasite Leishmania donovani. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1716–1720. doi: 10.1073/pnas.82.6.1716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zilberstein D., Philosoph H., Gepstein A. Maintenance of cytoplasmic pH and proton motive force in promastigotes of Leishmania donovani. Mol Biochem Parasitol. 1989 Sep;36(2):109–117. doi: 10.1016/0166-6851(89)90183-7. [DOI] [PubMed] [Google Scholar]
  37. ter Kuile B. H., Cook M. The kinetics of facilitated diffusion followed by enzymatic conversion of the substrate. Biochim Biophys Acta. 1994 Aug 3;1193(2):235–239. doi: 10.1016/0005-2736(94)90158-9. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES