Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Nov 1;351(Pt 3):717–722.

Soluble GPI8 restores glycosylphosphatidylinositol anchoring in a trypanosome cell-free system depleted of lumenal endoplasmic reticulum proteins.

D K Sharma 1, J D Hilley 1, J D Bangs 1, G H Coombs 1, J C Mottram 1, A K Menon 1
PMCID: PMC1221412  PMID: 11042127

Abstract

We previously established an in vitro assay for glycosylphosphatidylinositol (GPI) anchoring of proteins using trypanosome membranes. We now show that GPI anchoring is lost when the membranes are washed at high pH and restored to physiological pH prior to assay. We show that soluble component(s) of the endoplasmic reticulum that are lost in the high-pH wash are required for GPI anchoring. We reconstituted the high-pH extract with high-pH-treated membranes and demonstrated restoration of activity. Size fractionation of the high-pH extract indicated that the active component(s) was 30-50 kDa in size and was inactivated by iodoacetamide. Activity could also be restored by reconstituting the inactivated membranes with Escherichia coli-expressed, polyhistidine-tagged Leishmania mexicana GPI8 (GPI8-His; L. mexicana GPI8 is a soluble homologue of yeast and mammalian Gpi8p). No activity was seen when iodoacetamide-treated GPI8-His was used; however, GPI8-His could restore activity to iodoacetamide-treated membranes. Antibodies raised against L. mexicana GPI8 detected a protein of approx. 38 kDa in an immunoblot of the high-pH extract of trypanosome membranes. Our data indicate (1) that trypanosome GPI8 is a soluble lumenal protein, (2) that the interaction between GPI8 and other putative components of the transamidase may be dynamic, and (3) that GPI anchoring can be biochemically reconstituted using an isolated transamidase component.

Full Text

The Full Text of this article is available as a PDF (220.6 KB).

Selected References

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

  1. Abe Y., Shirane K., Yokosawa H., Matsushita H., Mitta M., Kato I., Ishii S. Asparaginyl endopeptidase of jack bean seeds. Purification, characterization, and high utility in protein sequence analysis. J Biol Chem. 1993 Feb 15;268(5):3525–3529. [PubMed] [Google Scholar]
  2. Amthauer R., Kodukula K., Gerber L., Udenfriend S. Evidence that the putative COOH-terminal signal transamidase involved in glycosylphosphatidylinositol protein synthesis is present in the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3973–3977. doi: 10.1073/pnas.90.9.3973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bangs J. D., Brouch E. M., Ransom D. M., Roggy J. L. A soluble secretory reporter system in Trypanosoma brucei. Studies on endoplasmic reticulum targeting. J Biol Chem. 1996 Aug 2;271(31):18387–18393. doi: 10.1074/jbc.271.31.18387. [DOI] [PubMed] [Google Scholar]
  4. Bangs J. D., Ransom D. M., McDowell M. A., Brouch E. M. Expression of bloodstream variant surface glycoproteins in procyclic stage Trypanosoma brucei: role of GPI anchors in secretion. EMBO J. 1997 Jul 16;16(14):4285–4294. doi: 10.1093/emboj/16.14.4285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bangs J. D., Uyetake L., Brickman M. J., Balber A. E., Boothroyd J. C. Molecular cloning and cellular localization of a BiP homologue in Trypanosoma brucei. Divergent ER retention signals in a lower eukaryote. J Cell Sci. 1993 Aug;105(Pt 4):1101–1113. doi: 10.1242/jcs.105.4.1101. [DOI] [PubMed] [Google Scholar]
  6. Benghezal M., Benachour A., Rusconi S., Aebi M., Conzelmann A. Yeast Gpi8p is essential for GPI anchor attachment onto proteins. EMBO J. 1996 Dec 2;15(23):6575–6583. [PMC free article] [PubMed] [Google Scholar]
  7. Clayton C., Adams M., Almeida R., Baltz T., Barrett M., Bastien P., Belli S., Beverley S., Biteau N., Blackwell J. Genetic nomenclature for Trypanosoma and Leishmania. Mol Biochem Parasitol. 1998 Nov 30;97(1-2):221–224. doi: 10.1016/s0166-6851(98)00115-7. [DOI] [PubMed] [Google Scholar]
  8. Doering T. L., Schekman R. Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system. Biochem J. 1997 Dec 1;328(Pt 2):669–675. doi: 10.1042/bj3280669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ferguson M. A. The structure, biosynthesis and functions of glycosylphosphatidylinositol anchors, and the contributions of trypanosome research. J Cell Sci. 1999 Sep;112(Pt 17):2799–2809. doi: 10.1242/jcs.112.17.2799. [DOI] [PubMed] [Google Scholar]
  10. Hamburger D., Egerton M., Riezman H. Yeast Gaa1p is required for attachment of a completed GPI anchor onto proteins. J Cell Biol. 1995 May;129(3):629–639. doi: 10.1083/jcb.129.3.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hilley J. D., Zawadzki J. L., McConville M. J., Coombs G. H., Mottram J. C. Leishmania mexicana mutants lacking glycosylphosphatidylinositol (GPI):protein transamidase provide insights into the biosynthesis and functions of GPI-anchored proteins. Mol Biol Cell. 2000 Apr;11(4):1183–1195. doi: 10.1091/mbc.11.4.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hiroi Y., Komuro I., Chen R., Hosoda T., Mizuno T., Kudoh S., Georgescu S. P., Medof M. E., Yazaki Y. Molecular cloning of human homolog of yeast GAA1 which is required for attachment of glycosylphosphatidylinositols to proteins. FEBS Lett. 1998 Jan 16;421(3):252–258. doi: 10.1016/s0014-5793(97)01576-7. [DOI] [PubMed] [Google Scholar]
  13. Kodukula K., Micanovic R., Gerber L., Tamburrini M., Brink L., Udenfriend S. Biosynthesis of phosphatidylinositol glycan-anchored membrane proteins. Design of a simple protein substrate to characterize the enzyme that cleaves the COOH-terminal signal peptide. J Biol Chem. 1991 Mar 5;266(7):4464–4470. [PubMed] [Google Scholar]
  14. Kuhelj R., Dolinar M., Pungercar J., Turk V. The preparation of catalytically active human cathepsin B from its precursor expressed in Escherichia coli in the form of inclusion bodies. Eur J Biochem. 1995 Apr 15;229(2):533–539. doi: 10.1111/j.1432-1033.1995.0533k.x. [DOI] [PubMed] [Google Scholar]
  15. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  16. Masterson W. J., Doering T. L., Hart G. W., Englund P. T. A novel pathway for glycan assembly: biosynthesis of the glycosyl-phosphatidylinositol anchor of the trypanosome variant surface glycoprotein. Cell. 1989 Mar 10;56(5):793–800. doi: 10.1016/0092-8674(89)90684-3. [DOI] [PubMed] [Google Scholar]
  17. Maxwell S. E., Ramalingam S., Gerber L. D., Brink L., Udenfriend S. An active carbonyl formed during glycosylphosphatidylinositol addition to a protein is evidence of catalysis by a transamidase. J Biol Chem. 1995 Aug 18;270(33):19576–19582. doi: 10.1074/jbc.270.33.19576. [DOI] [PubMed] [Google Scholar]
  18. Mayor S., Menon A. K., Cross G. A. Transfer of glycosyl-phosphatidylinositol membrane anchors to polypeptide acceptors in a cell-free system. J Cell Biol. 1991 Jul;114(1):61–71. doi: 10.1083/jcb.114.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Meyer U., Benghezal M., Imhof I., Conzelmann A. Active site determination of Gpi8p, a caspase-related enzyme required for glycosylphosphatidylinositol anchor addition to proteins. Biochemistry. 2000 Mar 28;39(12):3461–3471. doi: 10.1021/bi992186o. [DOI] [PubMed] [Google Scholar]
  20. Nicchitta C. V., Blobel G. Lumenal proteins of the mammalian endoplasmic reticulum are required to complete protein translocation. Cell. 1993 Jun 4;73(5):989–998. doi: 10.1016/0092-8674(93)90276-v. [DOI] [PubMed] [Google Scholar]
  21. Ohishi K., Inoue N., Maeda Y., Takeda J., Riezman H., Kinoshita T. Gaa1p and gpi8p are components of a glycosylphosphatidylinositol (GPI) transamidase that mediates attachment of GPI to proteins. Mol Biol Cell. 2000 May;11(5):1523–1533. doi: 10.1091/mbc.11.5.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Paver J. L., Hawkins H. C., Freedman R. B. Preparation and characterization of dog pancreas microsomal membranes specifically depleted of protein disulphide-isomerase. Biochem J. 1989 Feb 1;257(3):657–663. doi: 10.1042/bj2570657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sharma D. K., Vidugiriene J., Bangs J. D., Menon A. K. A cell-free assay for glycosylphosphatidylinositol anchoring in African trypanosomes. Demonstration of a transamidation reaction mechanism. J Biol Chem. 1999 Jun 4;274(23):16479–16486. doi: 10.1074/jbc.274.23.16479. [DOI] [PubMed] [Google Scholar]
  24. Udenfriend S., Kodukula K. How glycosylphosphatidylinositol-anchored membrane proteins are made. Annu Rev Biochem. 1995;64:563–591. doi: 10.1146/annurev.bi.64.070195.003023. [DOI] [PubMed] [Google Scholar]
  25. Vidugiriene J., Menon A. K. Soluble constituents of the ER lumen are required for GPI anchoring of a model protein. EMBO J. 1995 Oct 2;14(19):4686–4694. doi: 10.1002/j.1460-2075.1995.tb00150.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yu J., Nagarajan S., Knez J. J., Udenfriend S., Chen R., Medof M. E. The affected gene underlying the class K glycosylphosphatidylinositol (GPI) surface protein defect codes for the GPI transamidase. Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12580–12585. doi: 10.1073/pnas.94.23.12580. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES