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. 1992 Nov 2;119(4):763–772. doi: 10.1083/jcb.119.4.763

Proteins containing an uncleaved signal for glycophosphatidylinositol membrane anchor attachment are retained in a post-ER compartment

PMCID: PMC2289691  PMID: 1385443

Abstract

Glycophosphatidylinositol (GPI)-anchored membrane proteins are initially synthesized with a cleavable COOH-terminal extension that signals anchor attachment. Overexpression in COS cells of hGH-DAF fusion proteins containing the GPI signal of decay accelerating factor (DAF) fused to the COOH-terminus of human growth hormone (hGH), produces both GPI-anchored hGH-DAF and uncleaved precursors that retain the GPI signal. Using hGH-DAF fusion proteins containing a mutated, noncleavable GPI signal, we show that uncleaved polypeptides are retained inside the cell and accumulate in a brefeldin A-sensitive, Golgi-like juxtanuclear structure. Retention requires the presence of either a functional or a noncleavable GPI signal; hGH-DAF fusion proteins containing only the COOH-terminal hydrophobic domain (a component of the GPI signal) are secreted. Immunofluorescence analysis shows colocalization of the retained, uncleaved fusion proteins with both a Golgi marker and with p53, a marker of the ER-Golgi intermediate compartment. Since N-linked glycosylation is postulated to facilitate the transport of proteins to the cell surface, we engineered a glycosylation site into hGH-DAF. Glycosylation failed to completely override the transport block, but allowed some uncleaved hGH-DAF to pass through the secretory pathway and acquire endoglycosidase H resistance. The retained molecules remained endoglycosidase H sensitive. We suggest that the uncleaved fusion protein is retained in a sorting compartment between the ER and the medial Golgi complex. We speculate that a mechanism exists to retain proteins containing an uncleaved GPI signal as part of a system for quality control.

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Selected References

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  1. Anderson D. J., Blobel G. Immunoprecipitation of proteins from cell-free translations. Methods Enzymol. 1983;96:111–120. doi: 10.1016/s0076-6879(83)96012-3. [DOI] [PubMed] [Google Scholar]
  2. Boothroyd J. C., Paynter C. A., Cross G. A., Bernards A., Borst P. Variant surface glycoproteins of Trypanosoma brucei are synthesised with cleavable hydrophobic sequences at the carboxy and amino termini. Nucleic Acids Res. 1981 Sep 25;9(18):4735–4743. doi: 10.1093/nar/9.18.4735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Caras I. W. An internally positioned signal can direct attachment of a glycophospholipid membrane anchor. J Cell Biol. 1991 Apr;113(1):77–85. doi: 10.1083/jcb.113.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caras I. W., Weddell G. N., Davitz M. A., Nussenzweig V., Martin D. W., Jr Signal for attachment of a phospholipid membrane anchor in decay accelerating factor. Science. 1987 Nov 27;238(4831):1280–1283. doi: 10.1126/science.2446389. [DOI] [PubMed] [Google Scholar]
  5. Caras I. W., Weddell G. N., Williams S. R. Analysis of the signal for attachment of a glycophospholipid membrane anchor. J Cell Biol. 1989 Apr;108(4):1387–1396. doi: 10.1083/jcb.108.4.1387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cross G. A. Glycolipid anchoring of plasma membrane proteins. Annu Rev Cell Biol. 1990;6:1–39. doi: 10.1146/annurev.cb.06.110190.000245. [DOI] [PubMed] [Google Scholar]
  7. Ferguson M. A., Homans S. W., Dwek R. A., Rademacher T. W. Glycosyl-phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane. Science. 1988 Feb 12;239(4841 Pt 1):753–759. doi: 10.1126/science.3340856. [DOI] [PubMed] [Google Scholar]
  8. Ferguson M. A., Williams A. F. Cell-surface anchoring of proteins via glycosyl-phosphatidylinositol structures. Annu Rev Biochem. 1988;57:285–320. doi: 10.1146/annurev.bi.57.070188.001441. [DOI] [PubMed] [Google Scholar]
  9. Guan J. L., Machamer C. E., Rose J. K. Glycosylation allows cell-surface transport of an anchored secretory protein. Cell. 1985 Sep;42(2):489–496. doi: 10.1016/0092-8674(85)90106-0. [DOI] [PubMed] [Google Scholar]
  10. Guan J. L., Rose J. K. Conversion of a secretory protein into a transmembrane protein results in its transport to the Golgi complex but not to the cell surface. Cell. 1984 Jul;37(3):779–787. doi: 10.1016/0092-8674(84)90413-6. [DOI] [PubMed] [Google Scholar]
  11. Hsu V. W., Yuan L. C., Nuchtern J. G., Lippincott-Schwartz J., Hammerling G. J., Klausner R. D. A recycling pathway between the endoplasmic reticulum and the Golgi apparatus for retention of unassembled MHC class I molecules. Nature. 1991 Aug 1;352(6334):441–444. doi: 10.1038/352441a0. [DOI] [PubMed] [Google Scholar]
  12. Hurtley S. M., Helenius A. Protein oligomerization in the endoplasmic reticulum. Annu Rev Cell Biol. 1989;5:277–307. doi: 10.1146/annurev.cb.05.110189.001425. [DOI] [PubMed] [Google Scholar]
  13. Klausner R. D. Architectural editing: determining the fate of newly synthesized membrane proteins. New Biol. 1989 Oct;1(1):3–8. [PubMed] [Google Scholar]
  14. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  15. Lewis M. J., Pelham H. R. A human homologue of the yeast HDEL receptor. Nature. 1990 Nov 8;348(6297):162–163. doi: 10.1038/348162a0. [DOI] [PubMed] [Google Scholar]
  16. Lin A. Y., Devaux B., Green A., Sagerström C., Elliott J. F., Davis M. M. Expression of T cell antigen receptor heterodimers in a lipid-linked form. Science. 1990 Aug 10;249(4969):677–679. doi: 10.1126/science.1696397. [DOI] [PubMed] [Google Scholar]
  17. Lippincott-Schwartz J., Yuan L. C., Bonifacino J. S., Klausner R. D. Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell. 1989 Mar 10;56(5):801–813. doi: 10.1016/0092-8674(89)90685-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lodish H. F., Kong N., Hirani S., Rasmussen J. A vesicular intermediate in the transport of hepatoma secretory proteins from the rough endoplasmic reticulum to the Golgi complex. J Cell Biol. 1987 Feb;104(2):221–230. doi: 10.1083/jcb.104.2.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Low M. G. Glycosyl-phosphatidylinositol: a versatile anchor for cell surface proteins. FASEB J. 1989 Mar;3(5):1600–1608. doi: 10.1096/fasebj.3.5.2522071. [DOI] [PubMed] [Google Scholar]
  20. Machamer C. E., Florkiewicz R. Z., Rose J. K. A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface. Mol Cell Biol. 1985 Nov;5(11):3074–3083. doi: 10.1128/mcb.5.11.3074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. McClary J. A., Witney F., Geisselsoder J. Efficient site-directed in vitro mutagenesis using phagemid vectors. Biotechniques. 1989 Mar;7(3):282–289. [PubMed] [Google Scholar]
  23. Mellman I., Simons K. The Golgi complex: in vitro veritas? Cell. 1992 Mar 6;68(5):829–840. doi: 10.1016/0092-8674(92)90027-A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Micanovic R., Gerber L. D., Berger J., Kodukula K., Udenfriend S. Selectivity of the cleavage/attachment site of phosphatidylinositol-glycan-anchored membrane proteins determined by site-specific mutagenesis at Asp-484 of placental alkaline phosphatase. Proc Natl Acad Sci U S A. 1990 Jan;87(1):157–161. doi: 10.1073/pnas.87.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moran P., Caras I. W. A nonfunctional sequence converted to a signal for glycophosphatidylinositol membrane anchor attachment. J Cell Biol. 1991 Oct;115(2):329–336. doi: 10.1083/jcb.115.2.329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Moran P., Raab H., Kohr W. J., Caras I. W. Glycophospholipid membrane anchor attachment. Molecular analysis of the cleavage/attachment site. J Biol Chem. 1991 Jan 15;266(2):1250–1257. [PubMed] [Google Scholar]
  27. Munro S., Pelham H. R. A C-terminal signal prevents secretion of luminal ER proteins. Cell. 1987 Mar 13;48(5):899–907. doi: 10.1016/0092-8674(87)90086-9. [DOI] [PubMed] [Google Scholar]
  28. Pelham H. R. Evidence that luminal ER proteins are sorted from secreted proteins in a post-ER compartment. EMBO J. 1988 Apr;7(4):913–918. doi: 10.1002/j.1460-2075.1988.tb02896.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pelham H. R. Recycling of proteins between the endoplasmic reticulum and Golgi complex. Curr Opin Cell Biol. 1991 Aug;3(4):585–591. doi: 10.1016/0955-0674(91)90027-v. [DOI] [PubMed] [Google Scholar]
  30. Rose J. K., Doms R. W. Regulation of protein export from the endoplasmic reticulum. Annu Rev Cell Biol. 1988;4:257–288. doi: 10.1146/annurev.cb.04.110188.001353. [DOI] [PubMed] [Google Scholar]
  31. Saraste J., Palade G. E., Farquhar M. G. Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6425–6429. doi: 10.1073/pnas.83.17.6425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schweizer A., Fransen J. A., Bächi T., Ginsel L., Hauri H. P. Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus. J Cell Biol. 1988 Nov;107(5):1643–1653. doi: 10.1083/jcb.107.5.1643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schweizer A., Matter K., Ketcham C. M., Hauri H. P. The isolated ER-Golgi intermediate compartment exhibits properties that are different from ER and cis-Golgi. J Cell Biol. 1991 Apr;113(1):45–54. doi: 10.1083/jcb.113.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tse A. G., Barclay A. N., Watts A., Williams A. F. A glycophospholipid tail at the carboxyl terminus of the Thy-1 glycoprotein of neurons and thymocytes. Science. 1985 Nov 29;230(4729):1003–1008. doi: 10.1126/science.2865810. [DOI] [PubMed] [Google Scholar]
  35. Vaux D., Tooze J., Fuller S. Identification by anti-idiotype antibodies of an intracellular membrane protein that recognizes a mammalian endoplasmic reticulum retention signal. Nature. 1990 Jun 7;345(6275):495–502. doi: 10.1038/345495a0. [DOI] [PubMed] [Google Scholar]
  36. Warren G. Protein transport. Signals and salvage sequences. Nature. 1987 May 7;327(6117):17–18. doi: 10.1038/327017a0. [DOI] [PubMed] [Google Scholar]

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