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. 1999 Nov;11(11):2233–2248. doi: 10.1105/tpc.11.11.2233

Saturation of the endoplasmic reticulum retention machinery reveals anterograde bulk flow

AJ Crofts 1, N Leborgne-Castel 1, S Hillmer 1, DG Robinson 1, B Phillipson 1, LE Carlsson 1, DA Ashford 1, J Denecke 1
PMCID: PMC144130  PMID: 10559446

Abstract

We have studied the possible mechanisms of endoplasmic reticulum (ER) export and retention by using natural residents of the plant ER. Under normal physiological conditions, calreticulin and the lumenal binding protein (BiP) are efficiently retained in the ER. When the ER retention signal is removed, truncated calreticulin is much more rapidly secreted than truncated BiP. Calreticulin carries two glycans of the typical ER high-mannose form. Both glycans are competent for Golgi-based modifications, as determined from treatment with brefeldin A or based on the deletion of the ER retention motif. In contrast to BiP, calreticulin accumulation is strongly dependent on its retention signal, thereby allowing us to test whether saturation of the retention mechanism is possible. Overexpression of calreticulin led to 100-fold higher levels in dilated globular ER cisternae as well as dilated nuclear envelopes and partial secretion of both BiP and calreticulin. This result shows that both molecules are competent for ER export and supports the concept that proteins are secreted by default. This result also supports previous data suggesting that truncated BiP devoid of its retention motif can be retained in the ER by association with calreticulin. Moreover, even under these saturating conditions, cellular calreticulin did not carry significant amounts of complex glycans, in contrast to secreted calreticulin. This result shows that calreticulin is rapidly secreted once complex glycans have been synthesized in the medial/trans Golgi apparatus and that the modified protein does not appear to recycle back to the ER.

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Selected References

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  1. Balch W. E., McCaffery J. M., Plutner H., Farquhar M. G. Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum. Cell. 1994 Mar 11;76(5):841–852. doi: 10.1016/0092-8674(94)90359-x. [DOI] [PubMed] [Google Scholar]
  2. Barlowe C., Orci L., Yeung T., Hosobuchi M., Hamamoto S., Salama N., Rexach M. F., Ravazzola M., Amherdt M., Schekman R. COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell. 1994 Jun 17;77(6):895–907. doi: 10.1016/0092-8674(94)90138-4. [DOI] [PubMed] [Google Scholar]
  3. Boevink P., Oparka K., Santa Cruz S., Martin B., Betteridge A., Hawes C. Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network. Plant J. 1998 Aug;15(3):441–447. doi: 10.1046/j.1365-313x.1998.00208.x. [DOI] [PubMed] [Google Scholar]
  4. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  5. Ceriotti A., Colman A. Binding to membrane proteins within the endoplasmic reticulum cannot explain the retention of the glucose-regulated protein GRP78 in Xenopus oocytes. EMBO J. 1988 Mar;7(3):633–638. doi: 10.1002/j.1460-2075.1988.tb02857.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Connolly C. N., Futter C. E., Gibson A., Hopkins C. R., Cutler D. F. Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase. J Cell Biol. 1994 Nov;127(3):641–652. doi: 10.1083/jcb.127.3.641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cosson P., Letourneur F. Coatomer interaction with di-lysine endoplasmic reticulum retention motifs. Science. 1994 Mar 18;263(5153):1629–1631. doi: 10.1126/science.8128252. [DOI] [PubMed] [Google Scholar]
  8. Crofts AJ, Leborgne-Castel N, Pesca M, Vitale A, Denecke J. BiP and calreticulin form an abundant complex that is independent of endoplasmic reticulum stress . Plant Cell. 1998 May;10(5):813–824. doi: 10.1105/tpc.10.5.813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dean N., Pelham H. R. Recycling of proteins from the Golgi compartment to the ER in yeast. J Cell Biol. 1990 Aug;111(2):369–377. doi: 10.1083/jcb.111.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deblaere R., Bytebier B., De Greve H., Deboeck F., Schell J., Van Montagu M., Leemans J. Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants. Nucleic Acids Res. 1985 Jul 11;13(13):4777–4788. doi: 10.1093/nar/13.13.4777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Denecke J., Botterman J., Deblaere R. Protein secretion in plant cells can occur via a default pathway. Plant Cell. 1990 Jan;2(1):51–59. doi: 10.1105/tpc.2.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Denecke J., Carlsson L. E., Vidal S., Höglund A. S., Ek B., van Zeijl M. J., Sinjorgo K. M., Palva E. T. The tobacco homolog of mammalian calreticulin is present in protein complexes in vivo. Plant Cell. 1995 Apr;7(4):391–406. doi: 10.1105/tpc.7.4.391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Denecke J., De Rycke R., Botterman J. Plant and mammalian sorting signals for protein retention in the endoplasmic reticulum contain a conserved epitope. EMBO J. 1992 Jun;11(6):2345–2355. doi: 10.1002/j.1460-2075.1992.tb05294.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Denecke J., Goldman M. H., Demolder J., Seurinck J., Botterman J. The tobacco luminal binding protein is encoded by a multigene family. Plant Cell. 1991 Sep;3(9):1025–1035. doi: 10.1105/tpc.3.9.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Denecke J., Vitale A. The use of protoplasts to study protein synthesis and transport by the plant endomembrane system. Methods Cell Biol. 1995;50:335–348. doi: 10.1016/s0091-679x(08)61041-9. [DOI] [PubMed] [Google Scholar]
  16. Dorner A. J., Wasley L. C., Raney P., Haugejorden S., Green M., Kaufman R. J. The stress response in Chinese hamster ovary cells. Regulation of ERp72 and protein disulfide isomerase expression and secretion. J Biol Chem. 1990 Dec 15;265(35):22029–22034. [PubMed] [Google Scholar]
  17. Gomord V., Denmat L. A., Fitchette-Lainé A. C., Satiat-Jeunemaitre B., Hawes C., Faye L. The C-terminal HDEL sequence is sufficient for retention of secretory proteins in the endoplasmic reticulum (ER) but promotes vacuolar targeting of proteins that escape the ER. Plant J. 1997 Feb;11(2):313–325. doi: 10.1046/j.1365-313x.1997.11020313.x. [DOI] [PubMed] [Google Scholar]
  18. Hauri H. P., Schweizer A. The endoplasmic reticulum-Golgi intermediate compartment. Curr Opin Cell Biol. 1992 Aug;4(4):600–608. doi: 10.1016/0955-0674(92)90078-Q. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hohl I., Robinson D. G., Chrispeels M. J., Hinz G. Transport of storage proteins to the vacuole is mediated by vesicles without a clathrin coat. J Cell Sci. 1996 Oct;109(Pt 10):2539–2550. doi: 10.1242/jcs.109.10.2539. [DOI] [PubMed] [Google Scholar]
  20. Hong W. Protein transport from the endoplasmic reticulum to the Golgi apparatus. J Cell Sci. 1998 Oct;111(Pt 19):2831–2839. doi: 10.1242/jcs.111.19.2831. [DOI] [PubMed] [Google Scholar]
  21. Hunt D. C., Chrispeels M. J. The signal Peptide of a vacuolar protein is necessary and sufficient for the efficient secretion of a cytosolic protein. Plant Physiol. 1991 May;96(1):18–25. doi: 10.1104/pp.96.1.18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Johannes L., Goud B. Surfing on a retrograde wave: how does Shiga toxin reach the endoplasmic reticulum? Trends Cell Biol. 1998 Apr;8(4):158–162. doi: 10.1016/s0962-8924(97)01209-9. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Leborgne-Castel N., Jelitto-Van Dooren E. P., Crofts A. J., Denecke J. Overexpression of BiP in tobacco alleviates endoplasmic reticulum stress. Plant Cell. 1999 Mar;11(3):459–470. doi: 10.1105/tpc.11.3.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lee H. I., Gal S., Newman T. C., Raikhel N. V. The Arabidopsis endoplasmic reticulum retention receptor functions in yeast. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11433–11437. doi: 10.1073/pnas.90.23.11433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lewis M. J., Pelham H. R. Ligand-induced redistribution of a human KDEL receptor from the Golgi complex to the endoplasmic reticulum. Cell. 1992 Jan 24;68(2):353–364. doi: 10.1016/0092-8674(92)90476-s. [DOI] [PubMed] [Google Scholar]
  27. Lippincott-Schwartz J., Donaldson J. G., Schweizer A., Berger E. G., Hauri H. P., Yuan L. C., Klausner R. D. Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell. 1990 Mar 9;60(5):821–836. doi: 10.1016/0092-8674(90)90096-w. [DOI] [PubMed] [Google Scholar]
  28. Lord J. M., Roberts L. M. Retrograde transport: going against the flow. Curr Biol. 1998 Jan 15;8(2):R56–R58. doi: 10.1016/s0960-9822(98)70034-x. [DOI] [PubMed] [Google Scholar]
  29. Majoul I., Sohn K., Wieland F. T., Pepperkok R., Pizza M., Hillemann J., Söling H. D. KDEL receptor (Erd2p)-mediated retrograde transport of the cholera toxin A subunit from the Golgi involves COPI, p23, and the COOH terminus of Erd2p. J Cell Biol. 1998 Nov 2;143(3):601–612. doi: 10.1083/jcb.143.3.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Maliga P., Sz-Breznovits A., Márton L. Streptomycin-resistant plants from callus culture of haploid tobacco. Nat New Biol. 1973 Jul 4;244(131):29–30. doi: 10.1038/newbio244029a0. [DOI] [PubMed] [Google Scholar]
  31. Miesenböck G., Rothman J. E. The capacity to retrieve escaped ER proteins extends to the trans-most cisterna of the Golgi stack. J Cell Biol. 1995 Apr;129(2):309–319. doi: 10.1083/jcb.129.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Navazio L., Baldan B., Mariani P., Gerwig G. J., Vliegenthart J. F. Primary structure of the N-linked carbohydrate chains of Calreticulin from spinach leaves. Glycoconj J. 1996 Dec;13(6):977–983. doi: 10.1007/BF01053193. [DOI] [PubMed] [Google Scholar]
  34. Nishimura N., Balch W. E. A di-acidic signal required for selective export from the endoplasmic reticulum. Science. 1997 Jul 25;277(5325):556–558. doi: 10.1126/science.277.5325.556. [DOI] [PubMed] [Google Scholar]
  35. Pahl H. L., Baeuerle P. A. Endoplasmicreticulum-induced signal transduction and gene expression. Trends Cell Biol. 1997 Feb;7(2):50–55. doi: 10.1016/S0962-8924(96)10050-7. [DOI] [PubMed] [Google Scholar]
  36. Pedrazzini E., Giovinazzo G., Bielli A., de Virgilio M., Frigerio L., Pesca M., Faoro F., Bollini R., Ceriotti A., Vitale A. Protein quality control along the route to the plant vacuole. Plant Cell. 1997 Oct;9(10):1869–1880. doi: 10.1105/tpc.9.10.1869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Pelham H. R. Sorting and retrieval between the endoplasmic reticulum and Golgi apparatus. Curr Opin Cell Biol. 1995 Aug;7(4):530–535. doi: 10.1016/0955-0674(95)80010-7. [DOI] [PubMed] [Google Scholar]
  39. Pueyo J. J., Chrispeels M. J., Herman E. M. Degradation of transport-competent destabilized phaseolin with a signal for retention in the endoplasmic reticulum occurs in the vacuole. Planta. 1995;196(3):586–596. doi: 10.1007/BF00203660. [DOI] [PubMed] [Google Scholar]
  40. Rabouille C., Hui N., Hunte F., Kieckbusch R., Berger E. G., Warren G., Nilsson T. Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides. J Cell Sci. 1995 Apr;108(Pt 4):1617–1627. doi: 10.1242/jcs.108.4.1617. [DOI] [PubMed] [Google Scholar]
  41. Scheel J., Pepperkok R., Lowe M., Griffiths G., Kreis T. E. Dissociation of coatomer from membranes is required for brefeldin A-induced transfer of Golgi enzymes to the endoplasmic reticulum. J Cell Biol. 1997 Apr 21;137(2):319–333. doi: 10.1083/jcb.137.2.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Spang A., Schekman R. Reconstitution of retrograde transport from the Golgi to the ER in vitro. J Cell Biol. 1998 Nov 2;143(3):589–599. doi: 10.1083/jcb.143.3.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tatu U., Helenius A. Interactions between newly synthesized glycoproteins, calnexin and a network of resident chaperones in the endoplasmic reticulum. J Cell Biol. 1997 Feb 10;136(3):555–565. doi: 10.1083/jcb.136.3.555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Townsley F. M., Wilson D. W., Pelham H. R. Mutational analysis of the human KDEL receptor: distinct structural requirements for Golgi retention, ligand binding and retrograde transport. EMBO J. 1993 Jul;12(7):2821–2829. doi: 10.1002/j.1460-2075.1993.tb05943.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vitale A, Denecke J. The endoplasmic reticulum-gateway of the secretory pathway . Plant Cell. 1999 Apr;11(4):615–628. doi: 10.1105/tpc.11.4.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vollenweider F., Kappeler F., Itin C., Hauri H. P. Mistargeting of the lectin ERGIC-53 to the endoplasmic reticulum of HeLa cells impairs the secretion of a lysosomal enzyme. J Cell Biol. 1998 Jul 27;142(2):377–389. doi: 10.1083/jcb.142.2.377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wandelt C. I., Khan M. R., Craig S., Schroeder H. E., Spencer D., Higgins T. J. Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants. Plant J. 1992 Mar;2(2):181–192. doi: 10.1046/j.1365-313x.1992.t01-41-00999.x. [DOI] [PubMed] [Google Scholar]

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