Abstract
Soluble proteins that reside in the lumen of the endoplasmic reticulum are known to have at their carboxyterminus the tetrapeptides KDEL or HDEL. In yeast and mammalian cells, these tetrapeptides function as endoplasmic reticulum (ER)-retention signals. To determine the effect of an artificially-introduced KDEL sequence at the exact carboxyterminus of a plant secretory protein, we modified the gene of the vacuolar protein phytohemagglutinin-L (PHA) so that the amino-acid sequence would end in LNKDEL rather than LNKIL, and expressed the modified gene in transgenic tobacco with a seed-specific promoter. Analysis of the glycans of PHA showed that most of the control PHA had one endoglycosidase H-sensitive and one endoglycosidase H-resistant glycan, indicating that it had been processed in the Golgi complex. On the other hand, a substantial portion of the PHA-KDEL (about 75% at mid-maturation and 50% in mature seeds) had two endoglycosidase H-sensitive glycans. Phytohemagglutinin with two endoglycosidase H-sensitive glycans is normally found in the ER. Using immunocytochemistry we found that a substantial portion of the PHA-KDEL was present in the ER or accumulated in the nuclear envelope while the remainder was found in the protein storage vacuoles (protein bodies). We interpret these data to indicate that carboxyterminal KDEL functions as an ER retention-retardation signal and causes protein to accumulate in the nuclear envelope as well as in the ER. The incomplete ER retention of this protein which is modified at the exact carboxyterminus may indicate that structural features other than carboxyterminal KDEL are important if complete ER retention is to be achieved.
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Abbreviations
- endoH:
-
endoglycosidase H
- ER:
-
endoplasmic reticulum
- Mr:
-
relative molecular mass
- PHA:
-
phytohemagglutinin
- SDS:
-
sodium dodecyl sulfate
- PAGE:
-
polyacrylamide gel electrophoresis
- TBST:
-
Tris-buffered saline containing Tween 20
References
Bevan, M. (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 12, 8711–8721
Burgess, T.L., Kelly, R.B. (1987) Constitutive and regulated secretion of proteins. Annu. Rev. Cell. Biol. 3, 243–293
Ceriotti, A., Colman, A. (1988) Binding to membrane proteins within the endoplasmic reticulum cannot explain the retention of the glucose-regulated protein GRP 78 in Xenopus oocytes. EMBO J. 7, 633–638
Chrispeels, M.J. (1983) The Golgi apparatus mediates the transport of phytohemagglutinin to the protein bodies in bean cotyledons. Planta 158, 140–151
Doms, R.W., Russ, G., Yewdell, J.W. (1989) Brefelding A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum. J. Cell Biol. 109, 61–72
Hardwick, K.G., Lewis, M.J., Semenza, J., Dean, N., Pelham, H.R.B. (1990) ERD1, a yeast gene required for the retention of luminal endoplasmic reticulum proteins, affects glycoprotein processing in the Golgi apparatus. EMBO J. 9, 623–630
Herman, E.M. (1988) Immunocytochemical localization of macromolecules with the electron microscope. Annu. Rev. Plant Physiol. Mol. Biol. 39, 139–155
Herman, E.M., Shannon, L.M. (1984) Immunocytochemical localization of Concanavalin A in developing Jack bean cotyledons. Planta 161, 97–104
Herman, E.M., Chrispeels, M.J., Hoffman, L.M. (1989) Vacuole accumulation of storage protein and lectin expressed in transgenic tobacco seeds. Cell Biol. Int. Rep. 13, 37–45
Hoffman, L.M., Donaldson, D.D. (1987) Synthesis of mitogenic phytohemagglutinin-L in Escherichia coli. Biotechnology 5, 157–160
Hoffman, L.M., Hundt, H. (1988) Use of a gas Chromatograph oven for DNA amplification by the polymerase chain reaction. Biotechniques 6, 932–936
Hoffman, L.M., Donaldson, D.D., Bookland, R., Rashka, K., Herman, E.M. (1987) Synthesis and protein deposition of maize 15-kd zein in transgenic tobacco seeds. EMBO J. 6, 3213–3221
Horsch, R.B., Fry, J.E., Hoffman, N.L., Eichholtz, D., Rogers, S.G., Fraley, R.T. (1985) A simple and general method for transferring genes into plants. Science 227, 1229–1231
Inohara, N., Shimomura, S., Fukui, T., Futai, M. (1989) Auxinbinding protein located in the endoplasmic reticulum of maize shoots: Molecular cloning and complete primary structure. Proc. Natl. Acad. Sci. USA 86, 3564–3568
Klausner, R.D. (1989) Sorting and traffic in the central vacuolar system. Cell 57, 703–706
Kornfeld, S. (1987) Trafficking of lysosomal enzymes. FASEB J. 1, 462–468
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275
Munro, S., Pelham, H.R.B. (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, 899–907
Pelham, H.R.B. (1988) Evidence that luminal ER proteins are sorted from secreted proteins in a post-ER compartment. EMBO J. 7, 913–918
Pelham, H.R.B. (1989) Control of protein exit from the endoplasmic retriculum. Annu. Rev. Cell Biol. 5, 1–23
Pelham, H.R.B., Hardwick, K.G., Lewis, M.J. (1988) Sorting of soluble ER proteins in yeast. EMBO J. 7, 1757–1762
Rose, M.D., Misra, L.M., Vogel, J.P. (1989) KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP:GRP 78 gene. Cell 57, 1211–1221
Sengupta-Gopalan, C., Reichert, N.A., Barker, R.F., Hall, T.C., Kemp, J.D. (1985) Developmentally regulated expression in the bean β-phaseolin gene in tobacco seed. Proc. Natl. Acad. Sci. USA 82, 3320–3324
Sturm, A., Chrispeels, M.J. (1986) The high mannose oligosaccharide of phytohemagglutinin is attached to Asparagine 12 and the modified oligosaccharide to Asparagine 60. Plant Physiol. 81, 320–322
Sturm, A. Johnson, K.D., Szumilo, T., Elbein, A.D., Chrispeels, M.J. (1987) Subcellular localization of glycosidases and glycosyltransferases involved in the processing of N-linked oligosaccharides. Plant. Physiol. 85, 741–745
Sturm, A. Voelker, T.A., Herman, E.M., Chrispeels, M.J. (1988) Correct glycosylation, Golgi-processing, and targeting to protein bodies of the vacuolar protein phytohemagglutinin in transgenic tobacco. Planta 175, 170–183
Trimble, R.B., Maley, F. (1984) Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-β-N-acetylglucosaminidase H. Anal. Biochem. 141, 515–522
Vaux, D., Tooze, J., Fuller, S. (1989) Identification of an intracellular receptor for the KDEL retention signal. J Cell. Biol. 109, 99a
Vitale, A., Ceriotti, A., Bollini, R., Chrispeels, M.J. (1984a) Biosynthesis and processing of phytohemagglutinin in developing bean cotyledons. Eur. J. Biochem. 141, 97–104
Vitale, A., Warner, T.G., Chrispeels, M.J. (1984b) Phaseolus vulgaris phytohemagglutinin contains high-mannose and modified oligosaccharide chains. Planta 160, 256–263
Voelker, T., Sturm, A., Chrispeels, M.J. (1987) Differences in expression between two seed lectin alleles obtained from normal and lectin-deficient beans are maintained in transgenic tobacco. EMBO J. 6, 3571–3577
Voelker, T.A., Herman, E.M., Chrispeels, M.J. (1989) In vitro mutated phytohemagglutinin genes expressed in tobacco seeds: role of glycans in protein targeting and stability. Plant Cell 1, 95–104
Zagouras, P., Rose, J.K. (1989) Carboxyterminal SEKDEL sequences retard but do not retain two secretory proteins in the endoplasmic reticulum. J. Cell Biol. 109, 2633–2652
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We thank Debra Donaldson for her contribution to the PHA gene constructions. This work has been supported by grants from the National Science Foundation (Cell Biology) and the Department of Energy (DE-FG03-86ER13497) to Maarten J. Chrispeels. The assistance of the staff of the Electron Microscope Laboratory, USDA, Beltsville is gratefully acknowledged.
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Herman, E.M., Tague, B.W., Hoffman, L.M. et al. Retention of phytohemagglutinin with carboxyterminal tetrapeptide KDEL in the nuclear envelope and the endoplasmic reticulum. Planta 182, 305–312 (1990). https://doi.org/10.1007/BF00197126
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DOI: https://doi.org/10.1007/BF00197126