Abstract
We have investigated targeting to the endoplasmic reticulum (ER) of wild-type GUS and a modified form (GUS S358) by making an N-terminal fusion of the β-glucuronidase (GUS) enzyme with the wheat α-amylase signal peptide.In vitro studies demonstrated that the modified GUS (S358) lacked the glycosylation site present within the wild-type enzyme. Analysis of transgenic tobacco plants revealed that the modified GUS enzyme retained activity upon passage to the ER. When further experiments were carried out to determine the cellular location of the modified GUS enzyme, it was found that (contrary to expectation) the majority of GUS activity was retained within the cell and was not secreted to the cell surface via the default pathway. The data indicated that the modified GUS enzyme is an unsuitable reporter enzyme for studying protein secretion.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Bevan, M. (1984) Binary vectors for plant transformation.Nucl. Acids Res. 12, 8711–21.
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding.Anal. Biochem. 72, 248–54.
Deblaere, R., Bytebier, B., De Greve, H., Deboeck, F., Schell, J., Van Montagu M. and Leemans, J. (1985) Efficient octopine Ti plasmid derived vectors forAgrobacterium-mediated gene transfer.Nucl. Acids Res. 13, 4777–88.
Denecke, J., Botterman, J. and Deblaere, R. (1990) Protein secretion in plant cells can occur via a default pathway.Pl. Cell 2, 51–9.
Draper, J., Scott, R., Armitage, P. and Walden, R. (1988)Plant Genetic Transformation and Gene Expression: a Laboratory Manual. Oxford, UK: Blackwell Scientific Publications.
Farrell, B.L. and Beachy, R.N. (1990) Manipulation of β-glucuronidase for use as a reporter in vacuolar targeting studies.Pl. Mol. Biol. 15, 821–5.
Firek, S., Draper, J., Owen, M.R.L., Gandecha, A., Cockburn, B. and Whitelam, G.C. (1993) Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures.Pl. Mol. Biol (in press).
Guerineau, F., Lucy, A. and Mullineaux, P. (1992) Effect of two consensus sequences preceding the translation initiator codon on gene expression in plant protoplasts.Pl. Mol. Biol. 18, 815–8.
Hein, B.H., Tang, Y., McLeod, D.A., Janda, K.D. and Hiatt, A. (1991) Evaluation of immunoglobulins from plant cells.Biotechnol. Prog. 7, 455–61.
Iturriaga, G., Jefferson, R.A. and Bevan, M. (1989) Endoplasmic reticulum targeting and glycosylation of hybrid proteins in transgenic tobacco.Pl. Cell 1, 381–90.
Jefferson, R.A., Kavanagh, T.A. and Bevan, M. (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO J. 6, 3901–7.
Kavanagh, T.A., Jefferson, R.A. and Bevan, M.W. (1988) Targeting a foreign protein to chloroplasts using fusions to the transit peptide of a chlorophyla/b binding protein.Mol. Gen. Genet. 215, 38–45.
Kunkel, T.A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic effects.Proc. Natl Acad. Sci. USA 82, 488–92.
Ozcan, S., Firek, S. and Draper, J. (1993) Selectable marker genes engineered for specific expression in target cells for transformation.Bio/Technology 11, 218–21.
Restrepo, M.A., Freed, D.D. and Carrington, J.C. (1990) Nuclear transport of plant potyviral proteins.Pl. Cell 2, 987–8.
Rothstein, S.J., Lazarus, C.M., Smith, W.E., Baulcombe, D.C. and Gatenby, A.A. (1984) Secretion of a wheat α-amylase inSaccharomyces cerevisiae.Nature 308, 662–5.
Schmitz, U.K. and Lonsdale, D.M. (1989) A yeast mitocondrial presequence functions as a signal for targeting to plant mitocondriain vivo.Pl. Cell 1, 783–91.
Vitale, A., Ceriotti, A. and Denecke, J. (1993) The role of the endoplasmic reticulum in protein synthesis, modification and intracellular transport.J. Exp. Bot. 44, 1417–44.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Firek, S., Whitelam, G.C. & Draper, J. Endoplasmic reticulum targeting of active modified β-glucuronidase (GUS) in transgenic tobacco plants. Transgenic Research 3, 326–331 (1994). https://doi.org/10.1007/BF01973593
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01973593