Skip to main content
PMC home page
The Plant Cell logoLink to The Plant Cell
. 1989 Jan;1(1):95–104. doi: 10.1105/tpc.1.1.95

In vitro mutated phytohemagglutinin genes expressed in tobacco seeds: role of glycans in protein targeting and stability.

T A Voelker 1, E M Herman 1, M J Chrispeels 1
PMCID: PMC159740  PMID: 2535471

Abstract

Phytohemagglutinin is a glycoprotein that accumulates in the protein storage vacuoles of bean seeds. The mature glycoprotein has a high-mannose and a complex glycan. We describe here the use of site-directed mutagenesis and expression of the mutated genes in transgenic tobacco to study the role of glycans in intracellular targeting. The reading frame for phytohemagglutinin-L was mutated so that either one or both of the glycosylation signals were disrupted to specifically prevent the attachment of asparagine-linked glycans. Expression of these genes with the beta-phaseolin promoter in the seeds of transgenic tobacco plants showed that phytohemagglutinin-L with only one glycan or without glycans was correctly targeted to the protein storage vacuoles of the seeds. Furthermore, the absence of either the complex glycan or the high-mannose glycan did not alter the processing of the other glycan. On the basis of these results, we propose that the targeting signal of this vacuolar protein is contained in its polypeptide domain and not in its glycans.

Full Text

The Full Text of this article is available as a PDF (3.0 MB).

Selected References

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

  1. A simple and general method for transferring genes into plants. Science. 1985 Mar 8;227(4691):1229–1231. doi: 10.1126/science.227.4691.1229. [DOI] [PubMed] [Google Scholar]
  2. Beachy R. N., Chen Z. L., Horsch R. B., Rogers S. G., Hoffmann N. J., Fraley R. T. Accumulation and assembly of soybean beta-conglycinin in seeds of transformed petunia plants. EMBO J. 1985 Dec 1;4(12):3047–3053. doi: 10.1002/j.1460-2075.1985.tb04044.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bevan M. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 1984 Nov 26;12(22):8711–8721. doi: 10.1093/nar/12.22.8711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burgess T. L., Kelly R. B. Constitutive and regulated secretion of proteins. Annu Rev Cell Biol. 1987;3:243–293. doi: 10.1146/annurev.cb.03.110187.001331. [DOI] [PubMed] [Google Scholar]
  5. Chappell J., Chrispeels M. J. Transcriptional and Posttranscriptional Control of Phaseolin and Phytohemagglutinin Gene Expression in Developing Cotyledons of Phaseolus vulgaris. Plant Physiol. 1986 May;81(1):50–54. doi: 10.1104/pp.81.1.50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chrispeels M. J., Higgins T. J., Spencer D. Assembly of storage protein oligomers in the endoplasmic reticulum and processing of the polypeptides in the protein bodies of developing pea cotyledons. J Cell Biol. 1982 May;93(2):306–313. doi: 10.1083/jcb.93.2.306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dorner A. J., Bole D. G., Kaufman R. J. The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins. J Cell Biol. 1987 Dec;105(6 Pt 1):2665–2674. doi: 10.1083/jcb.105.6.2665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Faye L., Sturm A., Bollini R., Vitale A., Chrispeels M. J. The position of the oligosaccharide side-chains of phytohemagglutinin and their accessibility to glycosidases determines their subsequent processing in the Golgi. Eur J Biochem. 1986 Aug 1;158(3):655–661. doi: 10.1111/j.1432-1033.1986.tb09803.x. [DOI] [PubMed] [Google Scholar]
  9. Hall T. C., Ma Y., Buchbinder B. U., Pyne J. W., Sun S. M., Bliss F. A. Messenger RNA for G1 protein of French bean seeds: Cell-free translation and product characterization. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3196–3200. doi: 10.1073/pnas.75.7.3196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hoffman L. M., Donaldson D. D., Bookland R., Rashka K., Herman E. M. Synthesis and protein body deposition of maize 15-kd zein in transgenic tobacco seeds. EMBO J. 1987 Nov;6(11):3213–3221. doi: 10.1002/j.1460-2075.1987.tb02638.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hoffman L. M., Donaldson D. D. Characterization of two Phaseolus vulgaris phytohemagglutinin genes closely linked on the chromosome. EMBO J. 1985 Apr;4(4):883–889. doi: 10.1002/j.1460-2075.1985.tb03714.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Johnson L. M., Bankaitis V. A., Emr S. D. Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease. Cell. 1987 Mar 13;48(5):875–885. doi: 10.1016/0092-8674(87)90084-5. [DOI] [PubMed] [Google Scholar]
  13. Kelly R. B. Pathways of protein secretion in eukaryotes. Science. 1985 Oct 4;230(4721):25–32. doi: 10.1126/science.2994224. [DOI] [PubMed] [Google Scholar]
  14. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  16. Leavitt R., Schlesinger S., Kornfeld S. Impaired intracellular migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and Sindbis virus. J Biol Chem. 1977 Dec 25;252(24):9018–9023. [PubMed] [Google Scholar]
  17. 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]
  18. Merlie J. P., Sebbane R., Tzartos S., Lindstrom J. Inhibition of glycosylation with tunicamycin blocks assembly of newly synthesized acetylcholine receptor subunits in muscle cells. J Biol Chem. 1982 Mar 10;257(5):2694–2701. [PubMed] [Google Scholar]
  19. Pfeffer S. R., Rothman J. E. Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu Rev Biochem. 1987;56:829–852. doi: 10.1146/annurev.bi.56.070187.004145. [DOI] [PubMed] [Google Scholar]
  20. Prives J., Bar-Sagi D. Effect of tunicamycin, an inhibitor of protein glycosylation, on the biological properties of acetylcholine receptor in cultured muscle cells. J Biol Chem. 1983 Feb 10;258(3):1775–1780. [PubMed] [Google Scholar]
  21. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schwaiger H., Hasilik A., von Figura K., Wiemken A., Tanner W. Carbohydrate-free carboxypeptidase Y is transferred into the lysosome-like yeast vacuole. Biochem Biophys Res Commun. 1982 Feb 11;104(3):950–956. doi: 10.1016/0006-291x(82)91341-9. [DOI] [PubMed] [Google Scholar]
  23. Sengupta-Gopalan C., Reichert N. A., Barker R. F., Hall T. C., Kemp J. D. Developmentally regulated expression of the bean beta-phaseolin gene in tobacco seed. Proc Natl Acad Sci U S A. 1985 May;82(10):3320–3324. doi: 10.1073/pnas.82.10.3320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sly W. S., Fischer H. D. The phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes. J Cell Biochem. 1982;18(1):67–85. doi: 10.1002/jcb.1982.240180107. [DOI] [PubMed] [Google Scholar]
  25. Staswick P., Chrispeels M. J. Expression of lectin genes during seed development in normal and phytohemagglutinin-deficient cultivars of Phaseolus vulgaris. J Mol Appl Genet. 1984;2(6):525–535. [PubMed] [Google Scholar]
  26. Sturm A., Chrispeels M. J. The high mannose oligosaccharide of phytohemagglutinin is attached to asparagine 12 and the modified oligosaccharide to asparagine 60. Plant Physiol. 1986 May;81(1):320–322. doi: 10.1104/pp.81.1.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sturm A., Van Kuik J. A., Vliegenthart J. F., Chrispeels M. J. Structure, position, and biosynthesis of the high mannose and the complex oligosaccharide side chains of the bean storage protein phaseolin. J Biol Chem. 1987 Oct 5;262(28):13392–13403. [PubMed] [Google Scholar]
  28. Tague B. W., Chrispeels M. J. The plant vacuolar protein, phytohemagglutinin, is transported to the vacuole of transgenic yeast. J Cell Biol. 1987 Nov;105(5):1971–1979. doi: 10.1083/jcb.105.5.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Trimble R. B., Maley F. Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H. Anal Biochem. 1984 Sep;141(2):515–522. doi: 10.1016/0003-2697(84)90080-0. [DOI] [PubMed] [Google Scholar]
  30. Valls L. A., Hunter C. P., Rothman J. H., Stevens T. H. Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide. Cell. 1987 Mar 13;48(5):887–897. doi: 10.1016/0092-8674(87)90085-7. [DOI] [PubMed] [Google Scholar]
  31. Vitale A., Ceriotti A., Bollini R., Chrispeels M. J. Biosynthesis and processing of phytohemagglutinin in developing bean cotyledons. Eur J Biochem. 1984 May 15;141(1):97–104. doi: 10.1111/j.1432-1033.1984.tb08162.x. [DOI] [PubMed] [Google Scholar]
  32. Vitale A., Chrispeels M. J. Transient N-acetylglucosamine in the biosynthesis of phytohemagglutinin: attachment in the Golgi apparatus and removal in protein bodies. J Cell Biol. 1984 Jul;99(1 Pt 1):133–140. doi: 10.1083/jcb.99.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Voelker T. A., Florkiewicz R. Z., Chrispeels M. J. Secretion of phytohemagglutinin by monkey COS cells. Eur J Cell Biol. 1986 Dec;42(2):218–223. [PubMed] [Google Scholar]
  34. Voelker T., Sturm A., Chrispeels M. J. Differences in expression between two seed lectin alleles obtained from normal and lectin-deficient beans are maintained in transgenic tobacco. EMBO J. 1987 Dec 1;6(12):3571–3577. doi: 10.1002/j.1460-2075.1987.tb02687.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Plant Cell are provided here courtesy of Oxford University Press

RESOURCES