Abstract
The dynamics of intracellular transport and processing of one of the vacuolar chitinases of tobacco (Nic-otiana tabacum L.), chitinase A (CHN A; EC 3.2.1.14), was investigated with pulse-chase experiments in conjunction with cell fractionation and immunoprecipitation. Mature CHN A is composed of two domains, the N-terminal cysteine-rich chitin-binding domain and the catalytic domain, linked by a short peptide spacer containing several hydroxyprolines. It is synthetized as a preproprotein with a signal peptide for cotranslational transport into the endoplasmic reticulum (ER) and a C-terminal, vacuolar targeting peptide (VTP) required for targeting to the vacuole, which is removed by proteolytic cleavage. We investigated transformed N. sylvestris plants constitutively expressing CHN A or a mutant CHN A lacking the chitin-binding domain and spacer (ΔCS CHN A), as well as N. plumbaginifolia protoplasts transiently expressing the same constructs. Processing and transport in the two systems was very similar. A shift in the apparent molecular weight of chitinase, indicative of prolyl hydroxylation, was detectable only 30 min after appearance of newly synthesized prochitinase, indicating that it might occur in a post-ER compartment. In total, labelled chitinase was detected in the microsomal fraction for up to 90–120 min as a prochitinase, bearing the VTP. Later, it appeared only in the soluble fraction (comprising the vacuolar sap) as the mature CHN A without the VTP. In both systems, intracellular transport and processing of ΔCS CHN A was faster than that of the wildtype form, indicating that correct folding of the cysteine-rich chitin-binding domain and/or prolyl hydroxylation of the spacer delays transport to the vacuole.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- CBD:
-
chitin-binding domain
- CHN A:
-
chitinase A
- PBS:
-
phosphate-buffered saline
- S:
-
proline-rich spacer
- VTP:
-
vacuolar targeting peptide
- Δ CS:
-
deletion of CBD and S;
- Δ VTP:
-
deletion of VTP
References
Andreae, M., Blankenstein, P., Yu-Hua, Z., Robinson, D.G. (1988) Towards the subcellular localization of plant prolyl hydroxylase. Eur. J. Cell Biol. 47, 181–192
Bednarek, S.Y., Raikhel, N.V. (1992) Intracellular trafficking of secretory proteins. Plant Mol. Biol. 20, 133–150
Bednarek, S.Y., Wilkins, T.A., Dombrowski, J.E., Raikhel, N.V. (1990) A carboxyl-terminal propeptide is necessary for proper sorting of barley lectin to vacuoles of tobacco. Plant Cell 2, 1145–1155
Blum, H., Beier, H., Gross, H.J. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93–99
Boller, T. (1993) Antimicrobial functions of the plant hydrolases, chitinase and β-1,3-glucanase. In: Mechanisms of plant defense responses, pp. 391–400, Fritig, B., Legrand, M., edspp. Kluwer Academic Publishers, Dordrecht
Bonner, W.M., Laskey, R.A. (1974) A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83–88
Chrispeels, M.J. (1991) Sorting of proteins in the secretory system. Annu. Rev. Plant Physiol. 42, 21–53
Chrispeels, M.J., Raikhel, N. (1992) Short peptide domains target proteins to plant vacuoles. Cell 68, 613–616
Collinge, D.B., Kragh, K.M., Mikkelsen, J.D., Nielsen, K.K., Rasmussen, U., Vad, K. (1993) Plant chitinases. Plant J. 3, 31–40
Doolittle, R.F. (1976) Of URFs and ORFs: A primer on how to analyze derived amino acid sequences. University Science, Mill Valley, California
Gomez, L., Chrispeels, M.J. (1993) Tonoplast and soluble vacuolar proteins are targeted by different mechanisms. Plant Cell 5, 1113–1124
Goodall, G.J., Wiebauer, K., Filipowicz, W. (1990) Analysis of premRNA processing in transfected plant protoplasts. Methods Enzymol. 181, 148–161
Greenwood, F.C., Hunter, W.M., Glover, J.S. (1963) The preparation of 131I-labelled human growth hormone of high specific radioactivity. Biochem. J. 89, 114–123
Harlow, E., Lane, D. (1988) Antibodies. A laboratory manual. Cold Spring Harbor Laboratory, New york
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680–685
Meins, F. Jr, Neuhaus, J.-M., Sperisen, C., Ryals, J. (1992) The primary structure of plant pathogenesis-related glucanohyd-E. Freydl et al.: Transport and maturation of tobacco chitinase rolases and their genes. In: Plant gene research, vol. 8, pp. 245–282, Boller, T., Meins, F. Jr, eds. Springer, Wien
Neuhaus, J.-M., Sticher, L., Meins, F. Jr, Boller, T. (1991a) A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole. Proc. Natl. Acad. Sci. USA 88, 10362–10366
Neuhaus, J.-M., Ahl-Goy, P., Hinz, U., Flores, S., Meins, F. Jr (1991b) High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection. Plant Mol. Biol. 16, 141–151
Neuhaus, J.-M., Pietrzak, M., Boller, T. (1994) Mutation analysis of the C-terminal vacuolar targeting peptide of tobacco chitinase,low specificity of the sorting system, and gradual transition between intracellular retention and secretion into the extracellular space. Plant J. 5, 45–54
Raikhel, N.V., Lerner, D.R. (1991) Expression and regulation of lectin genes in cereals and rice. Dev. Genet. 12, 255–260
Satiat-Jeunemaitre, B., Hawes, C. (1993) Insights into the secretory pathway and vesicular transport in plant cells. Biol. Cell 79, 7–15
Sauer, A., Robinson, D.G. (1985) Intracellular localization of posttranslational modifications in the synthesis of hydroxyprolinerich glycoproteins. I. Peptidyl proline hydroxylation in maize roots. Planta 164, 287–294
Shinshi, H., Meins, F. Jr (1987) Regulation of a plant pathogenesisrelated enzyme, Inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin. Proc. Natl. Acad. Sci. 84, 89–93
Sticher, L., Hinz, U., Meyer, A.D., Meins, F. Jr (1992a) Intracellular transport and processing of tobacco vacuolar β-1,3-glucanase. Planta 188, 559–565
Sticher, L., Hofsteenge, J., Milani, A., Neuhaus, J.-M., Meins, F. Jr (1992b) Vacuolar chitinase of tobacco, A new class of hydroxyproline-containing proteins. Science 257, 655–657
Sticher, L., Hofsteenge, J., Neuhaus, J.-M., Boller, T., Meins, F. Jr (1993) Posttranslational processing of a new class of hydroxyproline-containing proteins, prolyl hydroxylation and C-terminal cleavage of tobacco (Nicotiana tabacum) vacuolar chitinase. Plant Physiol. 101, 1239–1247
Stüber, D., Matile, H., Garotta, G. (1990) System for high-level production in Escherichia coli and rapid purification of recombinant proteins. In: Immunological methods, pp. 121–152, Lefkovits, I., Pernis, B., eds. Academic Press, New York
Wright, C.S., Gavilines, F., Peterson, D.L. (1984) Primary structure of wheat germ agglutinin isolectin. 2. Peptide order deduced from X-ray structure. Biochemistry 23, 280–287
Author information
Authors and Affiliations
Corresponding author
Additional information
We thank M. Müller and T. Hohn, Friedrich Miescher-Institute, Basel, for the preparation of the protoplasts and F. Fischer, Friedrich Miescher-Institute, Basel, for the synthesis of the peptide. This work was supported by the Swiss National Science Foundation, Grants 31-26402.89 and 3100-037434.93.
Rights and permissions
About this article
Cite this article
Freydl, E., Meins, F., Boller, T. et al. Kinetics of prolyl hydroxylation, intracellular transport and C-terminal processing of the tobacco vacuolar chitinase. Planta 197, 250–256 (1995). https://doi.org/10.1007/BF00202644
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00202644