KINETICS OF PROLYL HYDROXYLATION, INTRACELLULAR-TRANSPORT AND C-TERMINAL PROCESSING OF THE TOBACCO VACUOLAR CHITINASE

The dynamics of intracellular transport and processing of one of the v acuolar chitinases of tobacco (Nicotiana tabacum L.), chitinase A (CHN A; EC 3.2.1.14), was investigated with pulse-chase experiments in con junction with cell fractionation and immunoprecipitation. Mature CHN A is composed of two domains, the N-terminal cysteine-rich chitin-bindi ng domain and the catalytic domain, linked by a short peptide spacer c ontaining several hydroxyprolines. It is synthetized as a preproprotei n with a signal peptide for cotranslational transport into the endopla smic reticulum (ER) and a C-terminal, vacuolar targeting peptide (VTP) required for targeting to the vacuole, which is removed by proteolyti c cleavage. We investigated transformed N. sylvestris plants constitut ively expressing CHN A or a mutant CHN A lacking the chitin-binding do main and spacer (Delta CS CHN A), as well as N. plumbaginifolia protop lasts transiently expressing the same constructs. Processing and trans port in the two systems was very similar. A shift in the apparent mole cular weight of chitinase, indicative of prolyl hydroxylation, was det ectable only 30 min after appearance of newly synthesized prochitinase , indicating that it might occur in a post-ER compartment. In total, l abelled chitinase was detected in the microsomal fraction for up to 90 -120min 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 proces sing of Delta CS CHN A was faster than that of the wildtype form, indi cating that correct folding of the cysteine-rich chitin-binding domain and/or prolyl hydroxylation of the spacer delays transport to the vac uole.