Kinetics of prolyl hydroxylation, intracellular transport and C-terminal processing of the tobacco vacuolar chitinase

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.