The Cleavable N-terminal Domain of Plant Endopolygalacturonases from Clade B May Be Involved in a Regulated Secretion Mechanism

Florence Dal Degan,Robin Child,Ib Svendsen,Peter Ulvskov

doi:10.1074/jbc.m102136200

Florence Dal Degan, Robin Child + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m102136200

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Abstract

Polygalacturonases represent the most abundant carbohydrate hydrolase family in the Arabidopsis thaliana genome, and they are thought to be involved in nearly all of the developmental processes requiring cell wall modifications during the life cycle of the plant. By phylogenetic analysis, plant polygalacturonases fall into at least three groups, one of which is distinguished from the others by the presence of an additional N-terminal domain. We have used RDPG1, the polygalacturonase involved in pod dehiscence in oilseed rape (Brassica napus), as a model to investigate the function of this domain. We have confirmed that this domain is absent in the mature protein by determination of the N-terminal sequence of mature RDPG1 purified from oilseed rape pod. We have furthermore investigated the accumulation and subcellular localization of the precursor containing the N-terminal domain and of the mature protein throughout the development and maturation of the pod. Using recombinant expression in Pichia pastoris, we have produced the RDPG1 precursor, and we present evidence that the N-terminal domain of plant polygalacturonases is not involved in folding or inactivation of the precursor but may play a role in the intracellular transport of this protein family via a novel regulated secretion pathway.

Highlights

Polygalacturonases (PGs)1 (EC 3.2.1.15) are one of the most important classes of enzymes associated with pectin degradation and cell wall rearrangements
We have revisited and confirmed the presence of a cleavable N-terminal domain, which is absent in the mature protein for PGs from clade B using RDPG1 as a model, and we have investigated the site and time of cleavage of this N-terminal domain from the precursor protein in the plant
The RDPG1 precursor is composed of a predicted 23-amino acid-long signal peptide (Met-1–Ala-23) [24], a 41-amino acid-long cleavable N-terminal domain (Leu-24 –Thr-64), and a 367-amino acid-long catalytic domain starting at Glu-65

Summary

EXPERIMENTAL PROCEDURES

Plant Material—Culture of oilseed rape cultivars Topas and Fido and flower tagging to determine the days after anthesis (DAA) at each harvest were carried out as described by Petersen et al [6]. Recombinant Expression of Pro-RDPG1 and ⌬Pro-RDPG1 in P. pastoris—The nucleotides 156 –1388 and 285–1388 regions of the cDNA clone RDPG1 [6], corresponding to the presumed proenzyme (L24-P433) and mature (S67-P433) forms of RDPG1, respectively, were amplified by PCR using the forward primers 5Ј-AAAGAATTCCTTTGAGTAGCAACGTAGAT-3Ј and 5Ј-AACTGCAGCATCAACTGTTAGTGTTTC-3Ј and the reverse primer 5Ј-GCTCTAGATCATTAAGGGCATTTAGG-3Ј. These primers had been designed to introduce an EcoRI or PstI restriction site at the 5Ј-end of the PCR product and a XbaI site at its 3Ј-end (underlined in the sequences), as well as two stop codons at the 3Ј-end (in italics in the sequence). The supernatant was ultrafiltrated to a final volume of 100 ml using a Minitan unit equipped with

52 Fully mature

RESULTS

DISCUSSION