The cleavable N-terminal domain of plant endopolygalacturonases from clade B may be involved in a regulated secretion mechanism.
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.
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Biotechnology Group, Danish Institute of Agricultural Sciences, Thorvaldsensvej 40, DK-1871 Frederiksberg C., Denmark. fdd@pharmexa.com
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Svendsen
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Ulvskov
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001937:Brassica / Q000201:enzymology*; D017931:DNA Primers; D006868:Hydrolysis; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011096:Polygalacturonase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D011096:Polygalacturonase