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RD21-like proteases are ubiquitous, plant-specific papain-like proteases typified by carrying a C-terminal granulin domain. RD21-like proteases are involved in immunity and associated with senescence and various types of biotic and abiotic stresses. Here, we interrogated Arabidopsis RD21 regulation and trafficking by site-directed mutagenesis, agroinfiltration, western blotting, protease activity profiling and protein degradation. Using an introduced N-glycan sensor, deglycosylation experiments and glyco-engineered N. benthamiana plants, we show that RD21 passes through the Golgi where it becomes fucosylated. Our studies demonstrate that RD21 is regulated at three post-translational levels. Prodomain removal is not blocked in the catalytic Cys mutant, indicating that RD21 is activated by a proteolytic cascade. However, RD21 activation in Arabidopsis does not require vacuolar processing enzymes (VPEs) or aleurain-like protease AALP. In contrast, granulin domain removal requires the catalytic Cys and His residues and is therefore autocatalytic. Furthermore, SDS can (re-)activate latent RD21 in Arabidopsis leaf extracts, indicating the existence of a third layer of post-translational regulation, possibly mediated by endogenous inhibitors. RD21 causes a dominant protease activity in Arabidopsis leaf extracts, responsible for SDS-induced proteome degradation.

Original publication

DOI

10.1371/journal.pone.0032422

Type

Journal article

Journal

PLoS One

Publication Date

2012

Volume

7

Keywords

Arabidopsis, Arabidopsis Proteins, Biotinylation, Catalysis, Cysteine, Cysteine Endopeptidases, Cysteine Proteases, Gene Deletion, Gene Expression Regulation, Plant, Glycosylation, Golgi Apparatus, Intercellular Signaling Peptides and Proteins, Mass Spectrometry, Mutation, Plant Extracts, Plant Leaves, Polysaccharides, Protein Processing, Post-Translational, Protein Structure, Tertiary