title: Post-translational regulation and trafficking of the granulin-containing protease RD21 of Arabidopsis thaliana.
abstract: 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.
authors.affiliation: The Plant Chemetics Lab, Chemical Genomics Centre of the Max Planck Society, Max Planck Institute for Plant Breeding Research, Cologne, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Gu
authors.forename: Mohammed
authors.initials: M
authors.lastname: Shabab
authors.forename: Richard
authors.initials: R
authors.lastname: Strasser
authors.forename: Pieter J
authors.initials: PJ
authors.lastname: Wolters
authors.forename: Takayuki
authors.initials: T
authors.lastname: Shindo
authors.forename: Melanie
authors.initials: M
authors.lastname: Niemer
authors.forename: Farnusch
authors.initials: F
authors.lastname: Kaschani
authors.forename: Lukas
authors.initials: L
authors.lastname: Mach
authors.forename: Renier A L
authors.initials: RA
authors.lastname: van der Hoorn
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism*; D019921:Biotinylation; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry; D057056:Cysteine Proteases / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D018506:Gene Expression Regulation, Plant; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D009154:Mutation; D010936:Plant Extracts / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D000077153:Progranulins; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary
chemical_list: D029681:Arabidopsis Proteins; D036341:Intercellular Signaling Peptides and Proteins; D010936:Plant Extracts; D011134:Polysaccharides; D000077153:Progranulins; D057056:Cysteine Proteases; C549570:RD21 protein, Arabidopsis; D003546:Cysteine Endopeptidases; C046726:aleurain; C089278:vacuolar processing enzyme; D003545:Cysteine