Post-translational regulation and trafficking of the granulin-containing protease RD21 of Arabidopsis thaliana

Journal article

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: Gu, C; Shabab, M; Strasser, R; Wolters, P; Shindo, T

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Public Library of Science
• Journal: PLoS ONE
• Volume: 7
• Issue: 3
• Pages: e32422
• Publication date: 2012-03-02
• Acceptance date: 2012-01-26
• DOI: 10.1371/journal.pone.0032422
• EISSN: 1932-6203
• Language: English
• Keywords: Mutation; Protein Processing, Post-Translational; Arabidopsis; Gene Deletion; Plant Leaves; Biotinylation; Catalysis; Glycosylation; Cysteine Endopeptidases; SBTMR; Gene Expression Regulation, Plant; Mass Spectrometry; Arabidopsis Proteins; Polysaccharides; Plant Extracts; Cysteine; Intercellular Signaling Peptides and Proteins; Cysteine Proteases; Golgi Apparatus; Protein Structure, Tertiary