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

PLoS One. 2012;7(3):e32422. doi: 10.1371/journal.pone.0032422. Epub 2012 Mar 2.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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


  • Arabidopsis Proteins
  • Intercellular Signaling Peptides and Proteins
  • Plant Extracts
  • Polysaccharides
  • Progranulins
  • Cysteine Proteases
  • RD21 protein, Arabidopsis
  • Cysteine Endopeptidases
  • aleurain
  • vacuolar processing enzyme
  • Cysteine