The KEEP ON GOING protein of Arabidopsis recruits the ENHANCED DISEASE RESISTANCE1 protein to trans-Golgi network/early endosome vesicles

Plant Physiol. 2011 Apr;155(4):1827-38. doi: 10.1104/pp.110.171785. Epub 2011 Feb 22.

Abstract

Loss-of-function mutations in the Arabidopsis (Arabidopsis thaliana) ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced resistance to powdery mildew infection, enhanced senescence, and enhanced programmed cell death under both abiotic and biotic stress conditions. All edr1-mediated phenotypes can be suppressed by a specific missense mutation (keg-4) in the KEEP ON GOING (KEG) gene, which encodes a multidomain protein that includes a RING E3 ligase domain, a kinase domain, ankyrin repeats, and HERC2-like (for HECT and RCC1-like) repeats. The molecular and cellular mechanisms underlying this suppression are poorly understood. Using confocal laser scanning microscopy and fluorescent protein fusions, we determined that KEG localizes to trans-Golgi network/early endosome (TGN/EE) vesicles. Both the keg-4 mutation, which is located in the carboxyl-terminal HERC2-like repeats, and deletion of the entire HERC2-like repeats reduced endosomal localization of KEG and increased localization to the endoplasmic reticulum and cytosol, indicating that the HERC2-like repeats facilitate the TGN/EE targeting of KEG. EDR1 colocalized with KEG to the TGN/EE when coexpressed but localized primarily to the endoplasmic reticulum when expressed alone. Yeast two-hybrid and coimmunoprecipitation analyses revealed that EDR1 and KEG physically interact. Deletion of the HERC2-like repeats abolished the interaction between KEG and EDR1 as well as the KEG-induced TGN/EE localization of EDR1, indicating that the recruitment of EDR1 to the TGN/EE is based on a direct interaction between EDR1 and KEG mediated by the HERC2-like repeats. Collectively, these data suggest that EDR1 and KEG function together to regulate endocytic trafficking and/or the formation of signaling complexes on TGN/EE vesicles during stress responses.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Arabidopsis / genetics
  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Endosomes / metabolism*
  • Gene Expression Regulation, Plant
  • Microscopy, Confocal
  • Mutagenesis, Site-Directed
  • Mutation
  • Plants, Genetically Modified / genetics
  • Plants, Genetically Modified / metabolism
  • Protein Interaction Domains and Motifs
  • Protein Structure, Secondary
  • Protein Transport
  • Protoplasts / metabolism
  • Recombinant Fusion Proteins / metabolism
  • Sequence Deletion
  • Transport Vesicles / metabolism
  • Two-Hybrid System Techniques
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*
  • trans-Golgi Network / metabolism*

Substances

  • Arabidopsis Proteins
  • EDR1 protein, Arabidopsis
  • Recombinant Fusion Proteins
  • KEG protein, Arabidopsis
  • Ubiquitin-Protein Ligases