The Heat Shock Protein 40-Type Chaperone MASH Supports the Endoplasmic Reticulum-Associated Degradation E3 Ubiquitin Ligase MAKIBISHI1 in Medicago truncatula

Abstract

Jasmonates (JA) are oxylipin-derived phytohormones that trigger the production of specialized metabolites that often serve in defense against biotic stresses. InMedicago truncatula, a JA-induced endoplasmic reticulum-associated degradation (ERAD)-type machinery manages the production of bioactive triterpenes and thereby secures correct plant metabolism, growth, and development. This machinery involves the conserved RING membrane-anchor (RMA)-type E3 ubiquitin ligase MAKIBISHI1 (MKB1). Here, we discovered two additional members of this protein control apparatus via a yeast-based protein–protein interaction screen and characterized their function. First, a cognate E2 ubiquitin-conjugating enzyme was identified that interacts with MKB1 to deliver activated ubiquitin and to mediate its ubiquitination activity. Second, we identified a heat shock protein 40 (HSP40) that interacts with MKB1 to support its activity and was therefore designated MKB1-supporting HSP40 (MASH).MASHexpression was found to be co-regulated with that ofMKB1. The presence of MASH is critical for MKB1 and ERAD functioning because the dramatic morphological, transcriptional, and metabolic phenotype ofMKB1knock-downM. truncatulahairy roots was phenocopied by silencing ofMASH. Interaction was also observed between theArabidopsis thaliana(Arabidopsis) homologs of MASH and MKB1, suggesting that MASH represents an essential and plant-specific component of this vital and conserved eukaryotic protein quality control machinery.