The ESCRT-III-Interacting Deubiquitinating Enzyme AMSH3 is Essential for Degradation of Ubiquitinated Membrane Proteins in Arabidopsis thaliana

Abstract

Post-translational modification by ubiquitin plays a key role in the regulation of endocytic degradation in which ubiquitinated plasma membrane cargos are transported to the vacuole for degradation dependent on the ESCRT (endosomal sorting complex required for transport) machinery. Arabidopsis AMSH3 (ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM 3) is a deubiquitinating enzyme that interacts with at least two subunits of the ESCRT-III machinery, VPS2.1 and VPS24.1. amsh3 null mutation causes seedling lethality, and amsh3 null mutants show defects in multiple intracellular trafficking pathways. In this study, we further analyzed the amsh3 mutant phenotype and showed that amsh3 accumulates membrane-associated ubiquitinated proteins, supporting the indication that AMSH3 functions in ubiquitin-mediated endocytic degradation. In accordance with this, an enzymatic inactive variant of AMSH3 inhibits the AvrPtoB-dependent endocytic degradation of CERK1 (CHITIN ELICITOR RECEPTOR KINASE 1). Furthermore, we showed that the interaction of AMSH3 with ESCRT-III is important for its function in planta. Together, our data indicate the importance of AMSH3 and the AMSH3-ESCRT-III interaction for deubiquitination and degradation of ubiquitinated membrane substrates in plants.

VPS36-Dependent Multivesicular Bodies Are Critical for Plasmamembrane Protein Turnover and Vacuolar Biogenesis

“…Notably, vps36-1 can still undergo embryogenesis and develop past the germination stage, which suggests that VPS36 is functionally different from VACUOLELESS, an Arabidopsis protein essential for vacuolar biogenesis during embryogenesis (Rojo et al, 2001). These developmental defects in seed maturation and postgermination development have previously been reported for the amsh3 mutant, in which a gene encoding an ESCRT-III-associated deubiquitination enzyme is disrupted (Isono et al, 2010;Katsiarimpa et al, 2011Katsiarimpa et al, , 2014. The convergence of phenotypes highlights the importance of the EE/TGN and the ESCRT targeting pathway in vacuolar biogenesis.…”

“…3) and revealed the abundant accumulation of membrane-associated ubiquitinated proteins in vps36-1 seedlings (Fig. 9), a result that was also observed in the amsh3 mutant (Isono et al, 2010;Katsiarimpa et al, 2014). In wild-type cells, plasma membrane proteins constantly cycle between the plasma membrane and the EE/TGN.…”

Arabidopsis ALIX is required for the endosomal localization of the deubiquitinating enzyme AMSH3

“…AMSH proteins were previously shown to be essential for plant growth and development (20,27). To investigate whether ALIX functions in the same biological pathway as AMSH3, we analyzed available T-DNA insertion lines of ALIX.…”

“…Defects in AMSH function impair a number of intracellular trafficking events, including degradation of endocytosis cargos (22,25,27), vacuolar transport, and vacuole biogenesis (20), as well as defects in autophagic degradation (18,20,21). Although AMSH function in intracellular trafficking and protein degradation is well established, the molecular framework surrounding its function is not yet completely understood.…”

Routes to and from the plasma membrane: bulk flow versus signal mediated endocytosis

“…Other dominant internalization signals may be constituted by AP2-interacting tyrosine motifs 16,17 or ubiquitination. 18,19 Besides its influence during endocytosis, long TMDs may also affect the early secretory pathway. Even though the shortest deletion (DCT) was shown to cycle via the plasma membrane 6 the dramatic difference between DCT and DCTLoTM cannot be explained by an inhibition of endocytosis alone as it would not explain the redistribution of the ER-retained portion.…”