The Arabidopsis Deubiquitinating Enzyme AMSH3 Interacts with ESCRT-III Subunits and Regulates Their Localization  

Abstract

Ubiquitination and deubiquitination regulate various cellular processes. We have recently shown that the deubiquitinating enzyme Associated Molecule with the SH3 domain of STAM3 (AMSH3) is involved in vacuole biogenesis and intracellular trafficking in Arabidopsis thaliana. However, little is known about the identity of its interaction partners and deubiquitination substrates. Here, we provide evidence that AMSH3 interacts with ESCRT-III subunits VPS2.1 and VPS24.1. The interaction of ESCRT-III subunits with AMSH3 is mediated by the MIM1 domain and depends on the MIT domain of AMSH3. We further show that AMSH3, VPS2.1, and VPS24.1 localize to class E compartments when ESCRT-III disassembly is inhibited by coexpression of inactive Suppressor of K+ transport Defect 1 (SKD1), an AAA-ATPase involved in the disassembly of ESCRT-III. We also provide evidence that AMSH3 and SKD1 compete for binding to VPS2.1. Furthermore, we show that the loss of AMSH3 enzymatic activity leads to the formation of cellular compartments that contain AMSH3, VPS2.1, and VPS24.1. Taken together, our study presents evidence that AMSH3 interacts with classical core ESCRT-III components and thereby provides a molecular framework for the function of AMSH3 in plants.

The dynamics of plant plasma membrane proteins: PINs and beyond

“…Interference with ESCRT-III activity, by contrast, was found to efficiently block the vacuolar sorting of constitutively ubiquitylated cargo protein (Herberth et al, 2012;Scheuring et al, 2012). Moreover, CHMP1 proteins, which are predicted to function as regulators of ESCRT-III, and the Arabidopsis AMSH3-type deubiquitinase (DUB) that acts in conjunction with ESCRT-III, were found to be required for the vacuolar sorting of plasma membrane protein cargo including PINs (Spitzer et al, 2009;Isono et al, 2010;Katsiarimpa et al, 2011) (Fig. 4B).…”

A BAR-Domain Protein SH3P2, Which Binds to Phosphatidylinositol 3-Phosphate and ATG8, Regulates Autophagosome Formation in Arabidopsis

“…However, there are only limited evidences for this model in plant cells (Rose et al, 2006;Toyooka et al, 2006;Katsiarimpa et al, 2011;Takatsuka et al, 2011;Hanamata et al, 2012). SH3P2-GFP signals were shown to translocate onto the autophagosome membrane upon autophagy induction in Arabidopsis cells (Figures 1B and 2B), indicating its involvement in membrane progression of autophagosome formation.…”

“…However, structures that are necessary for autophagosome formation in plants have remained elusive. Double-membrane structures, morphologically recognized as autophagosomes, had been reported in plants but had not been validated (Rose et al, 2006;Katsiarimpa et al, 2011;Reyes et al, 2011;Minibayeva et al, 2012). In our investigation, SH3P2-positive structures in Arabidopsis share several features with autophagosomes in mammalian cells: (1) They arise from the isolation membrane as well as from omegasome-like structures and subsequently wrap together into bi/multilayer-membrane autophagosome structures (Figures 4 and 5A; see Supplemental Figure 4 online); (2) they can expand and mature by fusing with endosomes or by engulfing cytoplasmic cargos ( Figures 5B and 5C); and (3) they reacted positively with the autophagosome marker ATG8 (Figures 2, 6A, and 6C).…”

“…Membrane sources, including the endoplasmic reticulum (ER), mitochondria, ER-mitochondria contact sites, ER-Golgi intermediate compartment, Golgi apparatus, and plasma membrane have been postulated to contribute to autophagosome formation in yeast and/or mammalian cells (Axe et al, 2008;Hayashi-Nishino et al, 2010a, 2010bHailey et al, 2010;Noda et al, 2011;Ohashi and Munro, 2010;Ravikumar et al, 2010;Ge et al, 2013;Hamasaki et al, 2013). In plants, however, most observations of autophagosome-related structures have been based on the classical features of autophagosomes: their isolated double-membrane appearance, enclosed autophagic cargos, and labeling with an autophagosome marker (Rose et al, 2006;Toyooka et al, 2006;Katsiarimpa et al, 2011;Takatsuka et al, 2011;Hanamata et al, 2012). So far, only one electron microscopy (EM) study has shown direct labeling of a double-membrane autophagosome structure in Arabidopsis thaliana with antibodies against the autophagosomal marker ATG8 (Reyes et al, 2011).…”

Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses

“…In the case of AGC1.8, this PCR was performed in two rounds due to the extended length of the polybasic motif. Protoplast transformation was performed as previously described (Katsiarimpa et al, 2011). Confocal analysis was carried out 14 h after transformation.…”