Interactome of the plant-specific ESCRT-III component AtVPS2.2 in Arabidopsis thaliana.

The endosomal sorting complexes required for transport (ESCRT) guides transmembrane proteins to domains that bud away from the cytoplasm. The ESCRT machinery consists of four complexes. ESCRT complexes 0-II are important for cargo recognition and concentration via ubiquitin binding. Most of the membrane bending function is mediated by the large multimeric ESCRT-III complex and associated proteins. Here we present the first in vivo proteome analysis of a member of the ESCRT-III complex which is unique to the plant kingdom. We show with LC-MS/MS, yeast-two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) that coimmunoprecipitated proteins from Arabidopsis thaliana roots expressing a functional GFP-tagged VACUOLAR PROTEIN SORTING 2.2 (AtVPS2.2) protein are members of the ESCRT-III complex and associated proteins. Therefore we propose that at least in plants the large ESCRT-III membrane scaffolding complex consists of a mixture of SNF7, VPS2 and the associated VPS46 and VPS60 proteins. Apart from transmembrane proteins, numerous membrane-associated but also nuclear and extracellular proteins have been identified, indicating that AtVPS2.2 might be involved in processes beyond the classical ESCRT role. This study is the first in vivo proteome analysis with a tagged ESCRT-III component demonstrating the feasibility of this approach and provides numerous starting points for the investigation of the biological process in which AtVPS2.2 is involved.

The Arabidopsis deubiquitinating enzyme AMSH3 interacts with ESCRT-III subunits and regulates their localization.

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.

AtEXO70A1, a member of a family of putative exocyst subunits specifically expanded in land plants, is important for polar growth and plant development.

The exocyst is a hetero-oligomeric protein complex involved in exocytosis and has been extensively studied in yeast and animal cells. Evidence is now accumulating that the exocyst is also present in plants. Bioinformatic analysis of genes encoding plant homologs of the exocyst subunit, Exo70, revealed that three Exo70 subgroups are evolutionarily conserved among angiosperms, lycophytes and mosses. Arabidopsis and rice contain 22 and approximately 39 EXO70 genes, respectively, which can be classified into nine clusters considered to be ancient in angiosperms (one has been lost in Arabidopsis). We characterized two independent T-DNA insertional mutants of the AtEXO70A1 gene (exo70A1-1 and exo70A1-2). Heterozygous EXO70A1/exo70A1 plants appear to be normal and segregate in a 1:2:1 ratio, suggesting that neither male nor female gametophytes are affected by the EXO70A1 disruption. However, both exo70A1-1 and exo70A1-2 homozygotes exhibit an array of phenotypic defects. The polar growth of root hairs and stigmatic papillae is disturbed. Organs are generally smaller, plants show a loss of apical dominance and indeterminate growth where instead of floral meristems new lateral inflorescences are initiated in a reiterative manner. Both exo70A1 mutants have dramatically reduced fertility. These results suggest that the putative exocyst subunit EXO70A1 is involved in cell and organ morphogenesis.