Abstract
The endosomal sorting complex required for transport (ESCRT) consists of several multi-protein subcomplexes which assemble sequentially at the endosomal surface and function in multivesicular body (MVB) biogenesis. While ESCRT has been relatively well characterized in yeasts and mammals, comparably little is known about ESCRT in plants. Here we explored the yeast two-hybrid protein interaction network and subcellular localization of the Arabidopsis thaliana ESCRT machinery. We show that the Arabidopsis ESCRT interactome possesses a number of protein–protein interactions that are either conserved in yeasts and mammals or distinct to plants. We show also that most of the Arabidopsis ESCRT proteins examined at least partially localize to MVBs in plant cells when ectopically expressed on their own or co-expressed with other interacting ESCRT proteins, and some also induce abnormal MVB phenotypes, consistent with their proposed functional role(s) as part of the ESCRT machinery in Arabidopsis. Overall, our results help define the plant ESCRT machinery by highlighting both conserved and unique features when compared to ESCRT in other evolutionarily diverse organisms, providing a foundation for further exploration of ESCRT in plants.
Highlights
In animal cells, plasma membrane proteins destined to be degraded are internalized via endocytosis and delivered through the endosomal pathway to the lysosome (Gruenberg and Stenmark, 2004)
First identified in the yeast Saccharomyces cerevisiae as class E vacuolar protein-sorting (Vps) proteins based on the observation that their mutations result in abnormal late endosomal structures termed class E compartments (Raymond et al, 1992), endosomal sorting complex required for transport (ESCRT) proteins have been since found in a wide range of other evolutionarily diverse species, suggesting they perform a variety of conserved functions (Leung et al, 2008)
The results from our yeast two-hybrid assays, as discussed below, revealed several new interactions for Arabidopsis ESCRT proteins, and reconfirmed almost all of the interactions previously published for selected Arabidopsis ESCRT proteins using the same or other [e.g., immunoprecipitations or biomolecular fluorescence complementation (BiFC)] interaction methods, validating the reliability of this assay for mapping the Arabidopsis ESCRT interactome overall
Summary
Plasma membrane proteins destined to be degraded are internalized via endocytosis and delivered through the endosomal pathway to the lysosome (Gruenberg and Stenmark, 2004) This sorting process is highly complex, involving the concentration of endocytosed membrane-bound, ubiquitinated cargo proteins within intralumenal vesicles of late endosomes or so-called multivesicular bodies (MVBs), which are derived from early (and recycling) endosomes by the progressive invagination and scission of their boundary membranes. The formation and scission of MVB intralumenal vesicles is unique, because, unlike other vesicle budding events (e.g., those mediated by clathrin or the coat protein complexes), it is directed away from the cytosol This process is mediated by a set of ∼30 soluble proteins collectively known as the endosomal sorting complex required for transport (ESCRT), which, in a sophisticated hierarchical- and stoichiometric-dependent manner, are assembled at the endosomal membrane into several multi-protein subcomplexes, termed ESCRT0, -I, -II, and -III. First identified in the yeast Saccharomyces cerevisiae as class E vacuolar protein-sorting (Vps) proteins based on the observation that their mutations result in abnormal late endosomal structures termed class E compartments (Raymond et al, 1992), ESCRT proteins have been since found in a wide range of other evolutionarily diverse species, suggesting they perform a variety of conserved functions (Leung et al, 2008)
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