ArabidopsisEPSIN1 Plays an Important Role in Vacuolar Trafficking of Soluble Cargo Proteins in Plant Cells via Interactions with Clathrin, AP-1, VTI11, and VSR1

Abstract

Epsin and related proteins play important roles in various steps of protein trafficking in animal and yeast cells. Many epsin homologs have been identified in plant cells from analysis of genome sequences. However, their roles have not been elucidated. Here, we investigate the expression, localization, and biological role in protein trafficking of an epsin homolog, Arabidopsis thaliana EPSIN1, which is expressed in most tissues we examined. In the cell, one pool of EPSIN1 is associated with actin filaments, producing a network pattern, and a second pool localizes primarily to the Golgi complex with a minor portion to the prevacuolar compartment, producing a punctate staining pattern. Protein pull-down and coimmunoprecipitation experiments reveal that Arabidopsis EPSIN1 interacts with clathrin, VTI11, γ-adaptin-related protein (γ-ADR), and vacuolar sorting receptor1 (VSR1). In addition, EPSIN1 colocalizes with clathrin and VTI11. The epsin1 mutant, which has a T-DNA insertion in EPSIN1, displays a defect in the vacuolar trafficking of sporamin:green fluorescent protein (GFP), but not in the secretion of invertase:GFP into the medium. Stably expressed HA:EPSIN1 complements this trafficking defect. Based on these data, we propose that EPSIN1 plays an important role in the vacuolar trafficking of soluble proteins at the trans-Golgi network via its interaction with γ-ADR, VTI11, VSR1, and clathrin.

Abscisic Acid Uridine Diphosphate Glucosyltransferases Play a Crucial Role in Abscisic Acid Homeostasis in Arabidopsis

“…2B), which confirms their cytosolic localization. As a control for the soluble fractionation, Arabidopsis aleurain-like protein (AALP; Song et al, 2006) was detected with anti-AALP antibody. It was specifically detected in the soluble fraction, confirming the fractionation.…”

EpsinR2 Interacts with Clathrin, Adaptor Protein-3, AtVTI12, and Phosphatidylinositol-3-Phosphate. Implications for EpsinR2 Function in Protein Trafficking in Plant Cells

“…Epsin 1 was originally identified as a protein that plays a critical role in endocytosis (Chen et al, 1998). Subsequently, a large number of epsin-related proteins were identified in a variety of organisms (De Camilli et al, 2002;Wendland, 2002;Overstreet et al, 2003;LegendreGuillemin et al, 2004;Song et al, 2006). A common feature of all epsin-related APs is the presence of a highly conserved epsin N-terminal homology (ENTH) domain.…”

“…Second, epsins recruit clathrin, as well as other accessory proteins, to the donor membrane compartment through protein-protein interactions. Epsins have been shown to bind to a number of proteins, including clathrin, AP complexes, and vesicle-associated SNARE (Rosenthal et al, 1999;Drake et al, 2000;Chidambaram et al, 2004;Song et al, 2006). Interestingly, rat epsin1, and yeast Ent1p and Ent2p, which participate in endocytosis, have binding motifs for AP-2, the hetrotetrameric adaptor complex for endocytosis (Chen et al, 1998;Legendre-Guillemin et al, 2004).…”

“…A common feature of all epsin-related APs is the presence of a highly conserved epsin N-terminal homology (ENTH) domain. Epsin-related proteins can be divided into two functional categories: group one is involved in endocytosis from the plasma membrane and includes animal epsin1 and Lqf, and yeast (Saccharomyces cerevisiae) Ent1p and Ent2p (Chen et al, 1998;De Camilli et al, 2002;Wendland, 2002); group two is involved in lysosomal/ vacuolar trafficking pathways from the TGN or endosomes and includes animal EpsinR/clint/enthoprotin, yeast Ent3p and Ent4p, and Arabidopsis (Arabidopsis thaliana) EPSIN1 (Kalthoff et al, 2002;Wasiak et al, 2002;Hirst et al, 2003;Chidambaram et al, 2004;Eugster et al, 2004;Saint-Pol et al, 2004;Song et al, 2006). Although the epsin proteins are divided into two functional groups, they may play essentially the same role in different pathways, that is, facilitation of CCV formation at donor membrane compartments.…”

TNO1 Is Involved in Salt Tolerance and Vacuolar Trafficking in Arabidopsis

“…Storage proteins with C-terminal sorting sequences are also bound by VSRs or by a second putative sorting receptor, RMR1 (Jiang et al, 2000;Shimada et al, 2003a;Park et al, 2005Park et al, , 2007Hinz et al, 2007). After the receptors recognize their cargo, vacuolar proteins are transported to the prevacuolar compartment (PVC; Sanderfoot et al, 1998;Happel et al, 2004;Song et al, 2006) before transport on to the vacuole. In mammalian cells, cargo receptors are recycled from their destination to the site of cargo binding (Seaman, 2005).…”