The A/ENTH Domain-Containing Protein AtECA4 Is an Adaptor Protein Involved in Cargo Recycling from the trans-Golgi Network/Early Endosome to the Plasma Membrane

Abstract

Endocytosis and subsequent trafficking pathways are crucial for regulating the activity of plasma membrane-localized proteins. Depending on cellular and physiological conditions, the internalized cargoes are sorted at (and transported from) the trans-Golgi network/early endosome (TGN/EE) to the vacuole for degradation or recycled back to the plasma membrane. How this occurs at the molecular level remains largely elusive. Here, we provide evidence that the ENTH domain-containing protein AtECA4 plays a crucial role in recycling cargoes from the TGN/EE to the plasma membrane in Arabidopsis thaliana. AtECA4:sGFP primarily localized to the TGN/EE and plasma membrane (at low levels). Upon NaCl or mannitol treatment, AtECA4:sGFP accumulated at the TGN/EE at an early time point but was released from the TGN/EE to the cytosol at later time points. The ateca4 mutant showed higher resistance to osmotic stress and more sensitive to exogenous abscisic acid (ABA) than the wild type, as well as increased expression of ABA-inducible genes RD29A and RD29B. Consistently, ABCG25, a plasma membrane-localized ABA exporter, accumulated at the prevacuolar compartment in ateca4, indicating a defect in recycling to the plasma membrane. However, the role of AtECA4 in cargo recycling is not specific to ABCG25, as it also functions in the recycling of BRI1. These results suggest that AtECA4 plays a crucial role in the recycling of endocytosed cargoes from the TGN/EE to the plasma membrane.

EPSIN1 Modulates the Plasma Membrane Abundance of FLAGELLIN SENSING2 for Effective Immune Responses

“…These six EPSs form a subfamily that is distinct from the divergent ENTH domain-containing MODIFIED TRANSPORT TO THE VACUOLE, with roles in TGN/ EE-to-vacuolar cargo trafficking (Sauer et al, 2013). The Arabidopsis ENTH subfamily also clusters away from the large ANTH protein family that includes EPSIN-LIKE CLATHRIN ADAPTOR4 (ECA4), PHOSPHATI-DYLINOSITOL BINDING CLATHRIN ASSEMBLY PROTEIN5A (PICALM5; also called ECA2), PICALM5b, and AP180, for which physiological roles have been recently described using loss-of-function mutants (Sauer et al, 2013;Zouhar and Sauer, 2014;Muro et al, 2018;Nguyen et al, 2018;Kaneda et al, 2019). Thus, our identification of EPS1 modulating immune responses expands the short list of Arabidopsis ENTH/ANTH/ VHS domain family members with known physiological functions.…”

“…For quantifying intracellular compared with PM pixel intensity, single-plane confocal images from 7-d-old seedling cotyledons were analyzed for pixel intensity using ImageJ (https://imagej.net/Fiji/Downloads) as performed previously (Kitakura et al, 2011;Nguyen et al, 2018). Briefly, the Oval Selection tool was used to select regions of the cell cytoplasm adjacent to the PM and measure the mean pixel intensity.…”

The roles of endomembrane trafficking in plant abiotic stress responses

“…Many important PM‐localized proteins, such as the auxin transporters PINs, the BR receptor BRI1, and the pattern recognition receptor FLS2, are subject to the CME for protein turnover (Fan et al 2015; Dubeaux and Vert 2017; Liu et al 2018). Recently, the ABA exporter ABCG25 has also been reported to undergo the CME for vacuolar degradation and its spatial regulation is important for ABA homeostasis under stress conditions (Figure 1E) (Kuromori et al 2010; Park et al 2016; Nguyen et al 2018). More endosomal localizations of ABCG25 were observed when treated with NaCl, indicating that salt stress enhances the endocytosis of ABCG25 to reduce the efflux of ABA and increase the ABA content inside the cells.…”

Global Identification of ANTH Genes Involved in Rice Pollen Germination and Functional Characterization of a Key Member, OsANTH3

“…Endocytosis processes include various steps, such as cargo collection, vesicle assembly and invagination from the PM, and trafficking to the trans-Golgi network (TGN)/early endosome (EE), that are mediated by coat proteins as well as adaptor and accessory proteins (Nguyen et al, 2018). Clathrin, which functions as a coat protein during vesicle formation, does not directly interact with cargoes; instead, it interacts via adaptor proteins.…”

“…AtAP180/PICALM6 has been extensively shown to interact with clathrin and accelerate cargo efficiency in vitro (Barth and Holstein, 2004). Arabidopsis epsin-like clathrin adaptor (AtECA) protein 1, AtECA2/PICALM5a, and PICALM4 have shown involvement in cytokinesis during cell plate expansion (Song et al, 2012;Nguyen et al, 2018). Recent studies demonstrated the role of ANTH domain-containing proteins in Arabidopsis pollen tube growth, such as acting as adaptors of CCV or engaging in pollen tube PM integrity (Muro et al, 2018;Kaneda et al, 2019).…”