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. Clathrin coated vesicle is the main type of vesicle trafficking route at the post Golgi compartment in eukaryotic cell. Clathrin subunits function as a mechanical scaffold for vesicle formation, however clathrin cannot directly bind to both membrane and cargo. In fact, vesicle formation is a stepwise processes required energy for membrane deformation. Because of these reasons, adaptor and accessory protein are vital for vesicle initiation. In this study, I investigated one type of clathrin adaptors in Arabidopsis: an A/ENTH protein. Here I provide evidence that the ENTH domain-containing protein AtECA4 plays a crucial role in recycling cargoes from the TGN/EE to the plasma membrane. AtECA4:sGFP primarily localized to the TGN/EE and the 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 Arabidopsis thaliana mutant ateca4 showed higher resistance to osmotic stress and more sensitive to exogenous ABA than wild type, as well as increased expression of the ABA-inducible genes RD29A and RD29B. Consistent with the phenotype of ateca4 plants, ABCG25, a plasma membrane-localized ABA exporter, accumulated at the TGN/EE, revealing a defect in recycling to the plasma membrane. However, the role of AtECA4 in recycling is not specific to ABCG25, as it also functions in the recycling of BRI1. These results suggest that AtECA4 plays a role in the recycling of endocytosed cargoes from the TGN/EE to the plasma membrane.