Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants.

Clathrin-mediated endocytosis is an essential cellular internalization pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. The evolutionarily ancient TSET-TPLATE complex (TPC) plays an essential, but ill-defined role in endocytosis in plants. Here we show that two highly disordered TPC subunits, AtEH1 and AtEH2, function as scaffolds to drive biomolecular condensation of the complex. These condensates specifically nucleate on the plasma membrane through interactions with anionic phospholipids, and facilitate the dynamic recruitment and assembly of clathrin, as well as early- and late-stage endocytic accessory proteins. Importantly, condensation promotes ordered clathrin assemblies. TPC-driven biomolecular condensation thereby facilitates dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, we show that a disordered region of AtEH1 controls the material properties of endocytic condensates in vivo. Alteration of these material properties disturbs the recruitment of accessory proteins, influences endocytosis dynamics and impairs plant responsiveness. Our findings reveal how collective interactions shape endocytosis.

Protein phase separation in plant membrane biology: more than just a compartmentalization strategy.

The formation of biomolecular condensates through phase separation is an important strategy to compartmentalize cellular functions. While it is now well established that condensates exist throughout eukaryotic cells, how condensates assemble and function on lipid membranes is only beginning to be understood. In this perspective, we highlight work from plant, animal, and yeast model systems showing that condensates assemble on many endomembrane surfaces to carry out diverse functions. In vesicle trafficking, condensation has reported roles in the formation of endocytic vesicles and autophagosomes and in the inactivation of secretory COPII vesicles. We briefly discuss how membranes and membrane lipids regulate the formation and function of membrane-associated condensates. This includes how membranes act as surfaces for condensate assembly, with lipids mediating the nucleation of condensates during endocytosis and other processes. Additionally, membrane-condensate interactions give rise to the biophysical property of "wetting", which has functional importance in shaping autophagosomal and vacuolar membranes. We also speculate on the existence of membrane-associated condensates during cell polarity in plants and discuss how condensation may help to establish functional plasma membrane domains. Lastly, we provide advice on relevant in vitro and in vivo approaches and techniques to study membrane-associated phase separation.

The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo.

Endocytosis controls the perception of stimuli by modulating protein abundance at the plasma membrane. In plants, clathrin-mediated endocytosis is the most prominent internalization pathway and relies on two multimeric adaptor complexes, the AP-2 and the TPLATE complex (TPC). Ubiquitination is a well-established modification triggering endocytosis of cargo proteins, but how this modification is recognized to initiate the endocytic event remains elusive. Here we show that TASH3, one of the large subunits of TPC, recognizes ubiquitinated cargo at the plasma membrane via its SH3 domain-containing appendage. TASH3 lacking this evolutionary specific appendage modification allows TPC formation but the plants show severely reduced endocytic densities, which correlates with reduced endocytic flux. Moreover, comparative plasma membrane proteomics identified differential accumulation of multiple ubiquitinated cargo proteins for which we confirm altered trafficking. Our findings position TPC as a key player for ubiquitinated cargo internalization, allowing future identification of target proteins under specific stress conditions.

NHX-type Na+(K+)/H+ antiporters are required for TGN/EE trafficking and endosomal ion homeostasis in Arabidopsis thaliana.

The regulation of ion and pH homeostasis of endomembrane organelles is critical for functional protein trafficking, sorting and modification in eukaryotic cells. pH homeostasis is maintained through the activity of vacuolar H+-ATPases (V-ATPases) pumping protons (H+) into the endomembrane lumen, and counter-action by cation/proton exchangers, such as the NHX family of Na+(K+)/H+ exchangers. In plants, V-ATPase activity at the trans-Golgi network/early endosome (TGN/EE) is important for secretory and endocytic trafficking; however, the role of the endosomal antiporters NHX5 and NHX6 in endomembrane trafficking is unclear. Here we show through genetic, pharmacological and live-cell imaging approaches that double knockout of NHX5 and NHX6 results in the impairment of endosome motility and protein recycling at the TGN/EE, but not in the secretion of integral membrane proteins. Furthermore, we report that nhx5 nhx6 mutants are partially insensitive to osmotic swelling of TGN/EE induced by the monovalent cation ionophore monensin, and to late endosomal swelling by the phosphatidylinositol 3/4-kinase inhibitor wortmannin, demonstrating that NHX5 and NHX6 function to regulate the luminal cation composition of endosomes.

Two Endosomal NHX-Type Na+/H+ Antiporters are Involved in Auxin-Mediated Development in Arabidopsis thaliana.

In Arabidopsis thaliana, the endosomal-localized Na+/H+ antiporters NHX5 and NHX6 regulate ion and pH homeostasis and are important for plant growth and development. However, the mechanism by which these endosomal NHXs function in plant development is not well understood. Auxin modulates plant growth and development through the formation of concentration gradients in plant tissue to control cell division and expansion. Here, we identified a role for NHX5 and NHX6 in the establishment and maintenance of auxin gradients in embryo and root tissues. We observed developmental impairment and abnormal cell division in embryo and root tissues in the double knockout nhx5 nhx6, consistent with these tissues showing high expression of NHX5 and NHX6. Through confocal microscopy imaging with the DR5::GFP auxin reporter, we identify defects in the perception, accumulation and redistribution of auxin in nhx5 nhx6 cells. Furthermore, we find that the steady-state levels of the PIN-FORMED (PIN) auxin efflux carriers PIN1 and PIN2 are reduced in nhx5 nhx6 root cells. Our results demonstrate that NHX5 and NHX6 function in auxin-mediated plant development by maintaining PIN abundance at the plasma membrane, and provide new insight into the regulation of plant development by endosomal NHX antiporters.