Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane.

The Arabidopsis EH proteins (AtEH1/Pan1 and AtEH2/Pan1) are components of the endocytic TPLATE complex (TPC) which is essential for endocytosis. Both proteins are homologues of the yeast ARP2/3 complex activator, Pan1p. Here, we show that these proteins are also involved in actin cytoskeleton regulated autophagy. Both AtEH/Pan1 proteins localise to the plasma membrane and autophagosomes. Upon induction of autophagy, AtEH/Pan1 proteins recruit TPC and AP-2 subunits, clathrin, actin and ARP2/3 proteins to autophagosomes. Increased expression of AtEH/Pan1 proteins boosts autophagosome formation, suggesting independent and redundant pathways for actin-mediated autophagy in plants. Moreover, AtEHs/Pan1-regulated autophagosomes associate with ER-PM contact sites (EPCS) where AtEH1/Pan1 interacts with VAP27-1. Knock-down expression of either AtEH1/Pan1 or VAP27-1 makes plants more susceptible to nutrient depleted conditions, indicating that the autophagy pathway is perturbed. In conclusion, we identify the existence of an autophagy-dependent pathway in plants to degrade endocytic components, starting at the EPCS through the interaction among AtEH/Pan1, actin cytoskeleton and the EPCS resident protein VAP27-1.

In plants, vacuolar H + -ATPase (V-ATPase) activity acidifies both the trans-Golgi network/early endosome (TGN/EE) and the vacuole. This dual V-ATPase function has impeded our understanding in how the pH homeostasis within the plant TGN/EE controls exo-and endocytosis.⋆ Staffan. Persson@unimelb.au.edu, karin.schumacher@cos.uni-heidelberg.de, and eugenia.russinova@psb.vib-ugent. Additional informationSupplementary information is available on line. Competing interestsThe authors declare no competing financial interests Europe PMC Funders GroupAuthor Manuscript Nat Plants. Author manuscript; available in PMC 2016 June 13. Here, we show that the weak V-ATPase mutant deetiolated3 (det3) displayed a pH increase in the TGN/EE, but not in the vacuole, strongly impairing secretion and recycling of the brassinosteroid receptor and the cellulose synthase complexes to the plasma membrane, in contrast to mutants lacking tonoplast-localized V-ATPase activity only. The brassinosteroid insensitivity and the cellulose deficiency defects in det3 were tightly correlated with reduced Golgi and TGN/EE motility. Thus, our results provide strong evidence that acidification of the TGN/EE, but not of the vacuole, is indispensable for functional secretion and recycling in plants.Plant exo-and endocytic pathways converge at the trans-Golgi network/early endosome (TGN/EE) compartment where different cargos are sorted to further destinations1,2. In animal and yeast cells, acidification of intracellular organelles is crucial for the function of the secretory and endocytic pathways and requires proton pumping activity of the vacuolar H + -ATPases (V-ATPase)3-5. The V-ATPase is conserved across species and consists of multiple subunits that are organized in a cytosolic V1 domain, which is important for the ATP hydrolysis (including A, B, C, D, E, F, G, and H subunits), and an integral membrane V0 domain, which forms the proton pore (including a, d, c, c" and e subunits)3. In Arabidopsis thaliana, the V-ATPase activity is associated with both the TGN/EEs and the tonoplast that are marked by the differential localization of the membraneVHA-a1, VHA-a2 and VHA-a3 isoforms1,6,7. The vha-a3 mutant and the vha-a2 vha-a3 double mutant that lack the tonoplast V-ATPase activity do not display severe defects in cell expansion, whereas the inducible inhibition of the TGN/EE-localized VHA-a1 isoform constrains it7,8. Treatment with the V-ATPase inhibitor concanamycinA (ConcA) resulted in loss of the TGN/EE identity and interfered with the trafficking of endocytic and secretory cargos1,2. Given the differential localization of the V-ATPases, the reduced cell expansion has been concluded to be caused by defects in TGN/EE compartments rather than in the vacuole8, but the nature of these defects has not been clarified. In contrast, the cytosolic V-ATPase subunit C (VHA-C), encoded by the single-copy VHA-C/DEETIOLATED3 (DET3) gene, is required for V-ATPase activity at the TGN/EEs and at the vacuole9. A knockdown allele of DET3 displayed pleiotropic phe...