V-ATPase-activity in the TGN/EE is required for exocytosis and recycling in Arabidopsis

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 phenotypes including impaired cell expansion, photomorphogenesis in the dark, ectopic lignification, reduced sensitivity to brassinosteroids (BRs) and reduced cellulose levels8–11. A clear explanation for those phenotypes is currently lacking. BRs are sensed at the plasma membrane by the BRASSINOSTEROID-INSENSITIVE1 (BRI1) receptor that transits through the TGN/EE during secretion and endocytosis en route to the vacuole and also undergoes recycling1,2,12,13. Cellulose is synthesized at the plasma membrane by cellulose synthase A (CesA) complexes that are assembled in the Golgi, or the endoplasmic reticulum, and delivered to the plasma membrane where they become activated14–17. Our results reveal that the det3 mutant has a reduced ability to acidify the TGN/EE, but not the Golgi and the vacuole, leading to secretion and recycling defects that contribute to BR insensitivity and cellulose deficiency.