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

Abstract

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...

The Plant Trans-Golgi Network: Not Just a Matter of Distinction

“…lower TGN pH values of 5.6 and 6.1 have been reported by other groups (Martinière et al, 2013;Luo et al, 2015).…”

“…In plant cells, interfering with the TGN-resident VHA-a1 isoform causes the swelling of Golgi cisternae and inhibits cell expansion and hypocotyl growth (Dettmer et al, 2006;Brüx et al, 2008). Mutants with reduced activity of the cytosolic VHA-c subunit alter the steady-state pH in the TGN/EE, leading to reduced motility of both Golgi and TGN and defects in secretion of the brassinosteroid receptor BRI1 and Cellulose Synthase (Luo et al, 2015). Curiously, the TGN/EE-localized V-ATPase seems to contribute via an undefined mechanism to vacuolar pH (Kriegel et al, 2015).…”

Functional Characterization of Propeptides in Plant Subtilases as Intramolecular Chaperones and Inhibitors of the Mature Protease

“…pH-dependent cleavage and degradation of the propeptide was also reported for some mammalian PCs and has been well studied in furin, where it provides a mechanism for compartment-specific activation of the protease (26,29). Similarly in SBT3, pH dependence of PP cleavage may prevent precocious activation and ensure that the active enzyme is not released before the pH drops along the secretory pathway from pH 6.3 in trans-Golgi cisternae to pH 5.6 in the trans-Golgi network/ early endosome (45).…”

Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase

“…An alternative explanation would be that vesicular trafficking from the TGN/EE to the vacuole delivers a substantial amount of protons and/or organic acids to the vacuolar lumen. The TGN/EE is the most acidic compartment of the plant endomembrane system (Martinière et al, 2013;Shen et al, 2013), and using a pH-sensor with suitable resolution at low pH, we have recently shown that in Arabidopsis roots pH in the TGN/EE is indeed lower than the vacuolar pH (Luo et al, 2015). At least three independent, parallel trafficking pathways exist from the TGN/EE to the vacuole (Pedrazzini et al, 2013;Rojas-Pierce, 2013), and it remains to be determined if the combined flux of protons delivered to the vacuolar lumen would be sufficient to maintain a proton gradient even in the absence of tonoplast-localized proton pumps.…”