Structure of the hexameric fungal plasma membrane proton pump in its auto-inhibited state

The fungal plasma membrane H+-ATPase Pma1 is a vital enzyme, generating a proton-motive force that drives the import of essential nutrients. Auto-inhibited Pma1 hexamers in starving fungi are activated by glucose signalling resulting in phosphorylation of the auto-inhibitory domain. As related P-type ATPases are not known to oligomerise, the physiological relevance of Pma1 hexamers remains unknown. We have determined the structure of hexameric Pma1 from Neurospora crassa by cryo-EM at 3.3 Å resolution, elucidating the molecular basis for hexamer formation and auto-inhibition, and providing a basis for structure-based drug development. Coarse-grained molecular dynamics simulations in a lipid bilayer suggest lipid-mediated contacts between monomers and a substantial protein-induced membrane deformation that could act as a proton-attracting funnel.

Dynamic measurement of cytosolic pH and [NO3−] uncovers the role of the vacuolar transporter AtCLCa in cytosolic pH homeostasis

Ion transporters are key players of cellular processes. The mechanistic properties of ion transporters have been well elucidated by biophysical methods. Meanwhile, the understanding of their exact functions in cellular homeostasis is limited by the difficulty of monitoring their activity in vivo. The development of biosensors to track subtle changes in intracellular parameters provides invaluable tools to tackle this challenging issue. AtCLCa (Arabidopsis thalianaChloride Channel a) is a vacuolar NO3−/H+exchanger regulating stomata aperture inA.thaliana. Here, we used a genetically encoded biosensor, ClopHensor, reporting the dynamics of cytosolic anion concentration and pH to monitor the activity of AtCLCa in vivo inArabidopsisguard cells. We first found that ClopHensor is not only a Cl−but also, an NO3−sensor. We were then able to quantify the variations of NO3−and pH in the cytosol. Our data showed that AtCLCa activity modifies cytosolic pH and NO3−. In an AtCLCa loss of function mutant, the cytosolic acidification triggered by extracellular NO3−and the recovery of pH upon treatment with fusicoccin (a fungal toxin that activates the plasma membrane proton pump) are impaired, demonstrating that the transport activity of this vacuolar exchanger has a profound impact on cytosolic homeostasis. This opens a perspective on the function of intracellular transporters of the Chloride Channel (CLC) family in eukaryotes: not only controlling the intraorganelle lumen but also, actively modifying cytosolic conditions.

Involvement of signalling molecules NO, H2O2 and H2S in modification of plasma membrane proton pump in cucumber roots subjected to salt or low temperature stress

In the present study we demonstrate that the signalling molecules NO, H2O2 and H2S are important for understanding the mechanisms of modification of plasma membrane H+-ATPase (EC 3.6.3.14) activity in conditions of both salt (50 mM NaCl) and low temperature (10°C, LT) stress. Plants were subjected to stress conditions for 1 or 6 days. After 3 days of exposure to stress some of the plants were transferred to control conditions for another 3 days: post-stressed plants (3 + 3). We measured the endogenous levels of signalling molecules in stressed plants. To determine the physiological significance of NO, H2O2 and H2S induced activity of plasma membrane H+-ATPase (PM H+-ATPase) in salt and LT stresses, we investigated the activity of the plasma membrane proton pump in stress conditions, and plants were additionally supplemented with PTIO (a scavenger of NO), ascorbic acid (a scavenger of H2O2) or hypotaurine (a scavenger of H2S). H2S contributed to increased activity of PM H+-ATPase in short-term salt stress (1 day) and in low temperature treated plants (both 6 days and post-stressed plants), by stimulation of expression of several genes encoding isoforms of the plasma membrane proton pump (CsHA2, CsH4, CsH8, CsH9 and CsHA10). In contrast, NO and H2O2 play a minor role in the regulation of ATPase activity at the genetic level, because they significantly increased the expression of only one isoform, CsHA1, the expression level of which was very low in the tissues of the control plants, and additionally they slightly increased the expression of the gene encoding the isoform CsHA2. However, NO plays an important role in stimulation of the plasma membrane proton pumps under salt stress and low temperature. NO participates in post-translational modifications because it leads to increased enzyme phosphorylation and an increased H+/ATP coupling ratio.