Differential response of vacuolar proton pumps to osmotica

2006 ◽  
Vol 33 (2) ◽  
pp. 195 ◽  
Author(s):  
Fan S. Chiu ◽  
Shen H. Hsu ◽  
Jiun H. Chen ◽  
Yi Y. Hsiao ◽  
Yih J. Pan ◽  
...  
Keyword(s):  
Osmotic Pressure ◽  
Proton Translocation ◽  
Proton Pumping ◽  
Enzymatic Activities ◽  
Proper Function ◽  
Proton Pumps ◽  
Higher Plant ◽  
Plant Vacuole ◽  
Total Cell Volume ◽  
High Concentrations

The vacuole is a fundamental and dominant organelle and occupies a large part of the total cell volume in most mature plant cells. The higher-plant vacuole contains two types of proton-translocating pumps, H+-ATPase (EC 3.6.1.3) and H+-pyrophosphatase (EC 3.6.1.1), residing on the same membrane. These two enzymes generate roughly equal proton gradients across the vacuolar membrane for the secondary transport of ions and metabolites. However, the pumps respond differentially to stress in order to maintain critical functions of the vacuole. In this work, tonoplasts from etiolated mung bean seedlings (Vigna radiata L.) were used to investigate the function of these two enzymes under high osmotic pressure. At high concentrations of sucrose or sorbitol, the light scattering and volume of isolated vesicles were progressively changed. Concomitantly, enzymatic activities, proton translocation, and coupling efficiencies of these two proton-pumping enzymes were inhibited to various extents under high osmotic pressure. No significant change in enzymatic activities of purified vacuolar H+-PPase and H+-ATPase under similar conditions was observed. We thus believe that the membrane structure is an important determinant for proper function of proton pumping systems of plant vacuoles. Furthermore, kinetic analysis shows different variation in apparent Vmax but not in KM values of vacuolar H+-PPase and H+-ATPase at high osmolarity of sucrose and sorbitol, respectively, suggesting probable alterations in substrate hydrolysis reactions but not substrate-binding affinity of the enzymes. A working model is proposed to interpret supplemental roles of vacuolar H+-PPase and H+-ATPase to maintain appropriate functions of plant tonoplasts.

scholarly journals Cryo-EM structure and dynamics of the green-light absorbing proteorhodopsin

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Stephan Hirschi ◽  
David Kalbermatter ◽  
Zöhre Ucurum ◽  
Thomas Lemmin ◽  
Dimitrios Fotiadis
Keyword(s):  
Proton Translocation ◽  
Functional Characterization ◽  
Green Light ◽  
Proton Donor ◽  
Molecular Organization ◽  
Primary Proton ◽  
Proton Pumps ◽  
Model Protein ◽  
Dynamics Simulations ◽  
And Function

AbstractThe green-light absorbing proteorhodopsin (GPR) is the archetype of bacterial light-driven proton pumps. Here, we present the 2.9 Å cryo-EM structure of pentameric GPR, resolving important residues of the proton translocation pathway and the oligomerization interface. Superposition with the structure of a close GPR homolog and molecular dynamics simulations reveal conformational variations, which regulate the solvent access to the intra- and extracellular half channels harbouring the primary proton donor E109 and the proposed proton release group E143. We provide a mechanism for the structural rearrangements allowing hydration of the intracellular half channel, which are triggered by changing the protonation state of E109. Functional characterization of selected mutants demonstrates the importance of the molecular organization around E109 and E143 for GPR activity. Furthermore, we present evidence that helices involved in the stabilization of the protomer interfaces serve as scaffolds for facilitating the motion of the other helices. Combined with the more constrained dynamics of the pentamer compared to the monomer, these observations illustrate the previously demonstrated functional significance of GPR oligomerization. Overall, this work provides molecular insights into the structure, dynamics and function of the proteorhodopsin family that will benefit the large scientific community employing GPR as a model protein.

CRISPR/Cas9-Mediated Gene Editing of Vacuolar ATPase Subunit D Mediates Phytohormone Biosynthesis and Virus Resistance in Rice

2021 ◽  
Author(s):  
Qinghua Lu ◽  
Xiangwen Luo ◽  
Xiao Yang ◽  
Tong Zhou ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Virus Resistance ◽  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Resistance Breeding ◽  
Rice Stripe Virus ◽  
Proton Pumps ◽  
Wild Type ◽  
Knockout Mutant ◽  
Atpase Subunit ◽  
Vacuolar Atpases

Abstract Background: Vacuolar ATPases (v-ATPases) are proton pumps for proton translocation across membranes that utilize energy derived from ATP hydrolysis; Previous research revealed Osv-ATPases mediates phytohormes levels and resistance in rice. Osv-ATPase subunit d (Osv-ATPase d) is part of an integral, membrane-embedded V0 complex of V-ATPases complex, whether Osv-ATPase d involves in phytohormes biosynthesis and resistance in rice remains unknown.Finding: The knockout mutant line (line 5) of Osv-ATPase d was generated using the CRISPR/Cas9 system, mutation of Osv-ATPase d did not show any detrimental effect on plant growth or yield productivity. Transcriptomic results showed Osv-ATPase d probably involved in mediating the biosynthesis of plant hormones and resistance in rice. Mutation of Osv-ATPase d significantly increased JA and ABA biosynthesis than wild type. Compared to wild type, mutation of Osv-ATPase d increased the resistance against Southern rice black-streaked dwarf virus (SRBSDV), however, decreased the resistance against Rice stripe virus (RSV) in rice. Conclusion: Taken together, our data reveal the Osv-ATPase d mediates phytohormone biosynthesis and virus resistance in rice, which can be selected as a potential target for resistance breeding in rice.

scholarly journals Mutation of a single residue in the ba3 oxidase specifically impairs protonation of the pump site

2015 ◽  
Vol 112 (11) ◽  
pp. 3397-3402 ◽  
Author(s):  
Christoph von Ballmoos ◽  
Nathalie Gonska ◽  
Peter Lachmann ◽  
Robert B. Gennis ◽  
Pia Ädelroth ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Time Constant ◽  
Thermus Thermophilus ◽  
Proton Translocation ◽  
Proton Pumping ◽  
Wild Type ◽  
Structural Variant ◽  
In The Wild ◽  
Membrane Bound ◽  
Proton Uptake

The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative “pump site” was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase.

scholarly journals Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Julia Steiner ◽  
Leonid Sazanov
Keyword(s):  
Electron Transfer ◽  
Electrostatic Interactions ◽  
Proton Translocation ◽  
Proton Pumps ◽  
Entire Family ◽  
Surface Charge Distribution ◽  
Large Protein Family ◽  
Anoxybacillus Flavithermus ◽  
Respiratory Complex I ◽  
Ph Adaptation

Multiple resistance and pH adaptation (Mrp) antiporters are multi-subunit Na+ (or K+)/H+ exchangers representing an ancestor of many essential redox-driven proton pumps, such as respiratory complex I. The mechanism of coupling between ion or electron transfer and proton translocation in this large protein family is unknown. Here, we present the structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. It is a dimer of seven-subunit protomers with 50 trans-membrane helices each. Surface charge distribution within each monomer is remarkably asymmetric, revealing probable proton and sodium translocation pathways. On the basis of the structure we propose a mechanism where the coupling between sodium and proton translocation is facilitated by a series of electrostatic interactions between a cation and key charged residues. This mechanism is likely to be applicable to the entire family of redox proton pumps, where electron transfer to substrates replaces cation movements.

scholarly journals A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast

2011 ◽  
Vol 437 (2) ◽  
pp. 269-278 ◽  
Author(s):  
José R. Pérez-Castiñeira ◽  
Agustín Hernández ◽  
Rocío Drake ◽  
Aurelio Serrano
Keyword(s):  
Fluorescent Dyes ◽  
Experimental System ◽  
Proton Pumping ◽  
Endocytic Pathway ◽  
Inorganic Pyrophosphatase ◽  
Proton Pumps ◽  
Specific Class ◽  
Ph Gradients ◽  
Atpase Subunit ◽  
Relative Contribution

V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.

Proton pumping pyrophosphatase from higher plant mitochondria

1999 ◽  
Vol 105 (4) ◽  
pp. 763-768 ◽  
Author(s):  
Angelo Vianello ◽  
Francesco Macrì
Keyword(s):  
Plant Mitochondria ◽  
Proton Pumping ◽  
Higher Plant

scholarly journals Five decades of research on mitochondrial NADH-quinone oxidoreductase (complex I)

2018 ◽  
Vol 399 (11) ◽  
pp. 1249-1264 ◽  
Author(s):  
Tomoko Ohnishi ◽  
S. Tsuyoshi Ohnishi ◽  
John C. Salerno
Keyword(s):  
Electron Transfer ◽  
Respiratory Chain ◽  
Complex I ◽  
Proton Translocation ◽  
Atp Synthesis ◽  
Mitochondrial Respiratory Chain ◽  
Proton Pumping ◽  
Entry Site ◽  
Quinone Oxidoreductase ◽  
Terminal Electron Acceptor

AbstractNADH-quinone oxidoreductase (complex I) is the largest and most complicated enzyme complex of the mitochondrial respiratory chain. It is the entry site into the respiratory chain for most of the reducing equivalents generated during metabolism, coupling electron transfer from NADH to quinone to proton translocation, which in turn drives ATP synthesis. Dysfunction of complex I is associated with neurodegenerative diseases such as Parkinson’s and Alzheimer’s, and it is proposed to be involved in aging. Complex I has one non-covalently bound FMN, eight to 10 iron-sulfur clusters, and protein-associated quinone molecules as electron transport components. Electron paramagnetic resonance (EPR) has previously been the most informative technique, especially in membranein situanalysis. The structure of complex 1 has now been resolved from a number of species, but the mechanisms by which electron transfer is coupled to transmembrane proton pumping remains unresolved. Ubiquinone-10, the terminal electron acceptor of complex I, is detectable by EPR in its one electron reduced, semiquinone (SQ) state. In the aerobic steady state of respiration the semi-ubiquinone anion has been observed and studied in detail. Two distinct protein-associated fast and slow relaxing, SQ signals have been resolved which were designated SQNfand SQNs. This review covers a five decade personal journey through the field leading to a focus on the unresolved questions of the role of the SQ radicals and their possible part in proton pumping.

scholarly journals Structural aspects of the effectiveness of bisphosphonates as competitive inhibitors of the plant vacuolar proton-pumping pyrophosphatase

1999 ◽  
Vol 337 (3) ◽  
pp. 373-377 ◽  
Author(s):  
Ruth GORDON-WEEKS ◽  
Saroj PARMAR ◽  
T. G. Emyr DAVIES ◽  
Roger A. LEIGH
Keyword(s):  
Nitrogen Atom ◽  
Active Site ◽  
General Structure ◽  
Specific Inhibitor ◽  
Carbon Chain ◽  
Proton Pumping ◽  
Azido Group ◽  
Higher Plant ◽  
Plant Vacuoles ◽  
Increased Activity

The bisphosphonates (general structure PO3-R-PO3) competitively inhibit soluble and membrane-bound inorganic pyrophosphatases (PPases) with differing degrees of specificity. Aminomethylenebisphosphonate (AMBP; HC(PO3)2NH2) is a potent, specific inhibitor of the PPase of higher plant vacuoles (V-PPase). To explore the possibility of constructing photoactivatable probes from bisphosphonates to label the active site of V-PPase we analysed the effects of different analogues on the hydrolytic and proton pumping activity of the enzyme. Bisphosphonates with a range of structures inhibited competitively and the effects on PPi hydrolysis correlated with the effects on proton pumping. Low-molecular-mass bisphosphonates containing hydrophilic groups (α-NH2 or OH) were the most effective, suggesting that the catalytic site is in a restricted polar pocket. Bisphosphonates containing a benzene ring were less active but the introduction of a nitrogen atom into the ring increased activity. Compounds of the general formula NH2(CH2)nC(PO3)2OH were more inhibitory than compounds of the H(CH2)nC(PO3)2NH2, NH2(CH2)nC(PO3)2NH2 or OH(CH2)nC(PO3)2NH2 series, with activity decreasing as n increased. A nitrogen atom in the carbon chain increased activity but activity was decreased by the presence of an oxygen atom. An analogue with a ring attached via a four-carbon chain, which included an amide linkage and a hydroxy group on the α-carbon atom, inhibited competitively (Ki = 62.0 µM), suggesting that it may be possible to design bisphosphonate inhibitors which contain a photoactivatable azido group for photoaffinity labelling of V-PPase active site.

scholarly journals Hydrostatic and osmotic pressure activated channel in plant vacuole

1991 ◽  
Vol 60 (6) ◽  
pp. 1326-1336 ◽  
Author(s):  
Joel Alexandre ◽  
Jean-Paul Lassalles
Keyword(s):  
Osmotic Pressure ◽  
Plant Vacuole
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