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16 pages, 2379 KB

Open AccessArticle

Na⁺ Translocation Dominates over H⁺-Translocation in the Membrane Pyrophosphatase with Dual Transport Specificity

by Alexander V. Bogachev, Viktor A. Anashkin, Yulia V. Bertsova, Elena G. Zavyalova and Alexander A. Baykov

Int. J. Mol. Sci. 2024, 25(22), 11963; https://doi.org/10.3390/ijms252211963 - 7 Nov 2024

Cited by 1 | Viewed by 1425

Abstract

Cation-pumping membrane pyrophosphatases (mPPases; EC 7.1.3.1) vary in their transport specificity from obligatory H⁺ transporters found in all kingdoms of life, to Na⁺/H⁺-co-transporters found in many prokaryotes. The available data suggest a unique “direct-coupling” mechanism of H⁺ transport, in which the transported proton is generated from nucleophilic water molecule. Na⁺ transport is best rationalized by assuming that the water-borne proton propels a prebound Na⁺ ion through the ion conductance channel (“billiard” mechanism). However, the “billiard” mechanism, in its simple form, is not applicable to the mPPases that simultaneously transport Na⁺ and H⁺ without evident competition between the cations (Na⁺,H⁺-PPases). In this study, we used a pyranine-based fluorescent assay to explore the relationship between the cation transport reactions catalyzed by recombinant Bacteroides vulgatus Na⁺,H⁺-PPase in membrane vesicles. Under appropriately chosen conditions, including the addition of an H⁺ ionophore to convert Na⁺ influx into equivalent H⁺ efflux, the pyranine signal measures either H⁺ or Na⁺ translocation. Using a stopped-flow version of this assay, we demonstrate that H⁺ and Na⁺ are transported by Na⁺,H⁺-PPase in a ratio of approximately 1:8, which is independent of Na⁺ concentration. These findings were rationalized using an “extended billiard” model, whose most likely variant predicts the kinetic limitation of Na⁺ delivery to the pump-loading site. Full article

(This article belongs to the Special Issue 25th Anniversary of IJMS: Advances in Biochemistry)

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10 pages, 1535 KB

Open AccessEditor’s ChoiceArticle

Generation of Two-Line Restorer Line with Low Chalkiness Using Knockout of Chalk5 through CRISPR/Cas9 Editing

by Gucheng Fan, Jiefeng Jiang, Yu Long, Run Wang, Famao Liang, Haiyang Liu, Junying Xu, Xianjin Qiu and Zhixin Li

Biology 2024, 13(8), 617; https://doi.org/10.3390/biology13080617 - 15 Aug 2024

Cited by 4 | Viewed by 2179

Abstract

Chalkiness is an important grain quality trait in rice. Chalk5, encoding a vacuolar H⁺-translocating pyrophosphatase, is a major gene affecting both the percentage of grains with chalkiness (PGWC) and chalkiness degree (DEC) in rice. Reducing its expression can decrease both PGEC and DEC. In this study, the first exon of Chalk5 was edited in the elite restorer line 9311 using the CRISPR/Cas9 system and two knockout mutants were obtained, one of which did not contain the exogenous Cas9 cassette. PGWC and DEC were both significantly reduced in both mutants, while the seed setting ratio (SSR) was also significantly decreased. Staggered sowing experiments showed that the chalkiness of the mutants was insensitive to temperature during the grain-filling stage, and the head milled rice rate (HMRR) could be improved even under high-temperature conditions. Finally, in the hybrid background, the mutants showed significantly reduced PGWC and DEC without changes in other agronomic traits. The results provide important germplasm and allele resources for breeding high-yield rice varieties with superior quality, especially for high-yield indica hybrid rice varieties with superior quality in high-temperature conditions. Full article

(This article belongs to the Special Issue Mining Favorable Alleles for Crop Yield, Biotic and Abiotic Stress Tolerance)

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22 pages, 3266 KB

Open AccessEditor’s ChoiceArticle

Evaluation of Pyrophosphate-Driven Proton Pumps in Saccharomyces cerevisiae under Stress Conditions

by Krishnan Sreenivas, Leon Eisentraut, Daniel P. Brink, Viktor C. Persson, Magnus Carlquist, Marie F. Gorwa-Grauslund and Ed W. J. van Niel

Microorganisms 2024, 12(3), 625; https://doi.org/10.3390/microorganisms12030625 - 20 Mar 2024

Cited by 3 | Viewed by 2853

Abstract

In Saccharomyces cerevisiae, pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H⁺-ATPase) which constitutes a major drain within cellular ATP supply. Here, an exogenous proton-translocating pyrophosphatase (H⁺-PPase) from Arabidopsis thaliana, which uses inorganic pyrophosphate (PP_i) rather than ATP, was evaluated for its effect on reducing the ATP burden. The H⁺-Ppase was localized to the vacuolar membrane or to the cell membrane, and their impact was studied under acetate stress at a low pH. Biosensors (pHluorin and mQueen-2m) were used to observe changes in intracellular pH (pH_i) and ATP levels during growth on either glucose or xylose. A significant improvement of 35% in the growth rate at a pH of 3.7 and 6 g·L⁻¹ acetic acid stress was observed in the vacuolar membrane H⁺-PPase strain compared to the parent strain. ATP levels were elevated in the same strain during anaerobic glucose and xylose fermentations. During anaerobic xylose fermentations, co-expression of pHluorin and a vacuolar membrane H⁺-PPase improved the growth characteristics by means of an improved growth rate (11.4%) and elongated logarithmic growth duration. Our study identified a potential method for improving productivity in the use of S. cerevisiae as a cell factory under the harsh conditions present in industry. Full article

(This article belongs to the Section Microbial Biotechnology)

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25 pages, 3028 KB

Open AccessArticle

H⁺-Translocating Membrane-Bound Pyrophosphatase from Rhodospirillum rubrum Fuels Escherichia coli Cells via an Alternative Pathway for Energy Generation

by Evgeniya A. Malykh, Liubov I. Golubeva, Ekaterina S. Kovaleva, Mikhail S. Shupletsov, Elena V. Rodina, Sergey V. Mashko and Nataliya V. Stoynova

Microorganisms 2023, 11(2), 294; https://doi.org/10.3390/microorganisms11020294 - 23 Jan 2023

Cited by 5 | Viewed by 3289

Abstract

Inorganic pyrophosphatases (PPases) catalyze an essential reaction, namely, the hydrolysis of PP_i, which is formed in large quantities as a side product of numerous cellular reactions. In the majority of living species, PP_i hydrolysis is carried out by soluble cytoplasmic PPase (S-PPases) with the released energy dissipated in the form of heat. In Rhodospirillum rubrum, part of this energy can be conserved by proton-pumping pyrophosphatase (H⁺-PPase^Rru) in the form of a proton electrochemical gradient for further ATP synthesis. Here, the codon-harmonized gene hppa^Rru encoding H⁺-PPase^Rru was expressed in the Escherichia coli chromosome. We demonstrate, for the first time, that H⁺-PPase^Rru complements the essential native S-PPase in E. coli cells. ¹³C-MFA confirmed that replacing native PPase to H⁺-PPase^Rru leads to the re-distribution of carbon fluxes; a statistically significant 36% decrease in tricarboxylic acid (TCA) cycle fluxes was found compared with wild-type E. coli MG1655. Such a flux re-distribution can indicate the presence of an additional method for energy generation (e.g., ATP), which can be useful for the microbiological production of a number of compounds, the biosynthesis of which requires the consumption of ATP. Full article

(This article belongs to the Special Issue Microbial Cell Factories: Production of Amino Acids Using Microorganisms)

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21 pages, 5270 KB

Open AccessArticle

A Lumenal Loop Associated with Catalytic Asymmetry in Plant Vacuolar H⁺-Translocating Pyrophosphatase

by Viktor A. Anashkin and Alexander A. Baykov

Int. J. Mol. Sci. 2021, 22(23), 12902; https://doi.org/10.3390/ijms222312902 - 29 Nov 2021

Cited by 6 | Viewed by 2531

Abstract

Membrane-integral inorganic pyrophosphatases (mPPases) couple pyrophosphate hydrolysis with H⁺ and Na⁺ pumping in plants and microbes. mPPases are homodimeric transporters with two catalytic sites facing the cytoplasm and demonstrating highly different substrate-binding affinities and activities. The structural aspects of the functional asymmetry are still poorly understood because the structure of the physiologically relevant dimer form with only one active site occupied by the substrate is unknown. We addressed this issue by molecular dynamics (MD) simulations of the H⁺-transporting mPPase of Vigna radiata, starting from its crystal structure containing a close substrate analog (imidodiphosphate, IDP) in both active sites. The MD simulations revealed pre-existing subunit asymmetry, which increased upon IDP binding to one subunit and persisted in the fully occupied dimer. The most significant asymmetrical change caused by IDP binding is a ‘rigid body’-like displacement of the lumenal loop connecting α-helices 2 and 3 in the partner subunit and opening its exit channel for water. This highly conserved 14–19-residue loop is found only in plant vacuolar mPPases and may have a regulatory function, such as pH sensing in the vacuole. Our data define the structural link between the loop and active sites and are consistent with the published structural and functional data. Full article

(This article belongs to the Collection Computational Studies of Biomolecules)

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15 pages, 2957 KB

Open AccessArticle

Vacuolar Proton Pyrophosphatase Is Required for High Magnesium Tolerance in Arabidopsis

by Yang Yang, Ren-Jie Tang, Baicong Mu, Ali Ferjani, Jisen Shi, Hongxia Zhang, Fugeng Zhao, Wen-Zhi Lan and Sheng Luan

Int. J. Mol. Sci. 2018, 19(11), 3617; https://doi.org/10.3390/ijms19113617 - 16 Nov 2018

Cited by 16 | Viewed by 5245

Abstract

Magnesium (Mg²⁺) is an essential nutrient in all organisms. However, high levels of Mg²⁺ in the environment are toxic to plants. In this study, we identified the vacuolar-type H⁺-pyrophosphatase, AVP1, as a critical enzyme for optimal plant growth under high-Mg conditions. The Arabidopsis avp1 mutants displayed severe growth retardation, as compared to the wild-type plants upon excessive Mg²⁺. Unexpectedly, the avp1 mutant plants retained similar Mg content to wild-type plants under either normal or high Mg conditions, suggesting that AVP1 may not directly contribute to Mg²⁺ homeostasis in plant cells. Further analyses confirmed that the avp1 mutant plants contained a higher pyrophosphate (PPi) content than wild type, coupled with impaired vacuolar H⁺-pyrophosphatase activity. Interestingly, expression of the Saccharomyces cerevisiae cytosolic inorganic pyrophosphatase1 gene IPP1, which facilitates PPi hydrolysis but not proton translocation into vacuole, rescued the growth defects of avp1 mutants under high-Mg conditions. These results provide evidence that high-Mg sensitivity in avp1 mutants possibly resulted from elevated level of cytosolic PPi. Moreover, genetic analysis indicated that mutation of AVP1 was additive to the defects in mgt6 and cbl2 cbl3 mutants that are previously known to be impaired in Mg²⁺ homeostasis. Taken together, our results suggest AVP1 is required for cellular PPi homeostasis that in turn contributes to high-Mg tolerance in plant cells. Full article

(This article belongs to the Section Molecular Plant Sciences)

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