Proton-driven sodium secretion in a saline water animal.

Aquatic animals residing in saline habitats either allow extracellular sodium concentration to conform to environmental values or regulate sodium to lower levels. The latter strategy requires an energy-driven process to move sodium against a large concentration gradient to eliminate excess sodium that diffuses into the animal. Previous studies of invertebrate and vertebrate species indicate a sodium pump, Na+/K+ ATPase, powers sodium secretion. We provide the first functional evidence of a saline-water animal, Aedes taeniorhynchus mosquito larva, utilizing a proton pump to power this process. Vacuolar-type H+ ATPase (VHA) protein is highly expressed on the apical membrane of the posterior rectal cells, and in situ sodium flux across this epithelium increases significantly in larvae held in higher salinity and is sensitive to Bafilomycin A1, an inhibitor of VHA. We also report the first evidence of splice variants of the sodium/proton exchanger, NHE3, with both high and low molecular weight variants highly expressed on the apical membrane of the posterior rectal cells. Evidence of NHE3 function was indicated with in situ sodium transport significantly inhibited by a NHE3 antagonist, S3226. We propose that the outward proton pumping by VHA establishes a favourable electromotive gradient to drive sodium secretion via NHE3 thus producing a hyperosmotic, sodium-rich urine. This H+- driven Na+ secretion process is the primary mechanism of ion regulation in salt-tolerant culicine mosquito species and was first investigated over 80 years ago.

Energetics of the H-Bond Network in Exiguobacterium sibiricum Rhodopsin.

Exiguobacterium sibiricum rhodopsin (ESR) functions as a light-driven proton pump utilizing Lys96 for proton uptake and maintaining its activity over a wide pH range. Using a combination of methodologies including the linear Poisson-Boltzmann equation and a quantum mechanical/molecular mechanical approach with a polarizable continuum model, we explore the microscopic mechanisms underlying its pumping activity. Lys96, the primary proton uptake site, remains deprotonated owing to the loss of solvation in the ESR protein environment. Asp85, serving as a proton acceptor group for Lys96, does not form a low-barrier H-bond with His57. Instead, deprotonated Asp85 forms a salt-bridge with protonated His57, and the proton is predominantly located at the His57 moiety. Glu214, the only acidic residue at the end of the H-bond network exhibits a pKa value of ∼6, slightly elevated due to solvation loss. It seems likely that the H-bond network [Asp85···His57···H2O···Glu214] serves as a proton-conducting pathway toward the protein bulk surface.

Overcoming Protein Orientation Mismatch Enables Efficient Nanoscale Light-Driven ATP Production.

Adenosine triphosphate (ATP)-producing modules energized by light-driven proton pumps are powerful tools for the bottom-up assembly of artificial cell-like systems. However, the maximum efficiency of such modules is prohibited by the random orientation of the proton pumps during the reconstitution process into lipid-surrounded nanocontainers. Here, we overcome this limitation using a versatile approach to uniformly orient the light-driven proton pump proteorhodopsin (pR) in liposomes. pR is post-translationally either covalently or noncovalently coupled to a membrane-impermeable protein domain guiding orientation during insertion into preformed liposomes. In the second scenario, we developed a novel bifunctional linker, trisNTA-SpyTag, that allows for the reversible connection of any SpyCatcher-containing protein and a HisTag-carrying protein. The desired protein orientations are verified by monitoring vectorial proton pumping and membrane potential generation. In conjunction with ATP synthase, highly efficient ATP production is energized by the inwardly pumping population. In comparison to other light-driven ATP-producing modules, the uniform orientation allows for maximal rates at economical protein concentrations. The presented technology is highly customizable and not limited to light-driven proton pumps but applicable to many membrane proteins and offers a general approach to overcome orientation mismatch during membrane reconstitution, requiring little to no genetic modification of the protein of interest.

Mitochondria at the Nanoscale: Physics Meets Biology-What Does It Mean for Medicine?

Mitochondria are commonly perceived as "cellular power plants". Intriguingly, power conversion is not their only function. In the first part of this paper, we review the role of mitochondria in the evolution of eukaryotic organisms and in the regulation of the human body, specifically focusing on cancer and autism in relation to mitochondrial dysfunction. In the second part, we overview our previous works, revealing the physical principles of operation for proton-pumping complexes in the inner mitochondrial membrane. Our proposed simple models reveal the physical mechanisms of energy exchange. They can be further expanded to answer open questions about mitochondrial functions and the medical treatment of diseases associated with mitochondrial disorders.

Plasmodium falciparum utilizes pyrophosphate to fuel an essential proton pump in the ring stage and the transition to trophozoite stage.

During asexual growth and replication cycles inside red blood cells, the malaria parasite Plasmodium falciparum primarily relies on glycolysis for energy supply, as its single mitochondrion performs little or no oxidative phosphorylation. Post merozoite invasion of a host red blood cell, the ring stage lasts approximately 20 hours and was traditionally thought to be metabolically quiescent. However, recent studies have shown that the ring stage is active in several energy-costly processes, including gene transcription, protein translation, protein export, and movement inside the host cell. It has remained unclear whether a low glycolytic flux alone can meet the energy demand of the ring stage over a long period post invasion. Here, we demonstrate that the metabolic by-product pyrophosphate (PPi) is a critical energy source for the development of the ring stage and its transition to the trophozoite stage. During early phases of the asexual development, the parasite utilizes Plasmodium falciparum vacuolar pyrophosphatase 1 (PfVP1), an ancient pyrophosphate-driven proton pump, to export protons across the parasite plasma membrane. Conditional deletion of PfVP1 leads to a delayed ring stage that lasts nearly 48 hours and a complete blockage of the ring-to-trophozoite transition before the onset of parasite death. This developmental arrest can be partially rescued by an orthologous vacuolar pyrophosphatase from Arabidopsis thaliana, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae, which lacks proton pumping activities. Since proton-pumping pyrophosphatases have been evolutionarily lost in human hosts, the essentiality of PfVP1 suggests its potential as an antimalarial drug target. A drug target of the ring stage is highly desired, as current antimalarials have limited efficacy against this stage.

Features of the Mechanism of Proton Transport in ESR, Retinal Protein from Exiguobacterium sibiricum.

Retinal-containing light-sensitive proteins - rhodopsins - are found in many microorganisms. Interest in them is largely explained by their role in light energy storage and photoregulation in microorganisms, as well as the prospects for their use in optogenetics to control neuronal activity, including treatment of various diseases. One of the representatives of microbial rhodopsins is ESR, the retinal protein of Exiguobacterium sibiricum. What distinguishes ESR from homologous proteins is the presence of a lysine residue (Lys96) as a proton donor for the Schiff base. This feature, along with the hydrogen bond of the proton acceptor Asp85 with the His57 residue, determines functional characteristics of ESR as a proton pump. This review examines the results of ESR studies conducted using various methods, including direct electrometry. Comparison of the obtained data with the results of structural studies and with other retinal proteins allows us to draw conclusions about the mechanisms of transport of hydrogen ions in ESR and similar retinal proteins.

Plastid-localized xanthorhodopsin increases diatom biomass and ecosystem productivity in iron-limited surface oceans.

Microbial rhodopsins are photoreceptor proteins that convert light into biological signals or energy. Proteins of the xanthorhodopsin family are common in eukaryotic photosynthetic plankton including diatoms. However, their biological role in these organisms remains elusive. Here we report on a xanthorhodopsin variant (FcR1) isolated from the polar diatom Fragilariopsis cylindrus. Applying a combination of biophysical, biochemical and reverse genetics approaches, we demonstrate that FcR1 is a plastid-localized proton pump which binds the chromophore retinal and is activated by green light. Enhanced growth of a Thalassiora pseudonana gain-of-function mutant expressing FcR1 under iron limitation shows that the xanthorhodopsin proton pump supports growth when chlorophyll-based photosynthesis is iron-limited. The abundance of xanthorhodopsin transcripts in natural diatom communities of the surface oceans is anticorrelated with the availability of dissolved iron. Thus, we propose that these proton pumps convey a fitness advantage in regions where phytoplankton growth is limited by the availability of dissolved iron.

TCA cycle tailoring facilitates optimal growth of proton-pumping NADH dehydrogenase-dependent Escherichia coli.

IMPORTANCE: Energy generation pathways are a potential avenue for the development of novel antibiotics. However, bacteria possess remarkable resilience due to the compensatory pathways, which presents a challenge in this direction. NADH, the primary reducing equivalent, can transfer electrons to two distinct types of NADH dehydrogenases. Type I NADH dehydrogenase is an enzyme complex comprising multiple subunits and can generate proton motive force (PMF). Type II NADH dehydrogenase does not pump protons but plays a crucial role in maintaining the turnover of NAD+. To study the adaptive rewiring of energy metabolism, we evolved an Escherichia coli mutant lacking type II NADH dehydrogenase. We discovered that by modifying the flux through the tricarboxylic acid (TCA) cycle, E. coli could mitigate the growth impairment observed in the absence of type II NADH dehydrogenase. This research provides valuable insights into the intricate mechanisms employed by bacteria to compensate for disruptions in energy metabolism.

An unusual conformation from Na+-sensitive non-gastric proton pump mutants reveals molecular mechanisms of cooperative Na+-binding.

The Na+,K+-ATPase (NKA) and non-gastric H+,K+- ATPase (ngHKA) share ~65 % sequence identity, and nearly identical catalytic cycles. These pumps alternate between inward-facing (E1) and outward-facing (E2) conformations and differ in their exported substrate (Na+ or H+) and stoichiometries (3 Na+:2 K+ or 1 H+:1 K+). We reported that structures of the NKA-mimetic ngHKA mutant K794S/A797P/W940/R949C (SPWC) with 2 K+ occluded in E2-Pi and 3 Na+-bound in E1·ATP states were nearly identical to NKA structures in equivalent states. Here we report the cryo-EM structures of K794A and K794S, two poorly-selective ngHKA mutants, under conditions to stabilize the E1·ATP state. Unexpectedly, the structures show a hybrid with both E1- and E2-like structural features. While transmembrane segments TM1-TM3 and TM4's extracellular half adopted an E2-like conformation, the rest of the protein assumed an E1 configuration. Two spherical densities, likely bound Na+, were observed at cation-binding sites I and III, without density at site II. This explains the E2-like conformation of TM4's exoplasmic half. In NKA, oxygen atoms derived from the unwound portion of TM4 coordinated Na+ at site II. Thus, the lack of Na+ at site II of K794A/S prevents the luminal portion of TM4 from taking an E1-like position. The K794A structure also suggests that incomplete coordination of Na+ at site III induces the halfway rotation of TM6, which impairs Na+-binding at the site II. Thus, our observations provide insight into the molecular mechanism of E2-E1 transition and cooperative Na+-binding in the NKA and other related cation pumps.

Quinone Catalysis Modulates Proton Transfer Reactions in the Membrane Domain of Respiratory Complex I.

Complex I is a redox-driven proton pump that drives electron transport chains and powers oxidative phosphorylation across all domains of life. Yet, despite recently resolved structures from multiple organisms, it still remains unclear how the redox reactions in Complex I trigger proton pumping up to 200 Å away from the active site. Here, we show that the proton-coupled electron transfer reactions during quinone reduction drive long-range conformational changes of conserved loops and trans-membrane (TM) helices in the membrane domain of Complex I from Yarrowia lipolytica. We find that the conformational switching triggers a π → α transition in a TM helix (TM3ND6) and establishes a proton pathway between the quinone chamber and the antiporter-like subunits, responsible for proton pumping. Our large-scale (>20 µs) atomistic molecular dynamics (MD) simulations in combination with quantum/classical (QM/MM) free energy calculations show that the helix transition controls the barrier for proton transfer reactions by wetting transitions and electrostatic effects. The conformational switching is enabled by re-arrangements of ion pairs that propagate from the quinone binding site to the membrane domain via an extended network of conserved residues. We find that these redox-driven changes create a conserved coupling network within the Complex I superfamily, with point mutations leading to drastic activity changes and mitochondrial disorders. On a general level, our findings illustrate how catalysis controls large-scale protein conformational changes and enables ion transport across biological membranes.

Light-driven Proton Pumps as a Potential Regulator for Carbon Fixation in Marine Diatoms.

Diatoms are a major phytoplankton group responsible for approximately 20% of carbon fixation on Earth. They perform photosynthesis using light-harvesting chlo-rophylls located in plastids, an organelle obtained through eukaryote-eukaryote endosymbiosis. Microbial rhodopsin, a photoreceptor distinct from chlo-rophyll-based photosystems, was recently identified in some diatoms. However, the physiological function of diatom rhodopsin remains unclear. Heterologous expression techniques were herein used to investigate the protein function and subcellular localization of diatom rhodopsin. We demonstrated that diatom rhodopsin acts as a light-driven proton pump and localizes primarily to the outermost membrane of four membrane-bound complex plastids. Using model simulations, we also examined the effects of pH changes inside the plastid due to rhodopsin-mediated proton transport on photosynthesis. The results obtained suggested the involvement of rhodopsin-mediated local pH changes in a photosynthetic CO2-concentrating mechanism in rhodopsin-possessing diatoms.

Evolution of the sodium pump.

Eukaryotic plasma membranes (PMs) are energized by electrogenic P-type ATPases that generate either Na+ or H+ motive forces to drive Na+ and H+ dependent transport processes, respectively. For this purpose, animal rely on Na+/K+-ATPases whereas fungi and plants employ PM H+-ATPases. Prokaryotes, on the other hand, depend on H+ or Na+-motive electron transport complexes to energize their cell membranes. This raises the question as to why and when electrogenic Na+ and H+ pumps evolved? Here it is shown that prokaryotic Na+/K+-ATPases have near perfect conservation of binding sites involved in coordination of three Na+ and two K+ ions. Such pumps are rare in Eubacteria but are common in methanogenic Archaea where they often are found together with P-type putative PM H+-ATPases. With some exceptions, Na+/K+-ATPases and PM H+-ATPases are found everywhere in the eukaryotic tree of life, but never together in animals, fungi and land plants. It is hypothesized that Na+/K+-ATPases and PM H+-ATPases evolved in methanogenic Archaea to support the bioenergetics of these ancestral organisms, which can utilize both H+ and Na+ as energy currencies. Both pumps must have been simultaneously present in the first eukaryotic cell, but during diversification of the major eukaryotic kingdoms, and at the time animals diverged from fungi, animals kept Na+/K+-ATPases but lost PM H+-ATPases. At the same evolutionary branch point, fungi did loose Na+/K+-ATPases, and their role was taken over by PM H+-ATPases. An independent but similar scenery emerged during terrestrialization of plants: they lost Na+/K+-ATPases but kept PM H+-ATPases.

Proton Pumps: Molecular Mechanisms, Inhibitors and Activators of Proton Pumping.

Protein molecular machines, also known as proton pumps, are the most important element of biological membranes [...].

Optogenetic cytosol acidification of mammalian cells using an inward proton-pumping rhodopsin.

Ion gradients are a universal form of energy, information storage and conversion in living cells. Advances in optogenetics inspire the development of novel tools towards control of different cellular processes with light. Rhodopsins are perspective tools for optogenetic manipulation of ion gradients in cells and subcellular compartments, controlling pH of the cytosol and intracellular organelles. The key step of the development of new optogenetic tools is evaluation of their efficiency. Here, we used a high-throughput quantitative method for comparing efficiency of proton-pumping rhodopsins in Escherichia coli cells. This approach allowed us to show that an inward proton pump xenorhodopsin from Nanosalina sp. (NsXeR) is a powerful tool for optogenetic control of pH of mammalian subcellular compartments. Further, we demonstrate that NsXeR can be used for fast optogenetic acidification of the cytosol of mammalian cells. This is the first evidence of optogenetic cytosol acidification by an inward proton pump at physiological pH values. Our approach offers unique opportunities to study cellular metabolism at normal and pathological conditions and might help to understand the role of pH dysregulation in cellular dysfunctions.

Oriented Insertion of ESR-Containing Hybrid Proteins in Proteoliposomes.

Microbial rhodopsins comprise a diverse family of retinal-containing membrane proteins that convert absorbed light energy to transmembrane ion transport or sensory signals. Incorporation of these proteins in proteoliposomes allows their properties to be studied in a native-like environment; however, unidirectional protein orientation in the artificial membranes is rarely observed. We aimed to obtain proteoliposomes with unidirectional orientation using a proton-pumping retinal protein from Exiguobacterium sibiricum, ESR, as a model. Three ESR hybrids with soluble protein domains (mCherry or thioredoxin at the C-terminus and Caf1M chaperone at the N-terminus) were obtained and characterized. The photocycle of the hybrid proteins incorporated in proteoliposomes demonstrated a higher pKa of the M state accumulation compared to that of the wild-type ESR. Large negative electrogenic phases and an increase in the relative amplitude of kinetic components in the microsecond time range in the kinetics of membrane potential generation of ESR-Cherry and ESR-Trx indicate a decrease in the efficiency of transmembrane proton transport. On the contrary, Caf-ESR demonstrates a native-like kinetics of membrane potential generation and the corresponding electrogenic stages. Our experiments show that the hybrid with Caf1M promotes the unidirectional orientation of ESR in proteoliposomes.

Phototrophy by antenna-containing rhodopsin pumps in aquatic environments.

Energy transfer from light-harvesting ketocarotenoids to the light-driven proton pump xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to abundant rhodopsin proton pumps3 from marine photoheterotrophs have thus far failed4-6. Here we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins using functional metagenomics combined with chromophore extraction from the environment. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet- or blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas may have a substantial effect on rhodopsin phototrophy in the world's lakes, seas and oceans. However, the functional implications of our findings are yet to be discovered.

Targeting ATP12A, a Nongastric Proton Pump α Subunit, for Idiopathic Pulmonary Fibrosis Treatment.

Idiopathic pulmonary fibrosis (IPF) is a pathological condition of unknown etiology that results from injury to the lung and an ensuing fibrotic response that leads to the thickening of the alveolar walls and obliteration of the alveolar space. The pathogenesis is not clear, and there are currently no effective therapies for IPF. Small airway disease and mucus accumulation are prominent features in IPF lungs, similar to cystic fibrosis lung disease. The ATP12A gene encodes the α-subunit of the nongastric H+, K+-ATPase, which functions to acidify the airway surface fluid and impairs mucociliary transport function in patients with cystic fibrosis. It is hypothesized that the ATP12A protein may play a role in the pathogenesis of IPF. The authors' studies demonstrate that ATP12A protein is overexpressed in distal small airways from the lungs of patients with IPF compared with normal human lungs. In addition, overexpression of the ATP12A protein in mouse lungs worsened bleomycin induced experimental pulmonary fibrosis. This was prevented by a potassium competitive proton pump blocker, vonoprazan. These data support the concept that the ATP12A protein plays an important role in the pathogenesis of lung fibrosis. Inhibition of the ATP12A protein has potential as a novel therapeutic strategy in IPF treatment.

Plant Plasma Membrane Proton Pump: One Protein with Multiple Functions.

In plants, the plasma membrane proton pump (PM H+-ATPase) regulates numerous transport-dependent processes such as growth, development, basic physiology, and adaptation to environmental conditions. This review explores the multifunctionality of this enzyme in plant cells. The abundance of several PM H+-ATPase isogenes and their pivotal role in energizing transport in plants have been connected to the phenomena of pleiotropy. The multifunctionality of PM H+-ATPase is a focal point of numerous studies unraveling the molecular mechanisms of plant adaptation to adverse environmental conditions. Furthermore, PM H+-ATPase is a key element in plant defense mechanisms against pathogen attack; however, it also functions as a target for pathogens that enable plant tissue invasion. Here, we provide an extensive review of the PM H+-ATPase as a multitasking protein in plants. We focus on the results of recent studies concerning PM H+-ATPase and its role in plant growth, physiology, and pathogenesis.

Arsenic inhibits citric acid accumulation via downregulating vacuolar proton pump gene expression in citrus fruits.

Citric acid content is a critical quality determinant in citrus (Citrus spp.) fruits. Although arsenic (As) can effectively reduce citric acid content to improve citrus fruit quality, it can have adverse environmental effects. The discovery of nontoxic substitutes is hampered by the incomplete elucidation of the underlying mechanisms of As action in citrus fruits. Metabolic, transcriptomic, and physiological analyses were employed to investigate As action on citric acid accumulation to discover the mechanisms of As action in citrus. The enzyme activity related to citrate biosynthesis was not inhibited and the content of the involved metabolites was not reduced in As-treated fruits. However, the proton pump genes CitPH5 and CitPH1 control the vacuolar citric acid accumulation and transcription factor genes CitTT8 and CitMYB5, which regulate CitPH5 and CitPH1, were downregulated. The oxidative stress-response genes were upregulated in As-treated fruits. The reactive oxygen species (ROS) treatment also downregulated CitTT8 and CitMYB5 in juice cells. The mitochondrial ROS production rate increased in As-treated fruits. AsIII was more potent in stimulating isolated mitochondria to overproduce ROS compared to AsV. Our results indicate that the As inhibition of citric acid accumulation may be primarily due to the transcriptional downregulation of CitPH5, CitPH1, CitTT8, and CitMYB5. As-induced oxidative stress signaling may operate upstream to downregulate these acid regulator genes. Mitochondrial thiol proteins may be the principal targets of As action in citrus fruits.