A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities.

Brine shrimp (Artemia) are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na+,K+-ATPase (NKA), a heterodimeric (αß) pump that usually exports 3Na+ in exchange for 2 K+ per hydrolyzed ATP. Artemia express several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2KK, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines (Xenopus α1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that Artemia express two salinity-independent canonical α subunits (α1NN and α3NN), as well as two ß variants, in addition to the salinity-controlled α2KK. These ß subunits permitted heterologous expression of the α2KK pump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2KK pumps and compared it to that of Xenopus α1 (and its α2KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2KK-like characteristics to Xenopus α1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2KK's Lys308 helps to maintain high affinity for external K+ when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K+-congener 86Rb+, we prove that double-lysine-substituted pumps transport 2Na+ and 1 K+ per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowing Artemia to maintain steeper Na+ gradients in hypersaline environments.

Structure and function of H+/K+ pump mutants reveal Na+/K+ pump mechanisms.

Ion-transport mechanisms evolve by changing ion-selectivity, such as switching from Na+ to H+ selectivity in secondary-active transporters or P-type-ATPases. Here we study primary-active transport via P-type ATPases using functional and structural analyses to demonstrate that four simultaneous residue substitutions transform the non-gastric H+/K+ pump, a strict H+-dependent electroneutral P-type ATPase, into a bona fide Na+-dependent electrogenic Na+/K+ pump. Conversion of a H+-dependent primary-active transporter into a Na+-dependent one provides a prototype for similar studies of ion-transport proteins. Moreover, we solve the structures of the wild-type non-gastric H+/K+ pump, a suitable drug target to treat cystic fibrosis, and of its Na+/K+ pump-mimicking mutant in two major conformations, providing insight on how Na+ binding drives a concerted mechanism leading to Na+/K+ pump phosphorylation.

Cryo-EM of the ATP11C flippase reconstituted in Nanodiscs shows a distended phospholipid bilayer inner membrane around transmembrane helix 2.

ATP11C is a member of the P4-ATPase flippase family that mediates translocation of phosphatidylserine (PtdSer) across the lipid bilayer. In order to characterize the structure and function of ATP11C in a model natural lipid environment, we revisited and optimized a quick procedure for reconstituting ATP11C into Nanodiscs using methyl-ß-cyclodextrin as a reagent for the detergent removal. ATP11C was efficiently reconstituted with the endogenous lipid, or the mixture of endogenous lipid and synthetic dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylserine (DOPS), all of which retained the ATPase activity. We obtained 3.4 Å and 3.9 Å structures using single-particle cryo-electron microscopy (cryo-EM) of AlF- and BeF-stabilized ATP11C transport intermediates, respectively, in a bilayer containing DOPS. We show that the latter exhibited a distended inner membrane around ATP11C transmembrane helix 2, possibly reflecting the perturbation needed for phospholipid release to the lipid bilayer. Our structures of ATP11C in the lipid membrane indicate that the membrane boundary varies upon conformational changes of the enzyme and is no longer flat around the protein, a change that likely contributes to phospholipid translocation across the membrane leaflets.

Transport Cycle of Plasma Membrane Flippase ATP11C by Cryo-EM.

ATP11C, a plasma membrane phospholipid flippase, maintains the asymmetric distribution of phosphatidylserine accumulated in the inner leaflet. Caspase-dependent inactivation of ATP11C is essential for an apoptotic "eat me" signal, phosphatidylserine exposure, which prompts phagocytes to engulf cells. We show six cryo-EM structures of ATP11C at 3.0-4.0 Å resolution in five different states of the transport cycle. A structural comparison reveals phosphorylation-driven domain movements coupled with phospholipid binding. Three structures of phospholipid-bound states visualize phospholipid translocation accompanied by the rearrangement of transmembrane helices and an unwound portion at the occlusion site, and thus they detail the basis for head group recognition and the locality of the protein-bound acyl chains in transmembrane grooves. Invariant Lys880 and the surrounding hydrogen-bond network serve as a pivot point for helix bending and precise P domain inclination, which is crucial for dephosphorylation. The structures detail key features of phospholipid translocation by ATP11C, and a common basic mechanism for flippases is emerging.

Crystal structure of a human plasma membrane phospholipid flippase.

ATP11C, a member of the P4-ATPase flippase, translocates phosphatidylserine from the outer to the inner plasma membrane leaflet, and maintains the asymmetric distribution of phosphatidylserine in the living cell. We present the crystal structures of a human plasma membrane flippase, ATP11C-CDC50A complex, in a stabilized E2P conformation. The structure revealed a deep longitudinal crevice along transmembrane helices continuing from the cell surface to the phospholipid occlusion site in the middle of the membrane. We observed that the extension of the crevice on the exoplasmic side is open, and the complex is therefore in an outward-open E2P state, similar to a recently reported cryo-EM structure of yeast flippase Drs2p-Cdc50p complex. We noted extra densities, most likely bound phosphatidylserines, in the crevice and in its extension to the extracellular side. One was close to the phosphatidylserine occlusion site as previously reported for the human ATP8A1-CDC50A complex, and the other in a cavity at the surface of the exoplasmic leaflet of the bilayer. Substitutions in either of the binding sites or along the path between them impaired specific ATPase and transport activities. These results provide evidence that the observed crevice is the conduit along that phosphatidylserine traverses from the outer leaflet to its occlusion site in the membrane and suggest that the exoplasmic cavity is important for phospholipid recognition. They also yield insights into how phosphatidylserine is incorporated from the outer leaflet of the plasma membrane into the transmembrane.

A single K+-binding site in the crystal structure of the gastric proton pump.

The gastric proton pump (H+,K+-ATPase), a P-type ATPase responsible for gastric acidification, mediates electro-neutral exchange of H+ and K+ coupled with ATP hydrolysis, but with an as yet undetermined transport stoichiometry. Here we show crystal structures at a resolution of 2.5 Å of the pump in the E2-P transition state, in which the counter-transporting cation is occluded. We found a single K+ bound to the cation-binding site of the H+,K+-ATPase, indicating an exchange of 1H+/1K+ per hydrolysis of one ATP molecule. This fulfills the energy requirement for the generation of a six pH unit gradient across the membrane. The structural basis of K+ recognition is resolved and supported by molecular dynamics simulations, establishing how the H+,K+-ATPase overcomes the energetic challenge to generate an H+ gradient of more than a million-fold-one of the highest cation gradients known in mammalian tissue-across the membrane.

Crystal structures of the gastric proton pump.

The gastric proton pump-the H+, K+-ATPase-is a P-type ATPase responsible for acidifying the gastric juice down to pH 1. This corresponds to a million-fold proton gradient across the membrane of the parietal cell, the steepest known cation gradient of any mammalian tissue. The H+, K+-ATPase is an important target for drugs that treat gastric acid-related diseases. Here we present crystal structures of the H+, K+-ATPase in complex with two blockers, vonoprazan and SCH28080, in the luminal-open state, at 2.8 Å resolution. The drugs have partially overlapping but clearly distinct binding modes in the middle of a conduit running from the gastric lumen to the cation-binding site. The crystal structures suggest that the tight configuration at the cation-binding site lowers the pK a value of Glu820 sufficiently to enable the release of a proton even into the pH 1 environment of the stomach.

D1-Asn-298 in photosystem II is involved in a hydrogen-bond network near the redox-active tyrosine YZ for proton exit during water oxidation.

In photosynthetic water oxidation, two water molecules are converted into one oxygen molecule and four protons at the Mn4CaO5 cluster in photosystem II (PSII) via the S-state cycle. Efficient proton exit from the catalytic site to the lumen is essential for this process. However, the exit pathways of individual protons through the PSII proteins remain to be identified. In this study, we examined the involvement of a hydrogen-bond network near the redox-active tyrosine YZ in proton transfer during the S-state cycle. We focused on spectroscopic analyses of a site-directed variant of D1-Asn-298, a residue involved in a hydrogen-bond network near YZ We found that the D1-N298A mutant of Synechocystis sp. PCC 6803 exhibits an O2 evolution activity of ∼10% of the wild-type. D1-N298A and the wild-type D1 had very similar features of thermoluminescence glow curves and of an FTIR difference spectrum upon YZ oxidation, suggesting that the hydrogen-bonded structure of YZ and electron transfer from the Mn4CaO5 cluster to YZ were little affected by substitution. In the D1-N298A mutant, however, the flash-number dependence of delayed luminescence showed a monotonic increase without oscillation, and FTIR difference spectra of the S-state cycle indicated partial and significant inhibition of the S2 → S3 and S3 → S0 transitions, respectively. These results suggest that the D1-N298A substitution inhibits the proton transfer processes in the S2 → S3 and S3 → S0 transitions. This in turn indicates that the hydrogen-bond network near YZ can be functional as a proton transfer pathway during photosynthetic water oxidation.

Genetically introduced hydrogen bond interactions reveal an asymmetric charge distribution on the radical cation of the special-pair chlorophyll P680.

The special-pair chlorophyll (Chl) P680 in photosystem II has an extremely high redox potential (Em ) to enable water oxidation in photosynthesis. Significant positive-charge localization on one of the Chl constituents, PD1 or PD2, in P680+ has been proposed to contribute to this high Em To identify the Chl molecule on which the charge is mainly localized, we genetically introduced a hydrogen bond to the 131-keto C=O group of PD1 and PD2 by changing the nearby D1-Val-157 and D2-Val-156 residues to His, respectively. Successful hydrogen bond formation at PD1 and PD2 in the obtained D1-V157H and D2-V156H mutants, respectively, was monitored by detecting 131-keto C=O vibrations in Fourier transfer infrared (FTIR) difference spectra upon oxidation of P680 and the symmetrically located redox-active tyrosines YZ and YD, and they were simulated by quantum-chemical calculations. Analysis of the P680+/P680 FTIR difference spectra of D1-V157H and D2-V156H showed that upon P680+ formation, the 131-keto C=O frequency upshifts by a much larger extent in PD1 (23 cm-1) than in PD2 (<9 cm-1). In addition, thermoluminescence measurements revealed that the D1-V157H mutation increased the Em of P680 to a larger extent than did the D2-V156H mutation. These results, together with the previous results for the mutants of the His ligands of PD1 and PD2, lead to a definite conclusion that a charge is mainly localized to PD1 in P680^.

Development of a novel cryogenic microscope with numerical aperture of 0.9 and its application to photosynthesis research.

A novel cryogenic optical-microscope system was developed in which the objective lens is set inside of the cryostat adiabatic vacuum space. Being isolated from the sample when it was cooled, the objective lens was maintained at room temperature during the cryogenic measurement. Therefore, the authors were able to use a color-aberration corrected objective lens with a numerical aperture of 0.9. The lens is equipped with an air vent for compatibility to the vacuum. The theoretically expected spatial resolutions of 0.39µm along the lateral direction and 1.3µm along the axial direction were achieved by the developed system. The system was applied to the observations of non-uniform distributions of the photosystems in the cells of a green alga, Chlamydomonas reinhardtii, at 94K. Gaussian decomposition analysis of the fluorescence spectra at all the pixels clearly demonstrated a non-uniform distribution of the two photosystems, as reflected in the variable ratios of the fluorescence intensities assigned to photosystem II and to those assigned to photosystem I. The system was also applied to the fluorescence spectroscopy of single isolated photosystem I complexes at 90K. The fluorescence, assigned to be emitted from a single photosystem I trimer, showed an intermittent fluctuation called blinking, which is typical for a fluorescence signal from a single molecule. The vibronic fluorescence bands at around 790nm were observed for single photosystem I trimers, suggesting that the color aberration is not serious up to the 800nm spectral region.

Clock-controlled and FLOWERING LOCUS T (FT)-dependent photoperiodic pathway in Lotus japonicus II: characterization of a microRNA implicated in the control of flowering time.

Plant circadian clock generates rhythms with a period close to 24 h, and it controls a wide variety of physiological and developmental events, including the transition to reproductive growth (or flowering). During the last decade, significant research progress in Arabidopsis thaliana has been made in defining the molecular mechanism by which the circadian clock regulates flowering time in response to changes in photoperiod. In Lotus japonicus, we have found that LjFTa, which encodes a ortholog of the Arabidopsis FLOWERING LOCUS T (FT), plays an important role in the promotion of flowering, but it is not clear how the expression of LjFTa is regulated in L. japonicus. Based on current knowledge of photoperiodic control of flowering time in A. thaliana, here we examined whether a microRNA is involved in the activation of LjFTa in L. japonicus. Two putative L. japonicus genes that are responsible for the production of miR172 (designated LjmiR172a and LjmiR172b) were cloned. Overexpression of LjmiR172a/b in A. thaliana resulted in markedly accelerated flowering through enhancement of the expression of FT, concomitantly reducing the expression level of TARGET OF EARLY ACTIVATION TAGGED 1 (TOE1) transcripts, the protein product of which functions as a transcriptional repressor of FT. These results suggest that LjmiR172 genes play a positive role in the LjFTa-mediated promotion of flowering in L. japonicus.

Functional roles of D2-Lys317 and the interacting chloride ion in the water oxidation reaction of photosystem II as revealed by fourier transform infrared analysis.

Photosynthetic water oxidation in plants and cyanobacteria is catalyzed by a Mn4CaO5 cluster within the photosystem II (PSII) protein complex. Two Cl(-) ions bound near the Mn4CaO5 cluster act as indispensable cofactors, but their functional roles remain to be clarified. We have investigated the role of the Cl(-) ion interacting with D2-K317 (designated Cl-1) by Fourier transform infrared spectroscopy (FTIR) analysis of the D2-K317R mutant of Synechocystis sp. PCC 6803 in combination with Cl(-)/NO3(-) replacement. The D2-K317R mutation perturbed the bands in the regions of the COO(-) stretching and backbone amide vibrations in the FTIR difference spectrum upon the S1 → S2 transition. In addition, this mutation altered the (15)N isotope-edited NO3(-) bands in the spectrum of NO3(-)-treated PSII. These results provide the first experimental evidence that the Cl-1 site is coupled with the Mn4CaO5 cluster and its interaction is affected by the S1 → S2 transition. It was also shown that a negative band at 1748 cm(-1) arising from COOH group(s) was altered to a positive intensity by the D2-K317R mutation as well as by NO3(-) treatment, suggesting that the Cl-1 site affects the pKa of COOH/COO(-) group(s) near the Mn4CaO5 cluster in a common hydrogen bond network. Together with the observation that the efficiency of the S3 → S0 transition significantly decreased in the core complexes of D2-K317R upon moderate dehydration, it is suggested that D2-K317 and Cl-1 are involved in a proton transfer pathway from the Mn4CaO5 cluster to the lumen, which functions in the S3 → S0 transition.

Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed.

Clock-controlled and FLOWERING LOCUS T (FT)-dependent photoperiodic pathway in Lotus japonicus I: verification of the flowering-associated function of an FT homolog.

During the last decade, significant research progress in the study of Arabidopsis thaliana has been made in defining the molecular mechanism by which the plant circadian clock regulates flowering time in response to changes in photoperiod. It is generally accepted that the clock-controlled CONSTANS (CO)-FLOWERING LOCUS T (FT)-mediated external coincidence mechanism underlying the photoperiodic control of flowering time is conserved in higher plants, including A. thaliana and Oryza sativa. However, it is also assumed that the mechanism differs considerably in detail among species. Here we characterized the clock-controlled CO-FT pathway in Lotus japonicus (a model legume) in comparison with that of A. thaliana. L. japonicus has at least one FT orthologous gene (named LjFTa), which is induced specifically in long-days and complements the mutational lesion of the A. thaliana FT gene. However, it was speculated that this legume might lack the upstream positive regulator CO. By employing L. japonicus phyB mutant plants, we showed that the photoreceptor mutant displays a phenotype of early flowering due to enhanced expression of LjFTa, suggesting that LjFTa is invovled in the promotion of flowering in L. japonicus. These results are discussed in the context of current knowledge of the flowering in crop legumes such as soybean and garden pea.

Exploring dynamics of molybdate in living animal cells by a genetically encoded FRET nanosensor.

Molybdenum (Mo) is an essential trace element for almost all living organisms including animals. Mo is used as a catalytic center of molybdo-enzymes for oxidation/reduction reactions of carbon, nitrogen, and sulfur metabolism. Whilst living cells are known to import inorganic molybdate oxyanion from the surrounding environment, the in vivo dynamics of cytosolic molybdate remain poorly understood as no appropriate indicator is available for this trace anion. We here describe a genetically encoded Förester-resonance-energy-transfer (FRET)-based nanosensor composed of CFP, YFP and the bacterial molybdate-sensor protein ModE. The nanosensor MolyProbe containing an optimized peptide-linker responded to nanomolar-range molybdate selectively, and increased YFP:CFP fluorescence intensity ratio by up to 109%. By introduction of the nanosensor, we have been able to successfully demonstrate the real-time dynamics of molybdate in living animal cells. Furthermore, time course analyses of the dynamics suggest that novel oxalate-sensitive- and sulfate-resistant- transporter(s) uptake molybdate in a model culture cell.

Molecular mechanisms of circadian rhythm in Lotus japonicus and Arabidopsis thaliana are sufficiently compatible to regulate heterologous core clock genes robustly.

Recent intensive studies of the model plant Arabidopsis thaliana have revealed the molecular mechanisms underlying circadian rhythms in detail. Results of phylogenetic analyses indicated that some of core clock genes are widely conserved throughout the plant kingdom. For another model plant the legume Lotus japonicus, we have reported that it has a set of putative clock genes highly homologous to A. thaliana. Taking advantage of the L. japonicus hairy root transformation system, in this study we characterized the promoter activity of A. thaliana core clock genes CCA1 and PRR5 in heterologous L. japonicus cells and found that the molecular mechanism of circadian rhythm in L. japonicus is compatible with that of A. thaliana.

Thiol modulation of the chloroplast ATP synthase is dependent on the energization of thylakoid membranes.

Thiol modulation of the chloroplast ATP synthase γ subunit has been recognized as an important regulatory system for the activation of ATP hydrolysis activity, although the physiological significance of this regulation system remains poorly characterized. Since the membrane potential required by this enzyme to initiate ATP synthesis for the reduced enzyme is lower than that needed for the oxidized form, reduction of this enzyme was interpreted as effective regulation for efficient photophosphorylation. However, no concrete evidence has been obtained to date relating to the timing and mode of chloroplast ATP synthase reduction and oxidation in green plants. In this study, thorough analysis of the redox state of regulatory cysteines of the chloroplast ATP synthase γ subunit in intact chloroplasts and leaves shows that thiol modulation of this enzyme is pivotal in prohibiting futile ATP hydrolysis activity in the dark. However, the physiological importance of efficient ATP synthesis driven by the reduced enzyme in the light could not be demonstrated. In addition, we investigated the significance of the electrochemical proton gradient in reducing the γ subunit by the reduced form of thioredoxin in chloroplasts, providing strong insights into the molecular mechanisms underlying the formation and reduction of the disulfide bond on the γ subunit in vivo.

Characterization of shade avoidance responses in Lotus japonicus.

Sessile plants must continuously adjust their growth and development to optimize photosynthetic activity under ever-fluctuating light conditions. Among such light responses in plants, one of the best-characterized events is the so-called shade avoidance, for which a low ratio of the red (R):far-red (FR) light intensities is the most prominent stimulus. Such shade avoidance responses enable plants to overtop their neighbors, thereby enhancing fitness and competitiveness in their natural habitat. Considerable progress has been achieved during the last decade in understanding the molecular mechanisms underlying the shade avoidance responses in the model rosette plant, Arabidopsis thaliana. We characterize here the fundamental aspects of the shade avoidance responses in the model legume, Lotus japonicus, based on the fact that its phyllotaxis (or morphological architecture) is quite different from that of A. thaliana. It was found that L. japonicus displays the characteristic shade avoidance syndrome (SAS) under defined laboratory conditions (a low R:FR ratio, low light intensity, and low blue light intensity) that mimic the natural canopy. In particular, the outgrowth of axillary buds (i.e., both aerial and cotyledonary shoot branching) was severely inhibited in L. japonicus grown in the shade. These results are discussed with special emphasis on the unique aspects of SAS observed with this legume.

Light-responsive double B-box containing transcription factors are conserved in Physcomitrella patens.

In the model seed plant Arabidopsis thaliana, a sub-family of B-box containing transcriptional factors (BBXs), which is classified in the BBX-IV group based on the domain structure, contains two tandem B-box domains and plays crucial roles in early photomorphogenesis under the control of blue light receptors, cry1 and cry2. The results of an examination of light responsiveness of representative Physcomitrella BBX-IV genes and their heterologous expression in Arabidopsis suggested that the light signaling-related characteristics of the BBX-IV subfamily are evolutionarily conserved in a moss, which is a basal lineage of land plants.

Functional characterization of HY5 homolog genes involved in early light-signaling in Physcomitrella patens.

The developmental programs of Physcomitrella patens, a basal lineage of land plants, are regulated by phytohormones and light-signaling responses. In this study, our attention was focused on the HY5-family of transcription factors, which are known to play important roles immediately downstream of photoreceptors during the early photomorphogenesis of Arabidopsis thaliana. We retrieved two HY5-homologs, named PpHY5a and PpHY5b, from the whole genome sequence database of P. patens. Arabidopsis transgenic plants overproducing the basic leucine zipper (bZIP) domain of PpHY5a exhibited a phenotype of short hypocotyls, suggesting a functional relationship between PpHY5 and Arabidopsis HY5. A loss-of-function Δhy5a Δhy5b double mutant was defective in the vigorous protrusion of caulonema cells from the protonema networks of P. patens under light and dark conditions. These results suggest that the function of HY5-homologs in P. patens is evolutionarily conserved, and is implicated in a process of caulonema development.