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. © 2012 The Author

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

Effect of over-expression and knockdown of <i>Hs</i>MoT2/MFSD5 in molybdate uptake rate. A

<p>, Time course of R_F530:F480 in control cells (for Panel B). HEK-293T was co-transfected with MolyProbe and mock vector by the polyethyleneimine method. Fluorescence was measured after addition of molybdate, and R_F530:F480 calculated. <b>B</b>, Time course of <i>Hs</i>MoT2/MFSD5 over-expressing cells. mRNA level of <i>Hs</i>MoT2/MFSD5 was about sixty-fold compared to the control cell. <b>C</b>, Time course of R_F530:F480 in control cells (for Panel D) transfected with MolyProbe by X-tremeGENE siRNA reagent. <b>D</b>, Time course of R_F530:F480 in <i>Hs</i>MoT2/MFSD5 knockdown cells. mRNA level of <i>Hs</i>MoT2/MFSD5 was 11–35% compared to the control cell. Concentrations of molybdate in working medium are follows: 0 µM (closed circle), 0.1 µM (cross), 0.3 µM (closed triangle), 1 µM (open triangle), 3 µM (closed diamond), 10 µM (open diamond), 30 µM (closed square), 100 µM (open square). Averages and SDs from triplicate samples are shown. n = 3.</p

Sensitivity and specificity of MolyProbe. A

<p>, Titration curve of MolyProbe to molybdate. Emission spectrum was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058175#pone-0058175-g001" target="_blank">Figure 1</a>. Emission intensity ratio (F530: F475) was calculated and plotted against molybdate concentration. The plot was fitted by the Hill equation. <b>B</b>, Titration curves for similar oxyanions. <b>C</b>, Inhibitory effect of chloride. Titration curves for molybdate were determined in the presence of 1–100 mM KCl. Apparent <i>K</i>_0.5 values for molybdate were calculated and plotted against concentrations of potassium chloride. <b>D</b>, Inhibitory effect of bicarbonate. <b>E</b>, Inhibitory effect of MolyProbe. Titration curves were determined at a concentration of 0.5–200 nM MolyProbe. Average data were obtained by triplicate assays. The SDs were small (<2% for A, B and <5% for C–E).</p

FRET-based genetically encoded nanosensor for molybdate. A

<p>, Primary structure of MolyProbe. CFP (Cerulean), two molybdate binding domains (MoBD) and YFP (cp157-Venus) are connected by optimized peptide linkers. MoBDs are from <i>E.coli</i> ModE factor. T272A/T444A is a loss-of-function mutant. <b>B</b>, Schematic representation of molybdate binding between two MoBDs, which increase FRET efficiency. <b>C</b>, Spectral property of MolyProbe <i>in vitro</i>. The emission spectrum of recombinant protein (20 nM) was measured at λ_Ex 430 nm (λ_max for CFP), with or without 10 µM molybdate. <b>D</b>, Emission spectral property of the T272A/T444A double mutant.</p

Real time imaging of molybdate level in living animal cells. A

<p>, Time course of R_F530:F475 in bulk HEK-293T cells transfected with MolyProbe after exposure to 1 mM molybdate at 37°C. <b>B</b>, Time course of R_F530:F475 in the bulk cells treated with 1 mM molybdate at 22°C. Averages and SDs from triplicate samples are shown. <b>C</b>, Confocal CFP(F475) and YFP(F535) images of the cells before and after treatment with 1 mM molybdate. <b>D</b>, Variation in expression levels of MolyProbe. A density image of MolyProbe (green) calculated from a pair of CFP and YFP image was merged with a transmission image (grey). Low-level expression cells (1), middle-level cells (2) and high-level cells (3) showed a different time course of the ratio change (Figure E). <b>E</b>, A series of ratio images of the cells accumulating molybdate. Ratio (F535:F475) images were calculated from pairs of CFP and YFP images taken every 15 sec, and the ratios are presented in pseudo-color. Intracellular molybdate increased by addition of 1 mM molybdate to the medium. Velocity of the molybdate increment in pseudopod was fast compared to the cell body (arrow), whereas nuclear space was slow (arrowhead).</p

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 <i>Synechocystis</i> 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 ¹⁵N 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 p<i>K</i>_a 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