Shin Ozawa

Ribosome rescue by Escherichia coli ArfA (YhdL) in the absence of trans-translation system

Although SsrA(tmRNA)‐mediated trans‐translation is thought to maintain the translation capacity of bacterial cells by rescuing ribosomes stalled on messenger RNA lacking an in‐frame stop codon, single disruption of ssrA does not crucially hamper growth of Escherichia coli. Here, we identified YhdL (renamed ArfA for alternative ribosome‐rescue factor) as a factor essential for the viability of E. coli in the absence of SsrA. The ssrA–arfA synthetic lethality was alleviated by SsrADD, an SsrA variant that adds a proteolysis‐refractory tag through trans‐translation, indicating that ArfA‐deficient cells require continued translation, rather than subsequent proteolysis of the truncated polypeptide. In accordance with this notion, depletion of SsrA in the ΔarfA background led to reduced translation of a model protein without affecting transcription, and puromycin, a codon‐independent mimic of aminoacyl‐tRNA, rescued the bacterial growth under such conditions. That ArfA takes over the role of SsrA was suggested by the observation that its overexpression enabled detection of the polypeptide encoded by a model non‐stop mRNA, which was otherwise SsrA‐tagged and degraded. In vitro, purified ArfA acted on a ribosome‐nascent chain complex to resolve the peptidyl‐tRNA. These results indicate that ArfA rescues the ribosome stalled at the 3′ end of a non‐stop mRNA without involving trans‐translation.

Biochemical and Structural Studies of the Large Ycf4-Photosystem I Assembly Complex of the Green Alga Chlamydomonas reinhardtii

Ycf4 is a thylakoid protein essential for the accumulation of photosystem I (PSI) in Chlamydomonas reinhardtii. Here, a tandem affinity purification tagged Ycf4 was used to purify a stable Ycf4-containing complex of >1500 kD. This complex also contained the opsin-related COP2 and the PSI subunits PsaA, PsaB, PsaC, PsaD, PsaE, and PsaF, as identified by mass spectrometry (liquid chromatography–tandem mass spectrometry) and immunoblotting. Almost all Ycf4 and COP2 in wild-type cells copurified by sucrose gradient ultracentrifugation and subsequent ion exchange column chromatography, indicating the intimate and exclusive association of Ycf4 and COP2. Electron microscopy revealed that the largest structures in the purified preparation measure 285 × 185 Å; these particles may represent several large oligomeric states. Pulse-chase protein labeling revealed that the PSI polypeptides associated with the Ycf4-containing complex are newly synthesized and partially assembled as a pigment-containing subcomplex. These results indicate that the Ycf4 complex may act as a scaffold for PSI assembly. A decrease in COP2 to 10% of wild-type levels by RNA interference increased the salt sensitivity of the Ycf4 complex stability but did not affect the accumulation of PSI, suggesting that COP2 is not essential for PSI assembly.

Photosystem II complex in vivo is a monomer

Photosystem II (PS II) complexes are membrane protein complexes that are composed of >20 distinct subunit proteins. Similar to many other membrane protein complexes, two PS II complexes are believed to form a homo-dimer whose molecular mass is ∼650 kDa. Contrary to this well known concept, we propose that the functional form of PS II in vivo is a monomer, based on the following observations. Deprivation of lipids caused the conversion of PS II from a monomeric form to a dimeric form. Only a monomeric PS II was detected in solubilized cyanobacterial and red algal thylakoids using blue-native polyacrylamide gel electrophoresis. Furthermore, energy transfer between PS II units, which was observed in the purified dimeric PS II, was not detected in vivo. Our proposal will lead to a re-evaluation of many crystallographic models of membrane protein complexes in terms of their oligomerization status.

Natural dissociation of olivine to (Mg,Fe)SiO3 perovskite and magnesiowüstite in a shocked Martian meteorite

We report evidence for the natural dissociation of olivine in a shergottite at high-pressure and high-temperature conditions induced by a dynamic event on Mars. Olivine (Fa34-41) adjacent to or entrained in the shock melt vein and melt pockets of Martian meteorite olivine-phyric shergottite Dar al Gani 735 dissociated into (Mg,Fe)SiO3 perovskite (Pv)+magnesiowüstite (Mw), whereby perovskite partially vitrified during decompression. Transmission electron microscopy observations reveal that microtexture of olivine dissociation products evolves from lamellar to equigranular with increasing temperature at the same pressure condition. This is in accord with the observations of synthetic samples recovered from high-pressure and high-temperature experiments. Equigranular (Mg,Fe)SiO3 Pv and Mw have 50–100 nm in diameter, and lamellar (Mg,Fe)SiO3 Pv and Mw have approximately 20 and approximately 10 nm in thickness, respectively. Partitioning coefficient, KPv/Mw = [FeO/MgO]/[FeO/MgO]Mw, between (Mg,Fe)SiO3 Pv and Mw in equigranular and lamellar textures are approximately 0.15 and approximately 0.78, respectively. The dissociation of olivine implies that the pressure and temperature conditions recorded in the shock melt vein and melt pockets during the dynamic event were approximately 25 GPa but 700u2009°C at least.

Chloroplast-encoded polypeptide PsbT is involved in the repair of primary electron acceptor QA of photosystem II during photoinhibition in Chlamydomonas reinhardtii

PsbT is a small chloroplast-encoded hydrophobic polypeptide associated with the D1/D2 heterodimer of the photosystem II (PSII) reaction center and is required for the efficient post-translational repair of photodamaged PSII. Here we addressed that role in detail in Chlamydomonas reinhardtii wild type and ΔpsbT cells by analyzing the activities of PSII, the assembly of PSII proteins, and the redox components of PSII during photoinhibition and repair. Strong illumination of cells for 15 min decreased the activities of electron transfer through PSII and QA photoreduction by 50%, and it reduced the amount of atomic manganese by 20%, but it did not affect the steady-state level of PSII proteins, photoreduction of pheophytin (pheoD1), and the amount of bound plastoquinone (QA), indicating that the decrease in PSII activity resulted mainly from inhibition of the electron transfer from pheoD1 to QA. In wild type cells, we observed parallel recovery of electron transfer activity through PSII and QA photoreduction, suggesting that the recovery of QA activity is one of the rate-limiting steps of PSII repair. In ΔpsbT cells, the repairs of electron transfer activity through PSII and of QA photoreduction activity were both impaired, but PSII protein turnover was unaffected. Moreover, about half the QA was lost from the PSII core complex during purification. Since PsbT is intimately associated with the QA-binding region on D2, we propose that this polypeptide enhances the efficient recovery of QA photoreduction by stabilizing the structure of the QA-binding region.

Discovery of coesite and stishovite in eucrite

Significance Quartz and/or cristobalite in eucrite were transformed into denser minerals, coesite and stishovite, under transient high-pressure and high-temperature conditions. Coesite and stishovite probably formed simultaneously under pressures of similar magnitudes but under different temperature conditions. The expected age of the dynamic event that formed coesite and stishovite is ca. 4.1 Ga ago, which is inconsistent with the predicted formation age (ca. 1.0 Ga) of the impact basins on 4 Vesta. Howardite–eucrite–diogenite meteorites (HEDs) probably originated from the asteroid 4 Vesta. We investigated one eucrite, Béréba, to clarify a dynamic event that occurred on 4 Vesta using a shock-induced high-pressure polymorph. We discovered high-pressure polymorphs of silica, coesite, and stishovite originating from quartz and/or cristobalite in and around the shock-melt veins of Béréba. Lamellar stishovite formed in silica grains through a solid-state phase transition. A network-like rupture was formed and melting took place along the rupture in the silica grains. Nanosized granular coesite grains crystallized from the silica melt. Based on shock-induced high-pressure polymorphs, the estimated shock-pressure condition ranged from ∼8 to ∼13 GPa. Considering radiometric ages and shock features, the dynamic event that led to the formation of coesite and stishovite occurred ca. 4.1 Ga ago, which corresponds to the late heavy bombardment period (ca. 3.8–4.1 Ga), deduced from the lunar cataclysm. There are two giant impact basins around the south pole of 4 Vesta. Although the origin of HEDs is thought to be related to dynamic events that formed the basins ca. 1.0 Ga ago, our findings are at variance with that idea.

Characterization of photosystem I antenna proteins in the prasinophyte Ostreococcus tauri

Prasinophyceae are a broad class of early-branching eukaryotic green algae. These picophytoplankton are found ubiquitously throughout the ocean and contribute considerably to global carbon-fixation. Ostreococcus tauri, as the first sequenced prasinophyte, is a model species for studying the functional evolution of light-harvesting systems in photosynthetic eukaryotes. In this study we isolated and characterized O. tauri pigment-protein complexes. Two photosystem I (PSI) fractions were obtained by sucrose density gradient centrifugation in addition to free light-harvesting complex (LHC) fraction and photosystem II (PSII) core fractions. The smaller PSI fraction contains the PSI core proteins, LHCI, which are conserved in all green plants, Lhcp1, a prasinophyte-specific LHC protein, and the minor, monomeric LHCII proteins CP26 and CP29. The larger PSI fraction contained the same antenna proteins as the smaller, with the addition of Lhca6 and Lhcp2, and a 30% larger absorption cross-section. When O. tauri was grown under high-light conditions, only the smaller PSI fraction was present. The two PSI preparations were also found to be devoid of the far-red chlorophyll fluorescence (715-730 nm), a signature of PSI in oxygenic phototrophs. These unique features of O. tauri PSI may reflect primitive light-harvesting systems in green plants and their adaptation to marine ecosystems. Possible implications for the evolution of the LHC-superfamily in photosynthetic eukaryotes are discussed.

Chloroplast-mediated regulation of CO2-concentrating mechanism by Ca2+-binding protein CAS in the green alga Chlamydomonas reinhardtii

Significance Ca2+ and CO2 are fundamental biological signaling molecules in microbes, animals, and plants. Although Ca2+ was proposed to act as a second messenger in CO2 signaling in guard cells of terrestrial plants, the role of Ca2+ in CO2 signal transduction pathways in aquatic photosynthetic organisms remains largely unknown. We show here that a chloroplast Ca2+-binding protein, CAS, changes its localization in response to environmental CO2 conditions and regulates the expression of nuclear-encoded limiting-CO2–induced genes, including two key bicarbonate transporters. These findings led us to propose a model for the participation of Ca2+ signals in chloroplast-regulated CO2 signal transduction of aquatic photosynthetic organisms and help us to further understand the role of Ca2+ in CO2 signal transduction in eukaryotes. Aquatic photosynthetic organisms, including the green alga Chlamydomonas reinhardtii, induce a CO2-concentrating mechanism (CCM) to maintain photosynthetic activity in CO2-limiting conditions by sensing environmental CO2 and light availability. Previously, a novel high-CO2–requiring mutant, H82, defective in the induction of the CCM, was isolated. A homolog of calcium (Ca2+)-binding protein CAS, originally found in Arabidopsis thaliana, was disrupted in H82 cells. Although Arabidopsis CAS is reported to be associated with stomatal closure or immune responses via a chloroplast-mediated retrograde signal, the relationship between a Ca2+ signal and the CCM associated with the function of CAS in an aquatic environment is still unclear. In this study, the introduction of an intact CAS gene into H82 cells restored photosynthetic affinity for inorganic carbon, and RNA-seq analyses revealed that CAS could function in maintaining the expression levels of nuclear-encoded CO2-limiting–inducible genes, including the HCO3– transporters high-light activated 3 (HLA3) and low-CO2–inducible gene A (LCIA). CAS changed its localization from dispersed across the thylakoid membrane in high-CO2 conditions or in the dark to being associated with tubule-like structures in the pyrenoid in CO2-limiting conditions, along with a significant increase of the fluorescent signals of the Ca2+ indicator in the pyrenoid. Chlamydomonas CAS had Ca2+-binding activity, and the perturbation of intracellular Ca2+ homeostasis by a Ca2+-chelator or calmodulin antagonist impaired the accumulation of HLA3 and LCIA. These results suggest that Chlamydomonas CAS is a Ca2+-mediated regulator of CCM-related genes via a retrograde signal from the pyrenoid in the chloroplast to the nucleus.

D1 fragmentation in photosystem II repair caused by photo-damage of a two-step model

Light energy drives photosynthesis, but it simultaneously inactivates photosynthetic mechanisms. A major target site of photo-damage is photosystem II (PSII). It further targets one reaction center protein, D1, which is maintained efficiently by the PSII repair cycle. Two proteases, FtsH and Deg, are known to contribute to this process, respectively, by efficient degradation of photo-damaged D1 protein processively and endoproteolytically. This study tested whether the D1 cleavage accomplished by these proteases is affected by different monochromic lights such as blue and red light-emitting-diode light sources, remaining mindful that the use of these lights distinguishes the current models for photoinhibition: the excess-energy model and the two-step model. It is noteworthy that in the two-step model, primary damage results from the absorption of light energy in the Mn-cluster, which can be enhanced by a blue rather than a red light source. Results showed that blue and red lights affect D1 degradation differently. One prominent finding was that D1 fragmentation that is specifically generated by luminal Deg proteases was enhanced by blue light but not by red light in the mutant lacking FtsH2. Although circumstantial, this evidence supports a two-step model of PSII photo-damage. We infer that enhanced D1 fragmentation by luminal Deg proteases is a response to primary damage at the Mn-cluster.

The stability of anhydrous phase B, Mg14Si5O24, at mantle transition zone conditions

The stability of anhydrous phase B, Mg14Si5O24, has been determined in the pressure range of 14–21xa0GPa and the temperature range of 1100–1700xa0°C with both normal and reversal experiments using multi-anvil apparatus. Our results demonstrate that anhydrous phase B is stable at pressure–temperature conditions corresponding to the shallow depth region of the mantle’s transition zone and it decomposes into periclase and wadsleyite at greater depths. The decomposition boundary of anhydrous phase B into wadsleyite and periclase has a positive phase transition slope and can be expressed by the following equation: P(GPa)u2009=u20097.5u2009+u20096.6u2009×u200910−3T (°C). This result is consistent with a recent result on the decomposition boundary of anhydrous phase B (Kojitani et al., Am Miner 102:2032–2044, 2017). However, our phase boundary deviates significantly from this previous study at temperaturesu2009<u20091400xa0°C. Subducting carbonates may be reduced at depthsu2009>u2009250xa0km, which could contribute ferropericlase (Mg, Fe)O or magnesiowustite (Fe, Mg)O into the deep mantle. Incongruent melting of hydrous peridotite may also produce MgO-rich compounds. Anh-B could form in these conditions due to reactions between Mg-richxa0oxides and silicates. Anh-B might provide axa0new interpretation for the origin of diamonds containing ferropericlase–olivine inclusions and chromitites which have been found to havexa0ultrahigh-pressure characteristics. We propose that directly touching ferropericlase–olivine inclusions found in natural diamonds might be the retrogressive products of anhydrous phase B decomposing via the reaction (Mg,Fe)14Si5O24 (Anh-B)xa0=xa0(Mg,Fe)2SiO4 (olivine)xa0+xa0(Mg,Fe)O (periclase). This decomposition may occur during the transportation of the host diamonds from their formation depths of <u2009500xa0km in the upper part of the mantle transition zone to the surface.