SummaryAlthough 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 SsrA DD , 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 DarfA background led to reduced translation of a model protein without affecting transcription, and puromycin, a codonindependent 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 ribosomenascent chain complex to resolve the peptidyl-tRNA. These results indicate that ArfA rescues the ribosome stalled at the 3Ј end of a non-stop mRNA without involving trans-translation.

The carbon-concentrating mechanism (CCM) is essential to support photosynthesis under CO2-limiting conditions in aquatic photosynthetic organisms, including the green alga Chlamydomonas reinhardtii. The CCM is assumed to be comprised of inorganic carbon transport systems that, in conjunction with carbonic anhydrases, maintain high levels of CO2 around ribulose-1, 5-bisphosphate carboxylase/oxygenase in a specific compartment called the pyrenoid. A set of transcripts up-regulated during the induction of the CCM was identified previously and designated as low-CO2 (LC)-inducible genes. Although the functional importance of one of these LC-inducible genes, LciB, has been shown recently, the biochemical properties and detailed subcellular localization of its product LCIB remain to be elucidated. Here, using yeast two-hybrid, immunoprecipitation and mass spectrometry analyses we provide evidence to demonstrate that LCIB interacts with the LCIB homologous protein LCIC in yeast and in vivo. We also show that LCIB and LCIC are co-localized in the vicinity of the pyrenoid under LC conditions in the light, forming a hexamer complex of approximately 350 kDa, as estimated by gel filtration chromatography. LCIB localization around the pyrenoid was dependent on light illumination and LC conditions during active operation of the CCM. In contrast, in the dark or under high-CO2 conditions when the CCM was inactive, LCIB immediately diffused away from the pyrenoid. Based on these observations, we discuss possible functions of the LCIB-LCIC complex in the CCM.

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The Chlamydomonas reinhardtii proton gradient regulation5 (Crpgr5) mutant shows phenotypic and functional traits similar to mutants in the Arabidopsis (Arabidopsis thaliana) ortholog, Atpgr5, providing strong evidence for conservation of PGR5-mediated cyclic electron flow (CEF). Comparing the Crpgr5 mutant with the wild type, we discriminate two pathways for CEF and determine their maximum electron flow rates. The PGR5/proton gradient regulation-like1 (PGRL1) ferredoxin (Fd) pathway, involved in recycling excess reductant to increase ATP synthesis, may be controlled by extreme photosystem I acceptor side limitation or ATP depletion. Here, we show that PGR5/PGRL1-Fd CEF functions in accordance with an ATP/redox control model. In the absence of Rubisco and PGR5, a sustained electron flow is maintained with molecular oxygen instead of carbon dioxide serving as the terminal electron acceptor. When photosynthetic control is decreased, compensatory alternative pathways can take the full load of linear electron flow. In the case of the ATP synthase pgr5 double mutant, a decrease in photosensitivity is observed compared with the single ATPase-less mutant that we assign to a decreased proton motive force. Altogether, our results suggest that PGR5/PGRL1-Fd CEF is most required under conditions when Fd becomes overreduced and photosystem I is subjected to photoinhibition. CEF is not a valve; it only recycles electrons, but in doing so, it generates a proton motive force that controls the rate of photosynthesis. The conditions where the PGR5 pathway is most required may vary in photosynthetic organisms like C. reinhardtii from anoxia to high light to limitations imposed at the level of carbon dioxide fixation.Photosynthesis is a highly regulated process that integrates different electron transfer pathways to convert light energy into ATP and NADPH and balance this production of chemical energy with its use in anabolic metabolism. Linear electron flow accounts for the major flux of electrons from the primary electron donor water to PSII and intersystem carriers to reduce NADP + , the terminal acceptor associated with PSI. Electron transfer is coupled to proton transfer through reactions involving plastoquinones/plastoquinols that are dependent on the activity of the cytochrome b 6 f complex (cyt f); the protons are transferred from the stroma into the thylakoid lumen. The proton motive force generated is used for ATP synthesis by the ATP synthase. The NADPH and ATP produced in the light serve as the energy/reductant that drives the fixation of carbon dioxide (CO 2 ) by Rubisco and the Calvin-Benson cycle and also supports other downstream metabolic reactions.The Calvin-Benson cycle has a stoichiometric requirement of 3 ATP and 2 NADPH per CO 2 molecule; this requirement is not fulfilled by linear electron flow, because it is slightly imbalanced in favor of NADPH production. Cyclic electron flow (CEF) pathways allow the cells to fulfill the energetic requirement for sustained CO 2 fixation through recycling or reoxidation of ...

Flaxseed lignan secoisolariciresinol diglucoside (SDG) has been reported to prevent and alleviate lifestyle-related diseases including diabetes and hypercholesterolaemic atherosclerosis. This study assesses the effect of SDG on the development of diet-induced obesity in mice and the effect of the SDG metabolite enterodiol (END) on adipogenesis in 3T3-L1 adipocytes. We compared body weight, visceral fat weight, liver fat content, serum parameters, mRNA levels of lipid metabolism-related enzymes and adiponectin in mice fed either a low-fat diet (5 % TAG), high-fat diet (30 % TAG) or high-fat diet containing 0·5 and 1·0 % (w/w) SDG for 4 weeks. Administration of SDG to mice significantly reduced highfat diet-induced visceral and liver fat accumulation, hyperlipaemia, hypercholesterolaemia, hyperinsulinaemia and hyperleptinaemia. SDG also suppressed sterol regulatory element binding protein 1c mRNA level in the liver and induced increases in the adiponectin mRNA level in the white adipose tissue and carnitine palmitoyltransferase I mRNA level in the skeletal muscle. Differentiated 3T3-L1 adipocytes were treated with 0, 5, 10 and 20 mmol/l END and then assayed for mRNA expression of adipogenesis-related genes and DNA binding activity of PPARg to the PPAR response element consensus sequence. END induced adipogenesis-related gene mRNA expression including adiponectin, leptin, glucose transporter 4 and PPARg, and induced PPARg DNA binding activity in 3T3-L1 adipocytes. In conclusion, SDG induced adiponectin mRNA expression and showed beneficial effects on lipid metabolism in diet-induced obesity in mice. Flaxseed lignans are suggested to regulate adipogenesis-related gene expressions through an increase in PPARg DNA binding activity. Flaxseed: Lignan: Obesity: AdiponectinObesity is a major risk factor for various diseases, and results from an imbalance between energy intake and expenditure. It is also known that altered lipid metabolism, including fat oxidation and fatty acid synthesis, is a related factor in obesity (1,2) . Furthermore, diet-induced obesity in man is often complicated by various lifestyle-related diseases, including diabetes, hyperlipaemia, hypertension and hyperinsulinaemia. Therefore, prevention of obesity is strongly encouraged in order to alleviate various lifestyle-related diseases.Lignans are found in various foodstuffs, such as plant seeds, whole grains, legumes, vegetables and fruits (3) . Flax (Linum usitatissimum) is one of the richest sources of plant lignans (4) . The concentration of lignans in flaxseed was shown to be more than 100 times higher than that in most other foods (3)

Disulfides, with a series of alkyl spacers containing porphyrins at both ends, were prepared to evaluate the effect of the spacer length on the interfacial structure and photoelectrochemical properties of selfassembled monolayers (SAMs) on a gold electrode. The structure of the SAMs was investigated using UV-visible absorption spectroscopy, cyclic voltammetry, and photoelectrochemical studies. These measurements showed that as the length of the spacers increases, the SAMs tend to form a highly ordered structure on the gold electrode. Photoelectrochemical studies, using modified Au and Pt electrodes, were carried out in the presence of methyl viologen as an electron carrier. The photocurrents decrease dramatically with a decrease in the spacer length, indicating that there are two competitive deactivation pathways for the excited porphyrin, i.e., the quenching by the electrode and electron carrier.

Photosystem I (PSI) is a multiprotein complex consisting of the PSI core and peripheral light-harvesting complex I (LHCI) that together form the PSI-LHCI supercomplex in algae and higher plants. The supercomplex is synthesized in steps during which 12-15 core and 4 -9 LHCI subunits are assembled. Here we report the isolation of a PSI subcomplex that separated on a sucrose density gradient from the thylakoid membranes isolated from logarithmic growth phase cells of the green alga Chlamydomonas reinhardtii. Pulse-chase labeling of total cellular proteins revealed that the subcomplex was synthesized de novo within 1 min and was converted to the mature PSI-LHCI during the 2-h chase period, indicating that the subcomplex was an assembly intermediate. The subcomplex was functional; it photooxidized P700 and demonstrated electron transfer activity. The subcomplex lacked PsaK and PsaG, however, and it bound PsaF and PsaJ weakly and was not associated with LHCI. It seemed likely that LHCI had been integrated into the subcomplex unstably and was dissociated during solubilization and/or fractionation. We, thus, infer that PsaK and PsaG stabilize the association between PSI core and LHCI complexes and that PsaK and PsaG bind to the PSI core complex after the integration of LHCI in one of the last steps of PSI complex assembly.

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 wildtype 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 3 185 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.