Search

Metabolic water, the water that is produced from O2 during respiration, carries an isotopic signature that can be different from that of the water the cell is growing in. It has been well known that for large land organisms, like birds and mammals, metabolic water contributes significantly to the water balance and has an important control on the signature of the oxygen-stable-isotopes of the water inside the organism. This isotopic signature is then carried over through isotopic equilibrium to other oxygen-bearing species like phosphate. However, for small organisms like bacteria, it has been widely assumed for decades, that the large surface area to volume ratio enables a fast exchange of the cell water with the ambient water. As a result, the isotopic signature of the metabolic water will be heavily diluted and erased. In contrast, a recent work reported indirect evidence of significant control of metabolic water on the oxygen isotopes inside microbial cells. This indirect evidence is based on deviations of oxygen isotopes in phosphate from the expected equilibrium with the ambient water. Here we report the results of experiments that directly tested the possible contribution of metabolic water to phosphate oxygen isotopes in bacteria. We found that ambient water did control the oxygen isotopes in the phosphate. However, there were large deviations from the expected equilibrium. Nevertheless, we found that these deviations were not correlated with the isotopic composition of metabolic water. Hence, other mechanisms, which will be discussed, are responsible for these deviations.

Partially charged chiral molecules act as spin filters, with preference for electron transport toward one type of spin ("up" or "down"), depending on their handedness. This effect is named the chiral induced spin selectivity (CISS) effect. A consequence of this phenomenon is spin polarization concomitant with electric polarization in chiral molecules. These findings were shown by adsorbing chiral molecules on magnetic surfaces and investigating the spin-exchange interaction between the surface and the chiral molecule. This field of study was developed using artificial chiral molecules. Here we used such magnetic surfaces to explore the importance of the intrinsic chiral properties of proteins in determining their stability. First, proteins were adsorbed on paramagnetic and ferromagnetic nanoparticles in a solution, and subsequently urea was gradually added to induce unfolding. The structural stability of proteins was assessed using two methods: bioluminescence measurements used to monitor the activity of the Luciferase enzyme, and fast spectroscopy detecting the distance between two chromophores implanted at the termini of a Barnase core. We found that interactions with magnetic materials altered the structural and functional resilience of the natively folded proteins, affecting their behavior under varying mild denaturing conditions. Minor structural disturbances at low urea concentrations were impeded in association with paramagnetic nanoparticles, whereas at higher urea concentrations, major structural deformation was hindered in association with ferromagnetic nanoparticles. These effects were attributed to spin exchange interactions due to differences in the magnetic imprinting properties of each type of nanoparticle. Additional measurements of proteins on macroscopic magnetic surfaces support this conclusion. The results imply a link between internal spin exchange interactions in a folded protein and its structural and functional integrity on magnetic surfaces. Together with the accumulating knowledge on CISS, our findings suggest that chirality and spin exchange interactions should be considered as additional factors governing protein structures.

SummaryMitochondria are semi-autonomous organelles that serve as hubs for aerobic energy metabolism. The biogenesis of the respiratory (OXPHOS) system relies on nuclear-encoded factors, which regulate the transcription, processing and translation of mitochondrial (mt)RNAs. These include proteins of primordial origin, as well as eukaryotic-type RNA-binding families recruited from the host genomes to function in mitogenome expression. Pentatricopeptide repeat (PPR) proteins constitute a major gene-family in angiosperms that is pivotal in many aspects of mtRNA metabolism, such as editing, splicing or stability. Here, we report the analysis ofMITOCHONDRIA STABILITY/PROCESSING PPR FACTOR1(MSP1, At4g20090), a canonical mitochondria-localized PPR protein that is necessary for mitochondrial biogenesis and embryo-development. Functional complementation confirmed that the phenotypes result from a disruption of theMSP1gene. As a loss-of-function allele ofArabidopsis MSP1leads to seed abortion, we employed an embryo-rescue method for the molecular characterization ofmsp1mutants. Our data show thatmsp1embryo-development fails to proceed beyond the heart-torpedo transition stage as a consequence of a severe nad1 pre-RNA processing-defect, resulting in the loss of respiratory complex I (CI) activity. The maturation ofnad1involves the processing of three RNA-fragments,nad1.1, nad1.2andnad1.3. Based on biochemical analyses and the mtRNA profiles in wild-type andmsp1plants, we concluded that through its association with a specific site innad1.1, MSP1 facilitates the generation of its 3’-terminus and stabilizes it -a prerequisite fornad1exons a-b splicing. Our data substantiate the importance of mtRNA metabolism for the biogenesis of the respiratory machinery during early-plant development.

The light environment in a mixing water column is arguably the most erratic condition under which photosynthesis functions. Shifts in light intensity, by an order of magnitude, can occur over the time scale of hours. In marine Synechococcus, light is harvested by massive, membrane attached, phycobilisome chromophore-protein complexes (PBS). We examined the ability of a phycobilisome-containing marine Synechococcus strain (WH8102) to acclimate to illumination perturbations on this scale. Although changes in pigment composition occurred gradually over the course of days, we did observe significant and reversible changes in the pigment's fluorescence emission spectra on a time scale of hours. Upon transition to ten-fold higher intensities, we observed a decrease in the energy transferred to Photosystem II. At the same time, the spectral composition of PBS fluorescence emission shifted. Unlike fluorescence quenching mechanisms, this phenomenon resulted in increased fluorescence intensities. These data suggest a mechanism by which marine Synechococcus WH8102 detaches hexamers from the phycobilisome structure. The fluorescence yield of these uncoupled hexamers is high. The detachment process does not require protein synthesis as opposed to reattachment. Hence, the most likely process would be the degradation and resynthesis of labile PBS linker proteins. Experiments with additional species yielded similar results, suggesting that this novel mechanism might be broadly used among PBS-containing organisms.

Mitochondria play key roles in cellular energy metabolism in eukaryotes. Mitochondria of most organisms contain their own genome and specific transcription and translation machineries. The expression of angiosperm mtDNA involves extensive RNA-processing steps, such as RNA trimming, editing, and the splicing of numerous group II-type introns. Pentatricopeptide repeat (PPR) proteins are key players in plant organelle gene expression and RNA metabolism. In the present analysis, we reveal the function of the MITOCHONDRIAL SPLICING FACTOR 2 gene (MISF2, AT3G22670) and show that it encodes a mitochondria-localized PPR protein that is crucial for early embryo development in Arabidopsis. Molecular characterization of embryo-rescued misf2 plantlets indicates that the splicing of nad2 intron 1, and thus respiratory complex I biogenesis, are strongly compromised. Moreover, the molecular function seems conserved between MISF2 protein in Arabidopsis and its orthologous gene (EMP10) in maize, suggesting that the ancestor of MISF2/EMP10 was recruited to function in nad2 processing before the monocot–dicot divergence ~200 million years ago. These data provide new insights into the function of nuclear-encoded factors in mitochondrial gene expression and respiratory chain biogenesis during plant embryo development.

Cyanobacteria of the genus Synechococcus play a key role as primary producers and drivers of the global carbon cycle in temperate and tropical oceans. Synechococcus use phycobilisomes as photosynthetic light-harvesting antennas. These contain phycoerythrin, a pigment-protein complex specialized for absorption of blue light, which penetrates deep into open ocean water. As light declines with depth, Synechococcus photo-acclimate by increasing both the density of photosynthetic membranes and the size of the phycobilisomes. This is achieved with the addition of phycoerythrin units, as demonstrated in laboratory studies. In this study, we probed Synechococcus populations in an oligotrophic water column habitat at increasing depths. We observed morphological changes and indications for an increase in phycobilin content with increasing depth, in summer stratified Synechococcus populations. Such an increase in antenna size is expected to come at the expense of decreased energy transfer efficiency through the antenna, since energy has a longer distance to travel. However, using fluorescence lifetime depth profile measurement approach, which is applied here for the first time, we found that light-harvesting quantum efficiency increased with depth in stratified water column. Calculated phycobilisome fluorescence quantum yields were 3.5% at 70 m and 0.7% at 130 m. Under these conditions, where heat dissipation is expected to be constant, lower fluorescence yields correspond to higher photochemical yields. During winter-mixing conditions, Synechococcus present an intermediate state of light harvesting, suggesting an acclimation of cells to the average light regime through the mixing depth (quantum yield of ~2%). Given this photo-acclimation strategy, the primary productivity attributed to marine Synechococcus should be reconsidered.

The sparsely distributed Limodorum abortivum is a European-Mediterranean orchid species, which grows on decomposing plant material. Although some chlorophyll-pigmentation is observed in the degenerated scales-shaped leaf and stems regions of the plant, its photosynthetic capacity is assumed to be insufficient to support the full energy requirements of an adult plant. In Israel, L. abortivum shows a patchy distribution patterns in the Galilee, Golan, Carmel and Judean regions. To gain more insights into the physiology and photosynthetic activity of L. abortivum, we analyzed the organellar morphologies, photosynthetic activities the chloroplast-DNA sequence by Illumina-HTS. Microscopic analyses indicated to the presence of mature chloroplasts with well-organized grana-thylakoids in the leaves and stems of L. abortivum. However, the numbers of chloroplasts per cell and the grana ultrastructure density within the organelles were notably lower than those of model plant species and fully photosynthetically-active orchids. The cpDNA of L. abortivum (154,954 bp) encodes 60 proteins, 34 tRNAs and 4 rRNAs. The coding-regions of 24 genes are interrupted by 26 group-II intron-sequences. While many genes related to photosynthesis (RuBisCo, PSI, PSII and cytochrome b 6 /f subunits) have remained intact in the cpDNA, the majority of the NADH-dehydrogenase (ndh) subunits were either lost or became nonfunctional (i.e. pseudogenized). In agreement with previous reports, the photosynthetic-rates of adult Limodorum plants were found to be very low, further indicating that carbon-assimilation activity is insufficient to support the energy requirements of an adult plant, and may suggest that L. abortivum have adopted nutritional strategies similar to that of mycoheterotrophic orchid species.

Pgr5 proteins play a major direct role in cyclic electron flow paths in plants and eukaryotic phytoplankton. The genomes of many cyanobacterial species code for Pgr5-like proteins but their function is still uncertain. Here, we present evidence that supports a link between the Synechocystis sp. PCC6803 Pgr5-like protein and the regulation of intracellular redox balance. The knockout strain, pgr5KO, did not display substantial phenotypic response under our experimental conditions, confirming results obtained in earlier studies. However, the overexpression strain, pgr5OE, accumulated 2.5-fold more chlorophyll than the wild type and displayed increased content of photosystems matching the chlorophyll increase. As a result, electron transfer rates through the photosynthetic apparatus of pgr5OE increased, as did the amount of energy stored as glycogen. While, under photoautotrophic conditions, this metabolic difference had only minor effects, under mixotrophic conditions, pgr5OE cultures collapsed. Interestingly, this specific phenotype of pgr5OE mutants displayed a tendency for reverting, and cultures which previously collapsed in the presence of glucose were now able to survive. DNA sequencing of a pgr5OE strain revealed a second site suppression mutation in slr1916, a putative esterase associated with redox regulation. The phenotype of the slr1916 knockout is very similar to that of the strain reported here and to that of the pmgA regulator knockout. These data demonstrate that, in Synechocystis 6803, there is strong selection against overexpression of the Pgr5-like protein. The pseudoreversion event in a gene involved in redox regulation suggests a connection of the Pgr5-like protein to this network.

Plants produce a myriad of specialized (secondary) metabolites that are highly diverse chemically, and exhibit distinct biological functions. Here, we focus on meta-tyrosine (m-tyrosine), a non-proteinogenic byproduct that is often formed by a direct oxidation of phenylalanine (Phe). Some plant species (e.g., Euphorbia myrsinites and Festuca rubra) produce and accumulate high levels of m-tyrosine in their root-tips via enzymatic pathways. Upon its release to soil, the Phe-analog, m-tyrosine, affects early post-germination development (i.e., altered root development, cotyledon or leaf chlorosis, and retarded growth) of nearby plant life. However, the molecular basis of m-tyrosine-mediated (phyto)toxicity remains, to date, insufficiently understood and are still awaiting their functional characterization. It is anticipated that upon its uptake, m-tyrosine impairs key metabolic processes, or affects essential cellular activities in the plant. Here, we provide evidences that the phytotoxic effects of m-tyrosine involve two distinct molecular pathways. These include reduced steady state levels of several amino acids, and in particularly altered biosynthesis of the phenylalanine (Phe), an essential α-amino acid, which is also required for the folding and activities of proteins. In addition, proteomic studies indicate that m-tyrosine is misincorporated in place of Phe, mainly into the plant organellar proteomes. These data are supported by analyses of adt mutants, which are affected in Phe-metabolism, as well as of var2 mutants, which lack FtsH2, a major component of the chloroplast FtsH proteolytic machinery, which show higher sensitivity to m-tyrosine. Plants treated with m-tyrosine show organellar biogenesis defects, reduced respiration and photosynthetic activities and growth and developmental defect phenotypes.

Photosynthetic microorganisms encounter an erratic nutrient environment characterized by periods of iron limitation and sufficiency. Surviving in such an environment requires mechanisms for handling these transitions. Our study identified a regulatory system involved in the process of recovery from iron limitation in cyanobacteria. We set out to study the role of bacterioferritin co-migratory proteins during transitions in iron bioavailability in the cyanobacterium Synechocystis sp. PCC 6803 using knockout strains coupled with physiological and biochemical measurements. One of the mutants displayed slow recovery from iron limitation. However, we discovered that the cause of the phenotype was not the intended knockout but rather the serendipitous selection of a mutation in an unrelated locus, slr1658. Bioinformatics analysis suggested similarities to two-component systems and a possible regulatory role. Transcriptomic analysis of the recovery from iron limitation showed that the slr1658 mutation had an extensive effect on the expression of genes encoding regulatory proteins, proteins involved in the remodeling and degradation of the photosynthetic apparatus and proteins modulating electron transport. Most significantly, expression of the cyanobacterial homologue of the cyclic electron transport protein PGR5 was upregulated 1000-fold in slr1658 disruption mutants. pgr5 transcripts in the Δslr1658 mutant retained these high levels under a range of stress and recovery conditions. The results suggest that slr1658 is part of a regulatory operon that, among other aspects, affects the regulation of alternative electron flow. Disruption of its function has deleterious results under oxidative stress promoting conditions.

Photosynthetic light harvesting is the first step in harnessing sunlight toward biological productivity. To operate efficiently under a broad and dynamic range of environmental conditions, organisms must tune the harvesting process according to the available irradiance. The marine cyanobacteria Synechococcus WH8102 species is well-adapted to vertical mixing of the water column. By studying its responses to different light regimes, we identify a new photo-acclimation strategy. Under low light, the phycobilisome (PBS) is bigger, with extended rods, increasing the absorption cross-section. In contrast to what was reported in vascular plants and predicted by Forster resonance energy transfer (FRET) calculations, these longer rods transfer energy faster than in the phycobilisomes of cells acclimated to a higher light intensity. Comparison of cultures grown under different blue light intensities, using fluorescence lifetime and emission spectra dependence on temperature at the range of 4-200 K in vivo, indicates that the improved transfer arises from enhanced energetic coupling between the antenna rods' pigments. We suggest two physical models according to which the enhanced coupling strength results either from additional coupled pathways formed by rearranging rod packing or from the coupling becoming non-classical. In both cases, the energy transfer would be more efficient than standard one-dimensional FRET process. These findings suggest that coupling control can be a major factor in photosynthetic antenna acclimation to different light conditions.

Summary A pathogen's ability to engage host receptors is a critical determinant of its host range and interspecies transmissibility, key issues for understanding emerging diseases. However, the identification of host receptors, which are also attractive drug targets, remains a major challenge. Our structural bioinformatics studies reveal that both bacterial and viral pathogens have evolved to structurally mimic native host ligands (ligand mimicry), thus enabling engagement of their cognate host receptors. In contrast to the structural homology, amino acid sequence similarity between pathogen molecules and the mimicked host ligands was low. We illustrate the utility of this concept to identify pathogen receptors by delineating receptor tyrosine kinase Axl as a candidate receptor for the polyomavirus SV40. The SV40-Axl interaction was validated, and its participation in the infection process was verified. Our results suggest that ligand mimicry is widespread, and we present a quick tool to screen for pathogen-host receptor interactions.

Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic Delta psbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in Delta psbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in Delta psbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in Delta psbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher Q(B)/Q(-)(B) ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the Q(A)/Q(B) sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the Q(B) site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II.

We have designed a site-specific drug colloidal carrier ultimately for improving pancreatic and lung cancer treatment. It is based on a nanoparticulate drug delivery system that targets tumors overexpressing H-ferritin. A monoclonal antibody, AMB8LK, specifically recognizing H-ferritin was thiolated and conjugated to maleimide-activated polylactide nanoparticles (NPs) resulting in the formation of immunonanoparticles (immunoNPs). The AMB8LK immunoNPs exhibited a mean diameter size of 112 ± 20 nm and a density of 76 antibody molecules per NP. AMB8LK immunoNPs were evaluated for uptake and binding properties on CAPAN-1 and A-549 cell lines, using confocal microscopy. ImmunoNPs demonstrated specific binding and increased uptake of the desired cells by means of monoclonal antibodies (MAbs), compared to nonconjugated NPs. A lipophilic paclitaxel derivative, paclitaxel palmitate (pcpl), was encapsulated within the various NP formulations, and their cytotoxic effect was evaluated on A-549 cells using MTT assay. Pcpl-loaded AMB8LK immunoNPs showed a significantly increased cytotoxic effect when compared to pcpl solution and pcpl NPs. Surface plasmon resonance (SPR) was used to determine quantitatively the affinity constants of native AMB8LK and AMB8LK immunoNPs to gain insight on the affinity of the MAbs following the conjugation process onto NPs. The results of the association/dissociation and affinity kinetics of the interaction between H-ferritin and native AMB8LK or AMB8LK immunoNPs revealed similar constant values, showing that the conjugation process of the MAb to the NPs did not alter the intrinsic specificity and affinity of the MAb to the antigen. In conclusion, at the cellular level, AMB8LK immunoNPs may carry drugs to desired overexpressing antigen cells with adequate affinity properties, potentially leading to improved drug therapy and reduced systemic adverse effects.

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the gene mutated in GNE myopathy. In an attempt to elucidate GNE functions that could account for the muscle pathophysiology of this disorder, the interaction of GNE with α-actinins has been investigated. Surface plasmon resonance and microscale thermophoresis analysis revealed, that in vitro, GNE interacts with α-actinin 2, and that this interaction has a 10-fold higher affinity compared to the GNE-α-actinin 1 interaction. Further, GNE carrying the M743T mutation, the most frequent mutation in GNE myopathy, has a 10-fold lower binding affinity to α-actinin 2 than intact GNE. It is possible that this decrease eventually affects the interaction, thus causing functional imbalance of this complex in skeletal muscle that could contribute to the myopathy phenotype. In vivo, using bi-molecular fluorescent complementation, we show the specific binding of the two proteins inside the intact cell, in a unique interaction pattern between the two partners. This interaction is disrupted in the absence of the C-terminal calmodulin-like domain of α-actinin 2, which is altered in α-actinin 1. Moreover, the binding of GNE to α-actinin 2 prevents additional binding of α-actinin 1 but not vice versa. These results suggest that the interaction between GNE and α-actinin 1 and α-actinin 2 occur at different sites in the α-actinin molecules and that for α-actinin 2 the interaction site is located at the C-terminus of the protein.

Reversible phosphorylation of chl a/b protein complex II (LHCII), the mobile light-harvesting antenna, regulates its association and energy transfer/dissipation to photosystem (PS) II or I (state transition). Excitation of LHCII induces conformational changes affecting the exposure of the phosphorylation site at the N-terminal domain to protein kinase(s) [Zer, H., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282; Zer, H., et al. (2003) Biochemistry 42, 728-738]. Thus, it was of interest to examine whether the pigment composition of LHCII affects the light-induced modulation of LHCII phosphorylation and state transition. To this end, we have used thylakoids of wild-type Chlamydomonas reinhardtii and xanthophyll deficient mutants npq1, lor1, npq2, npq1 lor1, and npq2 lor1. Phosphorylated protein bands P11, P13, and P17 are considered components of the mobile C. reinhardtii LHCII complex. The protein composition of these bands has been analyzed by mass spectrometry using Qtof-2 with a nanospray attachment. P11 and P13 contain C. reinhardtii light-harvesting chlorophyll a/b binding protein LhcII type I. P17 contains C. reinhardtii LhcII types III and IV. Illumination of isolated thylakoids inhibits the redox-controlled phosphorylation of polypeptide bands P13 and P17 and to a lower extent that of P11. The light-induced inhibition of LHCII phosphorylation and the state transition process are not influenced by extensive differences in the xanthophyll composition of the mutants. Thus, LHCII can be visualized as possessing two functionally distinct, independent domains: (i) the pigment binding transmembrane domain regulating the extent of energy transfer/dissipation and (ii) the surface-exposed phosphorylation site regulating the association of LHCII with PSII or PSI.

Photoinhibition is a state of physiological stress that occurs in all oxygen evolving photosynthetic organisms exposed to light. The primary damage occurs within the reaction center of Photosystem II (PS II). While irreversible photoinduced damage to PS II occurs at all light intensities, the efficiency of photosynthetic electron transfer decreases markedly only when the rate of damage exceeds the rate of its repair, which requires de novo PS II protein synthesis. Photoinhibition has been studied for over a century using a large variety of biochemical, biophysical and genetic methodologies. The discovery of the light induced turnover of a protein, encoded by the plastid psbA gene (the D1 protein), later identified as one of the photochemical reaction center II proteins, has led to the elucidation of the underlying mechanism of photoinhibition and to a deeper understanding of the PS II ‘life cycle.’

Iron and manganese are part of a small group of transition metals required for photosynthetic electron transport. Here, we present evidence for a functional link between iron and manganese homeostasis. In the unicellular cyanobacterium, Synechocystis sp. PCC 6803, Fe and Mn deprivation resulted in distinct modifications of the physiological status. The effect on growth and photosynthetic activity under Fe limitation were more severe than those observed under Mn limitation. Moreover, the intracellular elemental quotas of Fe and Mn were found to be linked. Fe limitation reduced the intracellular Mn quota. Mn limitation did not exert a reciprocal effect on Fe quotas. Microarray analysis comparing Mn and Fe limitation revealed a stark difference in the extent of the transcriptional response to the two limiting conditions, reflective of the physiological responses. The effects of Fe limitation on the transcriptional network are widespread while the effects on Mn limitation are highly specific. Our analysis also revealed an overlap in the transcriptional response of specific Fe and Mn transporters. This overlap provides a framework for explaining Fe limitation induced changes in Mn quotas.

Barrier to autointegration factor (BAF) is an essential mobile protein that binds lamins, LEM-domain proteins, histones, and DNA. Under environmental stress, BAF becomes immobile. This phenomenon is not shared with other chromatin-binding proteins. The ability of BAF mutants to be immobilized by heat shock in gut cells correlated with normal or increased affinity for emerin., Barrier to autointegration factor (BAF) is an essential component of the nuclear lamina that binds lamins, LEM-domain proteins, histones, and DNA. Under normal conditions, BAF protein is highly mobile when assayed by fluorescence recovery after photobleaching and fluorescence loss in photobleaching. We report that Caenorhabditis elegans BAF-1 mobility is regulated by caloric restriction, food deprivation, and heat shock. This was not a general response of chromatin-associated proteins, as food deprivation did not affect the mobility of heterochromatin protein HPL-1 or HPL-2. Heat shock also increased the level of BAF-1 Ser-4 phosphorylation. By using missense mutations that affect BAF-1 binding to different partners we find that, overall, the ability of BAF-1 mutants to be immobilized by heat shock in intestinal cells correlated with normal or increased affinity for emerin in vitro. These results show BAF-1 localization and mobility at the nuclear lamina are regulated by stress and unexpectedly reveal BAF-1 immobilization as a specific response to caloric restriction in C. elegans intestinal cells.

Light induces conformational changes in the CP43 chl-a-protein antenna complex in isolated PS II core-complexes exposing phosphorylation site(s) to PS II core-associated protein kinase(s), to added ...

Light-dependent activation of thylakoid protein phosphorylation regulates the energy distribution between photosystems I and II of oxygen-evolving photosynthetic eukaryotes as well as the turnover of photosystem II proteins. So far the only known effect of light on the phosphorylation process is the redox-dependent regulation of the membrane-bound protein kinase(s) activity via plastoquinol bound to the cytochrome bf complex and the redox state of thylakoid dithiols. By using a partially purified thylakoid protein kinase and isolated native chlorophyll (chl) a / b light-harvesting complex II (LHCII), as well as recombinant LHCII, we find that illumination of the chl-protein substrate exposes the phosphorylation site to the kinase. Light does not activate the phosphorylation of the LHCII apoprotein nor the recombinant pigment-reconstituted complex lacking the N-terminal domain that contains the phosphothreonine site. The suggested light-induced conformational change exposing the N-terminal domain of LHCII to the kinase is evidenced also by an increase in its accessibility to tryptic cleavage after light exposure. Light activates preferentially the trimeric form of LHCII, and the process is paralleled by chl fluorescence quenching. Both phenomena are slowly reversible in darkness. Light-induced exposure of the LHCII N-terminal domain to the endogenous protein kinase(s) and tryptic cleavage occurs also in thylakoid membranes. These results demonstrate that light may regulate thylakoid protein phosphorylation not only via the signal transduction chain connecting redox reactions to the protein kinase activation, but also by affecting the conformation of the chl-protein substrate.

Thylakoid protein phosphorylation regulates state transition and PSII protein turnover under light-dependent redox control via a signal transduction system. The redox-dependent activation/deactivation of the membrane-bound protein kinase(s), mostly localized in the grana partitions, differs for the various phosphoproteins. Reduction of the plastoquinone pool may be sufficient to activate phosphorylation of few of these proteins. Phosphorylation of LHCII, requires the presence of the cytochrome bf complex in an ‘activating mode’ characterized by the reduction of its high potential path components and ability to interact with a reduced plastoquinol without oxidizing it. Activation and maintenance of this kinase activity is considered to involve alternate interactions with a cytochrome bf in its activating mode and with the substrate PSII(LHCII). The segregation of the thylakoid components into grana and stroma partitions appears to be mandatory for the kinase activation process. The protein substrate specificity and kinetics differs for various kinases. The thylakoid redox-controlled kinase(s) have not yet been isolated. Preparations highly enriched in kinase activity capable to phosphorylate LHCII and PSII core proteins, contain two kinase active bands, resolved by denaturing electrophoresis and renaturation, and having apparent molecular masses of about 53 and 66 kDa. The roughly estimated abundance of these putative kinase(s) in the grana partitions may be compatible with a ratio of kinase(s): PSII(LHCII) dimers:cytochrome bf dimers in the range of 1:60:30 and a ratio of kinase:phosphorylation sites of about 1:2000. Only about 10–20% of these sites are phosphorylated during state transition. The low turnover rate of the LHCII kinase(s) (< 5) may be due to hindrance of the required random lateral migration within the grana domain rich in tightly packed PSII(LHCII) and cytochrome bf complexes.

The effect of 3-(3,4-dichlorophenyl)-1,1 -dimethyl urea (diuron) binding at the secondary quinone (QB) binding site of reaction center II (RCII), on the high-light-induced degradation of the RCII proteins D1 and D2, and the core proteins CP43 and CP47 was investigated in vivo in Chlamydomonas reinhardtii. The degradation of the RCII-D2 and the CP43 proteins shows a short lag relative to that of the RCII-D1 protein. Diuron retards but does not prevent the degradation of RCII-D1, D2 and CP43 proteins. The degradation of the CP47 protein is not retarded by diuron. The RCII-D1 protein present in cells photoinactivated in the presence of diuron is subsequently degraded in cells transferred to low light or to darkness. The protein can be replaced (turnover) at least partially under both conditions. The RCII-D1 protein is not degraded during photoinactivation of a cytochrome-bf-defective mutant. Degradation occurs however when the cells are returned to low light permitting slow reoxidation of plastoquinol [Zer, H., Prasil, O. & Ohad, I. (1994) J. Biol. Chem. 269, 17670–17676]. Addition of diuron does not prevent the degradation of the protein at this stage. Tryptic digestion of the RCII-D1 protein is partially inhibited by diuron in isolated thylakoids [Trebst, A., Depka, B., Kraft, B. & Johanningmeier, U. (1988) Photosynth. Res. 18, 163–177] but not in thylakoids obtained from photoinactivated cells. We conclude that photoinactivation induces a series of sequential changes in RCII exposing the cleavage site of the RCII-D1 protein to degradation and abolishing the regulatory role of the QB site occupancy by plastoquinone or analog ligands on the cleavage process. The degradation of the RCII-D2 and CP43 proteins may be a secondary process following modification and/or loss of the RCII-D1 protein.

Paraquat, a widely used herbicide, is photoreduced by photosystem I to the monovalent cation radical, which in turn, can react quickly and efficiently with molecular oxygen to produce superoxide anion radicals. In the presence of redox-active iron (or copper) superoxide radicals can serve as a source for the more active species such as hydroxyl radicals. The present sludv investigated the possible mediatory role of iron in paraquat to xicity. The results demonstrate that desferrioxamme (0–150μM) a highiy specific iron chelator, reduces the loss of proteins (by 34–69%) and lipid peroxidation (by 31–96%) in paraquat treated leaf cuts. Dcsferrioxamine also protects malate dehydrogenase (61–70%) hydroxvpyruvate reductase (54–100%), and Ca2+-dependent ATPase (25–34%) against the paraquat-induced loss of their activity. It also induces an increase in glutathione reductase activity (by 188%). These results, together with those from other experiments concerning the effect of desferrioxamine on paraquat uptake by the leaf cuts, suggest that the protection by desferrioxamine arises from its specific iron chelanon properties, and lead to the conclusion that nan-protein-bound and redoxactive forms of iron pluy a role in the manifestation of paraquat toxicity in plants.

The light-induced turnover of the D1 protein subunit of reaction center II (RCII) was investigated in Chlamydomonas reinhardtii y-1 (control) and D6, AC208, and B4 mutants lacking cytochrome b6/f, plastocyanin or photosystem I activity, respectively, and, thus, impaired in light-dependent plastoquinol (PQH2) oxidation. Charge recombination assayed by thermoluminescence measurements indicated similar RCII properties in control and mutant cells. The D1 protein is not degraded in the mutants during photoinactivation; however, RCII-D1 is irreversibly altered, and the protein is degraded when the cells are incubated in low light permitting slow reoxidation of the PQH2 pool. Photoinactivation precedes D1 degradation also in the control cells. Thus, in vivo under physiological conditions photoinactivation and "tagging" of RCII-D1 are resolved from the degradation process. RCII activity in photoinactivated cells may be recovered only following D1 degradation and replacement. Recovery may occur either in the light or dark in the absence of de novo chlorophyll synthesis. The degradation of the photoinactivated RCII-D1 protein is a prerequisite for the synthesis and stable integration of new D1 indicating that tagged D1 is still assembled in the inactive reaction centers. The physiological implication of these results is that oxidation of the PQH2 pool in photoinactivated cells affects RCII-D1 protein degradation and replacement, and, thus, D1 turnover in vivo is regulated by the turnover of PQ at the binding site of the secondary stable electron acceptor quinone of RCII.

The nectary of Rosmarinus officinalis L. has the form of a four-lobed, asymmetrical disc situated around the base of the ovary. The nectary lobe facing the lower flower lip is enlarged and is the only one to have modified stomata. Vascular strands consisting of phloem only occur in the nectariferous tissue. It is suggested that the pre-nectar originating in the phloem accumulates primarily as starch grains in plastids of the nectariferous cells. The number of grains is very large before anthesis and decreases considerably at anthesis. The transport of the pre-nectar to the various nectariferous cells appears to be mainly via the symplast

The effects of paraquat (PQ) on Pisum sativum L. proteins were investigated in vivo in a new experimental system utilizing 10-day-old plant cuts.A marked decrease in the specific activity of membrane-bound Ca2+-dependent ATPase was recorded, while that of Mg2+-dependent ATPase remained unchanged. Concurrently with a drop in the total plant protein, the specific activities of the three cytoplasmic enzymes, malate dehydrogenase, hydroxypyruvate reductase and triose-phosphate isomerase, were also found to decrease. The effect on various enzymes involved in cellular defense mechanisms was also studied: glutathione reductase and superoxide dismutase activities increased, while ascorbate peroxidase was not affected. These findings shed light on the selectivity of PQ-induced injurious processes, focusing on protein homeostasis mechanisms in the membrane and cytoplasmic compartments at the cellular level, as well as on the prominent role played by enzymatic defense systems against PQ poisoning.

NKp30 is a natural cytotoxicity receptor expressed by human NK cells and involved in NK lytic activity. We previously published that membranal heparan sulfate serves as a coligand for human NKp30. In the present study, we complement our results by showing direct binding of recombinant NKp30 to immobilized heparin. The heparan sulfate epitope(s) on target tumor cells and the heparin epitope(s) recognized by NKp30 share similar characteristics. Warren and colleagues (Warren HS, Jones AL, Freeman C, Bettadapura J, Parish CR. 2005. Evidence that the cellular ligand for the human NK cell activation receptor NKp30 is not a heparan sulfate glycosaminoglycan. J Immunol. 175:207-212) published that NKp30 does not bind to membranal heparan sulfate on target cells and that heparan sulfate is not involved in NKp30-mediated lysis. In the current study, we examine the binding of six different recombinant NKp30s to membranal heparan sulfate and conclude that NKp30 does interact with membranal heparan sulfate. Yet, two of the six recombinant NKp30s, including the commercially available recombinant NKp30 (employed by Warren et al.) did not show heparan sulfate-dependent binding. We demonstrate that this is due to an altered glycosylation of these two recombinant NKp30s. Upon removal of its N-linked glycans, heparan sulfate-dependent binding to tumor cells and direct binding to heparin were restored. Overall, our results emphasize the importance of proper glycosylation for analysis of NKp30 binding to its ligand and that membranal heparan sulfate could serve as a coligand for NKp30. At the cellular level, soluble heparan sulfate enhanced the secretion of IFNgamma by NK-92 natural killer cells activated with anti-NKp30 monoclonal antibody. We discuss the involvement of heparan sulfate binding to NKp30 in NKp30-mediated activation of NK cells.

Thylakoid membrane proteins are phosphorylated by different enzymes, which are subject to different control mechanisms. Activation of the light harvesting complex (LHCII) kinase is signaled by the redox state of plastoquinone and the cytochrome b/f complex and modulated by the thiol reduction state. Phosphorylation of Photosystem II (PS II) proteins may involve kinase(s) associated with the PS II core complex that do not involve the cytochrome b/f complex. Exposure of the phosphoprotein phosphorylation site(s) to protein kinases is regulated by light-induced conformational changes. Thus, thylakoid protein phosphorylation is regulated at both the enzyme and substrate levels. Thylakoid protein dephosphorylation is also under regulatory control, involving interaction between an immunophilin and a membrane-bound phosphatase. The physiological significance of thylakoid protein phosphorylation is not fully understood. Phosphorylation of LHCII is suggested to have a dual role: i) regulation of the LHCII/PSII/PS I interaction, underlying the mechanism of energy transfer balance and ii) prevention of the light-induced aggregation of LHCII or LHCII-PS II complexes. The formation of such macrodomains may affect the dynamics of the thylakoid membrane, which requires unhindered lateral diffusion of integral protein complexes. Phosphorylation of PS II subunits appear to be essential for the repair of photodamage to its reaction center occurring during light stress conditions.

Photoinhibition is a state of physiological stress that occurs in all oxygen evolving photosynthetic organisms exposed to light. The primary damage occurs within the reaction center of Photosystem II (PS II). While irreversible photoinduced damage to PS II occurs at all light intensities, the efficiency of photosynthetic electron transfer decreases markedly only when the rate of damage exceeds the rate of its repair, which requires de novo PS II protein synthesis. Photoinhibition has been studied for over a century using a large variety of biochemical, biophysical and genetic methodologies. The discovery of the light induced turnover of a protein, encoded by the plastid psbA gene (the D1 protein), later identified as one of the photochemical reaction center II proteins, has led to the elucidation of the underlying mechanism of photoinhibition and to a deeper understanding of the PS II 'life cycle.'

Two recent publications concerning the chloroplast membrane-protein phosphorylation and state transition might lead to further progress in the elucidation of the mechanism and role of this process. A thylakoid-bound protein TSP9 is released to the chloroplast matrix upon redox-dependent stepwise phosphorylation of three threonine sites and might signal redox-dependent gene transcription. The state-transition process and phosphorylation of the light-harvesting complex II require the activity of a novel protein kinase Stt7.

Redox-controlled, reversible phosphorylation of the thylakoid light harvesting complex II (LHCII) regulates its association with photosystems (PS) I or II and thus, energy distribution between the two photosystems (state transition). Illumination of solubilized LHCII enhances exposure of the phosphorylation site at its N-terminal domain to protein kinase(s) and tryptic cleavage in vitro [Zer et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282]. Here we report that short illumination (5-10 min, 15-30 micromol m(-2) s(-1)) enhances the accessibility of LHCII phosphorylation site to kinase(s) activity also in isolated thylakoids. However, prolonged illumination or higher light intensities (30 min, 80-800 micromol m(-2) s(-1)) prevent phosphorylation of LHCII in the isolated membranes as well as in vivo, although redox-dependent protein kinase activity persists in the illuminated thylakoids toward exogenous solubilized LHCII. This phenomenon, ascribed to light-induced inaccessibility of the phosphorylation site to the protein kinase(s), affects in a similar way the accessibility of thylakoid LHCII N-terminal domain to tryptic cleavage. The illumination effect is not redox related, decreases linearly with temperature from 25 to 5 degrees C and may be ascribed to light-induced conformational changes in the complex causing lateral aggregation of dephosphorylated LHCII bound to and/or dissociated from PSII. The later state occurs under conditions allowing turnover of the phospho-LHCII phosphate. The light-induced inaccessibility of LHCII to the membrane-bound protein kinase reverses readily in darkness only if induced under LHCII-phosphate turnover conditions. Thus, phosphorylation prevents irreversible light-induced conformational changes in LHCII allowing lateral migration of the complex and the related state transition process.

The photosystem II (PSII) complex of photosynthetic oxygen evolving membranes comprises a number of small proteins whose functions remain unknown. Here we report that the low molecular weight protein encoded by the psbJ gene is an intrinsic component of the PSII complex. Fluorescence kinetics, oxygen flash yield, and thermoluminescence measurements indicate that inactivation of the psbJ gene in Synechocystis 6803 cells and tobacco chloroplasts lowers PSII-mediated oxygen evolution activity and increases the lifetime of the reduced primary acceptor Q(A)(-) (more than a 100-fold in the tobacco DeltapsbJ mutant). The decay of the oxidized S(2,3) states of the oxygen-evolving complex is considerably accelerated, and the oscillations of the Q(B)(-)/S(2,3) recombination with the number of exciting flashes are damped. Thus, PSII can be assembled in the absence of PsbJ. However, the forward electron flow from Q(A)(-) to plastoquinone and back electron flow to the oxidized Mn cluster of the donor side are deregulated in the absence of PsbJ, thereby affecting the efficiency of PSII electron flow following the charge separation process.

In this chapter we briefly present and discuss recent progress in the elucidation of certain physiological and molecular aspects of the cyanobacterial inorganic carbon (Ci)-concentrating mechanism (CCM). The reader is referred to earlier chapters and reviews [1—14] for a comprehensive account of other important aspects, including the acclimation of cyanobacteria to changing CO2 concentration.

Photosynthetic microorganisms acclimate to large changes in the ambient CO2 concentrations. The cells undergo a syndrome of changes in response to the concentration of CO2 experienced during growth [1]. The most prominent aspect of this syndrome but certainly not the only one, is the modulation of the activity of the inorganic carbon (Ci)-concentrating mechanism (CCM) operating in these organisms.

Redox-controlled phosphorylation of thylakoid proteins plays a major role in the PSIl dynamic processes related to light-induced turnover of the reaction center D 1 (D2) proteins and the regulation of energy distribution between the two photosystems [1, 2]. The activation of the redox-controlled protein-kinase is regulated by the interaction of reduced plastoquinone with the quinol oxidation site of the cytochrome bf complex [3]. Despite many efforts to isolate the thylakoid protein kinase(s) and identify its molecular nature [2] the identity of the redox-controlled enzyme(s) is still unknown. Two putative kinase bands of 67 and 53 kDa were identified by renaturation of SDS-PAGE resolved proteins [4] of a crude thylakoid cytochrome bf preparation, that was shown before to be enriched in protein-kinase activity [5, 6]. Here we report the further purification of a solubilised protein kinase highly enriched in activity, that phosphorylates isolated LHCII as well as the CP43 protein of isolated PSII-core preparations.

The distribution of light energy between the two photosystems as well as the light-induced turnover of PSII proteins are regulated by the reversible phosphorylation of LHCII and the PSII-core proteins. The thylakoid protein kinase(s) is activated by a signal transduction system involving the interaction of reduced plastoquinone with the quinol oxidation site of the cytochrome bf complex [1]. Phosphorylation of the mobile pool of LHCII induces dissociation of this antenna from PSII and allows its interaction with the PSI in the stroma exposed membranes (state transition)[21. Dephosphorylation of LHCII by a membrane -bound phosphatase appears to be regulated by a cyclophilinlike protein located in the thylakoid lumen [1, 31. So far, it was assumed that the role of light in the above processes is only to drive the photosynthetic electron flow thus reducing plastoquinone and triggering the signal transduction system resulting in the protein kinase(s) activation. In this work we have used an in vitro reconstituted system consisting of partially purified, solubilized thylakoid protein-kinase(s) and isolated native as well as recombinant LHCII. We demonstrate that light induces conformational changes in the LHCII structure exposing the specific threonine phosphorylation site to the kinase activity, thus suggesting a regulation of protein phosphorylation also at the substrate level.

GNE Myopathy (formerly designated HIBM/DMRV) is a rare neuromuscular recessive disorder caused by missense mutations in GNE, the key enzyme of sialic acid biosynthesis. To date, over 60 different mutations in GNE have been reported to cause the disease worldwide, but a single homozygous missense mutation M712T, is common in the Middle East. The mechanisms leading from GNE mutations to the disease are not yet understood. In an attempt to elucidate the pathophysiological pathway of the disease, we have searched for proteins potentially interacting with GNE. We have previously determined that GNE interacts with α -actinin1, one of the 4 α -actinins isoforms, a family of proteins binding the actin filaments. We studied the interactions of recombinant human GNE protein with other members of the actinin family, in particular α -actinin2 which is located at the Z-disk of the muscle fiber. Using a Surface Plasmon Resonance (SPR)-Biosensor based assay, we found that GNE, in its wild type and M712T mutated forms, also binds α -actinin 2. Furthermore, GNE binds α -actinin1 and α -actinin 2 differently, either at different interaction sites on the GNE protein, or alternatively, by changing the conformational state of GNE. GNE exists in different oligomeric forms, dimers, tetramers and hexamers, which are in a dynamic interplay in the cell. To evaluate the potential effect of common mutations found in GNE myopathy patients, on the oligomeric state of GNE protein, we have investigated GNE–GNE interactions and affinity, and found the highest affinity between wtGNE and wtGNE, the lowest between mutGNE–mutGNE, and intermediate affinity between wt and mutated GNE proteins. These differences could have a direct effect on both the oligomeric state of GNE and on the dynamic interplay between the oligomeric forms of the protein. These studies could contribute new understandings on the mechanism of GNE in health and disease.

The essential mediatory role of copper or iron in the manifestation of paraquat toxicity has been demonstrated (Kohen and Chevion (1985) Free Rad. Res. Commun. 1, 79–88; Korbashi, P. et al. (1986) J. Biol. Chem. 261, 12472–12476). Several liver cell lines, characterized by their resistance to copper, were challenged with paraquat and their cross-resistance to paraquat and copper was studied. Cell growth and survival data showed that copper-resistant cells, containing elevated copper, are more sensitive towards paraquat than wild type cells. Copper-deprived resistant cells did not have this sensitivity. Paraquat was also shown to cause a marked degradation of cellular glutathione in all cell lines. Albeit the fact that the basal glutathione levels are higher in copper-resistant than in wild type cells, there is more paraquat-induced degradation of cellular glutathione (GSH + GSSG) in resistant cells. It is suggested that in copper-resistant cells which contain elevated levels of copper, paraquat-induced cellular injury is potentiated even where glutathione levels are elevated. Additionally, in vitro experiments are presented that support the in vivo findings demonstrating a role for copper in glutathione degradation.

BACKGROUND: Hereditary inclusion body myopathy (HIBM) is a rare neuromuscular disorder caused by mutations in GNE, the key enzyme in the biosynthetic pathway of sialic acid. While the mechanism leading from GNE mutations to the HIBM phenotype is not yet understood, we searched for proteins potentially interacting with GNE, which could give some insights about novel putative biological functions of GNE in muscle. METHODOLOGY/PRINCIPAL FINDINGS: We used a Surface Plasmon Resonance (SPR)-Biosensor based assay to search for potential GNE interactors in anion exchanged fractions of human skeletal muscle primary culture cell lysate. Analysis of the positive fractions by in vitro binding assay revealed alpha-actinin 1 as a potential interactor of GNE. The direct interaction of the two proteins was assessed in vitro by SPR-Biosensor based kinetics analysis and in a cellular environment by a co-immunoprecipitation assay in GNE overexpressing 293T cells. Furthermore, immunohistochemistry on stretched mouse muscle suggest that both GNE and alpha-actinin 1 localize to an overlapping but not identical region of the myofibrillar apparatus centered on the Z line. CONCLUSIONS/SIGNIFICANCE: The interaction of GNE with alpha-actinin 1 might point to its involvement in alpha-actinin mediated processes. In addition these studies illustrate for the first time the expression of the non-muscle form of alpha-actinin, alpha-actinin 1, in mature skeletal muscle tissue, opening novel avenues for its specific function in the sarcomere. Although no significant difference could be detected in the binding kinetics of alpha-actinin 1 with either wild type or mutant GNE in our SPR biosensor based analysis, further investigation is needed to determine whether and how the interaction of GNE with alpha-actinin 1 in skeletal muscle is relevant to the putative muscle-specific function of alpha-actinin 1, and to the muscle-restricted pathology of HIBM.