Regulatory factors for the assembly of thylakoid membrane protein complexes

Major multi-protein photosynthetic complexes, located in thylakoid membranes, are responsible for the capture of light and its conversion into chemical energy in oxygenic photosynthetic organisms. Although the structures and functions of these photosynthetic complexes have been explored, the molecular mechanisms underlying their assembly remain elusive. In this review, we summarize current knowledge of the regulatory components involved in the assembly of thylakoid membrane protein complexes in photosynthetic organisms. Many of the known regulatory factors are conserved between prokaryotes and eukaryotes, whereas others appear to be newly evolved or to have expanded predominantly in eukaryotes. Their specific features and fundamental differences in cyanobacteria, green algae and land plants are discussed.     

Genetic engineering of thylakoid protein complexes by chloroplast transformation in Chlamydomonas reinhardtii

Chloroplast transformation of Chlamydomonas reinhardtii has developed into a powerful tool for studying the structure, function and assembly of thylakoid protein complexes in a eukaryotic organism. In this article we review the progress that is being made in the development of procedures for efficient chloroplast transformation. This focuses on the development of selectable markers and the use of Chlamydomonas mutants, individually lacking thylakoid protein complexes, as recipients. Chloroplast transformation has now been used to engineer all four major thylakoid protein complexes, photosystem II, photosystem I, cytochrome b ₆/f and ATP synthase. These results are discussed with an emphasis on new insights into assembly and function of these complexes in chloroplasts as compared with their prokaryotic counterparts.Abbreviations ENDOR electron nuclear double resonance - ESEEM electron spin echo envelope modulation - LHC light harvesting complex - PSI Photosystem I - PS II Photosystem II - P₆₈₀ primary electron donor in PS II - P₇₀₀ primary electron donor in PS I     

Understanding protein translocation across chloroplast membranes: Translocons and motor proteins

Subcellular organelles in eukaryotes are surrounded by lipid membranes.In an endomembrane system,vesicle trafficking is the primary mechanism for the delivery of organellar proteins to specific organelles.However,organellar proteins for chloroplasts,mitochondria,the nucleus,and peroxisomes that are translated in the cytosol are directly imported into their target organelles.Chloroplasts are a plant-specific organelle with outer and inner envelope membranes,a dual-membrane structure that is simil...     

Role of Reactive Oxygen Species in the Initiation of Plant Retrograde Signaling

The interaction between the nucleus and the different organelles is important in the physiology of the plant. Reactive oxygen species (ROS) are a by-product of the oxidation of organic molecules to obtain energy by the need to carry out the electron transfer between the different enzymatic complexes. However, they also have a role in the generation of what is known as retrograde signaling. This signal comes from the different organelles in which the oxidation of molecules or the electron transference is taking place such as mitochondria and chloroplasts. Furthermore, ROS can also induce the release of signals from the apoplast. It seems that these signals plays a role communicating to the nucleus the current status of the different parts of the plant cell to induce a changes in gene expression. In this review, the molecular mechanism of ROS retrograde signaling is described.

     

Orientation of pigments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. I. Determination of optimal conditions for linear dichroism measurement

The nature and possible causes of polarized light-scattering artefacts in linear dichroism measurements are investigated. Using criteria described in this article, the available orientation techniques have been critically assessed in order to obtain the linear dichroism spectra of thylakoids and of pigment-protein complexes isolated from pea. It is demonstrated here that the polyacrylamide gel squeezing technique of Abdourakhmanov et al. (Abdourakhmanov, I.A., Ganago, A.O., Erokhim, Yu.E., Solov'ev, A.A. and Chugunov, V.A. (1979) Biochim. Biophys. Acta 546, 183–186) does not lead to pigment degradation and that the linear dichroism spectra obtained in these conditions are essentially free of scattering artefacts. The linear dichroism spectra of light-harvesting complex isolated in different states of aggregation or incorporated into phospholipid vesicles are compared to the spectra of thylakoids. This comparison indicates: (1) that the isolation procedure of Burke et al. (Burke, J.J., Ditto, C.L. and Arntzen, C.J. (1978) Arch. Biochem. Biophys. 187, 252–263) leads to light-harvesting complex in which the in vivo orientation of pigments is preserved; (2) that the antenna chlorophyll a molecules of this complex have a significant degree of orientation with respect to the plane of the thylakoid.     

Inclusion complexes of N-sulfamoyloxazolidinones with beta-cyclodextrin

A study of inclusion complexes of six N-sulfamoyloxazolidinone derivatives with beta-cyclodextrin is described. The inclusion complexes were prepared in solution and in solid state with stoichiometry host-guest 1:1, and characterized. In solution, the complexation was carried out by spectrophotometric measurements at 25 degrees C. The stoichiometries and stability constants of complexes at various pHs have been determined using second-derivative spectrophotometry UV-vis. Hydrophobic properties of N-sulfamoyloxazolidinones are improved following their inclusion into beta-CD.     

Function of 3′ non-coding sequences and stop codon usage in expression of the chloroplast psaB gene in Chlamydomonas reinhardtii

The rate of mRNA decay is an important step in the control of gene expression in prokaryotes, eukaryotes and cellular organelles. Factors that determine the rate of mRNA decay in chloroplasts are not well understood. Chloroplast mRNAs typically contain an inverted repeat sequence within the 3 untranslated region that can potentially fold into a stem-loop structure. These stem-loop structures have been suggested to stabilize the mRNA by preventing degradation by exonuclease activity, although such a function in vivo has not been clearly established. Secondary structures within the translation reading frame may also determine the inherent stability of an mRNA. To test the function of the inverted repeat structures in chloroplast mRNA stability mutants were constructed in the psaB gene that eliminated the 3 flanking sequences of psaB or extended the open reading frame into the 3 inverted repeat. The mutant psaB genes were introduced into the chloroplast genome of Chlamydomonas reinhardtii. Mutants lacking the 3 stem-loop exhibited a 75% reduction in the level of psaB mRNA. The accumulation of photosystem I complexes was also decreased by a corresponding amount indicating that the mRNA level is limiting to PsaB protein synthesis. Pulse-chase labeling of the mRNA showed that the decay rate of the psaB mRNA was significantly increased demonstrating that the stem-loop structure is required for psaB mRNA stability. When the translation reading frame was extended into the 3 inverted repeat the mRNA level was reduced to only 2% of wild-type indicating that ribosome interaction with stem-loop structures destabilizes chloroplast mRNAs. The non-photosynthetic phenotype of the mutant with an extended reading frame allowed us to test whether infrequently used stop codons (UAG and UGA) can terminate translation in vivo. Both UAG and UGA are able to effectively terminate PsaB synthesis although UGA is never used in any of the Chlamydomonas chloroplast genes that have been sequenced.     

Apomissia in “Ochna Serrulata„ Walp

Structural and functional characteristics of the photosynthetic apparatus in 15-day old Brassica rapa plants grown aboard the space shuttle Columbia (STS-87) have been studied. Maintaining of the same growth conditions for control plants was realized using the Orbiter Environmental Simulator in Kennedy Space Center. The main differences in spaceflight plants in comparison with control ones have been shown to be the following. An average volume of one mesophyll palisade cell increased approximately twice and the chloroplast number per cell by 69.8%. Partial volumes of stromal thylakoids, starch grains and plastoglobuli also increased by 19.4%, 20.6% and 2 times accordingly. At the same time, the grana number per chloroplast decreased. Greater diversity of the thylakoid length in grana and a decrease in thylakoid membrane stacking were revealed. A decrease of PSII and PSI light-harvesting antennae has been detected, for PSII by an increase of Chl a/b ratio and kinetics delay in chlorophyll fluorescence induction, and for PSI by a decrease of integral intensity in the excitation spectrum of fluorescence at 735?nm, which indicated a decline of PSI absorption cross-section. Some distortion of PSI complexes have been displayed by fluorescence spectra. A slight decrease in PSII photochemistry yield was detected for the spaceflight material. PSI is concluded to be more susceptible to the microgravity conditions.     

Identification and characterization of mixed disulphide complexes of E apoprotein in high density lipoprotein of subjects with acute alcoholic hepatitis.

Two E apoprotein complexes have been isolated from the plasma high density lipoprotein fraction of patients with alcoholic hepatitis. Both were mixed disulphide complexes and could be dissociated into subunits with β-mercaptoethanol but not with 1% sodium dodecyl sulphate or 8 M urea. One of the complexes (molecular weight 46,000) was identified as an E-AII unit and the other, of molecular weight 106,000 was consistent in properties with an E trimer. The latter has not been described before and the E-AII complex has been reported once previously in plasma of patients with Type III hyperlipo-proteinaemia. It is proposed that the presence of E complexes in abnormal disease states may affect the normal recognition of E monomer by cells thereby altering the subsequent fate of their host lipoproteins and their lipid constituents.     

Proposals for the naming of chloroplast genes. II. Update to the nomenclature of genes for thylakoid membrane polypeptides

The nomenclature for genes for components of the photosynthetic membranes has been reviewed and updated. Newly discovered genes have been added to the existing convention for gene nomenclature. Genes designatedpetA throughpetI are described for components of the photosynthetic electron transport systems,psaA throughpsaK for photosystem I components, andpsbA throughpsbR for photosystem II, including the extrinsic polypeptides of the oxygen-evolving complex. References for representative examples of each gene are given.     

Synthesis and assembly of a full‐length human monoclonal antibody in algal chloroplasts

Monoclonal antibodies can be effective therapeutics against a variety of human diseases, but currently marketed antibody‐based drugs are very expensive compared to other therapeutic options. Here, we show that the eukaryotic green algae Chlamydomonas reinhardtii is capable of synthesizing and assembling a full‐length IgG1 human monoclonal antibody (mAb) in transgenic chloroplasts. This antibody, 83K7C, is derived from a human IgG1 directed against anthrax protective antigen 83 (PA83), and has been shown to block the effects of anthrax toxin in animal models. Here we show that 83K7C heavy and light chain proteins expressed in the chloroplast accumulate as soluble proteins that assemble into complexes containing two heavy and two light chain proteins. The algal‐expressed 83K7C binds PA83 in vitro with similar affinity to the mammalian‐expressed 83K7C antibody. In addition, a second human IgG1 and a mouse IgG1 were also expressed and shown to properly assemble in algal chloroplast. These results show that chloroplasts have the ability to fold and assemble full‐length human mAbs, and suggest the potential of algae as a platform for the cost effective production of complex human therapeutic proteins. Biotechnol. Bioeng. 2009; 104: 663–673 © 2009 Wiley Periodicals, Inc.     

Chromatin-modifying and -remodeling complexes.

Nucleosomes have long been known to inhibit DNA transactions on chromosomes and a remarkable abundance of multiprotein complexes that either enhance or relieve this inhibition have been described. Most is known about chromatin-remodeling complexes that perturb nucleosome structure.     

Genetic analyses of adaptin function from yeast to mammals

Adaptor protein (AP) complexes are heterotetrameric assemblies of subunits named adaptins. Four AP complexes, termed AP-1, AP-2, AP-3, and AP-4, have been described in various eukaryotic organisms. Biochemical and morphological evidence indicates that AP complexes play roles in the formation of vesicular transport intermediates and the selection of cargo molecules for inclusion into these intermediates. This understanding is being expanded by the application of genetic interference procedures. Here, we review recent progress in the genetic analysis of the function of AP complexes, focusing on studies that make use of targeted interference or naturally-occurring mutations in various model organisms.     

Features of Grana Organization in Pea Chloroplasts

Two fractions of membrane fragments—the pellets precipitated at 1300 and 20000 g (fractions G1.3 and G20, respectively)—were isolated from pea (Pisum sativum L.) chloroplasts after solubilization with digitonin. These fragments assigned to grana displayed the following differences: (1) in spectra of low-temperature fluorescence, the ratio of short-wave and long-wave band intensities, as well as integrated intensity of the whole spectrum, were higher for G1.3 than for G20 fraction; (2) in excitation spectra of long-wave fluorescence, the ratio of peaks at 650 and 680 nm and integrated intensity of the spectrum were higher for G1.3 than for G20 fraction; and (3) the shapes of fluorescence excitation spectra differed for G1.3 and G20. These results indicate that the two fractions examined differed in proportion of photosystem I and photosystem II complexes, as well as in organization of these complexes. The size of light-harvesting antenna was larger in PSI complexes of G1.3 fraction, owing, in particular, to a higher content of chlorophyll a/b-protein complexes in this fraction. After repeated digitonin fragmentation of G1.3 and G20 preparations, more than 80% of G1.3 fraction was decomposed into lighter fragments, whereas G20 fraction was resistant to fragmentation (it lost about 10% of its material). Analysis of the data suggests the presence of two structurally different types of thylakoids in grana. The yield of G20 fraction (about 20%) is comparable to the ratio between the number of intergranal thylakoids, connected to granum in pea chloroplasts, and the total number of thylakoids in this granum. Based on these data, we assume that G20 fraction represent the fragments of intergranal thylakoids that extend into the granum.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 499–506.Original Russian Text Copyright © 2005 by Kochubei, Shevchenko, Bondarenko.     

Analogs of Eu3+ DOTAM-Gly-Phe-OH and Tm3+ DOTAM-Gly-Lys-OH: synthesis and magnetic properties of potential PARACEST MRI contrast agents

Chelated lanthanide ions, especially gadolinium, have found wide use as contrast agents in magnetic resonance imaging. A new paradigm for generating contrast, termed PARACEST, was recently described that requires the slow exchange of water or other exchangeable protons present in the ligand framework. In previous work, we have described a synthetic method for the preparation of dipeptide conjugates of DOTAM for use as PARACEST agents. Two compounds possessed interesting magnetic properties: the Eu(3+) complex of DOTAM-Gly-Phe-OH and the Tm(3+) complex of DOTAM-Gly-Lys-OH. To understand the relationship between the structure of these complexes and their magnetic properties, we have expanded our synthetic methodology and prepared several new complexes. Ligands have been prepared in which the terminal phenylalanine moieties have been replaced with tryptophan or tyrosine, the distance to the amino acid residue possessing an alpha-substituent has been changed, or phenylalanine and lysine have been combined in the peptide sequence. The preparation of lanthanide(III) complexes of these ligands has been achieved and their PARACEST properties have been determined.