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     

The PsbP Domain Protein 1 Functions in the Assembly of Lumenal Domains in Photosystem I

Photosystem I (PS I) is a multisubunit membrane protein complex that functions as a light-driven plastocyanin-ferredoxin oxidoreductase. The PsbP domain protein 1 (PPD1; At4g15510) is located in the thylakoid lumen of plant chloroplasts and is essential for photoautotrophy, functioning as a PS I assembly factor. In this work, RNAi was used to suppress PPD1 expression, yielding mutants displaying a range of phenotypes with respect to PS I accumulation and function. These PPD1 RNAi mutants showed a loss of assembled PS I that was correlated with loss of the PPD1 protein. In the most severely affected PPD1 RNAi lines, the accumulated PS I complexes exhibited defects in electron transfer from plastocyanin to the oxidized reaction center P₇₀₀⁺. The defects in PS I assembly in the PPD1 RNAi mutants also had secondary effects with respect to the association of light-harvesting antenna complexes to PS I. Because of the imbalance in photosystem function in the PPD1 RNAi mutants, light-harvesting complex II associated with and acted as an antenna for the PS I complexes. These results provide new evidence for the role of PPD1 in PS I biogenesis, particularly as a factor essential for proper assembly of the lumenal portion of the complex.     

Internal and external membrane proteins of the cyanobacterium, Synechococcus cedrorum

The protein composition and architecture of the photosynthetic membranes from the cyanobacterium, Synechococcus cedrorum, were analyzed with the aid of site-specific labels. Using membranes labeled with ³⁵S, about 50 membrane proteins can be detected by sodium dodecyl sulfate acrylamide gel electrophoresis. Approximately half of the proteins are accessible to modification by the impermeant probe, lactoperoxidase, indicating that they have surface-exposed domains. At least six of these external proteins can be removed by EDTA washing; the correspondence in molecular weights between five of these EDTA-extractable proteins and those of typical chloroplast coupling factor preparations may indicate that they are subunits of a membrane-bound ATPase. The photoactive, lipophilic compound, [¹²⁵I]iodonaphthyl azide, was used to label protein domains in contact with the lipid bilayer. Iodonaphthyl azide modification led to a labeling pattern significantly different from that seen with lactoperoxidase. In particular, proteins in the 13 000–20 000 dalton range that were labeled poorly or not at all by lactoperoxidase were heavily modified by iodonaphthyl azide.Photosystem I and II particles, extracted from the membrane by digitonin treatment, were iodinated by lactoperoxidase after isolation. The PS I particles acted as a relatively tight complex, with most of the proteins remaining inaccessible to surface modification. The PS II particles, on the other hand, responded as a more open structure, with most of the subunits yielding to lactoperoxidase iodination. Similar studies on a highly fluorescent, temperature-sensitive mutant of S. cedrorum revealed a different organization of the PS II complex. This mutant, when grown at 40°C, inserts a 51 kdalton polypeptide in place of a 53 kdalton protein. This protein also replaces the 53 kdalton species in the PS II complex of the mutant after 40°C growth. The structure of this complex is altered in that more sites become accessible to lactoperoxidase. This is particularly true of the 51 kdalton protein, which is barely labeled in wild-type PS II complexes.     

Structure and function of photosystem I: interaction with its soluble electron carriers and external antenna systems

Photosystem I (PS I) is a large membrane protein complex that catalyzes the first step of solar conversion, the light-induced transmembrane electron transfer, and generates reductants for CO2 assimilation. It consists of 12 different proteins and 127 cofactors that perform light capturing and electron transfer. The function of PS I includes inter-protein electron transfer between PS I and smaller soluble electron transfer proteins. The structure of PS I is discussed with respect to the potential docking sites for the soluble electron acceptors, ferredoxin/flavodoxin, at the stromal side and the soluble electron donors, cytochrome c6/plastocyanin, at the luminal side of the PS I complex. Furthermore, the potential interaction sites with the peripheral antenna proteins are discussed.     

Oxidizing side of the cyanobacterial Photosystem I: Mutational analysis of the luminal H loop of the PsaB subunit

PS I core proteins are expected to interact with the electron donor proteins plastocyanin or cytochrome c ₆. To investigate the role of the luminal H loop of PsaB in the assembly and function of the PS I complex, we generated 15 deletion and repetition mutations in the H loop of the PsaB protein from Synechocystis sp. PCC 6803. The mutant strains differed in their photoautotrophic growth. The PS I proteins could not be detected in the membranes of mutants in which the N438–E448, I453–T464, or S500–G512 region was deleted from the PsaB protein, indicating the essential role of these segments in proper folding of the PsaB protein. Mutants with partial or complete deletion of the L469–D496 segment contained the PS I proteins. These results indicate that the regions near the transmembrane helices are more important for the assembly of PsaB than the middle region of the H loop. The L469-D496 segment in the H loop of PsaB is dispensable in the interaction between the PS I complex and the soluble donor proteins. These results suggested that sections of the H loop of PsaB are crucial for the structural integrity of the PsaB protein.     

Negatively charged residues in the H loop of PsaB subunit in Photosystem I from Synechocystis sp. PCC 6803 appear to be responsible for electrostatic repulsions with plastocyanin*

Wild-type plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803 does not form any kinetically detectable transient complex with Photosystem I (PS I) during electron transfer, but the D44R/D47R double mutant of copper protein does [De la Cerda et al. (1997) Biochemistry 36: 10125–10130]. To identify the PS I component that is involved in the complex formation with the D44R/D47R plastocyanin, the kinetic efficiency of several PS I mutants, including a PsaF–PsaJ-less PS I and deletion mutants in the lumenal H and J loops of PsaB, were analyzed by laser flash absorption spectroscopy. The experimental data herein suggest that some of the negative charges at the H loop of PsaB are involved in electrostatic repulsions with mutant plastocyanin. Mutations in the J loop demonstrate that this region of PsaB is also critical. The interaction site of PS I is thus not as defined as first expected but much broader, thereby revealing how complex the evolution of intermolecular electron transfer mechanisms in photosynthesis has been. This revised version was published online in June 2006 with corrections to the Cover Date.     

Structural and functional organization of the peripheral light-harvesting system in Photosystem I

This review centers on the structural and functional organization of the light-harvesting system in the peripheral antenna of Photosystem I (LHC I) and its energy coupling to the Photosystem I (PS I) core antenna network in view of recently available structural models of the eukaryotic Photosystem I–LHC I complex, eukaryotic LHC II complexes and the cyanobacterial Photosystem I core. A structural model based on the 3D homology of Lhca4 with LHC II is used for analysis of the principles of pigment arrangement in the LHC I peripheral antenna, for prediction of the protein ligands for the pigments that are unique for LHC I and for estimates of the excitonic coupling in strongly interacting pigment dimers. The presence of chlorophyll clusters with strong pigment–pigment interactions is a structural feature of PS I, resulting in the characteristic red-shifted fluorescence. Analysis of the interactions between the PS I core antenna and the peripheral antenna leads to the suggestion that the specific function of the red pigments is likely to be determined by their localization with respect to the reaction center. In the PS I core antenna, the Chl clusters with a different magnitude of low energy shift contribute to better spectral overlap of Chls in the reaction center and the Chls of the antenna network, concentrate the excitation around the reaction center and participate in downhill enhancement of energy transfer from LHC II to the PS I core. Chlorophyll clusters forming terminal emitters in LHC I are likely to be involved in photoprotection against excess energy.     

光系统Ⅰ（ＰＳⅠ）的结构与功能研究进展

光系统Ⅰ(PSⅠ)是整合于光合膜上的由多个蛋白亚基组成的色素蛋白复合物,它在光合电子传递链中催化电子从PC经过一系列电子传递体到Fd的传递.近20年特别是近几年来,有关光合作用PSⅠ结构与功能的研究取得了显著的进展,获得了很多具有重要意义的结果.本文综合介绍了近年来在PSⅠ的蛋白亚基组成及其特性、PSⅠ介导的3种电子传递过程、PSⅠ特有的外周捕光色素蛋白复合物系统(LHCⅠ)以及最新的有关PSⅠ4?分辨率的三维晶体结构生物学研究的进展情况,并对未来的研究进行了展望.     

Lipid-transfer proteins in membrane trafficking at the Golgi complex

The Golgi complex (GC) represents the central junction for membrane trafficking. Protein and lipid cargoes continuously move through the GC in both anterograde and retrograde directions, departing to and arriving from diverse destinations within the cell. Nevertheless, the GC is able to maintain its identity and strict compartmentalisation, having a different composition in terms of protein and lipid content compared to other organelles. The discovery of coat protein complexes and the elucidation of their role in sorting cargo proteins into specific transport carriers have provided a partial answer to this phenomenon. However, it is more difficult to understand how relatively small and diffusible molecules like lipids can be concentrated in or excluded from specific subcellular compartments. The discovery of lipid-transfer proteins operating in the secretory pathway and specifically at the GC has shed light on one possible way in which this lipid compartmentalisation can be accomplished. The correct lipid distribution along the secretory pathway is of crucial importance for cargo protein sorting and secretion. This review focuses on what is now known about the putative and effective lipid-transfer proteins at the GC, and on how they affect the function and structure of the GC itself.     

Biomedical applications of protein chips

The development of microchips involving proteins has accelerated within the past few years. Although DNA chip technologies formed the precedent, many different strategies and technologies have been used because proteins are inherently a more complex type of molecule. This review covers the various biomedical applications of protein chips in diagnostics, drug screening and testing, disease monitoring, drug discovery (proteomics), and medical research. The proteomics and drug discovery section is further subdivided to cover drug discovery tools (on-chip separations, expression profiling, and antibody arrays), molecular interactions and signaling pathways, the identification of protein function, and the identification of novel therapeutic compounds. Although largely focused on protein chips, this review includes chips involving cells and tissues as a logical extension of the type of data that can be generated from these microchips.     

Quantifying and monitoring functional photosystem II and the stoichiometry of the two photosystems in leaf segments: approaches and approximations

Given its unique function in light-induced water oxidation and its susceptibility to photoinactivation during photosynthesis, photosystem II (PS II) is often the focus of studies of photosynthetic structure and function, particularly in environmental stress conditions. Here we review four approaches for quantifying or monitoring PS II functionality or the stoichiometry of the two photosystems in leaf segments, scrutinizing the approximations in each approach. (1) Chlorophyll fluorescence parameters are convenient to derive, but the information-rich signal suffers from the localized nature of its detection in leaf tissue. (2) The gross O(2) yield per single-turnover flash in CO(2)-enriched air is a more direct measurement of the functional content, assuming that each functional PS II evolves one O(2) molecule after four flashes. However, the gross O(2) yield per single-turnover flash (multiplied by four) could over-estimate the content of functional PS II if mitochondrial respiration is lower in flash illumination than in darkness. (3) The cumulative delivery of electrons from PS II to P700(+) (oxidized primary donor in PS I) after a flash is added to steady background far-red light is a whole-tissue measurement, such that a single linear correlation with functional PS II applies to leaves of all plant species investigated so far. However, the magnitude obtained in a simple analysis (with the signal normalized to the maximum photo-oxidizable P700 signal), which should equal the ratio of PS II to PS I centers, was too small to match the independently-obtained photosystem stoichiometry. Further, an under-estimation of functional PS II content could occur if some electrons were intercepted before reaching PS I. (4) The electrochromic signal from leaf segments appears to reliably quantify the photosystem stoichiometry, either by progressively photoinactivating PS II or suppressing PS I via photo-oxidation of a known fraction of the P700 with steady far-red light. Together, these approaches have the potential for quantitatively probing PS II in vivo in leaf segments, with prospects for application of the latter two approaches in the field.     

Immunogold localization of light-harvesting and photosystem I complexes in the thylakoids ofFucus serratus (Phaeophyceae)

Summary The repartition of light-harvesting complex (LHC) and photosystem I (PS I) complex has been examined in isolated plastids ofFucus serratus by immunocytochemical labelling. LHC is distributed equally all along the length of thylakoid membranes, without any special repartition in the appressed membranes of the three associated thylakoids ofFucus. PS I is present on all the thylakoid membranes, but the external membranes of the three associated thylakoids are largely enriched relatively to the inner ones. This specific repartition of PSI on non-appressed membranes can be compared to the localization of PSI on stroma thylakoid membranes of higher plants and green algae. Consequently, although they share some common features with those of higher plants and green algae, the appressions of thylakoids in brown algae has neither the same structure nor the same functional role as typical grana stacked membranes in the repartition of the harvested energy.Abbreviations BSA bovine serum albumin - GAR goat anti-rabbit immunoglobulin G - LHC light-harvesting complex - PBS phosphatebuffered saline - PS I photosystem I - PS II photosystem II     

Unusual Properties of Regulatory DNA from the Drosophila Engrailed Gene: Three ``pairing-Sensitive'''' Sites within a 1.6-Kb Region

We have previously shown that a 2-kb fragment of engrailed DNA can suppress expression of a linked marker gene, white, in the P element vector CaSpeR. This suppression is dependent on the presence of two copies of engrailed DNA-containing P elements (P[en]) in proximity in the Drosophila genome (either in cis or in trans). In this study, the 2-kb fragment was dissected and found to contain three fragments of DNA which could mediate white suppression [called ``pairing-sensitive sites' (PS)]. A PS site was also identified in regulatory DNA from the Drosophila escargot gene. The eye colors of six different P[en] insertions in the escargot gene suggest an interaction between P[en]-encoded and genome-encoded PS sites. I hypothesize that white gene expression from P[en] is repressed by the formation of a protein complex which is initiated at the engrailed PS sites and also requires interactions with flanking genomic DNA. Genes were sought which influence the function of PS sites. Mutations in some Polycomb and trithorax group genes were found to affect the eye color from some P[en] insertion sites. However, different mutations affected expression from different P[en] insertion sites and no one mutation was found to affect expression from all P[en] insertion sites examined. These results suggest that white expression from P[en] is not directly regulated by members of the Polycomb and trithorax group genes, but in some cases can be influenced by them. I propose that engrailed PS sites normally act to promote interactions between distantly located engrailed regulatory sites and the engrailed promoter.     

Requirement of PEN-2 for stabilization of the presenilin N-/C-terminal fragment heterodimer within the gamma-secretase complex

Gamma-secretase is a protease complex composed of presenilin (PS), nicastrin (NCT), APH-1, and PEN-2, which catalyzes intramembrane cleavage of several type I transmembrane proteins including the Alzheimer's disease-associated beta-amyloid precursor protein. We generated stable RNA interference-mediated PEN-2 knockdown cells to probe mutant PEN-2 variants for functional activity. Knockdown of PEN-2 was associated with impaired NCT maturation and deficient PS1 endoproteolysis, which was efficiently rescued by wild type or N-terminally tagged PEN-2 but not by C-terminally tagged PEN-2 or by the C-terminally truncated PEN-2-DeltaC mutant. Although the latter mutants rescued the PS1 holoprotein accumulation associated with the PEN-2 knockdown, they failed to restore normal levels of the PS1 N- and C-terminal fragments and to maturate NCT. PEN-2-DeltaC was highly unstable and rapidly turned over by proteasomal degradation consistent with its failure to become stably incorporated into the gamma-secretase complex. In addition, expression of PEN-2-DeltaC caused a selective instability of the PS1 N-/C-terminal fragment heterodimer that underwent proteasomal degradation, whereas NCT and APH-1 were stable. Interestingly, when we knocked down PEN-2 in the background of the endoproteolysis-deficient PS1 Deltaexon9 mutant, immature NCT still accumulated, demonstrating that PEN-2 is also required for gamma-secretase complex maturation when PS endoproteolysis cannot occur. Taken together, our data suggest that PEN-2 is required for the stabilization of the PS fragment heterodimer within the gamma-secretase complex following PS endoproteolysis. This function critically depends on the PEN-2 C terminus. Moreover, our data show that PEN-2 is generally required for gamma-secretase complex maturation independent of its activity in PS1 endoproteolysis.     

Is the 9 kDa thylakoid membrane phosphoprotein functionally and structurally analogous to the 'H' subunit of bacterial reaction centres?

Although the amino acid sequence of the 9 kDa (phospho)protein of chloroplasts has been determined, the function of this thylakoid membrane protein in photosynthetic electron transport and the reason for its physiological control remains unclear. In this paper, I briefly review the evidence which indicates that the phosphorylation of the 9 kDa protein results in a partial inhibition of photosynthetic oxygen evolution by increasing the stability of the semiquinone bound to QA the primary, plastoquinone-binding site of photosystem II (PS II). I propose that in its dephosphorylated state, the 9 kDa thylakoid membrane protein may serve PS II to ensure efficient photochemical charge separation by aiding the transfer of reducing equivalents out of the reaction centre to the attendant plastoquinone pool. This function is analogous to that proposed for the H-subunit of the reaction centre of photosynthetic eubacteria. Whether these two proteins have evolved from a common ancestral reaction centre protein is discussed in the light of a comparison of their amino acid sequences and predicted secondary structures.     

The detection,isolation and characterization of a light-harvesting complex which is specifically associated with Photosystem I

10% of the chlorophyll associated with a ‘native’ Photosystem (PS) I complex (110 chlorophylls/P-700) is chlorophyll (Chl) b. The Chl b is associated with a specific PS I antenna complex which we designate as LHC-I (i.e., a light-harvesting complex serving PS I). When the native PS I complex is degraded to the core complex by LHC-I extraction, there is a parallel loss of Chl b, fluorescence at 735 nm, together with 647 and 686 nm circular dichroism spectral properties, as well as a group of polypeptides of 24-19 kDa. In this paper we present a method by which the LHC-I complex can be dissociated from the native PS I. The isolated LHC-I contains significant amounts of Chl b (Chl $\text{a}\text{b ? 3.7}$). The long-wavelength fluorescence at 730 nm and circular dichroism signal at 686 nm observed in native PS I are maintained in this isolated complex. This isolated fraction also contains the low molecular weight polypeptides lost in the preparation of PS I core complex. We conclude that we have isolated the PS I antenna in an intact state and discuss its in vivo function.     

Some speculations concerning the evolution of photosynthetic function

Following a brief review of the light-driven reactions in photosynthetic membranes, two questions are addressed. (1) Why is the first charge separation reaction in photosynthetic reaction centers so fast; and (2) given what we know about the contemporary structure and function of reaction centers, can we develop a simple model for a much more primitive reaction center? It is proposed that the primary charge separation step in reaction centers is optimized to be ultra-fast principally in order to compete with detrapping into the antenna complex, rather than to compete with radiative and non-radiative losses in the special pair. This leads to a notion of kinetic perfection analogous to that developed for enzymes which operate under diffusion-limited conditions, but elaborated to permit even more ‘perfect’ function. This hypothesis is testable by changing components in photosynthetic membranes and subjecting them to selective pressures. We speculate that the reaction center is far too complex to have served as an early functional unit, and consider possible roles for the iron-quinone part of the reaction center as a very primitive photosynthetic unit. It is suggested that this working end later became associated with primitive antenna complexes, which then evolved into the elaborate structure we find today. The role of photosynthesis in the origin of life has been a topic of speculation for many years. It is evident that photosynthetic function is ancient and central. As a person who does not work in the field of evolution. I am not very familiar with much of the speculation that precedes this paper. Proposals and speculation by others are likely based on much firmer ground, and therefore I apologize in advance if some of these ideas have already been suggested by others. In this chapter I take the liberty to speculate on how a structure as complex as the contemporary photosynthetic reaction center (RC) could have evolved from more primitive units, and why it retains some of its remarkable properties and seemingly unnecessary components. Both subjects lead to specific predictions and testable hypotheses.     

Two-dimensional gel electrophoresis of the membrane-bound protein complexes, including photosystem I, of thylakoid membranes in the presence of sodium oligooxyethylene alkyl ether sulfate/dimethyl dodecylamine oxide and sodium dodecyl sulfate

Two-dimensional polyacrylamide gel electrophoresis (PAGE), using a mixture of sodium oligooxyethylene alkyl ether sulfate and dimethyl dodecylamine oxide as detergents (AES-DDAO mixture) in the first dimension and sodium dodecyl sulfate (SDS) in the second dimension, was developed and applied to an analysis of the photosystem I (PS I) complex in thylakoid membranes prepared from spinach chloroplasts. When thylakoid membranes of chloroplasts were solubilized directly in the AES-DDAO mixture and subjected to PAGE in the presence of these detergents as the first dimension, some protein complexes containing chlorophyll were observed. The protein components in these complexes separated into an array of polypeptide spots when the strip of gel after PAGE in the first dimension was subjected to PAGE in the presence of SDS as the second dimension. The main band of protein which separated in the first dimension was demonstrated to be the PS I complex. This complex retained the intrinsic photochemical activity of P700 even after it was subjected to one-dimensional PAGE. These results suggest that certain protein complexes can be separated, with the maintenance of their original structures, by electrophoresis in the presence of the AES-DDAO mixture, and this method appears to have valuable potential for analysis of the components of membrane-bound protein complexes.     

Regulation of chloroplast membrane function: protein phosphorylation changes the spatial organization of membrane components

A chlorophyll-protein complex of chloroplast membranes, which simultaneously serves as light-harvesting antenna and membrane adhesion factor, undergoes reversible, lateral diffusion between appressed and nonappressed membrane regions under the control of a protein kinase. The phosphorylation-dependent migration process regulates the amount of light energy that is delivered to the reaction centers of photosystems I and II (PS I and PS II), and thereby regulates their rate of turnover. This regulatory mechanism provides a rationale for the finding that the two photosystems are physically separated in chloroplast membranes (PS II in appressed, grana membranes, and PS I in nonappressed, stroma membranes). The feedback system involves the following steps: a membrane-bound kinase senses the rate of PS II vs. PS I turnover via the oxidation-reduction state of the plastoquinone pool, which shuttles electrons from PS II via cytochrome f to PS I. If activated, the kinase adds negative charge (phosphate) to a grana- localized pigment-protein complex. The change in its surface charge at a site critical for promoting membrane adhesion results in increased electrostatic repulsion between the membranes, unstacking, the lateral movement of the complex to adjacent stroma membranes, which differ in their functional composition. The general significance of this type of membrane regulatory mechanism is discussed.