Distribution of the extrinsic proteins as a potential marker for the evolution of photosynthetic oxygen-evolving photosystem II

Distribution of photosystem II (PSII) extrinsic proteins was examined using antibodies raised against various extrinsic proteins from different sources. The results showed that a glaucophyte (Cyanophora paradoxa) having the most primitive plastids contained the cyanobacterial-type extrinsic proteins (PsbO, PsbV, PsbU), and the primitive red algae (Cyanidium caldarium) contained the red algal-type extrinsic proteins (PsO, PsbQ', PsbV, PsbU), whereas a prasinophyte (Pyraminonas parkeae), which is one of the most primitive green algae, contained the green algal-type ones (PsbO, PsbP, PsbQ). These suggest that the extrinsic proteins had been diverged into cyanobacterial-, red algal- and green algal-types during early phases of evolution after a primary endosymbiosis. This study also showed that a haptophyte, diatoms and brown algae, which resulted from red algal secondary endosymbiosis, contained the red algal-type, whereas Euglena gracilis resulted from green algal secondary endosymbiosis contained the green algal-type extrinsic proteins, suggesting that the red algal- and green algal-type extrinsic proteins have been retained unchanged in the different lines of organisms following the secondary endosymbiosis. Based on these immunological analyses, together with the current genome data, the evolution of photosynthetic oxygen-evolving PSII was discussed from a view of distribution of the extrinsic proteins, and a new model for the evolution of the PSII extrinsic proteins was proposed.     

Glycinebetaine stabilizes the association of extrinsic proteins with the photosynthetic oxygen-evolving complex.

The photosynthetic oxygen-evolving activity of the photosystem 2 complex, prepared from spinach, was labile when the complex was exposed to high-salt conditions under which the extrinsic proteins were dissociated from the complex. Glycinebetaine prevented the dissociation of the 18-kDa and the 23-kDa extrinsic proteins from the photosystem 2 complex in the presence of 1 M NaCl. It also prevented the dissociation of the 33-kDa extrinsic protein from the complex in the presence of 1 M MgCl2 or 1 M CaCl2. The oxygen-evolving activity of the photosystem 2 complex was stabilized by glycinebetaine when the complex was subjected to treatment with NaCl and MgCl2.     

Integrators of the cytoskeleton that stabilize microtubules.

Sensory neurodegeneration occurs in mice defective in BPAG1, a gene encoding cytoskeletal linker proteins capable of anchoring neuronal intermediate filaments to actin cytoskeleton. While BPAG1 null mice fail to anchor neurofilaments (NFs), BPAG1/NF null mice still degenerate in the absence of NFs. We report a novel neural splice form that lacks the actin-binding domain and instead binds and stabilizes microtubules. This interaction is functionally important; in mice and in vitro, neurons lacking BPAG1 display short, disorganized, and unstable microtubules defective in axonal transport. Ironically, BPAG1 neural isoforms represent microtubule-associated proteins that when absent lead to devastating consequences. Moreover, BPAG1 can functionally account for the extraordinary stability of axonal microtubules necessary for transport over long distances. Its isoforms interconnect all three cytoskeletal networks, a feature apparently central to neuronal survival.     

Manganese-binding proteins of the oxygen-evolving complex

The extrinsic 33-kDa protein (P33) was cross-linked covalently to the binding site on P33-depleted PSII preparations which is responsible for reconstitution of photosynthetic water oxidation after PSII preparations have been washed with 1 M CaCl2. Conditions were found in which more than half of the cross-linked protein complexes formed in the PSII preparations retained the ability to catalyze the oxidation of water. The complex is composed of the P33 cross-linked to the D1 and D2 proteins and a 34-kDa protein, which is present in lower abundance than the other three proteins. After solubilization of the membranes with SDS and purification by preparative SDS-PAGE, the complex retains bound manganese and can catalyze the conversion of H2O2 to O2. Calcium and chloride increased the catalase activity of the purified cross-linked complex while lanthanum or hydroxylamine abolished the activity. By use of the specific activity of the H2O2-dependent reaction to follow the extent of purification of the cross-linked complex, the most highly purified complex was determined to contain 0.34 microgram of manganese/180 micrograms of protein. The mole ratio of Mn/protein was calculated to range from 3.6 to 4.5 depending on the assumed stoichiometry of the protein subunits. The results presented here provide direct evidence that one or more of the three proteins that have cross-linked to the P33 are responsible for binding the manganese of the oxygen-evolving complex.     

Physiologically active chloroplasts contain pools of unassembled extrinsic proteins of the photosynthetic oxygen-evolving enzyme complex in the thylakoid lumen

The oxygen-evolving complex (OEC) of photosystem II (PS II) consists of at least three extrinsic membrane-associated protein subunits, OE33, OE23, and OE17, with associated Mn2+, Ca2+, and Cl- ions. These subunits are bound to the lumen side of PS II core proteins embedded in the thylakoid membrane. Our experiments reveal that a significant fraction of each subunit is normally present in unassembled pools within the thylakoid lumen. This conclusion was supported by immunological detection of free subunits after freshly isolated pea thylakoids were fractionated with low levels of Triton X-100. Plastocyanin, a soluble lumen protein, was completely released from the lumen by 0.04% Triton X-100. This gentle detergent treatment also caused the release from the thylakoids of between 10 and 20%, 40 and 60%, and 15 and 50% of OE33, OE23, and OE17, respectively. Measurements of the rates of oxygen evolution from Triton-treated thylakoids, both in the presence and absence of Ca2+, and before and after incubation with hydroquinone, demonstrated that the OEC was not dissociated by the detergent treatment. Thylakoids isolated from spinach released similar amounts of extrinsic proteins after Triton treatment. These data demonstrate that physiologically active chloroplasts contain significant pools of unassembled extrinsic OEC polypeptide subunits free in the lumen of the thylakoids.     

Structure and evolution of calcium-modulated proteins

This review suggests that the intracellular functions of calcium are best understood in terms of calcium's functioning as a second messenger. Further, when functioning as a second messenger, calcium completes its mission not by transferring charge nor by binding to lipid but by binding to specific targets, calcium-modulated proteins. This concept is broadly interpreted to include proteins involved in calcium transport. There is strong evidence that many, if not all, of these calcium-modulated proteins are homologs. Their structures and properties are contrasted to those of extracellular calcium-binding proteins which are not homologous to one another or to the intracellular calcium-modulated proteins. Finally, this line of thought leads to a suggestion of the evolutionary reason for the choice of calcium as the sole inorganic second messenger.     

Structure and evolution of the actin crosslinking proteins

The actin crosslinking proteins exhibit marked diversity in size and shape and crosslink actin filaments in different ways. Amino acid sequence analysis of many of these proteins has provided clues to the origin of their diversity. Spectrin, alpha-actinin, ABP-120, ABP-280, fimbrin, and dystrophin share a homologous sequence segment that is implicated as the common actin binding domain. The remainder of each protein consists of repetitive and non-repetitive sequence segments that have been shuffled and multiplied in evolution to produce a variety of proteins that are related in function and in composition, but that differ significantly in structure.     

Protection of the oxygen-evolving machinery by the extrinsic proteins of photosystem II is essential for development of cellular thermotolerance in Synechocystis sp. PCC 6803

The oxygen-evolving machinery of photosystem II in cyanobacteria is associated with three extrinsic proteins: the manganese-stabilizing protein, cytochrome c(550), and PsbU. To elucidate the effect of the presence of these extrinsic proteins on the stabilization of the oxygen-evolving machinery against high-temperature stress, we inactivated the genes for these proteins individually in Synechocystis sp. PCC 6803 by targeted mutagenesis. The thermal stability of the oxygen-evolving machinery decreased in all mutated cells but the extent of the susceptibility to heat inactivation varied between the photosystems lacking the different extrinsic proteins. Cells that lacked either the manganese-stabilizing protein or cytochrome c(550) were unable to enhance the thermal stability of the oxygen-evolving machinery and, moreover, failed to increase cellular thermotolerance when grown at moderately high temperatures. Our findings indicate that the three extrinsic proteins stabilize the oxygen-evolving machinery independently against high-temperature stress and that the thermal stability of the machinery influences cellular thermotolerance.     

The extreme halophyte Salicornia veneta is depleted of the extrinsic PsbQ and PsbP proteins of the oxygen-evolving complex without loss of functional activity

Background and Aims

Photosystem II of oxygenic organisms is a multi-subunit protein complex made up of at least 20 subunits and requires Ca²⁺ and Cl⁻ as essential co-factors. While most subunits form the catalytic core responsible for water oxidation, PsbO, PsbP and PsbQ form an extrinsic domain exposed to the luminal side of the membrane. In vitro studies have shown that these subunits have a role in modulating the function of Cl⁻ and Ca²⁺, but their role(s) in vivo remains to be elucidated, as the relationships between ion concentrations and extrinsic polypeptides are not clear. With the aim of understanding these relationships, the photosynthetic apparatus of the extreme halophyte Salicornia veneta has been compared with that of spinach. Compared to glycophytes, halophytes have a different ionic composition, which could be expected to modulate the role of extrinsic polypeptides.

Methods

Structure and function of in vivo and in vitro PSII in S. veneta were investigated and compared to spinach. Light and electron microscopy, oxygen evolution, gel electrophoresis, immunoblotting, DNA sequencing, RT–PCR and time-resolved chlorophyll fluorescence were used.

Key Results

Thylakoids of S. veneta did not contain PsbQ protein and its mRNA was absent. When compared to spinach, PsbP was partly depleted (30 %), as was its mRNA. All other thylakoid subunits were present in similar amounts in both species. PSII electron transfer was not affected. Fluorescence was strongly quenched upon irradiation of plants with high light, and relaxed only after prolonged dark incubation. Quenching of fluorescence was not linked to degradation of D1 protein.

Conclusions

In S. veneta the PsbQ protein is not necessary for photosynthesis in vivo. As the amount of PsbP is sub-stoichiometric with other PSII subunits, this protein too is largely dispensable from a catalytic standpoint. One possibility is that PsbP acts as an assembly factor for PSII.Key words: Photosystem II, PsbQ, PsbP, halophytes, Salicornia veneta     

Inhibition of the oxygen-evolving complex of photosystem II and depletion of extrinsic polypeptides by nickel

The toxic effect of Ni²⁺ on photosynthetic electron transport was studied in a photosystem II submembrane fraction. It was shown that Ni²⁺ strongly inhibits oxygen evolution in the millimolar range of concentration. The inhibition was insensitive to NaCl but significantly decreased in the presence of CaCl₂. Maximal chlorophyll fluorescence, together with variable fluorescence, maximal quantum yield of photosystem II, and flash-induced fluorescence decays were all significantly declined by Ni²⁺. Further, the extrinsic polypeptides of 16 and 24 kDa associated with the oxygen-evolving complex of photosystem II were depleted following Ni²⁺ treatment. It was deduced that interaction of Ni²⁺ with these polypeptides caused a conformational change that induced their release together with Ca²⁺ from the oxygen-evolving complex of photosystem II with consequent inhibition of the electron transport activity.     

Structure, function and evolution of multidomain proteins

Proteins are composed of evolutionary units called domains; the majority of proteins consist of at least two domains. These domains and nature of their interactions determine the function of the protein. The roles that combinations of domains play in the formation of the protein repertoire have been found by analysis of domain assignments to genome sequences. Additional findings on the geometry of domains have been gained from examination of three-dimensional protein structures. Future work will require a domain-centric functional classification scheme and efforts to determine structures of domain combinations.     

Binding and functional properties of the extrinsic proteins in oxygen-evolving photosystem II particle from a green alga,Chlamydomonas reinhardtii having his-tagged CP47

Oxygen-evolving photosystem II (PSII) particles were purified from Chlamydomonas reinhardtii having His-tag extension at the C terminus of the CP47 protein, by a single-step Ni(2+)-affinity column chromatography after solubilization of thylakoid membranes with sucrose monolaurate. The PSII particles consisted of, in addition to intrinsic proteins, three extrinsic proteins of 33, 23 and 17 kDa. The preparation showed a high oxygen-evolving activity of 2,300-2,500 micro mol O(2) (mg Chl)(-1) h(-1) in the presence of Ca(2+) using ferricyanide as the electron acceptor, while its activity was 680-720 micro mol O(2) (mg Chl)(-1) h(-1) in the absence of Ca(2+) and Cl(-) ions. The activity was 710-820 micro mol O(2) (mg Chl)(-1) h(-1) independent of the presence or absence of Ca(2+) and Cl(-) when 2,6-dichloro-p-benzoquinone was used as the acceptor. These activities were scarcely inhibited by DCMU. The kinetics of flash-induced fluorescence decay revealed that the electron transfer from Q(A)(-) to Q(B) was significantly inhibited, and the electron transfer from Q(A)(-) to ferricyanide was largely stimulated in the presence of Ca(2+). These results indicate that the acceptor side, Q(B) site, was altered in the PSII particles but its donor side remained intact. Release-reconstitution experiments revealed that the extrinsic 23 and 17 kDa proteins were released only partially by NaCl-wash, while most of the three extrinsic proteins were removed when treated with urea/NaCl, alkaline Tris or CaCl(2). The 23 and 17 kDa proteins directly bound to PSII independent of the other extrinsic proteins, and the 33 kDa protein functionally re-bound to CaCl(2)-treated PSII which had been reconstituted with the 23 and 17 kDa proteins. These binding properties were largely different from those of the extrinsic proteins in higher plant PSII, and suggest that each of the three extrinsic proteins has their own binding sites independent of the others in the green algal PSII.     

Structural characterization of photosystem II complex from red alga Porphyridium cruentum retaining extrinsic subunits of the oxygen-evolving complex.

The structure of photosystem II (PSII) complex isolated from thylakoid membranes of the red alga Porphyridium cruentum was investigated using electron microscopy followed by single particle image analysis. The dimeric complexes observed contain all major PSII subunits (CP47, CP43, D1 and D2 proteins) as well as the extrinsic proteins (33 kDa, 12 kDa and the cytochrome c(550)) of the oxygen-evolving complex (OEC) of PSII, encoded by the psbO, psbU and psbV genes, respectively. The single particle analysis of the top-view projections revealed the PSII complex to have maximal dimensions of 22 x 15 nm. The analysis of the side-view projections shows a maximal thickness of the PSII complex of about 9 nm including the densities on the lumenal surface that has been attributed to the proteins of the OEC complex. These results clearly demonstrate that the red algal PSII complex is structurally very similar to that of cyanobacteria and to the PSII core complex of higher plants. In addition, the arrangement of the OEC proteins on the lumenal surface of the PSII complex is consistent to that obtained by X-ray crystallography of cyanobacterial PSII.     

Ribosomal proteins: Structure,function, and evolution

The question concerning reasons for the variety of ribosomal proteins that arose for more than 40 years ago is still open. Ribosomes of modern organisms contain 50–80 individual proteins. Some are characteristic for all domains of life (universal ribosomal proteins), whereas others are specific for bacteria, archaea, or eucaryotes. Extensive information about ribosomal proteins has been obtained since that time. However, the role of the majority of ribosomal proteins in the formation and functioning of the ribosome is still not so clear. Based on recent data of experiments and bioinformatics, this review presents a comprehensive evaluation of structural conservatism of ribosomal proteins from evolutionarily distant organisms. Considering the current knowledge about features of the structural organization of the universal proteins and their intermolecular contacts, a possible role of individual proteins and their structural elements in the formation and functioning of ribosomes is discussed. The structural and functional conservatism of the majority of proteins of this group suggests that they should be present in the ribosome already in the early stages of its evolution.     

Structure of photosystem II and molecular architecture of the oxygen-evolving centre

Photosynthesis utilizes light energy to oxidize water molecules to molecular oxygen at the oxygen-evolving centre of photosystem II. The structure of photosystem II from the cyanobacterium Thermosynechococcus elongatus has been reported at 3.5A resolution and, for the first time, the complete molecular structure of this 650 kDa complex, including the oxygen-evolving centre, has been revealed.     

Structure, function and evolution of the hemagglutinin-esterase proteins of corona- and toroviruses

Virus attachment to host cells is mediated by dedicated virion proteins, which specifically recognize one or, at most, a limited number of cell surface molecules. Receptor binding often involves protein-protein interactions, but carbohydrates may serve as receptor determinants as well. In fact, many different viruses use members of the sialic acid family either as their main receptor or as an initial attachment factor. Sialic acids (Sias) are 9-carbon negatively-charged monosaccharides commonly occurring as terminal residues of glycoconjugates. They come in a large variety and are differentially expressed in cells and tissues. By targeting specific Sia subtypes, viruses achieve host cell selectivity, but only to a certain extent. The Sia of choice might still be abundantly present on non-cell associated molecules, on non-target cells (including cells already infected) and even on virus particles themselves. This poses a hazard, as high-affinity virion binding to any of such “false' receptors would result in loss of infectivity. Some enveloped RNA viruses deal with this problem by encoding virion-associated receptor-destroying enzymes (RDEs). These enzymes make the attachment to Sia reversible, thus providing the virus with an escape ticket. RDEs occur in two types: neuraminidases and sialate-O-acetylesterases. The latter, originally discovered in influenza C virus, are also found in certain nidoviruses, namely in group 2 coronaviruses and in toroviruses, as well as in infectious salmon anemia virus, an orthomyxovirus of teleosts. Here, the structure, function and evolution of viral sialate-O-acetylesterases is reviewed with main focus on the hemagglutinin-esterases of nidoviruses.     

The extrinsic proteins of Photosystem II

Years of genetic, biochemical, and structural work have provided a number of insights into the oxygen evolving complex (OEC) of Photosystem II (PSII) for a variety of photosynthetic organisms. However, questions still remain about the functions and interactions among the various subunits that make up the OEC. After a brief introduction to the individual subunits Psb27, PsbP, PsbQ, PsbR, PsbU, and PsbV, a current picture of the OEC as a whole in cyanobacteria, red algae, green algae, and higher plants will be presented. Additionally, the role that these proteins play in the dynamic life cycle of PSII will be discussed.     

The extrinsic proteins of Photosystem II

In this review we examine the structure and function of the extrinsic proteins of Photosystem II. These proteins include PsbO, present in all oxygenic organisms, the PsbP and PsbQ proteins, which are found in higher plants and eukaryotic algae, and the PsbU, PsbV, CyanoQ, and CyanoP proteins, which are found in the cyanobacteria. These proteins serve to optimize oxygen evolution at physiological calcium and chloride concentrations. They also shield the Mn(4)CaO(5) cluster from exogenous reductants. Numerous biochemical, genetic and structural studies have been used to probe the structure and function of these proteins within the photosystem. We will discuss the most recent proposed functional roles for these components, their structures (as deduced from biochemical and X-ray crystallographic studies) and the locations of their proposed binding domains within the Photosystem II complex. This article is part of a Special Issue entitled: Photosystem II.