Detection of subtle abnormalities on chest radiographs after irreversible compression

In both oxidative phosphorylation and photo-phosphorylation, electron flow through a carrier is linked to the generation of ATP. The energy released by electron transport is converted to potential energy forming a proton gradient across the membranes in chloroplasts. The proton gradient can be measured by a pH microelectrode. In this report, pH changes produced by photo-induced proton transport through spinach chloroplast membranes were measured by a glass microelectrode. The effect of 5,5'-dithiobis(2-nitrobenzoate) (DTNB) on the kinetics of proton movement across the thylakoid membranes was studied. The results showed that the rate of proton uptake was reduced with increasing DTNB concentration. The rate of leakage of accumulated protons through thylakoid membranes also decreased. The results support the notion that cysteinyl residue is involved in proton translocation. The inhibition of proton transport would subsequently affect the chemical reactions of the Calvin Cycle that takes place in the stroma which is the soluble compartment surrounding the thylakoid membranes.     

Multiple pathways for protein transport into or across the thylakoid membrane

Many thylakoid proteins are cytosolically synthesized and have to cross the two chloroplast envelope membranes as well as the thylakoid membrane en route to their functional locations. In order to investigate the localization pathways of these proteins, we over-expressed precursor proteins in Escherichia coli and used them in competition studies. Competition was conducted for import into the chloroplast and for transport into or across isolated thylakoids. We also developed a novel in organello method whereby competition for thylakoid transport occurred within intact chloroplasts. Import of all precursors into chloroplasts was similarly inhibited by saturating concentrations of the precursor to the OE23 protein. In contrast, competition for thylakoid transport revealed three distinct precursor specificity groups. Lumen-resident proteins OE23 and OE17 constitute one group, lumenal proteins plastocyanin and OE33 a second, and the membrane protein LHCP a third. The specificity determined by competition correlates with previously determined protein-specific energy requirements for thylakoid transport. Taken together, these results suggest that thylakoid precursor proteins are imported into chloroplasts on a common import apparatus, whereupon they enter one of several precursor-specific thylakoid transport pathways.     

Anxiety and alcohol abuse in patients in treatment for depression

Oxygen transport in thylakoid membranes of spinach chloroplasts (Spinacia oleracea) has been studied by observing the collisions of molecular oxygen with spin labels, using line broadening electron paramagnetic resonance (EPR) spectroscopy. Stearic acid spin labels were used to probe the local oxygen diffusion-concentration product. The free radical moiety was located at various distances from the membrane surface, and collision rates were estimated from linewidths of the EPR spectra measured in the presence and absence of molecular oxygen. The profile of the local oxygen diffusion-concentration product across the membrane determined at 20 degrees C demonstrates that this product, at all membrane locations, is higher than the value measured in water. From the profile of the oxygen diffusion-concentration product, the membrane oxygen permeability coefficient has been estimated using the procedure developed earlier (W.K. Subczynski, J.S. Hyde, A. Kusumi, Proc. Natl. Acad. Sci. USA 86 (1989) 4474-4478). At 20 degrees C, the oxygen permeability coefficient for the lipid portion of the thylakoid membrane was found to be 39.5 cm s-1. This value is 20% higher than the oxygen permeability coefficient of a water layer of the same thickness as the thylakoid membrane. The high permeability coefficient implies that the oxygen concentration difference across the thylakoid membrane generated under the illumination of the leaf by saturating actinic light is negligible, smaller than 1 microM.     

Origin of a chloroplast protein importer

During evolution, chloroplasts have relinquished the majority of their genes to the nucleus. The products of transferred genes are imported into the organelle with the help of an import machinery that is distributed across the inner and outer plastid membranes. The evolutionary origin of this machinery is puzzling because, in the putative predecessors, the cyanobacteria, the outer two membranes, the plasma membrane, and the lipopolysaccharide layer lack a functionally similar protein import system. A 75-kDa protein-conducting channel in the outer envelope of pea chloroplasts, Toc75, shares approximately 22% amino acid identity to a similarly sized protein, designated SynToc75, encoded in the Synechocystis PCC6803 genome. Here we show that SynToc75 is located in the outer membrane (lipopolysaccharide layer) of Synechocystis PCC6803 and that SynToc75 forms a voltage-gated, high conductance channel with a high affinity for polyamines and peptides in reconstituted liposomes. These findings suggest that a component of the chloroplast protein import system, Toc75, was recruited from a preexisting channel-forming protein of the cyanobacterial outer membrane. Furthermore, the presence of a protein in the chloroplastic outer envelope homologous to a cyanobacterial protein provides support for the prokaryotic nature of this chloroplastic membrane.     

Energy-transducing thylakoid membranes remain highly impermeable to ions during protein translocation

We investigated the operation of a posttranslational protein translocation pathway to determine whether ions are excluded from the translocase during protein transport. The membrane capacitance during protein translocation across chloroplast thylakoid membranes was monitored via electric-field-indicating carotenoid electrochromic bandshift measurements. Evidence is presented that shows that the membrane ion conductance is not increased during the complete cycle of binding, transport, and substrate release by the DeltapH-dependent translocase; i.e., the membrane remains ion-tight during protein translocation. We further demonstrate that a synthetic targeting peptide that directs proteins across this membrane does not gate translocation pores. We conclude that protein transport across the thylakoid membrane does not compromise its ability to maintain ion gradients and is, thus, unlikely to affect its functions in energy transduction.     

Analysis of the psbU gene encoding the 12-kDa extrinsic protein of photosystem II and studies on its role by deletion mutagenesis in Synechocystis sp. PCC 6803

The gene encoding the 12-kDa extrinsic protein of photosystem II from Synechocystis sp. PCC 6803 was cloned based on N-terminal sequence of the mature protein. This gene, named psbU, encodes a polypeptide of 131 residues, the first 36 residues of which were absent in the mature protein and thus served as a transit peptide required for its transport into the thylakoid lumen. A psbU gene deletion mutant grew photoautotrophically in normal BG11 medium at almost the same rate as that of the wild type strain. This mutant, however, grew apparently slower than the wild type did upon depletion of Ca2+ or Cl- from the growth medium. Photosystem II oxygen evolution decreased to 81% in the mutant as compared with that in the wild type, and the thermoluminescence B- and Q-bands shifted to higher temperatures accompanied by an increase in the Q-band intensity. These results indicate that the 12-kDa protein is not essential for oxygen evolution but may play a role in optimizing the ion (Ca2+ and Cl-) environment and maintaining a functional structure of the cyanobacterial oxygen-evolving complex. In addition, a double deletion mutant lacking cytochrome c-550 and the 12-kDa protein grew photoautotrophically with a phenotype identical to that of the single deletion mutant of cytochrome c-550. This supports our previous biochemical results that the 12-kDa protein cannot bind to photosystem II in the absence of cytochrome c-550 (Shen, J.-R., and Inoue, Y. (1993) Biochemistry 32, 1825-1832).     

Investigation of the structure of thylakoid membranes (spinach) by means of small-angle neutron scattering

This paper presents experimental data on the determination of the thickness of thylakoid membranes by small-angle neutron scattering. The thylakoids were isolated from spinach chloroplasts. The partial volume of proteins and lipids in the "washed" and "unwashed" membranes was estimated. It is shown that the thickness of thylakoid membranes, measured with this techniques depends on the way the membranes were separated. When isolated thylakoids by the standard method, the membrane thickness amounted to 75 A but if the extracted thylakoids were additionally washed with the isolation medium, the measured thickness was 50 A. In this case a significant decrease in the protein partial volume of the membrane was observed. The obtained results make it possible to explain numerous data on X-ray and small-angle neutron scattering by thylakoid membranes of different origins, proceeding from the assumption that all these membranes have a unified structure and consist of a stable core with a thickness of about 50 A, and layers of peripheral, weakly bound proteins with a thickness which may depends on the nature of the object under investigation and extracting conditions.     

Complementation of Escherichia coli unc mutant strains by chloroplast and cyanobacterial F1-ATPase subunits

The genes encoding the five subunits of the F1 portion of the ATPases from both spinach chloroplasts and the cyanobacterium Synechocystis sp. PCC 6803 were cloned into expression vectors and expressed in Escherichia coli. The recombinant subunits formed inclusion bodies within the cells. Each particular subunit was expressed in the respective unc mutant, each unable to grow on non-fermentable carbon sources. The following subunits restored growth under conditions of oxidative phosphorylation: alpha (both sources, cyanobacterial subunit more than spinach subunit), beta (cyanobacterial subunit only), delta (both spinach and Synechocystis), and epsilon (both sources), whereas no growth was achieved with the gamma subunits from both sources. Despite a high degree of sequence homology the large subunits alpha and beta of spinach and cyanobacterial F1 were not as effective in the substitution of their E. coli counterparts. On the other hand, the two smallest subunits of the E. coli ATPase could be more effectively replaced by their cyanobacterial or chloroplast counterparts, although the sequence identity or even similarity is very low. We attribute these findings to the different roles of these subunits in F1: The large alpha and beta subunits contribute to the catalytic centers of the enzyme, a function rendering them very sensitive to even minor changes. For the smaller delta and epsilon subunits it was sufficient to maintain a certain tertiary structure during evolution, with little emphasis on the conservation of particular amino acids.     

Improved outcome in patients treated with postoperative radiation therapy for pathologic stage I/II endometrial cancer

OEE33, a component of the oxygen-evolving enzyme in chloroplasts, normally resides in the thylakoid lumen. In an attempt to study the fate of mistargeted proteins in chloroplasts, we substituted the bipartite transit peptide of OEE33 with that of CAB7, an integral thylakoid-membrane protein. As a result, when imported into isolated chloroplasts, the chimeric protein protein was targeted to the stroma instead of the thylakoid lumen. Whereas the wild-type OEE33 was totally stable for at least 2 h, the chimeric protein was rapidly degraded, with a half-life of 60 min. Degradation of the chimeric protein was stimulated by ATP supplementation. Degradation could also be observed in lysed chloroplasts, in an ATP-stimulated manner. When lysates were fractionated, the proteolytic activity was found to be associated mainly with the stromal fraction. This activity was very effectively inhibited by all tested inhibitors of serine proteases. Western blot analysis demonstrated that the stromal fraction active in degrading the chimeric OEE33 contains ClpC and ClpP, homologues of the regulatory and proteolytic subunits, respectively, of the bacterial, ATP-dependent, serine-type Clp protease.     

Old and new pathways of protein export in chloroplasts and bacteria

Targeting of chloroplast proteins to the thylakoid membrane is analogous to bacterial secretion, and much of what we know has been learned from secretory mechanisms in Escherichia coli. However, chloroplasts also use a delta pH-dependent pathway to target thylakoid proteins, at least some of which are folded before transport. Previously, this pathway seemed to have no cognate in bacteria, but recent results have shown that the HCF106 gene in maize encodes a component of this pathway and has bacterial homologues. This delta pH-dependent pathway might be an ancient conserved mechanism for protein translocation that evolved before the endosymbiotic origin of plastids and mitochondria.     

Chloroplast SRP54 interacts with a specific subset of thylakoid precursor proteins

Signal recognition particles (SRPs) have been identified in organisms as diverse as mycoplasma and mammals; in several cases these SRPs have been shown to play a key role in protein targeting. In each case the recognition of appropriate targeting signals is mediated by SRP subunits related to the 54-kDa protein of mammalian SRP (SRP54). In this study we have characterized the specificity of 54CP, a chloroplast homologue of SRP54 which is located in the chloroplast stroma. We have used a nascent chain cross-linking approach to detect the interactions of 54CP with heterologous endoplasmic reticulum-targeting signals. 54CP functions as a bona fide signal recognition factor which can discriminate between functional and non-functional targeting signals. Using a range of authentic thylakoid precursor proteins we found that 54CP discriminates between thylakoid-targeting signals, interacting with only a subset of protein precursors. Thus, the light-harvesting chlorophyll a/b-binding protein, cytochrome f, and the Rieske FeS protein all showed strong cross-linking products with 54CP. In contrast, no cross-linking to the 23- and 33-kDa proteins of the oxygen-evolving complex were detected. The selectivity of 54CP correlates with the hydrophobicity of the thylakoid-targeting signal and, in the case of light-harvesting chlorophyll a/b-binding protein, with previously determined transport/integration requirements. We propose that 54CP mediates the targeting of a specific subset of precursors to the thylakoid membrane, i.e. those with particularly hydrophobic signal sequences.     

Studies of Ca2+ binding in spinach photosystem II using 45Ca2+

The Ca(2+)-binding properties of photosystem II were investigated with radioactive 45Ca2+. PS II membranes, isolated from spinach grown on a medium containing 45Ca2+, contained 1.5 Ca2+ per PS II unit. Approximately half of the incorporated radioactivity was lost after incubation for 30 h in nonradioactive buffer. About 1 Ca2+/PS II bound slowly to Ca(2+)-depleted membranes in the presence of the extrinsic 16- and 23-kDa polypeptides in parallel with restoration of oxygen-evolving activity. The binding was heterogeneous with dissociation constants of 60 microM (0.7 Ca2+/PS II) and 1.7 mM (0.3 Ca2+/PS II), respectively, which could reflect different affinities of the dark-stable S-states for Ca2+. The reactivation of oxygen-evolving activity closely followed the binding of Ca2+, showing that a single exchangeable Ca2+ per PS II is sufficient for the water-splitting reaction to function. In PS II, depleted of the 16- and 23-kDa polypeptides, about 0.7 exchangeable Ca2+/PS II binds with a dissociation constant of 26 microM, while 0.3 Ca2+ binds with a much weaker affinity (Kd > 0.5 mM). The rate of binding of Ca2+ in the absence of the two extrinsic polypeptides was significantly higher than with the polypeptides bound. The rate of dissociation of bound Ca2+ in the dark, which had a half-time of about 80 h in intact PS II, increased in the absence of the 16- and 23-kDa polypeptides and showed a further increase after the additional removal of the 33-kDa protein and manganese. The rate of dissociation was also significantly faster in weak light than in the dark regardless of the presence or absence of the 16- and 23-kDa polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sec-independent protein translocation by the maize Hcf106 protein

The bacterial Sec and signal recognition particle (ffh-dependent) protein translocation mechanisms are conserved between prokaryotes and higher plant chloroplasts. A third translocation mechanism in chloroplasts [the proton concentration difference (DeltapH) pathway] was previously thought to be unique. The hcf106 mutation of maize disrupts the localization of proteins transported through this DeltapH pathway in isolated chloroplasts. The Hcf106 gene encodes a receptor-like thylakoid membrane protein, which shows homology to open reading frames from all completely sequenced bacterial genomes, which suggests that the DeltapH pathway has been conserved since the endosymbiotic origin of chloroplasts. Thus, the third protein translocation pathway, of which HCF106 is a component, is found in both bacteria and plants.     

Deletion of the structural gene for the NADH-dehydrogenase subunit 4 of Synechocystis 6803 alters respiratory properties

Chloroplasts and cyanobacteria contain genes encoding polypeptides homologous to some subunits of the mitochondrial respiratory NADH-ubiquinol oxidoreductase complex (NADH dehydrogenase). Nothing is known of the role of the NADH dehydrogenase complex in photosynthesis, respiration, or other functions in chloroplasts, and little is known about the specific roles of the perhaps 42 subunits of this complex in the mitochondrion. Inactivation of a gene for subunit 4 (ndhD-2, ndh4) of this complex in the cyanobacterium Synechocystis 6803 has no effect on photosynthesis, judging from the rate of photoautotrophic growth of mutant cells, but the mutant's respiratory rate is about 6 times greater than that of wild-type cells. Respiratory electron transport activity in cyanobacteria is associated both with photosynthetic thylakoid membranes and with the outer cytoplasmic membrane of the cell. Cytoplasmic membranes of mutant cells have much greater NADH-dependent cytochrome reductase activity than preparations from wild-type cells; this activity remains at wild-type levels in isolated thylakoid membranes. It is suggested that the 56.6-kD product of ndhD-2 is not essential for the activity of a cytoplasmic membrane-bound NADH dehydrogenase but that it regulates the rate of electron flow through the complex, establishing a link between this ndh gene and respiration. The activity of the molecularly distinct thylakoid-bound NADH dehydrogenase is apparently unaffected by the loss of ndhD-2.     

Comparison of cytochrome b-559 content in photosystem II complexes from spinach and Synechocystis species PCC 6803

Cytochrome b-559 is an integral component of photosystem II complexes from both plants and cyanobacteria. However, the number of cytochrome b-559 associated with the photosystem II reaction center has been the subject of controversy. Some studies have concluded that there is one heme equivalent of cytochrome b-559 per reaction center, some studies have found two, and some studies have reported intermediate values. Most of the previous experiments have used only one method to quantitate the antenna size of the preparation. In this study, we compare the cytochrome b-559 content in a cyanobacterial and a plant photosystem II preparation. The plant preparation is derived from spinach, and previous work has shown that it has an antenna size of approximately 100 chlorophylls [MacDonald, G. M., & Barry, B. A. (1992) Biochemistry 31, 9848-9856]. The cyanobacterial preparation is from Synechocystis sp. PCC 6803, and previous work has shown that it has an antenna size of approximately 60 chlorophylls [Noren, G. H., Boerner, R. J., & Barry, B. A. (1991) Biochemistry 30, 3943-3950]. Both preparations are isolated through the use of ion-exchange chromatography, and both preparations are monodisperse in the same nonionic detergent. In our comparative study, we quantitate antenna size by three different methods. Our work shows that, depending on the method used to estimate antenna size, the oxygen-evolving spinach photosystem II preparation contains 0.82-1.0 cytochrome b-559 per reaction center, while the oxygen-evolving cyanobacterial preparation contains 1.5-2.1 cytochrome b-559 per reaction center.     

Purification of a protein phosphatase from chloroplast stroma capable of dephosphorylating the light-harvesting complex-II

A protein phosphatase was purified from the stroma of Pea (Pisum sativum L.) chloroplasts that is capable of dephosphorylating synthetic phosphopeptides. Following chromatographic purification of greater than 400-fold, two-dimensional electrophoresis indicated that the stromal protein phosphatase is a 29-kD protein. A similar molecular size was determined for the protein-phosphatase activity using gel-permeation chromatography, indicating that the stromal protein phosphatase is probably a monomer. The purified enzyme was able to dephosphorylate synthetic phosphopeptides, which mimic the thylakoid light-harvesting complex II (LHC-II) N terminus, as well as LHC-II in thylakoid membranes, but did not dephosphorylate the major 64-kD phosphoprotein in the stroma. The stromal protein phosphatase did not discriminate between dephosphorylation of phosphothreonine and phosphoserine residues in synthetic peptide substrates, providing further evidence that this enzyme is distinct from the protein phosphatase localized in thylakoid membranes. The exact physiological role of the stromal protein phosphatase has yet to be determined, but it may function in the dephosphorylation of LHC-II.     

Substrate-binding lipoprotein of the cyanobacterium Synechococcus sp. strain PCC 7942 involved in the transport of nitrate and nitrite

Of the four genes (nrtABCD) required for active transport of nitrate in the cyanobacterium Synechococcus sp. strain PCC 7942, nrtBCD encode membrane components of an ATP-binding cassette transporter involved in the transport of nitrite as well as of nitrate, whereas nrtA encodes a 45-kDa cytoplasmic membrane protein, the biochemical function of which remains unclear. Characterization of the nrtA deletional mutants showed that the 45-kDa protein is essential for the functioning of the nitrate/nitrite transporter. A truncated NrtA protein lacking the N-terminal 81 amino acids, expressed in Escherichia coli cells as a histidine-tagged soluble protein, was shown to bind nitrate and nitrite with high affinity (Kd = 0.3 microM). Immunoblotting analysis using the antibody against the 45-kDa protein revealed a 48-kDa precursor of the protein, which accumulated in the cyanobacterial cells treated with globomycin, an antibiotic that specifically inhibits cleavage of the signal peptide of lipoprotein precursors. These findings indicated that the nrtA gene product is a nitrate- and nitrite-binding lipoprotein. The N-terminal sequences of putative cyanobacterial substrate-binding proteins suggested that lipoprotein modification of substrate-binding proteins of ATP-binding cassette transporters is common in cyanobacteria.     

Eubacterial origin of nuclear genes for chloroplast and cytosolic glucose-6-phosphate isomerase from spinach: sampling eubacterial gene diversity in eukaryotic chromosomes through symbiosis

Higher plants possess two distinct nuclear-encoded glucose-6-phosphate isomerase (GPI) isoenzymes, a cytosolic enzmye of the Embden-Meyerhof pathway and a chloroplast enzyme essential to storage and mobilization of carbohydrate fixed by the Calvin cycle. We have purified spinach chloroplast GPI to homogeneity, determined amino acid sequences from the active enzyme, and cloned cDNAs for chloroplast and cytosolic GPI isoenzymes from spinach. Sequence comparisons reveal three distantly related families of GPI genes that are non-uniformly distributed among contemporary eubacteria and archaebacteria, suggesting that ancient gene diversity existed for this glycolytic enzyme. Spinach chloroplast GPI is much more similar to its homologue from the cyanobacterium Synechocystis PCC6803 than it is to the enzyme from any other source, providing strong evidence that the gene for chloroplast GPI was acquired by the nucleus via endosymbiotic gene transfer from the cyanobacterial antecedants of chloroplasts. Eukaryotic nuclear genes for cytosolic GPI are more similar to eubacterial than to archaebacterial homologues, suggesting that these too were acquired by eukaryotes from eubacteria, probably during the course of the endosymbiotic origin of mitochondria. Chloroplast and cytosolic GPI provide evidence for a eubacterial origin of yet another component of the eukaryotic glycolytic pathway.     

Purification and properties of a novel chloroplast stromal peptidase. Processing of polyphenol oxidase and other imported precursors

Polyphenol oxidases (PPOs) are nuclear-encoded chloroplast proteins that are targeted to the thylakoid lumen by a bipartite presequence. The N-terminal part of this sequence is removed by a stromal processing peptidase (SPP), and the resulting intermediate is translocated across the thylakoid and processed to the mature protein. A 4800-fold-purified SPP processed a PPO precursor (pPPO) at a site identical to that occurring in organelle. The in vitro product of SPP action on pPPO was further processed and translocated by thylakoids. This SPP processed other precursors but was inactive toward those of light-harvesting chlorophyll binding proteins. The enzyme appeared to be a metalloendopeptidase, like previously reported SPPs. However, it differed in substrate specificity, apparent size, and, most significantly, cleavage site of pPPO. Whereas the processing sites of lumen proteins determined so far were relatively distant from the hydrophobic core of the thylakoid targeting domain, pPPO was cleaved immediately before this domain. Cleavage removed the twin arginine motif characteristic of thylakoid targeting domains of lumen proteins, which are translocated by the DeltapH-dependent pathway. The possible significance of these observations to PPO translocation mechanism is discussed. It is suggested that several SPPs may exist in chloroplasts with preferences for different subsets of precursors.