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1.
Insertion and assembly of the precursor of subunit II into the photosystem I complex may precede its processing.
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The biogenesis and assembly of subunit II of photosystem I (PSI) (psaD gene product) were studied and characterized. The precursor and the mature form were produced in vitro and incubated with intact plastids or isolated thylakoids. Following import of the precursor into isolated plastids, mostly the mature form of subunit II was found in the thylakoids. However, when the processing activity was inhibited only the precursor form was present in the membranes. The precursor was processed by a stromal peptidase and processing could occur before or after insertion of the precursor into the thylakoids. Following insertion into isolated thylakoids, both the precursor and the mature form of subunit II were confined to the PSI complex. Insertion of the mature form of subunit II was much less efficient than that of the precursor. Kinetic studies showed that the precursor was inserted into the membrane. Only at a later stage, the mature form began to accumulate. These results suggest that in vivo the precursor of subunit II is inserted and embedded in the thylakoids, as part of the PSI complex. Only later, it is processed to the mature form through the action of a stromal peptidase. 相似文献
2.
The PsaD subunit of photosystem I. Mutations in the basic domain reduce the level of PsaD in the membranes. 总被引:2,自引:0,他引:2
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The PsaD subunit of photosystem I (PSI) is a peripheral protein that provides a docking site for ferredoxin and interacts with the PsaB, PsaC, and PsaL subunits of PSI. We used site-directed mutagenesis to determine the function of a basic region in PsaD of the cyanobacterium Synechocystis sp. PCC 6803. We generated five mutant strains in which one or more charged residues were altered. Western blotting showed that replacement of lysine (Lys)-74 with glutamine or glutamic acid led to a substantial decrease in the level of PsaD in the membranes. The mutant PSI complexes showed reduced NADP+ photoreduction activity mediated by ferredoxin; the decrease in activity correlated with the reduced level of PsaD. Using protein synthesis inhibitors we showed that the degradation rates of the mutant and wild-type PsaD were similar, indicating a defect in the assembly of the mutant protein. Treatment of the mutant PSI complexes with a different concentration of NaI showed that the mutations decreased affinity between PsaD and the transmembrane components of PSI. With glutaraldehyde, the mutant and wild-type PsaD proteins could be cross-linked with PsaC, but the PsaD-PsaL cross-linked product was reduced drastically when arginine-72, Lys-74, and Lys-76 were mutated simultaneously. These studies demonstrate that the basic residues in the central region of PsaD, especially Lys-74, are crucial in the assembly of PsaD into the PSI complex. 相似文献
3.
Multiple functions for the C terminus of the PsaD subunit in the cyanobacterial photosystem I complex
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PsaD subunit of Synechocystis sp PCC 6803 photosystem I (PSI) plays a critical role in the stability of the complex and is part of the docking site for ferredoxin (Fd). In the present study we describe major physiological and biochemical effects resulting from mutations in the accessible C-terminal end of the protein. Four basic residues were mutated: R111, K117, K131, and K135, and a large 36-amino acid deletion was generated at the C terminus. PSI from R111C mutant has a 5-fold decreased affinity for Fd, comparable with the effect of the C terminus deletion, and NADP+ is photoreduced with a 2-fold decreased rate, without consequence on cell growth. The K117A mutation has no effect on the affinity for Fd, but decreases the stability of PsaE subunit, a loss of stability also observed in R111C and the deletion mutants. The double mutation K131A/K135A does not change Fd binding and reduction, but decreases the overall stability of PSI and impairs the cell growth at temperatures above 30 degrees C. Three mutants, R111C, K117A, and the C-terminal deleted exhibit a higher content of the trimeric form of PSI, in apparent relation to the removal of solvent accessible positive charges. Various regions in the C terminus of cyanobacterial PsaD thus are involved in Fd strong binding, PSI stability, and accumulation of trimeric PSI. 相似文献
4.
PsaD is a peripheral stromal-facing subunit of photosystem I (PSI), a multisubunit complex of the thylakoid membranes. PsaD plays a major role in both the function and assembly of PSI. Past studies with radiolabeled PsaD indicated that PsaD is able to assemble in vitro specifically into the PSI complex. To unravel the mechanism by which this assembly takes place, the following steps were taken. (i) Mature PsaD of spinach and PsaD of the prokaryotic caynobacterium Mastigocladus laminosus, both bearing a six-histidine tag at their C-termini, were overexpressed in Escherichia coli and purified to homogeneity. (ii) The purified recombinant protein was introduced into the isolated PSI complex. (iii) Following incubation, the PsaD that assembled into PSI was separated from the nonassembled PsaD by a sucrose gradient. Differential Western blot analysis was used to determine whether the native and the recombinant PsaD were present as free or assembled proteins of the PSI complex. Antibodies that can recognize only the recombinant PsaD (anti-his) or both the native and recombinant PsaD (anti-PsaD) were used. The findings indicated that an exchange mechanism enables the assembly of a newly introduced PsaD into PSI. The latter replaces the PsaD subunit that is present in situ within the complex. In vivo studies that followed the assembly of PsaD in Chlamydomonas reinhardtii cells supported this in vitro-characterized exchange mechanism. In C. reinhardtii, in the absence of synthesis and assembly of new PSI complexes, newly synthesized PsaD assembled into pre-existing PSI complexes. 相似文献
5.
This study concerns the assembly into a multisubunit enzyme complex of a small hydrophobic protein imported into isolated mitochondria. Subunit 8 of yeast mitochondrial ATPase (normally a mitochondrial gene product) was expressed in vitro as a chimaeric precursor N9L/Y8-1, which includes an N-terminal-cleavable transit peptide to direct its import into mitochondria. Assembly into the enzyme complex of the imported subunit 8 was monitored by immunoadsorption using an immobilized anti-F1-beta monoclonal antibody. Preliminary experiments showed that N9L/Y8-1 imported into normal rho+ mitochondria, with its complement of fully assembled ATPase, did not lead to an appreciable assembly of the exogenous subunit 8. With the expectation that mitochondria previously depleted of subunit 8 could allow such assembly in vitro, target mitochondria were prepared from genetically modified yeast cells in which synthesis of subunit 8 was specifically blocked. Initially, mitochondria were prepared from strain M31, a mit- mutant completely incapable of intramitochondrial biosynthesis of subunit 8. These mit- mitochondria however were unsuitable for assembly studies because they could not import protein in vitro. A controlled depletion strategy was then evolved. An artificial nuclear gene encoding N9L/Y8-1 was brought under the control of a inducible promoter GAL1. This regulated gene construct, in a low copy number yeast expression vector, was introduced into strain M31 to generate strain YGL-1. Galactose control of the expression of N9L/Y8-1 was demonstrated by the ability of strain YGL-1 to grow vigorously on galactose as a carbon source, and by the inability to utilize ethanol alone for prolonged periods of growth. The measurement of bioenergetic parameters in mitochondria from YGL-1 cells experimentally depleted of subunit 8, by transferring growing cells from galactose to ethanol, was consistent with the presence in mitochondria of a mosaic of ATPase, namely fully assembled functional ATPase complexes and partially assembled complexes with defective F0 sectors. These mitochondria demonstrated very efficient import of N9L/Y8-1 and readily incorporated the imported processed subunit 8 protein into ATPase. Comparison of the kinetics of import and assembly of subunit 8 showed that assembly was noticeably delayed with respect to import. These findings open the way to a new systematic analysis of the assembly of imported proteins into multisubunit mitochondrial enzyme complexes. 相似文献
6.
Photophosphorylation has been reconstituted in a liposomal system containing reaction centers of photosystem I and coupling-factor complex, both highly purified from spinach chloroplasts. This energy-converting model system was put together by diluting the preparation of the coupling-factor complex with an aqueous suspension of proteolipid vesicles, preformed from photosystem-I reaction centers and soybean phospholipids by sonication. In the presence of reduced N-methyl-phenazonium methosulfate the system catalyzed photophosphorylation with rates up to 50 mumol ATP formed x mg chlorophyll-1 x h-1, which was sensitive to uncouplers and to N,N'-dicyclohexyl-carbodiimide. The properties of the system in comparison to chloroplasts is discussed. 相似文献
7.
Photosystem I of higher plants functions in photosynthesis as a light-driven oxidoreductase producing reduced ferredoxin. Its peripheral subunit PsaD has been identified as the docking site for ferredoxin I. With the aim of elucidating the structure-function relationship and the role of this subunit, a recombinant form of the spinach protein was produced by heterologous expression in Escherichia coli. The PsaD protein was synthesized in soluble form and purified to homogeneity. The interaction of the PsaD subunit with ferredoxin I was investigated using three different approaches: chemical cross-linking between the two purified proteins in solution, affinity chromatography of the PsaD subunit on a ferredoxin-coupled resin, and titration with ferredoxin of the protein fluorescence of the subunit. All these studies indicated that the isolated PsaD in solution has a definite conformation and maintains the ability to bind ferredoxin I with high affinity and specificity. The Kd value of the complex of PsaD and ferredoxin is in the nanomolar range, which is consistent with reported Km values for ferredoxin I photoreduction by thylakoid membranes. The ionic strength dependence of the K(d) suggests that the protein-protein interaction is at least partially electrostatic in nature. Nevertheless, none of the glutamate residues of the acidic cluster of residues 92-94 of ferredoxin I, which have been reported to be involved in the interaction with the subunit, seems to be essential for PsaD binding, as borne out by experiments using ferredoxin I mutants in positions 92-94. 相似文献
8.
Tomitsuka E Goto Y Taniwaki M Kita K 《Biochemical and biophysical research communications》2003,311(3):774-779
Succinate-ubiquinone reductase (complex II) is an important enzyme complex in aerobic respiration and the tricarboxylic acid cycle. We recently identified two distinct cDNAs for the human flavoprotein subunit (Fp) from a single individual and demonstrated mRNAs of these two isoforms, Type I Fp and Type II Fp, in skeletal muscle, liver, brain, heart, and kidney. Type I Fp was expressed at higher levels than Type II Fp in all cases. In the present study, the biochemical properties of Type II Fp-containing complex II in Raji cells predominantly expressing Type II Fp were investigated. Complex II having Type II Fp was separated from that having Type I Fp by isoelectric focusing in the presence of sucrose monolaurate. Together with the fact that succinate-ubiquinone reductase activity of mitochondria prepared from Raji cell was almost identical to that from human liver, these results clearly indicate the presence of two distinct isoforms of active complex II in human mitochondria. 相似文献
9.
PsaD is a peripheral protein on the reducing side of photosystem I (PS I). We expressed the psaD gene from the thermophilic cyanobacterium Mastigocladus laminosus in Escherichia coli and obtained a soluble protein with a polyhistidine tag at the carboxyl terminus. The soluble PsaD protein was purified by Ni-affinity chromatography and had a mass of 16716 Da by MALDI-TOF. The N-terminal amino acid sequence of the overexpressed PsaD matched the N-terminal sequence of the native PsaD from M. laminosus. The soluble PsaD could assemble into the PsaD-less PS I. As determined by isothermal titration calorimetry, PsaD bound to PS I with 1.0 binding site per PS I, the binding constant of 7.7x10(6) M-1, and the enthalpy change of -93.6 kJ mol-1. This is the first time that the binding constant and binding heat have been determined in the assembly of any photosynthetic membrane protein. To identify the surface-exposed domains, purified PS I complexes and overexpressed PsaD were treated with N-hydroxysuccinimidobiotin (NHS-biotin) and biotin-maleimide, and the biotinylated residues were mapped. The Cys66, Lys21, Arg118 and Arg119 residues were exposed on the surface of soluble PsaD whereas the Lys129 and Lys131 residues were not exposed on the surface. Consistent with the X-ray crystallographic studies on PS I, circular dichroism spectroscopy revealed that PsaD contains a small proportion of alpha-helical conformation. 相似文献
10.
The photochemistry of the isolated Photosystem II reaction-centre core from pea and the green alga Scenedesmus was examined by e.s.r. Two types of triplet spectrum were observed in addition to the spin-polarized reaction-centre triplet previously identified. The additional triplet formed on continuous illumination at 4.2 K was attributed to a monomeric phaeophytin molecule. The second triplet, which was stable in the dark at 4.2 K following illumination, was assigned to the radical pair Donor+I-. This provides evidence that an electron donor to chlorophyll P680 is present on the polypeptide D1-polypeptide D2-cytochrome b-559 core complex. 相似文献
11.
Structural features of cytochrome f necessary for assembly into the cytochrome bf complex were examined in isolated pea chloroplasts following import of (35)S-labelled chimeric precursor proteins, consisting of the presequence of the small subunit of Rubisco fused to the turnip cytochrome f precursor. Assembly was detected by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. A cytochrome f polypeptide unable to bind haem because of mutagenesis of Cys21 and Cys24 to alanine residues was assembled into the complex and had similar stability to the wild-type polypeptide. This indicates that covalent haem binding to cytochrome f is not necessary for assembly of the protein into the cytochrome bf complex. A truncated protein lacking the C-terminal 33 amino acid residues, including the transmembrane span and the stroma-exposed region, was translocated across the thylakoid membrane, had a similar stability to wild-type cytochrome f but was not assembled into the complex. This indicates that the C-terminal region of cytochrome f is important for assembly into the complex. A mutant cytochrome f unable to bind haem and lacking the C-terminal region was also translocated across the thylakoid membrane but was extremely labile, indicating that, in the absence of the C-terminal membrane anchor, haem-less cytochrome f is recognized by a thylakoid proteolytic system. 相似文献
12.
Picosecond time-resolved energy transfer in Porphyridium cruentum. Part II. In the isolated light harvesting complex (phycobilisomes) 总被引:8,自引:0,他引:8
The transfer of excitation energy between phycobiliproteins in isolated phycobilisomes has been observed on a picosecond time scale. The photon density of the excitation pulse has been carefully varied so as to control the level of exciton interactions induced in the pigment bed. The 530 nm light pulse is absorbed predominantly by B-phycoerythrin, and the fluorescence of this component rises within the pulse duration and shows a mean 1/e decay time of 70 ps. The main emission band, centred at 672 nm, is due to allophycocyanin and is prominent because of the absence of energy transfer to chlorophyll. Energy transfer to this pigment from B-phycoerythrin via R-phycocyanin produces a risetime of 120 ps to the fluorescence maximum. The lifetime of the allophycocyanin fluorescence is found to be about 4 ns using excitation pulses of low photon densities (10(13) photons.cm-2), but decreases to about 2 ns at higher photon densities. The relative quantum yield of the allophycocyanin fluorescence decreases almost 10 fold over the range of laser pulse intensities, 10(13)--10(16) photons-cm-2. Fluorescence quenching by exciton-exciton annihilation is only observed in allophycocyanin and could be a consequence of the long lifetime of the single exciton in this pigment. 相似文献
13.
Assembly and disassembly of the Drosophila RNA polymerase II complex during transcription. 总被引:11,自引:0,他引:11
J T Kadonaga 《The Journal of biological chemistry》1990,265(5):2624-2631
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16.
Four double mutants in the epsilon subunit were generated, each containing two cysteines, which, based on the NMR structure of this subunit, should form internal disulfide bonds. Two of these were designed to generate interdomain cross-links that lock the C-terminal alpha-helical domain against the beta-sandwich (epsilonM49C/A126C and epsilonF61C/V130C). The second set should give cross-linking between the two C-terminal alpha-helices (epsilonA94C/L128C and epsilonA101C/L121C). All four mutants cross-linked with 90-100% efficiency upon CuCl(2) treatment in isolated Escherichia coli ATP synthase. This shows that the structure obtained for isolated epsilon is essentially the same as in the assembled complex. Functional studies revealed increased ATP hydrolysis after cross-linking between the two domains of the subunit but not after cross-linking between the C-terminal alpha-helices. None of the cross-links had any effect on proton pumping-coupled ATP hydrolysis, on DCCD sensitivity of this activity, or on ATP synthesis rates. Therefore, big conformational changes within epsilon can be ruled out as a part of the enzyme function. Protease digestion studies, however, showed that subtle changes do occur, since the epsilon subunit could be locked in an ADP or 5'-adenylyl-beta,gamma-imidodiphosphate conformation by the cross-linking with resulting differences in cleavage rates. 相似文献
17.
Pandit A Shirzad-Wasei N Wlodarczyk LM van Roon H Boekema EJ Dekker JP de Grip WJ 《Biophysical journal》2011,(10):2507-2515
Self-aggregation of isolated plant light-harvesting complexes (LHCs) upon detergent extraction is associated with fluorescence quenching and is used as an in vitro model to study the photophysical processes of nonphotochemical quenching (NPQ). In the NPQ state, in vivo induced under excess solar light conditions, harmful excitation energy is safely dissipated as heat. To prevent self-aggregation and probe the conformations of LHCs in a lipid environment devoid from detergent interactions, we assembled LHCII trimer complexes into lipid nanodiscs consisting of a bilayer lipid matrix surrounded by a membrane scaffold protein (MSP). The LHCII nanodiscs were characterized by fluorescence spectroscopy and found to be in an unquenched, fluorescent state. Remarkably, the absorbance spectra of LHCII in lipid nanodiscs show fine structure in the carotenoid and Qy region that is different from unquenched, detergent-solubilized LHCII but similar to that of self-aggregated, quenched LHCII in low-detergent buffer without magnesium ions. The nanodisc data presented here suggest that 1), LHCII pigment-protein complexes undergo conformational changes upon assembly in nanodiscs that are not correlated with downregulation of its light-harvesting function; and 2), these effects can be separated from quenching and aggregation-related phenomena. This will expand our present view of the conformational flexibility of LHCII in different microenvironments. 相似文献
18.
Phi6 is an enveloped dsRNA bacteriophage of Pseudomonas syringae. The viral envelope encloses a nucleocapsid, consisting of an RNA-dependent RNA polymerase complex within an icosahedral shell assembled from approximately 800 copies of a 16 kDa subunit (protein P8, encoded by viral gene 8). During infection, the nucleocapsid penetrates the host plasma membrane and enters the cytosol, whereupon the P8 shell disassembles and the polymerase complex is activated. To understand the molecular mechanisms of shell assembly and disassembly-processes that have counterparts in most viral infections-we have investigated the structure, stability, and dynamics of P8 in different assembly states using time-resolved Raman spectroscopy and hydrogen-isotope exchange. In the presence of Ca(2+), which promotes shell assembly, the highly alpha-helical conformation of the P8 subunit is stabilized by rapid assembly into shell-like structures. However, in the absence of Ca(2+), the P8 subunit is thermolabile and unstable, manifested by a slow alpha-helix --> beta-strand conformational change and the accumulation of aberrant aggregates. In both properly assembled shells and aberrant aggregates, the P8 subunit retains an alpha-helical core that is protected against deuterium exchange of amide NH groups. Surprisingly, no additional protection against amide exchange is conferred by the shell lattice. Time-resolved assembly and disassembly experiments in deuterated buffers indicate that the regions of P8 involved in subunit/subunit interactions in the intact shell undergo rapid exchanges, presumably due to local unfolding events that are characterized by low activation barriers. Such localized dynamics of P8 within the shell lattice may mediate the nucleocapsid/host membrane interactions that are required in the cytosol for particle assembly during maturation and disassembly during infection. 相似文献
19.
It has recently been reported that bilirubin forms a complex with Cu(II). In this paper we show that the formation of the complex results in the reduction of Cu(II) to Cu(I) and the redox cycling of the metal gives rise to the formation of reactive oxygen species, particularly hydroxyl radical. The bilirubin-Cu(II) complex causes strand breakage in calf thymus DNA and supercoiled plasmid DNA. Cu(I) was shown to be an essential intermediate in the DNA cleavage reaction by using the Cu(I) specific sequestering reagent neocuproine. Bilirubin-Cu(II) produced hydroxyl radical and the involvement of active oxygen species was established by the inhibition of DNA breakage by various oxygen radical quenchers. 相似文献
20.
Assembly of the precursor and processed light-harvesting chlorophyll a/b protein of Lemna into the light-harvesting complex II of barley etiochloroplasts 总被引:16,自引:2,他引:14
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P R Chitnis E Harel B D Kohorn E M Tobin J P Thornber 《The Journal of cell biology》1986,102(3):982-988
When the in vitro synthesized precursor of a light-harvesting chlorophyll a/b binding protein (LHCP) from Lemna gibba is imported into barley etiochloroplasts, it is processed to a single form. Both the processed form and the precursor are found in the thylakoid membranes, assembled into the light-harvesting complex of photosystem II. Neither form can be detected in the stromal fraction. The relative amounts of precursor and processed forms observed in the thylakoids are dependent on the developmental stage of the plastids used for uptake. The precursor as well as the processed form can also be detected in thylakoids of greening maize plastids used in similar uptake experiments. This detection of a precursor in the thylakoids, which has not been previously reported, could be a result of using rapidly developing plastids and/or using an heterologous system. Our results demonstrate that the extent of processing of LHCP precursor is not a prerequisite for its inclusion in the complex. They are also consistent with the possibility that the processing step can occur after insertion of the protein into the thylakoid membrane. 相似文献