RESUMEN

Photosystem (PSII) is a supramolecular polypeptide complex found in oxygenic photosynthetic membranes, which is capable of extracting electrons from water for the reduction of plastoquinone. An intriguing feature of this assembly is the fact that it includes more than a dozen low-mass polypeptides of generally unknown function. Using a transplastomic approach, we have individually disrupted the genes of the psbEFLJoperon in Nicotiana tabacum, which encode four such polypeptides, without impairing expression of downstream loci of the operon. All four mutants exhibited distinct phenotypes; none of them was capable of photoautotrophic growth. All mutants bleached rapidly in the light. Disruption of psbEand psbF, which code for the alpha and beta apoproteins of cytochrome b(559), abolished PSII activity, as expected; Delta psbL and Delta psbJ plants displayed residual PSII activity in young leaves. Controlled partial solubilisation of thylakoid membranes uncovered surprisingly severe impairment of PSII structure, with subunit and assembly patterns varying depending on the mutant considered. In the Delta psbL mutant PSII was assembled primarily in a monomeric form, the homodimeric form was preponderant in Delta psbJ, and, unlike the case in Delta psbZ, the thylakoids of both mutants released some PSII supercomplexes. On the other hand, Photosystem I (PSI), the cytochrome b(6)f complex, ATP synthase, LHCII, and CP24/CP26/CP29 antennae were present in near wild-type levels. The data are discussed in terms of their implications for structural, biogenetic and functional aspects of PSII.

Asunto(s)

Genes de Plantas , Nicotiana/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Secuencia de Bases , Cloroplastos/genética , Cloroplastos/metabolismo , ADN de Plantas/genética , Marcación de Gen , Microscopía Electrónica , Mutación , Operón , Fenotipo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Complejo de Proteína del Fotosistema I , Complejo de Proteína del Fotosistema II , Filogenia , Subunidades de Proteína , Nicotiana/metabolismo , Nicotiana/ultraestructura , Transformación Genética

RESUMEN

Regulation of manganese acquisition by bacteria occurs by both biochemical regulation of the activity of the transporters and transcriptional regulation of gene expression. Structural analysis suggests that calcium ions may regulate the function of an Mn ATP-binding cassette (ABC)-permease in Synechocystis 6803, a cyanobacterium, as well as in a number of other bacteria. The expression of genes encoding the manganese transporter in Synechocystis 6803 is regulated by a two-component signal-transduction mechanism that has not been previously observed for manganese and zinc transport in bacteria.

Asunto(s)

Bacterias/genética , Bacterias/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Manganeso/metabolismo , Secuencia de Aminoácidos , Bacterias/clasificación , Transporte Biológico , Proteínas Portadoras/química , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Estructura Secundaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética

RESUMEN

During oxygenic photosynthesis in cyanobacteria and chloroplasts of plants and eukaryotic algae, conversion of light energy to biologically useful chemical energy occurs in the specialized thylakoid membranes. Light-induced charge separation at the reaction centers of photosystems I and II, two multisubunit pigment-protein complexes in the thylakoid membranes, energetically drive sequential photosynthetic electron transfer reactions in this membrane system. In general, in the prokaryotic cyanobacterial cells, the thylakoid membrane is distinctly different from the plasma membrane. We have recently developed a two-dimensional separation procedure to purify thylakoid and plasma membranes from the genetically widely studied cyanobacterium Synechocystis sp. PCC 6803. Immunoblotting analysis demonstrated that the purified plasma membrane contained a number of protein components closely associated with the reaction centers of both photosystems. Moreover, these proteins were assembled in the plasma membrane as chlorophyll-containing multiprotein complexes, as evidenced from nondenaturing green gel and low-temperature fluorescence spectroscopy data. Furthermore, electron paramagnetic resonance spectroscopic analysis showed that in the partially assembled photosystem I core complex in the plasma membrane, the P700 reaction center was capable of undergoing light-induced charge separation. Based on these data, we propose that the plasma membrane, and not the thylakoid membrane, is the site for a number of the early steps of biogenesis of the photosynthetic reaction center complexes in these cyanobacterial cells.

Asunto(s)

Cianobacterias/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/biosíntesis , Membrana Celular/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Espectrometría de Fluorescencia

RESUMEN

The photosystem II (PSII) complex of photosynthetic oxygen evolving membranes comprises a number of small proteins whose functions remain unknown. Here we report that the low molecular weight protein encoded by the psbJ gene is an intrinsic component of the PSII complex. Fluorescence kinetics, oxygen flash yield, and thermoluminescence measurements indicate that inactivation of the psbJ gene in Synechocystis 6803 cells and tobacco chloroplasts lowers PSII-mediated oxygen evolution activity and increases the lifetime of the reduced primary acceptor Q(A)(-) (more than a 100-fold in the tobacco DeltapsbJ mutant). The decay of the oxidized S(2,3) states of the oxygen-evolving complex is considerably accelerated, and the oscillations of the Q(B)(-)/S(2,3) recombination with the number of exciting flashes are damped. Thus, PSII can be assembled in the absence of PsbJ. However, the forward electron flow from Q(A)(-) to plastoquinone and back electron flow to the oxidized Mn cluster of the donor side are deregulated in the absence of PsbJ, thereby affecting the efficiency of PSII electron flow following the charge separation process.

Asunto(s)

Proteínas Bacterianas , Cianobacterias/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Complejo de Proteína del Fotosistema II , Secuencia de Bases , Cianobacterias/genética , Cartilla de ADN , Electrones , Cinética , Proteínas de la Membrana/genética , Tilacoides/metabolismo , Nicotiana

RESUMEN

CtpA, a carboxyl-terminal processing protease, is a member of a novel family of endoproteases that includes a tail-specific protease from Escherichia coli. In oxygenic photosynthetic organisms, CtpA catalyzes C-terminal processing of the D1 protein of photosystem II, an essential event for the assembly of a manganese cluster and consequent light-mediated water oxidation. We introduced site-specific mutations at 14 conserved residues of CtpA in the cyanobacterium Synechocystis sp. PCC 6803 to examine their functional roles. Analysis of the photoautotrophic growth capabilities of these mutants, their ability to process precursor D1 protein and hence evolve oxygen, along with an estimation of the protease content in the mutants revealed that five of these residues are critical for in vivo activity of CtpA. Recent x-ray crystal structure analysis of CtpA from the eukaryotic alga Scenedesmus obliquus (Liao, D.-I., Qian, J., Chisholm, D. A., Jordan, D. B. and Diner, B. A. (2000) Nat. Struct. Biol. 7, 749-753) has shown that the residues equivalent to Ser-313 and Lys-338, two of the five residues mentioned above, form the catalytic center of this enzyme. Our in vivo analysis demonstrates that the three other residues, Asp-253, Arg-255, and Glu-316, are also important determinants of the catalytic activity of CtpA.

Asunto(s)

Carboxipeptidasas , Endopeptidasas/química , Endopeptidasas/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Complejo de Proteína del Fotosistema II , Proproteína Convertasas , Proteínas Algáceas , Secuencia de Aminoácidos , Aminoácidos/química , Western Blotting , Catálisis , División Celular , Secuencia Conservada , Cianobacterias/química , Electroforesis en Gel de Poliacrilamida , Lisina/química , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Oxígeno/metabolismo , Homología de Secuencia de Aminoácido , Serina/química , Temperatura , Factores de Tiempo , Agua/metabolismo

RESUMEN

In this study, the unicellular photosynthetic cyanobacterium Synechocystis sp. PCC 6803 was used as a model phototroph to study the contribution of enzymatic photoreactivation to the overall protection against UV irradiation. We have isolated genes encoding two DNA photolyase homologs, phrA and phrB, from Synechocystis 6803. phrA encodes an 8-hydroxy-5-deazariboflavin (HDF) type, Class I DNA photolyase. By complementing a photolyase-deficient mutant strain of Escherichia coli, we demonstrated that PhrA is a DNA photolyase. Analysis of a phrA knockout mutant strain suggested that this gene is responsible for the majority of the observed UV resistance in Synechocystis 6803. Similar studies on phrB demonstrated that it also contributes to photoreactivation, but to a much lesser degree. Based on these findings, we conclude that enzymatic photoreactivation is the primary process used for repairing UV-induced damage in Synechocystis 6803.

Asunto(s)

Transportadoras de Casetes de Unión a ATP , Cianobacterias/enzimología , Cianobacterias/efectos de la radiación , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Proteínas de Escherichia coli , Rayos Ultravioleta , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Cianobacterias/genética , Cartilla de ADN , Escherichia coli/enzimología , Escherichia coli/genética , Eliminación de Gen , Prueba de Complementación Genética , Proteínas Recombinantes/metabolismo

RESUMEN

The D1 protein is an integral component of the photosystem II reaction center complex. In the cyanobacterium Synechocystis sp. PCC 6803, D1 is synthesized with a short 16-amino acids-long carboxyl-terminal extension. Removal of this extension is necessary to form active oxygen-evolving photosystem II centers. Our earlier studies have shown that this extension is cleaved by CtpA, a specific carboxyl-terminal processing protease. The amino acid sequence of the carboxyl-terminal extension is conserved among D1 proteins from different organisms, although at a level lower than that of the mature protein. In the present study we have analyzed a mutant strain of Synechocystis sp. PCC 6803 with a duplicated extension, and a second mutant that lacks the extension, to investigate the effects of these alterations on the function of the D1 protein in vivo. No significant difference in the growth rates, photosynthetic pigment composition, fluorescence induction, and oxygen evolution rates was observed between the mutants and the control strain. However, using long-term mixed culture growth analysis, we detected significant decreases in the fitness of these mutant strains. The presented data demonstrate that the carboxyl-terminal extension of the precursor D1 protein is required for optimal photosynthetic performance.

Asunto(s)

Cianobacterias/fisiología , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Complejo de Proteína del Fotosistema II , Proteínas de Plantas/metabolismo , Precursores de Proteínas/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Cianobacterias/crecimiento & desarrollo , Cianobacterias/metabolismo , Immunoblotting , Luz , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas de Plantas/química , Reacción en Cadena de la Polimerasa , Precursores de Proteínas/química , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína

RESUMEN

A new broad-host-range plasmid, pSL1211, was constructed for the over-expression of genes in Synechocystis sp. strain PCC 6803. The plasmid was derived from RSF1010 and an Escherichia coli over-expression plasmid, pTrcHisC. Over-expressed protein is made with a removable N-terminal histidine tag. The plasmid was used to over-express the phrA gene and purify the gene product from Synechocystis sp. strain PCC 6803. PhrA is the major ultraviolet-light-resistant factor in the cyanobacterium. The purified PhrA protein exhibited an optical absorption spectrum similar to that of the cyclobutane pyrimidine dimer (CPD) DNA photolyase from Synechocuccus sp. strain PCC 6301 (Anacystis nidulans). Mass spectrometry analysis of PhrA indicated that the protein contains 8-hydroxy-5-deazariboflavin and flavin adenine dinucleotide (FADH2) as cofactors. PhrA repairs only cyclobutane pyrimidine dimer but not pyrimidine (6-4) pyrimidinone photoproducts. On the basis of these results, the PhrA protein is classified as a class I, HDF-type, CPD DNA photolyase.

Asunto(s)

Transportadoras de Casetes de Unión a ATP , Proteínas Bacterianas/genética , Cianobacterias/enzimología , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Proteínas de Escherichia coli , Dímeros de Pirimidina/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Cianobacterias/genética , Vectores Genéticos , Plásmidos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos

RESUMEN

The ndhD gene encodes a membrane protein component of NAD(P)H dehydrogenase. The genome of Synechocystis sp. PCC6803 contains 6 ndhD genes. Three mutants were constructed by disrupting highly homologous ndhD genes in pairs. Only the DeltandhD1/DeltandhD2 (DeltandhD1/D2) mutant was unable to grow under photoheterotrophic conditions and exhibited low respiration rate, although the mutant grew normally under photoautotrophic conditions in air. The DeltandhD3/DeltandhD4 (DeltandhD3/D4) mutant grew very slowly in air and did not take up CO(2). The results demonstrated the presence of two types of functionally distinct NAD(P)H dehydrogenases in Synechocystis PCC6803 cells. TheDeltandhD5/DeltandhD6 (DeltandhD5/D6) mutant grew like the wild-type strain. Under far-red light (>710 nm), the level of P700(+) was high in DeltandhD1/D2 and M55 (ndhB-less mutant) at low intensities. The capacity of Q(A) (tightly bound plastoquinone) reduction by plastoquinone pool, as measured by the fluorescence increase in darkness upon addition of KCN, was much less in DeltandhD1/D2 and M55 than in DeltandhD3/D4 and DeltandhD5/D6. We conclude that electrons from NADPH are transferred to the plastoquinone pool mainly by the NdhD1.NdhD2 type of NAD(P)H dehydrogenases.

Asunto(s)

Cianobacterias/enzimología , NAD(P)H Deshidrogenasa (Quinona)/clasificación , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Dióxido de Carbono/metabolismo , Respiración de la Célula , Clorofila/genética , Clorofila/metabolismo , Cianobacterias/genética , Cianobacterias/crecimiento & desarrollo , Cianobacterias/metabolismo , Oscuridad , Transporte de Electrón , Fluorescencia , Genes Bacterianos/genética , Genes Bacterianos/fisiología , Concentración de Iones de Hidrógeno , Rayos Infrarrojos , Isoenzimas/clasificación , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Luz , Mutación/genética , NAD(P)H Deshidrogenasa (Quinona)/genética , Oxidación-Reducción , Oxígeno/metabolismo , Plastoquinona/metabolismo , Homología de Secuencia de Aminoácido

RESUMEN

Specific inhibition of photosystem I (PSI) was observed under low-temperature conditions in the cyanobacterium Synechocystis sp. strain PCC 6803. Growth at 20 degrees C caused inhibition of PSI activity and increased degradation of the PSI reaction center proteins PsaA and PsaB, while no significant changes were found in the level and activity of photosystem II (PSII). BtpA, a recently identified extrinsic thylakoid membrane protein, was found to be a necessary regulatory factor for stabilization of the PsaA and PsaB proteins under such low-temperature conditions. At normal growth temperature (30 degrees C), the BtpA protein was present in the cell, and its genetic deletion caused an increase in the degradation of the PSI reaction center proteins. However, growth of Synechocystis cells at 20 degrees C or shifting of cultures grown at 30 degrees C to 20 degrees C led to a rapid accumulation of the BtpA protein, presumably to stabilize the PSI complex, by lowering the rates of degradation of the PsaA and PsaB proteins. A btpA deletion mutant strain could not grow photoautotrophically at low temperature, and exhibited rapid degradation of the PSI complex after transfer of the cells from normal to low temperature.

Asunto(s)

Proteínas Bacterianas , Cianobacterias/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Frío , Cianobacterias/genética , Cianobacterias/crecimiento & desarrollo , Hidrólisis , Complejo de Proteína del Fotosistema I , Complejo de Proteína del Fotosistema II

RESUMEN

Based on DNA sequence data a novel c-type cytochrome, cytochrome cM, has been predicted to exist in the cyanobacterium Synechocystis 6803. The precursor protein consists of 105 amino acids with a characteristic heme-binding motif and a hydrophobic domain located at the N-terminal end that is proposed to act as either a signal peptide or a membrane anchor. For the first time we report the detection of cytochrome cM in Synechocystis 6803 using Western blot analysis. The soluble portion cytochrome cM has been overexpressed in Escherichia coli in two forms, one with a poly histidine tag to facilitate purification and one without such a tag. The overexpressed protein has been purified and shown to bind heme, exhibiting an absorption peak in the Soret band near 416 nm and a peak in the alpha band at 550 nm. The extinction coefficient of cytochrome cM is 23.2 +/- 0.5 mM-1.cm-1 for the reduced minus oxidized alpha band peak (550-535 nm). The isoelectric point of cytochrome cM is 5.6 (without the histidine tag), which is significantly lower than the pI of 7.2 predicted from the amino acid sequence. The redox midpoint potential of cytochrome cM expressed in E. coli is 151 +/- 5 mV (pH 7.1), which is quite low compared to other c-type cytochromes in which a histidine and a methionine residue serve as the axial ligands to the heme. This work opens the way for determining the three-dimensional structure of cytochrome cM and investigating its function in cyanobacteria.

Asunto(s)

Proteínas Bacterianas , Cianobacterias/enzimología , Grupo Citocromo c/aislamiento & purificación , Grupo Citocromo c/metabolismo , Escherichia coli/genética , Secuencia de Aminoácidos , Western Blotting , Grupo Citocromo c/química , Grupo Citocromo c/genética , Hemo/metabolismo , Histidina/metabolismo , Punto Isoeléctrico , Metionina/metabolismo , Peso Molecular , Oxidación-Reducción , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia/genética , Solubilidad , Análisis Espectral , Volumetría

RESUMEN

A tetra-manganese cluster in the photosystem II (PSII) pigment-protein complex plays a critical role in the photosynthetic oxygen evolution process. PsbY, a small membrane-spanning polypeptide, has recently been suggested to provide a ligand for manganese in PSII (A.E. Gau, H.H. Thole, A. Sokolenko, L. Altschmied, R.G. Herrmann, E.K. Pistorius [1998] Mol Gen Genet 260: 56-68). We have constructed a mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 with an inactivated psbY gene (sml0007). Southern-blot and polymerase chain reaction analysis showed that the mutant had completely segregated. However, the DeltapsbY mutant cells grew normally under photoautotrophic conditions. Moreover, growth of the wild-type and mutant cells were similar under high-light photoinhibition conditions, as well as in media without any added manganese, calcium, or chloride, three required inorganic cofactors for the oxygen-evolving complex of PSII. Analysis of steady-state and flash-induced oxygen evolution, fluorescence induction, and decay kinetics, and thermoluminescence profiles demonstrated that the DeltapsbY mutant cells have normal photosynthetic activities. We conclude that the PsbY protein in Synechocystis 6803 is not essential for oxygenic photosynthesis and does not provide an important binding site for manganese in the oxygen-evolving complex of PSII.

Asunto(s)

Proteínas Bacterianas , Cianobacterias/metabolismo , Fotosíntesis , Ureohidrolasas/metabolismo , Clorofila/metabolismo , Cianobacterias/genética , Cianobacterias/crecimiento & desarrollo , Mediciones Luminiscentes , Proteínas de la Membrana , Mutagénesis Insercional , Sistemas de Lectura Abierta , Oxígeno/metabolismo , Fotosíntesis/genética , Reacción en Cadena de la Polimerasa/métodos , Mapeo Restrictivo , Transformación Bacteriana , Ureohidrolasas/genética

RESUMEN

The molecular basis for the transport of manganese across membranes in plant cells is poorly understood. We have found that IRT1, an Arabidopsis thaliana metal ion transporter, can complement a mutant Saccharomyces cerevisiae strain defective in high-affinity manganese uptake (smf1 delta). The IRT1 protein has previously been identified as an iron transporter. The current studies demonstrated that IRT1, when expressed in yeast, can transport manganese as well. This manganese uptake activity was inhibited by cadmium, iron(II) and zinc, suggesting that IRT1 can transport these metals. The IRT1 cDNA also complements a zinc uptake-deficient yeast mutant strain (zrt1zrt2), and IRT1-dependent zinc transport in yeast cells is inhibited by cadmium, copper, cobalt and iron(III). However, IRT1 did not complement a copper uptake-deficient yeast mutant (ctr1), implying that this transporter is not involved in the uptake of copper in plant cells. The expression of IRT1 is enhanced in A. thaliana plants grown under iron deficiency. Under these conditions, there were increased levels of root-associated manganese, zinc and cobalt, suggesting that, in addition to iron, IRT1 mediates uptake of these metals into plant cells. Taken together, these data indicate that the IRT1 protein is a broad-range metal ion transporter in plants.

Asunto(s)

Proteínas de Arabidopsis , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión , Metales Pesados/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Oligoelementos/metabolismo , Arabidopsis/metabolismo , Transporte Biológico , Cadmio/metabolismo , Proteínas Portadoras/genética , Prueba de Complementación Genética , Hierro/metabolismo , Manganeso/metabolismo , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Zinc/metabolismo

RESUMEN

The structure of the membrane protein MntB, a component of a manganese transporter system in Synechocystis sp. strain PCC 6803, was examined with a series of fusions to the reporter proteins alkaline phosphatase and beta-galactosidase. The results support a topological model for MntB consisting of nine transmembrane segments, with the amino terminus of the protein being in the periplasm and the carboxyl terminus being in the cytoplasm.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/química , Proteínas Bacterianas , Cianobacterias/química , Manganeso , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Western Blotting , Cianobacterias/enzimología , Cianobacterias/genética , Cianobacterias/metabolismo , Citoplasma/metabolismo , Escherichia coli/genética , Genes Reporteros , Manganeso/metabolismo , Modelos Moleculares , Periplasma/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo

RESUMEN

Photosystem I is a large pigment-protein complex embedded in the thylakoid membranes of chloroplasts and cyanobacteria. In the cyanobacterium Synechocystis sp. PCC 6803, the btpA gene encodes a 30-kDa polypeptide. Mutations in this gene significantly affect accumulation of the reaction center proteins of photosystem I in Synechocystis 6803 [Bartsevich, V. V. & Pakrasi, H. B. (1997) J. Biol. Chem. 272, 6372-6378]. We describe here the intracellular localization of the BtpA protein. Immunolocalization in Synechocystis 6803 cells demonstrated that the BtpA protein is tightly associated with the thylakoid membranes. Phase fractionation in the detergent Triton X-114 indicated that BtpA is a peripheral membrane protein. To determine which surface of the thylakoid membrane BtpA is exposed to, we used a two-phase polymer partitioning technique to develop a novel method to isolate inside-out and right-side-out thylakoid vesicles from Synechocystis 6803. Treatments of such vesicles with different salts and protease showed that the BtpA protein is an extrinsic membrane protein which is exposed to the cytoplasmic face of the thylakoid membrane.

Asunto(s)

Proteínas Bacterianas/metabolismo , Cianobacterias/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Bacterianas/genética , Cloroplastos/metabolismo , Cianobacterias/genética , Genes Bacterianos , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Fracciones Subcelulares/metabolismo

RESUMEN

The CtpA protein in the cyanobacterium Synechocystis 6803 is a C-terminal processing protease that is essential for the assembly of the manganese cluster of the photosystem II complex. When fused to different chloroplast-targeting transit peptides, CtpA can be imported into isolated spinach chloroplasts and is subsequently translocated into the thylakoid lumen. Thylakoid transport is mediated by the cyanobacterial signal peptide which demonstrates that the protein transport machinery in thylakoid membranes is functionally conserved between chloroplasts and cyanobacteria. Transport of CtpA across spinach thylakoid membranes is affected by both nigericin and sodium azide indicating that the SecA protein and a transthylakoidal proton gradient are involved in this process. Saturation of the Sec-dependent thylakoid transport route by high concentrations of the precursor of the 33-kDa subunit of the oxygen-evolving system leads to a strongly reduced rate of thylakoid translocation of CtpA which demonstrates transport by the Sec pathway. However, thylakoid transport of CtpA is affected also by excess amounts of the 23-kDa subunit of the oxygen-evolving system, though to a lesser extent. This suggests that the cyanobacterial protein is capable of also interacing with components of the deltapH-dependent route and that transport of a protein across the thylakoid membrane may not always be restricted to a single pathway.

Asunto(s)

Proteínas Bacterianas , Cloroplastos/enzimología , Cianobacterias/enzimología , Endopeptidasas/metabolismo , Endopeptidasas/biosíntesis , Membranas Intracelulares/enzimología , Cinética , Sustancias Macromoleculares , Biosíntesis de Proteínas , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Spinacia oleracea/metabolismo , Transcripción Genética

RESUMEN

Photosystem I (PSI) is a multisubunit pigment-protein complex in the thylakoid membranes of cyanobacteria and chloroplasts. BP26, a random photosynthesis-deficient mutant strain of the cyanobacterium Synechocystis 6803 has a severely reduced PSI content, whereas its photosystem II is present in normal amount. The BP26 mutant is complemented by a novel gene, btpA, that encodes a 30-kDa protein. In this strain, a missense mutation in the btpA gene, resulting in the replacement of a valine by a glycine residue, significantly affects the accumulation of the PSI reaction center proteins, PsaA and PsaB. Northern blot analysis revealed that the steady-state levels of the transcripts from the psaAB operon, encoding these proteins, remain unaltered in the mutant strain. Hence the BtpA protein regulates a post-transcriptional process during the life cycle of the PSI protein complex such as 1) translation of the psaAB mRNA, 2) assembly of the PsaA/PsaB polypeptides and their associated cofactors into a functional complex, or 3) degradation of the protein complex. Close relatives of the BtpA protein have been found in nonphotosynthetic organisms, viz. the archaebacterium Methanococcus jannaschii, the eubacterium Escherichia coli, and the nematode, Caenorhabditis elegans, suggesting that these proteins may regulate biogenesis of other protein complexes in these evolutionarily distant organisms.

Asunto(s)

Cianobacterias/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Complejo de Proteína del Fotosistema I , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cianobacterias/genética , Transporte de Electrón , Genes Bacterianos , Prueba de Complementación Genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Alineación de Secuencia , Análisis Espectral , Transcripción Genética

RESUMEN

Photosystem II is a reaction center protein complex located in photosynthetic membranes of plants, algae, and cyanobacteria. Using light energy, photosystem II catalyzes the oxidation of water and the reduction of plastoquinone, resulting in the release of molecular oxygen. A key component of photosystem II is cytochrome b559, a membrane-embedded heme protein with an unknown function. The cytochrome is unusual in that a heme links two separate polypeptide subunits, alpha and beta, either as a heterodimer (alphabeta) or as two homodimers (alpha2 and beta2). To determine the structural organization of cytochrome b559 in the membrane, we used site-directed mutagenesis to fuse the coding regions of the two respective genes in the cyanobacterium Synechocystis sp. PCC 6803. In this construction, the C terminus of the alpha subunit (9 kDa) is attached to the N terminus of the beta subunit (5 kDa) to form a 14-kDa alphabeta fusion protein that is predicted to have two membrane-spanning alpha-helices with antiparallel orientations. Cells containing the alphabeta fusion protein grow photoautotrophically and assemble functional photosystem II complexes. Optical spectroscopy shows that the alphabeta fusion protein binds heme and is incorporated into photosystem II. These data support a structural model of cytochrome b559 in which one heme is coordinated to an alpha2 homodimer and a second heme is coordinated to a beta2 homodimer. In this model, each photosystem II complex contains two cytochrome b559 hemes, with the alpha2 heme located near the stromal side of the membrane and the beta2 heme located near the lumenal side.

Asunto(s)

Grupo Citocromo b/química , Grupo Citocromo b/genética , Modelos Moleculares , Mutación , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Complejo de Proteína del Fotosistema II , Secuencia de Aminoácidos , Cianobacterias/química , Cianobacterias/genética , Cianobacterias/crecimiento & desarrollo , Grupo Citocromo b/metabolismo , Cartilla de ADN/genética , Hemo/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

We have inactivated the genes encoding components of MntABC, an ABC (ATP binding cassette) transporter system for manganese in the cyanobacterium Synechocystis sp. PCC 6803. The growth rates of these mutant strains were significantly lower in a manganese-deficient medium and were restored to near normal levels upon addition of micromolar concentrations of Mn2+, indicating the presence of a second transport system for manganese in this organism. 54Mn2+ uptake experiments indicated that the MntABC transporter was induced under manganese starvation conditions, whereas the second transporter system was induced in the presence of micromolar levels of manganese. Both of these systems were nonfunctional at low temperatures and could transport trace levels of 54Mn2+, reflecting high affinity active transport. The initial rates of Mn2+ uptake for cells grown with or without manganese exhibited biphasic saturation kinetics, suggesting that Mn2+ can also be accumulated by a low affinity system in these bacteria. The kinetic parameters for the MntABC transporter system are Km = 1-3 microM and Vmax = 3-8 pmol/min.10(8) cells. Accumulation of manganese by this system was competitively inhibited by Cd2+ (Ki = 4-8 microM), Co2+ and Zn2+ (Ki = 8-15 microM). In contrast, the second high affinity system was highly specific for manganese and was not inhibited by any tested metal ion. We have also demonstrated that in this organism, photosynthetic electron transport is necessary for optimal rates of manganese uptake.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/genética , Cianobacterias/metabolismo , Manganeso/metabolismo , Southern Blotting , División Celular/efectos de los fármacos , Frío , Sulfato de Cobre/farmacología , Cianobacterias/efectos de los fármacos , Cianobacterias/genética , ADN Bacteriano/química , Cinética , Fenotipo , Mapeo Restrictivo , Sulfato de Zinc/farmacología

RESUMEN

The D1 reaction center protein of the Photosystem II complex in green plants is synthesized with a short carboxyl-terminal extension. Proteolytic cleavage and removal of this extension peptide in the thylakoid lumen are necessary for the assembly of a manganese cluster that is essential for the oxygen evolution activity of Photosystem II. We have isolated cDNAs encoding CtpA, the carboxyl-terminal processing protease for the D1 protein, from two higher plants, spinach and barley. In each of these organisms, CtpA is encoded by a single copy nuclear gene, and its steady-state mRNA levels are light-regulated. The CtpA protein is detectable in etiolated material, and its level increases approximately 5-fold upon illumination. Moreover, the CtpA gene is expressed in shoot tissues and not in roots. In its precursor form, the CtpA protein harbors a bipartite transit sequence characteristic for thylakoid lumenal proteins. Cell fractionation studies demonstrated that CtpA is associated with thylakoid membranes and is resistant to treatments with thermolysin, consistent with its localization in the lumen of thylakoids. Comparisons of the sequence of the higher plant CtpA enzyme with those of other related carboxyl-terminal processing proteases suggest that these proteins constitute a new family of proteases.

Asunto(s)

Carboxipeptidasas , Endopeptidasas/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Proproteína Convertasas , Proteínas Algáceas , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario , ADN de Plantas , Endopeptidasas/metabolismo , Datos de Secuencia Molecular , Complejo de Proteína del Fotosistema II , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Spinacia oleracea/química , Spinacia oleracea/genética