Transport of CtpA Protein from the Cyanobacterium <i>Synechocystis</i> 6803 Across the Thylakoid Membrane in Chloroplasts

Karnauchov, Ivan; Herrmann, Reinhold G.; Pakrasi, Himadri B.; Klösgen, Ralf Bernd

doi:10.1111/j.1432-1033.1997.t01-1-00497.x

Cited by 17 publications

(16 citation statements)

References 49 publications

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“…Sec-dependent protein transport (which was analyzed in parallel) is not affected under these conditions (Ref. 39 and data not shown), confirming that competition was pathway-specific.…”

Section: Thylakoid Targeting Of the Rieske Protein Is Retarded In Thesupporting

confidence: 62%

The Rieske Fe/S Protein of the Cytochromeb /f Complex in Chloroplasts

Molik

Karnauchov²,

Weidlich

et al. 2001

Journal of Biological Chemistry

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125

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The Rieske Fe/S protein, a nuclear-encoded subunit of the cytochrome b 6 /f complex in chloroplasts, is retarded in the stromal space after import into the chloroplast and only slowly translocated further into the thylakoid membrane system. As shown by the sensitivity to nigericin and to specific competitor proteins, thylakoid transport takes place by the ⌬pH-dependent TAT pathway. The Rieske protein is an untypical TAT substrate, however. It is only the second integral membrane protein shown to utilize this pathway, and it is the first authentic substrate without a cleavable signal peptide. Transport is instead mediated by the NH 2 -terminal membrane anchor, which lacks, however, the twin-arginine motif indicative of ⌬pH/TAT-dependent transport signals. Furthermore, transport is affected by sodium azide as well as by competitor proteins for the Sec pathway in chloroplasts, demonstrating for the first time some cross-talk of the two pathways. This might take place in the stroma where the Rieske protein accumulates after import in several complexes of high molecular mass, among which the cpn60 complex is the most prominent. These untypical features suggest that the Rieske protein represents an intermediate or early state in the evolution of the thylakoidal protein transport pathways.

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“…Sec-dependent protein transport (which was analyzed in parallel) is not affected under these conditions (Ref. 39 and data not shown), confirming that competition was pathway-specific.…”

Section: Thylakoid Targeting Of the Rieske Protein Is Retarded In Thesupporting

confidence: 62%

The Rieske Fe/S Protein of the Cytochromeb /f Complex in Chloroplasts

Molik

Karnauchov²,

Weidlich

et al. 2001

Journal of Biological Chemistry

Self Cite

125

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“…This part of the protein is co-translationally translocated into the luminal space and then immediately removed by the action of an endopeptidase called the C-terminal processing protease (CtpA) (7)(8)(9)(10). In eukaryotic organisms, the enzyme is nuclear encoded (8 -10) and imported from the cytosol to thylakoidal lumen (11). When present on the D1 protein, this extension's removal is absolutely essential for the integration of the machinery for water oxidation, i.e.…”

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confidence: 99%

Overexpression and Characterization of Carboxyl-terminal Processing Protease for Precursor D1 Protein

Yamamoto

Inagaki

Satoh

2001

Journal of Biological Chemistry

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CtpA, which is classified as a novel type of serine protease with a Ser/Lys catalytic dyad, is responsible for the C-terminal processing of precursor D1 protein (pD1) of the photosystem II reaction center, a process that is indispensable for the integration of water-splitting machinery in photosynthesis. In this study, overexpression in Escherichia coli and one-step purification of spinach CtpA were carried out to analyze the characteristics of this new type of protease and to elucidate the molecular interactions in the C-terminal processing of pD1 on the thylakoid membrane. The successful accumulation of functional CtpA in E. coli may argue against the possibility, based on homology to E. coli Tsp, that the enzyme is involved in the degradation of incomplete proteins in chloroplasts, e.g. by utilizing the ssrA-tagging system. Analysis using a synthetic pD1 oligopeptide demonstrated that the enzymatic properties (including substrate recognition) of overexpressed CtpA with an extra sequence of GSHMLE at the N terminus were indistinguishable from those of the native enzyme. CtpA was insensitive to penem, which has been shown to inhibit some Ser/Lys-type proteases, suggesting that the catalytic center of CtpA is quite unique. By using the substrate in different molecular environments (i.e. synthetic pD1 oligopeptide in solution and pD1 in photosystem II-enriched thylakoid membrane), we observed a dramatic difference in the pH profile and affinity for the substrate, suggesting the presence of a specific interaction of CtpA with a factor(s) that modulates the pH dependence of proteolytic action in response to physiological conditions. The D1 protein is a membrane-spanning subunit constituting the core part of the photosystem II (PSII) 1 reaction center (1, 2). In the predicted secondary structure, the D1 protein consists of five transmembrane ␣-helices and a C-terminal extension protruding into the luminal space of thylakoids (reviewed in Ref. 2). A curious feature of this protein is the extraordinarily high rate of metabolic turnover in vivo, despite its fundamental importance in the structure and function of PSII (3). In the dynamic process, the D1 protein, which is encoded by the psbA gene in the plastid genome in the case of eukaryotic organisms, is synthesized on membrane-associated ribosomes on the cytosolic or stromal surface of thylakoids in a precursor form with a short C-terminal extension consisting of 8 -16 amino acids (4 -6). This part of the protein is co-translationally translocated into the luminal space and then immediately removed by the action of an endopeptidase called the C-terminal processing protease (CtpA) (7-10). In eukaryotic organisms, the enzyme is nuclear encoded (8 -10) and imported from the cytosol to thylakoidal lumen (11). When present on the D1 protein, this extension's removal is absolutely essential for the integration of the machinery for water oxidation, i.e. the manganese cluster in PSII (12-15). However, the absence of this extension by itself at least under the conditions exam...

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“…The predicted signal sequence indicates that CtpA is transported across the cytoplasmic membrane and thus suggests that the C-terminal processing by CtpA may occur in the periplasmic space. Other characterized CtpA proteases also contain N-terminal signal sequences (31,36). In eukaryotic organisms, the enzyme is encoded in the nucleus and is imported from the cytosol to the thylakoid lumen of chloroplasts (41).…”

Section: Discussionmentioning

confidence: 99%

Pleiotropic Effects of Inactivating a Carboxyl-Terminal Protease, CtpA, in Borrelia burgdorferi

et al. 2004

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A gene encoding a putative carboxyl-terminal protease (CtpA), an unusual type of protease, is present in the Borrelia burgdorferi B31 genome. The B. burgdorferi CtpA amino acid sequence exhibits similarities to the sequences of the CtpA enzymes of the cyanobacterium Synechocystis sp. strain PCC 6803 and higher plants and also exhibits similarities to the sequences of putative CtpA proteins in other bacterial species. Here, we studied the effect of ctpA gene inactivation on the B. burgdorferi protein expression profile. Total B. burgdorferi proteins were separated by two-dimensional gel electrophoresis, and the results revealed that six proteins of the wild type were not detected in the ctpA mutant and that nine proteins observed in the ctpA mutant were undetectable in the wild type. Immunoblot analysis showed that the integral outer membrane protein P13 was larger and had a more acidic pI in the ctpA mutant, which is consistent with the theoretical change in pI for P13 not processed at the carboxyl terminus. Matrix-assisted laser desorption ionization-time of flight data indicated that in addition to P13, the BB0323 protein may serve as a substrate for carboxyl-terminal processing by CtpA. Complementation analysis of the ctpA mutant provided strong evidence that the observed effect on proteins depended on inactivation of the ctpA gene alone. We show that CtpA in B. burgdorferi is involved in the processing of proteins such as P13 and BB0323 and that inactivation of ctpA has a pleiotropic effect on borrelial protein synthesis. To our knowledge, this is the first analysis of both a CtpA protease and different substrate proteins in a pathogenic bacterium.

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Transport of CtpA Protein from the Cyanobacterium Synechocystis 6803 Across the Thylakoid Membrane in Chloroplasts

Cited by 17 publications

References 49 publications

The Rieske Fe/S Protein of the Cytochromeb /f Complex in Chloroplasts

The Rieske Fe/S Protein of the Cytochromeb /f Complex in Chloroplasts

Overexpression and Characterization of Carboxyl-terminal Processing Protease for Precursor D1 Protein

Pleiotropic Effects of Inactivating a Carboxyl-Terminal Protease, CtpA, in Borrelia burgdorferi

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