| 1. |
- Baba, Kyoko, et al.
(författare)
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Organellar gene transcription and early seedling development are affected in the rpoT;2 mutant of Arabidopsis.
- 2004
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Ingår i: Plant J. - 0960-7412. ; 38:1, s. 38-48
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Tidskriftsartikel (refereegranskat)abstract
- An Arabidopsis mutant that exhibited reduced root length was isolated from a population of activation-tagged T-DNA insertion lines in a screen for aberrant root growth. This mutant also exhibited reduced hypocotyl length as well as a delay in greening and altered leaf shape. Molecular genetic analysis of the mutant indicated a single T-DNA insertion in the gene RpoT;2 encoding a homolog of the phage-type RNA polymerase (RNAP), that is targeted to both mitochondria and plastids. A second T-DNA-tagged allele also showed a similar phenotype. The mutation in RpoT;2 affected the light-induced accumulation of several plastid mRNAs and proteins and resulted in a lower photosynthetic efficiency. In contrast to the alterations in the plastid gene expression, no major effect of the rpoT;2 mutation on the accumulation of examined mitochondrial gene transcripts and proteins was observed. The rpoT;2 mutant exhibited tissue-specific alterations in the transcript levels of two other organelle-directed nuclear-encoded RNAPs, RpoT;1 and RpoT;3. This suggests the existence of cross-talk between the regulatory pathways of the three RNAPs through organelle to nucleus communication. These data provide an important information on a role of RpoT;2 in plastid gene expression and early plant development.
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| 2. |
- Blanco-Rivero, Amaya, et al.
(författare)
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Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii
- 2012
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Ingår i: PLOS ONE. - : Public library science. - 1932-6203. ; 7:11
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Tidskriftsartikel (refereegranskat)abstract
- Background: Cah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO2 fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO2 conditions. Results/Conclusions: In the present work we demonstrate that after transfer to low CO2, Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO2 conditions, the Cah3 activity increased about 5-6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO2 conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO2 conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO2 the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules. Significance: This is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.
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| 3. |
- Burén, Stefan, et al.
(författare)
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Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana
- 2011
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 6:6, s. e21021-
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundThe Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells.Methodology/Principal FindingsTransient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited.Conclusions/SignificanceWe show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.
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| 4. |
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| 7. |
- Buren, Stefan, et al.
(författare)
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Use of the foot-and-mouth disease virus 2A peptide co-expression system to study intracellular protein trafficking in arabidopsis
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:12, s. e51973-
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Tidskriftsartikel (refereegranskat)abstract
- Background: A tool for stoichiometric co-expression of effector and target proteins to study intracellular protein trafficking processes has been provided by the so called 2A peptide technology. In this system, the 16-20 amino acid 2A peptide from RNA viruses allows synthesis of multiple gene products from single transcripts. However, so far the use of the 2A technology in plant systems has been limited.Methodology/Principal Findings: The aim of this work was to assess the suitability of the 2A peptide technology to study the effects exerted by dominant mutant forms of three small GTPase proteins, RABD2a, SAR1, and ARF1 on intracellular protein trafficking in plant cells. Special emphasis was given to CAH1 protein from Arabidopsis, which is trafficking to the chloroplast via a poorly characterized endoplasmic reticulum-to-Golgi pathway. Dominant negative mutants for these GTPases were co-expressed with fluorescent marker proteins as polyproteins separated by a 20 residue self-cleaving 2A peptide. Cleavage efficiency analysis of the generated polyproteins showed that functionality of the 2A peptide was influenced by several factors. This enabled us to design constructs with greatly increased cleavage efficiency compared to previous studies. The dominant negative GTPase variants resulting from cleavage of these 2A peptide constructs were found to be stable and active, and were successfully used to study the inhibitory effect on trafficking of the N-glycosylated CAH1 protein through the endomembrane system.Conclusions/Significance: We demonstrate that the 2A peptide is a suitable tool when studying plant intracellular protein trafficking and that transient protoplast and in planta expression of mutant forms of SAR1 and RABD2a disrupts CAH1 trafficking. Similarly, expression of dominant ARF1 mutants also caused inhibition of CAH1 trafficking to a different extent. These results indicate that early trafficking of the plastid glycoprotein CAH1 depends on canonical vesicular transport mechanisms operating between the endoplasmic reticulum and Golgi apparatus.
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| 8. |
- Kupriyanova, Elena, et al.
(författare)
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Extracellular carbonic anhydrases of the stromatolite-forming cyanobacterium Microcoleus chthonoplastes.
- 2007
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Ingår i: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 153:Pt 4, s. 1149-56
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Tidskriftsartikel (refereegranskat)abstract
- Active extracellular carbonic anhydrases (CAs) were found in the alkaliphilic stromatolite-forming cyanobacterium Microcoleus chthonoplastes. Enzyme activity was detected in intact cells and in the cell envelope fraction. Western blot analysis of polypeptides from the cell envelope suggested the presence of at least two polypeptides cross-reacting with antibodies against both {alpha} and beta classes of CA. Immunocytochemical analysis revealed putative {alpha}-CA localized in the glycocalyx. This {alpha}-CA has a molecular mass of about 34 kDa and a pI of 3.5. External CAs showed two peaks of activity at around pH 10 and 7.5. The possible involvement of extracellular CAs of M. chthonoplastes in photosynthetic assimilation of inorganic carbon and its relationship to CaCO3 deposition during mineralization of cyanobacterial cells are discussed.
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| 9. |
- Moskvin, O V, et al.
(författare)
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Carbonic anhydrase activities in pea thylakoids. : A photosystem II core complex-associated carbonic anhydrase
- 2004
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Ingår i: Photosynthesis Research. - 0166-8595. ; 79:1, s. 93-100
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Tidskriftsartikel (refereegranskat)abstract
- Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 µmol H+ (mg Chl)–1 h–1 at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal beta-CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO3 – dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 µmol H+ (mg Chl)–1 h–1) were observed in the presence of detergents (Triton X-100 or n-octyl-beta-D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 µmol H+ (mg Chl)–1 h–1 which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.
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| 10. |
- Rouhier, Nicolas, et al.
(författare)
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Identification of plant glutaredoxin targets
- 2005
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Ingår i: Antioxidants and Redox Signaling. - Larchmont, NY : Mary Ann Liebert. - 1523-0864 .- 1557-7716. ; 7:7-8, s. 919-929
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Tidskriftsartikel (refereegranskat)abstract
- Glutaredoxins (Grxs) are small ubiquitous proteins of the thioredoxin (Trx) family, which catalyze dithiol–disulfide exchange reactions or reduce protein-mixed glutathione disulfides. In plants, several Trx-interacting proteins have been isolated from different compartments, whereas very few Grx-interacting proteins are known. We describe here the determination of Grx target proteins using a mutated poplar Grx, various tissular and subcellular plant extracts, and liquid chromatography coupled to tandem mass spectrometry detection. We have identified 94 putative targets, involved in many processes, including oxidative stress response [peroxiredoxins (Prxs), ascorbate peroxidase, catalase], nitrogen, sulfur, and carbon metabolisms (methionine synthase, alanine aminotransferase, phosphoglycerate kinase), translation (elongation factors E and Tu), or protein folding (heat shock protein 70). Some of these proteins were previously found to interact with Trx or to be glutathiolated in other organisms, but others could be more specific partners of Grx. To substantiate further these data, Grx was shown to support catalysis of the stroma β-type carbonic anhydrase and Prx IIF of Arabidopsis thaliana, but not of poplar 2-Cys Prx. Overall, these data suggest that the interaction could occur randomly either with exposed cysteinyl disulfide bonds formed within or between target proteins or with mixed disulfides between a protein thiol and glutathione.
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