| 1. |
- Kjellbom, Per, et al.
(författare)
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Cytochrome P-450/420 in plant plasma membranes : a possible component of the blue-light-reducible flavoprotein-cytochrome complex
- 1985
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Ingår i: Photochemistry and Photobiology. - : Wiley. - 0031-8655 .- 1751-1097. ; 42:6, s. 779-783
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Tidskriftsartikel (refereegranskat)abstract
- Carbon monoxide difference spectra and pyridine binding spectra indicate the presence of cytochrome P-450/420 in plasma membranes from cauliflower inflorescences. Mild lithium dodecylsulfate polyacrylamide gel electrophoresis shows only one heme staining band in the plasma membrane fraction at an apparent molecular weight of 93 kiloDalton. This band is suggested to be due to a cytochrome P-450/420 dimer, in view of the known molecular weights of animal cytochromes P-450/420. The plasma membrane-bound cytochrome P-450/420 is probably identical to the blue-light-reducible b-type cytochrome of plant plasma membranes, which has been inferred to have a role in photomorphogenesis.
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| 2. |
- Agemark, Maria, et al.
(författare)
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Reconstitution of water channel function and 2D-crystallization of human aquaporin 8.
- 2012
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 0005-2736. ; 1818:3, s. 839-850
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Tidskriftsartikel (refereegranskat)abstract
- Among the thirteen human aquaporins (AQP0-12), the primary structure of AQP8 is unique. By sequence alignment it is evident that mammalian AQP8s form a separate subfamily distinct from the other mammalian aquaporins. The constriction region of the pore determining the solute specificity deviates in AQP8 making it permeable to both ammonia and H(2)O(2) in addition to water. To better understand the selectivity and gating mechanism of aquaporins, high-resolution structures are necessary. So far, the structure of one human aquaporin (HsAQP5) has been solved at atomic resolution. For mammalian aquaporins in general, high-resolution structures are only available for those belonging to the water-specific subfamily (including HsAQP5). Thus, it is of interest to solve structures of other aquaporin subfamily members with different solute specificities. To achieve this the aquaporins need to be overexpressed heterologously and purified. Here we use the methylotrophic yeast Pichia pastoris as a host for the overexpression. A wide screen of different detergents and detergent-lipid combinations resulted in the solubilization of functional human AQP8 protein and in well-ordered 2D crystals. It also became evident that removal of amino acids constituting affinity tags was crucial to achieve highly ordered 2D crystals diffracting to 3Å.
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| 3. |
- Alexandersson, Erik, et al.
(författare)
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Arabidopsis Plasma Membrane Proteomics Identifies Components of Transport, Signal Transduction and Membrane Trafficking
- 2004
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Ingår i: Plant and Cell Physiology. - : Oxford University Press (OUP). - 1471-9053 .- 0032-0781. ; 45:11, s. 1543-1556
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Tidskriftsartikel (refereegranskat)abstract
- In order to identify integral proteins and peripheral proteins associated with the plasma membrane, highly purified Arabidopsis plasma membranes from green tissue (leaves and petioles) were analyzed by mass spectrometry. Plasma membranes were isolated by aqueous two-phase partitioning, which yields plasma membrane vesicles with a cytoplasmic-side-in orientation and with a purity of 95%. These vesicles were turned inside-out by treatment with Brij 58 to remove soluble contaminating proteins enclosed in the vesicles and to remove loosely bound contaminating proteins. In total, 238 putative plasma membrane proteins were identified, of which 114 are predicted to have transmembrane domains or to be glycosyl phosphatidylinositol anchored. About two-thirds of the identified integral proteins have not previously been shown to be plasma membrane proteins. Of the 238 identified proteins, 76% could be classified according to function. Major classes are proteins involved in transport (17%), signal transduction (16%), membrane trafficking (9%) and stress responses (9%). Almost a quarter of the proteins identified in the present study are functionally unclassified and more than half of these are predicted to be integral.
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| 4. |
- Alexandersson, Erik, et al.
(författare)
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Plasma membrane proteomics
- 2006
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Ingår i: Plant Proteomics. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 9783540726166 - 9783540726173 ; , s. 186-206
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Bokkapitel (refereegranskat)abstract
- Proteins residing in the plasma membrane have key functions in transport, signal transduction, vesicle trafficking and many other important processes. To better understand these processes it is necessary to reveal the identity of plasma membrane proteins and to monitor modifications and regulation of their expression. This chapter is an overview of the methods used in plant plasma membrane proteomic studies and the results obtained so far. It focuses on studies using mass spectrometry for identification and includes aspects of plasma membrane fractionation, extraction and washing treatments, assessment of purity, separation methods for plasma membrane proteins and choice of techniques for protein cleavage. Finally, the results of plasma membrane proteomic studies are compared and problems with contaminating proteins are discussed.
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| 5. |
- Alexandersson, Erik, et al.
(författare)
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Purification and proteomic analysis of plant plasma membranes.
- 2008
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 432, s. 161-173
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Tidskriftsartikel (refereegranskat)abstract
- All techniques needed for proteomic analyses of plant plasma membranes are described in detail, from isolation of plasma membranes to protein identification by mass spectrometry (MS). Plasma membranes are isolated by aqueous two-phase partitioning yielding vesicles with a cytoplasmic side-in orientation and a purity of about 95%. These vesicles are turned inside-out by treatment with Brij 58, which removes soluble contaminating proteins enclosed in the vesicles as well as loosely attached proteins. The final plasma membrane preparation thus retains all integral proteins and many peripheral proteins. Proteins are separated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein bands are excised and digested with trypsin. Peptides in tryptic digests are separated by nanoflow liquid chromatography and either fed directly into an ESI-MS or spotted onto matrix-assisted laser desorption ionization (MALDI) plates for analysis with MALDI-MS. Finally, data processing and database searching are used for protein identification to define a plasma membrane proteome.
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| 6. |
- Alexandersson, Erik, et al.
(författare)
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Transcriptional regulation of aquaporins in accessions of Arabidopsis in response to drought stress.
- 2010
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Ingår i: Plant Journal. - 1365-313X. ; 61, s. 650-660
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Tidskriftsartikel (refereegranskat)abstract
- Summary Aquaporins facilitate water transport over cellular membranes and are therefore believed to play an important role in water homeostasis. In higher plants aquaporin-like proteins, also called major intrinsic proteins (MIPs), are divided into 5 subfamilies. We have previously shown that MIP transcription in Arabidopsis thaliana generally is down-regulated in leaves upon drought stress, apart from two members of the Plasma membrane Intrinsic Protein (PIP) subfamily, AtPIP1;4 and AtPIP2;5, which are up-regulated. In order to assess if this regulation is general or accession-specific we monitored gene expression of all PIPs in five Arabidopsis accessions. Overall drought regulation of PIPs was well conserved for all five accessions tested suggesting a general and fundamental physiological role of this drought response. In addition, significant differences among accessions were identified for transcripts of three PIP genes. Principal component analysis showed that most of the PIP transcriptional variation during drought stress could be explained by one variable linked to leaf water content. Promoter-GUS constructs of AtPIP1;4, AtPIP2;5 and also AtPIP2;6, which is unresponsive to drought stress, had distinct expression patterns concentrated to the base of the leaf petioles and parts of the flowers. The presence of drought stress response elements within the 1.6 kb promoter regions of AtPIP1;4 and AtPIP2;5, was demonstrated by comparing transcription of the promoter reporter construct and the endogenous gene upon drought stress. Analysis by ATTED-II and other web-based bioinformatical tools showed that several of the MIPs down-regulated upon drought are strongly co-expressed, whereas AtPIP1;4, AtPIP2;5 and AtPIP2;6 are not co-expressed.
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| 7. |
- Alexandersson, Erik, et al.
(författare)
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Whole gene family expression and drought stress regulation of aquaporins
- 2005
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Ingår i: Plant Molecular Biology. - : Springer Science and Business Media LLC. - 1573-5028 .- 0167-4412. ; 59:3, s. 469-484
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Tidskriftsartikel (refereegranskat)abstract
- Since many aquaporins (AQPs) act as water channels, they are thought to play an important role in plant water relations. It is therefore of interest to study the expression patterns of AQP isoforms in order to further elucidate their involvement in plant water transport. We have monitored the expression patterns of all 35 Arabidopsis AQPs in leaves, roots and flowers by cDNA microarrays, specially designed for AQPs, and by quantitative real-time reverse transcriptase PCR (Q-RT-PCR). This showed that many AQPs are pre-dominantly expressed in either root or flower organs, whereas no AQP isoform seem to be leaf specific. Looking at the AQP subfamilies, most plasma membrane intrinsic proteins (PIPs) and some tonoplast intrinsic proteins (TIPs) have a high level of expression, while NOD26-like proteins (NIPs) are present at a much lower level. In addition, we show that PIP transcripts are generally down-regulated upon gradual drought stress in leaves, with the exception of AtPIP1;4 and AtPIP2;5, which are up-regulated. AtPIP2;6 and AtSIP1;1 are constitutively expressed and not significantly affected by the drought stress. The transcriptional down-regulation of PIP genes upon drought stress could also be observed on the protein level.
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| 8. |
- Ampah-Korsah, Henry, et al.
(författare)
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Single amino acid substitutions in the selectivity filter render NbXIP1;1α aquaporin water permeable
- 2017
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Ingår i: BMC Plant Biology. - : Springer Science and Business Media LLC. - 1471-2229. ; 17:1, s. 61-61
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Aquaporins (AQPs) are integral membrane proteins that facilitate transport of water and/or other small neutral solutes across membranes in all forms of life. The X Intrinsic Proteins (XIPs) are the most recently recognized and the least characterized aquaporin subfamily in higher plants. XIP1s have been shown to be impermeable to water but permeable to boric acid, glycerol, hydrogen peroxide and urea. However, uncertainty regarding the determinants for selectivity and lack of an activity that is easy to quantify have hindered functional investigations. In an effort to resolve these issues, we set out to introduce water permeability in Nicotiana benthamiana XIP1;1α (NbXIP1;1α), by exchanging amino acid residues of predicted alternative aromatic/arginine (ar/R) selectivity filters of NbXIP1;1α for residues constituting the water permeable ar/R selectivity filter of AtTIP2;1.RESULTS: Here, we present functional results regarding the amino acid substitutions in the putative filters as well as deletions in loops C and D of NbXIP1;1α. In addition, homology models were created based on the high resolution X-ray structure of AtTIP2;1 to rationalize the functional properties of wild-type and mutant NbXIP1;1α. Our results favour Thr 246 rather than Val 242 as the residue at the helix 5 position in the ar/R filter of NbXIP1;1α and indicate that the pore is not occluded by the loops when heterologously expressed in Pichia pastoris. Moreover, our results show that a single amino acid substitution in helix 1 (L79G) or in helix 2 (I102H) is sufficient to render NbXIP1;1α water permeable. Most of the functional results can be rationalized from the models based on a combination of aperture and hydrophobicity of the ar/R filter.CONCLUSION: The water permeable NbXIP1;1α mutants imply that the heterologously expressed proteins are correctly folded and offer means to explore the structural and functional properties of NbXIP1;1α. Our results support that Thr 246 is part of the ar/R filter. Furthermore, we suggest that a salt bridge to an acidic residue in helix 1, conserved among the XIPs in clade B, directs the orientation of the arginine in the ar/R selectivity filter and provides a novel approach to tune the selectivity of AQPs.
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| 9. |
- Ampah-Korsah, Henry, et al.
(författare)
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The aquaporin splice variant NbXIP1;1α is permeable to boric acid and is Phosphorylated in the N-terminal domain
- 2016
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Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 7:JUNE2016
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel.
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| 10. |
- Anderberg, Hanna, et al.
(författare)
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Annotation of Selaginella moellendorffii Major Intrinsic Proteins and the Evolution of the Protein Family in Terrestrial Plants.
- 2012
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Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- Major intrinsic proteins (MIPs) also called aquaporins form pores in membranes to facilitate the permeation of water and certain small polar solutes across membranes. MIPs are present in virtually every organism but are uniquely abundant in land plants. To elucidate the evolution and function of MIPs in terrestrial plants, the MIPs encoded in the genome of the spikemoss Selaginella moellendorffii were identified and analyzed. In total 19 MIPs were found in S. moellendorffii belonging to 6 of the 7 MIP subfamilies previously identified in the moss Physcomitrella patens. Only three of the MIPs were classified as members of the conserved water specific plasma membrane intrinsic protein (PIP) subfamily whereas almost half were found to belong to the diverse NOD26-like intrinsic protein (NIP) subfamily permeating various solutes. The small number of PIPs in S. moellendorffii is striking compared to all other land plants and no other species has more NIPs than PIPs. Similar to moss, S. moellendorffii only has one type of tonoplast intrinsic protein (TIP). Based on ESTs from non-angiosperms we conclude that the specialized groups of TIPs present in higher plants are not found in primitive vascular plants but evolved later in a common ancestor of seed plants. We also note that the silicic acid permeable NIP2 group that has been reported from angiosperms appears at the same time. We suggest that the expansion of the number MIP isoforms in higher plants is primarily associated with an increase in the different types of specialized tissues rather than the emergence of vascular tissue per se and that the loss of subfamilies has been possible due to a functional overlap between some subfamilies.
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