| 1. |
- Alsterfjord, Magnus, et al.
(författare)
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Plasma membrane H+-ATPase and 14-3-3 Isoforms of Arabidopsis leaves: Evidence for isoform specificity in the 14-3-3/H+-ATPase interaction
- 2004
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Ingår i: Plant and Cell Physiology. - : Oxford University Press (OUP). - 1471-9053 .- 0032-0781. ; 45:9, s. 1202-1210
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Tidskriftsartikel (refereegranskat)abstract
- The plasma membrane H+-ATPase is activated by binding of 14-3-3 protein to the phosphorylated C terminus. Considering the large number of 14-3-3 and H+-ATPase isoforms in Arabidopsis (13 and 11 expressed genes, respectively), specificity in binding may exist between 14-3-3 and H+-ATPase isoforms. We now show that the H'-ATPase is the main target for 14-3-3 binding at the plasma membrane, and that all twelve 14-3-3 istiforms tested bind to the H+-ATPase in vitro. Using specific antibodies for nine of the 14-3-3 isoforms, we show that GF14epsilon, mu, lambda, omega, chi, phi, nu, and upsilon are present in leaves, but that isolated plasma membranes lack GF14chi, phi and upsilon. Northern blots using isoform-specific probes for all 14-3-3 and H+-ATPase isoforms showed that transcripts were present for most of the isoforms. Based on mRNA levels, GF14epsilon, mu, lambda and chi are highly expressed 14-3-3 isoforms, and AHA1, 3, and 11 highly expressed H+-ATPase isoforms in leaves. However, mass peptide fingerprinting identified AHA1 and 2 with the highest score, and their presence could be confirmed by MS/MS. It may be calculated that under 'unstressed' conditions less than one percent of total 14-3-3 is attached to the H+-ATPase. However, during a condition requiring full activation of H+ pumping, as induced here by the presence of the fungal toxin fusicoccin, several percent of total 14-3-3 may be engaged in activation of the H+-ATPase.
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| 2. |
- Sehnke, PC, et al.
(författare)
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Evolution and isoform specificity of plant 14-3-3 proteins
- 2002
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Ingår i: Plant Molecular Biology. - 1573-5028. ; 50:6, s. 1011-1018
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Tidskriftsartikel (refereegranskat)abstract
- The 14-3-3 proteins, once thought of as obscure mammalian brain proteins, are fast becoming recognized as major regulators of plant primary metabolism and of other cellular processes. Their presence as large gene families in plants underscores their essential role in plant physiology. We have examined the Arabidopsis thaliana 14-3-3 gene family, which currently is the largest and most complete 14-3-3 family with at least 12 expressed members and 15 genes from the now completed Arabidopsis thaliana genome project. The phylogenetic branching of this family serves as the prototypical model for comparison with other large plant 14-3-3 families and as such may serve to rationalize clustering in a biological context. Equally important for ascribing common functions for the various 14-3-3 isoforms is determining an isoform-specific correlation with localization and target partnering. A summary of localization information available in the literature is presented. In an effort to identify specific 14-3-3 isoform location and participation in cellular processes, we have produced a panel of isoform-specific antibodies to Arabidopsis thaliana 14-3-3s and present initial immunolocalization studies that suggest biologically relevant, discriminative partnering of 14-3-3 isoforms.
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| 3. |
- Alsterfjord, Magnus
(författare)
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The Arabidopsis 14-3-3 Family - Evolution, Expression, Localization, and Target Specificity
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In plants, 14-3-3 proteins are key regulators of primary metabolism and membrane transport. It was previously thought that the 14-3-3 isoforms were not very specific with regard to target proteins, but more recent data suggest that the specificity may be high. Therefore, identification, localization, and characterization of all 14-3-3 isoforms in the model plant Arabidopsis are important. We identified all 14-3-3 genes in the fully sequenced Arabidopsis genome and partly characterized and localized the novel isoforms. We found three new 14-3-3 genes and determined the total number of genes to 15. Two of the novel genes were found to be transcribed and named grf11and 12, and the corresponding new 14-3-3 isoforms were named omicron and iota, respectively, and thereby 13 genes have now been shown to be expressed. Omicron is expressed in leaves, roots, and flowers, whereas the gene coding for iota is specifically expressed in flowers. We furthermore analysed the evolutionary history of the 14-3-3 family in plants, and particularly in Arabidopsis, which currently is the largest and most complete 14-3-3 family. We then used the interaction between 14-3-3 and the plasma membrane H+ pumping ATPase in Arabidopsis as a model system to elucidate potential isoform specificity in 14-3-3 binding. Considering the large number of 14-3-3 and H+-ATPase isoforms in Arabidopsis (13 and 11 expressed genes, respectively), specificity in binding may exist between 14-3-3 and H+-ATPase isoforms. We could show that the H+-ATPase is the main target for 14-3-3 binding at the plasma membrane, and that all twelve 14-3-3 isoforms tested bind to the H+-ATPase in vitro. Mass peptide fingerprinting identified two H+-ATPase isoforms, AHA1 and 2, as major isoforms in leaf plasma membranes. Using specific antibodies for nine of the 14-3-3 isoforms, we could show that most of the 14-3-3 isoforms are present in leaves, but that isolated plasma membranes lacked three of these isoforms, which suggests some specificity in the 14-3-3/ H+-ATPase interaction in vivo. We could also calculate that under ?unstressed? conditions less than one percent of total 14-3-3 in the cell is attached to the H+-ATPase. However, during a condition requiring full activation of H+ pumping several percent of total 14-3-3 may be engaged in activation of the H+-ATPase.
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| 4. |
- Butler, Éile, et al.
(författare)
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Proteins of novel lactic acid bacteria from Apis mellifera mellifera: an insight into the production of known extra-cellular proteins during microbial stress
- 2013
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Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Background: Lactic acid bacteria (LAB) has been considered a beneficial bacterial group, found as part of the microbiota of diverse hosts, including humans and various animals. However, the mechanisms of how hosts and LAB interact are still poorly understood. Previous work demonstrates that 13 species of Lactobacillus and Bifidobacterium from the honey crop in bees function symbiotically with the honeybee. They protect each other, their hosts, and the surrounding environment against severe bee pathogens, bacteria, and yeasts. Therefore, we hypothesized that these LAB under stress, i.e. in their natural niche in the honey crop, are likely to produce bioactive substances with antimicrobial activity. Results: The genomic analysis of the LAB demonstrated varying genome sizes ranging from 1.5 to 2.2 mega-base pairs (Mbps) which points out a clear difference within the protein gene content, as well as specialized functions in the honeybee microbiota and their adaptation to their host. We demonstrate a clear variation between the secreted proteins of the symbiotic LAB when subjected to microbial stressors. We have identified that 10 of the 13 LAB produced extra-cellular proteins of known or unknown function in which some are arranged in interesting putative operons that may be involved in antimicrobial action, host interaction, or biofilm formation. The most common known extra-cellular proteins secreted were enzymes, DNA chaperones, S-layer proteins, bacteriocins, and lysozymes. A new bacteriocin may have been identified in one of the LAB symbionts while many proteins with unknown functions were produced which must be investigated further. Conclusions: The 13 LAB symbionts likely play different roles in their natural environment defending their niche and their host and participating in the honeybee's food production. These roles are partly played through producing extracellular proteins on exposure to microbial stressors widely found in natural occurring flowers. Many of these secreted proteins may have a putative antimicrobial function. In the future, understanding these processes in this complicated environment may lead to novel applications of honey crop LAB proteins.
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| 5. |
- Olofsson, Tobias, et al.
(författare)
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Lactobacillus apinorum sp. nov., Lactobacillus mellifer sp. nov., Lactobacillus mellis sp. nov., Lactobacillus melliventris sp. nov., Lactobacillus kimbladii sp. nov., Lactobacillus helsingborgensis sp. nov., and Lactobacillus kullabergensis sp. nov., isolated from the honey stomach of the honeybee Apis mellifera.
- 2014
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Ingår i: International Journal of Systematic and Evolutionary Microbiology. - : Microbiology Society. - 1466-5026 .- 1466-5034. ; 64, s. 3109-3119
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Tidskriftsartikel (refereegranskat)abstract
- We discovered a symbiotic lactic acid bacterial (LAB) microbiota in the honey stomach of the honeybee Apis mellifera. The microbiota was composed of several phylotypes of Bifidobacterium and Lactobacillus. 16S ribosomal ribonucleic acid (rRNA) gene analyses and phenotypic and genetic characteristics revealed that the Lactobacillus phylotypes isolated represent seven novel species. One is grouped with Lactobacillus kunkeei and the others belong to the Lactobacillus buchneri and Lactobacillus delbrueckii subgroups of Lactobacillus. We propose the names Lactobacillus apinorum sp. nov., Lactobacillus mellifer sp. nov., Lactobacillus mellis sp. nov., Lactobacillus melliventris sp. nov., Lactobacillus kimbladii sp. nov., Lactobacillus helsingborgensis sp. nov., and Lactobacillus kullabergensis sp. nov., with the respective type strains being Fhon13NT ( = DSM 26257T = CCUG 63287T), Bin4NT ( = DSM 26254T = CCUG 63291T), Hon2NT ( = DSM 26255T = CCUG 63289T), Hma8NT ( = DSM 26256T = CCUG 63629T), Hma2NT ( = DSM 26263T = CCUG 63633T), Bma5NT ( = DSM 26265T = CCUG 63301T) and Biut2NT ( = DSM 26262T = CCUG 63631T).
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| 6. |
- Otterhag, L, et al.
(författare)
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Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase
- 2006
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Ingår i: Biochimie. - : Elsevier BV. - 1638-6183 .- 0300-9084. ; 88:1, s. 11-21
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Tidskriftsartikel (refereegranskat)abstract
- The A rabidopsis thaliana protein kinase AtPDK1 was identified as a homologue of the mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), which is involved in a number of physiological processes including cell growth and proliferation. We now show that AtPDK1 expressed in E. coli as a recombinant protein, undergoes autophosphorylation at several sites. Using mass spectrometry, three phosphorylated amino acid residues, Ser-177, Ser-276 and Ser-382, were identified, followed by mutational analyses to reveal their roles. These residues are not conserved in mammalian PDK1s. Mutation of Ser-276 in AtPDK1 to alanine resulted in an enzyme with no detectable autophosphorylation. Autophosphorylation was significantly reduced in the Ser177Ala mutant but was only slightly reduced in the Ser382Ala mutant. Other identified sites of importance for autophosphorylation and/or activity of AtPDK1 were Asp-167, Thr-176, and Thr-211. Sites in the mammalian PDK1 corresponding to Asp- 167 and Thr-211 are essential for PDK1 autophosphorylation and activity. Autophosphorylation was absent in the Asp167Ala mutant while the Thr176Ala and The211Ala mutants exhibited very low but detectable autophosphorylation, pointing to both similarity and difference between mammalian and plant enzymes. We also demonstrate that AtS6k2, an A. thaliana homologue to the mammalian S6 kinases, is an in vitro tar et of AtPDK1. Our data clearly show that Asp- 167, Thr-176, Ser-177, Thr-211, and Ser-276 in AtPDK1 are important for the downstream phosphorylation of AtS6k2. The results confirm that AtPDK1, like mammalian PDK1, needs phosphorylation at several sites for full downstream phosphorylation activity. Finally, we investigated A. thaliana 14-3-3 proteins as potential AtPDK1 regulatory proteins and the effect of phospholipids on the AtPDK1 activity. Nine of the 12 14-3-3 isoforms tested enhanced AtPDK1 activity whereas one isoform suppressed the activity. No significant effects on AtPDK1 activity by the various phospholipids (including phosphoinositides) were evident. (C) 2005 Elsevier SAS. All rights reserved.
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