Effect of K-/S- segments on subcellular localization and dimerization of wheat dehydrin WZY1-2.
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ABSTRACT:
SUBMITTER: Wang X
PROVIDER: S-EPMC7671062 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Dehydrins (DHNs), group 2 of late embryogenesis abundant (LEA) proteins, are up-regulated in most plants during cold, drought, heat, or salinity stress. All DHNs contain at least one K-segment, which is believed to play a significant role in DHN function by forming an amphipathic helix. In previous studies, wzy2, an YSK2-type DHN gene, was isolated from the Zhengyin 1 cultivar of Triticum aestivum under cold and drought stress treatment conditions. Four WZY2 truncated derivatives were constructed to knock out the K-, Y- or S-segment, which potentially affect the function of the protein. In vivo assays of Escherichia coli viability enhancement, in vitro lactate dehydrogenase (LDH) activity protection and ex vivo protein aggregation prevention assays revealed that WZY2 acted as a protectant and improved stress tolerance during temperature variation. The results also showed that unlike the truncated derivative without K-segments, the derivative containing two K-segments had remarkable effects that were similar to those of full-length WZY2, indicating that the K-segment is the major functional component of WZY2. Moreover, compared with the other segments, the first K-segment might be the most critical contributor to WZY2 functionality. In general, this work highlights the behavior of DHNs in relieving cold stress ex vivo and the contribution of the K-segment to DHN function.
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Project description:The C-terminal Eps 15 Homology Domain proteins (EHD1-4) play important roles in regulating endocytic trafficking. EHD2 is the only family member whose crystal structure has been solved, and it contains an unstructured loop consisting of two proline-phenylalanine (PF) motifs: KPFRKLNPF. In contrast, despite EHD2 having nearly 70% amino acid identity with its paralogs, EHD1, EHD3 and EHD4, the latter proteins contain a single KPF or RPF motif, but no NPF motif. In this study, we sought to define the precise role of each PF motif in EHD2's homo-dimerization, binding with the protein partners, and subcellular localization. To test the role of the NPF motif, we generated an EHD2 NPF-to-NAF mutant to mimic the homologous sequences of EHD1 and EHD3. We demonstrated that this mutant lost both its ability to dimerize and bind to Syndapin2. However, it continued to localize primarily to the cytosolic face of the plasma membrane. On the other hand, EHD2 NPF-to-APA mutants displayed normal dimerization and Syndapin2 binding, but exhibited markedly increased nuclear localization and reduced association with the plasma membrane. We then hypothesized that the single PF motif of EHD1 (that aligns with the KPF of EHD2) might be responsible for both binding and localization functions of EHD1. Indeed, the EHD1 RPF motif was required for dimerization, interaction with MICAL-L1 and Syndapin2, as well as localization to tubular recycling endosomes. Moreover, recycling assays demonstrated that EHD1 RPF-to-APA was incapable of supporting normal receptor recycling. Overall, our data suggest that the EHD2 NPF phenylalanine residue is crucial for EHD2 localization to the plasma membrane, whereas the proline residue is essential for EHD2 dimerization and binding. These studies support the recently proposed model in which the EHD2 N-terminal region may regulate the availability of the unstructured loop for interactions with neighboring EHD2 dimers, thus promoting oligomerization.
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Project description:Dehydrins are a family of plant proteins that accumulate in response to dehydration stresses, such as low temperature, drought, high salinity, or during seed maturation. We have previously constructed cDNA libraries from Rhododendron catawbiense leaves of naturally non-acclimated (NA; leaf LT50, temperature that results in 50% injury of maximum, approximately -7°C) and cold-acclimated (CA; leaf LT50 approximately -50°C) plants and analyzed expressed sequence tags (ESTs). Five ESTs were identified as dehydrin genes. Their full-length cDNA sequences were obtained and designated as RcDhn 1-5. To explore their functionality vis-à-vis winter hardiness, their seasonal expression kinetics was studied at two levels. Firstly, in leaves of R. catawbiense collected from the NA, CA, and de-acclimated (DA) plants corresponding to summer, winter and spring, respectively. Secondly, in leaves collected monthly from August through February, which progressively increased freezing tolerance from summer through mid-winter. The expression pattern data indicated that RcDhn 1-5 had 6- to 15-fold up-regulation during the cold acclimation process, followed by substantial down-regulation during deacclimation (even back to NA levels for some). Interestingly, our data shows RcDhn 5 contains a histidine-rich motif near N-terminus, a characteristic of metal-binding dehydrins. Equally important, RcDhn 2 contains a consensus 18 amino acid sequence (i.e., ETKDRGLFDFLGKKEEEE) near the N-terminus, with two additional copies upstream, and it is the most acidic (pI of 4.8) among the five RcDhns found. The core of this consensus 18 amino acid sequence is a 11-residue amino acid sequence (DRGLFDFLGKK), recently designated in the literature as the F-segment (based on the pair of hydrophobic F residues it contains). Furthermore, the 208 orthologs of F-segment-containing RcDhn 2 were identified across a broad range of species in GenBank database. This study expands our knowledge about the types of F-segment from the literature-reported single F-segment dehydrins (FSKn) to two or three F-segment dehydrins: Camelina sativa dehydrin ERD14 as F2S2Kn type; and RcDhn 2 as F3SKn type identified here. Our results also indicate some consensus amino acid sequences flanking the core F-segment in dehydrins. Implications for these cold-responsive RcDhn genes in future genetic engineering efforts to improve plant cold hardiness are discussed.
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Project description:BackgroundNitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme formed by an α- and a β₁-subunit. A splice variant (C-α₁) of the α₁-subunit, lacking at least the first 236 amino acids has been described by Sharina et al. 2008 and has been shown to be expressed in differentiating human embryonic cells. Wagner et al. 2005 have shown that the amino acids 61-128 of the α₁-subunit are mandatory for quantitative heterodimerization implying that the C-α₁-splice variant should lose its capacity to dimerize quantitatively.Methodology/principal findingsIn the current study we demonstrate preserved quantitative dimerization of the C-α₁-splice by co-purification with the β₁-subunit. In addition we used fluorescence resonance energy transfer (FRET) based on fluorescence lifetime imaging (FLIM) using fusion proteins of the β₁-subunit and the α₁-subunit or the C-α₁ variant with ECFP or EYFP. Analysis of the respective combinations in HEK-293 cells showed that the fluorescence lifetime was significantly shorter (≈0.3 ns) for α₁/β₁ and C-α₁/β₁ than the negative control. In addition we show that lack of the amino-terminus in the α₁ splice variant directs it to a more oxidized subcellular compartment.Conclusions/significanceWe conclude that the amino-terminus of the α₁-subunit is dispensable for dimerization in-vivo and ex-vivo, but influences the subcellular trafficking.
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Project description:Physiological parameters and expression levels of drought related genes were analyzed in early vegetative stage of two bread wheat cultivars (Sids and Gmiza) differ in drought tolerance capacity. Both cultivars were imposed to gradual water depletion started on day 17 till day 32 after sowing. Sids, the more tolerant cultivar to drought showed higher fresh and dry weights than the drought sensitive genotype, Gmiza. Under water stress, Sids had higher membrane stability index (MSI), lower accumulated H2O2 and higher activity of the antioxidant enzymes; catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX) and superoxide dismutase (SOD) than Gmiza. On the other hand, the differential expression patterns of the genes dhn, wcor and dreb were observed due to water deficit intensity according to cultivar's tolerance to drought. The DNA sequence alignment of dun showed high similarity of about 80-92% identities with other related plants. The most striking overall observed trend was the highly induction in the expression of dun, wcor and dreb in leaves of the tolerant genotype, Sids under severe water stress.
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Project description:1. The subcellular distribution of transglutaminase was investigated by using the analytical approach of differential and isopycnic centrifugation as applied to three organs of the rat: liver, kidney and lung. After differential centrifugation by the method of de Duve, Pressman, Gianetto, Wattiaux & Appelmans [(1955) Biochem. J. 63, 604-617], transglutaminase is mostly recovered in the unsedimentable fraction S and the nuclear fraction N. After isopycnic centrifugation of the N fraction in a sucrose density gradient, a high proportion of the enzyme remains at the top of the gradient; a second but minor peak of activity is present in high-density regions, where a small proportion of 5'-nucleotidase, a plasma-membrane marker, is present together with a large proportion of collagen recovered in that fraction. 2. Fractions where a peak of transglutaminase was apparent in the sucrose gradient were examined by electron microscopy. The main components are large membrane sheets with extracellular matrix and free collagen fibers. 3. As these results seem to indicate that some correlation exists between particulate transglutaminase distribution and those of collagen and plasma membranes, the possible binding of transglutaminase by collagen (type I) and by purified rat liver plasma membrane was investigated. 4. The binding studies indicated that collagen is able to bind transglutaminase and to make complexes with plasma-membrane fragments whose density is higher than that of plasma-membrane fragments alone. Transglutaminase cannot be removed from such complexes by 1% Triton X-100, but can be to a relatively large extent by 0.5 M-KCl and by 50% (w/v) glycerol. 5. Such results suggest that the apparent association of transglutaminase with plasma membrane originates from binding in vitro of the cytosolic enzyme to plasma membrane bound to collagen, which takes place during homogenization of the tissue, when the soluble enzyme and extracellular components are brought together.
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Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Piero Carninci mailto:carninci@riken.jp). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track shows 5' cap analysis gene expression (CAGE) tags and clusters in RNA extracts (http://hgwdev.cse.ucsc.edu/cgi-bin/hgEncodeVocab?type=rnaExtract) from different sub-cellular localizations (http://hgwdev.cse.ucsc.edu/cgi-bin/hgEncodeVocab?type=localization) in multiple cell lines (http://hgwdev.cse.ucsc.edu/cgi-bin/hgEncodeVocab?type=cellType). A CAGE cluster is a region of overlapping tags with an assigned value that represents the expression level. The data in this track were produced as part of the ENCODE Transcriptome Project. Release 2 has three new downloads only files per experiment (Clusters, TSS Gencode 7 and TSS HMM) and four new cell lines (A459, AG04450, BJ and SK-N-SH_RA). Release 1 on hg19 contained the original data on hg18 (http://hgwdev.cse.ucsc.edu/cgi-bin/hgTrackUi?db=hg18&g=wgEncodeRikenCage) that was remapped and indicated in this release as Generation 0 since that data had no replicates. If there is both old and new generation data available for a particular experiment, only the new generation data is displayed and the older data is available for download. The new data for this track was done with a different process and has standard replicate numbers. The replicate labeling in the genome browser view is a counter indicating the total number of replicates submitted. The producing lab has replicate numbers that correspond to their internal bio-replicate numbering. Where these two numbering systems conflict, both are listed in the long label of the specific track. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell). RNA molecules longer than 200 nt were isolated from each subcellular compartment and then were fractionated into polyA+ and polyA- fractions as described in these protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/general/rnaExtracts.txt). The CAGE tags were sequenced from the 5' ends of cap-trapped cDNAs produced using RIKEN CAGE technology (Kodzius et al. 2006; Valen et al. 2009). To create the tag, a linker was attached to the 5' end of polyA+ or polyA- reverse-transcribed cDNAs which were selected by cap trapping (Carninci et al. 1996). The first 27 bp of the cDNA were cleaved using class II restriction enzymes. A linker was then attached to the 3' end of the cDNA. After PCR amplification, the tags were sequenced (36 bp single reads) using Illumina's Genome analyzer. Tags were mapped to the human genome (hg19) using the program delve (T. Lassmann manuscript in preparation). Delve is a new probabilistic aligner focused on giving the best possible alignment of reads to a genome rather than focusing on speed. It calculates the mapping accuracy (probability of each alignment being true or not) for each alignment. There is no set limit on the number of errors allowed and therefore the mapping rate is commonly 100%. However, for analysis it is recommended to discard alignments with low mapping qualities. Exceptions to the above protocol are the polyA- RNA samples from K562 cytosol, K562 nucleus, and prostate whole cell which were sequenced using ABI SOLiD (http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing.html) technology. These reads were mapped using Bowtie using default parameters. Clusters were defined as regions of overlapping CAGE reads. The expression level was computed as the number of reads making up the cluster, divided by the total number of reads sequenced, times 1 million.
2011-12-16 | E-GEOD-34448 | biostudies-arrayexpress