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Characterization of a cDNA encoding a novel heat-shock protein that binds to calmodulin. 总被引:9,自引:0,他引:9 下载免费PDF全文
A cDNA clone (pTCB48) encoding a calmodulin-binding protein was isolated by screening a lambda ZAPII cDNA expression library constructed from cell cultures of heat-shocked tobacco (Nicotiana tabacum L. cv Wisconsin-38) with metabolically labeled [35S]calmodulin. Calmodulin gel overlay analysis indicated that pTCB48 generated major peptides of 53, 36, and 22 kD and two minor peptides of 37 and 16 kD that bound calmodulin in a Ca(2+)-dependent manner. Deletion analysis of pTCB48 indicated that these and the minor calmodulin-binding proteins resulted from the insert. A probe made from the cDNA insert recognized two bands with sizes of 2.1 and 1.8 kb on northern blot analysis. Both species of RNAs were undetectable in the control and were induced after 15 min of heat-shock treatment at 38 degrees C. The intensity of the two bands reached maximum after 1.5 h of heat-shock treatment. The cDNA clone was not full length; however, the complete sequence was determined by 5' rapid amplification of cDNA ends using nested antisense primers. The full-length cDNA contains 1648 bp and a single open reading frame of 1347 bp and is expected to encode a protein of approximately 50 kD. No significant homology with other reported genes and proteins was found. Structural predictions, deletion analysis, and gel overlay analysis suggested that the calmodulin-binding domain was a basic amphiphilic alpha-helix near the C terminus of the protein. The strong induction of the mRNA for this protein suggests a role for Ca2+/calmodulin-mediated process in the heat-shock response. 相似文献
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Kathare Praveen Kumar Dharmasiri Sunethra Arellano Israel Dharmasiri Nihal 《Plant Molecular Biology Reporter》2020,38(2):343-351
Plant Molecular Biology Reporter - Auxin plays crucial roles in modulating various aspects of plant growth and development throughout the plant life cycle. At the molecular level, auxin rapidly... 相似文献
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Dharmasiri N Dharmasiri S Weijers D Karunarathna N Jurgens G Estelle M 《The Plant journal : for cell and molecular biology》2007,52(1):114-123
Cullin-RING ubiquitin-protein ligases such as the Skp1, cullin, F-box protein (SCF) have been implicated in many growth and developmental processes in plants. Normal SCF function requires that the CUL1 subunit be post-translationally modified by related to ubiquitin (RUB), a protein related to ubiquitin. This process is mediated by two enzymes: the RUB-activating and RUB-conjugating enzymes. In Arabidopsis, the RUB-activating enzyme is a heterodimer consisting of AXR1 and ECR1. Mutations in the AXR1 gene result in a pleiotropic phenotype that includes resistance to the plant hormone auxin. Here we report that the AXL (AXR1-like) gene also functions in the RUB conjugation pathway. Overexpression of AXL in the axr1-3 background complements the axr1-3 phenotype. Biochemical analysis indicates that AXL overexpression restores CUL1 modification to the wild-type level, indicating that AXR1 and AXL have the same biochemical activity. Although the axl mutant resembles wild-type plants, the majority of axr1 axl-1 double mutants are embryo or seedling lethal. Furthermore, the axl-1 mutation reveals novel RUB-dependent processes in embryo development. We conclude that AXR1 and AXL function redundantly in the RUB conjugating pathway. 相似文献
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Heat shock modulates phosphorylation status and activity of nucleoside diphosphate kinase in cultured sugarcane cells 总被引:1,自引:0,他引:1
Sunethra Dharmasiri H. Michael Harrington Nihal Dharmasiri 《Plant cell reports》2010,29(11):1305-1314
Nucleoside diphosphate kinase (NDPK) is involved in the regeneration of nucleoside triphosphates (NTPs) through its phosphotransferase
activity via an autophosphorylating histidine residue. Additionally, autophosphorylation of serine and/or threonine residues
is documented for NDPKs from various organisms. However, the metabolic significance of serine/threonine phosphorylation has
not been well characterized. In this study we report the cloning and characterization of NDPKI from cultured sugarcane (Saccharum officinarum L. line H50-7209) cells, and modulation of serine autophosphorylation of NDPK1 in response to heat-shock (HS). Heat-shock
treatment at 40°C for 2 h resulted in a 40% reduction in labeled phosphoserine in NDPK1. This dephosphorylation was accompanied
by an increase in NDPK enzyme activity. In contrast, NDPK1 in cultured tobacco (cv. W-38) cells did not show changes in autophosphorylation
or increased enzyme activity in response to HS. The mRNA or protein level of NDPK1 did not increase in response to HS. Sugarcane
cells sustain the constitutive protein synthesis in addition to heat-shock protein synthesis during HS, while constitutive
protein synthesis is significantly reduced in tobacco cells during HS. Thus, HS modulation of NDPK1 activity and serine dephosphorylation
in sugarcane cells may represent an important physiological role in maintaining cellular metabolic functions during heat stress. 相似文献
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Auxin action in a cell-free system 总被引:24,自引:0,他引:24
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Mass spectrometric analysis of integral membrane proteins: application to complete mapping of bacteriorhodopsins and rhodopsin. 下载免费PDF全文
L. E. Ball J. E. Oatis Jr K. Dharmasiri M. Busman J. Wang L. B. Cowden A. Galijatovic N. Chen R. K. Crouch D. R. Knapp 《Protein science : a publication of the Protein Society》1998,7(3):758-764
Integral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC-electrospray ionization mass spectrometry (ESI-MS) and -tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has been applied to wild type (WT) bacteriorhodopsin (bR), cysteine containing mutants of bR, and the prototypical G-protein coupled receptor, rhodopsin (Rh). In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the protein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are separated by reversed phase HPLC using an isopropanol/acetonitrile/aqueous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of single amino acid fragments and the glycosylated fragment of Rh, which is observable by matrix assisted laser desorption ionization (MALDI)-MS, this system permits analysis of the entire protein in a single HPLC run. This methodology will enable pursuit of chemical modification and crosslinking studies designed to probe the three dimensional structures and functional conformational changes in these proteins. The approach should also be generally applicable to analysis of other integral membrane proteins. 相似文献
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Auxin signaling and regulated protein degradation 总被引:13,自引:0,他引:13