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Publikationen - Molekulare Signalverarbeitung

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Bürstenbinder, K.; Savchenko, T.; Müller, J.; Adamson, A.W.; Stamm, G.; Kwong, R.; Zipp, B.J.; Dhurvas Chandrasekaran, D. & Abel, S. Arabidopsis calmodulin-binding protein IQ67-domain 1 localizes to microtubules and interacts with kinesin light chain-related protein-1 J Biol Chem 288, 1871-1882, (2013) DOI: 10.1074/jbc.M112.396200

Calcium (Ca2+) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (Kd ∼ 0.6 μm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca2+/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins.

Ziegler, J.; Brandt, W.; Geißler, R.; Facchini, P. J. Removal of Substrate Inhibition and Increase in Maximal Velocity in the Short Chain Dehydrogenase/Reductase Salutaridine Reductase Involved in Morphine Biosynthesis J Biol Chem 284, 26758-26767, (2009) DOI: 10.1074/jbc.M109.030957

Salutaridine reductase (SalR, EC catalyzes the stereospecific reduction of salutaridine to 7(S)-salutaridinol in the biosynthesis of morphine. It belongs to a new, plant-specific class of short-chain dehydrogenases, which are characterized by their monomeric nature and increased length compared with related enzymes. Homology modeling and substrate docking suggested that additional amino acids form a novel -helical element, which is involved in substrate binding. Site-directed mutagenesis and subsequent studies on enzyme kinetics revealed the importance of three residues in this element for substrate binding. Further replacement of eight additional residues led to the characterization of the entire substrate binding pocket. In addition, a specific role in salutaridine binding by either hydrogen bond formation or hydrophobic interactions was assigned to each amino acid. Substrate docking alsorevealed an alternative mode for salutaridine binding, which could explain the strong substrate inhibition of SalR. An alternate arrangement of salutaridine in the enzyme was corroborated by the effect of various amino acid substitutions on substrate inhibition. In most cases, the complete removal of substrate inhibition was accompanied by a substantial loss in enzyme activity. However, some mutations greatly reduced substrate inhibition while maintaining or even increasing the maximal velocity. Based on these results, a double mutant of SalRwas created that exhibited the complete absence of substrate inhibition and higher activity compared with wild-type SalR.

Feussner, I.; Fritz, I.G.; Hause, B.; Ullrich, W.R.; Wasternack, C. Induction of a new lipoxygenase form in cucumber leaves by salicylic acid or 2,6-dichloroisonicotinic acid Bot. Acta 110, 101-108, (1997) DOI: 10.1111/j.1438-8677.1997.tb00616.x

Changes in lipoxygenase (LOX) protein pattern and/or activity were investigated in relation to acquired resistance of cucumber (Cucumis sativus L.) leaves against two powdery mildews, Sphaerotheca fuliginea (Schlecht) Salmon and Erysiphe cichoracearum DC et Merat. Acquired resistance was established by spraying leaves with salicylic acid (SA) or 2,6-dichloroisonicotinic acid (INA) and estimated in whole plants by infested leaf area compared to control plants. SA was more effective than INA. According to Western blots, untreated cucumber leaves contained a 97 kDa LOX form, which remained unchanged for up to 48 h after pathogen inoculation. Upon treatment with SA alone for 24 h or with INA plus pathogen, an additional 95 kDa LOX form appeared which had an isoelectric point in the alkaline range. For the induction of this form, a threshold concentration of 1 mM SA was required, higher SA concentrations did not change LOX-95 expression which remained similar between 24 h and 96 h but further increased upon mildew inoculation. Phloem exudates contained only the LOX-97 form, in intercellular washing fluid no LOX was detected. dichloroisonicotinic localization revealed LOX protein in the cytosol of the mesophyll cells without differences between the forms.

Abel, S.; Nguyen, M.D.; Theologis, A. The PS-IAA4/5-like family of early auxin-inducible mRNAs in Arabidopsis thaliana Journal of Biological Chemistry 270, 19093-19099, (1995)

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase is the key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene. The enzyme is encoded by a divergent multigene family in Arabidopsis thaliana, comprising at least five genes, ACS1-5 (Liang, X., Abel, S., Keller, J. A., Shen, N. F., and Theologis, A.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11046-11050). In etiolated seedlings, ACS4 is specifically induced by indoleacetic acid (IAA). The response to IAA is rapid (within 25 min) and insensitive to protein synthesis inhibition, suggesting that the ACS4 gene expression is a primary response to IAA. The ACS4 mRNA accumulation displays a biphasic dose-response curve which is optimal at 10 μM of IAA. However, IAA concentrations as low as 100 nM are sufficient to enhance the basal level of ACS4 mRNA. The expression of ACS4 is defective in the Arabidopsis auxin-resistant mutant lines axr1-12, axr2-1, and aux1-7. ACS4 mRNA levels are severely reduced in axr1-12 and axr2-1 but are only 1.5-fold lower in aux1-7. IAA inducibility is abolished in axr2-1. The ACS4 gene was isolated and structurally characterized. The promoter contains four sequence motifs reminiscent of functionally defined auxin-responsive cis-elements in the early auxin-inducible genes PS-IAA4/5 from pea and GH3 from soybean. Conceptual translation of the coding region predicts a protein with a molecular mass of 53,795 Da and a theoretical isoelectric point of 8.2. The ACS4 polypeptide contains the 11 invariant amino acid residues conserved between aminotransferases and ACC synthases from various plant species. An ACS4 cDNA was generated by reverse transcriptase-polymerase chain reaction, and the authenticity was confirmed by expression of ACC synthase activity in Escherichia coli.
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