Single amino acid variation in barley 14-3-3 proteins leads to functional isoform specificity in the regulation of nitrate reductase
| العنوان: | Single amino acid variation in barley 14-3-3 proteins leads to functional isoform specificity in the regulation of nitrate reductase |
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| المؤلفون: | Tom D Bunney, Ilja Roobeek, Antonie J. W. G. Visser, Albertus H de Boer, Jos N M Mol, Mark P. Sinnige |
| المصدر: | The Plant Journal. 44:1001-1009 |
| بيانات النشر: | Wiley, 2005. |
| سنة النشر: | 2005 |
| مصطلحات موضوعية: | Gene isoform, Protein structure, Biochemistry, Genetics, Post-translational regulation, Cell Biology, Plant Science, Hordeum vulgare, Binding site, Biology, Ligand (biochemistry), Peptide sequence, 14-3-3 protein |
| الوصف: | The highly conserved family of 14-3-3 proteins function in the regulation of a wide variety of cellular processes. The presence of multiple 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 suggest functional isoform specificity of 14-3-3 isoforms in the regulation of target proteins. Indeed, several studies observed differences in affinity and functionality of 14-3-3 isoforms. However, the structural variation by which isoform specificity is accomplished remains unclear. Because other reports suggest that specificity is found in differential expression and availability of 14-3-3 isoforms, we used the nitrate reductase (NR) model system to analyse the availability and functionality of the three barley 14-3-3 isoforms. We found that 14-3-3C is unavailable in dark harvested barley leaf extract and 14-3-3A is functionally not capable to efficiently inhibit NR activity, leaving 14-3-3B as the only characterized isoform able to regulate NR in barley. Further, using site directed mutagenesis, we identified a single amino acid variation (Gly versus Ser) in loop 8 of the 14-3-3 proteins that plays an important role in the observed isoform specificity. Mutating the Gly residue of 14-3-3A to the alternative residue, as found in 14-3-3B and 14-3-3C, turned it into a potent inhibitor of NR activity. Using surface plasmon resonance, we show that the ability of 14-3-3A and the mutated version to inhibit NR activity correlates well with their binding affinity for the 14-3-3 binding motif in the NR protein, indicating involvement of this residue in ligand discrimination. These results suggest that both the availability of 14-3-3 isoforms as well as binding affinity determine isoform-specific regulation of NR activity. |
| تدمد: | 1365-313X 0960-7412 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_________::13d97db84e10215ab8ad602ea6a21492 https://doi.org/10.1111/j.1365-313x.2005.02599.x |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi...........13d97db84e10215ab8ad602ea6a21492 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 1365313X 09607412 |
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