Vacuolar NHX antiporters: understanding structure-function relationships and regulation
Potassium (K) is an essential nutrient for plants and the most abundant cation in plant cells, comprising up to 10% of plant dry weight. While cytosolic K is kept at homeostatic concentrations close to 100 mM, surplus K is stored in vacuoles. The tonoplast-localized K+,Na+/H+ exchangers NHX1 and NHX2 proteins of Arabidopsis mediate this K+ accumulation in the vacuole, thereby increasing the osmotic potential, water uptake and the turgor pressure necessary for cell expansion and growth. Vacuolar remodeling during stomatal movements also depends on these proteins. Structural domains and essential amino acid residues putatively involved in ion transport, cation coordination and pH sensing, have been identified by phylogenetic analysis and computational modeling of the NHX1 protein. To determine the relevance of these residues in the biochemical activity of NHX1, and its pH dependence, point-mutation alleles have been generated. Mutant NHX1 proteins have been functionally tested in yeasts nhx1 mutants and in vitro ion transport assays. The presence of a calmodulin-binding domain comprising amphipathic ¿-helices at the C-termini of NHX1 and NHX2 have also been detected by computational and biochemical analyses. The importance of the putative calmodulin-binding domain for NHX1 activity has been demonstrated by functional analyses in yeast, whereas the interaction of NHX1 and NHX2 with CalModulin-Like18 (CML18) has been analyzed by BiFC and Y2H assays. Our results evidence the fine-tuning of NHX1 and NHX2 activity in response to developmental and environmental cues. In addition, we expect to unravel the biochemical mechanisms for pH sensing and regulation of these critical K transporters of Arabidopsis.
| Main Authors: | , , , , |
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| Format: | póster de congreso biblioteca |
| Published: |
Universidad de Oviedo
2016-06-22
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| Online Access: | http://hdl.handle.net/10261/160448 |
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| Summary: | Potassium (K) is an essential nutrient for plants and the most abundant cation in plant cells, comprising up to 10% of plant dry weight. While cytosolic K is kept at homeostatic concentrations close to 100 mM, surplus K is stored in vacuoles. The tonoplast-localized K+,Na+/H+ exchangers NHX1 and NHX2 proteins of Arabidopsis mediate this K+ accumulation in the vacuole, thereby increasing the osmotic potential, water uptake and the turgor pressure necessary for cell expansion and growth. Vacuolar remodeling during stomatal movements also depends on these proteins.
Structural domains and essential amino acid residues putatively involved in ion transport, cation coordination and pH sensing, have been identified by phylogenetic analysis and computational modeling of the NHX1 protein. To determine the relevance of these residues in the biochemical activity of NHX1, and its pH dependence, point-mutation alleles have been generated. Mutant NHX1 proteins have been functionally tested in yeasts nhx1 mutants and in vitro ion transport assays. The presence of a calmodulin-binding domain comprising amphipathic ¿-helices at the C-termini of NHX1 and NHX2 have also been detected by computational and biochemical analyses. The importance of the putative calmodulin-binding domain for NHX1 activity has been demonstrated by functional analyses in yeast, whereas the interaction of NHX1 and NHX2 with CalModulin-Like18 (CML18) has been analyzed by BiFC and Y2H assays. Our results evidence the fine-tuning of NHX1 and NHX2 activity in response to developmental and environmental cues. In addition, we expect to unravel the biochemical mechanisms for pH sensing and regulation of these critical K transporters of Arabidopsis. |
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