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Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-Directed Mutagenesis
[Image: see text] The anion transporter 1 (ANTR1) from Arabidopsis thaliana, homologous to the mammalian members of the solute carrier 17 (SLC17) family, is located in the chloroplast thylakoid membrane. When expressed heterologously in Escherichia coli, ANTR1 mediates a Na(+)-dependent active trans...
Autores principales: | , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2010
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911078/ https://www.ncbi.nlm.nih.gov/pubmed/20565143 http://dx.doi.org/10.1021/bi100239j |
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author | Ruiz-Pavón, Lorena Karlsson, Patrik M. Carlsson, Jonas Samyn, Dieter Persson, Bengt Persson, Bengt L. Spetea, Cornelia |
author_facet | Ruiz-Pavón, Lorena Karlsson, Patrik M. Carlsson, Jonas Samyn, Dieter Persson, Bengt Persson, Bengt L. Spetea, Cornelia |
author_sort | Ruiz-Pavón, Lorena |
collection | PubMed |
description | [Image: see text] The anion transporter 1 (ANTR1) from Arabidopsis thaliana, homologous to the mammalian members of the solute carrier 17 (SLC17) family, is located in the chloroplast thylakoid membrane. When expressed heterologously in Escherichia coli, ANTR1 mediates a Na(+)-dependent active transport of inorganic phosphate (P(i)). The aim of this study was to identify amino acid residues involved in P(i) binding and translocation by ANTR1 and in the Na(+) dependence of its activity. A three-dimensional structural model of ANTR1 was constructed using the crystal structure of glycerol 3-phosphate/phosphate antiporter from E. coli as a template. Based on this model and multiple sequence alignments, five highly conserved residues in plant ANTRs and mammalian SLC17 homologues have been selected for site-directed mutagenesis, namely, Arg-120, Ser-124, and Arg-201 inside the putative translocation pathway and Arg-228 and Asp-382 exposed at the cytoplasmic surface of the protein. The activities of the wild-type and mutant proteins have been analyzed using expression in E. coli and radioactive P(i) transport assays and compared with bacterial cells carrying an empty plasmid. The results from P(i)- and Na(+)-dependent kinetics indicate the following: (i) Arg-120 and Arg-201 may be important for binding and translocation of the substrate; (ii) Ser-124 may function as a transient binding site for Na(+) ions in close proximity to the periplasmic side; (iii) Arg-228 and Asp-382 may participate in interactions associated with protein conformational changes required for full transport activity. Functional characterization of ANTR1 should provide useful insights into the function of other plant and mammalian SLC17 homologous transporters. |
format | Text |
id | pubmed-2911078 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-29110782010-07-28 Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-Directed Mutagenesis Ruiz-Pavón, Lorena Karlsson, Patrik M. Carlsson, Jonas Samyn, Dieter Persson, Bengt Persson, Bengt L. Spetea, Cornelia Biochemistry [Image: see text] The anion transporter 1 (ANTR1) from Arabidopsis thaliana, homologous to the mammalian members of the solute carrier 17 (SLC17) family, is located in the chloroplast thylakoid membrane. When expressed heterologously in Escherichia coli, ANTR1 mediates a Na(+)-dependent active transport of inorganic phosphate (P(i)). The aim of this study was to identify amino acid residues involved in P(i) binding and translocation by ANTR1 and in the Na(+) dependence of its activity. A three-dimensional structural model of ANTR1 was constructed using the crystal structure of glycerol 3-phosphate/phosphate antiporter from E. coli as a template. Based on this model and multiple sequence alignments, five highly conserved residues in plant ANTRs and mammalian SLC17 homologues have been selected for site-directed mutagenesis, namely, Arg-120, Ser-124, and Arg-201 inside the putative translocation pathway and Arg-228 and Asp-382 exposed at the cytoplasmic surface of the protein. The activities of the wild-type and mutant proteins have been analyzed using expression in E. coli and radioactive P(i) transport assays and compared with bacterial cells carrying an empty plasmid. The results from P(i)- and Na(+)-dependent kinetics indicate the following: (i) Arg-120 and Arg-201 may be important for binding and translocation of the substrate; (ii) Ser-124 may function as a transient binding site for Na(+) ions in close proximity to the periplasmic side; (iii) Arg-228 and Asp-382 may participate in interactions associated with protein conformational changes required for full transport activity. Functional characterization of ANTR1 should provide useful insights into the function of other plant and mammalian SLC17 homologous transporters. American Chemical Society 2010-06-22 2010-08-03 /pmc/articles/PMC2911078/ /pubmed/20565143 http://dx.doi.org/10.1021/bi100239j Text en Copyright © 2010 American Chemical Society http://pubs.acs.org This is an open-access article distributed under the ACS AuthorChoice Terms & Conditions. Any use of this article, must conform to the terms of that license which are available at http://pubs.acs.org. |
spellingShingle | Ruiz-Pavón, Lorena Karlsson, Patrik M. Carlsson, Jonas Samyn, Dieter Persson, Bengt Persson, Bengt L. Spetea, Cornelia Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-Directed Mutagenesis |
title | Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-Directed Mutagenesis |
title_full | Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-Directed Mutagenesis |
title_fullStr | Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-Directed Mutagenesis |
title_full_unstemmed | Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-Directed Mutagenesis |
title_short | Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-Directed Mutagenesis |
title_sort | functionally important amino acids in the arabidopsis thylakoid phosphate transporter: homology modeling and site-directed mutagenesis |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911078/ https://www.ncbi.nlm.nih.gov/pubmed/20565143 http://dx.doi.org/10.1021/bi100239j |
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