Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-directed Mutagenesis
2010 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 30, 6430-6439 p.Article in journal (Other academic) Published
The anion transporter 1 (ANTR1) from Arabidopsis thaliana, homologous to the mammalian SLC17 family, has recently been localized to the chloroplast thylakoid membrane. When expressed heterologously in Escherichia coli, ANTR1 mediates a Na+-dependent active transport of inorganic phosphate (Pi). The aim of this study was to identify amino acids involved in substrate binding/translocation by ANTR1 and in the Na+-dependence of its activity. A threedimensional structural model of ANTR1 was constructed using the crystal structure of glycerol-3-phosphate/phosphate antiporter (GlpT) 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, Arg-228 and Asp-382 exposed at the cytosolic surface of the protein. The activities of the wild type and mutant proteins have been analyzed using expression in E. coli and radioactive transport assays, and compared with bacterial cells carrying an empty plasmid. Based on Pi- and Na+-dependent kinetics, we propose that Arg-120, Arg-201 and Arg-228 are involved in binding and translocation of the substrate, Ser-124 functions as a periplasmic gate for Na+ ions, and finally Asp-382 participates in the turnover of the transporter via ionic interaction with either Arg-228 or Na+ ions. We also propose that the corresponding residues may have a similar function in other plant and mammalian SLC17 homologous transporters.
Place, publisher, year, edition, pages
2010. Vol. 49, no 30, 6430-6439 p.
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:liu:diva-51119DOI: 10.1021/bi100239jOAI: oai:DiVA.org:liu-51119DiVA: diva2:272974
On the day of the defence day the status of this article was Manuscript2009-10-192009-10-192010-09-02Bibliographically approved