Abstract
Phosphate is an essential mineral in both prokaryotic and eukaryotic living cells. Phosphate is the major component of many bio-molecules such as nucleic acids, nucleoside triphosphate and many membrane lipids. Phosphate is also key components in enzyme activity regulation and signal transduction pathways. In plants, phosphate homeostasis is a complicated network, in which phosphate transportation across biological membranes is a major component. Several phosphate transporters are membrane proteins and mainly locate at plasma membrane or endomembrane system that in charge of phosphate acquisition and efflux. Phosphate transporters in plants can be classified into three groups, high affinity phosphate transporter, low affinity phosphate transporter and phosphate exporter. PHO1 is the first protein to be correlated with phosphate export. The detail molecular mechanism of PHO1 is still not clear and there is no three dimensional structure of PHO1. PHO1 contains the N-terminal soluble domain, SPX and the C-terminal transmembrane domain, EXS domain. EXS domain only is reported to have a function of phosphate export. The molecular weight of PHO1-EXS is about 46 kDa. It is predicted that PHO1-EXS might have 10 transmembrane helices. In this study, we successfully cloned, expressed Arabisopsis thaliana and Oryza sativa PHO1-EXS by yeast heterologous protein expression system. Arabisopsis thaliana and Oryza sativa PHO1-EXS was solubilized by detergent and purified by immobilized metal ion affinity chromatography (IMAC). Size-exclusion chromatography (SEC) indicates that purified PHO1-EXS form dimer in solution. Reverse Native PAGE indicate that purified PHO1-EXS is in a single conformation in solution. Also, the pre-crystallization trials are undergoing. However, the purity and quantity of PHO1-EXS still need to be improved in order to do the structural study by crystallography method in the future.