Functional and structural characterization of the membrane-integrated plastid phosphate translocators

Atomic structures of proteins provided a wealth of information about the functioning of proteins at the molecular level, e.g. the catalytic mechanisms of enzymes, pointing to the enormous impact of X-ray crystallography on modern biology. Because of techn ical hurdles almost all of the more than 40 000 atomic structures have been obtained from soluble proteins while only about 100 structures of membrane proteins have been determined. Thus, knowledge about transport processes across membranes, especially in eukaryotic cells, is quite limited to date. The proposed project is aimed at the structure determination of plastidic phosphate translocators (pPTs) which play an important role in the primary and secondary metabolism of plants by connecting metabolic pa thways in plastids and the sourrounding cytosol. The pPTs belong to the TPT/NST superfamily which mainly consists of nucleotide sugar transporters of Golgi membranes. All these proteins are proposed to share a common structure and transport mechanism so t hat the determined structure of a pPT will be a framework for the whole superfamily. The proposed project can be divided into five parts. First, pPT proteins will be heterologously expressed in yeast and E. coli and, second, the expressed proteins will be purified by affinity chromatography. The main, third part of the project will be a screening for optimal crystallization conditions. In the fourth part, crystalls will be obtained and analyzed by X-ray crystallography. In the last part, the structures wi ll be analyzed using bioinformatic tools. The atomic structure of the pPTs will add significantly to our current picture of the mechanism of transport of molecules across membranes.