Molecular cloning and structural analysis of the phosphate translocator from pea chloroplasts and its comparison to the spinach phosphate translocator

Using an 5-AvaII fragment of the spinach (Spinacia oleracea L.) phosphate translocator cDNA as a probe for a hybridization screening of a pea (Pisum sativum L.) cDNA library we have cloned and sequenced a cDNA clone coding for the phosphate translocator precursor protein from pea chloroplasts. The full-length cDNA clone comprises 42 base pairs (bp) at the 5-non-coding region, a 1206-bp coding region corresponding to a polypeptide of 402 amino-acid residues (relative molecular mass 43 671) and 244 bp at the non-coding 3-region. Determination of the N-terminal sequence of the phosphate translocator from both pea and spinach chloroplasts revealed that the transit peptides consist of 72 and 80 amino-acid residues, respectively. These transit peptides are different from those of other chloroplastic transit peptides in that they both contain an amphiphilic -helix which is located either in close proximity to the processing site in pea or at the N-terminus in spinach. The mature proteins from pea and spinach both contain about 87% identical amino-acid residues and about seven putative membrane-spanning -helices. Some of these -helices have an amphiphilic character and might serve to form a hydrophilic translocation channel through the membrane. The in-vitro synthesized pea precursor protein is directed to the chloroplast and inserted into the chloroplast envelope membrane.Abbreviations bp base pairs - kDa kilodaltons - M_r relative moleculas mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We wish to thank Dr D. Pappin and R. Jakes (AFRC Sequencing Laboratory, Department of Biochemistry, University of Leeds, UK) for performing the N-terminal sequence determinations and are greatful to Dr J. S. Gantt (Botany Department, University of Georgia, Athens, USA) for a pea leaf cDNA library and to Professor J. C. Gray (University of Cambridge, Department of Botany, Cambridge, UK) for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, the Science and Engineering Research Council and the Royal Society. D.L.W. was the recipient of the Royal Society Rosenheim research fellowship and K.F. was supported by a fellowship from the Studienstiftung des deutschen Volkes.