Purification and membrane topology of PSI-D and PSI-E, two subunits of the photosystem I reaction center.
Structural studies have been conducted on polypeptides PSI-D and PSI-E, which are extrinsic but firmly bound to the photosystem I reaction center. These subunits are predicted to be involved in the correct interaction with soluble electron acceptor(s), like ferredoxin. We designed an original method to extract both polypeptides directly from thylakoid membranes and to purify them: a stepwise extraction with NaSCN followed by size fractionation and reverse-phase HPLC. Investigation of the in situ topology of PSI-D and PSI-E was undertaken using monoclonal antibody binding, controlled proteolysis, peptide sequencing and electron microscopy. The precise identification of numerous proteolytic sites indicates that the entire N-terminal regions of PSI-E (up to Glu15) and PSI-D (up to Lys15) are exposed to the medium. Partial mapping of the exposed epitopes was possible using purified fragments of each polypeptide. In the case of PSI-E, this mapping confirmed the accessibility of the N-terminal part, and suggested the need for another exposed sequence, probably located after Met39 in the second half of the protein. For PSI-D, this mapping revealed that the sequence between Met74 and Met140, including the most basic amino acid clusters, is also partly accessible. These experiments provide the first detailed informations, although still partial, on the topology of these polypeptides. They give a preliminary basis for hypotheses concerning the sites of interaction with the soluble counterparts.
Lagoutte
B
B
Département de Biologie Cellulaire et Moléculaire, Saclay, Gif-sur-Yvette, France.
Vallon
O
O
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002462:Cell Membrane; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D045331:Photosystem I Protein Complex; D010940:Plant Proteins
D000939:Epitopes; D008565:Membrane Proteins; D010446:Peptide Fragments; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D010940:Plant Proteins