Two polypeptides of 10 kDa and 22 kDa, shown to be components of the higher plant photosystem 2, were purified and examined. A NaCl/Triton X-100 treatment was designed, which released these two polypeptides from the thylakoid membrane, in concert with the extrinsic 16-kDa and 23-kDa proteins, concomitant with a loss in oxygen-evolution activity. After this treatment the oxygen-evolving activity of the photosystem 2 membranes devoid of the 10-kDa and the 22-kDa polypeptides could be restored with CaC12, but not by readdition of the purified 23-kDa protein. This defiency was caused by an inability of the 23-kDa protein to rebind to the photosystem 2 membranes. In analogy, the oxygen-evolution activity of a highly purified photosystem 2 core preparation, devoid of the 10-kDa and 22-kDa polypeptides, was stimulated by CaC12, but not by the 23-kDa protein. We, therefore, suggest that the 1 0-kDa or the 22-kDa polypeptides provide a binding-site for the extrinsic 23-kDa protein to the thylakoid membrane.The 1 0-kDa and 22-kDa polypeptides were isolated through ion-exchange chromatography in the presence of detergents. They both displayed hydrophobic properties, verified by their low proportion of polar amino acid residues and their partition to the hydrophobic phase during Triton X-114 fractionation. The purified polypeptides did not contain metallic cofactors or substances with absorption in the visible region of the spectrum.In recent years extensive knowledge about biophysics, biochemistry and molecular biology of higher plant photosystem 2 has accumulated (reviewed in [l -31). The present concept of the photosystem 2 is of a supramolecular complex, with both integral and peripheral protein subunits, mainly located in the grana region of the thylakoid membrane. A number of studies have indicated that the catalytic photosystem 2 core complex is made up of at least five different hydrophobic proteins [4 -61 and a number of low-molecular-mass polypeptides [7]. In addition extrinsic proteins of 33 kDa, 23 kDa and 16 kDa have been shown to be located at the inner thylakoid surface and to be regulatory components of the oxygen-evolving complex of photosystem 2. The 33-kDa protein seems to have a stabilizing effect on the catalytic manganese, while the 23-kDa and the 16-kDa proteins are involved in the binding of the calcium and chloride necessary for oxygen evolution [2].In an immunoprecipitation study we have previously shown that a close structural association exists between the 33-kDa and 23-kDa proteins, and three polypeptides of 10 kDa, 22 kDa and 24 kDa [8]. Furthermore, the 10-kDa polypeptide was found to be released from everted photosystem 2 membranes by alkaline Tris [9], a treatment which inhibits oxygen evolution. A nearest-neighbor analysis, using chemical cross-linking, also revealed a close proximity between the 33-kDa protein and 22-kDa and 24-kDa polypeptides [lo].In the present study we report the isolation and chardcterization of the two polypeptides of 10 kDa and 22 kDa.
MATERIALS AND METHODS
Sub-thy...