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...

Recombine,It phages that encode the complete precursor polypeptide for the 22 kDa pol~cpeptide associated with photosystem it have been serologically selected fi'om two Agtll expression libraries made from polyadenylated RNA of spinach seedlings, The eDNAs hybridize to a 1.3 kb RNA species. The precursor protein is comprised of 274 amino acid residues and carries an N-terminal transit peptide of probably 69 amino acid residues, The mature protein exhibits Ibm' predicted transmembrane segments and is shown to be an integral component ofphotosystem It originating in a single-copy gene, The unique characteristics of this protein are: (i) it is the result of a eerie-internal duplication of an arJcestot with two ,rtembraqe spans, (it) a striking resemblance to LHC i/11, CP24/CP29 apoproteins, and ELIPs, although it does not bind chlorophyll and is p,'esent in cyanobacteria, mad. as these proteins, (iii) it integrates into the membrane with uncleared routing signals that display remarkable resemblance to patterns found in bipartite transit peptides, 22 kDa polypeptide

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Using lgtl 1 expression cloning and immunoscreening, CDNA-containing recombinant phages for subunits IV and V of the photosystem I reaction center were isolated, sequenced and used to probe Northern blots of polyadenylated RNA prepared from spinach seedlings. The mRNA sizes for both components are _ 1000 and 850 nucleotides, respectively. The 968 nucleotide cDNA sequence and derived amino acid sequence for subunit IV predict a single open reading frame of 231 amino acid residues (25.4 kDa). Comparison with a 13-residue N-terminal amino acid sequence determined for subunit IV suggests a mature protein of 17.3 kDa (154 residues) and a transit sequence of 77 amino acids (8.1 kDa). The corresponding data for subunit V are 677 bp (cDNA), 167 residues for the precursor protein (18.2 kDa), 98 residues for the mature polypeptide (10.8 kDa) and 69 residues for the transit peptide (7.4 kDa). Secondary structure predictions indicate that both proteins possess greatly different transit sequences and that none is membrane-spanning.

We have investigated the photosystem-I1 organization in differentiating-bundle-sheath cells of the three malate dehydrogenase (oxaloacetate decarboxylating) (NADPf)-type C4 species maize, Sorghum and Pennisetum. Using a set of nine different antisera raised against individual subunits of photosystem-11, we demonstrate that photosystem-I1 components constitute a substantial part of the thylakoid membranes of young bundle-sheath chloroplasts. The abundance of subunits of the photosystem-I1 core, i.e. the 47-and 43-kDa chlorophyll-a-binding proteins, polypeptides D1 and D2, cytochrome b S s 9 , and the 34-kDa polypeptide, varies with the developmental state of the plant. However, the levels of the 23-kDa, 16-kDa and 10-kDa extrinsic polypeptides of the wateroxidation complex are drastically reduced in bundle-sheath chloroplasts of all three species analyzed, regardless of their state of differentiation. The reduction in protein abundance is also reflected at the transcript level: only traces of the nuclear-encoded mRNAs are found in differentiating bundle-sheath cells of Sorghum, suggesting that the transcription of these genes has been switched off. Our data are compatible with the idea that the wateroxidation complex is a prime site for initiating or maintaining the process leading to photosystem-I1 depletion during differentiation of bundle-sheath cells.