Wolfgang P. Schröder

The intrinsic 22 kDa protein is a chlorophyll-binding subunit of photosystem II

The intrinsic 22 kDa polypeptide associated with photosystem II (psbS protein) was found to be able to bind chlorophyll. Extraction of isolated photosystem II membranes with octyl‐thioglucopyranoside, followed by repetitive electrophoresis under partially denaturing conditions gave only one green band. It contained both chlorophyll a and chlorophyll b, exhibited an absorption maximum at 674 nm and a 77 K fluorescence peak at 675 nm. The chlorophyll‐protein band contained a single polypeptide of 22 kDa. Based on these results and on previous protein sequence comparisons, it is suggested that the psbS protein is a chlorophyll a/b binding polypeptide and should thus be denoted CP22.

Photosynthetic water oxidation: The protein framework.

Approximately 20 protein subunits are associated with the PS II complex, not counting subunits of peripheral light-harvesting antenna complexes. However, it is not yet established which proteins specifically are involved in the water-oxidation process. Much evidence supports the concept that the D1/D2 reaction center heterodimer not only plays a central role in the primary photochemistry of Photosystem II, but also is involved in electron donation to P680 and in ligation of the manganese cluster. This evidence includes (a) the primary donor to P680 has been shown to be a redox-active tyrosyl residue (Tyr161) in the D1 protein, and (b) site-directed mutagenesis and computer-assisted modeling of the reaction center heterodimer have suggested several sites with a possible function in manganese ligation. These include Asp170, Gln165 and Gln189 of the D1 protein and Glu69 of the D2 protein as well as the C-terminal portion of the mature D1 protein. Also, hydrophilic loops of the chlorophyll-binding protein CP43 that are exposed at the inner thylakoid surface could be essential for the water-splitting process.In photosynthetic eukaryotes, three lumenal extrinsic proteins, PS II-O (33 kDa), PS II-P (23 kDa) and PS II-Q (16 kDa), influence the properties of the manganese cluster without being involved in the actual catalysis of water oxidation. The extrinsic proteins together may have multiple binding sites to the integral portion of PS II, which could be provided by the D1/D2 heterodimer and CP47. A major role for the PS II-O protein is to stabilize the manganese cluster. Most experimental evidence favors a connection of the PS II-P protein with binding of the Cl- and Ca2+ ions required for the water oxidation, while the PS II-Q protein seems to be associated only with the Cl- requirement. The two latter proteins are not present in PS II of prokaryotic organisms, where their functions may be replaced by a 10–12 kDa subunit and a newly discovered low-potential cytochrome c-550.

Characterisation of an Arabidopsis cDNA encoding a thylakoid lumen protein related to a novel `pentapeptide repeat' family of proteins

We have cloned an Arabidopsis cDNA encoding a novel thylakoid lumen protein, P17.4, that has been previously isolated from lumen extracts of spinach chloroplasts. The protein is synthesised with a bipartite presequence containing a Sec‐type lumen‐targeting signal peptide and the precursor protein is imported into the lumen of pea chloroplasts. The encoded protein is homologous to an Anabaena protein that is essential for correct glycolipid localisation, and is also related to at least 16 unassigned open reading frames in Synechocystis. This family of proteins is characterised by the presence of numerous pentapeptide repeats with the consensus structure AXLXX, and its members are predicted to be located in the cytosol, plasma membrane and periplasm/lumen. P17.4 is therefore the first higher plant member of an extended family of putative cyanobacterial proteins that may serve important roles in lipid transport or assembly.

Compositional and topological studies of the PsbW protein in spinach thylakoid membrane

The lateral distribution and transversal orientation of the nuclear encoded PsbW protein (psbW gene product) has been investigated. The main part (80%) of the PsbW protein was found in the grana region of the thylakoid membrane, corroborating earlier observations that the PsbW protein was closely associated with Photosystem II (PS II). The localisation within the PS II complex was analysed by a comparative quantification of the PsbW content between PS II membrane fragments (BBY) and various isolated PS II reaction centres. Our results showed that the PsbW protein could be detected in all PS II reaction centre preparations, whereas the chlorophyll a proteins CP47 and CP43 were not detectable. However, a careful analysis based on the number of reaction centres, revealed that the amount of the PsbW protein found in the PS II reaction centre preparation (Nanba-Satoh type) was lower than that in a BBY preparation. These results suggested that the PsbW protein was located close to the D1/D2 heterodimer, but the PsbW protein could, at least partially, be removed from the PS II reaction centre during isolation. Quantification of the amounts of the PsbW protein in various reaction centre preparations indicated that the presence of Triton X-100 throughout the isolation procedure appeared to be a crucial point for obtaining low amounts of the PsbW protein in the PS II reaction centre preparation. Trypsin digestion followed by SDS-PAGE, immunoblotting and Enzyme Linked Immunosorbent Assay (ELISA) revealed that the hydrophobic PsbW protein contained one transmembrane span with its C-terminus exposed on the stroma side while the N-terminus faced the lumen side of the thylakoid membrane. Thus, despite that the protein had a typical lumenal targeting presequence, it was an integral membrane protein. Moreover, it had its N-terminus on the opposite side of the membrane compared to other PS II reaction centre proteins.

Engineering of N-terminal threonines in the D1 protein impairs photosystem II energy transfer in Synechocystis 6803

Mutants of the cyanobacterium Synechocystis sp. PCC 6803 with N‐terminal changes in the photosystem (PSII) II D1 protein were analysed by flash‐induced oxygen evolution, chlorophyll a fluorescence decay kinetics and 77 K fluorescence emission spectra. The data presented here show that mutations of the Thr‐2, Thr‐3 and Thr‐4 in D1 do not influence the oxygen evolution. A perturbation on the acceptor side was observed and the importance of the N‐terminal threonines for an efficient energy transfer between the phycobilisome and PSII and for stability of the PSII complex was demonstrated.

Characterisation of an Arabidopsis thaliana cDNA encoding a novel thylakoid lumen protein imported by the ΔpH-dependent pathway

Abstract. An Arabidopsis thaliana (L.) Heynh. cDNA encoding a novel 16-kDa protein (P16) of the chloroplast thylakoid lumen has been characterised. The function of the protein is unknown but it shares some sequence similarity with alpha allophycocyanins. P16 is synthesised with a bipartite, lumen-targeting presequence, and import experiments demonstrated that this protein follows the ΔpH-dependent pathway. Analysis of the thylakoid transfer peptide revealed two unusual features. Firstly, the key targeting determinant is predicted to be a twin-arginine followed by a highly hydrophobic residue two residues later, rather than at the third position as in most transfer peptides. Secondly, the C-terminal domain of the transfer peptide contains multiple charged residues which may help to prevent mistargeting by the Sec-type protein translocase.

Isolation and Characterization of the Thylakoid Lumen from Spinach Chloroplasts

The thylakoid membrane of higher plants encloses a narrow, continuous compartment, designated the lumen. So far no isolation method has been available for obtaining a high yield of pure thylakoid lumen. The present knowledge of this chloroplast compartment is therefore from a compositional and functional point of view fragmentary.

PsbX Protein from Photosystem II in Higher Plants: Localization and Gene Regulation

Photosystem II complex (PSII) of higher plants consists of more than 25 different subunits (for reviews, see ref. 1 and 2). The D1 and D2 proteins have been found to bind most, if not all of the cofactors needed for the primary electron transfer reactions, and these two proteins constitute the PSII reaction center. However, no oxygen evolving active D1–D2 heterodimer has so far been isolated, which suggests that additional proteins are needed for the PSII complex to retain this activity. Some of these ancillary proteins have been identified and characterized to varying extents, including the newly discovered PsbW protein (3, 4, 5). However, the true complexity of PSII remains to be established. This applies particularly to a series of low molecular mass proteins.

Two Novel Lumen Proteins from Arabidopsis thaliana

Although the photosynthetic process of the thylakoid membranes has been studied in intricate detail, the knowledge of the lumen enclosed by the thylakoid membrane has emerged slowly. In order to gain insight into the resident proteins of the thylakoid lumen. and hence understand more about the functions of the thylakoid network, we recently devised a fractionation procedure which allows the preparation of lumen contents in a highly purified form [1]. At least 25 polypeptides could be identified in the lumen fraction by SDS-electrophoresis, of which only six corresponded to known proteins. Among those were the well characterized photosynthetic proteins plastocyanin, PsbO, PsbP and PsbQ as well as polyphenol oxidase and the recently purified and characterized prolyl cis/trans isomerase [2]. The majority of the remainder are of unknown identity. N-terminal sequencing of the unknown proteins led so far to the identification of three new lumenal proteins. These data we used to identify and characterize the Arabidopsis th. DNAs encoding two of these proteins. We showed that one clone encoded a novel 17.4 kDa lumenal protein (P17.4) with significant homology to a new protein family from Synechocystisis sp. and an Anabaena sp. protein that is suggested to be essential for correct localisation of glycolipids [3]. The other clone encoded a 16.5 kDa protein (P16.5) of unknown function, which is a new example for a protein that follows the ΔpH translocation pathway.