Jonathan B. Marder

Identification of psbA and psbD gene products, D1 and D2, as reaction centre proteins of photosystem 2

A recent report (Nanba O, Satoh K: Proc. Natl. Acad. Sci. USA 84: 109–112, 1987) described the isolation from spinach of a putative photosystem 2 reaction centre which contained cytochrome b-559 and three other electrophoretically resolvable polypeptide bands, two of which have molecular weights comparable to the D1 and D2 polypeptides. We have used in vivo labelling with radioactive methionine and probed with D1 and D2 monospecific antibodies (raised against synthetically expressed sequences of the psbA and psbD genes) for specific detection of these proteins in a similarly prepared photosystem 2 reaction centre preparation. These techniques identified a 32 000 dalton D1 band, a 30 000 dalton D2 band and a 55 000 dalton D1/D2 aggregate, the latter apparently arising from the detergent treatments employed. Digestions with a lysine-specific protease further confirmed the identification of the lysine-free D1 polypeptide and also confirmed that the D1 molecules in the 55 000 dalton band were in aggregation with other bands and not in self-aggregates. The D1 and D2 polypeptides (including the aggregate) are considerably enriched in the photosystem two reaction centre preparation compared to the other resolved fractions.

Identification and characterization of the psbA gene product: The 32-kDa chloroplast membrane protein

Publisher Summary This chapter presents the techniques that are useful for the identification and characterization of the 32-kDa protein. The rapidly metabolized 32-kDa chloroplast membrane protein is the product of the psbA gene. It is associated with the plastoquinone oxidoreductase function of photosystem II and seems to be the site of action of several classes of herbicides. It is a major product of chloroplast protein synthesis in the light. Its messenger RNA (mRNA) is one of the most abundant in the chloroplast. It is synthesized and degraded in the light with rates exceeding those for other known chloroplast proteins. A major problem encountered when studying the 32-kDa protein is its extremely low steady-state amount compared to other chloroplast proteins. The chapter presents a recommended general protocol for labeling green leaf tissue with high specific activity [ 35 S] methionine to obtain radioactive 32-kDa protein.

Localization and identification of phosphoproteins within the Photosystem II core of higher-plant thylakoid membranes

Photosystem II core particles were isolated, using the method of Gounaris and Barber (Gounaris, K. and Barber, J. (1985) FEBS Lett. 188, 68–72) from [32P]phosphate- and [35S]methionine-labelled thylakoids of pea (Pisum sativum) chloroplasts. SDS-polyacrylamide gel analysis of the PS II core particles showed diffuse Coomassie blue stained polypeptide bands centred at molecular mass of 43, 40, 33, 30 and 10 kDa. Subsequent fluorography of the SDS-polyacrylamide gels revealed 32P-containing bands at 55, 43, 40, 33 and 30 kDa and a 35S-containing band, representing the D1 (herbicide-binding) protein, at slightly higher than 33 kDa. The 30 kDa [32P]polypeptide which was the major 32P-labelled species was therefore assigned to be the D2 protein, a conclusion reinforced by its degradation by the lysine-specific endoprotease, Lys-C. The approx. 33 kDa D1 protein, however, did not appear to be phosphorylated, since the weaker 32P-containing 33 kDa band was degraded by the lysine specific enzyme, whilst the [35S]D1 band was unaffected. Estimation of cytochrome b-559 in different fractions of the sucrose density gradient used to prepare PS II core particles indicated that the cytochrome was confined to the PS II core fraction, whereas the 10 kDa 32P-labelled component was present in other fractions. Moreover, the 10 kDa 32P-labelled polypeptide was readily degraded by the lysine-specific endoprotease, a result not expected for the lysine free cytochrome b-559 10 kDa polypeptide. It is, therefore, also concluded that the 10 kDa [32P]component cannot represent the cytochrome b-559 apoprotein.

The D1 polypeptide subunit of the Photosystem II reaction centre has a phosphorylation site at its amino terminus

Abstract The D1 polypeptide subunit of the Photosystem II reaction centre was phosphorylated by incubating isolated pea and wheat thylakoids with radioactive ATP. The phosphate label was followed through defined enzymatic digestion procedures, using D1-specific antibodies and sequence information to detect and identify expected cleavages. The phosphorylation site was determined to be at the amino terminus of D1, most probably on the threonine residue at position 2.

Determination of catabolism of the photosystem II D 1 subunit by structural motifs in the polypeptide sequence.

Abstract Proteolytic mapping of the D 1 subunit of photosystem two and a degradation product which arises during its rapid catabolism shows that the latter is a result of proteolysis within the peptide motif QEEET. This motif is located in a portion of the D1 protein thought to form a stroma-exposed connection between fourth and fifth transmembrane segments. This connection domain also contains a “PEST”-like sequence and forms part of the QB/herbicide binding niche. The QEEET motif seems to provide a major epitope in immunological studies, as judged from reaction of D1 and its fragments with polyclonal antibodies. Antibodies against D1 were found to react with other animal and plant proteins which contain similar sequence motifs.

Photosystem II reaction centers isolated from phosphorylated pea thylakoids carry phosphate on the D1 and D2 polypeptide subunits

The reaction centre of Photosystem II, composed of the D1/D2/cytochrome b-559 polypeptides, has been isolated from pea thylakoids after phosphorylation by a redox-activated kinase. It is shown that both the D1 and D2 polypeptides are phosphorylated using a lysine-specific protease which digests D2 and not D1. Proteolysis of phospho-D2 yields a 27 kDa non-phosphorylated fragment. By considering the digestion pattern in relation to amino acid sequence information it is concluded that D2 is phosphorylated near the amino terminus. The chromatographic procedures adopted for isolation of the reaction centre also resolved a highly phosphorylated fraction of the light-harvesting chlorophyll ab complex.

Presence of the rapidly-labelled 32 000-dalton chloroplast membrane protein in triazine resistant biotypes

Introduction A large number of herbicides, including triazines, inhibit photosynthesis by blocking electron transport at the reducing side of photosystem II [l-3]. The triazines are presently considered to interfere with the electron flow between Q, the primary electron accep- tor of photosystem II, and the secondary quencher, B. Binding studies with radiolabelled atrazine

PROBING OF APOPROTEIN FUNCTION IN THE PHOTOSYSTEM II REACTION CENTRE BY PROTEOLYTIC MODIFICATION

The photochemically active pigments of photosystem II (PSII) are housed in an apoprotein environment provided by the Dl and D2 polypeptides which also contain binding sites for the acceptor quinones (QA and QB). The organisation of the polypeptides and chromophores has been inferred (1–3) from various similarities and homologies between PSII reaction centres and the photosynthetic reaction centres of purple bacteria, such as Rhodopseudomonas viridis, which have been structurally resolved in considerable detail (e.g. 4). By analogy with the L and M polypeptides of bacterial reaction centres, Dl and D2 each contain 5 transmembrane helical spans. There is strong sequence homology between all four polypeptides in Helix 4, which contains ligands for the (bacterio) chlorophyll photochemical electron donor (“special pair”) and a non-haem ferrous ion. QA and QB appear to be hydrogen bonded by residues in Helix 4 and the connection between Helices 4 and 5 of Dl/L and D2/M respectively. The two quinone binding sites serve slightly different functions. The QA site is permanently filled by quinone which undergoes photochemical reduction to semiquinone by pheophytin, and reoxidation by QB. The QB quinone, on the other hand, is released as a quinol following its two-electron reduction by a double turnover of QA. The emtpy QB site is then refilled by an oxidised quinone molecule. Other molecules are able to displace the quinone from the QB site and thereby block electron flow. These include herbicides such as dichlorophenyl dimethylurea (DCMU) which inhibits PSII, and certain triazine herbicides, which are effective on both PSII and bacterial photosystems.

Phosphorylated Polypeptides of the Photosystem 2 Core Complex

Several proteins of higher plant thylakoids can be phosphorylated through the catalytic action of endogenous, membrane-associated kinase (1). Most prominently phosphorylated are the polypeptide components of the chlorophyll a/b-containing light-harvesting complex (2–4). Reversible phosphorylation of these 23–27 kDa polypeptides is thought to provide a molecular mechanism whereby the distribution of quanta between the two photosystems can be regulated (4–7). The identities of other proteins which are phosphorylated by thylakoid-bound kinase have not yet been established, though it has been suggested that at least some of them are associated with photosystem 2 (PS2), in particular those polypeptides with apparent molecular masses of 40–45, 30–35 and 10 kDa (8). Indeed, the use of neutral detergents such as Triton X-100, has permitted the preparation of membrane subfractions highly enriched in PS2 (9,10) and allowed us, as reported in a previous paper (11), to show that the major [32 P] labelled polypeptides were present in the PS2-LHC-2 supramolecular complex. Recently there has been increased interest in isolating more purified PS2 preparations along lines pioneered by Satoh (12). Density gradient fractionation allows the removal of LHC-2 and other polypeptides from Triton X-1.00-solubilized PS2-enriched membranes, yielding PS2 ‘core’ particles (13), here called RCC2, which possess a small number of polypeptides.

The Role of Photosystem II D1 Apoprotein Metabolism in the Physiology of Photoinhibition

The reaction centre of photosystem II (PSII) contains several proteins including D2,D1, cytochrome b559 and the psbl gene product, and is closely associated with many others (proteins of the oxygen evolving complex, the 9kDa phosphoprotein, chlorophyll binding proteins and LHCII). Apart from the haem of cytochrome b559, D1 and D2 bind all the reaction centre pigments and also provide the quinone binding sites. Interestingly, only D1 consistently shows rapid light-driven turnover which may reflect a physiological need to replace photodamaged molecules of this polypeptide.