Kleoniki Gounaris

Isolation and characterisation of a photosystem II reaction centre lipoprotein complex

A photochemically active reaction centre complex has been isolated from photosystem II preparations of spinach chloroplasts by Triton X‐100 solubilisation and sucrose gradient fractionation. Electrophoresis of the complex revealed 5 bands indicating polypeptides of apparent molecular masses of 47, 43, 33, 30 and 10 kDa. Lipid analyses showed that polar, as well as neutral, lipids are associated with the complex. For approx. 40 chlorophyll a molecules there were 3.4 plastoquinone‐9, 3.3 pheophytin a, 2.9 β‐carotene, 29.3 monogalactosyldiacylglycerol and 12.4 sulphoquinovosyldiacylglycerol molecules. These results suggest that this photosystem II reaction centre complex, which most likely represents a minimum photochemically active unit, is a lipoprotein complex. A striking feature of the associated polar lipids is their very low degree of unsaturation.

Tightly bound sulpholipids in chloroplast CF0-CF1

Highly purified preparations of CF0-CF1 from chloroplasts contain a small amount of tightly bound lipids. Extraction and analysis of these lipids show that they are almost exclusively sulpholipids. The calculated amount of bound sulpholipids in spinach and in Dunaliella salina CF0-CF1 preparations are 5 and 20 mols/mol enzyme, respectively. Attempts to exchange the bound lipids with other lipids or with detergents have failed, indicating a very strong association with CF0-CF1.

The role of different thylakoid glycolipids in the function of reconstituted chloroplast ATP synthase

Abstract ATPase activity of CF 0 CF 1 from spinach chloroplasts is specifically stimulated by chloroplast lipids (Pick, U., Gounaris, K., Admon, A. and Barber, J. (1984) Biochim. Biophys. Acta 765, 12–20). The association of CF 0 -CF 1 with isolated lipids and their mixtures has been examined by analyzing the stimulation of ATPase and ATP-P i exchange activities, by binding studies and by measurement of proton conductance of reconstituted proteoliposomes. Monogalactosyldiacylglycerol is the only chloroplast lipid which by itself activates ATP hydrolysis. A mild saturation of the fatty acids of the lipid partially inhibits the activation. CF 0 -CF 1 has a higher binding capacity for monogalactosyldiacylglycerol (1.5 mg/mg protein) than for other thylakoid glycolipids. However, ATPase activation is not correlated with the amount of bound lipid but rather with its type. For the same amount of bound lipid, monogalactosyldiacylglycerol best activates ATP hydrolysis, while the acidic lipids phosphatidylglycerol and sulphoquinovosyldiacylglycerol inhibit ATPase activity. Optimal activation of ATP-P i exchange requires, in addition to monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulphoquinovosyldiacylglycerol at a ratio of 6:3:1, respectively. Correlations between proton conductance, ATP-P i exchange and uncoupler stimulation of ATPase activity indicate that sulphoquinovosyldiacylglycerol reduces the permeability of the proteoliposomes to protons. The results suggest that: (a) association of CF 0 -CF 1 with polyunsaturated monogalactosyldiacylglycerol greatly stimulates ATPase activity; (b) reconstitution of coupled CF 0 -CF 1 proteoliposomes requires a careful balance of the natural glycolipids of thylakoid membranes in similar proportions to their occurrence in chloroplasts, and (c) sulphoquinovosyldiacylglycerol may control the permeability of chloroplast membranes to protons.

What role does sulpholipid play within the thylakoid membrane

Sulphoquinovosyldiacylglycerol is a negatively charged lipid which exists in the thylakoid membrane. It is proposed that a large proportion of this acidic lipid does not form a part of the bulk lipid matrix but is closely associated with protein complexes where it is tightly bound and participates in either optimising catalytic activities, or maintaining the complexes in a functional conformation. Experimental evidence for this proposal is emerging from studies with isolated photosystem 2, and coupling factor complexes.

Activation of the CF0-CF1, ATP synthase from spinach chloroplasts by chloroplast lipids

Abstract The interactions of CF 0 -CF 1 with different lipids were studied by following the stimulation of Mg-ATPase and of P i -ATP exchange activities of reconstituted CF 0 -CF 1 proteoliposomes. The following results were obtained: (1) Both P i -ATP exchange and Mg-ATPase activities are stimulated by lipids. Furthermore, the inhibition of Mg-ATPase by N , N ′-dicyclohexylcarbodiimide is dependent on the interactions of CF 0 -CF 1 with lipids. (2) A polar lipid extract of thylakoid membranes stimulates Mg-ATPase activity of CF 0 -CF 1 more efficiently than phospholipids. The relative effectiveness of Mg-ATPase stimulation is: chloroplast lipids > soybean phospholipids > phosphatidylcholine/phosphatidylserine (4: 1) > phosphatidylcholine. The rate of P i -ATP exchange in chloroplast lipids CF 0 -CF 1 proteoliposomes is, however, lower than in soybean lipids CF 0 -CF 1 proteoliposomes, due to their higher permeability to protons. Addition of 10% phosphatidylserine to chloroplast lipids reduces their permeability to protons and stimulates P i -ATP exchange. (3) The kinetic mechanism of ATPase stimulation by chloroplast lipids is by decreasing the K m (ATP) and by increasing V max in comparison to soybean lipid proteoliposomes. This may explain the low affinity for ATP and the slow turnover rate of the purified enzyme in artificial lipids in comparison to the native enzyme in chloroplast thylakoids. (4) Chloroplast lipids lacking monogalactosyldiacylglycerols only poorly activate CF 0 -CF 1 . A large stimulation of P i -ATP exchange is obtained by a mixture of 60% monogalactosyldiacylglycerol and 40% of the rest of the chloroplast lipids, but not by mixtures of monogalactosyldiacylglycerol with phospholipids. Hydrogenation of the unsaturated fatty acids of monogalactosyldiacylglycerol inhibits the activation of CF 0 -CF 1 . (5) The results suggest that: (a) interactions of specific chloroplast lipids with CF 0 -CF 1 activates the enzyme by increasing its turnover and its affinity for ATP; (b) specific requirements for CF 0 -CF 1 activation are the presence of monogalactosyldiacylglycerols together with another chloroplast lipid component and of highly unsaturated fatty acids.

The interaction between the 33 kDa manganese-stabilising protein and the D1/D2cytochrome b-559 complex

Using affinity chromatography with the extrinsic 33 kDa protein as the immobilised ligand, it was demonstrated that the reaction centre complex of photosystem II, composed of the D1, D2 and cytochrome b‐559 polypeptides, can directly interact with the 33 kDa protein. By this approach it was possible to purify the reaction centre from solubilised photosystem II core complexes since neither the 47 kDa nor the 43 kDa protein would bind to the ligand.

Stoichiometry and turnover of photosystem II polypeptides

Using Triton X‐100 solubilization and sucrose gradient fractionation, a photosystem II core complex has been isolated from the green alga Dunaliella salina. The complex was similar to that previously isolated from spinach [(1985) FEBS Lett. 188, 68‐72] being composed of five main polypeptides of apparent molecular masses, 47, 40, 32, 30 and 10 kDa. Studies using 14C indicate that with either dark or illuminated cells the Stoichiometry of these polypeptides is 1:1:1:1:1 with about 20 chlorophyll a molecules per monomeric unit. According to 35S labelling only the 32 kDa polypeptide, identified as the D1 or QB binding protein by immunoblotting, is rapidly turning over at a rate greater than the growth rate. The level of labelled sulphur associated with each polypeptide is reasonably consistent with the amino acid content derived from gene sequences and identifies the 47, 40, 32, 30 and 10 kDa apoproteins as products of the psbB, psbC, psbA, psbD and psbE genes.

Isolation of the Photosystem Two Reaction Centre and the Location and Function of Cytochrome b-559

Although there have been numerous studies to understand the role of cytochrome b-559 in photosynthesis, no clear picture has yet emerged. The study of this cytochrome is complicated by the fact that there seems to be two distinctly different pools. One pool consists of a low potential form (E~ −30 mV) and is located in the unappressed stromal regions of higher plant chloroplasts (1). This low potential form may be in some way linked to electron and proton translocation through the cytochrome b6-f complex (2). The other pool is localized in the appressed regions which constitute the grana (3,4) and normally seems to exist in a high potential form (E~ 380 mV). In the earlier literature there was some confusion about the redox properties of the high potential form because its midpoint potential can be significantly modified after certain treatments which affect membrane structure especially by the action of detergents (5,6). In this case the Em changes from 380 mV to about 80 mV. Apparently this shift is due to a perturbation of the environment around the cytochrome and can be reversed by adding back polar lipids derived from the thylakoid, particularly digalactosyldiacylglycerol (7,8). Reconstitution of a certain extrinsic polypeptide (23 kDa) associated with water oxidation, has also been reported to revert the cytochrome to its high potential form (9).

The Sensitivity of Pisum Sativum Thylakoid Membrane Protein Kinase Activity to Inhibitors, Detergents and Heating

Several polypeptides of the thylakoid membrane become reversibly phosphorylated in excess photosystem two (PSII) light or under conditions in the dark when the plastoquinone pool is chemically reduced (1). Much work has been directed to the phosphorylation of the light harvesting chlorophyll a/b protein complex (LHCII) (2) but several polypeptides of PSII also become phosphorylated, notably at 10,30,33,43 kD (3). There have been efforts to isolate the protein kinase(s) involved (4) and Clark et al (5) claim to have isolated the kinase responsible for LHCII phosphorylation. Isolation and reconstitution of full LHCII phosphorylating activity with redox control has yet to be demonstrated. Although it is well known that the kinase is closely associated with the membrane it has to be established whether the enzyme is found in appressed or non-appressed thylakoids or in both. Attempts to investigate this question using Triton X-100 derived appressed membrane fragments emphasised the sensitivity of the kinase(s) to this detergent. Mechanical fractionation procedures were therefore used to show the presence of kinase activity in fractions enriched in appressed membranes. Further experiments with Triton X-100, (3-octylglucoside and sodium cholate have been performed to investigate the effects of detergents upon the phosphorylation of LHCII and PSII polypeptides.

Further Characterisation of the Isolated PS2 Reaction Centre

The reaction centre of photosystem two (PS2) consisting of the D1/D2/cyt b559 complex isolated from peas has been further characterised both in terms of its composition and electron transport properties. Experiments are presented which support the earlier claims (Barber et al. FEBS Lett. 1987, 67–83) that silicomolybdate (SiMo) can be used as a convenient artificial electron acceptor when suitable donors such as diphenylcarbazide (DPC), MnCl2, NH2OH or KI are present. However, when the Triton X-100 associated with the isolated complex is exchanged with beta-lauryl maltoside, a light-induced signal is observed on addition of decyl plastoquinone. This signal corresponds to the photoreduction of about 30% of the cytochrome b-559 within the complex. A similar proportion of the cytochrome is reduced in the dark by decyl plastoquinone when the reductant, sodium borohydride, is added. This result, coupled with the finding that the photoreduction of cytochrome b559 is inhibited by DCMU, suggests that the added quinone is also photoreduced possibly by the QA and/or OB sites.