Phosphatidylglycerol is involved in the dimerization of photosystem II.

Photosystem II core dimers (450 kDa) and monomers (230 kDa) consisting of CP47, CP43, the D1 and D2 proteins, the extrinsic 33-kDa subunit, and the low molecular weight polypeptides PsbE, PsbF, PsbH, PsbI, PsbK, PsbL, PsbTc, and PsbW were isolated by sucrose density gradient centrifugation. The photosystem II core dimers were treated with phospholipase A2 (PL-A2), which cuts phosphatidylglycerol (PG) and phosphatidylcholine molecules at the sn-2 position. The PL-A2-treated dimers dissociated into two core monomers and further, yielding a CP47-D1-D2 subcomplex and CP43. Thin layer chromatography showed that photosystem II dimers contained four times more PG than their monomeric counterparts but with similar levels of phosphatidylcholine. Consistent with this was the finding that, compared with monomers, the dimers contained a higher level of trans-hexadecanoic fatty acid (C16:1Delta3tr), which is specific to PG of the thylakoid membrane. Moreover, treatment of dimers with PL-A2 increased the free level of this fatty acid specific to PG compared with untreated dimers. Further evidence that PG is involved in stabilizing the dimeric state of photosystem II comes from reconstitution experiments. Using size exclusion chromatography, it was shown that PG containing C16:1Delta3tr, but not other lipid classes, induced significant dimerization of isolated photosystem II monomers. Moreover, this dimerization was observed by electron crystallography when monomers were reconstituted into thylakoid lipids containing PG. The unit cell parameters, p2 symmetry axis, and projection map of the reconstituted dimer was similar to that observed for two-dimensional crystals of the native dimer.

Membrane protein complexes often exist in oligomeric states in vivo despite the fact that when isolated as monomers, they can carry out their basic enzymatic activities. For example, the structure of mammalian cytochrome oxidase recently determined by x-ray crystallography (1,2) indicates that this complex is dimeric. Dimeric configurations have also been advocated for many other membrane proteins, including cytochrome b-c complexes (3,4), Na ϩ K ϩ ATPase (5), ethylene response mediator (6), and intercellular adhesion molecule 1 (7). In oxygenic photosynthetic organisms it is also widely believed that photosystem II (PSII) 1 normally functions as a dimer (8), whereas photosystem I in cyanobacteria is trimeric (9). Light harvesting complex II (LHCII) is also trimeric (10). In this paper, we focus on the stabilization of the dimeric form of PSII and reveal the importance of the thylakoid membrane lipid phosphatidylglycerol (PG) in maintaining this conformation.
PSII is the water-plastoquinone oxidase of oxygenic photosynthesis. It is a multisubunit complex located in the lipid matrix of the thylakoid membrane. The main protein subunits of the core complex are the reaction center proteins (D1 and D2), the inner antenna proteins (CP47 and CP43), the extrinsic protein of the oxygen evolving complex, 33-, 23-, and 17-kDa proteins, and a number of low molecular weight proteins. In higher plants and green algae, this central core is flanked by the outer chlorophyll a/b binding antenna composed of LHCII and CP29, CP26, and CP24 (11). The structure of the central core and the organization of the protein subunits within it have been revealed by electron microscopy to different levels of resolution (12)(13)(14)(15)(16)(17)(18)(19). These and other studies (20) have indicated that PSII normally exists as a dimeric complex. The functional role of the dimeric organization is as yet not understood, nor is its implication for conformational changes that must occur as a consequence of the light-induced turnover of the D1 reaction center protein (21). The dynamic process of D1 protein exchange is reported to involve the monomerization of the PSII core dimer followed by the release of CP43 (22). It is suggested that in this way, the damaged D1 protein is exposed, allowing its exchange for a new, fully functional D1 subunit. Little or nothing is known about the factors controlling these structural changes, nor do we have any knowledge of the role of lipids in the thylakoid membrane in stabilizing the different conformational states of PSII.
The thylakoid membrane in which PSII is embedded consists of PG, monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), sulfoquinovosyl diacylglycerol (SQDG), and traces of phosphatidylcholine (PC). The distribution of these lipids within the thylakoid membrane has been studied in considerable detail (23). Lipids can be divided into those making up the fluid matrix of bulk lipid phase, allowing molecular motion and rotational and lateral diffusion, and those that, for structural or functional purposes, are specifically bound to protein subunits. In particular, PG, with its unusual trans-⌬3-hexadecanoic fatty acid (C16:1-⌬3tr) at the sn-2 position, is reported to have a structural and functional role in PSII (24 -26) and is tightly bound to the D1 protein of PSII in cyanobacteria (27). Furthermore, it is reported to be involved in the trimerization of LHCII (28 -30) and the dimerization of LHCI (31).
Here, we report the possible involvement of specific PG molecules in PSII dimer-monomer interconversion and the release of CP43 from the D1-D2-CP47 subcomplex. The molecular dynamics of PSII dimer dissociation is discussed in the light of recently determined structural information obtained for the PSII core dimer (19) and the monomeric CP47-reaction center complex (17) by cryoelectron crystallography and with respect to D1 turnover studies (22).

Isolation of Particles of Grana Membrane Fraction Enriched in PSII,
PSII Core Monomers, and Dimers-The stacked granal regions of the thylakoid membrane enriched in PSII, as well as purified monomeric and dimeric PSII core complexes, were isolated from market spinach according to Hankamer et al. (8). Essentially, PSII-enriched membranes were solubilized with n-octyl-␤-D-glucopyranoside (Calbiochem) to detach the LHCII proteins from the PSII core, whereas sucrose gradients supplemented with n-dodecyl-␤-D-maltoside were employed to obtain oxygen evolving PSII core monomers and dimers. The polypeptide compositions of the isolated PSII preparations were analyzed by gradient SDS-PAGE (10 -17% polyacrylamide) containing 6 M urea (32). The gels were silver stained according to Kruse et al. (33).
HPLC Size Exclusion Analysis of Phospholipase A2-treated PSII Core Dimers-Samples containing 50 l of dimeric PSII complex (final concentration, 0.5 mg of Chl ml Ϫ1 ) were incubated with 10 l of PL-A2 (10 mg ml Ϫ1 stock solution, Sigma) in 0.1 M HEPES, pH 7.5, solution at room temperature and in the dark. Dissociation of PSII dimers was monitored by HPLC size exclusion chromatography (Zorbax GF450 column with a 200 mM Tris, 1 mM n-dodecyl-␤-D-maltoside mobile phase). The column was calibrated using ␤-amylase, apoferritin, and bovine serum albumin (Sigma) as molecular weight standards, and elution peaks were monitored at 280 and 415 nm.
Extraction and Purification of Lipids from PSII Preparations-Polar lipids (MGDG, DGDG, SQDG, PG, and PC) and nonpolar pigments (chlorophylls and carotenoids) were extracted from PSII-enriched membranes, PSII core monomers, and dimers. These PSII preparations were first washed in 20 mM Mes, pH 6.5, and concentrated either by centrifugation (PSII-enriched membranes) or using a Centricon 100 concentrator unit purchased from Amicon (PSII dimers and monomers) in order to reduce the detergent concentration. The thylakoid lipids were extracted from these PSII preparations by sequentially vortexing the samples in methanol, chloroform, and acetone followed by removal of the protein fraction by centrifugation. The solvent extracts were pooled and dried under vacuum, and the pellets containing the mixture of polar lipids and pigments were redissolved in chloroform:methanol (1:1, v/v) before being stored at Ϫ20°C. Purification of single lipid classes from PSII-enriched membranes was achieved by using Silica gel (Merck), DEAE (DE23, Whatman), and Florisil (Merck) column chromatography according to Schmid et al. (34) with minor modifications. Pure MGDG and DGDG fractions were obtained after separation on a DEAE column by applying chloroform:methanol mixtures (57:3 to 1:1) in a stepwise fashion. SQDG was subsequently separated on a Florisil column eluted with chloroform/methanol/dimethoxypropane. Finally, PG was eluted from the column with 100% methanol.
Qualitative Analysis of PSII Lipid Components by TLC-The purified lipid extracts from PSII-enriched membranes and PSII core monomer and dimer preparations were resolved into their constituent components by thin layer chromatography for qualitative analysis. Essentially, the lipid extracts (nonpolar and polar lipids) were dispensed (100 g of Chl) onto Silica Gel 60 TLC plates (Merck). The lipid components were then resolved using a 75% chloroform, 13% methanol, 9% acetic acid, and 3% water mobile phase (35). The separated phospholipids were stained with molybdenumoxide (36), whereas the galactolipids were detected using anthron. The anthron solution was prepared by dissolving 0.2 g of anthron (Merck) in 100 ml of H 2 SO 4 and diluting the solution (2 ml of anthron solution:1 ml of water). The TLC plates were then sprayed with the diluted anthron solution and incubated at 100°C for 5 min to stain the sugar-containing lipids MGDG, DGDG, and SQDG.
Quantitative Analysis of PSII Lipid Components by TLC-For quantitative analysis, the total lipid extracts were loaded (1 mg of Chl) onto laboratory-prepared Silica Gel 60 TLC plates and separated under the conditions described above. Subsequently, the plates were dried and stained with iodine gas in a closed chamber. Iodine reacts reversibly with the double bonds of unsaturated fatty acids (37). The stained galactolipid bands were marked, collected from the TLC plate after a recovery period of 15 min, and solubilized in 1 ml of 2% phenol and 4 ml of 100% H 2 SO 4 , and absorption was measured at 480 nm. Phospholipids were assayed at 815 nm according to Ref. 38 with ammonia-heptamolybdate and Fiske-Subarow reagent after incubation at 190°C for 30 min in 0.7 ml of 70% perchloric acid.
Qualitative Analysis of Fatty Acids-To determine differences in the composition of fatty acids extracted from PSII dimers by petroleum ether before and after PL-A2 treatment, the isolated extract was saponified and methylated for gas chromatography according to Ref. 35. This method predominantly extracts free fatty acids and nonpolar components. The final esterification step was carried out using an N,Ndimethylformamid-dimethyl acetate methylation reagent kit (Macherey and Nagel, Dü ren, Germany). The methyl ester derivatives of the fatty acid components were resolved on a HP-FFAP column using a Hewlett-Packard 5890 gas chromatograph and nitrogen as a carrier gas. The column temperature was adjusted to 190°C, with the detector and injection port temperatures set to 300°C. The areas under the peaks were calculated using a HP 3396 series II integrator. The retention time of the C16:1⌬3 trans-hexadecanoic (C16:1⌬3tr) fatty acid was identified by comparison with a standard C16:1⌬9 trans-hexadecanoic fatty acid (NuChekPrep).
Reconstitution of Dimeric PSII Complexes from Purified PSII Core Monomers-PSII core monomers isolated by sucrose density gradient centrifugation were mixed with purified lipids (PG, DGDG, and SQDG) that had been dried down previously under N 2 gas and solubilized with n-heptyl-␤-D-thioglucopyranoside (90 mM) to give a final concentration of 1 mg of Chl ml Ϫ1 . The monomer:detergent:lipid mix was then incubated on ice for 40 min and analyzed by size exclusion chromatography in 5 mM Mes, pH 6.0, 15% glycerol, 0.03% n-dodecyl-␤-D-maltoside, and 1 mM sodium azide, using a BIOSEP SEC3000 column and a flow rate of 0.5 ml min Ϫ1 . The column was calibrated using PSII monomers and dimers isolated by sucrose density gradient centrifugation.
Two-dimensional Crystallization-PSII core monomers (1 mg of Chl ml Ϫ1 ) were supplemented with purified thylakoid lipids and crystallized using the method of Morris et al. (16). Samples were harvested after a 16 h incubation at 19°C.
Electron Microscopy-The samples containing the two-dimensional crystals were applied to carbon-coated electron microscope grids, washed with distilled water, and negatively stained with 1% uranyl acetate. The grids were examined in a Philips CM100 electron microscope at an accelerating voltage of 80 kV. Images were recorded at calibrated magnifications of ϫ 51,100 on Agfa Scientia Film.
Image Processing-Electron micrographs were digitized using an Emil densitometer (Image Science) at an initial step size of 7.05 m. Regions of 2 ϫ 2 pixels were averaged, and further analysis was conducted at a sampling interval of 14.1 m, corresponding to 0.276 nm at the specimen level. Medical Research Council (39) image analysis programs together with locally developed software were used for twodimensional crystal analysis. Individual images were corrected for longrange disorder (40) and averaged projection maps, calculated by Fourier methods.

Phospholipid Depletion Leads to Dissociation of the Isolated
Dimeric PSII Complex-To investigate the possibility that PG and perhaps PC are involved in maintaining the oligomeric state of PSII, PSII dimers isolated by sucrose density gradient centrifugation were incubated with PL-A2. This enzyme cleaves phospholipids at the sn-2 position, yielding the sn-2 fatty acid and the corresponding lysolipid components. The PL-A2-treated and untreated dimeric cores were analyzed by HPLC size exclusion chromatography and SDS-PAGE. Fig. 1a shows the elution profile of untreated PSII core dimers that consists of a single peak corresponding to 450 kDa. Their subunit composition before and after HPLC treatment is shown in Fig. 2 (see lanes a and b). It can be seen in Fig. 2 that the PSII core dimers (Fig. 2, lane a) incubated for 2 h at room temperature in the absence of PL-A2 lost the 33-kDa extrinsic protein of the oxygen evolving complex (lane b) during the HPLC treatment. Despite this, the dimeric form of the PSII core was sustained, indicating that the 33-kDa extrinsic protein is not required to maintain this oligomeric state, a conclusion in agreement with Morris et al. (16). The population of dimers decreased, however, after 2 h of treatment with PL-A2 (see Fig.  1b), leading to the appearance of two new fractions with ap-proximate molecular masses of 230 and 170 kDa. A smaller peak having a molecular mass of about 30 kDa was also detected. As indicated by SDS-PAGE, the 230-kDa fraction consisted of CP47, CP43, D1, and D2 proteins (see Fig. 2, lane c), whereas the 170-kDa fraction was depleted of CP43 (see Fig. 2, lane d). The 230-kDa fraction represents a monomeric form of the PSII core and had the same retention time as PSII core monomers isolated by sucrose density gradient centrifugation. The fraction having a molecular mass of 170 kDa corresponds to the monomeric form of the CP47-D1-D2 subcore complex (41) (see Fig. 2, lane d). SDS-PAGE analysis and amino acid sequencing showed that the lower molecular mass fraction (30 kDa) was PL-A2.
Isolated PSII Core Dimers Have Significantly Higher Molar PG:Reaction Center Ratios Than Their Monomeric Counterparts-The PL-A2 treatment described above suggests that phospholipids play a role in stabilizing the PSII core structure. To establish which of the thylakoid phospholipids (PG or PC) is involved in the stabilization of the dimer, PSII complexes isolated by sucrose density centrifugation were subjected to lipid analyses using TLC. These TLC separations showed that PSIIenriched granal membranes contained MGDG, DGDG, PG, and low levels of PC (Fig. 3, lane c). SQDG was also present, as indicated by staining with anthron solution (not shown).
The isolated core dimers (Fig. 3, lane e) had lipid ratios similar to those of the PSII-enriched membranes (lane c). Of particular note is that the PG level is considerably higher than that of PC in both the PSII membranes and core dimers. Core monomers, however, isolated by sucrose density centrifugation, had considerably lower levels of PG (Fig. 3, lane d).
All lanes shown in Fig. 3 were loaded on the basis of equal amounts of Chl (100 g). To determine the molar ratios of lipid:reaction center, lipids resolved by TLC were analyzed using spectrometric methods (see under "Materials and Methods"). Solvent treatment of the isolated core dimers and monomers extracted both the nonpolar pigments and polar lipids. Using Chl:reaction center ratios of the PSII core dimers (40:1)

FIG. 2. SDS-PAGE analysis of PSII complexes resolved by size exclusion size exclusion HPLC after PL-A2 treatment. a, isolated
PSII core dimer before HPLC; b, PSII core dimers after size exclusion HPLC. Note the loss of the 33-kDa subunit (at room temperature for 7.75 min). c, fraction (at room temperature for 8.30 min) collected from HPLC size exclusion column after 2 h of PL-A2 treatment of dimers. d, fraction (at room temperature for 9.00 min) collected from HPLC size exclusion column after 2 h of PL-A2 treatment of dimers. and monomers (36:1) previously determined (8), it was possible to calculate lipid:reaction center ratios (Table I). Monomers contained lower levels of each lipid class compared with their dimeric counterparts. However, whereas MGDG and DGDG were depleted by only 8 and 18%, respectively, PG levels were depleted by 75% in monomers compared with dimers. Core dimers were calculated to contain 16 molecules of PG and 2 of PC per reaction center, whereas monomers contained 4 molecules of PG and approximately 1 of PC. Consequently, it seems that 12 molecules of PG and approximately 1 molecule of PC per reaction center are lost during dimer to monomer conversion.
Fatty Acid Analysis Suggests That Monomerization of Cores Involves Loss of C16:1⌬3tr PG-The fatty acid levels determined for isolated dimers and monomers is given in Table II. Of particular note is that the level of the C16:1⌬3tr fatty acid is lower in the monomeric form of PSII than in the dimer by approximately 40%. This drop is consistent with the 75% loss of PG as a consequence of dimer to monomer conversion, taking into account that PG contains other fatty acids at the sn-2 position as well as the unique C16:1⌬3tr. Another overall feature is that fatty acids associated with the PSII cores were significantly more saturated when considered as a whole compared with those that make up the bulk of the thylakoid membrane. This difference in the level of fatty acid saturation between lipids closely associated with PSII proteins compared with the bulk membrane lipids has been noted previously (23). Of particular interest is that the saturation level is higher in the dimer compared with the monomer reflecting the reduction in PG levels. In general, the fatty acids of PG are more saturated than those of the major thylakoid lipids (42).
Free fatty acids associated with the isolated core dimer before (control) and after treatment with PL-A2 were also determined. Table III shows that in the untreated core dimers, C16:0 and C18:0 fatty acids were readily detected, whereas levels of C16:1⌬3tr, C18:1, and C18:3 were low. However, when the free fatty acid profiles of four replicates of core dimers treated with PL-A2 for 2 h and control samples are compared, the PL-A2-treated samples were found to be greatly enriched in the free fatty acid C16:1⌬3tr (455.0%). The minor C18:1 component may also have been enriched (66.6%) but the signals were so low as to give a high level of uncertainty on this data, a point also applicable to the C18:3 data. Consequently we conclude that PG molecules binding the C16:1⌬3tr fatty acid at the sn-2 position are cleaved by the PL-A2 treatment. The cleavage of small quantities of PC molecules cannot be ex-cluded. However, overall, our results support the idea that PG plays a role in stabilizing the dimeric state of PSII.
Dimeric PSII Complexes Are Formed from PSII Monomers on Addition of Purified PG and Isolated Thylakoid Lipids-To determine whether the dimer to monomer conversion process is reversible, purified PSII monomers were mixed with detergentsolubilized PG, and the oligomeric state of the product was assayed by size exclusion HPLC. Fig. 4 shows that with increasing levels of PG (isolated from PSII-enriched membranes), a shoulder developed in the HPLC profile indicative of the formation of PSII core dimers. The position of the shoulder corresponds to a molecular mass of 450 kDa. The peak corresponding to the monomer (230 kDa) has been normalized in Fig. 4. Fig. 4 also shows one of the controls using a relatively high level of DGDG in which no significant dimerization was detected, a result also found over a range of concentrations of this lipid and with the sulfolipid, SQDG. The small yet relatively broad shoulder observed with DGDG (compare with 1 g of PG trace) did not seem to correspond with dimer formation but rather to some other, larger aggregation state. In the case of PG, the dimerization process was not 100% complete and saturated at about 3 g of PG⅐g of Chl Ϫ1 . Interestingly, when PG, consisting of C18:1 and C16:0 (Sigma P6956), was used, dimerization did not occur, indicating the importance of the C16:1⌬3tr fatty acid in the aggregation process. FIG. 4. HPLC size exclusion analyses to determine the effect of reconstituting PSII core monomers with different amounts of PG. PG was isolated from spinach PSII membranes, detergent-solubilized, and mixed with isolated PSII monomers (0.77 g of Chl ml Ϫ1 ). The effect of the addition of a high level of DGDG isolated from PSIIenriched membranes is also shown. The signals are normalized to the peak due to the elution of monomers.  To study further the PG-induced monomer to dimer conversion, isolated PSII monomers were incubated with a mix of detergent-solubilized thylakoid lipids containing all of the polar lipids (MGDG, DGDG, SQDG, PC, and PG). On detergent removal, protein-packed vesicles formed that contained twodimensional crystals (Fig. 5a). 2 These crystals were imaged by electron microscopy in negative stain. Fourier analysis of such crystals gave unit cell parameters of a ϭ 12.2 nm, b ϭ 17.4 nm, and cell angle ϭ 106.7 o . These parameters are almost identical to those reported previously for two-dimensional crystals of the isolated PSII dimer (16), suggesting that the monomers formed native dimers on reconstitution with thylakoid lipids containing PG. The Fourier-filtered images are shown before (Fig. 5b) and after (Fig. 5d) the imposition of p2 symmetry. Even in its unsymmetrized form, the projection map has a clear 2-fold rotational symmetry axis around the central stain filled region and is virtually identical to that obtained from negatively stained crystals of PSII core dimers (16). These results indicate that when PSII core monomers are reconstituted with thylakoid lipids containing PG, they are able to reassemble into dimers that are structurally similar to native dimers at a resolution of about 30 Å. DISCUSSION The above results suggest that PG containing C16:1⌬3tr plays an important role in maintaining the structural integrity of the PSII complex. As mentioned above, this lipid has been shown to be necessary for trimerization of LHCII (28 -30). Here we show for the first time that PG plays a role in the dimerization of PSII. Previously, PG had been implicated in the structural and functional properties of PSII (24 -27, 43-45), but none of these studies had implicated the role of this lipid in controlling the aggregation state of the complex. In the case of LHCII, it seems that the anionic PG molecule interacts with positively charged amino acids of the LHCII monomer between residues 9 and 49 (29,46). Specific sites for the binding of PG within PSII are not known, but it has been suggested by Kruse and Schmid (27) that one possible site is on the D1 protein between residues 27 and 225. The structural details of PSII are gradually emerging, and recently, ordered two-dimensional crystals of the dimeric core complex and CP47-D1-D2 subcomplex have been grown and analyzed by electron crystallography (17,19). The resulting structural model has identified the protein densities that form the interface between the two monomeric cores and the points of contact between the CP47-D1-D2 subcomplex and CP43. It is possible that PG molecules are located at these interfaces. Fig. 5c shows a projection map, obtained by cryoelectron microscopy, and a superimposed model of the PSII core dimer (19) that incorporates the transmembrane helices of the D1, D2, CP47, and CP43 proteins based on the three-dimensional structure of the CP47-D1-D2 complex (17). Also shown are seven further transmembrane helices, identified in the three-dimensional structure of the CP47-D1-D2 subcore complex, attributed to the small polypep-FIG. 5. Two-dimensional crystals and their analysis by electron microscopy. a, electron micrograph of a negatively stained twodimensional crystal obtained by reconstituting PSII core monomers into thylakoid lipids. b and d are projection maps derived from the two-dimensional crystal shown in a, before and after imposition of p2 symmetry, respectively. Note the strong 2-fold rotational symmetry axis around the central stain filled region. The unit cell parameters are a ϭ 12.2 nm, b ϭ 17.4 nm, and cell angle ϭ 106.7°, and so the parameters are virtually identical to that obtained from native PSII core dimers in negative stain (16). c, the subunit organization of the PSII core dimer reported by Hankamer et al. (19). Here, CP47 is placed next to D2 and CP43 adjacent to D1 based on cross-linking studies (53) and D1 turnover studies (22). Additional helices colored purple correspond to low molecular weight proteins identified in the 8-Å three-dimensional map of the CP47-reaction center subcomplex (17). The monomeric CP47-reaction center subcomplex contains the low molecular weight proteins PsbE, PsbF, PsbI, PsbTc, and PsbW (41), whereas the core dimer contains additionally PsbK, PsbL, and PsbH (Footnote 2). The two central regions of density colored purple and marked * are present in the core dimer projection map (19) but are absent from the CP47-reaction center projection map (17). Consequently a PsbH, PsbK, or PsbL subunit could be located within each of these regions of density.
tides PsbE, PsbF, PsbI, PsbK, PsbL, PsbTc, and PsbW (41). The densities at the interface of the two monomers (Fig. 5c, purple and *) that constitute the dimer could be the location of PsbH that is contained in the PSII core dimer but not in the CP47-D1-D2 subcore complex (19). Interestingly, PsbH contains a cluster of positively charged residues at its N terminus (Lys-14, Lys-23, Lys-28, Lys-31, and Arg-35) located at the stromal surface of its single transmembrane helix. Thus, this region could be a site for binding PG. In higher plants and algae, it is also the region on the PsbH protein where reversible N-terminal phosphorylation occurs (47). Also of note is that the deletion of the psbH gene from Chlamydomonas reinhardtii inhibited the accumulation of PSII in the membrane (48,49) and led Summers et al. (48) to conclude that perhaps the primary role of the PsbH protein was to facilitate PSII assembly/stability. Furthermore, according to the current structural model of PSII shown in Fig. 5c, the region linking the CP47-D1-D2 subcore with CP43 involves the D1 protein, particularly transmembrane helices 2, 3, and 5 (see Fig. 5c) and, according to Kruse and Schmid (27), this could also contain a PG binding site. In vivo, the interconversion between the dimer and monomers and the selective release of CP43 has been implicated with the D1 protein repair cycle (22). Because these organizational changes involve association as well as dissociation of proteins, it is unlikely that PG binding in itself is the only regulatory factor. One possibility is that these large structural changes are controlled by reversible N-terminal phosphorylation. In higher plants and algae, the D1 and D2 proteins, PsbH and CP43, can indeed be phosphorylated, and perhaps changes in the electrostatic interactions induced by their phosphorylation/dephosphorylation, coupled with the presence of specifically bound PG, bring about the macromolecular conformation changes that characterize the dynamics of PSII.
In conclusion, it seems that PG of the thylakoid membrane, with its unusual C16:1⌬3tr fatty acid, is important for the stabilization of the oligomeric states of PSII and its light harvesting systems. This conclusion is in part supported by gene deletion studies leading to mutants devoid of C16:1⌬3tr PG (50,51). In the case of C. reinhardtii, the mutation resulted in the loss of PSII activity and no trimerization of LHCII (50). A mutant of Arabidopsis thalina, lacking trans-hexadecanoic acid, also lost the ability to trimerize LHCII, but there was no apparent effect on PSII assembly and function (51). At this stage, it is difficult to reconcile the latter work with the findings presented in this paper and with the other, numerous results that implicate C16:1⌬3tr PG with PSII function. The controversy will hopefully be resolved by elucidating the precise binding sites of PG within PSII. This will required high resolution structural data of the type that has yielded details of lipid-protein interactions in other systems. Of particular note is recent work on the reaction center of Rhodobacter sphaeroides in which x-ray diffraction analysis at a 2.1-Å resolution has identified the specific binding sites of a cardiolipin molecule at Arg-267 and His-145 on the M subunit. 3 Binding sites for lipids have also been identified in the high resolution structure of cytochrome oxidase (3,4) and bacteriorhodopsin (52).