Báo cáo Y học: An alternative model for photosystem II/light harvesting complex II in grana membranes based on cryo-electron microscopy studies pptx

Eur J Biochem 269, 326±336 (2002) Ó FEBS 2002 An alternative model for photosystem II/light harvesting complex II in grana membranes based on cryo-electron microscopy studies Robert C Ford1, Svetla S Stoylova2 and Andreas Holzenburg3 Department of Biomolecular Sciences, UMIST, Manchester, UK; 2The Burnham Institute, La Joua, CA, USA; Department of Biology and Department of Biochemistry and Biophysics, Microscopy and Imaging Center, Texas A & M University, College Station, TX, USA The photosynthetic protein complexes in plants are located in the chloroplast thylakoid membranes These membranes have an ultrastructure that consists of tightly stacked ÔgranaÕ regions interconnected by unstacked membrane regions The structure of isolated grana membranes has been studied here by cryo-electron microscopy The data reveals an unusual arrangement of the photosynthetic protein complexes, staggered over two tightly stacked planes Chaotrope treatment of the paired grana membranes has allowed the separation and isolation of two biochemically distinct membrane fractions These data have led us to an alternative model of the ultrastructure of the grana where segregation exists within the grana itself This arrangement would change the existing view of plant photosynthesis, and suggests potential links between cyanobacterial and plant photosystem II light harvesting systems Photosynthesis, one of the most important biochemical processes, occurs in the thylakoid membranes of plants that are located inside specialized cell compartments (chloroplasts) The thylakoid membrane system is highly organized, with characteristic stacks of membranes that are termed grana, which are interconnected by unstacked regions of membrane (see Fig 1A) In the classical view of plant photosynthesis, one part of the photosynthetic electron transfer chain (photosystem II) is segregated into the grana, whilst other components of the system (photosystem I and the H+-ATPase) are located in the unstacked thylakoid membranes [1±3] The location of the fourth component of the photosynthetic system, the cytochrome b6/f complex, is not clear, and indeed it may exist in both areas of the membrane Light is trapped by chlorophyll and carotenoid (pigments) bound inside thylakoid membrane proteins For photosystem II (PSII) in plants, light energy is mainly trapped by the light harvesting complex II (LHCII), which consists of several related proteins of mass  25 kDa [4] One of the proteins, LHCIIb, is present in high stoichiometry (8±12 molecules per PSII complex), with the stoichiometry being in¯uenced by the illumination conditions at any given time [4±6] LHCII must transfer light energy to the core light harvesting proteins of PSII, which in turn, pass it to the reaction centre chlorophylls of PSII The latter convert the light energy into chemical potential energy via electron transfer [7] This chemical potential is eventually used to carry out the universally recognized functions of photosynthesis, i.e to ®x atmospheric carbon dioxide for biomass, liberate oxygen from water, and in general drive the energy requiring processes in the plant The complete PSII/LHCII complex is thought to consist of more than 20 different polypeptides and several hundred (250±350) pigment molecules [8,9] The total mass of PSII/ LHCII has been estimated at around MDa With such complexity, it is understandable that knowledge of the structure of PSII/LHCII proteins has largely come from studies of isolated components of the system [10] or subcomplexes that have been removed from the membrane by detergent extraction [11,12] However two-dimensional crystalline arrays of PSII/LHCII have sometimes been observed to form in situ in the grana membranes, and these can be studied using electron crystallography techniques [13±21] Such native crystals are inevitably smaller than crystals of isolated proteins [10] or puri®ed complexes of proteins [11] However it has been shown using experimental data [22] and by simulations [23] that averaging of cryoelectron microscopy data for several small crystalline areas is practical and results in structural data equivalent in quality to that obtained from much larger single crystalline arrays In this article, we describe cryo-electron microscopy studies of grana membranes, and show a projection Ê structure for PSII/LHCII to A as well as a threeÊ resolution for the complete dimensional structure to 30 A complex in situ This latter observation reveals an unexpected arrangement of the protein domains This, combined with new biochemical data has led us to an alternative model of the ultrastructure of the thylakoid grana where segregation exists within the grana itself, with LHCII and PSII core components segregated in alternate membranes within the stack This arrangement would change the existing view of plant photosynthesis in several areas, has implications for our understanding of Correspondence to R C Ford, Department of Biomolecular Sciences, UMIST, PO Box 88, Manchester M60 1QD, UK Fax: + 44 161 2360409, E-mail: r.ford@umist.ac.uk Abbreviations: Chl, chlorophyll; LHCII, light harvesting complex II; PSII, photosystem II (Received 20 July 2001, revised 18 August 2001, accepted November 2001) Keywords: photosynthesis, structure, photosystem II, lightharvesting, electron crystallography Ó FEBS 2002 PSII/LHCII structure in situ (Eur J Biochem 269) 327 Fig Thylakoid membrane morphology (a) Transmission electron micrograph of an ultrathin section of isolated barley chloroplast membranes (thylakoids) Note the tightly stacked membranes (grana) Scale bar ˆ 500 nm (b) Zoomed region of (a) showing a single grana stack, sectioned slightly obliquely (c) Explanation of the morphology shown in (b) with unstacked regions (u) and tightly appressed membrane pairs forming the stacked regions (s) A single membrane pair (dotted line) is indicated (d) Higher magni®cation of two membrane pairs in a stack in side view with the narrow partition gap between the membranes highlighted (white arrows) Scale bar ˆ 100 nm (e) Micrograph of isolated membrane pairs in face view, embedded in negative stain, and displaying two-dimensional crystals of PSII/ LHCII Scale bar ˆ 150 nm photosystem II structure/function and suggests potential links between cyanobacterial and plant PSII light harvesting systems EXPERIMENTAL PROCEDURES Barley viridis zb63 grana membranes were prepared, and electron microscopy was performed as described previously [19,21] Image processing was carried out with a group of programs developed mainly at the Medical Research Council Laboratory of Molecular Biology [26] After correction for lattice defects (lattice unbending) and for the contrast transfer function (CTF), data was merged The program PLOTALL was used to calculate the phase errors for the structure factors (kindly provided by W Kuhlbrand, MPI Biophysics, Frankfurt) Phase origins were determined using the program ORIGTILTD with restriction to the lower Ê resolution (to 20 A), high signal/noise (IQ3 or better) re¯ections [26,46] The IQ is an integer value determined by the peak-to-background ratio at a point in reciprocal space determined by the reciprocal lattice, with a value of IQ1 for a ÔgoodÕ ratio of : or more, with IQ values 2±8 identifying re¯ections with progressively worse peak to background ratios Finally IQ9 is assigned to re¯ections with amplitudes below background The phase origins were Ê then further re®ned using the lower resolution (to 20 A) averaged structure factors from the initial merging procedure as the starting reference Structure factors were vectorially averaged using the program AVGAMPHS using only re¯ections of signal/noise IQ7 or better Projection maps were calculated using an isotropic temperature factor Ê (500 A2) applied to all the averaged structure factors with ®gure of merit > 0.88 to compensate for resolutiondependent fading in image intensities This resulted in the enhancement of higher resolution features in the map, without the over-representation of these frequencies that can occur when very high temperature factors are applied Ê The same temperature factor of 500 A2 was employed in the study by Rhee & coworkers [11], suggesting that fading is not signi®cantly steeper for the in situ PSII crystals we have studied The image processing approach employed here for small crystalline areas was initially described by Perkins et al [22] An assessment of the procedure using simulated cryo-electron microscopy data was later carried out [23] These articles demonstrated that it was possible to greatly improve structural data obtained from cryo-electron microscopy of small crystals by averaging structure factors over several crystalline areas Each observed re¯ection in a Fourier transform consists of a noise component and a signal component, with the noise affecting the accuracy of both amplitude and phase components Amplitudes are generally noisier for cryo-electron microscopy, whilst phases are usually more reliable when compared to X-ray crystallography The approach developed by Perkins et al depends on oversampling (redundancy) followed by averaging by vector summation A measure of the redundancy for each structure factor is therefore an important indication of the reliability of its vector sum phase and amplitude values, with high redundancy correlating with better accuracy Within a data set derived from 21 crystal areas, 328 R C Ford et al (Eur J Biochem 269) Ó FEBS 2002 a mean redundancy of 5.6 shows that these data are convoluted with noise but not completely buried in the noise, with a probability of 0.28 of an individual high resolution structure factor being recorded for a single small crystal, with a raw peak-to-background ratio better than 1.6, i.e equivalent to IQ7 or better In comparison, control areas (noncrystalline) give IQ7 or better observations (by chance) with a probability of only 0.08 for a given (imaginary) high resolution reciprocal lattice point For a theoretical dataset of 21 separate control (noncrystalline) areas, a redundancy of > or > would arise by chance with a probability (see below) of only 0.022 and 0.004, respectively, for any given structure factor Thus a redundancy > in an experimental data set is indicative that signi®cant information is likely to be present for a given structure factor Probability is given as: P(r) ˆ nCr pr (1 ) p)n±r where n Cr ˆ n!/[r!(n ) r)!] and r is the number of observations of a structure factor in a data set of n crystals, with p being the individual (one-off) probability of observing data of IQ or better by chance alone A redundancy of 5.6 in this data set corresponds to standard errors for the mean (vector sum) phases of around 30° (see Tables 1±3) Standard error of the mean (vector sum) phase appears to be a more reasonable estimate for the phase errors for this image processing procedure because this measure includes a weighting for the number of observations, i.e the redundancy of the data is taken into account In comparison, unweighted interimage phase residuals not take into account the redundancy of the data and hence can give a misleading pessimistic impression of the reliablility of oversampled data The three-dimensional data set was obtained using the same approach as above and as described in Amos et al [26], but because of the very large body of data, we initially restricted the analysis to the lower resolution/higher amplitude components Table lists the number of ®les employed in the different tilt ranges, demonstrating that reciprocal space is reasonably evenly sampled by the data Nevertheless, the physical restriction imposed by the specimen holder in the microscope means that there is a Ômissing coneÕ of data corresponding to tilts beyond  60±70° The effect that this missing data has on the three-dimensional reconstruction has been discussed previously [26], with its main outcome being some loss of resolution perpendicular to the crystal plane A three-dimensional Coulomb density map for the cyanobacterial PSII core complex was calculated using the SPIDER image processing software package (Health Research Inc New York) and inputting the Protein Data Bank ®le 1fe1 [12] This ®le lacks the extramembraneous loops of the transmembrane protein subunits and one of the extrinsic subunits of the cyanobacterial PSII core complex, which remain to be identi®ed in the electron density map For a projection map, slices parallel to the predicted membrane plane were selected from the three-dimensional map and averaged together The resolution was arti®cially Ê curtailed to  A resolution for the projection map, or Ê 30 A resolution for the three-dimensional volume using a suitable Fourier ®lter Ê Table Crystallographic image processing statistics for the 8-A projection map Ê Table Crystallographic image processing statistics for the 30 A threedimensional map Scan step at the specimen level Plane group Lattice parameters No of crystalline areas No of observations (to IQ8) (to IQ7) No of structure factors No with FOM2 > 0.8 No used for map with FOM > 0.88 Ê Mean redundancy (250±8 A, to IQ7) Ê 2.6 A p1 Ê a 155.6 ‹ 1.5 A Ê b 230.6 ‹ 2.4 A aÄ 97.1° ‹ 1.7° 21 9824 4810 846 734 557 5.6 Scan step at the specimen level No of crystalline areas Maximum tilt angle No of ®les in tilt range Ê No of observations (to 30 A) No of structure factors used Ê Overall weighted phase residual to 30 A (where 90° is random) 0±30° 30±40° 40±50° 50±60° 60±66° Ê Ê 6.6 A or 8.9 A 168 ‹ 66° 69 14 28 54 5066 470 24° Ê Table Crystallographic image processing statistics for the A projection map over di€erent resolution ranges Resolution Rmergea Mean FOMb SE (°)c Redundancyd % Completee Ê 250±50 A Ê 50±30 A Ê 30±15 A Ê 15±10 A Ê 10±8 A 0.28 0.26 0.26 0.34 0.38 0.99 0.98 0.95 0.93 0.93 9.0 23.5 29.1 29.4 28.6 13.9 6.7 5.6 5.3 4.7 91% 85% 56% 63% 60% a Average amplitude variation for structure factors in the given resolution range For any individual structure factor Rmerge ˆ € € |Ii±Imean|/ Ii where Ii is each separate observation of the amplitude of the structure factor b Average ®gure of merit for structure factors in the resolution range FOM is the weight for each structure factor that gives the smallest r.m.s error in the Fourier synthesis [47] cStandard error of the mean phase was calculated for each structure factor and then averaged over the given resolution range dAverage number of observations of IQ7 or better, for structure factors within this resolution range e Number of structure factors used (with FOM > 0.88) for calculating the map vs the number of structure factors actually expected in this resolution range Ó FEBS 2002 The isolation of membrane fractions from grana membranes was carried out by sucrose density gradient centrifugation following disruption of the tightly stacked membrane pairs by chaotropic agents Treatment with Tris-base (1.5 M Tris/hydroxymethyl aminomethane, pH 8.8) for h in subdued light at 20 °C, was followed by one freeze-thaw cycle overnight, and then the treated membranes were washed and collected by centrifugation for h at 110 000 g in a Beckman SW41 rotor onto a sucrose cushion composed of M sucrose in buffer A (20 mM Mes, mM MgCl2, 15 mM NaCl, pH 6.3) The sharp green band at the 2-M sucrose interface was collected and then frozen at )20 °C and thawed once more before being loaded onto a linear sucrose gradient composed of 0±2 M sucrose in 0.75 M Tris-base, M urea, pH 8.8 After centrifugation for h at 110 000 g in a Beckman SW41 rotor, green bands corresponding to different membrane fractions were harvested Membranes were diluted : with distilled water and then centrifuged at 110 000 g for h to obtain pellets After resuspension in buffer A, the membranes were analysed by absorbance spectroscopy, SDS/PAGE and electron microscopy Absorbance spectra were recorded with a Kontron spectrophotometer (model Uvikon 943) with 1-cm pathlength cuvettes SDS/PAGE was carried out as described previously [19,21] RESULTS PSII/LHCII structure Figures 1a,b shows the morphology of the thylakoid membranes we employed, with the characteristic stacked membranes of the grana Isolation of tightly stacked membrane pairs (Fig 1c,d) is readily achieved, and twodimensionally ordered arrays of PSII/LHCII present in these membranes (Fig 1e) can be observed [13±21] Cryoelectron microscopy of such two-dimensional arrays generated projection and three-dimensional structures of the PSII/LHCII complex Figure 2a shows the signal-to-noise ratios of reciprocal lattice points after averaging 21 untilted crystalline arrays The data is relatively complete and the phase errors are acceptably low (Table 1) A three-dimensional data set was subsequently generated by tilting the two-dimensional crystals Lattice lines are displayed in Fig 2b with a low resolution (h,k ˆ 0, 2) and a higher resolution (1, )5) lattice line for comparison The phases are better clustered than the amplitudes for both lattice lines; this is expected for electron microscopy-derived structure factors Oversampling allows the improvement of the estimates for the interpolated values of the vector sum Ê phases along z* to  30 A resolution Table gives a more quantitative assessment of the quality of the structural data as a function of resolution Figure 3A shows a projection map of the crystal plane using contours to indicate protein density For comparison of general vs ®ne features, data is included up to a spatial Ê Ê resolution of 18 A (right) and A (left) See Table for an indication of the reliability of the structural information at these two resolution limits A high density region of a Ê roughly rectangular outline (140 ´ 100 A) is apparent, bisected by a lower density channel Approximately at the Ê Ê centre of the 140 ´ 100 A ÔcoreÕ domain in the 8-A map is a distinctive S-shaped region formed by several strong density PSII/LHCII structure in situ (Eur J Biochem 269) 329 peaks The S-shaped region could be the location of the reaction centre of PSII, which, on the basis of its predicted similarity to the bacterial reaction centre [24], has been observed in three-dimensional density maps obtained for PSII core complexes [11,12] (Fig 4B) The overall dimenÊ sions of the core domain (140 ´ 100 A) match very closely to the dimensions of one monomer of the cyanobacterial Ê PSII core complex (130 ´ 100 A) determined by X-ray crystallography [12], as shown in Fig 3B This supports the conclusion that the higher plant PSII complex is monomeric in vivo, as suggested previously [14,15,19±21,23] Clearly, caution must be exercised in a more detailed comparison of the two projection maps especially regarding the identi®cation of transmembrane helices, because in the native PSII structure, additional extrinsic proteins and loops will be superimposed (compare to Figure 4), whereas in the current deposition of the cyanobacterial structure, only the transmembrane regions and two of the extrinsic subunits are Ê de®ned [12] Similarly, the 8-A resolution projection map of the PSII core complex derived by Rhee & coworkers [25], did not allow the unambiguous identi®cation of transmembrane helices nor the reaction centre; but this was resolved Ê when the A three-dimensional structure became available Ê [11], as con®rmed by the 3.8-A three-dimensional structure [12] A roughly twofold rotational symmetry can be discerned in Fig 3A for the core domain, with a twofold axis in the middle of the S-shaped region as might be predicted for a heterodimeric complex Interestingly, the S-shape is echoed in the surrounding high density domains which arch around Ê Ê it in bands 30±40 A wide and  130 A long These bands terminate at two o'clock and eight o'clock positions on the periphery of the core, leaving gaps which are discussed below The high density core domains not directly contact each other, but each is surrounded by wide lanes of lower density, presumably corresponding to lipid Several small connecting densities appear to be responsible for forming bridges between the core domains in the lattice (arrows) In order to obtain further structural information, the three-dimensional structure of PSII/LHCII in the grana membranes was also obtained, using established methodology [11,26,27] Details regarding the image processing statistics are given in Table The three-dimensional Ê structure has been calculated to a resolution of  30 A This cut-off is suitable for comparison with earlier studies of negatively stained PSII/LHCII, which have a similar Ê resolution Three-dimensional data beyond 30 A have been collected and processed, but further crystals need to be included in the analysis to adequately oversample threeÊ dimensional reciprocal space to higher (8 A) resolution Figure shows different views of the PSII/LHCII complex, with a surface generated at a suitable threshold for discrimination of protein density The main features of the Ê 140 ´ 100 A core domain correspond closely to those described earlier for negatively stained specimens [14,15] The distinctive cavity on the lumenal side of the complex is apparent, surrounded by four prominent lumenal domains, some of which were previously assigned to extrinsic PSII proteins that enhance oxygen evolution Sequential removal of these extrinsic proteins, followed by structural analysis has identi®ed domains I, II and III as the approximate locations of oxygen evolution enhancing (OEE) proteins I, 330 R C Ford et al (Eur J Biochem 269) Ó FEBS 2002 Fig Quality of the electron crystallography data (a) Cryo-electron crystallography data after averaging over 21 separate untilted crystalline areas The size of the box and number indicate the standard error of the mean phase (SE) for the structure factor with ˆ SE < 8°, ˆ SE 8±14°, Ê ˆ SE 14±20°, ˆ SE 20±30° and boxes without a number ˆ SE 30±40° The rings correspond to 15, 10 and A resolution (inner to outer rings), and the principal crystallographic axes are indicated (b) Lattice lines within the three-dimensional data set showing the sampling of reciprocal space along z* (perpendicular to a*b*) Each data point represents a separate observation of the amplitude and phase (in degrees) for a given re¯ection, with the z* value given by the tilt angle and the angle between the tilt axis and a* The trend of the data for the continuous transform along z* is shown by the ®tted line A lattice line for a relatively low resolution re¯ection, with well clustered phases (h,k ˆ 0,2), is compared with a lattice line for a higher resolution re¯ection (h,k ˆ 1,5) Ó FEBS 2002 PSII/LHCII structure in situ (Eur J Biochem 269) 331 Fig Projection maps of the entire PSII/ LHCII complex (A) Maps are calculated to Ê Ê A (left) and 18 A resolution (right) The crystallographic a and b axes are indicated (lower left) Solid contours begin at a density level corresponding to 0.5 r above the mean level, and extend up in even steps to 3.5 r above the mean The two dotted contours are drawn at the mean density level and at 0.25 r above the mean The thick arrows indicate densities that appear to bridge the wide low density channel running approximately parallel to the a axis The repeat along a is Ê Ê 155.6 A, along b, 230.6 A (B) Comparison of Ê the main core region of the A map (left) and a projection map calculated from the protein data bank deposition 1fe1 for the cyanobacterial PSII core complex (right), which is composed mainly of the transmembrane helices identi®ed so far in the structure An S-shaped reaction centre domain consisting of the transmembrane helices of polypeptides D1 and D2 is highlighted in the cyanobacterial map (dashed ellipse) This region is tentatively assigned in the higher plant map (ellipse), and is centred on a rough twofold symmetry axis The transmembrane helices of the accessory polypeptides CP47 and CP43 can not be readily identi®ed in the higher plant map, however, as < 50% of the mass of these subunits is contained in the transmembrane helices, then their identi®cation in a projection map is unlikely because of convolution with overlying densities II and III, respectively, whilst domain IV was assigned to the large lumenal loops of core polypeptide CP47 [15] A further domain (V) underlying and contributing to domains II and I was assigned to the lumenal portion of CP43 In the X-ray structure of the core complex of cyanobacteria ([12], Fig 4, lower panels), density for part of OEE I (Psb O) is present, and occupies a lumenal position in the corner of the complex which would correspond to the location of domain I in the higher plant complex The cyanobacterial system does not have the OEE II polypeptide, but rather has an extrinsic cytochrome c550 subunit This sits in another lumenal corner of the cyanobacterial complex in a position equivalent to domain II in the higher plant three-dimensional structure The third (12 kDa) extrinsic polypeptide of the cyanobacterial complex was not resolved in the published structure [12], but is likely to appear in later density maps (P Orth, FU, Berlin, personal communication) The overall dimensions and shape of the cyanobacterial PSII core complex and Ê the higher plant PSII core region are very similar at 30 A resolution, again supporting the idea that the higher plant PSII complex is monomeric in situ The location of the connecting densities that bridge between the core domains was unexpected It is clear from Fig that the connecting domains lie in a separate plane to the main core region All these small domains align almost exactly along a single plane, which immediately suggests that they are not due to random noise or poor sampling of three-dimensional space The most likely explanation for this observation, given the double-layered nature of the crystals, is that the connecting domains occupy a membrane that is separate to the one housing the core domain A narrow but distinct gap between the two planes of density is  0.5±1 nm across, which would correspond closely to the width of the partition region that can be identi®ed between pairs of closely appressed grana membranes in ultrathin sections (Fig 1d) The overall size (4 nm height ´ nm 332 R C Ford et al (Eur J Biochem 269) Ó FEBS 2002 Ê Fig Upper panels show the three-dimensional structure of PSII/LHCII at 30 A resolution (green) Left panel shows a view from the lumenal side, with the characteristic four-lobed appearance (domains I±IV) and the central cavity Note the small interconnecting domains are still resolved at this resolution Right panel shows a side view, incorporating a slice through the closest PSII core complex revealing the extent of the central cavity Note Ê the interconnecting domains all lie in a separate plane to the core domains The putative boundaries of two closely appressed lipid bilayers 40 A thick are indicated by the white parallel lines Lower panel (blue) shows equivalent views generated from the protein data bank deposition 1fe1 for the cyanobacterial PSII core complex The extrinsic subunits PsbO and cyt-c550 in the cyanobacterial PSII complex are indicated Note: protein regions and loops external to the membrane and one extrinsic subunit are not included in 1fe1, explaining the apparent truncation of the volume when viewed along the membrane plane (right) The scale bar relates to all panels width ´ nm length) and number (4±5) of the connecting domains immediately suggested that they could be peripheral LHCII proteins [10], although the resolution was insuf®cient for unambiguous identi®cation, and one cannot exclude the possibility that these densities may be due to ordered peripheral proteins If the assignment to LHCII is correct, then the observation of only 4±5 densities rather than 8±12 implies that only a subset of the LHCII population is involved in the contacts between core complexes Biochemical evidence for two grana membrane fractions Biochemical evidence for the presence of two different membrane types in grana thylakoid membrane fractions is scant A search for conditions that would allow the disruption of the paired membranes without membrane solubilization was carried out Several procedures employing chaotropes and/or proteases were found to give some separation of the membrane pairs A procedure employing high concentrations of Tris-base combined with urea and freeze-thaw cycles was found to be the most effective, as judged by the separation of several different membrane fractions by sucrose density gradient centrifugation (Fig 5A) In control experiments, grana membranes migrated in the density gradient to a single location at around 1.1 M sucrose These membranes had an absorption spectrum that was typical for grana membranes, with a high content of chlorophyll (Chl) b as demonstrated by the Chl b absorption bands at about 650 and 480 nm (Fig 5B) For Ó FEBS 2002 PSII/LHCII structure in situ (Eur J Biochem 269) 333 Fig Characterization of grana membrane fractions after Tris/urea-treatment and separation by sucrose density gradient centrifugation (A) Control membranes migrated as a single band on the gradient whilst Tris/urea-treated membranes migrated as three bands, a±c (left) (B) The absorption spectra of the Tris/ureatreated membranes, a-c are shown (a ˆ solid line, b ˆ gray line, c ˆ dashed line) The spectrum of the control membranes was not signi®cantly di€erent to that shown by band b of the Tris/urea-treated material (C) Polypeptide composition of the sucrose density gradient fractions from Tris/urea-treated membranes as determined by SDS/PAGE and Coomassie staining The left lane shows band a and the right lane band c Molecular mass markers are indicated on the left of the panel Tris/urea-treated membranes, this band was also observed, but it contained less material when compared to the control (band b) Two further distinct chlorophyll-containing bands were observed for the Tris/urea-treated material: membranes separating as a broad band located at  1.4 M sucrose (band c) showed a radically changed absorption spectrum, being depleted in the Chl b absorption bands at 650 and 480 nm, consistent with a lack of light-harvesting Chl a/b proteins (LHCII) Membranes located slightly above the main band at  1.0 M sucrose (band a) had a similar absorption spectrum to the main band, but with a slightly increased Chl b absorption SDS/PAGE of the Tris-treated membranes is shown in Fig 5C The fraction isolated from around 1.4 M sucrose (band c in panel A) was signi®cantly depleted in LHCII Fig Electron microscopy of negatively stained Tris/urea-treated membranes after separation on a sucrose gradient (a) Core PSII-enriched membranes (band c from the sucrose gradient) contain tightly packed  14 nm diameter particles (inset) (b) and (c) LHCII-enriched membranes (band a from the sucrose gradient) are tubular in morphology with small particles The scale bar represents 500 nm polypeptides, but was enriched in the core polypeptides D1,D2, CP43 and CP47 (right track) No bands due to extrinsic polypeptides of PSII (33, 23, 17, 10 kDa) could be observed, but these polypeptides will be removed by the chaotrope treatment The fraction isolated at 1.0 M sucrose (band a in panel A) is signi®cantly depleted in the D1,D2, CP43 and CP47 polypeptides (left track) whilst retaining intensely staining LHCII polypeptides These data therefore suggest that separation of grana membranes into denser PSII core-enriched membranes and less dense LHCIIenriched membranes is possible after chaotrope treatment Electron microscopy of the two chaotrope-treated membrane fragments revealed two different membrane morphologies (Fig 6) The core PSII-enriched density gradient fraction consisted of larger ( 200 nm diameter) ¯at 334 R C Ford et al (Eur J Biochem 269) Ó FEBS 2002 membrane patches that contained large  14 nm diameter) particles (Fig 6a, insert) consistent with core PSII The packing of these complexes is very tight (2300 particlesálm)2), considerably higher than that observed for untreated grana membranes (1300±1500 particlesálm)2) The LHCII-enriched density gradient fraction contained rolled-up membrane ÔtubesÕ with small internal features (Fig 6b,c) DISCUSSION Interpretation of the three-dimensional data The data presented here provide information for the complete PSII/LHCII complex observed under conditions that preserve its native state [27] In earlier structural studies, negative stain was employed where dehydration and shrinkage are known to be problems [14,21] as well as differential staining of upper and lower surfaces of the specimen [28] These combined factors may explain why previous studies did not readily identify two planes of density Negatively stained PSII/LHCII crystals in spinach grana [14] display some small domains that lie in a lower plane than the main core of the complex [14], but there was no complete separation of these densities into two planes as observed in this work The data shown in Fig suggest that the physical separation of grana membranes into fractions differing in density is possible No detergent is involved in this separation process, and the density gradient fractions can be recovered (after dilution) by centrifugation This strongly suggests that the fractions are membranes and not detergent-solubilized PSII/LHCII complexes, as con®rmed by electron microscopy (Fig 6) Isolation of discrete membrane fractions enriched in either core PSII or LHCII has not been previously described, despite the widespread use of grana membranes reported in the literature This may be because harsh conditions (which will result in PSII inactivation) are required to disengage the two tightly appressed membranes, and therefore these conditions are unlikely to have been widely explored previously The use of such chaotropes is, however, undesirable, and a search for milder dissociation conditions is underway This should help to exclude any possibility that the chaotropes have artefactually induced the segregation we observe Diagrams to explain the structural models for PSII/ LHCII in situ are presented in Fig 7, with the currently accepted model shown in Fig 7a and an alternative model shown in Fig 7b In the new model the arrays are composed of large core PSII complexes that are connected to each other via small bridging light harvesting complexes that are located in a separate adjacent membrane This ®ts the structural and biochemical data, where PSII core complexes can be observed in one discrete plane and membrane fraction, and LHCII complexes can be observed in another membrane fraction A survey of previous structural studies of thylakoid membranes [13,16,17,21, 29±32] suggests that they may be newly interpreted in terms of the alternative model of thylakoid structure A review of these studies is beyond the scope of this paper and will be presented elsewhere The alternative model, if correct, has several implications for understanding PSII function ranging from light Fig Models for grana ultrastucture (a) Existing, widely accepted model of thylakoid ultrastructure PSII core (red) and LHCII (green) coexist in the same, tightly packed lipid bilayer (blue), with light energy transferred laterally from LHCII to PSII core The repeat distance in the stack is 16 nm, and some interdigitation is required in order to accommodate the large lumenal domains of PSII in this model (b) Alternative model of the ultrastructure of grana with LHCII and PSII located in separate lipid bilayers in the stack The boxed area represents a crystalline array viewed edge-on, i.e two tightly appressed membranes with lattice contacts along the crystal plane formed by LHCII harvesting control [33±38] to the optimization of diffusion of PSII and of components around PSII [39±44] A discussion of these implications is again beyond the scope of this paper, and will be addressed in a separate review However we note that migration of light energy to the PSII core in a direction perpendicular to the membrane plane would not be unique to plants The more ancient cyanobacterial PSII does not have LHCII proteins, but rather it depends on water-soluble light harvesting proteins that are attached as a ÔphycobilisomeÕ to the stromal surface of the PSII core [36] Other photosynthetic bacteria, such as the green sulphur bacteria, also move light excitation energy from chlorosomes to the membrane in which the reaction centre is found [37] Testing the model This paper highlights a discord between the structural data and the existing model of PSII/LHCII and grana architecture, and this should now open a debate on the merits of the alternative models We note that Ômacro-domainsÕ of LHCII in plants have already been proposed to explain data derived from several biophysical techniques [45], and that intercalation of LHCII and PSII core domains in paired grana membranes has recently been discussed [48] Thus some movement towards a revised view of grana ultrastructure has already been made However, it is important to stress that many questions remain unanswered for Ó FEBS 2002 the model that we have presented, and that several reports based on detergent solubilized complexes obtained from higher plant grana have proposed alternative arrangements for the interaction of LHCII with the PSII core [49±51] The ÔsupercoreÕ and ÔmegacoreÕ complexes identi®ed by Boekema & coworkers by single particle image processing are interpreted as showing LHCII and PSII core in close side-by-side association The number of LHCII molecules that are assigned in these large tetrameric complexes is, however, much less than the 8±12 required per PSII core, hence the two alternative interpretations of LHCII±PSII structural data might be compatible if a small subset of LHCII polypeptides associate more intimately with PSII core whilst the remaining occupy a separate membrane Progress is slowly being made towards processing a higher resolution 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PSII core complexes can be observed in one discrete plane and membrane fraction, and LHCII complexes can be observed in another membrane fraction A survey of previous structural studies of thylakoid... subunits is contained in the transmembrane helices, then their identi®cation in a projection map is unlikely because of convolution with overlying densities II and III, respectively, whilst domain IV