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].. LHCII must tr
Trang 1An alternative model for photosystem II/light harvesting complex II
in grana membranes based on cryo-electron microscopy studies
Robert C Ford1, Svetla S Stoylova2and Andreas Holzenburg3
1 Department of Biomolecular Sciences, UMIST, Manchester, UK; 2 The Burnham Institute, La Joua, CA, USA;
3 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 ƠgranaÕ
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
treat-ment of the paired grana membranes has allowed the
sepa-ration 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
Keywords: photosynthesis, structure, photosystem II, light-harvesting, electron crystallography
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
(photo-system 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
mem-brane 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 atmo-spheric 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 1 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 cryo-electron 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 structure for PSII/LHCII to 8 Ă as well as a three-dimensional structure to 30 Ă resolution for the complete 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 alter-nate 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 5 November
2001)
Trang 2photosystem II structure/function and suggests potential
links between cyanobacterial and plant PSII light
harvest-ing systems
E X P E R I M E N T A L P R O C E D U R E S
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
programPLOTALLwas used to calculate the phase errors for
the structure factors (kindly provided by W Kuhlbrand,
MPI Biophysics, Frankfurt)
using the programORIGTILTDwith restriction to the lower
resolution (to 20 AÊ), 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 8 : 1 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
then further re®ned using the lower resolution (to 20 AÊ)
averaged structure factors from the initial merging
proce-dure 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 (500 AÊ2) applied to all the averaged structure factors with
®gure of merit > 0.88 to compensate for resolution-dependent 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 The same temperature factor of 500 AÊ2was 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 mi-croscopy 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 crystal-lography The approach developed by Perkins et al depends on oversampling (redundancy) followed by aver-aging 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,
Fig 1 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 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 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 150 nm
Trang 3a 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 > 4 or > 5 would arise by chance
with a probability (see below) of only 0.022 and 0.004,
respectively, for any given structure factor Thus a
redun-dancy > 5 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) nCr pr (1 ) p)n±r where
nCr 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 7 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)
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 do 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 ampli-tude components Table 1 lists the number of ®les employed
in the different tilt ranges, demonstrating that reciprocal space is reasonably evenly sampled by the data Neverthe-less, the physical restriction imposed by the specimen holder
in the microscope means that there is a Ơmissing 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 curtailed to 8 Ă resolution for the projection map, or
30 Ă resolution for the three-dimensional volume using a suitable Fourier ®lter
Table 1 Crystallographic image processing statistics for the 8-Ă
pro-jection map.
Scan step at the specimen level 2.6 Ă
b 230.6 2.4 Ă
aÄ 97.1° 1.7°
No of observations (to IQ8) 9824
No of structure factors 846
No used for map with FOM > 0.88 557
Mean redundancy (250±8 Ă, to IQ7) 5.6
Table 2.
8 Crystallographic image processing statistics for the 8 Ă projection map over dierent resolution ranges.
a Average amplitude variation for structure factors in the given resolution range For any individual structure factor Rmerge P
|I i ±I mean |/PI i where I i 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] c Standard error of the mean phase was calculated for each structure factor and then averaged over the given resolution range d Average 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.
Table 3 Crystallographic image processing statistics for the
three-dimensional map.
Scan step at the specimen level 6.6 Ă or 8.9 Ă
No of ®les in tilt range 0±30° 69
30±40° 14 40±50° 28 50±60° 54 60±66° 2
No of observations (to 30 Ă) 5066
No of structure factors used 470 Overall weighted phase residual to 30 Ă 24°
(where 90° is random)
Trang 4The isolation of membrane fractions from grana
mem-branes was carried out by sucrose density gradient
centrif-ugation following disruption of the tightly stacked
membrane pairs by chaotropic agents Treatment with
Tris-base (1.5M Tris/hydroxymethyl aminomethane,
pH 8.8) for 2 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
2 h at 110 000 g in a Beckman SW41 rotor onto a sucrose
cushion composed of 2Msucrose in buffer A (20 mMMes,
5 mMMgCl2, 15 mMNaCl, pH 6.3) The sharp green band
at the 2-Msucrose interface was collected and then frozen at
)20 °C and thawed once more before being loaded onto a
linear sucrose gradient composed of 0±2Msucrose in 0.75M
Tris-base, 3Murea, pH 8.8 After centrifugation for 2 h at
110 000 g in a Beckman SW41 rotor, green bands
corre-sponding to different membrane fractions were harvested
Membranes were diluted 1 : 1 with distilled water and then
centrifuged at 110 000 g for 2 h to obtain pellets After
resuspension in buffer A, the membranes were analysed by
absorbance spectroscopy, SDS/PAGE and electron
micros-copy Absorbance spectra were recorded with a Kontron
spectrophotometer (model Uvikon 943) with 1-cm
path-length cuvettes SDS/PAGE was carried out as described
previously [19,21]
R E S U L T S
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
two-dimensionally ordered arrays of PSII/LHCII present in
these membranes (Fig 1e) can be observed [13±21]
Cryo-electron microscopy of such two-dimensional arrays
gener-ated 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-dimen-sional data set was subsequently generated by tilting the
two-dimensional crystals Lattice lines are displayed in
Fig 2b with a low resolution (h,k 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
phases along z* to 30 AÊ resolution Table 3 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
resolution of 18 AÊ (right) and 8 AÊ (left) See Table 2 for an
indication of the reliability of the structural information at
these two resolution limits A high density region of a
roughly rectangular outline (140 ´ 100 AÊ) is apparent,
bisected by a lower density channel Approximately at the
centre of the 140 ´ 100 AÊ ÔcoreÕ domain in the 8-AÊ map is a
distinctive S-shaped region formed by several strong density
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 dimen-sions of the core domain (140 ´ 100 AÊ) match very closely
to the dimensions of one monomer of the cyanobacterial PSII core complex (130 ´ 100 AÊ) 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®ca-tion 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 trans-membrane regions and two of the extrinsic subunits are de®ned [12] Similarly, the 8-AÊ resolution projection map of the PSII core complex derived by Rhee & coworkers [25], did not allow the unambiguous identi®cation of transmem-brane helices nor the reaction centre; but this was resolved when the 8 AÊ three-dimensional structure became available [11], as con®rmed by the 3.8-AÊ 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
it in bands 30±40 AÊ wide and 130 AÊ 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 do 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 method-ology [11,26,27] Details regarding the image processing statistics are given in Table 3 The three-dimensional structure has been calculated to a resolution of 30 AÊ This cut-off is suitable for comparison with earlier studies of negatively stained PSII/LHCII, which have a similar resolution Three-dimensional data beyond 30 AÊ have been collected and processed, but further crystals need to be included in the analysis to adequately oversample three-dimensional reciprocal space to higher (8 AÊ) resolution Figure 4 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
140 ´ 100 AÊ 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)
Trang 5Fig 2.
5 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 1 SE < 8°, 2 SE 8±14°,
3 SE 14±20°, 4 SE 20±30° and boxes without a number SE 30±40° The rings correspond to 15, 10 and 8 AÊ 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 0,2), is compared with a lattice line for a higher resolution re¯ection (h,k 1,5).
Trang 6II 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 c550subunit 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
cyanobac-terial complex was not resolved in the published structure
[12], but is likely to appear in later density maps (P Orth,
FU, Berlin, personal communication)
dimen-sions and shape of the cyanobacterial PSII core complex and
the higher plant PSII core region are very similar at 30 AÊ 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 4 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 ´ 3 nm
Fig 3.
6 Projection maps of the entire PSII/
LHCII complex (A) Maps are calculated to
8 AÊ (left) and 18 AÊ 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
par-allel to the a axis The repeat along a is
155.6 AÊ, along b, 230.6 AÊ (B) Comparison of
the main core region of the 8 AÊ map (left) and
a projection map calculated from the protein
data bank deposition 1fe1 for the
cyanobac-terial 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.
Trang 7width ´ 4 nm length) and number (4±5) of the connecting
domains immediately suggested that they could be
periph-eral 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 employ-ing 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 1.1Msucrose 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
Fig 4 Upper panels show the three-dimensional structure of PSII/LHCII at 30 AÊ 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 the interconnecting domains all lie in a separate plane to the core domains The putative boundaries of two closely appressed lipid bilayers
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-c 550 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.
Trang 8Tris/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:
mem-branes separating as a broad band located at 1.4M
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 1.0M 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 1.4Msucrose
(band c in panel A) was signi®cantly depleted in LHCII
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 1.0Msucrose (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 LHCII-enriched membranes is possible after chaotrope treatment Electron microscopy of the two chaotrope-treated mem-brane fragments revealed two different memmem-brane morpho-logies (Fig 6) The core PSII-enriched density gradient fraction consisted of larger ( 200 nm diameter) ¯at
Fig 6 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.
Fig 5 Characterization of grana membrane
fractions after Tris/urea-treatment and
separa-tion by sucrose density gradient centrifugasepara-tion.
(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/urea-treated membranes, a-c are shown (a solid
line, b gray line, c dashed line) The
spectrum of the control membranes was not
signi®cantly dierent to that shown by band b
of the Tris/urea-treated material (C)
Poly-peptide 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.
Trang 9membrane patches that contained large 14 nm diameter)
particles (Fig 6a, insert) consistent with core PSII The
packing of these complexes is very tight (2300
parti-clesálm)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)
D I S C U S S I O N
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] do 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 5 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
deter-gent-solubilized PSII/LHCII complexes, as con®rmed by
electron microscopy (Fig 6) Isolation of discrete
mem-brane 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
inacti-vation) 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
artefactu-ally 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
implica-tions for understanding PSII function ranging from light
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 archi-tecture, 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 impor-tant to stress that many questions remain unanswered for
Fig 7 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 repre-sents a crystalline array viewed edge-on, i.e two tightly appressed membranes with lattice contacts along the crystal plane formed by LHCII.
Trang 10the 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 three-dimensional data set for the PSII/
LHCII crystals When this is complete, the data should
reveal much more concerning the nature of the contacts in
the crystals and offer further insight into the interplay
between PSII structure and function in the thylakoid
membrane
A C K N O W L E D G E M E N T S
We would like to thank Dr M F Rosenberg for his assistance with
software and Dr S Prince, Dr S V Rue and Prof G Garab for
useful suggestions and debate T D Flint is thanked for plant growth
and specimen preparation as well as L Child and P McPhie for expert
technical assistance The data collection phase of this work was
supported by the UK Biotechnology and Biological Sciences Research
Council.
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