18
J. Cell Sd. 80, 57-73 (1986) 57 Printed in Great Britain © The Company of Biologists Limited 1986 FREEZE-FRACTURE ANALYSIS OF THYLAKOID MEMBRANES AND PHOTOSYSTEM I AND II ENRICHED FRACTIONS FROM PHORMIDIUM LAMINOSUM RICHARD E. GLICK 1 •, RICHARD E. TRIEMER 2 AND BARBARA A. ZILINSKAS^f ^Department of Biochemistry and Microbiology, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, NJ 08903, U.SA. 2 Bureau of Biological Research, Rutgers University, New Brunswick, NJ 08903, U.SA. SUMMARY Thylakoid membranes of the thermophilic cyanobacterium Phormidium laminosum have been fractionated into photosystem II and photosystem I particles. These fractions have been characterized by their partial electron transport activities, and biochemical and spectral properties. Exoplasmic fracture face and protoplasmic fracture face particles in the unfractionated thylakoid membranes were shown to correspond in size to particles in freeze-fractured photosystem II and photosystem I fractions, respectively. Differences between the histograms of the thylakoid mem- brane protoplasmic fracture face particles and the isolated photosystem I particles suggest that in addition to photosystem I complexes some of the particles on the thylakoid protoplasmic fracture face may be related to cytochrome b/f complexes, the hydrophobic component of the coupling factor, or respiratory complexes. INTRODUCTION The characterization of particles in thylakoid membranes revealed by freeze- fracture electron microscopy has been the subject of several studies, which have indicated a correlation between intramembranous particles and photosynthetic pro- cesses. Arntzen, Dilley & Crane (1969) demonstrated that chloroplast membrane fractions enriched in photosystem II (PS II) were also enriched in large exoplasmic fracture face (E-face) particles, while fractions containing mostly photosystem I (PS I) showed smaller protoplasmic fracture face (P-face) particles. Many subsequent studies of higher plant chloroplasts have shown that there is a correlation between the E-face particles and PS II complexes (Miller, Miller & Mclntyre, 1976; Armond & Arntzen, 1977; Armond, Staehelin & Arntzen, 1977; Simpson, 1978; Miller & Cushman, 1979), while the P-face contains the PS I particles as well as several other complexes (Kaplan & Arntzen, 1982). •Present address: Division of Molecular Plant Biology, University of California, Berkeley, CA 94720, U.S.A. •f Author for correspondence. Key words: cyanobacterium, freeze-fracture, Phormidium laminosum, photosystems I and II, thylakoid membrane.

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J. Cell Sd. 80, 57-73 (1986) 57Printed in Great Britain © The Company of Biologists Limited 1986

FREEZE-FRACTURE ANALYSIS OF THYLAKOIDMEMBRANES AND PHOTOSYSTEM I AND IIENRICHED FRACTIONS FROM PHORMIDIUM

LAMINOSUM

RICHARD E. GLICK1 •, RICHARD E. TRIEMER2

AND BARBARA A. ZILINSKAS^f^Department of Biochemistry and Microbiology, Cook College, New Jersey AgriculturalExperiment Station, Rutgers University, New Brunswick, NJ 08903, U.SA.2Bureau of Biological Research, Rutgers University, New Brunswick, NJ 08903, U.SA.

SUMMARY

Thylakoid membranes of the thermophilic cyanobacterium Phormidium laminosum have beenfractionated into photosystem II and photosystem I particles. These fractions have beencharacterized by their partial electron transport activities, and biochemical and spectral properties.Exoplasmic fracture face and protoplasmic fracture face particles in the unfractionated thylakoidmembranes were shown to correspond in size to particles in freeze-fractured photosystem II andphotosystem I fractions, respectively. Differences between the histograms of the thylakoid mem-brane protoplasmic fracture face particles and the isolated photosystem I particles suggest that inaddition to photosystem I complexes some of the particles on the thylakoid protoplasmic fractureface may be related to cytochrome b/f complexes, the hydrophobic component of the couplingfactor, or respiratory complexes.

INTRODUCTION

The characterization of particles in thylakoid membranes revealed by freeze-fracture electron microscopy has been the subject of several studies, which haveindicated a correlation between intramembranous particles and photosynthetic pro-cesses. Arntzen, Dilley & Crane (1969) demonstrated that chloroplast membranefractions enriched in photosystem II (PS II) were also enriched in large exoplasmicfracture face (E-face) particles, while fractions containing mostly photosystem I (PSI) showed smaller protoplasmic fracture face (P-face) particles. Many subsequentstudies of higher plant chloroplasts have shown that there is a correlation between theE-face particles and PS II complexes (Miller, Miller & Mclntyre, 1976; Armond &Arntzen, 1977; Armond, Staehelin & Arntzen, 1977; Simpson, 1978; Miller &Cushman, 1979), while the P-face contains the PS I particles as well as several othercomplexes (Kaplan & Arntzen, 1982).

•Present address: Division of Molecular Plant Biology, University of California, Berkeley, CA94720, U.S.A.

•f Author for correspondence.

Key words: cyanobacterium, freeze-fracture, Phormidium laminosum, photosystems I and II,thylakoid membrane.

58 R. E. Glick, R. E. Triemer and B. A. Zilinskas

In red algae and cyanobacteria, the basic photosynthetic apparatus is analogous tothat in higher plants and algae containing chlorophyll b; water is the electron donorand oxygen is evolved, there are two pigment systems with reaction centres based onchlorophyll a, and the electron transport components are comparable. The maindifference between higher plant and red algal/cyanobacterial photosynthesis is intheir respective light-harvesting apparatus; the chlorophyll a/b pigment proteincomplex is found in the former and phycobiliproteins in the latter. The phycobili-proteins are organized into highly ordered structures, phycobilisomes (PBsomes),which are attached to the stromal, or outer, surface of the thylakoid membrane.

Consistent with their function as light harvesters, PBsomes are energeticallycoupled to the chlorophyll proteins and reaction centres in the membrane. Studieshave indicated that light energy absorbed by PBsomes is transferred preferentially toPS II (Ley & Butler, 1976; Wang, Stevens & Myers, 1977). Diner (1979) showedthat in the case of the red alga, Cyanidium caldarium, as few as half of the PBsomeswere functionally connected with PS II centres. More recently, on the other hand, ithas been found that the PBsomes of the cyanobacterium, Anacystis nidulans, arefunctionally (and presumably structurally) connected to two PS II centres, resultingin a competition for excitation energy (Mandori & Melis, 1985).

Freeze-fracture studies of thylakoids from several cyanobacterial and red algalspecies have revealed, as is the case in higher plants, two basic types of intra-membranous particles associated with thylakoid membranes: somewhat larger E-f aceparticles and smaller, more densely packed P-face particles (Wollman, 1979;Golecki, 1979; Giddings & Staehelin, 1979; Cox, Benson & Dwarte, 1981;Giddings, Wassman & Staehelin, 1983). On the whole, size differences between E-face and P-face particles are greater in higher plants than in PBsome-containingorganisms, probably due to the chlorophyll a/b protein complex component ofhigher plant E-face particles. In the red alga, Spermothamnion turneri, the particleson both fracture faces are similar in size, but the spread of P-face particle sizes isgreater than that for the E-face, which suggested that several categories of similarlysized particles might be present in the P-face (Staehelin, Giddings, Badami &Krzymowski, 1978). An additional feature of the intramembranous particles of someof the organisms studied is that ordered rows of E-face particles have been ob-served (Neushul, 1971; Lefort-Tran, Cohen-Bazire & Pouphile, 1973; Lichtle& Thomas, 1976; Wollman, 1979; Golecki, 1979; Giddings & Staehelin, 1979;Giddings et al. 1983). Lichtle & Thomas (1976) and Giddings et al. (1983) havecorrelated the spacing of PBsome rows to E-face particle rows, suggesting that thePBsomes may be associated with the E-face particles in the thylakoid. Theseultrastructural findings, in conjunction with those showing that PBsomes transferexcitation energy preferentially to PS II, have supported the hypothesis that the E-face particles represent PS II units in the thylakoid membrane. Thus far, theevidence supporting this hypothesis is based on ultrastructural observations oforganisms deficient in some photosynthetic function (e.g. heterocysts, which lackPS II (Giddings & Staehelin, 1979), or mutants without PBsomes (Wollman, 1979))and comparison with the ultrastructure of vegetative cells or wild-type organisms.

Freeze-fracture of Phormidium laminosum 59

Furthermore, these studies have not addressed the possible identity or function(s) ofthe P-face particles.

This study was undertaken in order to correlate the intramembranous thylakoidparticles with photosynthetic functions by isolating fractions enriched in PS I andPS II from thylakoid membranes and characterizing the particles seen after freeze-fracture. The combination of biochemical characterization of the fractions coupledwith ultrastructural observations provides further evidence for the identity andfunction of intramembranous thylakoid particles.

MATERIALS AND METHODS

The culture of Phormidium laminosum (OH-l-p, clone 1) used in this study was a generous giftfrom Dr R. W. Castenholz. Cells were grown in medium D of Castenholz (1969) in a 14-1 NewBrunswick Scientific Co. fermentor at 45°C with cool white fluorescent light at an intensity of6Wm~2. The cells were aerated with 1 % CO2 in air.

Cells were harvested at late log phase with a CEPA continuous flow centrifuge and usedimmediately. PS I and PS II fractions were isolated as described by Stewart & Bendall (1979).Phycobilisomes were isolated from P. laminosum, using the procedure described for Nostoc sp.(Troxler, Greenwald & Zilinflkas, 1980).

Rates of electron transport from water to ferricyanide/2,6-dimethyl-/>-benzoquinone (FeCy/DMBQ) and from dichlorophenolindophenol (DCPIP)/ascorbate to methyl viologen (MV) weremeasured at 25°C at light saturation (106 ergscm~2s-1) with a Clark-type oxygen electrode.Chlorophyll concentration was determined from 80 % (v/v) acetone extracts according to Arnon(1949).

Absorption spectra were measured with a Cary 17D spectrophotometer. Corrected fluorescenceemission spectra at 77 K were measured with an SLM 4800s spectrofluorometer. The excitationand emission slits were 8 nm and 2 run, respectively. Samples were suspended in 'buffer C (10 mM-HEPES/NaOH, pH7-S, 5 mM-NaH2PO4/K2HPO.,, pH7-5, 10mM-MgCl2, 25% (v/v) glycerol(Stewart & Bendall, 1979)) at a chlorophyll concentration of 3/igml"1. For absorption measure-ments of chlorophyll proteins excised from a polyacrylamide gel, small pieces of gel containing thechlorophyll protein were placed in a 1 cm pathlength cuvette filled with distilled water and pressedflat against the front surface. A piece of gel containing no protein was placed in a matched cuvette toserve as a reference. The same gel slices were frozen in liquid N2, placed in a Dewar flask, and usedfor fluorescence measurements.

Chlorophyll protein complexes were separated by sodium dodecyl sulphate/polyacrylamide gelelectrophoresis (SDS/PAGE) according to Markwell, Reinman & Thornber (1978). Samples ofthylakoid membranes were adjusted to a chlorophyll concentration of l-Omgml"1 and treated with0-35 % (w/v) lauryldimethylamine oxide (LDAO) for 30 min at 4CC. The treated membranes andPS I and PS II enriched fractions were suspended to a chlorophyll concentration of 0-4mgml~' ina buffer containing 2-5mM-Tris, 20mM-glycine (pH8-4), and treated at 4°C with SDS at a finalconcentration of 0-8% (w/v) (SDS:chl = 20); 25/il (10^g chlorophyll) was applied to the gel.Electrophoresis was carried out at 4°C for 3h at a constant voltage of 100 V. The unstained gelswere scanned at 672 nm with a Gilford spectrophotometer equipped with a linear transport device.Total polypeptide composition of thylakoids and PS I and PS II fractions was determined asdescribed previously (Glick & Zilinskas, 1982). The resolving gel was a 10% to 20% lineargradient of acrylamide. Samples were treated with SDS at 25°C. A 15 /Jg sample of chlorophyll wasapplied to each lane of the gel.

Intact filaments of P. laminosum were prepared for freeze-fracture electron microscopy byadding 5 ml of 70 % (v/v) glycerol, in the growth medium (medium D), to an equal volume of cellsthat were washed and resuspended in medium D. The glycerol was added dropwise over a period of1 h with stirring at 4CC. After at least 1 h in 35 % glycerol, filaments were centrifuged at 3000 £ for5 min. Drops of PS I and PS II fractions in buffer C containing 35 % glycerol and treated filaments(all at 4°C) were placed on copper support discs, frozen in liquid Freon cooled by liquid N2, andstored under liquid N2. All of these samples were fractured at — 100cC and etched for 2-3 min in a

60 R. E. Glick, R. E. Triemer and B. A. Zilinskas

Balzers BAF-301 freeze-etch apparatus. In addition, buffer C containing 35% glycerol was alsofractured and etched as a control and, as expected, no particles were detected. Replicas wereexamined in a Siemens 1A electron microscope operated at 80 kV. Magnifications were calibratedwith a line grating replica. Micrographs for particle si2e measurements were made with a 7xmagnifier equipped with a scale calibrated in 0-1-mm units. Particle diameters were measuredaccording to Staehelin (1979). The width of a shadow of a given particle was measured over theshadowed half of the particle. Where the edge of the particle appeared fuzzy or irregular, aminimum width was always taken. In cases where shadows of adjacent particles overlapped,measurements were not made. Following particle size measurements, the mean particle diameter(Jc), standard deviation (s), and standard error of the mean (s^) were calculated for E-face and P-face particles and for particles in freeze-fractured PS I and PS II fractions. To determine whetherdifferences in the mean particle diameters were statistically significant, the 99% confidenceintervals for each mean were calculated.

RESULTS AND DISCUSSION

Determining the structural sites of the two pigment systems and two photo-chemical reactions in red algal and cyanobacterial thylakoids has been the aimof several ultrastructural studies (Wollman, 1979; Giddings & Staehelin, 1979;Giddings et al. 1983). While valuable information has been obtained from thesestudies, a more definitive approach would be to correlate specific biochemical andphysiological functions with ultrastructural observations. We have isolated PS I andPS II fractions from thylakoid membranes of Phormidium laminosum in order toshow a more direct relationship between photosynthetic electron transport activitiesand intramembranous thylakoid particles in cyanobacteria.

One of the problems that has prevented isolation of photochemically activefractions from algal thylakoid membranes is the sensitivity of some photosyntheticfunctions to membrane solubilization techniques. PS II enriched fractions inparticular have proven to be difficult to isolate without oxygen evolution. Recentdevelopments in membrane fractionation have led to improved methods for theseparation of photosynthetically active complexes from cyanobacterial thylakoidmembranes (Stewart & Bendall, 1979). Fractionation techniques applicable tothermophilic organisms have also been developed in the belief that the physiologicalprocesses of these organisms would be less labile and therefore increase the prob-ability of isolating membrane fractions that retain physiological functions (Newman& Sherman, 1978; Stewart & Bendall, 1979). It was for these reasons that thethermophilic cyanobacteriumP. laminosum was chosen for the studies reported here.The procedure of Stewart & Bendall (1979, 1980) used in this study was particularlyuseful as it yields a satisfactory separation of PS I and PS II, and each fraction can berecovered from the same experiment.

The photosynthetic electron transport activities of thylakoid membranes isolatedfrom P. laminosum and those of the PS I and PS II enriched fractions are shown inTable 1. Although there is some residual PS I activity in the PS II fraction, the rateof oxygen evolution was as much as fivefold higher in the PS II fraction relative tointact membranes. The PS I fraction was also greatly enriched in PS I activity. Theratio of PS II to PS I activity in the intact membranes was 0*65, while in the PS Ifraction the ratio was 013 (a fivefold difference relative to the membranes); in the

Freeze-fracture of Phormidium laminosum 61

Table 1. Photochemical activities of isolated thylakoids and the PS I and the PS IIenriched fractions

/jmol of O2 mg chl ' h"

H2O->FeCy/DMBQ DCPIP/Asc->MVO2 evolved/

O2 consumed

ThylakoidsPS I fractionPS II fraction

13621

448

21115885

0-650-135-27

Oxygen evolution was measured in buffer C (lOmM-Hepes/NaOH, pH7-5; 5 mM-NaH2PO4/K2HPO4, pH7-5; 10mM-MgCl2; 25% (v/v) glycerol) containing 2mM-potassiumferricyanide, 1 mM-2,6-dimethyl-£-benzoquinone and 10/Jgml"1 chlorophyll (chl). Oxygen evol-ution was inhibited 100% by the addition of 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU) ata final concentration of 10~5 M. Oxygen consumption with methyl viologen (MV) was measured inbuffer C containing 100/iM-2,6-dichlorophenol indophenol, 2-5mM-sodium ascorbate, 10 /iM-DCMU, 3 mM-NH4Cl, 30/igml"1 MV, 1 mM-NaN3> and lO^gml"1 chlorophyll. Values given arethe mean from four isolations.

700 650

Wavelength (nm)

700 750 800

Fig. 1. Spectral properties of isolated thylakoid membranes. PS I fraction, and PS IIfraction. A. Room temperature absorption spectra of isolated thylakoids ( ), PS Ifraction ( ), PS II fraction ( ). Chlorophyll concentrations of thylakoids, PS Ifraction, and PS II fraction were 13-6, 11-7 and 9-5 \vg chlorophyll ml"1, respectively, allsuspended in buffer C. B. LOW temperature (77 K) fluorescence spectra of isolatedthylakoids ( ), PS I fraction ( ), PS II fraction ( ). All samples contained

chlorophyll ml~~ , suspended in buffer C. Excitation was at 430.

62 R. E. Glick, R. E. Triemer and B. A. Zilinskas

PS II fraction the ratio was 5-27 (an eightfold difference relative to the membranes).In the best single experiment, the PS I and PS II activities constituted 89% and85 % of the total electron transport activity of their respective fractions.

The spectral properties of the membranes and isolated fractions are shown inFig. 1. The room temperature absorption maxima of membranes and the PS Ifraction are at 678-679 nm, while the PS II fraction has a maximum at 672 nm.These absorption maxima are consistent with published reports, which show thatchlorophylls that absorb longer wavelengths are characteristic of the PS I-associatedantenna, while PS II chlorophylls absorb maximally at wavelengths that are blue-shifted with respect to PS I chlorophylls (French, Brown & Lawrence, 1972).Furthermore, these absorption properties are similar to those reported for purifiedPS I and PS II fractions from spinach chloroplasts (Satoh & Butler, 1978).

The fluorescence emission at 77 K of isolated thylakoids has minor peaks at 684and 694 nm and a major peak at 727 nm. The fluorescence emission of the PS Ifraction is at 726 nm, and the PS II fraction has major peaks at 684 and 694 nm anda lower peak at 724 nm. As indicated by the fluorescence spectrum of isolatedmembranes, the 727 nm peak is the major emission peak, because at 77 K thefluorescence yield of and the energy transferred to the PS I chlorophyll is greaterthan that of PS II chlorophyll (Goedheer, 1972). Therefore, the emission at 724nmin the PS II fraction represents the contribution of only a small amount of PS Ichlorophyll present in the PS II fraction. In cells of red algae and cyanobacteria, thefluorescence emission peaks at 77 K due to chlorophyll a are usually observed at ornear 685, 695 and between 710 and 730nm (Rijgersberg & Amesz, 1980). Theemission at 685 and 695 nm has been attributed to PS II and the 710-730 nmemission to PS I (Diner & Wollman, 1979; Rijgersberg & Amesz, 1980).

When isolated thylakoids and the PS I and PS II fractions are solubilized withSDS (SDS: chJ (w/w) = 20) and electrophoresed in a 5 % polyacrylamide gel at 4°C,the chlorophyll remains non-covalently bound to its apoprotein, so that it is possibleto resolve the chlorophyll proteins and visualize them in the gel (Markwell et al.1978). The chlorophyll proteins may then be excised from the gel and their spec-tral properties investigated. Two chlorophyll proteins are resolved from isolatedthylakoid membranes. The spectral properties of each of them (Fig. 2) indicate thatthe chlorophyll protein of lower electrophoretic mobility is associated with PS I, asits room temperature absorption and 77 K fluorescence emission maxima, 676and 723 nm, respectively, are characteristic of PS I chlorophyll, while the fastermigrating chlorophyll protein (absorption and fluorescence maxima at 670 and686 nm, respectively) is characteristic of PS II chlorophyll.

The pattern of chlorophyll proteins that is resolved when the PS I and PS IIfractions are run in the 5 % gel at 4°C reveals the presence of predominantly the PS I-associated chlorophyll protein in the PS I fraction (Fig. 3), while the PS II sampleshows a fourfold enrichment of PS II chlorophyll protein relative to the amountpresent in the membranes. In the membranes, 85 % of the total chlorophyll appliedto the gel is associated with the PS I chlorophyll protein and 15% with the PS IIchlorophyll protein (Fig. 3). In the PS I fraction, 99% of the protein-associated

Freeze-fracture of Phormidium laminosum 63

8CCO

•e1

0-6

0-4

0-2

1 • 1 1 1 | 1 1 1 1 | 1 I 1

r

- y j

/ iI '

' 1 '/

• IVVv JV J

\ ycJ\ /•>/

1 • . i • 1 . \ < i 1 i • i

1 1A .

11. 1111 -I I

1M •

ili\\\-

; \

. • 1 . . . . ,

(iI I11

11

i ,

i ii ii \ /1 M/ J

Ai \i \\

\

\\\\

• i i i i i

B .

-

-

- 60

40

o3

20

400 500 600 700 650 700 750

Wavelength (nm)

Fig. 2. Spectral properties of chlorophyll proteins separated on a 5 % polyacrylamide gel.A. Room temperature absorption spectra of PS I ( ) and PS II ( ) chlorophyllproteins. Full scale was 0-5 for the PS II chlorophyll protein spectrum and 1-0 for thePS I chlorophyll protein spectrum. B. Low temperature (77 K) fluorescence spectra ofPS I ( ) and PS II ( ) chlorophyll proteins. Excitation was at 430 nm.

chlorophyll is PS I chlorophyll, while 58% of the chlorophyll of the PS II sample isassociated with PS II chlorophyll protein. These data may overestimate the amountof PS I chlorophyll protein relative to PS II, as the PS II chlorophyll proteincomplex is much more susceptible to dissociation of the chlorophyll from theapoprotein than is the PS I complex. The small peak in the gel scan at 4-5 cmrepresents detergent-solubilized chlorophyll that is not associated with protein andruns with the dye front during electrophoresis. These electrophoretic patterns arecomparable to those reported by Stewart (1980) using a similar gel system, exceptthat in the work reported here, very little PS II chlorophyll protein was detected inthe PS I sample.

Re-electrophoresis of the chlorophyll proteins in a Laemmli system (SDS:chl = 100) at room temperature has revealed that the PS I chlorophyll proteinconsists of a major polypeptide with a molecular weight of 65X103 and minorpolypeptides of molecular weight 10— 18X103 (data not shown). These polypeptidesare of similar size to those of a purified PS I reaction centre from a thermophiliccyanobacterium (Nechushtai et al. 1983). The PS II chlorophyll protein was madeup of two polypeptides of molecular weight 51X103 and 47 X103, which are similar insize to polypeptides of PS II chlorophyll protein from other cyanobacteria(Rusckowski & Zilinskas, 1980; Guikema & Sherman, 1983). In Fig. 4, it can be

64 R. E. Glick, R. E. Triemer and B. A. Zilinskas

015

010 -

005

2 3 4 5 6 7

Migration (cm)Fig. 3. Profiles of chlorophyll proteins separated on a 5 % polyacrylamide gel at 4°Caccording to Markwell et al. (1978). A lO^ig sample of each of: A, isolated thylakoids;B, PS I; and c, PS II fractions were applied to the gel (SDS: chl (w/w) = 20). Thylakoidswere pre-treated with LDAO at LDAO:chl (w/w) = 3-5 for 30min at 4°C. Unstainedgels were scanned at 672 nm.

Freeze-fracture of Phormidium laminosum 65

seen that the PS I fraction contains a 65XlO3Mr polypeptide and that the PS IIfraction shows an enrichment of two polypeptides of 52 and 48X10 MT. Theseresults indicate that the PS I fraction is free of polypeptides representing PS IIchlorophyll protein and that the PS II fraction is highly enriched in PS IIchlorophyll protein polypeptides.

Electron micrographs of freeze-fractured cells of P. laminosum (Fig. 5) reveal thetwo types of fracture faces present in the thylakoid membranes. Unlike the situationfound in higher plant chloroplasts, in P. laminosum the intra-thylakoid space isextremely narrow, making the steps between surfaces and fractures nearly im-perceptible. The arrowheads in Fig. 5 illustrate areas of transition between thesurface and the fracture face of the membrane. The E-face is characterized by fewer,widely spaced particles, while the P-face is densely packed with particles. Themean diameters of the particles on the E-face and P-face are 8-3 nm and 7-1 nm,respectively. The main size class of E-face particles is 8-0 nm, while most of theP-face particles are either 6-0 or 7-0nm in diameter. The E-face particles aresometimes seen to be organized into short rows a few particles long (Fig. 5), similarto these described by Giddings et al. (1983). Long rows of particles were seen onlyinfrequently.

Electron micrographs of freeze-fractured PS I and PS II fractions are shown inFigs 6 and 7. The particles in the PS I fraction are in packed arrays that appear to beassociated with membrane. The general distribution of the particles is qualitativelysimilar to the distribution of the particles on the P-face of the P. laminosum cells, i.e.many particles in close proximity to each other. The particles in the PS II fractionare not associated with membrane but are scattered freely in the suspending medium.Occasionally, groups of particles can be seen that appear to form a short row. Themean sizes of the particles in the PS I and PS II fractions are 7 8 and 8-7nm,respectively, and the range of particle sizes is shown in Fig. 8 in the form of ahistogram. The PS II particle histogram is superimposed over that of the E-faceparticle histogram, as are the PS I and P-face particle histograms. The maxima ofboth the E-face and PS II histograms are at 8*0 nm. The P-face particle histogramhas a broad maximum at 6-0-70 nm, while that of the PS I histogram is at 7 0 nm.There is good agreement between the E-face and PS II histograms. Statisticalanalysis shows (Table 2 and Fig. 9) that the difference between the mean diameter ofE-face and PS II particles is not significant, indicating that the particles seen in thefreeze-fractured PS II preparation are the same as those on the E-face of freeze-fractured P. laminosum cells. There were, however, significant differences betweenthe mean E-face and P-face particle sizes and the mean PS I and PS II particle sizes.The P-face and PS I particle histograms have the same basic shape; the significantdifference in their mean particle size is probably a result of the fact that the entire PSI particle population is 'shifted' 10nm higher than the P-face particle population ofthylakoid membranes. This suggests that in addition to PS I, there are othercomponents that contribute to the particles in the thylakoid P-face.

It is nearly certain that the P-face of cyanobacterial thylakoids contains more thanjust PS I particles. Arntzen (1978) proposed that on the P-face of spinach thylakoids

R. E. Glick, R. E. Triemer and B. A. Zilinskas

1 2 3 4 5

Fig. 4. Polypeptides of isolated thylakoids, PS I and PS II fractions separated on a lineargradient (10% to 20%) of acrylamide. Fifty microlitres of sample buffer (Glick &Zilinskas, 1982) was added to 15 )A of each sample (all at a chlorophyll concentration ofl-Omgml"1). The entire sample (15 y% chlorophyll) was applied to the gel. Samples weretreated with SDS at room temperature. Lane 1, P. laminosum PBsomes; lane 2, isolatedthylakoids; lane 3, PS I fraction; lane 4, PS II fraction; lane 5, molecular weightstandards: phosphorylase b (94X103), bovine serum albumin (68X103), ovalbumin(43X103), carbonic anhydrase (30X1O3), soybean trypsin inhibitor (21X10^), lysozyme(14X103).

Freeze-fracture of Phormidium laminosum 67

there are particles whose functions are not related to PS I. It was argued that thismust be the case since the ratio of P-face to E-face particles in higher plant maturegrana thylakoids is 3:1 (Staehelin, 1979), while the ratio of PS I to PS II reactioncentres is about 1:2 (Melis & Brown, 1980). In spinach thylakoids, the P-face hastwo size classes of particles that average 8*0— 8*5 nm and 10-5nm in diameter(Staehelin, 1979; Armond et al. 1977). Furthermore, purified coupling factorcomplexes and isolated cytochrome b/f complexes reconstituted into liposomes haveshown particles of diameters 9-5 nm (Mullet, Pick & Arntzen, 1981) and 8-5 nm(Morschel & Staehelin, 1983), respectively. Kaplan & Arntzen (1982) have thereforesuggested that the particles on the P-face of spinach thylakoids represent a mixture ofPS I complexes, cytochrome b/f complexes and the intrinsic membrane componentof chloroplast coupling factor (CF0).

Although less is known about the thylakoid ultrastructure in cyanobacteria,similarities between the complexes in cyanobacterial thylakoids and those in thechloroplast membranes have been documented (Staehelin et al. 1978). In addition, acytochrome b/f complex has been isolated from cyanobacteria (Krinner, Hauska,Hurt & Lockau, 1982), using a procedure similar to that used with spinachchloroplasts, implying that the complex probably exists as a discrete membraneparticle, as has been shown for spinach thylakoids (Morschel & Staehelin, 1983).Unfortunately, particle sizes for the cyanobacterial cytochrome ^//complex (or CF0)are not known.

A further indication that there are particles other than PS I complexes on theP-face of cyanobacterial thylakoids is seen when comparing the ratio of P-face toE-face thylakoid particles and the ratio of PS I to PS II reaction centres. The ratio ofP- to E-face particles in various cyanobacteria has been reported to be greater than 5(Armond & Staehelin, 1979; Giddings & Staehelin, 1979; Coxetal. 1981), while theratio of PS I to PS II reaction centres is 2-3—2-5 (Kawamura, Mimuro & Fujita,1979; Myers, Graham & Wang, 1980; Melis & Brown, 1980; Manodori & Melis,1984), indicating that not all P-face particles represent PS I complexes.

In addition to PS I, cytochrome b/f and CFQ complexes, there may be particles inthylakoid membranes that are related to respiratory functions. The site of respiratoryelectron transport in cyanobacteria has not been established, but most evidencesuggests that the respiratory chain is located in both the thylakoid and the plasmamembrane. If this is so, then some of the thylakoid P-face particles might functionin respiratory electron flow (Binder, 1982). The respiratory and photosyntheticelectron transport chains do contain some of the same components such as quinones,the cytochrome b/f complex, plastocyanin and cytochrome C553 (Binder, 1982).Peschek (1983) has shown that the cytochrome b/f complex participates in bothrespiratory and photosynthetic electron transport, implying a close physical re-lationship of respiration and photosynthesis in cyanobacteria. He proposed (andSandmann & Malkin (1983) have confirmed) that the cytochrome b/f complex candonate electrons to either P7Oo or cytochrome oxidase in cyanobacteria. In the light ofthese findings, it may be proposed that particles representing respiratory complexesin the thylakoid membrane are in close association with PS I complexes, and that the

R. E. Glick, R. E. Triemer and B. A. Zilinskas

Freeze-Jracture of Phormidium laminosum 69

P-face of the freeze-fractured thylakoid membranes may therefore contain particleswith dual roles in photosynthesis and respiration, as well as particles involved ineither function alone.

Consequently, as the P-face of thylakoid membranes probably contains more thanjust PS I complexes, one must assume that at least some of these complexes musthave been removed as a consequence of the PS I isolation procedure. This is the mostlogical explanation to account for differences in the mean particle sizes of thethylakoid P-face and the isolated PS I fraction. These smaller, non-PS I, P-faceparticles might have been partially solubilized by the detergent and separated fromthe PS II complexes during subsequent chromatography on Sepharose 6B. Un-fortunately, owing to dense packing, similar sizes, and diverse functions of P-faceparticles, their identification has been slow; it is hoped that, with further charac-terization of other detergent-solubilized functional complexes of cyanobacteria byboth biochemical and microscopic means, there will be less need for speculation.

200

160

S3 120ao

EZ 80

40

EF

PSII

L -

PFl

PSI ' 1

*

ii•

iI1

11

j

40 80 120 40

Particle diameter (A)

i r80 120

Fig. 8. Histograms of particle sizes on E- and P-faces, and in PS I and PS II fractions.Number of particles measured for all classes was 560.

Fig. 5. Freeze-fracture micrograph of a portion of a P. laminosum cell showing cross-fractured thylakoids (t), and exposed particles on the E- and P-faces of the membranes.Bar, 0-25 fan.Fig. 6. Freeze-fractured PS I fraction. Bar, 0-25 ^m.Fig. 7. Freeze-fractured PS II fraction. Bar, 025/im.

70 R. E. Glick, R. E. Triemer and B. A. Zilinskas

Table 2. Statistical analysis of freeze-fracture particles of P. laminosum thylakoidsand PS I and PS H-enriched fractions

Particletype

E-faceP-facePS IIPS I

The number of particles measured of each particle type was 560.

Mean particlediameter (nm)

8-277-108-697-76

Standarddeviation (nm)

1-721-362-241-43

Standard errorof mean (nm)

0-070060-100-06

PSII PFParticle class

PSI

Fig. 9. Comparison of the mean particle diameters of particles on the E- and P-faces ofP. laminosum thylakoids and particles in freeze-fractured PS I and PS II preparations.The 99 % confidence intervals for each mean particle size are indicated.

As noted above, the PS I freeze-fracture particles appear to be membrane-boundbut the PS II particles are not. Such being the case, an explanation may be offered asto how the separation of PS II from PS I occurs. In the membrane fractionationprocedure, the solubilization of the thylakoid membranes with LDAO is followed byultracentrifugation (100000^ for lh) . The pellet fraction of the centrifugation

Freeze-fracture of Phormidium laminosum 71

contains the bulk of the chlorophyll and the greater PS I activity (PS I fraction),while the clarified green supernatant shows the higher rate of oxygen evolution (PSII fraction). This supernatant can be pelleted after it has been passed through aSepharose 6B column, which removes the detergent and residual phycobiliprotein.The fact that ultracentrifugation separates PS II from PS I implies that the PS IIfraction consists of rather small particles, since they remain in the supernatant afterbeing subjected to 100 000 £ for 1 h and require centrifugation at 100 000 g for 16 h tosediment them. This also implies that LDAO is selectively solubilizing PS II fromthe membrane but leaves PS I in the membrane.

A small amount (10—20%) of membranous material was observed in freeze-fracture replicas of the PS II fraction. There were also particles present on some ofthe membranous material with diameters of about 7-5 nm (data not shown). In viewof the above discussion about the nature of the PS I particles being membrane-bound, it is probable that the membranous material in the PS II fraction is the sourceof the PS I contamination of this fraction. As these clearly different particles were notincluded in measurements made, a true representation of the PS II particles wasobtained.

The observation of rows of particles on the E-face of thylakoids in PBsome-containing organisms has led many investigators to propose that the E-face particlesare structural equivalents of PS II centres in the membrane (Neushul, 1971; Lefort-Tranefa/. 1973; Lichtle& Thomas, 1976; Wollman, 1979; Golecki, 1979; Giddings& Staehelin, 1979). A more rigorous study of the relationship between thylakoid E-face particles and PBsomes has been published (Giddings et al. 1983) in which thespacing both within and between E-face particle rows and PBsomes was compared.The authors concluded that there was a close correspondence between rows of E-faceparticles and PBsomes but noted that rows of E-face particles were not consistentlyseen in the thylakoids. The work presented here supports the observation that rowsof E-face particles are not a predominant feature of the E-face of cyanobacterialthylakoids. It seems likely that rows of E-face particles do occur in some cyano-bacterial species, as they have been observed by many investigators, but they are notuniversally found in cyanobacteria or red algae (Staehelin et al. 1978). The questionof their significance still exists.

This work was supported in part by the Science and Education Administration of the UnitedStates Department of Agriculture under grant 8S-CRCR-1-1562 from the Competitive ResearchGrants Office. New Jersey Agricultural Experiment Station, Publication no. D-01104-1-85,supported by State Funds and by the United States Hatch Act. Thanks are given to Charles Kupattfor assistance with the statistical analysis.

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{Received 29 April 1985 - Accepted 30 August 1985)