Biol Cell (1992) 74, 187-194 © Elsevier, Paris

187

Original article

Immunogoid localization of photosystem I and photosystem II light-harvesting complexes in cryptomonad thylakoids

Christiane Lichtl6 ~, R Michael L McKay 2, Sarah P Gibbs 2

* Laboratoire des Biomembranes et Surfaces cellulaires v~gdtales (UA CNRS D 0311), Ecole Normale Supdrieure, 46, rue d'Ulm, 75230 Paris Cedex 05, France; 2Department o f Biology,

McGill University, Montrdal, Quebec H3A 1B1, Canada (Received 22 April 1991 ; accepted 17 December 1991)

Summary - The molecular organization of the thylakoids of Cryptomonas rufescens was studied by immunoelectron microscopy employing antibodies against photosystem (PS)-I and two PS-II antenna proteins. The PS-I complex and the 19-kDa chlorophyll a/c light-harvesting (LH) protein are both localized along the length of the thylakoid membranes. The external membranes of the paired thylakoids are enriched in PS-I whereas the chlorophyll a/c LH protein is more concentrated in the internal or appressed mem- branes. However, unlike the situation in higher plants and Chlamydomonas, there is not a marked asymmetry in the concentration of PS-I and chorophyll a/c LH protein in the two types of membranes. Double labelling studies of sections and isolated PE-PS-II particles with anti-phycoerythrin and anti-LH confirmed that phycoerythrin is localized in the thylakoid lumen and that this pigment exists in two forms, a fraction closely associated with the thylakoid membranes and another fraction free in the lumen. These results confirm the uniqueness of cryptomonad thylakoids.

Cryptomonas rufescens I light-harvesting protein / photosystems / phyeoerythrin / thylakoids

Introduct ion

The organization of the photosynthetic apparatus of cryp- tomonads is unique among the chromophyte algae [36, 37]. In addition to possessing chlorophyll (chl) c, cryptophyte chloroplasts have phycobiliprotein localized in the thylak- oid lumen [6, 8, 15, 35]. Both pigments, chl c and phycobiliprotein (phycoerythrin (PE) or phycocyanin de- pending on the algal species) form antenna pigment-protein complexes which independently transfer light energy to photosystem (PS)-II [4, 17]. Treatment of isolated thylak- oids with detergent and separation on sucrose gradients has allowed the isolation of these two antenna complex- es, the chl a/c antenna [13, 19, 24] and the PE-PS-II an- tenna [19]. Immunocytochemist ry has confirmed the localization of PE inside the thylakoid lumen [20, 22, 28, 29] and the association of the chl a/c antenna with the thylakoid membranes [24]. However, it is not known whether there is a differential distribution of PS-I and PS- II on appressed and non-appressed thylakoid membranes of cryptomonads as there is in green algae and higher plants. In this study we have employed immunoelectron microscopy to determine the distribution of PS-I and of a PS-II-associated chl a/c antenna protein on the internal (appressed) and external (non-appressed) membranes of the paired thylakoids. We also confirm that the discs at- tached to isolated inside-out thylakoid vesicles of the PE- PS-II antenna fraction [19] are indeed phycoerythrin.

Abbreviations: chl, chlorophyll ; CP, chlorophyll-protein complex; GAM, goat anti-mouse immunoglobin G; GAR, goat anti-rabbit immunoglobin G; LH, light-harvesting protein; PE, phycoerythrin ; PS, photosystem.

Materials and methods

Culture conditions

Cryptomonas rufescens Skuja cells were grown in S2T 2 medium at 17°C as previously described [16]. Cells were harvested late in the logarithmic or early in the stationary phase of growth.

Chloroplast fractions and phycoerythrin extraction

The isolation of the PS-II antennae (fractions 2 and 3) and of PS-I (fraction 1) on a sucrose step gradient was performed as described by Lichtl~ et al [19]. The polypeptides of whole thylak- oids and of the different fractions were separated on polyacryl- amide slab gels [14] as described previously [19].

The phycoerythrin was partially extracted from thylakoids of algae placed for 30 min at 4°C before centrifugation (1500 g) [15].

A ntisera preparation

Phycoerythrin antiserum raised in rabbit against PE 566 from Cryptomonas ovata [10] was the gift of R MacColl (New York State Department of Health, Albany, NY). Antiserum to PS-I raised against chlorophyll-protein (CP) complex I of spinach was the gift of B Lagoutte (CEA, Service de Biophysique, Saclay, France). Spinach CPI was isolated according to Setif et al [26] and 50 tsg was mixed with Freund's adjuvant and injected into a mouse. A second injection was made three weeks later. An- tiserum to the 19-kDa protein of the chl a/c fight-harvesting com- plex (LH) of C rufescens, designated LH-antiserum, was prepared as follows. The polypeptides of fraction 2 were separated by gel electrophoresis and then transferred to a nitrocellulose sheet [32] and stained in Ponceau red to determine the exact position of the polypeptides. The 19-kDa band was cut out, ground in li- quid nitrogen, mixed with Freund's complete adjuvant, and in- jected into a rabbit. Four injections were given at weekly intervals and the serum tested from the third week on. Preimmune serum was taken before the first immunization.

188 c Lichtl~ et al

Immunoblotting

The separated polypeptides of whole thylakoids, fractions 1 and 2 were transferred electrophoretically to nitrocellulose sheets [32]. After blocking with 307o bovine serum albumin (BSA) in TBST (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1070 Tween-20) for 1 h, blots were incubated 2 h at room temperature in the anti-LH se- rum or in the anti-PS-I serum diluted with TBST plus 0.3070 BSA at different dilutions from l:10 ~ to l:10 ~. The blots were washed several times with TBST and then incubated for 45 min in a 1:5 x l0 ~ dilution of horseradish peroxidase-conjugated goat anti-rabbit for anti-LH or goat anti-mouse immunoglobulin G for anti-PS-I (Bio-Rad, Paris, France) in TBS. After several wash- es in TBST, immunoreactive bands were visualized by incubat- ing blots in the substrate paraphenylene diamine pyrocatechol in 50 mM Tris, pH 7.4, plus 0.02070 H20 2.

Postembedding immunocytochemistry

Cultures of C rufescens were grown to a density of 7.0 x 10 s cells/ml and harvested by gentle centrifugation. Cells were fixed at 4°C for 90 min in 1.0070 glutaraldehyde in 0. l M sodium phos- phate buffer, pH 7.4. Following three 10-min rinses in buffer alone, the cells were dehydrated in a graded ethanol series at 4°C and embedded in LR White medium grade resin (JB EM Serv- ices, Montr6al, Qu6bec) by the following protocol: 100O7o ethanol/resin, l : l , two changes at 4°C, 30 min and 1 h; pure resin, three changes at room temperature, 45 min, overnight and 2 h ; pure resin at 58°C for 20 h. Pale gold-coloured sections cut with a diamond knife were collected on formvar-coated nickel grids. Grids were floated successively on drops of the following solutions: phosphate-buffered saline (PBS), 10 min; PBS plus 207o BSA, 15 min; antisera diluted in PBS plus 207o BSA (LH- antiserum, 1:250 dilution, overnigth ; PE-antiserum, l : l0 ~ dilu- tion, for l h; PS-I-antiserum, 1:250 dilution overnight). After washing with PBS, the sections were placed for 1 h on drops of gold-labelled secondary antibody diluted i:30 in PBS plus 2°7o BSA either goat anti-rabbit (GAR), 5 nm or 10 nm gold parti-

cles, or goat anti-mouse (GAM), l0 nm gold particles (Janssen Pharmaceutica, Beerse, Belgium). For the double labelling ex- periments, the dilutions were the same as for single antiserum. The first labelling was made with PE-antiserum and GAR 5 nm, and the second labelling with LH-antiserum and GAR l0 nm. Improved contrast of the membranes was achieved by overnight exposure to OsO~ vapors followed by 2°70 uranyl acetate stain- ing. Sections were viewed in either a Philips EM 300 or a Philips EM 410 electron microscope operated at 80 kV.

The following control experiments were performed: incuba- tion in immunoglobulin G alone omitting the antibody step ; in- cubation with non-immune serum in place of the antiserum ; and incubation with PBS plus 207o BSA in place of the antiserum.

lmmunocytochemistry on negatively stained preparations

Fraction 3 was removed from the sucrose density gradient and placed on formvar-coated nickel grids as described by Lichtl~ et al [19]. The labelling with antiserum was performed accord- ing to the method of Vallon et al [34]. Grids were floated on drops of anti-LH 0:250 dilution in PBS plus 2070 BSA) for 30 min or anti-PE 0:500 dilution in PBS plus 2070 BSA) for 30 min, followed by GAR 10 nm for 20 min. For the double labelling, the antisera dilutions were the same and the grids were exposed successively to anti-PE and GAR 5 nm and to anti-LH and GAR 10 nm. The grids were post-stained 5 min with 2°7o uranyl acetate.

Quantification of labelling

The thylakoids of cryptomonad chloroplasts are loosely associat- ed in pairs, each pair thus has two external and two internal mem- branes. For the two antisera raised against membrane proteins, anti-PS-1 and anti-LH, gold particles located partially or entire- ly over the external membrane were counted as labelling the ex- ternal membrane (category 1, see fig 4). Gold particles lying between the two dense lumens of a thylakoid pair were counted as internal membrane labelling (category 2, fig 4). Particles ly- ing over the dense thylakoid lumen (category 3, fig 4) were count-

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Fig 1. Li-PAGE profiles and immunoblots of thylakoid fractions from Cryptomonas rufescens. Lane A : Li-PAGE profile of poly- peptide components of PS-I (fraction 1, see Materials and methods) ; lane B : immunoblot of A probed with PS-I-antiserum from spinach ; lane C : Li-PAGE profile of polypeptides associated with PS-II (fraction 2) ; lane D : immunoblot of C probed with LH- antiserum; lane E: Li-PAGE profile of polypeptide components of whole thylakoids; lane F: immunoblot of E probed with PS-I- antiserum from spinach ; lane G : immunoblot of E probed with LH-antiserum. Protein markers from Pharmacia kit : ph0sphorylase, 94 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 20.1 kDa; ~-lactalbumin, 14.4 kDa.

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190 c Lichtl~ et al

ed separately and then half were arbitrarily assigned to the ex- ternal membrane and half to the internal membrane. For anti- LH, 3102 gold particles were counted; for anti-PS-I, 1877 par- ticles were counted.

Results

Immunoblotting

The proteins associated with the PS-I of C rufescens (frac- tion 1 on a sucrose gradient ; fig 1, lane A), those of the chl a/c antenna (fraction 2 on a sucrose gradient; fig 1, lane C) and those of the whole thylakoids (fig 1, lane E) were separated on polyacrylamide gels and stained with Coomassie blue [19]. After immunoblotting, anti-LH recognized exclusively the 19-kDa protein of the chl a/c antenna (fig 1, lanes D and G), whereas anti-PS-I of spinach reacted with a 67-kDa protein corresponding to the PS-I centers of fraction 1 (fig 1, lane B) and whole thylakoids (fig 1, lane F). No reaction was observed with preimmune sera (not shown). Ludwig and Gibbs [20] have shown by immunoblotting that anti-PE 566 of C ovata cross-reacts with the/3 subunit of PE 545 of the crypto- phyte alga Rhodomonas lens.

Immunolabelling of sections

The thylakoids of Cryptomonas rufescens are associated in pairs and in cells grown in low light, the pairs are fre- quently piled up to form large stacks which fill most of the chloroplast (figs 2 - 4 ) . After fixation in glutaralde- hyde and embedding in LR White and in the absence of OsO 4, the PE-containing thylakoid lumens appear elec- tron dense, whereas the thylakoid membranes appear elec- tron translucent (figs 2 - 4). Even within the large stacks, it is easy to identify the thylakoid pairs, for a space of ap- proximately 4 - 7 nm separates the two dense lumens of a pair, whereas a larger space ( 1 0 - 1 5 nm) separates the dense lumens of adjacent pairs. In these preparations, the two internal membranes of a thylakoid pair appear as a single electron translucent area, whereas a thin dense line can be seen separating the two electron translucent exter- nal membranes of adjacent thylakoid pairs.

In a section of a chloroplast labelled with antibody to PS-I ofcspinach, gold particles are distributed along the length of the thylakoids (fig 2). Very few are observed over the chloroplast stroma or over the pyrenoid (not shown). Control sections labelled with preimmune serum or by in- cubation in PBS alone followed by gold-labelled secon- dary antibody showed no labelling (fig 3). To quantitate the distribution of PS-I labelling between the external and internal membranes of the paired thylakoids, regions where paired thylakoids had been cut perpendicularly were marked at each end with vertical lines and all gold parti- cles between the lines counted and categorized. Thus the length of external membrane analyzed exactly equaled the length of internal membrane analyzed. Of the 1877 parti- cles counted, 42.6°70 were localized over the external mem- branes, 29.6°70 over the internal membranes, and 27.7°70 were localized over the lumens. Since PS-I is a membrane localized, the lumenal counts were arbitrarily assigned half to each membrane. Figure 5 shows that anti-PS-I label- ling was slightly higher (56.5°70) over the external mem- branes than over the internal membranes (43.5070).

In a chloroplast section labelled with antiserum prepared against the 19-kDa chl a/c antenna protein of PS-II of C

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rufescens, the chloroplast thylakoids are highly labelled whereas the chloroplast stroma is unlabelled (fig 4). Par- ticle counts showed that 19.5070 of the anti-LH labelling was over the external membranes and 32.2°70 was over the internal membranes. Taking into account the particles localized over the lumen (48.2070), the LH protein is more concentrated on the internal (54.2°70) than on the external (45.8°70) membranes of the thylakoid pairs.

In a chloroplast which has been double labelled with anti-PE marked with 5 nm gold followed by anti-LH marked with 10 nm gold, the thylakoids are heavily labelled with each antibody (fig 6). A quantitative analy- sis of anti-PE labelling (3004 particles counted) showed that 13.7°70 of the particles were located over the external membrane not touching the dense lumen, 21.7°70 were over the external membrane touching the lumen, 34.9°70 were over the lumen, 22.9°70 were over the internal membrane touching the lumen and 6.9°70 were over the internal mem- branes not touching either lumen. Ludwig and Gibbs [20] have demonstrated in another cryptomonad species, Rhodomonas lens, that all PE is inside the thylakoid lu- men and that even those gold particles located over the membrane mark lumenal PE. They concluded that much of the lumenal PE is membrane bound. Our data demon- strate that much of the PE is also membrane associated in C rufescens (65070 of the labelling) and furthermore that PE is associated with both the internal and the external membranes of the thylakoid pairs. In a cell of C rufescens from which PE has been partially extracted [15], PE has been preferentially extracted from the center of the thylak- old lumen and regularly spaced rod-shaped structures are clearly seen attached to the inner (star) or outer (arrows) membranes of the thylakoid pairs (fig 7).

Immunolabelling of &olated PE-PS-H particles

Lichtl6 et al [19] isolated and characterized a photosyn- thetically active PE-containing subchloroplast fraction

Organization of cryptomonad thylakoids 191

Figs 6, 7. Double labelling of a chloroplast section with anti-LH marked with 10 nm gold and anti-PE marked with 5 nm gold. The small gold particles marking PE are observed both over the dense thylakoid lumen (lu) and the thylakoid membranes (me). The latter labelling is presumed to arise from membrane-associated lumenal PE. Bar = 0.2 gm; x 102000.7. Chloroplast of a cell of Crufescens from which PE has been preferentially extracted. Periodic rod-shaped structures are present in the lumen of the paired thylakoids. The rods may be attached to either the internal (star) or outer membrane (arrows) of the paired thylakoids, me, external membranes of a thylakoid pair; mi, internal membranes of a pair. Bar = 0.2/~m; x 150000.

(fraction 3) from C rufescens which is highly enriched in PS-II reaction centers and relatively deficient in LH chl molecules. In the electron microscope, these purified PE- PS-II particles consisted of small vesicles 25 - 40 nm in di- ameter to which were attached small discs 1 0 - 12 nm in diameter and 6 nm thick. At places rods of 3 - 4 stacked discs were observed attached to the vesicles. Lichtl6 et al [19] interpreted the vesicles to be inside-out fragments of thylakoids with attached discs of phycoerythrin. To prove this, we have isolated these PE-PS-11 particles (fraction 3) and labelled them with ant i-LH and anti-PE or double labelled them with both antisera prior to negative stain- ing. When fraction 3 was labelled by antibody against the 19-kDa LH protein of the thylakoid membrane followed by GAR-10 nm gold, the gold particles lie over the mem- brane vesicles (fig 8a). When fraction 3 was labelled with anti-PE marked with 5 nm gold, the labelling is associat- ed with the small 10-nm discs which are often arranged in rows (fig 8b). No labelling was observed when the an- tisera were omitted (fig 8c). When fraction 3 was double labelled by anti-LH marked with l0 nm gold and anti-PE marked with 5 nm gold, the large gold particles mark the membranes of the vesicles and the small gold particles the associated discs (figs 8 d - f ) . We conclude that the vesi- cles are indeed inside-out fragments of thylakoid mem-

branes with the 19-kDa chl a/c antenna protein located in the membrane and PE localized in the attached 10-nm discs.

Discussion

In higher plants, PS-I is localized almost entirely on the membranes of the stroma thylakoids and on the limiting membranes of the grana stacks, whereas PS-II is localized largely on the appressed membranes of the grana stacks [1, 2, 5, 30, 33, 34]. A similar distribution of PS-I and PS-II components has been found in the green alga Chlamydomonas reinhardtii [33,34]. However, almost nothing is known about the distribution of PS-I and PS- II in the chl c-containing chromophyte algae. In Chlamydomonas and in various other green algae, grana- like configurations of the thylakoid membranes are ob- served [9], but in the chromophyte algae, the thylakoids are organized into extended lamellae frequently travers- ing the length of the chloroplast. Each lamella consists of three appressed thylakoids, except in the cryptomonads where the lamellae consist of paired thylakoids. Freeze- etch studies of brown algae [3], dinoflagellates [31] and cryptomonads [7, 18, 23, 27] showed that the thylakoids

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Fig 8. Negatively-stained isolated PE-PS-II particles (fract ion 3) from C ru f e scens , The particles consist of small vesicles (v) to which small 10 nm discs (pe) are at tached• a. Particles labelled with an t i -LH and marked with GAR-10 nm gold• The gold particles (long arrow) are located over the small thylakoid vesicles• b. Particles labelled with an t i -PE and marked with GAR-5 nm gold• The small discs a t tached to the vesicles are labelled (short arrow)• e. Control• Both ant isera were omitted• d - f . Particles double- label led with ant i -LH (GAR-10 nm gold) and an t i -PE (GAR-5 nm gold)• The large gold particles are localized over the thylakoid vesicles (long arrows) and the small gold particles (short arrows) mark the small discs a t tached to the vesicles• Bar = 0 .2 /~m; x 120000.

Organization of cryptomonad thylakoids 193

of these chromophyte algae possess EF fracture faces resembling the EF fracture faces of the appressed and non- appressed thylakoid membranes of higher plants. This sug- gests that PS-II particles might be preferentially located on the appressed membranes of the thylakoid lamellae, but this has not been directly demonstrated.

In this study we show by immunoelectron microscopy that PS-I is slightly more concentrated on the external membranes of the thylakoid pairs, whereas the 19-kDa chl a/c LH protein associated with PS-II is more concentrat- ed on the loosely appressed internal membranes of the pair. First, however, the limitations of the immunolabelling technique must be discussed. In particular, we must ask if the resolution of the technique is sufficient to distinguish labelling on the external membranes from that on inter- nal membranes. In our fixed tissues, the thylakoids are only 20 nm wide. An antibody molecule is approximately 8 nm long [25] whereas the gold particles are 10 nm in di- ameter. The maximum distance the center of a gold parti- cle could be from the labelled antigen is 8 nm plus 8 nm (the secondary antibody) plus 5 nm (ie, 21 rim). Thus a gold particle localized over on the external membrane could sometimes have originated from an antigen in an internal membrane and vice versa, but because of the paired nature of the thylakoids, these errors should can- cel each other out. A bigger problem is the large number of gold particles located over the lumen (28°7o in the case of PS-I labelling and 48070 in the case of anti-LH label- ling). We arbitrarily assigned 50% of these labels to the internal membrane and 50070 to the external membrane. This procedure would make the labelling of the two types of membranes more equal than it actually is. If we simply discard the labels not definitely located on one membrane or another, we find that with anti-LH, 62.3% of the gold particles are over the internal membranes and with anti- PS-I, 59.0070 of the labelling is on the external membrane.

Thus it appears that there is a genuine difference be- tween the labelling observed with anti-PS-I and with anti- LH. However, compared with the situation in higher plants and green algae, it is clear that both PS-I and the 19-kDa chl a/c LH protein are found on both internal and exter- nal membranes and are distributed along the entire length of the thylakoids. It is not clear how this relates to the freeze-etch studies, although Staehelin [30] showed that in cryptomonads, areas of concentrated EF particles may occur in the same membrane as areas of sparsely distribut- ed EF particles. If these EF particles are PS-II reaction centers with associated chl a/c LH protein, then our data indicate that such patches of lateral heterogeneity would be present in both external and internal membranes.

In higher plants, the chl a/b LH protein is believed to play a role in membrane stacking [2, 12, 21]. Spear- Bernstein and Miller [29] have recently proposed a model for crypt9monad thylakoids in which the chl a/c LH pro- tein is located only in appressed internal membranes. Our data contradict this model for although anti-LH labelling is slightly more concentrated on internal membranes, it is also present on the external membranes. Also cryp- tomonad thylakoids are not tightly appressed as they are in higher plants, but have a 2 - 4 nm space between them, so true stacking may not exist.

Our results with anti-PE labelling confirm the results of previous workers [20, 22, 27, 28] showing that PE is localized in the lumen and that a part of the PE is preferen- tially associated with the thylakoid membrane. In addi- tion, we demonstrate for the first time that PE is associated with the internal and external thylakoid membrane. Since

PE transfers light excitation energy to PS-II [4, 17], this observation, like our anti-LH labelling results, indicates that PS-II is located on both membranes. We also show that when free PE is extracted from the cells, one sees small rods attached to the internal or to the external membrane. By immunolabelling negatively-stained preparations of a fraction containing the PE antenna attached to PS-II, we have confirmed the earlier suggestion of Lichtl~ et al [19] that the small 10-nm subunits associated to the membrane vesicles are discs of PE attached to inside-out thylakoid vesicles. These results support the observations of Hiller and Martin [1 l] that PE of cryptomonads has multiple forms, part of the pigment having a light-harvesting role whereas the other part associated with the membrane would transfer collected light energy to the PS-II reaction centers,

Acknowledgments

This research was supported in large part by a Franco-Qu~bec Exchange grant held by C Lichtl6 and S Gibbs. We thank A Spilar and C Passaquet for their skillful technical assistance, and F Puel for preparation of the LH-antibody.

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