Polypeptide composition, assembly and ... - Springer Link

Planta (1994)194:42-54

P l a n t a 9 Springer-Verlag 1994

Polypeptide composition, assembly and phosphorylation patterns of the photosystem II antenna system of Cblamydomonas reinhardtii Keith D. Allen*, L. Andrew Staehelin Department of Molecular, Cellular and Developmental Biology,University of Colorado, Boulder, CO 80309, USA Received: 24 June 1993 / Accepted: 9 September 1993 Abstract. In recent years major progress has been made in describing the gene families that encode the polypeptides of the light-harvesting antenna system of photosystem II (PSII). At the same time, advances in the biochemical characterization of these antennae have been hampered by the high degree of similarity between the apoproteins. To help interpret the molecular results, we have re-examined the composition, the assembly and the phosphorylation patterns of the light-harvesting antenna of PSII (LHCII) in the green alga Chlamydomonas reinhardtii Dang. using a non-Tris SDS-PAGE system capable of resolving polypeptides that differ by as little as 200 daltons. Research to date has suggested that in C. reinhardtii the LHCII comprises just four polypeptides (pll, p13, p16 and p17), and CP29 and CP26just one polypeptide each (p9 and pl0, respectively), i.e. a total of six polypeptides. We report here that these antenna systems contain at least 15 polypeptides, 10 associated with LHCII, 3 with CP29, and 2 with CP26. All of these polypeptides have been positively identified by means of appropriate antibodies. We also demonstrate substantial heterogeneity to the pattern of in-vitro phosphorylation, with major differences found among members of closely spaced and immunologically related polypeptides. Most intriguing is the fact that the polypeptides that cross-react with the anti-type 2 LHCII antibodies of higher plants (p16, and to a lesser extent pl 1) are not phosphorylated, whereas in higher plants these are the most highly phosphorylated polypeptides. Also, unlike in higher plants, CP29 is heavily phosphorylated. Phosphorylation does not appear to have any effect on the mobility of polypeptides on fully denaturing SDS-PAGE gels. To learn more about the accumulation and organization of the light-harvesting polypeptides, we have also investigated a chlorophyll b-less mutant, cbnl-48. The LHCII is almost completely lost in this mutant, along with at least

*Present address: Department of Plant Biology,111 KoshlandHall, University of California, Berkeley,CA 94720, USA Correspondence to: K.D. Allen; FAX: 1 (510) 642 4995; Tel. 1 (510) 642 7742; E-mail: [email protected]

some LHCI. But the accumulation of CP29 and CP26 and their binding to PSII core complexes, is relatively unaffected. As expected, the loss of antenna polypeptides is accompanied by a reduction of the size of large reaction-center complexes. Following in-vitro phosphorylation the number of phosphorylated proteins is greatly increased in the mutant thylakoids compared to wildtype thylakoids. We present a model of the PSII antenna system to account for the new polypeptide complexity we have demonstrated. Key words: Chlamydomonas (photosystem II) - Chlorophyll b deficiency - Chlorophyll-protein complex - Phosphorylation (light-harvesting antenna of PSII)

Introduction The light-harvesting chlorophyll pigments of higher plants and green algae are complexed with specific binding proteins embedded in the thylakoid membrane (see reviews by Staehelin 1986; Melis 1991). The reaction centers of photosystems I and II (PSI and PSII) bind chlorophyll a and carotenoids, and carry out the primary photochemistry of photosynthesis. Each photosystem has an associated antenna system which binds a mixture of chlorophylls a and b and xanthophylls. In Chlamydomonas reinhardtii, as in higher plants, there are five chlorophyll a/b-binding complexes in the membrane: LHCII, the major light-harvesting antenna for PSII; LHCI, the PSI antenna; and three minor PSII antenna complexes CP29, CP26 and CP24 (Bassi and Wollman 1991). The bulk of the chlorophyll b in the membrane is bound by LHCII, with a chlorophyll a/b ratio of 1.45 in C. reinhardtii. In this alga, it has been reported that LHCII contains four subunits in the molecular weight range 31 to 25 kDa, designated p11, p13, p16 and p17 (Bassi and Wollman 1991). The major apoproteins pl 1, p16 and p17 all have chloropyll a/b ratios below one. A minor component, p13, has a chlorophyll a/b ratio of 1.9. The LHCII

K.D. Allen and L.A. Staehelin: Photosystem II antenna in Chlamydomonas a p o p r o t e i n s n o r m a l l y a s s e m b l e i n t o a t r i m e r in the m e m b r a n e ( K i i h l b r a n d t a n d W a n g 1991), w h i c h reversibly a s o c i a t e s with P S I I . T h e c o m p l e x e s CP29 a n d CP26 h a v e e a c h been d e s c r i b e d in C. reinhardtii as h a v i n g single a p o p r o t e i n s , p9 a n d p l 0 , respectively, b o t h of w h i c h a r e l a r g e r t h a n the L H C I I a p o p r o t e i n s . A s in h i g h e r plants, b o t h of these c o m p l e x e s b i n d less c h l o r o p h y l l b t h a n d o e s L H C I I , w i t h a c h l o r o p h y l l a/b r a t i o of 3.0 for CP29, a n d 2.4 for CP26. B o t h CP29 a n d CP26 are t h o u g h t to be t i g h t l y b o u n d P S I I a n t e n n a e ( M o r r i s s e y et al. 1989; Bassi a n d D a i n e s e 1990). T h e c o m p l e x C P 2 4 is a m i n o r P S I I a n t e n n a c o m p o n e n t , with a c h l o r o p h y l l a/b r a t i o of 2.2 in C. reinhardtii (Bassi a n d W o l l m a n 1991). Chlamydomonas reinhardtii L H C I has a c h l o r o p h y l l a/b r a t i o of a b o u t 4.0 ( H e r r i n et al. 1987) a n d c o m p r i s e s as m a n y as ten subunits in the 20- to 3 1 - k D a range, at least three of which b i n d c h l o r o p h y l l s a a n d b (Bassi et al. 1992). O u r w o r k with s p i n a c h s h o w e d t h a t the p o l y p e p t i d e c o m p o s i t i o n of the P S I I a n t e n n a s y s t e m is m u c h m o r e c o m p l e x t h a n p r e v i o u s l y t h o u g h t (Allen a n d Staehelin 1992). This led us to r e - e x a m i n e the p o l y p e p t i d e c o m p o sition of the C. reinhardtii P S I I a n t e n n a system. S u b s t a n tial p r o g r e s s has been m a d e in u n d e r s t a n d i n g the m o l e c u l a r aspects of the gene families t h a t e n c o d e the l i g h t - h a r vesting p o l y p e p t i d e s of P S I I ( L h c b l - 6 , J a n s s o n et al. 1992, see also D e m m i n 1989; P i e c h u l l a et al. 1991 a n d K e l l m a n n et al. 1993). But we are j u s t b e g i n n i n g to relate the m u l t i p l i c i t y of these genes to the b a n d s r e s o l v e d b y S D S - P A G E ( G r e e n et al. 1992; Sigrist a n d Staehelin 1992). This is i m p o r t a n t b o t h to i n t e r p r e t the m o l e c u l a r d a t a a n d to e l u c i d a t e f u n c t i o n a l differences b e t w e e n l i g h t - h a r v e s t i n g p o l y p e p t i d e s . This s t u d y was u n d e r t a k e n first to p r o v i d e a m o r e d e t a i l e d a n a l y s i s of the a n t e n n a p o l y p e p t i d e s of C. reinhardtii b y m e a n s of a very high r e s o l u t i o n n o n - T r i s S D S - P A G E s y s t e m in c o n j u n c t i o n with i m m u n o b l o t t i n g . U s i n g this system we are a b l e to d i s t i n g u i s h p o l y p e p t i d e s differing in a p p a r e n t m o l e c u l a r weight b y as little as 200 D a . N e x t we e x a m i n e d the accum u l a t i o n a n d a s s e m b l y of c h l o r o p h y l l - p r o t e i n c o m p l e x e s in a c h l o r o p h y l l b-less m u t a n t c b n l - 4 8 (Steinmetz 1990, a n d references therein). Lastly, we s o u g h t to differentiate between light-harvesting polypeptides based on in-vitro phosphorylation.

Materials and methods Cultures and growth conditions. Cultures of Chlamydomonas reinhardtii Dang. wild-type strain 137c and the chlorophyll b-less mutant cbnl-48 were obtained from the Chlamydomonas Genetics Center (Duke University, Durham, NC, USA). Liquid cultures were grown mixotrophically in medium described by Holmes and Dutcher (1989). This is a mineral medium with sodium acetate as a carbon source. Cultures were grown with stirring under continuous illumination (300 ~tmol photons.m 2.s-l) from fluorescent lamps.

Preparation of thylakoid membranes. Cultures of C. reinhardtii were harvested in late logarithmic phase (5-106 cell/ml) by centrifugation. Pelleted cells were resuspended in a buffer containing 50 mM TrisHC1 (pH 7.0), 10 mM NaC1, 5 mM MgCI2, and protease inhibitors, 5 mM z-amino caproic acid and 1 mM benzamidine HC1. Cells were disrupted by two passages through a chilled French pressure cell (Aminco Instruments, Silver Spring, Md., USA) operated at

43

28 MPa. Whole cells and large debris were pelleted with a brief, low-speed spin, and membranes were pelleted at 5000.g for 10 min, 4~ The membrane pellet was washed twice with the same buffer. The resulting crude pellet, which contained primarily thylakoid membranes, was washed twice (12 000-g, 10 min, 4~ SS34 rotor; Sorvall, Newton, Conn., USA) in Hepes-EDTA [5 mM Hepes (pH 7.5), 10 mM EDTA, and protease inhibitors]. This pellet was resuspended with a Teflon homogenizer in 1.8 M sucrose in the same buffer, loaded into Beckman SW27 ultracentrifuge tubes, and overlaid with a layer of 1.3 M sucrose and a layer of 0.8 M sucrose in the same buffer. Step gradients were spun at 27000rpm (132 000-g max.) for 1.5 h, following which the 1.3 M sucrose layer, now containing the bulk of the chlorophyll, was collected. Sucrose was removed by washing in Hepes-EDTA buffer. Pellets were resuspended in the same buffer containing 10%o glycerol, frozen in liquid nitrogen, and stored at -80~

Native green gel electrophoresis. Native green gel electrophoresis was carried out as described by Allen and Staehelin (1991). Washed membrane pellets were solubilized in 0.45% octyl glucoside, 0.45% decyl maltoside, 0.1% lithium dodecyl sulfate, 10%o glycerol, and 2 mM Tris maleate (pH 7.0). Solubilization buffer was added to yield a ratio of total nonionic detergent to chlorophyll of 20:1 (w/w). Gels were run in precooled (about 4~ minigel apparatus (Hoefer Scientific Instruments, San Francisco, Cal., USA). Pigment determinations were performed on a model 330 spectrophotometer (Perkin Elmer, Norwalk, Conn., USA), in dimethyl formamide, using the simultaneous equations of Porra et al. (1989). Fully-denaturing SDS-PAGE. Fully denaturing SDS-PAGE was carried out using a modification of the ammediol (2-amino-2methyl-l,3-propanediol) buffer system (Bothe et al. 1985 and references therein). We have found that, regardless of gel format, this buffer system substantially increases the resolution of thylakoid polypeptides. Samples were run on 0.75-mm thick, 30-cm-long gels, containing 18 % acrylamide at an acrylamide to bisacrylamide ratio of 90:1. Because bromophenol blue runs well ahead of the chlorophyll front in this gel system, malachite green was used as a dye marker. When all proteins below about 20 kDa were to be run off the bottom of the gel, congo red, which runs with an apparent molecular weight of about 30 kDa in this system, was used. Gels were stained using the low-background silver-stain protocol described by Blum et al. (1987). For two-dimensional gels, 1.5-mm-thick strips were excised from green gel lanes, incubated for 15 min at 55~ in solubilization buffer containing 1X stacking gel buffer, 2% sodium dodecyl sulfate, 2%o 13-mercaptoethanol, and 10%o glycerol. Treated gel strips were loaded directly onto the stacking gel of a 1.5-mm gel containing 18% polyacrylamide with an acrylamide to bis ratio of 90:1. For immunoblots, proteins were transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, Bedford, Mass., USA). Membranes were blocked with nonfat dry milk, decorated with primary antisera, stained with goat anti-rabbit horseradish peroxidase conjugate secondary antibodies (BioRad Laboratories, Richmond, Cal., USA) and visualized according to the BioRad protocol. Phosphorylation experiments. Thylakoids were suspended at a chlorophyll concentration of 200 I~g per ml in 50 mM Tris-HC1 (pH 8.0), 100 mM sorbitol, 10 mM NaC1, 5 m M MgCI2, 4 mM ATP with 37 MBq per ml y-[32p]ATP (New England Nuclear, Wilmington, Del., USA). Samples were illuminated at 250 lamol photons.m 2's 1 for 15 min at room temperature with illumination from a fiber-optic light source equipped with heat filters (Schott Glaswerk, Wiesbaden, Germany). At the end of the illumination period, NaF was added to a concentration of 5 mM. Phosphorylated thylakoids were washed twice in 20 mM Tris-HC1 (pH 7.0), 5 mM NaF. Aliquots were solubilized and subjected to fully denaturing SDSPAGE using the ammediol buffer system already described. Autoradiography was carried out according to standard procedures.

44

K.D. Allen and L.A. Staehelin: Photosystem II antenna in Chlamydomonas

Results

Polypeptide compositions. The first phase of this study was a general re-examination of Chlamydomonas reinhardtii thylakoid polypeptide composition using a very high resolution non-Tris SDS-PAGE system. Figure 1 shows a comparison of thylakoid polypeptide compositions of the wild-type (strain 137c) and the chlorophyll b-less cbnl-48 mutant, as well as an isolated PSI-LHCI complex from the wild-type prepared by excising a band from a native green gel (Allen and Staehelin 1991). Some of the major landmarks of the polypeptide pattern, as identified by immunoblot and fractionation experiments, are indicated. The identification of all of the minor bands resolved with this gel system is beyond the scope of this paper, but it is clear that many of the thylakoid components previously thought to consist of single polypeptides are actually composed of several polypeptides. The PSI-LHCI complex, shown here for reference, contains, in addition to the diffusely staining P700 apoproteins at about 55 kDa, 13 main bands in the region from 27.5 kDa to 8.5 kDa (Fig. 1). Our LHCI antisera, prepared against spinach LHCI, did not cross-react to any of the polypeptides in this region. Nevertheless, we have been able to tentatively assign some of the PSI and L H C I subunits on the basis of molecular weight and fractionation experiments. The 27.5-kDa band is p14 of LHCI (Herrin et al. 1987). One or perhaps all of the triplet at 26.5, 26.25, and 26.0 kDa corresponds to p15 of LHCI (Herrin et al. 1987). The triplet at 24.75, 24.5 and 24.0 kDa most likely corresponds to LHCI p17.2 (Herrin et at. 1987; Bassi et al. 1992). On more heavily loaded gels, we also resolve subunits at 17, 13, 9.5, and 8.5 kDa. Only the 13-kDa subunit is visible in this photograph. On 30-cm gels on which proteins below about 20 kDa have been run off the bottom of the gel, we resolve ten L H C I I apoproteins (Fig. 2). Thus each of the four previously described L H C I I apoproteins, p l l , p13, p16 and p17 may be resolved into multiple bands. We also resolve the apoproteins of CP29 and CP26 (p9 and pl0, Bassi and Wollman 1991) into multiple bands. Analyses of the apoproteins of CP29, CP26 and LHCII are summarized in Table 1. Figure 2 also shows immunoblots stained with antisera raised against spinach LHCII, spinach CP29 (Dunahay and Staehelin 1987) and anti-peptide antibodies raised against the synthetic peptide sequence, EDRPKYLGPFSEQTPS, corresponding to an N-terminal portion of a type-2 petunia LHCII gene (Petunia Lhcb2*l; Sigrist and Staehelin 1992). CablI-1, the only cab gene reported to date for C. reinhardtii (Imbault et al. 1988), contains an analogous sequence that is identical at 9 out of these 16 amino acids. Anti-LHCII antisera recognize a triplet at 31.2, 31.0, and 30.7 kDa (these bands collectively comprise p l l described in earlier studies; Chua and Blomberg 1979; Delepelaire and Chua 1981), a doublet at 26.7 and 26.5 kDa (which comprise p16), and a triplet at 26.0, 25.7, and 25.5 kDa (which comprise p17). Note that the pl 1 triplet is difficult to discern in the photographic reproductions of these gels. By examination of the original gels through multiple repetitions of the experiment we have determined that there are at least three

Fig. 1. Polypeptide composition of thylakoid membranes of Chlamydomonas reinhardtii. Samples from the wild-type (137c), a chlorophyll b-less mutant (cbnl-48), and an isolated wild-type photosystem I complex (PSI-LHCI). Samples were separated on a 30cm gel and visualizedwith silver staining. Dots next to the PSI-LHCI lane indicate, from top to bottom, p14 (27.5 kDa), p15 (triplet at 26.51 26.25, and 26.0 kDa), p17.2 ((24.75, 24.5, and 24.0 kDa), and p22 (20.0 and 19.5kDa). Positions of soluble molecular-weight markers (kDa) are indicated


45

Fig. 2. The LHCII polypeptides of C. reinhardtii thylakoid membranes. Portion of a 30-cm gel on which all proteins below about 20 kDa have been run off the bottom in order to expand the LHCII region. The first panel shows the silverstain pattern. The remaining three panels are immunoblots from similar gels, with antiserum type shown at the bottom of each panel. Note that the anti-CP29 blot is a second stain on the same membrane shown in the third panel. The bands at 26.7 and 26.5 kDa are not recognized by the anti-CP29 antiserum. Dots in the second and fourth panels indicate the position of the novel 31.7-kDa band enriched in cbnl-48 thylakoids. Apparent molecular weights of the apoproteins of LHCII, CP29 and CP26, are indicated along the left edge. WE, wild type; cbn148, chlorophyll b-less mutant

Table 1. Properties of PSII antenna polypeptides in Chlamydomonas a Mr

Designation

Immunoreactivity anti-LHCII

34.5 34.2 34.0 33.0 31.7 31.2 31.0 30.7 28.5 28.2 26.7 26.5 26.0 25.7 25.5

p9

(CP29)

pl0

(CP26)

~

-~p13 -qpl6 j p17

++ -]-

++ ++ ++

pll

(LHCII)

anti-Type 2

Present in : anti CP29

137c

cbnl-48

137c

++ ++ ++ _+ +

+ + + +

+ + + + + _+ __ _+

+ + + __+

+ •

-]-

++ ++ + + +

Phosphorylation

++ ++

+ + + + + + + + + +

cbnl-48

++ ++ ++ ++ ++

a Blank space indicates no reaction or polypeptide not present

a n t i - L H C I I i m m u n o r e a c t i v e b a n d s in this region. It s h o u l d be b o r n e in mind, however, t h a t there m a y be m o r e p o l y p e p t i d e s in the p l 1 r e g i o n t h a t we are n o t yet a b l e to resolve. T h e d o u b l e t at 28.5 a n d 28.2 k D a corres p o n d s to the L H C I I c o m p o n e n t p13 ( D e l e p e l a i r e a n d C h u a 1981; Bassi a n d W o l l m a n 1991). O n m o r e h e a v i l y

l o a d e d gels, the p13 p o l y p e p t i d e s r e a c t w e a k l y with a n t i L H C I I a n t i s e r a (see below). T h e a n t i - t y p e 2 p e p t i d e a n t i s e r u m recognizes the p16 d o u b l e t , a n d , m o r e w e a k l y , the h i g h e r - m o l e c u l a r - w e i g h t p l 1 b a n d s . Bassi a n d W o l l m a n (1991) h a v e s h o w n t h a t each of the four g r o u p s o f L H C I I a p o p r o t e i n s (i.e. p l 1, p13, p16 a n d p17) b i n d s c h l o r o p h y l l

46


b, but in that study each of these resolved as only a single band. Hence it is not clear whether all or only some of the 10 L H C I I apoproteins that we have resolved bind chlorophyll b. We show here that each of the four previously described polypeptides may be resolved into multiple bands, and that all of these component bands are LHCII-related based on immunological similarity. The anti-CP29 antiserum recognizes five bands at 34.5, 34.2, 34, and a doublet at 33.0 kDa. The three largest of these are the apoproteins of CP29 (p9), whereas the 33.0-kDa doublet is the CP26 apoproteins (pl0, Bassi and Wollman 1991).

Polypeptide changes in a chlorphyll b-less mutant. In cbnl48 thylakoids, the loss of chlorophyll b is correlated with the loss of some, but not all of the chlorophyll a/b-binding proteins of PSI and PSII. These polypeptide changes are summarized in Table 1. Some, but apparently not all L H C I is lost in the mutant. The triplet at 24.75, 24.5 and 24.0 kDa seen in the PSI-LHCI lane of Fig. 1 most likely corresponds to L H C I p17.2 (Herrin et al. 1987). None of these bands are decreased in the mutant. P22, a doublet here at 20 and 19.5 kDa, is also not changed in the mutant. In contrast, p15.1 of LHCI, a doublet here at 26.25 and 26.0 kDa is missing in cbnl-48. The L H C I component p14, at 27.5 k D a of is also missing in the mutant. There is a dramatic reduction in the levels of LHCII in cbnl-48 thylakoids. Only the p l l polypeptides are still detectable on immunoblots. In contrast, accumulation of the CP29 polypeptides in the membrane is not affected in cbnl-48, and CP26 is only slightly reduced. Thus while some LHCI, and virtually all L H C I I is lost in the mutant, the accumulation of CP29 and CP26, which bind appreciably less chlorophyll b than does LHCII, is independent. In addition to the polypeptide losses, several novel bands occur in cbnl-48 thylakoids. Note that both the anti-LHCII and the anti-CP29 antisera recognize a 31.7kDa polypeptide (marked with dots in Fig. 2) that is enriched in cbnl-48 thylakoids. The anti-LHCII antiserum also recognizes a faint 15.5-kDa band in the mutant that is not present in the wild type (data not shown). Chlorophyll-protein complexes. The second goal of this study was to re-examine C. reinhardtii chlorophyllprotein complexes using a new native green gel system (Allen and Staehelin 1991). Figure 3 shows an unstained green gel (upper panel) and a long-wavelength UV-fluorescence image of this same gel (lower panel). Because the PSI reaction center is a more efficient sink for incoming excitation energy than is the PSII reaction center, PSI complexes are non-fluorescent at room temperature, whereas PSII complexes appear very faintly fluorescent. Light-harvesting antenna complexes not associated with reaction centers are highly fluorescent. A description of the pattern of chlorophyll-protein complexes resolved with this system has been given elsewhere (Allen and Staehelin 1991). Briefly, the wild-type pattern is divided into five zones (Fig. 3): RC-LHC, containing PSI-LHCI and PSII-LHCII complexes (including the low-mobility bands marked with an asterisk in Fig. 3); RC-Core, containing smaller PSII and PSI reac-

Fig. 3. Non-denaturing green gel electrophoresis. The upper panel shows the unstained green band pattern for chlorophyll-protein complexes from wild-type and cbnl-48 C. reinhardtii. The lower panel is a fluorescencepicture of this same gel on a long-wavelength UV transilluminator. See text for explanation of labels

tion-center core complexes; LHCII*, containing trimeric L H C I I complexes; SC, or small complexes, containing a number of small PSII-related complexes as well as some L H C I I material; and the free-pigment zone (FP). The free-pigment zone, which is largely carotenoid, typically contains about 5% of the total pigment. Multiple PSIL H C I and PSII-LHCII complexes are resolved, and are frequently interdigitated with each other. Molecular weights of chlorophyll-protein complexes were estimated using calibrated sucrose gradients with gel-filtration markers as standards, followed by green-gel analysis. We estimate the molecular weight of the LHCII* region at 110-200 kDa. The lower end of this range (i.e. LHCII*D) corresponds reasonably well with an estimated molecular weight of an LHCII* trimer of 124 kDa (based on an average polypeptide of 28 kDa and 15 chlorophylls per monomer, Butler and Kfihlbrandt 1988). The RC-Core region ranges from 200 to 400 kDa. This is the expected size range for the PSII-Core complexes and CP1 complexes that are found in this region. The molecular weight of an intact C. reinhardtii PSII complex with 50 bound chlorophylls has been estimated at 274 kDa in the absence of detergent and lipid, and up to 430 kDa in the presence of a lipid/detergent micelle, depending on the amount of associated detergent (De Vitry et al. 1991). A CPI complex, containing just the P700 apoproteins and attached pigments and cofactors, should be about 200 kDa. The R C - L H C region is from 400 kDa up to about 1200 kDa. A large PSII-LHCII


47

Fig. 4A-D. Two-dimensional SDS-PAGE analysis. This is an overview of polypeptide composition of chlorophyll-protein complexes from thylakoids of wild-type and cbnl-48 C. reinhardtii. First-dimension gels were similar those shown in Fig. 3. The second-dimension gels were 1.5 mm thick, to accommodate green gel strips, and 15 cm in length. Dotted lines indicate the origin of the cbnl-48 green gel dimension in each panel. A Silver stain. The apoproteins of CP47, D1 and D2 are indicated. CPI is a pigmented dimer of the P700 apoproteins of PSI. Arrowheads indicate PSI subunits in the cbnl-48 pattern. B Immunoblot stained with anti-LHCII antiserum. C Immunoblot stained with anti-type 2 LHCII antiserum. D Immunoblot stained with anti-CP29 antiserum. P9 and pl0 are the apoproteins of CP29 and CP26, respectively. Positions of soluble molecular-weight markers are indicated. Arrowheads in panels B and D indicate novel spots enriched in cbnl-48 thylakoids

complex would come out to more than 700 kDa. The group of bands labelled with an asterisk in Fig. 3 are in the range of 800 to 1200 kDa. Most of these bands are P S I I - L H C I I complexes, although there are also several large P S I - L H C I complexes in this region. These complexes are substantially larger than would be expected for monomeric P S I I - L H C I I complexes, and may correspond to the PSII-dimers observed by other workers (Peter and Thornber 1991; Bassi et al. 1992). The most striking difference in the mutant chlorophyll-protein complex pattern is the complete loss of the highly fluorescent L H C I I * complexes (corresponding to the trimeric form of the L H C I I antenna of PSII). In the wild type, L H C I I * is resolved into four green bands, all of which are missing in the mutant. This indicates that any residual L H C I I in the mutant either does not assemble into trimers, or fails to do so stably enough to survive solubilization and electrophoresis. Several partial PSII core complexes, all containing some CP29, are observed in cbnl-48 thylakoids. Some of these complexes are shifted down into what would be the L H C I I * region (Fig. 4D). In the mutant the SC band contains CP29,

CP26 and the residual p l l apoproteins, as well as several bands in the 45- to35-kDa range.

Assembly of chlorophyll-protein complexes. Assembly of thylakoid components can be assayed by two-dimensional (2D) electrophoresis with a native green gel in the first dimension and fully denaturing S D S - P A G E in the second (Fig. 4A). These second-dimension gels show an arc corresponding to material that ran as monomers in the green gel dimension. Anything that runs below this arc in the 2D pattern ran as part of a complex in the first dimension. Photosystem II complexes can be identied in the 2D pattern by the presence of the apoproteins of CP47, CP43, D1 and D2. Most of the P700 apoproteins ran as pigmented CPI on the second-dimension gel. The positions of the P S I - L H C I complexes are best judged by the positions of the PSI subunits in the range of 27-8.5 kDa. Note that the core subunits of PSI and PSII are more heavily loaded in the cbnl-48 pattern because the first-dimension green gels are loaded on a constant-chlorophyll basis. Because of the loss of the chlorophyll a/b binding proteins, which account for up to 50% of both total

48


chlorophyll and total protein in the membrane, the core reaction-center proteins appear to be disproportionately represented. This will be true whether samples are loaded on a constant-protein or on a constant-chlorophyll basis. The first major difference between the wild-type and the mutant 2D patterns is, as observed on the green gels in Fig. 3, the complete loss of trimeric LHCII. The second major change is the shift of PSI-LHCI and PSII-LHCII complexes to faster-migrating PSI and PSI! core complexes, coincident with the loss of bound antennae. This shift in mobility confirms that the LHCII in the upper parts of the gel is in fact connected to PSII complexes. A number of very large PSII-Core complexes are present in the cbnl-48 chlorophyll-protein complex pattern. These complexes are smaller than the PSII-LHCII complexes found in the wild type, but are still much larger than would be expected for PSII monomers. Based on size, then, these complexes appear to be PSII dimers, indicating that dimerization may occur in the absence of LHCII. Figure 4B D shows immunoblots of gels similar to that shown in Fig. 4A stained with antisera directed against spinach L H C I I (Fig. 4B), anti-type 2 LHCII (Fig. 4C), and CP29 (Fig. 4D). The anti-LHCII antiserum reacts primarily with p l l , p16 and p17, but at this loading (which is heavier than that in Fig. 2) there is also easily detectable cross-reactivity to the LHCII component p13, the CP26 apoproteins (pl0), and faintly to the CP29 apoproteins (p9). This immunoblot confirms that trimeric LHCII* is completely absent in the mutant, although there is some residual staining around 31 kDa (pl 1) in the small-complex region of the mutant. More interestingly, the mutant is enriched for several novel spots, running between pl0 and pl 1, that are absent in the wild type (arrowhead, Fig. 4B). These novel spots are the same apparent molecular weight as the novel band detected on the immunoblots in Fig. 2. The heavy loading on the anti-type 2 LHCII immunoblot (Fig. 4C) makes the labelling of p l l and p16 appear equivalent, due to saturation of the peroxidase reaction used to visualize antibody binding (compare with Fig. 2). In Fig. 4C, the anti-type 2 LHCII antibodies are seen to bind to the pl 1 and p16 polypeptides, but not to pl0 (CP26) or the p17 polypeptides. The p13 polypeptide is very weakly recognized by these antisera, and is visible in Fig. 4C because of the relatively heavy protein loading on these gels. All of these polypeptides are recognized by the general-purpose anti-LHCII antiserum used in Fig. 4B. It is interesting that the distribution of Type 2 immunoreactive material roughly matches that of the 'bulk' LHCII (compare Fig. 4B and 4C). This indicates that there is not a preferential distribution of type 2 LHCII, relative to bulk LHCII, among the chlorophyll-protein complexes resolved in the first-dimension green gel. There is very little detectable LHCII in the mutant, with the only staining being in the SC region. The anti-CP29 antiserum (Fig. 4D) primarily labels the CP29 polypeptides (p9), but there is also some crossreactivity to the CP26 doublet (pl0) and to the novel spots enriched in the mutant. Not only are the levels of CP29 not changed in the mutant, but the distribution of CP29 among various complexes is relatively unchanged

Fig. 5. In-vitro phosphorylation of thylakoid proteins from wild-

type and cbnl-48 C. reinhardii. Labelled samples were separated on a 30-cm gel. Positions of soluble molecular-weightmarkers are indicated. S, silver stain; A, autoradiogram

K.D. Allen and L.A. Staehelin: Photosystem II antenna in Chlamydomonas by the loss of the LHCII apoproteins, demonstrating that L H C I I is not required for the assembly of CP29 into any of the large PSI! complexes resolved here. The SC band contains CP29 and CP26. These appear to be a complex because they are shifted to the left of the arc, but it must be a dimeric rather than trimeric combination because the complex migrates substantially ahead of LHCII*D. In-vitro phosphorylation. Phosphorylation experiments were undertaken to improve our knowledge of C. reinhardtii phosphoproteins by taking advantage of the enhanced resolution of the ammediol gel system. Figure 5 shows the in-vitro phosphorylation pattern of wild-type and cbnl-48 thylakoids. In broad detail, the wild-type pattern is essentially the same as has been previously described for this species (Owens and Ohad 1982; Delepelaire and Wollman 1985; Ikeuchi et al. 1987). The CP29 polypeptides, which run as a single band on this gel, are highly labelled, as are p l l , p13 and p17 of LHCII. In contrast, CP26 is only lightly labelled, and the p16 LHCII polypeptide is virtually unlabelled. The broad, diffuse labelling above CP29 is a PSII component (Ikeuchi et al. 1987). In cbnl-48, the general spectrum of in-vitro kinase activity is greatly increased, perhaps as a result of the

49

absence of its major substrates, the LHCII apoproteins. CP29 is still labelled in the mutant, CP26 is more highly labelled in the mutant than in the wild type, and the residual p l l bands (barely visible in the anti-LHCII Western in Fig. 2) are also labelled. But the most striking labelling is of the P700 apoproteins, at about 55 kDa, and a 15.5-kDa band that is barely visible on the silverstained gel. The 15.5-kDa band accounts for about 40% of the total phosphate incorporation in cbnl-48 thylakoids. Using anti-LHCII antibodies, this band is detectable on immunoblots in the mutant but not in the wild type, suggesting that it may represent an L H C I I breakdown product that is an excellent substrate for the kinase. Phosphorylation of the PSII antenna apoproteins is quite heterogeneous, with some bands not labelling at all (Fig. 6). All three of the CP29 (34.5, 34.2 and 34.0 kDa) apoproteins are labelled. Only the bottom band of the 35-kDa doublet is labelled (determined by aligning the autoradiogram with the dried gel). This doublet does not label with any of the antisera used in this study (not shown), and so would appear to be a new phosphoprotein. CP26 (33 kDa) is faintly labelled in the wild type but heavily labelled in the mutant. Of the L H C I I polypeptides, the pl 1 group, resolved as a doublet here, has just the bottom band labelled (30.7 kDa). In the 28.5/ 28.2 kDa doublet (p13) only the 28.2-kDa band is labelled. P16 (26.7 and 26.5 kDa) is not labelled at all. Finally, all the bands in the p17 group (26.0, 25.7 and 25.5 kDa, note that the 25.7- and 25.5-kDa bands are not resolved on this gel) appear to be labelled. The phosphorylation results are summarized in Table 1.

Discussion

Fig. 6. In-vitro phosphorylation of LHCII apoproteins from wildtype (W/) and cbnl-48 C. reinhardtii. This is the LHCII region of an SDS-PAGE gel similar to that shown in Fig. 2. Proteins below about 20 kDa were run off the bottom of the gel in order to expand the LHCII region. Apparent molecular weights of components of LHCII, CP29, and CP26, are indicated along the left-hand margin

Complexity of the PSII antenna system. The LHCII antenna carries out a variety of functions in the thylakoid membrane. In addition to a light-harvesting role, L H C I I is involved in the formation and maintenance of grana stacks (and thus of lateral heterogeneity of the thylakoid membrane) and in the regulation of energy distribution between the two photosystems (Staehelin 1986). This functional complexity is mirrored by the complex gene family encoding LHCII and the other light-harvesting polypeptides (Green et al. 1991). In tomato, for which the most complete molecular description is available, thirteen PSII-antenna gene sequences and six PSI-antenna gene sequences are now available (Green et al. 1992). All of these are expressed in photosynthetic tissues, but steady-state m R N A levels for individual transcripts vary by more than 20-fold (Kellmann et al. 1993). The fact that all of these genes are expressed at some level implies that all are necessary for efficient light harvesting. But the variation in expression levels suggests different functional requirements for individual polypeptides. One indication of functional heterogeneity of L H C I I polypeptides is the fact that only some are phosphorylated (Figs. 5, 6; Islam 1987). Further, only a subset of phosphorylated L H C I I migrates from grana to stroma membrane domains during the state transition (Islam 1987; Larsson et al. 1987).

50


The one LHCII sequence reported to date for C. reinhardtii (Imbault et al. 1988) shares features of both type 1 and type 2 sequences. Thus the type 1/type 2 distinction may or may not apply readily in C. reinhardtii. That the C. reinhardtii LHCII polypeptides differ significantly from their higher-plant counterparts is indicated by the fact that most of our anti-peptide antibodies that distinguish type 1 and type 2 polypeptides in higher plants (Sigrist and Staehelin 1992) do not react with C. reinhardtii polypeptides. However, the anti-type 2 antisera shown in Figs. 2 and 4 did preferentially stain the p16 group, with weaker staining of the pl 1 bands, and no staining of the pl7 bands. Interestingly, in higher plants the type 2 LHCII polypeptides are generally the most highly phosphorylated, and migrate preferentially during the state transition. Yet the C. reinhardtii bands most strongly labelled by the anti-type 2 antisera appear not to be phosphorylated at all (Figs. 5, 6). We interpret this as an indication that the C. reinhardtii light-harvesting polypeptides may need to be classified differently than the higher-plant counterparts. Thus, more gene- and protein-sequence data are required. The biochemical description provided in this paper should facilitate the acquisition of protein-sequence data.

Differential stability of chlorophyll a/b-binding proteins. To better understand the organization and accumulation of the PSII antenna system in the thylakoid membrane, we have used a chlorophyll b-less mutant that we predicted would be missing some or all of these polypeptides. The cbnl-48 mutant was originally described as containing no detectable chlorophyll b (chlorophyll a/b ratio > 1000, as opposed to about 2.0 in the wild type, Chunayev et al. 1990, and references therein) and reduced levels of neoxanthin, a xanthophyll pigment normally associated with LHCII. These authors proposed, partly on the basis of chlorophyll-precursor measurements, that the wildtype cbn-1 gene encodes the enzyme that forms chlorophyllide b (Chunayev 1990, and references therein). Other workers have confirmed the absence of chlorophyll b and the reduction in neoxanthin content, and further shown that the level of lutein is reduced, violaxanthin is enriched, [3-carotene levels remain unchanged, but ~carotene, normally a minor component, is increased more than 30-fold (Steinmetz et al. 1990). The loss of chlorophyll b in cbn 1-48 is correlated with a dramatic reduction in the accumulation of LHCII and at least some of the L H C I apoproteins. Loss of the chlorophyll a/b-binding proteins is a common, although not universal, feature of chlorophyll b-less mutants. In the well studied chlorina f2 b-less barley mutant, it has been shown that the failure of the LHC apoproteins to accumulate in the thylakoid membrane is not due to failure of transcription or translation of Lhca/Lhcb mRNA, import into the chloroplast, or thylakoid insertion. Rather it appears that the L H C apoproteins are rapidly degraded after thylakoid insertion, presumably because of inability to reach a stable folding conformation in the absence of chlorophyll b (Bellemare et al. 1982). A requirement for chlorophyll b in producing the final folding conformation of LHCII seems reasonable since roughly

half of the 15 chlorophylls bound by an individual LHCII apoprotein are chlorophyll b (Butler and Kiihlbrandt 1988), and that these chlorophylls are bound at specific sites in the protein structure (K/ihlbrant and Wang 1991). That LHCII turnover is occurring in cbnl48, in a fashion similar to that in chlorina f2, is suggested by the detection of a novel low-molecular-weight band in cbnl-48 thylakoids, immunologically related to mature LHCII, that is not found in the wild type (data not shown). This band may represent an LHCII degradation product. Although the loss of chlorophyll b is correlated with the loss of most LHCII and at least some LHCI apoproteins in cbnl-48, there is not a complete loss of the entire range of chlorophyll a/b-binding proteins. The complexes CP29 and CP26 are largely unaffected by the cbnl-48 mutation. One possible explanation is that since CP29 and CP26 have a much lower chlorophyll b content (chlorophyll a/b ratios of 3.0 and 2.4, respectively, Bassi and Wollman 1991) than LHCII (generally around 1.0, Bassi and Wollman 1991), these complexes are less sensitive to the loss of chlorophyll b. It should also be noted that the effect on individual LHCII apoproteins is not uniform. The apoproteins of p l 3, p l 6, and p l 7 are essentially undetectable, whereas the p l l apoproteins are present in very small amounts. This kind of behaviour has also been seen in other chlorophyll b-less mutants of C. reinhardtii (De Vitry and Wollman 1988).

Assembly of chlorophyll-protein complexes. The decreased size of PSI and PSII complexes following the loss of bound antenna (Figs. 3, 4) was expected based on a variety of studies, including freeze-fracture examinations, with other photosynthetic mutants (Simpson 1986; Staehelin 1986; Allen et al. 1988). While a decrease in the size of the PSI complex, coincident with the loss of bound LHCI, has been observed with lower-resolution green gel techniques for other chlorophyll b-less mutants (Bassi et al. 1985; Allen et al. 1988), intact PSII-LHCII complexes are not retained in most native green gel systems, and so this shift has not previously been observed. The shift of the large PSII-LHCII complexes observed in the wild type (asterisk in Fig. 3) to smaller PSII-Core complexes in the mutant, indicates that LHCII is in fact bound to these complexes. The large PSII-LHCII complexes observed in the wild type probably correspond to the 18-nm EFs particles seen in freeze-fracture experiments (Staehelin 1986; Simpson 1986; Allen et al. 1988). The components of CP29 and CP26 remain attached to the PSII reaction center even following the complete loss of bound LHCII from these complexes (Fig. 4). This indicates that CP29 and CP26 do not require LHCII in order to bind to the reaction-center particle, and that they are located close to the reaction-center core (Morrissey et al. 1989; Bassi and Dainese 1990). Recently a complex of CP29, CP24 and LHCII has been isolated following isoelectric focussing of solubilized thylakoid components (Bassi et al. 1992), and it was suggested that this constitutes an independent entity in the membrane. Our results support the idea that CP29 and CP26 are parts of a PSII core complex to which LHCII attaches. Given that the three

K.D. Allen and L.A. Staehelin: Photosystem II antenna in Chlamydomonas minor PSII antenna components CP29, CP26 and CP24 are all proposed to bind specifically to LHCII, it is not surprising to find complexes of each of these with LHCII in solubilized fractions. The presence of large, apparently dimeric PSII-LHCII complexes in the wild-type samples is consistent with other biochemical evidence favoring PSI! dimerization (Peter and Thornber 1991; Bassi and Dainese 1992). De Vitry and coworkers (1991) found that in solution the predominant form of the C. reinhardtii reaction center was a monomer, with a small amount of dimer also present. Freeze-etch observations of tris-washed membranes (a treatment that removes the extrinsic OEE proteins) suggest a dimeric structure to the higher-plant PSII complex (Seibert et al. 1987; Bassi et al. 1989). There may be a similarity here to cyanobacteria in which dimeric PSII centers have also been observed by freeze fracture/freeze etch microscopy (Giddings et al. 1983) or by negative staining of isolated, solubilized reaction centers (Dekker et al. 1988). The presence of dimeric PSII centers in cbnl-48 thylakoids, even though LHCII is virtually undetectable, argues that dimerization cannot be mediated by LHCII. Thus, the apparent PSII dimerization observed here must be an interaction between core complexes. It is not clear from our study, or from previous biochemical observations of PSII dimerization (Bassi et al. 1989; Peter and Thornber 1991) whether this has any significance in vivo. Altered in-vitro phosphorylation of thylakoid polypeptides. Phosphorylation of thylakoid proteins plays an important role in the regulation of photosynthetic electron transport and photosynthesis (Allen 1992). Among the kinases responsible for the phosphorylation reactions, the redox-controlled kinase that phosphorylates primarily LHCII polypeptides and thereby controls State 1State 2 transitions has been most closely examined (Staehelin and Arntzen 1983; Allen 1992). The in-vitro light regulation of this kinase has been shown to be mediated by the binding of reduced quinone molecules to cytochrome b6/f complex (see references in Allen 1992), which occurs when PSII turns-over more rapidly than PSI due to a greater number of light-harvesting chlorophylls associated with PSII. This raises the interesting question of whether the redox-controlled kinase can be light-activated in mutants devoid of LHCII, since such mutants lack the primary substrate for the kinase, and have a radically altered antenna composition. In the case of the chlorophyll b-less chlorina f2 mutant of barley, which also lacks LHCII and LHCI, and which has 65% of its chlorophyll associated with PSI, no light-activation of the kinase is observed in the presence of ATP (Bhalla and Bennett 1987). These authors concluded that inability to light-activate the kinase results from PSI being permanently overstimulated with respect to PSII. But the kinase is activatable in BF3, a C. reinhardtii chlorophyll b-less mutant that is missing most LHCII (De Vitry and Wollman 1988), as well as in the chlorophyll b-less pg113 mutant of C. reinhardtii (Michel et al. 1983). These previous findings suggested that control of the kinase may be different in C. reinhardtii than in barley. When chloro-

51

phyll b is absent from the membrane, there is a much larger proportional loss of PSII antenna than of PSI antenna. But the thylakoid adapts to this imbalance by increasing the ratio of PSII to PSI up to twofold (Melis 1991). Further, different species display this adaptation to different extents. So even though the kinase is not lightactivatable in chlorina f2, it was not clear if it would be activatable in vitro in cbnl-48 thylakoids. The very highly enhanced kinase activity we observed in the cbnl-48 mutant (Figs. 5, 6) was completely unexpected. Among the more than thirty labelled bands, the most heavily labelled were the P700 apoproteins of PSI, and a novel 15.5-kDa band that is also labelled with anti-LHCII antisera. Phosphorylation of the P700 apoproteins, which does not seem to occur in higher plants, has been reported previously in C. reinhardtii (Ikeuchi et al. 1987). The functional significance of this observation is unclear. Even on autoradiograms of wild-type-thylakoid gels, we resolve many more LHCII phosphoproteins than have previously been described for C. reinhardtii. It is interesting that within a group of closely spaced bands, even though immunoreactivity may be very similar, all bands are not necessarily phosphorylated. For example, only the 28.2-kDa band of the LHCII p13 doublet is phosphorylated, but all three of the CP29 polypeptides (34.5, 34.2, and 34.0 kDa) are labelled. Comparison of unphosphorylated with phosphorylated thylakoid samples shows no appreciable differences in the position of individual bands (data not shown), leading us to believe that this gel system is not separating apoproteins based on phosphorylation state. We also see a new phosphoprotein at 35 kDa. This band is close enough to the CP29 apoproteins that it is very unlikely that it would have been detected in any earlier studies. Our results, along with the work of Bassi and Wollman (1991), indicate that there are important similarities between CP29 of higher plants and C. reinhardtii. These include a higher chlorophyll a/b ratio and a higher molecular weight than LHCII, immunological relatedness and proximity to the PSII core. But C. reinhardtii has the important difference that CP29 is highly phosphorylated by a thylakoid-associated kinase, (Figs. 5, 6; see also De Vitry and Wollman 1988; Ikeuchi et al. 1987), whereas higher-plant CP29 is only minimally phosphorylated (Dunahay et al. 1987; Peter and Thornber 1991). In addition, the distribution of CP29 in spinach thylakoids does not change during a statel-state 2 transition during which some proportion of LHCII migrates from appressed to non-appressed regions (Dunahay and Staehelin 1987). The functional significance of the differences in phosphorylation of C. reinhardtii and higher-plant CP29 remains to be elucidated. The general role of phosphorylation in the regulation of photosynthesis has recently been re-examined (Allen 1992). The widely held view is that phosphorylation of LHCII increases the net negative surface-charge density in the grana (Barber 1986), leading to the well-documented migration of a subset of LHCII from grana to stroma domains during the state transition (Staehelin and Arntzen 1983). But this idea does not explain why a sub-

K.D. Allen and L.A. Staehelin:PhotosystemII antenna in Chlamydomonas

52

stantial portion of phospho-LHCII remains in the grana membranes during state 2 (Islam 1987). It also does not explain the phosphorylation of PSII core components, or of CP29 which also remains in the grana during state 2. A more complete explanation of this phenomenon may come from a consideration of the effects of phosphorylation on other, more highly characterized proteins. There are several examples in which the addition of a phosphate group, apart from simply adding to the net surface charge of a molecule, changes three-dimensional structure such that protein-protein or enzyme-subtrate interactions are altered (see references in Allen 1992). Thus, phosphorylation of LHCII may serve to alter binding properties, perhaps favoring association with PSI over PSII. Further, phosphorylation of CP29 and CP26 in C. reinhardtii may serve to discourage LHCII binding.

Hypothetical organization of the PSII antenna system. Figure 7A-C depicts a hypothetical arrangement of the PSII antenna system. This model specifies three levels of antenna organization. The first level is the PSII core complex comprising CP47, CP43, the D1/D2/cytochrome complex and a number of smaller polypeptides (Fig. 7A). This particle, which binds about 50 chlorophyll a molecules (Bricker 1990; De Vitry et al. 1991), appears to be the predominant PSII complex in thylakoids of chlorina f2 barley and other higher-plant chlorophyll bless mutants (see for example Allen et al. 1988; Peter and Thornber 1991). The second level, comprising the chlorophyll a/b-binding proteins CP29, CP26 and CP24, is closely associated with the reaction-center core and is thought to be a linker between the reaction center and at least part of the LHCII population (Bassi and Dainese

1992; and this work). Addition of the linker antennae to the core particle yields the predominant PSII complex found in cbnl-48 thylakoids (Fig. 7B). Addition of a single LHCII trimer to this complex should yield PSIII3 with 130 chlorophyll a+b (assuming approximately 40 chlorophylls for CP29/26/24, and 45 chlorophylls per LHCII trimer, Butler and K/ihlbrandt 1988). The third level of organization is the peripheral LHCII antenna system. Addition of the peripheral antenna yields PSII~ (Fig. 7C) with approximately 210 chlorophylls a+b (Melis 1991). The precise arrangement of the core subunits is not completely known. However, it appears from biochemical dissection and chemical protection experiments that CP47 is very closely associated with the D1/D2/cytochrome complex, and CP43 is more peripherally located (Bricker 1990, Frankel and Bricker 1992; Odom and Bricker 1992). Photosystem II dimers have been visualized in a number of different grana-membrane preparations (Seibert et al. 1987; Bassi et al. 1989; Lyon et al. 1993). At least three biochemical studies have presented evidence that the PSII dimer is the predominant form of PSII in the membrane (Peter and Thornber 1991; Barbato et al. 1992; Bassi and Dainese 1992). We also observe dimeric PSII complexes in our preparations. In the twodimensional gel of cbnl-48 thylakoids in Fig. 4A there is a PSII complex with an apparent molecular weight in excess of 1000 kDa (near the origin of the green gel dimension), corresponding to a dimer of the complex shown in Fig. 7B. But it is not clear at this point whether the PSI! dimerization seen in our green gels has any invivo significance or is simply an artefact of preparation. Our model shows CP29 and CP24 bound to each oth-

A) WildType PSIl-Core

LHCII

Trime

I B) cbnl-48PSIIcomplex

plla p13a

p16b

FPlla ,

/p16a k p17a-P

,

FPllb

F p11c-P

k p17b-P

k p17b-P

|pllc-P, |p13b-P etc.

C) WildType PSII-LHCIIcomplex

Fig. 7. Proposed organization of the PSII antenna system. See text for details

K.D. Allen and L.A. Staehelin: Photosystem II antenna in Chlamydomonas er based on the finding of a C P 2 9 / 2 4 / L H C I I complex that dissociates on phosphorylation to yield a CP29/24 complex and L H C I I (Bassi and Dainese 1992 and references therein). Because of data indicating that CP29 and CP26 are very closely associated with the reaction center (Bassi et al. 1990), we do not show CP29 as part of a large L H C I I complex free in the membrane, as has been proposed (Bassi and Dainese 1992). The CP29/24 complex is shown bound to CP47 based on the biochemical evidence of Rigoni et al. (1992) for direct contact between CP29 and CP47. It should be noted that this point is also not resolved, and other models call for CP29 binding to CP43. This uncertainty is reflected in the diagram. The trimeric form of L H C I I appears to be predominant in the thylakoid membrane. Higher-order oligomers have been observed in solubilized preparations by other groups (Bassi and Dainese 1992), and we also observe very large L H C I I - c o n t a i n i n g particles that are either P S I I - L H C I I complexes or some sort of large L H C I I oligomer (Fig. 4, near the origin of the green gel dimension). But although higher- and lower-order oligomers m a y be found in solution, the trimer is by far the most stable (Butler and Kiihlbrandt 1988). Further, L H C I I trimers crystallized by Kfihlbrandt and Wang (1991) correspond in size to the 8-nm P-Face freeze-fracture particles associated with L H C I I in the thylakoid (Staehelin 1986). It is less clear whether L H C I I binds to PSII as a trimer, or if m o n o m e r s m a y also bind. The observed phosphorylation of CP29 (Figs. 5, 6) raises the possibility that in C. reinhardtii, interaction of L H C I I with the reaction center m a y be modified by phosphorylation of both L H C I I and CP29. In this view, phosphorylated CP29 would remain attached to the reaction center, decreasing L H C I I binding affinity during state 2. We suggest that the L H C I I polypeptide heterogeneity we have demonstrated reflects the functional heterogeneity of L H C I I in the membrane. Subpopulations of L H C I I are observed during the state transition, differing in phosphorylation rate and tendency to migrate away from PSII during state 2 (see, for example, Islam 1987). Thus, L H C I I has been divided into phosphorylated mobile, phosphorylated non-mobile, and non-phosphorylated non-mobile pools. A tightly b o u n d subpopulation of L H C I I is thought to bind directly to the chlorophyll a antenna proteins (Bassi and Dainese 1992). The improved resolution of antenna polypeptides we have demonstrated will allow us to examine the extent to which different L H C I I subpopulations are enriched for individual L H C I I apoproteins. For example, the presence of multiple immunologically related variants of p 11, some of which are phosphorylatable and some of which are not (Fig. 6) could reflect some feature of the p l l polypeptides that is required in more than one L H C I I pool.

This work was supported by National Institute of Health grant GM22912 to L.A.S.. We would like to thank Anastasios Melis for helpful discussions.

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References

Allen, J.F. (1992) Protein phosphorylation in regulation of photosynthesis. Biochim. Biophys. Acta 1098, 275-335 Allen, K.D., Duysen, M.E., Staehelin, L.A. (1988) Biogenesis of thylakoid membranes is controlled by light intensity in the conditional chlorophyll b-deficient CD3 mutant of wheat. J. Cell Biol. 107, 907-19 Allen, K.D., Staehelin, L.A. (1991) Resolution of 16 to 20 chlorophyll-protein complexes using a low ionic strength native green gel system. Anal. Biochem. 194, 214-222 Allen, K.D., Staehelin, L.A. (1992) Biochemical characterization of photosystem-II antenna polypeptides in grana and stroma membranes of spinach. Plant Physiol. 100, 1517-1526 Barbato, R., Friso, G., Rigoni, F., Vecchia, F. D., Giacometti, G. M. (1992) Structural changes and lateral redistribution of photosystern II during donor side photoinhibition of thylakoids. J. Cell Biol. 119, 325-335 Barber, J. (1986) Surface electrical charges and protein phosphorylation. In: Encyclopedia of plant physiology, N. S., vol. 19: Photosynthesis III, pp. 653-663, Staehelin, L.A., Arntzen, C., eds. Springer-Verlag, Berlin Bassi, R., Dainese, P. (1992) A supramolecular light-harvesting complex from chloroplast photosystem-II membranes. Eur. J. Biochem. 204, 317-326 Bassi, R., Dainese, P. (1990) The role of the light harvesting complex II and of the minor chlorophyll a/b-binding proteins in the organization of the photosystem II antenna system. Prog. Photosyn. Res. 2, 209-216 Bassi, R., Hinz, U., Barbarato, R. (1985) The role of the light harvesting complex and photosystem II in thylakoid stacking in the chlorina 12 barley mutant. Carlsberg Res. Commun. 50, 347-367 Bassi, R., Magaldi, A.G., Tognon, G., Giacometti, G.M., Miller, K.R. (1989) Two dimensional crystals of the photosytem II reaction center complex. Eur. J. Biochem. 50, 84-93 Bassi, R., Soen, S.Y., Frank, G., Zuber, H., Rochaix, J.-D. (1992) Characterization of chlorophyll-a/b proteins of photosystem-I from Chlamydomonas reinhardtii. J. Biol. Chem., 267, 2571425721 Bassi, R., Wollman, F.-A. (1991) The chlorophyll-a/b proteins of photosystem II in Chlamydomonas reinhardtii. Planta 183, 423433 Bellemare, G., Bartlett, S.G., Chua, N.-H. (1982) Biosynthesis of chlorophyll a/b-binding ploypeptides in wild type and the chlorina f2 mutant of barley. J. Biol. Chem. 257, 7762-7767 Bhalla, P., Bennett, J. (1987) Chloroplast phosphoproteins: phosphorylation of a 12-kDa stromal protein by the redox-controlled kinase of thylakoid membranes. Arch. Biochem. Biophys. 252, 97-104 Blum, H., Beier, H., Gross, H.J. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis. 8, 93-99 Bothe, D., Simonis, M., Doehren, H.V. (1985) A sodium dodecyl sulfate-gradient gel electrophoresis system that separates polypeptides in the molecular weight range of 1500 to 100,000. Anal. Biochem. 151, 49-54 Bricker, T.M. (1990) The structure and function of CPa-1 and CPa-2 in photosystem II. Photosyn. Res. 24, 1 13 Butler, P.J.G., Kiihlbrandt, W. (1988) Determination of the aggregate size in detergent solution of the light-harvesting chlorophyll-protein complex from thylakoid membranes. Proc. Natl. Acad. Sci. 85, 3797-3801 Chua, N.-H., Blomberg, F. (1979) Immunochemical studies of thylakoid membrane polypeptides from spinach and Chlamydomonas reinhardtii. A modified procedure for crossed immunoelectrophoresis of dodecyl sulfate protein complexes. J. Biol. Chem. 254, 215-23 Chunayev, A. (1990) Genetics of photosynthesis in Chlamydomonas. Photosynthetica 24, 283-308 De Vitry, C., Wollman, F.-A. (1988) Changes in the phosphorylation of thylakoid membrane proteins in light-harvesting com-

54


plex mutants from Chlamydomonas reinhardtii. Biochim. Biophys. Acta 933, 444-449 De Vitry, C., Diner B.A., Popot J.L. (1991) Photosystem-II particles from Chlamydomonas reinhardtii - purification, molecular weight, small subunit composition, and protein phosphorylation. J. Biol. Chem. 266, 16614-16621 Dekker, J.P., Boekema, E.J., Witt, H.T., Rogner, M. (1988) Refined purification and further characterization of the oxygen-evolving and tris-treated photosystem II particles from the thermophilic cyanobacterium Synechococus sp. Biochim. Biophys. Acta 936, 307-318 Delepelaire, P., Chua, N.-H. (1981) Electrophoretic purification of chlorophyll a/b-protein complexes from Chlamydomonas reinhardtii and spinach and analysis of their polypeptide compositions. J. Biol. Chem. 256, 9300-9307 Delepelaire, P., Wollman, F.-A. (1985) Correlations between fluorescence and phosphorylation changes in thylakoid membranes of Chlamydomonas reinhardtii in vivo: a kinetic analysis. Biochim. Biophys. Acta 809, 277-283 Demmin, D., Stockinger, E.J., Chang, Y.C., Walling, L.L. (1989) Phylogenetic relationships between the chlorophyll-a/b binding protein (cab) multigene family - an intraspecies and interspecies study. J. Mol. Evol. 29, 266 279 Dunahay, T.G., Staehelin, L.A. (1987) Immunolocalization of the chl a/b light harvesting complex and CP29 under conditions favoring phosphorylation and dephosphorylation of thylakoid membranes (state 1 state 2 transition). In: Progress in photosynthesis research, pp. 701 704, Biggins, J., ed. Martinus Nijhoff, the Netherlands Frankel, L.K., Bricker, T.M. (1992) Interaction of CPa-1 with the manganese-stabilizing protein of photosystem-II - Identification of domains on CPa-1 which are shielded from n-hydroxysuccinimide biotinylation by the manganese-stabilizing protein. Biochemistry 45, 11059-11064 Giddings, T.H., Wasman, C., Staehelin, L.A. (1983) Structure of the thylakoids and the envelope membrane of the cyanelles of Cyanophora paradoxa. Plant Physiol. 71, 409-419 Green, B.R., Pichersky, E., Kloppstech, K. (1991) Chlorophyll-a/bbinding proteins an extended family. Trends Biochem. Sci. 16, 181-186 Green, B.R., Shen, D.R., Aebersold, R., Pichersky, E. (1992) Identification of the polypeptides of the major light-harvesting complex of photosystem-II (LHCII) with their genes in tomato. FEBS Lett. 305, 18-22 Herrin, D.L., Plumley, F.G., lkeuchi, M., Michaels, A.S., Schmidt, G.W. (1987) Chlorophyll antenna proteins of photosystem I: topology, synthesis, and regulation of the 20-kDa subunit of Chlamydomonas light-harvesting complex of photosystem I. Arch. Biochem. Biophys. 254, 397-408 Holmes, J., Dutcher, S. (1989) Cellular asymmetry in Chlamydomonas reinhardtii. J. Cell Sci. 94, 273-285 Ikeuchi, M., Plumley, F., Inoue, Y., Schmidt, G. (1987) Identification of phosphorylated reaction center polypeptides in thylakoids of Chlamydomonas reinhardtii and Pisum sativum. Progr. Photosynth. Res. 2, 805-808 Imbault, H.P., Wittemer, C., Johanningmeier, U., Jacobs, J.D., Howell, S.H. (1988) Structure of the Chlamydomonas reinhardtii cablI-I gene encoding a chlorophyll-a/b-binding protein. Gene. 73, 397-407 Islam, K. (1987) The rate and extent of phosphorylation of the two light-harvesting chlorophyll a/b binding protein complex (LHCII) polypeptides in isolated spinach thylakoids. Biochim. Biophys. Acta 893, 333-341 Jansson, S., Pichersky, E., Bassi, R., Green, B.R., Ikeuchi, M., Melis, A., Simpson, D.J., Spangfort, M., Staehelin, L.A., Thornber, J.P. (1992) A nomenclature for the genes encoding the cholorphyll a/b-binding proteins of higher plants. Plant Mol. Biol. Rep. 10, 242 253 Kellmann, J.W., Merforth, N., Wiese, M., Pichersky, E., Piechulla, B. (1993) Concerted circadian oscillations in transcript levels of 19

lha/b (cab) genes in Lycopersicon esculentum (tomato) Mol. Gen. Genet. 237, 439-448 Kfihlbrandt, W., Wang, D. (1991) Three-dimensional structure of plant light-harvesting complex determined by electron crystallography. Nature 350, 130-134 Larsson, U., Sundby, C., Andersson, B. (1987) Characterization of two different subpopulations of spinach LHCII: polypeptide composition, phosphorylation pattern, and association with photosystem II. Biochim. Biophys. Acta 894, 59 68 Lyon, M.K., Marr, K.M., Furcinetti, P.S. (1993) Formation and characterization of two dimensional crystals of photosystem II. J. Struct. Biol. 110, 133-140 Melis, A. (1991) Dynamics of photosynthetic membrane composition and function. Biochim. Biophys. Acta 1058, 87-106 Michel, H., Tellenbach, M., Boschetti, A. (1983) A chlorophyll b-less mutant of Chlamydomonas reinhardtii lacking in the light-harvesting chlorophyll a/b protein complex but not its apoproteins. Biochim. Biophys. Acta 725, 417-424 Morrissey, P., Glick, R., Melis, A. (1989) Supramolecular assembly and function of subunits associated with the chlorophyll a/b light-harvesting complex II (LHCII) in soybean chloroplasts. Plant Cell Physiol. 30, 335-344 Odom, W.R., Bricker, T.M. (1992) Interaction of CPa-1 with the manganese-stabilizing protein of photosystem-II - Identification of domains cross-linked by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Biochemistry 31, 5616-5620 Owens, G., Ohad, I. (1982) Phosphorylation of Chlamydomonas reinhardtii chloroplast membrane proteins in vivo and in vitro. J. Cell Biol. 93, 712 718 Peter, G.F., Thornber, J.P. (1991) Biochemical composition and characterization of higher plant photosystem II light-harvesting pigment proteins. J. Biol. Chem 266, 16745-16754 Piechulla, B., Kellmann, J.W., Pichersky, E., Schwartz, E. (1991) Determination of steady-state mRNA levels of individual chlorophyll a/b binding protein genes of the tomato cab gene family. Mol. Gen. Genet. 230, 413-422 Porra, R., Thompson, W., Kriedemann, P. (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta 975, 384-394 Rigoni, F., Barbato, R., Friso, G., Giacometti, G.M. (1992) Evidence for direct interaction between the chlorophyll proteins CP29 and CP47 in photosystem II. Biochem. Biophys. Res. Comm. 184, 1094-1100 Seibert, M., Dewitt, M., Staehelin, L.A. (1987) Structural localization of the O2-evolving apparatus to multimeric (tetrameric) particles on the lumenal surface of freeze-etched photosynthetic membranes. J. Cell Biol. 105, 2257 2265 Sigrist, M., Staehelin, L.A. (1992) Identification of type-1 and type-2 light-harvesting chlorophyll-a/b-binding proteins using monospecific antibodies. Biochim. Biophys. Acta 1098, 191-200 Simpson, D. (1986) Freeze-fracture studies of mutant barley chloroplast membranes. In Encyclopedia of plant physiology, N. S., vol. 19: Photosynthesis III, pp. 665-674, Staehelin, L.A., Arntzen, C.J., eds. Springer-Verlag, Berlin Staehelin, L.A. (1986) Chloroplast structure and supramolecular organization of photosynthetic membranes. In: Encyclopedia of plant physiology, N. S., vol. 19: Photosynthesis III, pp. 1-84, Staehelin, L.A., Arntzen, C.J., eds. Springer-Verlag, Berlin Staehelin, L.A., Arntzen, C.J. (1983) Regulation of chloroplast membrane function : Protein phosphorylation changes the spatial organization of membrane components. J. Cell Biol. 97, 1327-1337 Steinmetz, D., Damm, I., Grimme, L. (1990) Reconstitution of the light-harvesting chlorophyll a/b protein complex of the chlorophyll b-less cbnl-48 mutant of Chlamydomonas reinhardtii with a pigment extract derived from wild type. In: Current Research in Photosynthesis, pp. 855 858, Baltcheffsky, M., ed. Ktuwer Academic, The Netherlands