THE JOURNAL 0 1987 by The American Society for Biochemistry

OF BIOLOGICAL CHEMISTRY and Molecular Biology, Inc.

Vol. 262, No. 27, Issue of September 25, pp. 13333-13341,1987 Printed in U. S. A.

Chlorophyll-Proteins of the Photosystem I1 Antenna System* (Received for publication, November 12,1986)

Roberto BassiSQ, Gunilla Hayer-HansenQ, Roberto BarbatoS, Giorgio M. Ciacometti$, and David J. Simpsong From the SDipartimento di Biologia, Universita di Padova via Loredan 10, 35100 Padova, Italy and the $Department of Physiology, Carlsberg Laboratory, Gamle Carlsberguej 10, 2500 Copenhagen Valby, Denmark

The chlorophyll-protein complexes of purified maize photosystem I1 membranes were separated by a new mild gel electrophoresis system under conditions which maintained all of the major chlorophyll ab-protein complex (LHCII) in the oligomeric form. This enabled the resolution of three chlorophyll ab-proteins in the 26-31-kDa region which are normally obscured by monomeric LHCII. All chlorophyll ab-proteins had unique polypeptide compositions and characteristic spectral properties. One of them (CP26) has not pre- viously been described, and another (CP24) appeared to be identical to the connecting antenna of photosys- tem I (LHCI-680). Both CP24 and CP29 from maize had at least one epitope in common with the light- harvesting antennae of photosystem I, as shown by cross-reactivity with a monoclonal antibody raised against LHCI from barley thylakoids. A complex des- ignated Chl,*P2*, which was capable of electron trans- port from diphenylcarbazide to 2,6-dichlorophenolin- dophenol, was isolated by nondenaturing gel electro- phoresis. It lacked CP43, which therefore can be ex- cluded as an essential component of the photosystem I1 reaction center core. Fractionation of octyl glucoside- solubilized photosystem I1 membranes in the presence and absence of Mg2+ enabled the isolation of the Chl,-P2* complex and revealed the existence of a light- harvesting complex consisting of CP29, CP26, and CP24. This complex and the major light-harvesting system (LHCII) are postulated to transfer excitation energy independently to the photosystem I1 reaction center via CP43.

The light reactions of photosynthesis are driven by the excitation energy absorbed by the antenna pigments and transferred to the reaction centers. In the photosynthetic apparatus of higher plants, as many as 210 molecules of chlorophyll are associated with photosystem I (PSI)’ and 235 with photosystem I1 (1). The primary chromophore respon- sible for light absorption in higher plants and algae is chlo- rophyll a. Accessory pigments such as chlorophyll b and carotenoids extend the spectral range of light absorption and transfer energy to chlorophyll a.

More and more chlorophyll-proteins have been discovered as electrophoretic techniques improve, enabling the retention

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

‘The abbreviations used are: PSI, photosystem I; DCPIP, 2,6- dichlorophenolindophenol; DPC, diphenylcarbazide; Hepes, 4-(2-hy- droxyethy1)-I-piperazineethanesulfonic acid; LHC, light-harvesting complex; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; Tricine, N-tris(hydroxymethy1)methylglycine.

of chlorophyll noncovalently attached to the various apopro- teins and facilitating the resolution of one chlorophyll-protein from another. The function of some of these chlorophyll- proteins is not known in detail, so they are often distinguished only by their electrophoretic mobility and named accordingly. Thus, CP47 refers to a chlorophyll-protein with an apparent molecular mass of 47 kDa. Photosystem I is composed of three different chlorophyll-proteins. CPI is the major one, contain- ing the reaction center (P700) and chlorophyll a. Two others (LHCI-680 and LHCI-730) serve as light-harvesting antennae (2-4). Photosystem I1 contains two chlorophyll-proteins, CP47 and CP43, which contain chlorophyll a but there is no direct evidence to show that they contain the photosystem I1 reaction center (P680). The major light-harvesting chloro- phyll ab-protein of PSII is called LHCII (or CPII or CP27), but at least two others are known: CP29 ( 5 , 6) and CP24 (7), which may function as minor or connecting antennae. The situation is further complicated by the ability of some of these chlorophyll-proteins to form homopolymers (e.g. LHCII** and LHCI-730*) under favorable conditions of solubilization and electrophoresis. Moreover, some chlorophyll-proteins form complexes with other chlorophyll-proteins and nonpigmented polypeptides (e.g. CPla and Chl,. P2*; see Fig. la).

Although the LHCII complex has been the subject of exten- sive investigation (8-lo), its polypeptide composition is still not well defined. Thus, while at least two polypeptides of 25 and 27 kDa are commonly associated with isolated LHCII, molecular genetic analysis has indicated the presence of at least 16 genes coding for chlorophyll a b proteins organized into five small multigene families (11, 12). In light of this, we have re-examined the composition of isolated PSII mem- branes in terms of their chlorophyll-protein complexes with the aim of obtaining detailed information about the functional connections between the component chlorophyll a b binding complexes. Up to seven different polypeptides can be resolved in the 26-31-kDa range, belonging to three distinct chloro- phyll ab-protein complexes, as the result of an improved electrophoretic gel system, which also reveals a PSII core complex (Chl,.P2*) that is the smallest known from higher plants capable of electron transport activity. We show that CP24 of PSII is almost identical to the LHCI-680 of PSI and propose a model for the organization of the chlorophyll- proteins of PSII based on fractionation patterns with the detergent octyl glucoside.

EXPERIMENTAL PROCEDURES

Preparation of Thylakoid Membranes-Zea mays L. seedlings (cul- tivar DeKalb DF28) were grown for 2-3 weeks in a growth chamber at 28/21 “C day/night at a light intensity of 10,000 lux. Thylakoids from mesophyll and bundle sheath chloroplasts were prepared as previously described (4). PSII membranes were obtained according to the method of Berthold et al. (13) using the modifications described by Dunahay et al. (14). Aliquots were frozen in liquid nitrogen and

13333

13334 Chlorophyll-proteins of the PSII Antenna System

stored in 5 mM MgCl2, 25 mM Hepes, pH 8.0, a t -80 "C until required. Barley PSI-200 preparations and thylakoids were made according to Bassi and co-workers (4, 35).

Gel Electrophoresis-Glycerol was added to a final concentration of 40% to PSII membranes, suspended in storage buffer (1 mg chlorophyll/ml), which were then solubilized using a 10% stock so- lution of octyl glucoside (octyl glucoside:chlorophylI, 401) and stirred on ice for 5 min. Nonsolubilized material was removed by centrifuga- tion a t 15,000 X g for 15 min and the supernatant was loaded onto acrylamide tube gels (4% stacking and 11% resolving). The buffer system and running conditions were as in Ref. 4, except for the incorporation of 10% glycerol into the gels. Green bands were excised and pooled from four to six gels, chopped into small pieces, and rerun in 9% acrylamide tube gels to avoid contamination by comigrating polypeptides that do not bind chlorophyll (15). For analytical electro- phoresis, green bands were macerated and loaded onto a gradient gel (14-20% acrylamide) containing 6 M urea and run a t 15 mA for 3 days using the buffer system described by Bassi et a/. (4). Gels were fixed in methano1:water:acetic acid, 55: l (v/v/v) containing 10% trichloroacetic acid and stained with Coomassie R-250.

Spectroscopy-For low temperature fluorescence spectroscopy, green bands were excised, macerated, loaded into a quartz tube, and frozen in liquid nitrogen as quickly as possible. Spectra were recorded using a Perkin-Elmer MPF 44 fluorescence spectrophotometer equipped with a low temperature accessory. For room temperature absorption spectroscopy, green bands were ground in distilled water and eluted overnight. The acrylamide debris were removed by cen- trifugation and the spectrum of the supernatant was recorded using a Perkin-Elmer Lambda-5 spectrophotometer. Chlorophyll concen- tration and a/b ratios were determined in 80% acetone according to Arnon (16). PSII electron transport measurements were made spec- trophotometrically with DPC as the donor, as described in Ref. 4.

Immunological Techniques-Monoclonal antibodies against LHCI were prepared as described in Ref. 17. For immunoblot assays, chlo- rophyll-proteins were prepared as described above, their apoproteins were resolved in urea-containing gels and transferred to a nitrocel- lulose filter (Millipore). The filters were then assayed with the mono- clonal antibody as in Ref. 17.

Sucrose Gradient Ultracentrifugation-PSI1 membranes (1 mg chlorophyll/ml) in 5 mM Tricine, pH 8.0, were solubilized with octyl glucoside (octyl glucoside:chlorophylI, 401) for 5 min on ice and then centrifuged a t 15,000 X g for 15 min. Samples (1 ml) were loaded onto a 0.1 to 1 M sucrose gradient containing 1% octyl glucoside and run a t 39,000 rpm in an SW41 rotor for 17 h a t 4 "C.

Phosphorylation of Thylakoids-Seedling leaves were dark-adapted for 1 h a t room temperature to ensure dephosphorylation of thylakoid chlorophyll-proteins. Thylakoids were then isolated according to the method of Bassi et al. (4) and resuspended a t 250 pg of chlorophyll/ ml in 100 mM sucrose, 10 mM KCI, 5 mM MgC12, 50 mM Hepes- NaOH, pH 7.5, and 5 mM NaF to inhibit phosphatase activity, as described by Farchaus et al. (18). To an aliquot of this suspension, 30 pCi/ml of [y"P]ATP was added to a final ATP concentration of 1 mM. After illumination for 10 min at 20 "C, the thylakoids were pelleted by centrifugation, solubilized in octyl glucoside as described above, and resolved into component chlorophyll-proteins by nonde- naturing SDS-PAGE. The chlorophyll-proteins were excised and rerun in the presence of 6 M urea (see above) and exposed for autoradiography.

RESULTS

Resolution and Characterization of Chlorophyll a/b Pro- teins-The results of nondenaturing electrophoretic analysis of thylakoids and PSII membranes from mesophyll chloro- plasts of maize are shown in Fig. 1. When purified PSII membranes were solubilized and resolved as described under "Experimental Procedures," seven green bands were pro- duced, four of which were chlorophyll a/b-protein complexes (Fig. lb). With the electrophoretic conditions used in this paper, the major green band was the oligomeric form of LHCII (LHCII**). This complex could be resolved into five distinct polypeptide bands (see Table I) by SDS-PAGE in the presence of 6 M urea (Fig. 2). By maintaining all the LHCII in this oligomeric state, the 26-31-kDa region was not superimposed by monomeric LHCII and could be resolved into three distinct chlorophyll-protein bands. They all contained chlorophyll a

Thylakoids

CPla - Chla-P1 - Chlalb- P2.O-

FP-

a

PSIL membranes "

-Chla-P2*(CP2-b)

- C P I

- LHCII"

- CP47 - CP43

- CP26 - CP29

- CP24

- FP

b FIG. 1. Unstained tube gels after nondenaturing SDS-

PAGE, showing the chlorophyll-protein complexes of maize mesophyll thylakoids (a) and purified PSII membranes (b) . The uppermost bands of each gel are different, inspite of having similar mobilities. CPla contains the PSI reaction center, LHCI antenna, and other polypeptides (4). Chl.-P2* is a complex of PSII, containing CP47, Dl , D2, and cytochrome b-559. LHCIT** is the major light-harvesting chlorophyll ab-protein complex of PSII. CP47 and CP43 have been correlated with the PSII reaction center and PSII internal antenna, respectively. In PSII membranes, CP47 is mainly organized in the oligomeric Chl..P2* form. CP29 is a PSII antennae and CP26 is a new PSII antennae complex. CP24 in PSII membranes appears to be identical to the PSI antenna called LHCI- 680. The nomenclature is that of Camm and co-workers (6, 7, 23) with that of Simpson and co-workers (4, 36) also shown. FP, free pigment.

TABLE I Characteristics of the chlorophyll a/b-protein complexes

of PSII membranes Name Molecular mass of Chlorophyll Absorption Fluorescence

UobeDtides a h ratio maximum maximum

kDa nm nm LHCII** 26, 28.5, 1.2 614 683

28.8, 29.5, 30

CP29 31 2.5 674 680 CP26 28,29 2.0 671 680 CP24 20.25 669 680

and b, and analysis by denaturing SDS-PAGE revealed unique polypeptide compositions (Fig. 2).

The uppermost band in the 26-31-kDa region contained a single polypeptide a t 31 kDa and was consistent in its spectral properties (see below) with one previously described (5) and called CP29 (6). The band below it contained two polypeptides of 28 and 29 kDa and was designated CP26. This complex comigrated with the monomeric form of LHCII, but differed from it in several respects, including the number and electro- phoretic mobility of its component polypeptides (see Table I). It had different spectral properties and, under conditions where all but the 26-kDa polypeptide of LHCII** became labeled with 32P, there was no significant labeling of the polypeptides of CP26 beyond what was due to a slight contam- ination of the four polypeptides of LHCII (Fig. 3).

The fastest migrating band had a chlorophyll a/b ratio (and consequently absorption spectrum) which varied with the isolation procedure. In particular, decreasing chlorophyll a/b

Chlorophyll-proteins of the PSII Antenna System 13335

0) N

V a a R

V

t N

V a

0 0

#o r -1

kD

u)

E x m h - 5 1

t N a

2 V

(0 N

0 a

3

0) N

0 a

4

0 . !=I r

5

V

-1

. U 0 3 5'

0) N

V a

4

(0 N

V a

3

a

2'

z V

kD

-36 45-

36- - 29

29-

24 -

14.2-

FIG. 2. Polypeptide composition of chlorophyll ab-protein complexes of PSII membranes (indicated by arrowheads). Chlorophyll-proteins were separated by mild SDS-PAGE of octyl glucoside-solubilized membranes. The excised green bands were rerun under similar conditions in a gel with a different acrylamide compo- sition, excised, and loaded onto a 14-20% acrylamide gradient gel containing 6 M urea. Those polypeptide bands not labeled with arrowhead9 in lanes 3-6 are regarded as contaminants.

ratios were measured after successive electrophoretic runs; thus the initial value of 1.2 decreased to 1.0 after the second and to 0.8 after the third run. This preferential loss of chlo- rophyll a is not unusual for chlorophyll ab-proteins, partic- ularly LHCI-680 which was recently isolated from PSI prep- arations and runs in the same region of the gel (4). This PSII chlorophyll-protein consisted of two polypeptides of 20 and 25 kDa (Table I) and appears to be identical to a chlorophyll- protein complex designated CP24, isolated from spinach PSII membranes (7).

The same three chlorophyll-protein bands in the 26-31- kDa range were also resolved when whole thylakoids rather than PSII membranes were used as the starting material (Fig. la). In some experiments an additional green band migrated very close to CP26, and a diffuse background was always found over the 26-31-kDa area. Analysis of the polypeptide compositions of the green bands in this region was compli- cated by the presence of the apoproteins of LHCII in all three bands.

The room temperature absorption spectra (600-720 nm) of the four chlorophyll ab-containing bands of Fig. l b are shown in Fig. 4. LHCII** had a red absorption maximum a t 674 nm with a shoulder at 650 nm due to chlorophyll b. CP29 was also characterized by a peak at 674 nm, but a smaller shoulder

Coomassie stain Autoradiography FIG. 3. Phosphorylation pattern of chlorophyll ab-proteins

of PSII membranes. Thylakoids were phosphorylated in vitro with [-y-'*P]ATP and the chlorophyll-proteins were resolved by nondena- turing gel electrophoresis. The chlorophyll ab-proteins indicated in the figure were excised and pooled from several gels and rerun in the presence of 6 M urea. Under conditions where four of the polypeptides of LHCII** were heavily labeled, there was no significant labeling of CP26 above that due to contamination by monomeric LHCII (as a result of using whole thylakoids).

at 650 nm clearly distinguished this complex from the former. Blue-shifted peaks a t 671 and 669 nm characterized CP26 and CP24, respectively. The 77°K fluorescence emission peak values are given in Table I. A single sharp emission peak at 680 nm was observed from all of the complexes except LHCII**, whose fluorescence emission peak was red-shifted to 683-4 nm.

Immunological Cross-reactivity-Monoclonal antibodies were raised against the barley PSI-200 preparation whose polypeptide composition is shown in Fig. 5. One of these antibodies (CMpLHCkl) recognized a number of polypep- tides in the 20-25-kDa range of PSI-200 plus another poly- peptide at 29 kDa present in barley thylakoids, but not in PSI-200 (Fig. 5). This antibody was tested against the chlo- rophyll-proteins of maize PSII membranes separated by non- denaturing electrophoresis as in Fig. lb , re-electrophoresed in 6 M urea SDS-PAGE, and blotted over to nitrocellulose (17).

13336 Chlorophyll-proteins of the PSII Antenna System

674

...... L”C” a

--- CP24

m - ?!

620 660 680 700 720

Wavelength (nm) FIG. 4. Room temperature absorption spectra of chlorophyll

ab-proteins from PSII membranes. Green bands were excised, ground in distilled water, and eluted for 2-3 h at 4 “C. While each sample displays a characteristic absorption spectrum, that of CP24 varied from one preparation to the next (see “Results”).

Lanes 7’ and 8’ of Fig. 6 show the reaction of authentic LHCI- 680 and LHCI-730 isolated from maize PSI-200, demonstrat- ing that the 20- and 21-kDa polypeptides share a common epitope. There was a significant reaction in this region with PSII membranes (lanes 6 and 6’) and with CP24 isolated from PSII membranes (lanes 2 and 2’). While there was absolutely no cross-reactivity with the polypeptides of LHCII** (lanes 5 and 5’), there was a very strong reaction with the 29-kDa apoprotein of CP29 (lanes 4 and 4 ‘ ) . These results show that CP29 and CP24 share at least one epitope with the light-harvesting chlorophyll ab-binding antennae of PSI (LHCI-680 and LHCI-730).

Characterization of Chlorophyll a Proteins-In addition to the four chlorophyll ab-proteins of PSII membranes, there were three chlorophyll a-binding proteins. Two of these, CP47 and CP43, are associated with the PSII reaction center (14). The slowest migrating band resolved by nondenaturing gel electrophoresis was designated Chl. - P2* and shown by SDS- PAGE in the presence of 6 M urea (Fig. 7) to consist of a single chlorophyll-protein (CP47) plus the quinone-binding proteins D l and D2 and cytochrome bSs9 (9 and 4 kDa). When eluted from the gel, it was capable of electron transport rates, from DPC to DCPIP, that were one-quarter that of octyl glucoside-solubilized PSII membranes (19). This rate was only partially susceptible to inhibition by 3-(3,4-dichlorophenyl)- 1,l-dimethylurea (Table 11).

The low temperature absorption and fluorescence emission spectra were recorded using Chl.. P2* in situ in acrylamide and analyzed by Gaussian deconvolution (Figs. 8 and 9). The component bands are summarized in Table 111. The fourth derivative of the fitted curves closely matched that of the data, indicating that the solution was reasonably good. The major absorption band was centered at 672.6 nm (k15.8 nm). A Gaussian band centered at 679 nm (k10.5 nm), which is a major component (about 15% of the total area) in the spec- trum of the octyl glucoside-solubilized reaction center and CP43,2 was not found in Chl..P2*. Analysis of the fluores- cence emission spectra (Fig. 9 and Table 111) revealed four components, the largest being centered at 689 nm (k16.0 nm). Chl.. P2* was very unstable and, if not measured immediately after electrophoresis, the fluorescence emission maximum shifted from 689 to 682 nm, due to an increase in the com- ponent at 680.4 nm, and decreases in the amplitudes of those at 689.0 and 700.8 nm.

* D. J. Simpson, unpublished data.

kC I

66 - Irl .Qcp

45-

1

36- - methylgreen .. .

29 - m-

- methylgreen

24 - 20.1 -

14.2-

a b FIG. 5. Polypeptide composition (a) and immunoblots (b) of

barley thylakoids and of a barley PSI particle with a chloro- phyll a b ratio of 6.0 (PSI-200) used as the antigen for the production of monoclonal antibodies against LHCI. The methyl green was added during the electrophoretic run as a marker to orient the blot. The blot in b was probed with the same clone CMpLHC1:l used in the cross-reactivity assay in Fig. 6.

Sucrose Gradient Ultracentrifugation-We have so far shown that there are at least four different chlorophyll a b - proteins associated with the maize PSII antenna and that they are composed of a t least 10 resolvable polypeptides

Chlorophyll-proteins of the PSII Antenna System 13337

FIG. 6. Immunoblots of chloro- phyll ab-proteins isolated from maize PSII membranes. Chlorophyll- proteins were obtained by SDS-PAGE under mild conditions, run in urea gels (14-20% acrylamide), and electroblotted onto nitrocellulose sheets. The blots were then assayed with a monoclonal antibody against LHCI, and the reacting bands were revealed with peroxidase- coupled anti-mouse antibody. Peroxi- dase staining was with 9-aminocarbazol. The gels were loaded with 10 pg of chlo- rophyll for thylakoids and PSII mem- branes, but no attempt was made to load equal amounts of chlorophyll for the iso- lated chlorophyll-proteins. Note that lanes 2 and 2' contain CP24 from PSII membranes, that CP26 is contaminated with CP29 (lanes 3 and 3'), and that CP29 is contaminated with CP26 (lones 4 and 4'). LHCII ( l o n e 10) is the mono- meric form of LHCII**.

kt

66

45

36

29

24

20.1

14.1

Coomassie stained SDS - urea - lmmunoblot assay with PAGE CMpLHCI: 1

(Table I). These findings confirm the complexity of LHCII, suggested from the genetic data, and raise the question of the organization of such a system. Is there, for example, a single linear excitation energy transfer chain or do these antenna complexes independently transfer energy to the PSII reaction center or its closely associated chlorophyll a-binding com- plexes? To answer this question, we solubilized the PSII membranes with octyl glucoside and fractionated the PSII membranes in the presence or absence of 5 mM M$+, as described under "Experimental Procedures." Three green bands were obtained in the presence of M$+, and four without Mg'. Fig. 10 shows this separation diagrammatically. The chlorophyll-protein composition of the different bands was determined by nondenaturing SDS-PAGE and confirmed by analysis of their polypeptide composition by SDS-PAGE in 6 M urea (Fig. 11 and Table IV).

In the absence of M ~ + , octyl glucoside disrupted the PSII complex to produce four distinct bands, two of which (bands I1 and IV) were complementary in their chlorophyll-protein composition (Table IV). Band IV was identical in polypeptide composition to the Chl..P2* complex isolated by nondena- turing SDS-PAGE (Fig. Id), with a slight contamination by CP43. CP47 was not present in band 11, which contained all the other chlorophyll-proteins of PSII. We believe these bands consist of a complex of the component chlorophyll-proteins, rather than several cosedimenting chlorophyll-proteins. This is based on the fact that discrete bands are formed in the gradient, that LHCII and CP43 are both present in bands I, 11, and 111, and that the sedimentation coefficient is consistent with a high molecular weight complex, rather than monomeric chlorophyll-proteins. A comparison of bands I and I1 (Table IV) shows that they differ in the amount of CP43, which we

therefore regard as being important in binding Chl. - P2* (band IV) to the rest of the PSII complex (band I).

The presence of Mg2' appeared to stabilize specific associ- ations between chlorophyll-proteins, causing a different de- tergent disruption pattern (Fig. 10). Bands I and I1 were complementary in their chlorophyll-protein composition (Ta- ble IV), and this leads us to propose that each band contains a single, specific complex. Thus CP29, CP26, and CP24 form one complex, and CP47, CP43, LHCII, and CP24 form a second. Since CP24 was a component of each band, we suggest that it links CP29 and CP26 with the rest of the PSII components.

DISCUSSION

We describe four chlorophyll ab-protein complexes asso- ciated with PSII in maize. They differ in electrophoretic mobility, polypeptide composition, spectral properties, and immunological reactivity. Their isolation and characteriza- tion was made possible by preventing the dissociation of the major chlorophyll ab-binding protein (LHCII**) into the monomeric form which comigrates with the other, minor chlorophyll-proteins, thus masking their presence.

LHCZI-LHCII represents the most abundant chlorophyll- protein complex found in higher plant thylakoids, and con- tains most of the chlorophyll b. Its oligomeric form (LHCII**), as obtained from preparative gel electrophoresis, corresponds to the LHCII preparation isolated by sucrose gradient ultra- centrifugation of Triton-solubilized thylakoids (20, 21). This is based on their identical polypeptide composition, spectral properties, and mobility under nondenaturing gel electropho- resis. The minimum molecular mass of the complex is 143 kDa, obtained by summation of the component polypeptides,

13338 Chlorophyll-proteins of the PSII Antenna System

9 hl n I

IP - 0 c

0 0 hl I 4 v) a

CP47-

CP43-

D l

D2. CP29 i LHCII

FIG. 7. Polypeptide composition of maize thylakoids, PSII membranes, maize PSI-200, and the PSII reaction center complex Chl..P2* resolved by denaturing SDS-PAGE in the presenceof 6 M urea. A comparison of the polypeptide compositions of the PSI and PSII preparations reveals no cross-contamination. Chl.. P2' contains CP47, the quinone-binding proteins Dl and D2, and cytochrome 6 , (cyt bss9) and is capable of electron transport from DPC to DCPIP.

assuming an equimolar composition, and not considering the contribution of the pigments. This is much higher than the 64 kDa estimated from the mobility in nondenaturing electro- phoresis and is presumably due to nonstoichiometric binding of SDS to the oligomeric complex, since values of 275 kDa have been obtained from gel filtration studies (22).

TABLE I1 Electron transport rates (DPC + DCPIP) of PSII preparations

pmol DCPIP/mg DCMU' chlorophyll/h inhibition Sample

?6 PSII membranes 77 79 PSII membranes + octyl glucoside 66 63 PSII reaction center 36 50 Chla*P2* 17 23 * DCMU, 3-(3,4-dichlorophenyI)-l,l-dimethylurea.

scak-lO.65

d0 &O 6b 6b & S O

WAVELENGTH

FIG. 8. Low temperature absorption spectrum of Chl..P2* with component Gaussians fitted by a least squares method. The lowest curve is the difference between the original data and the sum of the Gaussians. The parameters relating to the component curves are given in Table 111.

scale m15.14 h - 1 - v

670 690 710 730 750 770

WAVELENGTH

FIG. 9. Low temperature fluorescence emission spectrum of Chl..P2* with component Gaussian bands fitted as in Fig. 9. Parameters of the curves are given in Table 111, those centered a t 689.0 and 700.8 nm being characteristic of the native complex.

CP29-A complex with a higher chlorophyll a/b ratio and slightly lower electrophoretic mobility than monomeric LHCII, containing a single apoprotein, was first isolated by Machold and Meister ( 5 ) and was described as being an internal antenna of PSII by Camm and Green (6, 23). In our hands, the complex from maize has a chlorophyll a /b ratio of 2.5, a red absorption maximum of 674 nm, and a 77°K fluorescence emission maximum a t 680 nm. Under denaturing conditions, a single polypeptide of 31 kDa is resolved which shows immunological cross-reactivity with LHCI. The pres- ence of a common epitope would suggest amino acid sequence

Chlorophyll-proteins of the PSII Antenna System 13339

TABLE 111 Low temperature spectral properties of Chl,, . PP

Absorption (a = 3.62) Fluorescence emission (a = 2.32)

Wave- Half- Ampli- Area Wave- Half- Ampli- Area leneth width tude leneth width tude

nrn nrn '?6 nrn nrn 75 661.6 16.3 129.0 12.6 680.4 9.3 258.7 9.3 672.6 15.8 842.9 80.2 689.0 16.0 830.9 51.6 681.2 18.0 66.1 7.2 700.8 35.4 199.6 27.4

744.9 38.8 78.2 11.7

+ Mg++

H I ;;i;;;;;;;;;;; n ............ .. . .. . . . .::

..........._ ........... ...........

- Mg"

H F l r ............ ........... ........... ............

FIG. 10. Diagram of the separation of octyl glucoside-solu- bilized PSII membranes separated by sucrose gradient ultra- centrifugation in the presence or absence of M?'.

homology between CP29 and the two antenna proteins of PSI (LHCI-680 and LHCI-730).

CP26"As far as we know, this complex has not previously been described. It can only be resolved from purified PSII membranes under strictly controlled conditions of solubiliza- tion. It has a chlorophyll a b ratio of 2.0, a red absorption maximum a t 671 nm, and a 77°K fluorescence emission max- imum a t 680 nm. It is composed of two polypeptides of 28.5 and 29 kDa which are not antigenically related to LHCI or CP29. Its relationship to LHCII, which it resembles in many respects, is unclear since not only does it have a different polypeptide composition from that of LHCII**, but neither of its two polypeptides becomes phosphorylated.

CP24"The properties of the fastest moving antenna chlo- rophyll-protein of PSII appear to be identical with those of the PSI antenna known as LHCI-680. This includes immu- nological cross-reactivity with a monoclonal antibody raised against barley LHCI-680. The possibility of primary amino acid sequence differences between the polypeptides of LHCI- 680 associated with PSI and CP24 cannot be ruled out, especially in view of the weaker reaction of the antibody to CP24 versus whole thylakoids (Fig. 6, lanes 2' and 7'). All of the spectroscopic and polypeptide data of our CP24 are iden- tical to those of the CP24 of PSII membranes recently re- ported by Dunahay and Staehelin (7). These authors consid- ered it to be a linker between the reaction center and the antenna chlorophyll-proteins, analogously to the pigmented linker polypeptides in cyanobacteria and LHCI of PSI. They argue that it is unlikely to be related to LHCI, based on its fluorescence emission spectrum, but did not distinguish be- tween LHCI-730 and LHCI-680, the latter having an identical fluorescence emission spectrum to CP24 (Table I).

The use of a monoclonal antibody against LHCI from a barley PSI preparation showed that CP29 and CP24 from

CP47 - CP43-

32 kD OEC - CP29 - CP26 =

CP24{

+ Mg** -Mg**

I l T r n I I r r n E 1 2 'D

66

45

-36

-29

-24

-20.1

-14.2

FIG. 11. Polypeptide composition of the fractions from the sucrose gradient after SDS-PAGE in the presence of 6 M urea. The lanes correspond to the fractions described in Fig. 10 and Table IV, and lanes 1 and 2 contain PSII membranes and thylakoids, respectively.

TABLE IV Chlorophyll-protein composition of the green bands separated by

sucrose Rradient ultracentrifugation Band +MZ' "2'

I CP29, CP26, CP24 CP43 (minor), CP29, LHCII**,

I1 CP47,CP43, LHCII**, CP43, CP29, CP26, LHCII**,CP24

111 CP47,CP43,LHCII**, CP47,CP43, CP29,CP26,LHCII**,

IV CP47, CP43 (minor)

CP26, CP24

CP24 (trace)

CP24 CP24

maize PSII membranes share a common epitope with the LHCI of the PSI antenna. Camm and Green (23) have re- ported that CP29 was not recognized by polyclonal antibodies raised against LHCII. A lack of cross-reactivity between the polypeptides of LHCI and LHCII is a consistent finding of many studies using both monoclonal and polyclonal antibod- ies raised against LHCII (24-28). Recent observations (29) regarding cross-reactivity between LHCI and LHCII may have resulted from the use of polyclonal antibodies recogniz- ing previously undetected epitopes. This is supported by the work of Darr et al. (30) who raised monoclonal antibodies against LHCII and reported two classes of antibodies which cross-reacted with LHCI polypeptides.

The complex called Chl.. P2* is a functional PSII prepara- tion lacking CP43, the chlorophyll ab-antenna proteins, and the extrinsic components of the oxygen evolving complex. It is equivalent to a PSII complex isolated from Synechococcus, called CP2-b (31-33). This is apparently more stable than Chl.-P2*, which has not previously been isolated from higher plants. The presence of glycerol during solubilization and

13340 Chlorophyll-proteins of the PSII Antenna System

.. . .. ..

FIG. 12. Model of the organization of PSII as deduced from the fractionation experiments with maize PSII membranes. The diagram is drawn parallel to the plane of the membrane so that the proteins extend above and below the surface of the page. The dotted lines indicate bonds stabilized by M P . Arrows indicate the preferred sites of cleavage by octyl glucoside in the presence or absence of Mg2'. cyt b-559, cytochrome b,,,.

W M t

0 FIG. 13. Hypothetical model of excitation energy transfer

in PSII, based on the results of fractionation of octyl gluco- side-solubilized PSII membranes.

electrophoresis may play an important role in the stabilization of hydrophobic binding between the components of Chl,. P2*, as well as LHCII monomers. The functional properties of Chl,. P2* clearly show that CP43 is not an essential part of the PSII reaction center core (cf. Ref. 34), as emphasized by Yamagishi and Satoh (33).

A preliminary model for the organization of the antenna chlorophyll-proteins of PSI has been proposed from the re- sults of fractionation and reconstitution studies (4, 35), sug- gesting a linear energy transfer sequence. In PSII, four chlo- rophyll ab-antenna proteins have now been found in addition to at least two chlorophyll a-proteins close to the reaction center itself (CP47 and CP43). The results of sucrose gradient fractionation of octyl glucoside-solubilized PSII membranes in the presence or absence of M$+ indicates an important role for CP43. We conclude from these studies that CP43 is required for the binding of LHCII to the PSII reaction center complex Chl,-P2*. Furthermore, M$+ is important in stabi- lizing the association between CP43 and CP47, as well as

between CP43 and LHCII. In the presence of M$+, the weakest binding is that between CP43 and CP24, the latter functioning as a linker between CP43 and the CP29.CP26 complex. These results are summarized in Fig. 12. In this model, two antenna systems independently transfer excitation energy to the PSII reaction center. The major one in terms of the number of chlorophyll molecules consists of LHCII oligomers, and the other consists of CP29 and CP26, both systems being connected to the reaction center via CP43 (Fig. 13). Reconstitution studies involving the measurement of fluorescence lifetimes are in progress to test this model. Since so much of the light-harvesting chlorophyll is in LHCII, the role of the other light-harvesting complex (CP29, CP26, and CP24) may be to remove excess excitation energy arriving at the PSII reaction center from LHCII. Under photoinhibitory conditions, this would provide a means of energy dissipation without damaging the reaction center.

Acknowledgments-We wish to thank Prof. Diter von Wettstein for his encouragement and helpful discussions. Prof. S. Gennari is thanked for making available the use of the Perkin-Elmer MPF 44 spectrofluorimeter, and Giorgio Varotto for growing the maize seed- lings. We thank Dr. Jeannette Brown of the Carnegie Institute, Washington, for making available the program RESOL for the Gaus- sian deconvolution. We are grateful to Nina Rasmussen for drawing the figures.

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