Characterization of Chlorophyll <i>a/b</i> Proteins of Photosystem I from <i>Chlamydomonas reinhardtii</i>

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Reference

Characterization of Chlorophyll a/b Proteins of Photosystem I from Chlamydomonas reinhardtii

BASSI, Roberto, et al.

Abstract

In this study we have isolated the chlorophyll a/b-binding proteins from a photosystem I preparation of the green alga Chlamydomonas reinhardtii and characterized them by N-terminal sequencing, fluorescence, and absorption spectroscopy and by immunochemical means. The results indicate that in this organism, the light-harvesting complex of photosystem I (LHCI) is composed of at least seven distinct polypeptides of which a minimum number of three are shown to bind chlorophyll a and b. Both sequence homology and immunological cross-reactivity with other chlorophyll-binding proteins suggest that all of the LHCI polypeptides bind pigments. Fractionation of LHCI by mildly denaturing methods showed that, in contrast to higher plants, the long wavelength fluorescence emission typical of LHCI (705 nm in C. reinhardtii) cannot be correlated with the presence of specific polypeptides, but rather with changes in the aggregation state of the LHCI components. Reconstitution of both high aggregation state and long wavelength fluorescence emission from components that do not show these characteristics confirm this hypothesis.

BASSI, Roberto, et al . Characterization of Chlorophyll a/b Proteins of Photosystem I from Chlamydomonas reinhardtii . Journal of Biological Chemistry , 1992, vol. 267, no. 36, p.

25714-25721

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http://archive-ouverte.unige.ch/unige:138213

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Characterization of Chlorophyll a/b Proteins of Photosystem I from Chlamydomonas reinhardtii"

(Received for publication, June 24, 1992) Roberto BassiSQlI, Su Yin SoenQ, Gerhard Frank11

,

Herbert Zuberll

,

and Jean-David RochaixQ

From the SDipartimento di Biologia, Uniuersita di Padoua, via Trieste, 75 I-35121 Padoua, Italy, the SDepartement de Biologie Moleculaire, Universite de Geneue, 30, Quai Ernest Ansermet CH-1022 Geneue, and the IIZentralinstitut fur Molekularbiologie und Biophysik, Eidgenossische Technische Hochschule, Zurich, Switzerland

In this study we have isolated the chlorophyll a/b- binding proteins from a photosystem I preparation of the green alga Chlamydomonas reinhardtii and char- acterized them by N-terminal sequencing, fluores- cence, and absorption spectroscopy and by immuno- chemical means. The results indicate that in this or- ganism, the light-harvesting complex of photosystem I (LHCI) is composed of at least seven distinct polypep- tides of which a minimum number of three are shown to bind chlorophyll a and b. Both sequence homology and immunological cross-reactivity with other chloro- phyll-binding proteins suggest that all of the LHCI polypeptides bind pigments. Fractionation of LHCI by mildly denaturing methods showed that, in contrast to higher plants, the long wavelength fluorescence emis- sion typical of LHCI (705 nm in C. reinhardtii) cannot be correlated with the presence of specific polypep- tides, but rather with changes in the aggregation state of the LHCI components. Reconstitution of both high aggregation state and long wavelength fluorescence emission from components that do not show these char- acteristics confirm this hypothesis.

The chlorophyll-binding proteins of the photosynthetic ap- paratus of higher plants and green algae can be divided into two major groups. The first includes chlorophyll a-binding proteins which belong to the photosynthetic core complexes and which catalyze electron transport reactions. The second contains the chlorophyll a/b-binding proteins of the light- harvesting complexes (LHC),' which collect and transfer light energy to the reaction centers. The light-harvesting complex associated to PSI (LHCI) has been isolated from several species and shown to have a lower Chl b content and a fluorescence emission (at 77 K) shifted toward longer wave- lengths as compared with LHCII, its PSII counterpart (1-5).

However, the total number of polypeptides and the identity

* This work was supported by Grant 31.26345.89 from the Swiss National Fund and by Grant 4.7240.90 from the Italian Ministry of Agriculture and Forestry. 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.

7 To whom correspondence should be addressed Dipartimento di Biologia, via Trieste 75, 35121 Padova, Italy. Tel.: 39-49-8286345;

Fax: 39-49-8286374.

The abbreviations used are: LHC, light-harvesting complexes; a- CP29, antibody against CP29; Cab, chlorophyll a/b proteins; CP, chlorophyll-protein complex; PSII, photosystem 11; Chl, chlorophyll;

DM, dodecyl b-D-maltoside; IEF, isoelectrofocusing; LHCII, the ma- jor light-harvesting complex of PSII; LHCI, light-harvesting complex

of photosystem I; PAGE, polyacrylamide gel electrophoresis; RC, reaction center; Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]

glycine.

of chlorophyll-binding proteins of this complex are still un- clear. Higher plant LHCI has been shown to consist of two components called LHCI-680 and LHCI-730 according to their low temperature fluorescence emission peaks (3-5), whereas a single high molecular weight complex, called CPO, was described in Chlamydomonus reinhardtii (1). The long wavelength fluorescence emission, although less shifted in C.

reinhardtii, 705 versus 730 nm in higher plants, is still signif- icantly different from the fluorescence of LHCII (685 nm).

Here we report the isolation of the PSI.LHC1 complex of C. reinhardtii and the purification of its components. Two- dimensional electrophoresis resolved at least seven LHCI polypeptides as shown by N-terminal sequencing and immu- nological analysis. Three of these were purified as pigment- bound complexes, but immunological cross-reactions and se- quence homologies to known chlorophyll-binding proteins suggest that the whole set of polypeptides bind pigments. The mild isolation techniques used in this work allowed us to purify LHCI with spectral properties significantly different from those reported previously. The absorption spectra sup- port the previous proposal that the long wavelength absorp- tion components acting as a sink for the excitation energy belong to the pericentral chlorophyll a/b proteins rather than to the chlorophyll a binding core complex. Finally the long wavelength emission at 705 nm could not be attributed to a specific polypeptide but was shown instead to be a property of the higher molecular weight aggregation forms of LHCI. In agreement with this observation, a high molecular weight form of LHCI reconstituted from isolated LHCI components, also showed 705-nm fluorescence emission.

MATERIALS AND METHODS

Isolation of Thylakoids-C. reinhardtii wild type cells were grown in Tris acetate-phosphate medium at a light flux of 200 lux and used in the mid-eponential growth phase (3 X lo6 cells/ml"). Thylakoid membranes were prepared as in (1).

Photosystem I Preparation-Thylakoid membranes were fraction- ated following the procedure previously described for Lemna gibba by Bruce and Malkin (6) with some modifications. Briefly, the mem- branes were resuspended in 0.3 M sucrose, 0.05 M Tris-HC1, pH 7.8, 0.01 ^M NaCI, 5 mM MgC12 (TS) containing 2 M NaBr for 30 min, diluted with an equal volume of distilled water, and centrifuged at 12,000 X g for 10 min. NaBr washing was repeated once and mem- branes washed twice with TS and resuspended in the same buffer at 3 mg Chl/ml. An equal volume of TS containing 10% ammonium sulfate and 60 mM octyl glucoside and 1% sodium cholate was added, and after incubation in ice for 30 min, the suspension was centrifuged at 250,000 X g for 1 h. The pellet was resuspended in distilled water at 1 mg Chl/ml, and Triton X-100 (Serva) was added from a 20%

stock solution to a final concentration of 0.45%. The suspension was incubated at 25 "C for 1 h with slow agitation and centrifuged first at 1500 X g for 10 min to eliminate starch and insoluble debris and then at 48,000 X g for 20 min to eliminate the supernatant containing most of PSII and LHCII. The pellet was resuspended in water at 1 mg/ml and further solubilized for 1 h at 25 "C with 0.9% Triton X-

25714

LHCI Proteins

of

C. reinhardtii

25715 100. The solubilized material was loaded on a 0.1-1 M sucrose gradient

containing 0.06% Triton and centrifuged overnight at 110,000 X g.

The PSI-LHCI complex was harvested at the interface with the cushion, diluted four times with TS, and pelletted at 48,000 X g for 1 h. Aliquots in TM at 3 mg Chl/ml were frozen in liquid nitrogen and stored at -80 “C. Alternatively a more rapid procedure was used as described previously (7).

Preparation of LHCI-LHCI-680 and LHCI-705 were prepared as reported previously(5).

Flat-bed Isoelectrofocwing-This was performed as reported pre- viously (8).

SDS-PAGE and Immunoblotting-Analytical SDS-PAGE was per- formed with gradient gels (12-18% acrylamide, 350 X 350 X 1 mm) containing 6 M urea and run at 10 mA using the Tris sulfate buffer system as described previously (9). Alternatively, a Tris-Tricine sys- tem without urea (10-16% acrylamide gradient) was used (10). Two- dimensional electrophoresis was performed by using the Tris-sulfate- urea system in the first dimension and the Tris-Tricine system in the second dimension. For immunoblot assays, samples were separated by one of the gel systems described above and transferred to a nitrocellulose filter (Millipore, Bedford, MA).

The filters were then incubated with antibodies, and antibody binding was detected by using alkaline phosphatase coupled to anti- rabbit IgG (Sigma). Antibodies were raised in rabbits and character- ized as described previously but using poly(A)-poly(U) as adjuvant (11).

Quantitation of Coomassie-stained proteins in SDS-PAGE gels was carried out following the procedure of Ball (12) as described previously (13).

Other Methods-Chlorophyll and carotenoid determinations were made in 80% acetone, using the equations of Wellburn and Lichten- thaler (14) (see this reference for limitation in the determination of carotenoids) or Porra et al. (15). The two methods yielded very similar results for Chl a to Chl b ratios ranging from 1 to 3. Absorption spectra of chlorophyll a/b proteins were taken in 10 mM Hepes, pH 7.6,0.06% dodecyl maltoside using a Kontron 930 spectrophotometer.

Slit width was 1 nm. Fourth derivative analysis was obtained with a built-in microcomputer using a 4-nm span. Protein determination in solution was by the bicinchoninic acid method (16) or by quantitative amino acid analysis as reported previously (13).

RESULTS

Identification of LHCI Polypeptides-We first used antibod- ies to higher plant polypeptides to identify homologous pro- teins in C. reinhardtii. In Fig. 1 the result of probing C.

C.r. Thylakoids (KDa Mw

42.1

- 10 -9

31

21.5

-11113

-

¹⁵

-16

-

^14114.2

-14.1 -15.1/18/18.1

- 17.1 -171122

-22/221/21

- ^22.2

coomassie irnrnuno stain blot

FIG. 1. SDS-urea-PAGE of C. reinhardtii thylakoids and immunobbt with anti-maize PSI-LHCI antibodies which rec- ognize all chlorophyll &-binding proteins in higher plants.

p9, p10, pll, p13, p16, p17, and p22 have been shown previously to be enriched in PSII membranes (7). The remaining bands are tenta- tively assigned to LHCI.

reinhardtii thylakoids with a polyclonal antibody directed to the maize PSI.LHC1 complex, which recognizes all of the maize chlorophyll a/b proteins (11), is shown. Following SDS- urea-PAGE, 12 bands are revealed. Among these polypeptides, six, p9, p10, pll, p13, p16, and p17 have been attributed to the PSII antenna system, because they are enriched in isolated PSII membranes, and because their properties resemble those of the higher plant PSII components (7). At least six other bands, which show higher mobility on this gel system as compared with PSII chlorophyll a/b proteins, were shown to be depleted in PSII membranes (7) and can therefore be assigned to LHCI; these are p17.2, p14.1, p15.1, p18, p17.1, and p22.

Isolation and Characterization of PSI-LHCI Complexes from C. reinhardtii-Further insights into the composition of the LHCI of C. reinhardtii was obtained by the isolation of the complex by an adaptation of the procedure of Bruce and Malkin (6). This method is rather lengthy; we found, however, that in contrast to other methods (17), it does not result in the loss of LHCI polypeptides from the PSI ^e LHCI complex as determined by Western blot analysis of the different frac- tions during the preparation. The polypeptide composition of this preparation is shown in Fig. 2 as determined by two- dimensional SDS-PAGE.

Besides the two high molecular weight polypeptides p2a and p2b, the products of the psaA and psaB genes wich have been shown to bind P-700, two groups of polypeptides are present. The first, with sizes in the 18-30-kDa range, is heavily stained, whereas others are smaller and less abundant.

We have only examined the first group because it includes polypeptides resembling known cab polypeptides as judged by their immunological cross-reaction and their size range (for review cf. Ref. 18). The apoproteins were isolated by two- dimensional preparative SDS-PAGE and electroelution.

These were then transferred to “Immobilon-P” membranes for N-terminal sequencing. The results of sequence analysis are shown in Fig. 3. Out of 11 spots resolved by two-dimen- sional SDS-PAGE in the 18-30-kDa range, 8 yielded N- terminal sequences, one (p15) was N-terminally blocked, one (p17.2), yielded heterogeneous N-terminals, and p14.2 was obtained in too small amounts. The sequences of seven poly- peptides (p14, p14.1, p15.1, p.18, p.18.1, p.22, and p22.1) could be aligned and showed homology with each other and with cab proteins from C. reinhardtii and higher plants. An addi- tional polypeptide was not homologous to the others; its sequence was identical to that of the PSI-RC subunit p21 encoded by the psaF gene (19).

Stoichiometry of Polypeptides-We then determined the stoichiometry of the polypeptides in the PSI ^e LHCI complex relative to the 2a and 2b polypeptides. This was done by determining the specific binding of CBB to purified apopro- teins (Fig. 4). Because of comigrations, we first determined the binding of Coomassie to groups of polypeptides, namely 2a+2b, 17.2+14+14.1, 15.1+18+18.1, 21+22+22.1, and we then repeated the procedure after cutting the bands from the first dimension and rerunning them in a different gel system.

The results are shown in Table I. It appears that the polypep- tides in the molecular mass 17-30-kDa range are present in variable number of copies per PSI.RC ranging from 1 (p15, Immunological Analysis-Purified apoproteins were also used for immunological analysis. Antigens were injected into rabbits, and antisera were obtained against p15, p17.2, p14, p15.1, p18.1, p21, and p22.1. These were used to challenge both the purified polypeptides and the thylakoid membranes.

Results are summarized in Table 11.

The injection of purified proteins yielded rather specific antisera for p14, p15.1, p22.1, and p21. However, cross-reac- p21) h J 0-7 (111 7.2, IIlfi.1).

25716

C.

reinhardtii B

FIG. 2. Two-dimensional SDS- PAGE ^of C. reinhardtii thylakoid membranes and of the PSI.LHCI preparation. In the first dimension ( A ) , the samples were fractionated by SDS-6 M urea-PAGE (12-18% acrylam- ide) using the Tris sulfate buffer system (9). The lower part of the gel, including polypeptides with apparent molecular mass lower than 45 kDa, was excised and loaded into the second dimension ( B ) on a n SDS-PAGE (10% acrylamide) gel with the Tris-Tricine buffer system (10).

WT, whole thylakoids.

A PSI- LHCI WT

(Koa1 MVJ

9 7 4 - 662 -

4 2 7 -

P 2 1 DIAGLTPCIXEKKAYAKLXKKXLKTLXKRLK P21'" DIACLTPCISESKAYAKLEKKELKTLEKRLK P I 4 AAVPENVKEASEWDIYUKSK~CAKFDAG---L

P l 4 . 1 EEKSIAKVDRSKDo~VCASQSSUYLDCS----LffiDFCFDPLGL p15.I

P l B . 1

...

MDRKLUAPCWAPEYLKCD----~CDYCUDPLGLCANPT ASSRPLWLPGSTPXAHLKGD----l.PCDFGFD?L

RQSWLPCSQIPAHLDTPAAQAUCNFCFDPV.LGKDPVAL KA6HVLPGSDAPAWLPDD""LPGNYGFD~LSLGD~PASLK CP24" AAAAPKKSWPAVKCCCLFNDPEWLDCS----LPDCFCFDPLGLCKDPAFLK CabII-I"' APKSSCVEFYGPN~KWLGPYSeP*YLDTP----F~DYGUDTAGI.S*DP~F

FIG. 3. N-terminal sequence of the proteins from the PSI.

LHCI complex. The best alignment was obtained with the FASTA program. CP24 (24) and CABII-I sequences (35) are shown as refer- ence for the alignment. The highly homologous sequences are under- lined. The p21 sequence did not align with CAB proteins but with the plastocyanin-binding protein of PSI.RC (19) which is shown below. (I), from Franzkn et al. (19); (2), from Schwartz and Pichersky (24); (3), from Imbault et al. (35).

2.5

-

;

pg protein

FIG. 4. Determination of specific Coomassie binding to PSI.

LHCI polypeptides. Increasing amounts of purified polypeptides were loaded into a SDS-PAGE gel. After electrophoresis and staining, the Coomassie was quantified following elution from individual bands.

tions were clearly detected, since the antiserum directed against p17.2 also recognized p14, anti-pl8.1 recognized p17.2, p9, and p10, whereas anti-p22.2 recognized p22.1. A summary of the cross-reactions is shown in Table 11.

The purified antigens were also challenged with antibodies directed to C. reinhardtii cab proteins of PSI1 (7). The anti- serum directed against p10 (corresponding to higher plants CP26) recognized p15, p15.1, p17.2, and p18, whereas anti- p13 recognized p15, p17.2, and, faintly, p14.1.

PSI

-

, WT LHCI

,~

¹ -,

427 -

310 -

21.5 -

1 4 4 -

0 ^3"

p

^{\ nc,}

TABLE I

Stoichiometry of PSI-LHCI polypeptides as determined by the quantification of Coomassie binding

Polypeptide ^Coomassie Brilliant _Blue" Corrected*

gi:i

^Molecular massC

2a+2b 2.0 12.48 3.9 82

15 1 1.36 0.7 31

17.2 2.1 30

14 4.3

30 14.1 34.2 30 6.0 3.9

14.2 2.0

30

15.1 15.0 9.52 7.7 26

18 2.3

25 18.1 5.06 25 8.3 2.0

21 0.9 18

22.1 11.6

22.6 5.8

21 4.1 22.2

21

The less stained polypeptide was set to 1.

*

Correction was made for molecular mass and specific Coomassie Molecular mass values were used as obtained from urea gels unless Brilliant Blue binding.

the value deduced from the gene sequence was known.

LHCI polypeptides were also challenged with antibodies directed against the two moieties of LHCI from higher plants known as LHCI-680 and LHCI-730. These two complexes in higher plants contain different polypeptides (5, 11). The re- sults show that p18.1 is only recognized by anti-LHCI-730, whereas p15, p22.1, and p22.2 are only recognized by anti LHCI-680. Several others C. reinhardtii LHCI polypeptides are recognized by both higher plants LHCI antibodies.

As can be expected from the similarities of their N-terminal sequences, several C. reinhardtii LHCI polypeptides are rec- ognized by an antibody directed against higher plants CP24 (Fig. 3 and Table 11).

Fractionation of the PSZ.LHCZ Complex by Sucrose Gra- dient Ultracentrifugation-Previous studies on higher plant PSI

-

LHCI showed that it consists of two distinct complexes named LHCI-680 and LHCI-730 according to their fluores- cence emissions at 77 "C. However, in C. reinhurdtii, LHCI was isolated as a single high molecular weight complex (1).

To clarify this point, we have isolated the PSI. LHCI complex

LHCI

Proteins of

C.

reinhardtii 25717 TABLE I1

Immunological cross-reactions of C. reinhurdtii polypeptides belonging to PSI-LHCI complex Antigens

15 17.2 14 14.1

15.1 Antibodies 18 18.1 22.2 22.1 21 9 10 11

P9

+ + +

^+/-

++ + ++ ++

PI0

+++ ++ + ++ + ++ +

P22

+++ +

PI1

+++ +

P13

++ +

^+/-

LHCI-730

+ +++

LHCI-680

+ + +

+/-

+ ++ + ++ + ++

CP24

+ + + + + ++ ++

PSI-LHCI ND"

+ + ++ + + + ++ +++ + ++

P15

+++

^+/-

+ +++

p17.2

++ +

P14

+

p15.1

+

p18.1 +/-

++ + ++

P21

+

p22.2

+ ++

ND, not determined.

PSbRC.LHCP,IO,OEEI LHCl-680 LHCI-705 F'Sl,RC(+LHCI)

Mw kDa

97 L 56.2

L2.7

Mw kDa

97.4 66.2 L2.7

' 31.0

SDS-urea 6 M SDS

FIG. 5. Fractionation of PSI. LHCI complex by sucrose gra- dient ultracentrifugation and characterization of fractions. A, schematic drawing of the sedimentation pattern after solubilization of the crude PSIeLHCI complex (shown in B ) with 1% DM. Ultra- centrifugation was at 4 "C for 20 h at 39,000 rpm in a Bechman SW 41 rotor. The upper green band contained non-PSI-LHCI contami- nant polypeptides. B, polypeptide composition of the fractions from the PSI.LHCI complex as determined by SDS-urea-PAGE (left panel) or SDS-PAGE (right panel). PSI. RC, preparation depleted of Chl-binding polypeptides (see text); band 2 of A, LHCI-680; bund 3 of A, LHCI-705; band 4 of A, PSI.LHCI; PSI-TX-100, crude PSI preparation.

by a simplified procedure (see "Materials and Methods"), and after solubilization with 2% DM, we further fractionated this complex by sucrose gradient ultracentrifugation (Fig. 5A).

The results are shown in Fig. 5, B and C. Four green bands were present after centrifugation. The upper one contained contaminating polypeptides including pll, p10, p9, etc., the

1

LHCI-680 LHCI-705 LHCI-IEF

- *

P

l- I -

FIG. 6. Two-dimensional SDS-PAGE analysis of PSI-LHCI and LHCI preparations. The separation was obtained by combin- ing the two separation methods of Fig. 5B). For the sake of simplicity, only the gel region including LHCI polypeptides is shown. Abbrevi- ations are the same as in the legend of Fig. 5.

second and the third bands contained only LHCI polypep- tides, whereas the bottom band contained LHCI and the PSI RC complex. According to their fluorescence emission peak at 77 K (see later) the chlorophyll-proteins in bands 2 and 3 will be named LHCI-680 and LHCI-705, respectively, in agreement with previous work with higher plant LHCI. Poly- peptide analysis of these complexes, however, showed that they had a very similar polypeptide composition (Fig. 5) as further confirmed by two-dimensional electrophoresis (Fig.

6). The main difference between LHCI-705 and LHCI-680 did not concern LHCI polypeptides but rather p21, a colorless PSI

-

RC subunit, which was present in LHCI-680 but absent in LHCI-705 as shown by two-dimensional electrophoresis (Fig. 6). It was difficult to completely remove LHCI polypep- tides from PSI-RC. This was achieved by further pelleting and solubilization of the PSI. LHCI complex (sucrose band 4) with zwittergent and DM. The polypeptide composition of this PSI-RC preparation which has a very high (>20) Chl a/

b ratio, is shown in Fig. 5B, lane I, and 5C, lane 2.

Fractionation of LHCI by Flat-bed IEF-To determine the identity of chlorophyll-binding polypeptides within the LHCI complex, we have solubilized the PSI. LHCI complex in highly dissociating conditions (0.6% zwittergent 16 plus 1% DM) and fractionated it by flat-bed IEF. This yielded seven green bands in the pH range of 4.1-4.8 which only contained LHCI polypeptides as determined by SDS-urea-PAGE (Fig. 7).

Other green bands were present at higher pH values (PI = 5- 6.5), but these will not be further considered, since LHCI was still bound to the PSI

-

RC. As shown in Fig. 7B, IEF bands 4,

A

4.8

-

4.6

-

4.4

-

4.2

-

&.O1

_B ;o

Distance from the anode

&

$0

do

^{IiO crn}

’

Mw (kDa)

- ^-

^{Y I ” ” ”}

^-

-31 ^{- 20.5} - 14.1 I 2 3 L H C I 4 5 6 7

FIG. 7. Flat-bed IEF analysis of LHCI chlorophyll binding proteins. A, plot of the position of the green bands in the pH gradient within the gel. B, SDS-urea-PAGE analysis of the IEF fractions. The two central lanes were loaded with two LHCI preparations as a reference: LHCI-705 and LHCI-IEF. For the sake of simplicity, only bands containing, exclusively, LHCI polypeptides are shown, whereas bands including PSI. RC, which exhibited PI values higher than 4.8, are omitted.

5, and 7 contained a single polypeptide (p17, p15, and p22, respectively), thus showing that at least three chlorophyll- binding proteins are present in C. reinhardtii LHCI.

Spectroscopic Characterization-The LHCI of C. reinhardtii is characterized by its peculiar fluorescence emission at 77 K peaking at 705 nm (1). In an attempt to identify the LHCI subunit responsible for this emission, we have analyzed the fractions, obtained as described above, by low temperature fluorescence emission spectroscopy. Absorption spectra were also collected and analyzed by their fourth derivative to obtain information about the distribution of chlorophyll absorption forms within the PSI antenna. As shown in Fig. 8A, the two LHCI preparations isolated by sucrose gradient ultracentrif- ugation (bands 2 and 3 in Fig. 5) were each characterized by a single emission peaking at 685 and 705 nm, respectively.

Their absorption spectra showed maxima at 673 nm (LHCI- 680) and 680 nm (LHCI-705) (Fig. 8B), the shift being due to the increased contribution of a 669-nm absorption component in LHCI 680 with respect to the major form peaking at 681- 682 nm as revealed by fourth derivative analysis (Fig. 8C).

When PSI preparations without and with LHCI (Fig. 5B, lane I ) were analyzed, the higher wavelength absorption com- ponents (Fig. 9B) were enriched in the latter; PSI.RC and PSI. LHCI showed a major component at 677 and 682 nm, respectively. This yielded a red shift in the absorption spectra from 677 to 679 nm. (Fig. 9A). The 77 K fluorescence emission of the two preparations peaked at 705 nm (PSI. LHCI) and 720 nm (PSI. RC) in agreement with previous reports (data not shown). The subfractions obtained by flat-bed IEF of zwittergent/DM-solubilized LHCI were also analyzed. As shown in Fig. 7 (lanes 4, 5, and 7) p17, p15, and p22 were recovered as single chlorophyll-binding polypeptides. Other fractions containing these same polypeptides, together with others, have differences in their absorption spectra as also shown by their fourth derivative analysis (Fig. 10, A and B ) , thus suggesting that most or all of the LHCI proteins bind

600 750 650 700 Wavelengl h I nml

“LHCI-705 ....- LHCI-680

--.-.LHCI-IEF ₆₇₃ 680 .-

-

_yl

-

Wavelengl h Inml

1

682 A

, .

* .

I I 1’

600 630 660 690 720

Wavelenglh lnml

FIG. 8. Spectroscopic characterization of LHCI prepara- tions. Excitation was at 430 nm (10-nm slit), and emission was analyzed between 600 and 800 nm with a 4-nm slit. Chlorophyll concentration was 5 pg/ml. Before taking the spectra, the prepara- tions were ultracentrifuged through a sucrose gradient containing 0.06% DM to eliminate free pigments that may be released after freezing and thawing of the samples. A, 77 K fluorescence emission spectra of the two sucrose gradient bands (LHCI-680 and LHCI-705) and of the LHCI complex reconstituted from isolated chlorophyll- proteins (LHCI-IEF); B, absorption spectra of the three samples in A; C, fourth derivative analysis of absorption spectra.

pigments. The solubilization treatment, however, strongly affected the spectroscopic properties of LHCI chlorophyll- proteins, their absorption maxima being blue-shifted by up to 12 nm in comparison with the intact LHCI-705 complex. This is reflected by the 77 K fluorescence emission spectra which exhibits mainly a 678-nm peak. Only IEF fractions 1 and 4 showed a shoulder at higher wavelength (705 and 735 nm, respectively, Fig. 1OC).

Reconstitution of LHCI-The preceding analysis of LHCI subfractions suggests that the spectroscopic properties of the complex and (presumably) its “in U ~ U O ” function within the PSI antenna system is dependent on the integrity of the multiprotein complex. This is supported by the fact that the only IEF fraction showing a 705-nm emission component, reminiscent of the original situation, contains several poly- peptides. However, the IEF method used does not lead to the

LHCI Proteins of C. reinhardtii 25719

" P S I - R C 677 . - n 6 7 9 ."" PSI'LHCI

"""" ~

."

..-

0~ ^{~ ~} 630 690 660 720

Wavelength Inml

PSI-LHCI

I ^{. , . , I .} I

600 630 660 690 720

Wovelength Inml

FIG. 9. Absorption spectra of PSI.RC preparations before and after depletion of LHCI polypeptides. A, absorption spectra;

B, fourth derivative analysis.

loss of pigments. We have therefore attempted to reconstitute LHCI from its components separated by IEF; fractions 1-7, eluted in 1% octyl P-D-glucoside, 10 mM Hepes, pH 7.6, were pooled together and the detergent dialyzed overnight. After concentration and further dialysis, the mixture was centri- fuged in a sucrose gradient containing 0.06% DM. Two green bands, respectively, with low and high mobility, were analyzed by SDS-PAGE and spectroscopy. When a chlorophyll-protein not belonging to LHCI (such as p16, a LHCII component) was added to the mixture, it was found in the upper rather than in the lower sucrose gradient band (Fig. 11).

Interestingly, reconstituted LHCI, which will be indicated as LHCI-IEF, had a higher mobility in sucrose gradients as compared with both LHCI-680 and LHCI-705. Polypeptide analysis of the reconstituted LHCI-IEF, showed that it con- tains essentially the same polypeptides present in LHCI-680 and LHCI-705. However p15 which is absent from LHCI-680 and LHCI-705 is present in LHCI-IEF, whereas only traces of p17.2 are present in this complex. Also, the relative amounts of the polypeptides are different in the three LHCI preparations. The presence of p15 in LHCI-IEF may be responsible for its higher mobility in sucrose gradients. In spite of these differences, the spectroscopic properties of LHCI are essentially restored as shown by the absorption spectrum (Fig. 8B) and fourth derivative analysis (Fig. 8C).

The 77 K fluorescence emission spectrum showed a major 705-nm peak, but a 679-nm emission was also present as a smaller shoulder (Fig. 8A).

DISCUSSION

Polypeptide Composition and Stoichiometry of LHCI- Light-harvesting chlorophyll a/b proteins specifically associ- ated to PSI have been isolated from a number of plant species and shown to contain major polypeptides of 20-24 kDa. In higher plants the assessment of the number of polypeptides involved in LHCI yielded very different results: one in spinach

Wavelength lnrnl

0

~ " ~

C ^r-LHCI/L

- - .

- 0 : -

: :

LL -

600 650 700 750 800

Wavelength lnml

FIG. 10. Spectroscopic characterization of the LHCI subfractions obtained by flat-bed IEF. A , absorption spectra; B, fourth derivative analysis; C, 77 K fluorescence emission spectra.

FIG. 11. Reconstitution of LHCI complex from IEF frac- tions. The fractions from IEF (Fig. 7) were combined and the detergent (0.8% octyl glucoside) dialyzed. The sample was then loaded tubes on a 0.1-1 B and M C the mobility of sucrose gradient containing LHCI-680 and 0.06% DM LHCI-705 _{(tube A).} is shown for In comparison. When included in the mixture, p16, a PSII chlorophyll- protein, did not cosediment with the aggregation form of LHCI but was found instead in the upper band.

(3) and Lemna (20), three in spinach (21) and maize (22), five in maize and barley (4, 5). In this study we have resolved 11 polypeptides in the 20-31-kDa range from a PSI. LHCI prep- aration. These polypeptides are depleted in PSII membranes (7). One of these polypeptides has been identified as the

plastocyanin-binding subunit of the PSI. RC by N-terminal sequencing; the others appear to be chlorophyll-binding pro- teins, since they exhibit cross-reactions with each other and with PSII cab proteins of C. reinhardtii and higher plants.

Although the cross-reaction pattern is quite complex, groups of LHCI can be defined with respect to their immunological cross-reactions: p15, p17.2, p14, and p14.1 are recognized by antibodies directed against LHCII proteins having high Chl b levels (pll and p13); p22.1 and p22.2 are recognized by anti- bodies against both CP24 and p22 (the CP24 counterpart in C. reinhardtii), whereas p15.1, p18, and p18.1 are recognized by antibodies directed against the pericentral cab polypeptides p9 and p10 of PSII from C. reinhardtii (corresponding to higher plants CP29 and CP26). Absorption spectra of LHCI subfractions suggest that the pigment complement of the different LHCI polypeptides is not the same with respect to both Chl a/b ratio and Chl/carotenoid ratio (Fig. 10, B and C; Table 111).

The sequence analysis showed that p14, p14.1, p15.1, p18.1, p22.1, and p22.2 are homologous to each other and to Cab proteins. p18 contains 4 additional amino acids (Ala-Ala-Gln- Ala), starting at position 21, which are not present in the other sequences nor in other cab genes as assessed by a scan of gene and protein banks. However, consensus sequences for type I and type I1 LHCII genes of higher plants show that a sequence of 4 amino acids (Lys-Pro-Val-Ser) is present in type I but not in type I1 proteins near the N-terminal end.

p18.1 has a 72.4% amino acid identity in a 29-amino acid stretch with cab 7 of tomato (23) and with LHCll of petunia.

In general the higher homologies were obtained with LHCI sequences from higher plants, but CP24 proteins (24) were also very similar in the N-terminal region. The lowest ho- mology was observed for p14. Its N-terminal end is rather different and shows only few conserved amino acids. However, this protein could not be sequenced in the high homology region which starts after the 4-amino acid insertion in p18.

It is not yet clear which LHCI subunit actually binds chlorophyll. Nechustai et al. (20) suggested that LHCI of Lemna contains a single polypeptide with a molecular mass of 20 kDa. However, also in Lemna, Bruce and Malkin (6) measured two copies of this polypeptide in LHCI and sug- gested that there must be additional Chl-binding proteins associated with LHCI, since it is unlikely that two copies of the 23-kDa polypeptide could bind the 100 Chl molecules assigned to Lemna LHCI.

We propose that as many as 10 chlorophyll-binding poly- peptides are present in LHCI from C. reinhardtii LHCI (Table IV). This is based on (i) the isolation of three LHCI proteins as single chlorophyll-bindingpolypeptides, (ii) the N-terminal sequences of six polypeptides that are clearly related to each other and to other cab proteins, and (iii) the immunological cross-reactions between individual LHCI polypeptides and other LHCI and LHCII chlorophyll a/b-binding proteins.

Determination of the stoichiometry of polypeptides in the TABLE 111

Characteristics of the chlorophyll-protein complexes or C. reinhardtii PSI

Chl a/Chl b Chl/x+c PSI-LHCI"

4.8 12.2

PSI-LHCIb 15.3 5.4

LHCI-705 2.8

8.2 LHCI-680 2.1 4.5

LHCI-IEF 1.8

7.6 PSI-RC >20

>17

LHCII 1.45

13.8

Purified according to Ref. 6.

Purified according to Ref. 17.

PSI.LHC1 complex reveals that as many as 30-35 copies of LHCI polypeptides per PSI.RC are present in LHCI. Spec- troscopic measurements have shown that as many as 370 Chl molecules are associated with PSI (25) in low light conditions similar to those we have used for cell growth. Since 90 Chl a molecules are bound to CP1 (5), 280 Chl molecules should be attributed to LHCI.

Accordingly a Chl complement of 8-9 molecules should be attributed to each LHCI polypeptide, a value consistent with those previously measured for CP26 and CP29, two chloro- phyll a/b proteins which, as LHCI, contain little Chl b.

These results are consistent with an organization of the PSI antenna system in a complex structure where individual chlorophyll-proteins have precise locations with respect to both PSI. RC and the other antenna polypeptides as recently elucidated for PSII (13, 26, 27). We have obtained two prep- arations of LHCI: LHCI-680 and LHCI-705 according to their fluorescence emissions at 77 K. Both preparations are de- pleted of several polypeptides, in particular p15 is missing, suggesting that this polypeptide is easily dissociated from the rest of the LHCI complex. However, we consider p15 a genuine subunit of LHCI based on its absence in PSII membranes (7) and on its ability to be part of the LHCI complex reconstituted from isolated proteins. However, p15 was shown to be present in the y-lp mutant lacking other LHCI polypeptides (28), and its immunological characteristics as well as high Chl b content suggest that it is structurally similar to LHCII proteins. It was suggested that p15 forms the actual PSI antenna, whereas the role of the other polypeptides of the complex is to connect p15 to the PSI.RC (28). Although we confirm that p15 binds Chl a and b (Figs. 7 and 9) and that it is peripheral with respect to other LHCI polypeptides, a role as bulk antenna can hardly be supported due to its low abundance.

A similar case can be made for p22.2 which is present in the large PSI.LHC1 complex with lower Chl a/b ratio but absent in smaller complexes. p22.1 appears similar to p22.2 but is more tightly bound to the complex. Both of these polypeptides are closely related to p22 which, however, was shown to be enriched in PSII membranes and was identified as the C. reinhardtii counterpart of higher plants CP24.

However, a polypeptide with similar mobility in SDS-PAGE was attributed to LHCI (29).

This situation is reminiscent of the early results obtained with higher plants when it was postulated that an identical polypeptide could participate to both the CP24 complex in PSII and the LHCI-680 complex in PSI, thus acting in both cases as a connecting element between RC and outer antenna complexes (4). Later studies using monoclonal antibodies (11) or N-terminal sequencing showed that these polypeptides are similar but distinct (30).

We obtained a partial protein sequence of p22.1 and p22.2.

p22.1 and p22.2 showed identical N-terminal sequences, but immunological assays (Table IV) and electrophoresis suggest they are different polypeptides. Differences may occur in the inner part of the sequence. However, we cannot rule out the possibility of a small contamination of p22.2 by p22.1 in the bulk procedure used for purification of the proteins for se- quencing. If p22.2 was N-terminally blocked as p15, then the sequence obtained could arise from the p22.1 contamination.

Organization of LHCI Chlorophyll-proteins-LHCI poly- peptides have been shown to be organized into two chloro- phyll-protein complexes with different spectral properties and polypeptide composition in higher plants (3-5).

In C. reinhardtii, however, LHCI-705 and LHCI-680 have a very similar polypeptide composition (Figs. 5 and 6), al- though they are different with respect to Chl a/b and Chl/

carotenoid ratios, absorption spectra, low temperature fluo- rescence emission, and sedimentation rates (Figs. 5 and 7).

LHCI Proteins of C. reinhardtii 25721 TABLE IV

Apparent molecular mass (kDa) of the polypeptides of C. reinhardtii PSI-LHCI complex

Molecular mass in Molecular mass in

SDS-urea-PAGE SDS-PAGE

2a+2b 65-70

15 31 29

17.2 30

25 14

29 25.8

14.1 29 26

14.2 29 25

15.1 26 25.5

18 25 23.5

18.1 25 22.0

21 21.0

22.0

22.1 21.5

21.0

22.2 22.0

21.0

70-75

The major difference is the absence in LHCI-705 of p21, a PSI subunit previously shown to act as a plastocyanin-binding protein (31). The association of this subunit to the LHCI complex was reported previously in higher plants (32). Thus there is a clear difference between C. reinhardtii and higher plants where LHCI-730 and LHCI-680 were shown not to have common polypeptides (4, 5). In C. reinhardtii the spec- troscopic properties of LHCI appear to be strongly dependent on the aggregation state of the complex as shown by (i) the absence of 705-nm emission in LHCI complexes containing one or few polypeptides and (ii) the reconstitution of both long wavelength fluorescence and absorptions from compo- nents that do not show these characteristics. These results imply that the conformation of the individual chlorophyll- proteins participating in the complex derives from cooperative interactions which stabilize the structure, thus maintaining the peculiar conformation of the long wavelength absorbing Chl a molecule responsible for the 705-nm fluorescence emis- sion (33).

Considering the presence of the 705-nm emission in wild type cells and its absence in the ac40 mutant (1) lacking LHCI polypeptides, we suggest that the LHCI complex with high mobility in sucrose gradients, in the reconstitution experi- ment, is not an aggregation artifact but rather the result of specific interactions reflecting the in vivo organization.

The absorption spectra of LHCI and PSI preparations show that the spectral components absorbing at the longer wave- length and therefore at lower energy are located in LHCI rather than in the Chl a binding core of PSI. This is in agreement with the previous report that LHCI is the only fluorescence emitter at 77 K when PSI traps are closed. LHCI thus acts as an efficient sink for the excitation energy ab- sorbed by all the Chl serving PSI. However, in previous reports absorption peaks at lower wavelength were reported (1-5). The milder procedures reported in this work allowed

US to obtain the chlorophyll-proteins in a more native confor- mation and thus show that the components which emit fluo-

rescence are also absorbing at lower energy as should be expected.

It is not clear yet whether the pericentral antenna com- plexes act as energy sinks during the process of energy transfer to the reaction centers and are therefore essential for the photosynthetic energy transfer or if light is funneled to them only if reaction centers are closed and therefore pericentral antennae act as a protection guard against photodestruction (34).

Further studies are needed to elucidate the role of pericen- tral antennae in photosynthesis.

Acknowledgments-We thank Dr. Reto Strasser for help in meas- uring low temperature fluorescence spectra and Otto Jenni for pic- tures and photography.

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