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Directed disruption of the Chlamydomonas chloroplast psbK gene destabilizes the photosystem II reaction center complex

TAKAHASHI, Yuichiro, et al.

Abstract

Using particle gun-mediated chloroplast transformation we have disrupted the psbK gene of Chlamydomonas reihardtii with an aadA expression cassette that confers resistance to spectinomycin. The transformants are unable to grow photoautotrophically, but they grow normally in acetate-containing medium. They are deficient in photosystem II activity as measured by fluorescence transients and O2 evolution and they accumulate less than 10% of wild-type levels of photosystem II as measured by immunochemical means. Pulse-labeling experiments indicate that the photosystem II complex is synthesized normally in the transformants. These results differ from those obtained previously with similar cyanobacterial psbK mutants that were still capable of photoautotrophic growth (Ikeuchi et al., J. Biol. Chem.

266 (1991) 1111–1115). In C. reinhardtii the psbK product is required for the stable assembly and/or stability of the photosystem II complex and essential for photoautotrophic growth. The data also suggest that the stability requirements of the photosynthetic complexes differ considerably between C. reinhardtii and cyanobacteria.

TAKAHASHI, Yuichiro, et al . Directed disruption of the Chlamydomonas chloroplast psbK gene destabilizes the photosystem II reaction center complex. Plant Molecular Biology , 1994, vol. 24, no. 5, p. 779-788

DOI : 10.1007/BF00029859

Available at:

http://archive-ouverte.unige.ch/unige:135751

Disclaimer: layout of this document may differ from the published version.

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Plant Molecular Biology 24: 779-788, 1994.

© 1994 Kluwer Academic Publishers. Printed in Belgium. 779

Directed disruption of the Chlamydomonas chloroplast psbK gene destabilizes the photosystem II reaction center complex

Yuichiro Takahashi 1'2'*, Hideki Matsumoto 2, Michel Goldschmidt-Clermont 3 and Jean-David Rochaix 3

iT he Graduate School of Natural Science and Technology," 2Faculty of Science, Department of Biology, Okayama University, Tsushima-naka, Okayama 700, Japan (*author for correspondence); 3Departments of Molecular and Plant Biology, University of Geneva, 30 Quai Ernest Ansermet, CH 12Ii Geneva 4,

Switzerland

Received 5 October 1993; accepted in revised form 14 January 1994

Key words: Chlamydomonas reinhardtii,

chloroplast, transformation, photosystem II,

psbK

Abstract

Using particle gun-mediated chloroplast transformation we have disrupted the

psbK

gene of

Chlamy- domonas reinhardtii

with an

aadA

expression cassette that confers resistance to spectinomycin. The transformants are unable to grow photoautotrophically, but they grow normally in acetate-containing medium. They are deficient in photosystem II activity as measured by fluorescence transients and 02 evolution and they accumulate less than 10~o of wild-type levels of photosystem II as measured by immunochemical means. Pulse-labeling experiments indicate that the photosystem II complex is syn- thesized normally in the transformants. These results differ from those obtained previously with simi- lar cyanobacterial

psbK

mutants that were still capable of photoautotrophic growth (Ikeuchi

et al., J.

Biol. Chem. 266 (1991) 1111-1115). In

C. reinhardtii

the

psbK

product is required for the stable assembly and/or stability of the photosystem II complex and essential for photoautotrophic growth. The data also suggest that the stability requirements of the photosynthetic complexes differ considerably between

C. reinhardtii

and cyanobacteria.

Introduction

Photosystem II (PSII) is a multiprotein complex located in the thylakoid membranes which medi- ates electron transfer reactions from water to plastoquinone using light energy. In eukaryotes, the PSII complex consists of seven chloroplast encoded intrinsic subunits (see for reviews [8, 25]): the two reaction center subunits D1 and D2 encoded by the

psbA and psbD

genes, the apo- proteins of the two core antenna complexes CP47 and CP43 encoded by the

psbB

and

psbC

genes,

the two subunits of cytochrome b559 encoded by the

psbE

and

psbF

genes and a small subunit encoded by the

psbI

gene. In addition, the three extrinsic proteins involved in O2-evolving en- hancement, which are products of the nuclear

psbO, psbP

and

psbQ

genes, are associated with the PSII complex on the lumenal side.

Recent developments of amino acid microse- quencing techniques ofpolypeptides together with progress in separating small proteins with appar- ent molecular masses less than 10 kDa have led to the identification of several small polypeptides

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associated with the PSII preparations, encoded by the psbH, psbL psbK, psbL, psbM, psbN and psbR genes [13]. It was found that the 4kDa polypeptide encoded by the chloroplast psbK gene (K polypeptide) is present in O2-evolving PSII membrane fragments [ 16, 22]. This polypep- tide is hydrophobic and is predicted to contain a single membrane-spanning region. In addition, comparison of the N-terminal amino acid se- quence of the K polypeptide with that deduced from the nucleotide sequence of the psbK gene suggests that this polypeptide is synthesized as a precursor and is processed at its N-terminal end [22].

The functional role of the K polypeptide is not yet clear. Biochemical analysis of O2-evolving PSII preparations have demonstrated that the K polypeptide carries neither redox components nor pigments and that it is therefore not directly in- volved in the PSII reactions [16, 22]. Previous results have shown that the cyanobacterial mu- tants in which the psbK gene is inactivated grow photoautotrophically and retain PSII activity, confirming that the K polypeptide is dispensable for PSII function even in vivo in prokaryotic cells [14].

In the present study, we have used the eukary- otic organism C. reinhardtii to inactivate the chlo- roplast psbK gene in order to examine its func- tional role in the chloroplast. This green alga was chosen because chloroplast transformation has been developed [4] and directed chloroplast gene disruptions have been reported in this organism [ 10, 23, 32]. We show that the psbK mutants are unable to accumulate substantial amounts of PSII and to grow photoautotrophically, in clear con- trast with similar cyanobacterial mutants. It ap- pears therefore that the K polypeptide is involved in the stabilization of the PSII complex in C.

reinhardtii.

Materials and methods Strains and growth conditions

C. reinhardtii wild-type strain 137c, the chloro- plast PSII mutant FuD7 [3], the nuclear mutant

nac 2-26 [18], and a PSI mutant with defective psaC [32] were used in the present study. The growth media Tris-acetate-phosphate (TAP) and high-salt minimal (HSM) were prepared as de- scribed [ 11 ]. The transformants were maintained on TAP medium containing 25 #g/ml spectino- mycin.

DNA and plasmids

Using a tobacco psbK probe, the 2.4 kb Pst I and 1.3 Barn HI-PstI chloroplast restriction frag- ments were cloned from chloroplast DNA of C. reinhardtii in the Bluescript KS - plasmid pro- ducing the plasmids pPP and pBP, respectively.

For the latter the Sal I/Acc I site within the poly- cloning site of KS - had been previously removed.

The aadA cassette was subsequently inserted in either orientation at the unique Acc I site within the psbK gene in the pBP plasmids (pBP7 and pBP8 have the cassette oriented in opposite di- rection relative to each other; cf. Fig. 1). The 1.1 kb Pst I-Barn HI fragment from the upstream region of psbK in the pPP plasmid was added to the pBP7 and pBP8 plasmids, generating pP7 and pP8, respectively. Recombinant DNA plas- raids for transformation were prepared using standard methods [27].

Chloroplast transformation in C. reinhardtii Chloroplast transformation in C. reinhardtii wild type cells was performed as described previously

psbK ORF46 tufA

[ II ~ ~ri i

- - Avall/BamHI

E J pBP 200bp

~ pPP

Fig. 1. Physical map of the flanking regions of the psbK gene.

The locations of the tufA gene and ORF46 are indicated. The Ava II/Bam HI fragment containing the psbK gene was used as probe for the northern hybridization shown in Fig. 2. The psbK gene was disrupted by inserting the aadA cassette at the unique Acc I site (A). Other restriction sites indicated are: B, Barn HI; E, Eco RI; P, Pst I.

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[10, 32] with a particle gun (Nippon Zeon Co.).

The bombarded cells were plated on TAP plates containing 150 #g/ml spectinomycin. The trans- formants were recloned on TAP-spectinomycin plates three to four times until they were homoplasmic.

Isolation of nucleic acids

Total DNA was prepared from 100 ml TAP cul- tures containing 25 #g/ml spectinomycin shaken continuously at 25 °C under dim light (200 lux).

Total DNA and RNA were isolated as described [26, 34].

Isolation of the thylakoid membranes and analysis of proteins

Thylakoid membranes were isolated from the wild type, FuD7 and the transformants as described [6] from 1 liter TAP cultures. Chlorophyll con- centration was determined as described [1].

Analysis of the polypeptide profile of the thyla- koid membranes was performed by SDS-poly- acrylamide gel electrophoresis (SDS-PAGE) as described [9, 19]. The resolving gel contained 7.5 M urea and 16-22~o acrylamide. The pro- teins of the thylakoid membranes were solubi- lized with 2~o SDS and 0.1 M DTT at 100 °C for 1 min and 20 #g chlorophyll was loaded per lane.

Western analysis

Antibody raised against CPI from a thermophilic cyanobacterium, Synechococcus elongatus, was a gift from Dr I. Enami. Total cell proteins were separated by electrophoresis, electroblotted on nitrocellulose membranes, reacted with anti- sera and then visualized by the enhanced chemi- luminescence (ECL) method (Amersham). The resolving gel contained 6 M urea and 15Yo acry- lamide.

781 Pulse-labeling of cells

Cells were grown under dim light (200 lux) to exponential phase (1-2x 106 cells/ml) in TAP medium. The cells were washed once and then resuspended in TAP lacking sulfate (1 x 10 7 cells/

ml). Cultures were continuously shaken for 2 h and then cycloheximide was added (10 #g/ml) for 5 min as described previously [ 11 ]. Pulse labeling of cells was performed with carrier-free Na235S 0 4 (0.1 mCi/ml) for 15 min. Aliquots of cultures were immediately centrifuged and the cell pellets were frozen at -20 °C. The cells were subsequently washed once with 50 mM Tris-HC1 pH 7.5 and the total cell proteins were solubilized with 2~o SDS and 0.1 M dithiothreitol at 100 ° C for 1 min.

Proteins were separated by SDS-polyacrylamide gel electrophoresis containing 6 M urea and 15 ~o acrylamide in the resolving gel.

Measurements of PSII activities

Light-induced O2-evolving activity was measured with a Clark type electrode at 25 ° C. The reaction mixture contained 10/~g/ml chlorophyll, 0.2 mM phenyl-p-benzoquinone. Fluorescence induction transients of cells adapted in the dark for 10 min were measured as described [2].

Results

Directed disruption of the psbK gene

The psbK gene has been previously mapped on the largest Eco RI fragment, R26, in the chloro- plast genome of C. reinhardtii and its nucleotide sequence as well as that of its flanking region have been determined [28, 30]. A small open reading frame, ORF46 and the tufA gene encoding the initiation factor EF-Tu are located 530 and 725 nucleotides downstream of the psbK gene in the same orientation [28] (Fig. 1). Since RNA map- ping indicates that the 3' end of the psbK tran- script is located approximately 200 bp down- stream of the psbK gene [30], neither tufa nor

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ORF46 is cotranscribed with it. As shown in Fig. 2 by northern analysis of total RNA from the wild-type cells, a single psbK transcript of 0.58 kb is detectable. One can therefore estimate that the 5' end is located approximately 250 bp upstream of the initiation codon of the psbK gene.

To elucidate the role of the psbK polypeptide in vivo, we disrupted the psbK gene using chloro- plast transformation. In the initial experiments plasmid pBP containing the 1.3 kb Barn HI-Pst I fragment with psbK, ORF46 and the 5'-coding region of tufA was used (cf. Materials and meth- ods, Fig. 1). The aadA expression cassette which confers resistance against spectinomycin and streptomycin [10] was subsequently inserted in either orientation at the unique Acc I site within the psbK gene, thereby generating plasmids pB P7

Fig. 2. RNA analysis of wild type cells. Total RNA was frac- tionated on a denaturing 1.5% agarose gel, blotted onto a nitrocellulose membrane and hybridized with a probe specific to psbK (shown in Fig. 1). Signals were visualized by en- hanced chemiluminescence (ECL). The positions of marker RNAs are indicated and the estimated size of the psbK tran- script is 0.58 kb.

and pBP8. The insertion site of the cassette is located 67 nucleotides downstream of the initia- tion codon of psbK.

However, attempts to transform C. reinhardtii with these plasmids were unsuccessful, probably because the size of the region upstream of the inserted marker within the psbK gene is only 300 bp and not large enough for achieving effi- cient homologous recombination in the chloro- plast. We therefore extended the upstream region of psbK with the 1.1 kb Barn HI-Pst I fragment from plasmid pPP (Fig. 1) in both pBP7 and pBP8. The resulting plasmids were designated pP7 and pP8, respectively. C. reinhardtiiwild-type cells were then transformed with these two con- structs and transformants were obtained by se- lecting for spectinomycin resistance on medium containing acetate and by screening for the pres- ence of aadA by colony hybridization. To obtain homoplasmic transformants, the putative trans- formants were recloned four to five times in the presence of spectinomycin.

Total DNA was isolated from the aadA- positive transformants and subjected to Southern analysis. When total DNA was digested with Pst I and hybridized with the aadA probe, 2.4 kb and 2.9 kb signals were detected in the transformants in which the aadA cassette was in the opposite (P7) or in the same orientation (P8) relative to psbK, respectively, while no signal was observed in the wild type (Fig. 3A). When the blot was reprobed with the 2.4 kb Pst I-Pst I fragment con- taining psbK, the 2.4 kb signal in the wild type was replaced by two bands of 1.9 and 2.4 kb in the P7, and 1.4 and 2.9 kb in the P8 transfor- mants (Fig. 3B). These results were further con- firmed by hybridization of the blots with the 1.1 kb Barn HI-Pst I probe, which hybridized to only one band of 1.9 and 2.9 kb in the P7 and P8 trans- formants, respectively (Fig. 3C). Since a Pst I site is present in the aadA cassette, these results are as expected from the restriction map. Absence of the wild-type signal in the P7 and P8 transfor- mants indicates that all wild-type copies of the psbK gene have been replaced with mutant cop- ies.

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kb

(A) (B) (C)

1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7

kb

783

2.9 - -

2.4 m IIkm ,,.~ ... 2.9

• '-m ~ __ 2.4

2.4" ~ l l = ~ 2.4- ==I ~, -- 1.9

. . . . . . 1.4

(D) P AB A P

WT I II BIB 1

psbK

• =1 1,

1.1 1.3

p AB P p

P7 I II • ] aaclA cassette • I

1.9 2.4

p AB P p

P8 l [ I • aaclA cassette I [

• 1 _- ~ m

2.9 1.4

Fig. 3. DNA analysis of wild type and transformant DNA from wild-type (lane 1), transformants P7 (lanes 2-5) and transformants P8 (lanes 6, 7) were digested with Pst I, separated on 0.8~o agarose gels and subsequently blotted onto nitrocellulose filters. The blots were hybridized with an aadA probe (A), with the 2.4 kb Pst I-Pst I fragment containing the psbK gene (B) or with the 1.1 kb Barn HI-Pst I fragment upstream of the psbK gene (C). Restriction maps of the transforming vectors, pP7 and pP8 (D). A, Acc I;

B, Barn HI; P, Pst I. Signals were visualized by enhanced chemiluminescence (ECL). The estimated sizes in kb are indicated.

psbK transformants are deficient in PSH activity To test if the transformants P7 and P8 can grow photoautotrophically, small samples of cells were plated on TAP and HSM plates. After incubation under dim light (2001ux) and bright light (2000 lux), P7 and P8 cells produced dark green spots on TAP plates comparable to wild-type cells as shown in Fig. 4A and B. In contrast the PSI mutant was not able to grow under bright light on TAP medium confirming that it is highly photo- sensitive [ 32 ], a feature which is clearly not shared by the psbK mutants. The P7 and P8 transfor- mants produced no significant spots on HSM plates under bright and dim light (Fig. 4C and D).

These results indicate that inactivation of psbK prevents C. reinhardtii to grow photoautotrophi- cally.

The fluorescence induction transients of dark- adapted cells from the transformants and from wild type are compared in Fig. 5. In wild-type cells, a slow rise and subsequent decrease of the fluorescence yield is observed, which are ascribed to the PSII and PSI activities, respectively. How- ever, the P7 and P8 transformants showed no variable phase of the fluorescence yield. This phe- notype is typically observed in PSII-deficient mu- tants [2]. Measurements of the O2-evolving ac- tivity in the presence of phenyl-p-benzoquinone as an artificial electron acceptor confirmed these results. While wild-type cells had an O2-evolving activity of 92/~mol 02 per mg chlorophyll per hour, no activity was detected in the P7 and P8 transformants. It can therefore be concluded that expression of the psbK gene is required for PSII activity.

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Fig. 41 Spot tests. Cells from wild-type and the transformants were spotted on agar plates of either TAP (A and B) or HSM (C and D) plates under bright light (2000 lux) (A and C) or dim light (200 lux) (B and D). 1, wild type; 2, transformant with disrupted psaC gene; 3-6, P7; 7-8, P8.

activity suggests that either the K polypeptide is an essential component for PSII activity or that the absence of the K polypeptide prevents normal assembly and/or stabilization of the PSII com- plex.

To determine the amount of PSII complex present in the transformants, total cell proteins were fractionated by SDS-PAGE, electroblotted onto a nitrocellulose membrane and incubated with antibody raised against the D 1 protein, one of the PSII reaction center subunits. The level of D1 protein can be used as a measure of the ac- cumulation of the PSII complex since mutants such as FuD7 lacking D 1 are also deficient in the other PSII core subunits. It can be seen in Fig. 6A that the transformants accumulate greatly re- duced levels of D 1 protein. Based on experiments with diluted wild type samples, the D 1 protein in the transformants accumulates to less than 10~

of the wild-type level (data not shown). However, it can be seen that the P7 and P8 transformants

The K polypeptide is required for stable accumula- tion of the PSH complex

The P S II complex is a multiprotein complex com- posed of more than 15 subunits. The fact that the transformants P7 and P8 are deficient in PSII

o C o ~0

O 14.

psbK::aadA

WT

0 2sec

Fig. 5. Fluorescence induction transients. Cells on TAP plates were preincubated in the dark for 10 rain before the fluores- cence measurements.

Fig. 6. Western blotting analysis. Total cell proteins were separated by PAGE (cf. Materials and methods) and were blotted onto nitrocellulose filter. The blot was incubated with antibodies raised against D1 protein (A) and against CPI (B).

1, wild type; 2, nac 2-26; 3, FuD7; 4-6, P7; 7-8, P8. The signals were visualized by enhanced chemiluminescence (ECL).

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contain more D1 protein than the PSII nuclear mutant, nac 2-26, which is deficient in the ex- pression of the psbD gene. As expected for PSII mutants, the P7 and P8 transformants accumu- late wild-type levels of photosystem I (PSI) reac- tion center (Fig. 6B).

To determine whether the deficiency of the P S II complex in the transformants was due to the in- stability of PSII or to reduced synthesis of the PSII subunits, cells were pulse-labeled in the presence of cycloheximide which blocks cytoplas- mic protein synthesis. The two pulse-labeled bands of 32 and 34 kDa in wild type and the P7 transformant correspond to the mature and pre- cursor forms of the D1 protein, respectively (Fig. 7). These signals are totally missing in the FuD7 mutant as expected. The rate of synthesis of D 1 in the P7 transformant appears to be com- parable to that of wild type based on the inten- sities of label detected in both the precursor and mature forms of D 1. The D2 protein which mi- grates slightly slower than D1 in the gel system used is also synthesized at a similar rate in wild-

Fig. 7. Pulse labeling of chloroplast encoded polypeptides.

Cells were labeled with Na235SO4 (0.1 mCi/mi) for 15 min in the presence of 10 #g/ml cycloheximide. Total cell proteins were separated by PAGE and autoradiographed (cf. Materi- als and methods). 1, wild type; 2, FuD7; 3, P7. The positions of marker proteins are indicated in kDa.

785 type, FuD7 and P7 cells. It can be concluded that disruption of the psbK gene and loss of the K polypeptide results in destabilization of PSII rather than in reduction of synthesis of its sub- units.

Discussion

Loss of the psbK polypeptide leads to destabilization of the PSII complex in C. reinhardtii

In the present study, we have generated mutants of C. reinhardtii using biolistic transformation, in which the chloroplast psbK gene is disrupted.

The mutants are deficient in PSII activity and thus unable to grow photoautotrophically. Loss of the K polypeptide leads to the destabilization of the PSII complex, and thereby to the accumu- lation of the complex at greatly reduced levels as compared to wild type.

In tobacco [29], rice [12] and liverwort [24], the psbK gene is flanked by the psbI gene that encodes a small hydrophobic polypeptide tightly associated to the PSII reaction center [15]. In C. reinhardtii, however, the psbI gene is located far away from psbK on the chloroplast genome.

Instead, the psbK gene is followed 530 bp down- stream by a small open reading frame, ORF46, of unknown function oriented in the same direction.

The wild-type psbK transcript is not cotrans- cribed with ORF 46 and its 5' end is estimated to be located ca. 250 bp upstream of the initiation codon of psbK. It therefore appears unlikely that disruption of psbK affects the expression of nearby downstream genes. These results lead to the conclusion that the K polypeptide is an es- sential component of the PSII complex and is required for its stable accumulation in C. rein- hardtii.

It has previously been shown in both C. rein- hardtii and cyanobacteria that inactivation of the core PSII genes psbA, psbB, psbC, psbD, psbE or psbF (the latter two only in cyanobacteria) results in the destabilization, at least partially, of the PSII complex. Biochemical analyses indicate that these integral subunits are all tightly associated with

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each other to form the PSII core complex which can only be disassembled by strong treatment with detergent and/or chaotropic reagents [ 33, 35, 36 ].

It is therefore likely that loss of any of these sub- units greatly affects the structure of the PSII com- plex and consequently renders the remaining sub- units more vulnerable to proteolysis in vivo.

The deduced primary structure of the K polypeptide suggests that this polypeptide con- tains a single membrane spanning region and that it might be associated with the PSII core complex by hydrophobic interactions like other integral polypeptides of the PSII complex. However, bio- chemical data indicate that the association of the K polypeptide to the PSII core complex is weak in comparison to that of the other intrinsic polypeptides of the PSII complex [7, 16, 17, 22].

In higher plants, the K polypeptide is recovered only in the P S II Oa-evolving membrane fragments which are obtained by mild detergent treatments, and is lost in the PSII O2-evolving core complex which is purified from the membrane fragment after treatment with detergents [16, 22]. How- ever, the strength of the association of the K polypeptide to the PSII core complex varies be- tween species. The K polypeptide has been found in the O2-evolving core complex from a thermo- philic cyanobacterium [ 17] and this polypeptide has been recovered in the PSII core complex from C. reinhardtii, after extensive purification by col- umn chromatography [7]. These data suggest that the K polypeptide is hydrophobically but periph- erically associated to the PSII complex.

Inactivation of psbK in C. reinhardtii and cyano- bacteria results in different phenotypes

The mutant of C. reinhardtii whose psbK gene has been inactivated has a rather different phenotype than the same mutant produced in Synechocystis.

In contrast to C. reinhardtii the cyanobacterial mutant is still able to grow photoautotrophically at a 2-fold reduced rate as compared to wild type [14]. Hence the K polypeptide in cyanobacteria is not an essential component for PSII activity but is needed for optimal PSII function. Although

all PSII mutants of Synechocystis examined have wild-type growth rates in the presence of glucose, the growth rate of the psbK mutant is still re- duced under these conditions. This suggests that the role of psbK is not limited to PSII in cyano- bacteria. However, in C. reinhardtii the psbK mu- tant appears to grow at the same rate as wild-type on acetate containing medium.

The difference in phenotype observed between the psbK mutants of C. reinhardtii and Syn- echocystis could be due to minor structural differ- ences in the PSII complex. The sequence of the mature K polypeptide of C. reinhardtii has 76 ~o identity with the corresponding sequence of Syn- echocystis [14, 30]. Other differences between PSII from eukaryotic and prokaryotic organisms have been noted, in particular the presence in cyanobacteria of only one of the three oxygen- evolving enhancer proteins that are associated with the lumenal side of PSII from higher plants and algae. Since the K polypeptide appears to be more tightly associated with the core PSII com- plex in C. reinhardtii, loss of this subunit may cause more pronounced structural changes in PSII in this alga.

However, the K polypeptide can be removed from the O2-evolving PSII membranes from higher plants without significant loss of PSII ac- tivity, indicating that the PSII complex is stable in vitro in the absence of the K polypeptide [ 16, 17]. These differences may reflect different re- quirements for PSII stability in vivo and in vitro. It is therefore possible that the K polypeptide is not directly involved in the PSII reactions but that it may be required for the structural integrity of P S II which renders the complex resistant to proteoly- sis in vivo.

Differences in phenotype have also been ob- served between other C. reinhardtii and cyano- bacterial photosynthetic mutants. Disruption of the psbO gene encoding the 33 kDa oxygen- evolving enhancer protein leads to the rapid turn- over of the PSII complex in C. reinhardtii [21]

whereas loss of this protein in Synechocystis still allows photoautotrophic growth and Oz-evolving activity is retained although at a reduced level [ 5 ].

Similarly, loss of the psaC protein of PSI in

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C. reinhardtii prevents stable accumulation of this complex [32] whereas in the cyanobacterium Anabaena variabilis the PSI complex is stable, but deficient in photochemical activity, and it accu- mulates to wild-type levels in the absence of the psaC protein [20].

Taken together, these observations suggest the existence of a chloroplast clearing system in C.

reinhardtii which recognizes and degrades mis- folded protein complexes and that is more effi- cient than its cyanobacterial counterpart. The lat- ter may only be able to recognize and degrade protein complexes that are severely misfolded.

Finally, it is also possible that some of the other additional low-molecular-weight proteins associ- ated with the photosynthetic reaction centers are involved in stabilization of the complexes in vivo.

In this respect it may not necessarily be possible to elucidate the functional role of a protein in the chloroplast of eukaryotic cells from the pheno- types observed in photosynthetic mutants from cyanobacteria.

Acknowledgements

We thank M. Sugiura for the tobacco psbK probe, I. Enami and L. McIntosh for the antibodies raised against CPI and D 1. We also thank Nip- pon Zeon Co. for providing the particle gun ap- paratus used in this study. This work was sup- ported by the Monbusho International Scientific Research Program-Joint research (63044148) and the grant in aid for Scientific Research from the Ministry of Education, Science and Culture, Japan, and by grant 31-34014.92 from the Swiss National Fund for Scientific Research.

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