The Plant Cell, Vol. 11, 87–99, January 1999, www.plantcell.org © 1999 American Society of Plant Physiologists

A Chromodomain Protein Encoded by the Arabidopsis

CAO

Gene Is a Plant-Specific Component of the Chloroplast Signal Recognition Particle Pathway That Is Involved inLHCP Targeting

Victor I. Klimyuk,

a,1

Fabienne Persello-Cartieaux,

b,1

Michel Havaux,

b

Pascale Contard-David,

b

Danja Schuenemann,

c

Karin Meiherhoff,

d

Patrice Gouet,

e

Jonathan D. G. Jones,

a

Neil E. Hoffman,

c

and Laurent Nussaume

b,2

a

Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom

b

Département d’Ecophysiologie Végétale et de Microbiologie, Commissariat à l’Energie Atomique/Cadarache, F-13108St. Paul lez Durance Cedex, France

c

Carnegie Institution of Washington, Department of Plant Biology, Stanford University, 260 Panama Street, Stanford, California 94305

d

Institut für Entwicklungs und Molekularbiologie der Pflanzen, Heinrich-Heine Universität, Universitätsstrasse 1, D-40225 Düsseldorf, Germany

e

Institut de Pharmacologie, Centre National de la Recherche Scientifique/Institut de Pharmacologie et de Biologie Structurale, 29 rue Jeanne Marvig, 31055 Toulouse Cedex, France

A recessive mutation in Arabidopsis, named

chaos

(for chlorophyll

a/b

binding protein harvesting–organelle specific;designated gene symbol

CAO

), was isolated by using transposon tagging. Characterization of the phenotype of the

chaos

mutant revealed a specific reduction of pigment binding antenna proteins in the thylakoid membrane. These nu-clear-encoded proteins utilize a chloroplast signal recognition particle (cpSRP) system to reach the thylakoid mem-brane. Both prokaryotes and eukaryotes possess a cytoplasmic SRP containing a 54-kD protein (SRP54) and an RNA. Inchloroplasts, the homolog of SRP54 was found to bind a 43-kD protein (cpSRP43) rather than to an RNA. We cloned the

CAO

gene, which encodes a protein identified as Arabidopsis cpSRP43. The product of the

CAO

gene does not resem-ble any protein in the databases, although it contains motifs that are known to mediate protein–protein interactions.These motifs include ankyrin repeats and chromodomains. Therefore,

CAO

encodes an SRP component that is uniqueto plants. Surprisingly, the phenotype of the cpSRP43 mutant (i.e.,

chaos

) differs from that of the Arabidopsis cpSRP54mutant, suggesting that the functions of the two proteins do not strictly overlap. This difference also suggests that thefunction of cpSRP43 is most likely restricted to protein targeting into the thylakoid membrane, whereas cpSRP54 may beinvolved in an additional process(es), such as chloroplast biogenesis, perhaps through chloroplast–ribosomal associa-tion with chloroplast ribosomes.

INTRODUCTION

Higher plants require chloroplasts for essential functionsranging from photosynthesis to sugar, lipid, and amino acidbiosyntheses. Many of the proteins required for the light re-actions of photosynthesis, including the chlorophyll-con-taining antenna proteins, are localized in the thylakoidmembrane. Thylakoid proteins are encoded by both the nu-clear and chloroplast genomes. Nuclear-encoded proteinsdestined for the thylakoid membrane are synthesized with a

cleavable N-terminal extension (transit peptide) that targetsthe protein across the envelope membranes into the stroma(reviewed in Cline and Henry, 1996; Kermode, 1996; Lübecket al., 1997). Multiple mechanisms exist for targeting pro-teins from the stroma to the thylakoid membrane (reviewedin Cline and Henry, 1996; Klösgen, 1997). These include

spontaneous insertion, a chloroplast Sec

r

–dependent path-way, a chloroplast signal recognition particle (cpSRP) path-way, and a pathway requiring only an electrochemical

potential of

D

pH across the thylakoid membrane. Thesepathways can be distinguished by specific requirements forstromal proteins and/or by energetic requirements when in

1

These authors contributed equally to this work.

2

To whom correspondence should be addressed: E-mail lnussaume@cea.fr; fax 33-442-25-4656.

88 The Plant Cell

vitro reconstitution assays are performed. In vitro, precursorproteins are targeted exclusively via one of the aforemen-tioned pathways.

LHCIIb proteins are the most abundant of the thylakoidmembrane proteins. They are the major polypeptides of thelight-harvesting chlorophyll

a

/

b

binding protein complex ofphotosystem II (PSII). They constitute approximately one-third of the total thylakoid protein and bind half of the chlo-rophylls (Yamamoto and Bassi, 1996). They belong to alarge family of related polypeptides, which include the minorchlorophyll protein complexes of PSII and the chlorophyll

antenna of photosystem I (PSI). The members of this familyare collectively called LHCPs in this study. Many of the pro-teins targeted to the thylakoid require a bipartite transit pep-tide consisting of an N-terminal envelope transit sequencefollowed by a thylakoid transfer domain that directs the pro-tein across the thylakoid membrane. The transit peptides ofLHCPs, however, comprise only the envelope transit se-quences, which target the hydrophobic proteins to the stro-ma, where they are able to insert into the thylakoid membranein the absence of their transit peptides (Lamppa, 1988).Thus, the information for LHCP integration into the thylakoid

Figure 1. Mutant Phenotype.

(A) Seedling stage.(B) Rosette stage. WT, wild type.(C) Electron microscopic analysis of chloroplasts. g, grana thylakoids; s, starch grains; v, vesicles present in the mutant only.

Identification of a Plant-Specific cpSRP Component 89

resides in the mature protein (Hand et al., 1989). Attempts atlocalizing the targeting information have not been success-ful (Auchincloss et al., 1992; Huang et al., 1992).

LHCP trafficking requires the intervention of protein fac-tors in the stroma (Cline, 1986; Chitnis et al., 1987; Yuan et al.,1993) and GTP (Hoffman and Franklin, 1994). One of theseprotein factors is a chaperone that maintains the solubility ofLHCPs as they are transported through the stroma (Payanand Cline, 1991). In vitro studies have shown that the sub-unit of the chaperone that interacts with the LHCPs iscpSRP54 (Franklin and Hoffman, 1993; Li et al., 1995).CpSRP54 is a chloroplast homolog of the 54-kD protein ofthe mammalian SRP, which is required for the transport ofproteins across the endoplasmic reticulum membrane (re-viewed in Rapoport, 1992). SRPs are not restricted to eu-karyotes: they have been found in a wide range of organismsand may function in protein translocation in every living or-ganism.

When cpSRP54 was removed from stromal extract by im-munodepletion, activities required for in vitro trafficking ofLHCPs were inhibited. However, the addition of cpSRP54did not restore activity (Li et al., 1995). This observation sug-gests the possibility that an additional factor was coimmu-noprecipitated along with cpSRP54. Most organisms have acytoplasmic SRP that contains SRP54 bound to an

SRP

RNA (reviewed in Lütcke, 1995). However, in plants, no suchRNA that binds to cpSRP54 has been detected. Rather,cpSRP54 was found attached to a 43-kD protein namedcpSRP43 required for LHCP targeting to the thylakoid mem-brane (Schuenemann et al., 1998). However, the exact natureand functions of the 43-kD protein have yet to be deter-mined.

In this study, we have identified

chaos

(for chlorophyll

a/b

binding protein harvesting–organelle specific), a tagged mu-tant of Arabidopsis that is deficient in the production ofcpSRP43. By using transposon tagging and by cloning, wewere able to identify and characterize

CAO

, the gene encod-ing the cpSRP43 polypeptide, and to determine a role for

cpSRP in the biogenesis of PSII light-harvesting proteins.The three-letter gene symbol

CAO

is used for the

chaos

mu-tation, which is in keeping with Arabidopsis nomenclatureguidelines.

RESULTS

The

chaos

Mutant Is Depleted of Chlorophylland Carotenoids

The

chaos

mutant was identified in a population of 217 inde-pendent lines of Arabidopsis ecotype Landsberg

erecta

(L

er

-0) containing a transposed enhancer trap derivative ofthe maize

Dissociation

(

Ds

) transposable element. Self-polli-nation of a plant from line 348/74/A resulted in progeny ex-hibiting a recessive chlorotic mutant phenotype (Klimyuk etal., 1995). The mutant was named

chaos

(Figures 1A and1B). The pale coloration was observed throughout the vege-tative cycle of the plant and uniformly affected all of theaerial tissues.

The chlorotic phenotype suggested that major pigmentlevels in the mutant had been reduced. Therefore, we mea-sured the concentration of chlorophylls and carotenoids inthe leaves of the mutant and the wild type. Total chlorophylldeclined by 43%, and the chlorophyll

a

/

b

ratio increasedfrom 2.6 in the wild type to 3.2 in the

chaos

mutant. Mem-bers of the LHCP family are the only proteins that bind chlo-rophyll

b.

In addition, LHCPs bind carotenoids, such aslutein, neoxanthin, and violaxanthin (Bassi et al., 1993),which were significantly reduced in the mutant, whereascarotenoids that are not present in the LHCPs of PSII, suchas

b

-carotene, exhibited little if any decrease (Table 1).Thus, the mutant phenotype appeared to affect primarilyLHCP-associated pigments.

Electron microscopic analysis of chloroplasts revealed nodifferences between the ultrastructure of the mutant andwild-type organelles (Figure 1C). In both cases, we foundstarch grains, plastoglobuli (lipids), and typical thylakoidgrana. The size, shape, and number of plastids in the mutantchlorotic cells did not vary when compared with those of thewild type (data not shown). The only apparent difference

Table 1.

Carotenoid and Chlorophyll Content of Leaves

Wild Type

a

chaos

a

Antheraxanthin 0.6

6

0.2 0.6

6

0.2

b

-Carotene 19.2

6

3.0 15.7

6

3.1Lutein 34.6

6

3.5 20.5

6

3.6Neoxanthin 7.9

6

0.9 3.8

6

0.4Violaxanthin 12.6

6

2.4 8.7

6

1.3Zeaxanthin 0.6

6

0.5 0.5

6

0.2Chlorophyll

a

236.2

6

21.2 142.9

6

25.1Chlorophyll

b

91.3

6

9.0 44.1

6

8.2

a

The average of five independent measurements expressed as nan-ograms per square millimeter is given

6

SE

. Plants were grown undera photon flux density of 600

m

mol of photons m

2

2

sec

2

1

.

Table 2.

Photosynthetic Oxygen Production Measured with aClark Electrode

Rate of Oxygen Production Wild Type

a

chaos

a

Light-limiting conditions(85

m

mol of photons m

2

2

sec

2

1

)0.021

6

0.004 0.012

6

0.003

Light-saturating conditions(1200

m

mol of photons m

2

2

sec

2

1

)0.069

6

0.007 0.058

6

0.005

a

The average of three independent measurements expressed as mi-cromoles of O

2

per square meter per second is given

6

SE

.

90 The Plant Cell

was the presence of numerous vesicles in the

chaos

stroma(Figure 1C).

The Light-Harvesting Step of Photosynthesis Is Reduced in the Mutant

To investigate the effects of the

chaos

mutation on photo-synthesis, we measured oxygen production by leaves with aClark-type oxygen electrode under different light conditions(Table 2). The level of oxygen production was always lowerin the mutant when compared with the wild type on the ba-sis of leaf area. There was a 16% decrease in oxygen pro-duction under high light conditions, whereas the differenceincreased to 43% under low light. This suggests that themutation selectively affects the light-harvesting process.

The light distribution between PSI and PSII was evaluatedby using the Emerson effect (Canaani and Malkin, 1984).Under state 2 light conditions, a quinone of the cytochrome

b

6

/

f

complex becomes reduced and activates the kinasesystem, which in turn phosphorylates the LHCPs (Williamsand Allen, 1987). Phosphorylated LHCP complexes dissoci-ate from PSII and associate with PSI. Under state 1 lightconditions, the quinone is oxidized, thus inducing the re-verse reaction and leading to the return of LHCP to PSII.

For both the wild type and

chaos

, we observed, as ex-pected, a very low Emerson effect (

E

) value in state 2, indi-cating that light energy absorption was well balancedbetween the two photosystems (Table 3). In state 1, we sawan increase in the

E

value effect, indicating that LHCP mi-gration from PSII to PSI occurred in both genotypes. How-ever, the

E

value was significantly higher in the wild typewhen compared with

chaos

, and these differences are moresignificant for state 1 than for state 2. Although both

chaos

and wild-type chloroplasts are able to regulate light distribu-tion between PSII and PSI, light absorption in PSII is selec-tively reduced when compared with PSI in the

chaos

mutant.

Electron Transfer in Both Photosystems Is Not Inhibited

The maximal quantum yield of PSII photochemistry (

F

) wasquantified by in vivo measurements of chlorophyll fluores-cence. The

F

value obtained for the wild type was 0.80

6

0.01(mean value of 17 independent experiments

6

SE

). Thisis a typical value for healthy plants (Björkman and Demmig,1987). The

chaos

mutant had a similar value (0.82

6

0.01).This clearly demonstrates that the maximal photochemicalefficiency of PSII was not affected by the

chaos

mutation.We also measured the actual quantum yield of PSII photo-chemistry in white light (100

m

mol of photons m

2

2

sec

2

1

),and no difference was observed between

chaos

and thewild type (0.74

6

0.01). These findings indicate that PSII re-mained photochemically competent in

chaos

and that theefficiency of photosynthetic electron transfer (PSI plus PSII)was not affected by the mutation.

The Mutation Affects the Level of Nuclear-Encoded Antenna Proteins

To identify the possible changes in the composition of pho-tosystems in the chaos mutant, we solubilized the thylakoidpigment protein complexes and analyzed them by partiallydenaturing gel electrophoresis (Figure 2A). A clear differencebetween the wild type and the mutant could be observeddue to a strong reduction of the trimeric and monomericforms of the LHCPs. Figure 2B shows that the chaos mutantlost a significant number of major chloroplast proteins be-tween 24 and 29 kD. To identify the proteins affected, weperformed immunoblot analyses with representative pro-teins of PSI (PSA D, E, F, and L) and PSII (D1, D2, CP47,CP43, CP24, and LHCIIb). The results given in Figure 2Ccan be considered as quantitative (the amounts of proteinrequired to avoid saturation problems were investigated inpreliminary experiments). As already shown (Figure 2A), thelevels of the major LHCP (i.e., LHCIIb) were lower in the mu-tant. Densitometric analysis revealed a decrease of z50%for the LHCIIb proteins (the result was similar when the thy-lakoid-pigmented complexes of Figure 2A were analyzed).This result was also observed with CP24, another nuclear-encoded antenna protein of PSII, whereas all other compo-nents of PSI and PSII remained unchanged (Figure 2C). Im-munoblotting was also conducted with components of theCyt b6/f complex and ATP synthase. These did not revealany differences between the wild type and the mutant (datanot shown).

The CAO Locus Is Tagged by Using the Ds Transposon

Genetic analysis performed with selfed progeny of a plantfrom line 348/74/A demonstrated linkage of the chaos alleleto the Ds insertion (Klimyuk et al., 1995). To confirm that thechaos mutation was caused by the Ds insertion, we selected

Table 3. Photosynthetic Oxygen Production Measured by Photoacoustic Spectroscopy

Emerson Effecta Wild Typeb chaosb

State 1 27 6 4 11 6 2State 2 9 6 2 6 6 2

a The Emerson effect (E ) is an indication of the balance in light ab-sorption between PSI and PSII. A high E value indicates a strong im-balance of light distribution between the two photosystems in favorof PSII. Under state 1 light conditions, the pool of plastoquinones isoxidized, and LHCII molecules are attached to PSII molecules,whereas under state 2 light conditions, the pool of plastoquinones isreduced, and the LHCII molecules migrate to PSI.b The average of four independent measurements of E is given 6SE.

Identification of a Plant-Specific cpSRP Component 91

revertants from among the progeny of a plant homozygousfor Ds insertion and carrying the Activator (Ac) transposase.Three revertants were isolated from the z15,000 plantletsthat were screened. Reversion of the mutation to the wild-type phenotype was correlated with the excision of the Dselement (data not shown). Analysis of the excision footprintsin the three revertants revealed complete restoration of theopen reading frame and a substitution of the first two bases(G and A) flanking the 39 end of the Ds element (G and A be-ing converted to C and A or G and C, depending on the re-vertant). The probability of such events is extremely low,which explains the low frequency of reversions that were

observed. Nevertheless, this analysis demonstrated that thechaos mutation was due to the Ds insertion.

Cloning of the CAO Gene

A 300-bp DNA sequence flanking the right border of the Dselement was amplified by inverse polymerase chain reac-tion, cloned, and sequenced. This probe was used to isolatefour cDNA clones of 1.4 kb (pLMC C6, pLMC C8, pLMC F1,and pLMC F2) from an Arabidopsis seedling library. A ge-nomic cosmid DNA library was screened, and a 6-kb BamHI

Figure 2. Gel Electrophoretic and Immunoblot Analyses of the Photosynthetic Apparatus.

(A) Separation of pigment–protein complexes solubilized from thylakoids of the wild type (WT) and the chaos mutant. The pigment–protein com-plexes were solubilized with a mixture of SDS and octylglucoside and separated by gel electrophoresis. The green bands were visible withoutstaining. CCI, core complex I; CCII, core complex II; FP, free pigment; LHCII, light-harvesting complex II; (LHCII)3, trimeric form of LHCII.(B) Total proteins and thylakoid membrane polypeptides of the chaos mutant and the wild type (WT) after electrophoresis in a 15% polyacryla-mide–SDS gel and silver staining. Each lane contains 8 mg of protein. Molecular masses (given in kilodaltons at the left) were estimated by co-electrophoresis with commercially available size standards.(C) Immunoblot analysis of chaos mutant and wild-type (WT) proteins. Wild-type and mutant proteins of thylakoids were electrophoresed in a12% polyacrylamide–SDS gel and immunodetected with antisera, as described by Meurer et al. (1996). Thylakoid proteins used in the immuno-blot analysis are as follows: PSI; PSII; PSA D, E, F, and L (subunits of PSI); CP47 and CP43 (chlorophyll a apoproteins of the inner antennae of PSII);D1 and D2 (PSII reaction center proteins); CP24 (one of the minor light-harvesting complexes of PSII); and LHCIIb (major light-harvesting com-plex of PSII).

92 The Plant Cell

fragment hybridizing with the probe was subcloned into thepBluescript KS1 vector. The sequence of the entire ge-nomic region of the CAO gene (GenBank accession numberAF013115) was determined (Figure 3). No introns were de-tected.

The four cDNA clones differed only at their 39 ends. Twoalternative polyadenylation sites were found at positions1149 and 1328 of the genomic sequence (Figure 3). A nearupstream element (a sequence that can control the utiliza-tion of a polyadenylation site), AAUGAA (Wu et al., 1994),

was found at positions 1119 and 1123. These positions, 16and 20 bp before the first polyadenylation site, are charac-teristic of such signals (Li and Hunt, 1995). A putative TATAbox, TATAAA, was located 158 bp upstream of the ATGcodon.

The Ds insertion was found just after position 825 of thegenomic sequence. The typical 8-bp target site duplicationusually flanking the insertion of Ds elements (Fedoroff, 1989)was absent. In its place was one extra base (a cytidine) pre-ceeding the 59 end of the Ds element.

The CAO Gene Exists as a Single Copy on Chromosome 2 in the Arabidopsis Genome

Gel blot analyses of genomic DNA from three Arabidopsisecotypes (Columbia, Ler, and Wassilewskija) digested withdifferent restriction enzymes were performed using thecloned cDNA as a probe. The gene was found to exist as asingle copy in the Arabidopsis genome (data not shown).The CAO gene was mapped to the bottom of chromosome2. It shares residues identical to those of the open readingframe encoding an unknown protein and is positioned onbacterial artificial chromosome T30B22, which is part ofyeast artificial chromosome CIC06C03 and most likely lo-cated upstream of the recombinant inbred marker mi79a.

Sequence Analysis of the Protein Encoded by CAO

The nucleotide sequence of the CAO cDNA contained oneopen reading frame with the potential to encode a polypep-tide of 376 amino acid residues, with a predicted molecularmass of 41.5 kD and an isoelectric point of 4.3 (Figure 3).The deduced amino acid sequence of the protein showedseveral interesting features. It contains a domain similar tothose of typical transit peptides of chloroplast proteins(Kermode, 1996). The first 60 amino acids are rich in the hy-droxylated amino acid serine (25%) and in hydrophobicamino acids (valine [11%] and alanine [6%]) but contain veryfew acidic amino acids (5%). Based on the cleavage siteconsensus sequence (V/I)X(A/C)/A defined by Gavel and vonHeijne (1990), the cleavage site is predicted to occur afterA-60. The processing site of the pea protein has been deter-mined by N-terminal sequencing and suggests that pro-cessing occurs at either A-60 or C-59, depending on howthe peptide is aligned with the Arabidopsis sequence.

Database searches using the Blast program (Altschul etal., 1990) identified two segments of the protein encoded byCAO with similarity to motifs mediating protein–protein in-teractions. The first one corresponds to four tandem ankyrinrepeats located between residues 130 and 254. These fourankyrin repeats (Figure 4A) exhibit a conservation of theconsensus motif (defined by Zhang et al., 1992), rangingfrom 62 to 85%. This is in agreement with the 54 to 100%

Figure 3. Genomic Sequence of the CAO Gene and the DeducedAmino Acid Sequence.

The amino acids comprising the potential chloroplast transit peptideare underlined with dots, and an arrowhead marks the most likely posi-tion for the cleavage. The ankyrin repeats are boxed, and the chro-modomain is underlined. The two positions observed for the poly(A)tail on CAO transcripts are indicated by dots. Insertion of the trans-posable element is indicated with an asterisk in the coding se-quence.

Identification of a Plant-Specific cpSRP Component 93

identity observed among hundreds of ankyrin repeats (Bork,1993).

The second domain has similarity to the chromatin bind-ing domain (chromodomain) originally identified in Polycomband heterochromatin protein 1, which are two Drosophila pro-teins (Paro and Hogness, 1991). In these proteins, chromo-domains have been implicated in the regulation of chromatinstructure through protein–protein interactions. Chromoboxesare conserved motifs in both plants and animals, as demon-strated by gel blot analyses (Singh et al., 1991). They vary inlength from 30 to 70 amino acids (Aasland and Stewart,1995; Ball et al., 1997) and typically have three blocks ofconserved sequences. The protein encoded by CAO hastwo chromodomains in tandem spanning residues 271 to368 (see alignment in Figure 4B).

The CAO Transcript Is Expressed in Aerial Tissues

The CAO gene was tagged by using an enhancer trap Ds el-ement (Klimyuk et al., 1995). The b-glucuronidase (GUS)gene, which was used as an enhancer trap, was activated inthe chaos mutant, with GUS being very weakly expressed inthe aerial tissues of the plant (Figure 5A). To confirm theseobservations, we performed RNA gel blot analyses with thecloned CAO cDNA as a probe. Poly(A) mRNA purified from100 mg of total RNA for each sample was used. This stepwas necessary due to the very low abundance of the CAOmRNA (no signal was obtained with 10 mg of total RNA). Thetranscript was detected in leaves and not in roots (Figure

5B). Thus, consistent with the chlorotic phenotype, expres-sion of CAO appears to be confined to chlorophyll-contain-ing tissues. Circadian fluctuations of the CAO mRNA levelswere investigated. The amplitude of the oscillation was notvery marked and did not exceed 55 to 65% (data notshown). The level of mRNA accumulation was maximal dur-ing the later part of the night period and decreased through-out the day.

Identification of chaos as a Mutant in CpSRP43

The presence of a transit peptide suggested that the proteinencoded by CAO was localized in the chloroplast. To con-firm this, we probed blots of proteins prepared from wild-type and chaos leaves with an antiserum raised against therecombinant CAO-encoded protein. Immunoblot analysisrevealed that the antiserum detected a protein in wild-typeleaves that was absent in the chaos mutant (Figure 5C). Fur-thermore, a unique polypeptide in the stromal fraction ofpurified Arabidopsis (Figure 5D) and pea (Figure 6B) chloro-plasts, respectively, cross-reacted with the antiserum, con-firming the chloroplast localization of the CAO gene product.The use of silver-stained gel and protein mass markers indi-cates that the sizes of the detected polypeptides were, re-spectively, 42 kD for Arabidopsis and 43 kD for pea (datanot shown). We established that the stromal and thylakoidmembrane fractions were basically free of contamination byusing antisera raised against stromal heat shock protein

Figure 4. Domains of Significant Similarity to Known Proteins.

(A) Similarity to ankyrin consensus motifs (defined by Zhang et al., 1992).(B) Similarity to chromodomains (Aasland and Stewart, 1995).CHD1, helicase-domain; Dm, Drosophila melanogaster; HET, heterochromatin protein; HP1, heterochromatin protein 1; Mo, mouse; Pc, Plano-coccus citri; PC, polycomb; Sp, Schizosaccharomyces pombe; SWI6, repressor of mating-type loci. A secondary structure prediction generatedwith the PREDICT (Rost and Sander, 1994) and HCA (Lemesle-Varloot et al., 1990) programs is shown. H indicates a prediction for an a helixand E for a b sheet. The numbering of the sequences refers to their position in the protein. Amino acids strictly conserved or closely related inmany of the sequences are underlined in the CHAOS sequences. Dashes were introduced to optimize alignment.

94 The Plant Cell

Hsp70 and the LHCIIb. More than 90% of cpSRP43 isestimated to be in the stroma, because the protein was notdetected in 10 mg of thylakoid proteins but was clearly de-tected in 2 mg of stromal proteins.

The phenotype of the chaos mutant suggested that the pro-tein encoded by CAO might play a role in LHCP biogenesis.LHCP trafficking requires a complex of the proteins cpSRP54and cpSRP43 (Schuenemann et al., 1998). CpSRP54 hasbeen characterized (Franklin and Hoffman, 1993; Li et al.,1995). However, the nature of cpSRP43 has not yet beenelucidated. Three lines of evidence demonstrate that theprotein encoded by CAO corresponds to cpSRP43. First,peptide sequence data from pea cpSRP43 are similar to thepredicted sequence from the Arabidopsis cDNA (Figure 6A).Second, antibodies raised against the protein encoded by

CAO recognize, respectively, the pea 43-kD (Figure 6B) andthe Arabidopsis 42-kD (data not shown) bands coimmuno-precipitated by antibodies raised against cpSRP54. Asshown in Figure 6B, the 43-kD polypeptide in the peastroma recognized by the CAO antiserum was efficiently re-moved from the stroma by the anti-cpSRP54 antibody andwas recovered in the pellet fraction, whereas none of the 43-kDpolypeptide was precipitated by an unrelated antiserum.Thus, the anti-cpSRP54 antibody immunodepletes thestroma of both cpSRP54 and the CAO-encoded polypep-tide. Third, we have shown that in pea stroma immunode-pleted of the SRP transit complex (composed of cpSRP54and cpSRP43), LHCP integration in thylakoids is reduced by.90%. This integration could only be resaturated when bac-terially expressed cpSRP54 and CAO-encoded proteins wereadded to the immunodepleted pea stroma (Schuenemann etal., 1998). However, the same experiments performed withArabidopsis chloroplasts failed, presumably because it isdifficult to isolate chloroplasts competent for import in thisspecies. These data unambiguously demonstrate that theCAO-encoded protein is the pea cpSRP43 homolog.

DISCUSSION

In this study, we have described a leaf pigmentation mutant,chaos, that resulted from the insertion of an enhancer trapDs element at the CAO locus of Arabidopsis. Sequencing ofthe interrupted gene revealed one open reading frame con-taining two types of motifs involved in protein–protein inter-actions. Consistent with the phenotype and based onbiochemical studies, we have determined that CAO en-codes cpSRP43, an SRP protein that appears to be foundonly in the plastid. Indeed, the CAO-encoded Arabidopsishomolog of cpSRP43 has no apparent homology to anyother known SRP subunits.

What is the function of cpSRP43 and why is it unique tocpSRP? Because normal levels of cpSRP54 accumulate inchaos (data not shown), we can exclude the possibility thatcpSRP43 is required to stabilize cpSRP54. The presence ofan RNA component has been described for all cytoplasmicSRPs studied, including those from animals, plants, andprokaryotes (reviewed in Rapoport, 1992). Recently, puta-tive SRP RNAs have been identified in the plastid genome ofa red alga and a diatom, but no such candidates have beendetected in higher plants (Packer and Howe, 1998). Onepossibility is that cpSRP43, an acidic protein with a calcu-lated pI of 4.3, might be a functional equivalent of the SRPRNA. However, we note that cpSRP is not only structurallydistinct from cytoplasmic SRP but possesses the unusualfeature of being able to interact post-translationally with itssubstrate, LHCP. Chemical cross-linking revealed a directinteraction between LHCPs and cpSRP54 when the stromawas incubated with LHCPs, whereas cpSRP54 alone wasinactive (Li et al., 1995). These results are consistent with the

Figure 5. Analysis of CAO Transcript and Products.

(A) Pattern of GUS enzyme activity in the chaos mutant.(B) RNA gel blot analysis of the CAO transcript. Poly(A)1 RNAs cor-responding to 100 mg of total RNA for each sample were analyzedusing the CAO cDNA as probe. The blot was reprobed with a cDNAof a nuclear gene encoding the b subunit of mitochondrial ATPase tocontrol the loading. WT, wild type.(C) Protein gel blot analysis of the indicated amounts of total solubleprotein extracts from wild-type (WT) and chaos plants. The blot wasprobed with antibodies directed against the CAO-encoded protein.(D) Protein gel blot analysis of stromal and thylakoid proteins fromthe wild type. The blot at the top was probed with antibodies di-rected against the CAO-encoded protein. Controls were performedwith antibodies raised against light-harvesting complex II purifiedfrom pea thylakoids (bottom) and with antibodies raised againstchloroplast Hsp70 purified from pea stroma (middle; Yuan et al.,1993).

Identification of a Plant-Specific cpSRP Component 95

idea that cpSRP43 configures cpSRP54 into a form capableof binding LHCPs post-translationally.

The DNA sequence of cpSRP43 revealed two types ofmotifs that can mediate protein–protein interactions: fourankyrin repeats and two closely spaced chromodomains sit-uated at the C terminus. This makes cpSRP43 the secondplant chromoprotein candidate to be characterized (Henikoffand Comai, 1998). Although we expect plants to containchromoproteins that bind chromatin, as described in othereukaryotes, cpSRP43 is noteworthy because it is a novelexample of a nonnuclear chromoprotein. Chromoproteinsare not known in prokaryotes, suggesting that cpSRP43evolved from eukaryotic genes relatively recently. Chromo-domains frequently are found in pairs that function indepen-

dently (Platero et al., 1995). The two cpSRP43 chromodomainsare more closely spaced than usual (Aasland and Stewart,1995), possibly creating a single but more stable binding tar-get. Because chromodomains are known to be involved indimerization (Cowell and Austin, 1997), one possibility is thatthis motif mediates the self-association of cpSRP43, whichwas recently found to be a dimer (D. Schuenemann and N.E.Hoffman, unpublished results). Ankyrin was first describedas an animal protein that couples integral membrane pro-teins to spectrin. The occurrence of ankyrin motifs in func-tionally diverse proteins from animals, yeasts, plants, andeven prokaryotes is inconsistent with a highly specializedfunction. Indeed, ankyrin repeats have been implicated inprotein–protein interactions between heterologous se-quences (Thompson et al., 1991). As such, we speculatethat the ankyrin-repeat domain of cpSRP43 is the regionthat binds cpSRP54.

The salient characteristic of the phenotype of the chaosmutant is the specific effect on the light-harvesting appara-tus. The observed chlorosis was due to a partial lack of pho-tosynthetic pigments. Pigments most affected were thoseassociated with LHCPs. The mutation reduced light-har-vesting efficiency but did not alter the photochemical abili-ties of PSI and PSII; neither did it reduce the steady statelevels of core complex and PSII internal antenna proteins(CP47 and CP43). In contrast, at least two LHC proteinswere found at lesser amounts in the mutant. Other LHCPsmay also be affected because the reduction of the trimerproteins and CP24 appears to be insufficient to account forthe observed decreases in pigment levels (Table 1; Yamamotoand Bassi, 1996). In addition, in the expected size range forLHCPs, several thylakoid membrane polypeptides appear tobe depleted in chaos. Earlier biochemical studies have indi-cated that cpSRP54 is required for LHCP biogenesis in vitro(Li et al., 1995). More recent in vitro studies have also dem-onstrated a requirement for both cpSRP54 and cpSRP43(Schuenemann et al., 1998). Because of the abundance ofLHCPs in the thylakoid membrane, efficient targeting is im-portant. The reduced level of LHCP in the chaos mutant nowprovides genetic confirmation that cpSRP is utilized forLHCP trafficking.

In vitro, LHCPs require the cpSRP pathway for integrationinto the thylakoid membrane (Li et al., 1995). However, asubstantial amount of LHCP is integrated into the thylakoidmembranes of the chaos mutant. This may be due to partialactivity of cpSRP54 in the absence of cpSRP43 or to an al-ternative targeting pathway that compensates for the defectin cpSRP. Such an alternative pathway (reviewed in Clineand Henry, 1996; Klösgen, 1997) may require intact chloro-plast to be functional. In this case, it would not have beendetected with the in vitro experiments performed. In theseexperiments, chloroplast extracts were used to investigatethe role of the cpSRP pathway for LHCP integration into thethylakoid membrane (Li et al., 1995).

Plants containing reduced levels of cpSRP54 have beenobtained by introducing a transgenic copy of cpSRP54 with

Figure 6. The CAO-Encoded Protein Is an Arabidopsis Homolog ofthe Pea CpSRP43.

(A) Comparison of peptide sequence data from pea cpSRP43 andthe CAO-encoded protein (CHAOS). Amino acids conserved orclosely related are underlined. The numbering of the sequence re-fers to their positions in the protein. The pea peptide listed at the topof each pair of sequences was obtained by sequencing the N-termi-nal end of the pea cpSRP43.(B) Antisera against the CAO-encoded protein recognizes cpSRP43.CpSRP was immunoprecipitated with antibodies raised againstcpSRP54 (a-54) from pea stroma (input, 90 mg of chlorophyll), asdescribed in Methods. Immunoprecipitation was also performedwith an antiserum raised against a nucleotide binding protein(a-NBP) from Synechococcus sp strain PCC7942. Proteins remain-ing in the supernatant (spnt) were concentrated by trichloroaceticacid precipitation. The CAO-encoded protein in the supernatant andcoimmunoprecipitates (co-IP) was detected as a 43-kD band by im-munoblot analysis after SDS-PAGE and electrotransfer to nitrocellu-lose membranes.

96 The Plant Cell

a dominant negative mutation (Pilgrim et al., 1998). Surpris-ingly, these mutants have a phenotype different from that ofchaos. The cpSRP54 mutants produce yellow first trueleaves that gradually become green. The chlorophyll a/b ra-tio is identical to that of the wild type in the first true leavesthat are yellow, indicating that the mutation affects proteinsbesides LHCPs. These first true leaves were not observed inthe chaos mutant, and the chlorophyll a/b ratio was signifi-cantly increased. The recent isolation of true null cpSRP54mutants exhibiting the same phenotype (D. Sy, P. Amin, M.Pilgrim, and N.E. Hoffman, unpublished data) has confirmedthat the phenotype is due to cpSRP54 deficiency.

The fact that cpSRP54 and cpSRP43 mutants have differ-ent phenotypes suggests that the two proteins do not al-ways function as a complex. Although most of cpSRP43 isbound to cpSRP54 (Figure 6), none binds to ribosomes,which contain approximately half of the plastid cpSRP54(Franklin and Hoffman, 1993; Schuenemann et al., 1998).Thus, cpSRP54 may have a function on the ribosome that isindependent of cpSRP43. One idea currently being tested isthat ribosome-bound cpSRP54 mediates the cotranslationaltargeting of certain chloroplast-encoded thylakoid proteins,whereas the complex containing cpSRP43 functions prima-rily in LHCP biogenesis.

METHODS

Plant Material and Growth Conditions

Transgenic plants carrying an enhancer trap Dissociation (Ds) ele-ment were described previously (Klimyuk et al., 1995). Arabidopsisthaliana ecotype Landsberg erecta (Ler-0) self-progeny of the line348/74/A segregated a chlorotic mutant named chaos. After in vitrogermination of seeds at 23 and 178C with an 8-hr photoperiod, youngplantlets were transferred to soil in a growth chamber at 23 and 178Cwith an 8-hr photoperiod and 60 to 85% relative humidity. The effectof light on the mutant was investigated by using four different light in-tensities (100, 300, 600, and 1000 mmol m22 sec21) for growing theplants. All molecular and biochemical analyses were performed withleaves harvested at the rosette stage.

Electron Microscopy

Leaves were fixed as described by Lascève et al. (1987). The micro-graphs were taken with a JEM 1200 EXII electron microscope (JEOL,Tokyo, Japan).

Pigments Analysis

Leaf discs 0.8 cm in diameter were frozen in liquid nitrogen beforepigment analysis was performed according Havaux and Tardy(1996).

Chlorophyll Fluorescence and Photosynthetic OxygenEvolution Measurements

Photosystem II Efficiency

The chlorophyll fluorescence measurements were conducted withintact leaves by using a PAM-2000 fluorometer (Walz, Effeltrich, Ger-many), as previously described (Havaux and Davaud, 1994). Themaximal quantum yield of photosystem II (PSII) photochemistry (F)was estimated in dark-adapted leaves (30 min) with an Fv/Fmax ratio.Fv (variable chlorophyll fluorescence) 5 (Fmax 2 Fo), where Fo is theinitial level of fluorescence under red light (655 nm) modulated at 600Hz, and Fmax is the maximal level of chlorophyll fluorescence inducedby a 800-msec pulse of strong white light (photon flux density .4000mmol m22 sec21). The actual quantum yield of PSII in illuminatedleaves was measured as (Fs 2 Fmax)/Fmax, where Fs is the actual flu-orescence level. This parameter is a good indicator of the quantumyield for whole-chain electron transport (involving both PSI andPSII).

Measurement of the Emerson Effect

The quantum yield of photosynthetic oxygen evolution was mea-sured on leaf discs 0.8 cm in diameter by the photoacoustic tech-nique, as previously described by Havaux and Davaud (1994). Thesamples were illuminated with 26 mmol m22 sec21 blue-green light(400 to 500 nm) modulated at 20 Hz. The Emerson effect (E ) wasmeasured in samples adapted to far-red light (730 nm; 45 W m22) for10 min (state 1) or to blue-green light (state 2). E 5 O2 (1far-red light)/O2 (2far-red light). The absolute rate of oxygen evolution in whitelight was measured with a Clark-type oxygen electrode (LD2/2; Han-satech Instruments Ltd., King’s Lynn, UK), as described by Havauxand Davaud (1994).

Chloroplast Isolation and Pigment Protein Electrophoresis

The experimental procedures for pigment protein electrophoresis(chloroplast isolation, pigment–protein complex solubilization, andseparation by partially denaturing 11% PAGE) were conducted asdescribed by Dörmann et al. (1995).

For assaying the CAO-encoded protein in the stroma and thyla-koids, we first isolated Arabidopsis or pea chloroplasts as describedby Spector et al. (1998). Chloroplasts were resuspended in 20 mMHepes-KOH, pH 8.0, and 1 mM phenylmethylsulfonyl fluoride to a fi-nal chlorophyll concentration of 1.5 mg/mL. The lysed chloroplastswere centrifuged at 50,000 rpm for 5 min. The clear supernatant wasused as stroma. Thylakoids were washed two additional times in0.33 M sorbitol and 50 mM Hepes-KOH, pH 8.0, and resuspended ina solution of 20 mM Hepes-KOH, pH 8.0, 1% SDS, and 1 mM phe-nylmethylsulfonyl fluoride. Protein was measured using the bicincho-ninic acid reagent (Smith et al., 1985).

PAGE and Immunoblotting

PAGE and immunoblotting were performed as described by Meureret al. (1996).

Identification of a Plant-Specific cpSRP Component 97

Isolation of Ds Flanking Sequences and Genomic and cDNA Clones of CAO

One sequence flanking the 59 end of Ds was isolated by inverse poly-merase chain reaction (PCR). chaos DNA was digested by Sau3AIand self-ligated. Two PCRs (948C for 20 sec, 588C for 20 sec, and728C for 60 sec for 35 cycles) were performed, the first one with oli-gonucleotides B34 (59-ACGGTCGGTACGGGATTTTCCCA-39) andB35 (59-TATCGTATAACCGATTTTGTTAGTT-39) and the secondone with nested primers D73 (59-TTTCCCATCCTACTTTCATCC-CTG-39) and D74 (59-CCGATTTTGTTAGTTTTATCCCGC-39). The300-bp product generated was cloned into pBluescript KS1 (Strat-agene, La Jolla, CA). This probe was used to screen an Arabidopsisgenomic library (kindly provided by C. Lister and C. Dean, John InnesCentre). One positive clone was recovered, and its 6-kb BamHIfragment hybridizing with the inverse PCR probe was sequenced. A1.3-kb PstI-SalI subfragment of this genomic clone was used as aprobe to screen a l ZAPII cDNA library (Stratagene) constructedfrom poly(A)1 RNA from 20-day-old Ler-0 plants (Parker et al., 1997).Four clones (pLMC C6, pLMC C8, pLMC F1, and pLMC F2) were iso-lated and sequenced. The GenBank accession number for CAO isAF013115.

mRNA Isolation and Analysis

Total RNA was isolated from 0.5 g of Arabidopsis tissue, as de-scribed by Verwoerd et al. (1989). The purification of mRNA was per-formed with a Dynabeads mRNA purification kit (Dynal, Oslo,Norway), using 100 mg of total RNA for each sample. RNA gel blotanalyses were performed under stringent conditions (Sambrook etal., 1989) by using the 1.3-kb PstI-SalI fragment of the CAO gene andthe 1.6-kb EcoRI-SalI cDNA fragment of the nuclear-encoded b sub-unit of the mitochondrial ATPase from Nicotiana plumbaginifolia(Boutry and Chua, 1985). Quantification of the signals from the hy-bridized filter was performed using a Storm 840 PhosphorImager(Molecular Dynamics Inc., Sunnyvale, CA).

DNA Isolation and Analysis

Total DNA was isolated from the plant tissue and digested with dif-ferent restriction enzymes, as described previously (Klimyuk et al.,1995). DNA gel blots were performed according to Sambrook et al.,1989) by using the 1.3-kb PstI-SalI fragment of the CAO genomicclone as probe.

Expression of the CAO-Encoded Protein in Escherichia coli

The cloned CAO cDNA was used as the template for PCR withprimer L5 (59-AAGATCATTGGATCCCGAACGGCGGGGGAAGGA-GC-39), which contains a BamHI site and hybridizes 270 bp after theATG codon, and primer L4 (5 9-GTAATCATCGGATTCAATCAT-TCATTCATT-39), which introduced an EcoRI site after the stopcodon. The PCR product was then cloned into the BamHI and theEcoRI sites of the pGEX4T3 vector (Pharmacia, Uppsala, Sweden),and the resulting plasmid was named pLMC11. The recombinantprotein was produced in E. coli, purified with the bulk glutathioneS-transferase purification kit (Pharmacia), and named DCHAOS.

Immunodetection of the Protein Encoded by CAO

Two batches of polyclonal IgY raised against DCHAOS (antibodiesanti-CHAOS 30 and 39) were obtained from the eggs of two chickensinjected with the purified recombinant protein. The protein encodedby CAO was detected using antibodies raised to chicken IgY cou-pled with horseradish peroxidase (303-035-003; Jackson Immuno-Research Laboratories, West Grove, PA). Light-harvesting complex IIwas detected using antibodies to rabbit IgG coupled to alkalinephosphatase (170-6518; Bio-Rad).

Immunoprecipitation of the Signal Recognition Particle Complex

Protein A–Sepharose (Sigma) was swollen, washed in 1 3 TBS (10mM Tris-Cl, pH 7.5, and 150 mM NaCl), resuspended in TBS to a fi-nal volume of 10% (w/v), and rotated end over end overnight at 48Cwith the indicated antisera (4.8 mg of IgG and 3 mg of protein A–Sepharose). The beads were washed three times in 1 3 TBS andtwice in IP buffer (20 mM Hepes-KOH, pH 8.0, 5 mM MgCl2, and 150mM KCl). The beads were resuspended in 375 mL of IP buffer. Chlo-roplasts were lysed to 3.6 mg of chlorophyll per mL in lysis buffer (20mM Hepes-KOH, pH 8.0, 5 mM MgCl2, 1 mM DTT, and 1 mM phe-nylmethylsulfonyl fluoride) and centrifuged to pellet thylakoids at135,000g for 10 min. Ribosomes were removed by centrifuging thestroma (2.5 mL) over a 1 M sucrose cushion (0.5 mL) in lysis buffer at346,000g for 1 hr. Ribosome-free stroma (25 mL) was added to thetreated beads, and the solution was rotated end over end for 4 hr at48C. The supernatant was removed, and the beads were washedthree times in IP buffer. The bound protein was eluted by the additionof 200 mL of 2 3 sample buffer and heated for 3 min at 958C. Corre-sponding amounts of the diluted ribosome-free stroma, the superna-tant, and the immunoprecipitate were separated by SDS-PAGE in12% polyacrylamide gels, blotted onto nitrocellulose, and probedwith the anti-chaos antiserum (1:5000), as described previously(Pilgrim et al., 1998).

Peptide Sequencing

CpSRP43 was purified as described previously (Schuenemann et al.,1998). N-terminal sequence data were obtained using automatedEdman degradation chemistry at the Rockefeller University Protein/DNA Technology Center (New York, NY). Internal sequence datawere obtained at the Stanford University Protein and Nucleic Acid fa-cility (Stanford, CA), as described previously (Hwang et al., 1996).

ACKNOWLEDGMENTS

We thank Mark Coleman for the gift of the cDNA library, Ken Clinefor antibodies raised against cp hsp70, Christoph Benning and PeterDoermann for their help with the green gels, and Jocelyne Jappéfor advice on the electron microscopic analyses. We are very gratefulto Caroline Dean and Richard Macknight for their helpful discus-sions. We also thank Albert-Jean Dorne for help offered duringanalyses of the lipid content of the mutant and Philippe Lessard, RobMartienssen, Christophe Robaglia, Catherine Sarrobert, and Marie-Christine Thibault for critical reading of this manuscript. Protein se-quence analysis was provided by Richard Winant of the Protein and

98 The Plant Cell

Nucleic Acid Facility, Beckman Center, Stanford University and theRockefeller University Protein/DNA Technology Center, which issupported in part by the U.S. National Institutes of Health shared in-strumentation grants and by funds provided by the U.S. Army andNavy for purchase of equipment. D.S. and N.E.H. were supportedby grants from Deutsche Forschungsgemeinschaft, the NationalScience Foundation (Grant No. MCB-9507745), and the U.S. Depart-ment of Agriculture (Grant No. 96-35304-3703). Work at theSainsbury Laboratory (V.I.K. and J.D.G.J.) is supported by theGatsby Charitable Foundation.

Received August 12, 1998; accepted October 23, 1998.

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100 The Plant Cell

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 This information is current as of August 13, 2019

 

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