Canonical Signal Recognition Particle Components Can Be Bypassed for Posttranslational Protein

Canonical Signal Recognition Particle Components CanBe Bypassed for Posttranslational Protein Targetingin Chloroplasts W

Tzvetelina Tzvetkova-Chevolleau,a Claire Hutin,a Laurent D. Noel,a Robyn Goforth,b Jean-Pierre Carde,c

Stephano Caffarri,a Irmgard Sinning,d Matthew Groves,d Jean-Marie Teulon,e Neil E. Hoffman,f

Ralph Henry,b Michel Havaux,a and Laurent Nussaumea,1

a Unite Mixte de Recherche 6191, Centre National de la Recherche Scientifique–Commissariat a l’Energie Atomique-Universite

Aix-Marseille II, Direction des Sciences du Vivant, Institut de Biologie Environnementale et de Biotechnologie,

Service de Biologie Vegetale et de Microbiologie Environnementale, Commissariat a l’Energie Atomique Cadarache,

F-13108 Saint-Paul-lez-Durance Cedex, Franceb Biological Sciences Department, University of Arkansas, Fayetteville, Arkansas 72701c Unite Mixte de Recherche 619, Biologie du Fruit, Plateforme Imagerie Institut Federatif de Recherche 103, Institut de Biologie

Vegetale Moleculaire, Institut National de la Recherche Agronomique Centre de Bordeaux, 33883 Villenave d’Ornon Cedex,

Franced Biochemie-Zentrum der Universitat Heidelberg, D-69120 Heidelberg, Germanye Direction des Sciences du Vivant, Institut de Biologie Environnementale et de Biotechnologie, Service de Biochimie Post-

Genomique et Toxicologie Nucleaire, Commissariat a l’Energie Atomique-Valrho-BP 17171, 30207 Bagnols/Ceze Cedex, Francef Environmental Risk Analysis Division, Biotechnology Regulatory Services, Animal and Plant Health Inspection Service/U.S.

Department of Agriculture, Riverdale, Maryland 20737

The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) target proteins both cotranslationally and

posttranslationally to the thylakoids. This dual function enables cpSRP to utilize its posttranslational activities for targeting

a family of nucleus-encoded light-harvesting chlorophyll binding proteins (LHCPs), the most abundant membrane proteins

in plants. Previous in vitro experiments indicated an absolute requirement for all cpSRP pathway soluble components. In

agreement, a cpFtsY mutant in Arabidopsis thaliana exhibits a severe chlorotic phenotype resulting from a massive loss of

LHCPs. Surprisingly, a double mutant, cpftsy cpsrp54, recovers to a great extent from the chlorotic cpftsy phenotype. This

establishes that in plants, a new alternative pathway exists that can bypass cpSRP posttranslational targeting activities.

Using a mutant form of cpSRP43 that is unable to assemble with cpSRP54, we complemented the cpSRP43-deficient mutant

and found that this subunit is required for the alternative pathway. Along with the ability of cpSRP43 alone to bind the

ALBINO3 translocase required for LHCP integration, our results indicate that cpSRP43 has developed features to function

independently of cpSRP54/cpFtsY in targeting LHCPs to the thylakoid membranes.

INTRODUCTION

Thylakoids are the internal chloroplast membranes and contain

essential proteins for photoautotrophic growth. A vast majority of

thylakoid proteins are encoded by the nuclear genome, synthe-

sized in the cytoplasm, and translocated by a two-step process.

In the first step, precursors directed by a cleavable N-terminal

targeting domain are imported into the chloroplast across the

envelope membranes by a unique translocon system (Kessler

and Schnell, 2006). During or soon after import, the chloroplast-

targeting domain is cleaved by a stroma-localized processing

protease. In the second step, processed pathway intermediates

in the stroma enter one of four distinct thylakoid localization

pathways, which appear substrate-specific in studies that re-

constitute localization in vitro (for review, see Keegstra and Kline,

1999). The polypeptides that reside in the thylakoid lumen are

mainly targeted by the chloroplast Sec (cpSec) and chloroplast

TAT (cpTAT) pathways. Integral membrane proteins rely mainly

on the chloroplast signal recognition particle (cpSRP) pathway or

use a mechanism that lacks any known protein components,

termed the spontaneous pathway.

The only known nucleus-encoded proteins to utilize the cpSRP

pathway are the light-harvesting chlorophyll binding proteins

(LHCPs). They constitute up to 50% of the protein bulk in thyla-

koids (Green and Salter, 1996) and therefore are considered the

most abundant membrane proteins on earth. General steps of the

LHCP targeting mechanism appear conserved with targeting by

SRPs that function in the cytosol of prokaryotes and eukaryotes.

Cytosolic SRPs bind hydrophobic signal sequences at the N

terminus of newly synthesized polypeptides as they emerge from

the ribosome (for review, see Pool, 2003; Luirink et al., 2005).

SRP then directs the bound ribosome/nascent polypeptide chain

1 To whom correspondence should be addressed. E-mail [email protected]; fax 33-442-254597.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Laurent Nussaume([email protected]).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.106.048959

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complex to a SRP receptor that functions at the endoplasmic

reticulum or the cytoplasmic membrane in bacteria. GTP binding

by SRP and its receptor coordinate the release of the nascent

polypeptide/ribosome complex to a Sec translocase, while GTP

hydrolysis promotes the release of SRP from its receptor. A

distinct functional difference in LHCP targeting by cpSRP stems

from the absence of a ribosome. A cpSRP-LHCP complex

termed the transit complex assembles in the stroma and repre-

sents the targeted form of LHCP (Li et al., 1995; Delille et al.,

2000). Targeting culminates at the thylakoid owing to interactions

between cpSRP, cpFtsY, and ALBINO3 (ALB3) (Moore et al.,

2003), a thylakoid insertase required for LHCP integration into the

lipid bilayer (Moore et al., 2000, 2003; Bellafiore et al., 2002).

The unique posttranslational substrate binding function of

cpSRP stems from its peculiar subunit composition (for review,

see Schuenemann, 2004). Cytosolic SRPs are minimally com-

posed of an RNA moiety in both prokaryotes and eukaryotes

bound to a 54-kD GTPase (SRP54; Ffh in Escherichia coli ) that

functions in binding hydrophobic signal sequences as well as

the SRP receptor, SRa, or its homolog FtsY in E. coli. cpSRP

contains a conserved 54-kD subunit (cpSRP54) but lacks an

RNA moiety, reflected by the absence of an ancestral RNA

binding site in cpSRP54 (Groves et al., 2001; Rosenblad and

Samuelsson, 2004). The novel posttranslational binding activity

of cpSRP is partially based on a plant-specific 43-kD subunit

(cpSRP43; Schuenemann et al., 1998) that forms a heterodimer

with cpSRP54 (Groves et al., 2001). Whereas cpSRP54 in the

transit complex is thought to bind the C-terminal transmembrane

span in LHCPs (High et al., 1997), cpSRP43 binds an internal 18–

amino acid polar motif termed L18 that is conserved among

LHCPs (Delille et al., 2000; Tu et al., 2000).

Despite the lack of an RNA moiety, cpSRP also exhibits a

classical cotranslational function important to the biogenesis of

chloroplast-encoded proteins. Biochemical experiments dem-

onstrated cpSRP54 binding to ribosome-associated D1, a com-

ponent of the photosystem II (PSII) core complex (Nilsson and

van Wijk, 2002; Zhang and Aro, 2002), which is found at reduced

levels along with other chloroplast-encoded reaction center

proteins in cpSRP54-deficient Arabidopsis thaliana (Pilgrim

et al., 1998). The duality in cpSRP function is consistent with

the identification of two cpSRP54 pools in stroma, one bound to

cpSRP43 and the other bound to ribosomes. cpSRP43 appears

absent in the ribosome-bound pool of cpSRP54 (Franklin and

Hoffman, 1993). In this context, deletion mutants of cpSRP54

(ffc) and cpSRP43 (chaos) also support a posttranslational and

cotranslational function of cpSRP; the absence of cpSRP43 affects

the accumulation of LHCPs, whereas the loss of cpSRP54 alters

the accumulation of bothLHCPs and chloroplast-synthesized pro-

teins (Pilgrim et al., 1998; Amin et al., 1999; Klimyuk et al., 1999).

Comparison of the strongly chlorotic chaos ffc double mutant with

mutants lacking either cpSRP43 or cpSRP54 demonstrated that

each subunit contributes to LHCP localization in an independent

and additive manner (Hutin et al., 2002).

Discovery of the chloroplast SRP receptor homolog cpFtsY led

to the demonstration that, along with GTP and cpSRP, LHCP

integration into isolated thylakoids requires cpFtsY (Kogata et al.,

1999; Tu et al., 1999). A cpFtsY-cpSRP54 complex, stabilized at

the thylakoid membrane by nonhydrolyzable GTP, associates

with ALB3, indicating that neither substrates nor cpSRP43 are

required for cpSRP interaction with ALB3 (Moore et al., 2003).

In maize (Zea mays) mutants lacking cpFtsY, there is an

accumulation of the cpSRP-LHCP transit complex in the stroma,

although the extent to which different LHCPs rely on cpFtsY

varies among LHCP family members (Asakura et al., 2004).

Nevertheless, maize cpFtsY mutants also exhibited a seedling-

lethal phenotype, as plants died 2 weeks after germination

(Asakura et al., 2004). Such a drastic phenotype, compared

with the less severe mutant phenotypes deficient for cpSRP43 or

cpSRP54, supports a central role for cpFtsY in targeting LHCPs

and in thylakoid biogenesis.

In Arabidopsis, a viable cpftsy mutant was recently identified

(Durett et al., 2006), but the repercussions of this mutation on the

cpSRP pathway were not studied. In this article, we identified a

second mutant allele of Arabidopsis cpftsy. The phenotype of this

cpftsy mutant was compared with the phenotype of Arabidopsis

mutants affected in the other cpSRP components. Importantly,

the viability of the cpftsy mutant made it possible to combine

mutations, which led to the discovery that removal of both

cpSRP54 and cpFtsY relieves the severity of the cpftsy mutant

phenotype. These data demonstrate that in the absence of

conserved SRP targeting components, cpSRP54 and cpFtsY, a

second targeting mechanism accommodates LHCP localization

to the thylakoids and most likely relies on cpSRP43 and its ability

to bind both LHCP and ALB3.

RESULTS

cpFtsY Is Not Required for Arabidopsis Viability

An Arabidopsis mutant defective for cpFtsY production was

identified by protein gel blot screening of an ethyl methanesulfo-

nate (EMS)–mutagenized collection using a polyclonal antibody

directed against cpFtsY (Figures 1A and 1B). The cpftsy gene

present in the mutant was amplified by PCR and sequenced. This

revealed a C-to-A transversion in exon 6, creating a stop codon

that truncated half of the protein (Figure 1C). The absence of a

cpFtsY fragment in the mutant suggests that the identified allele

was most probably a null mutant. The mutant exhibited a strong

chlorotic phenotype, which remained linked with the cpFtsY

mutation after three backcrosses that were required to remove

another albino mutation present in this line. In order to confirm that

the observed phenotype of the backcrossed line resulted only

from lack of cpFtsY, the mutant was successfully complemented

with cpFtsY cDNA fused to a modified cauliflower mosaic virus

35S promoter from plasmid pKYLX71-35S2 (Figures 1A and 1B). It

is noteworthy that transformants overexpressing the cpFtsY

cDNA accumulated normal levels of cpFtsY protein compared

with the wild type (data not shown), which could stem from

posttranscriptional control of the cpFtsY level. Despite its marked

chlorosis, the Arabidopsis cpftsy mutant is fully viable and can

easily be grown in vitro on agarose or on soil.

The Arabidopsis cpftsy Mutant Exhibits a Very Similar

Phenotype to the ffc chaos Double Mutant

The very strong chlorosis exhibited by the cpftsy mutant (Figure

2A) was due to the loss of ;80% of the photosynthetic pigments

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(Figure 2B). More specifically, there was a 67% reduction of

xanthophylls, an 80% reduction of b-carotene, and an 84%

reduction of chlorophylls (Figure 2B). This pigment decrease did

not alter the chlorophyll a/b ratio, which was ;2.4 in the cpftsy

mutant and the wild-type control. Electron micrographs of cpftsy

mutant chloroplasts revealed important alterations in the ultra-

structure of this organelle, with a drastic reduction in the amount of

both appressed and unappressed thylakoid membranes (Figure

2C). Taken together, these traits are much like those of the ffc

chaos (cpSRP54�/cpSRP43�) double mutation (Hutin et al., 2002),

which exhibits a similar loss of photosynthetic pigments and

alteration of chloroplast ultrastructure (Figure 2B). Also like the ffc

chaos double mutant, the cpftsy mutation greatly reduced the

accumulation of various LHCPs relative to the wild type. However,

the extent of the reduction differs between the various LHCPs

(Figure 3A). The two most abundant antennae of PSII (Lhcb1 and

Lhcb2) have a similar size and could not be distinguished by the

antibody used, which recognizes both proteins (LHCII in Figure 3A).

In addition to the LHCPs, the cpftsy mutation affected chloroplast-

encoded proteins, which require the cpSRP54 subunit to reach

their thylakoid location (Amin et al., 1999; Nilsson et al., 1999;

Nilsson and van Wijk, 2002). These include the reaction center

proteins of PSI (PsaA and PsaB) and PSII (D1). As observed in

Figure 3B, both complexes are reduced in the cpftsy mutant

compared with the wild-type control. This decrease in reaction

center proteins also resembles closely the phenotype of the ffc

chaos double mutant (Figure 3B).

Figure 1. The Arabidopsis cpftsy Mutant Exhibits a Severe Chlorotic

Phenotype That Is Complemented by the Expression of a cpFtsY

Transgene.

(A) Phenotypic comparison of the wild type, the cpftsy mutant, and the

cpftsy mutant transformed with a cpFtsY expression construct (cpftsy/

35ScpFtsY) is shown for plants grown to the rosette stage.

(B) Total protein extracts from each plant shown in (A) were separated by

SDS-PAGE, blotted, and probed with antibody directed against cpFtsY.

(C) EMS mutation in the CS3171 line introduced a stop codon in the

coding region of the cpFtsY gene.

Figure 2. The cpftsy Mutant and ffc chaos Double Mutant Exhibit Similar

Severe Phenotypic and Ultrastructural Alterations.

(A) Wild-type, double mutant ffc chaos, and cpftsy mutant plants are

presented at the rosette stage.

(B) Pigments were extracted from the plant lines shown in (A) and

analyzed as described in Methods. Data are mean values of five

measurements 6 SD. The scale (ng/mm2) for minor carotenoids is shown

at right, and the scale for chlorophylls is given at left. antera, antherax-

anthin; bcar, b-carotene; chla, chlorophyll a; chlb, chlorophyll b; lut,

lutein; neo, neoxanthin; vio, violaxanthin; zea, zeaxanthin.

(C) Chloroplast ultrastructure was examined by electron microscopy

using leaf sections from the plant lines shown in (A). Representative

electron micrographs are shown. Bar ¼ 1 mm.

New Pathway for LHCP Targeting 1637

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As described previously for all of the cpSRP mutants, equal

loading of total membrane proteins for immunoblot analyses

resulted systematically in an overrepresentation of chloroplast

envelope membrane proteins from mutant plants relative to the

wild type, due to the lack of LHCP (main membrane proteins).

Therefore, in order to quantify the loading differences, we used

an antibody raised against the stromal protein ClpC, which

associates with the chloroplast inner membrane translocon

(Akita et al., 1997). As observed previously (Hutin et al., 2002)

for ffc chaos, an excess of ClpC protein was also found for the

cpftsy mutant (Figure 3C). Therefore, the differences in specific

protein levels observed between mutants and the wild-type

control in Figures 3A and 3B appear underrepresented, owing to

the fact that a greater number of chloroplast membranes are

present in the mutant-derived samples to achieve an equal

amount of total membrane protein in mutant and wild type

Figure 3. Photosystem Protein Accumulation and Photosystem Activity of cpftsy and chaos ffc Mutants Indicate Similar but Distinct Functions of

cpSRP and cpFtsY.

Total membrane proteins were extracted from leaves of wild-type and mutant (cpftsy and chaos ffc) plants as described in Methods and analyzed by

protein gel blotting ([A] to [C]) using antibodies directed against the proteins indicated at right. Samples were loaded at three different concentrations to

detect and avoid possible saturation of the immunodetection (quantification is indicated in Supplemental Table 1 online), and blotting was repeated at

least three times to ensure reproducibility. A representative blot is shown for each protein detected.

(A) Antenna proteins of PSI and PSII (Lhca and Lhcb, respectively). The antibody dilution used (in parentheses) and the amount of membrane proteins

loaded on each lane were as follows: Lhca1 (1:100), 3/5/7 mg; Lhca2 (1:200), 3/5/7 mg; Lhca3 (1:1000), 1.5/3/5 mg; Lhca4 (1:100), 5/7/10 mg; LHCII

(1:1000), 1/2/4 mg; Lhcb3 (1:30), 1.5/3/5 mg; Lhcb4 (1:30), 1/3/5 mg; Lhcb5 (1:100), 0.75/1.5/3 mg; and Lhcb6 (1:750), 1/2/4 mg.

(B) PSI and PSII reaction center binding proteins. The antibody dilution used (in parentheses) and the amount of membrane proteins loaded on each

lane were as follows: D1 (1:100), 2/4/6 mg; and PsaA/B (1:1000), 5/10/15 mg.

(C) Proteins targeted by thylakoid pathways distinct from cpSRP. Fibrilin C34 (Fibr.C34) is targeted by the DpH pathway; plastocyanin (PC) and the

33-kD subunit of the oxygen evolving complex (OE33) use the Sec pathway; and PsbS is targeted by the spontaneous insertion pathway. ClpC is

a component of the chloroplast envelope translocase included for comparison. The antibody dilutions used (in parentheses) and the protein amounts

loaded in each lane were as follow: ClpC (1:4000), 3/5/7 mg; fibrilin C34 (1:1000), 3/5/7 mg; PC (1:5000), 2/5/10 mg; OE33 (1:10,000), 0.5/1.25/3 mg; and

PsbS (1:4000), 2/5/10 mg.

(D) and (E) Photochemistry. Chlorophyll fluorescence characteristics were measured from isolated leaves as described in Methods to compare

maximum quantum yield of PSII (Fv/Fm) (D) and quantum yield of PSII-mediated electron transport (DF/Fm9) (E). Data are mean values of four separate

experiments 6 SD.

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samples. The function of the various other thylakoid targeting

pathways was investigated by immunodetection of proteins

localized specifically by the spontaneous (PsbS), Sec (OE33

and plastocyanin [PC]), and cpTAT (fibrilin CDSP34) pathways. In

all cases, with the exception of OE33, the proteins targeted by

these various pathways were found in greater amounts in both

mutants (Figure 3C). This confirmed that the mutation studied

here does not inhibit these pathways. The higher levels observed

are consistent with the observation that both mutant-derived

samples contain more chloroplast proteins than the wild-type

control (except of course for cpSRP-targeted proteins). The

slight decrease in the level of OE33 protein, which is a subunit of

the PSII oxygen-evolving complex, could be attributed to the

decreased PSII levels observed in these plants.

Chlorophyll Fluorescence Characteristics Distinguish

cpftsy from the ffc chaos Double Mutant

Previous analyses indicated that the ffc chaos double mutation

abolishes the cpSRP pathway required for thylakoid targeting

of LHCP in chloroplasts (Hutin et al., 2002). Similarity between

the cpftsy mutant phenotype and the ffc chaos double mutant

suggests that cpFtsY is also absolutely required for cpSRP

function. Yet, chlorophyll fluorescence analyses point to distinct

features between both mutants. The photochemical activity of

PSII was analyzed in vivo using chlorophyll fluorescence mea-

surements (Fv/Fm) in dark-adapted leaves. The Fv/Fm ratio is a

measure of the maximal photochemical efficiency of PSII when

all of the primary quinonic PSII electron acceptors are oxidized

(Maxwell and Johnson, 2000). As found previously (Hutin et al.,

2002), the ffc chaos double mutants did not differ significantly

from the wild-type control, whereas this value was noticeably

affected (28% decrease) in the cpftsy mutant (Figure 3D). This

suggests that, in contrast with ffc chaos, the proper assembly

and the photochemical competence of PSII reaction centers

were affected in the cpftsy mutant. The actual quantum yield of

PSII photochemistry (DF/Fm9) was measured at different photon

flux densities in cpftsy and ffc chaos. DF/Fm9 is a measure of the

efficiency of linear electron transport in the chloroplasts (Genty

et al., 1989). At illumination levels of <250 mmol�m�2�s�1, the

quantum yield was more reduced in the cpftsy mutant relative

to the ffc chaos double mutant, indicating that photosynthe-

tic electron transport was more affected and was more

rapidly saturated by light in the cpftsy mutant. There are many

factors that could contribute to the reduction of the efficiency

of photosynthetic electron transport in cpftsy, including the

inhibition of PSII (indicated by the reduced Fv/Fm value) or a

block of the electron transport chain after PSII (e.g., an inhibition

of PSI).

Lack of cpSRP54 Abolishes the cpFtsY Effect on LHCP

Targeting to Thylakoids

The Arabidopsis ffc mutant lacking cpSRP54 accumulates more

LHCP (Amin et al., 1999) than the cpftsy mutant. This may

indicate that cpSRP54 functions only to improve the efficiency of

protein targeting by a mechanism that relies on cpFtsY even in

the absence of cpSRP54. Alternatively, the requirement for

cpFtsY may be bypassed in the absence of cpSRP54, owing to

a need for cpFtsY only when targeting substrates are bound by

cpSRP54. These hypotheses may be genetically tested by

creating a ffc cpftsy double mutant lacking both cpSRP54 and

cpFtsY. The resulting double mutant would exhibit a severe

phenotype similar to that of the cpftsy mutant if cpSRP54 simply

improves the efficiency of a cpFtsY-dependent routing mecha-

nism. By contrast, the ffc cpftsy double mutant would exhibit a

less severe ffc-like phenotype if cpFtsY function were dedicated

to substrate targeting by cpSRP54. This scenario implicates the

function of a second LHCP localization pathway that is indepen-

dent of both cpSRP54 and cpFtsY and that operates only when

LCHP targeting substrates fail to interact with cpSRP54.

The cpftsy and ffc mutants were crossed to produce F1 plants.

As expected (both mutations are recessive), the resulting hybrids

exhibited a wild-type phenotype. In the progeny of these F1

plants, we analyzed various individuals using antibodies raised

against cpFtsY and cpSRP54. This allowed the identification of a

ffc cpftsy double mutant that exhibited a ffc mutant phenotype

(Figures 4A and 4B). The double mutant was also verified by

backcrosses with ffc or cpftsy mutants. The resulting F1 progeny

exhibited ffc or cpftsy phenotypes, respectively, as expected.

Pigment analyses of the ffc cpftsy double mutant revealed that

the levels of chlorophyll a and b, b-carotene, lutein, and neo-

xanthin were similar in the ffc and ffc cpftsy mutants. However, in

the ffc cpftsy double mutant, violaxanthin was almost completely

converted to zeaxanthin and antheraxanthin (Figure 4C), sug-

gesting the presence of a light stress in this mutant (Demmig-

Adams and Adams, 2000). Analysis of nucleus-encoded (LHCP;

Figure 4D) and chloroplast-encoded (reaction centers PSI and

PSII; Figure 4E) proteins targeted by cpSRP revealed no striking

difference between ffc and ffc cpftsy mutants. This suggests

that, for the proteins targeted by the cpSRP pathway, the ffc

mutation is epistatic to the cpftsy mutation. Examination of

proteins targeted by other thylakoid-targeting pathways did not

reveal striking differences between ffc and ffc cpftsy, with the

exception of plastocyanin, which accumulated to a higher level in

the double mutant (Figure 4F). Taken together, these data are in

agreement with a model in which cpFtsY activity requires the

presence of cpSRP54.

In assays that reconstitute LHCP integration into isolated

thylakoids, integration requires all cpSRP pathway soluble tar-

geting components (cpSRP43, cpSRP54, and cpFtsY). Yet,

much like the cpSRP54 mutant alone (ffc), eliminating both

cpSRP54 and cpFtsY in vivo (ffc cpftsy) affected LHCP accu-

mulation only moderately. This indicates the presence of an

alternative pathway for LHCP targeting that bypasses these two

proteins in plants when cpSRP54 is absent and unable to interact

with LHCP to form a cpSRP-LHCP transit complex. Since a

previously reported double mutant lacking both cpSRP54 and

cpSRP43 (ffc chaos) accumulates a much lower level of LHCP

than either the ffc mutant or the ffc cpftsy double mutant, it can

be assumed that cpSRP43 is required for this alternative path-

way. The cpSRP54-independent activity of cpSRP43 can be ex-

amined using a cpSRP43 mutant that lacks the ability to bind

cpSRP54, since cpSRP54-dependent LHCP integration activity

is lost in vitro when cpSRP54-cpSRP43 heterodimer formation is

disrupted (Goforth et al., 2004).


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cpSRP43-Deficient Mutants Can Be Fully Complemented by

Expression of a Modified cpSRP43 Protein That Does Not

Bind cpSRP54

Combined functional and interaction studies demonstrated that

CD2, the second of three chromodomains present in cpSRP43

(amino acids 270 to 320), is required for cpSRP43 binding to

cpSRP54 (Goforth et al., 2004). Arabidopsis cpSRP43 cDNA

deleted of the CD2 domain (DCD2cpSRP43) driven by the

cauliflower mosaic virus 35S promoter was introduced into the

cpSRP43-null mutant (chaos). Several independent transform-

ants were produced, and seven of them (named DC2cpSRP43-A

to -G) containing only one copy of the construct were selected by

DNA gel blot for further analysis (data not shown). As reported

previously, the chaos mutant exhibited a pale green phenotype

(Klimyuk et al., 1999), resulting from the loss of ;45% of the

photosynthetic pigments (Figure 5A). Interestingly, all of the

chaos transformants complemented with DC2cpSRP43 exhib-

ited increased pigmentation, reaching in most cases wild-type

levels (Figure 5A). The ability of DC2cpSRP43 expression to

complement the chaos mutant was independent of cpSRP

heterodimer formation, as indicated by the results of coprecip-

itation assays conducted with anti-cpSRP43 antibody, which

binds both native cpSRP43 and DC2cpSRP43 (Figure 5B).

Although cpSRP54 was found to coprecipitate with cpSRP43

using soluble leaf extract from wild-type plants, cpSRP54 was

absent when DC2cpSRP43 was immunoprecipitated from solu-

ble leaf extract derived from chaos transformants expressing

DC2cpSRP43 (Figure 5B). These results are consistent with

sizing chromatography assays, which showed that cpSRP54

coelutes with cpSRP43 using wild-type plant extracts but elutes

separately from DC2cpSRP43 using leaf extracts derived from

the chaos transformants (data not shown). Together, these

findings verify the results of in vitro studies showing that

DC2cpSRP43 is unable to bind cpSRP54 (Goforth et al., 2004).

Importantly, they also demonstrate that normal LHCP targeting

can take place in the absence of cpSRP43/cpSRP54 dimer for-

mation, adding further support for a cpSRP54/cpFtsY-independent

function of cpSRP43.

cpSRP43 Interacts with ALB3 in the Absence of cpSRP54

and cpFtsY

Genetic data supporting a cpSRP54/cpFtsY-independent tar-

geting pathway that relies on the function of cpSRP43 raises the

possibility that cpSRP43 is able to interact directly with ALB3 in

targeting LHCPs. To investigate this possibility, double immu-

nolabeling experiments were performed in various mutant

backgrounds to examine the colocalization of cpSRP43 and

ALB3 (Figure 6A). A cryofixation protocol was employed to

freeze the localization of proteins occurring in the cell, thereby

avoiding vagaries more common to classical fixation protocols.

The association between cpSRP54 and cpSRP43 in the stroma

of intact chloroplasts was previously reported using a sim-

ilar approach (Hutin et al., 2002). In micrographs from wild-

type and mutant backgrounds examined, immunolabeling of

cpSRP43 was observed about twice as often as ALB3. There-

fore, colocalization events were evaluated in relation to the less

Figure 4. The Double Mutant ffc cpftsy Exhibits an ffc Phenotype.

(A) ffc cpftsy and ffc mutants are shown at the rosette stage.

(B) Total leaf protein extracts from wild-type and mutant (ffc, cpftsy, and

ffc cpftsy) plants were examined by protein gel blotting using antibody

against cpSRP54 or cpFtsY as indicated.

(C) Pigments extracted from leaves of ffc cpftsy and ffc mutants were

characterized as described in Methods. Data are mean values of four

measurements 6 SD. The concentrations (ng/mm) of carotenoids are

indicated at left and those of chlorophylls are indicated at right. antera,

antheraxanthin; bcar, b-carotene; chla, chlorophyll a; chlb, chlorophyll b;

lut, lutein; neo, neoxanthin; vio, violaxanthin; zea, zeaxanthin.

(D) to (F) Relative levels of PSI and PSII antenna proteins (Lhca and Lhcb,

respectively) (D), reaction center binding proteins (D1 and PsaA/B) (E),

and proteins targeted by the DpH (fibrilin [Fibr.C34]), Sec (PC and OE33),

and spontaneous insertion (PsbS) pathways (F) were examined by

protein gel blotting as described in the legend to Figure 3.

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frequently detected protein ALB3 and were calculated for in-

dependent micrographs by counting the number of colocaliza-

tion events (Figure 6A). x2 analysis was used to confirm the

statistical validity of the results (data not shown).

In the wild-type background and in the cpftsy mutant, where

cpSRP54/cpSRP43 heterodimers persist, cpSRP43 did not or

only occasionally colocalized with ALB3 (0 and 1% of colocal-

ization events observed, respectively). These data are consistent

with previous biochemical studies showing that cpSRP interacts

poorly with ALB3 in the absence of cpFtsY and only takes place

efficiently in the presence of cpFtsY when nonhydrolyzable GTP

is added to stabilize cpSRP–cpFtsY interaction at the membrane

in a complex with ALB3 (Moore et al., 2003). Interestingly, in the

absence of cpSRP54 (ffc and ffc cpftsy backgrounds; Figure 6A),

colocalization of cpSRP43 with ALB3 can clearly be observed (18

and 16%, respectively). Taken together, these results support

the possibility that in both the ffc mutant and the ffc cpftsy double

mutant, cpSRP43 may act to target LHCPs to ALB3, owing to an

affinity of cpSRP43 for ALB3.

To understand whether cpSRP43 alone possesses the ability

to target LHCP substrates through interactions with ALB3, thy-

lakoids salt-washed to remove detectable cpSRP54, cpSRP43,

and cpFtsY were incubated with increasing amounts of His-

tagged cpSRP43. Following removal of the unbound cpSRP43,

thylakoid-bound cpSRP43 was repurified from maltoside-

solubilized membranes and examined by protein gel blotting for

the presence of copurified proteins (Figure 6B). Both OE23 and

cpSecY were undetectable in these coprecipitation assays.

LHCP was detectable at the same low level whether cpSRP43

was absent or present in amounts as high as 4 mg, suggesting

that the small amount of copurified LHCP observed in these

assays resulted from nonspecific binding to the metal affinity

beads.

When 10 mg of cpSRP43 was included in the assay, a slight

increase in the amount of copurified LHCP was observed relative

to levels seen in the absence of cpSRP43. However, this most

likely stemmed from nonspecific binding of cpSRP43 to thyla-

koids, since no additional LHCP signal resulted from incubation

of thylakoids with 30 mg of cpSRP43. This also indicates that

LHCP, once properly integrated and assembled into light-

harvesting complexes, is no longer available to interact specifically

with cpSRP43, in contrast with the specific cpSRP43–LHCP

interaction that takes place in solution as LHCP targeting sub-

strate crosses the stroma (Tu et al., 2000). By contrast, ALB3

copurified with cpSRP43 in a concentration-dependent manner,

revealing a clear ability of cpSRP43 to interact with ALB3. The

demonstration of an interaction between ALB3 and cpSRP43

without a requirement of cpFtsY or cpSRP54 supports the in situ

colocalization data and the putative bypass of these two com-

ponents by cpSRP43 in posttranslational targeting of LHCPs to

the thylakoid membrane.

DISCUSSION

cpFtsY Is Not Essential for the Survival of Arabidopsis Plants

To date, the impact of cpFtsY deficiency on the cpSRP protein

targeting pathway in vivo has been investigated only in maize

(Asakura et al., 2004), in which the absence (or reduction) of

cpFtsY produced seedling-lethal plants that died 2 weeks after

germination. The situation is clearly distinct in Arabidopsis, in

which the null mutant identified in this study exhibited a severe

chlorotic mutant phenotype similar to that in maize but is viable.

Figure 5. cpSRP43-cpSRP54 Heterodimer Formation Is Not Required

for cpSRP43 to Complement chaos Mutants.

(A) chaos mutants lacking cpSRP43 were transformed with DNA coding

for cpSRP43 that lacks the cpSRP54 binding domain. Pigment accu-

mulation (chlorophylls a and b and carotenoids) of the chaos mutant and

different T1 plants resulting from transformation of chaos (chaos

DC2cpSRP43-A to -G) was analyzed as described in Methods and

expressed as a percentage of wild-type values (ecotype Landsberg

erecta).

(B) Soluble protein extract was prepared from leaves of the wild type

corresponding to ffc (Col), ffc mutant, the wild type corresponding to

chaos (Landsberg erecta), chaos, and two of the chaos transformants

described for (A) (chaos DC2cpSRP43-F and -G) as indicated. The top

and middle panels (input) show the level of cpSRP43 (closed arrow-

heads), DC2cpSRP43 (open arrowhead), or cpSRP54 in each ex-

tract detected by protein gel blotting with the antibodies (a) indicated

at right. The bottom panel shows cpSRP43 (closed arrowhead) and

DC2cpSRP43 (open arrowhead) copurified with cpSRP54 following

immunoprecipitation with cpSRP54 antibody (IP a-cpSRP54).


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This observation is in agreement with the recent map-based

cloning of fro4, a mutation residing in cpFtsY (Durett et al., 2006).

The fro4 mutants were identified for their inability to increase root

Fe(III) chelatase activity when exposed to low iron availability. No

studies were conducted to examine the effect of cpFtsY defi-

ciency on the cpSRP pathway, despite fro4 mutants exhibiting a

similar severe chlorotic phenotype. It is noteworthy that in both

Arabidopsis and maize, chloroplast ultrastructure is similarly

perturbed during the first weeks after germination. However,

growing cpFtsY-deficient Arabidopsis under low light allowed

the recovery of normal chloroplast ultrastructure after 3 to 4

months of culture (data not shown), suggesting that the cpFtsY

mutant may be sensitive to photooxidative stress.

The Function of cpFtsY Extends beyond Its Role as a cpSRP

Receptor in the Biogenesis of Chloroplast-Synthesized

Thylakoid Proteins

It has been demonstrated that cpSRP54 binds nascent D1 poly-

peptides (Nilsson et al., 1999) and that ffc mutants lacking

cpSRP54 exhibit a reduction of D1 insertion into the thylakoid

membranes (Amin et al., 1999). This was accompanied by re-

duced accumulation of other PSI and PSII reaction center com-

ponents in the ffc mutants, indicating that cpSRP54 contributes

to the targeting of chloroplast-encoded proteins through a co-

translational process. Nevertheless, a substantial level of these

chloroplast-encoded proteins accumulated in the absence of

cpSRP54, suggesting that a less efficient cpSRP54-independent

routing mechanism functions in the localization of chloroplast-

synthesized thylakoid proteins (Figure 4) (Amin et al., 1999). The

comparison of cpftsy and chaos ffc phenotypes indicates that the

chloroplast-encoded proteins (D1, PsaA/B) are affected similarly

in both mutants, consistent with the role of cpFtsY as a receptor

for cpSRP in cotranslational targeting to the thylakoid. However,

two observations suggest that cpFtsY also functions in photosys-

tem assembly. First, PSII photochemical activity (Fv/Fm) was

affected in the cpftsy mutant but not in the chaos ffc double

mutant. Second, photosynthetic electron transport (DF/Fm9) was

diminished in the cpftsy mutant relative to the chaos ffc double

mutant, mainly at light levels of <250 mmol�m�2�s�1. Inhibition

of Fv/Fm and DF/Fm9 occurred despite the fact that these

two mutants accumulate similar pigment and antenna levels.

Similarly, photosynthetic electron transport was inhibited in ffc

cpftsy double mutants relative to ffc mutants (see Supplemental

Figure 1 online).Figure 6. cpSRP43 Interaction with ALB3 Takes Place in the Absence of

cpSRP54 and cpFtsY.

(A) cpSRP43 colocalizes with ALB3 in the absence of cpSRP54 and

cpFtsY. Immunolabeling of ALB3 (white arrowheads; 10-nm gold parti-

cle) and cpSRP43 (black arrowheads; 5-nm gold particle) in wild-type,

cpftsy, ffc, and ffc cpftsy backgrounds was conducted as described

in Methods. Representative micrographs are shown for each sample.

Bars ¼ 100 nm. Analysis of independent micrographs was performed

to evaluate the frequency of cpSRP43 and ALB3 colocalization events.

For each genotype, the percentage of ALB3 colocalized with cpSRP43,

the total amount of cpSRP43 immunolabeled observed, and the total

amount of ALB3 immunolabeled observed in all micrographs, respectively,

are indicated as follows: wild type, 0%/147/78; ffc, 18%/92/40; cpftsy,

1%/181/87; and ffc cpftsy, 16%/185/75.

(B) cpSRP43 specifically coprecipitates ALB3. Salt-washed thylakoids

(200 mg) were incubated with different amounts of cpSRP43-his protein

(0 to 30 mg) as indicated in the presence of 0.5 mM GMP-PNP. Treated

thylakoids were washed, solubilized in maltoside, and mixed with Talon

resin to repurify His-tagged cpSRP43 and all proteins bound to cpSRP43

as described previously (Moore et al., 2003). cpSRP43 and any cop-

urified proteins were eluted from the Talon resin as described in

Methods. The eluted proteins were then examined on protein gel blots

probed with antibodies directed against the proteins indicated at left. A

lane of salt-washed thylakoids was included to verify the specificity of

each antibody.

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Although we have not examined the accumulation of all pho-

tosystem core polypeptides, these differences do not correlate

with the level of photosystem core polypeptides examined. In this

context, it is possible that cpFtsY plays a role in photosystem

assembly beyond its receptor action in cpSRP targeting. This is

supported by recent results showing that cpftsy is linked to the

posttranslational reduction of Fe(III) chelate reductase activity

(Durett et al., 2006). The absence of cpFtsY may reduce iron

content in the very early stage of development. PSI and the

cytochrome b6/f complex contain iron–sulfur clusters; therefore,

iron is crucial for the function of those photosynthetic complexes.

Iron deficiency would lead to an inhibition of electron transfer

through PSI and/or cytochrome b6/f, thus explaining the fluores-

cence results described above, which showed a marked reduc-

tion of photosynthetic electron transport efficiency when cpFtsY

was lacking. Inhibition of PSII photochemistry, indicated by a

decreased Fv/Fm, could be a secondary effect of the impairment

of the PSI and cytochrome b6/f electron transfer activities.

Presumably, any additional function of cpFtsY, beyond its role

as a membrane receptor for cpSRP, relies on its ability to localize

to the thylakoid membrane, since immunolocalization failed to

detect cpFtsY in the stroma (data not shown). Despite the fact

that cpFtsY is distributed almost equally between stroma and

thylakoids prepared from pea (Pisum sativum) chloroplasts (Tu

et al., 1999), our immunolocalization data are in agreement with

biochemical results obtained with maize (Asakura et al., 2004)

showing that cpFtsY is almost entirely associated with the

thylakoid membrane.

cpSRP54 Activity Is Detrimental to Thylakoid Biogenesis

in the Absence of cpFtsY

Similar to maize mutants lacking cpFtsY (Asakura et al., 2004),

the Arabidopsis cpftsy mutant exhibited a stronger phenotype

than mutants lacking either cpSRP43 or cpSRP54. This obser-

vation supports the proposal formulated by Asakura and co-

workers (2004) that cpFtsY plays an essential role for LHCP

targeting as well as the assembly of other membrane compo-

nents of the photosynthetic apparatus. Nevertheless, analysis of

the ffc cpftsy double mutant clearly modifies our interpretation of

the cpftsy phenotype described above. Epistasy of the ffc

mutation on the cpftsy mutation (at least concerning proteins

targeted by cpSRP) indicates that both genes have a comple-

mentary action and that cpSRP54 must be present for cpFtsY to

function in the chloroplast SRP targeting pathway; the severe

phenotype of plants lacking cpFtsY (cpftsy mutant) is largely

reversed by additionally removing cpSRP54 (ffc cpftsy double

mutant). The resulting phenotype of the ffc cpftsy double mutant

closely resembles that of plants lacking cpSRP54 alone (ffc).

Taken together, our data indicate that an alternative sorting

pathway functions in the chloroplast independent of both

cpSRP54 and cpFtsY. Our data also suggest that targeting

substrates, normally targeted by cpSRP/cpFtsY, gain access to

the alternative pathway only in the absence of cpSRP54. In the

presence of cpSRP54, cpFtsY is strictly required.

In cotranslational protein targeting by mammalian and bacte-

rial SRPs, the targeting reaction is initiated in the cytosol by the

interaction of a cpSRP54 homolog (SRP54 and Ffh, respectively)

with N-terminal hydrophobic signal sequences in the targeting

substrates. SRP then directs its ribosome-associated targeting

substrate to an SRP receptor homolog of cpFtsY at the mem-

brane, owing to the affinity of SRP54 for the receptor, an

interaction that stimulates GTP binding by both SRP54 and its

receptor. In the presence of an available translocase, the

targeted substrate is released from SRP54 followed by GTP

hydrolysis to release SRP from its receptor (Pool, 2005). Bio-

chemical studies indicate that these interactions are conserved

in chloroplasts. A stable complex is formed between cpSRP54

and cpFtsY at the thylakoid in the presence of nonhydrolyzable

GTP to prevent their dissociation (Moore et al., 2003). The

cpSRP54-cpFtsY complex can form independently of cpSRP43

and associates with the ALB3 translocon. In this context, it would

be expected that targeting substrates would remain bound to

cpSRP54 in the absence of cpFtsY and thereby be unavailable

for membrane insertion during thylakoid biogenesis. In maize that

lacks cpFtsY, LHCP bound to cpSRP was found to accumulate

in the stroma and on the surface of the thylakoids, supporting the

need for cpFtsY to release targeting substrates from cpSRP

(Asakura et al., 2004). While the severe nature of the observed

cpftsy phenotype is consistent with substrates entering a dead-

end targeting pathway upon interaction with cpSRP54, the less

severe ffc-like phenotype exhibited by the cpftsy ffc double

mutant indicates that in the absence of substrate binding by

cpSRP54, substrates can be successfully routed to the thylakoid

by an alternative pathway that leads to their proper integration

and assembly. Although cpFtsY appears to play an additional

role in photosystem assembly, as discussed above, the similar

accumulation of LHCPs in ffc and cpftsy ffc mutants indicates

that once LHCP localization is independent of cpSRP54, it also

becomes independent of cpFtsY, even if cpFtsY is present.

Since the proper localization of LHCPs relies on the function of

ALB3 for proper integration into the thylakoid membrane, it

would be expected that a cpSRP54-independent (or cpSRP54/

cpFtsY-independent) routing mechanism that is active in both

the ffc and cpftsy ffc mutants would also possess the ability to

direct LHCP-targeting substrates to ALB3.

cpSRP43 Is a Component of a cpSRP54/cpFtsY-Independent

LHCP-Targeting Pathway

Insight into the nature of the cpSRP54-independent targeting of

LHCP comes from consideration of the ffc chaos double mutant,

which lacks both cpSRP54 and cpSRP43 (Hutin et al., 2002).

Unlike the ffc or cpftsy ffc mutants, the absence of both cpSRP

subunits results in a marked decrease in LHCP accumulation,

indicating that cpSRP43 is required for LHCP localization even in

the absence of cpSRP54. This theory has been tested by

expression in the chaos background of a cpSRP43 variant that

is unable to interact with cpSRP54 and to support the formation

of a cpSRP-LHCP transit complex (Goforth et al., 2004). Rever-

sion of transgenic plants with the cpSRP43 variant, which

contains its LHCP binding site, confirmed the possibility that

cpSRP43 can function independent of cpSRP54 in the delivery of

LHCP to thylakoids. Moreover, we found that cpSRP43 coloc-

alizes with ALB3 in the absence of cpSRP54 (Figure 6). While

colocalization data alone may not be interpreted as a direct


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indication of protein–protein interaction, they are consistent with

the finding that recombinant cpSRP43 alone exhibits affinity for

ALB3 (Figure 6). Taken together with the fact that ALB3 is an

essential component for thylakoid biogenesis and for posttrans-

lational integration of LHCPs by the cpSRP-targeting pathway

(Sundberg et al., 1997; Moore et al., 2000, 2003), it seems likely

that cpSRP43 functions in targeting LHCPs to ALB3 when

cpSRP54 is absent (ffc and cpftsy ffc mutants). We are currently

examining details of the cpSRP43–ALB3 interaction to under-

stand whether this interaction plays a role in the cpSRP54/

cpFtsY-dependent LHCP targeting mechanism.

In vitro (Schuenemann et al., 1998; Tu et al., 1999; Yuan et al.,

2002), all soluble components of the cpSRP pathway (cpSRP54,

cpSRP43, and cpFtsY) are required to achieve the integration of

LHCP into the thylakoids. The absence of only one component

reduces LHCP integration to levels that are barely detectable.

Prior to the studies reported here, this result was difficult to

reconcile with the partial LHCP targeting observed in both ffc and

chaos mutants and with the additive phenotype of the ffc chaos

double mutant (Hutin et al., 2002). The existence of an alternative

pathway based on cpSRP43 provides information necessary to

better understand the results of previous in vivo studies. The fact

that cpSRP43 alone appears unable to support LHCP integration

into isolated thylakoids suggests that cpSRP43 may function

along with other stroma proteins to mediate LHCP integration in

ffc and cpftsy ffc mutants. The use of chloroplast subfractions

from these mutants should aid in understanding details of this

new LHCP routing mechanism. While other components may be

required for such a pathway, sizing chromatography of cpSRP43

from soluble extracts of plants lacking cpSRP54 suggests that if

other components do function with cpSRP43, they are not bound

to cpSRP43 prior to interaction with LHCP-targeting substrates

(data not shown).

To summarize, we propose a model (Figure 7) whereby LHCP

binding to cpSRP43-cpSRP54 leads to the formation of a transit

complex in the stroma to target LHCP from the chloroplast

envelope to the thylakoid, owing to the ability of cpSRP54 to bind

cpFtsY at the membrane. Upon transfer of LHCP to ALB3, GTP

hydrolysis by both cpSRP54 and cpFtsY would release cpSRP54

from its receptor at the membrane. Thylakoid proteins encoded

by the chloroplast and routed using the cotranslational cpSRP

pathway (such as D1, PsaA, or PsaB) would use a similar mech-

anism that differs only by the absence of cpSRP43 and the

presence of the ribosome. Nevertheless, an alternative pathway

targeting nucleus-encoded antenna would coexist. Such a path-

way would use cpSRP43 to stabilize substrate in the stroma

and route proteins to ALB3, owing to the affinity of ALB3 for

cpSRP43. This alternative mechanism appears to be indepen-

dent of cpSRP54 and cpFtsY for protein integration. It should be

noted that ELIP proteins, LHCP-related proteins induced by

various stresses, have been described to require cpSRP43 but

not cpSRP54 for their targeting (Hutin et al., 2003), suggesting

that ELIP proteins are possible substrates for this alternative

pathway. Increased accumulation of ELIPs observed in the ffc

cpftsy double mutant compared with the ffc or cpftsy single

mutant (data not shown) adds support to this view. Additional

pathways also have to be considered, as the ffc chaos mutant

still exhibited ;20% of LHCP. We may assume that the alter-

native pathway described here may work without cpSRP43

less efficiently. Therefore, it would rely only on ALB3 proteins.

This would be in agreement with the alb3 mutant phenotype

(Sundberg et al., 1997), which has no pigments (suggesting that

LHCPs do not integrate in such mutants). Unfortunately, such a

hypothesis cannot be genetically tested, as the alb3 mutation is

lethal.

Eight years ago, Gunter Blobel received the Nobel Prize for his

work on SRP protein targeting. One of his major achievements

was the identification of signal peptides and the analysis of the

ribonucleotide protein complex needed to mediate protein tar-

geting to the endoplasmic reticulum. In chloroplasts, the cpSRP

pathway was named after the discovery that thylakoid targeting

of LHCP required cpSRP54 (Li et al., 1995), the canonical soluble

SRP protein conserved in bacteria and mammals. Nevertheless,

plants seem to have developed unique features for LHCP tar-

geting: posttranslational SRP activity, loss of an SRP RNA

component, the presence of a plant-specific SRP component

(cpSRP43), and a cpSRP43 binding motif in LHCPs. Now it

appears that the robust nature of LHCP targeting is maintained

Figure 7. Model Proposed for LHCP Targeting to Thylakoids, Which

Integrates the cpSRP54/cpFtsY-Dependent Pathway and the Indepen-

dent Alternative Pathway Identified Here.

Refer to Discussion for a detailed explanation. TIC, translocon of the

inner membrane of chloroplasts; TOC, translocon of the outer membrane

of chloroplasts.

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through the function of an alternative targeting system that can

bypass the evolutionarily conserved SRP soluble components,

cpSRP54 and cpFtsY, using cpSRP43, the only plant-specific

component. The increased number of recently published ge-

nomes has made it possible to identify putative homologs of

cpSRP43 in monocots (Os 03g0131900 in Oryza sativa), moss

(clone ppls42j07 from Physcomitrella), and even the green alga

Ostreoccocus tauri (Ot 02g03770). On the other hand, no obvious

homolog of cpSRP43 is present in cyanobacteria. These data

suggest that cpSRP43 is not restricted to higher plants and could

have appeared during the evolution of LHCPs. In this context, it is

noteworthy that the red algae possess an SRP RNA moiety that is

absent in green algae, moss, and higher plants (Rosenblad and

Samuelsson, 2004) and that to date no cpSRP43 has been found

in the red algae. This could indicate that cpSRP43 is specific to

green plants.

METHODS

Plant Materials and Growth Conditions

The Arabidopsis thaliana null mutant lacking cpSRP54 (ffc) and the double

mutant lacking both cpSRP54 and cpSRP43 (ffc chaos) have been

described previously (Klimyuk et al., 1999; Amin et al., 1999; Hutin et al.,

2002).

The Arabidopsis mutant lacking cpFtsY (cpftsy; CS3171) was isolated

from the George Redei chlorotic mutant collection deposited at the ABRC

and identified by protein gel blot screening using antibody directed

against cpFtsY (Tu et al., 1999). The mutation was identified by sequenc-

ing the PCR product obtained with primers (480) 59-CTCTAGCAC-

AACTGCCATGGCAACTTCT-39 and (489) 59-TAGTGAGACGAGACACA-

AGCAGTTCCTAT-39, which hybridize upstream and downstream, re-

spectively, of the cpFtsY coding sequence. The identified EMS cpftsy

mutant was backcrossed three times with wild-type Columbia plants

before conducting physiological and biochemical analyses. Plants were

grown in soil at 23/178C (day/night) with an 8-h illumination photoperiod

(300 mmol�m�2�s�1) and a RH of 60 to 85%. All studies were performed

using leaves harvested during the vegetative phase, before flowering.

Complementation of the EMS cpftsy Mutant

DNA Constructs

The plasmid pKYLX71-35S2 (Maiti et al., 1993) is a binary vector allowing

expression of subcloned cDNA under the control of a modified cauliflower

mosaic virus 35S promoter. cpFtsY cDNA digested from pTU1 (Tu et al.,

1999) at the NcoI and HindIII sites was blunt-ended with Klenow and

cloned into pKYLX71-35S2 at the XhoI site to form p35S2-cpFtsY. The

resulting DNA was amplified in Escherichia coli BL21 supplied with 10 mg/

mL tetracycline and subsequently introduced into Agrobacterium tume-

faciens strain GV3101 (Koncz et al., 1984) using tetracycline (2 mg/mL) for

selection.

Plant Transformation

Due to the severe cpftsy phenotype, transformation of plants containing

the cpftsy mutation was performed with heterozygous (cpFtsY/cpftsy)

plants. The binary plasmid described above containing the 35S2-cpFtsY

cassette was introduced into heterozygous plants by Agrobacterium

vacuum infiltration (Clough and Bent, 1998). Transformed seeds were

selected in the T2 generation from primary transformants on a medium

supplied with kanamycin (50 mg/mL). The presence of at least one copy of

35S2-cpFtsY in plants homozygous for the cpftsy genomic mutation was

confirmed by sequencing PCR products amplified with forward and

reverse primers that will hybridize to the intron sequence flanking the EMS

mutation, 59-CAAATAGGTGAGAAATTTTGCC-39 (FtMUT59) and 59-CAC-

CAAACACCGATACTGGCAAGCC-39 (FtMUT39).

Electron Microscopy and Immunocytochemistry

Leaf discs (3 mm in diameter) were taken from the lamina of detached

leaves and covered with hexadecene. The discs were rapidly degassed in

a syringe filled with hexadecene, transferred to a specimen carrier filled

with hexadecene, and flash-frozen with a high-pressure freezing system

(EMPACT; Leica Microsystems) prior to transfer in liquid nitrogen to

cryovials filled with dry acetone containing 0.2% uranyl acetate. Follow-

ing incubation for 3 d at�908C in an automatic freeze substitution system

(AFS; Leica Microsystems), the temperature of the substitution medium

was raised to�608C (by increasing the temperature at 48C/h), maintained

at �608C for 8 h, raised to �308C (48C/h over a period of 7.5 h),

and maintained at �308C for 12 h. Substitution medium was changed

(2 h at �308C) and then rinsed twice with pure acetone for 2 h at �208C

and 1 h at �158C prior to progressive infiltration of the specimens with

London Gold resin at 158C for 24 h. Finally, the resin was polymerized in

UV light, first at �158C and then at 208C.

Sections (60 nm thick) were collected on 200-mesh nickel grids (Gilder)

covered with a parlodion film and incubated with primary antibodies

against cpSRP43, cpSRP54, cpFtsY, and ALB3 proteins raised in rabbit,

mouse, or chicken. Colocalization was revealed by incubating sections

with mixtures of primary antibodies, then with mixtures of 1:45 diluted

secondary antibodies (Biocell International) conjugated to 10- or 5-nm

gold particles. The sections were contrasted with uranyl and lead prior to

observation with an FEI CM10 electron microscope operated at 80 kV.

Digital images were recorded as 6-megapixel files using an AMT XR 60

camera.

Pigment Analyses

Leaf discs (0.8 cm in diameter) were used for pigment quantification after

extraction in pure methanol. Photosynthetic pigments were identified

using HPLC (Lagarde et al., 2000).

Protein Extraction and Immunoblot Analyses

Immunodetection of cpSRP54, cpSRP43, and cpFtsY was performed

using total soluble protein extract from fresh leaves isolated as described

(Amin et al., 1999). Total membrane proteins were extracted as described

(Gillet et al., 1998). Protein concentration was determined using the

Lowry protein assay (Sigma-Aldrich). Membrane proteins (D1, PsaA/B,

ClpC, fibrilin C34, PC, OE33, and PsbS) were resolved by SDS-PAGE,

transferred to nitrocellulose membranes, and detected with alkaline

phosphatase–conjugated secondary antibody. Antiserum against LHCPs,

including Lhca1, Lhca2, Lhcb1, Lhcb2, Lhcb3, Lhcb5, Lhcb6, Lhca3,

Lhcb4, and Lhca4, have been described (Hoffman et al., 1987; Knoetzel

et al., 1992; Sigrist and Staehelin, 1992, 1994; Krol et al., 1995). Antisera

against D1, PsaA/B, OE33, PC, cpSRP54, cpSRP43, and PsbS were

used as described (Hutin et al., 2002). Antisera against fibrilin C34 was

used at 1:1000 dilution with 3, 5, and 7 mg of total membrane proteins

loaded in each lane. Protein gel blot analysis conducted with antiserum

directed against cpFtsY (Tu et al., 1999) was performed using 10 mg of

total soluble protein extract. The revelation of alkaline phosphate activity

was performed using nitroblue tetrazolium (Roche 1,087,479)/5-bromo-

4-chloro-3-indolyl phosphate (Interchim UP38047A) as described

(Sambrook et al., 1989). Densitometry analysis of the immunoblots was

performed using YabGelImageX1.0 (http://homepage.mac.com/yabyab/

rb/gelimage.html).


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Chlorophyll Fluorescence Measurements

In vivo chlorophyll fluorescence from attached or detached leaves was

measured at room temperature using a PAM-2000 fluorometer (Walz) as

described (Hutin et al., 2003). In brief, the maximum quantum yield of PSII

photochemistry was measured in dark-adapted leaves by subtracting the

initial level of chlorophyll fluorescence (Fo; induced by a dim red light

modulated at 600 kHz) from the maximum fluorescence level (Fm;

induced by an 800-ms pulse of intense white light): (Fm � Fo)/Fm ¼ Fv/

Fm. The actual quantum yield of PSII photochemistry was measured in

light-adapted leaves with the fluorescence parameter DF/Fm9 ¼ (Fm9 �Fs)/Fm9, where Fm9 and Fs are the maximal and steady state fluores-

cence levels, respectively, in the light.

Coimmunoprecipitation

Soluble protein extract (300 mL) prepared as described above was

incubated for 2 h at 48C with mouse anti-cpSRP54 antiserum (7.5 mL).

After 1 h of incubation with 20 mL of protein G–Sepharose (Amersham),

IgG beads were washed four times with 20 mM HEPES-KOH, pH 8.0,

5 mM MgCl2, and 180 mM NaCl. Bound proteins were eluted in 40 mL of

13 Laemmli buffer for SDS-PAGE and analyzed by protein gel blotting.

Binding of cpSRP43 to ALB3

Construction, Expression, and Purification of His-Tagged cpSRP43

The coding sequence for mature cpSRP43 beginning with AAVQRNY was

amplified from pGEX-4T-2 (Moore et al., 2003) using a forward primer that

coded for a 59 BamHI and a six-His tag in conjunction with a reverse

primer corresponding to the C terminus of cpSRP43 and incorporating an

EcoRI restriction site. The resulting PCR product was inserted into pGEX-

6P-2 (GE Healthcare) using BamHI and EcoRI restriction sites to create

his-cpSRP43-pGEX-6P-2. Sequence analysis confirmed the addition of

the N-terminal amino acids GSHHHHHH to cpSRP43.

BL21 Star (Invitrogen) cells containing his-cpSRP43-pGEX-6P-2 were

grown to mid log phase and induced with 0.5 mM isopropyl-b-D-

thiogalactopyranoside for 2 h at 378C to produce GST-cpSRP43-his.

Initial purification utilized Glutathione Sepharose Fast Flow (GE Health-

care). GST-cpSRP43-his was desalted into 50 mM Tris-HCl, pH 7.0 (at

258C), 150 mM NaCl, 1 mM EDTA, and 1 mM DTT utilizing a HiPrep 26/10

desalting column (GE Healthcare). The GST affinity tag was cleaved by

incubation with PreScission Protease (GE Healthcare) overnight at 48C

prior to desalting into PBS. Cleaved GST and PreScission Protease were

removed using Glutathione Sepharose Fast Flow resin. The resulting

cpSRP43-his was desalted into 10 mM HEPES and 10 mM MgCl2 as

described above, divided into aliquots, and stored at �808C for fur-

ther use.

Formation of the cpSRP43-ALB3 Complex

Salt-washed thylakoids were prepared as described (Yuan et al., 2002)

and analyzed by protein gel blot to confirm the absence of cpSRP43,

cpSRP54, and cpFtsY. The association between cpSRP43 and ALB3 was

examined by incubating salt-washed thylakoids (200 mg of chlorophyll)

with cpSRP43-his in the presence of 0.5 mM GMP-PNP for 30 min at

258C. Treated thylakoids were isolated by centrifugation, buffer-washed

with IBM (50 mM HEPES-KOH, pH 8, 330 mM sorbitol, and 10 mM

MgCl2), and reisolated by centrifugation. The washed membranes were

resuspended in 100 mL of 3% BSA. Subsequently, 100 mL of 2%

maltoside in IBM was added and the samples were incubated at room

temperature with gentle shaking for 10 min prior to centrifugation at

70,000g for 12 min. The soluble fraction was diluted to 0.1% maltoside

with IBM mixed with 50 mL of Talon resin (Clontech) for 30 min at 258C to

precipitate His-tagged cpSRP43 and all coprecipitating proteins. After

incubation, samples were washed three times with 0.1% maltoside in IBM

and once with IBM prior to elution with 100 mL of SDS solubilization buffer

containing b-mercaptoethanol and heating to 708C for 15 min as de-

scribed (Moore et al., 2003). Protein gel blots of the precipitates were

probed to identify the presence of the proteins indicated.

Accession Numbers

Arabidopsis Genome Initiative locus identifiers for the genes mentioned

in this article are At2g28800 (ALB3), At2g45770 (cpFtsY ), At2g47450

(cpSRP43), and At5g03940 (cpSRP54).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Table 1. Densitometry Analysis of the Immunoblots

Presented in Figures 3A, 3B, and 3C Made Using YabGelImageX1.0.

Supplemental Figure 1. Quantum Yield of PSII-Mediated Electron

Transport (DF/Fm9) of ffc, cpftsy, and ffc cpftsy Mutants and the Wild-

Type Control.

ACKNOWLEDGMENTS

We thank the team at the Groupe de Recherches Appliquees en Phyto-

technologie for skillful technical assistance in growing the plants. We

also thank Roberto Bassi for insightful scientific discussions and advice

regarding LHCP biogenesis and Nathan Lewis for his graphics contri-

butions. This work was supported by Department of Energy Grant BR-

15569 (R.H.), by National Institutes of Health Grant P20 RR-15569 from

the COBRE Program of the National Center for Research Resources

(R.G.), and by the Commissariat a l’Energie Atomique through a PhD

grant for T.T.-C.

Received November 17, 2006; revised April 3, 2007; accepted April 18,

2007; published May 18, 2007.

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