Chloroplast SRP54 Was Recruited for Posttranslational Protein Transport via Complex Formation with Chloroplast SRP43 during Land Plant Evolution

In bacteria, membrane proteins are targeted cotranslationally via a signal recognition particle (SRP). During the evolution of higher plant chloroplasts from cyanobacteria, the SRP pathway underwent striking adaptations that enable the posttranslational transport of the abundant light-harvesting chlorophyll-a/b-binding proteins (LHCPs). The conserved 54-kDa SRP subunit in higher plant chloroplasts (cpSRP54) is not bound to an SRP RNA, an essential SRP component in bacteria, but forms a stable heterodimer with the chloroplast-specific cpSRP43. This heterodimeric cpSRP recognizes LHCP and delivers it to the thylakoid membrane whereby cpSRP43 plays a central role. This study shows that the cpSRP system in the green alga Chlamydomonas reinhardtii differs significantly from that of higher plants as cpSRP43 is not complexed to cpSRP54 in Chlamydomonas and cpSRP54 is not involved in LHCP recognition. This divergence is attributed to altered residues within the cpSRP54 tail and the second chromodomain of cpSRP43 that are crucial for the formation of the binding interface in Arabidopsis. These changes are highly conserved among chlorophytes, whereas all land plants contain cpSRP proteins with typical interaction motifs. These data demonstrate that the coevolution of LHCPs and cpSRP43 occurred independently of complex formation with cpSRP54 and that the interaction between cpSRP54 and cpSRP43 evolved later during the transition from chlorophytes to land plants. Furthermore, our data show that in higher plants a heterodimeric form of cpSRP is required for the formation of a low molecular weight transit complex with LHCP.     

Structural diversity of signal recognition particle RNAs in plastids

One of the pathways for protein targeting to the plasma membrane in bacteria utilizes the co-translationally acting signal recognition particle (SRP), a universally conserved ribonucleoprotein complex consisting of a 54 kDa protein and a functional RNA. An interesting exception is the higher plant chloroplast SRP, which lacks the otherwise essential RNA component. Furthermore, green plant chloroplasts have an additional post-translational SRP-dependent transport system in which the chloroplast-specific cpSRP43 protein binds to imported substrate proteins and to the conserved 54 kDa SRP subunit (cpSRP54). While homologs to the bacterial SRP protein and RNA component previously have been identified in genome sequences of red algae and diatoms, a recent study investigated the evolution of the green plant SRP system.¹ Analysis of hundreds of plastid and nuclear genomes showed a surprising pattern of multiple losses of the plastid SRP RNA during evolution and a widespread presence in all non-spermatophyte plants and green algae. Contrary to expectations, all green organisms that have an identified cpSRP RNA also contain a cpSRP43. Notably, the structure of the plastid SRP RNAs is much more diverse than that of bacterial SRP RNAs. The apical GNRA tetraloop is only conserved in organisms of the red lineage and basal organisms of the green lineage, whereas further chloroplast SRP RNAs are characterized by atypical, mostly enlarged apical loops.     

A unique sequence motif in the 54-kDa subunit of the chloroplast signal recognition particle mediates binding to the 43-kDa subunit

Chloroplasts contain a novel type of signal recognition particle (cpSRP) that consists of two proteins, cpSRP54 and cpSRP43. cpSRP is involved in the post-translational targeting of the nuclear encoded light-harvesting chlorophyll-binding proteins (LHCPs) to the thylakoid membrane by forming a soluble cpSRP.LHCP transit complex in the stroma. Despite high sequence homology between chloroplast and cytosolic SRP54 proteins, the 54-kDa subunit of cpSRP is unique in its ability to bind cpSRP43. In this report, we identified a 10-amino acid long segment of cpSRP54 that forms the cpSRP43-binding site. This segment is located at position 530-539 close to the C terminus of cpSRP54. In addition, we demonstrate that arginine at position 537 is essential for binding cpSRP43 and that mutation of arginine 536 drastically reduced cpSRP43 binding. Mutations within the cpSRP43-binding site of cpSRP54 that reduced or completely abolished cpSRP complex formation also did inhibit transit complex formation and integration of LHCP into the thylakoid membrane, reflecting the importance of these residues for LHCP targeting. Alignment studies revealed that the cpSRP43-binding site is conserved in chloroplast SRP54 proteins and is not present in any SRP54 subunit of cytosolic SRPs.     

The L18 domain of light-harvesting chlorophyll proteins binds to chloroplast signal recognition particle 43

Chloroplast signal recognition particle (cpSRP) is a novel type of SRP that contains a homolog of SRP54 and a 43-kDa subunit absent from all cytoplasmic SRPs but lacks RNA. It is also distinctive in its ability to post-translationally interact with light-harvesting chlorophyll proteins (LHCP), hydrophobic proteins synthesized in the cytoplasm and targeted to the thylakoid via the stroma. LHCP integration into thylakoid membranes requires the two subunits of cpSRP, cpFtsY, GTP, and the membrane protein ALB3. It had previously been shown that the L18 domain, an 18-amino acid peptide between the second and third transmembrane domains, and a hydrophobic domain are required for interaction with cpSRP. In the present study we used a pull-down assay, with cpSRP43 or cpSRP54 fused to glutathione-transferase, to study interactions between cpSRP43, cpSRP54, LHCP, and cpFtsY. cpFtsY was not observed to form significant interactions with any of the proteins even in the presence of nonhydrolyzable GTP analogs. Our data indicate that cpSRP43 binds to the L18 domain, that cpSRP54 binds to the hydrophobic domain, and that LHCP and cpSRP54 independently bind to cpSRP43. These data confirm that the novel post-translational interaction between LHCP and cpSRP is mediated through binding to cpSRP43.     

Functional analysis of the protein-interacting domains of chloroplast SRP43

The chloroplast signal recognition particle (cpSRP) consists of an evolutionarily conserved 54-kDa subunit (cpSRP54) and a dimer of a unique 43-kDa subunit (cpSRP43). cpSRP binds light-harvesting chlorophyll proteins (LHCPs) to form a cpSRP/LHCP transit complex, which targets LHCP to the thylakoid membrane. Previous studies showed that transit complex formation is mediated through the binding of the L18 domain of LHCP to cpSRP43. cpSRP43 is characterized by a four-ankyrin repeat domain at the N terminus and two chromodomains at the C terminus. In the present study we used the yeast two-hybrid system and in vitro binding assays to analyze the function of different domains of cpSRP43 in protein complex formation. We report here that the first ankyrin repeat binds to the 18-amino acid domain on LHCP that binds to cpSRP43, whereas the third and fourth ankyrin repeats are involved in the dimerization of cpSRP43. We show further that the interaction of cpSRP43 with cpSRP54 is mediated via binding of the methionine-rich domain of cpSRP54 to the C-terminally located chromodomains of cpSRP43. Both chromodomains contain essential elements for binding cpSRP54, indicating that the closely spaced chromodomains together create a single binding site for cpSRP54. In addition, our data demonstrate that the interaction of cpSRP54 with the chromodomains of cpSRP43 is enhanced indirectly by the dimerization motif of cpSRP43.     

Three-dimensional solution structures of the chromodomains of cpSRP43

Chloroplasts contain a unique signal recognition particle (cpSRP). Unlike the cytoplasmic forms, the cpSRP lacks RNA but contains a conserved 54-kDa GTPase and a novel 43-kDa subunit (cpSRP43). Recently, three functionally distinct chromodomains (CDs) have been identified in cpSRP43. In the present study, we report the three-dimensional solution structures of the three CDs (CD1, CD2, and CD3) using a variety of triple resonance NMR experiments. The structure of CD1 consists of a triple-stranded beta-sheet segment. The C-terminal helical segment typically found in the nuclear chromodomains is absent in CD1. The secondary structural elements in CD2 and CD3 include a triple-stranded antiparallel beta-sheet and a C-terminal helix. Interestingly, the orientation of the C-terminal helix is significantly different in the structures of CD2 and CD3. Critical comparison of the structures of the chromodomains of cpSRP43 with those found in nuclear chromodomain proteins revealed that the diverse protein-protein interactions mediated by the CDs appear to stem from the differences that exist in the surface charge potentials of each CD. Results of isothermal titration calorimetry experiments confirmed that only CD2 is involved in binding to cpSRP54. The negatively charged C-terminal helix in CD2 possibly plays a crucial role in the cpSRP54-cpSRP43 interaction.     

An archaeal protein homologous to mammalian SRP54 and bacterial Ffh recognizes a highly conserved region of SRP RNA.

The gene encoding the 54 kDa protein of signal recognition particle (SRP54) in the hyperthermophilic archaeon Pyrococcus furiosus has been cloned and sequenced. Recombinant P. furiosus SRP54 (pf-SRP54) and the N-terminal G-domain and C-terminal M-domain (pf-SRP54M) of pf-SRP54 with an amino-terminal addition of six histidine residues were expressed in Escherichia coli and subjected to binding experiments for SRP RNA, non-conserved 213-nucleotide RNA (helices 1, 2, 3, 4 and 5) and conserved 107-nucleotide RNA (helices 6 and 8) from SRP RNA. The RNA binding properties of the purified protein were determined by filter binding assays. The histidine-tagged pf-SRP54M bound specifically to the conserved 107-nucleotide RNA in the absence of pf-SRP19, unlike the eukaryotic homologue, with an apparent binding constant (K) of 18 nM.     

A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)–SRP Receptor Interaction by the Chloroplast SRP Pathway

Cotranslational protein targeting by the signal recognition particle (SRP) requires the SRP RNA, which accelerates the interaction between the SRP and SRP receptor 200-fold. This otherwise universally conserved SRP RNA is missing in the chloroplast SRP (cpSRP) pathway. Instead, the cpSRP and cpSRP receptor (cpFtsY) by themselves can interact 200-fold faster than their bacterial homologues. Here, cross-complementation analyses revealed the molecular origin underlying their efficient interaction. We found that cpFtsY is 5- to 10-fold more efficient than Escherichia coli FtsY at interacting with the GTPase domain of SRP from both chloroplast and bacteria, suggesting that cpFtsY is preorganized into a conformation more conducive to complex formation. Furthermore, the cargo-binding M-domain of cpSRP provides an additional 100-fold acceleration for the interaction between the chloroplast GTPases, functionally mimicking the effect of the SRP RNA in the cotranslational targeting pathway. The stimulatory effect of the SRP RNA or the M-domain of cpSRP is specific to the homologous SRP receptor in each pathway. These results strongly suggest that the M-domain of SRP actively communicates with the SRP and SR GTPases and that the cytosolic and chloroplast SRP pathways have evolved distinct molecular mechanisms (RNA vs. protein) to mediate this communication.     

Identification of chloroplast signal recognition particle RNA genes

The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the ER membrane in eukaryotes, the plasma membrane in bacteria and the thylakoid membrane in chloroplasts. In higher plants two different SRP-dependent mechanisms have been identified: one post-translational for proteins imported to the chloroplast and one co-translational for proteins encoded by the plastid genome. The post-translational chloroplast SRP (cpSRP) consists of the protein subunits cpSRP54 and cpSRP43. An RNA component has not been identified and does not seem to be required for the post-translational cpSRP. The co-translational mechanism is known to involve cpSRP54, but an RNA component has not yet been identified. Several chloroplast genomes have been sequenced recently, making a phylogenetically broad computational search for cpSRP RNA possible. We have analysed chloroplast genomes from 27 organisms. In higher plant chloroplasts, no SRP RNA genes were identified. However, eight plastids from red algae and Chlorophyta were found to contain an SRP RNA gene. These results suggest that SRP RNA forms a complex in these plastids with cpSRP54, reminiscent of the eubacterial SRP.     

Transient interaction of cpSRP54 with elongating nascent chains of the chloroplast-encoded D1 protein; 'cpSRP54 caught in the act'

The signal recognition particle (SRP) in bacteria and endoplasmic reticulum is involved in co-translational targeting. Plastids contain cpSRP54 and cpSRP43, unique to plants, but lack a SRP RNA molecule. A role for cpSRP in biogenesis of plastid-encoded membrane proteins has not been firmly established yet. In this study, a transient interaction between cpSRP54 and elongating D1 protein was observed using a homologous chloroplast translation system. Using the novel approach of cross-linking at different time points during elongation of full-length D1 protein, we showed that cpSRP54 interacts strongly with the elongating nascent chain forming two distinct cross-linked products. However, this interaction did not lead to an elongation arrest and cpSRP54 was released from the nascent chains, once they were longer than approximately 14 kDa. Detailed mutant analysis showed that the cpSRP54 interaction occurred via the first transmembrane domain, which could be replaced by other hydrophobic domains of more than 10 amino acids.     

Structural and functional characterisation of the signal recognition particle-specific 54 kDa protein (SRP54) of tomato

Two representative genes for the 54 kDa protein subunit of the signal recognition particle (SRP54) of tomato were cloned. It was shown that both genes are expressed in the tomato cv. Rentita. SRP54 is encoded by nine exons distributed over 10 kb of genomic sequence. The amino acid sequences deduced for the two SRP54 genes are 92% identical and the calculated protein size is 55 kDa. Like the homologous proteins isolated from other eukaryotes, the tomato SRP54 is evidently divided into two domains. As deduced from sequence motif identity, the N-terminally located G-domain can be assumed to have GTPase activity. The C-terminal part of the protein is methionine rich (14% methionine) and represents the M-domain. In in vitro binding experiments, SRP54 of tomato was able to attach to the 7S RNA of tomato, its natural binding partner in the SRP. This interaction can only take place in a trimeric complex consisting of 7S RNA, SRP54 and SRP19. The latter protein subunit of the SRP complex is assumed to induce a conformational change in the 7S RNA. The human SRP19 was able to mediate the binding of the tomato SRP54 to the 7S RNA, irrespective of whether this latter originated from tomato or man.     

A chromodomain protein encoded by the arabidopsis CAO gene is a plant-specific component of the chloroplast signal recognition particle pathway that is involved in LHCP targeting.

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 nuclear-encoded proteins utilize a chloroplast signal recognition particle (cpSRP) system to reach the thylakoid membrane. Both prokaryotes and eukaryotes possess a cytoplasmic SRP containing a 54-kD protein (SRP54) and an RNA. In chloroplasts, 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 resemble 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 unique to plants. Surprisingly, the phenotype of the cpSRP43 mutant (i.e., chaos) differs from that of the Arabidopsis cpSRP54 mutant, suggesting that the functions of the two proteins do not strictly overlap. This difference also suggests that the function of cpSRP43 is most likely restricted to protein targeting into the thylakoid membrane, whereas cpSRP54 may be involved in an additional process(es), such as chloroplast biogenesis, perhaps through chloroplast-ribosomal association with chloroplast ribosomes.     

Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates

Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome. A multidomain chloroplast-specific subunit of the SRP, cpSRP43, is proposed to take on the role of coordinating the sequence of targeting events. Here, we demonstrate that cpSRP43 exhibits significant interdomain dynamics that are reduced upon binding its SRP binding partner, cpSRP54. We showed that the affinity of cpSRP43 for the binding motif of LHCP (L18) increases when cpSRP43 is complexed to the binding motif of cpSRP54 (cpSRP54_pep). These results support the conclusion that substrate binding to the chloroplast SRP is modulated by protein structural dynamics in which a major role of cpSRP54 is to improve substrate binding efficiency to the cpSRP.     

Interaction of protein SRP19 with signal recognition particle RNA lacking individual RNA-helices.

Derivatives of human SRP-RNA were constructed by site-directed mutagenesis and tested for their ability to interact with protein SRP19. An RNA missing helix 6 barely interacts with SRP19, while the helix 8-deletion mutant retains much binding capability. A mutant RNA consisting just of helix 6 also binds the protein, but not as well as the unaltered molecule. SRP19 interacts to a full extent with the fourth mutant RNA composed of helices 6, 7, 8 and a portion of helix 5. It is concluded that helix 6- and not helix 8- is the major SRP19 binding site. Helices 7, 8 and portions of helix 5 contribute to the formation of a functional site. These results agree with data suggesting a proximity of helix 6 and the conserved part of SRP-RNA.     

Assembly of chloroplast signal recognition particle involves structural rearrangement in cpSRP43

Signal recognition particle in chloroplasts (cpSRP) exhibits the unusual ability to bind and target full-length proteins to the thylakoid membrane. Unlike cytosolic SRPs in prokaryotes and eukaryotes, cpSRP lacks an RNA moiety and functions as a heterodimer composed of a conserved 54-kDa guanosine triphosphatase (cpSRP54) and a unique 43-kDa subunit (cpSRP43). Assembly of the cpSRP heterodimer is a prerequisite for post-translational targeting activities and takes place through interactions between chromatin modifier domain 2 (CD2) of cpSRP43 and a unique 10-amino-acid region in cpSRP54 (cpSRP54_pep). We have used multidimensional NMR spectroscopy and other biophysical methods to examine the assembly and structure of the cpSRP43-cpSRP54 interface. Our data show that CD2 of cpSRP43 binds to cpSRP54_pep in a 1:1 stoichiometry with an apparent K_d of ∼ 1.06 μM. Steady-state fluorescence and far-UV circular dichroism data suggest that the CD2-cpSRP54_pep interaction causes significant conformational changes in both CD2 and the peptide. Comparison of the three-dimensional solution structures of CD2 alone and in complex with cpSRP54_pep shows that significant structural changes are induced in CD2 in order to establish a binding interface contributed mostly by residues in the N-terminal segment of CD2 (Phe5-Val10) and an arginine doublet (Arg536 and Arg537) in the cpSRP54 peptide. Taken together, our results provide new insights into the mechanism of cpSRP assembly and the structural forces that stabilize the functionally critical cpSRP43-cpSRP54 interaction.     

Functional characterization of recombinant chloroplast signal recognition particle

The signal recognition particle (SRP) is a ubiquitous system for the targeting of membrane and secreted proteins. The chloroplast SRP (cpSRP) is unique among SRPs in that it possesses no RNA and is functional in post-translational as well as co-translational targeting. We have expressed and purified the two components of the Arabidopsis thaliana chloroplast signal recognition particle (cpSRP) involved in post-translational transport: cpSRP54 and the chloroplast-specific protein, cpSRP43. Recombinant cpSRP supports the efficient in vitro insertion of pea preLhcb1 into isolated thylakoid membranes. Recombinant cpSRP is a stable heterodimer with a molecular mass of approximately 100 kDa as determined by analytical ultracentrifugation, gel filtration analysis, and dynamic light scattering. The interactions of the components of the recombinant heterodimer and pea preLhcb1 were probed using an immobilized peptide library (pepscan) approach. These data confirm two previously reported interactions with the L18 region and the third transmembrane helix of Lhcb1 and suggest that the interface of the cpSRP43 and cpSRP54 proteins is involved in substrate binding. Additionally, cpSRP components are shown to recognize peptides from the cleavable, N-terminal chloroplast transit peptide of preLhcb1. The interaction of cpSRP43 with cpSRP54 was probed in a similar experiment with a peptide library representing cpSPR54. The C terminus of cpSRP54 is essential for the formation of the stable cpSRP complex and cpSPR43 interacts with distinct regions of the M domain of cpSRP54.     

The alpha-helix of the second chromodomain of the 43 kDa subunit of the chloroplast signal recognition particle facilitates binding to the 54 kDa subunit

Chloroplasts of higher plants contain a unique signal recognition particle (cpSRP) that consists of two proteins, cpSRP54 and cpSRP43. CpSRP43 is composed of a four ankyrin repeat domain and three functionally distinct chromodomains (CDs). In this report we confirm previously published data that the second chromodomain (CD2) provides the primary binding site for cpSRP54. However, quantitative binding analysis demonstrates that cpSRP54 binds to CD2 significantly less efficiently than it binds to full-length cpSRP43. Further analysis of the binding interface of cpSRP by mutagenesis studies and a pepscan approach demonstrates that the C-terminal alpha-helix of CD2 facilitates binding to cpSRP54.     

Structures of SRP54 and SRP19, the two proteins that organize the ribonucleic core of the signal recognition particle from Pyrococcus furiosus

In all organisms the Signal Recognition Particle (SRP), binds to signal sequences of proteins destined for secretion or membrane insertion as they emerge from translating ribosomes. In Archaea and Eucarya, the conserved ribonucleoproteic core is composed of two proteins, the accessory protein SRP19, the essential GTPase SRP54, and an evolutionarily conserved and essential SRP RNA. Through the GTP-dependent interaction between the SRP and its cognate receptor SR, ribosomes harboring nascent polypeptidic chains destined for secretion are dynamically transferred to the protein translocation apparatus at the membrane. We present here high-resolution X-ray structures of SRP54 and SRP19, the two RNA binding components forming the core of the signal recognition particle from the hyper-thermophilic archaeon Pyrococcus furiosus (Pfu). The 2.5 A resolution structure of free Pfu-SRP54 is the first showing the complete domain organization of a GDP bound full-length SRP54 subunit. In its ras-like GTPase domain, GDP is found tightly associated with the protein. The flexible linker that separates the GTPase core from the hydrophobic signal sequence binding M domain, adopts a purely alpha-helical structure and acts as an articulated arm allowing the M domain to explore multiple regions as it scans for signal peptides as they emerge from the ribosomal tunnel. This linker is structurally coupled to the GTPase catalytic site and likely to propagate conformational changes occurring in the M domain through the SRP RNA upon signal sequence binding. Two different 1.8 A resolution crystal structures of free Pfu-SRP19 reveal a compact, rigid and well-folded protein even in absence of its obligate SRP RNA partner. Comparison with other SRP19*SRP RNA structures suggests the rearrangement of a disordered loop upon binding with the RNA through a reciprocal induced-fit mechanism and supports the idea that SRP19 acts as a molecular scaffold and a chaperone, assisting the SRP RNA in adopting the conformation required for its optimal interaction with the essential subunit SRP54, and proper assembly of a functional SRP.     

A highly conserved nucleotide in the Alu domain of SRP RNA mediates translation arrest through high affinity binding to SRP9/14.

Binding of the signal recognition particle (SRP) to signal sequences during translation leads to an inhibition of polypeptide elongation known as translation arrest. The arrest activity is mediated by a discrete domain comprised of the Alu portion of SRP RNA and a 9 and 14 kDa polypeptide heterodimer (SRP9/14). Although very few nucleotides in SRP RNA are conserved throughout evolution, the remarkable conservation of G24, which resides in the region of SRP9/14 interaction, suggests that it is essential for translation arrest. To understand the functional significance of the G24 residue, we made single base substitutions in SRP RNA at this position and analyzed the ability of the mutants to bind SRP9/14 and to reconstitute functional SRPs. Mutation of G24 to C reduced binding to SRP9/14 by at least 50-fold, whereas mutation to A and U reduced binding approximately 2- and 5-fold respectively. The mutant RNAs could nevertheless assemble into SRPs at high subunit concentrations. SRPs reconstituted with mutant RNAs were not significantly defective in translation arrest assays, indicating that the conserved guanosine does not interact directly with the translational machinery. Taken together, these results demonstrate that G24 plays an important role in the translation arrest function of SRP by mediating high affinity binding of SRP9/14.     

Binding of chloroplast signal recognition particle to a thylakoid membrane protein substrate in aqueous solution and delineation of the cpSRP43-substrate interaction domain

A cpSRP [chloroplast SRP (signal recognition particle)] comprising cpSRP54 and cpSRP43 subunits mediates the insertion of light-harvesting proteins into the thylakoid membrane. We dissected its interaction with a full-length membrane protein substrate in aqueous solution by insertion of site-specific photo-activatable cross-linkers into in vitro-synthesized Lhcb1 (major light-harvesting chlorophyll-binding protein of photosystem II). We show that Lhcb1 residues 166-176 cross-link specifically to the cpSRP43 subunit. Some cross-link positions within Lhcb1 are in the 'L18' peptide required for targeting of cpSRP substrates, whereas other cross-linking positions define a new targeting signal in the third transmembrane span. Lhcb1 was not found to cross-link to cpSRP54 at any position, and cross-linking to cpSRP43 is unaffected by the absence of cpSRP54. cpSRP43 thus effectively binds substrates autonomously, and its ability to independently bind an extended 20+-residue substrate region highlights a major difference with other SRP types?where the SRP54 subunit binds to hydrophobic target sequences. The results also show that cpSRP43 can bind to a hydrophobic, three-membrane span, substrate in aqueous solution, presumably reflecting a role for cpSRP in the chloroplast stroma. This mode of action, and the specificity of the cpSRP43-substrate interaction, may be associated with cpSRP's unique post-translational mode of action.