Distal regulatory regions restrict the expression of cis-linked genes to the tapetal cells

Rapid light-dependent turnover of the chloroplast-encoded D1 protein maintains photosystem II (PS II) functional over a wide range of light intensities. Following initiation of psbA mRNA translation, the elongating D1 is targeted, possibly by chloroplast signal recognition particle 54 (cpSRP54), to the thylakoid cpSecY translocation channel. Transmembrane domains of nascent D1 start interacting with other PS II core proteins already during the translocation process to ensure an efficient assembly of the multiprotein membrane complex. Here we review the progress recently made concerning the synthesis, targeting, membrane insertion and assembly to PS II of the chloroplast-encoded D1 protein and discuss the possible convergence of targeting and translocation of chloroplast- and nuclear-encoded thylakoid proteins.     

Interactions of ribosome nascent chain complexes of the chloroplast-encoded D1 thylakoid membrane protein with cpSRP54.

The mechanisms of targeting, insertion and assembly of the chloroplast-encoded thylakoid membrane proteins are unknown. In this study, we investigated these mechanisms for the chloroplast-encoded polytopic D1 thylakoid membrane protein, using a homologous translation system isolated from tobacco chloroplasts. Truncated forms of the psbA gene were translated and stable ribosome nascent chain complexes were purified. To probe the interactions with the soluble components of the targeting machinery, we used UV-activatable cross-linkers incorporated at specific positions in the nascent chains, as well as conventional sulfhydryl cross-linkers. With both cross-linking approaches, the D1 ribosome nascent chain was photocross-linked to cpSRP54. cpSRP54 was shown to interact only when the D1 nascent chain was still attached to the ribosome. The interaction was strongly dependent on the length of the nascent chain that emerged from the ribosome, as well as the cross-link position. No interactions with soluble SecA or cpSRP43 were found. These results imply a role for cpSRP54 in D1 biogenesis.     

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.     

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.     

Chloroplast FtsY, chloroplast signal recognition particle, and GTP are required to reconstitute the soluble phase of light-harvesting chlorophyll protein transport into thylakoid membranes.

The integration of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membrane proceeds in two steps. First, LHCP interacts with a chloroplast signal recognition particle (cpSRP) to form a soluble targeting intermediate called the transit complex. Second, LHCP integrates into the thylakoid membrane in the presence of GTP, at least one other soluble factor, and undefined membrane components. We previously determined that cpSRP is composed of 43- and 54-kDa polypeptides. We have examined the subunit stoichiometry of cpSRP and find that it is trimeric and composed of two subunits of cpSRP43/subunit of cpSRP54. A chloroplast homologue of FtsY, an Escherichia coli protein that is critical for the function of E. coli SRP, was found largely in the stroma unassociated with cpSRP. When chloroplast FtsY was combined with cpSRP and GTP, the three factors promoted efficient LHCP integration into thylakoid membranes in the absence of stroma, demonstrating that they are all required for reconstituting the soluble phase of LHCP transport.     

Regulation of the GTPase cycle in post-translational signal recognition particle-based protein targeting involves cpSRP43

The chloroplast signal recognition particle consists of a conserved 54-kDa GTPase and a novel 43-kDa chromodomain protein (cpSRP43) that together bind light-harvesting chlorophyll a/b-binding protein (LHCP) to form a soluble targeting complex that is subsequently directed to the thylakoid membrane. Homology-based modeling of cpSRP43 indicates the presence of two previously identified chromodomains along with a third N-terminal chromodomain. Chromodomain deletion constructs were used to examine the role of each chromodomain in mediating distinct steps in the LHCP localization mechanism. The C-terminal chromodomain is completely dispensable for LHCP targeting/integration in vitro. The central chromodomain is essential for both targeting complex formation and integration because of its role in binding the M domain of cpSRP54. The N-terminal chromodomain (CD1) is unnecessary for targeting complex formation but is required for integration. This correlates with the ability of CD1 along with the ankyrin repeat region of cpSRP43 to regulate the GTPase cycle of the cpSRP-receptor complex.     

Insertion of leader peptidase into the thylakoid membrane during synthesis in a chloroplast translation system

The mechanisms of targeting and insertion of chloroplast-encoded thylakoid membrane proteins are poorly understood. In this study, we have used a translation system isolated from chloroplasts to begin to investigate these mechanisms. The bacterial membrane protein leader peptidase (Lep) was used as a model protein because its targeting and insertion mechanisms are well understood for Escherichia coli and for the endoplasmic reticulum. Lep could thus provide insight into the functional homologies between the different membrane systems. Lep was efficiently expressed in the chloroplast translation system, and the protein could be inserted into thylakoid membranes with the same topology as in E. coli cytoplasmic membranes, following the positive-inside rule. Insertion of Lep into the thylakoid membrane was stimulated by the trans-thylakoid proton gradient and was strongly inhibited by azide, suggesting a requirement for SecA activity. Insertion most likely occurred in a cotranslational manner, because insertion could only be observed if thylakoid membranes were present during translation reactions but not when thylakoid membranes were added after translation reactions were terminated. To halt the elongation process at different stages, we translated truncated Lep mRNAs without a stop codon, resulting in the formation of stable ribosome nascent chain complexes. These complexes showed a strong, salt-resistant affinity for the thylakoid membrane, implying a functional interaction of the ribosome with the membrane and supporting a cotranslational insertion mechanism for Lep. Our study supports a functional homology for the insertion of Lep into the thylakoid membrane and the E. coli cytoplasmic membrane.     

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.     

ATP stimulates signal recognition particle (SRP)/FtsY-supported protein integration in chloroplasts

The signal recognition particle (SRP) and its receptor (FtsY in prokaryotes) are essential for cotranslational protein targeting to the endoplasmic reticulum in eukaryotes and the cytoplasmic membrane in prokaryotes. An SRP/FtsY-like protein targeting/integration pathway in chloroplasts mediates the posttranslational integration of the light-harvesting chlorophyll a/b-binding protein (LHCP) into thylakoid membranes. GTP, chloroplast SRP (cpSRP), and chloroplast FtsY (cpFtsY) are required for LHCP integration into thylakoid membranes. Here, we report the reconstitution of the LHCP integration reaction with purified recombinant proteins and salt-washed thylakoids. Our data demonstrate that cpSRP and cpFtsY are the only soluble protein components required for LHCP integration. In addition, our studies reveal that ATP, though not absolutely required, remarkably stimulates LHCP integration into salt-washed thylakoids. ATP stimulates LHCP integration by a mechanism independent of the thylakoidal pH gradient (DeltapH) and exerts no detectable effect on the formation of the soluble LHCP-cpSRP-targeting complex. Taken together, our results indicate the participation of a thylakoid ATP-binding protein in LHCP integration.     

The C Terminus of the Alb3 Membrane Insertase Recruits cpSRP43 to the Thylakoid Membrane

The YidC/Oxa1/Alb3 family of membrane proteins controls the insertion and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here we describe the molecular mechanisms underlying the interaction of Alb3 with the chloroplast signal recognition particle (cpSRP). The Alb3 C-terminal domain (A3CT) is intrinsically disordered and recruits cpSRP to the thylakoid membrane by a coupled binding and folding mechanism. Two conserved, positively charged motifs reminiscent of chromodomain interaction motifs in histone tails are identified in A3CT that are essential for the Alb3-cpSRP43 interaction. They are absent in the C-terminal domain of Alb4, which therefore does not interact with cpSRP43. Chromodomain 2 in cpSRP43 appears as a central binding platform that can interact simultaneously with A3CT and cpSRP54. The observed negative cooperativity of the two binding events provides the first insights into cargo release at the thylakoid membrane. Taken together, our data show how Alb3 participates in cpSRP-dependent membrane targeting, and our data provide a molecular explanation why Alb4 cannot compensate for the loss of Alb3. Oxa1 and YidC utilize their positively charged, C-terminal domains for ribosome interaction in co-translational targeting. Alb3 is adapted for the chloroplast-specific Alb3-cpSRP43 interaction in post-translational targeting by extending the spectrum of chromodomain interactions.     

Identification and characterization of a locus (RTM1) that restricts long‐distance movement of tobacco etch virus in Arabidopsis thaliana

The chloroplast homolog of the 54 kDa subunit of signal recognition particle is required for the in vitro targeting of chlorophyll a/b binding proteins (LHCP) to the thylakoid membrane. To explore the function of cpSRP54 in vivo, plants that are mutated in cpSRP54 function were generated. Dominant negative forms of cpSRP54 altered in single amino acids within the conserved guanine nucleotide binding domain were expressed in Arabidopsis. Transformed plants contained less than 30% of the wild-type level of cpSRP54 protein. As a consequence of the reduced cpSRP54 protein content, the first emerging leaves were yellow and contained immature chloroplasts. Although the chlorophyll (chl) content of the leaves was reduced by 75%, the chl a/b ratio was unaffected, indicating a role of cpSRP54 in the biogenesis of proteins besides LHCP. Many chloroplast proteins were less abundant in the first emerging leaves, including non-pigmented proteins, thylakoid proteins known to be targeted by alternative pathways, and soluble proteins. These observations indicate that the cpSRP54 mutation also has a pleiotropic effect on chloroplast biogenesis. Whereas the level of cpSRP54 remained low as the plants aged, leaves emerging subsequently had a wild-type appearance, suggesting that the adult plants compensated for the reduction in cpSRP54 protein.     

Mechanisms of protein import into thylakoids of chloroplasts

The thylakoid membrane of chloroplasts contains the major photosynthetic complexes, which consist of several either nuclear or chloroplast encoded subunits. The biogenesis of these thylakoid membrane complexes requires coordinated transport and subsequent assembly of the subunits into functional complexes. Nuclear-encoded thylakoid proteins are first imported into the chloroplast and then directed to the thylakoid using different sorting mechanisms. The cpSec pathway and the cpTat pathway are mainly involved in the transport of lumenal proteins, whereas the spontaneous pathway and the cpSRP pathway are used for the insertion of integral membrane proteins into the thylakoid membrane. While cpSec-, cpTat- and cpSRP-mediated targeting can be classified as 'assisted' mechanisms involving numerous components, 'unassisted' spontaneous insertion does not require additional targeting factors. However, even the assisted pathways differ fundamentally with respect to stromal targeting factors, the composition of the translocase and energy requirements.     

Function of a chloroplast SRP in thylakoid protein export.

Protein export systems derived from prokaryotes are used to transport proteins into or across the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoid membrane. Signal recognition particle (SRP) and its receptor are essential components used exclusively for cotranslational export of endomembrane and secretory proteins to the endoplasmic reticulum in eukaryotes and export of polytopic membrane proteins to the cytoplasmic membrane in prokaryotes. An organellar SRP in chloroplasts (cpSRP) participates in cotranslational targeting of chloroplast synthesized integral thylakoid proteins. Remarkably, cpSRP is also used to posttranslationally localize a subset of nuclear encoded thylakoid proteins. Recent work has begun to reveal the basis for cpSRP's unique ability to function in co- and posttranslational protein localization, yet much is left to question. This review will attempt to highlight these advances and will also focus on the role of other soluble and membrane components that are part of this novel organellar SRP targeting pathway.     

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.     

Chloroplast Chaperonin-Mediated Targeting of a Thylakoid Membrane Protein

Posttranslational protein targeting requires chaperone assistance to direct insertion-competent proteins to integration pathways. Chloroplasts integrate nearly all thylakoid transmembrane proteins posttranslationally, but mechanisms in the stroma that assist their insertion remain largely undefined. Here, we investigated how the chloroplast chaperonin (Cpn60) facilitated the thylakoid integration of Plastidic type I signal peptidase 1 (Plsp1) using in vitro targeting assays. Cpn60 bound Plsp1 in the stroma. In isolated chloroplasts, the membrane integration of imported Plsp1 correlated with its dissociation from Cpn60. When the Plsp1 residues that interacted with Cpn60 were removed, Plsp1 did not integrate into the membrane. These results suggested Cpn60 was an intermediate in thylakoid targeting of Plsp1. In isolated thylakoids, the integration of Plsp1 decreased when Cpn60 was present in excess of cpSecA1, the stromal motor of the cpSec1 translocon that inserts unfolded Plsp1 into the thylakoid. An excess of cpSecA1 favored integration. Introducing Cpn60’s obligate substrate RbcL displaced Cpn60-bound Plsp1; then, the released Plsp1 exhibited increased accessibility to cpSec1. These in vitro targeting experiments support a model in which Cpn60 captures and then releases insertion-competent Plsp1, whereas cpSecA1 recognizes free Plsp1 for integration. Thylakoid transmembrane proteins in the stroma can interact with Cpn60 to shield themselves from the aqueous environment.     

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.     

Interaction studies between the chloroplast signal recognition particle subunit cpSRP43 and the full-length translocase Alb3 reveal a membrane-embedded binding region in Alb3 protein

Posttranslational targeting of the light-harvesting chlorophyll a,b-binding proteins depends on the function of the chloroplast signal recognition particle, its receptor cpFtsY, and the translocase Alb3. The thylakoid membrane protein Alb3 of Arabidopsis chloroplasts belongs to the evolutionarily conserved YidC/Oxa1/Alb3 protein family; the members of this family facilitate the insertion, folding, and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here, we analyzed the interaction sites of full-length Alb3 with the cpSRP pathway component cpSRP43 by using in vitro and in vivo studies. Bimolecular fluorescence complementation and Alb3 proteoliposome studies showed that the interaction of cpSRP43 is dependent on a binding domain in the C terminus of Alb3 as well as an additional membrane-embedded binding site in the fifth transmembrane domain (TMD5) of Alb3. The C-terminal binding domain was mapped to residues 374-388, and the binding domain within TMD5 was mapped to residues 314-318 located close to the luminal end of TMD5. A direct binding between cpSRP43 and these binding motifs was shown by pepspot analysis. Further studies using blue-native gel electrophoresis revealed that full-length Alb3 is able to form dimers. This finding and the identification of a membrane-embedded cpSRP43 binding site in Alb3 support a model in which cpSRP43 inserts into a dimeric Alb3 translocation pore during cpSRP-dependent delivery of light-harvesting chlorophyll a,b-binding proteins.     

Mutations in a signal sequence for the thylakoid membrane identify multiple protein transport pathways and nuclear suppressors

The apparatus that permits protein translocation across the internal thylakoid membranes of chloroplasts is completely unknown, even though these membranes have been the subject of extensive biochemical analysis. We have used a genetic approach to characterize the translocation of Chlamydomonas cytochrome f, a chloroplast-encoded protein that spans the thylakoid once. Mutations in the hydrophobic core of the cytochrome f signal sequence inhibit the accumulation of cytochrome f, lead to an accumulation of precursor, and impair the ability of Chlamydomonas cells to grow photosynthetically. One hydrophobic core mutant also reduces the accumulation of other thylakoid membrane proteins, but not those that translocate completely across the membrane. These results suggest that the signal sequence of cytochrome f is required and is involved in one of multiple insertion pathways. Suppressors of two signal peptide mutations describe at least two nuclear genes whose products likely describe the translocation apparatus, and selected second-site chloroplast suppressors further define regions of the cytochrome f signal peptide.     

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.