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.     

A Dynamic cpSRP43-Albino3 Interaction Mediates Translocase Regulation of Chloroplast Signal Recognition Particle (cpSRP)-targeting Components

The chloroplast signal recognition particle (cpSRP) and its receptor, chloroplast FtsY (cpFtsY), form an essential complex with the translocase Albino3 (Alb3) during post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCPs). Here, we describe a combination of studies that explore the binding interface and functional role of a previously identified cpSRP43-Alb3 interaction. Using recombinant proteins corresponding to the C terminus of Alb3 (Alb3-Cterm) and various domains of cpSRP43, we identify the ankyrin repeat region of cpSRP43 as the domain primarily responsible for the interaction with Alb3-Cterm. Furthermore, we show Alb3-Cterm dissociates a cpSRP·LHCP targeting complex in vitro and stimulates GTP hydrolysis by cpSRP54 and cpFtsY in a strictly cpSRP43-dependent manner. These results support a model in which interactions between the ankyrin region of cpSRP43 and the C terminus of Alb3 promote distinct membrane-localized events, including LHCP release from cpSRP and release of targeting components from Alb3.     

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.     

Non-identical contributions of two membrane-bound cpSRP components, cpFtsY and Alb3, to thylakoid biogenesis

The insertion of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membrane of the chloroplast is cpSRP-dependent, and requires the stromal components cpSRP54 and cpSRP43, the membrane-bound SRP receptor cpFtsY and the integral membrane protein Alb3. Previous studies demonstrated that the Arabidopsis mutant lacking both cpSRP54 and cpSRP43 had pale yellow leaves, but was viable, whereas the mutants lacking Alb3 exhibit an albino phenotype that is more severe and seedling lethality. We previously showed that a maize mutant lacking cpFtsY had a pale yellow-green phenotype and was seedling lethal. To compare the in vivo requirements of cpFtsY and Alb3 in thylakoid biogenesis in greater detail, we isolated Arabidopsis null mutants of cpftsY, and performed biochemical comparisons with the Arabidopsis alb3 mutant. Both cpftsY and alb3 null mutants were seedling lethal on a synthetic medium lacking sucrose, whereas on a medium supplemented with sucrose, they were able to grow to later developmental stages, but were mostly infertile. cpftsY mutant plants had yellow leaves in which the levels of LHCPs were reduced to 10-33% compared with wild type. In contrast, alb3 had yellowish white leaves, and the LHCP levels were less than or equal to 10% of those of wild type. Intriguingly, whereas accumulation of the Sec and Tat machineries were normal in both mutants, the Sec pathway substrate Cyt f was more severely decreased in the cpftsY mutant than in alb3, which may indicate a functional link between cpFtsY and Sec translocation machinery. These results suggest that cpFtsY and Alb3 have essentially similar, but slightly distinct, contributions to thylakoid biogenesis.     

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.     

Interplay between the cpSRP pathway components, the substrate LHCP and the translocase Alb3: An in vivo and in vitro study

The chloroplast signal recognition particle (cpSRP) and its receptor, cpFtsY, posttranslationally target the nuclear-encoded light-harvesting chlorophyll-binding proteins (LHCPs) to the translocase Alb3 in the thylakoid membrane. In this study, we analyzed the interplay between the cpSRP pathway components, the substrate protein LHCP and the translocase Alb3 by using in vivo and in vitro techniques. We propose that cpSRP43 is crucial for the binding of LHCP-loaded cpSRP and cpFtsY to Alb3. In addition, our data suggest that a direct interaction between Alb3 and LHCP contributes to the formation of this complex.

Structured summary

MINT-7992851: Alb3 (uniprotkb:Q8LBP4) physically interacts (MI:0915) with cpSRP43 (uniprotkb:O22265) by two hybrid (MI:0018)MINT-7992897: cpSRP43 (uniprotkb:O22265) and Alb3 (uniprotkb:Q8LBP4) physically interact (MI:0915) by bimolecular fluorescence complementation (MI:0809)MINT-7993251: SRP43 (uniprotkb:O22265) binds (MI:0407) to LHCP (uniprotkb:P27490) by pull down (MI:0096)MINT-7993207: cpSRP43 (uniprotkb:O22265) physically interacts (MI:0915) with ftsY (uniprotkb:O80842), LHCP (uniprotkb:P27490), SRP-54 (uniprotkb:P37106) and Alb3 (uniprotkb:Q8LBP4) by pull down (MI:0096)MINT-7993272: Alb3 (uniprotkb:Q8LBP4) and LHCB (uniprotkb:P27490) physically interact (MI:0915) by bimolecular fluorescence complementation (MI:0809)MINT-7992960: cpSRP43 (uniprotkb:O22265) binds (MI:0407) to Alb3 (uniprotkb:Q8LBP4) by pull down (MI:0096)MINT-7993236: Alb3 (uniprotkb:Q8LBP4) binds (MI:0407) to LHCP (uniprotkb:P27490) by pull down (MI:0096)MINT-7993166: cpSRP43 (uniprotkb:O22265) physically interacts (MI:0915) with LHCP (uniprotkb:P27490) and Alb3 (uniprotkb:Q8LBP4) by pull down (MI:0096)MINT-7993118: cpSRP43 (uniprotkb:O22265) physically interacts (MI:0915) with Alb3 (uniprotkb:Q8LBP4), SRP-54 (uniprotkb:P37106) and LHCP (uniprotkb:P27490) by pull down (MI:0096)MINT-7993046: cpSRP43 (uniprotkb:O22265) physically interacts (MI:0915) with ftsY (uniprotkb:O80842), SRP-54 (uniprotkb:P37106) and Alb3 (uniprotkb:Q8LBP4) by pull down (MI:0096)MINT-7993004: cpSRP43 (uniprotkb:O22265) physically interacts (MI:0915) with SRP54 (uniprotkb:P37106) and Alb3 (uniprotkb:Q8LBP4) by pull down (MI:0096)     

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.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Proteins of photosystem II

The senescence of leaves is characterized by yellowing as chlorophyll pigments are degraded. Proteins of the chloroplasts also decline during this phase of development. There exists a non-yellowing mutant genotype of Festuca pratensis Huds. which does not suffer a loss of chlorophyll during senescence. The fate of chloroplast membrane proteins was studied in mutant and wild-type plants by immune blotting and immuno-electron microscopy. Intrinsic proteins of photosystem II, exemplified by the light-harvesting chlorophyll a/b-binding protein (LHCP-2) and D1, were shown to be unusually stable in the mutant during senescence, whereas the extrinsic 33-kilodalton protein of the oxygen-evolving complex was equally lable in both genotypes. An ultrastructural study revealed that while the intrinsic proteins remained in the internal membranes of the chloroplasts, they ceased to display the heterogenous lateral distribution within the lamellae which was characteristic of nonsenescent chloroplasts. These observations are discussed in the light of possible mechanisms of protein turnover in chloroplasts.Abbreviations kDa kilodalton - LHCP-2 light-harvesting chlorophyll a/b-binding protein - M_r relative molecular mass - PSII photosystem II - SDS sodium dodecyl sulphate     

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.     

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.     

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.     

YidC/Alb3/Oxa1 Family of Insertases

The YidC/Alb3/Oxa1 family functions in the insertion and folding of proteins in the bacterial cytoplasmic membrane, the chloroplast thylakoid membrane, and the mitochondrial inner membrane. All members share a conserved region composed of five transmembrane regions. These proteins mediate membrane insertion of an assorted group of proteins, ranging from respiratory subunits in the mitochondria and light-harvesting chlorophyll-binding proteins in chloroplasts to ATP synthase subunits in bacteria. This review discusses the YidC/Alb3/Oxa1 protein family as well as their function in membrane insertion and two new structures of the bacterial YidC, which suggest a mechanism for membrane insertion by this family of insertases.     

Functional interaction of chloroplast SRP/FtsY with the ALB3 translocase in thylakoids: substrate not required

Integration of thylakoid proteins by the chloroplast signal recognition particle (cpSRP) posttranslational transport pathway requires the cpSRP, an SRP receptor homologue (cpFtsY), and the membrane protein ALB3. Similarly, Escherichia coli uses an SRP and FtsY to cotranslationally target membrane proteins to the SecYEG translocase, which contains an ALB3 homologue, YidC. In neither system are the interactions between soluble and membrane components well understood. We show that complexes containing cpSRP, cpFtsY, and ALB3 can be precipitated using affinity tags on cpSRP or cpFtsY. Stabilization of this complex with GMP-PNP specifically blocks subsequent integration of substrate (light harvesting chl a/b-binding protein [LHCP]), indicating that the complex occupies functional ALB3 translocation sites. Surprisingly, neither substrate nor cpSRP43, a component of cpSRP, was necessary to form a complex with ALB3. Complexes also contained cpSecY, but its removal did not inhibit ALB3 function. Furthermore, antibody bound to ALB3 prevented ALB3 association with cpSRP and cpFtsY and inhibited LHCP integration suggesting that a complex containing cpSRP, cpFtsY, and ALB3 must form for proper LHCP integration.     

A second thylakoid membrane-localized Alb3/OxaI/YidC homologue is involved in proper chloroplast biogenesis in Arabidopsis thaliana

The integral membrane proteins Alb3, OxaI, and YidC belong to an evolutionary conserved protein family mediating protein insertion into the thylakoid membrane of chloroplasts, the inner membrane of mitochondria, and bacteria, respectively. Whereas OxaI and YidC are involved in the insertion of a wide range of membrane proteins, the function of Alb3 seems to be limited to the insertion of a subset of the light-harvesting chlorophyll-binding proteins. In this study, we identified a second chloroplast homologue of the Alb3/OxaI/YidC family, named Alb4. Alb4 is almost identical to the Alb3/OxaI/YidC domain of the previously described 110-kDa inner envelope protein Artemis. We show that Alb4 is expressed as a separate 55-kDa protein and that Artemis was identified mistakenly. Alb4 is located in the thylakoid membrane of Arabidopsis thaliana chloroplasts. Analysis of an Arabidopsis mutant (Salk_136199) and RNA interference lines with a reduced level of Alb4 revealed chloroplasts with an altered ultrastructure. Mutant plastids are larger and more spherical in appearance, and the grana stacks within the mutant lines are less appressed than in the wild-type chloroplasts. These data indicate that Alb4 is required for proper chloroplast biogenesis.     

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.     

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.     

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.     

Double mutation cpSRP43--/cpSRP54-- is necessary to abolish the cpSRP pathway required for thylakoid targeting of the light-harvesting chlorophyll proteins

Biochemical and genetic studies have established that the light-harvesting chlorophyll proteins (LHCPs) of the photosystems use the cpSRP (chloroplast signal recognition particle) pathway for their targeting to thylakoids. Previous analyses of single cpSRP mutants, chaos and ffc, deficient in cpSRP43 and cpSRP54, respectively, have revealed that half of the LHCPs are still integrated into the thylakoid membranes. Surprisingly, the effects of both mutations are additive in the double mutant ffc/chaos described here. This mutant has pale yellow leaves at all stages of growth and drastically reduced levels of all the LHCPs except Lhcb 4. Although the chloroplasts have a normal shape, the thylakoid structure is affected by the mutation, probably as a consequence of reduction of all the LHCPs. ELIPs (early light-inducible proteins), nuclear-encoded proteins related to the LHCP family and inducible by light stress, were also drastically reduced in the double mutant. However, proteins targeted by other chloroplastic targeting pathways (DeltapH, Sec and spontaneous pathways) accumulated to similar levels in the wild-type and the double mutant. Therefore, the near total loss of LHCPs and ELIPs in the double mutant suggests that cpSRP is the predominant, if not exclusive, targeting pathway for these proteins. Phenotypic analysis of the double mutant, compared to the single mutants, suggests that the cpSRP subunits cpSRP43 and cpSRP54 contribute to antenna targeting in an independent but additive way.     

Canonical signal recognition particle components can be bypassed for posttranslational protein targeting in chloroplasts

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.     

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.