Chloroplasts contain a novel type of signal recognition particle (cpSRP) that consists of two proteins, cpSRP54 and cpSRP43. cpSRP is involved in the posttranslational targeting of the nuclear encoded lightharvesting chlorophyll-binding proteins (LHCPs) to the thylakoid membrane by forming a soluble cpSRP⅐LHCP 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 cytosolic signal recognition particle (SRP) 1 is part of a ubiquitous protein-targeting machinery that mediates the cotranslational insertion of membrane proteins into the endoplasmic reticulum of eukaryotes and the cytoplasmic membrane of prokaryotes. All of the known cytosolic SRPs are ribonucleoproteins, and their minimal functional core is formed by an RNA component and a conserved ϳ54-kDa protein (SRP54). SRP54 consists of an N-terminal NG-domain encoding a GTPase function and a C-terminal located M-domain, which binds to the signal sequence of the elongating substrate protein (1-3). Recently, it was demonstrated that chloroplasts contain an SRP that is involved in the posttranslational targeting of members of the nuclear encoded light-harvesting chlorophyll-binding protein family (LHCPs) to the thylakoid membrane (4 -6). LHCPs form the peripheral antenna of photosystems I and II and comprise approximately one-third of the thylakoid membrane proteins. Similar to all of the known cytosolic SRPs, chloroplast SRP contains a 54-kDa subunit (cpSRP54). Interestingly, in contrast to cytosolic SRPs, chloroplast SRP does not contain a RNA but rather a novel protein subunit of 43 kDa (cpSRP43) (5, 6). Although cpSRP54 and bacterial SRP54 (Ffh) show high sequence similarity, the chloroplast protein is clearly distinguishable from Ffh because Ffh cannot bind to cpSRP43 (7). In the current model of LHCP targeting to the thylakoid membrane of higher plants, the nuclear encoded LHCP is imported across the envelope membranes into the chloroplast stroma. Here, the transit peptide is cleaved off and LHCP is bound by cpSRP to form the soluble transit complex (6). Besides cpSRP (cpSRP54 and cpSRP43), a chloroplast homologue of the bacterial SRP receptor (cpFtsY) and GT...

Background: In chloroplasts of higher plants, a heterodimeric cpSRP43⅐cpSRP54 complex targets LHC proteins to the thylakoid membrane. Results: In green algae, cpSRP43 alone forms a targeting complex with LHC proteins. Conclusion: The coevolution of LHC proteins and cpSRP43 occurred independently of complex formation with cpSRP54. Significance: The results provide new insights into the evolution of cpSRP-dependent protein transport.

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.

Edited by Ulf-Ingo Flügge Keywords:Signal recognition particle Chloroplast Protein transport Alb3 Translocase Light-harvesting chlorophyll-binding protein a b s t r a c t 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.

The a-helix of the second chromodomain of the 43 kDa subunit of the chloroplast signal recognition particle facilitates binding to the 54 kDa subunit Abstract 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 a-helix of CD2 facilitates binding to cpSRP54.