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 posttranslationally 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. SRP1 is a ubiquitous cytoplasmic ribonucleoprotein that mediates the co-translational targeting of endomembrane and secretory proteins to the endoplasmic reticulum in eukaryotes and of polytopic membrane proteins to the cytoplasmic membrane in prokaryotes (reviewed in Refs. 1 and 2). All cytoplasmic forms of SRP contain an RNA and a 54-kDa GTPase, SRP54. SRP54 plays a major role in SRP-dependent targeting, where it binds to nascent chains via an interaction with hydrophobic domains of signal sequences. The bound ribosome-nascent, chain mRNA is piloted to the membrane in part because of the affinity of SRP for its membrane-bound receptor. Upon binding its receptor, SRP dissociates from the nascent chain, and translation resumes at the membrane. A specialized organellar SRP identified in chloroplasts (cpSRP) (3) contains an SRP54 homolog (cpSRP54) (4) but differs from cytoplasmic forms in that it lacks an RNA (5, 6), contains a novel 43-kDa subunit (7), and binds substrates post-translationally (8). The known substrates of cpSRP are the LHCPs, hydrophobic proteins that are synthesized in the cytoplasm and post-translationally transported to the internal membranes of the chloroplast via a soluble pathway that proceeds through the stroma (9, 10). The solubility of LHCP is maintained in the stroma by its binding to cpSRP to form the targeting intermediate termed the transit complex (8, 11). Localization of LHCP to the thylakoid membrane further requires two additional soluble components, GTP (12) and chloroplast FtsY. The latter is a homolog of the SRP receptor ␣ subunit (6, 13). Recent evidence indicates that the thylakoid membrane localized translocon needed for translocation of LHCP into the lipid bilayer is composed minimally of the integr...
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