The L18 Domain of Light-harvesting Chlorophyll Proteins Binds to Chloroplast Signal Recognition Particle 43

Abstract

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...

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

“…First, the cytosolic and chloroplast SRPs recognize significantly different forms of "cargo." The cytosolic SRP interacts with ribosome • nascent chain complexes bearing SRP signal sequences (Walter et al, 1981;Schaffitzel et al, 2006), whereas the cpSRP binds to its substrates, LHCPs, as fully translated proteins (Tu et al, 2000;Delille et al, 2000). Second, the cpSRP lacks the SRP RNA, which is otherwise universally conserved in all the other SRP pathways.…”

“…A unique feature of the cpSRP pathway is that it uses a posttranslational mode of targeting. Instead of recognizing ribosome • nascent chain complexes as cargo, the cpSRP recognizes light-harvesting chlorophyll-binding proteins (LHCPs) that are imported into the chloroplast as fully synthesized proteins and delivers LHCPs from the chloroplast stroma to the thylakoid membrane (Delille et al, 2000;Tu et al, 2000). Analogous to the cytosolic SRP pathways, the cpSRP pathway is mediated by two GTPases, cpSRP54 and cpFtsY, that are close homologues of the cytosolic SRP54 and SR GTPases, respectively.…”

Efficient Interaction between Two GTPases Allows the Chloroplast SRP Pathway to Bypass the Requirement for an SRP RNA

“…Therefore, cpSRP43 does not replace all of the functions of the SRP RNA. This novel chloroplast protein may have evolved to mediate other important roles of the SRP RNA in the protein targeting reaction, such as recognition of the cargo protein (Schuenemann et al, 1998;Tu et al, 2000). Analogously, the SRP RNA may have been evolved to interact with ribosomal RNAs during cotranslational protein targeting in the classical SRP pathways (Rinke-Appel et al, 2002;Halic et al, 2004Halic et al, , 2006Schaffitzel et al, 2006).…”

“…cpSRP54 and cpFtsY are the chloroplast homologues of SRP and SR GTPases, respectively (Franklin and Hoffman, 1993;Li et al, 1995;Tu et al, 1999). cpSRP54 recognizes its cargo, the light-harvesting chlorophyll-binding proteins (LHCP), via a protein adaptor cpSRP43 (Tu et al, 2000). Together, cpSRP54 and cpSRP43 deliver the cargo protein from the stroma to the thylakoid membrane via the GTP-dependent interaction between cpSRP54 and cpFtsY (Tu et al, 1999).…”

Regulation of the GTPase Cycle in Post-translational Signal Recognition Particle-based Protein Targeting Involves cpSRP43

“…The unique ability of cpSRP to bind substrates post-translationally is attributed to cpSRP43, which binds both cpSRP54 and a charged 18-amino acid element in LHCPs (L18) to bring about the formation of transit complex (18,19). In the absence of L18 binding, the transit complex fails to form, and LHCPs do not properly integrate into the thylakoid membrane (18).…”