A novel precursor recognition element facilitates posttranslational binding to the signal recognition particle in chloroplasts

Abstract

Signal recognition particles (SRPs) in the cytosols of prokaryotes and eukaryotes are used to target proteins to cytoplasmic membranes and the endoplasmic reticulum, respectively. The mechanism of targeting relies on cotranslational SRP binding to hydrophobic signal sequences. An organellar SRP identified in chloroplasts (cpSRP) is unusual in that it functions posttranslationally to localize a subset of nuclear-encoded thylakoid proteins. In assays that reconstitute thylakoid integration of the light harvesting chlorophyll-binding protein (LHCP), stromal cpSRP binds LHCP posttranslationally to form a cpSRP͞LHCP transit complex, which is believed to represent the LHCP form targeted to thylakoids. In this investigation, we have identified an 18-aa sequence motif in LHCP (L18) that, along with a hydrophobic domain, is required for transit complex formation. Fusion of L18 to the amino terminus of an endoplasmic reticulum-targeted protein, preprolactin, led to transit complex formation whereas wild-type preprolactin exhibited no ability to form a transit complex. In addition, a synthetic L18 peptide, which competed with LHCP for transit complex formation, caused a parallel inhibition of LHCP integration. Translocation of proteins by the thylakoid Sec and Delta pH transport systems was unaffected by the highest concentration of L18 peptide examined. Our data indicate that a motif contained in L18 functions in precursor recruitment to the posttranslational SRP pathway, one of at least four different thylakoid sorting pathways used by chloroplasts. Signal recognition particle (SRP) and its receptor comprise essential components of a signal peptide-based protein targeting mechanism that is conserved across evolutionary boundaries (1-3). SRPs in the cytosols of eukaryotes and Escherichia coli target proteins cotranslationally to the endoplasmic reticulum and cytoplasmic membrane, respectively. Targeting is initiated as a result of SRP binding to the hydrophobic domain of amino-terminal signal peptides or signal anchors as they emerge from the ribosome. The entire ribosome͞nascent polypeptide chain complex (RNC) then is piloted by SRP to an SRP receptor that functions at the membrane. GTP binding and hydrolysis by SRP and its receptor result in both the release of SRP from its receptor and the release of SRP from the RNC, whereupon the nascent chain enters a translocation pore that directs the translating polypeptide into or across the lipid bilayer.An organellar SRP, which exhibits striking structural and functional differences from cytosolic SRPs, also has been identified in chloroplasts (4, 5). Chloroplast SRP (cpSRP) is a soluble Ϸ200-kDa stromal particle that contains an evolutionary conserved 54-kDa subunit (cpSRP54) as well as a unique 43-kDa polypeptide (cpSRP43) (6). Unlike cytosolic SRPs, an RNA moiety is conspicuously lacking in cpSRP. Biochemical and genetic evidence have demonstrated that cpSRP functions posttranslationally to localize a subset of nuclear-encoded thylakoid proteins belonging to the chlorophyll...

LTD is a protein required for sorting light-harvesting chlorophyll-binding proteins to the chloroplast SRP pathway

“…As shown in Figure 6e,f, similar results were obtained using this system. As expected, cpSRP43 interacted with the pLHCP 163 − 206 region, which contains the L18 region 18,19 (Fig. 6e).…”

“…5b). As expected, the transit complex was formed when recombinant cpSRP54 and cpSRP43 were incubated with [ 35 S]pLHCP 18,36 . Incubation of [ 35 S]pLHCP with cpSRP43 resulted in the formation of a cpSRP43-pLHCP complex, which is consistent with previous reports that cpSRP43 alone prevents aggregation of LHCP by complex formation 38 .…”

“…cpSRP43 contains a chromodomain at the N terminus, which is followed by four ankyrin repeats and then two chromodomains 16 . The ankyrin domain of cpSRP43 provides the binding site for the L18 region of LHCP and is involved in posttranslational targeting of LHCP to the thylakoid membrane 18,36 . Another ankyrin protein, AKR2A, has been also shown to interact with the outer envelope membrane protein OEP7 and targets it to the chloroplast 46 .…”

“…As cpSRP43 specifically recognizes the L18 region in LHCP 18,19 , we next investigated whether LTD also recognizes a specific region in LHCP. An alignment of the amino-acid sequences of LHCP (Lhcb1-6 and Lhca1-4) revealed several strictly conserved amino-acid regions ( Supplementary Fig.…”

“…The ankyrin domain of cpSRP43 interacts with a conserved L18 region between the second and third transmembrane domains of LHCP 18,19 . Targeting of LHCP to the membrane also requires the chloroplast SRP receptor homologue cpFtsY and GTP 20 .…”