Chloroplast SRP54 Interacts with a Specific Subset of Thylakoid Precursor Proteins

High, Stephen; Henry, Ralph; Mould, Ruth M.; Valent, Quido A.; Meacock, Suzanna L.; Cline, Kenneth; Gray, John C.; Luirink, Joen

doi:10.1074/jbc.272.17.11622

Cited by 62 publications

(57 citation statements)

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“…They did not identify interactions between cpSecA and OEC33 or pCytf, although it is known that OEC33 requires cpSecA for thylakoid membrane translocation (39,40). The lack of interaction between cpSecA and Cytf in this study (38) is most likely due to the absence of thylakoid membranes in their experimental setup. The observed affinity of cpSRP54 for pCytf is most likely related to the heterologous nature of the ribosome (see Ref.…”

Section: Figmentioning

confidence: 43%

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In Vitro Reconstitution of Insertion and Processing of Cytochrome f in a Homologous Chloroplast Translation System

Röhl¹,

Wijk

2001

Journal of Biological Chemistry

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Using a homologous chloroplast translation system, we have reconstituted insertion and processing of the chloroplast-encoded thylakoid protein cytochrome f (pCytf). Cross-linking demonstrated that pCytf nascent chains when attached to the 70 S ribosome tightly interact with cpSecA, but this is strictly dependent on thylakoid membranes and a functional signal peptide. This indicates that cpSecA is only operative in pCytf biogenesis when it is bound to the membrane, most likely as part of the Sec translocon. No evidence for interaction between the 54-kDa subunit of the chloroplast signal recognition particle (cpSRP) and the pCytf nascent chain could be detected, suggesting that pCytf, in contrast to the polytopic D1 protein, does not require cpSRP for targeting. Insertion of pCytf occurred only co-translationally, resulting in processing and accumulation of both the processed signal peptide and the mature protein in the thylakoid. This co-translational membrane insertion and processing required a functional signal peptide and was inhibited by azide, demonstrating that cpSecA is essential for translocation of the soluble luminal domain. pCytf also associated posttranslationally with thylakoids, but the soluble N-terminal domain could not be translocated into the lumen. This is the first study in which synthesis, targeting, and insertion of a chloroplast-encoded thylakoid membrane protein is reconstituted from exogenous transcripts and using the chloroplast translational machinery.

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Section: Figmentioning

confidence: 43%

“…High et al (38) identified interactions between cpSRP54 and different thylakoid proteins using a wheat germ translation system with 80 S ribosomes. In their study, truncated transcripts were translated and stable 80 S RNCs were purified and incubated with purified chloroplast stroma devoid of thylakoid membranes.…”

Section: Figmentioning

confidence: 99%

In Vitro Reconstitution of Insertion and Processing of Cytochrome f in a Homologous Chloroplast Translation System

Röhl¹,

Wijk

2001

Journal of Biological Chemistry

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“…With the exception of a synthetic peptide (see below), all reagents, enzymes, and standards were purchased commercially. Previously described plasmids were used for in vitro transcription͞translation of pLHCP (psAB80XD͞4) (13), tOE23 (14), iOE33 (15), and TM3-PC (12). However, the TM3-PC translation product, which codes for a fusion protein between the carboxyl-terminal third of pLHCP and the mature domain of plastocyanin (PCm), is designated here as pLHCP to more clearly indicate the amino acids derived from pLHCP.…”

Section: Methodsmentioning

confidence: 99%

“…Comparison of cpSRP and eukaryotic SRP to cotranslationally bind RNCs revealed that the substrate requirement for SRP binding is similar (12); signal sequence overall hydrophobicity stimulates SRP binding to RNCs. In these assays, the signal peptide of an endoplasmic reticulum-targeted protein (e.g.…”

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confidence: 99%

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

DeLille

Peterson

Johnson

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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117

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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...

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“…These routes include the spontaneous insertion of proteins (18), and signal recognition particle-dependent (9), ⌬pH-dependent (6, 21), and SecA-dependent pathways (22,33). A twin-arginine motif is specifically recognized by components of the ⌬pH-dependent pathway, and hydrophobic sequences are favored by the signal recognition particle-dependent pathway (12,13). The specificity of cpSecA for the Sec-dependent chloroplast signals observed here provides a mechanism for avoiding precursor proteins with those features.…”

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confidence: 99%

Chloroplast SecA and Escherichia coli SecA Have Distinct Lipid and Signal Peptide Preferences

et al. 2007

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Like prokaryotic Sec-dependent protein transport, chloroplasts utilize SecA. However, we observe distinctive requirements for the stimulation of chloroplast SecA ATPase activity; it is optimally stimulated in the presence of galactolipid and only a small fraction of anionic lipid and by Sec-dependent thylakoid signal peptides but not Escherichia coli signal peptides.The chloroplast Sec pathway for protein transport is particularly interesting because it sits at the evolutionary crossroads between eukaryotic systems which have Sec-dependent pathways that do not involve SecA (e.g., endoplasmic reticulum [ER] transport) and prokaryotic Sec systems that do require SecA. In bacteria, the components of this pathway include the SecYEG complex, which constitutes the translocation channel (8, 11), and SecA, an ATPase that powers the translocation of the polypeptide across the cytoplasmic membrane (16,31,32). In Escherichia coli, the efficiency of protein translocation is directly proportional to the amount of anionic phospholipids (17,20,25) and this reflects the effect of these lipids on SecA ATPase activity (19,29). In plants, genes encoding chloroplast homologues of SecY (15) and SecE (10) have been identified, as has a SecA homologue from several chloroplast sources (22,24,26,28). The lipid composition of the thylakoid membrane is dominated by the neutral galactolipid digalactosyldiacylglycerol (DGDG) (70 to 80%) and anionic dioleylphosphatidylglycerol (DOPG) (30). The extent to which these lipids influence the activity of the thylakoid transport machinery is not known.Sec-dependent proteins that cross the eukaryotic ER or the bacterial cytoplasmic membrane are synthesized with a single amino-terminal signal peptide whose highly hydrophobic nature plays a critical role in facilitating protein transport (5) through interactions with SecA (4). Proteins destined for the Sec pathway in the chloroplast thylakoid differ in that they are synthesized with presequences that are bipartite; the most aminoterminal portion corresponds to a transit sequence for passage through the chloroplast envelope and the second region is reminiscent of bacterial and ER signals. Does chloroplast SecA (cpSecA) directly interact with this signal, and is it tailored to discriminate thylakoid Sec signals from the variety of targeting signals for other chloroplast transport pathways?Here, we address these issues by using cpSecA ATPase activity as an earmark of ligand interactions. In marked contrast to E. coli SecA, cpSecA ATPase activity is enhanced by a high concentration of DGDG and only a small amount of DOPG. Furthermore, cpSecA ATPase activity is preferentially stimulated by weakly hydrophobic chloroplast, but not bacterial, Sec signal peptides.Helical content and ATPase activity of cpSecA is retained in lipid vesicles. To set the stage for examining the preferences of cpSecA for two key ligands, lipids and signal peptides, we first established that SecA retained secondary structural elements and ATPase activity upon lipid vesicle integration...

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Chloroplast SRP54 Interacts with a Specific Subset of Thylakoid Precursor Proteins

Cited by 62 publications

References 61 publications

In Vitro Reconstitution of Insertion and Processing of Cytochrome f in a Homologous Chloroplast Translation System

In Vitro Reconstitution of Insertion and Processing of Cytochrome f in a Homologous Chloroplast Translation System

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

Chloroplast SecA and Escherichia coli SecA Have Distinct Lipid and Signal Peptide Preferences

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