SecA Homolog in Protein Transport Within Chloroplasts: Evidence for Endosymbiont-Derived Sorting

Yuan, Jianguo; Henry, Ralph; McCaffery, Michael; Cline, Kenneth

doi:10.1126/science.7973633

Cited by 170 publications

(141 citation statements)

References 32 publications

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“…Immunoblots with horseradish peroxidase-conjugated 2°an-tibody (Bio-Rad) were developed with the ECL (Amersham) procedure according to the manufacturer's guidelines. The following sources and dilutions of primary antibodies were used: anti-Rbcs, 1:7,000 (E. Tobin, University of California, Los Angeles); anti-LHCP, 1:20,000 (Payan and Cline, 1991); anti-SecA, 1:5,000 (Yuan et al, 1994); anti-Pftf, 1:10,000 (Hugueney et al, 1995); anti-OE23, 1:20,000 (Fincher et al, 1998); anti-Hsp70, 1:5,000 (Yuan et al, 1993); anti-cpSRP54, 1:10,000 (R. Henry and K. Cline, unpublished results), anti-Toc75, anti-Toc34, and anti-Tic110, all at 1:7,000 (D. Schnell, Rutgers University, Newark, NJ).…”

Section: Sds-page and Immunoblottingmentioning

confidence: 99%

“…OE33 is routed into the thylakoid lumen on the thylakoid Sec pathway in chloroplasts (Yuan et al, 1994) by a bipartite signal peptide consisting of a stroma-targeting and lumen-targeting domain that is cleaved in two steps in a similar fashion as the ⌬pH pathway signal peptide. Following import and fractionation of OE33 in chromoplasts, processed mOE33 was recovered in the membrane subfrac- Figure 5.…”

Section: Isolated Chromoplasts Exhibit Plastid and Subplastid Proteinmentioning

confidence: 99%

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Red Bell Pepper Chromoplasts Exhibit in Vitro Import Competency and Membrane Targeting of Passenger Proteins from the Thylakoidal Sec and ΔpH Pathways but Not the Chloroplast Signal Recognition Particle Pathway1

Summer

Cline

1999

Plant Physiology

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Chloroplast to chromoplast development involves new synthesis and plastid localization of nuclear-encoded proteins, as well as changes in the organization of internal plastid membrane compartments. We have demonstrated that isolated red bell pepper (Capsicum annuum) chromoplasts contain the 75-kD component of the chloroplast outer envelope translocon (Toc75) and are capable of importing chloroplast precursors in an ATP-dependent fashion, indicating a functional general import apparatus. The isolated chromoplasts were able to further localize the 33-and 17-kD subunits of the photosystem II O 2 -evolution complex (OE33 and OE17, respectively), lumen-targeted precursors that utilize the thylakoidal Sec and ⌬pH pathways, respectively, to the lumen of an internal membrane compartment. Chromoplasts contained the thylakoid Sec component protein, cpSecA, at levels comparable to chloroplasts. Routing of OE17 to the lumen was abolished by ionophores, suggesting that routing is dependent on a transmembrane ⌬pH. The chloroplast signal recognition particle pathway precursor major photosystem II light-harvesting chlorophyll a/b protein failed to associate with chromoplast membranes and instead accumulated in the stroma following import. The Pftf (plastid fusion/translocation factor), a chromoplast protein, integrated into the internal membranes of chromoplasts during in vitro assays, and immunoblot analysis indicated that endogenous plastid fusion/translocation factor was also an integral membrane protein of chromoplasts. These data demonstrate that the internal membranes of chromoplasts are functional with respect to protein translocation on the thylakoid Sec and ⌬pH pathways.Plastids are developmentally related organelles capable of interconversion among a variety of structurally and biochemically distinct forms in response to both environmental and tissue-specific cues (Whatley, 1978;Thomson and Whatley, 1980). Formation of chromoplasts in many fruits is one striking example of this plasticity. Heavily pigmented, photosynthetically inactive chromoplasts frequently develop from chloroplasts during ripening. This conversion involves dramatic changes in the organization and composition of the internal plastid compartment, which include the loss of proteins involved in carbon fixation in the stroma and replacement with chromoplastspecific proteins, the breakdown of the photosynthetic thylakoid membranes and loss of proteins involved in light capture and electron transfer, and, in some cases, the formation of new membranes (Spurr and Harris, 1968; Camara and Brangeon, 1981;Piechulla et al., 1987;Kuntz et al., 1989).Chromoplast formation is an active rather than simply a degradative process. New proteins, specific to or enhanced in chromoplasts, are synthesized and compartmentalized in the plastid (Camara et al., 1995;Price et al., 1995). Most chromoplast proteins are predicted to be nuclear encoded, translated on cytoplasmic ribosomes, and posttranslationally imported into the plastid, as are nuclear-encoded chloroplast protein...

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Section: Sds-page and Immunoblottingmentioning

confidence: 99%

Section: Isolated Chromoplasts Exhibit Plastid and Subplastid Proteinmentioning

confidence: 99%

Red Bell Pepper Chromoplasts Exhibit in Vitro Import Competency and Membrane Targeting of Passenger Proteins from the Thylakoidal Sec and ΔpH Pathways but Not the Chloroplast Signal Recognition Particle Pathway1

Summer

Cline

1999

Plant Physiology

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“…Transthylakoid translocation mechanisms that have so far been identified are believed to each be specific for a subset of lumenal nuclear-encoded proteins. The isolation and characterization of chloroplastic homologues of bacterial SecA Yuan et al, 1994) has conclusively shown that one of these mechanisms is related to the bacterial Sec system. This translocation system is primarily responsible for the transport into the lumen of, among other proteins, plastocyanin (PC) and the 33-kDa subunit of the oxygen-evolving complex (OE33).…”

Section: Introductionmentioning

confidence: 99%

“…This translocation system is primarily responsible for the transport into the lumen of, among other proteins, plastocyanin (PC) and the 33-kDa subunit of the oxygen-evolving complex (OE33). Translocation via this pathway requires both nucleoside triphosphates and the chloroplastic SecA (Yuan et al, 1994). A transthylakoidal ApH is not required for this process but has been shown to stimulate the rate of translocation (Cline et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

A folded protein can be transported across the chloroplast envelope and thylakoid membranes.

Clark

Theg

1997

MBoC

161

109

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Many thylakoid lumenal proteins are nuclear encoded, cytosolically synthesized, and reach their functional location after posttranslational targeting across two chloroplast envelope membranes and the thylakoid membrane via proteinaceous transport systems. To study whether these transmembrane transport machineries can translocate folded structures, we overexpressed the 17-kDa subunit of the oxygen-evolving complex of photosystem II (prOE17) that had been modified to contain a unique C-terminal cysteine. This allowed us to chemically link a terminal 6.5-kDa bovine pancreatic trypsin inhibitor (BPTI) moiety to prOE17 to create the chimeric protein prOE17-BPTI. Redox reagents and an irreversible sulfhydryl-specific cross-linker, bis-maleimidohexane, were used to manipulate the structure of BPTI. Import of prOE17-BPTI into isolated chloroplasts and thylakoids demonstrates that the small tightly folded BPTI domain is carried across both the chloroplast envelopes and the ApH-dependent transmembrane transporter of the thylakoid membrane when linked to the correctly targeted OE17 precursor. Transport proceeded even when the BPTI moiety was internally cross-linked into a proteaseresistant form. These data indicate that unfolding is not a ubiquitous requirement for protein translocation and that at least some domains of targeted proteins can maintain a nonlinear structure during their translocation into and within chloroplasts.

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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).…”

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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...

show abstract

SecA Homolog in Protein Transport Within Chloroplasts: Evidence for Endosymbiont-Derived Sorting

Cited by 170 publications

References 32 publications

Red Bell Pepper Chromoplasts Exhibit in Vitro Import Competency and Membrane Targeting of Passenger Proteins from the Thylakoidal Sec and ΔpH Pathways but Not the Chloroplast Signal Recognition Particle Pathway1

Red Bell Pepper Chromoplasts Exhibit in Vitro Import Competency and Membrane Targeting of Passenger Proteins from the Thylakoidal Sec and ΔpH Pathways but Not the Chloroplast Signal Recognition Particle Pathway1

A folded protein can be transported across the chloroplast envelope and thylakoid membranes.

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

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