Chloroplast SecY Is Complexed to SecE and Involved in the Translocation of the 33-kDa but Not the 23-kDa Subunit of the Oxygen-evolving Complex*

Danja Schuenemann, Pinky Amin, Enno HartmannDagger , and Neil E. Hoffman§

From the Department of Plant Biology, Carnegie Institution of Washington, Stanford, California 94305 and the Dagger  Zentrum Biochemie und Molekulare Zellbiologie, Gosslerstrasse 12d, D-37073 Goettingen, Germany

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

SecY is a component of the protein-conducting channel for protein transport across the cytoplasmic membrane of prokaryotes. It is intimately associated with a second integral membrane protein, SecE, and together with SecA forms the minimal core of the preprotein translocase. A chloroplast homologue of SecY (cpSecY) has previously been identified and determined to be localized to the thylakoid membrane. In the present work, we demonstrate that a SecE homologue is localized to the thylakoid membrane, where it forms a complex with cpSecY. Digitonin solubilization of thylakoid membranes releases the SecY/E complex in a 180-kDa form, indicating that other components are present and/or the complex is a higher order oligomer of the cpSecY/E dimer. To test whether cpSecY forms the protein-conducting channel of the thylakoid membrane, translocation assays were conducted with the SecA-dependent substrate OE33 and the SecA-independent substrate OE23, in the presence and absence of antibodies raised against cpSecY. The antibodies inhibited translocation of OE33 but not OE23, indicating that cpSecY comprises the protein-conducting channel used in the SecA-dependent pathway, whereas a distinct protein conducting channel is used to translocate OE23.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Thylakoid membranes consist of proteins synthesized by both nuclear and chloroplast genomes. Nuclear encoded thylakoid proteins are first targeted to the chloroplast by means of the transit peptide, which initiates the translocation of the protein across the envelope membranes into the stroma (1). Translation initiation of chloroplast encoded thylakoid proteins appears to occur in the stroma (2, 3), and then synthesis appears to continue on thylakoid bound ribosomes through a co-translational targeting mechanism (4). Considerable progress has been made in defining mechanisms by which nuclear encoded thylakoid proteins insert or translocate posttranslationally across the membrane. One class of proteins insert into the membrane in the absence of an energy supply, soluble factors, or membrane components (5-8). A second class of proteins does not require any soluble factors but requires a trans-thylakoid pH gradient and the membrane protein encoded by the gene hcf106 (9-13). A third class of proteins requires ATP and a chloroplast homologue of the bacterial protein, SecA (14, 15). Finally, a fourth class of proteins requires GTP (16), chloroplast homologues of the bacterial proteins SRP541 (17, 18) and FtsY,2 and a novel protein, cpSRP43 (19, 20). Little is known about the targeting of chloroplast encoded proteins; however, it is likely that they share many of the same translocation components described above (21-24). In two of the cases mentioned above, no soluble factors are required (6, 10); in two other cases, reconstitution has been achieved in the presence of purified soluble components instead of stroma (14),2 thereby defining the minimum soluble-factor requirements. However, membrane components remain to be elucidated for the Delta pH, cpSec, and cpSRP pathways.

Protein export across the bacterial inner membrane is catalyzed by a membrane-embedded translocation apparatus consisting of SecY, SecE, SecG, SecF, SecD, and YajC in conjunction with the peripheral protein, SecA (25, 26). The essential core of the translocase is SecY/SecE and SecA (27, 28). SecY/E form a transmembrane channel through which the exported protein is threaded (29). SecA is thought to act like a piston, pushing the protein through the membrane channel (30, 31). Bacteria also contain an SRP, and recently it was shown that polytopic membrane proteins are dependent on this complex for insertion into the cytoplasmic membrane (32). Furthermore, it was shown that the Sec translocase is utilized in the bacterial SRP pathway. Thus, the SRP- and SecY-dependent pathways converge at SecY (33).

Bacteria also contain a pathway for exporting proteins across the inner membrane independently of SecY (34). Proteins that utilize this pathway have a twin arginine motif at the N terminus (34, 35), as do proteins that utilize the Delta pH pathway in chloroplasts (36). After Hcf106 was identified as a membrane component of the Delta pH pathway (13, 21), it became clear that two bacterial homologues, now designated TatA and TatE, also exist (13, 37, 38). Deletion of tatA/E inactivates transport of proteins containing a twin arginine motif but has no affect on Sec-dependent proteins (37). Thus, it was shown that the Delta pH pathway first described in chloroplasts also exists in bacteria (13, 34, 37, 38).

In addition to SecA, chloroplasts contain a thylakoid protein related to SecY (39). However, the cpSec translocase is largely uncharacterized. It has been assumed, although it remains to be shown, that cpSecY is part of the translocase that translocates SecA-dependent substrates. Furthermore, it is not known whether there is convergence of the SRP- and Delta pH-dependent targeting pathways at the level of SecY. Genetic experiments with maize mutants support the idea of pathway convergence, as SecY mutants have a more severe phenotype than SecA/Hcf106 double mutants (40). In this report, we have characterized a putative cpSecE homologue, and we establish that this protein indeed is a chloroplast protein that forms a complex with cpSecY. Furthermore, we have raised antibodies against cpSecY and have used these antibodies to address whether SecA and Delta pH-dependent proteins both utilize SecY for their translocation across thylakoid membranes.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Arabidopsis thaliana (ecotype Columbia) was grown in a growth chamber in a 16 h light/22 °C temperature versus 8 h dark/18 °C temperature cycle. The light intensity during the light period was 60 µE m-2 s-2. Digitonin (Calbiochem, high purity) was solubilized as a 10% stock solution in boiling ddH2O and kept at 95 °C for 15 min. After cooling the solution was spun for 10 min in a microcentrifuge, and the supernatant was used as stock solution.

Radiolabeled Arabidopsis cpSecE precursor (pcpSecE), the intermediate form of wheat OE33 (iOE33), and wheat OE23 precursor (pOE23) were prepared by in vitro transcription and translation by using SP6 polymerase and [35S]methionine as described (41).

CpSecE Cloning-- The forward primer 5'-CCACATGTCACTAACCGCACAATTC-3' and the reverse primer 5'-CCCAAGCTTCACATCATGCTGAAGAAGTCTTGAAC-3' were used to amplify the gene encoding cpSecE (Cse) from Arabidopsis genomic DNA by PCR using Pfu polymerase (Stratagene). To enhance radiolabeling, the amplified Cse PCR product was designed to contain two additional methionine residues at the C terminus. The PCR product was digested with AflIII and HindIII and cloned into the NcoI-HindIII site of the translation vector pGem4SS6.5NcoI (17), resulting in the plasmid pGem4SS6.5NcoIcpSecE.

For overexpression of cpSecE, a N-terminal hexahistidine-tagged version was constructed. Cse was amplified from pGem4SS6.5NcoIcpSecE by using the forward primer 5'-CCACATGTCACTAACCGCACAATTC-3' and the reverse primer 5'-CGGGATCCATGTCACTAACCGCACAATTC-3'. The PCR product was digested with HindIII and BamHI and cloned into the HindIII-BamHI site of the expression vector pQE30 (Qiagen). The resulting plasmid (pQE30cpSecE) was transformed into SG13009 cells.

Antibodies and Immunoblot Analysis-- SG13009 cells containing pQE30cpSecE were grown to an A600 of ~0.6 and incubated with 1 mM isopropyl-beta -D-thiogalactoside overnight. Cells were harvested, frozen and lysed in Buffer B (8 M urea, 100 mM Na2HPO4/NaH2PO4, 10 mM Tris, pH 8.0) + 1 mM phenylmethylsulfonyl fluoride. The histidine-tagged cpSecE was bound to Ni2+-NTA agarose; the column was washed three times with Buffer B and one time with Buffer C (8 M urea, 100 mM Na2HPO4/NaH2PO4, 10 mM Tris, pH 6.3) and eluted with Buffer C + 250 mM imidazole. The eluted cpSecE was further purified by SDS-polyacrylamide gel electrophoresis. The major 26-kDa band was excised from acrylamide gels and used to raise antibodies in chicken (Cocalico Biologicals, Inc., Reamstown, PA).

Antibodies against cpSecY were raised in rabbits injected with the synthetic peptide CYKNIEFYELDKYDP, corresponding to the C terminus of Arabidopsis cpSecY, fused to keyhole limpet hemocyanin.

Immunoblot analysis was done as described in Ref. 42. For cpSecE detection, crude antiserum was used at a dilution of 1:750. For cpSecY detection, the IgG fraction purified by ammonium sulfate precipitation and diethyl aminoethyl (DEAE)-Sephadex (43) was used at a dilution of 1:3500. Proteins were detected by enhanced chemiluminescence (42).

Thylakoid Isolation-- Arabidopsis leaf tissue (10 g of fresh weight, 4-6 weeks old) was ground in 400 ml of 50 mM Hepes-KOH, pH 8.0, 330 mM sorbitol, 10 mM EDTA, 5 mM sodium ascorbate, 0.05% bovine serum albumin in a polytron (Calbiochem) and the homogenate was filtered through two layers of Miracloth. The filtrate was centrifuged for 5 min at 2600 × g. The pellets were resuspended in 30 ml of 50 mM Hepes-KOH, pH 8.0, 330 mM sorbitol and centrifuged for 5 min at 2600 × g. Afterward, the pellet was resuspended in 10 ml of 10 mM Hepes-KOH, pH 8.0, 5 mM MgCl2 (HM buffer) and kept on ice for 10 min. Thylakoids were pelleted by centrifuging for 5 min at 2600 × g and washed two times in HM buffer. Finally the thylakoids were resuspended at 1 mg of chl/ml in HM buffer for translocation experiments or at 2 mg of chl/ml in 20 mM Hepes, pH 8.0, for other experiments, respectively.

Immunoprecipitation-- 150 µl of thylakoids (2 mg of chl/ml in 20 mM Hepes-KOH, pH 8.0) and an equal volume of detergent solution (4% digitonin in 20 mM Hepes-KOH, pH 8.0, 400 mM NaCl, 2 mM phenylmethylsulfonyl fluoride) were mixed and incubated on ice for 30 min. Solubilized thylakoid membrane proteins were separated from membranes by centrifuging for 10 min at 100,000 × g. The supernatant was incubated with 1.6 mg of anti-cpSecY IgGs cross-linked to 10 mg of protein A-Sepharose beads (43) for 2 h at 4 °C. The beads were transferred into Wizard minicolumns (Promega) and washed with 1 ml of 1% digitonin in 20 mM Hepes-KOH, pH 8.0, 200 mM NaCl followed by 4 ml of 20 mM Hepes-KOH, pH 8.0, 200 mM NaCl. Excess fluid was removed by centrifugation in a microcentrifuge, and the proteins were eluted with 30 µl of 8 M urea in 2× sample buffer.

Import and Translocation Assays-- Import of cpSecE into pea chloroplasts, treatment of intact chloroplasts with 0.1 mg/ml thermolysin, and fractionation of chloroplasts into thylakoids and stroma were done according to Ref. 44. Translocation of iOE33 and pOE23 was essentially done as described (45). Arabidopsis thylakoids were prepared as described above. For inhibition of the translocation with anti-SecY antibodies, thylakoids (45 µg chl) were incubated with the indicated amounts of purified total IgGs (12 µg protein/µl) for 1.5 h at 4 °C. The membranes were washed one time in HM buffer and resuspended at 1 mg of chl/ml in HM buffer (for pOE23) or pea stroma (for iOE33). Pea stroma was prepared as described (44) by lysing chloroplasts containing 2 mg of chl in 1 ml of HM buffer. 45 µl of the thylakoid suspension were incubated with 5 µl in vitro translated iOE33 or pOE23 and incubated for 30 min at 25 °C under illumination (100 µmol m-2 s-2). Assays for iOE33 translocation additionally contained 4 mM ATP. After incubation samples were digested with 0.2 mg/ml thermolysin for 1 h on ice. Thylakoids were washed with 1 ml of HM buffer and resuspended in 25 µl of 4× SDS sample buffer. For documentation, gels were developed by fluorography using either autofluor (National Diagnostics, Manville, NJ) or 20% 2,5-diphenyloxazole in acetic acid (46). For quantification gels were scanned by a PhosphorImager and quantitated using Imagequant software from Molecular Dynamics.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ArabidopsisContains a Homologue of Bacterial SecE-- SecE is an essential protein in bacteria (47, 48). Most forms of SecE contain a single transmembrane domain at the C terminus, unlike Escherichia coli, which contains three transmembrane domains (49). The highest sequence conservation between homologues occurs at the cytoplasmic domain just preceding the transmembrane domain (49, 50). Mutational analysis in E. coli has revealed that the conserved region followed by a generic transmembrane domain is essential for SecE function (50, 51). Recently, Bevan et al. (52) deposited into the GenBankTM data base, 1.9 mB of contiguous sequence from chromosome 4 of Arabidopsis. They noted that one of the hypothetical open reading frames has similarity to SecE preprotein translocase (GenBankTM accession number, Z97337). Fig. 1 shows an alignment of the Arabidopsis hypothetical protein and bacterial SecE sequences. The putative protein most resembles SecE from Thermotoga maritimus, in which the overall similarity is 28%, and similarity is 69% between residues 111 and 173. Like other SecE proteins, the Arabidopsis sequence predicts a protein with a single transmembrane domain at the C terminus with type II topology.


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Fig. 1.   Alignment of A. thaliana cpSecE with bacterial SecE sequences. Sequences of SecE homologues from Thermotoga maritimus, Bacillus subtilis, Synechocystis sp. strain PCC 6803, and Staphylococcus aureus were aligned to A. thaliana cpSecE sequence using Clustal W 1.7 and shaded with Boxshade. Residues conserved in all sequences are shaded in black, and residues conserved in four or five sequences are shaded in gray. Predicted transmembrane domains are underlined. The putative cleavage site for the stromal processing peptidase is indicated by an arrow.

ArabidopsisSecE Is a Chloroplast Protein-- The putative SecE protein is predicted to have a chloroplast transit peptide with a processing site between residues 38 and 39 based on the ChloroP transit peptide prediction program (53). To test this prediction, radiolabeled putative SecE protein (Fig. 2, lane 1) was incubated with isolated pea chloroplasts for 30 min. Nonimported protein was degraded by protease treatment, and chloroplasts were fractionated into stroma and thylakoids. As shown in Fig. 2, lane 3, a smaller, protease-resistant 16-kDa protein was present in the thylakoid fraction consistent with the size of the product predicted by ChloroP (15 kDa).


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Fig. 2.   CpSecE is an integral membrane protein of the thylakoid membrane. A, in vitro translated [35S]methionine-labeled cpSecE precursor (pcpsecE) (lane 1) was imported into isolated pea chloroplasts. After the import reaction, chloroplasts were thermolysin-treated, repurified by centrifugation over a Percoll cushion, and separated into the stromal compartment (str.) (lane 2) and thylakoid membranes (thyl.) (lane 3). B, Arabidopsis thylakoid membrane proteins (equivalent to 20 µg of chl) were separated by SDS-polyacrylamide gel electrophoresis (15% acrylamide) and subjected to immunoblot analysis with anti-cpSecE antibodies (lanes 4-6) and preimmune serum (PI-serum) (lanes 7-9). Integral membrane proteins (P) (lanes 5 and 8) were separated from peripheral membrane proteins (S) (lanes 6 and 9) by incubating thylakoid membranes with 0.1 N NaOH for 15 min at 4 °C followed by a 5 min centrifugation in a microcentrifuge.

That the putative SecE clone encodes a chloroplast protein was further established by immunoblot analysis. Antibodies that were raised against recombinant protein expressed from the putative SecE clone cross-reacted with a 16-kDa thylakoid protein that had the same apparent molecular weight as the imported protein (Fig. 2, lanes 3 and 4). The protein could not be extracted from the thylakoid membrane by incubation of the membranes with 0.1 N NaOH (Fig. 2, lanes 5 and 6), indicating that cpSecE is an integral membrane protein, as predicted from the sequence analysis (Fig. 1). Together, these experiments indicate that the putative SecE protein is encoded as a precursor containing a functional chloroplast transit peptide and the mature protein is localized in the thylakoid membrane.

cpSecE Is Bound to cpSecY-- To test whether cpSecE forms a complex with cpSecY, we examined whether the two proteins co-chromatographed and co-immunoprecipitated after detergent solubilization of thylakoid membranes. To facilitate this analysis, polyclonal antibodies were raised against a C-terminal peptide of Arabidopsis cpSecY. These antibodies reacted with a single protein in wheat germ translation extracts containing cpSecY precursor (Fig. 3) but did not cross-react with any proteins in wheat germ extract (data not shown) and reacted with a single 44-kDa protein found in the thylakoid membrane fraction after alkali extraction (Fig. 3), consistent with the fact that SecY is an integral membrane protein with 10 putative transmembrane helices (54).


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Fig. 3.   Antibodies against cpSecY recognize a 44-kDa integral membrane protein of the thylakoid membrane. In vitro translated cpSecY precursor (pcpSecY) or Arabidopsis thylakoid membrane proteins (equivalent to 2 µg of chl. were separated by SDS-polyacrylamide gel electrophoresis (12% acrylamide) and subjected to immunoblot analysis with anti-cpSecY antibodies. Integral membrane proteins (P) were separated from peripheral membrane proteins (S) by incubating thylakoid membranes (thyl.) with 0.1 N NaOH for 15 min at 4 °C, followed by a 5-min centrifugation in a microcentrifuge.

Thylakoid membrane proteins were solubilized with 2% digitonin, at approximately 70% efficiency, and the extracted proteins were separated by gel filtration chromatography. The relative amount of cpSecY and cpSecE in the various fractions was determined by immunoblot analysis using antibodies against cpSecY and cpSecE, respectively. As shown in Fig. 4, both proteins co-eluted in a single peak as higher molecular mass species of approximately 180 kDa. These data suggest either that other subunits are present or multiple copies of SecY and SecE are present in each complex. Furthermore, these data indicate that most, if not all, cpSecY and cpSecE are associated.


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Fig. 4.   CpSecY and cpSecE cofractionate during gel filtration analysis. 100 µl of solubilized thylakoid membrane proteins (equivalent to 200 µg of chlorophyll; see under "Experimental Procedures" for details) were fractionated on a Superose 6HR column (Amersham Pharmacia Biotech) in 20 mM Hepes-KOH, pH 8.0, 0.1% digitonin, 200 mM NaCl at a flow rate of 0.5 ml/min. The column was calibrated using the following proteins as standards: ferritin dimer (1, 880 kDa), bovine thyroglobulin (2, 670 kDa), ferritin monomer (3, 440 kDa), sweet potato amylase (4, 200 kDa), bovine serum albumin (5, 66 kDa), and cytochrome c (6, 14 kDa). The fractions were precipitated with 10% trichloroacetic acid, and cpSecY (closed circles) and cpSecE (open triangles) were detected by immunoblot analysis. Films were scanned and quantitated using Imagequant software from Molecular Dynamics. Vo, void volume.

A similar conclusion is reached by the co-immunoprecipitation experiment. Antibodies raised against cpSecY were used to immunoprecipitate the digitonin-solubilized complex, and cpSecY and cpSecE in the supernatant and precipitate were detected by immunoblot analysis. As shown in Fig. 5, cpSecY and cpSecE were quantitatively removed from the solubilized thylakoid proteins by the anti-cpSecY antibody and were recovered in the immunoprecipitate, whereas none of the proteins were precipitated by an irrelevant antiserum. cpSecY/E complex was not stable in 1% octylglucoside/dodecylmaltoside (1:1) or 1% Triton X-100 (data not shown). Where reconstitution of the translocase has been successful, digitonin has also been the detergent of choice (25, 55).


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Fig. 5.   Coimmunoprecipitation of cpSecY and cpSecE. Thylakoid membrane proteins were solubilized with 2% digitonin, and an immunoprecipitation was performed using anti-cpSecY antibodies or antibodies against an irrelevant protein as a control (see under "Experimental Procedures" for details). The solubilized thylakoid proteins (L), the flow through (FT), and the immunoprecipitate (IP) were subjected to immunoblot analysis with anti-cpSecY and anti-cpSecE antibodies.

cpSecY Is Sensitive to Trypsin-- It is well established that trypsin treatment of thylakoid membranes inhibits the translocation of proteins across the thylakoid membrane (6, 56). A likely target of trypsin action is the Sec translocase. To examine whether trypsin cleaves SecY, we performed immunoblot analysis on trypsin treated thylakoids. Fig. 6 reveals that SecY is indeed cleaved by levels of trypsin that efficiently inactivate translocation or integration of proteins into the thylakoid membrane.


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Fig. 6.   CpSecY is sensitive to trypsin digestion. Thylakoid membranes (2.5 µg of chlorophyll) were digested with the indicated amounts of trypsin for 30 min on ice in 50 mM Hepes-KOH, pH 8.0, 330 mM sorbitol. After stopping the reaction by the addition of 2 mM phenylmethylsulfonyl fluoride, the membranes were washed in 50 mM Hepes-KOH, pH 8.0, 330 mM sorbitol, 25 mM EDTA. The membrane proteins were solubilized in 4× sample buffer for 30 min at 37 °C and analyzed by immunoblot using antibodies against cpSecY.

CpSecY Translocates OE33 but Not OE23 across the Thylakoid Membrane-- To address whether cpSecY is a component of the translocon mediating the translocation of substrates on the Sec or Delta pH pathways, we took advantage of the fact that the anti-cpSecY antibody recognized the native cpSecY (Fig. 5). Assuming that the C terminus of SecY faces the stroma, these antibodies should bind and conceivably inhibit SecY activity. Antibodies against cpSecY and cpSecE failed to recognize the corresponding proteins in pea and spinach, necessitating the use of Arabidopsis thylakoids in the assays. To improve yields, Arabidopsis thylakoids were isolated directly from leaf tissue and not from intact chloroplasts. Pea stroma was used as the source of SecA. The substrate for the sec pathway, wheat iOE33, was efficiently translocated into the lumen of Arabidopsis thylakoids and processed to the mature size (Fig. 7A, lanes 1 and 2). Little to no translocation occurred in the absence of stroma, as shown in lane 3. When thylakoids were preincubated with increasing amounts of anti-cpSecY antibodies, translocation was progressively inhibited (Fig. 7A, lanes 4-7). However, the inhibition could be relieved by adding an excess of cpSecY peptide antigen during the antibody pretreatment (Fig. 7A, lane 8). Furthermore, antibodies against an irrelevant protein, cpSRP54, were not inhibitory (Fig. 7A, lane 9). Thus, the antibody effect was specific for cpSecY. These data provide the first direct demonstration that the Sec pathway substrate, iOE33, utilizes cpSecY for translocation. These data also suggest that cpSecY has a similar topology as the bacterial homologue, where the N and C termini are in the stroma (or the cis side of the membrane).


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Fig. 7.   Anti-cpSecY antibodies inhibit the translocation of iOE33 but not the translocation of pOE23. Arabidopsis thylakoids were incubated with iOE33 (lane 1) (A) or pOE23 (lane 1) (B) in the absence (lane 2) or presence of 0.05, 0.1, 0.3, and 1 µl of anti-cpSecY antibodies (12 µg of IgGs/µl) (lanes 4-7, respectively) as detailed under "Experimental Procedures." Control assays (lane 3) were performed in the absence of stroma for iOE33 or the presence of 15 µM CCCP for pOE23. Controls shown in lane 8 and 9 were done in presence of 1 µl of anti-cpSecY antibodies (12 µg of IgGs) and 100 ng of cpSecY-peptide (lane 8) or anti-cpSRP54 antibodies (12 µg of IgGs) as an irrelevant antiserum (lane 9). TP, translation product; mOE33 and mOE23, mature OE33 and OE23.

The substrate for the Delta pH pathway, wheat pOE23, was also efficiently translocated into the thylakoid lumen and was processed to the mature form (Fig. 7B, lanes 1 and 2). The translocation of pOE23 was dependent on the Delta pH, as demonstrated by the complete inhibition resulting from CCCP addition (Fig. 7B, lane 3). In contrast to the results seen with the Sec substrate, preincubation of the thylakoids with increasing amounts of anti-cpSecY antibodies had no affect on pOE23 translocation (Fig. 7B, lanes 4-7). This result demonstrates that pOE23 and probably other substrates of the Delta pH pathway are translocated via a translocon lacking cpSecY. Thus, the Delta pH and Sec pathways are parallel and do not converge at cpSecY in the thylakoid membrane.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This work clearly establishes the existence of a chloroplast localized SecE protein that is tightly associated with cpSecY. Thus, we can conclude that at a minimum, chloroplasts contain all the core elements of the Sec translocase: SecY, SecA, and SecE. The core elements of the Sec related translocase of the ER include Sec61alpha (a homologue of SecY), Sec61gamma (a homologue of SecE), and Sec61beta (49). Three to four of these heterotrimers form a pore-like structure in the ER (57, 58) that remains stable after solubilization with digitonin. Solubilization of the thylakoid membrane using the same detergent releases a 180-kDa complex that contains SecY and SecE. This complex may consist of multiple copies of SecY/E dimers forming a ring-like structure, like those seen after purification of the ER-complex, and may also include additional subunits, e.g. a SecG homologue. An important goal for future work will be to establish the subunit composition and stoichiometry of the proteins in the complex.

Several lines of evidence have indicated that there are multiple pathways for targeting proteins to the thylakoid membrane (reviewed in Refs. 1, 59, and 60). First, in vitro studies indicated that substrates fall into distinct classes with regard to their ability to act as competitors of protein targeting to the thylakoid membrane (11). Second, each of these classes has distinct energetic requirements for protein targeting (10, 16). Third, genetic studies largely corroborate the in vitro studies; loss of Hcf106, SecA, or cpSRP43 resulted in selective reductions in the proteins shown to be substrates for the Delta pH, Sec, and cpSRP pathways, respectively (12, 20, 61, 62). However, these studies did not exclude the possibility that SecY was common to all pathways. It has been observed that the targeting information specifying the Delta pH versus the Sec pathway is present in the transit peptide (36, 63-66), and when a Sec transit peptide is used to direct a Delta pH protein to the Sec translocase, the protein fails to be translocated across the thylakoid membrane (63, 65). Based on these findings, it has been postulated that substrates using the Delta pH pathway are unable to translocate through the Sec system, and hence a distinct translocase may be employed by the Delta pH pathway (65). The results from this paper clearly establish the validity of this hypothesis, as convergence at the level of the Sec translocase does not occur for the Delta pH pathway.

To test whether convergence occurs for the cpSRP pathway, considerable effort was made to reconstitute LHCP integration in Arabidopsis thylakoids supplemented with pea stroma. Unfortunately, thylakoids that translocated OE33 and OE23 failed to integrate LHCP. Arabidopsis thylakoids added to pea thylakoids efficiently inhibited LHCP integration into the pea thylakoids, and the inhibition could be overcome by treatment of the Arabidopsis thylakoids with alkylating agents. Thus, it appears that the Arabidopsis thylakoids possess an inhibitory activity that may act on either the pea stroma or thylakoids to prevent LHCP integration.

Plants that lack cpSRP are viable and contain elevated levels of cpSecY,3 suggesting the possibility that the increases observed in the mutant compensate for the loss of targeting efficiency resulting from the absence of cpSRP. Alternatively, the elevated level of cpSecY could indicate that cpSecY forms an alternative pathway for the cpSRP-dependent substrates. However, if the cpSRP delivers its substrate to cpSecY, it must use the translocase independently of SecA, as LHCP integration is not inhibited by azide, which inhibits SecA activity (11), LHCP integration is not competed by SecA-dependent substrates (11), and LHCP levels are not reduced in SecA mutants (12). These observations suggest the possibility that the SecY/E core has activity in the absence of SecA. Whereas loss of either SecA and SecY is lethal, the phenotype of the SecY mutant is more severe than the SecA mutant or even the SecA/Hcf106 double mutant (40). This observation is also consistent with the notion that cpSecY/E has a residual activity in the absence of SecA.

    ACKNOWLEDGEMENTS

We thank Colin Robinson and Ken Cline for providing the wheat pOE23 and iOE33 clones and Alexandra Mant for advice concerning the translocation assays.

    FOOTNOTES

* This work was supported by grants from the United States Department of Agriculture (to N. E. H.) and Deutsche Forschungsgemeinschaft (to D. S.). This article is Carnegie Institution of Washington Publication No. 1410.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Plant Biology, Carnegie Institution of Washington, 260 Panama St., Stanford, CA 94305. Tel.: 650-325-1521, ext. 214; Fax: 650-325-6857; E-mail: hoffman{at}andrew2.stanford.edu.

2 C.-J. Tu, D. Schuenemann, and N. E. Hoffman, manuscript in preparation.

3 P. Amin, D. Sy, M. Pilgrim, D. Parry, L. Nussaume, and N. E. Hoffman, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: SRP, signal recognition particle; cpSRP, chloroplast SRP; iOE33, intermediate form of the 33-kDa oxygen evolving protein; pOE23, precursor of the 23-kDa oxygen evolving protein; chl, chlorophyll; HM buffer, 10 mM Hepes-KOH, pH 8.0, 5 mM MgCl2.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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