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*

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.

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.
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)(6)(7)(8). A second class of proteins does not require any soluble factors but requires a transthylakoid 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 SRP54 1 (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)(22)(23)(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 ⌬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 ⌬pH pathway in chloroplasts (36). After Hcf106 was identified as a membrane component of the ⌬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 ⌬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 translo-cates SecA-dependent substrates. Furthermore, it is not known whether there is convergence of the SRP-and ⌬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 ⌬pH-dependent proteins both utilize SecY for their translocation across thylakoid membranes.

EXPERIMENTAL PROCEDURES
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 E m Ϫ2 s Ϫ2 . Digitonin (Calbiochem, high purity) was solubilized as a 10% stock solution in boiling ddH 2 O 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.
CpSecE Cloning-The forward primer 5Ј-CCACATGTCACTAACCG-CACAATTC-3Ј and the reverse primer 5Ј-CCCAAGCTTCACATCAT-GCTGAAGAAGTCTTGAAC-3Ј 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 5Ј-CCACATGTCACTAACCGCACAATTC-3Ј and the reverse primer 5Ј-CGGGATCCATGTCACTAACCGCACAATTC-3Ј. 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 A 600 of ϳ0.6 and incubated with 1 mM isopropyl-␤-D-thiogalactoside overnight. Cells were harvested, frozen and lysed in Buffer B (8 M urea, 100 mM Na 2 HPO 4 /NaH 2 PO 4 , 10 mM Tris, pH 8.0) ϩ 1 mM phenylmethylsulfonyl fluoride. The histidinetagged cpSecE was bound to Ni 2ϩ -NTA agarose; the column was washed three times with Buffer B and one time with Buffer C (8 M urea, 100 mM Na 2 HPO 4 /NaH 2 PO 4 , 10 mM Tris, pH 6.3) and eluted with Buffer C ϩ 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 ϫ 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 ϫ g. Afterward, the pellet was resuspended in 10 ml of 10 mM Hepes-KOH, pH 8.0, 5 mM MgCl 2 (HM buffer) and kept on ice for 10 min. Thylakoids were pelleted by centrifuging for 5 min at 2600 ϫ 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 l 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 ϫ 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 l of 8 M urea in 2ϫ 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 g chl) were incubated with the indicated amounts of purified total IgGs (12 g protein/l) 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 l of the thylakoid suspension were incubated with 5 l in vitro translated iOE33 or pOE23 and incubated for 30 min at 25°C under illumination (100 mol m Ϫ2 s Ϫ2 ). 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 l of 4ϫ 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
Arabidopsis Contains 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 GenBank TM 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 (GenBank TM 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.
Arabidopsis SecE 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).
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).
Thylakoid membrane proteins were solubilized with 2% dig-itonin, 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. 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).
cpSecY Is Sensitive to Trypsin-It is well established that trypsin treatment of thylakoid membranes inhibits the trans-  3). B, Arabidopsis thylakoid membrane proteins (equivalent to 20 g 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 (PIserum) (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.

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 g 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. location 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.
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 ⌬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).
The substrate for the ⌬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 ⌬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 ⌬pH pathway are translocated via a translocon lacking cpSecY. Thus, the ⌬pH and Sec pathways are parallel and do not converge at cpSecY in the thylakoid membrane. DISCUSSION 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 Sec61␣ (a homologue of SecY), Sec61␥ (a homologue of SecE), and Sec61␤ (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 mul- tiple 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 ⌬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 ⌬pH versus the Sec pathway is present in the transit peptide (36,(63)(64)(65)(66), and when a Sec transit peptide is used to direct a ⌬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 ⌬pH pathway are unable to translocate through the Sec system, and hence a distinct translocase may be employed by the ⌬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 ⌬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.