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Address correspondence to Kenneth Cline, Horticultural Sciences Department, Fifield Hall, University of Florida, Gainesville, Florida 32611. Tel.: (352) 392-4711 ext. 219. Fax: (352) 392-5653. E-mail: kcline{at}ufl.edu
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Abstract |
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Key Words: chloroplast; receptor; Tat pathway, signal peptide; twin arginine
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Introduction |
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Three different components of the pH-dependent and Tat systems have been identified: Hcf106 (Settles et al., 1997) and its bacterial orthologue TatB (Sargent et al., 1999); Tha4 (Mori et al., 1999; Walker et al., 1999) and its orthologue TatA (Sargent et al., 1998); cpTatC (Mori et al., 2001) and its ortholog TatC (Bogsch et al., 1998). Hcf106 and Tha4 have similar structures; they are anchored in the membrane by an NH2-proximal transmembrane domain and expose a predicted amphipathic helix and an acidic COOH-terminal domain to the stroma. Hcf106 and Tha4 also possess significant sequence similarity in their NH2-proximal regions, especially in the transmembrane domain (
65% identity), where a prolineglutamate motif is strictly conserved. cpTatC is a membrane protein with six predicted transmembrane segments, exposing its NH2 and COOH termini to the stroma (Mori et al., 2001). In the case of thylakoids, all three components directly participate in the transport process because antibody to any single component disables the pathway (Mori et al., 1999; Summer et al., 2000; Mori et al., 2001). Several models have been presented for the operation of these systems; each has suggested that a different component serves as precursor receptor, i.e., Hcf106/TatB and Tha4/TatA because of their receptor-like topology (Settles et al., 1997; Chanal et al., 1998), and TatC/cpTatC because it is the most conserved component among different species (Berks et al., 2000). However, evidence on this point has been lacking.
Previous studies showed that pH-dependent pathway transport can be divided into two steps,
pH-independent binding of precursors to the membrane, and
pH-dependent translocation across the membrane (Ma and Cline, 2000, Musser and Theg, 2000a). Binding is productive, or on pathway, because >80% of bound precursor chases into the lumen upon reestablishment of the pH gradient (Ma and Cline, 2000). Furthermore, binding is specific as it can be competitively inhibited by
pH-dependent pathway precursors but not a Sec pathway precursor (Ma and Cline, 2000). Here, we have characterized the precursor binding site and determined essential features of precursors for binding. The combination of blue native PAGE (BN-PAGE), coimmunoprecipitation, and chemical cross-linking demonstrated that precursors specifically bind to an
700-kD complex of cpTatC and Hcf106 and are in close contact with both components. Tha4 is absent from the precursor-bound and unbound cpTatCHcf106 complex. Nevertheless, Tha4 participation is required for progression of precursors beyond the cpTatCHcf106 bound state even in the presence of the pH gradient. These observations lead to a model for regulated assembly in which the cpTatCHcf106 complex serves as the core receptor complex for precursors, and subsequent recruitment of Tha4 reorganizes the components into a translocation channel.
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Results |
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To determine which components are involved in the binding step, antibodies to the pea components were preincubated with pea thylakoids, and the washed thylakoids were assayed for their ability to bind precursor. Anti-cpTatC IgG or anti-Hcf106 IgG inhibited binding (Fig. 1
, lanes 4, 5, 7, and 8), whereas anti-Tha4 IgG (lanes 11, 12) and preimmune IgG (lane 3) had no effect on binding. In the experiment shown in Fig. 1, anti-cpTatC or anti-Hcf106 IgGs reduced DT23 binding to 40% of control. In a similar experiment (data not shown), anti-cpTatC or anti-Hcf106 reduced tOE17 binding to
25% of control. Inhibition of binding was suppressed by including antigen during the antibody preincubation step (Fig. 1, lanes 6 and 9), demonstrating the specificity of the inhibition. Although antibodies to Hcf106 and cpTatC inhibited binding, neither antibody eliminated binding, nor did a mixture of antibodies (Fig. 1, lane 10). Parallel assays for these experiments showed that Tha4 antibody treatments were sufficient to inhibit
85% of
pH-dependent pathway transport (data not shown). These results confirm those of Ma and Cline (2000) that Hcf106 is involved in precursor binding and that Tha4 is required for transport, but not for binding. They further implicate cpTatC in the binding step.
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This tentative conclusion was confirmed by coimmunoprecipitation analysis performed with digitonin-solubilized thylakoids under nondenaturing conditions (Fig. 3)
. All of the cpTatC was immunoprecipitated by either anti-cpTatC (lane 6) or anti-Hcf106 (lane 8). Hcf106 was coimmunoprecipitated with anti-cpTatC (lane 6), but a portion of the Hcf106 was found in the unbound fraction (lane 5). Tha4 was not coimmunoprecipitated with either anti-cpTatC or anti-Hcf106 (lanes 6 and 8). Likewise, anti-Tha4 immunoprecipitated only Tha4 (lane 10). As negative controls for this experiment, immunoprecipitation was conducted with antibodies to cpSecY (lanes 11 and 12) and to cpOxa1p (lanes 13 and 14), components of the thylakoid Sec and SRP translocation pathways, respectively (Mori et al., 1999; Moore et al., 2000). These antibodies did not coimmunoprecipitate any pH-dependent pathway component, ruling out nonspecific associations. These results demonstrate that cpTatC and a portion of the Hcf106 form a stable
700-kD complex. They further suggest that a portion of Hcf106 and all of Tha4 are present in separate pools in the membrane.
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Electrophoresis of BN-PAGE lanes in a second dimension SDS polyacrylamide gel verified that radiolabel in the 700-kD band was due to precursor (Fig. 4 C). DT23 appeared on the second dimension gel as a single spot at the relative position of the 700-kD BN-PAGE complex. tOE17 appeared as a spot at the
700-kD position and also streaked down towards the bottom of the BN-PAGE lane, suggesting that some tOE17 dissociates from the complex during electrophoresis. In three experiments that were quantified, averages of 20% of the tOE17 and 70% of the DT23 applied to the blue native gel were recovered in the
700-kD band.
Coimmunoprecipitation under nondenaturing conditions confirmed that bound precursor was in a complex with cpTatC and Hcf106. Digitonin solubilized precursor-bound thylakoids were subjected to immunoprecipitation and analyzed by SDS-PAGE and fluorography (Fig. 4 D). Either anti-Hcf106 IgG or anti-cpTatC IgG immunoprecipitated nearly all of the tOE17 and DT23 (lanes 69). In contrast, all detectable precursors were recovered in the unbound fraction when anti-Tha4 (lanes 10 and 11), anti-cpSecY (lanes 12 and 13), or preimmune IgGs (lanes 4 and 5) were used for immunoprecipitation. An immunoblot of the bound and unbound samples verified that Tha4 was quantitatively removed from the supernatant by the anti-Tha4 beads (data not shown). This data, combined with the antibody inhibition experiment (Fig. 1), indicates that the 700-kD cpTatCHcf106 complex is the target for productive binding of precursors.
Chemical cross-linking of intact thylakoids confirms in situ associations of cpTatC and Hcf106, but not Tha4
Because detergent could disrupt labile interactions with Tha4, chemical cross-linking of intact thylakoids before and after precursor binding was undertaken to examine interactions that exist in the membrane. Conditions for cross-linking of thylakoids before precursor binding were designed to yield extensive cross-linking, such that interactions would be detected if present. Various cross-linkers were tested; homobifunctional amine-reactive cross-linkers proved effective for thylakoid proteins, and thiol-cleavable derivatives were used to facilitate identification of cross-linked partners. The membrane-permeable dithiobis (succinimidyl propionate) (DSP) was used in experiments shown in Fig. 5
. Extensive cross-linking of thylakoid proteins was apparent by SDS-PAGE and Coomassie staining in the absence of reducing agents (Fig. 5 A, lanes 13). Reversibility of cross-linking was verified by treating samples with ß-mercaptoethanol before SDS-PAGE (lanes 46). Immunoblotting confirmed that cpTatC, Hcf106, and Tha4 were extensively cross-linked into larger complexes, some of which must have failed to either migrate into the gels or transfer to the nitrocellulose membrane (Fig. 5 B, lanes 19).
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The specificity of interactions in these experiments was routinely monitored by immunoprecipitation of samples treated without cross-linker (Fig. 5 C, lanes 15) and by immunoprecipitation of cross-linked samples with preimmune IgG (lanes 6 and 11) or anti-cpOxa1p (lanes 10 and 15). In the experiment in Fig. 5, nonspecific cross-linking was not observed. We occasionally observed that a minor amount of Hcf106 (<<1%) was immunoprecipitated with anti-Tha4, anti-cpOxa1p, and even preimmune IgGs. That these latter interactions represent nonspecific association is further supported by the fact that reciprocal interactions were not detected (data not shown). Together, the cross-linking results support conclusions of BN-PAGE and nondenaturing coimmunoprecipitation that before precursor binding, cpTatC and Hcf106 are members of the same membrane complex and that Tha4 is not present in this complex.
Chemical cross-linking of precursor-bound thylakoids demonstrates a direct interaction between precursor and both cpTatC and Hcf106
The primary objective for cross-linking of precursor-bound thylakoids was to identify components that directly interact with the precursor. The precursor DT17 (see below) was used as it yielded a high level of cross-linking products in preliminary experiments with DSP and DTSSP. A concentration series of each cross-linker identified amounts that produced distinctive bands representing putative 1:1 adducts between precursors and nearest neighbors (Fig. 6
A, lanes 24 and 68). Notable products were those at 39,
48, and
75 kD. The relative amounts of these products varied with the cross-linker, but the band at
48 kD was uniformly present. Because Hcf106 migrates on SDS gels at
32 kD, cpTatC at
33 kD, and DT17 at
20 kD, the 48-kD band could conceivably represent a cpTatC-DT17 or Hcf106-DT17 cross-linking product. In addition to distinct bands, very large cross-linking products were also apparent (Fig. 6 A, lanes 24).
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Precursor binding to the cpTatCHcf106 complex requires both the RR and the hydrophobic core of the signal peptide
The above studies analyzed the nature of the precursor-binding site. The features of the precursors important for the interaction were determined by analyzing binding of precursors with altered signal peptides. Recovered thylakoids were subjected to BN-PAGE to assess the amount of precursor associated with the 700-kD complex (Fig. 7
B) and by SDS-PAGE to monitor the amount of precursor that associated with thylakoids (Fig. 7 C). As expected, deletion of the signal peptide eliminated binding to the cpTatCHcf106 complex. Although tOE17 showed significant binding to the
700-kD complex (Fig. 7 B, lane 9), mOE17 was not associated with the complex (lane 8). Similarly, the intermediate precursor iPftf also bound to the cpTatCHcf106 complex (lane 6); Pftf without its signal peptide (mPftf) did not (lane 7). iPftf is a membrane protein with an RR-containing signal peptide that is integrated into thylakoids by the
pH-dependent pathway (Summer et al., 2000).
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Precursors are not released from the 700-kD complex unless Tha4 can participate in the transport reaction
Results presented here and previously (Mori et al., 1999; Ma and Cline, 2000) show that Tha4 is required for transport but not binding of precursor. To determine the fate of precursors in the presence of pH when Tha4 is prevented from entering the reaction, thylakoid membranes were preincubated with anti-Tha4 IgG and then subjected to binding, chase, and transport reactions with DT23 (Fig. 8)
. Thylakoids recovered from assays were examined by SDS-PAGE/fluorography to assess the amount of precursor or mature protein associated with the membranes (Fig. 8 B) and by BN-PAGE/fluorography to assess specific binding (Fig. 8 A). As expected, antibody-treated membranes were as active as preimmune IgGtreated membranes in specifically binding DT23 in a standard binding assay on ice (Fig. 8, A and B, compare lanes 1 and 5). However, anti-Tha4treated membranes were unable to transport bound precursor (Fig. 8 B, lane 6), which remained associated with the
700-kD complex (Fig. 8 A, lane 6). In the control preimmune treated membranes, nearly all of the bound precursor was transported to the lumen, as assessed by the appearance of the mOE23 (Fig. 8 B, lane 2) and the absence of the
700-kD band on BN-PAGE (Fig. 8 A, lane 2). When anti-Tha4treated thylakoids were incubated at 25°C with freshly added DT23 in a protein transport assay, i.e., with a
pH, a substantially greater amount of precursor bound to the cpTatCHcf106 complex (Fig. 8, A and B, lane 7). Protease treatment of the membranes showed that the associated precursor was still exposed to the stromal face (data not shown). The pH gradient apparently contributed to the increased binding because considerably less precursor was bound in assays conducted with the protonophore nigericin (Fig. 8, A and B, lanes 8).
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Discussion |
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Several lines of evidence support the conclusion that precursors bind to a cpTatCHcf106 complex. First, antibodies to Hcf106 and to cpTatC inhibited the binding of at least two different precursors, whereas antibodies to Tha4, the third known component, had no effect on binding (Fig. 1; Ma and Cline, 2000). Second, cpTatC and Hcf106 exist in thylakoids as an 700-kD complex that is devoid of Tha4 (Figs. 2, 3, and 5). Upon detergent solubilization of precursor-bound thylakoids, precursors were associated with the cpTatCHcf106 complex as determined by BN-PAGE and coimmunoprecipitation analysis (Fig. 4). Finally, chemical cross-linking studies verified that precursors are in direct contact with both Hcf106 and cpTatC (Fig. 6), but not Tha4. These analyses also indicate that precursor binding per se did not alter the organization of components in the membrane. In particular, the precursor-bound complex contains cpTatC and Hcf106, but is still devoid of Tha4 (Figs. 4 and 6). In fact, an examination of component organization by BN-PAGE/immunoblotting subsequent to binding of saturating amounts of DT23 showed no significant changes in the banding pattern of any component, other than the expected slight shift in molecular mass of the 700-kD cpTatC and Hcf106 bands (unpublished data).
Analysis of precursor requirements for binding suggests characteristics of the binding site. The signal peptide appears to be the primary determinant of binding. Notably, precursor binding to the cpTatCHcf106 complex was strictly dependent on the twin arginine motif and the hydrophobic core of the signal peptide (Fig. 7), both of which are crucial factors for protein translocation on the pH-dependent pathway (Henry et al., 1997). This suggests that the binding site consists of a hydrophobic pocket and acidic or polar residues to bind the arginine pair. Because the detergent-solubilized precursor cpTatCHcf106 complex is very stable, it is reasonable to suspect that the entire binding site is made up of protein domains of the complex. Several considerations suggest that the binding site resides either on cpTatC or is formed by the interaction of Hcf106 and cpTatC. First, bacterial Tat components also exist in a large
600-kD complex (Bolhuis et al., 2000), which contains only TatB (Hcf106 orthologue) and TatC in 1:1 stoichiometry, and TatA (Tha4 orthologue) in substoichiometric amounts (Bolhuis et al., 2001). Similarly, we have partially purified an
700-kD cpTatCHcf106DT23 complex by affinity chromatography and found that cpTatC and Hcf106 are the major, if not only, thylakoid proteins present in the complex (unpublished data). Second, as precursors apparently fail to bind to the free pool of Hcf106 (Fig. 4), it is unlikely that Hcf106 possesses the entire binding site. Finally, the fact that bound precursor formed 1:1 cross-linking products with both Hcf106 and cpTatC demonstrates its proximity to each component (Fig. 6). Nevertheless, further analysis is required to identify the signal peptidebinding domain of the component(s). It will also be important to determine how many binding sites are present per
700-kD complex, as it appears to be a multimer of cpTatCHcf106 heterodimers.
An important goal will be to elucidate the events that occur after precursor binding and establishment of the pH gradient. Several observations persuade us that Tha4 joins the large complex and that this leads to formation of an active translocon. First, results presented here (Fig. 8) and in Ma and Cline (2000) demonstrate that Tha4 functions after the binding step and that, when antibodies sequester Tha4, the precursor cpTatCHcf106 remains intact even in the presence of a pH. Second, kinetic analysis of precursor transport from the bound state indicates that a rate-limiting step occurs on the membrane after the pH gradient is formed (Ma and Cline, 2000; Musser and Theg, 2000a). Musser and Theg (2000b) additionally showed that proton transfer limits events on the membrane leading to translocation. This would be consistent with such a hypothetical
pH-triggered assembly process. Finally, the fact that the purified
600-kD Escherichia coli Tat complex contains a small amount of TatA (Tha4 orthologue) in addition to the large amounts of TatC and TatB (Hcf106 orthologue) suggests that, under appropriate conditions, all three components can be present in one complex (Bolhuis et al., 2001). Analysis of the components in the process of protein translocation will be necessary to test this assembly model. This might be achieved by trapping a translocation intermediate. Berghöfer and Klösgen (1999) reported on a putative translocation intermediate of the
pH-dependent pathway, wherein the precursor protein appeared to be largely translocated into the lumen but still remained associated with the membrane. This intermediate migrated on BN-PAGE at
560 and 620 kD, which is similar in size to the complex that we have seen. If these truly represent proteins in the process of translocation, an analysis of their composition will be valuable for determining the components present in a translocation apparatus.
The nature of the active translocon is currently unknown. Berks et al. (2000) speculate that multiples of TatB/Hcf106 and TatA/Tha4 assemble to form a flexible channel that accommodates protein substrates of varied size. Our recent isolation of a membrane-spanning protein substrate arrested in the process of translocation (unpublished data) supports the notion that a channel structure is involved, as does the recent imaging of overexpressed TatA and TatB as ring-like structures (Sargent et al., 2001). The 510-fold excess of Hcf106 and Tha4 over translocation sites (Mori et al., 1999, 2001) is consistent with a mechanism in which the cpTatCHcf106 complex forms a core complex that recruits channel components for the translocation step. One advantage of a dynamic structure for the pH-dependent pathway would be to prevent premature formation of channels that would likely dissipate ion and pH gradients. Just as the studies reported here reveal insight into the precursor recognition step for this pathway, determining the nature of the active translocon will undoubtedly provide insight into the translocation step.
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Materials and methods |
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Isolation of chloroplasts and thylakoids and assay for import into chloroplasts and binding to thylakoid membranes
Chloroplasts and thylakoids were isolated from pea seedlings as described (Cline et al., 1993). Thylakoids were washed once with import buffer before use. Pretreatment of thylakoids with IgG was previously described (Mori et al., 1999). Chloroplast protein import assays were as described (Cline et al., 1993), except that incubations were for 20 min. Thylakoids were recovered from repurified chloroplasts by lysis and centrifugation and were washed with import buffer (Cline et al., 1993). Precursor binding assays were as described by Ma and Cline (2000). Typical assays contained radiolabeled precursor protein and thylakoids equivalent to 100 µg chlorophyll in a total volume of 300 µl of import buffer. After incubation on ice in darkness for 15 min, precursor-bound thylakoids were recovered by centrifugation, washed twice with import buffer, and transferred to a new microcentrifuge tube. Transport of precursor from the bound state (chase) was accomplished by resuspending precursor-bound thylakoids in import buffer, 5 mM MgCl2, 5 mM Mg-ATP, 5 mM dithiothreitol, and 500 µg stromal protein in a total volume of 300 µl and incubating in the light for 15 min at 25°C.
BN-PAGE
BN-PAGE was carried out as described (Schägger and von Jagow, 1991) with the following modifications. Washed thylakoids were suspended in resuspension buffer (20% [wt/vol] glycerol, 25 mM BisTris-HCl, pH 7.0) at 1.5 mg chlorophyll/ml or as otherwise indicated. An equal volume of resuspension buffer containing twice the final detergent concentration was added to the thylakoid suspension in a drop-wise manner. After incubation at 4°C for 1 h with end-over-end mixing, insoluble material (primarily appressed thylakoids) was removed by centrifugation at 200 kg for 20 min. The supernatant was combined with 1:10 volume of 5% Serva blue G (100 mM BisTris-HCl, pH 7.0, 0.5 M 6-amino-n-caproic acid, 30% glycerol) and applied to 0.75-mm-thick 513.5% acrylamide gradient gels in a Hoefer Mighty Small vertical electrophoresis unit connected to a cooling circulator. Electrophoresis was for a total of 35 h at 100150 V and 24°C. The cathode buffer was exchanged with buffer lacking dye after the top 1/31/2 of the gel was covered with dye (2 h). Gels to be analyzed by fluorography were treated with DMSO and PPO as described (Cline, 1986). Gels to be used for immunoblotting were incubated in 25 mM Tris, 192 mM glycine, 20% methanol, 0.1% SDS for 10 min at room temperature and then blotted to PVDF membranes (Millipore) at 50 V for 45 min. Membranes were destained with 20% methanol and 7% acetic acid overnight and then immunodecorated. To verify the specificity of antibody reactions, antiserum was first incubated with antigen or 20 mM HEPES-KOH, pH 8.0, for 1 h on ice and centrifuged at 20 kg for 20 min to remove antigenantibody complexes.
For two-dimensional analysis, excised BN-PAGE lanes were soaked in SDS sample buffer, 2.5% ß-mercaptoethanol for 10 min and were layered onto 1-mm-thick 11.5% SDS polyacrylamide gels.
Coimmunoprecipitation under nondenaturing conditions
Washed thylakoids were solubilized with digitonin as described above, except that resuspension buffer also contained 150 mM NaCl, 0.05% BSA, and 1 mM PMSF, or as stated in the figure legends. The 200,000 g supernatant was mixed with a 60% slurry of protein ASepharose that had been cross-linked to IgG by dimethylpimelimidate at 3:1 (vol/vol), and the suspension was incubated for 1 h at 4°C with end-over-end mixing. The unbound proteins were recovered by centrifugation, and the beads were washed with resuspension buffer containing 0.5% digitonin and 150 mM NaCl, or as stated in the legend. Bound proteins were recovered by incubating the beads in 8 M urea, 5% SDS, 125 mM Tris-HCl, pH 6.8, for 1 h at room temperature followed by centrifugation.
Cross-linking and immunoprecipitation under denaturing conditions
Thylakoids or precursor-bound thylakoids, at 0.33 mg chlorophyll/ml in import buffer, were treated with DSP (Pierce Chemical Co.) from a stock solution in dimethyl sulfoxide or with DTSSP (Pierce Chemical Co.) from a stock solution in import buffer. Control treatments for DSP contained dimethyl sulfoxide. Reactions were quenched with Tris-glycine, pH 8.0, at a final concentration of 50 mM for 30 min on ice. Thylakoids were recovered by centrifugation, washed with import buffer containing 5 mM EDTA, and then solubilized with 1% SDS in TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) for 10 min at 37°C at 0.5 mg chlorophyll/ml. Insoluble material was removed by centrifugation, and 50 µl of supernatant was combined with 1 ml of 1% BSA, 1% Triton X-100, 1 mM EDTA, TBS, and 1020 µl packed volume of protein ASepharose that had been cross-linked to IgG. After incubation for 1 h at 4°C with end-over-end mixing, the beads were recovered by centrifugation, washed with 1% Triton X-100 and TBS, and then with TBS alone. Bound proteins were eluted from the beads with SDS sample buffer containing 8 M urea, but lacking ß-mercaptoethanol, for 1 h at room temperature. ß-mercaptoethanol was then added to aliquots of the eluates at a final concentration of 2.5% to cleave the cross-linker. Immunoprecipitated proteins were analyzed by SDS-PAGE and immunoblotting with horseradish peroxidaseconjugated antirabbit Fc as a secondary antibody. Radiolabeled proteins were analyzed by SDS-PAGE and fluorography.
Miscellaneous
Antibodies to pea Hcf106 and pea cpTatC are as described (Mori et al., 2001). Antibodies to pea SecY and pea Tha4 are described in Mori et al. (1999). For antibodies to cpOxa1p, the COOH-terminal domain (residues 305442) was amplified by RT-PCR with pea mRNA based on the published sequence (EMBL/GenBank/DDBJ under accession no. Y12618), cloned into pETH3c, and overexpressed as an NH2-terminal histidinetagged fusion protein in E. coli BL21 (DE3) (Cline et al., 1993). The COOH-terminal peptide was purified from inclusion bodies with Ni-chelating Sepharose in the presence of 6 M urea. Antibodies were produced in rabbits by Cocalico Biological. Digitonin was purified as described (Mori et al., 1999). Protein was determined by BCA (Pierce Chemical Co.). Chlorophyll was determined according to Arnon (1949). Radiolabeled proteins were quantified by scintillation counting of proteins extracted from gel bands (Cline, 1986). For cpTatC analyses, all samples were dissolved in an SDS buffer containing 4 M urea at 25°C for 3060 min to avoid aggregation. Immunoblots were developed by the ECL procedure (Pierce Chemical Co.).
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Footnotes |
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Acknowledgments |
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This work was supported in part by National Institutes of Health grant R01 GM46951 to K. Cline. This manuscript is Florida Agricultural Experiment Station Journal series no. R-08261.
Submitted: 31 May 2001
Revised: 6 July 2001
Accepted: 9 July 2001
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