Thylakoid DeltapH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport

Abstract

The thylakoid DeltapH-dependent pathway transports folded proteins with twin arginine-containing signal peptides. Identified components of the machinery include cpTatC, Hcf106, and Tha4. The reaction occurs in two steps: precursor binding to the machinery, and transport across the membrane. Here, we show that a cpTatC-Hcf106 complex serves as receptor for specific binding of twin arginine-containing precursors. Antibodies to either Hcf106 or cpTatC, but not Tha4, inhibited precursor binding. Blue native gel electrophoresis and coimmunoprecipitation of digitonin-solubilized thylakoids showed that Hcf106 and cpTatC are members of an approximately 700-kD complex that lacks Tha4. Thylakoid-bound precursor proteins were also associated with an approximately 700-kD complex and were coimmunoprecipitated with antibodies to cpTatC or Hcf106. Chemical cross-linking revealed that precursors make direct contact with cpTatC and Hcf106 and confirmed that Tha4 is not associated with precursor, cpTatC, or Hcf106 in the membrane. Precursor binding to the cpTatC-Hcf106 complex required both the twin arginine and the hydrophobic core of the signal peptide. Precursors remained bound to the complex when Tha4 was sequestered by antibody, even in the presence of DeltapH. These results indicate that precursor binding to the cpTatC-Hcf106 complex constitutes the recognition event for this pathway and that subsequent participation by Tha4 leads to translocation.

Figure 1.

Antibodies to Hcf106 and cpTatC inhibit precursor binding to thylakoids. Pea thylakoids were preincubated with buffer (lane 2), preimmune (PI) IgG (lane 3), or two concentrations of anti-pea cpTatC IgG (lanes 4–6), anti-pea Hcf106 IgG (lanes 7–9), or anti-pea Tha4 IgG (lanes 11–13) in the absence or presence of 20 μM of the respective antigens as shown above the panel, or with a combination of 1 mg/ml anti-cpTatC and 0.2 mg/ml anti-Hcf106 (lane 10) (see Materials and methods). The concentrations chosen for IgG treatment (shown in mg/ml, top) were those previously determined to produce maximum inhibition of ΔpH-dependent transport and 50% of that concentration for each antibody. After washing, treated thylakoids were assayed for binding and transport (not shown) of radiolabled DT23. Thylakoids recovered from assays were analyzed by SDS-PAGE and fluorography. DT23 translation product (tp) is shown in lane 1. The amounts of bound DT23 are presented in the chart below the figure. All values are plotted relative to binding of the preimmune-treated thylakoids, which was 6% of the added precursor and is arbitrarily set at 100% for the bar chart.

Figure 2.

Blue native gel analyses of components of the ΔpH-dependent protein transport pathway. Pea thylakoids were solubilized with digitonin and subjected to BN-PAGE (Materials and methods). The final digitonin concentration (%) and the final thylakoid concentration (mg chlorophyll/ml) are designated (top). Proteins were detected by immunoblotting with anti-cpTatC (A), anti-Hcf106 (B), and anti-Tha4 (C). (D) Pea thylakoids were solubilized with 0.5% digitonin at 0.75 mg chlorophyll/ml. Proteins were immunodetected with anti-cpTatC (lanes 1 and 2), anti-Hcf106 (lanes 3 and 4), or anti-Tha4 (lanes 5 and 6) that was preincubated in the absence (lanes 1, 3, and 5) or presence (lane 2, 4, and 6) of the corresponding antigen. Molecular mass markers are ferritin (880 and 440 kD), β-amylase (200 kD), and bovine serum albumin (132 and 66 kD).

Figure 3.

Coimmunoprecipitation under nondenaturing conditions shows that cpTatC and Hcf106 are present in the same complex. Pea thylakoids (lane 1) were solubilized with 0.5% digitonin at 0.75 mg chlorophyll/ml. After centrifugation to remove insoluble materials, the resulting supernatant (lane 2) was incubated with protein A–Sepharose to which preimmune (lanes 3 and 4), anti-cpTatC (lanes 5 and 6), anti-Hcf106 (lanes 7 and 8), anti-Tha4 (lanes 9 and 10), anti-cpSecY (lanes 11 and 12), or anti-cpOxa1p (lanes 13 and 14) IgG had been cross-linked. After 1 h at 4°C, proteins unbound (lanes 3, 5, 7, 9, 11, and 13) and bound (lanes 4, 6, 8, 10, 12, and 14) to IgG protein A–Sepharose were analyzed by SDS-PAGE and immunoblotting. Proteins bound to IgG protein A–Sepharose were equivalent to 5 μg chlorophyll of starting thylakoids; all other samples were equivalent to 2.5 μg chlorophyll of starting thylakoids. Antibodies used for the immunoprecipitations are designated (top). Antibodies used for immunoblotting are designated (left).

Figure 4.

Precursors bind to the ∼700-kD cpTatC and Hcf106 complex. (A) Pea thylakoids were assayed for binding (b; lanes 2 and 4) or binding and then transport chase (c; lanes 3 and 5) with in vitro translated tOE17 and DT23. Recovered thylakoids were solubilized with 1% digitonin, 20% glycerol, and import buffer and analyzed by BN-PAGE and fluorography (see Materials and methods). Radiolabled Hcf106 from an import assay (see text) was used as a marker for the cpTatC–Hcf106 complex (lane 1). (B) Recovered thylakoids (A) were also subjected to SDS-PAGE and fluorography. The precursors used for the assays (tp) are in lanes 1 and 4. (C) BN-PAGE lanes of bound DT23 and bound tOE17 were subjected to second dimension SDS-PAGE (Materials and methods). An aliquot of the translation product mixed with prestained molecular weight markers was run on the left side of the SDS-PAGE gels. (D) Recovered thylakoids from binding assays with in vitro translated tOE17 or DT23 were solubilized with 1% digitonin, 20% glycerol, 0.5 M aminocaproic acid, and 1/2 × import buffer (total; lane 2), and the soluble 200 g supernatant (super; lane 3) was subjected to immunoprecipitation with IgGs cross-linked to protein A–Sepharose as designated top (as in Materials and methods, except that recovered beads were washed with solubilization buffer, 0.5% digitonin). Bound (B) and unbound (U) proteins were adjusted to 1:1 stoichiometry with respect to the original thylakoid sample and analyzed by SDS-PAGE and fluorography. tp; an aliquot of translation product used for the assay.

Figure 5.

Cross-linking of ΔpH-dependent pathway components in intact thylakoids. Cross-linking experiments were conducted as described in Materials and methods. (A) Thylakoids were treated without (lanes 1 and 4) or with 0.5 mM (lanes 2 and 5) or 2.0 mM DSP (lanes 3 and 6). Thylakoid proteins were separated by SDS-PAGE and stained with Coomassie blue. Protein samples were not reduced (lanes 1–3) or reduced with β-mercaptoethanol (4–6) before SDS-PAGE. DSP concentrations are shown at the top of the figures. (B) Proteins were analyzed by nonreducing SDS-PAGE and immunoblotting with anti-cpTatC (lanes 1–3), anti-Hcf106 (lanes 4–6), or anti-Tha4 (lanes 7–9). (C) Cross-linked thylakoids were solubilized with SDS and subjected to immunoprecipitation with preimmune (lanes 1, 6, and 11), anti-cpTatC (lanes 2, 7, and 12), anti-Hcf106 (lanes 3, 8, and 13), anti-Tha4 (lanes 4, 9, and 14), or anti-cpOxa1p (lanes 5, 10, and 15). Bound proteins were eluted, reduced with β-mercaptoethanol, and analyzed by SDS-PAGE and immunoblotting. Antibodies used for the immunoprecipitations are designated (top); antibodies used for immunoblotting are designated (left).

Figure 6.

Chemical cross-linking of precursor-bound thylakoids. (A) In vitro translated DT17 was incubated with thylakoid membranes in a binding assay. The membranes were washed and treated with varying concentrations of DSP or DTSSP as described in Materials and methods. Concentrations of cross-linker (mM) are depicted at top. The membranes were then analyzed by SDS-PAGE and fluorography. (B) A 0.1-mM DSP-treated sample, equivalent to 0.33 mg total chlorophyll, was denatured with SDS and divided into six aliquots, five of which were subjected to immunoprecipitation under denaturing conditions with IgGs cross-linked to beads as designated above the panel. Immunoprecipitated samples were released with SDS and analyzed by SDS-PAGE and fluorography as described in Materials and methods. (C) A portion of each sample in B was treated with 2.5% mercaptoethanol to cleave the cross-linking agent and was then subjected to SDS-PAGE and fluorography. Each sample in B is equivalent to 6 μg chlorophyll, and each sample in C is equivalent to 4 μg chlorophyll of starting thylakoids.

Figure 7.

Precursor binding to the cpTatC–Hcf106 complex requires both the RR and the hydrophobic core of the signal peptide. In vitro translated proteins were incubated with thylakoids in binding assays. Recovered thylakoids were then analyzed by BN-PAGE and SDS-PAGE (Materials and methods). (A) The sequence of the DT signal peptide from the amino terminus to the thylakoidal processing protease cleavage site (arrow). The hydrophobic core is underlined. Mutations are shown as the substituted amino acids below the DT sequence and the resulting precursors designated as shown to the right. (B) Thylakoids from binding assays were solubilized with 1% digitonin, 20% glycerol, and import buffer and analyzed by BN-PAGE/fluorography. Chloroplast import of pcpTatC and pTha4 was conducted, and the recovered thylakoids were analyzed in adjacent lanes as markers for these components. The precursors used are designated (top). Each lane was loaded with sample equivalent to 8% of the assay, and gels were exposed to film for 7 d. (C) Aliquots of the translation products (tp) equivalent to 0.125% of that added to each assay, total solubilized thylakoids (T) equivalent to 3% of the assay, and 200,000 g supernatants (S; i.e., the BN-PAGE samples) equivalent to 3% of the assay were analyzed by SDS-PAGE and fluorography on identical gels in parallel, which were exposed to the same piece of film for 4 d. The precursors are designated (bottom).

Figure 8.

Precursors remain bound to the cpTatC–Hcf106 complex under transport conditions unless Tha4 is free to participate. Thylakoids were pretreated with 0.4 mg/ml of preimmune or anti-Tha4 IgGs, washed, and then incubated with in vitro–translated DT23 in a binding assay (b) or a binding and chase assay (c), or they were incubated with DT23 under transport conditions (t), i.e., 5 mM Mg-ATP, 5 mM dithiothreitol, ∼500 μg stromal protein, and light at 25°C for 20 min, in the absence or presence of 0.4 μM nigericin as shown (top). Chase and transport assays were diluted 4.5- and 10-fold, respectively, with import buffer before removal from light and 25°C. (A) Recovered thylakoids were solubilized in 1% digitonin, 20% glycerol, and import buffer and analyzed by BN-PAGE and fluorography. Each lane contains sample equivalent to 6% of the assay. The minor band migrating just below the ∼700-kD band is occasionally observed and presumably represents a breakdown product of the ∼700-kD complex. It appears enhanced in overexposed lanes (3 and 7) but is present in the other lanes and in immunoblots (Fig. 3). (B) Recovered thylakoids (A) were analyzed by SDS-PAGE and fluorography. Lanes contain sample equivalent to 5% of the assay. DT23 translation product, equivalent to 0.25% of that added to each assay, is shown in the lane marked tp. (Lanes 1 and 5) Binding assays; (lanes 2 and 6) chase assays; (lanes 3, 4, 7, and 8) transport assays.