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A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid [Delta]pH/Tat translocase.

Mori H, Cline K - J. Cell Biol. (2002)

Bottom Line: In contrast, Tha4 was only associated with cpTatC and Hcf106 in the presence of a functional precursor and the DeltapH.Such an assembly-disassembly cycle could explain how the DeltapH/Tat system can assemble translocases to accommodate folded proteins of varied size.It also explains in part how the system can exist in the membrane without compromising its ion and proton permeability barrier.

View Article: PubMed Central - PubMed

Affiliation: Horticultural Sciences and Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL 32611, USA. Britta.J.Eickholt@kcl.ac.uk

ABSTRACT
The thylakoid DeltapH-dependent/Tat pathway is a novel system with the remarkable ability to transport tightly folded precursor proteins using a transmembrane DeltapH as the sole energy source. Three known components of the transport machinery exist in two distinct subcomplexes. A cpTatC-Hcf106 complex serves as precursor receptor and a Tha4 complex is required after precursor recognition. Here we report that Tha4 assembles with cpTatC-Hcf106 during the translocation step. Interactions among components were examined by chemical cross-linking of intact thylakoids followed by immunoprecipitation and immunoblotting. cpTatC and Hcf106 were consistently associated under all conditions tested. In contrast, Tha4 was only associated with cpTatC and Hcf106 in the presence of a functional precursor and the DeltapH. Interestingly, a synthetic signal peptide could replace intact precursor in triggering assembly. The association of all three components was transient and dissipated upon the completion of protein translocation. Such an assembly-disassembly cycle could explain how the DeltapH/Tat system can assemble translocases to accommodate folded proteins of varied size. It also explains in part how the system can exist in the membrane without compromising its ion and proton permeability barrier.

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Tha4 associates with cpTatC and Hcf106 under transport conditions to form cpTatC–Hcf106–Tha4 complex. Thylakoids were incubated in the light to generate a pH gradient. Where indicated, reactions were supplemented with 0.5/1.0 μM nigericin/valinomycin (Nig/Val) to dissipate the ΔpH and/or with 1.0 μM of tOE23. Cross-linking reactions were performed with 1 mM DSP final concentration as in the Materials and methods. Cross-linked thylakoids were recovered by centrifugation, dissolved with SDS, and subjected to immunoprecipitation with anti-cpTatC (A), anti-Hcf106 (B), anti-Tha4 (C), or anti-cpOxa1p (D). Immunoprecipitated proteins, after release from cross-linked partners with β-mercaptoethanol, were analyzed by SDS-PAGE and immunoblotting. Antibodies used for immunoblotting are designated just to the left of immunoblots. Immunoprecipitates equivalent to 10 μg Chl thylakoids were loaded (lanes 1–4). A thylakoid control (lane T) contained 2.5 μg Chl of untreated thylakoids, except for the OE23 immunoblots, which contained 0.1 μg Chl. (E) Cross-linked and solubilized thylakoid proteins were first subjected to immunoprecipitation with anti-cpTatC. The resulting immunoprecipitates were eluted by incubating the antigen–IgG–protein A–Sepharose complexes with 4 M urea, 2% SDS, 125 mM Tris-HCl, pH 6.8, for 1 h at room temperature. Eluates, diluted eightfold with immunoprecipitation buffer lacking SDS, were then subjected to a second immunoprecipitation with anti-Hcf106. Immunoprecipitates were then analyzed by immunoblotting, as in A–D.
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fig1: Tha4 associates with cpTatC and Hcf106 under transport conditions to form cpTatC–Hcf106–Tha4 complex. Thylakoids were incubated in the light to generate a pH gradient. Where indicated, reactions were supplemented with 0.5/1.0 μM nigericin/valinomycin (Nig/Val) to dissipate the ΔpH and/or with 1.0 μM of tOE23. Cross-linking reactions were performed with 1 mM DSP final concentration as in the Materials and methods. Cross-linked thylakoids were recovered by centrifugation, dissolved with SDS, and subjected to immunoprecipitation with anti-cpTatC (A), anti-Hcf106 (B), anti-Tha4 (C), or anti-cpOxa1p (D). Immunoprecipitated proteins, after release from cross-linked partners with β-mercaptoethanol, were analyzed by SDS-PAGE and immunoblotting. Antibodies used for immunoblotting are designated just to the left of immunoblots. Immunoprecipitates equivalent to 10 μg Chl thylakoids were loaded (lanes 1–4). A thylakoid control (lane T) contained 2.5 μg Chl of untreated thylakoids, except for the OE23 immunoblots, which contained 0.1 μg Chl. (E) Cross-linked and solubilized thylakoid proteins were first subjected to immunoprecipitation with anti-cpTatC. The resulting immunoprecipitates were eluted by incubating the antigen–IgG–protein A–Sepharose complexes with 4 M urea, 2% SDS, 125 mM Tris-HCl, pH 6.8, for 1 h at room temperature. Eluates, diluted eightfold with immunoprecipitation buffer lacking SDS, were then subjected to a second immunoprecipitation with anti-Hcf106. Immunoprecipitates were then analyzed by immunoblotting, as in A–D.

Mentions: To examine transient associations that might exist during protein transport, we used a chemical cross-linking approach with intact thylakoids (Fig. 1). Thylakoids were treated with the thiol-cleavable homobifunctional cross-linker dithiobis (succinimidyl propionate) (DSP) under a variety of conditions related to protein transport. After quenching of the reaction, thylakoids were solubilized and denatured with SDS and then subjected to immunoprecipitation with specific antibodies. Bound proteins were analyzed by SDS-PAGE and immunoblotting after cross-linker cleavage to release partners.


A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid [Delta]pH/Tat translocase.

Mori H, Cline K - J. Cell Biol. (2002)

Tha4 associates with cpTatC and Hcf106 under transport conditions to form cpTatC–Hcf106–Tha4 complex. Thylakoids were incubated in the light to generate a pH gradient. Where indicated, reactions were supplemented with 0.5/1.0 μM nigericin/valinomycin (Nig/Val) to dissipate the ΔpH and/or with 1.0 μM of tOE23. Cross-linking reactions were performed with 1 mM DSP final concentration as in the Materials and methods. Cross-linked thylakoids were recovered by centrifugation, dissolved with SDS, and subjected to immunoprecipitation with anti-cpTatC (A), anti-Hcf106 (B), anti-Tha4 (C), or anti-cpOxa1p (D). Immunoprecipitated proteins, after release from cross-linked partners with β-mercaptoethanol, were analyzed by SDS-PAGE and immunoblotting. Antibodies used for immunoblotting are designated just to the left of immunoblots. Immunoprecipitates equivalent to 10 μg Chl thylakoids were loaded (lanes 1–4). A thylakoid control (lane T) contained 2.5 μg Chl of untreated thylakoids, except for the OE23 immunoblots, which contained 0.1 μg Chl. (E) Cross-linked and solubilized thylakoid proteins were first subjected to immunoprecipitation with anti-cpTatC. The resulting immunoprecipitates were eluted by incubating the antigen–IgG–protein A–Sepharose complexes with 4 M urea, 2% SDS, 125 mM Tris-HCl, pH 6.8, for 1 h at room temperature. Eluates, diluted eightfold with immunoprecipitation buffer lacking SDS, were then subjected to a second immunoprecipitation with anti-Hcf106. Immunoprecipitates were then analyzed by immunoblotting, as in A–D.
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fig1: Tha4 associates with cpTatC and Hcf106 under transport conditions to form cpTatC–Hcf106–Tha4 complex. Thylakoids were incubated in the light to generate a pH gradient. Where indicated, reactions were supplemented with 0.5/1.0 μM nigericin/valinomycin (Nig/Val) to dissipate the ΔpH and/or with 1.0 μM of tOE23. Cross-linking reactions were performed with 1 mM DSP final concentration as in the Materials and methods. Cross-linked thylakoids were recovered by centrifugation, dissolved with SDS, and subjected to immunoprecipitation with anti-cpTatC (A), anti-Hcf106 (B), anti-Tha4 (C), or anti-cpOxa1p (D). Immunoprecipitated proteins, after release from cross-linked partners with β-mercaptoethanol, were analyzed by SDS-PAGE and immunoblotting. Antibodies used for immunoblotting are designated just to the left of immunoblots. Immunoprecipitates equivalent to 10 μg Chl thylakoids were loaded (lanes 1–4). A thylakoid control (lane T) contained 2.5 μg Chl of untreated thylakoids, except for the OE23 immunoblots, which contained 0.1 μg Chl. (E) Cross-linked and solubilized thylakoid proteins were first subjected to immunoprecipitation with anti-cpTatC. The resulting immunoprecipitates were eluted by incubating the antigen–IgG–protein A–Sepharose complexes with 4 M urea, 2% SDS, 125 mM Tris-HCl, pH 6.8, for 1 h at room temperature. Eluates, diluted eightfold with immunoprecipitation buffer lacking SDS, were then subjected to a second immunoprecipitation with anti-Hcf106. Immunoprecipitates were then analyzed by immunoblotting, as in A–D.
Mentions: To examine transient associations that might exist during protein transport, we used a chemical cross-linking approach with intact thylakoids (Fig. 1). Thylakoids were treated with the thiol-cleavable homobifunctional cross-linker dithiobis (succinimidyl propionate) (DSP) under a variety of conditions related to protein transport. After quenching of the reaction, thylakoids were solubilized and denatured with SDS and then subjected to immunoprecipitation with specific antibodies. Bound proteins were analyzed by SDS-PAGE and immunoblotting after cross-linker cleavage to release partners.

Bottom Line: In contrast, Tha4 was only associated with cpTatC and Hcf106 in the presence of a functional precursor and the DeltapH.Such an assembly-disassembly cycle could explain how the DeltapH/Tat system can assemble translocases to accommodate folded proteins of varied size.It also explains in part how the system can exist in the membrane without compromising its ion and proton permeability barrier.

View Article: PubMed Central - PubMed

Affiliation: Horticultural Sciences and Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL 32611, USA. Britta.J.Eickholt@kcl.ac.uk

ABSTRACT
The thylakoid DeltapH-dependent/Tat pathway is a novel system with the remarkable ability to transport tightly folded precursor proteins using a transmembrane DeltapH as the sole energy source. Three known components of the transport machinery exist in two distinct subcomplexes. A cpTatC-Hcf106 complex serves as precursor receptor and a Tha4 complex is required after precursor recognition. Here we report that Tha4 assembles with cpTatC-Hcf106 during the translocation step. Interactions among components were examined by chemical cross-linking of intact thylakoids followed by immunoprecipitation and immunoblotting. cpTatC and Hcf106 were consistently associated under all conditions tested. In contrast, Tha4 was only associated with cpTatC and Hcf106 in the presence of a functional precursor and the DeltapH. Interestingly, a synthetic signal peptide could replace intact precursor in triggering assembly. The association of all three components was transient and dissipated upon the completion of protein translocation. Such an assembly-disassembly cycle could explain how the DeltapH/Tat system can assemble translocases to accommodate folded proteins of varied size. It also explains in part how the system can exist in the membrane without compromising its ion and proton permeability barrier.

Show MeSH