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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.

Show MeSH
A twin arginine signal peptide induces the association of Tha4 with cpTatC–Hcf106. Reactions included 1.0 μM of the functional precursors (tOE23 and DT23), the nonfunctional precursor (KK-DT23), the Sec pathway precursor (pOE33), or the 33-residue synthetic signal peptide (tpOE17), as indicated in the figure, and were conducted in the light as described in Fig. 1. Antibodies used for the immunoprecipitation are designated to the right of each panel and antibodies used for immunoblotting are to the left. IP, immunoprecipitation.
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fig2: A twin arginine signal peptide induces the association of Tha4 with cpTatC–Hcf106. Reactions included 1.0 μM of the functional precursors (tOE23 and DT23), the nonfunctional precursor (KK-DT23), the Sec pathway precursor (pOE33), or the 33-residue synthetic signal peptide (tpOE17), as indicated in the figure, and were conducted in the light as described in Fig. 1. Antibodies used for the immunoprecipitation are designated to the right of each panel and antibodies used for immunoblotting are to the left. IP, immunoprecipitation.

Mentions: Thylakoid cross-linking assays were conducted with functional and nonfunctional precursors in the presence of the ΔpH in order to explore precursor requirements for Tha4 association with cpTatC–Hcf106 (Fig. 2). DT23 (lane 2) as well as tOE23 (lane 1) induced Tha4 association with cpTatC and Hcf106, as determined by reciprocal immunoprecipitations with antibodies to the three components. DT23 and tOE23 are both very efficient ΔpH/Tat pathway precursors (Henry et al., 1997). Neither KK-DT23, a nonfunctional precursor in which a twin lysine replaces the essential twin arginine (Cline and Mori, 2001), nor pOE33, a Sec pathway precursor protein induced Tha4 association (lanes 3 and 4). Remarkably, a 33-residue synthetic peptide (tpOE17) corresponding to the lumen-targeting signal peptide of maize OE17 precursor induced Tha4 interaction with cpTatC–Hcf106 (Fig. 2, lane 5). Interaction of tpOE17 with the translocation machinery was previously indicated by its ability to competitively inhibit protein transport on the ΔpH/Tat pathway (Teter and Theg, 1998). Taken together, these results suggest that binding of a twin arginine signal peptide and energizing the membrane with the proton gradient induce structural changes that promote Tha4 association with the cpTatC–Hcf106 receptor complex.


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)

A twin arginine signal peptide induces the association of Tha4 with cpTatC–Hcf106. Reactions included 1.0 μM of the functional precursors (tOE23 and DT23), the nonfunctional precursor (KK-DT23), the Sec pathway precursor (pOE33), or the 33-residue synthetic signal peptide (tpOE17), as indicated in the figure, and were conducted in the light as described in Fig. 1. Antibodies used for the immunoprecipitation are designated to the right of each panel and antibodies used for immunoblotting are to the left. IP, immunoprecipitation.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199252&req=5

fig2: A twin arginine signal peptide induces the association of Tha4 with cpTatC–Hcf106. Reactions included 1.0 μM of the functional precursors (tOE23 and DT23), the nonfunctional precursor (KK-DT23), the Sec pathway precursor (pOE33), or the 33-residue synthetic signal peptide (tpOE17), as indicated in the figure, and were conducted in the light as described in Fig. 1. Antibodies used for the immunoprecipitation are designated to the right of each panel and antibodies used for immunoblotting are to the left. IP, immunoprecipitation.
Mentions: Thylakoid cross-linking assays were conducted with functional and nonfunctional precursors in the presence of the ΔpH in order to explore precursor requirements for Tha4 association with cpTatC–Hcf106 (Fig. 2). DT23 (lane 2) as well as tOE23 (lane 1) induced Tha4 association with cpTatC and Hcf106, as determined by reciprocal immunoprecipitations with antibodies to the three components. DT23 and tOE23 are both very efficient ΔpH/Tat pathway precursors (Henry et al., 1997). Neither KK-DT23, a nonfunctional precursor in which a twin lysine replaces the essential twin arginine (Cline and Mori, 2001), nor pOE33, a Sec pathway precursor protein induced Tha4 association (lanes 3 and 4). Remarkably, a 33-residue synthetic peptide (tpOE17) corresponding to the lumen-targeting signal peptide of maize OE17 precursor induced Tha4 interaction with cpTatC–Hcf106 (Fig. 2, lane 5). Interaction of tpOE17 with the translocation machinery was previously indicated by its ability to competitively inhibit protein transport on the ΔpH/Tat pathway (Teter and Theg, 1998). Taken together, these results suggest that binding of a twin arginine signal peptide and energizing the membrane with the proton gradient induce structural changes that promote Tha4 association with the cpTatC–Hcf106 receptor complex.

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