Limits...
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
Tha4 dissociates from cpTatC–Hcf106 upon completion of translocation. Thylakoids were preincubated with radiolabeled tOE17 in the dark on ice for 15 min as previously described (Ma and Cline, 2000). After washing and resuspension in transport buffer at 0.33 mg Chl/ml, transport from the bound state was initiated by transfer of the thylakoids to a 25°C illuminated water bath. After a 15-min transport reaction, radiolabeled tOE23 was added to the reaction. 0.5 μM nigericin was added to the reactions shown in lanes 1 and 9 at the same time as transfer to light (lane 1) or addition of tOE23 (lane 9). Thylakoids were withdrawn at the indicated time points, recovered by centrifugation, and analyzed by SDS-PAGE/fluorography. Chemical cross-linking was performed on duplicate aliquots at the indicated time points with 0.75 mM DSP for 5 min followed by 5 min quenching with 50 mM Tris-HCl, pH 8.0. SDS-solubilized thylakoids were subjected to immunoprecipitation with anti-Hcf106, and immunoprecipitates were analyzed by immunoblotting with anti-Tha4. The reaction scheme is shown in A. (B) Immunoblot of anti-Hcf106 immunoprecipitates with anti-Tha4 and fluorograms of samples depicting tOE17 and tOE23 transport. The positions of precursor and mature forms are designated by p and m, respectively, on the left side of the blots. A thylakoid control (lane T) contained 2.5 μg Chl of untreated thylakoids. (C) Quantification of Tha4, tOE17, and mOE17 shown in B, lanes 2–7. The density of scanned Tha4 bands on X-ray film was determined using AlphaEase software (Alpha Innotech Corp.). tOE17 and mOE17 were quantified by scintillation counting of excised gel bands and adjusted for the different leucine contents of tOE17 and mOE17. Amounts of mOE17 were corrected by subtracting the amount of mOE17 present at zero time. The amounts displayed are relative quantities; the amount of tOE17 in lane 2 and the amount of Tha4 in lane 4 were arbitrarily set to 100%.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199252&req=5

fig3: Tha4 dissociates from cpTatC–Hcf106 upon completion of translocation. Thylakoids were preincubated with radiolabeled tOE17 in the dark on ice for 15 min as previously described (Ma and Cline, 2000). After washing and resuspension in transport buffer at 0.33 mg Chl/ml, transport from the bound state was initiated by transfer of the thylakoids to a 25°C illuminated water bath. After a 15-min transport reaction, radiolabeled tOE23 was added to the reaction. 0.5 μM nigericin was added to the reactions shown in lanes 1 and 9 at the same time as transfer to light (lane 1) or addition of tOE23 (lane 9). Thylakoids were withdrawn at the indicated time points, recovered by centrifugation, and analyzed by SDS-PAGE/fluorography. Chemical cross-linking was performed on duplicate aliquots at the indicated time points with 0.75 mM DSP for 5 min followed by 5 min quenching with 50 mM Tris-HCl, pH 8.0. SDS-solubilized thylakoids were subjected to immunoprecipitation with anti-Hcf106, and immunoprecipitates were analyzed by immunoblotting with anti-Tha4. The reaction scheme is shown in A. (B) Immunoblot of anti-Hcf106 immunoprecipitates with anti-Tha4 and fluorograms of samples depicting tOE17 and tOE23 transport. The positions of precursor and mature forms are designated by p and m, respectively, on the left side of the blots. A thylakoid control (lane T) contained 2.5 μg Chl of untreated thylakoids. (C) Quantification of Tha4, tOE17, and mOE17 shown in B, lanes 2–7. The density of scanned Tha4 bands on X-ray film was determined using AlphaEase software (Alpha Innotech Corp.). tOE17 and mOE17 were quantified by scintillation counting of excised gel bands and adjusted for the different leucine contents of tOE17 and mOE17. Amounts of mOE17 were corrected by subtracting the amount of mOE17 present at zero time. The amounts displayed are relative quantities; the amount of tOE17 in lane 2 and the amount of Tha4 in lane 4 were arbitrarily set to 100%.

Mentions: The above results imply that Tha4 dissociates from the cpTatC–Hcf106 complex upon completion of translocation. To directly test this possibility, we initiated protein transport with precursor-bound thylakoids and simultaneously monitored transport and component association as a function of time (Fig. 3 A for experimental schedule). For this experiment, we employed tOE17, a ΔpH/Tat pathway substrate that stably binds to the cpTatC–Hcf106 complex (Cline and Mori, 2001). Prebound tOE17 was transported to the lumen during the first 10 min of the transport reaction, as assessed by processing of precursor to mature form. After 10 min, no further transport occurred (Fig. 3, B and C). Association of components, assessed by immunoprecipitation with anti-Hcf106 and immunoblotting with anti-Tha4, rose to a peak at 2 min and was no longer detected after 10 min (Fig. 3). Addition of fresh tOE23 at 15 min resulted in restoration of protein transport and reassociation of Tha4 with Hcf106 (Fig. 3 B), indicating that Tha4 dissociation was not due to inactivation of the machinery or dissipation of the ΔpH. In a similar experiment, we verified that Tha4 also dissociated from cpTatC upon substrate depletion, but cpTatC and Hcf106 remained associated (unpublished data). These results demonstrate that Tha4 disassociates from the cpTatC–Hcf106 complex upon the completion of protein translocation, even in the presence of the ΔpH, and that the components then appear to return to their pretransport organization (Cline and Mori, 2001).


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 dissociates from cpTatC–Hcf106 upon completion of translocation. Thylakoids were preincubated with radiolabeled tOE17 in the dark on ice for 15 min as previously described (Ma and Cline, 2000). After washing and resuspension in transport buffer at 0.33 mg Chl/ml, transport from the bound state was initiated by transfer of the thylakoids to a 25°C illuminated water bath. After a 15-min transport reaction, radiolabeled tOE23 was added to the reaction. 0.5 μM nigericin was added to the reactions shown in lanes 1 and 9 at the same time as transfer to light (lane 1) or addition of tOE23 (lane 9). Thylakoids were withdrawn at the indicated time points, recovered by centrifugation, and analyzed by SDS-PAGE/fluorography. Chemical cross-linking was performed on duplicate aliquots at the indicated time points with 0.75 mM DSP for 5 min followed by 5 min quenching with 50 mM Tris-HCl, pH 8.0. SDS-solubilized thylakoids were subjected to immunoprecipitation with anti-Hcf106, and immunoprecipitates were analyzed by immunoblotting with anti-Tha4. The reaction scheme is shown in A. (B) Immunoblot of anti-Hcf106 immunoprecipitates with anti-Tha4 and fluorograms of samples depicting tOE17 and tOE23 transport. The positions of precursor and mature forms are designated by p and m, respectively, on the left side of the blots. A thylakoid control (lane T) contained 2.5 μg Chl of untreated thylakoids. (C) Quantification of Tha4, tOE17, and mOE17 shown in B, lanes 2–7. The density of scanned Tha4 bands on X-ray film was determined using AlphaEase software (Alpha Innotech Corp.). tOE17 and mOE17 were quantified by scintillation counting of excised gel bands and adjusted for the different leucine contents of tOE17 and mOE17. Amounts of mOE17 were corrected by subtracting the amount of mOE17 present at zero time. The amounts displayed are relative quantities; the amount of tOE17 in lane 2 and the amount of Tha4 in lane 4 were arbitrarily set to 100%.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Tha4 dissociates from cpTatC–Hcf106 upon completion of translocation. Thylakoids were preincubated with radiolabeled tOE17 in the dark on ice for 15 min as previously described (Ma and Cline, 2000). After washing and resuspension in transport buffer at 0.33 mg Chl/ml, transport from the bound state was initiated by transfer of the thylakoids to a 25°C illuminated water bath. After a 15-min transport reaction, radiolabeled tOE23 was added to the reaction. 0.5 μM nigericin was added to the reactions shown in lanes 1 and 9 at the same time as transfer to light (lane 1) or addition of tOE23 (lane 9). Thylakoids were withdrawn at the indicated time points, recovered by centrifugation, and analyzed by SDS-PAGE/fluorography. Chemical cross-linking was performed on duplicate aliquots at the indicated time points with 0.75 mM DSP for 5 min followed by 5 min quenching with 50 mM Tris-HCl, pH 8.0. SDS-solubilized thylakoids were subjected to immunoprecipitation with anti-Hcf106, and immunoprecipitates were analyzed by immunoblotting with anti-Tha4. The reaction scheme is shown in A. (B) Immunoblot of anti-Hcf106 immunoprecipitates with anti-Tha4 and fluorograms of samples depicting tOE17 and tOE23 transport. The positions of precursor and mature forms are designated by p and m, respectively, on the left side of the blots. A thylakoid control (lane T) contained 2.5 μg Chl of untreated thylakoids. (C) Quantification of Tha4, tOE17, and mOE17 shown in B, lanes 2–7. The density of scanned Tha4 bands on X-ray film was determined using AlphaEase software (Alpha Innotech Corp.). tOE17 and mOE17 were quantified by scintillation counting of excised gel bands and adjusted for the different leucine contents of tOE17 and mOE17. Amounts of mOE17 were corrected by subtracting the amount of mOE17 present at zero time. The amounts displayed are relative quantities; the amount of tOE17 in lane 2 and the amount of Tha4 in lane 4 were arbitrarily set to 100%.
Mentions: The above results imply that Tha4 dissociates from the cpTatC–Hcf106 complex upon completion of translocation. To directly test this possibility, we initiated protein transport with precursor-bound thylakoids and simultaneously monitored transport and component association as a function of time (Fig. 3 A for experimental schedule). For this experiment, we employed tOE17, a ΔpH/Tat pathway substrate that stably binds to the cpTatC–Hcf106 complex (Cline and Mori, 2001). Prebound tOE17 was transported to the lumen during the first 10 min of the transport reaction, as assessed by processing of precursor to mature form. After 10 min, no further transport occurred (Fig. 3, B and C). Association of components, assessed by immunoprecipitation with anti-Hcf106 and immunoblotting with anti-Tha4, rose to a peak at 2 min and was no longer detected after 10 min (Fig. 3). Addition of fresh tOE23 at 15 min resulted in restoration of protein transport and reassociation of Tha4 with Hcf106 (Fig. 3 B), indicating that Tha4 dissociation was not due to inactivation of the machinery or dissipation of the ΔpH. In a similar experiment, we verified that Tha4 also dissociated from cpTatC upon substrate depletion, but cpTatC and Hcf106 remained associated (unpublished data). These results demonstrate that Tha4 disassociates from the cpTatC–Hcf106 complex upon the completion of protein translocation, even in the presence of the ΔpH, and that the components then appear to return to their pretransport organization (Cline and Mori, 2001).

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