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

Cline K, Mori H - J. Cell Biol. (2001)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Horticultural Sciences and Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL 32611, USA. kcline@ufl.edu

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.

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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.
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fig1: 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.

Mentions: 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 ΔpH-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.


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

Cline K, Mori H - J. Cell Biol. (2001)

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.
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Related In: Results  -  Collection

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fig1: 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.
Mentions: 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 ΔpH-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.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Horticultural Sciences and Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL 32611, USA. kcline@ufl.edu

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.

Show MeSH
Related in: MedlinePlus