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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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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).
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fig3: 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).

Mentions: This tentative conclusion was confirmed by coimmunoprecipitation analysis performed with digitonin-solubilized thylakoids under nondenaturing conditions (Fig. 3) . All of the cpTatC was immunoprecipitated by either anti-cpTatC (lane 6) or anti-Hcf106 (lane 8). Hcf106 was coimmunoprecipitated with anti-cpTatC (lane 6), but a portion of the Hcf106 was found in the unbound fraction (lane 5). Tha4 was not coimmunoprecipitated with either anti-cpTatC or anti-Hcf106 (lanes 6 and 8). Likewise, anti-Tha4 immunoprecipitated only Tha4 (lane 10). As negative controls for this experiment, immunoprecipitation was conducted with antibodies to cpSecY (lanes 11 and 12) and to cpOxa1p (lanes 13 and 14), components of the thylakoid Sec and SRP translocation pathways, respectively (Mori et al., 1999; Moore et al., 2000). These antibodies did not coimmunoprecipitate any ΔpH-dependent pathway component, ruling out nonspecific associations. These results demonstrate that cpTatC and a portion of the Hcf106 form a stable ∼700-kD complex. They further suggest that a portion of Hcf106 and all of Tha4 are present in separate pools in the membrane.


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

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

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).
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Related In: Results  -  Collection

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fig3: 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).
Mentions: This tentative conclusion was confirmed by coimmunoprecipitation analysis performed with digitonin-solubilized thylakoids under nondenaturing conditions (Fig. 3) . All of the cpTatC was immunoprecipitated by either anti-cpTatC (lane 6) or anti-Hcf106 (lane 8). Hcf106 was coimmunoprecipitated with anti-cpTatC (lane 6), but a portion of the Hcf106 was found in the unbound fraction (lane 5). Tha4 was not coimmunoprecipitated with either anti-cpTatC or anti-Hcf106 (lanes 6 and 8). Likewise, anti-Tha4 immunoprecipitated only Tha4 (lane 10). As negative controls for this experiment, immunoprecipitation was conducted with antibodies to cpSecY (lanes 11 and 12) and to cpOxa1p (lanes 13 and 14), components of the thylakoid Sec and SRP translocation pathways, respectively (Mori et al., 1999; Moore et al., 2000). These antibodies did not coimmunoprecipitate any ΔpH-dependent pathway component, ruling out nonspecific associations. These results demonstrate that cpTatC and a portion of the Hcf106 form a stable ∼700-kD complex. They further suggest that a portion of Hcf106 and all of Tha4 are present in separate pools in the membrane.

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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