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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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Blue native gel analyses of components of the ΔpH-dependent protein transport pathway. Pea thylakoids were solubilized with digitonin and subjected to BN-PAGE (Materials and methods). The final digitonin concentration (%) and the final thylakoid concentration (mg chlorophyll/ml) are designated (top). Proteins were detected by immunoblotting with anti-cpTatC (A), anti-Hcf106 (B), and anti-Tha4 (C). (D) Pea thylakoids were solubilized with 0.5% digitonin at 0.75 mg chlorophyll/ml. Proteins were immunodetected with anti-cpTatC (lanes 1 and 2), anti-Hcf106 (lanes 3 and 4), or anti-Tha4 (lanes 5 and 6) that was preincubated in the absence (lanes 1, 3, and 5) or presence (lane 2, 4, and 6) of the corresponding antigen. Molecular mass markers are ferritin (880 and 440 kD), β-amylase (200 kD), and bovine serum albumin (132 and 66 kD).
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fig2: Blue native gel analyses of components of the ΔpH-dependent protein transport pathway. Pea thylakoids were solubilized with digitonin and subjected to BN-PAGE (Materials and methods). The final digitonin concentration (%) and the final thylakoid concentration (mg chlorophyll/ml) are designated (top). Proteins were detected by immunoblotting with anti-cpTatC (A), anti-Hcf106 (B), and anti-Tha4 (C). (D) Pea thylakoids were solubilized with 0.5% digitonin at 0.75 mg chlorophyll/ml. Proteins were immunodetected with anti-cpTatC (lanes 1 and 2), anti-Hcf106 (lanes 3 and 4), or anti-Tha4 (lanes 5 and 6) that was preincubated in the absence (lanes 1, 3, and 5) or presence (lane 2, 4, and 6) of the corresponding antigen. Molecular mass markers are ferritin (880 and 440 kD), β-amylase (200 kD), and bovine serum albumin (132 and 66 kD).

Mentions: Among the possibilities suggested by the antibody inhibition studies is that Hcf106 and cpTatC are members of a complex that binds precursors. To examine the organization of ΔpH-dependent pathway components, detergent solubilized thylakoids were subjected to BN-PAGE and immunoblotting. BN-PAGE is a high-resolution method for fractionating membrane protein complexes and simultaneously determining their molecular weights (Schägger and von Jagow, 1991). Several detergents were examined as solubilizing agents. Triton X-100 and dodecyl-maltoside proved too harsh, yielding a complex pattern of immunoreactive bands that varied with the detergent concentration. Importantly, banding patterns among cpTatC, Hcf106, and Tha4 did not overlap, and there was no association of components as determined by coimmunoprecipitation studies (data not shown). Digitonin, a milder detergent used to isolate complexes of protein translocation components (Görlich and Rapoport, 1993; Künkele et al., 1998), was ultimately used as it produced a simple and reproducible banding pattern (Fig. 2) .


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

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

Blue native gel analyses of components of the ΔpH-dependent protein transport pathway. Pea thylakoids were solubilized with digitonin and subjected to BN-PAGE (Materials and methods). The final digitonin concentration (%) and the final thylakoid concentration (mg chlorophyll/ml) are designated (top). Proteins were detected by immunoblotting with anti-cpTatC (A), anti-Hcf106 (B), and anti-Tha4 (C). (D) Pea thylakoids were solubilized with 0.5% digitonin at 0.75 mg chlorophyll/ml. Proteins were immunodetected with anti-cpTatC (lanes 1 and 2), anti-Hcf106 (lanes 3 and 4), or anti-Tha4 (lanes 5 and 6) that was preincubated in the absence (lanes 1, 3, and 5) or presence (lane 2, 4, and 6) of the corresponding antigen. Molecular mass markers are ferritin (880 and 440 kD), β-amylase (200 kD), and bovine serum albumin (132 and 66 kD).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196467&req=5

fig2: Blue native gel analyses of components of the ΔpH-dependent protein transport pathway. Pea thylakoids were solubilized with digitonin and subjected to BN-PAGE (Materials and methods). The final digitonin concentration (%) and the final thylakoid concentration (mg chlorophyll/ml) are designated (top). Proteins were detected by immunoblotting with anti-cpTatC (A), anti-Hcf106 (B), and anti-Tha4 (C). (D) Pea thylakoids were solubilized with 0.5% digitonin at 0.75 mg chlorophyll/ml. Proteins were immunodetected with anti-cpTatC (lanes 1 and 2), anti-Hcf106 (lanes 3 and 4), or anti-Tha4 (lanes 5 and 6) that was preincubated in the absence (lanes 1, 3, and 5) or presence (lane 2, 4, and 6) of the corresponding antigen. Molecular mass markers are ferritin (880 and 440 kD), β-amylase (200 kD), and bovine serum albumin (132 and 66 kD).
Mentions: Among the possibilities suggested by the antibody inhibition studies is that Hcf106 and cpTatC are members of a complex that binds precursors. To examine the organization of ΔpH-dependent pathway components, detergent solubilized thylakoids were subjected to BN-PAGE and immunoblotting. BN-PAGE is a high-resolution method for fractionating membrane protein complexes and simultaneously determining their molecular weights (Schägger and von Jagow, 1991). Several detergents were examined as solubilizing agents. Triton X-100 and dodecyl-maltoside proved too harsh, yielding a complex pattern of immunoreactive bands that varied with the detergent concentration. Importantly, banding patterns among cpTatC, Hcf106, and Tha4 did not overlap, and there was no association of components as determined by coimmunoprecipitation studies (data not shown). Digitonin, a milder detergent used to isolate complexes of protein translocation components (Görlich and Rapoport, 1993; Künkele et al., 1998), was ultimately used as it produced a simple and reproducible banding pattern (Fig. 2) .

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