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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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Precursor binding to the cpTatC–Hcf106 complex requires both the RR and the hydrophobic core of the signal peptide. In vitro translated proteins were incubated with thylakoids in binding assays. Recovered thylakoids were then analyzed by BN-PAGE and SDS-PAGE (Materials and methods). (A) The sequence of the DT signal peptide from the amino terminus to the thylakoidal processing protease cleavage site (arrow). The hydrophobic core is underlined. Mutations are shown as the substituted amino acids below the DT sequence and the resulting precursors designated as shown to the right. (B) Thylakoids from binding assays were solubilized with 1% digitonin, 20% glycerol, and import buffer and analyzed by BN-PAGE/fluorography. Chloroplast import of pcpTatC and pTha4 was conducted, and the recovered thylakoids were analyzed in adjacent lanes as markers for these components. The precursors used are designated (top). Each lane was loaded with sample equivalent to 8% of the assay, and gels were exposed to film for 7 d. (C) Aliquots of the translation products (tp) equivalent to 0.125% of that added to each assay, total solubilized thylakoids (T) equivalent to 3% of the assay, and 200,000 g supernatants (S; i.e., the BN-PAGE samples) equivalent to 3% of the assay were analyzed by SDS-PAGE and fluorography on identical gels in parallel, which were exposed to the same piece of film for 4 d. The precursors are designated (bottom).
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fig7: Precursor binding to the cpTatC–Hcf106 complex requires both the RR and the hydrophobic core of the signal peptide. In vitro translated proteins were incubated with thylakoids in binding assays. Recovered thylakoids were then analyzed by BN-PAGE and SDS-PAGE (Materials and methods). (A) The sequence of the DT signal peptide from the amino terminus to the thylakoidal processing protease cleavage site (arrow). The hydrophobic core is underlined. Mutations are shown as the substituted amino acids below the DT sequence and the resulting precursors designated as shown to the right. (B) Thylakoids from binding assays were solubilized with 1% digitonin, 20% glycerol, and import buffer and analyzed by BN-PAGE/fluorography. Chloroplast import of pcpTatC and pTha4 was conducted, and the recovered thylakoids were analyzed in adjacent lanes as markers for these components. The precursors used are designated (top). Each lane was loaded with sample equivalent to 8% of the assay, and gels were exposed to film for 7 d. (C) Aliquots of the translation products (tp) equivalent to 0.125% of that added to each assay, total solubilized thylakoids (T) equivalent to 3% of the assay, and 200,000 g supernatants (S; i.e., the BN-PAGE samples) equivalent to 3% of the assay were analyzed by SDS-PAGE and fluorography on identical gels in parallel, which were exposed to the same piece of film for 4 d. The precursors are designated (bottom).

Mentions: The above studies analyzed the nature of the precursor-binding site. The features of the precursors important for the interaction were determined by analyzing binding of precursors with altered signal peptides. Recovered thylakoids were subjected to BN-PAGE to assess the amount of precursor associated with the ∼700-kD complex (Fig. 7 B) and by SDS-PAGE to monitor the amount of precursor that associated with thylakoids (Fig. 7 C). As expected, deletion of the signal peptide eliminated binding to the cpTatC–Hcf106 complex. Although tOE17 showed significant binding to the ∼700-kD complex (Fig. 7 B, lane 9), mOE17 was not associated with the complex (lane 8). Similarly, the intermediate precursor iPftf also bound to the cpTatC–Hcf106 complex (lane 6); Pftf without its signal peptide (mPftf) did not (lane 7). iPftf is a membrane protein with an RR-containing signal peptide that is integrated into thylakoids by the ΔpH-dependent pathway (Summer et al., 2000).


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

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

Precursor binding to the cpTatC–Hcf106 complex requires both the RR and the hydrophobic core of the signal peptide. In vitro translated proteins were incubated with thylakoids in binding assays. Recovered thylakoids were then analyzed by BN-PAGE and SDS-PAGE (Materials and methods). (A) The sequence of the DT signal peptide from the amino terminus to the thylakoidal processing protease cleavage site (arrow). The hydrophobic core is underlined. Mutations are shown as the substituted amino acids below the DT sequence and the resulting precursors designated as shown to the right. (B) Thylakoids from binding assays were solubilized with 1% digitonin, 20% glycerol, and import buffer and analyzed by BN-PAGE/fluorography. Chloroplast import of pcpTatC and pTha4 was conducted, and the recovered thylakoids were analyzed in adjacent lanes as markers for these components. The precursors used are designated (top). Each lane was loaded with sample equivalent to 8% of the assay, and gels were exposed to film for 7 d. (C) Aliquots of the translation products (tp) equivalent to 0.125% of that added to each assay, total solubilized thylakoids (T) equivalent to 3% of the assay, and 200,000 g supernatants (S; i.e., the BN-PAGE samples) equivalent to 3% of the assay were analyzed by SDS-PAGE and fluorography on identical gels in parallel, which were exposed to the same piece of film for 4 d. The precursors are designated (bottom).
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fig7: Precursor binding to the cpTatC–Hcf106 complex requires both the RR and the hydrophobic core of the signal peptide. In vitro translated proteins were incubated with thylakoids in binding assays. Recovered thylakoids were then analyzed by BN-PAGE and SDS-PAGE (Materials and methods). (A) The sequence of the DT signal peptide from the amino terminus to the thylakoidal processing protease cleavage site (arrow). The hydrophobic core is underlined. Mutations are shown as the substituted amino acids below the DT sequence and the resulting precursors designated as shown to the right. (B) Thylakoids from binding assays were solubilized with 1% digitonin, 20% glycerol, and import buffer and analyzed by BN-PAGE/fluorography. Chloroplast import of pcpTatC and pTha4 was conducted, and the recovered thylakoids were analyzed in adjacent lanes as markers for these components. The precursors used are designated (top). Each lane was loaded with sample equivalent to 8% of the assay, and gels were exposed to film for 7 d. (C) Aliquots of the translation products (tp) equivalent to 0.125% of that added to each assay, total solubilized thylakoids (T) equivalent to 3% of the assay, and 200,000 g supernatants (S; i.e., the BN-PAGE samples) equivalent to 3% of the assay were analyzed by SDS-PAGE and fluorography on identical gels in parallel, which were exposed to the same piece of film for 4 d. The precursors are designated (bottom).
Mentions: The above studies analyzed the nature of the precursor-binding site. The features of the precursors important for the interaction were determined by analyzing binding of precursors with altered signal peptides. Recovered thylakoids were subjected to BN-PAGE to assess the amount of precursor associated with the ∼700-kD complex (Fig. 7 B) and by SDS-PAGE to monitor the amount of precursor that associated with thylakoids (Fig. 7 C). As expected, deletion of the signal peptide eliminated binding to the cpTatC–Hcf106 complex. Although tOE17 showed significant binding to the ∼700-kD complex (Fig. 7 B, lane 9), mOE17 was not associated with the complex (lane 8). Similarly, the intermediate precursor iPftf also bound to the cpTatC–Hcf106 complex (lane 6); Pftf without its signal peptide (mPftf) did not (lane 7). iPftf is a membrane protein with an RR-containing signal peptide that is integrated into thylakoids by the ΔpH-dependent pathway (Summer et al., 2000).

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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