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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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Precursors bind to the ∼700-kD cpTatC and Hcf106 complex. (A) Pea thylakoids were assayed for binding (b; lanes 2 and 4) or binding and then transport chase (c; lanes 3 and 5) with in vitro translated tOE17 and DT23. Recovered thylakoids were solubilized with 1% digitonin, 20% glycerol, and import buffer and analyzed by BN-PAGE and fluorography (see Materials and methods). Radiolabled Hcf106 from an import assay (see text) was used as a marker for the cpTatC–Hcf106 complex (lane 1). (B) Recovered thylakoids (A) were also subjected to SDS-PAGE and fluorography. The precursors used for the assays (tp) are in lanes 1 and 4. (C) BN-PAGE lanes of bound DT23 and bound tOE17 were subjected to second dimension SDS-PAGE (Materials and methods). An aliquot of the translation product mixed with prestained molecular weight markers was run on the left side of the SDS-PAGE gels. (D) Recovered thylakoids from binding assays with in vitro translated tOE17 or DT23 were solubilized with 1% digitonin, 20% glycerol, 0.5 M aminocaproic acid, and 1/2 × import buffer (total; lane 2), and the soluble 200 g supernatant (super; lane 3) was subjected to immunoprecipitation with IgGs cross-linked to protein A–Sepharose as designated top (as in Materials and methods, except that recovered beads were washed with solubilization buffer, 0.5% digitonin). Bound (B) and unbound (U) proteins were adjusted to 1:1 stoichiometry with respect to the original thylakoid sample and analyzed by SDS-PAGE and fluorography. tp; an aliquot of translation product used for the assay.
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fig4: Precursors bind to the ∼700-kD cpTatC and Hcf106 complex. (A) Pea thylakoids were assayed for binding (b; lanes 2 and 4) or binding and then transport chase (c; lanes 3 and 5) with in vitro translated tOE17 and DT23. Recovered thylakoids were solubilized with 1% digitonin, 20% glycerol, and import buffer and analyzed by BN-PAGE and fluorography (see Materials and methods). Radiolabled Hcf106 from an import assay (see text) was used as a marker for the cpTatC–Hcf106 complex (lane 1). (B) Recovered thylakoids (A) were also subjected to SDS-PAGE and fluorography. The precursors used for the assays (tp) are in lanes 1 and 4. (C) BN-PAGE lanes of bound DT23 and bound tOE17 were subjected to second dimension SDS-PAGE (Materials and methods). An aliquot of the translation product mixed with prestained molecular weight markers was run on the left side of the SDS-PAGE gels. (D) Recovered thylakoids from binding assays with in vitro translated tOE17 or DT23 were solubilized with 1% digitonin, 20% glycerol, 0.5 M aminocaproic acid, and 1/2 × import buffer (total; lane 2), and the soluble 200 g supernatant (super; lane 3) was subjected to immunoprecipitation with IgGs cross-linked to protein A–Sepharose as designated top (as in Materials and methods, except that recovered beads were washed with solubilization buffer, 0.5% digitonin). Bound (B) and unbound (U) proteins were adjusted to 1:1 stoichiometry with respect to the original thylakoid sample and analyzed by SDS-PAGE and fluorography. tp; an aliquot of translation product used for the assay.

Mentions: As a first approach to identifying the precursor-binding site, radiolabeled tOE17 and DT23 were bound to thylakoids, and the recovered and solubilized thylakoids were analyzed by BN-PAGE and fluorography (Fig. 4 A). Both precursors migrated at ∼700 kD on the blue native gel (Fig. 4 A, lanes 2 and 4). If bound precursors were chased into the lumen before solubilization, the radioactivity at ∼700 kD was absent or reduced (Fig. 4 A, lanes 3 and 5). For example, all of the bound tOE17 was chased to the lumen, as evidenced by the disappearance of precursor and appearance of mature OE17 on the SDS-PAGE fluorogram (Fig. 4 B, lane 3). Correspondingly, there was no detected band at 700 kD on the blue native gel (Fig. 4 A, lane 3). In addition, appearance of radioactivity at ∼700 kD depended on precursor interaction with intact thylakoids. When tOE17 and DT23 were combined with digitonin solubilization buffer or even solubilized thylakoids, they migrated as smears on the blue native gel (data not shown).


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

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

Precursors bind to the ∼700-kD cpTatC and Hcf106 complex. (A) Pea thylakoids were assayed for binding (b; lanes 2 and 4) or binding and then transport chase (c; lanes 3 and 5) with in vitro translated tOE17 and DT23. Recovered thylakoids were solubilized with 1% digitonin, 20% glycerol, and import buffer and analyzed by BN-PAGE and fluorography (see Materials and methods). Radiolabled Hcf106 from an import assay (see text) was used as a marker for the cpTatC–Hcf106 complex (lane 1). (B) Recovered thylakoids (A) were also subjected to SDS-PAGE and fluorography. The precursors used for the assays (tp) are in lanes 1 and 4. (C) BN-PAGE lanes of bound DT23 and bound tOE17 were subjected to second dimension SDS-PAGE (Materials and methods). An aliquot of the translation product mixed with prestained molecular weight markers was run on the left side of the SDS-PAGE gels. (D) Recovered thylakoids from binding assays with in vitro translated tOE17 or DT23 were solubilized with 1% digitonin, 20% glycerol, 0.5 M aminocaproic acid, and 1/2 × import buffer (total; lane 2), and the soluble 200 g supernatant (super; lane 3) was subjected to immunoprecipitation with IgGs cross-linked to protein A–Sepharose as designated top (as in Materials and methods, except that recovered beads were washed with solubilization buffer, 0.5% digitonin). Bound (B) and unbound (U) proteins were adjusted to 1:1 stoichiometry with respect to the original thylakoid sample and analyzed by SDS-PAGE and fluorography. tp; an aliquot of translation product used for the assay.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196467&req=5

fig4: Precursors bind to the ∼700-kD cpTatC and Hcf106 complex. (A) Pea thylakoids were assayed for binding (b; lanes 2 and 4) or binding and then transport chase (c; lanes 3 and 5) with in vitro translated tOE17 and DT23. Recovered thylakoids were solubilized with 1% digitonin, 20% glycerol, and import buffer and analyzed by BN-PAGE and fluorography (see Materials and methods). Radiolabled Hcf106 from an import assay (see text) was used as a marker for the cpTatC–Hcf106 complex (lane 1). (B) Recovered thylakoids (A) were also subjected to SDS-PAGE and fluorography. The precursors used for the assays (tp) are in lanes 1 and 4. (C) BN-PAGE lanes of bound DT23 and bound tOE17 were subjected to second dimension SDS-PAGE (Materials and methods). An aliquot of the translation product mixed with prestained molecular weight markers was run on the left side of the SDS-PAGE gels. (D) Recovered thylakoids from binding assays with in vitro translated tOE17 or DT23 were solubilized with 1% digitonin, 20% glycerol, 0.5 M aminocaproic acid, and 1/2 × import buffer (total; lane 2), and the soluble 200 g supernatant (super; lane 3) was subjected to immunoprecipitation with IgGs cross-linked to protein A–Sepharose as designated top (as in Materials and methods, except that recovered beads were washed with solubilization buffer, 0.5% digitonin). Bound (B) and unbound (U) proteins were adjusted to 1:1 stoichiometry with respect to the original thylakoid sample and analyzed by SDS-PAGE and fluorography. tp; an aliquot of translation product used for the assay.
Mentions: As a first approach to identifying the precursor-binding site, radiolabeled tOE17 and DT23 were bound to thylakoids, and the recovered and solubilized thylakoids were analyzed by BN-PAGE and fluorography (Fig. 4 A). Both precursors migrated at ∼700 kD on the blue native gel (Fig. 4 A, lanes 2 and 4). If bound precursors were chased into the lumen before solubilization, the radioactivity at ∼700 kD was absent or reduced (Fig. 4 A, lanes 3 and 5). For example, all of the bound tOE17 was chased to the lumen, as evidenced by the disappearance of precursor and appearance of mature OE17 on the SDS-PAGE fluorogram (Fig. 4 B, lane 3). Correspondingly, there was no detected band at 700 kD on the blue native gel (Fig. 4 A, lane 3). In addition, appearance of radioactivity at ∼700 kD depended on precursor interaction with intact thylakoids. When tOE17 and DT23 were combined with digitonin solubilization buffer or even solubilized thylakoids, they migrated as smears on the blue native gel (data not shown).

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