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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 remain bound to the cpTatC–Hcf106 complex under transport conditions unless Tha4 is free to participate. Thylakoids were pretreated with 0.4 mg/ml of preimmune or anti-Tha4 IgGs, washed, and then incubated with in vitro–translated DT23 in a binding assay (b) or a binding and chase assay (c), or they were incubated with DT23 under transport conditions (t), i.e., 5 mM Mg-ATP, 5 mM dithiothreitol, ∼500 μg stromal protein, and light at 25°C for 20 min, in the absence or presence of 0.4 μM nigericin as shown (top). Chase and transport assays were diluted 4.5- and 10-fold, respectively, with import buffer before removal from light and 25°C. (A) Recovered thylakoids were solubilized in 1% digitonin, 20% glycerol, and import buffer and analyzed by BN-PAGE and fluorography. Each lane contains sample equivalent to 6% of the assay. The minor band migrating just below the ∼700-kD band is occasionally observed and presumably represents a breakdown product of the ∼700-kD complex. It appears enhanced in overexposed lanes (3 and 7) but is present in the other lanes and in immunoblots (Fig. 3). (B) Recovered thylakoids (A) were analyzed by SDS-PAGE and fluorography. Lanes contain sample equivalent to 5% of the assay. DT23 translation product, equivalent to 0.25% of that added to each assay, is shown in the lane marked tp. (Lanes 1 and 5) Binding assays; (lanes 2 and 6) chase assays; (lanes 3, 4, 7, and 8) transport assays.
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fig8: Precursors remain bound to the cpTatC–Hcf106 complex under transport conditions unless Tha4 is free to participate. Thylakoids were pretreated with 0.4 mg/ml of preimmune or anti-Tha4 IgGs, washed, and then incubated with in vitro–translated DT23 in a binding assay (b) or a binding and chase assay (c), or they were incubated with DT23 under transport conditions (t), i.e., 5 mM Mg-ATP, 5 mM dithiothreitol, ∼500 μg stromal protein, and light at 25°C for 20 min, in the absence or presence of 0.4 μM nigericin as shown (top). Chase and transport assays were diluted 4.5- and 10-fold, respectively, with import buffer before removal from light and 25°C. (A) Recovered thylakoids were solubilized in 1% digitonin, 20% glycerol, and import buffer and analyzed by BN-PAGE and fluorography. Each lane contains sample equivalent to 6% of the assay. The minor band migrating just below the ∼700-kD band is occasionally observed and presumably represents a breakdown product of the ∼700-kD complex. It appears enhanced in overexposed lanes (3 and 7) but is present in the other lanes and in immunoblots (Fig. 3). (B) Recovered thylakoids (A) were analyzed by SDS-PAGE and fluorography. Lanes contain sample equivalent to 5% of the assay. DT23 translation product, equivalent to 0.25% of that added to each assay, is shown in the lane marked tp. (Lanes 1 and 5) Binding assays; (lanes 2 and 6) chase assays; (lanes 3, 4, 7, and 8) transport assays.

Mentions: Results presented here and previously (Mori et al., 1999; Ma and Cline, 2000) show that Tha4 is required for transport but not binding of precursor. To determine the fate of precursors in the presence of ΔpH when Tha4 is prevented from entering the reaction, thylakoid membranes were preincubated with anti-Tha4 IgG and then subjected to binding, chase, and transport reactions with DT23 (Fig. 8) . Thylakoids recovered from assays were examined by SDS-PAGE/fluorography to assess the amount of precursor or mature protein associated with the membranes (Fig. 8 B) and by BN-PAGE/fluorography to assess specific binding (Fig. 8 A). As expected, antibody-treated membranes were as active as preimmune IgG–treated membranes in specifically binding DT23 in a standard binding assay on ice (Fig. 8, A and B, compare lanes 1 and 5). However, anti-Tha4–treated membranes were unable to transport bound precursor (Fig. 8 B, lane 6), which remained associated with the ∼700-kD complex (Fig. 8 A, lane 6). In the control preimmune treated membranes, nearly all of the bound precursor was transported to the lumen, as assessed by the appearance of the mOE23 (Fig. 8 B, lane 2) and the absence of the ∼700-kD band on BN-PAGE (Fig. 8 A, lane 2). When anti-Tha4–treated thylakoids were incubated at 25°C with freshly added DT23 in a protein transport assay, i.e., with a ΔpH, a substantially greater amount of precursor bound to the cpTatC–Hcf106 complex (Fig. 8, A and B, lane 7). Protease treatment of the membranes showed that the associated precursor was still exposed to the stromal face (data not shown). The pH gradient apparently contributed to the increased binding because considerably less precursor was bound in assays conducted with the protonophore nigericin (Fig. 8, A and B, lanes 8).


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

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

Precursors remain bound to the cpTatC–Hcf106 complex under transport conditions unless Tha4 is free to participate. Thylakoids were pretreated with 0.4 mg/ml of preimmune or anti-Tha4 IgGs, washed, and then incubated with in vitro–translated DT23 in a binding assay (b) or a binding and chase assay (c), or they were incubated with DT23 under transport conditions (t), i.e., 5 mM Mg-ATP, 5 mM dithiothreitol, ∼500 μg stromal protein, and light at 25°C for 20 min, in the absence or presence of 0.4 μM nigericin as shown (top). Chase and transport assays were diluted 4.5- and 10-fold, respectively, with import buffer before removal from light and 25°C. (A) Recovered thylakoids were solubilized in 1% digitonin, 20% glycerol, and import buffer and analyzed by BN-PAGE and fluorography. Each lane contains sample equivalent to 6% of the assay. The minor band migrating just below the ∼700-kD band is occasionally observed and presumably represents a breakdown product of the ∼700-kD complex. It appears enhanced in overexposed lanes (3 and 7) but is present in the other lanes and in immunoblots (Fig. 3). (B) Recovered thylakoids (A) were analyzed by SDS-PAGE and fluorography. Lanes contain sample equivalent to 5% of the assay. DT23 translation product, equivalent to 0.25% of that added to each assay, is shown in the lane marked tp. (Lanes 1 and 5) Binding assays; (lanes 2 and 6) chase assays; (lanes 3, 4, 7, and 8) transport assays.
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getmorefigures.php?uid=PMC2196467&req=5

fig8: Precursors remain bound to the cpTatC–Hcf106 complex under transport conditions unless Tha4 is free to participate. Thylakoids were pretreated with 0.4 mg/ml of preimmune or anti-Tha4 IgGs, washed, and then incubated with in vitro–translated DT23 in a binding assay (b) or a binding and chase assay (c), or they were incubated with DT23 under transport conditions (t), i.e., 5 mM Mg-ATP, 5 mM dithiothreitol, ∼500 μg stromal protein, and light at 25°C for 20 min, in the absence or presence of 0.4 μM nigericin as shown (top). Chase and transport assays were diluted 4.5- and 10-fold, respectively, with import buffer before removal from light and 25°C. (A) Recovered thylakoids were solubilized in 1% digitonin, 20% glycerol, and import buffer and analyzed by BN-PAGE and fluorography. Each lane contains sample equivalent to 6% of the assay. The minor band migrating just below the ∼700-kD band is occasionally observed and presumably represents a breakdown product of the ∼700-kD complex. It appears enhanced in overexposed lanes (3 and 7) but is present in the other lanes and in immunoblots (Fig. 3). (B) Recovered thylakoids (A) were analyzed by SDS-PAGE and fluorography. Lanes contain sample equivalent to 5% of the assay. DT23 translation product, equivalent to 0.25% of that added to each assay, is shown in the lane marked tp. (Lanes 1 and 5) Binding assays; (lanes 2 and 6) chase assays; (lanes 3, 4, 7, and 8) transport assays.
Mentions: Results presented here and previously (Mori et al., 1999; Ma and Cline, 2000) show that Tha4 is required for transport but not binding of precursor. To determine the fate of precursors in the presence of ΔpH when Tha4 is prevented from entering the reaction, thylakoid membranes were preincubated with anti-Tha4 IgG and then subjected to binding, chase, and transport reactions with DT23 (Fig. 8) . Thylakoids recovered from assays were examined by SDS-PAGE/fluorography to assess the amount of precursor or mature protein associated with the membranes (Fig. 8 B) and by BN-PAGE/fluorography to assess specific binding (Fig. 8 A). As expected, antibody-treated membranes were as active as preimmune IgG–treated membranes in specifically binding DT23 in a standard binding assay on ice (Fig. 8, A and B, compare lanes 1 and 5). However, anti-Tha4–treated membranes were unable to transport bound precursor (Fig. 8 B, lane 6), which remained associated with the ∼700-kD complex (Fig. 8 A, lane 6). In the control preimmune treated membranes, nearly all of the bound precursor was transported to the lumen, as assessed by the appearance of the mOE23 (Fig. 8 B, lane 2) and the absence of the ∼700-kD band on BN-PAGE (Fig. 8 A, lane 2). When anti-Tha4–treated thylakoids were incubated at 25°C with freshly added DT23 in a protein transport assay, i.e., with a ΔpH, a substantially greater amount of precursor bound to the cpTatC–Hcf106 complex (Fig. 8, A and B, lane 7). Protease treatment of the membranes showed that the associated precursor was still exposed to the stromal face (data not shown). The pH gradient apparently contributed to the increased binding because considerably less precursor was bound in assays conducted with the protonophore nigericin (Fig. 8, A and B, lanes 8).

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