Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC–Hcf106 complex before Tha4-dependent transport

Abstract

The thylakoid ΔpH-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 ∼700-kD complex that lacks Tha4. Thylakoid-bound precursor proteins were also associated with an ∼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 ΔpH. 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.

Functional assembly of thylakoid ΔpH‐dependent/Tat protein transport pathway components in vitro

“…Imported Tha4 migrated at 240 kDa (lane 3). These are the same profiles obtained for endogenous components solubilized under comparable conditions [8].…”

“…Lanes were loaded with enriched fractions on an equal protein basis (lanes 1-4) and also in the approximate stoichiometric ratio that these membranes are present in chloroplasts (lanes 5-8). Both Tha4 and Hcf106 are primarily localized in thylakoids (lanes [5][6][7][8] but are also present in envelope fractions. This is especially apparent when equal amounts of protein are compared (lanes [1][2][3][4].…”

“…Antibodies to pea Hcf106, Tha4, cpTatC, cpSecY and cpOxa1p have been described [8,10,14]. Antibodies to maize Hcf106 were as described [14] and antibodies to maize Tha4 were the generous gift of A. Barkan [15].…”

“…Washed thylakoids were dissolved in 1% digitonin and subjected to blue native (BN) PAGE as described by Cline and Mori [8]. Gels were analyzed by fluorography or subjected to immunoblotting as described [8].…”

“…Gels were analyzed by fluorography or subjected to immunoblotting as described [8]. Molecular markers used for blue native gels were ferritin (880 kDa and 440 kDa) and BSA (132 kDa and 66 kDa).…”