Thylakoid Delta pH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport

The thylakoid Delta pH-dependent pathway transports folded proteins with tw in arginine-containing signal peptides. Identified components of the machin ery include cpTatC, Hcf106, and Tha4. The reaction occurs in two steps: pre cursor binding to the machinery, and transport across the membrane. Here, w e show that a cpTatC-Hcf106 complex serves as receptor for specific binding of twin arginine-containing precursors. Antibodies to either Hcf106 or cpT atC, but not Tha4, inhibited precursor binding. Blue native gel electrophor esis and coimmunoprecipitation of digitonin-solubilized thylakoids showed t hat Hcf106 and cpTatC are members of an similar to 700-kD complex that lack s Tha4. Thylakoid-bound precursor proteins were also associated with an sim ilar to 700-kD complex and were coimmunoprecipitated with antibodies to cpT atC or Hcf106. Chemical cross-linking revealed that precursors make direct contact with cpTatC and Hcf106 and confirmed that Tha4 is not associated wi th precursor, cpTatC, or Hcf106 in the membrane. Precursor binding to the c pTatC-Hcf106 complex required both the twin arginine and the hydrophobic co re of the signal peptide. Precursors remained bound to the complex when Tha 4 was sequestered by antibody, even in the presence of Delta pH. These resu lts indicate that precursor binding to the cpTatC-Hcf106 complex constitute s the recognition event for this pathway and that subsequent participation by Tha4 leads to translocation.