Precursor recognition and translocation by the thylakoid D pH-dependent/Tat protein translocation pathway

Abstract

Thylakoids employ four distinct pathways for translocation of newly synthesized proteins: the spontaneous, Sec, SRP, and D pH-dependent pathways. The D pH-dependent/Tat pathway is a novel system that transports folded proteins with twin arginine-containing signal peptides. Three proteins, Hcf106, Tha4, and cpTatC, comprise the transport machinery. Each component directly participates in the transport process in vitro because antibody to any single component disables the pathway. The transport reaction occurs in two steps, D pH-independent precursor binding to the machinery and D pH-dependent translocation across the membrane. A combination of antibody inhibition, blue native gel analysis, co-immunoprecipitation, and chemical crosslinking shows that precursors bind to a large ~700 kDa complex that contains Hcf106 and cpTatC. Precursor binding to the cpTatC-Hcf106 complex requires both the essential twin arginine and the hydrophobic core of the signal peptide. Tha4 is absent from the ~700 kDa complex regardless of the presence of precursor. Nevertheless Tha4 participation is required for progression of precursors beyond the cpTatC-Hcf106 bound state. Specifically, when antibodies sequestered Tha4, precursors remained bound to the large complex even in the presence of the D pH. These observations lead to a model for regulated assembly in which the cpTatC-Hcf106 complex serves as the core receptor for precursors, and subsequent Tha4 recruitment reorganizes the components into a translocation channel.