The chloroplast twin arginine transport (Tat) component, Tha4, undergoes conformational changes leading to Tat protein transport.

Twin arginine transport (Tat) systems transport folded proteins using proton-motive force as sole energy source. The thylakoid Tat system comprises three membrane components. A complex composed of cpTatC and Hcf106 is the twin arginine signal peptide receptor. Signal peptide binding triggers assembly of Tha4 for the translocation step. Tha4 is thought to serve as the protein-conducting element, and the topology it adopts during transport produces the transmembrane passageway. We analyzed Tha4 topology and conformation in actively transporting translocases and compared that with Tha4 in nontransporting membranes. Using cysteine accessibility labeling techniques and diagnostic protease protection assays, we confirm an overall N(OUT)-C(IN) topology for Tha4 that is maintained under transport conditions. Significantly, the amphipathic helix (APH) and C-tail exhibited substantial changes in accessibility when actively engaged in protein transport. Compared with resting state, cysteines within the APH became less accessible to stromally applied modifying reagent. The APH proximal C-tail, although still accessible to Cys-directed reagents, was much less accessible to protease. We attribute these changes in accessibility to indicate the Tha4 conformation that is adopted in the translocase primed for translocation. We propose that in the primed translocase, the APH partitions more extensively and uniformly into the membrane interface and the C-tails pack closer together in a mesh-like network. Implications for the mode by which the substrate protein crosses the bilayer are discussed.