Beschreibung:
<jats:title>Abstract</jats:title><jats:p>The chloroplast twin arginine transport (cpTat) system distinguishes itself as a protein transport pathway by translocating fully folded proteins, using the proton‐motive force (PMF) as the sole source of energy. The cpTat pathway is evolutionarily conserved with the Tat pathway found in the plasma membrane of many prokaryotes. The cpTat (<jats:italic>Escherichia coli</jats:italic>) system uses three proteins, Tha4 (TatA), Hcf106 (TatB), and cpTatC (TatC), to form a transient translocase allowing the passage of precursor proteins. Briefly, cpTatC and Hcf106, with Tha4, form the initial receptor complex responsible for precursor protein recognition and binding in an energy‐independent manner, while a separate pool of Tha4 assembles with the precursor‐bound receptor complex in the presence the PMF. Analysis by blue‐native polyacrylamide gel electrophoresis (BN‐PAGE) shows that the receptor complex, in the absence of precursor, migrates near 700 kDa and contains cpTatC and Hcf106 with little Tha4 remaining after detergent solubilization. To investigate the role that Hcf106 may play in receptor complex oligomerization and/or stability, systematic cysteine substitutions were made in positions from the N‐terminal transmembrane domain to the end of the predicted amphipathic helix of the protein. BN‐PAGE analysis allowed us to identify the locations of amino acids in Hcf106 that were critical for interacting with cpTatC. Oxidative cross‐linking allowed us to map interactions of the transmembrane domain and amphipathic helix region of Hcf106. In addition, we showed that in vitro expressed, integrated Hcf106 can interact with the precursor signal peptide domain and imported cpTatC, strongly suggesting that a subpopulation of the integrated Hcf106 is participating in competent cpTat complexes.</jats:p>