Models for the association of cpTatC, Tha4, and substrate during assembly of the Tat translocase. (A) A cartoon model for pea chloroplast TatC structure based on homology with the A. aeolicus TatC structure (see Materials and methods) with all Cys substitutions tested in this study colored yellow on the cpTatC backbone and labeled by residue number; yellow highlighted residue numbers produced disulfide cross-links with Tha4. Residues that, when mutated to alanine, impair twin arginine binding are colored orange. Glutamine 234, proposed to mediate docking of Tha4 E10, is colored magenta. The carboxyl-proximal helix of cpTatC TM5 is colored cyan. (B) Proposed arrangement of cpTatC, bound substrates, and the TMs of Tha4 (red cylinders) and Hcf106 (green cylinders) in a dimer of cpTatC. A ribbon depiction of the proposed cpTatC dimer as viewed from the chloroplast stroma with cpTatC subunits colored blue and purple, respectively, is shown. The suggested substrate arrangement is shown in yellow with the signal peptide hydrophobic helix associated with the Hcf106 TM and the carboxyl-proximal signal peptide and N-terminal residues of the substrate mature domain in the central cavity. A stippled yellow line depicts the segment that directs disulfide-linked substrate dimers. (C and D) Interpretation of the positions of Tha4 and Hcf106 TMs with cpTatC constitutively (C) and in the translocase (D) are based on disulfide cross-linking and the position of the E. coli TatB (Hcf106 orthologue; Rollauer et al., 2012). The N-terminal segments of Tha4 and Hcf106 were modeled onto the E. coli TatA structure (see Materials and methods).