Substrate protein signal peptides insert between cpTatC subunits. Thylakoid membranes containing radiolabeled or unlabeled cpTatC E73C (domain S1, shown below the panels) were incubated in a binding reaction with radiolabeled or unlabeled single and double Cys variants of the full-length tOE17-20F substrate as shown above the panels. Disulfide cross-linking was conducted as described in the Materials and methods. The identification of B3 as substrate × cpTatC is inferred from the migration of the cross-linking product between cpTatC E73C and substrate −25C (lanes 6 and 11). The identification of band B2 as substrate dimer is inferred from the migration of the bands from the reaction containing radiolabeled substrate −3C (lane 12). Identification of the B4 band as substrate₂ × cpTatC and the B5 band as substrate₂ × cpTatC₂ was based on their M_r and the substrate/cpTatC abundance in the band. The ratio of abundance was determined in an experiment in which substrate was labeled with ³H-leucine and cpTatC was labeled with ³⁵S-methionine (lane 13). The ratio of tritium to ³⁵S for the B3 band was set as 1:1 (asterisk) and the ratios of B4 and B5 calculated normalized to that value. Ratios are the average and standard deviation obtained from six individual cross-linking assays.