The H-domain of the substrate signal peptide makes contact with the cpTatC concave surface at TM5. Thylakoid membranes containing radiolabeled cpTatC Cys variants were incubated in a binding reaction with tOE17-20F substrate Cys variants as shown above the panels. After washing, thylakoids were subjected to disulfide cross-linking at 15°C. (A) Diagram of the signal peptide and early mature domain of the substrate showing numbering convention. The H-domain is underlined. (B) Survey of cpTatC Cys with substrate containing Cys in the signal peptide or early mature domain. (C, left) Demonstration that the ∼48-kD band is a cross-linking product between substrate −7C and cpTatC V270C. Cross-linking reactions were conducted in which substrate or cpTatC-Cys was absent, or in which only one or both were radiolabeled as shown above the panel. (Right) The cpTatC TM5 V270C was combined with substrate containing a range of single Cys substitutions. The ∼46-kD band produced from substrate −10C (asterisk) is a nonspecific cross-linking product.