Substrate-enhanced contacts between the Tha4 TM and cpTatC TM4 and TM5 also require the proton gradient. Thylakoid membranes containing radiolabeled cpTatC Cys variants in TM4 (L231C), TM5 (Y259C or V270C), or L3 (T275C) were incubated with unlabeled Tha4 Cys variants and mock translation extract or SpF12, and were subjected to disulfide cross-linking. All reactions were preincubated in light during Tha4 integration (see Materials and methods). Incubations for cross-linking in A were in darkness. Incubations in B and D were in the light. As designated by Nig/Val +, certain reactions received 1 µM nigericin and 2 µM valinomycin immediately after SpF12 addition to dissipate the proton gradient. (C) The cpTatC x Tha4 cross-linking products in A and B were quantified by densitometric analysis of films scanned by light transmission and analyzed with ImageJ software. The averages and differences from the mean were from two repeats of the experiment. All values from each film were normalized to the density of the cpTatC V270C × Tha4 P9C plus SpF12 band, which was assigned an arbitrary value of 100.