Figure 7.

Photocross-linking of pre-SufI single cysteine mutants with Tat components. (A) Shown here is cross-linking of pre-SufI with TatABC. The indicated single cysteine mutants of pre-SufI were modified with 4-(maleimido)benzophenone, incubated with Tat⁺⁺ IMVs, and irradiated with UV light for 5 min. Membrane-bound pre-SufI was recovered by centrifugation (16,200 × g, 30 min). The samples were resolved via SDS-PAGE and immunoblotted with TatA, TatB, TatC, and 6×His antibodies as indicated. Pre-SufI-TatB and pre-SufI-TatC adducts are both ∼80 kDa. Major (solid red box) and occasional (dashed red box) cross-links were observed in 100% and <50% of reactions, respectively (N ≥ 3). (B) Shown here is a cartoon of the interaction of the C-terminal half of the pre-SufI signal peptide hairpin with TatB. Highlighted residues (yellow) strongly cross-link to TatB (blue), as shown in (A). The helical domain of the signal peptide is colored as in Fig. 6A, and its interaction with the TatC structure (gray) is reproduced from Fig. 6 B. The globular pre-SufI mature domain (green; PDB: 2UXV) remains cytoplasmic when bound to TatBC in the absence of a pmf.