Mapping the Interactions of the Substrate Signal Peptide and cpTatC.
(A) S1 is closer to the amino-proximal side of RR and S2 closer to the carboxyl-proximal side. Radiolabeled precpTatC Cys variants were imported into chloroplasts and recovered thylakoids incubated with radiolabeled tOE17-25C-20F (top panels) or tOE17-20F-18C (bottom panels). Washed thylakoids were subjected to cross-linking with bis-maleimide agents that vary in spacer length: BMOE (left panels), BMH (middle panels), or BM(PEO)3 (right panels).
(B) S1 and S2 do not contact downstream residues of the signal peptide. Radiolabeled precpTatCaaa D78C (S1; lanes 1 to 7), precpTatCaaaT164C (S2; lanes 8 to 14), and precpTatCaaa (lanes 15 to 21) were imported into chloroplasts and recovered thylakoids incubated with radiolabeled substrate tOE17-20F with single Cys substitutions at varying positions as indicated below the panels. Washed thylakoids were then subjected to cross-linking with BM(PEO)3. The cpTatC-substrate adducts are marked with an asterisk.