Stability of the disulphide bond formed within the SecA₇₉₅–Y₂₆₈EG complex. (A) Before translocation, the purified SecA₇₉₅–Y₂₆₈EG complex (2 µg), untreated or reduced with 50 mM DTT, was analyzed by SDS-PAGE and stained with Coomassie brilliant blue dye. SecYEG is used as a marker on the left, the quantities of which have no relation to those used in the cross-linking experiment. (B) Equivalent quantities of spent translocation reactions (before proteinase K treatment) containing the cross-linked (SecA₇₉₅–Y₂₆₈EG; 1 µg) or uncross-linked (wild-type SecYEG; 0.43 µg) complexes were analyzed by SDS-PAGE and Western blotting for SecY. The lower molecular weight form of SecY (SecY′) in the latter sample is a result of a well known C-terminal cleavage product (Brundage et al., 1990).