Figure 5
YidC and SecY show cross-links between the hydrophobic slide and the lateral gate. Single cysteine mutants of YidC and SecY were generated and co-expressed in E. coli MK6. YidC432C was coexpressed with SecYEG that had a single cysteine at position 123 in TM3 of SecY (lanes 1, 2). The cells were pulse-labelled with 35S-methionine and treated with 200 µM DTNB (all lanes) followed by 1 mM DTT (lanes 1, 3, 6, 8, 10, 12) in some cases and analysed by immunoprecipitation with an antiserum to YidC and phosphorimaging. For a control, SecYC0 was coexpressed with YidC432C (lanes 3, 4). SecY that has a single cysteine at residue 328 in TM8 was coexpressed with the YidC mutants that have a single cysteine either at 498, 499, 500 or 501 in TM5. A cross-link band was observed between SecY328C and YidC499C (red asterisk). The expression of SecY was monitored on a Western blot (Fig. S7).
