FIGURE 3.

Detection of distinct SecYEG dimer states using in vivo photo-cross-linking. Each strain carrying the indicated amber mutation was grown in the presence of 1 mm pBpA until A₆₀₀ reached 0.3, when SecYEG was induced with IPTG at a final concentration of 1 mm for 2 h. Cells were harvested, resuspended in PBS buffer, and treated with UV irradiation for 20 min as indicated. Cell membranes were isolated and analyzed by Western blotting using c-Myc antibody as described under “Experimental Procedures.” Analysis of A and B, secY amber mutations in the front-to-front (FTF) dimer interface; C and D, secY and secE mutations, respectively, in the back-to-back (BTB) dimer interface; and E, secY mutations that lie at neither interface. Certain samples (boil) were heated at 100 °C for 5 min to induce SecY aggregation. YY indicates a genetically fused SecY dimer used as a marker (48). The star indicates a SecY-SecG cross-linked complex. Single letter amino acids are indicated.