FIGURE 6.

Analysis of the secY Ser¹¹¹ mutant during OmpA-GFP-induced translocon jamming by in vivo photo-cross-linking. A, view of T. maritima SecYEG docked in the front-to-front conformation with the same color scheme as described in the legend to Fig. 1. Residue Ser¹¹¹ is shown as magenta spheres. B–E, the secY Ser¹¹¹ mutant was grown in the presence of 1 mm pBpA, 30 μm IPTG, and 0.2% maltose until A₆₀₀ reached 0.15, when the OmpA-GFP chimera was induced by adding arabinose to a final concentration of 0.2%. Cells were harvested at the indicated time points post jamming, and exposed to UV irradiation as indicated. A wild-type (WT) strain was used in parallel as a control. B, Western blot of cell membranes probed with c-Myc antibody. C, Western blot of cell membranes probed with GFP antibody. D, Western blot of cells divided into cytoplasm/membrane (C) and periplasmic (P) fractions, compared with total (T) cell input, probed with MBP antibody. E, quantification of SecY dimer to monomer ratio (D/M) or cytoplasm/membrane MBP to total MBP ratio (C/T) during jamming. The average results from three experiments are plotted with standard error measurements.