FIGURE 10.

Analysis of the secY Lys²⁰ mutant during kasugamycin treatment by in vivo photo-cross-linking. A, kinetics of kasugamycin inhibition. BL26.1(λDE3) containing pSup-pBpARS-6TRN, pCDFT7secYmycEG, and pBAD-lacZ was grown to an A₆₀₀ of 0.2, when kasugamycin was added to a final concentration of 2 mg/ml at time 0; arabinose was added to a final concentration of 0.2% at the indicated times, and 40 min later β-galactosidase activity was measured. The average Miller units (41) of β-galactosidase activity of duplicate or triplicate samples are shown. The basal level of β-galactosidase activity in the untreated culture lacking both kasugamycin and arabinose was subtracted as background from all assays. B, the secY Lys²⁰ mutant was grown in the presence of 1 mm pBpA and 30 μm IPTG until A₆₀₀ reached 0.2, when kasugamycin was added to a final concentration of 2 mg/ml. Cells were harvested at the indicated times and exposed to UV irradiation as indicated. Cell membranes were isolated and analyzed by Western blotting using c-Myc antibody.