Figure 3.

Insertion and stability of deletion mutants in the membrane. (A) [³⁵S]Met labeling. Membranes prepared from cells expressing wild-type permease or a given mutant were labeled for 15 min with [³⁵S]methionine, subjected to NaDodSO₄/PAGE, and autoradiographed. Aliquots containing the same amount of membrane protein (30 μg) were applied to each lane, and prestained molecular size markers were used as indicated by the arrow at the left. (B) Lifetime studies. E. coli T184 harboring a plasmid encoding a given mutant was diluted 1:10 from overnight cultures and grown for 2 h at 37°C. IPTG was then added, and growth was continued for 2 h. Chloramphenicol (34 μg/ml) was added, and samples were collected at given times and flash frozen in liquid N₂. Membranes were prepared, subjected to NaDodSO₄/PAGE, and autoradiographed. WT, wild type; Δ5, deletion of residues 206–210; Δ12, deletion of residues 199–210; Δ20, deletion of residues 195–214.