Fig. 2.
Transport function of wild-type LacY in whole cells. Uphill transport of TMG (A) or downhill transport of lactose (B) normalized to total cell protein was determined as a function of time by AL95/pDD72 (closed squares, +PE), AL95/pAC-PCSlp-Sp-Gm (open squares -PE + PC), or AL95 (open circles -PE), all induced for expression of a plasmid-borne copy of the wild-type lacY gene. Uphill transport in -PE + PC cells was verified by addition of a protonophore (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) to the assay of TMG uptake (open diamonds), which reduced the uptake to the level of the -PE results (open circles). No uptake of TMG or lactose by AL95/pAC-PCSlp-Sp-Gm (closed diamonds, -PE + PC ΔLacY) demonstrated that uphill and downhill transport in -PE + PC cells (open squares) were mediated by LacY. Standard deviations for the average of two determinations are shown except for lines where uptake was essentially zero (open diamonds, open circles, and closed diamonds). The reduced level (one-fifth) of LacY in -PE + PC cells relative to the level of LacY in +PE cells (25 μg of protein per lane) is shown by Western blot analysis (B Inset) using anti-LacY polyclonal antibody.