FIGURE 8.
Time-delayed in vivo assembly system of subunit δ into preformed FOF1 subcomplexes yielding functional FOF1. A–D, DK8 carrying plasmids pBAD33.atp, pET-22b, and pT7POL26 (FOF1) or pBAD33.Δδ, pET22-atpH-TTG, and pT7POL26 (FOF1−δ; FOF1−δ+δ) was grown as described under “Experimental Procedures.” Seven independent cell batches were prepared in parallel containing the additives indicated. The data represent average values of three independent measurements. A, level of atp mRNA. The amount of atp mRNA was determined via rt-RT-PCR using primer pairs atpE′F (dark gray) and atpA (light gray). The amount of atp mRNA present in the FOF1 sample grown with Ara was set to 100%. B, immunoblot analysis of membrane vesicles (20 μg of protein/lane) as indicated. C, ATPase activity of membrane vesicles. Gray and white portions of the bars represent DCCD-sensitive and DCCD-insensitive fractions of ATP hydrolysis, respectively. D, ATP-driven proton translocation of membrane vesicles. The relative magnitude of ACMA fluorescence quenching induced by ATP is shown. Error bars, S.E.