Effects of epitope tags

 Since Fl has little affinity for the membrane in the absence of intact Fo, total

membrane associated F1-ATP hydrolysis activity was used as a test of F1Fo ATP synthase

complex assembly. Under conditions of high pH, Fl can be released from the influence

of Fo (146), so the amount of ATPase activity in the solution was used as a measure of

the amount of intact enzyme complex located in the membrane vesicles. The V5-epitope

tag did not have a significant affect on enzyme assembly. Membrane preparations with a

bwt-vs or ba7ses incorporated into the F1Fo ATP synthase complex had specific activities of

about 95% and 82% of the wild type strain, respectively (Table I). The latter value was

comparable to the effect of the seven amino acid deletion in the absence of the epitope

(194). A slightly greater decrease in specific activity was reproducibly observed in

membrane vesicles isolated from cells when a histidine epitope tag was incorporated onto

the bwt or b 7 subunit, about 90% and 79% of wild type, respectively. Nevertheless,

abundant activity was retained, suggesting very limited effects resulting from addition of

the epitope tags. Furthermore, comparable activities were observed in samples when the

histidine-tagged and V5-tagged b subunits were coexpressed.

 F1Fo ATP synthase-mediated ATP-driven proton pumping activity in membrane

vesicles prepared from the epitope-tagged mutants was used as an indication of coupled

activity. Acidification of inverted membrane vesicles was examined by fluorescence of

ACMA (Figure 2-9). The level of NADH-driven fluorescence quenching was monitored

for all membrane preparations to demonstrate that the vesicles were intact and closed.

The levels of NADH-driven fluorescence quenching were strong and directly comparable

in every case (Figure 2-10). Membranes isolated from cells with a V5 epitope tag