KM2/pTAM37/pTAM53 or KM2/pTAM37/pTAM54 specific activities of about 94%

and 91%, respectively, were observed indicating a quantity of intact F1Fo ATP synthase

complexes approaching the wild type strain levels.

 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 3-3). Membranes isolated from cells with a V5 epitope tag incorporated

onto the bwt subunit, KM2/pTAM46 (bwt-vs), reproducibly displayed a very small

reduction in coupled activity, correlating very well with the F1-ATP hydrolysis activity.

Consistent with previous observations, a larger reduction in coupled activity of about

20% was observed in membrane vesicles isolated from strain KM2/pTAM37 (bwt-his).

The coupled activities observed in membranes isolated from cells coexpressing histidine-

tagged and V5-tagged b subunits were intermediary between the V5-tagged species and

the histidine-tagged species. Strains expressing either barg364ile-V5 Or barg364glu-V5

displayed no ATP-dependent proton pumping activity as expected. Significantly, when

either one of the barg-36 mutants was coexpressed with bwt-his, KM2/pTAM3 7/pTAM53

(bwt-his/barg36,ile-V5) Or KM2/pTAM3 7/pTAM54 (bwt-hisbarg364glu-V5), the coupled activity

observed was essentially the same as strain KM2/pTAM3 7/pTAM46 (bwt-his/bwt-vs).

When expressed alone, the strains with barg-36-V5 mutants had no activity and the strains

expressing bwt-his displayed less activity than the bwt-his/barg-36-V5 mutant enzyme. As a

consequence, it was most likely that the F1Fo ATP synthase complexes with either the bwt-

his/barg364ile-V5 Or bwt-his/barg364glu-V5 peripheral stalks were active. To demonstrate that the