generated by site-directed mutagenesis. Again, the epitope tags were needed to facilitate

enzyme purification and subunit detection on a Western blot, respectively. In order to

express two different b subunits in the same cell, we first used the two-plasmid

expression system (Figure 2-8A). A total of four plasmids were constructed expressing

bwt-his (Cmr), b+7-his (Cmr), bwt-vs (Apr) or ba7.vs (Apr) (Table I). The deletion removed the

segment from Leu54-Ser60, and the insertion resulted in duplication of the same series of

amino acids.

 Previous work had shown that F1Fo ATP synthase complexes with b subunits

shortened and lengthened by 7 amino acids were essentially wild-type (193, 194).

However, it was possible that the epitope tags would affect enzyme assembly or function.

This was particularly a concern at the C-terminus where small deletions at the extreme C-

terminal end of the subunit had been shown to inhibit F1Fo ATP synthase function (206,

288). Addition of the fourteen amino acid V5-epitope tag to the C-terminus of b might

have impinged on enzyme assembly. The effects of the added epitope tags were studied

by the ability of the plasmids to complement the E. coli strain KM2 (Ab) (218). Growth

on succinate minimal medium was used as an initial qualitative gauge of enzyme activity

in vivo since E. coli strains lacking F1Fo ATP synthase cannot derive energy from

nonfermentable carbon sources. In each case, the strains expressing the epitope-tagged b

subunits grew comparably to the wild type strain (Table I). Hence, even though deletion

of as few as two amino acids affected the ability of the b dimer to interact with the Fl 6

subunit (41,42), addition of fourteen amino acids to the C-terminus did not interfere with

the interaction of the b and 6 subunits.