(bAl534end-his) and pTAM46 (bwt-vs) resulted in a specific activity of about 81% of the wild

type strain (Table 3-1). An indication of coupled activity was shown by F1Fo ATP

synthase-mediated ATP-driven proton pumping activity in membrane vesicles prepared

from the epitope-tagged mutants (Figure 3-5). Membranes containing only the bal534end-

his subunit exhibited no coupled activity as expected. Membranes isolated from cells

coexpressing pTAM5 1 (bAl534end-his) and pTAM46 (bwt-vs) displayed a slight reduction in

coupled activity, of about 20%, compared to membranes from cells coexpressing bwt-vs

and bwt-his. It is feasible that the observed reductions in enzymatic activities were entirely

due to formation of the mutant homodimeric species; this could not be directly

demonstrated by these methods due to background activity from bwt-vs homodimeric F1Fo

ATP synthases present in the membranes.

Heterodimer Formation of b+124-130-his COmplemented with bwt-vs

 It has been shown by Dr. Deepa Bhatt that insertions or deletions constructed in a

hydrophobic stretch corresponding to amino acids 124-130 (VAILAVA) of the b subunit

results in loss of enzyme function (Bhatt et al., manuscript in preparation). Immunoblot

analysis was performed in order to detect a heterodimer interaction between b+124-130-his

and bwt-vs (Figure 3-6A and B). The b subunit was detected only in membranes prepared

from strains expressing a functional b subunit (Figure 3-6A, Lanes 1-6). No b subunit

was present in membranes when KM2 (Ab) was complemented with plasmid pTAM3 8

(b+124-130-his). Immunoblot analysis using an anti-V5 antibody was performed on the

membrane preparations and Ni-purified products (Figure 3-6B). Interestingly,

coexpression of pTAM3 8 (b+124-130-his) and pTAM46 (bwt-vs) in the same cell resulted in

the appearance of a signal representing bwt-vs in a Ni-resin purified sample (Figure 3-6B,