dimerization with bwt-his indicating formation of heterodimeric F1Fo ATP synthase

complexes. Therefore, coexpression of the two different b subunits in the same cell led

to three distinct interactions within an intact F1Fo ATP synthase complex (Figure 3-2C):

1) a homodimer of bwt-his, 2) a homodimer of barg364ile-V5 Or barg364glu-V5, and 3) a

heterodimer consisting of both the defective and wild type b subunit.

 Since Fl displays reduced 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.

Previous data indicated minimal affects on specific activity due to the epitope tags (195).

Confirming these observations, the V5-epitope tag did not have an apparent affect on

enzyme assembly. Membrane preparations with a bwt-vs incorporated into the F1Fo ATP

synthase complex had virtually the same specific activity of the wild type strain (Table 3-

1). A small decrease in specific activity was reproducibly observed in membrane vesicles

isolated from cells when a histidine epitope tag was incorporated onto the bwt, about 89%

of the wild type strain. Furthermore, comparable activities were observed in samples

when the histidine-tagged and V5-tagged bwt subunits were coexpressed. Also verifying

previous data (215), when barg364ile-V5 Or barg364glu-V5 WAS expressed alone in strains

KM2/pTAM53 and KM2/pTAM54, the membranes retained abundant activity, about

60% and 54%, of the wild type strain, respectively (Table 3-1), indicating considerable

amounts of intact F1Fo ATP synthase complexes found in the membranes. When

barg364ile-V5 Or barg364glu-V5 WeTO COexpressed with bwt-his by construction of strains