membrane vesicles were intact and closed, the level of NADH-driven fluorescence

quenching was monitored for all membrane preparations. The levels ofNADH-driven

fluorescence quenching were strong and directly comparable in every case (data not

shown, for a representative figure, see Figure 2-10).

Heterodimer formation of bA1534end-his COmplemented with bwt-vs

 Deletion of the last four amino acids at the C-terminus has been shown to

dramatically affect the ability of the b dimer to form a stable interaction with the 6

subunit Fl resulting in a maj or F1Fo ATP synthase assembly defect (205, 206).

Immunoblot analysis was performed in order to detect a heterodimer interaction between

bal534end and bwt (Figure 3-4A and B). Coexpression of pTAM5 1 (bAl534end-his) and

pTAM46 (bwt-vs) in the same cell resulted in the appearance of a weak signal representing

bwt-vs in Ni-resin purified material (Figure 3-4B, Lane 8). Although there was no attempt

to be quantitative in this assay, the appearance of the band suggested relatively inefficient

assembly of these heterodimers. Nevertheless, when the two different b subunits are

expressed in the same cell, three distinct F1Fo complexes were assembled (Figure 3-4C).

These included a homodimer of bwt-vs in an intact F1Fo ATP synthase complex, a

homodimer of bAl534end-his in a partially assembled defective enzyme, and a heterodimeric

F1Fo ATP synthase consisting of both subunits. Apparently, the bwt-vs subunit stabilized

the bAl534end-his subunit within an intact F1Fo ATP synthase complex.

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

ATP synthase complex assembly (Table 3-1). As expected, membranes with enzyme

complexes incorporated with bAl534end-his displayed a specific activity similar to the

negative control, indicating virtually no interaction with Fl. Coexpression of pTAM5 1