alone in the cells no growth was detected. In each case, the strains coexpressing the

mutant epitope-tagged b subunits with a wild-type epitope-tagged b subunit grew

comparably to the wild type strain.

Heterodimer Formation of bargas Defective Subunits with bwt

 Although two unequal length b subunits formed a heterodimer in an intact F1Fo

ATP synthase complex (195), it was possible that a single amino acid substitution could

affect dimerization, preventing assembly of a complex. To consider whether a V5

epitope-tagged barg36 Subunit, barg364ile-V5 Or barg364glu-V5, COuld form a heterodimer with a

wild type histidine epitope-tagged b subunit, bwt-his each pair of subunits were expressed

in strains KM2/pTAM3 7/pTAM53 and KM2/pTAM3 7/pTAM54 (Ab) (Figure 3 -2A and

B). The b subunits were detected in membranes prepared from strains expressing a b

subunit by immunoblot analysis (Figure 3-2A, Lanes 1-9). However, only b subunits in

complexes with at least one histidine tag were retained by Ni-resin purification (Figure 3-

2A, lanes 10-13). Immunoblot analysis using an anti-V5 antibody was performed on the

membrane preparations and Ni-purified products (Figure 3-2B). The V5-epitope tag was

detected only in membrane vesicles derived from KM2 strains expressing either the V5-

tagged b subunit or the coexpressed V5-tagged and his-tagged b subunits (Figure 3-2B,

Lanes 1-9). When only V5-tagged b subunits were expressed alone in a cell, no F 1Fo

complexes were recovered from the Ni-resin purification (Figure 3-2B, Lane 10).

Finally, we investigated the ability of bwt-his to dimerize to form intact F1Fo ATP synthase

complexes with barg364ile-V5 Or barg364glu-V5. Both barg364ile-V5 and barg364glu-V5 were

detected with anti-V5 antibodies after Ni-resin purification (Figure 3-2B, Lanes 12-13).

The only mechanism for recovery of defective V5-tagged b subunits was through