amino acid deletion in the absence of the epitope tag. Similar activities were observed in

membrane vesicles isolated from cells with a histidine epitope tag was incorporated onto

the enzyme. F1Fo ATP synthase complexes with a bwt-his or b+7-his dimer had specific

activities of about 90% and 79% of the wild type strain, respectively (Table 2-1).

Expression of different b subunits in the same cell

 The ability to express two different b subunits in the same cell in roughly equal

quantities was crucial to the success of the experiment. Therefore it was necessary to

establish whether two separate plasmids could be used to direct production of two

different b subunits. Two b subunits, expressed from pTAM35 (b+7-his) and pTAM34

(bA7-HA), were expressed in the same KM2 (Ab) cell and their ability to exist in the same

cell was determined by Western blot analysis (Figure 2-4A). As expected, immunoblot

analysis of crude membrane preparations using anti-b antibodies showed the presence of

the b subunit in all strains complemented with an epitope-tagged b subunit (Figure 2-4A,

Lanes 3-6). As shown in the first lane, KM2 cells did not express b subunit. After

transforming with plasmid pKAM14 (be), the KM2 cells expressed the wild type b

subunit (Lane 2). The third and forth lanes represented KM2 cells expressing the bA7-HA

and b+7-his subunits, respectively. In lane 5, individual membrane preparations

representing lanes 3 and 4 were mixed together. Finally, lane 6 represented the KM2

expressing both the short and long b subunits in the same cell. This figure demonstrated

our ability to separate the short and long subunits enough to be sufficiently

distinguishable. Notably, the KM2 cells successfully expressed the two different b

subunits in roughly equal quantities from the same cell.