Formation of mixed length b subunits in F1Fo ATP synthase

 The two plasmid expression system was used to study whether unequal length b

subunits could form a dimer or, alternatively, whether the b subunits dimerize and

incorporate into enzyme complexes in a segregated manner. In order to determine if

there is a direct protein-protein interaction between the different length b subunits, we

investigated the ability of b+7-his to retain an interaction with a bwt-vs or a ba7~vs subunit

following Ni-purification (Figure 2-12). All membranes prepared from strains expressing

a b subunit were readily detectable and distinguishable by size on an immunoblot (Figure

2-12, Lanes 1-7). Only b subunits with a histidine tag were retained by Ni-resin

purification (Figure 2-12A, Lanes 8-12). Immunoblot analysis using an anti-V5 antibody

was performed on the membrane preparations and Ni-purified products (Figure 2-12B).

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 2-12B, Lanes 1-7). As expected, upon Ni-resin purification, the V5

epitope was not detected in samples containing only histidine-tagged b subunit (Figure 2-

12B, Lane 8). Likewise, samples containing only the V5-tagged b subunits were not

detected upon Ni-resin purification, signifying that they were efficiently removed from

the resin during wash steps (Figure 2-12B, Lanes 9-10). Finally, we investigated the

ability of b+7-his to dimerize to form intact F1Fo ATP synthase complexes with bwt-vs or

ba7~vs. The bwt-vs was detected with anti-V5 antibodies and was readily observed to

dimerize with the b+7-his indicating a bwt-his/bwt-vs interaction (Figure 2-12B, Lane 11). To

a much lesser extent, the ba7-vs subunit was also distinguishable with anti-V5 antibodies

(Figure 2-12B, Lane 12). The data indicated that the F1Fo ATP synthase enzyme