treating with 1mM BS53 for 30 minutes at room temperature. The cross-linking reaction

was stopped by addition of 100 mM ethanolamine HC1, pH 7.5, for 10 minutes.

 For the purposes of densitometric analysis, a total of 15 mg membrane protein

was solubilized and clarified as described above. The clarified protein was divided into

10 samples and then Ni-resin purified (1 ml packed resin volume divided into 10

microcentrifuge tubes) as described previously, and the Einal eluate was concentrated

with a Microcon YM-10. Attempts to purify the heterodimers with the batch method (15

mg membrane protein to 1 mL packed resin volume in a 15 mL corning conical tube,

undivided) proved unsuccessful; therefore division into several microcentrifuge tubes

was necessary.

Assays of F1Fo ATP Synthase Activity

 Growth on a minimal succinate medium was used as an initial, in vivo, assay for

enzyme viability. ATP hydrolysis activity was assayed by the acid molybdate method

(146), which measures the release of Pi from ATP, in order to determine the specific

activity of the epitope tagged F1Fo ATP synthase enzyme complexes. Membranes were

assayed to determine the linearity with respect to time and enzyme concentration. Prior

to the assay, all supplies and reagents were distributed, labeled and placed on ice or 37 oC

as specified below. For each membrane sample, duplicates of 60 Clg membrane protein

were incubated in 4.0 mL of the reaction buffer (50 mM Tris-HC1, 1 mM MgCl2, pH9. 1)

at 37 oC. Stop buffer, which acts to prevent further hydrolysis of ATP, was prepared as

needed and consisted of 1.3 part ddH20 to 0.6 part HCl/molybdate solution (2.5%

NH4Mo4024-4H20, 4.0 N HC1) to 0.4 part 10% SDS. The stop buffer was divided into 2

mL aliquots in 13x100 mm disposable borosilicate glass tubes and kept on ice throughout