driven proton pumping activity in inverted membrane vesicles prepared from the epitope-

tagged mutants was used as an indication of coupled activity. Acidification of the

inverted membrane vesicles upon addition of ATP was directly examined by fluorescence

quenching. Membrane protein (250 Gig) was suspended in 3 ml of assay buffer (50 mM

MOPS, 10 mM MgCl2, pH 7.3) in a quartz cuvette. The fluorescent dye ACMA was

added to a final concentration of 1 C1M, and fluorescence was monitored with a Perkin-

Elmer LS-3B Fluorescence Spectrometer with excitation at 410 nm and emission at 490

nm and directly recorded with a Perkin-Elmer GP-100 Graphics Printer. After several

seconds, the fluorescent emission stabilized and was manually set to 85% for the purpose

of the scale. The emission was graphically recorded for 1.5 minutes, ATP was added to a

final concentration of 0.4mM and the emission was recorded for and additional 10-12

minutes.

 The level of P-nicotinamide adenine dinucleotide, reduced form, (NADH)-driven

fluorescence quenching was monitored for all membrane preparations to demonstrate that

the vesicles were intact and closed. Membrane protein (250 Gig) was suspended in 3 ml

of assay buffer (50 mM MOPS, 10 mM MgCl2, pH 7.3). ACMA was added to a final

concentration of 1 CIM, and fluorescence was recorded excitation set at 410 nm and

emission set at 490 nm. After several seconds, the fluorescent emission stabilized and

was manually set to 95%. The emission was recorded for 1 minute, 5 CIL NADH (0.1

mM) was added and the emission was continually recorded. Over time, membrane

vesicle acidification peaked and the fluorescence quenching reached a maximum. As the

fluorescence began to rise, 5 CIL of 0.3 mM KCN was added and the emission was

recorded for a total time of about 10-15 minutes.