Effects of amino terminal mutations

 Since Fl has little affinity for the membrane in the absence of intact Fo, total

membrane associated F1-ATP hydrolysis activity was used as a test of F1Fo ATP synthase

complex assembly. Under conditions of high pH, Fl can be released from the influence

of Fo (146), so the amount of ATPase activity in the solution was used as a measure of

the amount of intact enzyme complex located in the membrane vesicles. The basn24ala

substitution, expressed from KM2/pTAM43, had no effect on enzyme assembly (Table 5-

1). The bthr64ala and bginioala,, expressed from KM2/pTAM44 and KM2/pTAM45, had a

slight, but not vitally significant effect on enzyme assembly. Membranes with a bthr64ala

or bginioala incorporated into the F1Fo ATP synthase complex had specific activities of

about 77% of the wild type strain.

 F1Fo ATP synthase-mediated ATP-driven proton pumping activity in membrane

vesicles prepared from the mutants was used as an indication of coupled activity.

Acidification of inverted membrane vesicles was examined by fluorescence of ACMA

(Figure 5-3). The level ofNADH-driven fluorescence quenching was monitored for all

membrane preparations to demonstrate that the vesicles were intact and closed. The

levels ofNADH-driven fluorescence quenching were strong and directly comparable in

every case (data not shown, for a representative figure, see Figure 2-10). Membranes

isolated from cells expressing basn24ala, KM2/pTAM43, displayed an insignificant

reduction, approximately 1.6 %, in coupled activity, correlating with the wild-type-like

F1-ATP hydrolysis activity (Figure 5-2 and Table 5-1). A larger reduction in coupled

activity, of about 1 1%, was observed in membrane vesicles isolated from cells in which a

bthe64ala Or bginlonala, expressed from KM2/pTAM44 or KM2/pTAM45, respectively, was