TABLE 1.

Functional properties of strains and membranes containing mutations perturbing the β_DP/α_DP hydrogen-bonding network Growth yield in limiting (3 mm) glucose and growth on succinate plates were determined as in (13). Growth yield data were measured via the turbidity (A₅₉₀) and are expressed as percentage of the value for the positive control. Quenching of acridine orange fluorescence by NADH and ATP was measured as in (16). The determination of the amount of F₁ on the membranes is based on the quantitative evaluation of western blots by densitometric analysis (see “Experimental Procedures”). ATPase activities were determined in 50 mm Tris/H₂SO₄, 10 mm ATP, 4 mm MgSO₄, pH 8.0, at 23 °C. The relative ATPase activity in the last column is expressed as percentage of the activity of the positive control, corrected for the different amounts of F₁ on the membrane. Strain pSN6/DK8 served as positive control; it expresses ATP synthase containing a βY331W mutation. βY331W mutant ATP synthase is a normal, active enzyme (10, 15, 46). All mutant strains described in the table were derived from strain pSN6/DK8. Strain pUC118/DK8 does not express ATP synthase and served as negative control.

Strain/mutation	Growth yield in limiting glucose	Growth on succinate	Acridine orange quenching		Amount of F1 on membranes	Membrane ATPase activity
			NADH-induced	ATP-induced
	%		%	%	%	Units/mg	%
A) Controls
pSN6/DK8	100	++++	94	85	100	0.55	100
pUC118/DK8 (unc–)	42	–	93	0	0	<0.01	NDa
B) Single mutations
αQ399N	88	+++	84	81	112	0.45	73
αQ399C	46	+	68	0	82	0.02	4
αQ399A	38	–	84	0	0	<0.01	ND
αE402D	76	++	90	54	111	0.31	50
αE402Q	94	+++	92	75	122	0.49	72
αE402A	51	++	90	15	121	0.06	9
αF406Wb	101b
αF406Cb	77b
αF406A	48	+	93	6	104	0.18	31
βR394K	58	++	93	21	93	0.13	25
βR394Q	54	++	93	19	88	0.13	27
βR394A	52	++	87	22	107	0.10	17
βR398K	96	+++	91	80	90	0.44	89
βR398Hc	92c	+++					71c
βR398Q	92	+++	93	66	107	0.45	76
βR398Wc	102c	++++	91c	89c			100c
βR398Cc	100c	++++	88c	85c			88c
βR398A	89	+++	92	68	88	0.37	76
βQ441N	94	+++	92	79	115	0.40	63
βQ441C	68	++	93	53	151	0.35	42
βQ441A	50	+	88	4	68	0.10	27
C) Triple mutation
βR394A/βR398A/βQ441A	46	+	91	11	104	0.06	10
S.D.d	5		5	10	20	0.05

^aND, not determinable (correction for amount of F₁ leads to division by 0)

^bMutations were generated previously.⁴ The plasmid containing the αF406W mutation was derived from plasmid pBOW1 (47), encoding ATP synthase with Trp-free F₁. The plasmid containing the αF406C mutation was derived from pBWU13.4 (48), encoding wild-type ATP synthase. Growth yields are given as percentage of the growth yield of the wild-type control (pBWU13.4/DK8). From both mutant strains an active F₁ could be isolated

^cMutations were described previously (34, 35). In all cases, wild-type enzyme was used as background. Growth yields and membrane ATPase activities are expressed as percentage of the respective values for the wild-type control. βR398W mutant F₁ was isolated and showed normal function (49)

^dMaximum standard deviation for the values in the respective column. All assays were run at least in triplicate, except for the determination of the amount of F₁ on the membrane which was done at least in duplicate