TABLE 1.

Analysis of subunit a mutants used in this study

Mutation	Growth on succinate minimal medium1	Growth yield on limiting glucose-minimal medium2	ATP Hydrolysis (µmoles/min/mg protein) 3	Sensitivity to DCCD4
wild type	++++	100	0.80 ± 0.04	70 ± 7 %
P204T/R210Q/Q252R	++	63± 4	0.21 ± 0.01	11 ± 3 %
P204A/R210Q/Q252R	++	61± 1	ND5	ND
P204S/R210Q/Q252R	++	59 ± 2	ND	ND
R210Q/Q252R	+	57 ± 2	ND	ND
P204T/R210G/Q252R	+++	79 ± 2	0.51 ± 0.02	53 ± 3 %
E219K	+++	80 ± 2	0.81 ± 0.19	ND
E219Q	−	52 ± 2	0.23 ± 0.02	ND
E219K (P204T/R210Q/Q252R)	+	55 ± 2	0.23 ± 0.03	ND
E219Q (P204T/R210Q/Q252R)	−	51 ± 2	0.23 ± 0.02	ND
wild type	++++	100	0.80 ± 0.04	70 ± 7 %
null	−	49 ± 1	ND	ND

¹For growth on succinate minimal medium, the mutations were constructed in the pTW1-HisHA plasmid, which encodes only subunit a, and the background strain was RH305, which fails to express subunit a. RH305 without a plasmid represents the null sample, and RH305 with pTW1-HisHA represents the wild type. The size of the colonies after growth for 48 h at 37 °C is indicated by the number of + signs. The – sign indicates no growth.

²Growth yield measurements were done in Minimal A medium with limiting glucose (6 mM), as measured by absorbance of liquid culture at 600 nm. For these measurements, the mutations were transferred to the pFV2-HA plasmid, which encodes all of the ATP synthase structural genes including an HA epitope-tagged subunit a. The background strain was DK8, which is deleted for the 8 structural genes of the atp operon. DK8 with plasmid pFV2-HA that has an internal deletion in the gene for subunit a from the 2 BamH I sites, represents the null sample, and DK8 with pFV2-HA represents the wild type.

³Rates were determined from two independent membrane preparations. The mean and the standard error are indicated.

⁴DCCD sensitivity reflects the remaining fraction of activity after an incubation with 20 µM DCCD for 30 minutes at 23°C.

⁵ND, Not determined.