tag are indicated along with the plasmid name for clarity, for example plasmid pTAM5 1

(bAl534end-his). Each plasmid and the control plasmids pKAM14 (b) and pBR322 were

expressed in the E. coli cell line KM2 (Ab) for study, so that the only b subunits in the

cells were the product of the plasmid genes. The two plasmid expression system allowed

expression of various combinations of histidine tagged and V5-tagged b subunits in the

same cell (Figures 2C, 4C, 6C, 8C). Appropriate antibiotics were added to the growth

medium, and in the case of the coexpressed plasmids, both ampicillin and

chloramphenicol were added to select for cells expressing both plasmids.

Preparative Procedures

 Inverted membrane vesicles from KM2 (Ab) strains expressing the desired b

subunits were prepared essentially as described previously (194). Protein concentrations

were determined by the bicinchoninic acid (BCA) assay (289). Ni-resin purification was

achieved using the High Capacity Nickel Chelate Affinity Matrix (Ni-CAM) purchased

from Sigma. A total of 5 mg of membrane protein was brought up to 1 mL with final

concentrations of 0.2% tegamineoxide WS-35, 0. 15 M NaC1, and 1 mM imidazole. The

purification procedure was accomplished using the batch method as described by the

manufacturer.

Immunoblot Analysis

 Proteins were loaded on a 15% tris-glycine SDS gel and transferred onto

nitrocellulose by electroblot. The b subunit antibody incubation was performed

essentially as described previously (195), using a 1:25,000 dilution of the anti-b subunit

antibodies. Secondary antibody incubation was performed with horseradish peroxidase-

linked donkey anti-rabbit antibody (1:50,000), and the antibody was detected by