FIG. 6.

Coisolation assay of MotB_C variants with MotB. MotB_C-His₆ designed to be exported into the periplasm (expressed from plasmid pNSK7, lane 1) and expressed in the cytoplasm (pNSK8, lane 2) was coexpressed with MotA and MotB (expressed from compatible plasmid pNSK31) (lanes 3 and 4). The expression levels of MotB and MotB_C were similar in both cases. The membranes were then isolated, solubilized with the detergent DPC, and mixed with Ni-NTA resin. MotB_C-His₆ and its associated proteins were eluted by imidazole, and samples prepared from each step were analyzed by immunoblotting with anti-MotB_C antibody. A monomeric variant of MotB_C [MotB_C(Δ197-210) expressed from pNSK7-Δ(197-210)] was also examined in the same way (lane 5). An alternative combination (MotA/MotB-His₈ and tagless MotB_C expressed from pNSK32 and pNSK28, respectively) was also examined (lane 6). Top panel, whole-cell extracts; middle panel, membrane fraction; bottom panel, eluted products. The 32.5-kDa marker position is shown at the left of each panel.