FIG. 3.
Effect of DcuSCys− concentration on dimer and tetramer formation in proteoliposomes (A) and in bacterial membranes of living cells (B). (A) Purified recombinant DcuSCys− (8 μg) was reconstituted in liposomes in the following protein/phospholipid ratios: 1:10 (lanes 1 and 2), 1:20 (lanes 3 and 4), 1:50 (lanes 5 and 6), and 1:100 (lanes 7 and 8). Samples were subjected to SDS-PAGE in the presence of DTT before and after cross-linking with disuccinimidyl suberate (DSS), blotted to nitrocellulose membranes (Protran), and immunostained with anti-DcuS. For calibration, PageRuler Plus prestained protein ladder (Fermentas) was used. (B) E. coli JM109(pMW967) (expressing DcuSCys−) was grown aerobically in LB broth in the presence of 0 μM (lane 2), 10 μM (lane 3), 50 μM (lane 4), 90 μM (lane 5), 133 μM (lanes 6 and 8), or 333 μM (lane 7) arabinose and cross-linked with DSS. Sixty micrograms (lanes 4 to 8), 180 μg (lane 3), or 230 μg (lane 2) of cell lysates were subjected to SDS-PAGE. DcuS was detected by Western blotting with antiserum against the periplasmic domain of DcuS. Lane 1 contains 1 μg of purified DcuSCys−. The anti-DcuS-positive bands of 61, 120, and 240 kDa correspond to monomeric, dimeric, and tetrameric DcuS, respectively.