Fig. 4.

Association of UncI protein with c-subunit and release of c₁₁-ring from UncI by Na⁺. (A) SDS/PAGE analysis of UncI_His-bound components. Lane 1, hF₁F_o⁽⁺ⁱ⁾. Membrane fractions of cells expressing pTR-I_Hisc (lane 2) were solubilized with Triton X-100. Solubilized proteins were isolated by ultracentrifugation (lane 3) and loaded on a Ni-NTA column. After washing the column, the proteins were eluted (lane 4), and the eluate was further treated with 10% trichloroacetic acid (lane 5). All of the buffers did not contain Na⁺. nc₁₁, aggregated c₁₁-ring; 2UncI_His, dimer UncI_His; digests, proteolytic products of UncI_His. Proteins on the gel were stained by Coomassie brilliant blue (CBB). (B) Effect of Na⁺ on the interaction between UncI_His and c-subunit. The eluate from the Ni-NTA column was directly subjected to SDS/PAGE without prior concentration procedures, and the proteins were stained with silver staining. UncI_His was only poorly stained by silver. Lane 6, the same as lane 4. Lane 7, the loaded Ni-NTA column was washed with the Na⁺-containing washing buffer (containing 100 mM NaCl) and eluted. (C) Detection of UncI in hF₁F_o^(−i)(+I). Membrane fractions of cells expressing pTR-hF₁F_o⁽⁻ⁱ⁾ ± pST-I were solubilized and loaded on a Ni-NTA column. The column was washed with the (Na⁺-free) washing buffer (lanes 8 and 10) or with the Na⁺-containing washing buffer (lanes 9 and 11). The hF₁F_o^(−i)(+I) was eluted, reacted with tetramethylrhodamine maleimide to label cysteine residues in UncI, and subjected to SDS/PAGE analysis. The proteins were visualized by CBB staining (lanes 8 and 9) or by fluorescence (excited at 302 nm, emission at >400 nm) (lanes 10 and 11). UncI proteins are indicated with arrowheads. Different from the gel in Fig. 1B, the band of UncI was visible even by CBB staining because the fluorescent dye labeling enhanced the sensitivity of UncI to the CBB staining.