Figure 3.

Juxtaposition of Pre6 and Pre8 in pre9Δ proteasomes revealed by disulfide engineering. (A) Control disulfide crosslinking in aqueous iodine of Pre8-HF(His/Flag)–Pre9-T7 for wild-type proteasomes analyzed by anti-Flag immunoblot. +DTT, reducing agent added prior to electrophoresis. (B) Anti-Flag immunoblot of CuCl₂-induced crosslinking of Pre8-HF to Pre6 in extracts from strains MHY2863 (lanes 5–9), MHY2865 (lanes 10–12), and MHY2867 (lanes 13–15). A parallel Pre8-HF–Pre9 (MHY1839; lanes 1–4) crosslinking reaction allowed direct comparison of crosslinking efficiency. Asterisk, an unknown cross-reacting band. (C) Crosslinking (CuCl₂) of two engineered His₆-tagged Pre6 subunits in pre9Δ proteasomes visualized by anti-His immunoblotting. Strains used were MHY2900 (lanes 1–3), MHY2901 (lanes 5–6), MHY2896 (lanes 7–8), and MHY2897 (lanes 9–10).