FIGURE 5.

Translocation activity of the SecY complex in the presence of Syd. A, SecY complex, wild-type (WT) or carrying the mutation secY24(G240D) or SecEΔ7-67, was overproduced in E. coli, and the IMVs were immunostained with anti-SecY antibodies. The SecY mutant complex cannot be overproduced to the same level as the wild type, probably due to a higher instability. B, IMVs were urea-stripped, and their concentration was adjusted so that a comparable amount of SecY complex is tested in the corresponding in vitro translocation assays. The IMVs were premixed with the indicated amount of purified Syd before addition of the translocation substrate ¹²⁵I-proOmpA and SecA (0.25 μg). Translocation was initiated with ATP (1 mm) for 10 min at 37 °C. After proteinase K digestion (10 min on ice), samples were analyzed by SDS-PAGE and phosphorimaging. The results were quantified by densitometry and expressed on the graph curve (right panel).