FIGURE 7:

YidC binds to E. coli ribosomes. (A) Purified and salt-washed 70S ribosomes (500 nM) were incubated in binding buffer (pH 7.6). After 30 min of incubation on ice, ribosomes were centrifuged through a sucrose cushion. Pellet (P) and supernatant fractions (S) were separated on SDS gels and analyzed by Coomassie staining (top) and immunodetection using antibodies against L23 (bottom). (B) Increasing concentrations of salt-washed ribosomes were incubated with either 100 nM detergent-purified SecYEG (top) or detergent-purified YidC (bottom) in binding buffer (pH 7.6) and subjected to the same conditions as described in A. Pellet and supernatant fractions were immunodetected using antibodies against the N-terminal His tags of SecY or YidC. The percentage of binding was calculated using ImageJ software. (C) Quantification of at least three independent ribosome-binding experiments, using varying concentrations of SecYEG or YidC, respectively. The amount of ribosome-bound SecY/YidC was plotted against the total ribosome concentration.