FtsY-depleted inner membrane vesicles are unable to support co-translational targeting. (A) MtlA189 RNCs were incubated with wild-type (wt) INV, FtsY-containing IY28 INV (FtsY+) and FtsY-depleted IY28 INV (FtsY) (1 μl, 50 μg protein) and subjected to flotation gradient centrifugation. Subsequently, the gradient was separated into five fractions, which were analysed on SDS-PAGE. Fraction 2 and 3 of the gradient correspond to the membrane fraction, while fraction 4 and 5 reflect the ribosome-nascent chains that did not bind to the inner membrane vesicles [INV]. The sum of the radioactive material in all fractions was set as 100% and the amount of radioactive material in the individual fractions was quantified using a phosphor imager. (B) Flotation gradient analyses as in A, but FtsY-depleted IY28 INV were pre-incubated with purified FtsY (2 μg/25 μl; reconst. IY28). (C) Western blot analyses of the INV (5 μl; 250 μg protein) analysed in A and B using polyclonal antibodies against FtsY and against the integral membrane protein YidC. (D) Western blot analyses of the in vitro transcription/translation system used in this study using polyclonal anti-FtsY antibodies. Two equivalents of the in vitro system components were loaded. One equivalent corresponds to the amount required for in vitro protein synthesis. As control wild-type INV (200 μg protein) and purified FtsY (0.1 μg) were loaded.
Mircheva et al. BMC Biology 2009 7:76 doi:10.1186/1741-7007-7-76