FIG 6.

In vivo, the majority of SecA is not in contact with the ribosome. (A) In vivo cross-linking of E. coli cells expressing either wild-type uL23 or uL23 variants containing pBpa either at position 52 or 71. Exponentially growing cells were harvested, and one-half of each culture was exposed to UV light, while the other was kept in the dark. After cell breakage, the cytosolic fraction was centrifuged in high-salt buffer through a 50% sucrose cushion for ribosome enrichment. After SDS-PAGE and Western blotting, the membrane was decorated with anti-SecA (top blots) and anti-Ffh (bottom blots) antibodies. (B) As in panel A, but after cell breakage, the membrane fraction was enriched via sucrose gradient centrifugation and analyzed as in panel A. Representative blots of at least three independent replicates are shown.