FIG 3.
The interaction of SecB with nascent substrates is dependent on the interaction of SecA with the ribosome in vivo. (A) Cells expressing wild-type L23 and wild-type SecA-biotin (DRH847) or (B) cells expressing L23FEVEVE/E89A and SecAKK-biotin (DRH933) were grown in M63 maltose containing IPTG and pulse-labeled with 35S-methionine, and SecB was immunoprecipitated from the cell lysates using specific antiserum. The coprecipitating proteins were separated by size using SDS-PAGE, gel slices of the lanes were subjected to in-gel proteolysis using the V8 protease, and the fragments were resolved in a second dimension in a second round of SDS-PAGE. The first dimension is depicted horizontally above. The running positions of full-length preLamB, mature LamB, preMBP, mature MBP, and SecB in the first dimension are indicated by arrows. The running positions of N-terminal proteolytic fragments of preMBP (p) and mature MBP (m) are indicated by arrowheads in the second dimension.