FIG 4 .

Identification of MepM as a putative substrate of the LbcA-CtpA proteolytic complex. (A) Tandem affinity tag purification. Proteins were purified from detergent-solubilized lysates of ΔctpA strains. All strains had a plasmid encoding LbcA-FLAG, as well as a second plasmid encoding untagged CtpA (negative control), CtpA-His₆, or the proteolytically inactive CtpA-S302A-His₆. Purification was done with nickel agarose followed by anti-FLAG M2 agarose resin. Samples were separated on a 12.5% SDS-polyacrylamide gel, which was stained with silver. Molecular mass marker proteins (in kilodaltons) are labeled on the left-hand side. An irrelevant region of the gel between the marker and sample lanes has been removed. (B) Scatterplot of proteins identified by mass spectrometry after purification from strains encoding CtpA-S302A-His₆. Data are the average peptide spectrum matches (PSMs) and sequence coverage from duplicate purifications from strains with CtpA-S302A-His₆. Each point represents a different protein. All proteins plotted were detected in both of the LbcA–CtpA-S302A purifications but neither of duplicate LbcA–CtpA purifications. (C) Immunoblot analysis of equivalent amounts of whole-cell lysates of the wild-type or ΔctpA and ΔlbcA mutant strains. All strains contained an arabinose-inducible expression plasmid encoding MepM-FLAG and were grown in medium containing 0.2% (wt/vol) arabinose. Approximate positions of molecular mass marker proteins (in kilodaltons) are indicated on the left-hand side. MepM-FLAG was detected with anti-FLAG monoclonal antibodies, and CtpA and LbcA were detected with polyclonal antisera. (D) Detection of endogenous MepM. Shown are results from immunoblot analysis of equivalent amounts of whole-cell lysates of the wild-type and ΔctpA strains. Strains contained the tac promoter expression plasmid pVLT35 (−) or derivatives encoding CtpA or CtpA-S302A, as indicated. Approximate positions of molecular mass marker proteins (in kilodaltons) are indicated on the left-hand side. MepM, CtpA, and LbcA were detected with polyclonal antisera.