FIGURE 4: Formylation-dependent selective destabilization of PpiB-based reporter proteins.

(A) Diagram of the expression cassette in which two identical tandem P_ara promoters express the C-terminally flag-tagged PpiB reference protein ^MVTF_wtPpiB_f and the reporter protein ^MYFYPpiB_f‑Ub (reporter-1). The latter differs from ^MVTF_wtPpiB_f by three amino acid residues adjacent to N‑terminal fMet, and by the presence of C-terminal Ub moiety, added to make the two proteins distinguishable by size.

(B) Lanes 1-6, formylation-lacking fmt E. coli were pulse-labeled with ³⁵S-methionine/cysteine for 1 min, followed by a chase (in the absence of chloramphenicol) for indicated times, extraction of proteins, immunoprecipitation with a monoclonal anti-flag antibody, SDS-PAGE, and autoradiography. Lanes 7-12, same but in wild‑type E. coli.

(C) Same as in (A), with the reference ^MVTF_wtPpiB_f and the reporter ^MDDDPpiB_f‑Ub (reporter-2), which differed from ^MYFYPpiB_f‑Ub in (A) and (B) by three residues (Asp‑Asp-Asp) downstream from N-terminal fMet.

(D) Same as in (B) but with ^MVTF_wtPpiB_f and ^MDDDPpiB_f‑Ub (reporter-2). See the main text for the logic and details of these experiments.