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. 2020 Dec 15;16(12):e9530. doi: 10.15252/msb.20209530

Figure 4. Validation of selected Lon and FtsH candidate substrates.

Figure 4

  • A
    Transcription profiles of ΔIndLon mutants grown in inducing (+) or depleting (−) conditions across the cell division and the MPN525 (DnaB) operons. The respective estimated protein copy numbers based on MS data are shown below.
  • B
    Time course experiments showing depletion levels of Lon over time and the corresponding accumulation of N‐terminal FLAG‐tagged derivatives of candidate substrates associated with cell division, including FtsA, FtsZ, and DnaB. Protein levels of Lon and candidate substrates were assessed by immunoblot analysis using anti‐Lon and anti‐FLAG antibodies. LC, loading control.
  • C
    In vivo degradation assays of FtsA, FtsZ, and DnaB. ΔIndLon mutants expressing the different N‐terminal FLAG‐tagged derivatives were grown in depleting conditions for 36 h. Then, Lon expression was transiently induced for 3 h before translation was blocked with gentamicin (Gm). Samples were taken at the indicated time points after gentamicin treatment, and processed for immunoblot analysis using anti‐FLAG antibodies. LC, loading control. As controls, non‐induced cells were also treated with gentamicin and processed at the same time points. Immunoblots are representative of two independent experiments.
  • D
    Estimated protein copy numbers based on MS data of components associated with the R‐M system.
  • E, F
    Similar to panel B (E) and C (F) but for a C‐terminal FLAG‐tagged derivative of MPN201 as a representative of an unstable HsdS subunit.
  • G
    Estimated protein copy numbers based on MS data of FtsH candidate substrates.
  • H
    Similar to panel B but for C‐terminal FLAG‐tagged derivatives of FtsH candidate substrates. Protein levels of FtsH and candidate substrates were assessed by immunoblot analysis using anti‐FtsH and anti‐FLAG antibodies. LC, loading control.
  • I
    Similar to panel C, but for FtsH candidate substrates. In this case, C‐terminal FLAG‐tagged derivatives were grown in depleting conditions for 60 h before transient expression of FtsH for 3 h and gentamicin treatment.