Figure 5.

In strain Newman, the L18P mutation transforms SaeS into an FtsH substrate. (A) Effect of the ftsH deletion on the protein level of SaeS L18P. Cells were grown in TSB; then SaeS was detected by Western blot analysis. WT, wild type; ΔftsH, ftsH deletion mutant; saeRS^P, the sae-deletion mutant of Newman expressing SaeR and SaeS L18P (i.e., NMΔsae[pCL-RS^P]); saeRS^L, the sae-deletion mutant of Newman expressing SaeR and the wild type SaeS (i.e., NMΔsae[pCL-RS^L]); Δsae, the sae-deletion mutant of Newman. Sortase A (SrtA), a membrane protein, was used as a loading control. The full-length blots are presented in Supplementary Fig. 9A. (B) Role of the FtsH enzymatic activities on the degradation of SaeS L18P. Proteins were detected by Western blot analysis. To detect the FtsH protein expressed from the complementation plasmids, anti-His₆-tag antibody was used. Sortase A (SrtA) was used as a loading control. WT, wild type Newman; ΔftsH, the ftsH-deletion mutant of Newman; -, no plasmid; VC, the vector control pCL55; pftsH, pCL55 carrying the wild type copy of ftsH; pK211N, pCL55 carrying the ftsH gene with K211N mutation at the ATPase domain; pH431A, pCL55 carrying the ftsH gene with H431A mutation at the protease domain; Δsae, the sae-deletion mutant of Newman. The full-length blots are presented in Supplementary Fig. 9B. (C) Proteolysis of various forms of SaeS proteins by membrane vesicles containing FtsH. The membrane vesicles were mixed with the purified SaeS proteins; then the proteolysis was initiated by addition of ATP. Quantification results are presented in Supplementary Fig. 9C.