Fig. 6: Porin binding is required for PapS localization.

a Model of the interaction interfaces of R. rubrum PapS (turquoise) and Por39 (light grey) or Por41 (dark grey), generated with AlphaFold-Multimer⁴⁸. Amino acids predicted to be involved in the interaction are shown in stick representation and labeled. b Colocalization of PapS_W22A/W58A-mNG with mCh-Por39 in the ΔpapS background. Cells (SP146) were cultivated in the absence (untreated) or presence (+) of cefalexin prior to imaging. The images show representative cells analyzed by widefield epifluorescence microscopy (untreated) and 3D-SIM (+ Cefalexin) (n = 3 biological replicates). Bar: 5 µm. The area overlap of the PapS_W22A/W58A-mNG and mCh-Por39 signals in the 3D-SIM images is 60.7% (see also Supplementary Data 4). c Superplots showing the distribution of cell sinuosities in populations of ΔpapS cells producing wild-type PapS (JR54), the porin-binding-deficient variant PapS_W22A/W58A (SP186), or the porin- and peptidoglycan-binding-deficient variant PapS_{W22A/W58A/R223A} (SP187) from a low-copy plasmid. The data are presented as described for Fig. 1f (n = 100 cells per replicate). The statistical significance (p values) of differences between cells producing the wild-type and the mutant proteins is indicated (unpaired two-sided Welch’s t-test). d Superplots showing a quantification of the outer-versus-inner-curve mNG signal ratios in ΔpapS cells producing wild-type PapS-mNG (SP136), the porin-binding-deficient variant PapS_W22A/W58A-mNG (SP197) or the porin- and peptidoglycan-binding-deficient variant PapS_{W22A/W58A/R223A}-mNG (SP174) from a low-copy plasmid. The data are presented as described for Fig. 1f (n = 100 cells per replicate). The results for wild-type PapS-mNG are replotted from Fig. 3e as a reference. The statistical significance (p values) of differences between cells producing the wild-type and the mutant proteins is indicated (unpaired two-sided Welch’s t-test). e SDS gel showing the presence or absence of porin-PapS complexes in Triton extracts of R. rubrum wild-type (S1) and ΔpapS (JR52) cells and of cells producing wild-type PapS (JR54) or PapS_W22A/W58A (SP186) from a low-copy plasmid. f Bubble plots showing the apparent single-molecule diffusion rates of different PapS-PAmCh variants in the ΔpapS background. Cells producing wild-type PapS-PAmCh (SP165), the porin-binding-deficient variant PapS_W22A/W58A-PAmCh (SP168), or the porin- and peptidoglycan-binding-deficient variant PapS_{W22A/W58A/R223A}-PAmCh (SP175) from a low-copy plasmid were subjected to SPT analysis. The results for cells producing wild-type PapS-PAmCh are replotted from Fig. 3f as a reference. The diffusive and immobile populations have mean apparent diffusion coefficients of 0.0657 µm² s^-1 and 0.0191 µm² s^-1, respectively. The number of cells analyzed is given in Supplementary Data 3. Source data are provided as a Source Data file.