Extended Data Fig. 1: Identification of Glp as a cell division protein.
(a) Venn diagram showing the overlap between 3 independent SepF interactomes using Cglu or Cglu_SepF-Scarlet strains. Proteins only detected in each interactome were identified by comparison with control condition and using the probability mode (p value < 0.05) of Patternlab Venn diagram module following a bayesian model68. Proteins enriched in SepF Co-IPs when compared to controls were identified using pairwise comparison module of Patternlab V based on XIC intensities. 20, 22 and 98 proteins were detected as SepF interactors in Cglu/α-SepF (strain/antibody), Cglu_SepF-Scalet/α-SepF and Cglu_SepF-Scalet/α-Scarlet respectively. 12 proteins were common to all of Co-IPs, and for 11 of them an enrichment factor in relation to the total proteome could be calculated, and thus represent the core SepF interactome (Supplementary Table 1,a and Fig. 1a). One additional interactor, the hypothetical protein Cgl1805, could not be detected in the proteome and no enrichment factor could thus be reported. (b) Western blots of whole cell extracts (120 μg) from Cglu (lane 1) and Cglu_Δglp strains transformed with the empty plasmid (lane 2) or mNeon-Glp (lane 3). Glp and mNeon-Glp levels were revealed using the α-Glp antibody. Left: molecular weight markers (kDa) (c) Cellular localization of Cglu MoeA homologs MoeA1/Cgl0212 (25% aa sequence identity) and MoeA3/Cgl1196 (27% aa sequence identity). Representative images in phase contrast and mNeon fluorescent signal for Cglu_mNeon-MoeA1 and Cglu_mNeon-MoeA3. Both MoeA1 and MoeA3 are cytosolic, which contrasts with the mid-cell localization of mNeon-Glp shown in Fig. 1e. All Scale bars 5μm. (d) the cytoplasmic distribution was not due to fusion protein degradation as shown by Western blots of whole cell extracts (120 μg) from Cglu carrying mNeon-MoeA1, mNeon-Glp or mNeon-MoeA3 plasmids and revealed using an α-mNeon antibody. Left: molecular weight markers (kDa); Lanes 1: mNeon-MoeA1 (sucrose); 2: mNeon-MoeA1 (gluconate); 3: mNeon-Glp (sucrose); 4: mNeon-Glp (gluconate); 5: mNeon-MoeA3 (sucrose); 6: mNeon-MoeA3 (gluconate).
