Figure 3. SRP-dependent co-translational insertion of signal peptides plays a major role in the membrane localization of inner-membrane-protein mRNAs.

(A) Fusion constructs between different signal peptides and mMaple3. (B) Stacked phase contrast (gray) and STORM cross-section images (color) of example E. coli cells expressing mMaple3 fused to the signal peptide from an SRP-dependent protein FhuB. The cells were stained with FISH probes against mMaple3. (C) Left: Average long-axis cross-section images of cells expressing mMaple3 fused to SRP-dependent signal peptides derived from FhuB, CcmH, AcrB, and TolB. Right: Average long-axis cross-section images of cells expressing mMaple3 fusions to AcrB and TolB signal peptides without the start codon (-AUG). (D) Density profiles derived from the average long-axis cross-section images of mMaple3 fusions to the AcrB signal peptide with (red) and without (blue) the start codon. Density profile is as defined in Figure 1E. (E) Stacked phase contrast and STORM cross-section images of example E. coli cells expressing mMaple3 fused to a signal peptide derived from a SecB-dependent protein GlpQ. The cells were stained with FISH probes against mMaple3. (F) Average long-axis cross-section images of cells expressing mMaple3 fused to SecB-dependent signal peptides derived from GlpQ, LivJ, PhoA and MalE. (G) Stacked phase contrast and STORM cross-section images for example E. coli cells treated with the translation-initiation-inhibitor kasugamycin. The cells were stained with the FISH probe set against inner-membrane-protein mRNAs in the abundance range of 3–30 copies per cell. (H) Average long-axis cross-section images of cells in the presence (+Kas) and absence (-Kas) of kasugamycin. The cells were stained with the FISH probes against inner-membrane-protein mRNAs in the abundance range of 3–30 copies per cell. Average long-axis cross-section images in C, F and H were derived from all measured cells, tens to hundreds of cells in each case. Scale bars: 2 µm.

DOI: http://dx.doi.org/10.7554/eLife.13065.007

Figure 3—figure supplement 1. mRNA for the inner-membrane protein BglF is enriched at the membrane in a translation-dependent fashion.

(A) Cartoon depiction of an mRNA expressing a fusion between full length, wild-type bglF and mMaple3. The green hashed region represents a putative SRP signal, i.e. the first transmembrane domains in bglF. (B) Left panel: Stacked phase contrast (gray) and STORM cross-section images (color) of example E. coli cells stained with FISH probes to mMaple3. Right panel: Average long-axis cross section images derived from hundreds of cells. (C) Density profile for the cross section in (B). Density profile is as defined in Figure 1E. (D–F) As in (A–C) but for bglF construct in which the start codon has been replaced by a stop codon (TAA). (G–I) As in (A–C) but for a bglF construct in which the codon at position 202 has been replaced with a stop codon (TAA). The resulting construct expresses only the portion of bglF upstream of the putative SRP signal sequence. This blgF derivative is identical to a previously published derivative that was reported to be membrane enriched (Nevo-Dinur et al., 2011). All constructs were expressed from the AttB site on the chromosome. Scale bars: 2 µm.

DOI: http://dx.doi.org/10.7554/eLife.13065.008