Fig. 4. PopZ recruits SpmX to the stalked pole through a periplasmic intermediary.

A. Localization of PopZ-YFP in ΔspmX cells (strain GB507).

B. Localization of SpmX-mCherry in wild-type (strain GB378) and ΔpopZ cells (strain GB387).

C. Quantification of the data in (B), as described in Experimental procedures. Peak refers to the intensity of the localized signal; Deloc refers to the median intensity of the delocalized signal across the cell body.

D. A Western blot showing the cellular levels of SpmX-mCherry protein in native popZ and ΔpopZ backgrounds.

E. Localization of SpmX-mCherry in FtsZ depletion strain GB574. Cells were grown in PYE medium supplemented with 0.03% xylose, then washed and grown for 3.5 hours in the same medium (upper panel) or PYE without xylose (lower panel).

F. A schematic of the spmX coding sequence (upper panel), with transmembrane domains (TM) indicated in black. The N-terminal localization domain (blue) is predicted to be exported to the periplasm. Localization (lower panel) of SpmX(1–350)-mCherry in wild-type (strain GB440) and ΔpopZ cells (strain GB447). SpmX(1–350)-mCherry expression was induced by growth in 50 µM vanillate for two hours prior to analysis.

G. Effects of PopZ overproduction on the localization of polar proteins. Top panel: DivJ-GFP localization during mCherry-PopZ overproduction (strain GB449). Middle panels: SpmX-mCherry and SpmX(1–350)-mCherry localization during PopZ-GFP overproduction (Strains GB430 and GB453). Lower panel: TipN-GFP localization during mCherry-PopZ overproduction (strain GB450). To achieve PopZ overproduction, cells were stimulated by growth in 0.3% xylose for 5 h prior to analysis.

H. DivJ-mCherry remains delocalized in a ΔspmX mutant in the context of PopZ-GFP overproduction (strain GB514). For PopZ-GFP overproduction, cells were stimulated as in (F). For all experiments, strains were grown in PYE medium and coloured fluorescence images are placed on a phase-contrast background.