Geometric cue for protein localization in a bacterium

Abstract

Proteins in bacteria often deploy to particular places within the cell, but the cues for localization are frequently mysterious. We found that the peripheral membrane protein SpoVM (VM) recognizes a geometric cue when localizing to a particular membrane during sporulation in Bacillus subtilis. Sporulation involves an inner cell maturing into a spore and an outer cell nurturing the developing spore. VM is produced in the outer cell, where it embeds in the membrane that surrounds the inner cell but not in the cytoplasmic membrane of the outer cell. We found that VM localized by discriminating between the positive curvature of the membrane surrounding the inner cell and the negative curvature of the cytoplasmic membrane. Membrane curvature could be a general cue for protein localization in bacteria.

Figure 1. VM-GFP localizes selectively to the surface of the forespore

Procedures (11). (A) Stages of sporulation. Top, division creates a mother cell (left) and a forespore (right). Middle, the mother cell engulfs the forespore. Bottom, the forespore is pinched off as a protoplast. (B) α-helical model of VM. Shown on the left is the hydrophobic, membrane-embedded face and on the right a 180° rotation of the helix along its long axis with positively charged residues labeled green. (C) VM-GFP localizes to the surface of the forespore, whereas VM^P9A-GFP localizes to all membranes (D). (E-F) Membrane stained cells in (C-D). Arrowheads identify the cell depicted in the cartoon. (G-L) Localization of VM-GFP or VM^P9A-GFP produced after topological isolation. Membrane surrounding the forespore was stained by a membrane-permeable (G, J) but not by a membrane impermeable dye (H, K). VM-GFP (I), but not VM^P9A-GFP (L), localized selectively to the surface of the forespore. Scale bars are 2 μm.

Figure 2. Positive curvature is necessary and sufficient for localization

Localization of VM-GFP in wild type (A) and in mutant cells (ΔD/M/P) with a straight septum (B). (C) Localization of VM-GFP in cells of the ΔD/M/P mutant with bulges. (D-F) Membrane stained cells in (A-C). (G-J) Localization of VM-GFP (G and I) and VM^P9A-GFP (H and J) in mutant E. coli cells (ΔmreBCD) that produce internal vesicles. (I and J) Vesicles were visualized by phase contrast microscopy. (K-N) Localization of VM-GFP (K and M) and VM^P9A-GFP (L and N) in mutant yeast cells (ΔVPH1) that produced fragmented vacuoles. (I and J) Vacuoles were stained with FM4-64. Scale bars are 2 μm.

Figure 3. VM-GFP detects curvature in vitro

(A) Confocal fluorescence micrographs of purified VM-GFP (left) or VM^P9A-GFP (right) incubated with phospholipid vesicles. Scale bars are 20 μm. (B) Membrane fluorescence intensity as a function of vesicle diameter. Vesicles were incubated with similar concentrations of VM-GFP (closed circles) or VM^P9A-GFP (open squares) The data points are mean +/- SD measured on N vesicles (N=3-18) and were fit with an exponential decay (11). (C): Size distribution of fluorescent vesicles whose intensity is at least 2.5-fold above background.

Figure 4. Preferential adsorption of VM-GFP onto smaller vesicles is concentration dependent

(A) Membrane fluorescence intensity as a function of vesicle diameter for increasing bulk concentrations of VM-GFP. At higher concentrations, vesicles with a diameter below a critical value (D_c) of ∼ 4 μm were preferentially labeled. (B) Membrane fluorescence intensity as a function of VM-GFP concentration for different vesicle diameters (indicated diameters are +/- 0.5 μm). Above D_c, all data points were similar and well approximated by a linear fit (dotted line), whereas below D_c, the isotherms were steeper and deviated from linear. (C) Typical behaviors for small (1.5 +/- 0.5 μm) and large (6.5 +/- 0.5 μm) vesicles, and theoretical curves obtained using a model for cooperativity (11). Data points are mean +/- SD measured on N vesicles (N=3-26), resulting from 3 independent experiments.