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 recognizes a geometric cue in 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. SpoVM 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 SpoVM 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.Proteins often localize to particular positions within bacteria, sometimes in a dynamic manner. A striking but mysterious example of subcellular localization occurs during spore formation in Bacillus subtilis when SpoVM (VM) localizes to a particular patch of membrane (1). How VM discriminates between different membrane surfaces in the same cell is unknown.During sporulation, the cell divides asymmetrically to create mother cell and forespore compartments. Next, the mother cell engulfs the forespore, enveloping it with inner and outer membranes (Fig. 1A). Following engulfment a protein coat is deposited around the outer forespore membrane (2). Coat assembly depends on VM, a 26-residue peptide that is produced in the mother cell (3). VM is an amphipathic α-helix (4) that inserts into the membrane with its long axis parallel to the membrane and its hydrophobic face buried in the lipid bilayer (5). During engulfment, VM localizes to the membrane that tracks around the forespore, eventually decorating the entire surface of the forespore, as visualized using a fusion to the Green Fluorescent Protein (VM-GFP; Fig 1C) (1). Proline 9 (P9; Fig. 1B) is critical for this localization (1), as substitution of P9 with alanine (VM P9A -GFP) resulted in localization to both the cytoplasmic and the outer forespore membranes (Fig. 1D).Following engulfment, the outer forespore membrane becomes topologically isolated from the cytoplasmic membrane. We wondered if VM would adhere to the outer forespore membrane after isolation. We engineered cells to produce VM-GFP in response to an inducer and triggered synthesis of the fusion protein after engulfment. To monitor topological isolation, we stained the membranes with a membrane permeable dye, which stains all membranes, and a membrane impermeable dye, which can only access the engulfment membrane before membrane fusion (6). VM-GFP localized almost exclusively to the outer forespore membrane even when the forespore was topologically isolated (Fig. 1G-I). As a control, VM P9A -GFP synthesized after * Publisher's Disclaimer: This manuscript has been accepted for publication in Science. This version has not undergone final editing.Please refer to the complete version of record at http://www....

Structural basis for the geometry-driven localization of a small protein

“…However, the mechanisms by which landmark proteins initially achieve their own proper localization are frequently unclear. Previously, we proposed that during sporulation in B. subtilis, the slightly convex surface of the developing forespore provides a geometric cue for the initial localization of the shape-sensing amphipathic α-helical protein SpoVM (24). In this report, we describe a previously unidentified structure-motivated mechanism by which SpoVM discriminates between differently curved membrane surfaces.…”

“…To determine the biochemical basis for the preferential adsorption of SpoVM onto slightly convex membrane surfaces, we first sought to generate a saturation-binding curve by incubating membrane surfaces of a given curvature with a range of concentrations of purified SpoVM-GFP. Previously, we demonstrated that purified SpoVM-GFP selectively bound to lipid vesicles similar in size to the forespore in a mixture of giant unilamellar vesicles of various sizes (24). In the current investigation, we eliminated limitations associated with giant unilamellar vesicles (variability in vesicle size and associated variability in membrane stiffness) by using SSLBs, in which a single phospholipid bilayer is assembled on the surface of silica beads of defined size (29,30).…”

“…Previously, we demonstrated that the landmark recognized by SpoVM is the slightly convex membrane surface of the forespore, the only convex surface in the mother cell cytosol (24). Membrane curvature recognition depends on a complex interplay of protein-lipid, protein-protein, and lipid-lipid interactions.…”

Cardiolipin microdomains localize to negatively curved regions of Escherichia coli membranes

“…Several observations suggest that cell curvature influences the distribution of cytoplasmic, amphiphilic proteins in bacteria. For example, in the rod-shaped bacterium B. subtilis, membrane curvature affects the localization of the sporulation factor protein SpoVM (via positive curvature) and multimers of the division protein DivIVA (via negative curvature) (13)(14)(15). The mismatch in length scales between a single molecule of DivIVA or SpoVM and the curvature of the membranes with which these proteins interact makes it unclear how individual proteins sense curvature.…”

Enterohemorrhagic Escherichia coli raises the I-BAR

“…In contrast, the IMDs fall within the I-BAR subfamily of BAR domains and cause convex deformation of membranes, inducing membrane protrusions; members of this subfamily, including IRSp53, have been shown to be involved in filopodia formation. Although BAR domain proteins have not been identified in bacteria, a recent report (9) describes the ability of the Bacillus subtilis peripheral membrane protein SpoVM to recognize the outer surface of spherical bacterial membranes or vesicles; although the structure of SpoVM has not yet been determined, the protein differs from known BAR domain proteins in that it forms an amphipathic ␣-helix with the hydrophobic face buried in the membrane and the positively-charged surface exposed (10).…”