Membrane localization of MinD is mediated by a C-terminal motif that is conserved across eubacteria, archaea, and chloroplasts

Abstract

MinD is a widely conserved ATPase that has been demonstrated to play a pivotal role in selection of the division site in eubacteria and chloroplasts. It is a member of the large ParA superfamily of ATPases that are characterized by a deviant Walker-type ATP-binding motif. MinD localizes to the cytoplasmic face of the inner membrane in Escherichia coli, and its association with the inner membrane is a prerequisite for membrane recruitment of the septation inhibitor MinC. However, the mechanism by which MinD associates with the membrane has proved enigmatic; it seems to lack a transmembrane domain and the amino acid sequence is devoid of hydrophobic tracts that might predispose the protein to interaction with lipids. In this study, we show that the extreme C-terminal region of MinD contains a highly conserved 8- to 12-residue sequence motif that is essential for membrane localization of the protein. We provide evidence that this motif forms an amphipathic helix that most likely mediates a direct interaction between MinD and membrane phospholipids. A model is proposed whereby the membrane-targeting motif mediates the rapid cycles of membrane attachment-release-reattachment that are presumed to occur during pole-to-pole oscillation of MinD in E. coli.

Fig 1.

Localization of EcMinD truncation mutants. (A) Illustration of the EcMinD constructs used in this study. EcMinDΔX indicates a C-terminal deletion of X residues. (B–G) Fluorescence micrographs showing localization in E. coli of unfused GFP, GFP-EcMinD, and GFP-tagged C-terminal truncation mutants of EcMinD (see text for details). (H) Western blot comparing the cellular levels of EcMinD and various C-terminal truncation mutants. The intense upper band is MinD and the weak lower band is presumed to be a MinD degradation product. (I) Far-UV CD spectra of His₆-EcMinD (○) and His₆-EcMinDΔ19 (•). MRE is the mean residue ellipticity in degrees⋅cm²⋅dmol⁻¹.

Fig 2.

The C terminus of MinD contains a widely conserved sequence motif. An alignment of the extreme C-terminal region of EcMinD with the corresponding region of MinD from various organisms is shown. Residues identical to the E. coli sequence are boxed in yellow and conservative substitutions are pink. The C-terminal residue is numbered (blue text to the right of each sequence). The consensus helical region (see text) is indicated by the cylinder above the sequences. The putative MTS, which is boxed in red, is conserved across eubacteria, archaea, and chloroplasts.

Fig 3.

B. subtilis MinD localizes to the membrane in E. coli. Fluorescence micrographs show localization in E. coli of BsMinD (A), BsMinDΔ24 (B), BsMinDΔ5 (C), and BsMinDΔ3 (D). (E) Illustration of two of the BsMinD constructs used in this study.

Fig 4.

The helicity and amphipathicity of the MTS is important for its membrane-targeting function. (A–F) Helical-wheel representations of the putative MinD MTS from the Gram-positive eubacterium B. subtilis (BsMinD) (A), the hyperthermophilic archaeon A. fulgidus (AfMinD) (B), the chloroplast of the unicellular green flagellate Mesostigma viride (MvMinD) (C), the Gram-negative eubacterium E. coli (EcMinD) (D); a mutant of EcMinD containing a three-residue insertion (Ala-Lys-Ile) between residues Leu-264 and Lys-265 of the MTS (EcMinDIns3) (E); and a mutant of EcMinD containing a two-residue insertion (Ala-Lys) between residues Leu-264 and Lys-265 of the MTS (EcMinDIns2) (F). Strongly hydrophobic residues are shown in red and positively charged residues are shown in blue. The N-terminal residue of each helix is numbered. Note the pronounced amphipathic nature of the WT sequences; one face of the helix is highly hydrophobic, whereas the other face is strongly polar and usually comprises several cationic residues. (G–I) Fluorescence micrographs showing localization in E. coli of EcMinDIns3 (G), EcMinDIns2 (H), and an L267E mutant of EcMinD (I).

Fig 5.

Model of the MinD membrane attachment–detachment cycle. In the absence of MinE (Lower), the C-terminal MTS of MinD forms an amphipathic helix (shaded cylinder) that interacts with the lipid bilayer. The helix most likely orients parallel to the membrane surface. Partial insertion of the helix into the cytoplasmic monolayer would allow residues such as Phe (F) and Leu (L) on the hydrophobic face of the amphipathic helix to interact with lipid acyl chains, whereas residues on the opposing polar face of the helix could interact with lipid headgroups. The numerous cationic residues (indicated by +) on the polar face of the MinD MTS probably make specific contacts with the headgroups of anionic phospholipids (indicated by −). We presume that MinE causes detachment of the MTS, thus releasing MinD from the membrane (Upper). It is unclear at present whether this release involves a direct interaction between MinE and the MTS, a conformational change in MinD provoked by MinE-induced ATP hydrolysis, or some other mechanism.