Abstract
The peripheral membrane ATPase MinD is a component of the Min system responsible for correct placement of the division site in Escherichia coli cells. By rapidly migrating from one cell pole to the other, MinD helps to block unwanted septation events at the poles. MinD is an amphitropic protein that is localized to the membrane in its ATP-bound form. A C-terminal domain essential for membrane localization is predicted to be an amphipathic alpha-helix with hydrophobic residues interacting with lipid acyl chains and cationic residues on the opposite face of the helix interacting with the head groups of anionic phospholipids (Szeto, T. H., Rowland, S. L., Rothfield, L. I., and King, G. F. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 15693-15698). To investigate whether E. coli MinD displays a preference for anionic phospholipids, we first examined the localization dynamics of a green fluorescent protein-tagged derivative of MinD expressed in a mutant of E. coli that lacks phosphatidylethanolamine. In these cells, which contain only anionic phospholipids (phosphatidylglycerol and cardiolipin), green fluorescent protein-MinD assembled into dynamic focal clusters instead of the broad zones typical of cells with normal phospholipid content. In experiments with liposomes composed of only zwitterionic, only anionic, or a mixture of anionic and zwitterionic phospholipids, purified MinD bound to these liposomes in the presence of ATP with positive cooperativity with respect to the protein concentration and exhibited Hill coefficients of about 2. Oligomerization of MinD on the liposome surface also was detected by fluorescence resonance energy transfer between MinD molecules labeled with different fluorescent probes. The affinity of MinD-ATP for anionic liposomes as well as liposomes composed of both anionic and zwitterionic phospholipids increased 9- and 2-fold, respectively, relative to zwitterionic liposomes. The degree of acyl chain unsaturation contributed positively to binding strength. These results suggest that MinD has a preference for anionic phospholipids and that MinD oscillation behavior, and therefore cell division site selection, may be regulated by membrane phospholipid composition.
Highlights
MinD protein, along with MinC and MinE, is required for selection of the correct placement of the division site in bacterial cells [1]
Because MinC binds to MinD, the movement of MinC from pole to pole with relatively long polar dwell times and a short transit time blocks the formation of polar Z-rings but not medial rings [10, 11]
GFP-MinD in PE-lacking Cells—We demonstrated previously that E. coli cells lacking PE were filamentous because of inhibition of cell division
Summary
Strains and Growth Conditions—To construct a phosphatidylethanolamine (PE)1-deficient strain expressing a green fluorescent protein. Curing of plasmid pDD72 as described previously [18] resulted in strain AD90/pWM1255, which lacks PE (contains only phosphatidylglycerol (PG) and cardiolipin (CL)) but maintains the same membrane protein-to-phospholipid ratio as wild-type cells made up of solely PG and CL [18]. Fractions containing His-MinD were desalted on Sephadex G-25 columns equilibrated with 50 mM HEPES, pH 7.2, 150 mM KCl, 0.1 mM EDTA, and 10% glycerol (storage buffer) and were frozen in 0.2-ml aliquots at Ϫ80 °C. MinD (0.2–20 M) was mixed with 1 mM ATP or ADP and liposomes (160 g/ml) in 100 l of buffer containing 25 mM Tris-HCl, pH 7.5, 50 mM KCl, and 5 mM MgCl2 and was incubated for 10 min at 30 °C. Samples contained 1 M total MinD protein in the same buffer that was used for the liposome sedimentation assay. A sample was excited at 322 nm (slit width 4 nm), and emitted light was scanned from 350 to 600 nm (slit width 4 nm, scan rate 100 nm/min)
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