title: Effects of phospholipid composition on MinD-membrane interactions in vitro and in vivo.
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.
authors.affiliation: Department of Biochemistry, The University of Texas Medical School, Houston, Texas 77030, USA.
authors.forename: Eugenia
authors.initials: E
authors.lastname: Mileykovskaya
authors.forename: Itzhak
authors.initials: I
authors.lastname: Fishov
authors.forename: Xueyao
authors.initials: X
authors.lastname: Fu
authors.forename: Brian D
authors.initials: BD
authors.lastname: Corbin
authors.forename: William
authors.initials: W
authors.lastname: Margolin
authors.forename: William
authors.initials: W
authors.lastname: Dowhan
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry*; D008164:Luminescent Proteins / Q000235:genetics; D010743:Phospholipids / Q000737:chemistry* / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000737:chemistry
chemical_list: D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008081:Liposomes; D008164:Luminescent Proteins; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D013439:Sulfhydryl Reagents; D049452:Green Fluorescent Proteins; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D000251:Adenosine Triphosphatases; C072069:MinD protein, E coli