Fluid membranes with acidic domains activate DnaA, the initiator protein of replication in Escherichia coli.
Acidic phospholipids in a fluid phase dissociate ADP or ATP tightly bound to DnaA protein and, in the presence of ATP and DNA, can restore an inactive ADP form to full activity (Sekimizu, K., and Kornberg, A. (1988) J. Biol. Chem. 263, 7131-7135). Further studies of the interactions between DnaA protein and lipids have used two functional assays: 1) release of ADP or ATP from DnaA and 2) DNA replication upon rejuvenation of an inactive ADP-DnaA protein complex. Among a variety of phospholipids tested were pure synthetic compounds and the mixtures from Escherichia coli auxotrophs (fabA), which are unable to synthesize unsaturated fatty acids and can be supplemented with different acyl derivatives. Fatty acid composition was determined by gas-liquid chromatography and membrane fluidity by fluorescence spectroscopy using 1,6-diphenyl-1,3,5-hexatriene as a probe. Lipid requirements of DnaA protein were shown to be: 1) phospholipids in a fluid phase (i.e. above the transition temperature), 2) a charged polar head group, 3) a lamellar phase (i.e. hexagonal II structures were inactive), and 4) a certain degree of fluidity imparted by the fatty acids esterified to the glycerol backbone. This conclusion was based on the incorporation of: 1) cholesterol, known to increase the packing of lipids, or 2) a branched fatty acyl derivative, which exhibits a fluidizing effect similar to that of a cis double bond. Both agents demonstrated that membrane fluidity is required for DnaA protein function in vitro, consistent with early studies of chromosome initiation in growing cells.
Castuma
C E
CE
Department of Biochemistry, Stanford University School of Medicine, California 94305-5307.
Crooke
E
E
Kornberg
A
A
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D004951:Esterification; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers; D008560:Membrane Fluidity; D010743:Phospholipids / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D005231:Fatty Acids, Unsaturated; D008051:Lipid Bilayers; D010743:Phospholipids; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate