GTPase-dependent signaling in bacteria: characterization of a membrane-binding site for era in Escherichia coli.
Era is an Escherichia coli GTPase that is essential for cell viability and is peripherally associated with the cytoplasmic membrane. Both immunoelectron microscopy and subcellular-fractionation experiments have shown that Era is present in cytoplasmic as well as membrane-associated pools. These data led to speculation that the mechanism of action of Era may require cycling between membrane and cytoplasmic sites. In order to investigate this possibility, an in vitro binding assay was developed to characterize the binding of Era to membrane fractions. Competition and saturation binding experiments suggest that a site that is specific for Era and capable of binding up to 5 ng of Era per microgram of membrane protein is present in membrane preparations. The binding curve is complex, indicating that multiple equilibria describe the interaction. The binding of Era to this putative receptor is dependent on guanine nucleotides; binding cannot be measured in the absence of nucleotide, and neither ATP nor UTP can substitute. Subfractionation of cell walls showed that the guanine nucleotide-dependent binding site was present in fractions enriched in cytoplasmic membrane. These data provide evidence that Era may be involved in a GTPase-receptor-coupled membrane-signaling pathway that is essential for growth in E. coli.
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Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854-5635.
Sharer
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March
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D001667:Binding, Competitive; D015153:Blotting, Western; D002458:Cell Fractionation; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D008954:Models, Biological; D011485:Protein Binding / Q000187:drug effects; D016601:RNA-Binding Proteins; D015398:Signal Transduction
D029968:Escherichia coli Proteins; D006150:Guanine Nucleotides; D016601:RNA-Binding Proteins; C068027:era protein, E coli; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins