Functional reconstitution of the putative Escherichia coli osmosensor, KdpD, into liposomes.
Expression of the Escherichia coli kdpABC operon, which is responsible for a high-affinity potassium-uptake system, is regulated in response to a change in the medium osmolarity. We present the first in vivo evidence that the putative osmosensor (kinase), KdpD, plays a crucial role in the osmotic regulation of kdpABC expression. We then isolated the intact form of KdpD from the cytoplasmic membrane with a mild non-ionic detergent, dodecyl maloside, as a soluble form. The solubilized KdpD itself did not exhibit the ability to phosphorylate the cognate response-regulator, KdpE. Upon reconstitution into liposomes, however, KdpD exhibited its kinase activity. Thus, using this partially purified form of KdpD, we succeeded in reconstituting KdpD-bearing liposomes that are fully functional in terms of their ability to phosphorylate the cognate positive regulator, KdpE. By adopting this in vitro method, some in vivo conditions were mimicked in order to address the molecular mechanism underlying signal transduction mediated by KdpD. Some catalytic properties of KdpD in liposomes are also presented.
Nakashima
K
K
Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University.
Sugiura
A
A
Mizuno
T
T
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D009876:Operon; D009994:Osmolar Concentration; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008081:Liposomes; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C075615:kdpE protein, E coli; C489580:KdpD protein, E coli; D011494:Protein Kinases; C075616:kdpD protein, Bacteria; D001616:beta-Galactosidase