Subunit organization in a soluble complex of tar, CheW, and CheA by electron microscopy.
The Salmonella and Escherichia coli aspartate receptor, Tar, is representative of a large class of membrane receptors that generate chemotaxis responses by regulating the activity of an associated histidine protein kinase, CheA. Tar is composed of an NH(2)-terminal periplasmic ligand-binding domain linked through a transmembrane sequence to a COOH-terminal coiled-coil signaling domain in the cytoplasm. The isolated cytoplasmic domain of Tar fused to a leucine zipper sequence forms a soluble complex with CheA and the Src homology 3-like kinase activator, CheW. Activity of the CheA kinase in the soluble complex is essentially the same as in fully active complexes with the intact receptor in the membrane. The soluble complex is composed of approximately 28 receptor cytoplasmic domain chains, 6 CheW chains, and 4 CheA chains. It has a molecular weight of 1,400,000 (Liu, I., Levit, M., Lurz, R., Surette, M.G., and Stock, J.B. (1997) EMBO J. 16, 7231-7240). Electron microscopy reveals an elongated barrel-like structure with a largely hollow center. Immunoelectron microscopy has provided a general picture of the subunit and domain organization of the complex. CheA and CheW appear to be in the middle of the complex with the leucine zippers of the receptor construct at the ends. These findings show that the receptor signaling complex forms higher ordered structures with defined geometric architectures. Coupled with atomic models of the subunits, our results provide insights into the functional architecture by which the receptor regulates CheA kinase activity during bacterial chemotaxis.
Francis
Noreen R
NR
W. M. Keck Institute for Cellular Visualization, Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454, USA.
Levit
Mikhail N
MN
Shaikh
Tanvir R
TR
Melanson
Linda A
LA
Stock
Jeffry B
JB
DeRosier
David J
DJ
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000648:ultrastructure; D002628:Chemoreceptor Cells; D002633:Chemotaxis; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism / Q000648:ultrastructure*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D000071677:Histidine Kinase; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008854:Microscopy, Electron; D016253:Microscopy, Immunoelectron; D008954:Models, Biological; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction
D001426:Bacterial Proteins; C489103:CheW protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D011956:Receptors, Cell Surface; C057331:Tar protein, E coli; C051236:CheW protein, Bacteria; D000071677:Histidine Kinase; C000607608:cheA protein, E coli