Transmembrane signaling. Mutational analysis of the cytoplasmic linker region of Taz1-1, a Tar-EnvZ chimeric receptor in Escherichia coli.
Taz1-1 is Tar-EnvZ chimeric receptor that is able to induce ompC-lacZ expression in response to aspartate. Previous studies indicated that aspartate binding to the receptor domain of the Taz1-1 receptor modulated the ratio of kinase and phosphatase activities of the cytoplasmic signaling domain. The 80-residue segment of chemoreceptors that is located between the second transmembrane domain and the signaling domain was defined as the linker region. The Taz1-1 chimeric receptor contains 43 amino acid residues of the Tar linker region. In order to understand further the function of the linker region in transmembrane signaling, site-directed random mutagenesis was carried out on the conserved Ala231 in the linker region. Substitution mutations with Val, Glu, Gly, Thr, Lys and His gave the locked "off-mode" form (low ompC-lacZ expression), and substitution mutations with Ile and Leu resulted in the locked "on-mode" form (constitutive ompC-lacZ expression). All the mutant Taz1-1 receptors still retained both OmpR kinase and phospho-OmpR phosphatase activities. Interestingly Taz1N6, a kinase defective mutant, was able to complement with Taz1H1, a phosphatase defective mutant, carrying an off-mode mutant at position 231 to restore Asp-inducible ompC-lacZ expression, but not with Taz1H1 carrying an on-mode mutation. These results suggest that the residue at position 231 in Taz1-1 plays a key role in signal transduction.
Jin
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Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
Inouye
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D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D002628:Chemoreceptor Cells; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011494:Protein Kinases; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C057331:Tar protein, E coli; C072091:Taz1 protein, E coli; D011494:Protein Kinases; C057843:envZ protein, E coli; D010749:Phosphoprotein Phosphatases