Localization and membrane topology of EnvZ, a protein involved in osmoregulation of OmpF and OmpC in Escherichia coli.
OmpR and EnvZ, the protein products of the ompB locus, are regulatory components required for osmoexpression of outer membrane porin proteins, OmpF and OmpC, in Escherichia coli. EnvZ is considered to be an osmosensor which transmits signals across the membrane to OmpR, a transcriptional activator for ompF and ompC. We inserted the envZ gene into a high expression vector, pIN-III. Following cellular fractionation, EnvZ was found to be localized in the inner membrane. Sequence analysis revealed that the signal peptide-like N-terminal sequence was not removed from the purified EnvZ. A genetic approach using EnvZ/beta-lactamase fusion proteins was taken to determine the topology of EnvZ in the inner membrane. When beta-lactamase was fused after the N-terminal signal peptide-like sequence, ampicillin resistance, conferred by the beta-lactamase moiety of the fusion protein, was expressed. However, when beta-lactamase was fused after the second downstream apolar sequence, the cells showed very poor ampicillin resistance indicating that the enzyme was localized on the cytoplasmic side of the inner membrane. The results of this approach reveal that the hydrophilic region of EnvZ between the two apolar sequences is periplasmically localized and that the hydrophilic region downstream of the second apolar sequence is cytoplasmically directed. These results were confirmed by partial proteolysis of the fusion proteins in intact cells.
Forst
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Department of Biochemistry, Robert Wood Johnson Medical School, Rutgers, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
Comeau
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Norioka
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Inouye
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D000595:Amino Acid Sequence; D000667:Ampicillin / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis*; D001483:Base Sequence; D002462:Cell Membrane / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D010447:Peptide Hydrolases / Q000378:metabolism; D021382:Protein Sorting Signals / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014882:Water-Electrolyte Balance
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000667:Ampicillin; D010447:Peptide Hydrolases