Genetic analysis of the Escherichia coli FtsZ.ZipA interaction in the yeast two-hybrid system. Characterization of FtsZ residues essential for the interactions with ZipA and with FtsA.
The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division in Escherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZ(D373G)) identified eight different changes at two residues within this sequence. In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZ(D373G) failed to interact. Two mutant proteins examined restored this interaction significantly. In vivo, the alleles tested are significantly more toxic than the wild type ftsZ and cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZ(D373G), no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.
Haney
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SA
Department of Infectious Disease, Wyeth-Ayerst Research, Pearl River, New York 10965 and the Department of Molecular Biology and Microbiology, Case Western Reserve University Medical School, Cleveland, Ohio 44106-4960.
Glasfeld
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E
Hale
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Keeney
D
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He
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Z
de Boer
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P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division / Q000235:genetics; D003598:Cytoskeletal Proteins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D029968:Escherichia coli Proteins; C071525:FtsZ protein, Bacteria; C103795:ZipA protein, E coli