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
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
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DOI：
10.1074/jbc.m009810200
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发表时间：
2001-04-13
影响因子：
4.8
通讯作者：
de Boer, P
中科院分区：
文献类型：
--
作者：
Haney, SA;Glasfeld, E;de Boer, P
通过FtsZ将ZipA募集到隔膜是大肠杆菌细胞分裂的早期重要步骤。我们已经使用聚合酶链反应介导的随机诱变在酵母双杂交系统来分析这种相互作用，并确定了一个高度保守的序列内的C末端的FtsZ作为ZipA结合位点的残基。对导致相互作用丧失的突变（ftsZ（D373 G））的抑制子的搜索在该序列内的两个残基处鉴定了八个不同的变化。在体外，野生型FtsZ在酶联免疫吸附测定中以高亲和力与ZipA相互作用，而FtsZ（D373 G）未能相互作用。两个突变蛋白检测恢复这种相互作用显着。在体内，测试的等位基因比野生型ftsZ毒性显著更大，并且不能补充缺失。我们已经证明，在双杂交系统中编码FtsZ的最后70个残基的融合体足以与FtsA和ZipA相互作用。然而，当将野生型序列与编码FtsZD 373 G的序列进行比较时，未观察到与任一蛋白质的相互作用。Asp-373周围的突变差异影响FtsZ与ZipA和FtsA的相互作用，表明这些蛋白质与FtsZ的C末端结合不同。
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 FtsZD373G, 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.