Hale C A, Rhee A C, de Boer P A
Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4960, USA.
J Bacteriol. 2000 Sep;182(18):5153-66. doi: 10.1128/JB.182.18.5153-5166.2000.
FtsZ and ZipA are essential components of the septal ring apparatus, which mediates cell division in Escherichia coli. FtsZ is a cytoplasmic tubulin-like GTPase that forms protofilament-like homopolymers in vitro. In the cell, the protein assembles into a ring structure at the prospective division site early in the division cycle, and this marks the first recognized event in the assembly of the septal ring. ZipA is an inner membrane protein which is recruited to the nascent septal ring at a very early stage through a direct interaction with FtsZ. Using affinity blotting and protein localization techniques, we have determined which domain on each protein is both sufficient and required for the interaction between the two proteins in vitro as well as in vivo. The results show that ZipA binds to residues confined to the 20 C-terminal amino acids of FtsZ. The FtsZ binding (FZB) domain of ZipA is significantly larger and encompasses the C-terminal 143 residues of ZipA. Significantly, we find that the FZB domain of ZipA is also required and sufficient to induce dramatic bundling of FtsZ protofilaments in vitro. Consistent with the notion that the ability to bind and bundle FtsZ polymers is essential to the function of ZipA, we find that ZipA derivatives lacking an intact FZB domain fail to support cell division in cells depleted for the native protein. Interestingly, ZipA derivatives which do contain an intact FZB domain but which lack the N-terminal membrane anchor or in which this anchor is replaced with the heterologous anchor of the DjlA protein also fail to rescue ZipA(-) cells. Thus, in addition to the C-terminal FZB domain, the N-terminal domain of ZipA is required for ZipA function. Furthermore, the essential properties of the N domain may be more specific than merely acting as a membrane anchor.
FtsZ和ZipA是隔膜环装置的重要组成部分，该装置介导大肠杆菌中的细胞分裂。FtsZ是一种细胞质微管蛋白样GTP酶，在体外形成原丝样同聚物。在细胞中，该蛋白在分裂周期早期于预期分裂位点组装成环形结构，这标志着隔膜环组装中第一个被识别的事件。ZipA是一种内膜蛋白，通过与FtsZ的直接相互作用在非常早期阶段被招募到新生的隔膜环。使用亲和印迹和蛋白质定位技术，我们已经确定了每种蛋白质上的哪个结构域在体外以及体内对于两种蛋白质之间的相互作用既是充分的又是必需的。结果表明，ZipA与FtsZ的20个C末端氨基酸限定的残基结合。ZipA的FtsZ结合（FZB）结构域明显更大，包含ZipA的C末端143个残基。重要的是，我们发现ZipA的FZB结构域在体外诱导FtsZ原丝的显著成束也是必需的和充分的。与ZipA结合和捆绑FtsZ聚合物的能力对其功能至关重要的观点一致，我们发现缺乏完整FZB结构域的ZipA衍生物在耗尽天然蛋白的细胞中无法支持细胞分裂。有趣的是，确实包含完整FZB结构域但缺乏N末端膜锚或其中该锚被DjlA蛋白的异源锚替代的ZipA衍生物也无法拯救ZipA（-）细胞。因此，除了C末端FZB结构域之外，ZipA的N末端结构域对于ZipA功能也是必需的。此外，N结构域的基本特性可能比仅仅作为膜锚更具特异性。
