Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), 28049 Madrid, Spain.
J Biol Chem. 2013 Feb 1;288(5):3219-26. doi: 10.1074/jbc.M112.434944. Epub 2012 Dec 11.
In Escherichia coli, the cell division protein FtsZ is anchored to the cytoplasmic membrane by the action of the bitopic membrane protein ZipA and the cytoplasmic protein FtsA. Although the presence of both ZipA and FtsA is strictly indispensable for cell division, an FtsA gain-of-function mutant FtsA* (R286W) can bypass the ZipA requirement for cell division. This observation casts doubts on the role of ZipA and its need for cell division. Maxicells are nucleoid-free bacterial cells used as a whole cell in vitro system to probe protein-protein interactions without the need of protein purification. We show that ZipA protects FtsZ from the ClpXP-directed degradation observed in E. coli maxicells and that ZipA-stabilized FtsZ forms membrane-attached spiral-like structures in the bacterial cytoplasm. The overproduction of the FtsZ-binding ZipA domain is sufficient to protect FtsZ from degradation, whereas other C-terminal ZipA partial deletions lacking it are not. Individual overproduction of the proto-ring component FtsA or its gain-of-function mutant FtsA* does not result in FtsZ protection. Overproduction of FtsA or FtsA* together with ZipA does not interfere with the FtsZ protection. Moreover, neither FtsA nor FtsA* protects FtsZ when overproduced together with ZipA mutants lacking the FZB domain. We propose that ZipA protects FtsZ from degradation by ClpP by making the FtsZ site of interaction unavailable to the ClpX moiety of the ClpXP protease. This role cannot be replaced by either FtsA or FtsA*, suggesting a unique function for ZipA in proto-ring stability.
在大肠杆菌中，细胞分裂蛋白 FtsZ 通过双拓扑膜蛋白 ZipA 和细胞质蛋白 FtsA 的作用锚定在细胞质膜上。尽管 ZipA 和 FtsA 的存在对细胞分裂是严格必需的，但 FtsA 的功能获得突变体 FtsA*（R286W）可以绕过 ZipA 对细胞分裂的要求。这一观察结果使人对 ZipA 的作用及其对细胞分裂的必要性产生了怀疑。巨细胞是无核细菌细胞，作为一种整体细胞体外系统，用于探测蛋白质-蛋白质相互作用，而无需蛋白质纯化。我们表明，ZipA 保护 FtsZ 免受在大肠杆菌巨细胞中观察到的 ClpXP 定向降解，并且 ZipA 稳定的 FtsZ 在细菌细胞质中形成膜附着的螺旋状结构。FtsZ 结合的 ZipA 结构域的过表达足以保护 FtsZ 免受降解，而缺乏它的其他 C 端 ZipA 部分缺失则不行。单体过表达 FtsA 或其功能获得突变体 FtsA并不会导致 FtsZ 保护。FtsA 或 FtsA的过表达与 ZipA 一起不会干扰 FtsZ 的保护。此外，当与缺乏 FZB 结构域的 ZipA 突变体一起过表达时，FtsA 或 FtsA都不能保护 FtsZ。我们提出，ZipA 通过使 FtsZ 的相互作用位点无法与 ClpXP 蛋白酶的 ClpX 部分接触，从而保护 FtsZ 免受 ClpP 的降解。这一作用不能被 FtsA 或 FtsA取代，这表明 ZipA 在原环稳定性中具有独特的作用。
