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Site-specific mutations of FtsZ - effects on GTPase and in vitro assembly

机译：FtsZ的位点特异性突变-对GTPase和体外组装的影响

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FtsZ, the major cytoskeletal protein in bacterial cytokinesis, assembles in vitro into protofilaments, which can further associate into sheets, bundles or tubes. We have constructed 16 site-directed mutants of E. coli ftsZ, and tested them for GTP hydrolysis and assembly in vitro, and for their ability to complement the temperature sensitive ftsZ84 mutation in E. coli. The mutants were grouped into three classes. Benign mutants, which mapped mostly to the front and back surface of the protofilament, were able to complement ftsZ84 in vivo and showed normal assembly in vitro. GTP contact mutations had less than 10% of wild type GTPase activity. They could all assemble in vitro, and several of these mutants could complement ftsZ84. A third, and newly discovered, class of mutations mapped to the sides of the protofilaments. These lateral mutants had mostly normal GTPase and assembly in vitro, but none of them complemented ftsZ84. The non-complementing mutants showed greatly reduced expression from the pBS58 vector, suggesting possible dominant negative effects. Several mutants with greatly reduced GTPase could still complement ftsZ84, suggesting that the high level of GTPase observed in vitro is not essential for in vivo function. All of the lateral mutants failed to complement ftsZ84, which suggests that these surfaces of the protofilaments are important for function in cell division. These lateral surfaces may mediate association of FtsZ protofilaments into pairs or small sheets, although their structure is apparently different from the sheets assembled in DEAE dextran or calcium.
机译：FtsZ是细菌胞质分裂的主要细胞骨架蛋白，在体外组装成原丝，原丝可以进一步结合成片，束或管。我们已经构建了大肠杆菌ftsZ的16个定点突变体，并测试了它们在体外的GTP水解和组装，以及它们对大肠杆菌中温度敏感的ftsZ84突变的互补能力。突变体分为三类。良性突变体主要定位在原丝的前表面和后表面，能够在体内补充ftsZ84并在体外显示正常组装。 GTP接触突变的野生型GTPase活性不足10％。它们都可以在体外组装，并且其中一些突变体可以补充ftsZ84。第三类是新发现的突变类别，映射到原丝的侧面。这些侧向突变体在体外具有大多数正常的GTPase和装配，但是它们都不能补充ftsZ84。非互补突变体显示从pBS58载体表达大大减少，表明可能存在显性负效应。 GTPase大大降低的几个突变体仍可以补充ftsZ84，这表明在体外观察到的高水平的GTPase对体内功能不是必需的。所有的侧向突变体都不能补充ftsZ84，这表明原丝的这些表面对于细胞分裂的功能很重要。这些侧面可将FtsZ原型丝的缔合介导成对或成小片，尽管它们的结构显然不同于组装在DEAE葡聚糖或钙中的片。

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