R174 of Escherichia coli FtsZ is involved in membrane interaction and protofilament bundling, and is essential for cell division
R174 of Escherichia coli FtsZ is involved in membrane interaction and protofilament bundling, and is essential for cell division
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DOI：
10.1046/j.1365-2958.2003.03876.x
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发表时间：
2004-02-01
影响因子：
3.6
通讯作者：
den Blaauwen, T
中科院分区：
文献类型：
--
作者：
Koppelman, CM;Aarsman, MEG;den Blaauwen, T
我们使用内向外囊泡研究了 FtsZ 与细胞质膜之间的相互作用。纯化的 FtsZ 和细胞质膜结合的 FtsZ 的胰蛋白酶可及性的比较表明，螺旋 6 和螺旋 7 之间的突出环受到保护，免受后者的胰蛋白酶消化。该疏水环在位置 174 处含有精氨酸残基。为了研究 R174 的作用，该残基被天冬氨酸取代，并将 FtsZ-R174D 与绿色荧光蛋白 (GFP) 融合。 FtsZ-R174D-GFP在允许和非允许温度下都可以定位在FtsZ和FtsZ84(Ts)背景中，并且与野生型FtsZ相比，其对细胞质膜的亲和力降低。 FtsZ-R174D 也可以定位于 FtsZ 耗尽菌株中。然而，与野生型 FtsZ 相比，FtsZ-R174D 无法补充 ftsZ84 突变或耗尽菌株并诱导丝状化。体外聚合实验表明，FtsZ-R174D 能够聚合，但这些聚合物在 10 mM CaCl2 存在下不能形成束。这是对 FtsZ 突变体的首次描述，该突变体对细胞质膜的亲和力降低，不支持细胞分裂，但仍能够定位。该突变体能够在体外形成原丝，但无法成束。这表明膜相互作用和成束都不是细胞分裂启动的必要条件。
We investigated the interaction between FtsZ and the cytoplasmic membrane using inside-out vesicles. Comparison of the trypsin accessibility of purified FtsZ and cytoplasmic membrane-bound FtsZ revealed that the protruding loop between helix 6 and helix 7 is protected from trypsin digestion in the latter. This hydrophobic loop contains an arginine residue at position 174. To investigate the role of R174, this residue was replaced by an aspartic acid, and FtsZ-R174D was fused to green fluorescent protein (GFP). FtsZ-R174D-GFP could localize in an FtsZ and in an FtsZ84(Ts) background at both the permissive and the non-permissive temperature, and it had a reduced affinity for the cytoplasmic membrane compared with wild-type FtsZ. FtsZ-R174D could also localize in an FtsZ depletion strain. However, in contrast to wild-type FtsZ, FtsZ-R174D was not able to complement the ftsZ84 mutation or the depletion strain and induced filamentation. In vitro polymerization experiments showed that FtsZ-R174D is able to polymerize, but that these polymers cannot form bundles in the presence of 10 mM CaCl2. This is the first description of an FtsZ mutant that has reduced affinity for the cytoplasmic membrane and does not support cell division, but is still able to localize. The mutant is able to form protofilaments in vitro but fails to bundle. It suggests that neither membrane interaction nor bundling is a requirement for initiation of cell division.