Interactions of FliJ with the Salmonella type III flagellar export apparatus
Interactions of FliJ with the Salmonella type III flagellar export apparatus
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DOI：
10.1128/jb.185.18.5546-5554.2003
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发表时间：
2003-09-01
影响因子：
3.2
通讯作者：
Macnab, RM
中科院分区：
文献类型：
--
作者：
Fraser, GM;González-Pedrajo, B;Macnab, RM
FliJ 是一种 17 kDa 蛋白，是沙门氏菌 III 型鞭毛蛋白输出系统的可溶性成分，具有抗聚集特性和其他几个特性，表明它可能具有类似伴侣的功能。我们现在已经详细检查了这种蛋白质。测试了覆盖整个 147 个氨基酸序列的 10 个氨基酸扫描缺失，以与 fliJ 无效菌株互补；仅对第一个和最后一个删除进行了补充。一些缺失，尤其是 C 末端的缺失，对野生型细胞产生了显着的负面影响，表明它们正在积极干扰功能。 Flij 的两个截短版本（代表其 N 端和 C 端一半）未能互补且不占优势。我们通过多种技术测试了 Flij 自联想。尺寸排阻色谱 (Superdex 200) 显示表观分子量约为 50 kDa，这可以反映多聚化或拉长形状或两者兼而有之。多角度光散射的峰值为 20 kDa，接近单体的分子量。分析超速离心提供了作为三聚体或四聚体的弱自缔合的证据。从之前的研究中得知，Flij 与 FliH（ATPase FliI 的负调节因子）的 N 末端区域相互作用。使用 Flij 的截断和删除版本，我们现在表明是它的 C 末端区域负责这种相互作用。我们还表明 FliJ 与输出装置最大膜成分 FlhA 的可溶性细胞质结构域相互作用；尽管 FliJ 中的小缺失不会干扰关联，但两个截短版本均未能关联，表明 Flij 序列的大量中心区域参与了关联。我们提出了一个总结这些多重相互作用的模型。
FliJ, a 17-kDa protein, is a soluble component of the Salmonella type III flagellar protein export system that has antiaggregation properties and several other characteristics that suggest it may have a chaperone-like function. We have now examined this protein in detail. Ten-amino-acid scanning deletions covering the entire 147-amino-acid sequence were tested for complementation of a fliJ null strain; only the first and last deletions complemented. A few of the deletions, especially towards the C terminus, exerted a dominant negative effect on wild-type cells, indicating that they were actively interfering with function. Two truncated versions of Flij, representing its N- and C-terminal halves, failed to complement and were not dominant. We tested for Flij self-association by several techniques. Size-exclusion chromatography (Superdex 200) indicated an apparent molecular mass of around 50 kDa, which could reflect either multimerization or an elongated shape or both. Multiangle light scattering gave a peak value of 20 kDa, close to the molecular mass of the monomer. Analytical ultracentrifugation gave evidence for weak self-association as a trimer or tetramer. It was known from previous studies that Flij interacts with the N-terminal region of FliH, a negative regulator of the ATPase FliI. Using both truncation and deletion versions of Flij, we now show that it is its C-terminal region that is responsible for this interaction. We also show that FliJ interacts with the soluble cytoplasmic domain of the largest membrane component of the export apparatus, FlhA; although small deletions in FliJ did not interfere with the association, both truncated versions failed to associate, indicating that a substantial amount of the central region of the Flij sequence participates in the association. We present a model summarizing these multiple interactions.