Intermolecular Cross-linking between the Periplasmic Loop3–4 Regions of PomA, a Component of the Na+-driven Flagellar Motor of Vibrio alginolyticus *
Intermolecular Cross-linking between the Periplasmic Loop3–4 Regions of PomA, a Component of the Na+-driven Flagellar Motor of Vibrio alginolyticus *
复制标题
PomA 周质环 3-4 区域之间的分子间交联，PomA 是溶藻弧菌 Na+ 驱动鞭毛马达的一个组成部分*
DOI：
10.1074/jbc.m000848200
复制
发表时间：
2000
期刊：
影响因子：
--
通讯作者：
M. Homma
中科院分区：
文献类型：
--
作者：
Tomohiro Yorimitsu;Y. Asai;Ken Sato;M. Homma
Poma和Pomb形成一种复合体，它传导钠离子，并为Na+驱动的溶藻弧菌极地鞭毛马达产生扭矩。POMA有四个跨膜片段。一个周质环(loop1-2)连接节段1和2，另一个(loop3-4)进行半胱氨酸扫描诱变连接节段3和4。当在C末端周质环(loop3-4)中引入半胱氨酸残基(Cys-POMA)的POMA在不暴露于还原剂的情况下检测时，观察到一条43 kDa的条带，而在还原条件下仅观察到一条对应于单体POMA的25 kDa条带。氧化剂CuCl2使大多数突变体的43 kDa条带强度增强。突变体P172C的43 kDa条带最强。突变体P172C的运动能力严重降低，表现为显性负效应，而用Ala、Ile或Ser替换Pro不影响运动能力。在DTT存在的情况下，小鼠游泳能力部分恢复，43 kDa蛋白含量减少。这些结果表明，二硫键交联干扰了POMA的功能。当突变的半胱氨酸残基被N-乙基马来酰亚胺修饰时，只标记了25 kDa的POMA条带，表明43 kDa的形式是一种交联型同源二聚体，表明POMA相邻亚基的3-4环在组装的马达中彼此接近。我们认为这个环区对于二聚体的形成和运动功能是重要的。
PomA and PomB form a complex that conducts sodium ions and generates the torque for the Na+-driven polar flagellar motor of Vibrio alginolyticus. PomA has four transmembrane segments. One periplasmic loop (loop1–2) connects segments 1 and 2, and another (loop3–4), in which cysteine-scanning mutagenesis had been carried out, connects segments 3 and 4. When PomA with an introduced Cys residue (Cys-PomA) in the C-terminal periplasmic loop (loop3–4) was examined without exposure to a reducing reagent, a 43-kDa band was observed, whereas only a 25-kDa band, which corresponds to monomeric PomA, was observed under reducing conditions. The intensity of the 43-kDa band was enhanced in most mutants by the oxidizing reagent CuCl2. The 43-kDa band was strongest in the P172C mutant. The motility of the P172C mutant was severely reduced, and P172C showed a dominant-negative effect, whereas substitution of Pro with Ala, Ile, or Ser at this position did not affect motility. In the presence of DTT, the ability to swim was partially restored, and the amount of 43-kDa protein was reduced. These results suggest that the disulfide cross-link disturbs the function of PomA. When the mutated Cys residue was modified withN-ethylmaleimide, only the 25-kDa PomA band was labeled, demonstrating that the 43-kDa form is a cross-linked homodimer and suggesting that the loops3–4 of adjacent subunits of PomA are close to each other in the assembled motor. We propose that this loop region is important for dimer formation and motor function.
DOI：
10.1073/pnas.96.10.5740
发表时间：
1999-05-11
影响因子：
11.1
作者：
Jaques, S;Kim, YK;McCarter, LL
通讯作者：
McCarter, LL