Genetic screen suggests an alternative mechanism for azide-mediated inhibition of SecA
Genetic screen suggests an alternative mechanism for azide-mediated inhibition of SecA
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遗传筛选提出了叠氮化物介导的 SecA 抑制的替代机制
DOI：
10.1101/173039
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Chandler R
中科院分区：
文献类型：
--
作者：
Chandler R
叠氮化钠通过抑制SecA的活性来抑制细菌生长，SecA是蛋白质跨细胞质膜易位所需的ATP酶20。为了研究叠氮化物的作用机制，我们使用转座子定向插入位点测序（TraDIS）来筛选转座子插入突变体的高密度文库，以寻找影响大肠杆菌易感性的突变。大肠杆菌转化为叠氮化物。在编码Sec 24机器的大多数组件的基因中插入增加了对叠氮化物的易感性。然而，截短SecA的C-末端25延伸（CTE）的插入降低了E.大肠杆菌转化为叠氮化物。编码许多金属结合蛋白的基因26中的插入也增加了对叠氮化物的易感性，并且转录谱27表明用叠氮化物治疗破坏了铁稳态。28种培养基中铁的存在降低了大肠杆菌的易感性。在secA基因中，29个突变赋予叠氮化物抗性，改变了大肠杆菌对叠氮化物的反应。大肠杆菌对铁的限制，提示铁代谢和蛋白质易位之间的联系。虽然先前的工作表明SecA与锌结合，但SecA在生理水平表达时与铁共纯化，并且叠氮化物破坏了C末端金属结合结构域（MeBD）与体内铁的相互作用。使用等温滴定量热法34和1H-核磁共振对MeBD的金属结合进行生物物理分析表明，与Zn 2+相比，Fe 2+具有明显的结合偏好。这些结果表明SecA的生理配体是铁，叠氮化物通过破坏铁结合来抑制SecA 36。37
Sodium azide inhibits bacterial growth by inhibiting the activity of SecA, an ATPase 20 required for translocation of proteins across the cytoplasmic membrane. To investigate the 21 mechanism of action of azide, we used transposon directed insertion-site sequencing (TraDIS) to 22 screen a high-density library of transposon insertion mutants for mutations that affect the 23 susceptibility of E. coli to azide. Insertions in genes encoding most components of the Sec 24 machinery increased susceptibility to azide. However, insertions truncating the C-terminal 25 extension (CTE) of SecA decreased susceptibility of E. coli to azide. Insertions in genes 26 encoding many metal binding proteins also increased susceptibility to azide, and transcriptional 27 profiling suggested that treatment with azide disrupted iron homeostasis. The presence of iron in 28 the media decreased the susceptibility of E. coli to azide, and mutations in the secA gene that 29 confer resistance to azide altered the response of E. coli to iron limitation, suggesting a 30 connection between iron metabolism and protein translocation. Although previous work suggests 31 that SecA binds to zinc, SecA copurified with iron when expressed at physiological levels, and 32 azide disrupted the interaction of the C-terminal metal-binding domain (MeBD) with iron in 33 vivo. Biophysical analysis of metal binding by the MeBD using isothermal titration calorimetry 34 and 1 H-nuclear magnetic resonance indicated a clear binding preference for Fe 2+ over Zn 2+. 35 These results indicate that the physiological ligand of SecA is iron and that azide inhibits SecA 36 by disrupting iron binding. 37
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DOI：
10.1073/pnas.87.21.8227
发表时间：
1990-11-01
影响因子：
11.1
作者：
OLIVER, DB;CABELLI, RJ;JAROSIK, GP
通讯作者：
JAROSIK, GP
影响因子：
7
作者：
Langridge, Gemma C.;Phan, Minh-Duy;Turner, A. Keith
通讯作者：
Turner, A. Keith
带负电荷的磷脂可恢复 prePhoE 跨磷脂酰甘油耗尽的大肠杆菌内膜的易位。
DOI：
10.1016/s0021-9258(18)31493-5
发表时间：
1991
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
R. Kusters;W. Dowhan;B. Kruijff
通讯作者：
B. Kruijff
影响因子：
11.4
作者：
Duong, F;Wickner, W
通讯作者：
Wickner, W
影响因子：
3.2
作者：
Huber, D;Boyd, D;Beckwith, J
通讯作者：
Beckwith, J