Effects of inorganic polyphosphate on the proteolytic and DNA-binding activities of Lon in Escherichia coli
Effects of inorganic polyphosphate on the proteolytic and DNA-binding activities of Lon in Escherichia coli
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DOI：
10.1074/jbc.m404725200
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发表时间：
2004-08-13
影响因子：
4.8
通讯作者：
Kuroda, A
中科院分区：
文献类型：
--
作者：
Nomura, K;Kato, J;Kuroda, A
Lon属于一组独特的蛋白酶，与DNA结合，参与调节几种重要的细胞功能，包括适应营养下调。以前，我们发现，无机聚磷酸盐（聚P）增加大肠杆菌在响应氨基酸饥饿，它刺激降解游离核糖体蛋白的Lon。在这项工作中，我们研究了聚P对Lon的蛋白水解和DNA结合活性的影响。添加顺序实验表明，聚P首先结合Lon，这刺激了Lon介导的核糖体蛋白降解。使用Lon缺失突变体的polyP结合测定表明，Lon的polyP结合位点位于ATP酶结构域中。由于相同的ATP酶结构域也包含DNA结合位点，聚P可以与DNA竞争结合Lon。事实上，等摩尔量的聚P几乎完全抑制DNA-Lon复合物的形成，表明Lon以比其结合DNA更高的亲和力结合聚P。总的来说，我们的结果表明，聚P可以控制Lon的细胞活性，不仅作为蛋白酶，而且作为DNA结合蛋白。
Lon belongs to a unique group of proteases that bind to DNA and is involved in the regulation of several important cellular functions, including adaptation to nutritional downshift. Previously, we revealed that inorganic polyphosphate ( polyP) increases in Escherichia coli in response to amino acid starvation and that it stimulates the degradation of free ribosomal proteins by Lon. In this work, we examined the effects of polyP on the proteolytic and DNA-binding activities of Lon. An order-of-addition experiment suggested that polyP first binds to Lon, which stimulates Lon-mediated degradation of ribosomal proteins. A polyP-binding assay using Lon deletion mutants showed that the polyP-binding site of Lon is localized in the ATPase domain. Because the same ATPase domain also contains the DNA-binding site, polyP can compete with DNA for binding to Lon. In fact, an equimolar amount of polyP almost completely inhibited DNA-Lon complex formation, suggesting that Lon binds to polyP with a higher affinity than it binds to DNA. Collectively, our results showed that polyP may control the cellular activity of Lon not only as a protease but also as a DNA-binding protein.