Mechanism of action of the ammonia gas channel AmtB in E. coli

I.项目摘要： 氨转运蛋白（Amt）和恒河猴（Rh）蛋白是目前仅有的两种已鉴定的生物气体 生物体之间的通道。Rh蛋白是最为人所知的人红细胞上的抗原， 二氧化碳气体通道。它们是绿藻莱茵衣藻最佳生长所必需的 在高CO2浓度下。Amt蛋白被认为是Rh蛋白的前体同源物，是Rh的气体通道。 氨。它们是肠道细菌大肠杆菌和鼠伤寒沙门氏菌最佳生长所必需的 在低NH3下。由于细胞膜的磷脂双层对气体物质是可渗透的， 电池为什么需要气体通道还不清楚。对E. coli和革兰氏阳性菌S.鼠伤寒 表明在低NH3浓度下，AmtB转运NH3似乎与谷氨酰胺合成酶偶联 其将NH3同化为谷氨酰胺。因此，在本提案中，我们试图了解AmtB在E. coli中的AmtB与谷氨酰胺合成酶功能性偶联，并假设AmtB和谷氨酰胺合成酶 物理上联系在一起。AmtB和谷氨酰胺合成酶之间的物理接触可能允许直接 NH3从AmtB的孔递送到谷氨酰胺合成酶的活性位点，并因此增加速率 NH3的同化作用。该提议的两个具体目的之一是鉴定在人乳腺癌细胞中的氨基酸突变。 AmtB在低NH3下损害生长和铵吸收活性。这些amtB突变不应 导致AmtB的整体蛋白质错误折叠或AmtB孔功能丧失，而是局部影响 是谷氨酰胺合成酶的结合位点的蛋白质的细胞质区域。第二个具体目标 是利用分子生物学和遗传学方法检测AmtB和GS之间的关联。 二.相关性： 本文对E. coliAmtB将使我们能够更好地了解气体通道是如何工作的 以帮助维持健康的细胞生理。了解气路的作用，有助于我们识别气路的基础 人类疾病如Rh无效疾病中功能障碍。

I. Project Summary: Ammonia transport (Amt) proteins and Rhesus (Rh) proteins are the only two identified biological gas channels among organisms. Rh proteins, which are best known as antigens on human red blood cells, are gas channels for CO2. They are required for optimal growth of the green alga Chlamydomonas reinhardtii at high C02. Amt proteins, which are known as the-ancestralhomolog to Rh proteins, are gas channels for NH3. They are required for optimal growth of enteric bacteria Escherichia coli and Salmonella typhimurium at low NH3. Since phospholipid bilayers of the cell membrane are permeable to the gas species, the reason why gas channels are needed by cells is not known. Physiological studies in E. coli and S. typhimurium show that at low NH3 concentrations NH3 transport by AmtB appears to be coupled to glutamine synthetase which assimilates NH3 into glutamine. Therefore, in this proposal we seek to understand how AmtB in E. coli is functionally coupled to glutamine synthetase and hypothesize that AmtB and glutamine synthetase associate physically. The physical contact between AmtB and glutamine synthetase may allow the direct delivery of NH3 from the pore of AmtB to the active site of glutamine synthetase and hence increase the rate of assimilation of NH3. One of the two specific aims of this proposal is to identify amino acid mutations in AmtB that impair growth and ammonium uptake activity at low NH3. These amtB mutations should not cause global protein misfolding of AmtB or loss of function of the pore of AmtB but rather locally affect cytoplasmic regions of the protein that are its binding site for glutamine synthetase. The second specific aim is to test the association between AmtB and GS using molecular biology and genetics approaches. II. Relevance: This proposed study of E. coli AmtB will enable us to gain a better understanding of how gas channels work to help maintain healthy cell physiology. Knowing gas channel function will help us identify the basis of dysfunction in human disease such as Rh null disease.