Lin Y P, Sharer J D, March P E
Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854-5635.
J Bacteriol. 1994 Jan;176(1):44-9. doi: 10.1128/jb.176.1.44-49.1994.
Era is an Escherichia coli GTPase that is essential for cell viability and is peripherally associated with the cytoplasmic membrane. Both immunoelectron microscopy and subcellular-fractionation experiments have shown that Era is present in cytoplasmic as well as membrane-associated pools. These data led to speculation that the mechanism of action of Era may require cycling between membrane and cytoplasmic sites. In order to investigate this possibility, an in vitro binding assay was developed to characterize the binding of Era to membrane fractions. Competition and saturation binding experiments suggest that a site that is specific for Era and capable of binding up to 5 ng of Era per microgram of membrane protein is present in membrane preparations. The binding curve is complex, indicating that multiple equilibria describe the interaction. The binding of Era to this putative receptor is dependent on guanine nucleotides; binding cannot be measured in the absence of nucleotide, and neither ATP nor UTP can substitute. Subfractionation of cell walls showed that the guanine nucleotide-dependent binding site was present in fractions enriched in cytoplasmic membrane. These data provide evidence that Era may be involved in a GTPase-receptor-coupled membrane-signaling pathway that is essential for growth in E. coli.
Era是一种大肠杆菌GTP酶，对细胞活力至关重要，且与细胞质膜外周相关。免疫电子显微镜和亚细胞分级分离实验均表明，Era存在于细胞质以及与膜相关的部分中。这些数据引发了一种推测，即Era的作用机制可能需要在膜和细胞质位点之间循环。为了研究这种可能性，开发了一种体外结合测定法来表征Era与膜组分的结合。竞争和饱和结合实验表明，膜制剂中存在一个对Era特异且每微克膜蛋白能够结合多达5 ng Era的位点。结合曲线很复杂，表明多种平衡描述了这种相互作用。Era与这个假定受体的结合依赖于鸟嘌呤核苷酸；在没有核苷酸的情况下无法检测到结合，并且ATP和UTP都不能替代。细胞壁的亚分级分离表明，鸟嘌呤核苷酸依赖性结合位点存在于富含细胞质膜的组分中。这些数据提供了证据，表明Era可能参与了一种对大肠杆菌生长至关重要的GTP酶-受体偶联膜信号通路。
