Analysis of Kef C : a glutathione gated potassium efflux system in Escherichia coli
The KefC system in E.coli is a transport system mediating efflux of potassium from the cell. The KefC protein is located within the membrane and forms the transmembrane route enabling potassium movement. The two major aims of the project were to investigate the biochemical mechanisms responsible for regulating KefC activity and to analyse the two dimensional structure of KefC membrane protein. In the course of these studies I have shown that KefC activity is regulated by glutathione (GSH) and specific S-linked metabolites of this tripeptide. Under normal physiological conditions KefC is negatively regulated by GSH and the system is inactive. Addition of the sulphydryl reagent N-ethylmaleimide (NEM) leads to rapid formation of the GSH adduct N-ethylsuccinimido-S-glutathione (ESG) which is a positive regulator of KefC. When ESG is formed KefC is strongly activated leading to rapid and complete loss of internal potassium. Reversal of ESG elicited efflux, allowing potassiumre-accumulation, occurs primarily through the breakdown of the activator molecule to liberate reduced GSH. Additional physiological factors such as anaerobiosis have been demonstrated to influence ESG elicited KefC activity. Based upon this work a model for the different states of activation has been proposed. The structural studies undertaken have proved problematical using the gene fusion technology employed. Possible reasons for this are discussed.