No abstract

The smallest membrane protein shown to catalyze ion-coupled transport is documented in this report. A gene coding for a small 110-amino acid membrane protein (emrE or mvrC) has been previously identified and cloned and shown to render Escherichia coli cells resistant to methyl viologen and to ethidium. In this report, it is shown that the resistance is due to extrusion of the toxic compounds in a process that requires a proton electrochemical gradient rather than ATP. For this purpose, cells in which the unc gene was inactivated were used so that the interconversion between the proton gradient and ATP is not possible, and the effect of agents, which specifically affect either of them, was tested on transport of ethidium in the intact cell. In addition, EmrE has been overexpressed and metabolically labeled with [35S]methionine. Strikingly, the protein can be quantitatively extracted with a mixture of organic solvents such as chloroform:methanol and is practically pure after this extraction. Moreover, after addition of E. coli lipids to the chloroform:methanol extract, EmrE has been reconstituted in proteoliposomes loaded with ammonium chloride. Upon dilution of the proteoliposomes in ammonium-free medium, a pH gradient was formed that drove transport of ethidium and methyl viologen into the proteoliposome. Both substrates compete with each other and exchange with previously transported solute. EmrE is a multidrug transporter of a novel type, and, because of its size and its solubility properties, it provides a unique model to study structure-function aspects of transport reactions in ion-coupled processes.

EmrE is an

No abstract

The small multidrug resistance family of transporters is widespread in bacteria and is responsible for bacterial resistance to toxic aromatic cations by proton-linked ef¯ux. We have determined the threedimensional (3D) structure of the Escherichia coli multidrug transporter EmrE by electron cryomicroscopy of 2D crystals, including data to 7.0 A Ê resolution. The structure of EmrE consists of a bundle of eight transmembrane a-helices with one substrate molecule bound near the centre. The substrate binding chamber is formed from six helices and is accessible both from the aqueous phase and laterally from the lipid bilayer. The most remarkable feature of the structure of EmrE is that it is an asymmetric homodimer. The possible arrangement of the two polypeptides in the EmrE dimer is discussed based on the 3D density map.

No abstract

A method for the measurement of the internal pH of chloroplasts is described. The method is based on the uptake of a fluorescent amine by chloroplasts, which can be observed by the lowering of the fluorescence emitted from a suspension of chloroplasts.It is shown that this uptake is dependent, as expected, on the dissociation constant of the amine and also on the number of ionisable amines in the molecule. Amines attached to two types of chromophores, acridine and naphthalene have been investigated. As previously shown for other amines, this uptake was found to be a prerequisite for their acting as uncouplers. The technique provides a simple way to continuously follow changes in ApH in chloroplasts.A relation between energy generation and the quenching of the fluorescence of the uncoupler atebrin has been described by Kraayenhof [i]. He showed that the required energy could be provided by electron transport, ATP hydrolysis or a pH gradient and it was therefore suggested that the extent of quenching may be used to measure the "energy state" of the chloroplast [1,2]. A similar effect was described in chromatophores and was suggested to be related to proton movements, rather than to the "energy state" [3].We have recently shown that atebrin distributes between the inside of the chloroplast and the solution in accordance with the ratio of proton concentrations [4], in a manner similar to other amines [5,6]. The quenching was shown to be a consequence of its uptake and probably due to several factors [a]. Thus, as was shown with 5,5-dimethyl-2,4-oxazolidinadione in mitochondria [7] or methylamine [5,8] and NH, [9] in chloroplasts this uptake could be used as a measure of the H+ gradient across the membrane. Thus, an amine of a high pK will be distributed across the membrane in accordance with the proton concentration gradient.I n the case of diamine,

No abstract