The membrane-bound CzcA protein, a member of the resistance-nodulation-cell division (RND) permease superfamily, is part of the CzcCB 2 A complex that mediates heavy metal resistance in Ralstonia sp. CH34 by an active cation efflux mechanism driven by cation/proton antiport. CzcA was purified to homogeneity after expression in Escherichia coli, reconstituted into proteoliposomes, and the kinetics of heavy metal transport by CzcA was determined. CzcA is composed of 12 transmembrane ␣-helices and two large periplasmic domains. Two conserved aspartate and a glutamate residue in one of these transmembrane spans are essential for heavy metal resistance and proton/cation antiport but not for facilitated diffusion of cations. Generalization of the resulting model for the function of CzcA as a two-channel pump might help to explain the functions of other RND proteins in bacteria and eukaryotes.Multiple drug resistant bacteria poses a threat to man's fight against infectious diseases. Some multiple drug resistance systems may detoxify their substrates by transport across the complete cell wall of Gram-negative bacteria, across cytoplasmic membrane, periplasm, and outer membrane. These assumed transenvelope transporters are composed of a pump protein that energizes the transport, in addition to a membrane fusion and an outer membrane-associated protein (1, 2). The pump protein may be an ATP-binding cassette transporter (3, 4), a transporter of the major facilitator superfamily (5), or a resistance-nodulation-cell division (RND) 1 protein (4, 6, 7). The archetype of the RND permease superfamily family is CzcA from the Gram-negative bacterium Ralstonia sp. CH34 (formerly Alcaligenes eutrophus strain CH34) (8 -12).This bacterium contains at least seven heavy metal resistance determinants, located either on the bacterial chromosome or on one of the two indigenous plasmids pMOL28 (163 kilobase pairs) and pMOL30 (238 kb) (8, 13-16). One of them, the czc-determinant of plasmid pMOL30, mediates inducible resistance to millimolar concentrations of Co 2ϩ , Zn 2ϩ , and Cd 2ϩ in strain CH34 (8, 17). The products of the genes czcA, czcB, and czcC form a membrane-bound protein complex catalyzing an energy-dependent efflux of these three metal cations (9, 11), probably across the complete envelope. The mechanism of action of CzcCB 2 A is that of a proton/cation antiporter, and the K m values of the efflux system for the substrate heavy metal cations are also in the millimolar range (10). Although indirect evidence led to the assumption that CzcA is the central cation/proton antiporter of the CzcCB 2 A complex (10), this has not been shown directly. This paper demonstrates that CzcA is a cation/proton antiporter, and develops the model of CzcA as a two-channel pump based on topology studies and the function of CzcA mutant proteins. This model sheds some light on other RND proteins involved in multiple drug resistance of bacteria or with previously unknown functions in mammals.
EXPERIMENTAL PROCEDURESBacterial Strains, Plasmids, and Growt...
