Copper Pump Research Articles

Escherichia coli CopA N-Terminal Cys(X)2Cys Motifs Are Not Required for Copper Resistance or Transport

Escherichia coli CopA is a Cu(I)-translocating P-type ATPase that is involved in copper export and resistance. It is an orthologue of the human Menkes and Wilson disease-related proteins. Each of those two human copper pumps has six N-terminal Cys(X)2Cys sequences, but their function in transport is unclear. CopA has two N-terminal Cys(X)2Cys sequences, GLSC14GHC17 and GMSC110ASC113. The requirement of these cysteine motifs was investigated by mutagenesis of the codons for all four cysteine residues, singly and in combination. Cells of a copA deletion strain expressing genes for the mutant genes were nearly as resistant to copper as the wild type. In addition, everted membrane vesicles from cells expressing the mutant copA genes exhibited ATP-coupled accumulation of copper similar to that of the wild type. The results indicate that neither of two N-terminal Cys(X)2Cys motifs is required for either resistance or transport.

Role of a Candida albicans P1-type ATPase in resistance to copper and silver ion toxicity.

Copper ion homeostasis is complicated in that copper is an essential element needed for a variety of cellular processes but is toxic at excess levels. To identify Candida albicans genes that are involved in resistance to copper ion toxicity, a library containing inserts of C. albicans genomic DNA was used to complement the copper sensitivity phenotype of a Saccharomyces cerevisiae cup1Delta strain that is unable to produce Cup1p, a metallothionein (MT) responsible for high-level copper ion resistance. A P1-type ATPase (CPx type) that is closely related to the human Menkes and Wilson disease proteins was cloned. The gene encoding this pump was termed CRD1 (for copper resistance determinant). A gene encoding a 76-amino-acid MT similar to higher eukaryotic MTs in structure was also cloned, and the gene was termed CRD2. Transcription of the CRD1 gene was found to increase upon growth with increasing copper levels, while the CRD2 mRNA was expressed at a constant level. Strains with the CRD1 gene disrupted were extremely sensitive to exogenous copper and failed to grow in medium containing 100 microM CuSO(4). These crd1 strains also exhibited increased sensitivity to silver and cadmium, indicating that Crd1p is somewhat promiscuous with respect to metal ion transport. Although strains with the CRD2 gene disrupted showed reduced growth rate with increasing copper concentration, the crd2 mutants eventually attained wild-type levels of growth, demonstrating that CRD2 is less important for resistance to copper ion toxicity. Crd1p is the first example of a eukaryotic copper pump that provides the primary source of cellular copper resistance, and its ability to confer silver resistance may enhance the prevalence of C. albicans as a nosocomial pathogen.

CopA: An Escherichia coli Cu(I)-translocating P-type ATPase.

The copA gene product, a putative copper-translocating P-type ATPase, has been shown to be involved in copper resistance in Escherichia coli. The copA gene was disrupted by insertion of a kanamycin gene through homologous recombination. The mutant strain was more sensitive to copper salts but not to salts of other metals, suggesting a role in copper homeostasis. The copper-sensitive phenotype could be rescued by complementation by a plasmid carrying copA from E. coli or copB from Enterococcus hirae. Expression of copA was induced by salts of copper or silver but not zinc or cobalt. Everted membrane vesicles from cells expressing copA exhibited ATP-coupled accumulation of copper, presumably as Cu(I). The results indicate that CopA is a Cu(I)-translocating efflux pump that is similar to the copper pumps related to Menkes and Wilson diseases and provides a useful prokaryotic model for these human diseases.

Molecular genetics of intracellular copper transport

Copper is essential for life processes as cofactor for many vital cuproenzymes, yet it is extremely toxic in excess. Efficient mechanisms have been developed to recruit and deliver copper to enzymes and for excretion of surplus metal. Imbalance of copper homeostasis is manifested in two human illnesses, Menkes disease and Wilson disease, which are both caused by defective export mechanisms that involve ATP7A and ATP7B, respectively. Severe deficiency of copper is mimicked by Menkes disease, whereas Wilson disease leads to toxic accumulations of the metal. The copper export pumps are energy requiring transporters possessing a number of unique molecular features. Besides domains typical for P-type ATPases, several heavy metal specific sites have been described in the copper subfamily of the ATPases. Identification of disease-causing mutations will help to define structural determinants of normal copper pump function. In this review we discuss the current knowledge about the human copper pumps, ATP7A and its homologue ATP7B. J. Trace Elem. Exp. Med. 12:297–313, 1999. © 1999 Wiley-Liss, Inc.

Reconstitution of Ceruloplasmin by the Cu(I)-Glutathione Complex: EVIDENCE FOR A ROLE OF Mg2+ AND ATP

The copper-glutathione complex (Cu(I)-GSH) efficiently acted in vitro as the source of Cu(I) in the reconstitution of apoceruloplasmin. Copper was found to reinstate in the various sites in a multistep process, with metal entry into the protein in a first phase, and a second step involving conformational changes of the protein leading to the recovery of the native structural and functional properties. This latter phase was found to be strongly facilitated by Mg2+ or Ca2+ and by ATP. Both Mg2+ and ATP had to be present for optimal reconstitution. These results may shed some light on the mechanisms governing the biosynthesis of ceruloplasmin in vivo. Cu(I)-GSH was the only complex able to reconstitute ceruloplasmin at neutral pH. Glutathione may thus function to shuttle the metal from the membrane copper pump, as the Wilson disease ATPase, and ceruloplasmin in the secretory compartments of the cell. The finding that ceruloplasmin acquires the native conformation after metal entry through a complex pathway triggered by Mg2+ and ATP suggests that they may act as physiological modulators of this process in vivo.

Copper and Silver Transport by CopB-ATPase in Membrane Vesicles of Enterococcus hirae

The P-type ATPase, CopB, of Enterococcus hirae is required for the copper resistance displayed by this organism and thus was postulated to be a copper pump. Using 64Cu+ and 110mAg+, we here show ATP-driven copper and silver accumulation catalyzed by CopB in native inside-out membrane vesicles of E. hirae. CopB ATPase exhibited an apparent Km for Cu+ and Ag+ of 1 microM and for ATP of 10 microM. Transport was maximal at pH 6 and had an apparent Vmax of 0.07 nmol.min-1.mg-1 for both copper and silver transport. Vanadate displayed a biphasic effect on transport: maximal inhibition was observed at 40 microM vanadate for copper transport and 60 microM for silver transport, respectively. At higher vanadate concentrations, these inhibitions were reversed. The CopB ATPase of E. hirae is thus a pump for the extrusion of monovalent copper and silver ions, with copper probably being the natural substrate.

Copper pumping ATPases: Common concepts in bacteria and man

Recently, four genes encoding putative copper pumping ATPases have been cloned from widely different sources: two genes from Enterococcus hirae that are involved in copper metabolism and two human genes that are defective in the copper-related Wilson and Menkes disease. The predicted gene products are P-type ATPases. They exhibit extensive sequence similarity and appear to be members of a new class of ATP driven copper pumps involved in the regulation of cellular copper.