A P-type ATPase importer that discriminates between essential and toxic transition metals

Abstract

Transition metals, although being essential cofactors in many physiological processes, are toxic at elevated concentrations. Among the membrane-embedded transport proteins that maintain appropriate intracellular levels of transition metals are ATP-driven pumps belonging to the P-type ATPase superfamily. These metal transporters may be differentiated according to their substrate specificities, where the majority of pumps can extrude either silver and copper or zinc, cadmium, and lead. In the present report, we have established the substrate specificities of nine previously uncharacterized prokaryotic transition-metal P-type ATPases. We find that all of the newly identified exporters indeed fall into one of the two above-mentioned categories. In addition to these exporters, one importer, Pseudomonas aeruginosa Q9I147, was also identified. This protein, designated HmtA (heavy metal transporter A), exhibited a different substrate recognition profile from the exporters. In vivo metal susceptibility assays, intracellular metal measurements, and transport experiments all suggest that HmtA mediates the uptake of copper and zinc but not of silver, mercury, or cadmium. The substrate selectivity of this importer ensures the high-affinity uptake of essential metals, while avoiding intracellular contamination by their toxic counterparts.membrane proteins ͉ transition-metal homeostasis ͉ heavy metal transporters ͉ P 1B ATPases M aintaining transition-metal homeostasis presents a unique challenge to all living organisms. Transition metals such as zinc, copper, and iron are essential to many physiological processes but are also toxic at elevated concentrations. Other metals, such as cadmium, silver, and mercury, exhibit acute toxicity by binding to macromolecules and perturbing their physiological interactions (1, 2). A diversity of membrane-embedded transporters translocate metals across the cell membrane, underlining the importance and the delicacy of this process. In prokaryotes, the concerted action of members of the RND, ABC, CDF, and P-type ATPase superfamilies functions to maintain appropriate intracellular concentrations of essential and toxic transition metals (3). In humans, perturbations in metal homeostasis lead to degenerative syndromes, cancer, and Wilson's and Menkes' diseases (4, 5).P-type ATPases constitute a superfamily of transporters characterized by unique signature motifs. The hallmark of this family of pumps is the formation of a phosphoenzyme intermediate (hence the name P-type ATPase), by the transfer of the ␥-phosphate from ATP to the highly conserved DKTGT motif (6). A family of P-type ATPases catalyzing the translocation of transition metals (also referred to as heavy-metal or type P 1B ATPases) has been identified (7,8), with members present in all kingdoms of life. Such P-type ATPases harbor a Cys-Pro-Xaa (or Xaa-Pro-Cys) motif, with Xaa ϭ Cys, Ser, or His, in their sixth transmembrane helix (TM6) that is essential for transport activity (7, 9, 10). Sequence homology of transition-metal P-type pump...

CtpA, a Copper-translocating P-type ATPase Involved in the Biogenesis of Multiple Copper-requiring Enzymes

“…In Pseudomonas aeruginosa, which possesses two operons encoding cbb 3 and a homologue of ctpA (PA1549), the PA1549 mutant did not show any phenotype in excess copper (27). More recently, expression of ctpA (PA1549) in the copper-sensitive strain of E. coli (GG44) did not restore resistance to copper (28). Similar results were previously obtained in R. capsulatus; the ccoI Ϫ mutant was neither resistant nor hypersensitive to exogenous copper (8).…”

The ATPases CopA and CopB both contribute to copper resistance of the thermoacidophilic archaeon Sulfolobus solfataricus

“…In contrast with many P IB -ATPases (Rensing et al, 2000;Lewinson et al, 2009), both S. solfataricus ATPases were incapable of compensating for the copper transport defect of the E. coli copA mutant. The small increase in growth rate at elevated temperatures may indicate weakly developing complemention.…”

“…2, because both ATPases of S. solfataricus belong to the other subgroup CopA-1, which comprises ATPases in charge of copper homeostasis. CopA-1-type ATPases are assigned as resistance factors exporting copper at high rates (Argüello et al, 2007;Osman & Cavet, 2008), whereas a few ATPases have been previously considered to be copper importers due to their phenotypic characteristics (Odermatt et al, 1993;Tottey et al, 2001;Lewinson et al, 2009). These apparently conflicting views have been lately reconciled by the statement that ''all Cu + -ATPases drive cytoplasmic Cu + -efflux, albeit at quite different transport rates'' .…”

“…The lack of full restoration may be explained by reduced catalytic activities of the thermophilic proteins at the relatively low-temperature living conditions of E. coli. However, this does not hold for all thermophiles, because the P IB -ATPase of Pyrococcus furiosus has been reported to achieve functional complementation at 37 u C (Lewinson et al, 2009). Although Pyrococcus can grow at 100 u C (Fiala & Stetter, 1986), the different natural di-and tetra-ether compositions of S. solfataricus and Pyrococcus membranes may prescribe specific lipid requests for CopA and CopB to attain functional complementation in E. coli membranes.…”

Mutagenesis of a Copper P-Type ATPase Encoding Genein Methylococcus capsulatus (Bath) Results inCopper-Resistance

“…They explained the mutant phenotype by the possibility of defects in copper uptake [26]. Furthermore, Lewinson et al [27] reported a copper-importing P-type ATPase, HmtA (heavy metal transporter A), in P. aeruginosa Q9I147 and confirmed their claim by many in vivo assays including, intracellular metal measurements. These results supported the proposal that Mc.…”