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.

Fig. 1.

Metal sensitivity assays. (A) Escherichia coli wild-type cells (black traces) or E. coli metal-sensitive strain (all other traces) were cultured in the absence (Left) or presence (Right) of 300 μM ZnCl₂. Optical density at 600 nm was continuously monitored. Cells were transformed with an empty, control plasmid (purple and black traces) or with a plasmid encoding R. radiobacter Q7D0J8 (green), Pseudomonas aeruginosa Q9HXVO (cyan), S. pneumoniae Q97PQ2 (blue), P. aeruginosa Q9HUY5 (red), or P. aeruginosa Q9HX93 (magenta). (B) Optical density of cells after 12-h growth in the presence of the indicated CuCl₂ concentrations. E. coli wild-type cells insensitive to metals (filled squares) or E. coli metal-sensitive strain (all other traces) were transformed with an empty plasmid (circles), plasmid encoding R. radiobacter A9CJE3 (open squares), P. aeruginosa Q913G8 (triangles), or P. aeruginosa Q9HXVO (crosses). Such plots were used to calculate minimal inhibitory concentrations and generate the data in Table 1.

Fig. 2.

HmtA-associated copper hypersensitivity. E. coli metal-sensitive cells were transformed with a control plasmid (Left) or HmtA-encoding plasmid (Right). Growth in the presence of the indicated CuCl₂ concentrations was continuously monitored. (Inset) Time-dependent HmtA expression by immunoblot detection.

Fig. 3.

Metal selectivity of HmtA. (A) The optical density at 600 nm of Escherichia coli cells cultured for 12 h in the presence of the indicated concentrations of CuCl₂. The filled squares represent E. coli wild-type cells transformed with an empty plasmid. All other traces depict an E. coli metal-sensitive strain transformed with different plasmids: an empty plasmid (filled circles), native HmtA in the absence of the inducer l-arabinose (open squares), an N-terminal His-tagged version of HmtA (full triangles), a C-terminal His-tagged version of HmtA (crosses), and native HmtA (open triangles). (Inset) SDS/PAGE immunoblot detection of the relative amounts of C-His HmtA and N-His HmtA in the membrane fraction. (B) CuCl₂ sensitivity in Pseudomonas aeruginosa. The optical density at 600 nm of wild-type P. aeruginosa (filled circles) or a mutant strain deleted of HmtA (filled squares) grown for 12 h in the presence of the indicated CuCl₂ concentrations is shown. (C) CdCl₂, AgNO₃, and ZnCl₂ sensitivity in E. coli. The optical density at 600 nm of E. coli cells cultured for 12 h in the presence of the indicated metal concentrations is shown. The filled squares represent E. coli wild-type cells transformed with an empty plasmid. The other traces depict an E. coli metal-sensitive strain transformed with either an empty plasmid (filled circles) or a C-terminal His-tagged version of HmtA (open triangles). The growth of the latter cells in the absence of the inducer l-arabinose is shown only for ZnCl₂ sensitivity (filled triangles).

Fig. 4.

Expression of HmtA results in increased intracellular metal concentrations. (A) E. coli metal-sensitive strains transformed with control plasmid (open bars) or HmtA-encoding plasmid (full bars) were cultured in the presence of 2.5 μM AgNO₃, 2.5 μM CdCl₂, 75 μM CuCl₂, or 75 μM ZnCl₂. Cells were harvested and washed with metal-free buffer, and total internal metal concentrations were measured by inductively coupled plasma mass spectroscopy. The asterisk denotes cells that were harvested early in the growth, before initiation of HmtA expression. Error bars represent standard deviations of three repeats. (B) Internal copper concentrations as a function of external copper in control cells (circles) or HmtA-expressing cells (triangles). Bars represent standard deviations of three repeats.

Fig. 5.

Time-dependent copper uptake. Cells transformed with empty plasmid (squares) or HmtA-encoding plasmid (all other traces) were cultured in the absence of metals, washed with metal-free buffer, and allowed to recover in the presence of glucose. Transport was initiated by the addition of 250 nM CuCl₂, and samples were withdrawn at the indicated times. Where indicated, 0.5 mM DTT or 0.25 mM cysteine was included in the reaction mixture. Total internal metal concentrations were measured by inductively coupled plasma mass spectroscopy. Error bars represent standard deviations of three repeats.

Fig. 6.

Amino acid sequence alignment of transmembrane domains 4–8 of Cu⁺/Ag⁺, Cu²⁺, Zn²⁺/Cd²⁺, or Cu²⁺/Zn²⁺ P-type ATPase pumps. Conserved amino acids are in bold, and numbers in superscript represent amino acid positions. Proteins identified in this work are denoted by an asterisk. Boxes highlight positions where HmtA residues are conserved with residues of either Cu⁺/Ag⁺ pumps or Zn²⁺/Cd²⁺ pumps.

Fig. 7.

Topological predictions using TMHMM (35) for Cu⁺/Ag⁺ (A), Zn²⁺/Cd²⁺ (B), Mg²⁺ (C), or Cu²⁺/Zn²⁺ (D) P-type pumps. Red bars indicate transmembrane domains, blue lines indicate intracellular loops, and magenta lines indicate extracellular loops.