P(1B)-type ATPases transport heavy metals (Cu+, Cu2+, Zn2+, Co2+, Cd2+, Pb2+) across membranes. Present in most organisms, they are key elements for metal homeostasis. P(1B)-type ATPases contain 6-8 transmembrane fragments carrying signature sequences in segments flanking the large ATP binding cytoplasmic loop. These sequences made possible the differentiation of at least four P(1B)-ATPase subgroups with distinct metal selectivity: P(1B-1): Cu+, P(1B-2): Zn2+, P(1B-3): Cu2+, P(1B-4): Co2+. Mutagenesis of the invariant transmembrane Cys in H6, Asn and Tyr in H7 and Met and Ser in H8 of the Archaeoglobus fulgidus Cu+-ATPase has revealed that their side chains likely coordinate the metals during transport and constitute a central unique component of these enzymes. The structure of various cytoplasmic domains has been solved. The overall structure of those involved in enzyme phosphorylation (P-domain), nucleotide binding (N-domain) and energy transduction (A-domain), appears similar to those described for the SERCA Ca2+-ATPase. However, they show different features likely associated with singular functions of these proteins. Many P(1B)-type ATPases, but not all of them, also contain a diverse arrangement of cytoplasmic metal binding domains (MBDs). In spite of their structural differences, all N- and C-terminal MBDs appear to control the enzyme turnover rate without affecting metal binding to transmembrane transport sites. In addition, eukaryotic Cu+-ATPases have multiple N-MBD regions that participate in the metal dependent targeting and localization of these proteins. The current knowledge of structure-function relationships among the different P(1B)-ATPases allows for a description of selectivity, regulation and transport mechanisms. Moreover, it provides a framework to understand mutations in human Cu+-ATPases (ATP7A and ATP7B) that lead to Menkes and Wilson diseases.

As in other P-type ATPases, metal binding to transmembrane metal-binding sites (TM-MBS) in Cu ؉ -ATPases is required for enzyme phosphorylation and subsequent transport. However, Cu ؉ does not access Cu ؉ -ATPases in a free (hydrated) form but is bound to a chaperone protein. CopA ͉ CopZ ͉ Cu homeostasis ͉ Cu-ATPase ͉ metal binding C opper is an essential cofactor in many biological processes (1). However, it also participates in harmful Fenton reactions. Consequently, Cu is ''buffered'' at a ''no-free Cu'' level by metallothioneins and chaperones with binding constants for Cu ϩ in the picomolar-femtomolar range (2, 3). Within these constraints, Cu ϩ chaperones route Cu ϩ to various intracellular targets, and Cu ϩ transmembrane transport systems maintain the total copper quota within the 10-100 M range (1-4). How the Cu ϩ chaperones transfer the metal to and from transmembrane transport sites is a central feature of transmembrane Cu ϩ transport. To better understand these phenomena, we have studied the delivery of Cu ϩ by the Archaeoglobus fulgidus Cu ϩ chaperone, CopZ, to the corresponding Cu ϩ -ATPase, CopA.CopA is a member of the P 1B subgroup of P-type ATPases (5-7). Cu ϩ -ATPases are essential to maintain Cu ϩ homeostasis. For instance, mutations in the two Cu ϩ -ATPase genes present in humans, ATP7A and ATP7B, lead to Menkes syndrome and Wilson's disease, respectively (8, 9). The Cu ϩ -ATPases transport cycle follows the classical E1/E2 Albers-Post model (10-12). Catalytic phosphorylation of the enzyme in the E1 conformation occurs upon binding of cytoplasmic metal to transmembrane metal-binding sites (TM-MBS) and ATP binding with high affinity (l M) to the ATP-binding domain (ATP-BD) (Fig. 1). It is assumed that upon phosphorylation, Cu ϩ is occluded within the transmembrane region. The subsequent conformational change allows metal deocclusion and release to the extracellular (vesicular/luminal) compartment followed by enzyme dephosphorylation and return to the E1 form (10). Functional studies of various Cu ϩ -ATPases have characterized the Cu ϩ transport, Cu ϩ -dependent ATPase activity, phosphorylation, and dephosphorylation partial reactions (5,(13)(14)(15)(16)(17)(18)(19).Cu ϩ -ATPases consist of eight transmembrane segments, two large cytosolic loops comprising the A-domain and the ATP-BD, and regulatory metal-binding domains (MBDs) in their N terminus (6, 8-10, 20, 21) (Fig. 1). A. fulgidus CopA has an atypical

SummaryIn bacteria, most Cu + -ATPases confer tolerance to Cu by driving cytoplasmic metal efflux. However, many bacterial genomes contain several genes coding for these enzymes suggesting alternative roles. Pseudomonas aeruginosa has two structurally similar Cu + -ATPases, CopA1 and CopA2. Both proteins are essential for virulence. Expressed in response to high Cu, CopA1 maintains the cellular Cu quota and provides tolerance to this metal. CopA2 belongs to a subgroup of ATPases that are expressed in association with cytochrome oxidase subunits. Mutation of copA2 has no effect on Cu toxicity nor intracellular Cu levels; but it leads to higher H2O2 sensitivity and reduced cytochrome oxidase activity. Mutation of both genes does not exacerbate the phenotypes produced by single-gene mutations. CopA1 does not complement the copA2 mutant strain and vice versa, even when promoter regions are exchanged. CopA1 but not CopA2 complements an Escherichia coli strain lacking the endogenous CopA. Nevertheless, transport assays show that both enzymes catalyse cytoplasmic Cu + efflux into the periplasm, albeit CopA2 at a significantly lower rate. We hypothesize that their distinct cellular functions could be based on the intrinsic differences in transport kinetic or the likely requirement of periplasmic partner Cu-chaperone proteins specific for each Cu + -ATPase.

The thermophilic, sulfur metabolizing Archaeoglobus fulgidus contains two genes, AF0473 and AF0152, encoding for PIB-type heavy metal transport ATPases. In this study, we describe the cloning, heterologous expression, purification, and functional characterization of one of these ATPases, CopA (NCB accession number AAB90763), encoded by AF0473. 50 ‫؍‬ 24 M). This is the first Ag ؉ /Cu ؉ -ATPase expressed and purified in a functional form. Thus, it provides a model for structurefunctional studies of these transporters. Moreover, its characterization will also contribute to an understanding of thermophilic ion transporters.

Copper is an important micronutrient required as a redox co-factor in the catalytic centers of enzymes. However, free copper is a potential hazard because of its high chemical reactivity. Consequently, organisms exert a tight control on Cu+ transport (entry-exit) and traffic through different compartments, ensuring the homeostasis required for cuproprotein synthesis and prevention of toxic effects. Recent studies based on biochemical, bioinformatics, and metalloproteomics approaches, reveal a highly regulated system of transcriptional regulators, soluble chaperones, membrane transporters, and target cuproproteins distributed in the various bacterial compartments. As a result, new questions have emerged regarding the diversity and apparent redundancies of these components, their irregular presence in different organisms, functional interactions, and resulting system architectures.

Bacterial copper (Cu) homeostasis enables both precise metallation of diverse cuproproteins and control of variable metal levels. To this end, protein networks mobilize Cu to cellular targets with remarkable specificity. However, the understanding of these processes is rather fragmented. Here, we use genome-wide transcriptomic analysis by RNA-Seq to characterize the response of to external 0.5 mm CuSO, a condition that did not generate pleiotropic effects. Pre-steady-state (5-min) and steady-state (2-h) Cu fluxes resulted in distinct transcriptome landscapes. Cells quickly responded to Cu stress by slowing down metabolism. This was restored once steady state was reached. Specific Cu homeostasis genes were strongly regulated in both conditions. Our system-wide analysis revealed induction of three Cu efflux systems (a P-ATPase, a porin, and a resistance-nodulation-division (RND) system) and of a putative Cu-binding periplasmic chaperone and the unusual presence of two cytoplasmic CopZ proteins. Both CopZ chaperones could bind Cu with high affinity. Importantly, novel transmembrane transporters probably mediating Cu influx were among those largely repressed upon Cu stress. Compartmental Cu levels appear independently controlled; the cytoplasmic Cu sensor CueR controls cytoplasmic chaperones and plasma membrane transporters, whereas CopR/S responds to periplasmic Cu Analysis of Δ and Δ mutant strains revealed a CopR regulon composed of genes involved in periplasmic Cu homeostasis and its putative DNA recognition sequence. In conclusion, our study establishes a system-wide model of a network of sensors/regulators, soluble chaperones, and influx/efflux transporters that control the Cu levels in compartments.

Zn 21 plays a critical role in plants as an essential component of key enzymes (Cu-Zn superoxide dismutase, alcohol dehydrogenase, RNA polymerase, etc.) and DNA-binding proteins (Marschner, 1995;Guerinot and Eide, 1999). Zn 21 deficiency leads to a reduction of internodal growth with a consequent rosette-like development and also produces an impaired response to oxidative stress, likely due to a reduction in superoxide dismutase levels (Hacisalihoglu et al., 2003). Thus, Zn 21 deficiency is a significant agricultural problem, particularly in cereals, limiting crop production and quality (Guerinot and Eide, 1999;Hacisalihoglu et al., 2003 Silva and Williams, 2001;Hall, 2002). Consequently, plants and other organisms have developed molecular chaperones, chelators, and specific transmembrane transporters to (1) absorb and distribute metal micronutrients throughout the entire organism and (2) prevent high cytoplasmic concentrations of free heavy metals ions (Fox and Guerinot, 1998;Rauser, 1999;Guerinot, 2000;Williams et al., 2000;Clemens, 2001;Cobbett and Goldsbrough, 2002;Hall, 2002). These processes require the metal to be transported through permeability barriers and compartments delimited by lipid membranes. Several types of heavy metal transmembrane transporters have been identified in plants (Rea, 1999;Guerinot, 2000;Maser et al., 2001;Baxter et al., 2003). Since metal ions must be transported against electrochemical gradients at some point during plant distribution, metal pumps involved in contragradient transport should play key roles in metal homeostasis. The presence of plant genes encoding proteins that specifically perform this function (mainly P IB -ATPases) is known and their potential importance has been repeatedly noted (Williams et al., 2000;Clemens, 2001;Hall, 2002 (Lutsenko and Kaplan, 1995;Axelsen and Palmgren, 1998;Argü ello, 2003). Initial reports named these proteins CPxATPases (Solioz and Vulpe, 1996). They confer metal tolerance to microorganisms (Solioz and Vulpe, 1996;Rensing et al., 1999) and are essential for the absorption, distribution, and bioaccumulation of metal micronutrients by higher organisms (Bull and Cox, 1994; 1 This work was supported by the U.S. Department of Agriculture (grant no. 2001-35106-10736) and by the National Science Foundation (grant no. MCM-0235165).* Corresponding author; e-mail arguello@wpi.edu; fax 508-831-5933.Article, publication date, and citation information can be found at www.plantphysiol.org/cgi

Cu؉ -ATPases drive metal efflux from the cell cytoplasm. Paramount to this function is the binding of Cu ؉ within the transmembrane region and its coupled translocation across the permeability barrier. Copper is an essential micronutrient (1, 2). It has critical catalytic and electron transfer roles in a number of key proteins (tyrosinase, lysyl oxidase, ferroxidase ceruloplasmin, plastocyanin, etc.). However, when free, copper participates in the production of reactive oxygen species leading to cellular damage. Toward sustaining intracellular copper balance, transmembrane transport systems maintain the copper cell quota, Cu ϩ chaperone proteins traffic the bound metal to specific cellular targets, and metal-sensing transcription factors control copper dependent protein expression (3-5). The metal coordination geometry in these proteins is central to the efficiency of the Cu ϩ mobilization processes. In this direction, the coordination should ensure the specificity and prevent the release of free Cu ϩ to the cytoplasm. Canonical copper metalloproteins have long been characterized and classified based on spectroscopic and magnetic properties (Types I, II, and III) (6 -8). Their study has provided great detail on copper coordination in "permanent" sites where copper is bound during the functional life of the proteins. Cu ϩ linear coordination by invariant Cys residues of chaperone proteins has been described, providing insight into the mechanism of copper trafficking and exchange among similar domains (9, 10). More recently, trigonal coordination by Cys 2 -His sites has been observed, for instance, in Mycobacterium tuberculosis transcription factor CsoR (11). Alternatively, Met n His was found in several Cu ϩ -trafficking proteins located in the oxidizing periplasm of prokaryotes (12)(13)(14). Despite this progress, Cu ϩ distribution and balance cannot be understood without describing the selective coordination during compartmental transmembrane transport.In eukaryotic cells, members of the Ctr family of proteins transport Cu ϩ inside the cell (15). Ctr1 organizes as homotrimers forming transmembrane pores that facilitate Cu ϩ transmembrane translocation by an apparently energy-independent undefined mechanism (16, 17). Although relevant Cu ϩ -binding Met have been observed in the extracellular loops of Ctr1 (15); none of the invariant transmembrane residues appear to be required for transport, and no direct coordination is evident (17).As a counterpart to influx systems, Cu ϩ -ATPases are responsible for cytoplasmic Cu ϩ efflux. Mutations of the human Cu ϩ -ATPase genes, ATP7A and ATP7B, lead to Menkes syndrome and Wilson disease, respectively (18,19). Cu ϩ -ATPases are members of the superfamily of P-type ATPases (20, 21). These couple Cu ϩ transport to the hydrolysis of ATP, following a classical Post catalytic/transport cycle (19,21). In this mechanism, transmembrane metal-binding sites (TMMBSs) 4 are responsible for handling the ion during transmembrane translocation (22). These transmembrane sites are expos...