CPx-type ATPases: a class of P-type ATPases that pump heavy metals.

ATP-driven heavy metal pumps represent a newly defined class of proteins that translocate toxic and essential metals across biological membranes. These transporters form a separate evolutionary branch of the ion-transporting P-type ATPases. We propose to call these enzymes CPx-type ATPases, based on the common novel feature of a conserved intramembranous cysteine-proline-cysteine or cysteine-proline-histidine motif.

Bacterial genetics by electric shock.

Subjecting bacteria to a high-voltage electric discharge renders the cells permeable to DNA. This powerful method allows the genetic manipulation of bacterial species that cannot easily be transformed by conventional techniques.

Intracellular copper routing: the role of copper chaperones.

Copper is required by all living systems. Cells have a variety of mechanisms to deal with this essential, yet toxic trace element. A recently discovered facet of homeostatic mechanisms is the protein-mediated, intracellular delivery of copper to target proteins. This routing is accomplished by a novel class of proteins, the 'copper chaperones'. They are a family of conserved proteins present in prokaryotes and eukaryotes, which suggests that copper chaperones are used throughout nature for intracellular copper routing.

Na/H antiporter mRNA expression in single nephron segments of rat kidney cortex.

Renal cortical tubules consist of polarized epithelial cells where Na/H antiport activity has been demonstrated on the apical and/or basolateral membrane. Apical Na/H antiport activity plays an important role in transcellular bicarbonate (HCO3-) reabsorption, whereas basolateral Na/H antiport activity could be involved in transcellular HCO3- secretion as well as cell volume and pH control. To determine whether this heterogeneity in both localization and function is due to the existence of more than one Na/H antiporter, we studied the tissue distribution of Na/H antiporter mRNA by use of reverse transcription (RT) and polymerase chain reaction (PCR) in isolated nephron segments from rat renal cortex. The primers used were directed against the rat renal cortical Na/H antiporter cDNA which is homologous to the human growth factor-activatable Na/H antiporter. RT/PCR of beta-actin mRNA were performed as positive controls. Na/H antiporter mRNA expression in the proximal tubule was not detectable in S1 and S2 segments from superficial and most midcortical nephrons, which exhibit exclusively luminal Na/H antiport activity. It was expressed in S1 and S2 segments from juxtamedullary nephrons which have also basolateral Na/H antiport activity. Beta-actin mRNA was expressed uniformly in all segments of the proximal tubule. Na/H antiporter mRNA was also expressed in cortical thick ascending limb and cortical collecting duct, segments with basolateral Na/H antiport activity as well as in the glomeruli. In conclusion, at least two different Na/H antiporters exist in the renal cortex, i.e., the proximal tubule. The close correlation between functional localization of basolateral Na/H antiport activity and mRNA expression suggests that the rat kidney Na/H antiporter DNA homologous to the human growth factor activatable Na/H antiporter encodes a basolateral exchanger. The observed expression in a minority of midcortical proximal tubules could reflect a certain heterogeneity in these nephron segments.

Electrogenic transport by the Enterococcus hirae ATPase.

A transport ATPase from Enterococcus hirae was reconstituted in lipid vesicles and its electrogenic action investigated with the fluorescent dye oxonol VI as membrane potential probe. Reconstitution in bacterial and in soybean phospholipid mixtures led to transport-active vesicle preparations. Inside-out oriented ATPase molecules were activated by the addition of ATP to the extravesicular medium, generating in all experiments an intravesicularly positive potential. The extravesicular pH strongly influenced the initial pumping rate and the duration of the pumping activity. At neutral pH, transient pumping activity was observed, lasting for 1-2 min, while at pH 5.6, pumping was continuous. The transport activity was not dependent on the ionic composition of the buffer on either side of the membrane. These findings can be interpreted as the action of a proton ATPase, regulated by the cytoplasmic proton concentration and electrogenically translocating protons from the cytoplasm to the extracellular space.

Acylphosphate formation by the Menkes copper ATPase.

The Menkes ATPase is the product of the MNK gene, defective in some inherited human disorders of copper metabolism. We here show the formation of an acylphosphate intermediate by the murine MNK homologue in membranes from normal and copper resistant Chinese hamster ovary cells. In the latter, fivefold higher levels of acylphosphate were formed. Challenging these cells with copper, which induces relocation of the MNK ATPase from the trans-Golgi network to the plasma membrane, did not influence acylphosphate formation. The kinetics of phosphorylation, metal dependence, and sensitivity to inhibitors were investigated. The results show that the MNK ATPase is an active P-type ATPase and provide a direct functional test for this enzyme.

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.

Phosphoenzyme formation by purified, reconstituted copper ATPase of Enterococcus hirae.

The Enterococcus hirae CopB ATPase serves in the secretion of excess copper from cells and belongs to the recently discovered, new class of heavy metal transport ATPases. We here report the affinity purification of CopB to near homogeneity and its reconstitution into phospholipid vesicles. In these proteoliposomes, the ATPase formed an acylphosphate reaction intermediate with the gamma-phosphate of ATP. ATPase activity and phosphoenzyme formation were inhibited by vanadate with an I(50) of 0.1 mM. Our results suggest that heavy metal and non-heavy metal ATPases operate by the same underlying mechanism.

Tetrathiomolybdate inhibition of the Enterococcus hirae CopB copper ATPase.

Tetrathiomolybdate (TTM) avidly interacts with copper and has recently been employed to reduce excess copper in patients with Wilson disease. We found that TTM inhibits the purified Enterococcus hirae CopB copper ATPase with an IC(50) of 34 nM. Dithiomolybdate and trithiomolybdate, which commonly contaminate TTM, inhibited the copper ATPases with similar potency. Inhibition could be reversed by copper or silver, suggesting inhibition by substrate binding. These findings for the first time allowed an estimate of the high affinity of CopB for copper and silver. TTM is a new tool for the study of copper ATPases.

The Enterococcus hirae copper chaperone CopZ delivers copper(I) to the CopY repressor.

Expression of the cop operon which effects copper homeostasis in Enterococcus hirae is controlled by the copper responsive repressor CopY. Purified Zn(II)CopY binds to a synthetic cop promoter fragment in vitro. Here we show that the 8 kDa protein CopZ acts as a copper chaperone by specifically delivering copper(I) to Zn(II)CopY and releasing CopY from the DNA. As shown by gel filtration and luminescence spectroscopy, two copper(I) are thereby quantitatively transferred from Cu(I)CopZ to Zn(II)CopY, with displacement of the zinc(II) and transfer of copper from a non-luminescent, exposed, binding site in CopZ to a luminescent, solvent shielded, binding site in CopY.

Efficient electrotransformation of Enterococcus hirae with a new Enterococcus-Escherichia coli shuttle vector.

In the present study, an Enterococcus-Escherichia coli shuttle vector, pC3, was constructed that allows efficient transformation by electroporation of Enterococcus hirae ATCC9790. 5 x 10(6) transformants per microgram of plasmid DNA were obtained, using a commercial capacitor discharge device with an improved circuitry and a home-made electrode assembly, delivering pulses of 24 kV/cm across the cell suspension. The transformants were stable without selective pressure and plasmid DNA reisolated from transformed cells displayed no alterations in restriction enzyme analysis. Chromosomal DNA from E coli or E hirae, carried by pC3, was stably maintained in E hirae, making cloning and genetic manipulation in this organism feasible.

Technical report. Video imaging of ethidium bromide-stained DNA gels with surface UV illumination.

We describe here the use of surface UV illumination to record ethidium bromide-stained DNA gels with a video camera. This mode of illumination allows the use of a standard video camera equipped with a red filter and results in a high signal strength. The assembly of a low-cost video system on this basis is described. It uses the public domain software called Image on a Macintosh computer and PostScript laser printer or a thermal printer to generate hard copies. The setup is sensitive enough to detect 500 pg of DNA on an ethidium bromide-stained DNA gel. The UV illumination method described here can also greatly improve the sensitivity of existing video recording equipment.

Role of proteolysis in copper homoeostasis.

The cop operon of Enterococcus hirae controls cytoplasmic copper levels. It encodes two copper ATPases, a repressor, and the CopZ metallochaperone. Transcription of these genes is induced by copper. However, at higher copper concentrations, CopZ is degraded by a copper-activated proteolytic activity. This specific proteolysis of CopZ can also be demonstrated in vitro with E. hirae extracts. Growth of the cells in copper increases the copper-inducible proteolytic activity in extracts. Zymography reveals the presence of a copper-dependent protease in crude cell lysates. Copper-stimulated proteolysis of CopZ appears to play an important role in copper homoeostasis by E. hirae.

Two trans-acting metalloregulatory proteins controlling expression of the copper-ATPases of Enterococcus hirae.

Enterococcus hirae possesses two P-type ATPases, CopA and CopB, that are involved in copper homeostasis. These enzymes are induced by extracellular copper concentrations that are either too low or too high for optimal growth. To identify the regulatory proteins involved in induction, the DNA upstream of copA was cloned and sequenced. Following a putative promoter region, it contains two genes, copY and copZ, that encode proteins of 145 and 69 amino acids, respectively. Both proteins contain metal binding motifs and exhibit significant sequence similarity to known regulatory proteins. Gene disruption of copY by reverse genetics caused constitutive overexpression of CopA and CopB, generating a copper-dependent phenotype. In contrast, disruption of copZ suppressed the expression of the two copper-ATPases, rendering the cells copper-sensitive. Both null mutations could be complemented in trans with plasmids bearing copY or copZ. Thus, copY and copZ encode trans-acting metalloregulatory proteins that are required for induction of the cop operon by copper. In this mechanism, CopY apparently acts as a metal-fist type repressor and CopZ as an activator.

Operon of vacuolar-type Na(+)-ATPase of Enterococcus hirae.

The Gram-positive bacteria Enterococcus hirae expel sodium by two systems: a Na+/H(+)-antiporter and a vacuolar-type Na(+)-ATPase. We isolated a mutant, NalkA, defective in the Na(+)-ATPase. NalkA grew normally at neutral pH but was unable to grow in the presence of > 100 mM sodium at pH 9.5. By functional complementation at high pH, we cloned pES1, a plasmid from an E. hirae gene bank containing a 5.2-kilobase pair region of genomic DNA. The genomic DNA in pES1 contains five complete open reading frames, ntpM, -N, -O, -P, and -Q, encoding proteins of 75, 16, 23, 38, and 11 kDa. A sixth incomplete open reading frame, ntp 'L, precedes ntpM. The 3'-end of the cloned DNA overlaps with a previously published sequence encoding the ntpA and ntpB subunits of the E. hirae Na(+)-ATPase (Takase, K., Yamato, I., and Kakinuma, Y. 1993) J. Biol. Chem. 268, 11610-11616). The insert of pES1 therefore represents the upstream region of the ntp operon that encodes the E. hirae Na(+)-ATPase. Complementation analysis with various deletions derived from pES1 suggest that the original mutation is in the ntpM gene. Of the new genes described here, three exhibited significant sequence similarity to known proteins; ntpM shares 24% identical amino acid residues with the "116-kDa" subunits of eukaryotic vacuolar ATPases, ntpN exhibits 28% sequence identity with the 16-kDa proteolipid of human vacuolar ATPase, and ntpO has sequence homology to the 31-kDa subunit of the bovine kidney vacuolar ATPase. No known proteins with sequence similarity to ntp'L, -P, or -Q could be identified. Disruption of either ntpM, -N, or -O in wild-type cells by cassette mutagenesis resulted in mutants unable to effect ATP-driven sodium extrusion. NtpM, -N, and -O therefore represent three new gene products involved in sodium extrusion by the vacuolar-type Na(+)-ATPase of E. hirae, and three more gene products, NtpL, -P, and -Q, may also be constituents of this enzyme. The ntp operon thus contains at least eight genes.

The vanadate-sensitive ATPase of Streptococcus faecalis pumps potassium in a reconstituted system.

The vanadate-sensitive ATPase of Streptococcus faecalis, purified to homogeneity, was reconstituted into soybean phospholipid vesicles in a functional state. Freeze-fracture electron micrographs revealed a relatively uniform population of unilamellar liposomes of 50-100 nm in diameter, with particles protruding from both fracture faces. Transport studies with 42K+ and with a K+-selective electrode showed that the ATP-ase catalyzes electrogenic potassium extrusion in proteoliposomes. The following parameters for potassium transport in the reconstituted system were determined: K+/ATP stoichiometry = 1, Km for potassium = 1.4 mM, Vmax = 0.1 mumol/min/mg. The ATPase could be activated by an electrical membrane potential, vesicle interior positive. This ATPase thus appears to function as a potential regulated, ATP-driven pump that serves in electrogenic potassium accumulation by the bacterial cell.