From the
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
Copper is an essential heavy metal ion by its function as a
cofactor of many enzymes. Since copper can be very toxic to both
eukaryotic and prokaryotic cells, homeostatic mechanisms have evolved
to balance the intracellular copper concentration. We have recently
described one such mechanism in Enterococcus hirae that is
dependent on the activity of two ATPases, CopA and CopB, that are
regulated in their expression by the copper concentration
(1, 2) . Studies with whole cells had suggested that
CopA serves in the uptake and CopB in the extrusion of copper
(3) . Based on their primary structure, CopA and CopB were
considered to be novel members of the family of P-type ATPases.
Typical features of P-type ATPases, for which
Ca
Several putative copper ATPases similar to CopA
and CopB of E. hirae have since been described from other
organisms. Most notably, in humans, two ATPases with over 30% amino
acid sequence identity to CopB were identified in connection with the
copper metabolic diseases Menkes
(7, 8, 9) and
Wilson
(10, 11, 12) . Two related ATPases, PacS
and CtaA, were described for the cyanobacteria Synechococcus 7942
(13, 14) . Also, hpCopA of Helicobacter
pylori (15) and ccc2 of Saccharomyces
cerevisiae (GenBank
Of
particular interest in this context are the cadmium resistance ATPases
that have been cloned from three different microorganisms
(17, 18, 19) . These proteins have metal binding
motifs similar to those of the putative copper ATPases and thus appear
to form, together with these, a family of heavy metal ATPases. The
cadmium ATPases are the only members of this family for which heavy
metal ion transport has actually been demonstrated. Native inside-out
membrane vesicles of Bacillus subtilis harboring the
Staphylococcus aureus cadA ATPase gene were shown to catalyze
ATP-driven
To demonstrate copper transport by the CopB ATPase of E.
hirae, native membrane vesicles proved to be a suitable
preparation. Such vesicles are a mixed population of inside-out and
right-side-out vesicles. However, since the bacterial membrane is
impermeable to ATP, only vesicles with the ATP binding site of the
ATPase facing outside (inside-out vesicles) will respond to added ATP.
Fig. 1
shows ATP-stimulated copper accumulation into vesicles. The
most rapid copper accumulation was observed in the complete reaction,
which contained 5 m
M dithiothreitol. Since dithiothreitol
reduces the added copper(II) to copper(I), the transported species
under these conditions is copper(I). Very little copper was taken up
without ATP. If dithiothreitol was omitted from the reaction, a basal
level of copper uptake and high background binding were observed. 40
µ
M vanadate, a specific inhibitor of P-type ATPases,
inhibited the dithiothreitol-stimulated copper uptake (see also below).
E. hirae membrane vesicles thus display ATP-stimulated,
vanadate-sensitive accumulation of Cu
We had previously shown that whole cells can
extrude silver and suggested that the enzyme involved in the process is
the CopB ATPase
(3) . To corroborate these in vivo observations, we investigated silver transport by native membrane
vesicles. Fig. 3 shows that vesicles containing only the CopB ATPase
showed ATP-stimulated accumulation of
Fig. 4
shows the analysis of kinetic parameters of copper
transport by CopB. The reaction had an apparent V
We here show that CopB of E. hirae is an ATP-driven
pump that can expel Cu
That copper(I) and not
copper(II) is the substrate of CopB suggests that in the cytoplasm,
copper not sequestered by copper enzymes occurs in the copper(I) form,
either due to the reducing environment of the cytoplasm or through the
action of reductases. The V
P-type ATPases, which evidently have evolved from a common
precursor, have developed into a versatile group of ion pumps capable
of transporting H
The proline residue of the purported ion channel is
located in a putative transmembranous helix and is invariant in all
known P-type ATPases. Site-directed mutagenesis of this amino acid in
the Ca
The other structural element that is
being considered typical for heavy metal ATPases is the heavy metal ion
binding domain in the polar N-terminal region of these enzymes. The
consensus sequence G XXC XXC, present in the N terminus
of CadA, occurs in all conjectured copper ATPases, but is absent from
CopB of E. hirae. In the proposed copper ATPases encoded by
the human Wilson and Menkes disease genes, the motif
G XXC XXC is even repeated five and six times,
respectively, and forms part of a reiterated building block of
60-70 amino acids
(31) . The notion that this motif forms
a general heavy metal ion binding site originates from the observation
that the same structural element also occurs in mercury reductases and
periplasmic mercury binding proteins
(32) .
Rather than the
consensus sequence G XXC XXC, the N terminus of CopB
features three repeats of the type
M XH XXMSGM XHS. Similar motifs are also found
in a periplasmic copper binding protein of P. syringae and has
been postulated to form copper binding domains
(5) . Also, CopB
is the only ATPase known to date that possesses the sequence CPH at the
site of the proposed ion channel. Why CopB differs from the other heavy
metal ATPases in these regards is at present not clear. A possibility
that should not be discarded at this point is that the substrates for
at least some of the putative ``copper'' ATPases is in fact
not copper, but another heavy metal ion.
Since radioactive copper is not readily available in
Europe, M. S. performed the copper transport experiments at the Howard
Hughes Medical Center of the University of California, San Francisco.
We are grateful to Jane Gitschier and Seymour Packman for their very
generous hospitality that made this work possible. Thanks are also due
to Christopher Vulpe for his help and very stimulating discussions and
to Susan Whitney and Thomas Weber for excellent technical support.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Cu
and
Ag
, 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 K
for Cu
and
Ag
of 1 µ
M and for ATP of 10
µ
M. Transport was maximal at pH 6 and had an apparent
V
of 0.07
nmol
min
mg
for both
copper and silver transport. Vanadate displayed a biphasic effect on
transport: maximal inhibition was observed at 40 µ
M vanadate for copper transport and 60 µ
M 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.
-ATPases are paradigms, are a protein subunit of
around 100 kDa, sensitivity to vanadate, and the formation of an
acylphosphate intermediate in the transport reaction. The relatedness
of P-type ATPases is also apparent by sequence features that are
conserved in these enzymes form bacteria to man, such as the
``phosphatase'' domain, TGES, or the
``phosphorylation'' domain, DKTGT
(4) . Besides the
presence of such conserved sequence elements in the CopB-ATPase, this
protein also exhibits two features that are known from other heavy
metal-binding proteins. These are (i) cysteine flanking the
intramembranous conserved proline that forms the ion channel in P-type
ATPases, and (ii) the N-terminal motif
M XH XXMSGM XHS that is also found in a
copper-binding protein of Pseudomonas syringae (5) .
Based on these observations, we had suggested that CopB is a copper
ATPase
(6) .
/EMBL Data Bank accession number
L36317) were postulated to be copper-pumping ATPases. Similar genes
that have as yet not been associated with any function are
fixI of Rhizobium meliloti (16) , open reading
frame YBR295w of S. cerevisiae, and open reading frame o732 of
Escherichia coli (GenBank
/EMBL Data Bank
accession numbers Z36164, L33259, and U00039, respectively).
Cd
accumulation
(20) . In contrast, the evidence that any of the postulated
copper ATPases is indeed a copper pump has so far been indirect and
rested on the behavior of whole cells in response to copper and on the
special structural features of these pumps
(1, 21) .
Here, we show ATP-driven
Cu
and
Ag
accumulation by the CopB ATPase in
native membrane vesicles, thus providing direct evidence that CopB of
E. hirae is a heavy metal ATPase.
Strains and Culture Conditions
E. hirae wild-type (ATCC 9790, formerly called Streptococcus faecalis or faecium) was obtained from the American Type Culture
Collection. The generation of the gene-disrupted strains copA,
copB, copAB, and copY have been described
previously
(2, 22) . Cells were grown semi-anaerobically
(the cultures were sealed but not made anaerobic) in 1%
NaHPO
2H
O, 1% trypticase
peptone (BBL), 0.5% yeast extract (BBL), 1% glucose. Growth was
monitored by measuring the absorption of a 1:10 dilution in water at
546 nm.
Vesicle Preparation
5-Liter cultures were
inoculated from frozen stocks of logarithmically growing cells and
grown in a fermentor at 37 °C. The cultures were induced with 500
µ
M CuSO, added at an OD of 1.5-2, and
grown for an additional 30 min. Cells were harvested by centrifugation
for 10 min at 2000
g and washed once with 1.5 liters
and once with 0.5 liter of 2 m
M MgSO
. Protoplasts
were obtained by suspending 10 g of cells in 60 ml of 250 m
M K
SO
, 50 m
M glycylglycine, 2
m
M MgSO
, pH 7.2, and treating with 40 mg of
lysozyme (Sigma) in the presence of 1 m
M phenylmethylsulfonyl
fluoride (Merck, Darmstadt, Germany) for 1 h at 37 °C in an orbital
shaking water bath. The protoplasts were centrifuged for 25 min at
23,000
g and the pellet suspended in 20 ml of CT
buffer (100 m
M Tris-Cl, 150 m
M KCl, 50 m
M NaCl, 5 m
M MgSO
, pH 6.5) for copper transport
experiments or ST buffer (100 m
M Tris-SO
, 25
m
M K
SO
, 25 m
M Na
SO
, 5 m
M MgSO
, pH
7) for silver transport experiments. The protoplasts were supplemented
with 1 mg of DNase I (Sigma) and homogenized in a Potter-Elvehjem-type
homogenizer before passing them through a French press at 70
Megapascal. Unlysed cells were collected by centrifugation for 12 min
at 23,000
g and the membrane fraction collected by
ultracentrifugation for 1 h at 90,000
g. The membrane
pellets were washed by resuspension in the same buffer and centrifuging
as before. The final pellet was suspended in 2.5 ml of the same buffer
and frozen in aliquots at -70 °C. The protein concentration
of these preparations was 3-4 mg/ml as determined with the
Bradford protein assay
(23) . For all experiments, frozen
membranes were thawed just before use. No loss in activity of frozen
membranes was observed over 2 months.
Copper Transport Measurements
0.1 mg of membrane
protein was suspended in 1 ml of CT buffer, supplemented with 5 m
M dithiothreitol, 0.5-5 µCi of Cu
(University of Missouri Research Reactor, Columbia, MO; specific
activity: 1-300 Ci/mg, depending on the time of use) and
0.05-10 µ
M cold copper, added as CuSO
,
as specified under ``Results.'' After 30 min preincubation at
37 °C with rotary shaking, transport was initiated by adding 0.5
m
M Na-ATP, pH 6.5. 100-µl samples were taken at intervals
and filtered through 25-mm filter disks of 0.22-µm nitrocellulose
membranes (Millipore) that had been stored for at least 1 h in CS
buffer containing 1 m
M CuSO
. The vesicle deposits
on the filters were immediately washed twice with 1 ml of CT buffer
containing 1 m
M CuSO
, the moist filters dissolved
in scintillation mixture, and the radioactivity determined in a
-counter. For the measurements of the pH dependence of transport,
CT buffer was adjusted to the corresponding pH values with Tris base.
Incubations at low pH were performed in phosphate buffer of the
following composition: 50 m
M NaP
, 50 m
M Tris-Cl, 75 m
M KCl, 25 m
M NaCl, 5 m
M MgSO
, adjusted to the desired pH with NaOH. The wash
steps were performed with CT buffer of the corresponding pH and
containing 1 m
M CuSO
. For the measurement of
vanadate inhibition, VO
was added to the
vesicles at the onset of the preincubation.
Silver Transport Measurements
Silver transport
experiments were conducted in the absence of dithiothreitol. 0.1 mg of
membrane protein was preincubated for 10 min at 37 °C with 1
µ
M tetrachlorosalicylanilide and 0.5 µ
M valinomycin in ST buffer. 0.2 µCi of
Ag
(Amersham Corp., 2 mCi/mg) and
0.2-10 µ
M AgNO
, as required by the
experiment, were added and preincubation continued for 10 min. Other
details of the procedure were as described for copper transport, except
that the filters were preincubated and washed with ST buffer containing
1 m
M AgNO
.
. Approximately
the same basal level of transport under nonreducing conditions or in
the presence of 40 µ
M vanadate was observed in all
experiments. It was not sensitive to uncouplers, and its origin is not
clear. Conceivably, it is due to nonspecific secondary transport or
binding. For calculation, we subtracted the basal
dithiothreitol-independent rate of transport.
Figure 1:
Copper
accumulation by native membrane vesicles. At time 0, transport was
initiated by the addition of ATP. The copper concentration was 50
n
M. Other details were as described under ``Experimental
Procedures.'' , complete reaction;
, without ATP;
, without dithiothreitol;
, in presence of 40 µ
M VO
.
The experiments of
Fig. 1
were conducted with a strain that overexpresses both the
CopA and the CopB ATPase due to disruption of the regulatory gene
copY
(22) . To ascertain weather CopA or CopB was
responsible for the observed copper accumulation, we employed strains
that were null mutants in either CopA ( copA), CopB
(
copB), or both (
copAB). Fig. 2 shows that
wild-type and a strain only possessing CopB exhibited high copper
transport activity, whereas strains deficient in either CopB, or both
CopA and CopB, only displayed the basal, dithiothreitol-independent
level of transport activity. We thus conclude that copper(I) is
accumulated in vesicles by the action of the CopB ATPase. This is in
line with the proposed in vivo function of CopB in copper
resistance by exporting copper from the cytoplasm. Further
investigation of CopB was conducted in the
copA strain
only expressing CopB.
Ag
. In the absence of ATP or in
membranes devoid of CopB, there were basal levels of silver
accumulation similar to those observed in copper transport experiments.
These basal rates were somewhat, but not completely, suppressed by the
K
and H
ionophores valinomycin and
tetrachlorosalicylanilide. Dithiothreitol, which proved essential to
measure Cu
transport, was not required for silver
transport; in fact, it interfered with the Ag
measurements, probably by interacting with Ag
.
These results show that silver(I) is a substrate for the CopB ATPase
and that dithiothreitol employed in the copper transport experiments is
not required by the vesicles, but strictly serves to reduce copper(II)
to copper(I). That Ag
can replace Cu
had also been observed in other instances, such as for the
copper-dependent yeast transcription factor ACE1
(24) .
of 0.07 nmol
min
mg
and a K
for Cu
of
1 µ
M. However, these values must be interpreted
cautiously; the reaction contains the copper binding entities Tris,
dithiothreitol, and the membrane vesicles. We thus cannot know the
exact concentration of free Cu
ions, and the value we
derived for the K
is most likely an
overestimate. Nevertheless, the enumeration of kinetic parameters is
useful in connection with the analysis of mutant forms of CopB. The
K
for ATP was 10 µ
M, and the
kinetic parameters for silver transport by CopB were essentially the
same as those for copper, although the same uncertainty regarding the
free silver concentration applies (data not shown).
Figure 4:
Plot of the copper transport rate, v,
versus the copper concentration, S. Transport rates at varying
copper concentrations were measured with copA membrane
vesicles as outlined in the legend to Fig. 2. Inset,
Lineweaver-Burk plot of the same data.
Fig. 5
shows the pH dependence of the transport by the CopB
ATPase. Maximal activity was observed at pH 6. At more acidic pH
values, the ATPase activity dropped rapidly, whereas at increasing pH,
only a gradual decline in activity was observed. We previously showed
that the Na-ATPase that is present in E. hirae is also active up to pH 9. At high pH, E. hirae can have
a very small or no proton motive force and can no longer rely on
symporters and antiporters for the ionic control of the cytoplasm and
becomes dependent on the operation of primary transport systems
(25) . If the regulation of cytoplasmic copper is vital at high
pH, the cell needs a copper pump that can operate under these
conditions.
Figure 5:
pH dependence of CopB activity. The rate
of copper transport as a function of pH was determined with
copA membrane vesicles. The copper concentration was 1
µ
M; other details of the experiment were as described
under ``Experimental Procedures.''
, measurements
conducted in phosphate buffer;
, measurements conducted in CT
buffer.
Vanadate is a specific inhibitor of P-type ATPases and
therefore is a diagnostic tool for these enzymes
(26) . The
vanadate inhibition of copper and silver transport was analyzed in more
detail in Fig. 6. An unusual, biphasic inhibition curve was observed.
Copper transport was progressively inhibited by up to 40 µ
M VO and silver transport by up to 60
µ
M VO
. A maximal level of
85% inhibition was observed for copper and 76% for silver transport.
These inhibitions were reversed at higher vanadate concentrations. We
have no obvious explanation for this behavior. The different minima in
the inhibition curves for copper and silver transport may be due to the
presence of dithiothreitol in only the copper experiments. Vanadium has
a complex chemistry with many different oxidation states and polymeric
forms
(27) . Conceivably, under the reducing conditions of the
copper transport reaction, species of vanadium other than
VO
act on CopB. Indeed, it has been
observed that the combination
VO
/glutathione leads to the formation of
a vanadium species that activates adiposite cyclic nucleotide
phosphodiesterase
(28) . Overall, both silver and copper
transport show the same unusual, biphasic vanadate inhibition curve.
The meaning of this inhibition/activation pattern remains to be
elucidated.
and Ag
from
the cytoplasm. It is the first example of a P-type ATPase for which
copper (and silver) transport has been shown. Wild-type E. hirae can tolerate up to 8 m
M copper in the media, and we have
shown by genetic analysis that CopB is required for this resistance
(2) . The physiological role of the CopB ATPase is thus the
control of cytoplasmic copper by extruding it from the cell. The growth
requirement of microorganisms for copper is satisfied by 1-10
µ
M copper
(29) ; these concentrations are not toxic
to E. hirae and do not induce the cop-operon.
Cytoplasmic copper concentrations may therefore be in this micromolar
range and a K
of 1 µ
M for
copper transport by CopB appears reasonable. Silver, in contrast to
copper, is not essential for cell growth and is extremely toxic to
E. hirae: 5 µ
M Ag
inhibits cell
growth. Silver transport catalyzed by CopB is probably fortuitous and
does not serve a physiological role.
of 0.07
nmol
min
mg
for copper
transport by vesicles is very low. The only comparable system, the CadA
ATPase of S. aureus, displayed transport rates for
Cd
that were roughly 100-fold higher
(20) . However, CadA of S. aureus is a plasmid-encoded
resistance mechanism only present in some strains, whereas the CopB
ATPase of E. hirae is a chromosomally coded function of copper
homeostasis. The observed transport rates may be sufficient to deal
with excess cytoplasmic copper, although we cannot entirely rule out
that our experimental conditions do not result in full activity.
, Na
,
K
, Ca
, Mg
,
Cd
and, as shown here, Cu
and
Ag
. Of this collection of ions, Cd
,
Cu
, and Ag
are distinct in that they
are heavy metal ions and thus have special properties. In particular,
they strongly interact with the side chains of cysteine and histidine
residues. This must be reflected by the primary structures of heavy
metal ion ATPases. When inspecting the amino acid sequences of the
S. aureus Cd
-ATPase and the E. hirae Cu
(Ag
)-ATPase, the only two
heavy metal ATPases of known ion specificity, two features are
particularly apparent: one or two cysteines flanking the proline
residue that is believed to be part of the ion channel through the
membrane, and the presence of heavy metal binding motifs in the N
terminus.
-ATPase of the sarcoplasmic reticulum showed
that this proline is essential for transport
(30) . It is
embedded in the sequence IPE-309 in the sarcoplasmic
Ca
-ATPase. Although no cysteines are found adjacent
to this proline in any of the non-heavy metal ATPases, it is flanked by
two cysteines in the Cd
-ATPase (CPC-373) and a
cysteine and a histidine (CPH-398) in the CopB ATPase. All the
postulated copper ATPases display the motif CPC in corresponding
positions (CPSC in the putative H. pylori ATPase). This
clearly suggests that the presence of the CPC motif is indicative of
heavy metal ion transport.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.