Dissecting the role of the N-terminal metal-binding
domains in activating the yeast copper ATPase in vivo
Isabelle Morin
1,2,3,
*, Simon Gudin
1,2,3
, Elisabeth Mintz
1,2,3
and Martine Cuillel
1,2,3
1 CEA, DSV, iRTSV, Laboratoire de Chimie et Biologie des Me
´
taux, Grenoble, France
2 CNRS, UMR 5249, Grenoble, France
3 Universite
´
Joseph Fourier, Grenoble, France
Introduction
Copper is an essential element for life because of its
ability to switch between Cu(I) and Cu(II) in vivo. Yet,
because of its redox properties, copper can also be
toxic when present in excess. Accordingly, the intracel-
lular concentration of this metal is tightly regulated by
a cascade of cytosolic and membrane proteins that
interplay to deliver copper to specific targets, namely
the cytosolic superoxide dismutase, the mitochondrial
cytochrome c oxidase and secreted enzymes maturing
in the trans-Golgi network (TGN) [1]. In humans, the
Menkes and Wilson proteins are copper ATPases
embedded in the TGN membranes that play an essen-
tial role in copper delivery to the secretory pathway

strain allowed us to study in vivo Atx1–Ccc2 and intra-Ccc2 domain–
domain interactions, leading to active copper transfer into the trans-Golgi
network. The Ccc2 N-terminus encloses two copper-binding domains, M1
and M2. We show that in vivo Atx1–M1 or Atx1–M2 interactions activate
Ccc2. M1 or M2, expressed in place of the metallo-chaperone Atx1, were
not as efficient as Atx1 in delivering copper to the Ccc2 N-terminus. How-
ever, when the Ccc2 N-terminus was truncated, these independent metal-
binding domains behaved like functional metallo-chaperones in delivering
copper to another copper-binding site in Ccc2 whose identity is still
unknown. Therefore, we provide evidence of a dual role for the Ccc2
N-terminus, namely to receive copper from Atx1 and to convey copper to
another domain of Ccc2, thereby activating the ATPase. At variance with
their prokaryotic homologues, Atx1 did not activate the Ccc2-derived
ATPase lacking its N-terminus.
Abbreviations
MBD, metal-binding domain; TGN, trans-Golgi network.
FEBS Journal 276 (2009) 4483–4495 ª 2009 The Authors Journal compilation ª 2009 FEBS 4483
highlight the need for copper, as well as its toxicity
[3,4].
The yeast Saccharomyces cerevisiae provides an
excellent model with which to characterize copper
delivery to the secretory pathway in further detail
because the proteins involved in copper homeostasis
are highly conserved from yeasts to humans. The first
step occurs at the membrane level where a reductase
converts extracellular Cu(II) into Cu(I); the latter then
enters the cell via the high-affinity transporter Ctr1 [5]
and is distributed to specific intracellular targets.
Delivery to the secretory pathway is mediated by the
metallo-chaperone Atx1, the homologue of Atox1, and

Several aspects of copper delivery into the TGN
have been studied. Under conditions where only high-
affinity transporters can ensure uptake, i.e. in copper-
and iron-limited media, deletion of ATX1 results in a
deficient high-affinity iron uptake and this leads to
growth arrest [15]. Consequently, atx1 D deletion
strains have been used as a tool to investigate whether
homologous proteins would replace Atx1 and ensure
copper transport into the TGN [18–22]. Deletion of
the CCC2 gene also results in a deficient high-affinity
iron uptake and leads to growth arrest in copper- and
iron-limited media. Thus, ccc2D deletion strains have
been used in complementation assays to assess the
transport of copper inside the TGN by Ccc2 [8] and
human homologues [23,24], or to investigate the role
of various amino acids in these proteins [25–28].
Although these studies provide useful information
about the functionality of the ATPase, they are all
interpreted independently of the metallo-chaperone.
Indeed, studying the Atx1–Ccc2 route in vivo remains
challenging, because of the existence of an Atx1-inde-
pendent pathway for copper delivery to the TGN [15].
This pathway was first discovered in atx1D , in which
overexpression of Ccc2 allows growth in a copper- and
iron-limited medium. The current hypothesis involves
the plasma membrane transporter Ctr1 which would
undergo endocytosis and deliver copper directly to
Ccc2, bypassing the need for Atx1 (Fig. 1A).
We designed a sensitive complementation assay to
study in vivo copper-dependent interactions between

Domain–domain interactions in Ccc2 I. Morin et al.
4484 FEBS Journal 276 (2009) 4483–4495 ª 2009 The Authors Journal compilation ª 2009 FEBS
(0.1–1 lm copper and 100 lm iron chelated by 1 mm
ferrozine). Such low concentrations of copper and iron
in the selective medium ensure that Ctr1 and the Fet3–
Ftr1 complex are the only effective transporters for
copper and iron uptake by the cells [5,8]. Bearing in
mind that Fet3 is essential for cell growth and that its
activity requires copper, the atx1Dccc2D deletion strain
cannot grow unless Atx1 and Ccc2 are coexpressed
and exchange copper, which then reaches Fet3 in the
TGN (Fig. 1A, plan arrows). In this section, we report
the design of a new complementation assay to dissect
the role of Atx1, M1 and M2 in activating Ccc2 in vivo
(the strains and plasmids needed for this assay are
described in Table 1).
Studying interactions between Atx1 and Ccc2 in vivo
was challenging because of the presence of an Atx1-
independent copper-delivery pathway to the TGN,
activated by Ccc2 overexpression [15] (Fig. 1A, dotted
arrows). Accordingly, expression of Ccc2 using a
multicopy vector pY–Ccc2 (PMA1 promoter) restored
act
cat
Cytoplasm
Golgi lumen
C
D
E
B

Ccc2
Ctr1
Ctr1
Ctr1
Golgi
Plasma
membrane
ATP
ADP
Fet3
Atx1
Fet3
Ftr1
Ccc2
Fig. 1. Ccc2 function, enzymatic cycle and schematic representations. (A) Copper delivery to the secretory pathway in yeast. Black dots rep-
resent Cu(I), white dots represent Fe(II). Both Cu(I) and Fe(II) are produced by Fre1, a membrane reductase that is not represented here to
simplify the scheme. Ctr1, high-affinity Cu(I) transporter at the plasma membrane; Atx1, cytosolic metallo-chaperone; Ccc2, copper ATPase
at the TGN membrane; Ftr1, high-affinity Fe(II) transporter; Fet3, multicopper ferroxidase. The Fet3–Ftr1 complex is secreted to the plasma
membrane. Under conditions where only high-affinity transporters are active, i.e. in copper- and iron-limited media, the physiological pathway
comprises Atx1, which delivers Cu(I) to Ccc2 (plain arrow). However, when Ccc2 is overexpressed, an endocytosis-dependent pathway
brings copper to Ccc2 independent of Atx1 (dotted arrow). (B) The enzymatic cycle of Ccc2. (1) The ATPase binds Cu(I) at its transport site,
a step which induces a conformational change and (2) phosphorylation from ATP; (3) the energy is used to open the transport site on the
other side of the membrane and to release Cu(I) into the TGN lumen; (4) the aspartyl-phosphate bound is hydrolyzed. (C) Wild-type Ccc2. A
schematic representation of Ccc2 structure that comes from the calcium ATPase structure [40]. Empty circles represent known Cu(I)-binding
sites. N-terminus includes M1 [white hexagon with the Cu(I)-binding sequence CASC] and M2 (grey hexagon with CGSC). The membrane
domain comprises eight helices and the transport site (CPC) is on helix 6. The catalytic loop (cat), where phosphoryl transfer occurs from
ATP to the phosphorylation site (Asp627 indicated by the star) when Cu(I) is bound to the transport site, is localized between helices 6 and
7. The actuator domain (act), between helices 4 and 5, participates in dephosphorylation of the catalytic loop. (D) Ccc2-like proteins bearing
only one functional domain, either M1 or M2. (E) A Ccc2-like protein unable to bind copper at its N-terminus. In (D) and (E), all the constructs
are named according to their N-terminus. Intact copper-binding motifs are denoted CXXC. SXXS refers to domains whose cysteines are

interacts with both domains of the Ccc2 N-terminus
(M1 and M2). Our new complementation assay using
pX was further assessed by studying copper transfer
from Atx1 to each MBD. Accordingly, we chose to
express Atx1 together with Ccc2 proteins bearing only
one intact MBD. We first generated pX–M1ssM2Ccc2
in which the copper-binding cysteines of M1 are chan-
ged to serines (i.e. M1 cannot bind copper) (Fig 1D).
Coexpression of Atx1 with M1ssM2Ccc2 restored the
growth of atx1Dccc2D in the selective medium, suggest-
ing that copper transfer occurred in vivo between Atx1
and M2 (Fig. 2C, lane 3). Growth restoration was dra-
matically reduced when Atx1 was not coexpressed
Table 1. Yeast strains and plasmids.
YPH499 Mata his3-D200 leu2-D1 trp1-D63 ura3-52 lys2-801 ade2-101
atx1Dccc2D Mata his3-D200 leu2-D1 lys2-801 ade2-101 atx1::SRP1 ccc2::URA3 This study
BY4741 Mata his3D1 leu2D0 met15D0 ura3D0
BY4741ccc2D Mata his3D1 leu2D0 met15D0 ura3D0 ccc2D ATCC
Derived from Plasmid name Promoter Terminator
Multicopy pYEp181 pY– PMA1 ADC1
Monocopy pRS313, pRS315 pC– PMA1 ADC1
Extra-low-copy pRS313 pX– Leak ADC1
Plasmid name Expressed protein Origin Ref.
Ccc2 Wild-type Ccc2 Saccharomyces cerevisiae [25]
M1ssM2Ccc2 C13S,C16S in Ccc2 This study
M1M2ssCcc2 C91S,C94S in Ccc2 This study
M1Ccc2 del (E81–G151) from Ccc2 This study
M1ssM2ssCcc2 C13S,C16S,C91S,C94S in Ccc2 This study
DMBDCcc2 del (M1–G151) This study
Ccc2GFP Green fluorescent protein tag in C-ter This study

dent interactions activate the ATPase in vivo, leading
to copper transfer in the TGN lumen and growth res-
toration in the selective medium. Our findings also
agree with those obtained with both human ATPases,
whose fifth or sixth MBD (of six) is sufficient for
normal transport activity of the protein in Chinese
hamster ovary cells [29,30].
Atx1 homologues and copper transfer to the
Ccc2 N-terminus
In a previous study, some structural homologues of
Atx1 (namely MerP, a mercury-binding protein from
Cupriavidus metallidurans CH34, Ntk, the MBD of the
cadmium ATPase from Listeria monocytogenes, and
CopZ, the metallo-chaperone from Bacillus subtilis),
were found to be able to deliver copper to endogenous
Ccc2 in an atx1D strain [22]. This study also showed
that M1 and M2, expressed as independent proteins
denoted Mbd1 and Mbd2, could also play the role of
Atx1. Given the higher sensitivity of our new expres-
sion system, we reasoned that expressing these Atx1
homologues would allow us to compare their ability to
transfer copper to Ccc2. For this purpose, Mbd1,
Mbd2, Mbd3 (M1 in tandem with M2 expressed as an
independent protein), MerP, CopZ and Ntk were coex-
pressed in atx1Dccc2D with Ccc2 and growth was
assessed in the selective medium (Fig. 3A). On the
whole, Mbd1, Mbd2 and Mbd3 were less efficient than
the other homologues in transferring copper to Ccc2, a
feature that was not observed in our previous study
[22]. Coexpression of Mbd1 or Mbd3 with Ccc2

+
pC-
pC-
pC-
pC-
pC-
pC-
atx1Δ ccc2Δ
YPH499
*
12
atx1Δ ccc2Δ
Cell dilution
Iron (µ
M)
pC-Atx1 pX-Ccc2+
pX-Ccc2
pC-Atx1 pX-M1ssM2Ccc2+
pX-M1ssM2Ccc2
pX-M1Ccc2
pX-M1Ccc2
+
+
+
+
pC-Atx1
1
2
3
4

pattern was similar (Fig. 3B). Interestingly, when
M1Ccc2 was expressed in place of Ccc2 neither Mbd1
nor Mbd2 seemed efficient in restoring copper transfer
(Fig. 3C). Taken together, these experiments suggest
that interactions between the independent MBDs and
one or the other MBD tethered to the membrane pro-
tein are poorly efficient or even not efficient in restor-
ing copper transfer. Recalling that copper transfer
from Atx1 to M1 or M2 is thought to occur via a
copper-mediated interaction [6,32], one possible expla-
nation is that M1 and M2 do not form homo- or
hetero-dimers in the presence of copper. This may be a
special feature of copper ATPase MBDs, as opposed
to the metallo-chaperones, because Atox1, Atx1 and
CopZ are known to form dimers in the presence of
copper [33–35]. Such an inability to form a dimer
would be beneficial to wild-type Ccc2, avoiding a
dead-end heterodimer between M1 and M2 and
favouring their interaction with Atx1 to collect copper.
These results prompted us to investigate further the
role of the N-terminus in the overall function of Ccc2.
In particular, we investigated whether Mbd1 or Mbd2
could deliver copper to some other site in Ccc2, apart
from the N-terminus.
Is the N-terminus dispensable in vivo?
In vitro studies of several ATPases have shown that
their N-terminus modulates their activity [36,37]. The
current hypothesis involves domain–domain interac-
tions between the N-terminus and other domains of
the ATPase. Indeed, in vitro studies of the purified

DKTGT in the catalytic loop. A tight coupling
between this chemical reaction and ion transport
across the protein is ensured by phosphoryl transfer
requiring copper to be bound to the so-called transport
site [43]. Once phosphorylated, the ATPase undergoes
a conformational change and copper can dissociate
towards the TGN lumen. The aspartyl-phosphate is
then hydrolysed and the ATPase is ready for a new
cycle. According to the known structures of other
P-type ATPases, the transport site is located among
the transmembrane helices, as indicated in Fig. 1C
Iron (µM)
Cell dilution
100 100 100
Metallo-chaperones
pC-Atx1
pC-
pC-Mbd1
pC-Mbd2
pC-Mbd3
pC-CopZ
pC-Ntk
pC-MerP
pX-Ccc2 pX-M1ssM2Ccc2 pX-M1Ccc2
ATPases
ABC
atx1
Δ
ccc2
Δ

parison with the background, evaluated with
Asp627Ala (Fig. 4C). In conclusion, DMBDCcc2 was
well localized in the cell and active in vitro but could
not deliver copper to the TGN because the N-terminus
was missing and Atx1–MBD copper-dependent inter-
actions could no longer occur to activate the ATPase.
We propose that, as described for the Wilson pro-
tein, the N-terminus interacts with another domain of
Ccc2 (the catalytic loop for example) and this interac-
tion inhibits the ATPase activity. Truncation of the
N-terminus alleviated these interactions in
DMBDCcc2, thereby facilitating the access of copper
at the transport site in vitro, as shown previously for
CadA [36,40]. Consequently, because Cu(I) is not
available in the cytosol [45], a suitable copper carrier
may be necessary to deliver copper to DMBDCcc2
in vivo. This is why we investigated whether Mbd1 or
Mbd2 could directly deliver copper to DMBDCcc2.
A role for M1 and M2 in delivering copper to
another binding site in Ccc2
In the first section of this study, we showed that Mbd1
and Mbd2 (M1 and M2 expressed as independent
proteins) were barely able to deliver copper to the
N-terminus. To evaluate the possibility that a native
N-terminal MBD delivers copper to a domain other
A
B
C
Fig. 4. DMBDCcc2 is active in vitro but does not restore copper
transport to the TGN. (A) The growth of atx1Dccc2D cotransformed

cysteines from the copper-binding site have been chan-
ged to serines (i.e. Mbd1ss cannot bind copper). Unlike
Atx1 (Fig. 5A, lane 2), Mbd1 and Mbd2 coexpressed
with DMBDCcc2 restored the growth of atx1Dccc2D in
the selective medium (Fig. 5A, lanes 4,5). No growth
restoration was observed with Mbd1ss, showing that
copper binding to Mbd1 or Mbd2 is a necessary step
to allow copper translocation into the TGN by
DMBDCcc2 (Fig. 5A, lane 3). In addition, no growth
restoration was obtained when CopZ or MerP was
coexpressed with DMBDCcc2 (data not show). Inter-
estingly, CopZ has an electrostatic potential surface
similar to that of Mbd1 and Mbd2 [22], but was still
unable to transfer copper to DMBDCcc2, suggesting
that electrostatic interactions are not the driving force
for copper transfer to DMBDCcc2. HA-tagged Mbd1,
Mbd1ss, Mbd2 and Atx1 were also coexpressed with
DMBDCcc2 to evidence their expression by immunode-
tection (Fig. 5B). Thus, our data disclose special prop-
erties of Mbd1 and Mbd2 which enable both to
transfer copper to DMBDCcc2, although this transfer
does not occur with Atx1.
Therefore, we can conclude that both M1 and M2,
when expressed as independent cytosolic proteins, are
able to bind copper in the cytosol and transfer this
metal to a copper-binding site in Ccc2, out of the
N-terminus. Although the site that receives copper
from Mbd1 or Mbd2 has not yet been identified in
DMBDCcc2, we can assume that when tethered to
Ccc2, M1 and M2 deliver copper to the same site.

First, we hypothesized the presence of inhibitory
domain–domain interactions involving the Ccc2 N-ter-
minus, as represented in Fig. 6A1, and similar to those
demonstrated for the Wilson ATPase in vitro [38,39].
These interactions were functionally evidenced by the
pY-Atx1
pY-Mbd1
pY-Mbd1ss
pY-Mbd2
pC-ΔMBDCcc2
pC-ΔMBDCcc2
pC-ΔMBDCcc2
pC-ΔMBDCcc2
pC-Atx1 pX-Ccc2
Cell dilution
A
+
+
+
+
+
B
p
YAH
1db
M
-
Yp
AH2d
bM

M) and iron-supplemented
(350 l
M) media. (B) Immunodetection of HA-tagged proteins
expressed in atx1Dccc2D using pY–Atx1HA, pY–Mbd1HA,
pY–Mbd2HA or pY–Mbd1ssHA together with pC–DMBDCcc2.
Domain–domain interactions in Ccc2 I. Morin et al.
4490 FEBS Journal 276 (2009) 4483–4495 ª 2009 The Authors Journal compilation ª 2009 FEBS
Cys to Ser mutations in all MBDs. The -SH to -OH
substitutions made the N-terminus unable to bind cop-
per, but still able to interact with the catalytic loop,
leading to a conformation that was inactive. Recent
structural data from the Archaeoglobus fulgidus copper
ATPase are in agreement with such interactions [47].
To alleviate these inhibitory domain–domain interac-
tions in wild-type Ccc2, copper transfer from Atx1 or
any other efficient metallo-chaperones to M1 or M2
domains of Ccc2 is essential (Fig. 6A1 and A2). We
demonstrate here that one, and only one, functional
copper-binding site on the Ccc2 N-terminus is required
for efficient copper transfer from Atx1 to Ccc2
(Fig. 6B where M1 is impaired and unable to bind
copper). Another way to disrupt these inhibitory
domain–domain interactions in Ccc2 is to delete the
N-terminus, as in DMBDCcc2. In vitro, DMBDCcc2 is
active and phosphorylatable by ATP in the presence of
copper. Copper binding at the membrane transport site
CPC (i.e. distinct from the N-terminus copper-binding
sites) induces phosphoryl transfer from ATP to
Asp627 (Fig. 1B) (for a description of the Ca
2+

tein–protein and intraprotein domain–domain interac-
tions leading to copper transfer from Atx1 to the
core of the copper ATPase. These interactions ensure
that no release of free copper occurs in the cytosol
during the transfer of the ion to the secretory path-
way. The assay designed here should be useful for
designing a functional Atox1–Menkes ATPase or
Atox1–Wilson ATPase pathway for copper delivery
into the TGN in yeast. This could enable further
investigation of the effect of mutations in Atox1 or
in the Menkes or Wilson ATPase N-terminus that
may impair copper transfer and result in disorders of
copper homeostasis.
Experimental procedures
Yeast strains
Genetic modifications were performed on the YPH499
strain of S. cerevisiae (a gift from Pierre Thuriaux, CEA,
Gif ⁄ Yvette, France). The chromosomal ATX1 gene was
disrupted in YPH499 using a deletion cassette bearing the
A
B
C
Fig. 6. Model for the copper route to the TGN. (A) Wild-type Ccc2:
(1) Atx1 can deliver Cu(I) to M1 or M2; (2) M2 has bound Cu(I); (3)
Cu(I) at the transport site (CPC) induces phosphorylation and trans-
port into the TGN. (B) One MBD is necessary and sufficient to
receive copper from Atx1 and to activate the ATPase. In this repre-
sentation, Atx1 delivers Cu(I) to the M2 domain of M1ssM2Ccc2.
(C) DMBDCcc2 receives Cu(I) from Mbd1 (or Mbd2) (1) and trans-
ports Cu(I) into the TGN (2).

Plasmids encoding the modified Ccc2 proteins described in
Fig. 1 were generated with the QuickChange site-mutagen-
esis kit (Stratagene, Agilent Technologies, Massy, France)
using pSP72CCC2 as the template [25]. In all cases, the
presence of designed mutations and the absence of fortu-
itous mutations were verified by automated DNA sequenc-
ing. CCC2 was first cloned in a multicopy plasmid (pY)
and in a monocopy plasmid (pC) under the control of the
PMA1 promoter and the ADC1 terminator and all bear-
ing HIS3 as selection marker (Table 1). The monocopy
plasmid was derived from pRS313 [50], the multicopy
plasmid from pYep181 in which the selection marker was
changed to HIS3 [51]. In an attempt to clone the CCC2
promoter in place of the PMA1 promoter in pC–Ccc2, a
fortuitous ligation occurred between SacII and StuI sites
generating a plasmid with neither CCC2 nor PMA1 pro-
moters. This plasmid was called pX–Ccc2, where pX
stands for extra-low-copy expression plasmid, and used
for extra-low expression of other constructs (Table 1).
When the enzymatic activity of DMBDCcc2 protein had
to be evaluated, the mutant was cloned in the pFastBac
plasmid for expression in Sf9 cells [25]. When the location
of the copper ATPase had to be verified, site-directed
mutagenesis was used to fuse the green fluorescent pro-
tein-coding sequence at the end of Ccc2 and DMBDCcc2
in the monocopy plasmid pC.
Expression in yeast and complementation assay
atx1Dccc2D was co-transformed with one plasmid express-
ing Atx1 or an Atx1-like protein and another expressing
wild-type or modified Ccc2. In some experiments, empty

O and
1mm ferrozine. Higher concentrations of iron in this
medium [350 lm NH
4
(FeSO
4
)
2
.6H
2
O] allow yeast cells with
an Atx1–Ccc2-deficient pathway to incorporate iron and
therefore to grow. The iron-supplemented medium was
systematically used as a control after each transformation of
atx1Dccc2D with modified Atx1 and ⁄ or Ccc2 proteins. For
each complementation assay, we used three different clones
obtained from at least two different transformations. Plates
were photographed after 3 days of growth at 30 °C.
Yeast proteins extracts: expression level analysis
Cells were spun down for 10 min at 1000 g and 4 °C and
washed twice with ice-cold water. They were then suspended
in buffer A (50 mm Tris ⁄ HCl, pH 8.0, 300 mm sorbitol,
2mm EDTA) supplemented with antiproteases [one tablet
of complete EDTA-free protease inhibitor (Roche Diagnos-
tics, Meylan, France) and 1 mm phenylmethanesulfonyl
fluoride]. After disruption with glass beads, the lysate was
centrifuged for 10 min at 1000 g to pellet cell debris and
nuclei. The resulting supernatant was spun down for 60 min
at 100 000 g and 4 °C and either the pellet was suspended in
buffer A for membrane protein determination or the super-

discussions and Naima Belgareh-Touze for her advice
on performing fluorescence microscopy with yeast.
Funding of this work was provided in part by the
Programme de Toxicologie Nucle
´
aire Environnemen-
tale. IM received support from a fellowship from the
Programme de Toxicologie Nucle
´
aire du CEA.
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