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J Biol Chem, Vol. 274, Issue 34, 23719-23725, August 20, 1999
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From the Departments of Environmental Health Sciences
and Biochemistry, The Johns Hopkins University School of Public Health,
Baltimore, Maryland 21205 and the Departments of ¶ Chemistry and
** Biochemistry, Molecular Biology and Cell Biology, Northwestern
University, Evanston, Illinois 60208-3113
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
The copper chaperone for superoxide dismutase
(SOD1) inserts the catalytic metal cofactor into SOD1 by an unknown
mechanism. We demonstrate here that this process involves the
cooperation of three distinct regions of the copper chaperone for SOD1
(CCS): an amino-terminal Domain I homologous to the Atx1p
metallochaperone, a central portion (Domain II) homologous to SOD1, and
a short carboxyl-terminal peptide unique to CCS molecules (Domain III). These regions fold into distinct polypeptide domains as revealed through proteolysis protection studies. The biological roles of the
yeast CCS domains were examined in yeast cells. Surprisingly, Domain I
was found to be necessary only under conditions of strict copper
limitation. Domain I and Atx1p were not interchangeable in
vivo, underscoring the specificity of the corresponding
metallochaperones. A putative copper site in Domain II was found to be
irrelevant to yeast CCS activity, but SOD1 activation invariably
required a CXC in Domain III that binds copper. Copper
binding to purified yeast CCS induced allosteric conformational changes
in Domain III and also enhanced homodimer formation of the polypeptide. Our results are consistent with a model whereby Domain I recruits cellular copper, Domain II facilitates target recognition, and Domain
III, perhaps in concert with Domain I, mediates copper insertion into
apo-SOD1.
Recently, a new class of eukaryotic proteins that act in the
intracellular trafficking of copper ions has been identified. These
copper chaperones have been characterized in both yeast and humans (1)
and are typically defined as soluble copper receptor proteins that
function to deliver copper ions to specific intracellular targets (2).
Whereas the copper enzymes that serve as targets for these molecules
exhibit a high affinity for copper in vitro, the copper
chaperone functions are critical in vivo because free
intracellular copper concentrations are quite restricted (3).
One of the first genes identified in copper trafficking, yeast
ATX1, encodes a protein that binds copper and docks with a specific partner protein (2, 4-6). This partner for Atx1p is the
P-type copper-transporting ATPase in the secretory pathway, yeast Ccc2p
(7), or in the case of human ATX1 (HAH1 or ATOX1), the human Wilson and
Menkes gene products (8, 9). The metal delivered in this pathway is
incorporated into copper-requiring enzymes destined for the cell
surface or extracellular milieu. Cox17p represents another well
conserved copper protein that acts in a distinct pathway to help
deliver copper to the mitochondria, where the metal is ultimately
incorporated into cytochrome oxidase (10-13). More recently, we
identified a third eukaryotic copper chaperone that targets the
cytosolic copper- and zinc-requiring superoxide dismutase
(SOD1).1 This copper
chaperone for SOD1 (CCS) was originally discovered as the yeast
LYS7 gene product, and a functional homologue has been
identified in humans (14-17). CCS is specific for its target and is
particularly crucial for activating SOD1 in vivo, where intracellular free copper is limiting (3). Yeast CCS (yCCS) protein has
been purified to homogeneity and been shown to be necessary and
sufficient for direct copper insertion into SOD1 (3); however, the
mechanism by which CCS recognizes SOD1 as its target and inserts the
metal into the enzyme is unknown.
To begin to understand the nature of CCS specificity and its mechanism
of action, we have conducted a structure/function analysis on the yeast
protein both in vivo and in vitro. We find that
this copper chaperone consists of three structurally distinct domains that carry out independent functions in activating SOD1 with copper: (i) an amino-terminal Atx1p-like region that appears critical for
capturing copper under metal starvation conditions, (ii) a central
SOD1-like region that may facilitate target recognition, and (iii) a
small, conserved COOH-terminal region that has the potential to bind
copper and is indispensable for CCS function under all conditions. Our
results are consistent with an allosteric model in which the tertiary
structure of the CCS protein, in particular the COOH-terminal domain,
undergoes metal-induced conformational changes that ultimately
facilitate direct insertion of copper into the active site of SOD1.
Yeast Strains and Media--
The yeast strains SY2950
(lys7 Biochemical Analyses--
Cell lysates were prepared for
immunoblot studies and for analysis of SOD1 activity by glass bead
homogenization as described (22) from cells grown overnight to
confluency in selecting synthetic dextrose medium. For immunoblot
analysis, 10 µg of cell protein was applied to a precast 14%
polyacrylamide gel (Novex) and subjected to SDS gel electrophoresis
followed by Western blot using either an anti-hemagglutinin (HA)
antibody (Babco) followed by a secondary anti-goat IgG (for detection
of yCCS-HA fusions) or an anti-yCCS antibody as described (3). For
analysis of SOD activity, 20 µg of the same extracts was subjected to
electrophoresis on a non-denaturing 12% precast gel (Novex), followed
by nitro blue tetrazolium staining for SOD activity as described
(14).
The yCCS protein was purified as described previously with substitution
of Ala for Met1 and Thr2 (3). Gel filtration
chromatography was carried out at 4 °C using a Superdex 75 HR 10/30
column (Amersham Pharmacia Biotech) equilibrated with 1×
phosphate-buffered saline, pH 7.2. The flow rate was 0.5 ml/min with UV
detection at 280 nm. A standard curve of molecular mass
versus retention volume was generated by analysis of known
globular proteins (bovine serum albumin, 66 kDa; ovalbumin, 43 kDa;
carbonic anhydrase, 29 kDa; cytochrome c, 12.4 kDa;
aprotinin, 6.5 kDa; and vitamin B-12, 1.35 kDa). The column void volume
(7.9 ml) was determined by blue dextran exclusion, and the bed volume of the column (24 ml) was provided by the manufacturer. Typically, ~100 µM yCCS protein solutions in 100 µl were
analyzed. To ensure the integrity of the copper protein, buffer was
purged rigorously with argon before equilibration of the column.
Varying experimental conditions such as pH, ionic strength, or protein
concentration did not alter the results of the apoprotein analysis.
Copper loading of yCCS for proteolysis and analytical gel filtration
studies was carried out essentially as described previously (3).
Full-length yCCS (50 µM) in 50 mM Tris, pH
7.8, with 200 mM NaCl and 10 mM each
dithiothreitol, histidine, and glutathione (as competing copper
ligands) was slowly charged with 150 µM
Cu(I)(CH3CN)4PF6 and 150 µM ZnSO4. After incubation in an anaerobic
chamber at 14 °C for 20 h, the protein was exchanged repeatedly
with the same buffer and competing ligands in an Amicon ultrafiltration cell. The competing ligands were then removed from the protein with
repeated exchanges of 50 mM Tris, pH 8.0, and 10 mM dithiothreitol only. Copper loading of the synthetic
yCCS-dIII (Ala216-Lys249) peptide (100 µM) was carried out under similar conditions, except that
20 mM Mes, pH 6.0, was used as buffer, and copper binding was achieved with 200 µM
Cu(I)(CH3 CN)4PF6. For
assessment of metal binding capacities, protein concentrations were
determined by the Bradford method, using IgG standards with appropriate
correction factors applied. Bradford correction factors for the
yCCS-dIII synthetic peptide and full-length yCCS were obtained from
both a measured mass of the lyophilized peptide and from the predicted
For limited trypsin proteolysis of yCCS, a solution containing 800 µg
of apo-yCCS was proteolyzed with 1 µg of trypsin for 30 min at
25 °C, after which phenylmethylsulfonyl fluoride was added to
terminate the reaction. The digest fragments were separated by
reverse-phase HPLC through a Vydac 214TP54 column with a
H2O/CH3CN/0.1% trifluoroacetic acid gradient.
Isolated fragments were dried under vacuum and resuspended in
H2O with 1% formic acid for ESI MS analysis.
For time-resolved chymotrypsin proteolysis, solutions containing 400 µg (37 µM) of either apo-yCCS or Cu-yCCS in 400 µl of 50 mM Tris, pH 8.0, were subjected to proteolysis by 2 µg
of chymotrypsin at 14 °C under anaerobic atmosphere. For each time
point, 20-µl aliquots of the proteolysis solution were extracted and
terminated with the addition of phenylmethylsulfonyl fluoride and were
analyzed by 15% SDS-polyacrylamide gel electrophoresis with Coomassie
Blue staining. A 200-µl aliquot was also extracted at the 30-min time point and was terminated by phenylmethylsulfonyl fluoride for analysis
by reverse-phase HPLC and ESI MS, as described above.
Plasmids--
The pHAL7 LEU2 plasmid expressing the
HA epitope-tagged yCCS has been described earlier (14). To construct
plasmids for the expression of yCCS-dII/dIII and the ATX1-CCS-dII/dIII
chimera, pHAL7 was used as a template in a polymerase chain reaction to amplify yCCS-HA sequences +216 (with respect to the yCCS start codon)
to +400 (with respect to the COOH-terminal HA) using upstream and
downstream primers engineered with SalI and BamHI
sites. This product was integrated at these same sites in pRS426 (23).
The resultant construct was then digested with KpnI and
SalI and ligated to one of two
KpnI-SalI ATX1-containing fragments
spanning sequences Structurally Distinct Domains of yCCS--
The amino acid sequence
of Saccharomyces cerevisiae Lys7 (referred to herein as
"yCCS") is similar to two yeast proteins, Atx1p and Cu,Zn-SOD (Fig.
1A). The
NH2-terminal 70 amino acids are homologous to Atx1p, and
the central region exhibits significant homology with yeast Cu,Zn-SOD.
The most COOH-terminal 30 amino acids of yCCS are not homologous to
available sequences in the data bases, although this segment is highly
conserved among CCS molecules from diverse species (e.g. the
yeast and human CCS molecules share nearly 50% identity over this
region (14)). The limited homologies noted with the three regions of
yCCS led us to test a multidomain model for this metallochaperone.
The existence of independent structural domains was established by
limited trypsin digests on purified apo-yCCS (Fig. 1B). Separation of peptide fragments by reverse-phase HPLC and analysis of
molecular mass by ESI MS revealed two stable units. The larger of these
units consisted of the NH2 terminus through residue
Lys226 (ESI MS, 24,609.9 Da; predicted mass, 24,609.3 Da),
which encompasses both the Atx1p-like and SOD1-like sequences. The
smaller fragment corresponded to the peptide spanning residues
Gly72 through Lys226 (ESI MS, 16,952.5 Da;
predicted mass, 16,954.1 Da), which includes only the SOD1-like
sequence (Fig. 1A and B). Time course experiments revealed that this latter peptide was quite resistant to tryptic digestion relative to the two terminal regions (data not shown). The
final 30 COOH-terminal amino acids of apo-yCCS were much more rapidly
digested by trypsin than was the remainder of the protein, suggesting
that this peptide segment has relatively little structure in the
absence of copper. Similar results with trypsin proteolysis were
observed with human CCS (data not shown). These results establish that
the CCS protein consists of at least three structurally distinct domains: an Atx1p-like NH2-terminal region, referred to as
Domain I; a central SOD1-like region, referred to as Domain II, and at the extreme COOH terminus, a short region denoted as Domain III. The
correlations between these proteolytically defined domains and the SOD1
and Atx1p homologous regions raise the possibility that each domain
serves an independent physiological function.
The ATX1-like Domain I of yCCS--
To examine the function of the
three CCS domains in vivo, specific truncation mutants of
yCCS were expressed in yeast cells. Polypeptides spanning Domains I and
II (yCCS-dI/dII), Domains II and III (yCCS-dII/dIII), and isolated
Domain I (yCCS-dI) accumulated to levels similar to that of native yCCS
when expressed in a lys7 null strain (Fig.
2A). lys7
Considering the homology between yCCS Domain I and the Atx1p
metallochaperone, we addressed whether these protein domains were
interchangeable. Domain I can cooperate with yCCS-dII/dIII in
trans; therefore, it was possible that the weak activity
observed with yCCS-dII/dIII involved endogenous Atx1p. However,
complementation by yCCS-dII/dIII was identical in
ATX1-expressing and atx1
The weak activation of SOD1 observed with isolated yCCS-dII/dIII
prompted us to examine the requirement for Domain I under varying
copper conditions. When the growth medium was treated with the
Cu(I)-specific chelator, bathocuproine sulfonate,yCCS-dII/dIII lost all
complementation activity, whereas intact yCCS retained full activity
under these copper starvation conditions (Fig.
4A). In comparison, treatment
of cells with additional copper dramatically enhanced the activity
obtained with yCCS-dII/dIII. Normally, the level of SOD1 activation by
yCCS-dII/dIII is too weak to be detected by an in situ gel
assay for SOD1 (Fig. 4B), which has a limit of detection of
2 ng of active SOD1 (3). Yet when cells are grown in the presence of
somewhat elevated, but non-toxic, copper concentrations (10 µM), yCCS-dII/dIII exhibited nearly wild type activation
of SOD1 as monitored by the gel assay (Fig. 4B). In comparison, this copper treatment failed to rescue the defect of
yCCS-dI/dII lacking Domain III (Fig. 4B). Hence, the
Atx1p-like Domain I of yCCS is only necessary under strict copper
limitation conditions. These observations are consistent with a role
for the Atx1p-like NH2-terminal domain in the recruitment
of copper to the yCCS molecule and a role for the remainder of the
protein in target recognition and direct transfer of copper to
SOD1.
Domains II and III of yCCS--
The surprising activity observed
with isolated yCCS-dII/dIII suggested that this region may contain a
functional copper-binding site that is involved in copper transfer to
SOD1. We have recently demonstrated that copper can be directly
transferred to SOD1 in vitro from yCCS loaded with a single
Cu(I) ion (3). When treated with an excess of metal ion in the presence
of stringent competitors, yCCS can tightly bind an additional copper
ion and retain activity. Although no binding of Zn(II) was observed
under these conditions, yCCS appears to have more than one high
affinity copper site; one possible site was noted in the SOD1-like
Domain II.
As is the case with SOD1, Domain II of yCCS contains four histidines
(Fig. 1A). Although the spacing between these His side chains at positions 130, 134, 151, and 198 does not completely correspond to the highly conserved pattern in SOD1 and hCCS, it is
possible that the three-dimensional fold of yCCS could accommodate a
functional copper transfer site. Each histidine in Domain II was
mutated to alanine. As seen in Fig. 4C, wild type levels of SOD1 activation were obtained with each corresponding mutant of full-length yCCS. Furthermore, the same histidine substitutions in
isolated yCCS-dII/dIII did not impair activity of this truncated yCCS
(not shown). Thus the potential copper site of Domain II is not
necessary for copper activation of SOD1.
The most highly conserved region among CCS molecules is Domain III
(Fig. 5A). As described above
(Figs. 2B and 4B), this domain is indispensable
for CCS activity in vivo. Domain III contains an invariant
CXC motif that is found in all members of the CCS family,
including that of mammals, plants, fungi, and insects (Fig.
5A). To assess the role of this motif in metallochaperone function, Cys to Ser mutations were introduced singly and in
combination at positions 229 and 231 in yCCS. The mutant derivatives
were stably expressed in lys7 null yeast (Fig.
5B). These mutations had a dramatic effect on yCCS activity.
All three Cys to Ser derivatives failed to activate SOD1 to levels that
could be detected by the in situ gel assay (Fig.
5B). In the more sensitive assay of lysine-independent growth, variant C231S was completely inactive, whereas yCCS C229S exhibited weak activity that was estimated to be less than 10% of wild
type yCCS (Fig. 5B). Therefore, the CXC motif in
Domain III of CCS is critical for copper chaperoning activity. Although the role of these cysteines in activation of SOD1 is not known, one
possibility is that they directly participate in copper transfer.
To test whether Domain III is capable of binding copper, a peptide
corresponding to residues Ala216-Lys249 of CCS
was synthesized and treated with Cu(I) under anaerobic conditions
similar to those used to load Atx1p with metal (2). The Cu(I)-peptide
complex was exposed to several buffer exchanges against metal-binding
competitor molecules to remove metal from low affinity sites on the
peptide. Removal of unbound metal and competitors revealed a copper
peptide stoichiometry of 0.53:1, demonstrating that Domain III indeed
has the capacity to bind Cu(I), most likely forming a copper-bridged
peptide dimer under this condition. Taken together, these observations
are consistent with a role of the COOH-terminal domain of CCS in
copper-handling steps during activation of SOD1.
Copper-mediated Changes in yCCS Conformation--
The cooperation
of multiple CCS domains may proceed through copper-mediated changes in
protein structure. Two biochemical assays were employed to probe the
effects of Cu(I) binding on yCCS conformation. First, analytical gel
filtration revealed that apo-yCCS migrates as a monomer (molecular
mass, 29 ± 2 kDa) under physiological buffer conditions at all
protein concentrations examined (100-750 µM) (Fig.
6). In comparison, the copper-loaded form
of yCCS exists as a mixture of monomers and dimers (molecular mass,
54 ± 4 kDa) in the 10-100 µM protein concentration
range. In both the one-copper and two-copper loaded states, the dimeric form of yCCS was slightly more abundant than the monomer (Fig. 6).
These results are consistent with a model in which copper induces a
conformational change in yCCS that significantly increases the
monomer-dimer equilibrium constant.
To further evaluate whether metal binding induces conformational
changes in yCCS, the apo and copper forms of the metallochaperone were
subjected to a proteolysis time course. As seen in Fig.
7, copper binding to yCCS resulted in a
significantly slower rate of chymotrypsin cleavage at Domain III.
Estimated half-lives of Domain III with full-length yCCS under these
conditions were 15 min for apo-yCCS versus 50 min for
Cu-yCCS (Fig. 7). Thus, the binding of one copper to yCCS attenuates
chymotrypsin digestion at sites distant from the proposed
copper-binding sites. Together with the gel filtration data of Fig. 6,
these observations indicate that copper binding induces allosteric
conformational changes in yCCS, and one important consequence of these
changes is an alteration at the dimerization interface.
The in vivo insertion of copper into SOD1 is shown here
to be a complex allosteric process that involves the concerted actions of three distinct domains of the metallochaperone, CCS. At the amino
terminus of CCS, an Atx1p-like Domain I appears responsible for
capturing copper under conditions of metal starvation, whereas a
central SOD1-like region is proposed to serve in target recognition. Copper insertion into SOD1 then requires a small copper-binding peptide
at the COOH terminus of CCS. Although our studies do not directly test
a role for the metallochaperone in loading of zinc into SOD1, our
experiments with purified protein indicate that Zn(II) binding to yCCS
is weak. Hence, CCS may be specifically designed for the copper
transfer process.
The NH2-terminal domain of CCS was originally suspected to
be the key player in copper activation of SOD1. This domain harbors the
same high affinity MXCXXC copper-binding site
that, in the case of Atx1p, directly transfers the metal from the
metallochaperone to its target (2). Yet with CCS, the Atx1p-like Domain
I is only required under conditions of strict copper limitation. We have recently demonstrated that the intracellular level of free copper
available to SOD1 is in the attomolar range (less than one free atom
per yeast cell), despite the micromolar quantities that are typically
accumulated by the cell (3). The Atx1p-like Domain I in CCS ensures
that the metallochaperone recruits the metal regardless of copper
availability. It is noteworthy that the mere presence of
MXCXXC in Domain I is not sufficient, since Atx1p
fails to substitute for Domain I in promoting CCS activity under low
copper conditions. Our finding that Atx1p and yCCS Domain I are not
interchangeable in vivo demonstrates that sequences unique
to these polypeptides must facilitate recognition of, and copper
transfer to, the cognate targets.
The central portion of CCS that exhibits homology to SOD1 (Domain II)
is proposed to participate in target recognition rather than in direct
transfer of copper. A possible set of copper-binding histidine side
chains was recognized in Domain II, yet all of these residues were
found to be irrelevant to metallochaperone activity in vivo.
Since copper loading of SOD1 by yCCS has been shown to involve direct
transfer from one protein to the other without release of the free
metal (3), formation of a specifically docked yCCS-SOD1 complex is a
likely prelude to the copper transfer step. Based on its homology to
SOD1, Domain II may facilitate this docking by heterodimer formation
with SOD1. We show here that yCCS homodimer formation in
vitro is stimulated upon copper binding to yCCS. Yet given the
copy number of intracellular yCCS (3) and estimates of the dimerization
constants, we suspect that the amount of yCCS homodimer in the cell is
relatively low. It is therefore conceivable that copper loading of yCCS
in vivo facilitates heterodimer formation with SOD1. Gitlin
and co-workers (15) recently obtained evidence for in vivo
docking between mammalian SOD1 and CCS, where hCCS regions
corresponding to yCCS Domains II and III were seen by
immunoprecipitation studies to interact with SOD1. The interactions
between metallochaperone and target are expected to be transient.
Steady state SOD1 exists as a homodimer, and because holo-SOD1 is
present in manyfold excess over CCS (3, 26), the metallochaperone must
be released from SOD1 after metal transfer in order to carry out
subsequent copper transfer cycles. An attractive idea is that
metallochaperone release from SOD1 is facilitated by metal-induced
allosteric changes involving Domains I and III.
The carboxyl-terminal Domain III of CCS emerges from these studies with
a central role in CCS function. This domain is indispensable for
in vivo activation of SOD1 even under conditions of excess copper. Domain III appears unique to CCS molecules and represents the
most highly conserved region by comparison of fungal, mammalian, insect, and plant polypeptides. We propose a model in which a copper-binding CXC in Domain III cooperates with the
amino-terminal domain of CCS in directing insertion of copper into
SOD1. As evidence for interaction between the amino and carboxyl
regions of the metallochaperone, Domain I was found to act in
trans with a yCCS molecule spanning Domains II and III.
Perhaps the most intriguing insight that arises from these studies is
the complexity of the mechanism by which copper is inserted into SOD1
in vivo. In comparison to our results shown here for CCS,
the delivery of copper by the Atx1p metallochaperone involves a single
metallochaperone domain and a lone copper site (2). The basis for these
differential requirements can be best explained in terms of the
respective protein targets for the two metallochaperones. In the case
of Atx1p, copper is delivered to Atx1p homologous metal-binding domains
present on the Ccc2p copper transporter target (2, 5, 6). This homology
should obviate the requirement for a separate metallochaperone domain
involved in target recognition. The Ccc2p target for Atx1p harbors
precisely the same surface-exposed MXCXXC copper
site employed by the metallochaperone (5), and copper transfer can
readily proceed through an associative exchange mechanism involving
dual cysteines on the two molecules (2). Thus the Atx1p
MXCXXC copper site serves a 2-fold purpose in
initially capturing copper and in direct metal transfer. By comparison, the MXCXXC copper site on CCS Domain I may not be
amenable to direct insertion of the metal into SOD1. The copper site in
SOD1 is buried within the enzyme, and only a few square angstroms are solvent-exposed. This copper in SOD1 is coordinated to three or four
histidines, depending on the oxidation state of the metal (27-29).
Based on studies with Atx1p, the MXCXXC copper
site is predicted to transfer the metal to solvent-exposed sulfur
ligands (2, 5). We therefore propose a model in which copper in CCS can
undergo direct intramolecular ligand exchange between cysteines of
Domain I and Domain III. The insertion of copper into SOD1 then poses
two major challenges for the metallochaperone: the change in
coordination environment upon metal transfer and insertion of the metal
at a position deep within the enzyme. The COOH-terminal copper-binding
peptide of CCS may be uniquely designed to accommodate this process.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
::LEU2 (18)), SL215
(atx1::LEU2 (4)), and KS107
(sod1::TRP1 (19)) have been previously described. The lys7:: LEU2
atx1::HIS3 strain, PS113, was constructed by deleting
the ATX1 gene of SY2950. Stocks of strains were maintained on enriched YPD medium (20) in anaerobic culture jars (BBL GasPak). All
yeast transformations were conducted by the method of electroporation (21). Spot tests for complementation of lys7 aerobic
lysine auxotrophy utilized a synthetic dextrose-based medium (20). Ferrozine plate tests for complementation of atx1 were
conducted as described (4).
280 and were in good agreement. Total metal
concentrations for each protein solution were determined by inductively
coupled plasma emission spectroscopy.
420 to either +3 (for pTH007) or +209 (for pTH008)
relative to the ATX1 start codon. Ligation resulted in the
in-frame fusion (via the GTC GAC SalI site) of yCCS amino
acid 75 either to the start codon of ATX1, generating
plasmid pTH007 for expression of yCCS-dII/dIII, or to ATX1
amino acid 70, generating plasmid pTH008 for expression of the
ATX1-CCS-dII/dIII chimera. Although gene transcription was driven by
the ATX1 promoter with these constructs, the yields of
steady state protein were virtually identical to those obtained with
genes driven by the LYS7 promoter (as in Fig.
2A). Three constructs expressing yCCS-dI were obtained; pTH006 (URA3) expressed a Domain I-HA fusion, and pTH005
(LEU2) and pPS022 (HIS3) expressed native Domain
I. To obtain pTH006, yCCS DNA sequences +1 to +224 were amplified using
upstream and downstream primers engineered with SalI and
NdeI sites, and the product was used to replace the
SalI-NdeI yCCS-dI/dIII fragment of pTH008,
resulting in the in-frame fusion of Domain I to the start site of
ATX1 and to HA at the COOH terminus. Construct pTH005 was
obtained by inserting a stop codon at yCCS amino acid 74 in pHAL7 by
site-directed mutagenesis using the QuikChangeTM
mutagenesis kit according to the manufacturer's specifications (Stratagene). pPS024 expressing yCCS-dI/dII (no HA tag) was obtained by
introducing a stop codon at yCCS amino acid 217 using as template the
HIS3 yCCS-expressing vector pHAL7-413. pHAL7-413 was also employed as a template for site-directed mutagenesis construction of
yCCS alleles H130A/H134A, H151A, H198A, C229S, C231S, and C229S/C231S. All constructs were subjected to DNA sequence analysis to ensure lack
of fortuitous mutations.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Definition of yCCS multidomain structure by
limited trypsin proteolysis. A, schematic alignments of
human and yeast CCS with yeast Atx1p and yeast Cu,Zn-SOD (SOD1) based
on amino acid sequence comparisons (14, 15, 17, 30). yCCS domain
regions are specified: large cross-hatched bars, sequence
regions of CCS with homology to yAtx1p (21% identity, 40% similarity
for yCCS); striped bars, sequence regions with CCS homology
to ySOD1 (30% identity, 37% similarity for yCCS); and small
cross-hatched bars, sequence regions with homology only to other
CCS molecules. Potential histidine (H), cysteine
(C), and aspartate (D) metal-binding ligands are
noted. Residues targeted for mutagenesis are denoted by numbering.
Arrows mark trypsin cleavage positions between Arg-71 and
Gly-72 and between Lys-226 and Gly-227. B, Coomassie
Blue-stained 12% SDS-polyacrylamide gel of proteolyzed yCCS digested
with trypsin for the indicated time points. C, electrospray
ionization mass spectrum of Domain II fragment isolated from
trypsin-proteolyzed yCCS. Expected mass of the
Gly72-Lys226 fragment is 16,954.1 Da.
strains are typically lysine auxotrophs when grown in air because
copper-SOD1 is essential for aerobic lysine biosynthesis (24, 25).
Expression of full-length yCCS complemented this defect, whereas no
complementation was observed with yCCS-dI/dII lacking domain III (Fig.
2B). Surprisingly, removal of the Atx1p-like Domain I did
not eliminate yCCS activity, as yCCS-dII/dIII complemented the
lys7 defect (Fig. 2B). However, this activity is
weak compared with intact yCCS, demonstrating that Domain I is required
for maximal yCCS activity. To test whether Domain I can act in
trans with the downstream segments of yCCS, we co-expressed
yCCS-dI and yCCS-dII/dIII as separate molecules. As seen in Fig.
2C, Domain I effectively enhanced the complementation obtained with yCCS-dII/dIII alone, suggesting that Domain I may physically interact with the carboxyl-terminal portion of yCCS to
activate SOD1.
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Fig. 2.
Expression of yCCS domains in a yeast
lys7 null strain. The lys7 strain
SY2950 (A, B, and C, ATX1
+) or the lys7 atx1 strain PS113 (C,
ATX1 ) was transformed with vector pRS426 (V)
or with plasmids expressing polypeptides that spanned the indicated
domains of yCCS or the ATX1-CCS-dII/dIII chimera. A, Western
blot analysis of yCCS derivative polypeptides using an anti-yCCS
polyclonal antibody (left) or an anti-HA antibody
(right) to recognize the HA epitope at the COOH terminus of
each polypeptide. The presence of this tag does not alter
complementation activity (data not shown). Numbers on
left indicate positions of molecular mass markers (Bio-Rad).
B and C, complementation of the aerobic lysine
auxotrophy of the lys7 null strain. Transformants expressing
polypeptides spanning the indicated domains of yCCS were spotted onto
medium containing (+Lys) or lacking lysine (Lys
and C) and were allowed to grow aerobically for 4 days.
Dilutions represent 2 × 105, 4 × 104, and 4 × 103 cells plated. Plasmids
utilized were: I, II, and III, pHAL7
expressing full-length yCCS (14); I and II,
pPS024; I, pTH006 (A) or pTH005 (B,
C); II and III, pTH007; II,
III, + I, strain co-transformed with pTH007 and
pPS022.
strains (Fig.
2C), suggesting that Atx1p could not mimic the action of yCCS Domain I. To address this issue further, we constructed a chimeric
gene in which Domain I of yCCS was replaced by Atx1p. This
ATX1-CCS-dII/dIII fusion protein was stably expressed in a
lys7 null strain (Fig. 2A), and complemented the
lysine auxotrophy of this cell; however, the activity obtained was not
substantially different from that seen with yCCS-dII/dIII alone (Fig.
3A). Hence, Atx1p cannot
substitute for yCCS Domain I. To test whether the opposite was true,
isolated Domain I was expressed in an atx1 strain. These
cells are defective for delivering copper to Fet3p copper oxidase (4)
and, as such, fail to grow on medium supplemented with the iron
chelator, ferrozine (Fig. 3B). This defect is complemented by expression of ATX1 but not by isolated yCCS Domain I. Thus, Domain I cannot substitute for Atx1p in delivering copper to
Ccc2p and Fet3p in the secretory pathway. It is noteworthy that the ATX1-CCS-dII/dIII chimera did complement the atx1 mutant
defect (Fig. 3B), indicating that this fusion copper
chaperone molecule can deliver copper to both SOD1 and to Ccc2p.
Presumably, specificity for Ccc2p is mediated by the amino-terminal
Atx1p portion, whereas activation of SOD1 involves the
carboxyl-terminal yCCS-dII/dIII region of the chimera molecule.
View larger version (66K):
[in a new window]
Fig. 3.
Expression of the chimera metallochaperone,
Atx1p fused to yCCS-dII/dIII. A, complementation of
aerobic lysine auxotrophy was tested in lys7 strains
expressing the indicated derivatives of yCCS as described in Fig. 2.
B, the atx1 strain SL215 was transformed with
either pRS425 vector, pRS-A1 expressing ATX1 (31), or with pTH008,
pTH005, or pTH007 for expression of ATX1-CCS-dII/dIII, yCCS-dI, or
yCCS-dII/dIII, respectively. The indicated strains were plated onto
ferrozine-containing medium as described (4) to test for
complementation of the iron starvation phenotype of atx1
mutant strains.
View larger version (37K):
[in a new window]
Fig. 4.
Effects of copper treatment on
yCCS-dII/dIII. The lys7 null strain SY2950 was
transformed with plasmids expressing the indicated domains of yCCS
(described in Figs. 2, 3) or with pRS425 vector (vec) or
with plasmids expressing the indicated His mutant derivatives of
full-length yCCS. A, spot test for complementation of
aerobic lysine auxotrophy as in Fig. 2, using lysine-deficient medium
that was supplemented where indicated (+BCS) with 150 µM Cu(I) chelator, bathocuproine sulfonate. B
and C, cells lysates were prepared from transformants, and
SOD activity was detected by the gel nitro blue tetrazolium assay as
described under "Experimental Procedures." Where indicated
(+Cu), cells were grown in minimal medium supplemented with
10 µM CuSO4 prior to lysis. The positions of
the mitochondrial manganese-containing SOD2 and Cu,Zn-SOD are
indicated.
View larger version (62K):
[in a new window]
Fig. 5.
Domain III of yCCS. A, an
alignment of the COOH-terminal regions of various homologues to yCCS.
Amino acid sequences that are identical or similar across unrelated
species are boxed. The conserved Cys are marked with
asterisks. Arrows mark predicted positions of
chymotrypsin cleavage. Numbers indicate amino acid
numbering of wild type yCCS. Sequences were derived as follows: yeast
(18); human (14); mouse (32); rat (EST accession no. AA996543);
Drosophila (EST accession no. AFO83312); tomato (cDNA
accession no. AAD12307; A. Nersissian and J. S. Valentine,
unpublished data); Arabidopsis (EST accession #AFO83312).
B, expression of yCCS Domain III mutants in lys7
null yeast and metallochaperone activity was monitored by Western blot
(top left), SOD activity (bottom left), and
complementation of aerobic lysine auxotrophy as in Fig. 4.
View larger version (24K):
[in a new window]
Fig. 6.
Gel filtration analyses of yCCS
oligomerization. Protein samples (100 µM) were
subjected to gel filtration chromatography as described under
"Experimental Procedures." Cu1-yCCS (solid
line) eluted as two peaks, whereas apo-yCCS gave a single peak
(dashed line). The inset shows the standard curve
generated under the experimental conditions with molecular mass
standards (bovine serum albumin, ovalbumin, carbonic anhydrase,
cytochrome c, aprotinin, and vitamin B-12; molecular masses
given "Experimental Procedures") indicated by the open
circles. Calculated results of yCCS molecular masses are shown by
the solid lines. Kav = [Ve Vo]/[Vb Vo], where Ve is elution
volume of protein, V0 is void volume of
column, and Vb is bed volume of
column.
View larger version (20K):
[in a new window]
Fig. 7.
Copper-induced conformational changes in yCCS
as monitored by time-resolved proteolysis. Purified yCCS protein
(400 µg) was incubated with chymotrypsin at a 200:1 w/w ratio at
14 °C, pH 8.0, for the indicated time periods prior to analysis by
15% SDS-polyacrylamide gel electrophoresis and Coomassie Blue
staining. A, apo-yCCS; B, Cu(I)-yCCS containing
1.1 mol of copper/mol of protein. MW, broad range molecular
mass standards; d-I,II, 5 µg of a purified yCCS spanning
Domains I and II (M1-A216). Sites of chymotrypsin proteolysis in both
apo- and Cu-yCCS were determined by ESI MS of HPLC-purified fragments
to be after Trp222 and Trp237.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
ACKNOWLEDGEMENTS |
|---|
We thank A. Rosenzweig, A. Torres, D. Huffman, and M. Portnoy for invaluable discussions.
| |
Note Added in Proof |
|---|
Crystallographic characterization of yCCS also reveals distinct folds for Domains I and II of the apo-form of this protein and is consistent with the functional roles described here (Lamb, A. L., Wernimont, A. K., Pufahl, R. A., Culotta, V. C., O'Halloran, T. V., and Rosenzweig, A. C. (1999) Nat. Struct. 6, in press).
| |
FOOTNOTES |
|---|
* This work was supported in part by The Johns Hopkins University National Institute of Environmental Health Sciences Center, by National Institutes of Health Grants GM 50016 and ES 08996 (to V. C. C.) and GM 54111 (to T. V. O.), and by a grant from the ALS association (to T. V. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Institutes of Health Training Grant ES 07141.
Supported by National Institutes of Health Postdoctoral
Fellowships GM 19457 and DK 09395.
To whom correspondence may be addressed: 2145 Sheridan Rd.,
Northwestern University, Evanston, IL 60208-3113. Tel.: 847-491-5060; Fax: 847-491-7713; E-mail: t-ohalloran@nwu.edu.
§§ To whom correspondence may be addressed: Dept. of Environmental Health Sciences and Biochemistry, The Johns Hopkins University School of Public Health, 615 N. Wolfe St., Rm. 7032, Baltimore, MD 21205. Tel.: 410-955-3029; Fax: 410-955-0116; E-mail: vculotta@jhsph.edu.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: SOD, superoxide dismutase; CCS, copper chaperone for superoxide dismutase; yCCS, yeast CCS; SD, synthetic dextrose; HPLC, high pressure liquid chromatography; ESI MS, electrospray ionization mass spectrophotometry; HA, hemagglutinin.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. |
Valentine, J. S.,
and Gralla, E. B.
(1997)
Science
278,
817-818 |
| 2. |
Pufahl, R. A.,
Singer, C. P.,
Peariso, K. L.,
Lin, S. J.,
Schmidt, P. J.,
Fahrni, C. J.,
Culotta, V. C.,
Penner-Hahn, J. E.,
and O'Halloran, T. V.
(1997)
Science
278,
853-856 |
| 3. |
Rae, T. D.,
Schmidt, P. J.,
Pufahl, R. A.,
Culotta, V. C.,
and O'Halloran, T. V.
(1999)
Science
284,
805-808 |
| 4. |
Lin, S.-J.,
Pufahl, R. A.,
Dancis, A.,
O'Halloran, T. V.,
and Culotta, V. C.
(1997)
J. Biol. Chem.
272,
9215-9220 |
| 5. | Rosenzweig, A. C., Huffman, D. L., Hou, M. Y., Wernimont, A. K., Pufahl, R. A., and O'Halloran, T. V. (1999) Structure 7, 605-617[Medline] [Order article via Infotrieve] |
| 6. |
Portnoy, M. E.,
Rosenzweig, A. C.,
Rae, T.,
Huffman, D. L.,
O'Halloran, T. V.,
and Culotta, V. C.
(1999)
J. Biol. Chem.
274,
15041-15045 |
| 7. |
Yuan, D. S.,
Stearman, R.,
Dancis, A.,
Dunn, T.,
Beeler, T.,
and Klausner, R. D.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
2632-2636 |
| 8. |
Klomp, L. W. J.,
Lin, S. J.,
Yuan, D.,
Klausner, R. D.,
Culotta, V. C.,
and Gitlin, J. D.
(1997)
J. Biol. Chem.
272,
9221-9226 |
| 9. | Bull, P. C., and Cox, D. W. (1994) Trends Genet. 10, 246-252[CrossRef][Medline] [Order article via Infotrieve] |
| 10. | Amaravadi, R., Glerum, D. M., and Tzagoloff, A. (1997) Hum. Genet. 99, 329-333[CrossRef][Medline] [Order article via Infotrieve] |
| 11. | Srinivasan, C., Posewitz, M. C., George, G. N., and Winge, D. R. (1998) Biochemistry 37, 7572-7577[CrossRef][Medline] [Order article via Infotrieve] |
| 12. |
Glerum, D. M.,
Shtanko, A.,
and Tzagoloff, A.
(1996)
J. Biol. Chem.
271,
14504-14509 |
| 13. |
Beers, J.,
Glerum, D. M.,
and Tzagoloff, A.
(1997)
J. Biol. Chem.
272,
33191-33196 |
| 14. |
Culotta, V. C.,
Klomp, L.,
Strain, J.,
Casareno, R.,
Krems, B.,
and Gitlin, J. D.
(1997)
J. Biol. Chem.
272,
23469-23472 |
| 15. |
Casareno, R. L.,
Waggoner, D.,
and Gitlin, J. D.
(1998)
J. Biol. Chem.
273,
23625-23628 |
| 16. |
Corson, L. B.,
Strain, J.,
Culotta, V. C.,
and Cleveland, D. W.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
6361-6366 |
| 17. | Gamonet, F., and Lauquin, G. J.-M. (1998) Eur. J. Biochem. 251, 716-723[Medline] [Order article via Infotrieve] |
| 18. | Horecka, J., Kinsey, P. T., and Sprague, G. F. (1995) Gene (Amst.) 162, 87-92[CrossRef][Medline] [Order article via Infotrieve] |
| 19. |
Slekar, K. H.,
Kosman, D.,
and Culotta, V. C.
(1996)
J. Biol. Chem.
271,
28831-28836 |
| 20. | Sherman, F., Fink, G. R., and Lawrence, C. W. (1978) Methods in Yeast Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
| 21. | Becker, D., and Guarente, L. (1991) Methods Enzymol. 194, 182-187[Medline] [Order article via Infotrieve] |
| 22. | Lin, S. J., and Culotta, V. C. (1996) Mol. Cell. Biol. 16, 6303-6312[Abstract] |
| 23. |
Sikorski, R. S.,
and Hieter, P.
(1989)
Genetics
122,
19-27 |
| 24. |
Liu, X. F.,
Elashvili, I.,
Gralla, E. B.,
Valentine, J. S.,
Lapinskas, P.,
and Culotta, V. C.
(1992)
J. Biol. Chem.
267,
18298-18302 |
| 25. | Bilinski, T., Krawiec, Z., Liczmanski, L., and Litwinska, J. (1985) Biochem. Biophys. Res. Commun. 130, 533-539[CrossRef][Medline] [Order article via Infotrieve] |
| 26. | Rothstein, J. D., Dykes-Hoberg, M., Corson, L. B., Becker, M., Cleveland, D. W., Price, D., Culotta, V. C., and Wong, P. C. (1999) J. Neurochem. 72, 422-429[CrossRef][Medline] [Order article via Infotrieve] |
| 27. | Djinovic, K., Gatti, G., Coda, A., Antolini, L., Pelosi, G., Desideri, A., Falconi, M., Marmocchi, F., Rotilio, G., and Bolognesi, M. (1992) J. Mol. Biol. 225, 791-809[CrossRef][Medline] [Order article via Infotrieve] |
| 28. | Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. (1982) J. Mol. Biol. 160, 181-217[CrossRef][Medline] [Order article via Infotrieve] |
| 29. | Ogihara, N. L., Parge, H. E., Hart, P. J., Weiss, M. S., Goto, J. J., Crane, B. R., Tsang, J., Slater, J., Ja, J. A. R., Valentine, J. S., Eisenberg, D., and Tainer, J. A. (1996) Biochemistry 35, 2316-2321[CrossRef][Medline] [Order article via Infotrieve] |
| 30. | Lyons, T. J., Nersissian, A., Goto, J. J., Zhu, H., Gralla, E. B., and Valentine, J. S. (1998) J. Biol. Inorg. Chem. 3, 650-662 [CrossRef] |
| 31. |
Lin, S.,
and Culotta, V. C.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
3784-3788 |
| 32. | Nishihara, E., Furuyama, T., Yamashita, S., and Mori, N. (1998) Mol. Neurosci. 9, 3259-3263 |
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