Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606
Received for publication, March 5, 2001, and in revised form, March 23, 2001
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ABSTRACT |
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Copper is an essential nutrient that
serves as a co-factor for enzymes involved in critical cellular
processes including energy generation, peptide hormone maturation,
oxidative stress protection, and iron homeostasis. Although genes have
been identified from yeast and mammals encoding a homologous subunit of
a plasma membrane high affinity copper transporter, the presence
of additional subunits that function as part of a copper transport
complex has not been reported. We observed that
ctr4+, a previously identified copper transport
protein from the fission yeast Schizosaccharomyces pombe,
fails to complement bakers' yeast cells defective in high affinity
copper transport and fails to be targeted to the plasma membrane.
However, selection for S. pombe genes, which, when
co-expressed with Ctr4, confer high affinity copper transport to
S. cerevisiae cells resulted in the identification of
ctr5+. Both Ctr4 and Ctr5 are integral membrane
proteins, are co-regulated by copper levels and the copper-sensing
transcription factor Cuf1, physically associate in vivo,
are interdependent for secretion to the plasma membrane, and are each
essential for high affinity copper transport. These studies in S. pombe identify Ctr4 and Ctr5 as components of a novel eukaryotic
heteromeric plasma membrane complex that is essential for high affinity
copper transport.
Copper is as an essential metal ion for life. Due to its ability
to adopt both oxidized (Cu(II)) and reduced (Cu(I)) states, copper is
an important redox co-factor for many copper-dependent enzymes, including cytochrome oxidase, copper/zinc superoxide dismutase, ceruloplasmin, and lysyl oxidase (1-3). Many human genetic
diseases have been linked to aberrant copper homeostasis, including
Menkes syndrome, Wilson disease, neurodegenerative diseases such as
amyotrophic lateral sclerosis, and iron deficiency anemia (4, 5).
Consequently, it is important to identify the components, and
understand the mechanisms for how organisms acquire, distribute, and
utilize copper.
The bakers' yeast Saccharomyces cerevisiae has been a
valuable model system to study eukaryotic copper homeostasis due to its
powerful genetics (2, 6). Cu(II) in yeast growth medium is thought to
be reduced to Cu(I) by cell surface Fe3+/Cu2+
reductases encoded by several FRE genes (7-11), before
being transported into the cell by two high affinity plasma membrane copper transporters, Ctr1 (12, 13) and Ctr3 (14, 15). Although Ctr1 and
Ctr3 are functionally redundant, and both possess three potential
transmembrane domains, they are otherwise dissimilar structurally. Ctr1
harbors eight copies of the "Mets" motif
MX2MXM (where M represents methionine
and X represents any amino acid) in its putative
amino-terminal extracellular domain, whereas Ctr3 is rich in cysteine
residues but lacks the Mets motif. Recently, the isolation of genes
encoding proteins involved in high affinity copper transport in the
fission yeast S. pombe, mice, and humans indicates the
presence of variable numbers of amino-terminal Mets motifs and sequence
homology to both S. cerevisiae Ctr1 and Ctr3 (2, 16-18).
Both S. cerevisiae Ctr1 and Ctr3 are thought to self-associate (12, 15); however, whether the copper transporters are
present in a high affinity copper transport complex with other functional subunits is not known.
The fission yeast Schizosaccharomyces pombe is also of
particular interest in studies of copper homeostasis since fission yeast exhibit similarity to mammals in several respects (19). Interestingly, a high affinity copper transport protein from S. pombe, Ctr4 (17), resembles a chimera between the S. cerevisiae Ctr1 and Ctr3 proteins at the primary sequence level.
Ctr4 harbors five MX2MXM repeats in
the predicted amino-terminal extracellular region similar to Ctr1, but
transmembrane domains homologous to Ctr3 (Fig. 1A). Like the
CTR1 and CTR3 genes in S. cerevisiae, ctr4+ transcription is controlled by
environmental copper levels and Cuf1, a copper-sensing transcription
factor functionally analogous to Mac1 in S. cerevisiae (17,
20-23).
In this work we report the identification of a new S. pombe
gene encoding a transmembrane high affinity copper transport protein, denoted ctr5+. We demonstrate that like Ctr4,
Ctr5 is essential for high affinity copper transport and is subject to
transcriptional control by the copper-sensing transcription factor
Cuf1. Furthermore, Ctr4 and Ctr5 both localize to the plasma membrane,
exhibit an interdependence for this localization, and are present in a
complex. These data describe the first example of a heteroprotein
complex that forms a high affinity copper transport activity at the
cell surface.
Strains, Plasmids, Media, and Reagents--
The wild type
S. pombe strain used in this study was FY254
(h
The multicopy S. pombe plasmids pSP1 and pSP2 (28) were used
to express ctr4+ and
ctr5+. The coding regions of these genes, with
~1 kilobase pair of 5' upstream sequences, were amplified from FY254
genomic DNA, cloned into pSP1 and pSP2, and sequenced. The expression
of ctr4+ and ctr5+ in
S. cerevisiae was from plasmids p423GPD and p426GPD (29), respectively, under the control of the strong constitutive GPD promoter. The GFP, myc, and FLAG epitope tags were added to
the carboxyl termini of Ctr4 and Ctr5, as described previously (15, 17).
Isolation of the ctr5+ Gene--
E. coli
strain BNN132 was transfected with an S. pombe Fluorescence Microscopy--
For localization of Ctr4-GFP fusion
proteins in S. cerevisiae, MPY17 cells harboring a
ctr4+-GFP-p423GPD plasmid were grown
in SC-His to early log phase. Cells were resuspended in SC-His with 100 µM bathocuproinedisulfonate (BCS) to
A600 = 2, and immobilized on slides with 1% low
melting agarose. Ctr4-GFP was visualized under a Zeiss Axioskop
photomicroscope with filters observing green fluorescence. Photos were
taken on Kodak TMAX 400 film and digitalized to CD-ROM, then processed by Adobe Photoshop 5.5 software. For Ctr4-GFP and Ctr5-GFP localization in S. pombe, cells were grown in EMM and treated as above.
The fluorescence signal was visualized by a Nikon Eclipse E800
fluorescent microscope with a Hamamatsu ORCA-2 cooled CCD camera.
Images were obtained using ESee and ISee software from Innovision
(Raleigh-Durham, NC) and then processed with Photoshop 5.5 software.
RNA Blot Analysis and Protein Co-immunoprecipitation
Experiments--
For RNA blotting S. pombe cells were
harvested at late log phase (A595 = 0.8) after
growth in YES. Either BCS or CuCl2 treatment was provided
for 1 h before harvesting cultures. Total RNA extraction and RNA
blotting was performed as described (17). 32P-Labeled
cDNAs were used as probes.
For co-immunoprecipitation experiments, FY254 cells were co-transformed
with ctr4+-FLAG-pSP1 and
ctr5+-myc-pSP2 and grown in
EMM-Leu-Ura and harvested at A595 = 0.8. Total
protein extraction and immunoprecipitation were performed as described
(15). Specifically, immunoprecipitation was carried out in the presence
of 0.5% Triton X-100, by using anti-FLAG M2 (Sigma) and
anti-c-myc 9E10 (Roche Molecular Biochemicals) antibodies. The immunoprecipitates were heated at 37 °C for 5 min before
fractionation by SDS-polyacrylamide gel electrophoresis.
Electrophoresis and immunoblotting were performed using standard
protocols (30). For biochemical fractionation of Ctr4 and Ctr5, FY254
cells were transformed and grown as above. Cells were broken with glass
beads in 10 mM Tris·Cl (pH 7.4), 250 mM
sucrose, and 2 mM EDTA, and the membrane fraction was
isolated by centrifugation at 100,000 × g at 4 °C
for 30 min. Membranes were treated in the same buffer with 0.2 M Na2CO3 or 1% Triton X-100 for 30 min on ice, and re-fractionated at 100,000 × g for
2 h. Pellets were solubilized in 1% Triton X-100, and the
resultant supernatants precipitated with 10% trichloroacetic acid and
re-dissolved in lysis buffer. Electrophoresis and immunoblotting were
carried out as described above.
In Vivo Copper Uptake Measurements--
S. pombe
cells were grown in YES medium to A595 = 0.6-1.0. 64CuCl2 was added to 2 ml of culture
to concentrations specified in Fig. 5A and incubated for 10 min at room temperature. Duplicate samples were incubated on ice to
control for nonspecific binding of 64Cu on the cell
surface. After incubation ice-cold EDTA was added to a final
concentration of 1 mM to terminate 64Cu uptake.
Cells were washed twice with ice-cold phosphate-buffered saline (pH
7.4) and collected in tubes to be counted with a Cobra II The S. pombe Ctr4 High Affinity Copper Transporter Is Nonfunctional
and Mislocalized in S. cerevisiae--
Due to the structural
similarity between the S. pombe Ctr4 high affinity copper
transporter and the S. cerevisiae Ctr1 and Ctr3 proteins
(Fig. 1A, and Ref. 17), we
assessed the ability of Ctr4 to complement the inability of an S. cerevisiae ctr1 Identification of the S. pombe Copper Transport Co-factor
Ctr5--
It is possible that S. pombe Ctr4 is mislocalized
when expressed in S. cerevisiae due to incorrect folding,
the presence of an inhibitory activity in S. cerevisiae, or
the lack of a functionally interacting partner protein in S. cerevisiae whose association is important for Ctr4 folding or
trafficking through the secretory pathway. To test this, we devised a
genetic screen for potential co-factor(s) that could facilitate
S. pombe Ctr4 function in S. cerevisiae cells
lacking high affinity copper transporters. S. cerevisiae
ctr1 Ctr5 Has Structural and Regulatory Properties Consistent with a
Role in High Affinity Copper Transport--
The amino acid sequence
encoded by the ctr5+ open reading frame (Fig.
3A) predicts a small protein
(173 amino acids) with three potential transmembrane domains (Fig. 3,
B and C) and 41% amino acid sequence identity
with Ctr4 throughout the two proteins. Interestingly, Ctr5 has a
predicted membrane topology that is similar to other microbial and
metazoan copper transporters (Fig. 1A). The Ctr5 amino acid
sequence also predicts, in the amino-terminal putative extracellular
domain, the presence of two copies of partially overlapping
MX2MXM sequences ("Mets" motif),
a putative copper binding motif that occurs in the amino-terminal
predicted extracellular domain of many high affinity copper
transporters identified to date. A similar sequence
(CXMXM) lies downstream of this region, with a
cysteine, another potential copper ligand, replacing methionine in the
motif (Fig. 3, A and C).
In addition to the shared structural homology of fungal high affinity
copper transport proteins, S. cerevisiae CTR1 and
CTR3, and S. pombe ctr4+ gene
expression is regulated as a function of copper availability by the
copper-sensing transcription factors Mac1 and Cuf1, respectively (17,
20-23). To ascertain the potential role of the
ctr5+ gene in S. pombe copper
homeostasis, we examined its possible regulation by extracellular
copper concentrations. As shown in Fig.
4, ctr5+ mRNA
was co-regulated with that of ctr4+ in response
to copper availability. Both ctr4+ and
ctr5+ had low basal levels of mRNA
expression in rich media, which was further repressed by the addition
of 10 µM CuSO4 to wild type S. pombe cells. In contrast, addition of the copper chelator BCS to
100 µM significantly induced the expression of both
genes. In a ctr4
The S. pombe cuf1+ gene encodes a nuclear
copper-sensing transcription factor that activates
ctr4+ gene expression under conditions of copper
limitation (17). In a cuf1 Inactivation of the Ctr5 Gene Results in Defective Copper
Transport--
The functional interactions of Ctr4 and Ctr5 in
S. cerevisiae, together with copper dependent regulation of
gene expression in S. pombe, all point to a role for Ctr5 in
copper acquisition in S. pombe. To ascertain whether Ctr5
plays a role in copper transport in S. pombe, the single
chromosomal ctr5+ gene was deleted by homologous
recombination and growth on nonfermentable carbon sources and high
affinity copper uptake evaluated. As shown in Fig.
5 (A and B), the
isogenic parental wild type strain transported 64Cu with
high affinity and was able to utilize nonfermentable carbon sources for
growth. In contrast, deletion of either the
ctr4+ or ctr5+ gene
resulted in a dramatic reduction in high affinity copper transport and,
consistent with the 64Cu transport defects observed in
these mutants, neither strain was able to grow on medium containing
glycerol/ethanol as sole carbon sources (Fig. 5B).
Furthermore, an isogenic strain harboring a deletion of both the
ctr4+ and ctr5+ genes
exhibited equivalent defects in high affinity 64Cu uptake
as compared with either single deletion, suggesting that the Ctr4 and
Ctr5 proteins function in the same pathway for copper acquisition in
S. pombe.
Ctr4 and Ctr5 Are Co-dependent for Localization to the
Plasma Membrane--
The structural, functional, and regulatory
features of Ctr5 suggest that it participates in the same pathway as
the Ctr4 protein in high affinity copper transport. Since previous
studies (17) established that Ctr4 resides on the plasma membrane, we
envision at least two possibilities for the functional relationship
between Ctr4 and Ctr5 in copper uptake in S. pombe. First,
both proteins could be present in a high affinity copper transport
complex, the presence of both of which is needed for the proper
function and or trafficking of the complex to the plasma membrane.
Alternatively, Ctr5 could be a resident in the secretory pathway and
facilitate the folding of Ctr4 and or its packaging into transport
vesicles. To distinguish between these two possibilities, we
ascertained the localization of the Ctr5 protein in S. pombe. Functional Ctr4-GFP and Ctr5-GFP fusion proteins were
expressed in S. pombe cells from multicopy plasmids under
their native promoters (Fig.
6A), and the localization of
each GFP fusion was determined by fluorescence microscopy. As shown in
Fig. 6B (panel 1), the Ctr4-GFP fusion was present on both the plasma membrane, and in a perinuclear region
that might coincide to the early secretory compartment. Because
episomal plasmids in S. pombe are present in multiple copies
with nonequivalent segregation to progeny cells (28), cell variability
in Ctr4-GFP expression is likely to underlie its presence in both
plasma membrane and perinuclear locations in many, but not all cells. A
similar perinuclear and plasma membrane distribution was observed for
the Ctr5-GFP fusion in wild type S. pombe cells (Fig.
6B, panel 4).
Because the levels of Ctr4-GFP protein expressed from high copy
plasmids in these cells are likely to be much higher than Ctr5
expressed from the single endogenous gene, the partial mislocalization of Ctr4-GFP could be due to inadequate levels of Ctr5 to act in concert
with Ctr4. To test this possibility wild type S. pombe cells
were co-transformed with multicopy plasmids expressing both the
Ctr4-GFP fusion and the wild type Ctr5 protein. As shown in Fig. 6B
(panel 2), in the presence of elevated levels of Ctr5, the
bulk of Ctr4-GFP fusion protein was found at the plasma membrane, with
little remaining in the perinuclear region. Similarly, in the presence
of elevated levels of Ctr4, the Ctr5-GFP fusion protein was
predominantly localized to the plasma membrane, with only a small
amount of fluorescence in the perinuclear compartment (Fig.
6B, panel 5). These results suggest
that specific levels of both Ctr4 and Ctr5 may be required for their
proper localization in S. pombe. To further test this
hypothesis, the ctr5+ gene was insertionally
inactivated in a wild type S. pombe strain by homologous
recombination. As shown in Fig. 6B (panel
3), ctr5 Ctr4 and Ctr5 Are Integral Membrane Proteins That Form a Copper
Transport Complex--
The observations that both Ctr4 and Ctr5 are
essential for high affinity copper transport, localize to the plasma
membrane, and exhibit an interdependence for this localization suggest
that these two proteins function in the same copper acquisition
pathway, perhaps as a complex. To test the possibility that Ctr4 and
Ctr5 may form a functional copper transport complex at the plasma
membrane, we first assessed whether Ctr4 and Ctr5 are integral membrane proteins as predicted by the presence of three potential
membrane-spanning domains within their primary sequences (Fig. 3). Two
copies of the FLAG epitope were added to the Ctr4 coding region, and
Ctr5 was tagged with four tandem copies of the myc epitope
to generate functional Ctr4 and Ctr5 epitope-tagged derivatives (Fig.
6A). As shown in Fig.
7A, functional epitope-tagged
versions of both Ctr4 and Ctr5 were detected by immunoblotting, in the
total cell lysate (lane 1), but not in the
soluble fraction after centrifugation of native membrane preparations
at 100,000 × g (lane 2).
Furthermore, Ctr4 and Ctr5 were efficiently extracted by 1% Triton
X-100 from the membrane fraction (lane 8), a
nonionic detergent that solubilizes membranes, but not by 0.2 M Na2CO3, which dissociates
peripheral membrane proteins from the membrane (lane
6). These data demonstrate that both Ctr4 and Ctr5 are
integral membrane proteins in S. pombe.
Because both Ctr4 and Ctr5 are integral membrane proteins that
co-localize to the plasma membrane in an obligate fashion and likely
function in the same pathway for copper uptake, we ascertained whether
Ctr4 and Ctr5 are physically associated. S. pombe cells expressing epitope-tagged Ctr4 and Ctr5 were used to prepare Triton X-100-solubilized total cell extracts. The data in Fig. 7B
show that Ctr4-FLAG2 and Ctr5-myc4 were
detected as a single band of ~58 kDa, and a doublet of ~30 and 36 kDa in whole total extracts, respectively (Fig. 7B,
lanes 2-4). Both proteins migrated in
SDS-polyacrylamide gel electrophoresis at a larger apparent molecular
weight than predicted by their primary sequences, possibly due to
glycosylation or other post-translational modifications.
Immunoprecipitation of Ctr5-myc4 resulted in the
co-immunoprecipitation of Ctr4-FLAG2 (Fig. 7B,
top panel, lane 8) and
immunoprecipitation of Ctr4-FLAG2 co-fractionated with
Ctr5-myc4 (Fig. 7B, bottom
panel, lane 8). Taken together, these
data support the hypothesis that S. pombe Ctr4 and Ctr5 are
directly or indirectly physically associated, and form a high affinity
copper transport complex at the plasma membrane.
Previous work on the mechanisms for copper transport across the
plasma membrane has resulted in the identification of related proteins
from bakers' yeast, fission yeast, mice, and humans that play a key
role in this process. Although experimental evidence suggests that Ctr1
and Ctr3 from S. cerevisiae (12-15) and human Ctr12 homomultimerize when
isolated from cells, no reports to date demonstrate that high affinity
copper transport requires a heteromeric protein complex. In this work,
we identify a new protein denoted Ctr5 that acts in concert with Ctr4
in copper uptake in S. pombe. Ctr4 and Ctr5 form a dual
partner complex, and the presence of both subunits is required for
appropriate localization to the plasma membrane and function of the
complex in copper uptake. This represents the first such example of a
copper transport complex containing at least two distinct partners. The
existence of Ctr4 and Ctr5 proteins distinguishes the S. pombe copper transport machinery from that previously established
in S. cerevisiae, where two high affinity transporters work
independently and appear to be functionally redundant (14).
The involvement of both Ctr4 and Ctr5 in high affinity copper uptake is
supported by a number of observations. First, both the
ctr4+ (17) and ctr5+
genes are regulated by copper availability. As may be expected for
proteins involved in copper uptake, these genes are activated in
response to copper scarcity and repressed in response to copper excess.
Both previously reported genetic experiments for
ctr4+ (17) and data presented here for
ctr5+ demonstrate that this regulation requires
the function of the S. pombe copper-sensing transcription
factor Cuf1. Furthermore, both the ctr4+ and
ctr5+promoters harbor Cuf1-responsive
cis-acting DNA sequences
(22).3 Second, the Ctr4 and
Ctr5 proteins have homology throughout their primary sequence and
specifically harbor amino-terminal Mets motifs that may play a role in
copper uptake. Third, growth assays on respiratory carbon sources and
64Cu uptake experiments demonstrate that both Ctr4 and Ctr5
must be functional for S. pombe cells to carry out high
affinity copper transport. Finally, the Ctr4 and Ctr5 proteins are both
polytopic membrane proteins that physically associate and are
interdependent for co-localization to the plasma membrane. Whether
Ctr4 and Ctr5 engage in direct or indirect interactions is currently
unknown. Although our results suggest that specific levels of Ctr4 and Ctr5 are important for the complex to properly localize to the plasma
membrane, the exact stoichiometry of these two partners in the complex
is unknown. Nor is it clear yet whether there are other components in
the high affinity copper transport complex.
The Ctr5 protein was identified based on its ability to alleviate a
block in Ctr4 secretion to the plasma membrane, and complement high
affinity copper uptake defects when expressed in S. cerevisiae cells. Although this is the first such example of a
protein complex involved in high affinity copper uptake, previous
studies have identified components of a high affinity iron transport
complex at the plasma membrane in S. cerevisiae (32-35).
Within this complex, the Fet3 protein serves as a multi-copper
ferroxidase, whereas the Ftr1 protein is thought to comprise an
iron-binding permease component. Indeed, similar to the features of
Ctr4 and Ctr5 in S. pombe, the Fet3-Ftr1 proteins are both
required for high affinity iron uptake, are regulated at the level of
gene transcription by iron, and are interdependent for secretion to the
S. cerevisiae plasma membrane. A homologous iron transport
complex, containing the Fet3-Ftr1-related proteins Fet5 and Fth1, has
been found on the S. cerevisiae vacuolar membrane and is
thought to pump iron from the vacuolar lumen to the cytosol (36). Most
relevant to the S. pombe Ctr4-Ctr5 copper transport complex,
expression of the S. pombe Fet3 homologue Fio1 in S. cerevisiae fails to rescue the iron uptake defect of a
fet3 The existence of Ctr4 and Ctr5 in a high affinity copper transport
complex at the S. pombe plasma membrane raises a number of
interesting questions. First, what are the respective roles of these
two proteins in the complex? Thus far we have not identified any
functional domains in Ctr4 or Ctr5 except the potential copper-binding Mets motifs. However, it is possible that Ctr4 and Ctr5 serve different
functional roles in the complex during copper uptake, in addition to
their interdependent roles in protein trafficking. Second, are there
homologous subunits in a high affinity copper transport complex in
other eukaryotes? Indeed, data base searching with both Ctr4 and Ctr5
from S. pombe has revealed the presence of multiple open
reading frames, in several organisms, with varying degrees of homology
to both proteins. However, whether homologous subunits exist in other
eukaryotes will require a structure-function analysis of Ctr4 and Ctr5
to identify critical functional residues that may be conserved across
species, as well as in vivo functional assays for copper
transport. Furthermore, it will be important to ascertain whether there
are additional components present in the Ctr4-Ctr5 complex, to
determine the precise role of each component in the function and
regulation of high affinity copper transport, and to explore the
possibility that defects in these subunits are associated with
pathophysiological states in humans.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
can1-1 leu-32
ade6-M210 ura44-D18). The isogenic
ctr4
(HZY3), ctr5
(HZY2), and
ctr4
ctr5
(HZY4) strains were constructed by replacing the coding region of each gene with a
hisG-URA4-hisG cassette through homologous recombination as
described (24). The genotypes of disruption strains were confirmed by
polymerase chain reaction, using gene-specific primers. The
cuf1
S. pombe strain SPY1 has been described
previously (17). The S. cerevisiae ctr1
ctr3
strain MPY17 was described previously (25). S. pombe cells
were grown at 30 °C in rich media
(YES),1 or synthetic media
(EMM) as described (26). The nonfermentable carbon source media YES-EG
was made by substituting the glucose in YES with 2% glycerol/3%
ethanol. S. cerevisiae cells were grown at 30 °C in rich
media (YPD) or synthetic media (SC), as previously described (27). YPEG
media was made by substituting the glucose in YPD with 2% glycerol/3% ethanol.
cDNA library in
YES phage vector (ATCC no. 87284, deposited by S. Elledge). The
resulting yeast expression library generated by self-recombination was
retrieved by a large scale plasmid preparation from 432,000 independent
transformants. S. cerevisiae strain MPY17 harboring plasmid
ctr4+-p413GPD was transformed with
the S. pombe cDNA library and plated on SC-His-Ura
medium. A total 360,000 transformants were pooled and re-plated on YPEG
plates. Among hundreds of colonies growing on YPEG, 24 were isolated,
their phenotype verified, plasmids retrieved, and inserts classified by
restriction endonuclease digestion and agarose gel electrophoresis. The
cDNAs from eight representative isolates were sequenced and were
all found to contain the same open reading frame, designated
ctr5+. The flanking sequences of the
ctr5+ gene were obtained from the S. pombe genome data base at the Sanger Center (Hinxton Hall, United Kingdom).
counter
(Packard). The counts of the samples on ice were subtracted from the
respective counts of room temperature samples to give the final uptake
values. The values were normalized to 1 A cells and
converted to amount of copper taken up, according to a standard curve.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ctr3
strain to grow on
nonfermentable carbon sources due to copper insufficiency. The S. pombe ctr4+ open reading frame was placed under the
control of the strong GPD promoter (29) and S. cerevisiae
ctr1
ctr3
cells expressing S. pombe
Ctr4 were tested for growth on the respiratory carbon sources
glycerol/ethanol, for which high affinity copper transport is required
for delivery of copper to cytochrome oxidase. As shown in Fig.
1B, although S. cerevisiae
ctr1
ctr3
cells expressing S. pombe
Ctr4 were able to grow on glucose, they were unable to utilize
glycerol/ethanol carbon sources. To explore the underlying reasons for
the inability of Ctr4 to complement the S. cerevisiae copper
uptake defect, a Ctr4-GFP fusion protein that is functional in S. pombe (Fig. 6A and Ref. 17) was localized in S. cerevisiae ctr1
ctr3
cells by fluorescence
microscopy. As shown in Fig. 1C, the Ctr4-GFP fusion protein
was not localized to the S. cerevisiae plasma membrane, but
rather appeared to be trapped within cells in both perinuclear and
other regions that might correspond to the secretory compartments. As a
control, the S. cerevisiae Ctr1-GFP fusion protein was
localized to the plasma membrane in these cells (Fig. 1C).
These observations suggest that at least one reason for the inability
of the S. pombe Ctr4 protein to function in high affinity
copper transport in S. cerevisiae is due to
mislocalization.
View larger version (39K):
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Fig. 1.
The S. pombe
(S.p.) copper transport protein Ctr4 is
mislocalized and nonfunctional in S. cerevisiae
(S.c.) A, structural
comparison of yeast and mammalian high affinity copper
transporters identified to date. The putative copper binding motif
MX2MXM is shown by
vertical ovals. The predicted transmembrane
(TM) domains are shown as horizontal
ovals. Regions with high sequence similarity are shown in
the same color. h/m, human/mouse. B, Ctr4
cannot complement the respiratory carbon source growth defect of
S. cerevisiae high affinity copper transport mutants. MPY17
(ctr1 ctr3
) was transformed with the p423GPD
(vector) or ctr4+-p423GPD, and plated on SC-His
(glucose as carbon source) and YPEG (glycerol/ethanol as carbon
source), respectively. Wild type (WT) strain DTY1 (harboring
vector control) were also tested. Cells were plated as 10-fold
dilutions and incubated for 2 days (YPD) or 5 days (YPEG) at 30 °C,
respectively. C, Ctr4-GFP is mislocalized in S. cerevisiae. MPY17 cells were transformed with
ctr4+-GFP-p423GPD or
pRS413-CTR1-GFP, respectively, and grown in SC-His with 100 µM BCS. Fluorescence micrographs and Nomarski optical
images were captured as described under "Experimental
Procedures."
ctr3
cells were co-transformed with a high
copy plasmid in which the Ctr4 coding region was expressed from the GPD
promoter, and an S. pombe cDNA library under the control
of the S. cerevisiae ADH1 promoter. Plasmids harboring
S. pombe cDNAs were recovered from eight
glycerol/ethanol-positive colonies and the cDNA inserts sequenced.
Of these eight independent isolates, all harbored a cDNA encoding a
single open reading frame, hereafter referred to as
ctr5+. Inspection of the S. pombe
genome data base indicates that, although the
ctr4+ gene is located on chromosome 3, the
ctr5+ gene is located on chromosome 1. As shown
in Fig. 2A, although expression of either S. pombe Ctr4 or Ctr5 alone in S. cerevisiae ctr1
ctr3
cells did not restore high
affinity copper transport, as ascertained by growth on glycerol/ethanol
media, co-expression of Ctr4 and Ctr5 allowed growth under these
conditions. Furthermore, although the S. pombe Ctr4-GFP
fusion protein was mislocalized in these cells, co-expression of
S. pombe Ctr5 facilitated the localization of the Ctr4-GFP
fusion protein to the S. cerevisiae plasma membrane (Fig.
2B). These data support the notion that S. pombe
Ctr5 is a co-factor required for Ctr4 localization to the plasma
membrane and function in high affinity copper transport in S. cerevisiae.
View larger version (44K):
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Fig. 2.
Expression of S. pombe Ctr5
complements S. cerevisiae ctr1 ctr3
growth defects and localizes Ctr4-GFP to the plasma
membrane. A, Ctr4 and Ctr5 demonstrate co-dependence
for complementation in S. cerevisiae. MPY17
(ctr1
ctr3
) was transformed with a
combination of ctr4+-p423GPD,
ctr5+-p426GPD, or vector alone. Growth was
tested on SC-His-Ura (glucose) for 2 days or YPEG (glycerol/ethanol)
for 5 days at 30 °C. WT, wild type. B,
localization of Ctr4-GFP in the presence and absence of Ctr5. MPY17 was
co-transformed with ctr4+-GFP-p423GPD
and either ctr5+-p426GPD or the empty vector
p426GPD and images captured as described for Fig. 1.
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Fig. 3.
The amino acid sequence of Ctr5 and its
predicted topology. A, the amino sequence of Ctr5. The
two partially overlapped MX2MXM
motifs and a similar CXMXM sequence are shown in
bold letters. Three predicted transmembrane
domains are boxed. B, the hydrophobicity plot of
Ctr5. The Toppred program was used to generate the plot using the GES
scale. The lines labeled Certain and
Putative indicate the possibility of the existence of
transmembrane domains. C, a topological model for Ctr5
predicted by Toppred. The positions of the "Mets" motifs and the
CXMXM sequence are indicated with
rectangles. N and C indicate amino and
carboxyl termini, respectively.
strain, which had previously been shown
to be copper-deficient (17), basal levels of
ctr5+ mRNA expression were dramatically
increased and as much as 100 µM CuSO4 was
required to significantly extinguish ctr5+
mRNA levels to that found at 10 µM in wild type
cells.
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Fig. 4.
The ctr4+ and
ctr5+ genes are co-regulated by copper and
the copper-sensing transcription factor Cuf1. S. pombe
strain FY254 (WT), HZY3 (ctr4 ), and SPY1
(cuf1
) were grown in YES medium to
A595 = 0.8. One hour before harvesting,
CuCl2 or BCS was added to the culture to the indicated
final concentration. Total RNA was extracted and fractionated on a
1.2% agarose gel, and RNA blot analysis was performed using the
indicated cDNAs as probes. The positions of the S. pombe
ctr4+, ctr5+, and
act1+ mRNAs are shown with
arrows.
strain, both
ctr4+ and ctr5+ gene
expression was severely diminished, regardless of the exogenous copper
levels (Fig. 4), indicating tight co-regulation of both genes by Cuf1.
Taken together, both protein structural features and regulation by
copper and the copper-sensing transcription factor Cuf1 suggest that
Ctr5 plays a role in copper homeostasis in S. pombe.
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Fig. 5.
Ctr4 and Ctr5 are required for growth on
nonfermentable carbon sources and high affinity 64Cu
transport. A, in vivo copper uptake by
S. pombe cells. FY254 (wt), HZY3
(ctr4 ), HZY2 (ctr5
), and HZY4
(ctr4
ctr5
) cells were grown in YES medium
to A595 = 0.6-1.0.
64CuCl2 was added to the culture to a final
concentration of 10 µM, and cultures were incubated
either at room temperature or on ice for 10 min. The values were
normalized to culture density, and counts on ice were subtracted from
the counts from room temperature incubation to give the final values.
B, S. pombe cells defective for
ctr4+ or ctr5+ are
defective for growth on nonfermentable carbon sources. Cells were
spotted on YES (glucose) or YES-EG (glycerol/ethanol), incubated for 5 or 7 days, respectively, and photographed.
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Fig. 6.
S. pombe Ctr4 and Ctr5
co-localized to the plasma membrane in an interdependent manner.
A, the epitope-tagged Ctr4 and Ctr5 proteins are functional.
Strains with indicated genotypes were transformed with plasmids
expressing the indicated Ctr4 and Ctr5 epitope-tagged proteins and
grown either 5 days on EMM-Leu (glucose) medium or 7 days on YES-EG
(glycerol/ethanol) medium and photographed. B, localization
of Ctr4-GFP and Ctr5-GFP in wild type and ctr4 or
ctr5
cells. FY254 (wild type (WT)), HZY2
(ctr5
), and HZY3 (ctr4
) cells were
transformed with ctr4+-GFP-pSP1
(Ctr4-GFP), ctr5+-pSP2
(Ctr5), ctr5+-GFP-pSP2
(Ctr5-GFP), or
ctr4+-pSP1(Ctr4), as indicated.
Nuclei were stained by Hoechst 33342. C, the deletion of
Ctr5 does not affect the localization of an unrelated membrane protein,
Liz1, fused to GFP. FY254 and HZY2 were transformed with pTE667
(Liz1-GFP) and photographed as described under "Experimental
Procedures."
cells accumulated the Ctr4-GFP fusion
protein almost exclusively in intracellular compartments, with little
or none detected on the plasma membrane. This effect appears to be
specific, since an S. pombe Liz1-GFP fusion protein (31) was
localized mainly to the plasma membrane in both wild type and
ctr5
cells (Fig. 6C). Furthermore, as shown in
Fig. 6B (panel 6), ctr4
cells accumulated Ctr5-GFP fusion protein in a perinuclear compartment,
and with punctate distribution, with little if any detected on the
plasma membrane. Taken together, these observations suggest that the Ctr4 and Ctr5 proteins are interdependent for localization to the
plasma membrane in S. pombe.
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Fig. 7.
Ctr4 and Ctr5 are integral membrane proteins
found in a complex. A, Ctr4 and Ctr5 are integral
membrane proteins. FY254 (wild type) S. pombe cells were
transformed ctr4+-FLAG-pSP1 and
ctr5+-myc-pSP2 plasmids. Cell lysate
was made by vortexing the cells with glass beads and centrifugation was
performed by 100,000 × g for 30 min. The supernatant
was loaded in lane 2, and the pellet was re-suspended and
treated with buffer, 0.2 M Na2CO3,
or 1% Triton X-100 for 30 min on ice. After re-centrifugation by
100,000 × g for 2 h, each of the pellets were
re-dissolved and loaded with supernatants as indicated (P
indicates pellet and S supernatant). 20 µg of protein was
loaded for each lane except lane 4, where all
protein was loaded. B, co-immunoprecipitation of Ctr4 and
Ctr5. FY254 was transformed with the indicated combination of
ctr4+-FLAG-pSP1,
ctr5+-myc-pSP2 (as indicated by + above the corresponding lanes) or control vectors (indicated
by ). Cells were harvested and Triton X-100-solubilized membrane
preparations subjected to immunoprecipitation (lanes marked
IP) and immunoblotting as described under "Experimental
Procedures." 15 µg of total lysate protein was loaded as comparison
(lanes labeled Total). Immunoprecipitation and
immunoblotting were performed using 9E10 anti-c-myc or M2
anti-FLAG antibody, as indicated. Numbers on the
left indicate molecular size markers (in kDa).
Asterisks indicate the position of the immunoglobulin heavy
chain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain. However, co-expression of Fip1, the S. pombe Ftr1 homologue, can reconstitute high affinity iron
transport in fet3
cells (37), suggesting that, although these are functionally homologous proteins, they cannot engage in
critical interactions across species. Perhaps a protein functionally, but not structurally, homologous to Ctr5 exists in S. cerevisiae, which is not capable of engaging in productive
interactions with the S. pombe Ctr4 transport subunit.
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ACKNOWLEDGEMENTS |
---|
We thank Robert Fuller, Jaekwon Lee, Sergi Puig, and Yasuhiro Nose for comments on the manuscript and for helpful advice, and Simon Labbé and Marj Peña for advice during the formulation of this project. Tamar Enoch kindly provided the Liz1-GFP plasmid. We thank Chen Kuang for excellent technical assistance and other Thiele laboratory members for helpful suggestions.
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FOOTNOTES |
---|
* This work was supported in part by Grant GM41840 from the National Institutes of Health (to D. J. T.).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.
Recipient of the Anthony and Lillian Lu Fellowship from the
Department of Biological Chemistry, University of Michigan Medical School.
§ To whom correspondence should be addressed. Tel.: 734-763-5717; Fax: 734-763-7799; E-mail: dthiele@umich.edu.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M102004200
2 J. Lee and D. J. Thiele, manuscript in preparation.
3 Beaudoin, J., and Labbé, S. (2001) J. Biol. Chem. 276, 15472-15480.
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ABBREVIATIONS |
---|
The abbreviations used are: YES, yeast extract with supplements; EMM, Edinburgh minimal medium; YPD, yeast extract with peptone and dextrose; YPEG: yeast extract with peptone, ethanol, and glycerol; SC, synthetic complete; GPD, glyceraldehyde-3-phosphate dehydrogenase; BCS, bathocuproinedisulfonate; GFP, green fluorescent protein.
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