From the Department of Neuroscience, University of
Connecticut Health Center, Farmington, Connecticut 06030-3401 and ¶ Department of Environmental Health Sciences, The Johns
Hopkins University School of Public Health, Baltimore, Maryland
21205
Received for publication, November 8, 2002, and in revised form, January 10, 2003
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ABSTRACT |
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We explored the role of known copper
transporters and chaperones in delivering copper to
peptidylglycine- Copper, a trace element required by most organisms, is
indispensable as a cofactor in a number of oxygen-processing proteins involved in diverse biological processes. For example, cytochrome c oxidase is essential for respiration, superoxide dismutase
is essential for free radical detoxification, lysyl oxidase is
essential for maturation for connective tissue, ceruloplasmin is
essential for iron uptake, tyrosinase is essential for melanin
synthesis, and dopamine Production of amidated peptides by PAM requires copper and molecular
oxygen and two reducing equivalents, usually supplied by two molecules
of ascorbate. The PHM domain of PAM binds copper at two non-equivalent
sites (CuA and CuB), both of which are critical for activity (8). Each copper atom is reduced by transfer of a single
electron from ascorbate, generating semidehydroascorbate. Copper is
bound by three His residues at the CuA site
(His107, His108, His172) and by two
His residues and a Met residue at the CuB site
(His242, His244, Met314) (9). The
process by which copper is delivered to the PHM domain of PAM in the
secretory pathway is unknown.
P-type ATPases are likely to play a role in copper transport into the
secretory pathway. Two copper transporting P-type ATPase genes,
ATP7A and ATP7B (10, 11), were identified in
studies of inherited disorders of human copper metabolism (12).
Mutations in the ATP7A gene lead to Menkes disease, a
disorder characterized by copper deficiency. ATP7A is expressed in all
tissues except the liver, and mutations in ATP7A prevent normal
intestinal absorption of copper and distribution of copper throughout
the body. The resulting lack of copper compromises the function of many
tissues, resulting in death in early childhood. Mutations in the ATP7B gene cause Wilson disease, which is characterized by failure of the
liver to excrete copper into the biliary tract or to deliver copper to
ceruloplasmin, a multi-copper oxidase needed for serum iron homeostasis
(13). The prevalence of ATP7B in the liver means that protein mutations
result in liver toxicity because of the accumulation of copper.
Although tissue-specific, both disorders involve a defect in P-type
ATPase-mediated export of copper from the cytosol into the
secretory/endocytic pathways (14).
Due in large part to the clarity that yeast genetics brings to the
analysis of protein function, Saccharomyces cerevisiae is an
excellent model organism for studying many fundamental processes of
eukaryotic cells (15). The importance of oxidative metabolism to
eukaryotic cells has led to conservation of mechanisms of copper metabolism from yeast to human cells. Indeed, it has been shown that
the molecular components of copper trafficking pathways are highly
conserved between yeast and humans. In S. cerevisiae, the pathway begins with Cu+ uptake through the action of copper
transporters Ctr1 and Ctr3 (16, 17). In the cytoplasm, copper is bound
by cytoplasmic copper chaperones that deliver the metal to specific
target enzymes (18). The Atx1 copper chaperone delivers copper to Ccc2,
the only yeast homologue of the mammalian Wilson and Menkes ATPases (19). Ccc2 is required for transport of copper from the cytosol into
the lumen of the trans-Golgi network, where the multi-copper ferroxidase Fet3, the yeast homolog of ceruloplasmin, is loaded with
copper (20, 21).
The yeast genome does not encode a PHM or a PAL homolog. In the present
study, we determined whether active PHM could be produced in the yeast
secretory pathway and whether its production requires the assistance of
known copper transporters and chaperones. In particular, the roles of
the P-type ATPase Ccc2 and the cytosolic copper chaperone Atx1 were
investigated. We expressed soluble rat PHMcc (PHM
catalytic core) using the yeast Yeast Strains and Growth Conditions--
Strains of S. cerevisiae used in this study are isogenic to wild-type YPH252
(Mat Plasmids--
We expressed PHMcc protein using a yeast
Mammalian Cell Culture--
The murine mottled fibroblast cell
line 802-1 (Mo Cell Extracts and Immunoblotting--
For Western blot analysis
of PAM-expressing S. cerevisiae cells, the yeast strains
transformed with pSM703 vector or vector encoding PHM Assays--
PHM activity was measured in yeast and mammalian
cell extracts as described previously (8) using
Immunofluorescence Microscopy--
Yeast strain YPH252
transformed with pSM703 vector (26), pSM703. Subcellular Fractionation--
The procedure for subcellular
fractionation of yeast cells is based on sedimentation through sucrose
density gradients, as described previously (32), but with some
modifications. Yeast (YPH252) transformed with pSM703. Organelle Marker Enzyme Assays--
Reactions (25 µl of each
fraction) were carried out in a total volume of 100 µl, except for
NADPH-cytochrome c reductase assays, which were
carried out in a volume of 1 ml. Vacuolar Expression of Active PHM in Yeast--
The yeast genome does not
encode a protein homologous to PHM or to any of the other members of
this family of copper-containing monooxygenases; the amino acid
sequence of rat PHMcc was compared with the S. cerevisiae
genome using BLASTN 2.2.3 (Apr-24-2002) (NCBI) (36). To determine
whether we could use S. cerevisiae to identify proteins
involved in the delivery of copper to PHM, we first expressed soluble
PHMcc in S. cerevisiae and analyzed cell extracts and medium
for PHM activity. We fused the
Extracts prepared from wild-type yeast contained no detectable PHM
activity (<0.01 units/µg protein). Control experiments demonstrated
complete recovery of exogenous rat PHMcc activity added to yeast
extract (1 µg protein) and assayed in the presence of 0.5 µM exogenous copper (data not shown). Extracts prepared from yeast expressing PHMcc Partially Co-localizes with Ccc2-HA in the Yeast Secretory
Pathway--
Indirect immunofluorescence microscopy was utilized to
determine the subcellular localization of PHMcc in yeast. Cells
expressing PHMcc were fixed and visualized with a PHM antiserum (Fig.
3A). PHM staining was observed
in punctate structures reminiscent of Golgi-like vesicles. The punctate
PHM staining pattern was similar to that obtained for proteins thought
to be associated with Golgi or post-Golgi vesicles
(38-40), with some puncta significantly larger than others. The
staining is specific for PHMcc, as only very faint homogeneous staining
was observed in cells transformed with empty vector (Fig.
3A). The 4',6'-diamidino-2-phenylindole staining profile
(Fig. 3A, middle panel) demonstrated that PHMcc is neither nuclear nor perinuclear and is not associated with mitochondria. Comparison of the distribution of PHMcc to the
differential interference contrast optics images (Fig.
3A, right panel) demonstrated that PHMcc is not
vacuole-associated.
The yeast copper transporter Ccc2 is known to reside in post-Golgi
vesicles (21, 41). To evaluate a functional connection between Ccc2 and
PHMcc, we compared the subcellular localization of expressed PHMcc with
that of an epitope-tagged version of Ccc2 (Fig. 3A). Yeast
of the wild-type strain, transformed with vectors encoding both PHMcc
and HA-tagged Ccc2, were stained simultaneously with a rabbit
polyclonal antiserum to PHM and a mouse monoclonal antibody to the HA
tag and examined using confocal microscopy (Fig. 3B).
PHM staining (in green) and Ccc2-HA staining (in red) were
superimposed. As shown in the merged image (Fig. 3B), PHMcc and Ccc2 were sometimes localized to the same vesicular structures (Fig. 3B, arrows). However, the distributions of
Ccc2 and PHMcc were not identical, with some vesicles staining more
intensely for PHM (arrowheads) and others staining more
intensely for Ccc2 (lines). Based on their subcellular
localization, Ccc2 and PHMcc have access to the same subcellular
compartments. As might be expected for an integral membrane protein and
a soluble, secretory protein, transient co-localization of Ccc2 and
PHMcc was observed.
PHMcc Co-migrates with a Golgi Marker during Subcellular
Fractionation--
To confirm our conclusion that PHMcc is at least
transiently localized to the Golgi compartment of the secretory
pathway, subcellular fractionation was carried out. Extracts prepared
from wild-type yeast expressing PHMcc were subjected to velocity
sedimentation through sucrose gradients. The various subcellular
compartments were identified in gradient fractions by measuring
enzymatic markers characteristic of each organelle (Fig.
4). The distribution of PHMcc protein was
determined by Western blot analysis. The bulk of the PHM was found in
fractions 8 though 11. The higher molecular mass cross-reactive
protein localized in fractions 1 through 5 corresponds to a
nonspecific band visualized in the same gradient fractions of yeast
cells transformed with expression vector alone. The Golgi marker GDPase
(34) was also localized to fractions 8 through 11. PHMcc was clearly
separated from the endoplasmic reticulum marker (35), NADPH-cytochrome
reductase, which was highly localized to fraction 13, and from the
vacuolar marker (33), Effects of CCC2 and ATX1 Gene Mutations on PHM Enzyme
Activity--
Because active PHM can be produced in wild-type yeast,
mutant yeast were used to assess the role of specific copper
transporters and chaperones in providing copper to PHM. In yeast, the
cytosolic copper chaperone Atx1 shuttles copper to Ccc2, an
intracellular copper transporter that is localized to the membranes of
the Golgi compartment (18, 19, 42, 43). This transporter pumps copper into the lumen of the Golgi complex, making it available for insertion into copper-dependent lumenal enzymes. To assess the role
of Ccc2 and the cytosolic copper chaperone Atx1 in providing copper to PHM, we expressed PHMcc in yeast bearing mutations in the
CCC2 or ATX1 genes (Fig.
5). As shown by Western blot analysis
(Fig. 5A), equal expression of PHMcc protein was observed in
wild-type yeast and in ccc2 and atx1 mutant
cells.
We next compared the catalytic activity of PHMcc produced in wild-type,
ccc2, and atx1 mutant cells. Because the addition of exogenous copper or the binding of copper made accessible following homogenization could obscure any deficits present in PHMcc expressed in
ccc2 or atx1 mutant cells, we varied the assay
conditions. Reduced ascorbate was added to the extraction buffer to
promote retention of bound copper (by keeping it reduced), and
bathocuproinedisulfonic acid was supplemented to ensure that any free
copper was made inaccessible (20). When assayed without added copper
ions, no PHM activity was found in yeast cells lacking either Ccc2 or
Atx1; in contrast, the specific activity of PHMcc in wild-type cell extracts was 2.04 ± 0.11 units/µg of protein. The PHMcc protein expressed in ccc2 and atx1 mutant yeast could be
fully reconstituted by in vitro addition of 0.5 µM copper (2.09 ± 0.18 and 2.31 ± 0.3 units/µg of protein, respectively) (Fig. 5B). Although
expression of PHMcc protein does not require Ccc2 or Atx1, expression
of active PHM does depend on provision of copper through the
trafficking pathway involving Ccc2 and Atx1.
ATP7A Supports the Production of Active PAM Enzyme in
Mammalian Cells--
Although yeast have a single copper-transporting
P-type ATPase, mammalian cells have two, ATP7A and ATP7B. We used a
murine cell line defective in the mouse homologue of the Menkes disease gene as a test system for assessing the role of ATP7A in providing copper to PAM (44). The 802-1 mottled cell line was derived from mutant
male mice carrying the ATP7a Mo-br allele of ATP7A; the
ATP7aMo-br gene has a 6-bp deletion that
eliminates two amino acids in a conserved region of the protein,
resulting in a severe phenotype similar to classical Menkes disease
(29, 45, 46). In addition, 802-1 mottled cells are heterozygous for
deletion of the metallothionein I and II genes, shown previously to
increase the sensitivity of these cells to copper, permitting a rapid
functional assay for the Menkes gene (44). A control cell line, 802-5, derived from wild-type male mice, has wild-type ATP7A and is also
heterozygous for deletion of the metallothionein I and II genes.
Neither the 802-1 nor the 802-5 cells express ATP7B, the other
mammalian copper-transporting P-type ATPase (29, 45, 46).
To determine whether active PAM enzyme can be produced in cells
lacking functional ATP7A and ATP7B, we used a recombinant adenovirus
encoding full-length PAM-1 to infect both mottled
802-1 cells and wild-type 802-5 cells. The 120-kDa PAM-1 protein was identified in cell extracts using the PHM antibody for Western blot
analysis (Fig. 6A). The same
level of PAM protein expression was achieved in the mutant and
wild-type cell lines. When the expressed enzyme was assayed for
activity in the absence of additional copper, the PHM activity in
wild-type 802-5 cells was 0.9 ± 0.06 units/µg of protein,
whereas no PHM activity was detected in extracts of the
mottled 802-1 cells (<0.01 units/µg protein) (Fig.
6B). Because expression of PAM-1 protein did not
require the presence of active ATP7A, we added exogenous copper to try
to activate the PAM-1 protein produced in mottled 802-1 cells. In vitro addition of 0.5 µM
CuSO4 to wild-type 802-5 cell extracts increased PAM activity almost 2-fold (1.73 ± 0.12 units/µg of protein),
suggesting that not all of the enzyme in the cells is fully metallated.
In vitro addition of 0.5 µM CuSO4
to mottled 802-1 cell extracts revealed substantial amounts
of PHM activity (0.94 ± 0.01 units/µg of protein) in the mutant
cells. It is not clear why the PHM activities in extracts of mutant and
wild-type cells are not identical after addition of copper. The
magnitude of the stimulation by copper suggests that none of the PHM
expressed in the mutant cell line has copper and that a functional
ATP7A protein is required for production of active PHM in these mouse
fibroblasts, which lack ATP7B.
Secretion of Cuproproteins--
Highly conserved
copper trafficking pathways have evolved to control levels of copper
and provide copper to the limited number of enzymes that need it while
protecting the rest of the cell (47). Several cytosolic and
mitochondrial cuproproteins acquire copper only when it is provided by
a specific chaperone protein. Most of these copper chaperones share
sequence homology with their target protein, facilitating a specific
metallochaperone-cuproprotein interaction (18). Delivery of copper to
secreted proteins like Fet3 and ceruloplasmin, multi-copper oxidases
needed for high affinity iron uptake in yeast and mammals, respectively
(41), requires cytosolic chaperone-mediated delivery of copper to a P-type ATPase. Hah1 (or Atox1), like its yeast homologue, Atx1 (47-49), shares an MTCXXC metal binding site with its
target P-type ATPases. Mice genetically engineered to lack
Atox1
The number of copper-dependent secretory pathway proteins
is small, but growing. Fet3/ceruloplasmin, laccases (multi-copper enzymes that catalyze the oxidation of phenolic and non-phenolic compounds), and secretory pathway enzymes like tyrosinase, PAM, dopamine Expression of Active PHM in Yeast--
When expressed in yeast
using the rat PAM signal sequence, both membrane and soluble forms of
PAM remained cytosolic and were not directed to the secretory pathway
(data not shown). In contrast, the secretion signal from yeast
Based on both immunofluorescence and subcellular fractionation, PHMcc
is partially localized at steady state to the Golgi, where Kex2 and
Ccc2 are located. Intact
Transient co-localization of PHMcc and Ccc2 in a post-Golgi compartment
is consistent with an important function for Ccc2 in providing copper
to PHM (see below). Cytosolic copper export is restricted
to a late Golgi compartment in the S. cerevisiae secretory
pathway (21). Yeast sec mutants blocked at pre-Golgi and
Golgi sites in the secretory pathway fail to deliver copper to Fet3. In
addition, yeast mutants like vps33, with defects in a
post-Golgi compartment, are also deficient in Fet3 activity. Thus, Ccc2
distribution is compromised when either early secretory pathway
integrity and/or post-Golgi sorting are disturbed, leading to
copper transport deficiency and Fet3 inactivity.
Directed to the secretory pathway, PHMcc expressed in wild-type yeast
is at least as active as PHMcc secreted by mammalian CHO cells. Despite
the fact that yeast S. cerevisiae does not express a protein
homologous to PAM or to any of the members of the family of
copper-containing monooxygenases, copper is made available to the newly
synthesized PHMcc protein traveling through the yeast secretory pathway.
Ccc2 and Atx1 Are Required for Production of Active PHMcc in S. cerevisiae--
We expressed PHMcc in wild-type, ccc2, and
atx1 mutant yeast to determine whether production of active
PHM were dependent on the copper trafficking pathways involving these
genes. Some newly synthesized metalloenzymes require the presence of
metal for proper folding and subsequent exit from the endoplasmic
reticulum (66). However, expression of PHMcc protein was unaltered in yeast lacking secretory pathway copper. These data agree with the fact
that an inactive PHMcc mutant unable to bind copper at CuA
or CuB was still efficiently synthesized and secreted by
CHO cells (8). When assayed in the absence of exogenous copper, no PHM
enzymatic activity is detected in yeast lacking Ccc2 or Atx1.
Nevertheless, following addition of exogenous copper, PHMcc produced in
ccc2 or atx1 mutant yeast was as active as PHMcc
expressed in wild-type yeast. Thus production of active PHMcc in
yeast requires functional Atx1 and Ccc2.
ATP7A and Production of PAM in Mammalian Cells--
Expression of
membrane PAM-1 protein was similar in fibroblast lines lacking
functional ATP7A and ATP7B or having functional ATP7A. Although PAM
enzyme produced in cells expressing ATP7A was partially metallated, PAM
enzyme produced in cells lacking both copper-transporting P-type
ATPases was inactive until addition of exogenous copper. ATP7A was
shown previously (49) to complement the ccc2 yeast mutant
and provide copper to Fet3, the yeast ceruloplasmin homologue. ATP7A is
also required for the production of active tyrosinase, a
copper-dependent enzyme involved in melanogenesis within
the secretory pathway (52). Thus copper delivered to the secretory
pathway by ATP7A is available to PAM.
Consistent with these data, our research group demonstrated
recently that the ability of PAM to produce bioactive amidated peptides
was compromised in the mottled brindled male mouse (Atp7a mouse). Although normal levels of PAM protein were found in
Atp7a mice, levels of amidated Joining Peptide and
Models--
Although our findings demonstrate that ATP7A delivers
copper into the secretory pathway, allowing PAM to acquire copper, the precise mechanism by which this process occurs is unknown. Two models
for the incorporation of copper into copper-dependent
enzymes of the secretory pathway need to be considered. In the first
model, ATP7A transfers copper into the lumen of the secretory pathway, where the metal is directly incorporated into apo-PHM and other copper-dependent lumenal enzymes. Alternatively, copper
pumped into the secretory pathway by ATP7A equilibrates with low and high affinity binding sites in the lumenal compartment, allowing secretory pathway cuproenzymes like PHM to bind copper. Based on the
fact that yeast, which do not produce a protein homologous to PHM, can
synthesize fully active enzyme, we argue against the existence of a
cuproenzyme-specific lumenal chaperone system similar to that described
in the cytosol (26, 68). However, a copper-binding protein capable of
retaining copper in the Golgi and making it available to lumenal
cuproenzymes could play a role. The lower pH and oxidizing environment
of the lumenal compartment may dictate different schemes for handling
cytosolic and lumenal copper.
In summary, our data suggest that loading of copper onto secretory
pathway enzymes like PAM is fundamentally different from loading of
copper onto cytosolic and mitochondrial enzymes. The sequestered
environment of the secretory pathway may eliminate the need for
specific copper chaperones. Measurement of total and free copper levels
within the secretory pathway will be informative.
-hydroxylating monooxygenase (PHM), a
copper-dependent enzyme that functions in the secretory pathway lumen. We examined the roles of yeast Ccc2, a P-type ATPase related to human ATP7A (Menkes disease protein) and ATP7B (Wilson disease protein), as well as yeast Atx1, a cytosolic copper chaperone. We expressed soluble PHMcc (catalytic core) in
yeast using the yeast pre-pro-
-mating factor leader region to target
the enzyme to the secretory pathway. Although the yeast genome encodes
no PHM-like enzyme, PHMcc expressed in yeast is at least as active as
PHMcc produced by mammalian cells. PHMcc partially co-migrated with a
Golgi marker during subcellular fractionation and partially co-localized with Ccc2 based on immunofluorescence. To determine whether production of active PHM was dependent on copper trafficking pathways involving the CCC2 or ATX1 genes, we
expressed PHMcc in wild-type, ccc2, and atx1
mutant yeast. Although ccc2 and atx1 mutant
yeast produce normal levels of PHMcc protein, it lacks catalytic
activity. Addition of exogenous copper yields fully active PHMcc.
Similarly, production of active PHM in mouse fibroblasts is impaired in
the presence of a mutant ATP7A gene. Although
delivery of copper to lumenal cuproproteins like PAM involves ATP7A,
lumenal chaperones may not be required.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-monooxygenase is essential for
catecholamine formation (1). Another important
copper-dependent enzyme, peptidylglycine
-amidating monooxygenase
(PAM),1 catalyzes the
C-terminal amidation of glycine-extended peptide precursors, a
modification essential for the bioactivity of numerous hormones and
neuropeptides (2-5) (see Fig. 1). Peptide amidation occurs in two
consecutive steps that require the peptidylglycine
-hydroxylating monooxygenase (PHM) and
peptidyl-
-hydroxyglycine
-amidating lyase (PAL) domains of the
bifunctional PAM protein (see Fig. 1). This reaction can be initiated
in the trans-Golgi network but occurs primarily within secretory
granules (6, 7).
-mating
factor leader sequence to target the enzyme to the yeast secretory
pathway (Fig. 1). We compared the
activity of PHMcc produced in yeast to that of PHMcc produced in
Chinese hamster ovary (CHO) cells. Using immunofluorescence microscopy and subcellular fractionation, we established that PHMcc was partially co-localized with Golgi markers and Ccc2 in yeast cells. Using gene
knockouts, we established an essential role for the secretory pathway
copper transporter Ccc2 and the cytosolic copper chaperone Atx1 in
producing active PHMcc in yeast. Finally, we demonstrated that active
Menkes protein, a mammalian homologue of Ccc2, can support production
of active PHM in fibroblasts.
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Fig. 1.
PAM-catalyzed reactions and PAM
proteins. The enzymatic reactions catalyzed by the PHM and PAL
domains of PAM are shown. PAM protein structure is compared with that
of the PHM catalytic core (cc) used in this study. The PHMcc
protein expressed in mammalian CHO cells and the -mating
factor-PHMcc fusion protein expressed in wild-type yeast cells (YPH252)
are shown. The yeast
-mating factor precursor is diagrammed with
-factor peptides (
F; clear boxes) separated
by spacer peptides consisting of KR followed by two or three EA or DA
pairs (horizontally cross-hatched boxes) that are used as
endopeptidase cleavage sites (60).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ura3-52 lys2-801 ade2-101 trp1-
1
his3-
200 leu2-
1) (22). In ccc2
mutant cells (ccc2
::URA3,
URA- by 5-fluoroorotic acid, strain MP103 (23)), the
CCC2 gene was replaced with the URA3 gene. The
atx1
::LEU2 cells (strain SL215 (19))
were in the same background but derived from the opposite mating type parental strain (YPH250). Strain stocks were maintained on
standard YPD (yeast
extract-peptone-dextrose) medium.
Cultures for experimental analysis were obtained by growth in a
synthetic minimal medium containing dextrose (SD) (24) or in low copper
defined medium prepared by using a metal-depleted nitrogen base
supplemented with all essential metals except copper (i.e.
1.4 mM CaCl2, 3.2 µM
MnSO4, 2.9 µM ZnSO4, 4.2 mM MgSO4, 1.6 µM
FeCl2) (Difco Manual).
-mating factor signal sequence. Soluble and integral membrane PAM
proteins (PAM-3 and PAM-2) expressed in yeast with the rat PAM signal
sequence were not directed to the secretory pathway, remained
cytosolic, and exhibited no enzyme activity, even in the presence of
added copper (data not shown). pSM703.
MF-PHMcc was generated by PCR amplification of PHMcc (amino acids 42-356 of rat PAM-1; nucleotides 421-1365) from pBS.PAM-1, which encodes full-length PAM-1 (25). The
PCR product was cloned into the pICK9 vector (Invitrogen) using the
unique XhoI and NotI sites; pICK9.PHMcc,
containing the PHM fragment with the
MF pre-pro-sequence (amino
acids 1-81) and a LEKR linker, was digested with BamHI and
NotI. The digested PHMcc product was ligated into the same
sites of the pICK9K vector (Invitrogen). A multi-copy
MF-PHMcc
plasmid, pSM703.
MF-PHMcc, was constructed by inserting the
BamHI-NotI
MF-PHMcc fragment of pICK9K.PHMcc
into the 2µ URA3 plasmid pSM703 (26). As described by Yuan
et al. (21), the pDY207.Ccc2-HA vector fused the 3'-end of
the CCC2 coding sequence with sequences encoding three
tandem copies of the influenza hemagglutinin (HA) epitope tag followed by two stop codons and the CYC1 terminator. The PAM-1
recombinant adenovirus (PAM-1 virus) encodes full-length rat PAM-1
(nucleotides 293-3245); its preparation was described previously (27,
28).
/Y, Mt
/+), and the corresponding wild-type cell line
802-5 (Mo+/Y, Mt
/+) were a gift from Dr. Jonathan D. Gitlin
(Washington University School of Medicine, St. Louis, MO). Cell lines
were maintained in Dulbecco's modified Eagle's medium containing 10%
fetal clone III bovine serum (Hyclone, Logan, UT) as described
previously (29). Cells were used 48 h after PAM-1 virus infection
(28).
MF-PHMcc were
harvested at mid-logarithmic phase (A600 = 1) in
regular SD medium or low copper SD medium (less than 0.25 µM copper) minus uracil. Yeast cell extracts were
prepared by glass bead homogenization in 20 mM sodium
(N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid)
(TES)/TMT (10 mM mannitol, 1% Triton X-100, pH 7.4)
containing protease inhibitors, 1 mM sodium ascorbate, and
1 mM bathocuproinedisulfonate to limit the availability of
copper during extraction as described previously (20). To analyze PAM
expression in mammalian fibroblasts, the 802-1 and 802-5 cells, grown
as described above, were scraped into TMT buffer containing protease
inhibitors, frozen and thawed three times, and centrifuged for 5 min to
remove cell debris (27). With either yeast or mammalian expression
systems, samples were resolved by 8 or 12% SDS-PAGE, transferred to
nitrocellulose membranes, and analyzed by Western blot using rabbit
polyclonal antiserum to PHM (JH1761) and the ECL kit (Amersham
Biosciences).
-N-acetyl-Tyr-Val-Gly as substrate. Protein (30 to 50 ng)
was assayed in the presence or absence of 0.5 µM
CuSO4 as indicated, in duplicate, and reactions were
carried out for 2 h. PHM specific activity is expressed as picomoles of product formed per h (units) per microgram of protein.
MF-PHMcc, pDY207 vector,
pDY207.CCC2-HA (21), or both pSM703.
MF-PHMcc and
pDY207.CCC2-HA were used for indirect immunofluorescence staining according to standard procedures (30, 31). Yeast cells were
grown to an A600 of 1 in selecting SD medium,
fixed with 4% formaldehyde, digested with zymolase, and incubated
overnight with PHM antiserum (JH1761) or purified mouse HA antibody
(HA.11; BabCo, Richmond, CA). The antigen-antibody complexes were
visualized using fluorescein isothiocyanate-conjugated goat anti-rabbit
IgG (Caltag, San Francisco, CA) or Cy3-conjugated donkey anti-mouse IgG
(Jackson ImmunoResearch, West Grove, PA). Nucleic acids were stained by
4',6'-diamidino-2-phenylindole (Sigma). Fluorescence images and
Nomarski differential interference contrast images were obtained with a
Zeiss Axiovert 135TV microscope (Microscopy Facility, Johns Hopkins
Medical Institutions). Where indicated, a Noran confocal laser scanning
fluorescence microscope was used. Two signals, fluorescein
isothiocyanate (green) and Cy3 (red), were collected simultaneously
from yeast cells in optical sections of 0.2 µm.
MF-PHMcc were
grown to log phase (A600 = 1) in SD medium minus
uracil. After being chilled on ice, cells (500 optical density
units) were harvested by centrifugation at 3700 × g
for 10 min. The pellet was resuspended in 30 ml of cold 10 mM sodium azide and centrifuged for 10 min at 2000 × g. Cells were resuspended to 50 A600
units/ml in spheroplast buffer (1.4 M sorbitol, 50 mM potassium phosphate, pH 7.5, 10 mM sodium azide, 40 mM mercaptoethanol), and zymolase 20T (ICN
Biomedicals) was added to 1.5 mg/ml; cells were converted to
spheroplasts by incubating for 45 min at 30 °C with gentle shaking.
All further steps were carried out on ice. The spheroplast suspension
was adjusted to 1 mM EDTA, layered on top of 8 ml of 1.8 M sorbitol, and spheroplasts were centrifuged through the
sorbitol cushion by centrifugation at 500 × g for 10 min. The pellet was resuspended in 5 ml of lysis buffer (0.8 M sorbitol, 10 mM MOPS, pH 7.2, 1 mM EDTA) containing a mixture of the following protease
inhibitors: 2 µg/ml leupeptin, 16 µg/ml benzamidine, and 300 µg/ml phenylmethylsulfonyl fluoride. The spheroplast lysate was
homogenized with a tissue grinder (Kontes Scientific Glassware) and
then centrifuged for 10 min at 500 × g to remove
unlysed cells and debris. The resulting supernatant (1 ml) was layered
onto a step gradient containing 1 ml each of 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54% sucrose (w/v) in 10 mM HEPES, pH 7.5, 1 mM MgCl2. Gradients were centrifuged for
2.5 h at 174,000 × g in an SW41 rotor (Beckman
Instruments) at 4 °C. Fractions (660 µl) were collected from the
top, and an equal aliquot of each fraction was analyzed. For Western
blot analysis, 50 µl of aliquot of each fraction was used for
SDS-PAGE as described above.
-mannosidase was assayed
through the generation of p-nitrophenol from
p-nitrophenyl-
-mannopyranoside, which leads to an
increase in A400. The reaction mixture consisted of 0.4 mM p-nitrophenyl-
-mannopyranoside, 40 mM sodium acetate, pH 6.5 (33). GDPase was assayed as
described by Abeijon et al. (34). Incubation tubes contained
20 mM imidazole, pH 7.4, 2 mM
CaCl2, 7 mM GDP, and 0.1% Triton X-100.
Liberated phosphate was measured with the Fiske Subbarow kit (Sigma).
Nonspecific NDPase plus free Pi were determined using CDP
as a nonspecific substrate. These values were subtracted from those
obtained with the substrate GDP to give GDPase activity.
NADPH-cytochrome c reductase was assayed by the absorbance
increase at 550 nm according to Feldman et al. (35). Assay
mixtures (1 ml) contained 1.0 mg/ml cytochrome c, 100 µM NADPH, 50 mM
KH2PO4, pH 7.4, 0.4 mM KCN, 1.0 µM FMN, and 0.6 M sorbitol. Reaction rates
were determined from the initial linear phase of the assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MF signal/pro-region to the catalytic
core of rat PHM (
MF-PHMcc) to direct the foreign monooxygenase into
the yeast secretory pathway. With its pro-region,
MF-PHMcc should
have a mass of 41 kDa. Transformation of wild-type yeast with vector
encoding
MF-PHMcc led to expression of a 34-kDa protein recognized
by antiserum to rat PHMcc (Fig. 2),
indicating that the
MF pro-region had been removed. Vector alone
yielded only a smaller, nonspecific cross-reactive protein. PHMcc
secreted by stably transfected CHO cells consistently had a slightly
higher apparent molecular mass (36 kDa) than the cross-reactive
product detected in yeast extracts. Although an LEKR sequence separated the
MF pro-region from PHMcc, the exact site of endoproteolytic cleavage was not determined, and this difference in mass may reflect different trimming or nicking of PHMcc in yeast and CHO cells.
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Fig. 2.
PHMcc expression in yeast S. cerevisiae. Strain YPH252 transformed with either
pSM703- MF-PHMcc (Wt-PHMcc) or empty vector (pSM703) was
extracted with TES-mannitol/Triton X-100. Equal amounts of PHMcc
enzymatic activity (25 or 125 units) expressed in yeast and purified
from CHO cells were subjected to Western blot analysis and visualized
with antiserum to rat PHM. The molecular mass markers are shown on the
left.
MF-PHMcc contained 5.4 ± 0.5 units/µg
of protein when assayed under the same conditions. To compare the specific activity of PHMcc expressed in yeast with that of
PHMcc secreted by stably transfected CHO cells (with PHM specific
activity of 9000 ± 500 units/µg of protein) (8) (Fig. 2), the
amount of PHMcc protein was estimated based on cross-reactivity with an
antiserum specific to rat PHMcc (37). PHMcc expressed in yeast is at
least as active as PHMcc secreted by mammalian cells; for the same
amount of PHM enzyme activity (25 or 125 units), less PHM protein is
present in the yeast lysate than in the mammalian cell medium. Despite
the fact that the
-mating factor-PHMcc construct yields a soluble,
active protein, PHMcc is secreted very poorly, accumulating in
post-Golgi vesicles. Secreted PHM could be detected in lyophilized
yeast growth medium by Western blot analysis; however PHM activity was
not detectable (data not shown).
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Fig. 3.
PHM and Ccc2 proteins partially co-localize
in yeast cells. YPH252 cells expressing PHMcc, Ccc2-HA, or
both proteins were examined by fluorescence microscopy (×1000
magnification). A, PHM was visualized with rabbit antibody
to rat PHM and a fluorescein isothiocyanate-tagged antibody to rabbit
IgG. Ccc2-HA was visualized with antibody to the HA epitope (HA.11)
followed by a Cy3-tagged antibody. Control cells were transformed with
an empty vector and subjected to the same staining protocol. In
parallel, cells were stained with 4',6'-diamidino-2-phenylindole
(DAPI) for nucleic acid detection and analyzed using
Nomarski optics to visualize the indentations of the yeast vacuole.
DIC, differential interference contrast optics.
B, simultaneous immunostaining of cells expressing PHMcc and
Ccc2-HA. PHMcc (in green) and Ccc2-HA (in red)
were visualized using two fluorescently tagged secondary antisera (as
described above) and confocal laser scanning fluorescence microscopy.
In the superimposed images shown on the right, vesicles
co-immunostained with antisera to PHMcc and to Ccc2-HA appear
yellow and are indicated by arrows; PHM staining
alone is indicated by arrowheads, and vesicles stained only
by Ccc2 are indicated by lines.
-mannosidase, which was concentrated in
fraction 3 (Fig. 4B). Based on both immunofluorescence and
subcellular fractionation, the bulk of PHMcc is localized to the Golgi
complex in S. cerevisiae at steady state.
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Fig. 4.
Subcellular localization of PHMcc in
S. cerevisiae. A spheroplast lysate
prepared from YPH252 cells expressing PHMcc was centrifuged at 500 × g for 10 min. The supernatant was layered onto a 18-54%
sucrose gradient, and subcellular organelles were separated during a
2.5-h spin at 174,000 × g (see "Experimental
Procedures"). A, aliquots of the gradient fractions were
separated by 8% SDS-PAGE, and the distribution of PHMcc was analyzed
by immunoblotting. B, for enzymatic assays, NADPH-cytochrome
c reductase was measured as an endoplasmic reticulum marker,
GDPase was measured as a Golgi marker, and -mannosidase was measured
as a vacuolar marker. Enzymatic activities are expressed on a per
fraction basis, for GDPase (1 unit = nmol phosphate produced per
20 min), NADPH-cytochrome c reductase (1 unit = nmol
cytochrome C reduced per min), and
-mannosidase (1 unit = nmol
of p-nitrophenyl-
-mannopyranoside cleaved per 15 min).
Fractions were harvested from the top (fraction 1) to the
bottom (fraction 16).
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Fig. 5.
Role of Ccc2 and Atx1 in producing active
PHMcc. Wild-type YPH252 cells (wt), ccc2
mutant cells (MP103 strain), and Atx1 mutant cells (SL215
strain) were transformed with pSM703. MF-PHMcc plasmid or empty
vector as an expression control. A, equal amounts of protein
(10 µg) from the different strains were analyzed by Western blot
using the PHM antibody. B, cells were extracted in TMT
buffer containing bathocuproinedisulfonate and ascorbate, and PHM
enzymatic activity was assayed in the absence or presence of 0.5 µM CuSO4 as indicated; activity was measured
in duplicate samples of cell extracts and is expressed as units of
amidated product formed per microgram of protein. Data are the
mean ± S.D. for triplicate cultures. Similar results were
obtained in two additional experiments.
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Fig. 6.
Menkes P-type ATPase is essential for
production of active PHM enzyme in mammalian cells. A,
immunoblot of extracts from three independent cultures of PAM-1
virus-infected mottled fibroblasts (802-1) and
wild-type fibroblasts (802-5); single cultures of
non-infected mottled and wild-type cells were also analyzed.
PAM protein was visualized using a PHM antiserum. B, cells
were extracted in TMT, and PHM enzyme activity was measured in the
absence or presence of 0.5 µM CuSO4. Data are
the mean ± S.D. (n = 3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
failed to thrive immediately after
birth, with surviving animals exhibiting growth failure, skin laxity,
hypopigmentation, and seizures (50). The activity of cuproenzymes such
as cytochrome oxidase and tyrosinase was decreased significantly in
Atox1
/
mice. Although PAM function was not
analyzed, decreased levels of amidated neuropeptides in the
Atox1
/
mice may contribute to the
neurodegeneration and growth retardation observed.
-monooxygenase, and lysyl oxidase are catalytically inactive in the absence of copper (8, 20, 51-53). Other secretory pathway proteins like Alzheimer precursor protein (APP) and prion protein (PrP) bind copper, but the function of the bound
copper is unknown (54-56). A role for secretory pathway chaperones in delivering lumenal copper provided by Ccc2, ATPA, or ATP7B to secretory
pathway cuproproteins has not been explored. We reasoned that
expression in yeast of a mammalian secretory pathway enzyme with no
yeast homolog would allow us to address this issue. Yeast lack genes
encoding any of the members of the PHM/dopamine
-monooxygenase family of monooxygenases. To take advantage of the genetic tools available, we expressed PHMcc in S. cerevisiae.
MF
targeted PHMcc to the secretory pathway. Secretion of biologically
active epidermal growth factor (57),
-endorphin, calcitonin (58),
and somatostatin (59) by yeast also required use of the
MF signal.
In S. cerevisiae, cleavage of pre-pro-
MF occurs in two
steps. The N-terminal 20-amino acid signal sequence is removed in the
endoplasmic reticulum by the signal peptidase complex (60, 61).
After transit to the Golgi, a membrane-bound endoprotease, Kex2,
removes the 60-amino acid pro-region and separates the four copies of
MF peptide (62, 63). Although expression of PHMcc was readily
detected in yeast cell extracts, secreted PHMcc could only be detected
after lyophilization of a large volume of medium and Western blot
analysis (data not shown). A soluble insulin-containing fusion protein
encoding the
MF leader fused to a single chain insulin variant was
also secreted inefficiently (64). Intracellular retention of insulin
required endoproteolytic cleavage of the fusion protein by Kex2 in the Golgi. Expression of an
MF leader/glycosylated erythropoietin fusion
protein in yeast identified several rate-limiting steps, including
cleavage of the
MF-erythropoietin precursor protein by Kex2 and
transport of the protein through the secretory pathway (65).
MF-PHMcc is not detected by Western blot
analysis, suggesting that cleavage of the precursor is not a
rate-limiting step. The transient co-localization of Ccc2 and PHMcc in
the same subcellular compartment should facilitate delivery of copper
to PHM. The PHMcc localized to non-Ccc2-containing vesicles (Fig.
3B) may represent enzyme in the process of being secreted or degraded. The multi-copper oxidase, Fet3, and Ftr1, a
permease involved in high affinity iron uptake in yeast, assemble into
a complex in a cellular compartment early in the secretory pathway. The
complex progresses to a post-Golgi compartment (21), where Ccc2
mediates copper delivery to Fet3. Finally, the copper-loaded Fet3
protein-permease complex is delivered to the plasma membrane and
becomes competent for iron transport (41).
-melanocyte stimulating hormone were diminished in the
pituitary, and levels of amidated cholecystokinin were diminished in
the cerebral cortex (67). Peptide amidation was reduced, but not
eliminated, in the Atp7a mouse. Taken together with our data
on fibroblasts lacking both ATP7A and ATP7B, this suggests that ATP7B
may also support the delivery of copper to PAM.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. D. Yuan for the pDY207.Ccc2-HA plasmid and J. Gitlin for the 802-1 and 802-5 mouse fibroblast cell lines. We thank Dr. F. Ferraro for critically reading the manuscript and M. Delannoy for help with confocal microscopy.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK-32949 (to B. A. E.) and GM-50016 (to V. C. C.).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 in part by a research grant from Roche Diagnostics (Penzberg, Germany).
To whom correspondence should be addressed: Dept. of
Neuroscience, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3401. Tel.: 860-679-8898; Fax: 860-679-1885; E-mail: eipper@uchc.edu.
Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M211413200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PAM, peptidylglycine
-amidating monooxygenase;
PHM, peptidylglycine-
-hydroxylating
monooxygenase;
PAL, peptidyl-
-hydroxyglycine
-amidating lyase;
CHO, Chinese hamster ovary;
-MF,
-mating factor;
HA, hemagglutinin;
TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid;
MOPS, 4-morpholinepropanesulfonic acid.
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