Supplying Copper to the Cuproenzyme Peptidylglycine alpha -Amidating Monooxygenase*

Rajaâ El MeskiniDagger §, Valeria Cizewski Culotta, Richard E. MainsDagger , and Betty A. EipperDagger ||

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We explored the role of known copper transporters and chaperones in delivering copper to peptidylglycine-alpha -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-alpha -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -monooxygenase is essential for catecholamine formation (1). Another important copper-dependent enzyme, peptidylglycine alpha -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 alpha -hydroxylating monooxygenase (PHM) and peptidyl-alpha -hydroxyglycine alpha -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).

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 alpha -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 alpha -mating factor-PHMcc fusion protein expressed in wild-type yeast cells (YPH252) are shown. The yeast alpha -mating factor precursor is diagrammed with alpha -factor peptides (alpha 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Yeast Strains and Growth Conditions-- Strains of S. cerevisiae used in this study are isogenic to wild-type YPH252 (Matalpha ura3-52 lys2-801 ade2-101 trp1-Delta 1 his3-Delta 200 leu2-Delta 1) (22). In ccc2 mutant cells (ccc2Delta ::URA3, URA- by 5-fluoroorotic acid, strain MP103 (23)), the CCC2 gene was replaced with the URA3 gene. The atx1Delta ::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).

Plasmids-- We expressed PHMcc protein using a yeast alpha -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.alpha 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 alpha 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 alpha MF-PHMcc plasmid, pSM703.alpha MF-PHMcc, was constructed by inserting the BamHI-NotI alpha 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).

Mammalian Cell Culture-- The murine mottled fibroblast cell line 802-1 (Mo-/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).

Cell Extracts and Immunoblotting-- For Western blot analysis of PAM-expressing S. cerevisiae cells, the yeast strains transformed with pSM703 vector or vector encoding alpha 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).

PHM Assays-- PHM activity was measured in yeast and mammalian cell extracts as described previously (8) using alpha -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.

Immunofluorescence Microscopy-- Yeast strain YPH252 transformed with pSM703 vector (26), pSM703.alpha MF-PHMcc, pDY207 vector, pDY207.CCC2-HA (21), or both pSM703.alpha 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.

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.alpha 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.

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 alpha -mannosidase was assayed through the generation of p-nitrophenol from p-nitrophenyl-alpha -mannopyranoside, which leads to an increase in A400. The reaction mixture consisted of 0.4 mM p-nitrophenyl-alpha -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha MF signal/pro-region to the catalytic core of rat PHM (alpha MF-PHMcc) to direct the foreign monooxygenase into the yeast secretory pathway. With its pro-region, alpha MF-PHMcc should have a mass of 41 kDa. Transformation of wild-type yeast with vector encoding alpha MF-PHMcc led to expression of a 34-kDa protein recognized by antiserum to rat PHMcc (Fig. 2), indicating that the alpha 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 alpha 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-alpha 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.

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 alpha 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 alpha -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).

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.


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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.

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), alpha -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 alpha -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 alpha -mannosidase (1 unit = nmol of p-nitrophenyl-alpha -mannopyranoside cleaved per 15 min). Fractions were harvested from the top (fraction 1) to the bottom (fraction 16).

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.


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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.alpha 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.

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.


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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

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-/- 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.

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 beta -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 beta -monooxygenase family of monooxygenases. To take advantage of the genetic tools available, we expressed PHMcc in S. cerevisiae.

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 alpha MF targeted PHMcc to the secretory pathway. Secretion of biologically active epidermal growth factor (57), beta -endorphin, calcitonin (58), and somatostatin (59) by yeast also required use of the alpha MF signal. In S. cerevisiae, cleavage of pre-pro-alpha 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 alpha 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 alpha 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 alpha MF leader/glycosylated erythropoietin fusion protein in yeast identified several rate-limiting steps, including cleavage of the alpha MF-erythropoietin precursor protein by Kex2 and transport of the protein through the secretory pathway (65).

Based on both immunofluorescence and subcellular fractionation, PHMcc is partially localized at steady state to the Golgi, where Kex2 and Ccc2 are located. Intact alpha 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).

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 alpha -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.

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.

    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 alpha -amidating monooxygenase; PHM, peptidylglycine-alpha -hydroxylating monooxygenase; PAL, peptidyl-alpha -hydroxyglycine alpha -amidating lyase; CHO, Chinese hamster ovary; alpha -MF, alpha -mating factor; HA, hemagglutinin; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid.

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DISCUSSION
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