From the Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
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ABSTRACT |
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A full-length E(ecto)-ATPase (Plesner, L. (1995)
Int. Rev. Cytol. 158, 141-214) cDNA was cloned from a
human brain cDNA library; it encodes a 610-amino acid protein that
contains two putative transmembrane domains. Heterologous expression of
this protein in COS-7 cells caused a significant increase in
intracellular membrane-bound nucleoside phosphatase activity. The
activity was highest with UDP as substrate and was stimulated by
divalent cations in the following order: Ca2+ Mg2+ > Mn2+. The results of immunofluorescence
staining indicate that this protein is located in the Golgi apparatus.
UDP hydrolysis was increased in the presence of Triton X-100 or
alamethicin, an ionophore that facilitates movement of UDP across the
membrane, suggesting that the active site of this UDPase is on the
luminal side of the Golgi apparatus. This is the first identification
of a mammalian Golgi luminal UDPase gene. Computer-aided sequence
analysis of the EATPase superfamily indicates that the human UDPase
is highly similar to two hypothetical proteins of the nematode
Caenorhabditis elegans and to an unidentified 71.9-kDa
yeast protein and is less related to the previously identified yeast
Golgi GDPase.
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INTRODUCTION |
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There is evidence that a UDPase is present on the luminal side of the Golgi apparatus of mammalian cells (1, 2). Proteins and lipids are glycosylated in the lumen of the Golgi apparatus. UDP-Gal, a sugar donor in glycosylation, is synthesized in the cytosol and translocated into the Golgi lumen via specific membrane carriers (3). After transfer of sugar residues to proteins and lipids by galactosyltransferases, the resulting UDP is hydrolyzed to UMP by UDPase (4). In this way, UDP, which is highly inhibitory to galactosyltransferases (5), does not accumulate in the lumen of the Golgi apparatus. UMP then exits the Golgi lumen by exchange with cytosolic UDP-galactose (for review, see Ref. 6).
A highly specific Saccharomyces cerevisiae Golgi GDPase has been described and purified to homogeneity (7). This enzyme appears to be involved in protein and lipid mannosylation, as the GDP is generated from the mannose donor, GDP-mannose, by mannosyltransferase. Null mutants of yeast GDPase accumulate GDP in the Golgi lumen and decrease mannosylation of proteins and lipids in this compartment (8). These mutant cells also have defects in the transfer of GDP-mannose into the Golgi lumen (9) because GMP exit is required for the entry of GDP-mannose. The gene encoding the yeast GDPase, GDA1 (8), is a member of the E-ATPase protein family (10) since it is similar in amino acid sequence to potato apyrase (11) and to animal ectoapyrase CD39 (12, 13). The genes encoding mammalian Golgi UDPase and GDPase have not been identified yet. Through a homology search of the GenBankTM Data Bank, we found a newly identified human partial cDNA (Kiaa0392) that encodes a novel E-ATPase (14). In this study, we show that this new E-ATPase is a Golgi luminal UDPase.
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EXPERIMENTAL PROCEDURES |
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Materials-- Nucleoside phosphates and Trichoderma viride alamethicin were purchased from Sigma. RPMI 1640 medium, fetal bovine serum, penicillin/streptomycin, Dulbecco's modified Eagle's medium, and L-glutamine were purchased from Life Technologies, Inc. Brefeldin A (BFA)1 was purchased from Molecular Probes, Inc. (Eugene, OR). Anti-Myc monoclonal antibody (9E10) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p58 monoclonal antibody that recognizes an epitope located on the 58-kDa Golgi peripheral membrane protein (15) was purchased from Sigma.
Cloning of a Full-length UDPase cDNA--
The rapid
amplification of 5'-cDNA ends (5'-RACE) method (16) was used to
amplify the 5'-end upstream sequence of the human Kiaa0392 cDNA
(GenBankTM accession number AB002390) (14) from a human brain
Marathon-Ready cDNA library (CLONTECH, Palo
Alto, CA). The sense PCR primer AP1 (5'-CCATCCTAATACGACTCACTATAGGGC) was provided by the manufacturer for amplifying the 5'-end adapter sequence (5'-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT). The antisense primer A1 (5'-GTGGCACATGCTCTGCAGCAAAGTTCAA) was
complementary to nucleotides 308-335 of the human brain Kiaa0392
cDNA (14). The PCR product was subcloned into pGEM3zf
(Promega, Madison, WI) with a blunt end at the 5'-end and a
PstI site at the 3'-end (underlined above).
Northern Analysis--
Polyadenylated RNA (2 µg/lane) from
human cell lines was subjected to electrophoresis on a
formaldehyde-containing 1.2% agarose gel and blotted by capillary flow
onto a charge-modified nylon membrane (NEN Life Science Products). A
human multiple-tissue Northern blot (2 µg of polyadenylated RNA/lane;
CLONTECH) was used to determine tissue specificity
of UDPase mRNA. The probes were generated from UDPase cDNA by
PCR and then labeled with [-32P]dATP using a random
priming kit (Life Technologies, Inc.). Prehybridization, hybridization,
and washing of membranes were carried out following the rapid
hybridization protocol from CLONTECH. Final washes
were at 68 °C in 0.1% sodium saline citrate and 0.1% SDS. The blot was exposed to Biomax Mr film (Eastman Kodak Co.) with intensifying screens at
80 °C for 48 h. A human
-actin cDNA probe
was used as a control.
Genomic Southern Analysis-- Human genomic DNA (10 µg) was digested with either EcoRI or NdeI, electrophoresed on a 0.8% agarose gel, and transferred to a charge-modified nylon membrane. The membrane blot was then prehybridized and hybridized under high stringency conditions (42 °C, 50% formamide and 5× SSC). For the probe, a 404-base pair DNA fragment (nucleotide sequence 1024-1428) was generated by PCR. The primer sequences were 5'-ATGGGCGGCGTGTCGACT and 5'-TTAGAGGGAAGTCTGGGTCTC. This DNA fragment has neither EcoRI nor NdeI restriction sites. PCR amplification of human genomic DNA with these two primers resulted in a 400-base pair product (data not shown), indicating that there is no intron between these two primers. Final washes were at 65 °C in 0.5% sodium saline citrate and 0.1% SDS.
Cell Culture and Transfection-- COS-7 cells were grown in a humidified incubator with 5% CO2 at 37 °C in Dulbecco's modified Eagle's medium supplemented with penicillin, streptomycin, glutamine, and 10% fetal bovine serum. COS-7 cells were transfected with cDNA encoding wild-type or Myc-tagged protein by LipofectAMINE reagent (Life Technologies, Inc.). The cells were harvested for immunoblotting or for enzymatic assay 48 h after transfection.
Preparation of COS-7 Crude Membranes-- Crude membranes of the transfected COS-7 cells were prepared by the method of Coppi and Guidotti (17). Protein concentration was determined by the method of Peterson (18).
Electrophoresis and Immunoblotting--
COS-7 cells transfected
with control vector or Myc-UDPase cDNA were harvested, solubilized
in reducing sample buffer (2% SDS, 10% glycerol, 1%
-mercaptoethanol, and 65 mM Tris-HCl, pH 6.8), and
boiled for 5 min prior to loading. Samples were analyzed on 9%
SDS-polyacrylamide gel, followed by immunoblotting with anti-Myc monoclonal antibody 9E10. Immunoreactive bands were visualized with
horseradish peroxidase-conjugated goat anti-mouse antibody and the
Renaissance chemiluminescence reagent (DuPont).
Measurement of Nucleoside Phosphatase Activity-- To measure nucleoside phosphatase activity, COS-7 crude membranes (12 µg at 0.24 mg/ml) were preincubated for 5 min at 37 °C with 1 mM NaN3, 0.5 mM Na3VO4, and 5 mM CaCl2 in 45 µl of buffer A (20 mM HEPES/Tris, pH 7.4, 120 mM NaCl, 5 mM KCl, and 0.2 mM EDTA). The nucleoside phosphatase reaction was initiated by addition of 0.1 volume (5 µl) of the same buffer containing nucleoside phosphate substrates, pH 7.0, to give a final concentration of 0.1-1 mM. Nucleoside phosphatase activities were linear for at least 30 min (see Fig. 3C). The divalent cation-stimulated nucleoside phosphatase activity was determined by measuring the inorganic phosphate released as described by Lanzetta et al. (19) or by Ames (20) and by subtracting values obtained with 0.2 mM EDTA alone from those obtained with various concentrations of divalent cation (e.g. CaCl2, MgCl2, or MnCl2) plus chelator. To examine the membrane orientation of the UDPase activity, alamethicin (final concentration of 0.2 mg/ml) or Triton X-100 (final concentration of 0.1% (v/v)) was added to facilitate transmembrane diffusion of UDP.
Intact or lysed COS-7 cells were used to determine whether the active site of this protein is located intracellularly or extracellularly. Transfected COS-7 cells were incubated with 10 mM EDTA to detach them from culture dishes and washed twice with buffer A. The intact cells were resuspended in buffer A with 1 mM NaN3 and 0.5 mM Na3VO4 to a final density of 106 cells/ml. Triton X-100 was added to a final concentration of 0.1% together with protease inhibitors including 1 mM phenylmethylsulfonyl fluoride and 2 µg/ml each aprotinin, chymostatin, pepstatin A, and leupeptin. The cells were allowed to lyse for 30 min on ice. In a separate experiment, cells were disrupted by homogenization with a Dounce homogenizer in the same buffer without Triton X-100. Both intact cells and cell lysates were then preincubated for 5 min at 37 °C in 180 µl of buffer A with 1 mM NaN3 and 0.5 mM Na3VO4, with or without 5 mM CaCl2. The nucleoside phosphatase reaction was initiated by addition of 0.1 volume (20 µl) of the same buffer containing UDP, pH 7.0, to give a final concentration of 1 mM. At the end of the incubation, cells that had not been homogenized or treated with Triton X-100 were still intact (>90%) as demonstrated by trypan blue exclusion. The calcium-activated UDPase activities were determined by measuring the inorganic phosphate released as described by Ames (20) and by subtracting values obtained with 1 mM EDTA alone from those obtained with 5 mM CaCl2.Immunofluorescence Staining-- COS-7 cells grown on polylysine-coated culture chamber slides (Nunc Inc., Naperville, IL) were transfected at ~40-60% confluence with Myc-UDPase cDNA using the LipofectAMINE method. After exposure of cells to BFA (20 µg/ml) for 30 min at 37 °C, cells were washed twice with phosphate-buffered saline, pH 7.4, containing 1 mM MgCl2 and 0.1 mM CaCl2. The immunofluorescence staining was done by modifying the method described by Yoon and Guidotti (21). Cells were fixed with 2% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with primary antibody. For staining Myc-UDPase, monoclonal antibody 9E10 (1 µg/ml) was used to recognize the Myc epitope. Monoclonal antibodies against the 58-kDa Golgi peripheral membrane protein (1:50 dilution) were used as markers of Golgi complexes (15, 22). Fluorescein isothiocyanate-labeled secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used at a dilution of 1:1000. Immunofluorescence was viewed with a Zeiss Axioskop microscope.
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RESULTS |
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DNA Cloning-- We cloned the full-length coding sequence of a novel cDNA (Fig. 1) from a human adult brain cDNA library by the 5'-RACE method (16). The protein product of this cDNA is a new E-ATPase protein (14) since it contains apyrase conserved regions (ACR1-4) (Fig. 1) that are present in all known E-ATPase proteins (11). The initiation codon (ATG) at nucleotide sequence 199-201 is the most likely translational initiation site because there are two stop codons in the upstream sequence of the same reading frame (Fig. 1). The deduced polypeptide sequence from this initiation codon contains 610 amino acids. Hydrophobicity analysis using the Kyte and Doolittle algorithm (23) predicts two hydrophobic stretches in the polypeptide (amino acid residues 33-55 and 551-573), both sufficiently long to transverse the membrane (Fig. 1). There are two potential N-glycosylation sites between the two putative transmembrane domains (indicated in boldface and underlined in Fig. 1), suggesting that this protein may be a membrane glycoprotein.
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Northern Blot Analysis-- To determine whether this new E-ATPase is another ectoapyrase or ecto-ATPase, we examined its mRNA expression pattern in cells with or without ectoapyrase activity. The mRNA is expressed in cells with ectoapyrase activity (human B lymphoblast LG2) and without ectoapyrase activity (human T lymphoma Jurkat cells) (13) as determined by Northern blot analysis (Fig. 2A). We were able to detect at least three mRNA transcripts (3.0, 3.2, and 7.5 kilobases in length) reacting with the same probe (Fig. 2A). Reverse transcription-PCR analysis of human breast cancer MCF-7 cell lines (with ectoapyrase activity) and their multidrug-resistant derivatives, MCF-7ADR cells (ectoapyrasedeficient), indicated that both cells express this mRNA (data not shown). The presence of mRNA in these four cell lines suggests that this new E-ATPase is probably not a new ectoapyrase or ecto-ATPase.
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Genomic Southern Analysis-- To determine the copy number of this new E-ATPase gene, human genomic DNA was digested with EcoRI (Fig. 2B, lane 1) or NdeI (lane 2) and then analyzed by Southern blotting with a cDNA probe (nucleotide sequence 1024-1428) under high stringency hybridization and wash conditions. The sequence of this cDNA probe has neither EcoRI nor NdeI sites and does not contain any intron within the fragment at the level of the genomic DNA (see "Experimental Procedures"). The presence of at least two bands in both digests (Fig. 2B) indicates that there might be two or more copies of this new E-ATPase gene in the human genome.
Functional Analysis-- To study the function of this new E-ATPase, a control expression vector or the cDNA encoding a Myc-tagged protein (Myc-UDPase) was transfected into COS-7 cells. Transfected COS-7 cells expressed an ~69-kDa protein recognized with anti-Myc monoclonal antibody 9E10 (Fig. 3A). No protein was detected in the immunoblot of COS-7 cells transfected with control vector.
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Cellular Localization-- To study the cellular localization of this human UDPase, we compared the enzymatic activity of intact and disrupted cells to determine whether it is located intracellularly or extracellularly. The rational of the experiment is that if the enzyme is located inside the plasma membranes, the UDPase activity will increase after the cells are disrupted. On the other hand, the UDPase activity will not change if the enzyme is an ectoprotein. In fact, the UDPase activity of Myc-UDPase cDNA-transfected COS-7 cells increased significantly after the cells were treated with 0.1% Triton X-100 (~5-fold) (Fig. 5A) or were homogenized with a Dounce homogenizer (~4-fold) (data not shown). At the end of the incubation, cells not treated with detergent were still intact (>90%), as demonstrated by trypan blue exclusion. These results suggest that human UDPase is located inside the cell. The UDPase activity of COS-7 cells transfected with GW1 control vector increased after these cells were solubilized with 0.1% Triton X-100. Since UDPase is likely present in all mammalian cells, it is not surprising to find that COS-7 cells have endogenous UDPase activities. Interestingly, the UDPase activity of Triton-treated mock-transfected cells was even higher that of intact cells transfected with Myc-UDPase cDNA (Fig. 5A). These results further support the conclusion that UDPase is an intracellular enzyme.
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Relatedness within the E-ATPase Protein Family-- Sequence alignment of the E-ATPase family members suggests the existence of four major subgroups in the E-ATPase protein superfamily (Fig. 7). Human Golgi UDPase is highly related to two nematode hypothetical proteins (C33H5.14 and R07E4.4) and to a functionally unidentified 71.9-kDa yeast protein, suggesting that the enzymes of Caenorhabditis elegans and yeast might be functionally similar to human Golgi UDPase. Yeast GDPase (8), pea NTPase (26), and potato apyrase (11) are less related to human UDPase, as are vertebrate ectoapyrases (27) and ecto-ATPases (28). Finally, the NTPases of the protozoan parasite Toxoplasma gondii (29) are more distant from all other E-ATPases in the dendrogram, suggesting an ancient and independent line of evolution of the T. gondii NTPases.
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DISCUSSION |
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We have cloned a new human E-ATPase gene, as the protein sequence
contains the four apyrase conserved regions (ACR1-4) (11). Expression
of the protein in COS-7 cells resulted in an increase in
Ca2+-dependent nucleoside phosphatase activity.
This activity was highest with UDP as substrate and was simulated by
divalent cations in the following order: Ca2+ Mg2+ > Mn2+. By immunofluorescence staining
and enhancement of UDP hydrolysis in the presence of alamethicin, the
protein was determined to be a Golgi luminal UDPase. Since rat liver
UDPase (1) and yeast GDPase (8) are involved in protein and lipid
glycosylation in the Golgi apparatus, it is likely that the human
UDPase might have the same function.
UDPase mRNA is present in all human tissues and all four cell lines examined here, suggesting that this UDPase is widely expressed in many human cells. Recently, the gene encoding the S. cerevisiae GDP-mannose transporter (Vrg4p) was identified; the lethal phenotype of a null vrg4 mutant suggests that glycosylation of protein and lipid in the Golgi apparatus is essential for viability (30). Therefore, Golgi UDPase might be essential for most, if not all, cells. It will be of interest to know whether there are genetic diseases affecting nucleoside phosphate transport or metabolism in the lumen of the Golgi apparatus, in a manner analogous to diseases involving cysteine and sialic acid transport into lysosomes.
Genomic Southern analysis under high stringency hybridization conditions indicates that there might be at least two copies of the UDPase gene in the human genome. In fact, the presence of another UDPase activity in the rough endoplasmic reticulum (RER) has been described in rat liver cells. Like the Golgi UDPase, the RER UDPase also preferentially hydrolyzes UDP and GDP. However, these two enzymes are slightly different in at least two respects. First, the RER UDPase (which actually should be named RER GDPase) hydrolyzes nucleotides in the order GDP > UDP. Second, the activity of Golgi UDPase is preferentially activated by Ca2+, whereas the activity of RER UDPase is stimulated by both Ca2+ and Mg2+ (1). Further study is required to know whether the second gene is indeed a RER UDPase. Interestingly, C. elegans also has two copies of a UDPase-like gene (Figs. 7 and 8). In the genome of S. cerevisiae, there are only two copies of an E-ATPase gene. The hypothetical 71.9-kDa protein of chromosome V (Figs. 7 and 8) also shares high similarity in amino acid sequence with human UDPase. It is likely that the two C. elegans proteins and this yeast protein may have functions similar to that of human UDPase. The second E-ATPase gene in S. cerevisiae encodes the Golgi GDPase. Although yeast GDPase is not essential for cell viability and growth, null mutants of GDPase have a partial block in O- and N-glycosylation of secreted proteins as well as a dramatic decrease in mannosylinositol phosphorylceramide (8) and a decrease in transport of GDPase-mannose into the Golgi lumen (9). Since glycosylation of proteins and lipids in the Golgi apparatus is essential for viability (30), we wonder whether the activity of the hypothetical 71.9-kDa protein may account for the viability of gda1 null mutants. It will be interesting to know whether the yeast GDPase and the 71.9-kDa protein have different biological functions.
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Members of the E-ATPase protein family all share high sequence
similarity in four apyrase conserved regions (ACR1-4) (11). ACR1 and
ACR4 sequences are similar to actin-hsp70-hexokinase - and
-phosphate-binding motifs, indicating a possible role in nucleotide
binding; however, the structures and functions of these ACRs are not
yet known. To understand the catalytic mechanism of E-ATPase, it is
important to point out the differences between various E-ATPase
enzymes. Ectoapyrase (CD39) is an ectoenzyme that hydrolyzes nucleoside
tri- or diphosphates (13, 31, 32); ecto-ATPase (CD39L1) is also an
ectoenzyme, but it preferentially hydrolyzes adenosine triphosphate
(28, 33, 34). Ca2+ and Mg2+ have almost the
same stimulatory effect on ectoapyrase (13). T-tubule ecto-ATPase is
more active with Mg2+ than Ca2+ (35). The
activity of human Golgi luminal UDPase is highest with UDP and GDP as
substrates and is stimulated by divalent cations in the following
order: Ca2+
Mg2+ > Mn2+
(Fig. 4). Interestingly, amino acid sequence alignments between UDPase
and other E-ATPases (Fig. 8) revealed that some regions of these
E-ATPases may be important in determining substrate specificity. First,
the sequence of the ACR1 region of UDPase/GDPase,
DXGS(S/T)G(S/T)RXXX, is
different from that of ecto-ATPase/ectoapyrase,
DXGSSHT(N/S)XXX, in two
amino acid residues (underlined). Since ACR1 is similar to the
-phosphate-binding motif of actin-hsp70-hexokinase, these two amino
acid residues may be responsible for distinguishing nucleoside
diphosphates from nucleoside triphosphatases. Second, at least two
other regions, X1 and X2, have significant differences between
UDPase/GDPase and ecto-ATPase/ectoapyrase; however, the functions of
these regions are still unknown.
In summary, we have cloned a new human E-ATPase gene and shown that it encodes a Golgi luminal UDPase. This is the first identification of a mammalian Golgi UDPase gene. The wide distribution of UDPase mRNA in human tissues and cells makes it evident that metabolism of nucleoside phosphates in the Golgi lumen is essential for the functions of the Golgi apparatus, e.g. protein and lipid glycosylation. Finally, we have shown that human UDPase is highly related to one yeast and two worm hypothetical proteins.
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ACKNOWLEDGEMENTS |
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We are very grateful to Yvonne Ou for help in subcloning and sequencing the full-length UDPase cDNA and Dr. Yi-Ping Hsueh for excellent assistance with the immunofluorescence microscopy.
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FOOTNOTES |
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* This work was supported in part by Grant HL08893 from the National Institutes of Health.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF016032.
Supported in part by a Maria Moors Cabot fellowship.
§ To whom correspondence should be addressed: Dept. of Molecular and Cellular Biology, Harvard University, 7 Divinity Ave., Cambridge, MA 02138. Tel.: 617-495-2301; Fax: 617-495-8308; E-mail: guidotti{at}fas.harvard.edu.
1 The abbreviations used are: BFA, brefeldin A; 5'-RACE, rapid amplification of 5'-cDNA ends; PCR, polymerase chain reaction; ACR, apyrase conserved region; RER, rough endoplasmic reticulum; EATPase, ecto-ATPase.
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REFERENCES |
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