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(The FASEB Journal. 2000;14:345-354.)
© 2000 FASEB

Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells

STEVE BESSET, JEAN-BAPTISTE VINCOURT, FRANÇOIS AMALRIC and JEAN-PHILIPPE GIRARD1

Laboratoire de Biologie Vasculaire, Institut de Pharmacologie et de Biologie Structurale du CNRS, 31077 Toulouse Cedex 4, France

1Correspondence: Institut de Pharmacologie et de Biologie Structurale du CNRS, 205 route de Narbonne, 31077 Toulouse Cedex 4, France. E-mail: girard{at}ipbs.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sulfation is a major modification of many molecules in eukaryotes that is dependent on the enzymatic synthesis of an activated sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). While sulfate activation has long been assumed to occur in the cytosol, we show in this study that human PAPS synthetase 1 (PAPSS1), a bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase enzyme sufficient for PAPS synthesis, accumulates in the nucleus of mammalian cells. Nuclear targeting of the enzyme is mediated by its APS kinase domain and requires a catalytically dispensable 21 amino acid sequence at the amino terminus. Human PAPSS1 and Drosophila melanogaster PAPSS localize to the nucleus in yeast and relieve the methionine auxotrophy of ATP sulfurylase- or APS kinase-deficient strains, suggesting that PAPSS1 is fully functional in vivo when targeted to the nucleus. A second PAPS synthetase gene, designated PAPSS2, has recently been described, mutations of which are responsible for abnormal skeletal development in human spondyloepimetaphyseal dysplasia and murine brachymorphism. We found that PAPSS2, which localizes to the cytoplasm when ectopically expressed in mammalian cells, is relocated to the nucleus when coexpressed with PAPSS1. Taken together, these results indicate that a sulfation pathway might exist in the nucleus of eukaryotic cells.—Besset, S., Vincourt, J.-B., Amalric, F., Girard, J.-P. Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells.


Key Words: sulfation • sulfate activation • PAPS synthesis • subcellular localization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SULFATION IS AN essential modification of proteins, carbohydrates, and lipids that controls many biological processes (1) . For instance, the symbiosis between the bacteria Rhizobium meliloti and legumes is determined by the sulfation of lipo-oligosaccharide signals (2) . In vertebrates, sulfation of the chemokine receptor CCR5, the principal HIV-1 coreceptor, facilitates HIV-1 entry into target cells (3) whereas sulfation of mucin-like glycoproteins regulates their binding to selectins, cell adhesion molecules that mediate the initial interaction of leukocytes with endothelium (4 5 6 7 8) . Sulfation also controls the interaction of proteoglycans with growth factors (9) and is therefore necessary for proper cartilage development (10) .

In animals, sulfation is catalyzed by a myriad of sulfotransferases that transfer sulfate onto a wide variety of biomolecules (1) . Cytosolic sulfotransferases (11 , 12) are responsible for sulfation of estrogen and steroid hormones, neurotransmitters, drugs, and diverse xenobiotics (phenol derivatives, hydroxylamines, hydroxamates). Many other sulfotransferases reside in the Golgi apparatus, where they catalyze the addition of sulfuryl groups on tyrosine residues (13 , 14) and sugars (15 , 16) . Sulfation of the corresponding glycoproteins and glycolipids is critical to their functions in various biological processes such as brain development (17) , platelet aggregation (18) , blood clotting (reviewed in ref 19 ), and leukocyte trafficking (reviewed in refs 20 , 21 ).

A prerequisite for all sulfation reactions is the conversion of the sulfate ion (SO42-) into a high-energy donor, an activation process catalyzed by two consecutive enzymatic activities (22) . ATP sulfurylase first combines an ATP molecule with SO42-, leading to its adenylylated form APS, which is subsequently phosphorylated by APS kinase to yield PAPS. PAPS then acts as an activated substrate for sulfate transfer by sulfotransferases (1) , and in lower organisms for sulfate reduction into sulfite by PAPS reductase (19 , 23) . In bacteria, yeasts, algae, fungi, protozoa, and plants, ATP sulfurylase and APS kinase activities are generally encoded by separate genes (19 , 23) , whereas in humans (24) , mice (25) , the marine worm Urechis caupo (26) , and the fruit fly Drosophila melanogaster (27) both activities are present on a single polypeptide, named PAPS synthetase 1 (PAPSS1). A cDNA encoding a second PAPS synthetase (PAPSS2) has recently been cloned in mice (28) and humans (29 , 30) , indicating that PAPS synthetases constitute a family of enzymes.

The critical role of sulfotransferases in the regulation of various biological processes is greatly evidenced by their high specificity toward a given sulfate acceptor. However, their strict dependence on PAPS availability as a sulfate donor (31) implies a key regulatory role for PAPS synthesis in the biosynthesis of sulfated compounds. The identification of mutations in PAPS synthetase genes as the underlying cause of two orthologous inherited chondrodysplasias has recently highlighted this critical role of PAPS synthesis in the regulation of sulfation in vivo (28 , 29) . Brachymorphic mice harbor a single point mutation in the PAPSS2 nucleotide sequence, resulting in abnormal hepatic detoxification and bleeding times as well as dramatically reduced postnatal growth due to profound undersulfation of cartilage proteoglycans (28) . Strikingly, similar skeletal abnormalities were observed in human spondyloepimetaphyseal dysplasia, a dwarfing condition caused by a stop codon in the human PAPSS2 gene (29) . Evidence for a key role of PAPS synthesis in the control of sulfation has also been provided in many other studies using chlorate, a selective inhibitor of the ATP sulfurylase activity of PAPS synthetase (24 , 32) . For instance, treatment with chlorate has been shown to abrogate tyrosine sulfation of the leukocyte mucin-type glycoprotein PSGL-1 and its recognition by P-selectin, thus inhibiting leukocyte-leukocyte and leukocyte–endothelium interactions (6 7 8) .

PAPS synthesis has long been assumed to take place exclusively in the cytosol (for a review, see ref 31 ). Indeed, cytosol and Golgi apparatus are the only sites of PAPS utilization by known sulfotransferases (11 , 12 , 16) . The identification of a cytosol-to-Golgi PAPS translocase further supports this hypothesis (33 , 34) , but no experiment has directly demonstrated that PAPS synthetases are cytosolic enzymes. In this study, we report that human PAPSS1 is a nuclear protein. PAPSS1 is targeted to the nucleus, via its APS kinase domain, in various mammalian cell lines as well as in yeast, where it functionally complements ATP-sulfurylase- and APS kinase-deficient strains. We show that PAPSS2, which accumulates in the cytoplasm when expressed ectopically in mammalian cells, is dramatically relocalized to the nucleus when coexpressed with PAPSS1. Altogether, these results suggest that PAPS synthetases might catalyze sulfate activation in the nucleus of eukaryotic cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian cell culture, transfection, and assay of PAPS synthesis
CHO F1 cells (kindly provided by R. P. McEver, Oklahoma City) and LS180 human colorectal carcinoma cells (ATCC CL-187) were cultured in minimal Eagle’s medium (Life Technologies, Inc.-BRL, Grand Island, N.Y.) supplemented with 1% glutamine. COS-7 cells (ATCC CRL-1651) and ECV304, spontaneously transformed human umbilical vein endothelial cells (ATCC CRL-1998), were grown in Dulbecco’s modified Eagle’s medium with Glutamax (Life Technologies). Cells were transfected using Lipofectamine (Life Technologies) and analyzed 2–4 days after transfection. For stable expression of hPAPSS1-HA, CHO F1 cells cotransfected with pEF-hPAPSS1-HA and pREP-Hygro were selected in medium containing 200 µg/ml hygromycin (Roche Diagnostics, Nutley, N.J.). CHO F1 cells were also stably transfected with empty pEF and pREP-Hygro vectors and the hygromycin-resistant cells obtained served as mock-transfected cells. Metabolic labeling of stable transfectants and quantification of PAPS production by high-voltage paper electrophoresis were performed as described previously (24) .

Vectors for expression of epitope-tagged proteins in mammalian cells
The pBSK-hPAPSS1 and pcDNA3-hPAPSS1 constructs, consisting of the entire hPAPSS1 cDNA (2511 pb) inserted in the pBluescript II SK(-) vector (Stratagene, San Diego, Calif.) or in the pcDNA3 expression vector (Invitrogen, San Diego, Calif.), have been described previously (24) . To obtain plasmid pBSK-hPAPSS1-HA, a 100 bp HindIII fragment containing the triple HA epitope tag (35) was amplified by polymerase chain reaction (PCR) and inserted in plasmid pBSK-hPAPSS1. To obtain plasmid pBSK-hPAPSS1-cMyc, 36 bp complementary oligonucleotides encoding the cMyc epitope sequence (EQKLISEEDL) were cloned into the HindIII site of plasmid pBSK-hPAPSS1. The XhoI/XhoI fragments excised from pBSK-hPAPSS1-HA and pBSK-hPAPSS1-cMyc were then used to replace the 0.6 kb XhoI/XhoI fragment from pcDNA3-hPAPSS1, resulting in constructs pcDNA3-hPAPSS1-HA and pcDNA3-hPAPSS1-cMyc, respectively. Plasmid pEGFPN3-hPAPSS1 was obtained by inserting the entire hPAPSS1 coding sequence (accession no. Y10387, nt 37–1907) between EcoRI and BamHI sites of the pEGFPN3 vector (Clontech, Palo Alto, Calif.) using PCR with primers 5'EcohAPSK and 3'BamhPAPSS. The pcDNA3-HA vector was obtained by insertion of the triple HA sequence (35) between the NotI and ApaI sites of pcDNA3. To generate pcDNA3-hAPSkinase-HA vector, the sequence encoding the APS kinase domain of hPAPSS1 (accession no. Y10387, nt 37–724) was amplified by PCR from plasmid pcDNA3-hPAPSS1 (using primers 5'EcohAPSK and 3'NothAPSK) and inserted between the EcoRI and NotI sites of the pcDNA3-HA vector. Green fluorescent protein (GFP) -fused full-size or deleted hPAPSS1 constructs were obtained by inserting the corresponding sequences, amplified by PCR from pcDNA3-hPAPSS1 plasmid with primers 5'EcohAPSK, 5'EcohATPS, 5'Ecoh2MSK, 3'BamhPAPSS or 3'BamhAPSK, between the EcoRI and BamHI sites of the pEGFPN3 vector. Plasmid pEGFPN3-DmPAPSS was similarly obtained by cloning into pEGFPN3, the Drosophila melanogaster PAPSS coding sequence (accession no. Y12861, nt 239-2129), amplified by PCR from plasmid pcDNA3-DmPAPSS (kindly provided by E. Käs, Toulouse, France) using primers 5'EcoDmPAPSS and 3'BamDmPAPSS. For construction of the pEGFPN3-mPAPSS2 plasmid, the murine PAPSS2 coding sequence (accession no. AF052453, nt 131-1996) was amplified by PCR from total mouse brain cDNAs (kindly provided by M. Nicoloso, Toulouse, France), using primers 5'KpnmPAPSS2 and 3'ApamPAPSS2, and inserted between the KpnI and ApaI sites of pEGFPN3.

All PCR reactions were performed with an Advantage cDNA PCR kit (Clontech), using 1 ng of plasmid template and 10 pmol of each primer, in a PTC-150 MiniCycler (MJ Research Inc.), with 25 cycles consisting each of 30 s at 94°C and 1 to 3 min at 68°C. PCR fragments were purified using an Advantage PCR-Pure kit (Clontech), digested with restriction enzymes, and cloned into the corresponding expression vectors. All constructs were sequenced over the entire coding sequence. We found that the mPAPSS2 cDNA was identical to that described by ul Haque et al. (29) , but was 15 bp shorter than that described by Kurima et al. (28) , with an in-frame deletion of residues 290–294, suggesting alternative splicing or polymorphism of the murine PAPSS2 gene.

Oligonucleotides used for the constructions are listed below with cloning sites underlined.

5'EcohAPSK: 5'-GCGGAATTCCACCATGGAGATCCCCGGGAGCTTGTGC-3'

5'EcohATPS: 5'-GCGGAATTCCACCATGGAACGGGATATTGTACCTGTGGATGC-3'

5'Ecoh2MSK: 5'-GCGGAATTCCACCATGCAGAGAGCAACCAATGTCACCTACC-3'

5'KpnmPAPSS2: 5'-GCGGGTACCAATTATCATGTCTGCAAATTCCAAAATGAACC-3'

5'EcoDmPAPSS: 5'-GCGGAATTCCACCATGCCGAATCCGCCAATTGGATTTTATCC-3'

3'NothAPSK: 5'-CGCGCGGCCGCATCCACAGGTACAATATCCCGTTCC-3'

3'BamhPAPSS: 5'-CGCGGATCCGGCTTTCTCCAAGGATTTGTAGTATTCTGT-3'

3'BamhAPSK: 5'-CGCGGATCCATCCACAGGTACAATATCCCGTTCCTG-3'

3'ApamPAPSS2: 5'-CGCGGGCCCGCATTGGTCTTCTCCAGAGACCTGTAGTAATC-3'

3'BamDmPAPSS: 5'-CGCGGATCCCGACTGCGGCAGGTTCTGGTAGTAGG-3'

Generation of polyclonal antibodies against recombinant human PAPSS1 and Western blot analysis
6 x His-tagged hPAPSS1 protein was expressed and purified using a QIAexpressionist kit (Qiagen, Chatsworth, Calif.) according to the manufacturer’s instructions. Briefly, the entire hPAPSS1 coding sequence was inserted between the BamHI and HindIII sites of pQE30 vector (Qiagen), and the resulting pQE30-HishPAPSS1 plasmid was transformed in Escherichia coli strain M15 (Qiagen). His-tagged protein was purified from inclusion bodies on a Ni-agarose column (Qiagen) under denaturing conditions and the eluate was used as an immunogen to produce polyclonal antiserum in rabbits (Eurogentec).

For Western blot analysis, 105 transfected CHO or COS-7 cells were trypsinized, washed with phosphate-buffered saline (PBS), and incubated for 30 min at 37°C in PBS containing 10 µg/ml DNaseI (Sigma). ECV human endothelial cell extracts were kindly provided by F. Soulet. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%) and transferred onto a PVDF Immobilon-P membrane (Millipore). After incubation for 1 h at room temperature in blocking buffer (Tris-buffered saline containing 0.5% Tween 20 and 5% non-fat milk), membranes were incubated 2 h at room temperature with rabbit polyclonal antibodies anti-GFP (Clontech, 5 µg/ml) or anti-hPAPSS1 (crude antiserum, 1/50000) or overnight at 4°C with mouse anti-HA mAb 12CA5 (Roche Diagnostics, 5 µg/ml), all diluted in blocking buffer. After three washing steps in TBST buffer (Tris-buffered saline with 0.5% Tween 20), membranes were incubated for 1 h with the secondary HRP-conjugated goat anti-mouse immunoglobulin G (IgG) or anti-rabbit IgG antibodies (Promega, 1/10000 in blocking buffer). After extensive washing in TBST, proteins were revealed using an ECL+ detection kit (Amersham Pharmacia Biotech, Arlington Heights, Ill.).

Immunocytochemistry and fluorescence microscopy on mammalian cells
Nontransfected cells and cells transfected with HA- or cMyc-tagged constructs were allowed to grow for 36 h on coverslips. Cells were washed twice with PBS, fixed for 15 min at room temperature in PBS containing 3.7% paraformaldehyde, and washed again three times with PBS prior to neutralization with 50 mM NH4Cl in PBS for 5 min at room temperature. After three more PBS washes, cells were permeabilized 5 min at room temperature in PBS containing 0.1% Triton-X100 and washed twice with PBS. Permeabilized cells were then incubated 2 h at room temperature with the anti-hPAPSS1 crude polyclonal serum diluted 1/50 in PBSA (PBS with 1% bovine serum albumin) or with mouse monoclonal antibodies directed against either the HA epitope (mAb 12CA5) or the cMyc epitope (anti-cMyc, Invitrogen) diluted in PBSA to a final concentration of 2 µg/ml and 7 µg/ml, respectively. Cells were then washed three times for 5 min at room temperature in PBSA and incubated for 1 h with FITC-labeled anti-mouse IgG (Zymed Laboratories Inc.), anti-rabbit IgG (Immunotech), or TRITC-labeled anti-mouse IgG or anti-rabbit IgG (Amersham Pharmacia Biotech) secondary antibodies diluted 1/40 in PBSA. After extensive washing in PBS, samples were air dried and mounted in Mowiol. Cells cotransfected with GFP-tagged and HA-tagged constructs were analyzed as described above. Cells transfected with GFP-tagged constructs alone were allowed to grow for 36 h on coverslips. The coverslips were then washed with PBS and mounted in PBS cell side down on a slide. Fluorescence of live GFP-expressing cells or fixed immunostained cells was viewed with a Zeiss confocal laser scanning microscope using a 63x oil immersion objective.

Yeast strains, expression vectors, complementation assays, and fluorescence microscopy
The Saccharomyces cerevisiae strains S5 (Mat{alpha}, met3, ura3–52, his3, leu 2–1) and X6 (Mat{alpha}, met14, ura3–52, his3, leu2–1, ade1, trp1, lys2), which carry the met3 and met14 mutations in the genes encoding ATP sulfurylase and APS kinase, respectively (kindly provided by T. Leustek, New Brunswick, N.J.), were used as host strains for all experiments. To generate the {lambda}YES-DmPAPSS-GFP expression vector, a fragment encoding DmPAPSS-GFP was amplified by PCR from plasmid pEGPFN3-DmPAPSS (see above) with primers 5'EcoDmPAPSS and 3'XbaGFP and used to replace the untagged Urechis caupo PAPSS sequence of plasmid pCN31 (a gift from T. Leustek) between the EcoRI and XbaI sites of the {lambda}YES vector. Full-length hPAPSS1 amplified by PCR with primers 5'EcohAPSK and 3'BamhPAPSS, as well as sequences encoding S. cerevisiae APS kinase (met14, accession no S55315, nt 429-1029) and ATP sulfurylase (met3, accession no X59735, nt 2744–4309), amplified from S. cerevisiae genomic DNA with primers 5'EcoScAPSK or 5'EcoScATPS and 3'BamScAPSK or 3'BamScATPS, were then inserted in place of DmPAPSS between EcoRI and BamHI sites of plasmid {lambda}YES-DmPAPSS-GFP to generate expression vectors {lambda}YES-hPAPSS1-GFP, {lambda}YES-ScAPSkin-GFP, and {lambda}YES-ScATPsul-GFP. PCR reactions were performed as described above, using oligonucleotides listed below.

5'EcoScAPSK: 5'-GCGGAATTCCACCATGGCTACTAATATTACTTGGCATCCTAATC-3

5'EcoScATPS: 5'-GCGGAATTCCACCATGCCTGCTCCTCACGGTGGTATTC-3'

3'XbaGFP: 5'-GCGTCTAGATTACTTGTACAGCTCGTCCATGCCGAG-3'

3'BamScAPSK: 5'-CGCGGATCCCTTACGGATGATTTTTTTTTCACTGATTAAG-3'

3'BamScATPS: 5'-CGCGGATCCGTACTTTTGAGATGGGAGCATTTTATGACG-3'

After transformation using the Li-acetate/polyethylene glycol method, yeast colonies were streaked onto minimal YNB media (Bio 101) plates containing either glucose or galactose as the sole carbon source, with or without methionine, to test for successful methionine auxotrophy complementation. For fluorescence microscopy analysis, single colonies from the different transformants were picked from the YNB-Gal+ plates used for complementation studies (see above) and grown in 10 ml liquid YNB-Gal+ medium to an A600 nm of 0.4–0.8. The 500 µl cells were then washed with PBS, fixed for 20 min at room temperature in PBS containing 3.7% paraformaldehyde, washed with culture medium, incubated for 20 min at 30°C in medium containing 0.5 µg/ml DAPI, and washed again with PBS. Cells were then visualized with a Zeiss fluorescence microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Human PAPSS1 localizes to the nucleus
Our recent cloning of human PAPSS1 (24) allowed us to investigate its subcellular localization. Our aim was to determine whether the PAPSS1 enzyme, believed to exhibit an exclusive cytosolic localization, could be associated with the Golgi apparatus or other intracellular organelles. We first designed constructs in which the human PAPSS1 (hPAPSS1) coding sequence was placed in pcDNA3 expression vector under control of CMV promoter and fused to sequences encoding either the triple HA (pcDNA3-hPAPSS1-HA) or the cMyc epitope tag (pcDNA3-hPAPSS1-cMyc). Unexpectedly, indirect immunofluorescence analysis after transient transfection of both constructs in CHO cells revealed a preferential nuclear localization of epitope-tagged hPAPSS1 (Fig. 1 Accumulation of hPAPSS1 in the nucleus did not appear to depend on the cellular context, since similar nuclear localization was observed after transfection of the hPAPSS1-HA and hPAPSS1-cMyc constructs in COS-7 and ECV cells (Fig. 1D-F ) as well as in LS180, a human colorectal carcinoma cell line that constitutively express sulfated mucin-type L-selectin ligands (data not shown). Confocal sectioning revealed that in all cell lines, the epitope-tagged hPAPSS1 exhibited an intranuclear localization. We also investigated the subcellular localization of hPAPSS1 fused to a human codon-optimized version of the green fluorescent protein by direct analysis of fluorescence in living cells. The hPAPSS1-GFP fusion protein localized to the nucleus of transiently transfected CHO cells, whereas GFP alone was localized throughout the cell (see below, Fig. 4 ). Together, these results indicate that ectopically expressed hPAPSS1 exhibits a preferential nuclear localization in mammalian cells that is independent of the nature of the epitope tag or the cell line used. We then wanted to determine whether the epitope-tagged hPAPSS1, which accumulates in the nucleus of transfected cells, was functionally active. For this purpose, we analyzed PAPS synthetase activity in stable CHO cell transfectants expressing HA-tagged hPAPSS1 and found a 12-fold increase in PAPS synthesis over mock-transfected cells (Fig. 1G ). This latter result indicates that the HA-tagged hPAPSS1 enzyme that localizes to the nucleus is actively involved in PAPS synthesis.



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  		Figure 1. Nuclear localization and functional activity of epitope-tagged human PAPSS1 ectopically expressed in various cell types. A–F) Confocal microscopy analysis. Confocal laser scanning (A, C–F) and Nomarsky interference microscopy (B) of CHO cells (A–C), human ECV endothelial cells (D), and COS-7 cells (E, F) transfected with expression vectors for HA-tagged (A, B, E) or cMyc-tagged (C, D, F) hPAPSS1. Permeabilized cells were analyzed by indirect immunofluorescence using monoclonal anti-HA (A, E) or anti-cMyc (C, D, F) antibodies. Bar = 10 µm. G) Assay of PAPS synthesis and Western blot analysis of hPAPSS1-HA expression in transfected CHO cells. Stably mock-transfected (lane 1) or hPAPSS1-HA-transfected (lane 2) CHO cells were sulfate-depleted for 60 min and then incubated for 30 min in medium containing ATP and Na235SO4. Cell extracts were prepared and analyzed by high-voltage paper electrophoresis and autoradiography (left panel). This system allows separation of PAPS from sulfate and higher molecular weight sulfated compounds that remain at the origin. Total extracts of the same stably mock-transfected (lane 1) or hPAPSS1-HA-transfected (lane 2) CHO cells were analyzed by immunoblotting with monoclonal anti-HA antibody (right panel).



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  		Figure 4. The APS kinase domain targets human PAPSS1 to the nucleus. A) Schematic summary of the structure and localization of the various GFP fusion proteins. The black box represents the APS kinase domain whereas the ATP sulfurylase domain is represented as an open box. The hatched box denotes the small linker region located between the two domains and the checkered box represents the C-terminal GFP tag. The names of the constructs are given on the left, with letters referring to the corresponding photographs. The corresponding subcellular localizations are given on the right (N, nuclear, C, cytoplasmic). B) Western blot analysis. Total extracts of CHO cells transiently transfected with either pEGFPN3 (lane 1), pEGFPN3-hPAPSS1 (lane 2), pEGFPN3-hAPSkinase (lane 3), pEGFPN3-hATPsulfurylase (lane 4), or pEGFPN3-h2MSK (lane 5) were analyzed by immunoblotting with a polyclonal anti-GFP antibody. C–L) Confocal microscopy analysis. Live CHO cells transiently transfected with either pEGFPN3 (C, D), pEGFPN3-hPAPSS1 (E, F), pEGFPN3-hAPSkinase (G, H), pEGFPN3-hATPsulfurylase (I, J), or pEGFPN3-h2MSK (K, L) expression vectors were analyzed by confocal laser scanning (C, E, G, I, K) and Nomarsky interference microscopy (D, F, H, J, L). Bar = 10 µm.

To confirm the findings obtained with epitope-tagged hPAPSS1 in transfected cells, we studied the localization of endogenous hPAPSS1 in human endothelial cells. Rabbit polyclonal antibodies to the hPAPSS1 protein were raised using purified recombinant hPAPSS1 as an immunogen. Western blot analysis of human endothelial cell extracts (ECV cell line) using the anti-hPAPSS1 polyclonal serum revealed a prominent protein band with a molecular mass of 70 kDa, consistent with the predicted molecular mass of the protein and similar to that of the purified recombinant hPAPSS1 (Fig. 2A ) and that of the in vitro translation product of the hPAPSS1 cDNA in rabbit reticulocyte lysate (data not shown). The hPAPSS1 antiserum was then used to perform immunocytochemical studies on ECV endothelial cells. In all cells, the hPAPSS1 immunoreactivity was found mainly in the nucleus (Fig. 2B ). Horizontal sections obtained by confocal laser scanning microscopy demonstrated that both the nucleoplasm and its surrounding membrane, but not the nucleoli, exhibited a strong fluorescent signal. No nuclear staining was observed in control experiments using crude preimmune antiserum from the same rabbit (data not shown), thus ruling out any possibility of nonspecific reactivity of the anti-hPAPSS1 polyclonal serum with other nuclear proteins. Conversely, the faint perinuclear and cytoplasmic staining observed with the anti-hPAPSS1 antibodies (Fig. 2B ) was also detected with the preimmune serum (data not shown), indicating that the hPAPSS1 protein accumulates primarily, if not exclusively, in the nucleus of ECV endothelial cells. Similarly, PAPSS1 immunoreactivity was also observed in the nuclei of CHO and COS-7 cells (data not shown). These results indicate that the endogenous hPAPSS1, similar to the epitope-tagged enzyme, accumulates preferentially in the nucleus of mammalian cells.



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  		Figure 2. Nuclear localization of endogenous PAPSS1 in human endothelial cells. A) Western blot analysis. Human ECV endothelial cell extracts (lane 1) and purified His-tagged hPAPSS1 recombinant protein (lane 2) were analyzed by immunoblotting with the anti-hPAPSS1 serum. The minor bands detected in the cell extract in addition to the major 70 kDa protein species were detected with both anti-hPAPSS1 and preimmune sera. Molecular mass markers are indicated on the left. B) Confocal microscopy image of hPAPSS1 immunostaining. Human ECV endothelial cells were analyzed by indirect immunofluorescence staining using the anti-hPAPSS1 serum. The faint perinuclear and cytoplasmic staining observed with the anti-hPAPSS1 antibodies is nonspecific since it is also detected with preimmune sera. Bar = 10 µm.

Human PAPSS1 and Drosophila PAPSS localize to the nucleus in yeast and complement the methionine auxotrophy of ATP sulfurylase- and APS kinase-deficient strains
To determine whether hPAPSS1 is fully functional when localized to the nucleus, we performed in vivo complementation assays in the budding yeast, S. cerevisiae. We used strains harboring mutations in the two genes required for PAPS synthesis, MET3 and MET14, which separately encode ATP sulfurylase and APS kinase activities. Due to the lack of availability of activated sulfate for PAPS reductase, met3 and met14 mutants are unable to synthesize sulfur-containing amino acids (23) and thus require the activity of an ectopically expressed enzyme (ATP sulfurylase, APS kinase, or PAPS synthetase) to complement their methionine auxotrophy.

We first determined the subcellular localization of hPAPSS1 ectopically expressed in yeast. hPAPSS1 was tagged at its carboxyl terminus with GFP and expressed into ATP sulfurylase- or APS kinase-deficient strains. In both strains, the hPAPSS1-GFP fusion protein was found to accumulate in the nucleus (Fig. 3A, B, E, F ). Similar results were obtained with Drosophila melanogaster PAPS synthetase (DmPAPSS) (Fig. 3C, D, G, H ), which exhibits 60% identity with human PAPSS1 and localizes to the nucleus in transfected CHO and COS-7 cells (data not shown).



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  		Figure 3. Nuclear localization and functional activity of human and Drosophila PAPS synthetases in yeast. A–H) Nuclear localization. Yeast strains S5 (A–D) and X6 (E–H) transformed with expression constructs for GFP-fused human PAPSS1 (A, B, E, F) or Drosophila PAPSS (C, D, G, H) were grown in minimal medium containing galactose and live yeast cells were observed by fluorescence microscopy (right column). Nuclear DNA was visualized by DAPI staining (left column). Bar = 10 µm. I) Complementation assay. Saccharomyces cerevisiae strains S5 (left column) and X6 (right column), which exhibit deficiencies in ATP sulfurylase and APS kinase activities, respectively, were transformed with expression vectors for GFP-fused human PAPSS1 (hPAPSS-GFP), Drosophila PAPSS (DmPAPSS-GFP), and S. cerevisiae ATP sulfurylase (ScATPsul) or APS kinase (ScAPSkin). Cells were grown on minimal medium with or without methionine, and the expression of GFP-tagged proteins was either repressed or induced using glucose or galactose as the sole carbon sources, respectively.

We then investigated the ability of these animal PAPS synthetases to rescue the methionine auxotrophy of the ATP sulfurylase- and APS kinase-deficient yeast strains (S5 and X6, respectively). We found that both hPAPSS1 and DmPAPSS were able to restore the ability of met3 and met14 yeast mutants to grow on media lacking methionine (Fig. 3I ). This relief of methionine auxotrophy was observed on media containing galactose, but not on media containing glucose, which represses transcription of the PAPS synthetases expression constructs. These results demonstrate that hPAPSS1 and DmPAPSS are bifunctional ATP sulfurylase/APS kinase enzymes possessing both ATP sulfurylase and APS kinase activities. More important, the success of these complementations in yeast suggests that human PAPSS1 and Drosophila PAPSS are fully active in vivo when localized to the nucleus.

The APS kinase domain of human PAPSS1 is necessary and sufficient for its nuclear localization
To define the sequences involved in nuclear targeting of the enzyme, a series of deletions and substitutions in the hPAPSS1 sequence were fused to the GFP tag (summarized in Fig. 4A ), and expressed in CHO cells (Fig. 4B ). Based on amino acid sequence homology to bacterial, plant, and fungal ATP sulfurylase and APS kinase proteins, the hPAPSS1 sequence could be divided into three parts: an APS kinase domain extending from residues 1 to 220; a linker region extending from residues 221 to 240 that shares very low homology with bacterial, plant, fungal, and even other animal sequences; and an ATP sulfurylase domain extending from residues 241 to 624. Similar to HA- or cMyc-tagged hPAPSS1 (Fig. 1) , the wild-type hPAPSS1-GFP protein was found to accumulate in the nucleus (Fig. 4E, F ), whereas GFP alone appeared to be distributed more diffusely throughout the cell (Fig. 4C, D ). We first determined whether a sequence resembling the SV40 T antigen-type nuclear localization sequence (NLS) found near the carboxyl terminus of the ATP sulfurylase domain (residues 567–571, KKKKR) played a role in nuclear targeting of hPAPSS1. When lysine residues 568–570 were replaced with alanines by site-directed mutagenesis, the resulting construct still accumulated in the nucleus (data not shown), indicating that the hPAPSS1 putative NLS is not critical for nuclear localization of the enzyme. We then analyzed the localization of a hAPSkinase-GFP mutant, resulting from the deletion of the whole ATP sulfurylase sequence (residues 241–624) and part of the linker sequence (residues 226–240). After expression of this hAPSkinase-GFP deletion mutant in CHO cells, fluorescence was detected exclusively in the nucleus, showing that the APS kinase domain of hPAPSS1 is sufficient for nuclear targeting of the enzyme (Fig. 4G, H ). In contrast, a hATPsulfurylase-GFP mutant resulting from the deletion of the APS kinase domain (residues 1 to 221) accumulated preferentially in the cytoplasm (Fig. 4I, J ), indicating that the APS kinase domain, but not the ATP sulfurylase part, plays a key role in nuclear targeting of hPAPSS1. The integrity of the APS kinase domain appears to be critical since we found that deletion of the first 21 amino acids [mutant h2MSK analogous to mutant 2MSK from Deyrup et al. (36) ] abrogates nuclear localization. The resulting h2MSK-GFP fusion protein localized to the cytoplasm (Fig. 4K, L ), indicating that the amino terminus of the APS kinase domain (aa 1–21), although not essential for catalytic activity (36) , is required for nuclear localization of hPAPSS1. However, the first 21 residues of hPAPSS1 do not function as an NLS since we found they are not sufficient to promote nuclear localization of GFP (data not shown). Essentially identical results were obtained when the different constructs were expressed in COS-7 cells instead of CHO cells, and these findings were further corroborated by using HA-tagged proteins expressed in either CHO or COS-7 cells (data not shown). Together, these results demonstrate that the hPAPSS1 enzyme is targeted to the nucleus by its APS kinase domain.

PAPSS2 relocates from the cytosol to the nucleus when coexpressed with PAPSS1
We then wanted to determine whether PAPSS2, which exhibits 76% identity at the amino acid level with PAPSS1 and plays a major role in the control of proteoglycan sulfation in cartilage (28 , 29) , localized to the nucleus similar to PAPSS1. To investigate the subcellular localization of PAPSS2 in mammalian cells, we expressed murine PAPSS2, which exhibits 93% identity with its human ortholog at the protein level (29) , in fusion with GFP (mPAPSS2-GFP). Unlike PAPSS1, ectopically expressed mPAPSS2-GFP was found to accumulate in the cytoplasm of the majority of transiently transfected COS-7 cells (Fig. 5A ) and CHO cells (Fig. 5B ). However, transient cotransfection of hPAPSS1-HA and mPAPSS2-GFP in COS-7 cells dramatically shifted the latter from a cytosolic to a nuclear localization (Fig. 5C, D ). The APS kinase domain of hPAPSS1, which is responsible for nuclear targeting of hPAPSS1, appeared to be sufficient for relocalization of mPAPSS2 from the cytosol to the nucleus since most of the cells coexpressing hAPSkinase-HA exhibited a predominant nuclear localization of mPAPSS2-GFP (Fig. 5E, F ). Altogether, these data indicate that PAPSS1 induces relocalization of ectopically expressed PAPSS2 from the cytosol to the nucleus and that the APS kinase domain of PAPSS1 plays a key role in this process.



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  		Figure 5. PAPSS1 induces PAPSS2 relocalization from the cytosol to the nucleus. COS-7 cells (A, C–F) or CHO cells (B) were transiently transfected with expression vector for mPAPSS2-GFP (A, B) or cotransfected with expression vectors for mPAPSS2-GFP and hPAPSS1-HA (C, D) or mPAPSS2-GFP and hAPSkinase-HA (E, F). Live (A, B) or fixed cells immunostained using monoclonal anti-HA primary antibody and TRITC-labeled anti-mouse IgG secondary antibody (C–F) were analyzed by confocal laser scanning microscopy (panels A, B, C, E: green fluorescence, mPAPSS2-GFP; panels D, F: red fluorescence, hPAPSS1-HA or hAPSkinase-HA). In panels C and D, arrowheads indicate a cell expressing only mPAPSS2-GFP and arrows denote a cell coexpressing mPAPSS2-GFP and hPAPSS1-HA. In panels E and F, all four cells coexpress mPAPSS2-GFP and hAPSkinase-HA, including the two cells indicated by arrows.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Sulfate activation into PAPS is a prerequisite for all sulfation reactions. PAPS synthesis has always been assumed to occur exclusively in the cytosol, since sulfotransferases that use PAPS as cosubstrate reside in the cytosol or Golgi apparatus and cytosol-to-Golgi PAPS translocases have been identified. Contrary to the current dogma, we show in this study that PAPSS1, one of the two mammalian PAPS synthetases, localizes to the nucleus of eukaryotic cells. We identify the APS kinase domain of the bifunctional PAPSS1 enzyme as an essential player in its nuclear localization. We found that deletion of a catalytically dispensable 21 amino acids sequence at the amino terminus is sufficient to abrogate nuclear accumulation, indicating that the integrity of the APS kinase domain is required for nuclear localization of PAPSS1. In addition, we demonstrate that the subcellular localization of a second PAPS synthetase (PAPSS2) can be modulated by PAPSS1, the coexpression of which allows PAPSS2 relocalization from the cytosol to the nucleus. Together, our results suggest that sulfate activation by PAPS synthetases might occur in the nucleus of eukaryotic cells and play a role in a nuclear sulfation pathway that remains to be characterized.

To demonstrate nuclear localization of PAPSS1, we used a combination of immunocytochemical and fluorescence methods. Both the endogenous and the ectopically expressed enzymes were found to accumulate preferentially in the nucleus. Human PAPSS1 fused to three different tags and expressed in four different mammalian cell lines localized to the nucleus in all cases, indicating that the subcellular distribution of the ectopically expressed enzyme was independent of the epitope tag or the recipient cell line used. The use of a GFP-tagged hPAPSS1 allowed direct analysis of fluorescence in live cells, thus excluding the possibility that nuclear localization of PAPSS1 may be due to formaldehyde fixation and detergent permeabilization of the transfected cells or cross-reactivity of antibodies with unrelated nuclear antigens. The use of confocal microscopy and confocal sectioning allowed us to show that hPAPSS1 exhibits a preferential intranuclear localization, including the nucleoplasm and nuclear membrane but excluding the nucleolus. Therefore, all the evidence obtained indicated that hPAPSS1 is a nuclear protein. These findings apparently disagree with current dogma that describes PAPS synthesis as an exclusive cytosolic event. However, although PAPS synthesis had always been assumed to occur in the cytosol, few attempts were made to confirm these assumptions and none of them directly localized the enzyme itself. For instance, in most of these earlier studies, biochemical procedures were used to show that upon subcellular fractionation, PAPS synthetase activity is recovered in the cytosolic fractions of rat brain, rat liver, or insect gut homogenates (37 38 39) . Our observations indicate that the results of fractionation studies must be considered cautiously if they are not taken together with more reliable microscopy analysis.

Our results suggest that the nucleus is a major site of PAPS production in eukaryotic cells. We observed a strong correlation between nuclear localization of hPAPSS1 and its functional activity in CHO cells or yeast. PAPS assays in CHO cells revealed that overexpression of hPAPSS1-HA in the nucleus markedly increased overall PAPS synthetase activity. Complementation assays in yeast showed that hPAPSS1, which accumulates in the yeast nucleus, is functionally active and able to complement the methionine auxotrophy of ATP sulfurylase- and APS kinase-deficient strains. Although it cannot be excluded that in vivo complementation in yeast may be due to the activity of small amounts of enzyme localized in the cytoplasm, these results strongly suggest that hPAPSS1 is actively synthesizing PAPS in the nucleus. It should be pointed out, however, that although nuclear accumulation of PAPS synthetases is likely to result in high levels of PAPS production in the nucleus, it may not be absolutely required for PAPS synthesis in vivo: purified recombinant PAPSS1 produced in E. coli is fully competent for PAPS production (36) , and both PAPSS2 and the 2MSK PAPSS1 deletion mutant, which localize in the cytoplasm, are known to be catalytically active in vitro (28 , 36) . Nuclear localization of PAPSS1 may function rather to enhance PAPS cellular levels by partially sequestering PAPS from the cytoplasm and protecting it from degradation by PAPS sulfohydrolase or PAPS 3'-phosphorylase (31 , 40) . An analogous role has recently been proposed for nuclear localization of cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) synthetase, the enzyme responsible for production of CMP-Neu5Ac, the activated sialic acid donor used by Golgi sialyltransferases (41 , 42) . The possibility that nuclear localization of PAPS and CMP-Neu5Ac synthetases may fulfill similar roles is supported by the fact that the sulfation and sialylation pathways share many features. For instance, sulfation and sialylation are both critical terminal modifications of many cell surface glycoconjugates (16 , 43) that result in highly anionic structures, such as the 6-sulfo-sialyl-LewisX [Sia({alpha}2–3)Gal(ß1–4)(Fuc({alpha}1–3))(6-SO3-)GlcNAc] motif controlling lymphocyte recruitment into lymph nodes (44 , 45) . Sulfotransferases and sialyltransferases involved in these processes are essentially Golgi resident, but sulfated and/or sialylated glycoconjugates are found in the nucleus (46 47 48 49) , and the existence of nuclear sulfation or sialylation pathways has not been ruled out. A precedent exists for a glycosylation pathway in the nucleus. While glycosylation reactions were assumed for a long time to occur exclusively in the Golgi apparatus, Hart and co-workers discovered that O-glycosylation is a major posttranslational modification of nuclear and cytoplasmic proteins (50) . The enzyme that catalyzes the transfer of single GlcNAc moieties on nuclear polypeptides, named O-GlcNAc transferase, has recently been cloned from rat liver and shown to localize to the nucleus and cytoplasm of mammalian cells (51) . Therefore, the nucleus, which appears to be a privileged subcellular compartment for activation of anionic metabolites such as sulfate and Neu5Ac into 5'-monophosphonucleoside high-energy donors, may harbor novel sulfation and glycosylation pathways not yet characterized.

Our results demonstrate that expression of PAPSS1 can modify PAPSS2 subcellular distribution. PAPSS1 and PAPSS2 are likely to be expressed in the same tissues or cell types, since transcripts for the two PAPS synthetases have been detected in a wide variety of human and mouse tissues (24 , 28 , 29) . Accordingly, we have found that PAPSS1 and PAPSS2 are coexpressed in endothelial cells from human umbilical vein and high endothelial venules (J. P. Girard, unpublished results). Therefore, variations in the expression levels of PAPSS1 in a given tissue may dramatically affect PAPSS2 localization. Thus, although PAPSS1 probably localizes to the nucleus of most cells, PAPSS2 may localize to the cytoplasm only in tissues with low PAPSS1 levels, such as liver and cartilage (24) , and localize to the nucleus in other tissues. It will be important to determine whether PAPSS1 and PAPSS2 can physically interact and whether the APS kinase domain of PAPSS1, which is sufficient for relocalization of PAPSS2 from the cytosol to the nucleus, contains a PAPSS2 binding site. Further studies will also be required to determine the precise subcellular localization of PAPS synthetases in animal tissues and their relative contribution to various biological processes. However, our results suggest that subcellular localization of PAPS synthetases is likely to provide an additional level of regulation of the sulfation pathways in eukaryotes.


   ACKNOWLEDGMENTS
 
We thank Philippe Bouvet (IPBS-CNRS, Toulouse) and Espen Baekkevold (LIIPAT, Oslo) for critical reading of the manuscript and other members of our group for fruitful discussions. We gratefully acknowledge Emmanuel Käs (LBME-CNRS, Toulouse) and Tom Leustek (New Brunswick, N.J.) for the gift of plasmids and yeast strains, Fabienne Soulet for providing ECV cell extracts, and Ali Hamiche for preparation of S. cerevisiae genomic DNA. Special thanks to Jacques Féliu for DNA sequencing, Yvette de Préval for oligonucleotide synthesis, and Stéphane Roga for technical assistance. S.B. had a fellowship from the Ministère de l’Education Nationale, de la Recherche et de la Technologie. This work was supported by grants from CNRS, Université Paul Sabatier, Région Midi-Pyrénées, Fondation de France, and Association pour la Recherche sur le Cancer.


   FOOTNOTES
 
Received for publication June 1, 1999. Revised for publication September 13, 1999.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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