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• W2134084751 abstract "Article15 June 1999free access Glycoprotein reglucosylation and nucleotide sugar utilization in the secretory pathway: identification of a nucleoside diphosphatase in the endoplasmic reticulum E.Sergio Trombetta Corresponding Author E.Sergio Trombetta Department of Cell Biology, Yale Medical School, PO Box 208002, New Haven, CT, 06520-8002 USA Search for more papers by this author Ari Helenius Ari Helenius Department of Biochemistry, Swiss Federal Institute of Technology, Universitaestrasse 16, CH-8092 Zürich, Switzerland Search for more papers by this author E.Sergio Trombetta Corresponding Author E.Sergio Trombetta Department of Cell Biology, Yale Medical School, PO Box 208002, New Haven, CT, 06520-8002 USA Search for more papers by this author Ari Helenius Ari Helenius Department of Biochemistry, Swiss Federal Institute of Technology, Universitaestrasse 16, CH-8092 Zürich, Switzerland Search for more papers by this author Author Information E.Sergio Trombetta 1 and Ari Helenius2 1Department of Cell Biology, Yale Medical School, PO Box 208002, New Haven, CT, 06520-8002 USA 2Department of Biochemistry, Swiss Federal Institute of Technology, Universitaestrasse 16, CH-8092 Zürich, Switzerland *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:3282-3292https://doi.org/10.1093/emboj/18.12.3282 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info UDP is generated in the lumen of the endoplasmic reticulum (ER) as a product of the UDP-glucose-dependent glycoprotein reglucosylation in the calnexin/calreticulin cycle. We describe here the identification, purification and characterization of an ER enzyme that hydrolyzes UDP to UMP. This nucleoside diphosphatase is a ubiquitously expressed, soluble 45 kDa glycoprotein devoid of transmembrane domains and KDEL-related ER localization sequences. It requires divalent cations for activity and hydrolyzes UDP, GDP and IDP but not any other nucleoside di-, mono- or triphosphates, nor thiamine pyrophosphate. By eliminating UDP, which is an inhibitory product of the UDP-Glc:glycoprotein glucosyltransferase, it is likely to promote reglucosylation reactions involved in glycoprotein folding and quality control in the ER. Introduction Several mechanisms operate in the cell to assist the folding and assembly of newly synthesized proteins. The endoplasmic reticulum (ER) is unique in that it contains a special molecular chaperone system devoted to the folding of glycoproteins with N-linked oligosaccharides (for a recent review see Helenius et al., 1997). The system is centered around two lectins, calnexin and calreticulin, which recognize monoglucosylated oligosaccharides present on newly synthesized and incompletely folded glycoproteins. By binding to these oligosaccharide trimming intermediates, calnexin and calreticulin promote proper folding and assembly of glycoproteins. They are also part of a quality control system that restricts exit of newly synthesized proteins to those that have reached their native conformations. The monoglucosylated structures recognized by calnexin and calreticulin arise by two mechanisms. They are generated by the sequential removal of two of the three glucose residues present on core oligosaccharides. They are also formed by reglucosylation of completely deglucosylated oligosaccharides through the action of a UDP-Glc:glycoprotein glucosyltransferase (GT) (Parodi et al., 1983). GT is a lumenal ER enzyme that uses UDP-Glc transported from the cytosol as the glucose donor (Pérez and Hirschberg, 1986; Vanstapel and Blanckaert, 1988; Trombetta et al., 1991) Studies in vitro and in vivo have shown that only glycoproteins that have failed to reach their native conformation are substrates for GT (Sousa et al., 1992; Trombetta and Parodi, 1992; Fernandez et al., 1994; Hammond et al., 1994; Van Leeuwen and Kearse, 1997; Wada et al., 1997; Cannon and Helenuis, 1999). Due to its capacity to distinguish between glycoprotein conformers, it thus serves as a folding sensor in the calnexin/calreticulin pathway (see Helenius et al., 1997). Reglucosylation by GT and removal of the added glucose by glucosidase II regulate a cycle of lectin binding and release that is repeated until substrate glycoproteins are correctly folded. Since glycoproteins typically contain more than one oligosaccharide chain, and given the cyclic nature of the de- and reglucosylation process, several molecules of UDP-Glc can be consumed during the folding of a glycoprotein. For the de- and reglucosylation cycle to work, UDP-Glc has to be transported from the cytosol where it is synthesized into the ER lumen, where it is used by GT (Perez et al., 1986; Vanstapel et al., 1988). A similar topological arrangement exists in the Golgi apparatus, where nucleotide sugars made in the cytosol are utilized by lumenally oriented glycosyltransferases. In this case, antiporters present in the Golgi membranes import nucleotide sugars in exchange for the corresponding nucleoside monophosphates (Hirschberg and Snider, 1987). Biochemical and morphological studies described a nucleoside diphosphatase in the trans-Golgi compartment that hydrolyzes the nucleoside diphosphates generated as products of glycosylation reactions into nucleoside monophosphates. It prevents the accumulation of diphosphonucleosides that would otherwise cause product inhibition of the glycosyltransferases, and at the same time generates the nucleoside monophosphates needed for import of new sugar nucleotides (Allen and Slater, 1961; Novikoff and Goldfischer, 1961; Allen, 1963a,b; Friend, 1969; Cheetham et al., 1971; Kuhn and White, 1977; Abeijon et al., 1993). We report here the identification and further characterization of a ubiquitous nucleoside diphosphatase from the ER of mammalian cells. It hydrolyzes UDP and GDP to UMP and GMP. We propose that by preventing accumulation of UDP in the ER lumen it may promote reglucosylation reactions and thus support the calnexin/calreticulin chaperone cycle. Results ER and Golgi complexes contain distinct UDPase activities To determine whether the ER possesses an enzyme capable of cleaving UDP to UMP different from the one present in the Golgi complex, we prepared ER- and Golgi-enriched organelle fractions from rat liver. As expected, efficient hydrolysis of UDP and thiamine pyrophosphate (TPP) was detected in the Golgi fractions (see total values ‘Tot.’ in Table I). The ER fractions also contained potent nucleoside diphosphatase activity capable of degrading UDP and GDP, although TPP was degraded much more efficiently by Golgi fractions. This suggested that the ER contained one or more nucleoside diphosphatases with substrate specificities distinct from those present in the Golgi complex. Table 1. Solubility of ER and Golgi UDPases Substrate Phosphatase activity No detergent 1% Triton 0.2% OG ER Golgi ER Golgi ER Golgi Man-6-P Tot. 42 5 38 6 44 5 SN 1 0 3 1 1 0 TPP Tot. 18 96 25 130 28 119 SN 5 16 25 102 22 46 UDP Tot. 810 1507 693 1390 705 1427 SN 31 88 418 1199 409 263 GDP Tot. 749 1531 642 1154 687 1307 SN 52 84 405 949 413 223 ER and Golgi complex derived organelle fractions were pre-treated with or without detergents as indicated. An aliquot was preserved (Tot.) and the remainder was ultracentrifugated to separate the solubilized material (SN). Phosphatase activity was meassured on total (Tot.) and solubilized (SN) fractions using the following substrates: mannose-6-phosphate (Man-6-P), thiamine pyrophosphate (TPP), uridine diphosphate (UDP) and guanosine diphosphate (GDP). Incubations were done in a total volume of 50 μl containing 2 mM CaCl2, 50 mM NaCl, 20 mM HEPES buffer pH 7.5, 0.1% Triton X-100, 2 mM substrate and 5 μl of membrane fraction to be assayed. Activity is expressed as nanomoles of inorganic phosphate released in 10 min. OG, octylglucoside. To compare further the activities detected in ER and Golgi, we compared their solubilization with detergents. As shown in Table I, both Golgi- and ER-associated nucleoside diphosphatase activities sedimented with the organelles in the absence of added detergent. When solubilizing amounts of a nonionic detergent were added (1% Triton X-100), both activities were recovered in the supernatant. However, the activity present in the ER could also be efficiently extracted in soluble form by low amounts of detergent (0.2% octylglucoside) under conditions that did not solubilize the activity from the Golgi membranes. As illustrated by the persistent membrane association of glucose-6-phosphatase, this mild detergent treatment caused membrane lysis without solubilization of integral membrane proteins. The ER nucleoside diphosphatase thus behaved as a soluble lumenal protein while the Golgi enzyme behaved like an integral membrane protein. To pursue the apparent solubility difference, the ER and Golgi fractions were submitted to Triton X-114 partition, which allows distinction between soluble/peripheral and integral membrane proteins (Bordier, 1981). The UDPase activity associated with the ER partitioned almost exclusively in the aqueous phase, indicating that the ER-nucleoside diphosphatase was hydrophilic and did not bind detergent (Table II). In contrast, the UDP- and TPP-hydrolyzing activities associated with the Golgi complex partitioned preferentially into the detergent phase, consistent with the nucleoside diphosphatase behaving as an integral membrane protein. The low TPPase activity detected in the ER partitioned like the one associated with the Golgi complex, suggesting that it represented a contamination of the ER fractions with Golgi-derived fractions. Table 2. Triton X-114 partition of ER and Golgi UDPase activities Phosphatase activity ER Golgi Fraction UDP TPP UDP TPP Total 422 47 794 103 SN 284 3 141 39 Pellet 31 36 235 54 Triton X-114 partition of ER and Golgi derived organelle fractions was performed as described in the Materials and methods. For measurement of phosphatase activities, incubations were done in a total volume of 50 μl containing 2 mM CaCl2, 50 mM NaCl, 20 mM HEPES buffer pH 7.5, 0.1% Triton X-100, 2 mM substrate and 5 μl of membrane fraction to be assayed. Activity is expressed as nanomoles of inorganic phosphate released in 5 min. The subcellular fractionation, solubility and Triton X-114 partitioning data indicated that the ER of mammalian cells contains a soluble UDP activity different from the membrane bound enzyme present in the trans-Golgi. We will hereafter refer to it as the ER-UDPase. Purification of ER-UDPase Isolated ER fractions were used as the starting material for the purification of the ER-UDPase. Initial attempts were made with ER fractions prepared from rat liver, but very little protein was left at later stages of the purification. In order to scale up, we switched to bovine liver, where the behavior of the ER-UDPase activity was indistinguishable from that detected in rat liver (not shown). The use of bovine tissue provided sufficient material to purify the ER-UDPase to homogeneity (see Table III; Figure 1B). Figure 1.Purification of the ER-UDPase. (A) DEAE–cellulose chromatography. The soluble extract from bovine liver ER-derived organelle fractions was applied to a DEAE–cellulose column as described in the Materials and methods. Fractions of 20 ml were collected and 5 μl aliquots were assayed for UDPase activity in the presence (●) or absence (○) of 10 mM CaCl2. (B) SDS–PAGE analysis of the ER-UDPase containing fractions from the eluate of the hydrophobic column. Aliquots containing 15 μl from each fraction were run on a 10% SDS–PAGE and protein was detected with Coomassie Blue R-250. Numbers on top of each lane indicate the phosphatase activity detected using UDP as substrate. (C) Purified ER-UDPase (0.5 ml) was run on a Superdex S-75 column equilibrated and developed at 0.5 ml/min in 5 mM 2-mercaptoethanol, 150 mM NaCl, 10 mM HEPES pH 7.5. The arrow denotes the elution volume of the 45 kDa standard ovoalbumin. (D) Purified ER-UDPase was digested overnight at 37°C with Endo H (lane +) or mock-treated under the same conditions (lane −). Samples were run on a 10% SDS–PAGE and protein detected with Coomassie Blue R-250. Download figure Download PowerPoint Table 3. Purification of ER-UDPase from bovine liver Fraction Volume (ml) Activity (U) Protein (mg) Specific activity (U/mg) Yield (%) ER memb. 1600 8000 6400 1.25 100 Soluble 1500 10480 2760 3.8 110 DEAE 60 5600 240 23 70 Con A 45 3800 9 420 47 Mono Q 1 1700 0.7 2428 21 S-75 1 560 0.1 5600 7 Alkyl S 2 82 0.015 5460 1 The ER-UDPase was first released from the membranes by lysis with low concentrations of detergent. Upon DEAE–cellulose chromatography of the soluble extract, the major UDPase activity present in ER membranes was recovered in a single peak (Figure 1A) which was pooled and applied to a Concanavalin A (Con A)–Sepharose column. The α-methyl-mannopyranoside eluate from the Con A–Sepharose column was subjected to anion exchange, gel filtration and hydrophobic chromatography. When analyzed by SDS–PAGE, the eluate from the last step showed a band of 45 kDa that accompanied the UDPase activity and could thus be identified as the ER-UDPase (Figure 1B). Characterization of the ER-UDPase The purified ER-UDPase eluted from gel filtration columns as a symmetric peak comigrating with a 45 kDa protein standard, indicating it was a monomer (Figure 1C). All the UDPase activity bound to the Con A–Sepharose column and was eluted with α-methyl-mannopyranoside, suggesting the presence of high mannose type oligosaccharides. Furthermore, digestion by Endo H reduced its molecular weight by 2–3 kDa (Figure 1D), a shift compatible with the loss of one or two high mannose oligosaccharides. The homogeneous ER-UDPase had a neutral optimum pH (not shown). Since no activity could be detected in the absence of divalent cations and since the enzyme was almost irreversibly inactivated by EDTA, the activity was routinely measured in the presence of 10 mM CaCl2 (Figure 1A). However, the purified enzyme was also active in the presence of MgCl2 and MnCl2, with half maximal activity reached at 0.3–0.5 mM CaCl2 or MgCl2 (Figure 2B). Figure 2.Characterization of ER-UDPase. (A) Inhibition of glycoprotein reglucosylation by UDP and UMP. Glycoprotein reglucosylation using homogeneous UDP-Glc:glycoprotein glucosyltransferase was assayed as described in Trombetta et al. (1992). Reactions contained 20 μM UDP [14C]-Glc and the indicated concentrations of UDP (○) or UMP (●). (B–D) Characterization of ER-UDPase activity. Homogeneous ER-UDPase from the hydrophobic chromatography step was used for the UDPase activity measurements and incubations were performed as described in the Materials and methods. (B) The effect of CaCl2 (○) or MgCl2 (●) on the activity of homogeneous ER-UDPase in the presence of 1 mM UDP. (C) ER-UDPase activity as a function of UDP (●) or GDP (○) in the presence of 1 mM CaCl2 (C) or MgCl2 (D). Download figure Download PowerPoint The availability of homogenous ER-UDPase enabled us to analyze its substrate specificity. As shown in Table IV, it hydrolyzed nucleoside diphosphates specifically, i.e. it did not act on the mono- or triphosphates tested (Table IV). It was also inactive on TPP, indicating that the low levels of TPPase detected in ER fractions (Table I) reflected contamination with Golgi. Of the pyrimidine diphosphates tested, only UDP was hydrolyzed, while thymidine or cytidine diphosphate were not. Among the purine diphosphates, the enzyme hydrolyzed guanine and inosine diphosphates but not adenosine or xantosine diphosphates. The enzyme was thus dependent on the base for activity, and required a terminal α–β pyrophosphate linkage. Table 4. Specificity of homogeneous ER-UDPase Phosphatase activity Base Monophosphate Diphosphate Triphosphate Adenosine <1 <1 <1 Guanosine 2 127 34a Xanthosine <1 1 1 Uridine 2 153 31a Thymidine 2 1 1 Cytosine <1 1 1 Inosine 1 131 21 Thiamine <1 <1 N.D. Phosphatase activity was measured as described in the Materials and methods using the indicated substrates at 1 mM concentration in the presence of 2 mM CaCl2. a In these cases, the phosphate released comes from contaminant nucleoside diphosphates, as detected by HPLC analysis of the substrates and the reaction and products (see Figure 3). N.D., not determined. High performance liquid chromatography (HPLC) analysis of the products formed by the ER-UDPase demonstrated that the enzyme converted the nucleoside diphosphates to nucleoside monophosphates (Figure 3). The low activity detected on UTP and GTP (Table IV) was clearly due to contaminating nucleoside diphosphate in the corresponding triphosphate preparations (Figure 3). The peaks corresponding to UTP and GTP were not affected by the ER-UDPase, whereas the UDP and GDP peaks were converted to UMP and GMP. Figure 3.HPLC analysis of the reaction products formed by homogeneous ER-UDPase. Incubations and HPLC analysis were performed as described in the Materials and methods. All samples contained 1 mM CaCl2 and homogeneous ER-UDPase. The substrates used were as follows: UDP (A, B), UTP (C, D), GDP (E, F) and GTP (G, H). For inactivated samples (A, C, E and G), 2 mM EDTA was present before the addition of ER-UDPase. In the digested samples (B, D, F and H), EDTA was added at the end of the incubations. Download figure Download PowerPoint ER-UDPase activity on UDP and GDP in the presence of saturating concentrations of CaCl2 or MgCl2 was similar, characterized by a hyperbolic behavior with a nearly identical Vmax and an apparent Km of 0.2–0.5 mM for both nucleoside diphosphates (Figure 2C and D). In the presence of 1 mM MgCl2 the activity on UDP and GDP was essentially the same. In the presence of 1 mM CaCl2, however, the enzyme seemed to hydrolyze UDP more rapidly than GDP. UDP inhibits GT A possible reason why cells have a nucleoside diphosphatase in the ER is that UDP accumulated as a product of reglucosylation reactions might inhibit GT, similar to the product inhibition of glycosyltransferases in the Golgi complex. Glycoprotein reglucosylation was measured in vitro using homogeneous GT and acceptor glycoproteins, in the presence or absence of UDP and UMP. Glycoprotein reglucosylation was more strongly inhibited by UDP than by similar concentrations of UMP (Figure 2A), indicating that conversion of UDP into UMP may serve to alleviate product inhibition of GT. Characterization of ER-UDPase cDNA Amino acid sequence information was obtained from the N-terminus and from internal peptides of the purified bovine ER-UDPase (Figure 4). Based on codon usage preferences in mammals, and amino acid sequence of peptide 27 (vpgqlpvlegeifdsvkpglsafv) and peptide 9 (ihvytfvqk), pools of degenerate primers were used to amplify a first strand cDNA library from mouse liver. A combination of primers S92 (5′-athcaygtntayacntty-3′) and A273 (5′-gartcraanatytcnccytc-3′) yielded a short PCR product that revealed an amino acid sequence (ihvytfvqktagqlpflegeifd) comprised of peptide 9 immediately followed by that of peptide 27, and containing an amino acid sequence corresponding to peptides 9 and 27 not included in the primers, indicating that it corresponded to a portion of the ER-UDPase. Comparison of the peptide sequence with EST databases matched a fragment in entries aa116990 and aa120757. Complete sequencing on both strands of these two cDNAs showed that the two clones contained the same open reading frame (ORF) shown in Figure 4. Differences were only found in the 5′ and 3′ flanking regions: Clone aa116990 had an insertion of 70 bp in the 5′ end, while clone aa120757 had an insertion of 231 bp at the 3′ end. The following characteristics indicated that the cDNA identified indeed coded for the ER-UDPase: (i) all six peptide sequences obtained from the purified enzyme could be found in the cDNA; (ii) the cDNA sequence showed a putative hydrophobic N-terminal signal sequence, followed by the sequence found in the N-terminus of the purified enzyme; (iii) the overall sequence coded for a hydrophilic protein of 43 kDa, in agreement with the biochemical properties of the isolated enzyme; (iv) the presence of two consensus triplets for N-glycosylation was consistent with the presence of glycans in the purified enzyme (Figure 1D); and (v) the sequence showed significant homology with enzymes that cleave α–β pyrophosphate linkages in nucleoside di- and triphosphates (Figure 5). A mouse cDNA sequence described as homologous to NTPases (Chadwick et al., 1998; DDBJ/EMBL/GenBank accession No. AF006482) is identical to most of the ER-UDPase presented here but differs from it at the C-terminus. We are confident the cDNA sequence presented in Figure 4 belongs to the purified ER-UDPase since we found a peptide in that region that precisely matches our cDNA sequence (see Figure 4) and does not appear in the mouse cDNA (Chadwick et al., 1998). Figure 4.Mouse liver ER-UDPase cDNA sequence. (A) Predicted ORF for mouse liver ER-UDPase (DDBJ/EMBL/GenBank accession No. AJ238636). Underlined portions correspond to peptides sequenced from the purified bovine ER-UDPase. The actual amino acid sequence of peptides obtained from the bovine enzyme are shown under the corresponding positions in the mouse sequence. The peptide MCPVWVSAGT corresponds to the N-terminus of the purified ER-UDPase. (B) Hydrophobicity plot of mouse liver ER-UDPase. Download figure Download PowerPoint Figure 5.Sequence comparison of mouse liver ER-UDPase with related pyrophosphatases. Pyrophosphatases homologous to mammalian ER-UDPase were aligned using a CLUSTAL_W algorithm, and conserved residues boxed. Numbering on left and right denotes amino acid position in the corresponding sequence. Letters on the left refer to the cDNA compared: A, mouse ER-UDPase; B, Pea NTPase (DDBJ/EMBL/GenBank accession No. Z32743) (Hsieh et al., 1996); C, S.cerevisiae GDPase (L19560) (Abeijon et al., 1993); D, potato ATP-diphosphohydrolase (U18778) (Handa and Guidotti, 1996); E, chicken ecto-ATP-diphosphohydrolase (AF041355) (Nagy et al., 1998). Download figure Download PowerPoint It is noteworthy that we found the ER-UDPase to be broadly expressed in mouse tissues. Using RT–PCR, we detected the mRNA in every tissue tested (Figure 6). Furthermore, numerous EST clones from diverse tissues were identified in databases. Figure 6.Tissue distribution of ER-UDPase. First strand cDNA synthesis was primed with oligo(dT) using total RNA from the indicated tissues. The mRNA for ER-UDPase was amplified as described in the Materials and methods. Lanes A, B and C contain controls: in lane A, the PCR template was a first strand synthesis devoid of RNA. In lane B, mouse liver RNA was subjected to a mock first strand cDNA synthesis lacking reverse transcriptase. In lane C, 10 ng of a plasmid containing ER-UDPase cDNA was used as template. The asterisk denotes the position of the 1 kb standard. Download figure Download PowerPoint Intracellular localization To analyze in more detail the subcellular localization of the enzyme, antibodies were raised against a peptide corresponding to amino acids 108–131 (EVAKDSIPRSHWERTPVVLKATAG) of the mouse ER-UDPase. By immunoblotting, the rabbit antibodies detected a single 45 kDa band that comigrated with the purified enzyme (Figure 7). The same 45 kDa band was detected in rat (Figure 7, lanes 1, 2, 5 and 6) and mouse (not shown) liver microsomes, and in lysates from mouse lymphocyte cell line A20, where the antigen was present in the organelle fraction (lane 7) but not in the cytosol (lane 8). That the polypeptide recognized by the antibody was indeed the ER-UDPase was confirmed by the shift of the reactive band upon Endo H digestion (Figure 7, lanes 5 and 6), consistent with the effect seen on the purified enzyme. Figure 7.Characterization anti ER-UDPase antibodies. Protein samples were run on 10% SDS–PAGE, transferred to nitrocellulose and probed with anti-peptide antibody against mouse ER-UDPase (lanes 1–11) or anti-myc monoclonal antibody 9E10 (lanes 12 and 13). Lanes 1–4 and 9 contain rat liver subcellular fractions: lanes 1 and 9, partially purified ER-UDPase (1 μg of protein, DEAE step); lane 2, ER-derived membranes (10 μg); lane 3, Golgi derived membranes (10 μg); lane 4, cytosol (20 μg). Lanes 5 and 6 contain 2 μg of the same partially purified ER-UDPase sample used in lane 1, mock-digested (lane 5) or digested (lane 6) with Endo H. Lane 7 contained 10 μg of microsomal protein, and lane 8 contained 20 μg of cytosolic protein extracted from the A20 mouse lymphoma cell line. Lanes 10–13 contain 30 μg of a lysate from control transfected CHO cells (lanes 10 and 12) or myc-tagged ER-UDPase cDNA transfected CHO cells (lanes 11 and 13). Download figure Download PowerPoint Antibodies were also used to investigate the distribution of the ER-UDPase in subcellular fractionations of rat liver prepared by isopycnic density-gradient centrifugation. The ER-UDPase was detected in fractions corresponding to the ER, but not in low density fractions containing the Golgi complex marker mannosidase II (Figure 8). Figure 8.Subcellular distribution of ER-UDPase. Rat liver post nuclear supernatant (400 μl) was prepared and analyzed on Optiprep gradients as described in the Materials and methods. Twelve fractions (350 μl each) were collected and analyzed for (A) the ER marker glucose-6-phosphatase; (B) the Golgi marker mannosidase II; or (C) UDPase activity. (D) ER-UDPase was detected in the same fractions by Western blotting using anti-peptide antibodies. (E) As a control to compensate for the low abundance of Golgi membranes in the post-nuclear supernatant, Golgi-enriched membranes (400 μl at 0.5 mg/ml) were loaded at the top of a gradient, which was run, fractionated and analyzed as in (D). The asterisks show the band detected in a lane containing purified ER-UDPase. Download figure Download PowerPoint Since attempts to study the intracellular localization of the ER-UDPase by immunocytochemical techniques using the above-described anti-peptide antibodies failed, we introduced an epitope tag at the C-terminus of the ER-UDPase and transiently expressed the epitope-tagged ER-UDPase cDNA in COS, HeLa, Chinese hamster ovary (CHO) and normal rat kidney (NRK) cells. Indirect immunofluorescence showed that the c-myc tagged ER-UDPase accumulated in intracellular compartments that also stained for the ER marker calnexin in all four cell lines (Figure 9A–H). Moreover, the distribution observed in NRK cells clearly differed from that of the Golgi apparatus marker mannosidase II (Figure 9I and J). The c-myc-tagged ER-UDPase expressed in CHO cells had the expected molecular weight and organelle association as detected by Western blot using anti-mouse ER-UDPase (Figure 7, lanes 10 and 11) or anti myc antibodies (Figure 7, lanes 12 and 13). Moreover, it appeared to be biologically active, since microsomal fractions from transfected cells showed a 3.5-fold increase in UDPase activity over control-transfected cells (not shown). Together with the presence of an N-terminal signal sequence and Endo H-sensitive oligosaccharides, these observations indicated that the ER-UDPase is a resident protein of the ER. Figure 9.Intracellular localization of c-myc-tagged ER-UDPase. COS (A, B), HeLa (C, D), CHO (E, F) and NRK (G–J) cells were transiently transfected with ER-UDPase cDNA containing a c-myc epitope at the C-terminus. Twenty-four hours after transfection, cells were fixed, permeabilized and processed for immunofluorescence. Calnexin was detected with polyclonal rabbit antiserum (A, C, E and I) and the c-myc epitope was detected with monoclonal antibody 9E10 (B, D, F, H and J). Golgi mannosidase II is shown in (G). Download figure Download PowerPoint Discussion We report the identification, isolation, characterization and subcellular localization of a nucleoside diphosphatase ubiquitously expressed in the ER of mammalian cells. In isolated form, the protein is a soluble monomer of 45 kDa. It is synthesized with a cleaved signal sequence and carries one or two high mannose N-linked glycans. While of low abundance, it is easily detected in purified microsomes and isolated ER fractions by its enzymatic activity, and may represent the so called ‘microsomal’ nucleoside diphosphatase (Kuriyama, 1972; Ohkubo et al., 1980; RayChaudhuri et al., 1985; Sano et al., 1988). We found that homogeneous ER-UDPase is able to cleave UDP, GDP and IDP to their corresponding nucleoside monophosphates. It is inactive on nucleoside mono- or triphosphates as well as ADP, CDP and TDP. While the Golgi complex associated nucleoside diphosphatase efficiently cleaves TPP, the ER-UDPase is inactive on this compound. The enzyme seems to require a terminal α–β pyrophosphate group and some specific base for activity. It also requires divalent cations with half maximal activation occurring at 0.3–0.5 mM for MgCl2 and CaCl2. Thus the enzyme should be fully active in its proposed location within the ER lumen. The cDNA sequence revealed that the ER-UDPase belongs to a family of enzymes that cleave α–β pyrophosphate bonds in nucleoside di- and triphosphates. Members of this fami" @default.
• W2134084751 created "2016-06-24" @default.
• W2134084751 creator A5031638977 @default.
• W2134084751 date "1999-06-15" @default.
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• W2134084751 title "Glycoprotein reglucosylation and nucleotide sugar utilization in the secretory pathway: identification of a nucleoside diphosphatase in the endoplasmic reticulum" @default.
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