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• W1990480314 abstract "Nucleotide sugar transporters form a family of distantly related membrane proteins of the Golgi apparatus and the endoplasmic reticulum. The first transporter sequences have been identified within the last 2 years. However, information about the secondary and tertiary structure for these molecules has been limited to theoretical considerations. In the present study, an epitope-insertion approach was used to investigate the membrane topology of the CMP-sialic acid transporter. Immunofluorescence studies were carried out to analyze the orientation of the introduced epitopes in semipermeabilized cells. Both an amino-terminally introduced FLAG sequence and a carboxyl-terminal hemagglutinin tag were found to be oriented toward the cytosol. Results obtained with CMP-sialic acid transporter variants that contained the hemagglutinin epitope in potential intermembrane loop structures were in good correlation with the presence of 10 transmembrane regions. This building concept seems to be preserved also in other mammalian and nonmammalian nucleotide sugar transporters. Moreover, the functional analysis of the generated mutants demonstrated that insertions in or very close to membrane-spanning regions inactivate the transport process, whereas those in hydrophilic loop structures have no detectable effect on the activity. This study points the way toward understanding structure-function relationships of nucleotide sugar transporters. Nucleotide sugar transporters form a family of distantly related membrane proteins of the Golgi apparatus and the endoplasmic reticulum. The first transporter sequences have been identified within the last 2 years. However, information about the secondary and tertiary structure for these molecules has been limited to theoretical considerations. In the present study, an epitope-insertion approach was used to investigate the membrane topology of the CMP-sialic acid transporter. Immunofluorescence studies were carried out to analyze the orientation of the introduced epitopes in semipermeabilized cells. Both an amino-terminally introduced FLAG sequence and a carboxyl-terminal hemagglutinin tag were found to be oriented toward the cytosol. Results obtained with CMP-sialic acid transporter variants that contained the hemagglutinin epitope in potential intermembrane loop structures were in good correlation with the presence of 10 transmembrane regions. This building concept seems to be preserved also in other mammalian and nonmammalian nucleotide sugar transporters. Moreover, the functional analysis of the generated mutants demonstrated that insertions in or very close to membrane-spanning regions inactivate the transport process, whereas those in hydrophilic loop structures have no detectable effect on the activity. This study points the way toward understanding structure-function relationships of nucleotide sugar transporters. Nucleotide sugar transporters form a family of structurally related multimembrane-spanning proteins of the Golgi apparatus and the endoplasmic reticulum (ER). 1The abbreviations used are:ER, endoplasmic reticulum; CHO, Chinese hamster ovary; Tr, transporter; CMP-Sia-Tr, CMP-sialic acid transporter; HA, hemagglutinin; mAb, monoclonal antibody; PSA, polysialic acid; TM, transmembrane domain; UDP-Gal-Tr, UDP-galactose transporter; UDP-GlcNAc-Tr, UDP-N-acetylglucosamine transporter; BSA, bovine serum albumin; PBS, phosphate-buffered saline; DTAF, dichlorotriazinylamino fluorescein.Their function resides in translocating activated sugars from the cytosol into the lumen of the ER and Golgi apparatus (1Kuhn N.J. White A. Biochem. J. 1976; 154: 243-244Crossref PubMed Scopus (42) Google Scholar, 2Capasso J.M. Hirschberg C.B. Biochim. Biophys. Acta. 1984; 777: 133-139Crossref PubMed Scopus (41) Google Scholar, 3Cecchelli R. Cacan R. Verbert A. Eur. J. Biochem. 1985; 153: 111-116Crossref PubMed Scopus (8) Google Scholar). Transporters therefore provide essential components of the glycosylation pathways in eukaryotic cells (for review, see Ref. 4Hirschberg C.B. Berger E.G. Roth J. The Golgi Apparatus. Birkhäuser, Basel, Switzerland1997: 163-178Crossref Google Scholar). Recently, the first nucleotide sugar transporters have been identified at the molecular level. Complementation cloning in mutant cells lacking specific nucleotide sugar transport activities identified the mammalian transporters (Tr) for CMP-sialic acid (5Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (149) Google Scholar, 6Eckhardt M. Gerardy-Schahn R. Eur. J. Biochem. 1997; 248: 187-192Crossref PubMed Scopus (40) Google Scholar), UDP-galactose (UDP-Gal) (7Miura N. Ishida N. Hoshino M. Yamauchi M. Hara T. Ayusawa D. Kawakita M. J. Biochem. 1996; 120: 236-241Crossref PubMed Scopus (120) Google Scholar, 8Ishida N. Miura N. Yoshioka S. Kawakita M. J. Biochem. 1996; 120: 1074-1078Crossref PubMed Scopus (92) Google Scholar), and UDP-N-acetylglucosamine (UDP-GlcNAc) (9Guillen E. Abeijon C. Hirschberg C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7888-7892Crossref PubMed Scopus (82) Google Scholar), the yeast transporters for UDP-GlcNAc (10Abeijon C. Robbins P.W. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5963-5968Crossref PubMed Scopus (111) Google Scholar) and UDP-Gal (11Tabuchi M. Tanaka N. Iwahara S. Takegawa K. Biochem. Biophys. Res. Commun. 1997; 232: 121-125Crossref PubMed Scopus (77) Google Scholar), and theLeishmania GDP-mannose transporter (12Descoteaux A. Luo Y. Turco S.J. Beverly S.M. Science. 1995; 269: 1869-1872Crossref PubMed Scopus (144) Google Scholar, 13Ma D. Russell D.G. Beverly S.M. Turco S.J. J. Biol. Chem. 1997; 272: 3799-3805Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Additional putative nucleotide sugar transporter sequences have been identified using sequence homologies (8Ishida N. Miura N. Yoshioka S. Kawakita M. J. Biochem. 1996; 120: 1074-1078Crossref PubMed Scopus (92) Google Scholar, 10Abeijon C. Robbins P.W. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5963-5968Crossref PubMed Scopus (111) Google Scholar, 13Ma D. Russell D.G. Beverly S.M. Turco S.J. J. Biol. Chem. 1997; 272: 3799-3805Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Surprisingly high sequence homology has been found between the mammalian transporters for CMP-sialic acid, UDP-Gal, and UDP-GlcNAc (8Ishida N. Miura N. Yoshioka S. Kawakita M. J. Biochem. 1996; 120: 1074-1078Crossref PubMed Scopus (92) Google Scholar, 9Guillen E. Abeijon C. Hirschberg C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7888-7892Crossref PubMed Scopus (82) Google Scholar), whereas the conservation between transporters of identical specificity in different biological kingdoms can be low (9Guillen E. Abeijon C. Hirschberg C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7888-7892Crossref PubMed Scopus (82) Google Scholar, 10Abeijon C. Robbins P.W. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5963-5968Crossref PubMed Scopus (111) Google Scholar). As mentioned above, cloning of transporters was achieved by complementation, and the cDNAs isolated were demonstrated to correct the mutant phenotype. Transport activity, however, has only been proven for the murine CMP-Sia-Tr, which could be functionally expressed in Saccharomyces cerevisiae (14Berninsone P. Eckhardt M. Gerardy-Schahn R. Hirschberg C.B. J. Biol. Chem. 1997; 272: 12616-12619Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Because yeast cells lack sialic acids, this result clearly demonstrates that the cloned cDNA in fact encodes the CMP-Sia-Tr and not an accessory protein required in the transport process. Moreover, the canine UDP-GlcNAc-Tr, although only 22% identical to the yeast orthologue, has been isolated by expression cloning in the UDP-GlcNAc-Tr negative mutant of Kluyveromyces lactis. This result allows us to speculate that structural elements involved in specific substrate recognition are formed via the tertiary and/or quaternary organization of the transport proteins. Limited information is available on the regulation of CMP-sialic acid translocation, or on how variations in the translocation rates influence sialylation reactions in the Golgi lumen. It is well established that lumenal CMP stimulates CMP-sialic acid uptake by Golgi vesicles, indicating antiport of CMP and CMP-sialic acid (15Milla M.E. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1786-1790Crossref PubMed Scopus (39) Google Scholar). However, CMP-sialic acid is also translocated in the absence of CMP (14Berninsone P. Eckhardt M. Gerardy-Schahn R. Hirschberg C.B. J. Biol. Chem. 1997; 272: 12616-12619Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 15Milla M.E. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1786-1790Crossref PubMed Scopus (39) Google Scholar), and CMP and derivatives of CMP-sialic acid have been shown to compete with CMP-sialic acid translocation if added onto the cytosolic side of the Golgi membrane (16Capasso J.M. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7051-7055Crossref PubMed Scopus (110) Google Scholar). These reagents could therefore be used to block the sialylation of cell surfaces. Artificial reduction of cell surface sialylation via application of CMP-sialic acid derivatives has been demonstrated to reduce growth and metastasis of tumor cells (17Harvey B.E. Thomas P. Biochem. Biophys. Res. Commun. 1993; 190: 571-575Crossref PubMed Scopus (34) Google Scholar). Moreover, because sialic acids provide recognition and receptor structures for viral and bacterial pathogens (for review, see Ref. 18Kelm S. Schauer R. Jeon K.W. Jarvik J.W. International Review of Cytology: A Survey of Cell Biology. Academic Press Inc., San Diego, CA1997: 137-240Google Scholar), reversible inhibition of cell surface sialylation has been discussed as a perspective to protect healthy cells against these invasive organisms. Understanding structure-function relationships of nucleotide sugar transporters requires knowledge of the three dimensional organization in the plane of the lipid bilayer. The first important step toward this aim is the determination of the membrane topology. Hydrophobicity analyses of the nucleotide sugar transporters cloned to date suggest between 6 and 10 transmembrane domains and the use of secondary structure prediction algorithms proposed models with eight transmembrane domains for both CMP-Sia-Tr and UDP-galactose transporter (6Eckhardt M. Gerardy-Schahn R. Eur. J. Biochem. 1997; 248: 187-192Crossref PubMed Scopus (40) Google Scholar, 7Miura N. Ishida N. Hoshino M. Yamauchi M. Hara T. Ayusawa D. Kawakita M. J. Biochem. 1996; 120: 236-241Crossref PubMed Scopus (120) Google Scholar). This study was undertaken to develop a detailed topological model of the CMP-Sia-Tr. An epitope-insertion approach was used to map membrane orientation. This approach, in contrast to other methods (e.g. methods using truncated proteins fused to reporter proteins), takes into account that even small changes in sequences surrounding transmembrane domains can alter the membrane topology (19Zhang J.T. Lee C.H. Duthie M. Ling V. J. Biol. Chem. 1995; 270: 1742-1746Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and that it is therefore important to confirm unaltered membrane orientation of the analyzed protein by the remaining activity. The results of this study strongly suggest a 10-transmembrane domain topology in which amino and carboxyl termini are oriented toward the cytosol. Monoclonal antibody (mAb) 12CA5, directed against the hemagglutinin (HA) epitope YPYDVPDYASL, was purchased from Boehringer Mannheim, and mAb M5, directed against the FLAG sequence MDYKDDDDK, was from Eastman Kodak, New Haven. Polysialic acid (PSA)-specific mAb 735 has been described (20Frosch M. Görgen I. Boulnois G.J. Timmis K.N. Bitter-Suermann D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1194-1198Crossref PubMed Scopus (341) Google Scholar). A rabbit antiserum against the catalytic domain of α-mannosidase II (21Moremen K.W. Touster O. Robbins P.W. J. Biol. Chem. 1991; 266: 16876-16885Abstract Full Text PDF PubMed Google Scholar) was a kind gift of Dr. K. Moremen (University of Georgia, Athens, GA). Secondary antibodies anti-mouse Ig-alkaline phosphatase-conjugate, anti-mouse Ig (DTAF)-conjugate and anti-rabbit Ig-tetramethylrhodamine isothiocyanate conjugate were from Dianova. Chinese hamster ovary (CHO) mutant 8G8 had been isolated from ethylmethane sulfonate-treated CHO-K1 cells (22Eckhardt M. Mühlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar) and was found to belong to the Lec2 complementation group, comprising cells with defects in the CMP-Sia-Tr gene (5Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (149) Google Scholar, 23Deutscher S.L. Nuwayhid N. Stanley P. Briles E.I.B. Hirschberg C.B. Cell. 1984; 39: 295-299Abstract Full Text PDF PubMed Scopus (198) Google Scholar). CHO cells were maintained in Ham's F-12 medium (Seromed) supplemented with 10% fetal calf serum, 1 mm sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin. COS cells were grown in Dulbecco's modified Eagle's medium (Seromed) supplemented with 5% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Mouse CMP-Sia-Tr, with carboxyl-terminal HA tag and amino-terminal FLAG sequence, respectively, were generated as described previously (5Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (149) Google Scholar, 6Eckhardt M. Gerardy-Schahn R. Eur. J. Biochem. 1997; 248: 187-192Crossref PubMed Scopus (40) Google Scholar). Using the FLAG-tagged construct as template, overlapping extension polymerase chain reaction (24Higiuchi R. Krummel B. Saiki R. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2104) Google Scholar) was used to generate two series of insertion mutants, the N constructs (N1–N15) and the HA constructs. For the production of N construct, primers were designed that introduced the sequence GGATCCAACGCTAGC at selected sites of the CMP-Sia-Tr (see Table I). This sequence, which encodes the pentapeptide GSNAS, introduces BamHI and NheI restriction sites and harbors a potential N-glycosylation site. The newly introduced restriction sites were then used to produce HA constructs by ligating a linker encoding the HA epitope (HA1-HA15). The linker was prepared by annealing the 5′-phosphorylated oligonucleotides HA sense (5′-GATCCTACCCTTATGACGTCCCCGATTACGCCAGCCTGG-3′) and HA antisense (5′-CTAGCCAGGCTGGCGTAATCGGGGACGTCATAAGGGTAG-3′). In the final constructs, the peptide sequence GSYPYDVPDYASLAS was inserted after the amino acid residue indicated in Table I (the epitope of mAb 12CA5 is underlined). Construct HA16 was generated by overlap extension polymerase chain reaction, directly introducing the HA encoding sequence (see above) between nucleotides 270 and 271 of the CMP-Sia-Tr coding sequence. A carboxyl-terminal truncated version of construct HA12 was prepared by digestion with NheI, fill-in with Klenow enzyme, and religation, resulting in HA12STOP with a stop codon immediately downstream of the HA epitope. All constructs were verified by DNA sequencing.Table ISummary of the results obtained with HA epitope-tagged CMP-Sia-Tr analyzed in this studyConstructInsertion after amino acidHA epitope orientationComplementation of CHO 8G8 cellsSubcellular localizationN1YesHA1Ala-38LumenalYesGolgiN10YesHA10Gly-68CytosolicYesGolgiHA16Ala-75CytosolicYesGolgiN2YesHA2Gly-83CytosolicYesGolgiN9NoGolgi + ERHA9Asn-102NDaND, not detectable.NoGolgi + ERN14NoHA14Leu-109LumenalNoGolgiN3NoHA3Ala-114NDNoGolgiN15NoHA15Gln-118NDNoGolgiN11NoHA11Lys-124NDNoGolgiN13YesHA13Asn-137CytosolicYesGolgiN4YesHA4Ala-165LumenalYesGolgiN5YesHA5Ser-202CytosolicYesGolgiN6YesHA6Ile-233LumenalYesbcomplementation of 8G8 cells by transient transfection of construct HA6 was less efficient and varied between different experiments, strongly depending on transfection efficiency.GolgiN7NoHA7Tyr-266CytosolicNoGolgiN8NoHA8Ser-287NDNoGolgiN12NoHA12Gln-294NDNoGolgiHA12STOPGln-294NDNoGolgia ND, not detectable.b complementation of 8G8 cells by transient transfection of construct HA6 was less efficient and varied between different experiments, strongly depending on transfection efficiency. Open table in a new tab Cells were seeded at 2.5 × 105 cells per 35-mm cell culture dish or at 1.5 × 106 per 10-cm dish. For immunofluorescence analysis, cells were seeded onto glass coverslips. Epitope-tagged CMP-Sia-Tr cDNAs were transfected using LipofectAMINE (Life Technologies, Inc.) following the instructions of the manufacturer. Briefly, 1 μg cDNA was mixed with 6 μl (24 μl) LipofectAMINE in 1 ml Opti-MEM medium (Life Technologies, Inc.) added to the cells that had been washed twice with Opti-MEM and incubated for 6–8 h. Transfections were stopped by adding 2 volumes of Dulbecco's modified Eagle's medium/Ham's F-12 medium (supplemented with 10% fetal calf serum, 1 mm sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin), and cells were analyzed 48 h later. Cells were washed twice with PBS, fixed in 4% paraformaldehyde for 15 min, again washed in PBS, and incubated for 20 min in 50 mm NH4Cl in PBS to neutralize residual paraformaldehyde. Thereafter, cells were permeabilized with 0.2% saponin, 0.1% BSA in PBS for 15 min and incubated with the respective primary antibody for 2 h at room temperature or overnight at 4 °C. Antibodies used were anti-FLAG mAb M5 (5.4 μg/ml), anti-HA mAb 12CA5 (2.6 μg/ml), and rabbit anti-α-mannosidase II antiserum (1:2000) in 0.2% saponin, 0.1% BSA in PBS. After washing four times in 0.1% BSA/PBS, cells were incubated with anti-mouse Ig-DTAF (1:100) and anti-rabbit Ig-TRITC (1:100) for 1 h at room temperature. The incubation was stopped by washing three times in 0.1% BSA/PBS. After a final wash in PBS, slides were briefly rinsed in water and mounted in moviol. Samples were visualized under a Zeiss Axiophot Epifluorescence microscope. To selectively permeabilize the plasma membrane of transfected CHO cells, we used low concentrations of digitonin (25Diaz R. Stahl P.D. Methods Cell Biol. 1989; 31: 25-43Crossref PubMed Scopus (24) Google Scholar). Cells were fixed in paraformaldehyde as described above and washed three times with PBS, and the plasma membrane was permeabilized by incubating the cells for 15 min at 4 °C in 5 μg/ml digitonin, 0.3 m sucrose, 0.1 m KCl, 2.5 mm MgCl2, 1 mm EDTA, and 10 mm Hepes, pH 6.9. Thereafter, cells were washed three times with PBS, and nonspecific binding sites were blocked with 1% BSA in PBS at room temperature for 30 min. Control cells were treated in the same way, but 0.1% saponin was added to the blocking solution to achieve complete permeabilization. For staining, cells were processed as described above, but saponin was omitted from solutions containing primary antibodies. Microsomal fractions were isolated from postnuclear supernatants of transiently transfected cells by centrifugation for 1 h at 150,000 × g. Cells or microsomal fractions were lysed in 20 mm Tris-HCl (pH 8.0), 150 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride and mixed with an equal volume of 120 mm Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 5% β-mercaptoethanol, and 0.01% bromphenol blue. Lysates were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting onto nitrocellulose as described (26Mühlenhoff M. Eckhardt M. Bethe A. Frosch M. Gerardy-Schahn R. EMBO J. 1996; 15: 6943-6950Crossref PubMed Scopus (96) Google Scholar). In case chemiluminescence detection was applied, samples were transferred onto polyvinylidene difluoride membranes (Boehringer Mannheim). Membranes were incubated with mAb 12CA5 (2.6 μg/ml), M5 (5.4 μg/ml), or 735 (10 μg/ml), in 2% nonfat dry milk in PBS. Primary antibodies were detected with anti-mouse Ig-alkaline phosphate conjugate (Dianova) using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in alkaline phosphatase buffer (100 mm Tris-HCl, pH 9.5, 100 mm NaCl, 5 mm MgCl2) as substrates. Alternatively, alkaline phosphatase was detected by chemiluminescence using disodium-3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo-[3.3.1.13,7]decan}-4-yl)phenylphosphate (Boehringer Mannheim). Two days after transfection, cells were fixed in 50% methanol/50% acetone. Polysialic acid was detected by sequential incubation with mAb 735 (10 μg/ml) and anti-mouse Ig-alkaline phosphate conjugate in 2% nonfat dry milk in PBS, as described for Western blotting. Bound secondary antibodies were revealed using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in alkaline phosphatase buffer. Putative transmembrane-spanning regions and helix ends of CMP-Sia-Tr, human and yeast UDP-Gal-Tr and a related putative nucleotide sugar transporter of Caenorhabditis elegans were estimated by using the programs PredictProtein (27Rost B. Casadio R. Fariselli P. Sander C. Protein Sci. 1995; 4: 521-533Crossref PubMed Scopus (643) Google Scholar) and interfacial hydrophobicity analysis (28White S.H. Jacobs R.E. J. Membr. Biol. 1990; 115: 145-158Crossref PubMed Scopus (29) Google Scholar). The murine CMP-Sia-Tr sequence was aligned to the human UDP-Gal-Tr, theSchizosaccharomyces pombe UDP-Gal-Tr and the relatedC. elegans protein ZK370.7 using CLUSTAL (29Higgins D.G. Sharp P.M. Gene. 1988; 73: 237-244Crossref PubMed Scopus (2885) Google Scholar). Helix ends were estimated by assuming that helix ends in corresponding transmembrane domains of different transporters are identical, if the sequences surrounding the helix ends are highly conserved. When different results were obtained for different transporter sequences or with different algorithms, the most plausible distribution of charged and polar residues was fitted to the experimental results. The murine wild-type CMP-Sia-Tr, with either amino-terminal FLAG or carboxyl-terminal HA tag, was transiently expressed in the Lec2 clone 8G8 (5Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (149) Google Scholar), and the orientation of the epitope tags was determined by immunofluorescence using digitonin-permeabilized cells. Low concentrations of digitonin selectively permeabilize the plasma membrane because of its higher concentration of cholesterol compared with intracellular membranes (30Lange Y. J. Lipid Res. 1991; 32: 329-339Abstract Full Text PDF PubMed Google Scholar). Cells were simultaneously stained with M5 and polyclonal α-mannosidase II antibodies or with mAb 12CA5 and polyclonal α-mannosidase II antibodies. Because the α-mannosidase II antiserum recognizes the catalytic domain of the enzyme exclusively (31Velasco A. Hendricks L. Moremen K.W. Tulsiani D.R. Touster O. Farquhar M.G. J. Cell Biol. 1993; 122: 39-51Crossref PubMed Scopus (281) Google Scholar), this staining could be used to control the integrity of Golgi membranes after permeabilization. As shown in Figs.1 A and2 A, both mAb 12CA5 and mAb M5 recognize their epitopes in digitonin-treated cells, whereas no α-mannosidase II staining was detectable (Figs. 1 B and2 B). After saponin permeabilization, all antibodies reached their epitopes, but the intensity of the staining with mAbs 12CA5 and M5 was not increased (Figs. 1, A and C, and 2,A and C), demonstrating that HA and FLAG epitopes were fully accessible in semipermeabilized cells. These results clearly demonstrate that CMP-Sia-Tr is composed of an even number of transmembrane domains, with amino and carboxyl termini oriented toward the cytosol.Figure 2Orientation of the amino terminus of CMP-Sia-Tr. CHO cells were grown on glass coverslips and transiently transfected with the amino-terminally FLAG-tagged mouse CMP-Sia-Tr using LipofectAMINE. Two days after transfection, cells were fixed in paraformaldehyde and incubated in 5 μg/ml digitonin to selectively permeabilize the plasma membrane (A andB) or in saponin (C and D). Thereafter cells were subjected to indirect immunofluorescence using the anti-FLAG mAb M5 (A and C). Simultaneously, all cells were stained with α-mannosidase II antiserum (B D). Bound primary antibodes were visualized using anti-mouse Ig-DTAF and anti-rabbit Ig-TRITC conjugates. Again, inaccessibility of the α-mannosidase II antiserum to its antigen revealed that the Golgi membrane was not permeabilized by digitonin treatment. Bar, 20 μm.View Large Image Figure ViewerDownload (PPT) The orientation of amino and carboxyl termini as evaluated in the first experiment was in agreement with the predicted model of the CMP-Sia-Tr (6Eckhardt M. Gerardy-Schahn R. Eur. J. Biochem. 1997; 248: 187-192Crossref PubMed Scopus (40) Google Scholar). Therefore, we used this model to select putative intermembrane loops for the generation of insertion mutants (Fig. 3). In the first set of mutants, a short nucleotide sequence that encodes the pentapeptide GSNAS was introduced at various positions of the amino-terminally FLAG-tagged murine CMP-Sia-Tr. The resulting mutants received the name N constructs, because the inserted pentapeptide contained the consensus motif for N-glycosylation. The foreign nucleotide sequence, in addition, introduced unique endonuclease restriction sites, which in a second step could be used to ligate a linker that encodes the HA epitope consisting of the amino acid sequence GSYPYDVPDYASLAS. The latter constructs received the name HA constructs. All insertion mutants are summarized in Table I. To evaluate the membrane topology of the CMP-Sia-Tr, the HA constructs were transiently expressed in CHO 8G8 cells (5Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (149) Google Scholar), and the orientation of the HA epitope was determined by immunofluorescence with the mAb 12CA5 in permeable (saponin-treated) and semipermeable (digitonin-treated) cells. Simultaneous staining with the α-mannosidase II antiserum served as a control for the integrity of Golgi membranes in digitonin-treated cells. The HA epitopes inserted at amino acid positions 38 (HA1), 109 (HA14), 165 (HA4), and 233 (HA6) could be detected after saponin but not after digitonin treatment of the cells (Fig. 4, 12CA5), indicating that the tags in these constructs are part of lumenal loops. In contrast, the HA epitopes introduced in mutants HA2, HA13, HA5, and HA7 (insertions were at amino acid positions 83, 137, 202, and 266, respectively) were detectable in both semipermeabilized and saponin-treated cells, demonstrating cytosolic orientation. Together, these results identified the hydrophobic regions I, II, IIIa, IIIb, IV, V, and VI (see Fig. 3 C) as membrane-spanning domains. Moreover, the cytosolic orientation of the HA epitope in construct HA2 and the lumenal orientation of the tag in construct HA14 clearly show that amino acids between these regions, despite of the relatively weak hydrophobic character, span the Golgi membrane. The co-localization of the transporter mutants with α-mannosidase II (Fig. 4, comparethird and fourth columns) indicates that Golgi targeting was not affected. The transporter variants HA9, HA3, HA15, HA11, HA8, and HA12 (see Figs.3 C and 8 A) were undetectable in immunofluorescence studies with mAb 12CA5; however, expression and localization could be displayed with the anti-FLAG mAb M5 (examples are displayed in Fig. 5). In the case of HA9, only few transfected cells were detectable by immunofluorescence (see Fig. 5), and the protein was not visible in Western blot (see Fig. 7). All other variants were stained with mAb 12CA5 and migrated with an apparent molecular mass of 31 kDa (see Fig. 7). The inaccessibility of the HA tags HA9, HA3, HA15, HA11, HA8, and HA12 in immunofluorescence suggests that these epitopes are inserted into transmembrane domains, or, as in the case of construct HA3, they are in close proximity to the membrane. Co-localization with α-mannosidase II was found for all constructs, but HA9 was partially retained in the ER. Correct targeting of the transporter mutants is consistent with their correct folding.Figure 5Localization of HA-tagged CMP-Sia-Tr.HA-tagged CMP-Sia-Trs that were undetectable with mAb 12CA5 were transiently transfected into CHO cells. Two days after transfection, cells were saponin permeabilized and subjected to immunofluorescence analysis using anti-HA mAb 12CA5 or anti-FLAG mAb M5. All cells stained with mAb M5 were simultaneously stained with α-mannosidase II antiserum. Results similar to those for HA11 and HA3 were obtained for constructs HA15, HA8, and HA12 (data not shown). Bar, 20 μm.View Large Image Figure ViewerDownload (PPT)Figure 7Functional analysis of HA and N constructs. Total cell lysates and microsomal fractions were prepared from CHO 8G8 cells transiently transfected with the indicated HA constructs (A) and N constructs (B) or with the empty vector pcDNA3. Samples of total cell lysates (50 μg/lane) were resolved by 7% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Functional complementation of 8G8 cells was determined by probing the membranes with polysialic acid specific mAb 735 (anti-PSA; upper panels in A and B). Membrane fractions (50 μg/lane) were resolved by 15% SDS-polyacrylamide gel electrophoresis, blotted onto polyvinylidene difluoride membranes," @default.
• W1990480314 created "2016-06-24" @default.
• W1990480314 creator A5008057473 @default.
• W1990480314 creator A5065723059 @default.
• W1990480314 creator A5089900216 @default.
• W1990480314 date "1999-03-01" @default.
• W1990480314 modified "2023-09-29" @default.
• W1990480314 title "Membrane Topology of the Mammalian CMP-Sialic Acid Transporter" @default.
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