The Saccharomyces cerevisiae DPM1 Gene Encoding Dolichol-

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Vol. 10, No. 9

MOLECULAR AND CELLULAR BIOLOGY, Sept. 1990, p. 4612-4622 0270-7306/90/094612-11$02.00/0 Copyright © 1990, American Society for Microbiology

The Saccharomyces cerevisiae DPM1 Gene Encoding DolicholPhosphate-Mannose Synthase Is Able To Complement a Glycosylation-Defective Mammalian Cell Line P. J. BECK,1* P. ORLEAN,2 C. ALBRIGHT,2 P. W. ROBBINS,2 M.-J. GETHING,13 AND J. F. SAMBROOK'

Department of Biochemistry' and Howard Hughes Medical Institute,3 University of Texas Southwestern Medical Center, Dallas, Texas 75235, and The Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 021392 Received 20 April 1990/Accepted 12 June 1990

The Saccharomyces cerevisiae DPMI gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescenceactivated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPMI CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPMJ protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines.

One of the earliest steps in the eucaryotic protein secretory pathway is the translocation of the nascent polypeptide

(51-53). Additionally, many conditionally lethal mutants of cerevisiae with defects in secretion and glycosylation have been isolated and characterized in detail (15, 46, 56). Such mutants are amenable to genetic manipulation and in many circumstances offer a potentially strong selection for the cloning of the affected genes. However, to take full advantage of the knowledge of both systems, it will be necessary to determine how similar the yeast and mammalian systems are at the molecular level. Do organisms possessing shared or homologous enzymatic functions necessarily possess similarity in organelle and protein structure at primary, secondary, tertiary, or quaternary levels? How much of what is now known about the yeast reticular and glycosylation enzymes and their localization may be correctly applied to the extensive mammalian ER network and glycosylation systems? To date, two mammalian genes, human and hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase (3) and mouse BiP (40), are known to complement mutants of S. cerevisiae that encode resident proteins of the ER. Several other examples of maminalian genes that complement S. cerevisiae cytoplasmic or nuclear functions have also been reported (for example, see references 2 and 7). Additionally, the S. cerevisiae RADIO gene is able to partially complement a DNA excision repair-defective Chinese hamster ovary (CHO) cell line (31), and certain yeast RASsc-] alleles have been shown to have transforming ability when expressed in mouse NIH 3T3 cells (9). We have expressed the S. cerevisiae gene (41) that encodes dolichol-phosphate-mannose (Dol-P-Man) synthase (EC 2.4.1.83) in B4-2-1 cells, a CHO cell line that is deficient in this enzyme activity (43, 54). The S. cerevisiae Dol-P-Man S.

chain into the lumen of the rough endoplasmic reticulum (ER). Many secretory proteins are modified shortly after translocation when a lipid-linked core oligosaccharide with the structure GlcNAc2-Man9-Glc3 is transferred from its lipid carrier, dolichol pyrophosphate, to an asparagine residue found in the consensus amino acid acceptor sequence Asn-X-Ser/Thr (28, 30). Glycoproteins are further modified when this core oligosaccharide unit is subsequently processed to form a complex or mannose-rich structure in the Golgi apparatus prior to the final transport of the protein to the cell surface or to intracellular organelles. While glycoprotein modification in the Golgi apparatus of Saccharomyces cerevisiae differs from that occurring in mammalian cells, the biochemical or enzymatic modifications and processing that occur in the ER are believed to be essentially identical in yeast and mammalian cells (25, 30). In all eucaryotic organisms, the initial N-linked glycosylation and subsequent processing of secretory proteins by various glycosyltransferases and glycosidases are part of a complex enzymatic pathway that is not only essential for the correct folding and stability of many proteins (4, 17, 21, 24, 36, 37) but is in fact coupled with and necessary for the correct routing of some of these proteins to their final destinations (16, 26). Much of our current knowledge concerning glycoprotein processing in mammalian cells has come from the isolation and characterization of lectin-resistant mutant cell lines

*

Corresponding author. 4612

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FIG. 1. DPMI expression vector. The DPMJ gene was inserted into the BgIll site in the polylinker of the pCMV vector ilustrated. The same vector was used to express DPMJ in COS-1 cells. KB, Kilobases; SV40, simian virus 40; CMV, cytomegalovirus.

synthase gene is essential for cell viability (41). The enzyme catalyzes the conversion of the nucleotide sugar GDPmannose to the lipid-linked sugar Dol-P-Man and may also effect the transfer of this product, Dol-P-Man, from the cytoplasmic face to the luminal face of the yeast ER (23, 25). Within both the mammalian and yeast ER, Dol-P-Man is believed to be the substrate of mannosyl transferases VI to IX, which catalyze the attachment of the final four mannose residues to the lipid-linked core oligosaccharide in the lumen of the ER (48, 49). It is not currently known whether the mammalian gene encoding Dol-P-Man synthase is an essential gene. B4-2-1 cells, which are deficient in Dol-P-Man synthase activity, grow well in culture and demonstrate no gross phenotypic changes caused by the absence of this enzyme activity (54). However, this cell line has previously been shown to secrete elevated levels of acid hydrolases because of an aberrant glycosylation-induced alteration of the mannose-6-phosphate receptor (43, 44, 54), and it is likely that many other B4-2-1 glycoproteins exhibit similar glycosylation-induced defects. Additionally, several proteins are now known to be anchored to cellular membranes by glycosyl-phosphatidylinositol anchors (35), and Dol-P-Man appears to play a role in the synthesis of at least some of these glycophospholipid attachments (13, 14, 59). It has also been reported that rat and hamster Dol-P-Man synthases catalyze the synthesis of mannosyl-phosphoryl-retinol (55). Consequently, it seems highly probable that the absence of Dol-P-Man synthase activity during mammalian growth and development would be lethal. In this paper, we show that the S. cerevisiae Dol-P-Man synthase gene can be expressed in a functional form in mammalian cells and that the yeast enzyme is capable of completely correcting the glycosylation defect in CHO B42-1 cells. We present evidence that the yeast gene product is

predominantly located in the ER membrane in mammalian cells. Our results suggest that the dynamics of the ER membrane environment and the molecular biology and biochemistry of N-linked protein glycosylation are highly conserved between yeast and mammalian cells. MATERIALS AND METHODS Recombinant DNA techniques. Recombinant DNA procedures and Northern (RNA) and Southern transfers were performed as described elsewhere (reference 47 and the references therein). Transformation of Escherichia coli XL1Blue (Stratagene, La Jolla, Calif.) was by the method of Hanahan (22). The cytomegalovirus promoter plasmid used for both CHO and COS-1 (18) transfections was a derivative of pCMV1 (1). Transfection procedures. The B4-2-1 cell line was transfected by the calcium phosphate method (19). Briefly, 5 x 105 cells were transfected with 1 ,ug of plasmid DNA. The cells were subsequently propagated for 48 h, at which time they were trypsinized and 10' cells were plated onto each of two 100-mm-diameter plates in medium containing 800 ,ug of G418 (GIBCO Laboratories, Grand Island, N.Y.) per ml. The G418-resistant cells (typically 5 to 50 colonies per plate) grew to confluency in approximately 3 weeks, at which time they were subjected to fluorescence-activated cell sorter (FACS) analysis and sorting. Prior to Southern analysis, the transfected populations were sequentially sorted on at least two separate occasions to ensure that the population was homogeneous. No attempt was made to clone cells from the final sorted population because these cells were repeatedly analyzed by FACS over a period of several months and no change in the phenotype of the culture was evident during that time. COS-1 cells were transfected with DEAE dextran (39, 50).

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FIG. 2. FACS analysis. Transfected cells were grown in media with (right panels) or without (left panels) swainsonine (2 ,ug/ml) for 60 h and then harvested by mild trypsinization. The cultures were suspended in medium containing 40 p.g of FITC-conjugated PHA-E per ml, incubated on ice for 1 h, washed, and analyzed by flow cytofluorometry. The y axis denotes the number of cells counted, and the x axis shows

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Cells. Parental CHO cells (CHO-Kl or wild-type CHO) and the B4-2-1 mutant cell line were kindly supplied by Pamela Stanley. Cells were maintained in culture at 37°C and 5% CO2 in RPMI 1640 supplemented with 10% fetal calf serum. G418 was added at 800 ,ug/ml to the growth media of all transfected B4-2-1 cells. COS-1 cells were grown at 37°C with 5% CO2 in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum. Antisera. The anti-Dol-P-Man synthase serum used for both immunoprecipitations and immunofluorescence was prepared as described previously (C. F. Albright, Ph.D. dissertation, Massachusetts Institute of Technology, Cambridge, 1989). Anti-hemagglutinin (HA) serum, aHA (17), was used to immunoprecipitate ihfluenza virus HA from infected cells. Immunofluorescence. Indirect immunofluorescence of DolP-Man synthase was performed after cells had been fixed and permeabilized with a -70°C solution of 50% acetone50% methanol. Cells were routinely treated with a rhodamine-conjugated goat anti-rabbit second antibody (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and a rhodamine-conjugated rabbit anti-goat third antibody (ICN Biochemicals Inc., Irvine, Calif.). Labeling and immunoprecipitations. Transfected COS-1 or CHO cells were radiolabeled for 2 h with [35S]methionine at a final concentration of 250 ,uCi/ml (Trans "S-label; ICN). Cells infected with influenza virus were infected and labeled, and the immunoprecipitates were treated with endoglycosidase H (endo H; ICN) as described elsewhere (24). All immunoprecipitations were performed as previously described (17). Oligosaccharide labeling and HPLC fractionation. Lipidlinked oligosaccharides for high-performance liquid chromatography (HPLC) analysis were labeled with 0.5 ml of 150-,uCi/ml [2-3H]mannose per 100-mm-diameter plate (American Radiolabeled Chemicals Inc., St. Louis, Mo.) as described previously (45). Oligosaccharides were extracted and prepared for chromatography by the method of Turco (58). HPLC was performed on an H.T. 1090 liquid chromatograph (Hewlett Packard Co., Palo Alto, Calif.) by the modified methods of Turco (42, 58). Presaturation, guard, and separation columns were purchased from Alltech Associates, Inc., Deerfield, Ill. Dol-P-Man synthase assays were performed on crude cell lysates as described elsewhere (27, 29). GDP-[3,4-3H]mannose was from Dupont, NEN Research Products, Boston, Mass. 3H-labeled oligosaccharide standards were prepared as described previously (32) and kindly provided by Mark Lehrman. Flow cytofluorometry. Cells analyzed by flow cytofluorometry were grown in RPMI 1640 with or without swainsonine (Sigma Chemical Co., St. Louis, Mo.) at a concentration of 2 ,ug/ml (32) for 60 to 70 h prior to being harvested by mild trypsinization. Cell suspensions were washed with RPMI 1640 supplemented with 10% fetal calf serum, and 2 x 106 cells were suspended in 200 RId of 40-,ug/ml fluorescein isothiocyanate (FITC)-conjugated lectin, phytohemagglutinin E (PHA-E) (Sigma), in serum-free RPMI 1640 and incubated on ice for 1 h. Cells were then washed three times, suspended in serum-free RPMI 1640, and subjected to cyto-

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fluorometric analysis and sorting with an Epics C FACS (Coulter Electronics, Inc., Hialeah, Fla.). RESULTS Expression of DPM1. As previously described, the DPMJ gene from S. cerevisiae (41) contains an 801-base-pair open reading frame that is capable of encoding a protein of 267 amino acids with a deduced molecular mass of 30 kilodaltons (kDa). To optimize the expression of the DPMJ gene in CHO cells, 5' and 3' nontranslated flanking sequences were removed by creating a unique BamHI site 10 bases 5' to the ATG initiation codon by oligonucleotide-generated site-directed mutagenesis and by utilizing a unique MaeIII site that occurs 15 base pairs downstream of the DPMJ termination codon. The BamHI-MaeIII fragment was ligated into the cytomegalovirus vector illustrated in Fig. 1 and transfected into B4-2-1 cells. As controls, the vector with no insert and the vector containing the DPMJ gene in a reverse orientation were also used to transfect B4-2-1 cells. The transfected cultures were subjected to selection with G418, and drugresistant cells were amplified in a bulk culture over a 3-week period. Each transfection was repeated on at least three separate occasions, and at no time did the transfection frequencies, which ranged from 10-3 to 10-4, vary significantly between the DPMJ expression construct and the vector controls. To identify cells that expressed the transfected DPMI gene and to initially characterize the N-linked glycoproteins being synthesized in these cells, we used flow cytofluorometry to quantitate cells that bound FITC-conjugated PHA-E before or after treatment with swainsonine (P. Beck, M. J. Gething, J. Sambrook, and M. Lehrman, submitted for publication), an alkaloid inhibitor of Golgi mannosidase II (10, 12). Swainsonine inhibits the a-mannosidase-catalyzed removal of two mannose residues from N-linked oligosaccharides, a reaction that occurs in the Golgi apparatus. As a result, the subsequent addition of terminal glycosyl residues to the oligosaccharide side chains does not occur on the al,6 branch of the trimannosyl "core" and swainsonine-treated wild-type cells primarily produce hybrid, mannose-rich glycoproteins (11, 57). Because B4-2-1 cells are deficient in Dol-P-Man synthase activity, they synthesize glycoproteins with truncated N-linked oligosaccharides (54). Although this truncated GlcNAc2-Man5 compound is a different structural isomer from the normal GlcNAc2-Man5 intermediate produced by a-mannosidase cleavage (34), it nevertheless serves as a substrate for the glycosyl transferases in the Golgi apparatus (32). Consequently, GlcNAc2-Man5blocked, mutant CHO glycoproteins are efficiently processed to complex glycoproteins in the presence and absence of swainsonine (32). To distinguish between cells that make wild-type oligosaccharides (GlcNAc2-Man9-Glc3) and those that are defective in Dol-P-Man synthase activity and consequently synthesize truncated glycolipids (GlcNAc2-Man5), transfected and control cultures were grown for 60 h in media supplemented with swainsonine. These cells were harvested and treated with FITC-conjugated PHA-E, a lectin that binds specifi-

the green fluorescence (measured in logarithm units) of the cells. (A) B4-2-1 cells, no swainsonine; (B) B4-2-1 cells with swainsonine; (C) wild-type CHO cells, no swainsonine; (D) wild-type CHO cells with swainsonine; (E) B4-2-1 cells transfected with the vector alone, no swainsonine; (F) B4-2-1 cells transfected with the vector alone, with swainsonine; (G) B4-2-1 cells transfected with pDPM, no swainsonine; (H) B4-2-1 cells transfected with pDPM, with swainsonine; (I) sorted and subsequently analyzed population of B4-2-1 cells transfected with pDPM with swainsonine. Vertical lines are for alignment.

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S~ FIG. 3. Expression of DPMJ in mammalian cells. (A) Southern analysis of sorted, transfected cell populations. DNA was isolated from cell cultures, digested with NdeI and BamHI, and electrophoresed on a 0.8% agarose gel. The DNA was transferred to a nylon membrane by vacuum filtration and UV cross-linked. The filter was hybridized in 6x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 1% Blotto at 65°C for 20 h to a DPMI probe prepared by polymerase chain reaction, subsequently washed in 2x SSC at 650C, and autoradiographed. Lanes: 1 and 2, 10 jig (each) of genomic DNA isolated from DPMI-transfected and sorted B4-2-1 population 1 and 2, respectively; 3, 10 ,ug of genomic DNA isolated from the B4-2-1 parental cell line; 4, 100 pg of the DPMI gene. kb, Kilobases. (B) Northern analysis of transfected cells. RNA was isolated from transfected cultures and electrophoresed on a 1.4% agarose-formaldehyde gel. The RNA was transferred to a nylon filter by electrophoresis, UV cross-linked, and hybridized in Denhardt solution with 50% formamide at 42°C for 20 h. The filter was subsequently washed in 2x SSC at 65°C and autoradiographed. Lanes 1 and 2, 50 ,ug of total RNA isolated from B4-2-1 DPMI and mock transfectants, respectively. (C) Immunoprecipitation of "S-labeled proteins from COS-1 transfectants. COS-1 cells were transfected with 2 jig of pDPM or vector sequences alone. At 47 h after transfection, each 100-mm plate was washed and pulse-labeled for

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cally to complex oligosaccharide structures (8, 20). The surface expression of complex or mannose-rich glycoproteins as detected by the presence or absence of bound fluoresceinated PHA-E was then measured by quantitative flow cytofluorometry. The FACS profiles in Fig. 2 demonstrate that wild-type CHO cells that have been grown in swainsonine bind little PHA-E when compared with control wild-type CHO cells that were propagated without swainsonine (Fig. 2C and D). Untransfected B4-2-1 cells as well as those containing vector sequences alone (Fig. 2A, B, E, and F) or the DPMJ gene in the reversed, unexpressed orientation (data not shown) were unaffected by treatment with swainsonine and display identical PHA-E-binding characteristics. However, swainsoninetreated B4-2-1 cells transfected with the pDPM construct showed a distinctive FACS profile that indicates the presence of a mixed population of cells. Approximately 65% of the cells did not bind FITC-PHA-E and therefore displayed wild-type sensitivity to swainsonine. The remaining 35% displayed the swainsonine-resistant, PHA-E-binding phenotype typical of the parental B4-2-1 cell line. The minority of transfectants that retained the parental phenotype should contain pDPM sequences because they are G418 resistant. However, it is probable that these cells do not express a functional DPMJ gene product, since indirect immunofluorescence with a primary polyclonal antiserum specific for S. cerevisiae Dol-P-Man synthase indicated that many of the cells in the original G418-resistant mixed culture contained very small or undetectable amounts of Dol-P-Man synthase (see Fig. 7). Those cells which appeared to be complemented by the DPMJ gene were sorted from the population (Fig. 21) and further characterized. S. cerevisiae Dol-P-Man synthase is present in transfected cells. The sorted population of transfected cells that displayed swainsonine-sensitive glycoprotein processing was further characterized. Genomic DNA was digested with restriction endonucleases to generate a 1,550-base-pair fragment that contained the intact gene. Southern analyses, performed on two independently transfected and sorted populations, are shown in Fig. 3A. The results indicate that cells from the first experiment contained an average of five integrated copies of the DPMJ gene per genome (Fig. 3A, lane 1). A more slowly migrating fragment of -4 kilobases probably resulted from a separate integration event in these cells that disrupted the plasmid somewhere within the 1,550base-pair fragment. The second independently transfected and sorted population (Fig. 3A, lane 2) contained only one copy of the integrated DPMJ gene. This second population was used to perform all further experiments discussed in this paper. Northern analysis indicated that only one primary DPMJ transcript, 1.8 kilobases in length, was synthesized in these transfectants (Fig. 3B). It should be noted that while the DPMI probe hybridized to an -12-kilobase genomic DNA fragment endogenous to CHO cells (Fig. 3A, lanes 1 to 3), no hybridization to an endogenous B4-2-1 (Fig. 3B) or wild-type

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(data not shown) CHO cell mRNA was observed, even after prolonged exposures of the autoradiographs. Immunoprecipitation of proteins from 35S-labeled transfectants demonstrated that anti-Dol-P-Man synthase polyclonal serum (C. F. Albright, Ph.D. dissertation) specifically recognized the 30-kDa S. cerevisiae Dol-P-Man synthase made in COS-1 cells transfected with pDPM (Fig. 3C, lane 2). Likewise, the 30-kDa product was also immunoprecipitated from DPMItransfected B4-2-1 cells (Fig. 3D, lane 2) but not from mock-transfected cells (Fig. 3D, lane 1). A 28-kDa Dol-PMan species, which appears to result from proteolysis, was also immunoprecipitated only from extracts of DPMI-transfected cells (Fig. 3D, lane 2). To further demonstrate that S. cerevisiae Dol-P-Man synthase was enzymatically active in the DPMI-transfected cells, crude membrane preparations were assayed for DolP-Man synthase activity (27, 29). Membranes from B4-2-1 DPMJ transfectants incorporated 3H-labeled GDP-mannose into lipid-linked product at a rate similar to that of membranes isolated from an equal number of wild-type CHO cells (Fig. 4). After a 25-min pulse-labeling, 15- to 20-fold more label was converted into lipid-linked product in membranes from DPMI transfectants than in membranes prepared from B4-2-1 cells transfected with the vector sequences alone. It should be possible to distinguish between the yeast and mammalian enzymes by performing more detailed and precise enzymatic characterizations. However, this was not done because no Dol-P-Man synthase activity was ever detected in the mock-transfected control populations or in B4-2-1 transfectants that contained but did not express the DPMJ gene. Glycoproteins synthesized in DPMI-transfected cells are sensitive to endo H digestion. To measure the efficiency of synthesis and subsequent transfer of complete, GlcNAc2Mang-Glc3, core oligosaccharides to a specific protein in cells transfected with the DPMJ gene, transfected and wild-type CHO cultures were infected with the AIJapan/305/ 57 strain of influenza virus. At 5 h after infection, the cells were pulse-labeled with [35S]methionine for 30 min and lysed, and the viral HA was immunoprecipitated from the lysates with a polyclonal antiserum (17). These immunoprecipitates were subsequently mock treated or digested with endo H. Endo H is able to distinguish between the truncated GlcNAc2-Man5 core oligosaccharide that is transferred to protein in mutant B4-2-1 cells (endo H resistant) and the full-length, GlcNAc2-Man9-Glc3 core unit that is transferred to protein in wild-type mammalian cells (endo H sensitive) (34, 54). After a 30-min pulse-labeling with [35S]methionine, most of the HA molecules immunoprecipitated from wildtype cells still possessed unmodified core oligosaccharides and were endo H sensitive, displaying an increased mobility on sodium dodecyl sulfate-polyacrylamide gels after digestion with the glycosidase (Fig. 5, lane 2). By contrast, the glycosylated HA molecules synthesized by B4-2-1 mock transfectants were completely endo H resistant, and no shift in gel migration was evident after glycosidase treatment.

2 h with 0.5 ml of methionine-free medium containing 250 ,uCi of [35S]methionine per ml. S. cerevisiae Dol-P-Man synthase was subsequently immunoprecipitated from cell lysates, electrophoresed on an 8% sodium dodecyl sulfate-polyacrylamide gel, and autoradiographed. Lanes: 1, "C molecular size markers (200, 92.5, 69, 46, and 30 kDa); 2, DPMI-transfected cells; 3, mock-transfected cells. (D) Immunoprecipitation of 35S-labeled proteins from B4-2-1 transfectants. CHO cell transfectants were washed and then pulse-labeled for 2 h with 0.5 ml of methionine-free medium containing 250 ,uCi of [35S]methionine per ml. S. cerevisiae Dol-P-Man synthase was subsequently immunoprecipitated from cell lysates, electrophoresed on an 8% sodium dodecyl sulfate-polyacrylamide gel, and autoradiographed. Lanes: 1, mock transfectants; 2, DPMI transfectants; 3, 30-kDa molecular size marker.

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MINUTES FIG. 4. Dol-P-Man synthase activity in transfected cells. Equivalent amounts of crude cell lysates were pulse-labeled with GDP-[3,43H]mannose. At the times indicated, portions were removed, the reactions being stopped by the addition of water-saturated N-butanol, and extracted. The organic phase containing the reaction product, mannosyl phosphoryl dolichol, was added to a xylene-based scintillant, and the incorporation of [3H]mannose into lipid-linked product was determined. Symbols: O, wild-type CHO cells; A, mock-transfected B4-2-1 cells; U, DPMI-transfected B4-2-1 cells.

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However, HA molecules immunoprecipitated from the pDPM-transfected B4-2-1 cells showed the same endo H sensitivity and gel migration pattern as did those from wild-type cells, confirming that expression of the S. cerevisiae DPMJ gene in the mutant cells completely corrects their glycosylation defect. DPMI transfectants synthesize full-length lipid-linked core oligosaccharides. To characterize the oligosaccharides generated following complementation of the glycosylation de-

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fect by the S. cerevisiae DPMJ gene, the transfected and wild-type CHO cells were pulse-labeled with [3H]mannose. The lipid-linked oligosaccharides were then extracted and analyzed by HPLC. These results (Fig. 6) show that the predominant lipid-linked mannosylated oligosaccharide synthesized in B4-2-1 mock transfectants had a GlcNAc2-Man5 structure. After an extended 30-min pulse, little or no completed core oligosaccharide, GlcNAc2-Man9-Glc3, was synthesized by B4-2-1 cells, an observation consistent with previous reports (6, 54). Inspection of the oligosaccharides present in pDPM transfectants indicates that while GlcNAc2-Man5 is the major lipid-linked oligosaccharide species found in the cells after a 10-min labeling, most of the radioactivity incorporated during a 30-min pulse is concentrated in the full-length oligosaccharide core structure, GlcNAc2-Man9-Glc3. Comparison FIG. 5. Glycoproteins made in DPMI-transfected cells are sensitive to endo H digestion. Confluent plates were infected with influenza virus, grown for 5 h at 37°C, and then pulse-labeled for 30 min with 0.5 ml of 250-,uCi/ml ["S]methionine. Cultures were lysed with Nonidet P40 buffer, and the influenza HA was immunoprecipitated. The immunoprecipitates were halved and mock treated (-) or digested with endo H (+) for 12 h followed by trichloroacetic acid precipitation and three successive acetone washes. The trichloroacetic acid-precipitated pellets were suspended in loading buffer, boiled, electrophoresed on an 8% sodium dodecyl sulfate-polyacrylamide gel, and autoradiographed. CHO WT, Wild-type CHO cells; B4-2-1 mock, mock-transfected B4-2-1 cells; B4-2-1 pDPM, DPMJtransfected B4-2-1 cells. Lane 7, 14C-labeled molecular mass markers (200, 92.5, 69, 46, and 30 kDa).

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FIG. 6. HPLC fractionation of [3H]mannose-labeled lipid-linked oligosaccharides from CHO cells. Confluent plates (100 mm) of cells were pulsed with 0.5 ml of 150-.Ci/ml [2-3H]mannose. Tritium-labeled lipid-linked oligosaccharides were extracted, and the lipid was removed by mild acid hydrolysis. The oligosaccharides were reduced with sodium borohydride and subsequently chromatographed on a silica column. Fractions (0.5 ml) were collected, and the incorporated radioactivity was determined by scintillation counting. (A and B) Wild-type CHO cultures pulsed for 10 and 30 min, respectively; (C and D) B4-2-1 mock transfectant cultures labeled for 10 and 30 min, respectively; (E and F) B4-2-1 DPMI transfectant cultures labeled for 10 and 30 min, respectively.

with the 3H-labeled oligosaccharides extracted from wildtype CHO cells demonstrates that the kinetics of tritiated mannose incorporation are similar but not identical to those measured for the pDPM transfectants. It appears that the endogenous CHO Dol-P-Man synthase may synthesize DolP-Man more efficiently or that this product may be utilized more efficiently than that made by the transfected yeast synthase. This would result in the faster accumulation of labeled mannose into GlcNAc2-Man9-Glc3 oligosaccharides that was observed for the wild-type cells. However, it should be noted that it is not possible to precisely quantitate the relative concentrations of endogenous and yeast enzymes present in the cells. Therefore, this difference in completed core oligosaccharide synthesis may simply be due to lower quantities of the S. cerevisiae enzyme. Localization of S. cerevisiae Dol-P-Man synthase in mammalian cells. Enzymes with Dol-P-Man synthase activity have been localized to the ER of S. cerevisiae (38) and mammalian cells (29). To investigate whether the transfected

yeast enzyme is associated with the ER of mammalian cells, stable CHO and transient COS-1 transfectants were grown on cover slips, fixed with methanol and acetone, and stained by indirect immunofluorescence with anti-S. cerevisiae DolP-Man synthase serum as the primary antibody. The fluorescence patterns which resulted (Fig. 7) demonstrate that in both the COS-1 and CHO cell lines, the yeast enzyme has a distinctly reticular localization.

DISCUSSION In this study, we have shown that the yeast DPMJ gene is active in mammalian cells and can fully complement the glycosylation defect in a Dol-P-Man synthase-deficient mutant CHO cell line, B4-2-1. It is notable that while the kinetics of lipid-linked oligosaccharide synthesis differ between the wild-type and complemented transfectants, the glycoprotein endo H sensitivity (Fig. 5) and lectin-binding patterns (Fig. 2) indicate that only full-length GlcNAc2-

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FIG. 7. Indirect immunofluorescence of S. cerevisiae Dol-P-Man

synthase in mammalian cells. Stably transfected B4-2-1 CHO cells (1) or transiently transfected COS-1 cells (2) were grown on cover slips, fixed with methanol and acetone (1:1), and adsorbed with anti-Dol-P-Man synthase rabbit serum. This was followed by adsorption with a rhodamine-conjugated goat anti-rabbit second antibody and a rhodamine-conjugated rabbit anti-goat third antibody.

Mang-Glc3 oligosaccharides are attached to protein in the DPMI-transfected cells. These results suggest that the biochemistry of the ER and of lipid-linked core oligosaccharide synthesis is highly conserved between S. cerevisiae and mammalian cells. For the yeast enzyme to restore GlcNAc2Mang-Glc3 core synthesis in B4-2-1 cells, several factors must be common to the ERs of both organisms. These include substrate and product concentration and availability, the absence of inhibitors, membrane fluidity, and protein localization. Expression of the DPMJ gene in CHO cells was not straightforward. The final vector depicted in Fig. 1 utilized the cytomegalovirus promoter because it was known to be transcriptionally active in most mammalian cell lines (1, 5). We found that although S. cerevisiae Dol-P-Man synthase could be immunoprecipitated from COS-1 cells transfected with a similar construct that lacked the 3' simian virus 40 t intron, no Dol-P-Man synthase protein or activity was detectable in transfected CHO cells. It seems likely that the t intron sequences in the final pDPM construct increase the mRNA stability or ensure proper processing of the DPMJ transcript in CHO cells. This was not investigated further. However, similar dependence upon the presence of 3' non-

MOL. CELL. BIOL.

translated sequences has previously been reported for the expression of the CHO thymidine kinase gene (33). The use of swainsonine to inhibit glycoprotein processing and affect lectin sensitivity was first described by Lehrman and Zeng (32). This procedure coupled with fluoresceinated lectin and FACS is a remarkably simple, sensitive, and reproducible method for the detection of cells with altered lipid-linked oligosaccharides. It also offers a potentially strong selection for the cloning of a transfected mammalian Dol-P-Man synthase gene. Additionally, this procedure, with the same or different inhibitors and lectins, may be used to clone or identify other affected genes. It is interesting that the cells which were subjected to swainsonine treatment, lectin adsorption, and sorting procedures showed no growth abnormalities when they were later plated and passaged. The results of the Southern analysis presented here indicate that the DPMJ gene hybridizes to CHO genomic DNA under stringent conditions. This may mean that the gene is highly conserved. However, we have no evidence to confirm that this hybridizing genomic sequence is an actual gene. The absence of a second band arising from hybridization of the yeast gene probe to an endogenous CHO mRNA on Northern autoradiographs prepared from CHO wild-type or B4-2-1 cells implies that it is not or that the gene is not constitutively expressed. In either case, the ability of S. cerevisiae DPMI to efficiently complement the CHO defect is somewhat surprising. Our data indicate that the yeast gene is not only able to express a functional enzymatic activity, but much further, that this enzyme is directed to its proper location in the cell, where it is able to correct a mutant phenotype and restore normal glycoprotein synthesis. Furthermore, the presence and expression of the DPMJ gene does not appear to disrupt the ER membrane environment or to have any detrimental effect upon the cells. Although yeast genes have previously been expressed in mammalian cells, we believe that these results are the first to show complete, functional, in vivo complementation of a mutant mammalian cell line by a yeast gene. This complementation indicates that there is a high degree of conservation between the yeast and mammalian ER and N-linked glycosylation systems. While many mammalian mutants have been defined biochemically, genetic analysis of these cell lines is frequently expensive and time-consuming. Often, there are no obvious selections or conditionally lethal phenotypes available to manipulate or enable cloning or identification of the affected genes. Our results confirm that expression shuttling of yeast genes with known enzymatic functions and activities into mammalian cell mutants may offer a powerful genetic tool for the identification, study, and manipulation of mammalian genes in the future. ACKNOWLEDGMENTS We thank Mark Lehrman for the generous gift of [3H]mannoselabeled oligosaccharide markers used for HPLC analysis. We thank Laura Roman for the kind gift of influenza virus and Colleen Brewer for the cytomegalovirus vector with G418 resistance. We thank Mark Segal for HPLC and Karen McCammon for FACS expertise and assistance. We thank Mark Lehrman and Ed Madison for helpful and illuminating discussions, and Mark Lehrman for critical reading of the manuscript. This work was supported by Public Health Service grants from the National Institutes of Health and a grant from Welch Foundation to J. Sambrook, M.-J. Gething, and P. W. Robbins and by an American Cancer Society Postdoctoral Fellowship (P.F. 3181) to P. Beck.

VOL. 10, 1990

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