The 48-kDa subunit of the mammalian oligosaccharyltransferase complex is homologous to the essential yeast protein WBP1.

Oligosaccharyltransferase has been purified from canine microsomal membranes as a protein complex with three nonidentical subunits of 66, 63/64, and 48 kDa. The 66- and 63/64-kDa subunits were found to be identical to ribophorins I and II, respectively. The ribophorins are integral membrane glycoproteins that were previously shown to be localized exclusively to the rough endoplasmic reticulum. The 48-kDa subunit (OST48) of the oligosaccharyltransferase complex is not a glycoprotein and is not recognized by antibodies to either ribophorin. Here, we describe the characterization of a cDNA clone that encodes OST48. Like ribophorins I and II, OST48 was found to be an integral membrane protein, with the majority of the polypeptide located within the lumen of the endoplasmic reticulum. OST48 does not show significant amino acid sequence homology to either ribophorin I or II. A 45-kDa integral membrane protein, designated WBP1, from the yeast Saccharomyces cerevisiae was found to be 25% identical in sequence to OST48. Recently, WBP1 was shown to be essential for in vivo and in vitro expression of oligosaccharyltransferase activity in yeast. We conclude that OST48 and WBP1 are homologous gene products.

Oligosaccharyltransferase has been purified from canine microsomal membranes as a protein complex with three nonidentical subunits of 66, 63/64, and 48 kDa. The 66-and 63/64-kDa subunits were found to be identical to ribophorins I and 11, respectively. The ribophorins are integral membrane glycoproteins that were previously shown to be localized exclusively to the rough endoplasmic reticulum. The 48-kDa subunit (OST48) of the oligosaccharyltransferase complex is not a glycoprotein and is not recognized by antibodies to either ribophorin. Here, we describe the characterization of a cDNA clone that encodes OST48. Like ribophorins I and II,OST48 was found to be an integral membrane protein, with the majority of the polypeptide located within the lumen of the endoplasmic reticulum. OST48 does not show significant amino acid sequence homology to either ribophorin I or 11. A 45-kDa integral membrane protein, designated WBP1, from the yeast Saccharomyces cerevisiae was found to be 25% identical in sequence to OST48. Recently, W B P l was shown to be essential for in vivo and in vitro expression of oligosaccharyltransferase activity in yeast. We conclude that OST48 and W B P l are homologous gene products.
Asparagine-linked glycosylation of proteins occurs within the lumen of the rough endoplasmic reticulum (RER)' during or shortly after transport of the nascent polypeptide across the membrane bilayer (1,2). The enzyme oligosaccharyltransferase catalyzes the transfer of a high mannose oligosaccharide (GlcNAcpMan9Glc3) onto asparagine acceptor sites within an Asn-X-Ser/Thr consensus motif, with X denoting any amino acid with the exception of proline (3). The dolichollinked oligosaccharide donor (dolichol-P-P-GlcNAc,Mang-Glc3) for the transferase reaction is preassembled by the addition of monosaccharides to dolichol phosphate by the sequential action of a series of membrane-bound glycosyltransferases (3).
Oligosaccharyltransferase has been purified from canine pancreas microsomal membranes as a protein complex consisting of three polypeptide subunits: ribophorins I and I1 and * This work was supported in part by National Institutes of Health Grant GM 43768 (to R. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M98392.
$ Performed this work during the tenure of an established investigatorship of the American Heart Association.
The abbreviations used are: RER, rough endoplasmic reticulum; PCR, polymerase chain reaction; MDCK, Madin-Darby canine kidney; kb, kilobase(s). a 48-kDa polypeptide (4). Oligosaccharyltransferase activity, as well as the protein complex consisting of ribophorins I and I1 and the 48-kDa polypeptide, can be immunodepleted from partially purified enzyme preparations by antibodies that recognize the cytoplasmic domain of ribophorin I (4). Ribophorins I and I1 are integral membrane glycoproteins of the rough, but not smooth, endoplasmic reticulum ( 5 ) that can be cross-linked to endogenous membrane-bound ribosomes (6). A functionally significant relationship between the ribophorins and ribosomes engaged in protein translocation was suggested by the intriguing discovery that the RER contains roughly equimolar amounts of the ribophorins and membranebound ribosomes (7). Although several subsequent reports suggest that the ribophorins do not participate directly in ribosome binding (8,9), antibodies directed against the cytoplasmic domain of ribophorin I inhibit protein translocation by interfering with ribosome targeting to the membrane (10). Ribophorin I, and by analogy the oligosaccharyltransferase complex, is presumably located in the immediate vicinity of the protein-conducting channels through which polypeptides are transported across the RER (11).
The protein sequences of ribophorins I and I1 have been deduced by analysis of rat and human cDNA clones (12)(13)(14). Both ribophorins contain an amino-terminal cleavable signal sequence for initiating translocation across the RER (15).
The membrane-spanning segments of ribophorins I and I1 are located toward the carboxyl terminus of the polypeptide; hence, the majority of both proteins is located within the lumen of the RER (12,13). The nonglycosylated 48-kDa subunit of the oligosaccharyltransferase (OST48) was not recognized by antibodies to ribophorins I and 11, suggesting that OST48 was not derived from either ribophorin by proteolysis (4). The finding that the oligosaccharyltransferase is a protein complex rather than a single polypeptide raised several questions concerning the structure of the complex and the function of the individual subunits. The membrane-spanning segment of ribophorin I contains a sequence that matches a proposed recognition element for the dolichol moiety of the lipid-linked oligosaccharide substrate of the enzyme (4,16). To date, biochemical or genetic data supporting a direct interaction between the proposed dolichol recognition element and a lipid-linked oligosaccharide have not been presented for any glycosyltransferase, so the functional significance of such a site within ribophorin I remains to be evaluated. Here, we describe the isolation of a cDNA clone that encodes the 48-kDa subunit of the oligosaccharyltransferase. Analysis of the amino acid sequence of OST48 revealed hydrophobic segments near the amino and carboxyl termini of the deduced protein sequence. The former hydrophobic sequence resembles a typical RER signal sequence, whereas the latter sequence resembles a membrane-spanning segment or stoptransfer sequence. The sequence of OST48 did not show homology to either ribophorin I or 11. A single significant homology was revealed by comparison of the protein sequence of OST48 to previously analyzed sequences within DNA and protein sequence data bases. WBP1, a 45-kDa yeast integral membrane protein (17), was found to be 25% identical to OST48 throughout the entire protein coding sequence.

EXPERIMENTAL PROCEDURES
Purification of 48-kDa Subunit of Oligosaccharyltransferase for Peptide Sequence Analysis-Oligosaccharyltransferase was purified from canine pancreas rough microsomal membranes as described previously using the nonionic detergent Nikkol in all purification buffers (4). Approximately 50 pmol of the purified complex was resolved into individual subunits by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred onto a nitrocellulose membrane (Schleicher & Schuell), and the subunits were localized by staining with Ponceau S (18). The stained band (-0.5 cm2) corresponding to the 48-kDa subunit of the oligosaccharyltransferase was excised from the nitrocellulose sheet, destained, and subjected to in situ trypsin digestion as described by Aebersold et al. (18) to produce peptides that were resolved by narrow-bore reverse phase high pressure liquid chromatography using a Vydac C18 column (18). The sequences of four tryptic peptides were determined by gas-phase sequencing. The initial yields of phenylthiohydantion-derivatives ranged from 5 to 10 pmol. In situ proteolysis, peptide purification, and gas-phase sequencing of tryptic peptides were performed by the Harvard University Microchemistry Facility (18).
Polymerase Chain Reaction (PCR)-PCR (19) was used to amplify DNA encoding a 25-residue tryptic peptide derived from the 48-kDa subunit of the oligosaccharyltransferase. Two degenerate oligonucleotide primers were synthesized based on the amino-terminal (primer 1) and carboxyl-terminal (primer 2) sequences of the 25-residue tryptic peptide (Fig. 1). PCR was performed in a 25-pl reaction volume with 125 pmol of each oligonucleotide primer, 0.4 unit of Taq DNA polymerase (Perkin-Elmer Cetus Instruments), and 0.1 pg of plasmid DNA isolated from a Madin-Darby canine kidney (MDCK) cell cDNA library. The MDCK cell cDNA library in the cloning vector pTEX was prepared by Herz et al. (20) and was the kind gift of Dr. Bernhard Dobberstein (EMBL). To amplify DNA encoding the 25-residue peptide from OST48, 25 cycles of denaturation (94 "C, 1 min), annealing (50 "C, 1 min), and extension (72 "C, 1.5 min) were carried out in an automatic heating/cooling cycler (Programmable Thermal Controller, MJ Research). The PCR products were resolved by gel electrophoresis using a 8% polyacrylamide gel and visualized by staining with ethidium bromide. The 91-nucleotide fragment was eluted from the gel, and ragged ends were filled using T4 DNA polymerase. The PCR product was digested with EcoRI and BamHI, cloned into EcoRI/BamHI-digested M13mp18, and sequenced by the dideoxy chain termination procedure (21). The PCR amplification product encoded the peptide used for the preparation of PCR primers (Fig. 1).
Isolation of OST48 cDNA Clones-Approximately 200,000 colonies bearing recombinant plasmids from the MDCK cell cDNA library were screened by in situ colony hybridization (22) with a 32P-labeled hybridization probe corresponding to the PCR product. The singlestranded hybridization probe was prepared from the M13mp18 clone of the PCR product by primer extension using a universal primer (23). Filters were hybridized overnight with the probe in 35% formamide, 5 x SSC, 5 x Denhardt's solution, 100 pg/ml denatured salmon sperm DNA, 0.1% SDS at 42 "C; washed in 2 X SSC, 0.1% SDS at 55 "C; and exposed for 4 h at -70 "C (22). Several hybridizationpositive colonies were selected, and plasmid DNA was isolated and digested with restriction endonucleases to determine the size of the cDNA insert. Two positive clones with 1.6-kb inserts were isolated in the initial screen (pOST48-llB and pOST48-6A). Additional OST48 clones were obtained by rescreening the cDNA library with a hybridization probe derived from the 5'-end of pOST48-llB. The 5'ends of six additional positive clones were sequenced, and one (pOST48-2) was found to contain 18 additional nucleotides.
Northern Blot Analysis-RNA was denatured with glyoxal/Me,SO, fractionated on a 1.2% agarose gel, and transferred to a Hybond N+ membrane (Amersham Corp.) by capillary action in 20 X SSC for 20 h (22). RNA was immobilized on the membrane by baking for 2 h at 80 "C under vacuum. The membranes were prehybridized, hybridized, and washed as described above using a 32P-labeled hybridization probe that corresponded to the antisense strand of the PCR product. The membrane was exposed to XAR-5 film for 3 days at -70 "C. DNA Sequencing-PstI-PstI and BamHI-PstI subclones in M13mp18 and M13mp19 were prepared from plasmid pOST48-llB. DNA sequencing of both strands of pOST48-llB was by the dideoxy chain termination method using deoxyadenosine 5'-a-[35S]thiotriphosphate (Du Pont-New England Nuclear), Sequenase (United States Biochemical Corp.), and either single-stranded M13 templates (21) or double-stranded pTEX plasmid templates (24). The M13 universal primer and synthetic oligodeoxynucleotides complementary to internal sequences were used as sequencing primers. Oligonucleotides were synthesized using an Applied Biosystems Model 392 DNA/ RNA synthesizer. The sequence shown in Fig. 2 is a composite of the sequence of the complete 1.6-kb insert in pOST48-llB (nucleotides 19-1597) and the 5"sequence from the 1.6-kb insert in pOST48-2 (nucleotides 1-18). Both strands of the cDNA insert in pOST48-llB were sequenced. Both strands of pOST48-2 were sequenced between the 5'-multiple cloning sequence and the first internal sequencing primer. DNA sequence analysis and protein sequence comparisons were done with MacVector (IBI), MicrogenieTM (Beckman Instruments), and DNA Star software programs.
Transcription, Translation, and in Vitro Integration of OST48 into Membranes-To permit transcription of an OST48 mRNA, the cDNA inserts in plasmids pOST48-2 and pOST48-llB were excised with NotI, the ends of the fragments were filled in using the Klenow fragment of DNA polymerase, and the 1.6-kb inserts were subcloned into SmaI-digested pGEM-4 (Promega Biotec) to obtain pGOST48-2 and pGOST48-11B. Full-length mRNA transcripts encoding OST48 were prepared using T7 RNA polymerase after linearization of the plasmid with EcoRI using standard procedures (25). The mRNA transcripts were isolated by extraction with phenol/chloroform and by successive precipitations with ethanol and with lithium chloride. In vitro translations of the mRNA transcripts utilized a wheat germ 523 (26) or a rabbit reticulocyte lysate (Promega Biotec) translation system (27) supplemented with placental RNase inhibitor (RNasin, Promega Biotec). Rough microsomal membranes isolated from canine pancreas (28) were added to in vitro translation reactions at a final concentration of 2 eq/25 p1. Analyses of in vitro integration experiments by proteolysis (29) and by sucrose gradient fractionation after sodium carbonate extraction have been described previously (30).

RESULTS
cDNA Cloning of OST48-Oligosaccharyltransferase was purified from canine pancreas microsomal membranes as a complex consisting of three polypeptide subunits (4). Preparative polyacrylamide gel electrophoresis in the presence of SDS resolved the 48-kDa subunit (OST48) from the 67-kDa (ribophorin I) and 63/64-kDa (ribophorin 11) subunits. After electrophoretic transfer to a nitrocellulose membrane, the 48-kDa subunit was localized by staining the nitrocellulose sheet with Ponceau S. Internal tryptic peptides released by in situ digestion of the immobilized 48-kDa subunit with trypsin were resolved by high pressure liquid chromatography (see "Experimental Procedures"). The amino acid sequences of four large internal peptides from OST48 were determined by gas-phase sequencing (Table I). Together, the four peptides correspond to -16% of the OST48 protein. Two degenerate oligonucleotide PCR primers were synthesized based on the amino acid sequence of the largest tryptic peptide (Fig. 1A). We obtained a PCR product of the anticipated size (91 base pairs) using plasmid DNA from a MDCK cell cDNA library as the template. The PCR product was eluted from a polyacrylamide gel and cloned into M13mp18 for DNA sequencing and for preparation of hybridization probes. DNA sequencing  A, the amino acid sequence (one-letter code) of a 25-residue tryptic peptide derived from the 48-kDa subunit of the oligosaccharyltransferase was used to design two degenerate oligonucleotides: primer 1 (32-fold degenerate) and primer 2 (72-fold degenerate). Inosine was used to reduce the degeneracy of primers. The valine in parentheses was not obtained in sufficient yield during peptide sequencing to allow unequivocal assignment. The 5'-extensions on primers 1 and 2 are EcoRI and BamHI linkers, respectively. Primers 1 and 2 were used in a PCR reaction with plasmid DNA from a MDCK cell cDNA library (20) serving as the template. The nucleotide sequence encoding residues 6-19 of the tryptic peptide (boned) was determined from the PCR reaction product as described under "Experimental Procedures." B, a schematic diagram of a cDNA clone (pOST48-llB) encoding the 48-kDa subunit of the oligosaccharyltransferase. The coding region is hatched, and the 5'-and 3'-ends are designated. Restriction sites for PstI used to generate subclones for sequencing are indicated. The arrows denote the direction and region sequenced using each primer. Arrows with dots designate sequences obtained using M13 sequencing primers. Additional sequencing primers were derived from internal sequence. The primer designated by an X was used to sequence the 5"portion of several other cDNA clones of OST48.
confirmed that the 91-base pair band was an authentic amplification product of an OST48 cDNA (Fig. 1A). A cDNA encoding the 48-kDa subunit of the oligosaccharyltransferase was obtained by colony hybridization screening of the MDCK cell cDNA library using a single-stranded hybridization probe derived from the PCR product. Two hybridization-positive colonies were identified in the initial screen. The size of the cDNA insert was estimated by agarose gel electrophoresis after digestion of positive plasmids with restriction enzymes (BamHI, NotI, SphI, or KpnI) that recognize sites within the linkers used in the construction of the cDNA library (20). Both plasmids (pOST48-llB and pOST48-6A) contained inserts of -1.6-1.65 kb. Preliminary Northern blot analysis of MDCK cell mRNA indicated that the predominant mRNA species for OST48 was -1.7 kb. The complete sequence of the pOST48-llB cDNA was determined using the sequencing strategy outlined in Fig. 1B. Nucleic Acid and Protein Sequences of OST48"The sequence of the pOST48-llB cDNA insert corresponds to nucleotides 19-1597 shown in Fig. 2. The cDNA library was rescreened with a hybridization probe derived from the 5'end of the 11B insert. Five additional positive colonies were obtained that harbored plasmids containing inserts of -1.6 kb. DNA sequencing of the region adjacent to the multiple cloning site in the latter plasmids revealed that the insert in one plasmid (pOST48-2) contained an additional 18 bases of the 5"sequence and a second plasmid was identical to pOST48-llB, whereas the inserts in three plasmids contained several fewer nucleotides than pOST48-llB. The sequencing primers used for the latter analysis were the M13 universal primer and the internal primer designated by the X in Nucleotide sequence, predicted amino acid sequence, and hydropathy analysis of 48-kDa subunit of oligosaccharyltransferase. Nucleotide residues are numbered on the right; amino acid residues are numbered on the left. The termination codon is designated as TER. The first 10 adenosines of a 3'-poly(A) sequence are shown. The deduced amino acid sequence is shown using the one-letter code. The four tryptic peptides derived from OST48 are underlined with solid lines. Residues denoted with asterisks were not obtained in sufficient yield to assign an amino acid. Hydropathy analysis was performed as described by Kyte and Doolittle (32) using a 20-amino acid averaging window. The dashed line beneath residues 417-436 designates a predicted membrane-spanning segment. The predicted signal peptidase cleavage site between residues 31 and 32 is designated by an arrow (34). of 49,631. In addition to the first AUG codon (nucleotides 4-6), there is a second in-frame AUG codon near the 5'-end of the cDNA insert (nucleotides 22-24). The context of both AUG codons is favorable for initiation of translation in that purines are present at positions -3 and +4 relative to the A of the AUG codon (31). Assuming that the 5"sequence obtained from pOST48-2 is derived from an authentic OST48 mRNA, then it is likely that initiation of translation occurs at the AUG codon that is closest to the 5'-end of the mRNA (31). Exceptions to this rule occur when the first AUG codon is located within 10 nucleotides of the mRNA cap site. Further analysis will be required to determine the length of the 5'noncoding region of the OST48 mRNA.
Northern blot analysis using the antisense strand of the PCR product as a probe revealed a mRNA of -1.7 kb in the total RNA isolated from COS, Chinese hamster ovary, and MDCK cells (Fig. 3, lanes a, b, and d, respectively). OST48 mRNA of a similar size was detected in poly(A)-selected mRNA isolated from canine pancreas (lane c ) . The migration position of a 1.7-kb T 7 RNA polymerase transcription product of the OST48 cDNA is designated by the asterisk. The 3'noncoding segment of the cDNA insert in pOST48-llB contained 40-45 adenosine residues in addition to those shown in Fig. 2. Assuming that the cellular RNA contains a typical polyadenosine tract of -200 nucleotides, we can conclude that

In Vitro
Membrane Integration of OST48"The membrane orientation of OST48 was investigated by in vitro translation of an OST48 mRNA transcript. The cDNA inserts from plasmids pOST48-llB and pOST48-2 were subcloned into pGEM-4 to allow transcription of mRNA with T7 RNA polymerase. Translation of the mRNA transcripts in a rabbit reticulocyte lysate translation system yielded mRNA-dependent translation products with apparent M , values of 50 (Fig.   4A, lanes a and c ) . The translation product encoded by the OST48-2 RNA transcript migrated slightly less rapidly than that encoded by the OST48-llB transcript, consistent with the presence of 6 additional amino-terminal amino acids. As expected, both AUG codons appear to be adequate for initiation of translation in the in vitro system. The inclusion of canine pancreas microsomal membranes increased the mobility of the translation product, consistent with proteolytic the cDNA sequence determined here is derived from a nearly full-length mRNA.
The locations of the four tryptic peptides from which amino acid sequence was obtained are indicated in Fig. 2 (solid  underlining). The agreement between the cDNA-derived sequence and the peptide sequence data was exact for all amino acid residues within the tryptic peptides that were unambiguously assigned. Hydropathy analysis using the method of Kyte and Doolittle (32) revealed the presence of two major hydrophobic protein segments, which are indicated by the negative hydrophilicity values in the graph in Fig. 2. The amino-terminal hydrophobic segment located between residues 15 and 31 resembles a cleavable signal sequence for initiating translocation across the endoplasmic reticulum (33,34). Eukaryotic signal sequences range in length from 13 to 36 amino acids and are composed of three structural regions: an amino-terminal basic region (n region), a central hydrophobic segment (h region), and a more polar carboxyl-terminal region (c region) (33). These three regions are readily identified within the first 31 residues of OST48. Signal peptidase cleavage sites are located in the c region, with small uncharged amino acids located at positions -1 and -3 relative to the processing site (33). The signal peptidase processing site in OST48 is predicted to be between residues 31 and 32 using the weight-matrix method of von Heijne (34). The calculated M , of 46,141 for the processed protein is in reasonable agreement with the M, of 48,000 estimated from mobility on SDS-polyacrylamide gels. A second nonpolar segment located between residues 417 and 436 is of sufficient length and hydrophobicity to be a membrane-spanning segment. Based upon this arrangement of hydrophobic segments, we would anticipate that the majority of OST48 is located within the endoplasmic reticulum lumen, with residues 437-445 located in the cytoplasm. The predicted cytoplasmic domain of OST48 is 9 amino acids in length and is highly charged. Membrane proteins that contain a single spanning segment and a carboxyl-terminal cytoplasmic domain are typically designated as type I integral membrane proteins. OST48 does not contain an Asn-X-Ser/Thr consensus site for Asn-linked glycosylation, consistent with our previous observation that the 48-kDa subunit of the oligosaccharyltransferase complex is not glycosylated (4). (lanes b and  d ) . The processed forms derived from the two precursors had identical mobilities, indicating that the 6 amino-terminal residues encoded by the pOST48-2 insert were not essential for the function of the signal sequence. The processed form of the in vitro translation product co-migrated with the Coomassie Blue-stained 48-kDa subunit of the oligosaccharyltransferase complex (data not shown).

removal of an amino-terminal signal sequence
Translation products produced in the absence and presence of microsomal membranes were subjected to extraction with sodium carbonate (pH 11.5) to determine whether OST48 behaves as an integral membrane protein or as a peripheral membrane protein or luminal content protein (Fig. 4A, lanes  e-h). Luminal content proteins, as well as peripheral mem-  2 (lanes a, b, and eh) and pGOST48-11B (lanes c and d ) were translated in a rabbit reticulocyte lysate translation system in the absence (lanes a, c, e,  and f ) or presence (lanes b, d, g, and h) of rough microsomal membranes (RM). Two samples were separated into supernatant (S) and pellet (P) fractions by Airfuge centrifugation after adjustment to 0.1 M Na2C03 (see "Experimental Procedures"). Translation products were resolved by polyacrylamide gel electrophoresis in SDS and visualized by fluorography. The precursor (pOST48) and mature (OST48) forms of the 48-kDa subunit are designated by arrows. In vitro translated OST48 co-migrated with purified canine OST48.
Lanes e-h were taken from a separate gel. B, T7 RNA polymerase transcripts of pGOST48-2 were translated in a wheat germ translation system in the absence (lanes a-c) or presence (lanes d-h) of rough microsomal membranes. After translation, several samples were digested with trypsin (lanes e andf) or proteinase K ( P K ) (lanes  a, b, g, and h) either in the absence (lanes a, e, and g) or presence  (lanes b, f, and h) of 1% Triton X-100 (TX-100).

P K + + ---"
+ + brane proteins, are extracted from RER-derived vesicles by sodium carbonate because the membrane vesicles are converted into sheets (35). The precursor form of OST48 (pOST48) was recovered in the supernatant (S) fraction irrespective of whether membranes were included in the i n vitro translation reaction (lanes e andg). A significant fraction of OST48 sedimented with the membrane pellet ( P ) after alkaline extraction (lane h). The incomplete recovery of proteolytically processed OST48 in the pellet fraction suggests that the i n vitro integrated form of OST48 may be less stably associated with the membrane bilayer than several other integral proteins that have been analyzed by this procedure (30,36). Nonetheless, the selective recovery of OST48 in the pellet fraction supports the identification of the carboxylterminal hydrophobic sequence of OST48 as a membranespanning segment.
The i n vitro translation products were digested with proteases to examine the membrane orientation of i n vitro integrated OST48 (Fig. 4B). Trypsin was initially selected for these protease protection experiments due to the presence of 3 lysyl residues within the putative 9-amino acid cytoplasmic domain of OST48. Although no increase in migration of OST48 was observed following trypsin digestion (lanes d and e ) , cleavage at all but the most amino-terminal of these three sites would probably not cause a detectable alteration in the mobility of OST48 on an SDS-polyacrylamide gel. Because an incomplete digestion of OST48 in detergent-permeabilized vesicles was routinely observed (lane f ) , additional protease protection experiments were conducted using proteinase K.
As expected, the 50-kDa precursor form of OST48 was completely digested by proteinase K when translated in the absence (lanes a-c) or presence (lanes d, g, and h )  Homology between OST48 and WBPl-A search of DNA (GenBank'" and EMBL) and protein (National Biomedical Research Foundation) sequence data bases disclosed a homology between OST48 and WBP1, a 45-kDa membrane protein from the yeast Saccharomyces cerevisiae. The WBPl gene was initially cloned as a possible nuclear pore protein based upon the identification of WBPl as a 45-kDa wheat germ agglutinin-binding protein (17). The WBPl gene encodes a protein of 430 amino acids, the first 20 of which correspond to an amino-terminal signal sequence (17). A second hydrophobic sequence near the carboxyl terminus of the protein was shown to function as a membrane-spanning segment i n vivo, thereby indicating that W B P l is a type I integral membrane protein (17). Immunofluorescence localization and cell fractionation experiments revealed that WBPl is localized to the nuclear envelope and endoplasmic reticulum i n vivo (17). Optimal alignment of the two protein sequences was obtained by introducing two large gaps in the W B P l sequence (Fig. 5). The sequence similarity between OST48 and WBPl extends throughout the luminal domains of the two proteins, as shown by sequence identities, which are boxed, and by chemically similar amino acids, which are designated by colons. Overall sequence identity between the two proteins is 25%, whereas sequence similarity was estimated to be 50%. The region of greatest sequence identity between OST48 and WBPl extends between the second large gap introduced in the WBPl sequence (Alaza1 of OST48) and the predicted membrane-spanning segment of both proteins (Ile411 of OST48). The two sequences are 34% identical within this 131-amino acid region. Interestingly, W B P l contains two consensus sites for Asn-linked glycosylation that are not conserved in OST48. Although the membrane-spanning and cytosolic domains of OST48 and WBPl contain chemically similar amino acids, similar levels of homology in these regions would probably be observed if two unrelated type I integral membrane proteins were compared. The membranespanning segments of both proteins do not match the proposed dolichol recognition element (4,16).

DISCUSSION
The protein sequences of ribophorins I and I1 had been determined (12)(13)(14) before these polypeptides were identified as subunits of the mammalian oligosaccharyltransferase complex. The objective of this study was to determine the sequence of the 48-kDa subunit. Several types of experimental evidence demonstrate that the cDNA clone isolated here encodes the 48-kDa protein present in the oligosaccharyltransferase preparation. First, the protein sequence encoded by the cDNA clone contains the sequences of three tryptic peptides in addition to the one used to design the PCR primers. Second, the i n vitro synthesized and membraneintegrated form of OST48 co-migrates with purified OST48 on SDS-polyacrylamide gels. Finally, the absence of consensus sites for Asn-linked glycosylation and the presence of a membrane-spanning segment are consistent with the available data concerning OST48.
Hydropathy analysis of OST48 revealed the presence of two hydrophobic segments located near the extreme amino and carboxyl termini of OST48. The amino-terminal hydrophobic segment was shown by in vitro translation experiments to function as a cleavable signal sequence for initiating translocation across the endoplasmic reticulum. The amino terminus of endogenous canine OST48 corresponds to serine 32.' Thus, the authentic signal peptidase cleavage site for OST48 agrees with the predicted signal peptidase cleavage site. The in vitro integration experiments suggest that the second hydrophobic segment functions as a membrane-spanning segment. Although further experimentation will be required to analyze the membrane topology of endogenous canine OST48, the current evidence suggests that OST48 is a type I integral membrane protein with the majority of the protein located within the endoplasmic reticulum lumen. The predicted 9-amino acid cytoplasmic domain of OST48 is significantly smaller than the cytoplasmic domains of ribophorins I and I1 (150 and 70 amino acid residues, respectively) (12)(13)(14). The sequence of OST48 was compared to those of both ribophorins to determine whether there was any homology between the three subunits of the oligosaccharyltransferase complex. Previous investigators have determined that the two ribophorins are not homologous (13,14), and we did not detect significant homology between OST48 and either ribophorin.
Protein immunoblot experiments using antibodies to ribophorins I and I1 have demonstrated that the ribophorins are present in many tissues from different vertebrate species (13,37). The latter observations are consistent with the proposed function of the ribophorins as subunits of an oligosaccharyltransferase complex since asparagine-linked glycosylation is a ubiquitous protein modification reaction in eukaryotic organisms. The broad distribution of the ribophorins raised the question of whether OST48 is present in tissues other than canine pancreas. Northern blot analysis revealed that the OST48 mRNA can be detected in primate, rodent, and canine tissue culture cells. More extensive Northern analysis of other vertebrate mRNA preparations was precluded by our finding that OST48 was homologous to a protein that had recently been identified in S. cereuisiae (17).
The 25% identity in sequence between canine OST48 and S. cereuisiue W B P l suggests that these two proteins perform analogous functions in these two diverse organisms. Confirmation of such a hypothesis was obtained while this work was in progress in an elegant series of yeast molecular genetics experiments (17,38). The WBPl gene was found to be required for viability of yeast (17). Based upon the hypothesis that WBPl was required for an essential function of the endoplasmic reticulum, te Heesen et al. (38) constructed a yeast strain bearing a single copy of the WBPl gene under control of the glucose-repressible Gall promoter. Depletion of WBPl was accompanied by underglycosylation of the vacuolar protein carboxypeptidase Y (38). A mutation in the W B P l gene was isolated (wbpl-1) that results in underglycosylation of proteins and a temperature-sensitive growth phenotype (38). Extracts prepared from the WBPl-depleted cells or wbpl-1 cells showed greatly reduced oligosaccharyltransferase activity relative to wild-type extracts when assayed using exogenous dolichol-P-P-GlcNAcz as the oligosaccharide donor and a synthetic peptide as the acceptor (38). Furthermore, overexpression of the WBPl protein does not increase detectable oligosaccharyltransferase activity (38). T. A. Rapoport, personal communication.
Likewise, overexpression of OST48 in a mammalian cell line would not be expected to enhance oligosaccharyltransferase activity.
The conclusions that have been drawn from the analysis of W B P l are in good agreement with our findings concerning the mammalian oligosaccharyltransferase complex. The finding that overexpression of the WBPl protein does not increase enzyme activity suggests that the yeast oligosaccharyltransferase is a protein complex (38). A compelling argument supporting a role for OST48 (WBP1) in Asn-linked glycosylation has now been provided by the fact that two entirely independent approaches led to the identification of this protein as a subunit of the oligosaccharyltransferase complex. Current studies in our laboratory are directed toward the purification of the yeast oligosaccharyltransferase complex. It will be of interest to determine whether the yeast enzyme has subunits that are homologous to the ribophorins.