Cell

Vol. 69. 55-65,

April 3, 1992, Copyright

0 1992 by Cell Press

Oligosaccharyltransferase Activity Is Associated with a Protein Complex Composed of Ribophorins I and II and a 48 kd Protein Daniel J. Kelleher,’ Gert Kreibich,f and Reid Gilmore’ *Department of Biochemistry and Molecular Biology University of Massachusetts Medical School Worcester, Massachusetts 01655 TDepartment of Cell Biology New York University Medical Center New York, New York 10016

Summary Oligosaccharyltransferase catalyzes the N-linked glycosylation of asparagine residues on nascent polypeptides in the lumen of the rough endoplasmic reticulum (RER). A protein complex composed of 66,63, and 46 kd subunits copurified with oligosaccharyltransferase from canine pancreas. The 66 and 63 kd subunits were shown by protein immunoblotting to be identical to ribophorin I and II, two previously identified RERglycoproteins that colocalize with membrane-bound ribosomes. The transmembrane segment of ribophorin I was found to be homologous to a recently proposed dolichol recognition consensus sequence. Based on a revision of the consensus sequence, we propose a model for the interaction of dolichol with the glycosyltransferases that catalyze the assembly and transfer of lipid-linked oligosaccharides. Introduction The biosynthesis of N-linked glycoproteins is initiated in the rough endoplasmic reticulum (RER) by the en bloc transfer of a high mannose (GlcaMansGlcNAc,) oligosaccharide onto asparagine residues of newly synthesized polypeptides. Sequence analysis of N-linked glycopeptides has established that the acceptor site is an asparagine within the sequence motif Asn-X-SerIThr, where X can be any amino acid other than proline. The enzyme that catalyzes transfer of high mannose oligosaccharide to Asn-X-Ser/Thr sites is dolicholdiphosphoryloligosaccharide:protein oligosaccharyltransferase, or simply oligosaccharyltransferase. Membrane-permeable synthetic tripeptides containing an Asn-X-Ser/Thr site can be glycosylated by the oligosaccharyltransferase when added to tissue culture cells (Wieland et al., 1987; Geetha-Habib et al., 1990) or intact microsomal membranes (Lau et al., 1983; Welply et al., 1983). Glycosylated tripeptide products are trapped within the microsomal membrane vesicle, indicating that the active site of the enzyme faces the lumen of the RER (Welply et al., 1983). Glycosylation of asparagine residues is a cotranslational reaction, with transfer occurring immediately after transport of the nascent polypeptide into the RER lumen (Rothman and Lodish, 1977; Glabe et al., 1980). The oligosaccharide donor for the transferase reaction is the dolichol-linked compound GlcsMansGlcNAcnPP-dolichol (OS-PP-Dol) (Liu et al., 1979). Synthesis of the lipid-linked oligosaccharide donor is initiated by glycosyl-

transferases that face the cytoplasmic side of the RER and completed by transferases that face the RER lumen (Snider and Rogers, 1984; Hirschberg and Snider, 1987; Abeijon and Hirschberg, 1990). When the initial reaction in lipid-linked oligosaccharide assembly is blocked by treatment of cells with tunicamycin, malfolded nonglycosylated proteins accumulate in the lumen of the ER (Elbein, 1987). Thus, the oligosaccharyltransferase functions at a point where the protein translocation and oligosaccharide assembly pathways converge. The identification of the oligosaccharyltransferase has proven to be difficult. Photoaffinity probes based upon synthetic tripeptide acceptors were used to identify a 60 kd protein from hen oviduct as the oligosaccharyltransferase (Welply et al., 1985). Subsequent experiments demonstrated that the glycosylated tripeptide photolabel was attached to a luminal 57 kd protein that was then named glycosylation site-binding protein (GSBP). The sequence of chicken GSBP was found to be 90% identical to rat or human protein disulfide isomerase (PDI), an abundant luminal RER protein (Geetha-Habib et al., 1988). Luminal proteins, including GSBP, were subsequently shown to be dispensable for glycosylation of in vitro translocated proteins (Vu et al., 1989; Noiva et al.. 1991). Purifications of the oligosaccharyltransferase have been reported by previous investigators who utilized glycosylation site (NXT/S)-containing peptides as affinity chromatography reagents (Das and Heath, 1980; Aubert et al., 1982). Although a 2000-fold purification was reported by one laboratory, the preparation was not characterized by SDSpolyacrylamide gel electrophoresis (Das and Heath, 1980). The preparation obtained by the other laboratory contained a predominant 69 kd polypeptide and minor polypeptides of 61 and 54 kd (Aubert et al., 1982). More recent reports indicate that the enzyme has yet to be purified to sufficient homogeneity to allow identification of the protein (Chalifour and Spiro, 1988). Here we report the copurification of oligosaccharyltransferase with a protein complex composed of 66, 63, and 48 kd subunits. The 66 and 63 kd subunits of the protein complex werefound to be ribophorin I and ribophorin II, respectively. The ribophorins are relatively abundant integral membrane glycoproteins that are restricted to the RER (Kreibich et al., 1978b; Marcantonio et al., 1984). The protein sequences of ribophorins I and II have been determined by sequencing rat (Harnik-Ort et al., 1987; Pirozzi et al., 1991) and human (Crimaudo et al., 1987) cDNA clones. The membrane-spanning segment of ribophorin I appears to be homologous to a protein sequence motif that has been proposed to be a recognition site for the polyisoprenoid dolichol (Albright et al., 1989). Results Purification of Oligosaccharyltransferase Enriched preparations of the oligosaccharyltransferase were obtained from canine pancreas rough microsomal

Cell 56

Table

1. Purification

of Oligosaccharyltransferase

from Canine

Protein Fraction”

(ms)

Rough microsomes Stripped rough microsomes Detergent extractC (c) Glycerol gradient (d) OAE-Sephadex (e) Mono-S eluate (9

17.2 7.6 5.02 0.198 0.079 0.014

(b)

Pancreas

Total ActivityD (pmollmin)

Specific Activity (pmollmin per mg)

Yield PM

Enrichment

172.0 177.0 91.2 72.7 42.6 13.8

10.0 23.3 18.2 367.2 539.2 985.0

100 103 53 42 25 8

1 2.3 1.8 36.7 53.9 98.5

a The letter in parentheses following selected fractions designates the corresponding gel lane in Figure 1. b Activity assays for rough microsomes and stripped rough microsomes utilized intact membranes. Activity assays for all other fractions were in detergent solution. c The detergent extract refers to a 1% Nikkol, 500 mM NaCl extract prepared from 2100 eq of membranes that had been depleted of extrinsic and luminal proteins as described in the Experimental Procedures.

membranes by combining the following purification steps: selective removal of peripheral and luminal proteins, detergent solubilization of the enzyme, glycerol gradient centrifugation, and anion- and cation-exchange chromatography. Previous purification attempts have been hindered by the lability of the detergent-solubiiized activity (Welply et al., 1986) and by the complex and relatively insensitive assay for the oligosaccharyltransferase. Technical developments from several laboratories provided solutions to these problems (Welply et al., 1983; Wieland et al., 1987; Chalifour and Spiro, 1988). We confirmed the results of Chalifour and Spiro (1988) showing that phosphatidylcholine stabilizes the detergent-solubilized activity when added to assays and chromatography buffers. To enhance

a

b

c

d

e

f

212-e

.*.

+66 4-63 t46

30+

21 + 14A

Figure 1. SDS-Polyacrylamide transferase Purification

Gel Analysis

of the Oligosaccharyl-

The samples are: lane a, molecular weight markers; lane b, saltstripped microsomal membranes (7.5 eq); lane c, detergent extract of intrinsic membrane proteins (7.5 eq); lane d, glycerol gradient pool (29 eq); lane e, QAE-Sephadex pool (26 eq); lane f, Mono-S eluate (148 eq). The quantities of protein loaded are defined with respect to the number of equivalents (eq) of microsomal membranes from which they are derived. The photograph of lane e is from a different gel and has been aligned to show an identical migration of the 48, 63, and 66 kd polypeptides.

the sensitivity of the oligosaccharyltransferase assay, we used the iodinated synthetic tripeptide Na-Ac-Asn-[1251] Tyr-Thr-NH* as the acceptor substrate (Wieland et al., 1987; Geetha-Habib et al., 1990). Protein purification was monitored by activity assays (Table l), and the polypeptide composition of selected fractions from the purification was evaluated by Coomassie blue staining of an SDS-polyacrylamidegel (Figure 1, lanes b-f). Peripheral membrane proteins were removed from rough microsomal membranes by extraction with 0.5 M NaCl to obtain the stripped microsomal membranes shown in Figure 1 (lane b). Luminal proteins were selectively removed by permeabilizing the stripped membranes with 0.05% Nikkol, a nonionic detergent, in the presence of 100 mM NaCI, followed by centrifugation. The permeabilized membranes were then solubilized with 1% Nikko1 in the presence of high salt (0.5 M NaCI) to prepare the detergent extract shown in lane c of Figure 1. Glycerol gradient centrifugation of the detergent extract separated the oligosaccharyltransferase activity from the majority of RER membrane proteins (Figure 1, compare lanes c and d). The 20-fold enrichment achieved by glycerol gradient centrifugation (Table 1) was due to the sedimentation of the oligosaccharyltransferase activity as a 1 OS species. Further purification of the activity was achieved by chromatography on a quaternary aminoethyl (QAE)-Sephadex ion-exchange column (lane e) and a Mono-S fast protein liquid chromatography (FPLC) column (lane f). Three major polypeptides with molecular masses of 66, 63, and 48 kd copurified with the activity (Figure 1, lane f). The eluate from the Mono-S column was enriched approximately 1OO-fold relative to the rough microsomal membrane fraction (Table 1). The detergent extract of the stripped and permeabilized microsomal membranes contained 82% of the oligosaccharyltransferase activity present in a detergent extract of intact rough microsomal membranes (data not shown). These results indicate that extrinsic and luminal RER proteins are not essential for oligosaccharyltransferase activity. Only 53% of the enzyme activity present in the intact membrane was recovered in the detergent extract (Table l), despite the lack of detectable activity in the detergentinsoluble membrane residue (data not shown). However, the 53% yield during detergent solubilization may be subin the assay conditions ject to error, owing to differences

Purification 57

of Oligosaccharyltransferase

A

a

b

c

d

e

212-b -

--

*

C PDI -w

-

Figure 2. ConA and Protein ryltransferase Purification

lmmunoblot

Analysis

of the Oligosaccha-

Nitrocellulose blots were probed with (A) ConA peroxidase. (6) a mixture of monoclonal antibodies to ribophorin I and ribophorin II, and(C) rabbit antiserum to protein disulfide isomerase. The fractions analyzed were: lane a, salt-stripped microsomal membranes (1.9 eq); lane b, detergent extract of intrinsic membrane proteins (1.9 eq); lane c. glycerol gradient pool (7.3 eq); lane d, OAE-Sephadex pool (6.5 eq); lane e, Mono-S eluate (39.6 eq). The arrows on the righthand side of (A) designate the migration positions of the 66,63, and 46 kd polypeptides as detected by colloidal gold staining of the nitrocellulose blot. Ribophorin I (RI), ribophorin II @II), and PDI are designated by the labeled arrows adjacent to (6) and (C).

for intact and detergent-solubilized samples. From Figure 1, we conclude that the oligosaccharyltransferase preparation contains three major polypeptides that could potentially be responsible for the enzyme activity. Identification of the 66 and 63 kd Polypeptides The glycoproteins present in the samples analyzed in Figure 1 were identified by probing a nitrocellulose blot of a similar gel with horseradish peroxidase-conjugated concanavalin A (ConA) (Figure 2). This experiment was conducted because calf thyroid oligosaccharyltransferase activity binds to ConA-Sepharose; hence, the bovine enzyme presumably contains high mannose oligosaccharide (Chalifour and Spiro, 1988). Canine oligosaccharyltransferase also binds to ConA-Sepharose, and can be eluted with a-methyl mannoside (data not shown). A 66 kd glycoprotein that was enriched during the purification comi-

grated precisely with the 66 kd protein present in the Mono-S eluate (Figure 2, lane e; designated by the upper arrow). The blot also showed that the 63 and 48 kd proteins did not bind ConA (Figure 2A, lower two arrows on right). A less abundant 64 kd glycoprotein comigrated precisely with a polypeptide that was faintly visible in the Coomassie blue-stained gel (see Figure 1, lanes d-f). Two less abundant glycoproteins (70-80 kd) were detected in the Mono-S eluate by ConA blotting (Figure 2A, lane e). We determined whether the 64 and 66 kd polypeptides were identical to known RER glycoproteins by immunoblotting. Canine ribophorin I migrates as a 66 kd glycoprotein on SDS-polyacrylamide gels (Marcantonio et al., 1982). Antibodies raised against rat liver ribophorin II recognize 63 and 64 kd polypeptides from canine microsomal membranes (Marcantonio et al., 1982). The more abundant 63 kd protein is presumed to be a nonglycosylated variant of the 64 kd ribophorin II (Crimaudo et al., 1987). Based upon the similarity between the molecular weights of the canine ribophorins and the glycoproteins detected in Figure 2A, a second nitrocellulose blot was probed with a mixture of monoclonal antibodies raised against rat liver ribophorins I and II (Figure 28). The anti-ribophorin I monoclonal antibody recognized the 66 kd glycoprotein in the oligosaccharyltransferase preparation, while two polypeptides were recognized by the anti-ribophorin II monoclonal antibody (Figure 28). The more abundant 63 kd form of ribophorin II comigrated with the nonglycosylated 63 kd protein detected by Coomassie blue staining in Figure 1, while the less abundant 64 kd form comigrated with the 64 kd glycoprotein detected with ConA in Figure 2A. We can conclude that the 63, 64, and 66 kd polypeptides in the oligosaccharyltransferase preparation are ribophorins I and II. lmmunoblot analysis showed that PDI is not present in the oligosaccharyltransferase preparation (Figure 2C); hence, the luminal protein PDI, which is homologous to GSBP, is not a subunit of the oligosaccharyltransferase. Luminal proteins, including PDI, were separated from the oligosaccharyltransferase activity when the membranes were permeabilized with 0.05% Nikko1 (Figure 2C, lane b). Oligosaccharyltransferase Activity Is Specifically Depleted by Antibody to Ribophorin I Although the preceding experiment identified two of the three major proteins in the oligosaccharyltransferase preparation as ribophorins I and II, the immunoblot experiment did not provide evidence that the ribophorins are responsible for the oligosaccharyltransferase activity. To establish a more direct link between the ribophorins and the enzyme, we asked whether oligosaccharyltransferase activity could be immunodepleted from detergent solution by an immobilized anti-peptide antibody raised against the cytoplasmically exposed C-terminal 20 residues of rat ribophorin I (Vu et al., 1990). For this experiment, a QAESephadex pool from an oligosaccharyltransferase preparation was incubated with the RI& antiserum immobilized on protein A-Sepharose beads. Enzyme samples were incubated in duplicate for each quantity of immobilized IgG. Depletion of the oligosaccharyltransferase activity

Cell 58

A

a

A

b

c

212+

B a

Antiserum

b

30+#@&

( p I)

8 Antiserum

0

0

6

6

12

12

18

18

24

24

24'

24'

Figure 4. Specific the RI& Antibody

*I+

._. gG -e 48kD+

Figure 3. lmmunodepletion Anti-Ribophorin I Antibody

of Oligosaccharyltransferase

Activity

by

(A) Aliquots of a QAE-Sephadex eluate were incubated in duplicate for 90 min at 4OC with protein A-Sepharose beads that had been precoated with 0 to 24 ul of WCs antiserum (closed squares, closed triangles) or 24 ul of nonimmune rabbit serum (open square, open triangle) as described in the Experimental Procedures. After centrifugation the unbound fraction (closed squares, open square) and the immunoaffinity bead bound fraction (closed triangles, open triangle) were assayed for oligosaccharyltransferase activity. The total activity recovered (bound plus unbound) was determined to allow calculation of the percent recovery in the bound (closed triangles, open triangle) and unbound (closed squares, open square) fractions. (6) A Coomassie blue-stained polyacrylamide gel showing unbound fractions from the duplicate incubations with the immunoaffinity beads. The volume of RICs antiserum (O-24 ul) or nonimmune rabbit antiserum (24’) is designated above each gel lane. Ribophorin I (RI), ribophorin II (RII), the 48 kd protein (48 kD), and IgG are designated by labeled arrows. The polypeptide that migrates directly above ribophorin I is rabbit serum albumin, Recovery of between 1% and 2% of the protein A-Sepharose beads in the unbound fraction is responsible for the presence of IgG. The samples for electrophoresis were not boiled in DTT, so the majority of the IgG migrated as a heavy chain-light chain heterodimer.

from solution by the immunoaffinity beads increased when the amount of WCs antiserum was increased (Figure 3A, closed squares). The activity that had been removed from solution was detected when the immunoaffinity beads were assayed for oligosaccharyltransferase activity (Figure 3A, closed triangles). As shown by the open symbols in Figure 3A, incubation of the oligosaccharyltransferase with nonimmune IgG did not result in immunodepletion or inhibition of the enzyme. Nonimmune IgG from three different rabbits gave comparable results. Aliquots of the unbound fraction from each of the duplicate incubations were analyzed by Coomassie blue staining after SDSpolyacrylamide gel electrophoresis to assess the correlation between the depletion of oligosaccharyltransferase activity and the depletion of ribophorin I, ribophorin II, and

lmmunoabsorption

of the Oligomeric

Complex

with

Anti-ribophorin I immunoaffinity columns were loaded with the active pool from a preparative glycerol gradient. After extensive washing, the immunoabsorbed proteins were eluted (see Experimental Procedures for column preparation and chromatography methods). (A) A Coomassie blue-stained gel of the load (lane b) and eluate (lane c) fractions from the immunoaffinity column. Molecular weight markers are shown in lane a. (6) Protein immunoblots using a mixture of monoclonal antibodies to ribophorins I and II. The lanes correspond to: lane a, load; lane b, eluate fractions from the immunoaffinity column. Ribophorin I (RI), the glycosylated and nonglycosylated forms of ribophorin II (RII), and the 48 kd protein (48) are designated by the labeled arrows between (A) and (6). The asterisk to the right of (A) indicates the migration position of the IgG heavy chain (lane c).

the 48 kd protein (Figure 36). The specific immunodepletion of the three proteins can be most easily observed by comparison of the samples that were treated with equalsized aliquots (24 ul) of the WCs antiserum and the nonimmune serum (Figure 38). Supernatants from duplicate incubations with the RICs antiserum are labeled 24, while supernatants from the duplicate incubations with the nonimmune sample are labeled 24’. Other proteins in the QAE-Sephadex pool, including one designated by two asterisks, were not removed from solution by the immunoaffinity beads. The proteins that bind to the immunoaffinity matrix were examined in a separate experiment. The activity pool from a preparative glycerol gradient was applied to an immunoaffinitycolumn prepared using the RICsantiserum. After extensive washing to remove unbound proteins, acid pH elution of the column yielded three polypeptides that correspond to ribophorin I, ribophorin II, and the 48 kd protein (Figure 4A, lane c). The identification of the 63 and 66 kd proteins as ribophorins I and II was confirmed by protein immunoblot analysis of the column load and eluate fractions (Figure 48, lanes a and b). The retention of ribophorin I, ribophorin II, and the48 kd protein bythe immunoaffinity column suggests that all three polypeptides are subunitsof an oligomeric complex, as the other proteins in the sample applied to the column (Figure 4A, lane b) were not bound by the antibody and as a consequence were not recovered in the eluate (Figure 48, lane c). lmmunoblots demonstrated that the RI& antibody reacts with ribophorin I and no other protein in the oligosaccharyltransferase preparation (data not shown). This latter result confirms previous

Purlftcation 59

of Oligosaccharyltransferase

data showing that the RI& antiserum (Vu et al., 1990).

was monospecific

Copurification of the Oligosaccharyltransferase and the Oligomeric Complex As noted earlier, only 53% of the oligosaccharyltransferase activity detected in the intact membrane was recovered in the detergent extract (Table 1). lmmunoblots of fractions from a preparative glycerol gradient demonstrated the presence of slowly sedimenting forms of ribophorins I and II in gradient fractions that lacked detectable oligosaccharyltransferase activity (data not shown). We postulated that the solubilization of the enzyme with 1% Nikko1 in the presence of high salt disrupted a significant fraction of the oligomeric complexes, resulting in a loss of enzyme activity. If this hypothesis were correct, then purification conditions that do not induce dissociation of the oligomeric complex should enhance the recovery of enzyme activity and improve the correlation between the three polypeptides and the oligosaccharyltransferase activity. As shown below, both of these predictions were fulfilled when the microsomal membranes were solubilized with the nonionic detergent digitonin. A digitonin extract of permeabilized and stripped microsomal membranes contained 3.6-fold more oligosaccharyltransferase activity than a Nikko1 high salt extract prepared from a similar

Figure

5. Cofractionation

of Oligosaccharyltransferase

Activity,

Flibophorin

membrane preparation. Lipid-linked oligosaccharide (OSPP-Dol) is insoluble in digitonin, so the OS-PP-Dol substrate was added to the oligosaccharyltransferase assay after being dissolved in dimethylsulfoxide (DMSO). A direct comparison of enzyme activities in the two detergent extracts cannot be made, since the assays contain different detergents and differ in DMSO content. After correction for the 1.5fold stimulation of activity that is observed when the Nikkol-purified enzyme is assayed in the digitonin-DMSO-based assay, the 3.6-fold higher activity of the digitonin-solubilized enzyme is roughly equivalent to a 2.4-fold increase in the total yield of solubilized activity. Glycerol gradient centrifugation was used to determine whether the ribophorins cosediment with the oligosacchatyltransferase activity in digitonin solution (Figures 5A and 5B). The oligosaccharyltransferase activity was primarily recovered in fractions 9-l 1 of a glycerol gradient that contained digitonin instead of Nikko1 (Figure 5A). Coomassie blue staining of an SDS-polyacrylamide gel revealed three major polypeptides that cosediment with the oligosaccharyltransferase activity (Figure 58). The three polypeptides designated by arrows correspond to ribophorin I, ribophorin II, and the 48 kd protein. The relative abundance of the three proteins in the individual gradient fractions appeared to correlate with the oligosaccharyltransferase activity. Fractions 9-11 from the glycerol gradient were pooled,

I, Ribophorin

Ii, and the 48 kd Protein

(A) Glycerol gradient centrifugation of a digitonin-high salt extract prepared from detergent-permeabilized microsomal membranes (see Experimental Procedures). Duplicate 5 ~1 aliquots from each of 14 gradient fractions were assayed for oligosaccharyltransferase activity. Fraction 1 is the top of the gradient. (ES)A Coomassie blue-stained gel of the glycerol gradient fractions. (C and D) The most active fractions from a preparative Mono-Q column were reapplied to a Mono-Q FPLC column and eluted with a 20 ml linear NaCl gradient (see Experimental Procedures for chromatography conditions). Twenty 1 ml fractions were collected. (C) Aliquots of the 12 ml load fraction (L) and the eluted fractions from the Mono-Q column were assayed for oligosaccharyltransferase activity. (D) A Coomassie blue-stained gel of the load (L) and eluate fractions from the Mono-Cl column. Ribophorin I (RI), the glycosylated and nonglycosylated forms of ribophorin II (WI). and the 48 kd protein (48) are designated by the labeled arrows adjacent to (6) and (D).

Cell 60

and further purification was achieved using a Mono-Q FPLC column. Oligosaccharyltransferase activity eluted from a preparative Mono-Q column at an NaCl concentration of approximately 250 mM, and the activity peak corresponded to the elution peak of the three major polypeptides. An aliquot of the Mono-Q eluate (Figure 5D, lane L) was reapplied to a Mono-Q column and subsequently eluted with a linear NaCl gradient (Figures 5C and 5D). The oligosaccharyltransferase activity eluted in fractions 9-l 1 (Figure 5C), as did ribophorin I and the glycosylated and nonglycosylated forms of ribophorin II and the 48 kd protein (Figure 5D). We noted that fraction 9 was slightly more active per unit of stainable protein than fraction 11. Significant amounts of the three proposed subunits of the oligosaccharyltransferase were not resolved from the enzyme activity at any stage during purification in digitonin solution. Although ribophorin I stained more intenselythan ribophorin II and the 48 kd protein with Coomassie blue, the staining intensity of the three proteins appeared equivalent when a silver staining procedure was used (data not shown). The 48 kd protein is not a glycoprotein (see Figure 2A), and it is not recognized by the RI& polyclonal antiserum or by monoclonal antibodies (see Figure 28) raised against ribophorin I and II. Several peptides derived from the 48 kd protein have been sequenced and were found to be nonhomologous to human and rat ribophorins I and II (data not shown). Thus, the 48 kd protein is not a proteolytic fragment derived from either ribophorin I or II. Discussion Several lines of evidence suggest that the oligosaccharyltransferase activity is mediated by a complex composed of ribophorin I, ribophorin II, and a 48 kd protein. Purification of the activity by conventional protein purification methods yielded these three polypeptides in approximately equal stoichiometry. An antibody directed against the cytoplasmic domafn of ribophorin I was able to immunodeplete the oligosaccharyltransferase activity from detergent solution and could be used to immunopurify the three polypeptides. Purification of theoligosaccharyltransferase in digitonin solution was accompanied by an increased yield of enzyme activity and a direct correlation between oligosaccharyltransferase activity and the chromatographic behavior of the three polypeptides. Taken together, these results suggest that the oligomeric complex is responsible for the oligosaccharyltransferase activity. Purification of glycosyltransferasesfrom the Golgi apparatus often requires a 1 O,OOO- to 1 OO,OOO-fold enrichment relative to a crude tissue homogenate. In contrast, the ribophorins are abundant proteins that are present in RER membranes in nearly 1:l stoichiometry with membranebound ribosomes (Marcantonio et al., 1984). This raises the question of whether the complex purified here is too abundant to be responsible for the oligosaccharyltransferase activity. We propose that the cotranslational nature of the glycosylation reaction provides a rationale for the presence of an abundant oligosaccharyltransferase. The protein substrate for the oligosaccharyltransferase is pre-

sumed to be the unfolded nascent polypeptide, in contrast to the Golgi enzymes that act upon folded proteins. Protein-folding reactions that commence upon entry of a nascent polypeptide into the lumen of the ER may interfere with subsequent glycosylation. Support for the latter conjecture is provided by the observation that in vitro glycosylation of proteins containing unutilized NXSIT sites requires prior denaturation of these protein substrates (Pless and Lennarz, 1977). One solution for this temporal constraint would be to position an oligosaccharyltransferase complex in the immediate vicinity of each translocation site, so that a nascent polypeptide can be monitored for the presence of NXT/S sites as it emerges into the RER lumen. A physical proximity between the ribophorins and membrane-bound ribosomes was first shown by chemical cross-linking experiments (Kreibich et al., 1978a) and later confirmed by the demonstration that antibodies raised against the cytoplasmic domain of ribophorin I inhibit protein translocation by blocking ribosome targeting to the membrane (Vu et al., 1990). The results described here raise several questions concerning the stoichiometry and the function of the individual subunits of the oligomeric complex. Although silver staining procedures suggest that the subunits of the complex are present in 1:l:l stoichiometry, the Coomassie bluestained gels suggest that the 48 kd protein may be less abundant. Additional experimentation will be required to resolve this issue. A turnover number (0.22 tripeptides glycosylated per min) for the oligosaccharyltransferase was calculated from the data in Table 1, assuming that the oligosaccharyltransferase preparation was 80% pure. The low turnover number may reflect the inability of the mixed detergent-phospholipid micelle to mimic accurately the conditions of the intact membrane. Higher turnover numbers may be obtained when the oligomeric complex is reconstituted into liposomes. Now that enriched preparations of the oligosaccharyltransferase are available, photoaffinity labeling studies using glycosylation site tripeptides may reveal the subunit that recognizes the NXTlS consensus glycosylation site. Ribophorins I and II have been detected by protein immunoblotting in a wide variety of eukaryotic species, indicating that at least two of the three subunits of this complex are highly conserved (Marcantonio et al., 1982; Crimaudo et al., 1987). Further research will be needed to determine whether the 48 kd protein is also present in other species. Additional support for the conclusion that oligosaccharyltransferase activity is mediated by the oligomeric complex was provided by comparing the protein sequence of ribophorin I with a sequence that was recently proposed to be a recognition site for dolichol (Table 2). We must stress that the proposed dolichol-binding sequence was derived by protein sequence comparison and not from biochemical or genetic data, so the following discussion is somewhat speculative. A 13 amino acid dolichol-binding site consensus sequence was derived by comparison of the transmembrane segments of several yeast glycosyltransferases (Albright et al., 1989). Two additional sequences from a mammalian glycosyltransferase allowed a revision of the consensus sequence (Zhu and Lehrman,

Purifrcahon 61

of Oligosaccharyltransferase

Table 2. Sequence

Homology

between

Ribophorin

I and Dolichol-Binding

Proteins

Protern

Location

Sequence”

1. ALG7 2 ALGl 3. DPMI 4. SEC59 5. GPTl 6 GPT2 7. Ribophorin I Consensus #l Consensus #2

79-92 19-32 245-258 332-345 66-79 221-234 423-436

YLFVMFIYIPFIFY IPLVVYYVIPYLFY ILFITFWSILFFYV WHFIIFLLIIPSFQ FLIILFCFIPFPFL HVFSLYFMIPFFFT FYILFFTVIIYVRL LFVXFXXIPFXFY FXFXXFXXIPFXF I I Y Y L 1 3 5 7

Resfdue

number

identity to Consensus #2 _-~. (717) (7/7) (7/7) (5/7) (7171 (6/7) (617)

(I)Y CL) 9

y 11

13

’ Protein sequences are in the one letter code for amino acids. In concensus #l, X designates amino acids normally found in membrane-spanning segments. In consensus sequence #2, X designates any amino acid normally found in membrane-spanning segments excluding alanine and glycine. Sequences 1-4 and consensus sequence #l are taken from Albright et al. (1989). Sequences 5 and 6 are from Zhu and Lehrman (1990). The sequence of rat and human ribophorin I are identical in this region (Crimaudo et al., 1987; Harnik-Ort et al., 1987). Conservative replacements are listed below concensus sequence #2. Nonconservative but allowed replacements of fsoleucine or leucine for proline are shown in parentheses. Residues in the sequences that match consensus sequence #2 are shown in bold type.

1990). The predicted transmembrane segment of ribophorin I (residues 416-434) is identical in the rat and human proteins, and this segment of ribophorin I can be aligned with the proposed dolichol-binding site (Table 2). The presence of this sequence motif suggests that ribophorin I may be the dolichol-binding subunit of the oligosaccharyltransferase complex. Direct experimental evidence for dolichol binding to ribophorin I will be needed to address this question. We propose a revision of the consensus sequence that allows conservative substitutions of tyrosine, isoleutine, or leucine for phenylalanine at several positions based on the amino acid replaceability, PAM 250 matrix (Dayhoff, 1978), and the observed variation within the seven currently available sequences (Table 2). The frequency of amino acids within the apolar segments of 62 eukaryotic membrane proteins has been calculated, and alanine and glycine residues account for 19% of the membrane-embedded residues (Von Heijne and Gavel, 1988). Interestingly, the putative dolichol-binding sequences in Table 2 are deficient in these two apolar amino acids that lack large side chains. As noted previously (Albright et al., 1989) the conserved proline at residue 10 is unusual given the low frequency of proline in membranespanning segments (Brand1 and Deber, 1986; Von Heijne and Gavel, 1988). The dolichol-binding consensus sequence is not homologous to the predicted transmembrane span of ribophorin II (Crimaudo et al., 1987; Pirozzi et al., 1991). A consideration of the amino acids in the consensus sequence suggests that the binding affinity of dolichol for these sequences might be enhanced by an interaction between the isoprene double bond and an ordered array of phenylalanine and tyrosine side chains. A possible mode of interaction between dolichol and a glycosyltransferase was obtained by the construction of helical net diagrams for the seven protein sequences (Figure 6). Membrane-spanning segments of proteins are typically in an a-helical conformation (Engelman et al., 1986), so the heli-

cal net diagrams should provide information on the disposition of the consensus sequence residues within the dolichol-binding proteins. The consensus sequence residues would not be located on the same face of the a helix, but instead could define a left-handed helix that appears to wrap 1.5 times around the right-handed protein a helix in several of the glycosyltransferases. The Scarbon isoprene unit is 4.7 A, so a typical dolichol molecule is 90100 A in length. The number of isoprene units that could be accommodated within the proposed dolichol-binding site was estimated by using 11.5A as the minimum diameter of the cylinder defined by the hydrogen atoms of dolichol. The 65 A length of the proposed consensus binding sequence could then accommodate 14 of the 18-20 isoprene units in a typical dolichol molecule (Chojnacki and Dallner, 1988). This model for dolichol binding, if valid, could provide an answer for how a 95 A long dolichol molecule could be incorporated into the 45 A thick membrane bilayer. A direct interaction between dolichol compounds and the phospholipid core of the membrane appears to be unfavorable, as dolichol phosphate induces the formation of nonbilayer structures when added to pure phospholipid vesicles (as reviewed by Chojnacki and Dallner [1988]). Interestingly, molecular mechanics methods predict that the most stable conformation of the dolichol molecule in solution corresponds to a central coiled region flanked by two short arms (Murgolo et al., 1989). Assembly of lipid-linked oligosaccharide is initiated on the cytoplasmic face of the membrane and is completed on the luminal face of the membrane (Snider and Rogers, 1984; Hirschberg and Snider, 1987; Abeijon and Hirschberg, 1990). Both the dolichol molecule and the putative dolichol-binding site lack dyad symmetry; hence, the polarity of substrate binding might be defined with respect to the protein. A consideration of the membrane orientation of ribophorin I (Harnik-Ort et al., 1987), DPMl (Orlean et al., 1988), and the proposed orientation of the GPT-1 (Zhu and Lehrman, 1990) suggests that the NH*-terminal end

Cell 62

ALG7

(yeast) -

GPTP (CHO)

Ribophorin

I (rat) A

SEC59

(yeast)

Figure 6. A Possible Binding Site

Model

for

a Dolichol-

Helical net diagrams were constructed for the sequences in Table 2. The conserved hydrophobic residues that correspond to the consensus sequence are connected by stippling. The conserved proline (residue 10) in ALG-7, GPT-1. GPT-2, and ALG-1 is circled in bold. The structure of a typical dolichol phosphate molecule (adapted from Albright et al. [1969]) is shown below the helical net diagrams. A potential interaction between dolichol phosphate and the consensus dolichol recognition site is shown in the bottom right diagram.

GPTl (CHO) ,___...______...._*

ALGl (yeast) ;-.------..-~

DPMl

Dolichol phosphate

(yeast) A

o-

of the consensus sequence is oriented toward the active site of these enzymes (Figure 6, bottom right). It should be noted that the simplest possible model for the membrane orientation of the ALG-1 protein would not be consistent with this alignment (Albright and Robbins, 1990). Those portions of the glycosyltransferases that recognize the carbohydrate portion of the dolichol-linked oligosaccharide should also contribute significantly to the polarity, affinity, and fidelity of substrate recognition. Biochemical and biophysical experiments will be required to determine whether the dolichol compounds are in direct contact with the transmembrane spans of the glycosyltransferases. Experimental

Procedures

Peptide Synthesis and lodination The tripeptide Asn-Tyr-Thr-NH2 was prepared using methoxybenzhydrylamine-Thr-resin (Peninsula Laboratories) as described (Stewart and Young, 1964). The N-terminal Boc protecting group was removed, and the peptide, while still on the resin with the side chain protective groups still in place to prevent 0-acetylation, was acetylated as described (Stewart and Young, 1964). The acetylation mixture containing peptidyl-resin, acetic anhydride, and triethylamine in N,N-dimethylformamide was filtered, washed with methylene dichloride, and then dried undervacuum at room temperature. Sidechain protectivegroups were removed, and the acetylated tripeptide amide (Na-AC-NYT-NHz) was cleaved from the resin by solvolysis using anhydrous hydrogen fluoride.Thepeptidewaspurified usingC18reversephasechromatography, and the amino acid composition, as well as the concentration

of the high pressure liquid chromatography-purified peptide solution, was determined by automated amino acid analysis using a-amino butyric acid as a recovery marker. Fifty microliters of 0.5 M sodium phosphate (pH 7.5) (NaPi) was added to 2 mCi of carrier-free [i*51]Nal (Amersham Searle, Arlington Heights, IL) followed by 100 nmol of tripeptide dissolved in 25 WI of 0.4 M NaPi and 50 ftl of chloramine-T (0.36 ftmol) in 0.05 M NaPi. After 2 min at room temperature, sodium metabisulfite (1.6 umol) in 100 PI of 0.05 M NaPi was added to stop the iodination reaction. The iodinated tripeptide was resolved from unincorporated lz51, chloramine-T, and buffer salts by chromatography on a 0.5 ml Dowex 1 anion-exchange column (AGl X6200-400; Bio-Rad, Richmond, CA) equilibrated in 0.05 M NaPi (pH 7.5). The iodinated tripeptide sample (245 ~1) was applied, and the column was washed with 500 ftl of H20. Elution of the column with 6 ml of 10% acetonitrile in Hz0 yielded 35%-45% of the original ‘? in a peptide-bound form. The eluate contained no detectable unincorporated ?, as assessed by thin layer chromatography in butanol: acetic acid:water (5:2:2) on Merck 5721 silica gel plates as previously described (Wieland et al., 1967). The recovery of peptide during the iodination procedure was estimated to be 70% using an extinction coefficient of 6.33 x 103 at 220 nm for the tripeptide. The eluate from the Dowex 1 column was dried in a Savant Speed-Vat, dissolved in H20, and stored as a 50 uM solution at 4%. The initial specific activity based on approximately 40 preparations was lE.OOO-26,000 cpmlpmol.

Preparation of Lipid-Linked Oligosaccharide Lipid-linked oligosaccharide (OS-PP-Dol) was isolated from 200 g of bovine pancreas as described previously (Das and Heath, 1960). The volumes of solvent used for all extractions were increased by 600-fold, owing to the greater quantity of starting tissue. The molar concentra-

Purification 63

of Ollgosaccharyltransferase

tion of functional OS-PP-Dol of glycosylated ‘251-labeled tranS.feraSe assay containing and the enzyme wasallowed OS-PP-Dol was approximately a molecular weight of 4000

was estimated by determining the amount peptide formed when an oligosaccharylan excess of both 1251-labeled peptide to go to completion. The yield of functional 3.5 ug per g of bovine pancreas using for OS-PP-Dol.

Oligosaccharyltransferase Assay Oligosaccharyltransferase was assayed in intact membranes using the membrane-permeable tripeptide Na-Ac-Asn-j’“I]Tyr-Thr-NH1 as the oligosaccharide acceptor and endogenous OS-PP-Dol as the oligosaccharide donor (Welply et al., 1983). The standard 100 ul assay for intact membranes contained 5 HIM Na-Ac-Asn-[‘251]Tyr-Thr-NHz (7,00028,000 cpmlpmol), 75 ug of membrane protein, 20 mM Tris-Cl (pH 7.4) 150 mM NaCI. 2 mM MnCb, 1 mM MgCI,, 0.1 mM EDTA, 1 mM dithlothreitol (DTT). and 3% glycerol. The standard 100 ul assay for the Nikko1 solubilizedenzymecontained5pM Na-Ac-Asn-(‘251]Tyr-Thr-NH2 (7,000-28,000 cpmlpmol), 1.8 uM OS-PP-Dol, 32 mM Tris-Cl (pH 7.4), 50 mM NaCI, 3.6 mM MnCb, 3.1 mM MgC12, 0.5% Nikko1 (NikkoChemical Co., Ltd., Tokyo, Japan), 0.675 mM egg yolk phosphatidylcholine (egg PC; Sigma Chemical Co.), 0.04 mM EDTA, I mM DTT, 10% glycerol. The OS-PP-Dol was added to empty assay tubes and the CHCb:CH30H:Hz0 (10:10:3) storage solvent was evaporated using a Savant Speed Vat. The dried OS-PP-Dot was dissolved in 42 ul of 0.75% Nikkol, 1 mM egg PC, 10 mM Tris-Cl (pH 7.4) 8% glycerol, 0.1 mM EDTA, 0.8 mM DTT by vortexing. For the digitonin-solubilized enzyme, the dried OS-PP-Dol substrate was dissolved in 15 ~1 of DMSO, as OS-PP-Dol is insoluble in digitonin. The OS-PP-Dol solution was diluted with 63 pl of 8% glycerol, 2.14 mM egg PC, 0.3 mM MnCb, 0.2 mM EDTA, 48mMTris-CI(pH 7.4),followed bytheenzymesolution and the iodinated tripeptide. A complete 100 ul assay for the digitonin-solubilized enzyme contained 5 uM Na-Ac-Asn-[‘ZSI]Tyr-Thr-NH2 (7,000-28,000 cpmlpmol), 1.8 uM OS-PP-Dol, 50 mM Tris-Cl (pH 7.4) 0.04 mM sodium-HEPES (pH 8.0), 2.5 mM NaCI, 2 mM MnCb, 3 mM MgCI,, 0.13 mM EDTA, 0.025% digitonin (Sigma Chemical Co.), 1.35 mM egg PC, 1 mM DTT, 6% glycerol, and 15% DMSO. Assaysof intact membranes and detergent-solubilized samples were incubated for 10 minor 60 min at 25OC, respectively. Oligosaccharyltransferase assays were terminated by the addition of 0.1 ml of ice-cold 3.2% Nikko1 followed by 1 ml of ice-cold 50 mM Tris-Cl (pH 7.4) 1 M NaCI, 1 mM MgCb, 1 mM MnC12, 1 mM CaCb, 0.02% Nikko1 (buffer A). ConASepharose beads (100 ul of a I:1 suspension in buffer A) were added, and the assay tubes were rotated for 20 min at 4°C. The beads were washed three times with 1 ml of bufferA to remove any nonglycosylated lodinated tripeptide. The quantity of glycosylated Na-Ac-Asn-[YjTyrThr-NH2 was determined by gamma counting of the ConA-Sepharose beads. Nonspecific binding of the tripeptide to the ConA beads accounted for less than 0.05% of the input radioactivity. Assay results were corrected by subtraction of an appropriate blank value (lOOO4000 cpm) determined in a control assay lacking enzyme. The oligosaccharyltransferase activity of intact canine rough microsomal membranes was found to be 10 pmollmin per mg when the lodinated tripeptide was used as the oligosaccharide acceptor. This level of activity is roughly 3-fold higher than that reported for hen oviduct microsomal membranes (Welplyet al., 1983). The estimated affinity of the enzyme for OS-PP-Dol under our assay conditions (K, = 0.85 uM) is in reasonable agreement with the value reported for the calf thyroid enzyme (K, = 0.3 uM) in an assay containing 0.15% NP40 and 1.35 mM egg PC (Chalifour and Spiro, 1988). Formation of the glycosylated tripeptide was proportional to the enzyme concentration, linear with respect to time, and dependent on Mn2+ as previously reported (Das and Heath, 1980; Lau et al., 1983; Welply et al., 1983). The glycosylated tripeptide product from assays of the oligosaccharyltransferase was characterized by thin layer chromatography before and after digestion with N-glycosidase F (Boehringer-Mannheim) as described by Wieland et al. (1987). The migration of the iodinated tripeptide was reduced by attachment of asparagine-linked oligosaccharide. Incubation of the glycosylated tripeptide product with glycopeptidase F caused the product to comigrate with the stock iodinated tripeptide, while mock digestion of the glycosylated tripeptide did not alter the mobility of the product (data not shown). These results demonstrate that the carbohydrate was attached via the asparagine side chain.

Purification of Oligosecchatyltransferase Rough microsomal membranes were isolated from canine pancreas as described (Walter and Blobsl, 1983). Twenty milliliters of rough microsomal membranes(A280 = 50,8.2 mg/ml) was thawed and diluted to 100 ml with 20 mM Tris-Cl (pH 7.4). 1 mM DTT, protease inhibitor cocktail (PIG), 1 mM phenylmethylsulfonyl fluoride. PIC corresponds to 0.1 jrglml each of pepstatin A, chymostatin. leupeptin, and antipain, and 1 uglml aprotinin. The membranes were sedimented bycentrifugation for 45 min at 184,000 gm in a Beckman Ti50.2 rotor. The membrane pellet was resuspended in 100 ml of 20 mM Tris-Cl (pH 7.4) 500 mM NaCI, 1 mM DTT, 1 mM PMSF, and PIC and then centrifuged as above and resuspended in 10 ml of 20 mM Tris-Cl (pH 7.4) 1 mM DTT, and PIC to obtain salt-stripped membranes at 2 eqArl(7.2 mglml protein). Salt-stripped membranes (5000 eq) were adjusted to a concentration of 1 eqlul by dilution with an equal volume of 20 mM Tris-Cl (pH 7.4), 0.2 M NaCl, 2 mM MgCb, 1 mM DTT, 0.1% Nikko1 detergent, and PIC. After 20 min at O’C, the detergent-permeabilized membranes were recovered by centrifugation for 30 min at 122,000 g.” in a Beckman Type 50 rotor. Integral membrane proteins were solubilized by resuspension in 5 ml of 20 mM Tris-Cl (pH 7.4) 0.5 M NaCI, 4 mM MnCb, 1 mM MgCb, 1 mM DTT, 1% Nikkol, and PIC. After 20 min at OOC, the detergent extract was clarified by centrifugation for 30 min at 122,000 g.” in a Beckman Type 50 rotor. The clarified detergent extract (2.1 ml) was layered onto each of two 36 ml 8%-30% glycerol gradients poured in Beckman quick seal tubes. The glycerol gradients contained 20 mM Tris-Cl (pH 7.4) 4 mM MnCb, 0.3 mM MgCb, 1 mM DTT, 0.5% Nikkol, PIC, 0.675 mM egg PC (buffer B) adjusted to 500 mM NaCI. The gradientswerecentrifugedfor 16.5 hrat86,000g.,ina BeckmanVTi50 rotor at 4OC. Fourteen fractions of 2.7 ml were collected using an ISCO gradient fractionator. The three fractions with the highest oligosaccharyltransferase activity from each gradient were combined to obtain a 16 ml glycerol gradient pool. The glycerol gradient pool was applied to a 145 ml QAE-Sephadex A-25 column equilibrated with buffer B adjusted to 100 mM NaCl and 20% glycerol. Chromatography on the OAE-Sephadexcolumn utilized thegradient-sievorptivechromatography method (Kirkegaard, 1973). The column was eluted by ascending flow with buffer B containing 20% glycerol and 400 mM NaCl at a rate of 1 mllmin. Fractions of 6 ml were collected and assayed. Active fractions eluting between 0.2 and 0.4 column volumes were pooled. A 1 .O ml Mono-S FPLC column was washed with 50 ml of 20 mM TrisCl (pH 7.4) 100 mM NaCI, 5 mM MnCb and equilibrated with buffer B adjusted to 100 mM NaCI. 20% glycerol. After the QAE-Sephadex pool (24 ml) was loaded, the Mono-S column was washed with 4 ml of buffer B containing 200 mM NaCI, 20% glycerol and then eluted with buffer B containing 20% glycerol and 500 mM NaCl. Purification of the Oligosaccharyltransferase in Digitonin Solution A stock solution of digitonin was prepared by dissolvmg 6 g of crude digitonin (Sigma, D-1407) in 100 ml of 10 mM sodium-HEPES (pH 8.0) at 100°C. The digitonin stock solution was stored for 4 days at 4OC. Insoluble material was then removed from the digitonin stock solution by centrifugation for 20 min at 8000 g*” in a Sorvall HB-4 rotor. A 6% digitonin, 4 mM egg PC solution was prepared by dissolving dried egg PC in the digitonin stock solution. After a 12 hr end-over-end rotation, insoluble material was removed by centrifugation as above. Salt-stripped, detergent-permeabilized membranes (2500 eq) were prepared from rough microsomal membranes as described above and were solubilized by resuspension of the membrane pellet in 2.5 ml of 20 mM Tris-Cl (pH 7.4) 5 mM sodium-HEPES (pH 8.0) 500 mM NaCI, I mM MgCl,, 1 mM MnCb, 1 mM DTT, 1.5% digitonin, and PIC. After 20 min at OOC, the detergent extract was clarified by centrifugation for 30 min at 122,000 9.” in a Beckman Type 50 rotor. The clarified extract (2.1 ml) was layered onto a 36 ml 8%-30% glycerol gradient poured in a Beckman quick seal tube. The glycerol gradients contained 20 mM Tris-Cl (pH 7.4) 0.8 mM sodium-HEPES (pH 8.0) 50 mM NaCI, 1 mM MnCb, 1 mM MgCb, 1 mM DTT, 0.5% digitonin, 0.33 mM egg PC, and PIC. The gradients were centrifuged for 3.2 hr at 206,000 g.” in a Beckman VTi50 rotor at 4OC. Fourteen fractions of 2.7 ml were collected using an ISCO gradient fractionator. The three fractions with the highest oligosaccharyltransferase activity from the gradient were combined and applied to a 1 ml Mono-Q FPLC column equilibrated with 20 mM Tris-Cl (pH 7.4) 0.8 mM sodium-HEPES (pH 8.0) 1 mM

Cell 64

MnCh, 1 mM MgCh, 1 mM DTT, 0.5% digitonin, 0.33 mM egg PC, 20% glycerol, and PIC (buffer C) adjusted to 25 mM NaCI. After sample application, the column was washed with 4 ml of buffer C containing 25 mM NaCl and eluted with a 20 ml linear gradient of 25-500 mM NaCl in buffer C. Twenty 1 ml fractions were collected and assayed. The majority of the oligosaccharyltransferase activity typically eluted in two fractions at approximately 250 mM NaCI. Analytical 8%-300/o glycerol gradients in digitonin solution (4.7 ml), identical in composition to those described above, were loaded with samples of the Mono-Cl eluate that had been diluted 4-fold with buffer C lacking glycerol. The gradients were centrifuged for 15.5 hr at 84,200 gm in a Beckman SW50.1 rotor at 4OC.

Acknowledgments We thank Dr. Steve Fuller for providing the antibody that recognizes protein disulfide isomerase. This work was supported by National Institutes of Health grants PHS GM 43768 (R. G.) and PHS GM 21971 (G. K.). This work was done during the tenure of an Established Investigatorship of the American Heart Association (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 “advetiisemen9’ in accordance with 18 USC Section 1734 solely to indicate this fact. Received

lmmunoabsorption and lmmunoaffinity Chromatography with RICs Antiserum RI& is a polyclonal rabbit antiserum raised against a synthetic peptide corresponding to the C-terminal 20 amino acid residues of rat ribophorin I (Harnik-Ort et al., 1987; Yu et al., 1990). For immunoabsorption experiments, O-24 ul of the RI& antiserum, or 24 ul of nonimmune antiserum, was incubated for 90 min at 4OC with 20 ul of protein ASepharose beads in 1 ml of 20 mM Tris-Cl (pH 7.4) and 150 mM NaCI. The beads were washed several times with 1.4 ml of the above buffer. Aliquots (170 ul) of partially purified oligosaccharyltransferase from the QAE-Sephadex ion-exchange column were incubated with the immunoaffinity beads in buffer B containing 100 mM NaCl and 20% glycerol for 90 min at 4OC. Incubations of the enzyme with protein ASepharose-immobilized antibodies were done in either duplicate or triplicate for each quantity of antiserum. Unbound proteins were separated from the immunoaffinity beads by a 30 s centrifugation in a microcentrifuge. The beads were washed with an additional 50 ul of buffer B adjusted to 100 mM NaCI, 20% glycerol, and this wash was combined with the supernatant for oligosaccharyltransferase assays, The beads were resuspended in 200 ul of the above buffer, and 40 ul aliquots were withdrawn for oligosaccharyltransferase assays. To prepare an immunoaffinity matrix containing covalently immobilized antibodies, the RI& antiserum (768 ~1) was rotated end over end for 90 min at 4’C with 960 pi of protein A-Sepharose CL-48 beads in 12 ml of 20 mM Tris-Cl (pH 7.4) 150 mM NaCI. The protein A-Sepharose beads were transferred to a column and washed with 200 ml of 200 mM triethanolamine-Cl (pH 8.2) to remove unbound proteins. Protein A-bound immunoglobulins were immobilized by cross-linking with 20 mM dimethylpimelimidate dihydrochloride (Pierce, Rockford, IL) as described (Schneider et al., 1982). The immunoaffinity matrix (RI& beads) was stored in 20 mM Tris-Cl (pH 7.4) 150 mM NaCI. Aliquots (900 ul) of fractions from a preparative glycerol gradient were incubated for 90 min at 4°C with 480 ul of packed RICe beads. Glycerol gradient fractions were in buffer B containing 500 mM NaCl and 20% glycerol. The RI& beadswere transferred toa0.9cm diameter column using buffer B adjusted to 0.1 mM DTT. 200 mM NaCI, 20% glycerol, and the column was washed with 25 ml of the above buffer to remove nonspecifically bound protein. The immunoaffinity column was eluted with 1.6 ml (3.3 column volumes) of 0.2 M glycine-Cl (pH 2.3) 0.025% Nikkol, 300 mM NaCI. lmmunoblot and ConA Blot Detection of Proteins Proteins were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose sheets (0.45 pm; Schleicher and Schuell, Inc., Keene, NH). The nitrocellulose blots were probed with either polyclonal rabbit antiserum or monoclonal antibodies that recognize ribophorin I, ribophorin II, or protein disulfide isomerase. After washing to remove unbound primary antibodies, the nitrocellulose sheets were probed with horseradish peroxidase-coupled second antibodies specific for mouseor rabbit immunoglobulins. Bound secondantibodies werevisualized using enhanced chemiluminescence (ECL Western blotting detection kit, Amersham-Searle. Arlington Heights, IL) following the manufacturers recommendations. Nitrocellulose blots were also probed with ConA-peroxidase as described (Evans et al., 1986). except that the bound ConA-peroxidase was visualized by the enhanced chemiluminescence detection method. Proteins recognized by antibodies or ConAwere aligned with polypeptides on the nitrocellulose blot by staining the transferred proteins with a colloidal gold stain (Auro Dye Forte, Amersham, Arlington Heights, IL).

August

12, 1991; revised

January

17, 1992

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Oligosaccharyltransferase activity is associated with a protein complex composed of ribophorins I and II and a 48 kd protein.

Oligosaccharyltransferase catalyzes the N-linked glycosylation of asparagine residues on nascent polypeptides in the lumen of the rough endoplasmic re...
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