THE JOURNAL OF BIOLOGICAL CHEMISTRY (c? 1986 by The American Society of Biological Chemists, Inc

Vol. 261, No. 31, Issue of November 5. pp. 14725-14732,1986 Printed in U. S . A.

Control of N-Linked Carbohydrate Unit Synthesis in Thyroid Endoplasmic Reticulum b,y Membrane Organization and Dolichyl Phosphate Availability*

(Received for publication, April 21, 1986)

Mary Jane Spiro and Robert G. Spirol From the Departments of Biological Chemistry and Medicine, Harvard Medical School and the Elliott P. J o s h Research Laboratory, Boston, Massachus&s 02215

Thyroid rough endoplasmic reticulum (ER) has been shown to contain a highly organized multienzyme sys- tem capable of carrying out the N-glycosylation of newly synthesized proteins. These reactions were stud- ied in isolated ER vesicles and found to be controlled to a large extent by the availability of a key substrate, dolichyl phosphate (Dol-P), as well as by the amount of endogenous polypeptide acceptor present. Although in intact vesicles UDP-Glc was utilized in an efficient manner to form Dol-P-Glc and glucosylated oligosac- charide-lipid, after disruption of vesicle integrity, even with low concentrations of Triton X-100, the coupling of Dol-P-Glc formation to lipid-linked oligo- saccharide assembly and subsequent N-glycosylation was substantially impaired, Increased incubation tem- peratures also resulted in a decreased effectiveness of glucose transfer from Dol-P-Glc to lipid-oligosaccha- ride, presumably because of a decline in the extent of structural organization of the ER membranes.

The limited availability of endogenous Dol-P was demonstrated by the pronounced stimulation in Dol-P- Glc formation resulting from the addition of this lipid acceptor to Triton-disrupted ER membranes as well as by its generation in intact vesicles. The latter was accomplished by stimulating recycling of endogenous Dol-P through the addition of a peptide (Tyr-Asn-Leu- Thr-Ser-Val) which is an N-glycosylation substrate. The inhibition of Dol-P-Glc synthesis from UDP-Glc observed in the presence of elevated levels of GDP- Man which could be relieved in Triton-disrupted or intact ER vesicles by the addition or generation, re- spectively, of Dol-P, is considered to be the result of a competing requirement for Dol-P by the mannosyl- transferase. Moreover GTP, by selectively inhibiting the mannosyltransferase, prevented the decrease of Dol-P-Glc formation caused by GDP-Man. Since addi- tion of the acceptor peptide to intact vesicles stimulated Dol-P-P-GlcNAc as well as Dol-P-Glc and Dol-P-Man synthesis it would appear that a pool of Dol-P available in common to all three enzymes responsible for doli- chol-linked monosaccharide synthesis exists in the ER membranes.

* This work was supported by Grant AM 17477 from the National Institutes of Health and Grant 82R335 from the Juvenile Diabetes Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

80th birthday. This paper is dedicated to Luis F. Leloir on the occasion of his

$ To whom correspondence should be addressed: Elliott P. Joslin Laboratory, One Joslin Place, Boston, MA 02215.

The formation of N-glycosidically linked carbohydrate units is to a large measure carried out in the membranes of the ER’ which contain the enzymes involved in the assembly of the lipid-bound oligosaccharide, its transfer to nascent polypeptide, and its initial processing by the removal of outer sugar residues (1-3). It is likely that the numerous reactions leading to N-glycosylation are controlled by the disposition of the enzymes and substrates within the ER membranes and that these interrelationships can best be explored in intact vesicles in which the physiological orientations are preserved.

Thyroid ER vesicles represent an attractive system for investigating the regulation of this multienzyme system as they carry out lipid-saccharide synthesis and N-glycosylation in an integrated manner (4). In the present study we have probed the organization of these vesicles with the help of radiolabeled sugar nucleotides (UDP-Glc, GDP-Man, UDP- GlcNAc) by modifying the state of the membranes with graded amounts of detergent and altered temperatures, as well as by circumventing potential control points through the addition of Dol-P and a peptide containing the Asn-X-Thr sequence. These experiments indicated that the reactions leading from sugar nucleotides to N-linked carbohydrate units by way of dolichol-linked mono- and oligosaccharides occur in a tightly coupled manner which depends on membrane integrity for optimal function. Furthermore, the activity of the vesicle system was found to be restricted by limited Dol-P and polypeptide acceptor pools. Our observations indicated that all three sugar nucleotide (UDP-Glc, GDP-Man, UDP- GlcNAc): Dol-P glycosyltransferases appear to have access to the same membrane associated Dol-P pool as their activities could be similarly stimulated by increasing the endogenous level of this substrate through the addition of a peptide acceptor for the oligosaccharyltransferase. Moreover inhibi- tion of UDP-Glc utilization brought about by GDP-Man in both intact and disrupted vesicles could be relieved by increas- ing the level of Dol-P.

EXPERIMENTAL PROCEDURES

Preparation of ER Vesicles-Calf thyroid glands brought to the laboratory on ice from a local slaughter house were used to prepare rough ER vesicles by the procedures previously described (4). The membranes obtained by sucrose density gradient centrifugation were washed twice with 0.25 M sucrose, 5 mM Tris acetate, pH 7.0 (100,000 X g for 60 min), and were then resuspended in this buffer. The protein cont.ent of the membranes was determined by the method of Lowry et al. (5) using bovine serum albumin as a standard.

Incubation ofER Vesicles-The incubations of vesicles with radio-

’ The abbreviations used are: ER, endoplasmic reticulum; Dol-P, dolichyl phosphate; Dol-P-P, dolichyl pyrophosphate; Dol-P-P-Oligo, glucosylated dolichol pyrophosphate-linked oligosaccharide; endo H, endo-0-N-acetylglucosaminidase H.

14725

14726 Control of N-Linked Carbohydrate Synthesis in ER Vesicles

labeled sugar nucleotides were carried in a manner similar to that previously reported (4). Unless otherwise specified the membranes

bated at 27 "C for 10 min in 80 p l of 50 mM Tris acetate, pH 7.0, (180 pg protein) together with 1 pCi of sugar nucleotide were incu-

buffer containing 25 mM potassium acetate, 10 mM magnesium ace- tate, 0.1 M sucrose, 1 mM ATP, 0.5 mM creatine phosphate and 12 pg creatine phosphokinase. The "standard assay" consisted of incuba- tions with 1 pCi UDP-[3H]glucose (1.45 p ~ ) under the aforemen- tioned conditions. When Triton X-100 was included in the incuba- tions, it was added to the tubes in an ice bath and allowed to react with the membranes at this temperature for 5 min prior to the addition of the radiolabeled substrate.

The radiolabeled sugar nucleotides employed included UDP-[6-3H] glucose (8.6 Ci/mmol) from Amersham and GDP-[3,4-3H]mannose (33 Ci/mmol) as well as UDP-N-a~ety1-[6-~H]glucosamine (6.6 Ci/ mmol) from New England Nuclear, while unlabeled GDP-Man and UDP-Glc, which were added in certain incubations, were purchased from Behring Diagnostics.

The commercially obtained Dol-P (Sigma) which was used as an exogenous acceptor was standardized against tritium-labeled Dol-P of known specific activity (0.6 pCi/pmol) (prepared by phosphoryla- tion (6 ) of a mixture of unlabeled dolichol (Sigma) and [l-3H]dolichol (New England Nuclear) and purified by thin layer chromatography) by comparing the dose-response curve for the enhancement of Dol- P-Man formation from GDP-[3H]Man in a thyroid microsomal sys- tem (7).

Measurement of Lipid- and Protein-linked Saccharides-The in- cubations were terminated by the addition of 2 ml of methanol, and lipid extractions were performed with chloroform/methanol/uffer (3:2:1) and chloroform/methanol/water (10103), in a manner previ- ously described, to yield dolichol-linked monosaccharide and oligo- saccharide fractions, respectively (8). The protein-bound oligosaccha- rides were obtained as glycopeptides after Pronase digestion of the delipidated membrane proteins; Dowex 50 and Dowex 1 columns were employed to separate the glycopeptides from glucan-derived neutral components (4). Radioactivity incorporated into the various fractions was determined by scintillation counting.

Incubation with Exogenous Peptides-To evaluate the effect of ex- ogenous peptide on the transferases, Tyr-Asn-Leu-Thr-Ser-Val (pep- tide A) or Tyr-Asn-Pro-Thr-Ser-Val (peptide B), supplied by Bio- search Laboratories, was incubated at a concentration of 150 p M with the ER vesicles for 15 min at 27 "C before the addition of radiolabeled sugar nucleotide. After a further 10-min incubation at 27 "C, the

phase from the chloroform/methanol/water (3:2:1) extract from samples were fractionated in the usual manner to provide an upper

which the glycosylated peptide was recovered after adsorption on a column of Dowex 50-X2 (H' form) 200-400 mesh and elution with 1.5 N NH,OH (9). Radioactivity incorporated into the peptides was determined by scintillation counting after removal of the solvent by lyophilization.

Endo H Digestion+-Oligosaccharides were released from the gly- cosylated exogenous hexapeptides or from the Pronase-prepared gly- copeptides from endogenous vesicle proteins by treatment with endo H from Streptomyces griseus (Miles Laboratories) as previously re- ported (10); digestion of oligosaccharide-lipids with this enzyme was carried out in 0.1% Triton X-100 as recently described (9). After passage of the digests through coupled columns of Dowex 50 (H' form) and Dowex 1 (acetate form), the liberated oligosaccharides were submitted to thin layer chromatography.

Evaluation of Vesicle Integrity-After radiolabeling vesicle com- ponents by incubation with UDP-[3H]Glc (5 pCi/tube) under stand- ard conditions five tubes were pooled, diluted with ice-cold 0.25 M sucrose, and pelleted by centrifugation at 100,000 X g for 60 min at 2 "C. Subsequent to a wash with the 0.25 M sucrose the radiolabeled vesicles (125 +g protein) were incubated for 10 min at 27 "C in 250 pl of 0.1 M sucrose, 50 mM Tris acetate buffer, pH 7.0, containing 25 mM potassium acetate, 5 mM magnesium acetate, and varying con- centrations of Triton X-100, The incubations were terminated by placing the tubes in ice and after centrifugation (100,000 X g for 60 min, 2 "C) the radioactivity in the solubilized material was determined by scintillation counting. The residual pellets were dissolved in 4% (w/v) sodium dodecyl sulfate and 5% 2-mercaptoethanol (v/v) in 80 mM Tris/HCl, pH 6.8, for measurement of radioactivity and exami- nation by polyacrylamide gel electrophoresis in a manner previously described (4).

Thin Layer Chromatography-Oligosaccharides were resolved by chromatography in 1-propanol/acetic acid/water, (3:3:2), for 40 h on plastic sheets precoated with silica gel 60 (0.2-mm thickness, Merck)

with a wick of Whatman No. 3 paper clamped to the top of the thin layer plate (11). The individual components, after detection by fluo- rography, were scraped from the plates and quantitated by scintilla- tion counting. Acid-labile saccharides from the lower phase of the chloroform/methanol/uffer (3:2:1) extract were identified by chro- matography on cellulose-coated plates (0.1 mm, Merck) in a pyridine/ ethyl acetate/water/acetic acid (5:5:3:1) system (4).

Radioactivity Measurements-Liquid scintillation counting was performed in Ultrafluor (National Diagnostics) with a Beckman LS 7500 instrument. Radioactive components on thin layer plates and electrophoresis gels were detected by fluorography at -70 "C after spraying with a scintillation cocktail containing 2-methylnaphthalene (4) or treating with ENHANCE (New England Nuclear), respectively. For all fluorography, X-Omat AR film (Eastman Kodak) was em- ployed.

RESULTS

Integrated Utilization of Glucose by Thyroid ER Vesicles- We have previously reported that intact thyroid ER vesicles incubated with UDP-[3H]Glc bring about a rapid incorpora- tion of glucose into a lower phase lipid characterized as Dol- P-Glc (4). This transfer, which has a K,,, of 0.54 PM, is maximal at 10 min under the conditions of our assay and is tightly coupled to reactions leading to the formation of oligosaccha- ride-lipid and the N-glycosylation of endogenous protein (Fig. 1). Indeed these two products reached plateaus at lower UDP- Glc concentrations than the Dol-P-Glc itself (Fig. l), although the latter component is known to be responsible for the glucosylation of the lipid-linked oligosaccharide (12), which in turn is transferred to protein. The amount of radioactivity recovered in the lipid-linked oligosaccharide which is almost exclusively present as Glc3MangGlcNAc2-P-P-Dol in this sys- tem (4), was close to three times that observed in the Dol-P- Glc (Fig. 1) under the usual conditions of assay (1.45 PM UDP-Glc). The radioactivity associated with the protein was endo H releasable and occurred, as revealed by thin layer chromatography, in the form of Glc3MangGlcNAc as well as GlczMangGlcNAc and Glc,Man,GlcNAc indicating that some processing had taken place.

Effect of Vesicle Integrity on Coupling of Lipid-Sacchnride Formation and N-glycosylation of Protein-To determine the

Dol-P-P-Oligo

n W

I 1 I

0 I 2 3

UDP-GLUCOSE IrM) FIG. 1. Effect of UDP-Glc concentration on the transfer of

glucose to endogenous lipid and protein acceptors by thyroid ER vesicles. Incubations were carried out with 1.0 pCi UDP-l3H] glucose (1.45 p ~ ) as described under "Experimental Procedures" (standard assay) for 10 min at 27 "C with 180 pg of membrane protein. Radioactivity was measured in the lower phase after chloroform/ methanol/water (3:2:1) extraction (Dol-P-Glc), in the chloroform/ methanol/water (1010:3) extract (Dol-P-P-Oligo), and in the glyco- peptides after Pronase digestion (Protein).

Control of N-Linked Carbohydrate Synthesis in ER Vesicles 14727

effect of vesicle disruption on the reactions leading to the N- glycosylation of protein incubations with increasing amounts of Triton X-100 were carried out (Fig. 2). Since it has been previously shown that permeabilized vesicles require exoge- nous lipid acceptor for Dol-P-Glc formation (4), these assays were carried out in the presence of 70 pM Dol-P to assure adequate synthesis of this first reaction product and thereby permit study of the subsequent transfer steps. A comparison of the intact (Fig. 1) and Triton-treated (Fig. 2) vesicles indicates that while there is an efficient transfer of glucose from Dol-P-Glc to oligosaccharide-lipid in the former situa- tion, this is no longer evident in the latter case; indeed in the presence of the detergent a 7-fold increase in Dol-P-Glc was required to achieve the maximal enhancement (1.7-fold) of the oligosaccharide-lipid. Furthermore, the glycosylation of protein showed a decline with increasing concentrations of Triton despite the presence of elevated levels of oligosaccha- ride-lipid donor (Fig. 2). These findings suggested that the coupling of the reactions involving the transfers of glucose from Dol-P-Glc to oligosaccharide-lipid and oligosaccharide to protein was substantially impaired by the action of Triton on vesicle integrity. This cannot be attributed to an adverse effect of Triton on the enzymes themselves as previous studies have shown that the Dol-P-G1c:oligosaccharide-lipid gluco- syltransferase (12) and the oligosaccharide-lipidpeptide oli- gosaccharyltransferase (13) are actually stimulated by this detergent in the concentration range used in the present study. Thin layer chromatography of endo H-released oligo- saccharides indicated that the Triton had no qualitative effect on the carbohydrate units associated with the lipid or protein.

To investigate the state of the ER membranes at various detergent concentrations the solubilization of membrane com- ponents from preradiolabeled vesicles was determined. Disas- sembly of the vesicles, already evident at 0.025% Triton, was essentially complete at 0.1% of the detergent (Fig. 3). Electro- phoresis of the material remaining after the Triton treatment indicated by fluorographic examination that the glucosylated proteins and oligosaccharide-lipids were solubilized in a non- selective fashion.

Effect of Membrane Fluidity on Lipid-Saccharide S y n t h - sis-In order to further examine the importance of membrane organization on the formation of Dol-P-Glc and glucosylated

0 0.025 0.05 0.075 0.10

TRITON ( 1 6 1 FIG. 2. Effect of Triton X-100 concentration on the forma-

tion of lipid-saccharides and the N-glycosylation of protein by thyroid ER vesicles in the presence of exogenous Dol-P. Incubations were carried out with UDP-[3H]Glc under standard conditions as described under “Experimental Procedures.” Each tube also contained Dol-P (70 p ~ ) which was found on the basis of assays with [3H]Dol-P to be completely in solution at Triton concentrations above 0.025%; at 0.01% Triton the solubility was 75%. The incorpo- ration of radioactivity is expressed relative to the values obtained in the absence of detergent (control) which were 18,000, 43,000, and 16,000 dpm for Dol-P-Glc, Dol-P-P-0120, and Protein, respectively.

TRITON (%)

FIG. 3. Effect of Triton X-100 concentration on the integ- rity of thyroid ER vesicles. After incubation of vesicles with UDP- [3H]Gle followed by centrifugation and washing the solubilization of the radiolabeled membrane components was evaluated at various Triton concentrations as described under “Experimental Procedures.”

(C.1 45 35 25 15 I I I I 0 1 I I

Dol-P-Glc - M Dol-P-P-Oligo

> c I a 0 a a a

I- O

(3 s

- 4.4 -

U I

4.2 - I

“p a 4.0 -

- 1 1 1 , -L , I I I I I I I I

3.1 3.2 3.3 3.4 3.5

I / T x IO3 ( K l

FIG. 4. Effect of temperature on the formation of lipid- saccharides by thyroid ER vesicles. Incubations with UDP-[3H] Glc were carried out at the indicated temperatures under the standard conditions described under “Experimental Procedures.” The radio- activity incorporated into Dol-P-Glc and Dol-P-P-Oligo is presented in the form of an Arrhenius plot as well as in a direct manner (inset).

oligosaccharide-lipid the effect of incubation temperature was determined. An Arrhenius plot of the data indicated that while Dol-P-Glc synthesis responded to temperature in a linear manner up to 37 “C, oligosaccharide-lipid glucosylation showed a transition point already at 22 “C (Fig. 4). This suggested that greater mobility of the membrane components at higher temperature brought about an uncoupling of reac- tions involving the transfer of glucose which was comparable to that observed with increasing Triton concentrations (Fig. Z), although we cannot exclude the possibility that the indi- vidual enzymes may have different temperature responses.

Effect of Exogenous Dol-P on Lipid-Saccharide Formation and N-Glycosylation of Protein-The addition of Dol-P to partially disassembled ER vesicles (0.04% Triton) resulted in a linear increase in Dol-P-Glc formation with a maximal stimulation at 80 g M (Fig. 5 ) . Although Dol-P-Glc synthesis responded in a sensitive manner to increasing Dol-P concen- tration, the formation of oligosaccharide-lipid again lagged behind and did not reach its maximum (2-fold stimulation) until a 10-fold increase in Dol-P-Glc had been attained (Fig. 5).

14728 Control of N-Linked Carbohydrate Synthesis i n ER Vesicles

4 -

3 -

2 -

I -

t

Dol-P-P-Oligo

Protein

0 0 0 40 80 120 160 200

Dol-P (pM) FIG. 5. Effect of Dol-P concentration on the formation of

lipid-saccharides and the N-glycosylation of protein by thy- roid ER vesicles. Incubations were carried out with UDP-[3H]Glc under standard conditions as described under “Experimental Proce- dures” except that 0.04% Triton X-100 and varying amounts of Dol- P were included in each sample. The values plotted represent the radioactivity incorporated into Dol-P-Glc, Dol-P-P-Oligo, and endog- enous Protein.

I I I I I I 1 0 10 20

GDP-Man (pM) FIG. 6. Effect of GDP-Man on the utilization of UDP-Glc

by intact thyroid ER vesicles. Incubations were carried out under standard conditions as described under “Experimental Procedures” with UDP-[3H]Glc (1.45 p ~ ) and varying amounts of unlabeled GDP- Man. The values are expressed relative to those obtained in the absence of GDP-Man (control) which were 28,000 and 57,000 dpm for Dol-P-Gk and Dol-P-P-Oligo, respectively.

Competition of Glycosyltransferases for Dol-P Pool-Since the formation of lipid-monosaccharide appears to be limited by the Dol-P content of the vesicle it was of interest to determine whether the ER UDP-Glc and GDP-Man:Dol-P glycosyltransferases compete for this lipid acceptor. When the vesicles were incubated with UDP-[3H]Glc (1.45 p ~ ) in the presence of increasing concentrations of unlabeled GDP- Man pronounced inhibition in the transfer of glucose to both Dol-P and to oligosaccharide-lipid were observed (Fig. 6), with almost complete blockage evident at 10 p~ GDP-Man. Although this inhibition by GDP-Man of glucose transfer was also evident in the presence of various concentrations of Triton (Fig. 7), it could be overcome by the addition of Dol- P; this relief which progressed with increasing concentration of the detergent became complete a t 0.2% Triton (Fig. 7) at which complete disruption of the vesicle membranes occurs (Fig. 3).

The inhibition imposed by GDP-Man on Dol-P-Glc syn- thesis could also be relieved by GTP (Fig. 8); since the addition of this nucleotide to the vesicle incubations selec- tively inhibited the GDP-Man:Dol-P mannosyltransferase, it apparently stimulated Dol-P-Glc production by in effect mak-

1 100

GDP-Man z 0

5 0 -

7 -

6 -

5 - I-

s 0 4 -

2 2 -

t- 3 - 0

a I -

GDP-Hon

-4 0 0.01 0.02 0.03 0.04 0.05 0.2

TRITON (X) FIG. 7. Effect of exogenous Dol-P on the inhibition by GDP-

Man of Dol-P-Glc formation in thyroid ER at various Triton X-100 concentrations. Incubations were performed with UDP- [3H]Glc (1.45 NM) under standard conditions as described under

the presence of unlabeled GDP-Man (10 p ~ ) with and without Dol- “Experimental Procedures” with varying concentrations of Triton in

P (70 p ~ ) as well as without either of these additions. The percent inhibition of Dol-P-Glc synthesis by GDP-Man in the absence and presence of Dol-P is shown in the upper panel while in the lower panel Dol-P-Glc formation is expressed as ratio to the value obtained

32,700 dpm. in the absence of GDP-Man, Dol-P, and Triton (control) which was

I Dol-P- GIC

0 c

I 20 0 x

0 W

g IO 2 $ 5

n o

c

n - 0

Dol-P-Man

GTP - + “ + + GDP-Mon - + - + FIG. 8. Effect of GTP on the inhibition by GDP-Man on Dol-

P-Glc formation in thyroid ER vesicles. Incubations with UDP- [3H]Glc (1.0 pCi, 1.45 p ~ ) to measure Dol-P-Glc formation were carried out under standard conditions as described under “Experi- mental Pracedures” in the presence of unlabeled GDP-Man (5 p ~ ) and/or GTP (1 mM). For comparison Dol-P-Man synthesis was measured by incubating the vesicles with GDP-[3H]Man (0.27 YCi, 0.10 p M ) in presence or absence of GTP (1 mM); similar inhibitions of Dol-P-Man synthesis were observed at GTP concentrations of 0.125 mM and 0.25 mM.

ing more of the endogenous Dol-P available to the glucosyl- transferase.

In contrast to the inhibitory effect of GDP-Man on UDP- Glc utilization, the latter nucleotide did not interfere with

Control of N-Linked Carbohydrate Synthesis in ER Vesicles 14729

Dol-P-Man synthesis in ER vesicles; addition of increasing amounts of unlabeled UDP-Glc (up to 100 PM) to incubations containing 0.38 PM GDP-[3H]Man resulted in no discernible decrease in mannose incorporation (data not shown). The absence of a mutual inhibition by the two sugar nucleotides in the Dol-P: glycosyltransferase reactions is most likely attributable to different affinities of the two enzymes for their substrates; indeed we find that the K,,, for the sugar nucleotide of the GDP-Man:Dol-P mannosyltransferase (0.052 PM) in the intact vesicles is one order of magnitude lower than that of the UDP-G1c:Dol-P glucosyltransferase (0.54 pM).

Effect of Exogenous Peptides on Lipid-Saccharide Formation and N-Glycosylation-Since exogenous Dol-P can not be made available to the transferases of the ER without vesicle disrup- tion, we explored the limitation in glycosylation imposed by the level of this substrate by generating endogenous Dol-P through a stimulation of Dol-P-P-oligosaccharide utilization. This was accomplished by adding a membrane permeable N- glycosylation substrate, namely Tyr-Asn-Leu-Thr-Ser-Val (peptide A), to the vesicles prior to carrying out the incuba- tions with UDP-[3H]Glc. The presence of peptide A substan- tially promoted N-glycosylation by the ER vesicles so that despite a progressive decrease in transfer of oligosaccharide to endogenous protein, the total saccharide recovered in as- paragine-linked carbohydrate (peptide A plus protein) was about 4-fold greater than in the absence of this peptide acceptor (Fig. 9).

Thin layer chromatography of the oligosaccharides released by endo H from the N-glycosylated peptide A indicated the presence of Glc3MangGlcNAc, GlcpMansGlcNAc and GlclMansGlcNAc (Fig. 10). Since the oligosaccharide-lipid formed by the ER vesicles is almost exclusively in the fully glucosylated form (4) processing of the transferred carbohy- drate units must have taken place and indeed this seemed to be more rapid than for the endogenous protein (Fig. 10).

I , , , , , I 0 80 1 6 0 0 80 1 6 0

PEPTIM (pM)

FIG. 9. Effect of various concentrations of exogenous pep- tides on the formation of lipid-saccharides and the transfer of oligosaccharide to the peptides as well as endogenous protein by thyroid ER vesicles. After preincubation (15 min, 27 “C) with the indicated amounts of peptide A (0) or peptide B (A) as described under “Experimental Procedures” the vesicles were incubated with UDP-[3H]Glc under standard conditions. The radioactivity trans-, ferred to lipid-saccharides (Dol-P-Glc and Dol-P-P-Oligo), Peptide and Protein was determined by the procedures described under “Ex- perimental Procedures” and the data are expressed relative to a control in which the vesicles were preincubated in the absence of exogenous peptide; the control values were Dol-P-Glc, 31,300 dpm; Dol-P-P-Oligo, 77,800 dpm; Peptide, 4,000 dpm; and Protein 14,800 dpm.

FIG. 10. Thin layer chromatographic examination of endo H-released oligosaccharides obtained from glycosylated ex- ogenous peptide and endogenous protein radiolabeled by in- cubations of thyroid ER vesicle with UDP-[3H]Glc. The oligo- saccharides released from the peptide fraction (Pept) after incubation in the presence ( 1 ) or absence (2) of 150 pM peptide A (Tyr-Asn-Leu- Thr-Ser-Val) are compared with those obtained from endogenous pronase digested protein (Prot). The incubations of thyroid vesicles with UDP-[3H]Glc were carried out under standard conditions with a 15-min preincubation with and without the exogenous peptide. The conditions for the thin layer chromatography are specified under “Experimental Procedures” and the components were visualized by fluorography. The migration of standard Glc3MansGlcNAc (G3), GlczMansGlcNAc (G2), and GlclMansGlcNAc ( G I ) are indicated.

The enhanced oligosaccharide transfer was accompanied by an almost complete depletion of oligosaccharide-lipid and more than a %fold increase in the synthesis of Dol-P-Glc (Fig. 9). These changes did not occur (Fig. 9) in the presence of Tyr-Asn-Pro-Thr-Ser-Val (peptide B) which differed from peptide A in containing proline in the place of leucine within the Asn-X-Thr sequence, an alteration which is known to make it unsuitable as an an acceptor for N-glycosylation (14).

Under the conditions of increased oligosaccharide transfer resulting from the addition of peptide A, enhanced synthesis of the other lipid-linked monosaccharides, namely Dol-P-Man and Dol-P-P-GlcNAc, was also brought about (Fig. 11); in the incubations with GDP-[3H]Man and UDP-[3H]GlcNAc un- labeled UDP-Glc (1 PM) was added to permit completion of the oligosaccharide-lipid and thereby assure efficient transfer to the peptide. The increases in the three Dol-linked mono- saccharide products were similar in magnitude and were not observed in the presence of the proline-containing peptide B, suggesting that a supply of endogenous Dol-P, accessible to the three transferases was produced due to the enhanced generation of Dol-P-P from the stimulated N-glycosylation reaction.

Since the increased oligosaccharide transfer engendered by addition of a peptide acceptor to ER vesicles appears to result in the formation of a larger Dol-P pool it should tend to relieve the GDP-Man inhibition of Dol-P-Glc formation from UDP-Glc. To test this possibility, we incubated the vesicles with UDP-[3H]Glc in the presence of inhibiting amounts of

14730 Control of N-Linked Carbohydrate Synthesis in ER Vesicles

Dol-P-Oh Dol-P-Man Dol-P-P-GlcNAc

FIG. 11. Effect of exogenous peptides on the formation of Dol-linked monosaccharides from several sugar nucleotides. Thyroid ER vesicles were incubated under standard conditions with 1 pCi of 3H-labeled sugar nucleotide (UDP-Glc, GDP-Man, or UDP- GlcNAc) after a 15-min preincubation at 27 “C with 150 p~ Tyr-Asn- Leu-Thr-Ser-Val (Peptide A) or Tyr-Asn-Pro-Thr-Ser-Val (Peptide B ) as well as in the absence of added peptide (Control). The amount of Dol-linked monosaccharide formed was calculated from the radio- activity recovered in the lower phase of the chloroform/methanol/ water (3:2:1) extract and the specific activity of the sugar nucleotide precursors (see “Experimental Procedures”). +

- 0 0

5 IO 20

GDP-Man (pM)

inhibition by GDP-Man of Dol-P-Glc formation in thyroid ER FIG. 12. Effect of an exogenous peptide acceptor on the

vesicles. Incubations with UDP-[3H]Glc (1 pCi, 1.45 p ~ ) to measure Dol-P-Glc formation were carried out under standard conditions as described under “Experimental Procedures” in the presence of vary- ing concentrations of unlabeled GDP-Man after a 15-min preincu- bation at 27 “C with or without peptide A acceptor (150 pM).

unlabeled GDP-Man both with and without peptide A and found that in the presence of the peptide acceptor there was a stimulation of Dol-P-Glc synthesis (Fig. 12); not unexpect- edly this stimulation was inversely related to the concentra- tion of the GDP-Man.

DISCUSSION

As our understanding of the intermediates and enzymatic reactions required for the addition of asparagine-linked car- bohydrate to newly formed peptide chains has enlarged, in- terest has grown in the manner in which this process is controlled. Since protein N-glycosylation appears to occur at the luminal face of the ER concomitant with the passage of the newly synthesized peptide through the membrane (15-17) while the initial steps of the pathway involving formation of dolichol-linked monosaccharides occur cytosolically (4,15,17,

18), a high level of organization of the various enzymes and substrates must be required.

Indeed our present studies indicate that the thyroid ER vesicles provide an integrated multienzyme system in which N-glycosylation of endogenous proteins from lipid-saccharide precursors takes place in an efficient manner. Disruption of the integrity of the vesicles by detergent led to an uncoupling of the reactions leading to protein glycosylation. Even in the presence of a small amount of Triton (0.025%), insufficient to solubilize the membrane components, the oligosaccharide transfer step was separated from the preceding reactions and transfer of glucose from Dol-P-Glc to oligosaccharide-lipid became much less effective. Since supplementation of the permeabilized vesicles with Dol-P stimulated Dol-P-Glc pro- duction without bringing about a corresponding effect on oligosaccharide-lipid synthesis or N-glycosylation, it appeared that a tight membrane structural organization is required to assure the concerted action of the various transferases. A similar type of uncoupling effect on the transfer of glucose was observed at incubation temperatures above 22 ”C where less rigidity of membrane structure would be expected.

A major locus of control for N-glycosylation has been postulated to be the level of Dol-P in ER membranes based on our findings with tissue slices (8) and those of others with cultured cells (19, 20) that when incubations are carried out in the presence of inhibitors of protein synthesis the amount of oligosaccharide-lipid remained constant presumably be- cause the Dol-P required for the formation of new lipid-linked oligosaccharide was limiting. The modulation of glycosyl- transferase activity which has been observed during organ development (21) or after hormone administration (22) has been attributed to elevated tissue levels of Dol-P, and more- over, addition of this lipid to pancreas slices has been found to increase the degree of glycosylation of ribonuclease (23).

In the present investigation with thyroid ER vesicles the limitation of the Dol-P pool was demonstrated in several ways, including the stimulation of Dol-P-Glc formation upon supplementation of permeabilized vesicles with exogenous Dol-P; the inability of the endogenous supply of Dol-P to support glucosyltransferase activity in the face of a competing requirement for this lipid acceptor by mannosyltransferase at elevated GDP-Man concentrations; and the enhancement of lipid-monosaccharide synthesis from UDP-Glc, GDP-Man, and UDP-GlcNAc as a result of vesicular Dol-P generation from Dol-P-P-oligosaccharide in the presence of exogenous peptide acceptor.

Although addition of Dol-P to permeabilized ER vesicles brought about a substantial stimulation of Dol-P-Glc synthe- sis (10-fold at 150 PM Dol-P) this could only be achieved in an extensively disrupted membrane system. A much more effective stimulation of Dol-P-Glc formation could be achieved in intact vesicles by promoting the recycling of endogenous Dol-P through the addition of a peptide acceptor for the oligosaccharyltransferase reaction. In accord with previous studies employing small peptide acceptors in oviduct microsomes (24) the hexapeptide (peptide A) which we used penetrated the intact ER vesicles to serve as a substrate for N-glycosylation and this permitted a pronounced enhance- ment of oligosaccharide-lipid utilization beyond that which can occur with the limited endogenous polypeptides which are present in ER membranes.

In order for Dol-P to be made available from the Dol-P-P released during the oligosaccharyltransferase reaction cleav- age by a Dol-P-P phosphatase must occur; such enzymes have been found in a variety of cell types (25-27) and indeed studies with liver microsomes employing the inhibitor bacitracin have

Control of N-Linked Carbohydrate Synthesis in ER Vesicles 14731

suggested that a large portion of Dol-P which serves as substrate for glycosylation originates from the pyrophosphate derivative (26). The approximate amount of Dol-P which can be generated and recycled by the addition of saturating amounts of exogenous peptide can be calculated, from the decrease in the radioactivity associated with oligosaccharide lipid (70,000 dpm; Fig. 10) and the specific activity of the UDP-Glc employed (19,000 dpm/pmol) to be about 2.5 pmol per tube based on our previous finding that the molecules of the endogenous thyroid oligosaccharide-lipid pool have an average of 1.5 sites per molecule available for attachment of additional glucose residues (28). Making this additional amount of Dol-P available by incubating with exogenous peptide acceptor resulted in about a 3.3 pmol stimulation of Dol-P-Glc synthesis (Fig. 10) while in contrast 1 5 p ~ (1,200 pmol per tube) of exogenous Dol-P had to be added to a permeabilized ER vesicle to achieve a similar enhancement in Dol-P-Glc formation (Fig. 4). The greater effectiveness (about 500-fold) of endogenous Dol-P is presumably a func- tion of its insertion into the membrane in a position appro- priate for optimal glucosyltransferase action.

Our data indicate that the inhibiting effect of GDP-Man on Dol-P-Glc synthesis from UDP-Glc, which has previously been observed by others (29, 30), is a function of a limited endogenous Dol-P pool. This inhibition was observed in intact as well as in Triton-disassembled ER vesicles and in case of the latter situation could be relieved by the addition of Dol- P. In the closed vesicles a reduction in the inhibiting effect of GDP-Man could be brought about by the additional Dol-P generated in the presence of an exogenous N-glycosylation substrate. Moreover, addition of GTP, which inhibits the mannosyltransferase without affecting the transfer of glucose, relieved the inhibition by GDP-Man providing further evi- dence for the limitation imposed by a shared Dol-P pool and arguing against the possibility that the mannose nucleotide produces its effect by reacting directly with the glucosyltrans- ferase as has been suggested (29). Indeed a study of UDP- G1c:Dol-P glucosyltransferase which had been purified from a plasmacytoma cell line so as to remove mannosyltransferase activity indicated that GDP-Man even at high concentrations had no inhibiting effect on the action of the enzyme (31).

The lack of inhibition by UDP-Glc on Dol-P-Man synthesis from GDP-Man which we noted suggests that the mannosyl- transferase may be more favorably positioned in respect to the endogenous lipid acceptor pool and/or may have a greater synthetic capacity. Indeed physiologically one might antici- pate such a situation as in most tissues the concentration of UDP-Glc is many times that of GDP-Man (32, 33) and in thyroid we find that the UDP-Glc level is approximately 25 pmol/g compared to 1.6 pmol/g for GDP-Man.' Despite this difference in the level of the two sugar nucleotides similar amounts of Dol-P-Glc and Dol-P-Man would have to be produced by the ER membranes in order to assemble a com- plete lipid-linked oligosaccharide (1-3). The K,,, values which we find for the two transferases in intact ER vesicles (UDP- Glc = 0.54 pM; GDP-Man = 0.052 p ~ ) are in keeping with the different concentrations of the two nucleotides in the tissue; other published reports of K, values for Dol- P:glucosyl- (31, 34, 35) and mannosyltransferases (7, 21, 36, 37) also indicate lower values for the latter enzyme.

Our study indicates that the Dol-P made available by recycling of Dol-P-P released by oligosaccharide transfer to protein becomes part of a common pool which is available to the three enzymes responsible for the synthesis of Dol-linked monosaccharides (Dol-P-Glc, Dol-P-Man, and Dol-P-P-

M. J. Spiro and R. G. Spiro, unpublished observations.

GlcNAc), a situation which apparently also applies to Dol-P formed through the dolichol kinase reaction (38). Since it is believed that oligosaccharide transfer to protein takes place on the luminal face of the ER (16, 17) while Dol-linked monosaccharide synthesis occurs on the cytoplasmic side (4, 15, 17, 18) the Dol-P generated by N-glycosylation and Dol- P-P phosphatase action must translocate before it can again be available at the outer membrane surface to react with the various Dol-P:glycosyltransferases. Although the mechanism of movement of Dol-P and its saccharide derivatives has not yet been defined, and represents one of the more challenging aspects of transmembrane movement of polar molecules, re- cent studies (39, 40) indicate that Dol-P molecules by locally inducing a nonbilayer phase in membranes can produce a more fluid environment in which the translocations required for the assembly of the N-linked carbohydrate units could take place.

Acknowledgment-We would like to thank Kathleen M. Gorski for her skillful assistance.

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