Determination of the Distance between the Oligosaccharyltransferase ...

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Active Site and the Endoplasmic Reticulum Membrane*. (Received for ... was from Promega. DNA Technique-Site-specific mutagenesis was performed ac-.
THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 268, No. 8, Issue of March 15, pp. 57965801,1993 Printed in U.S.A.

Q 1993 by The American Society forBiochemistry and MolecularBiology, Inc.

Determination of the Distance between the Oligosaccharyltransferase Active Site and the Endoplasmic Reticulum Membrane* (Received for publication, October 27, 1992)

IngMarie Nilsson and Gunnar von HeijneS From the Department of Molecular Biolom.Karolinska Institute Center for Structural Biochemistry, NOVUM, S-141 57 Huddinge, Sweden .

By in vitro transcription/translation of model proteins in the presence of dog pancreas microsomes, we have measured the minimum distance of an acceptor site from the lumenal end of a transmembranesegment required for N-linked glycosylation, both when the acceptor site isplaced N- and C-terminally to the membrane anchor. We observe a sharp threshold at a distance of 12-14 residues, suggesting that the oligosaccharyltransferase activesite is 30-40 A above the membrane andis oriented roughly parallel to the membrane surface.

The attachment of N-linked carbohydrate to eukaryotic secretory and membrane-bound proteins takes place during or soon after translocation of the nascent polypeptide across the endoplasmic reticulum membrane (1, 2). Thisinitial transfer of high-mannose oligosaccharides from a dolicholcarrier to Asn-X-Thr/Ser acceptor sites is catalyzed by the oligosaccharyltransferase enzyme, which is located in the endoplasmic reticulum membrane and has its catalytic site facing the lumenal compartment (3).While the characteristics of the acceptor sites are quite well understood (4, 5), little is known about the oligosaccharyltransferase itself. Recently, however, two abundant endoplasmic reticulum proteins, ribophorins I and11, together with a third48 kDa protein, were shown to co-purify with the oligosaccharyltransferase activity, and the activity could be specifically depleted by antibodies directed against ribophorin I (6). It thus seems that the ribophorins are intimately involved in the glycosylation reaction.In the present study, we have tried to further characterize the oligosaccharyltransferase by designing substrate proteins where the acceptor site is presented to thetransferase enzyme at defined distances from the lumenal surface of the endoplasmic reticulum membrane. We find that there is a precise distance constraint that only allows glycosylation of acceptor sites when the Asn residue is located a minipum of 12-14 residues (corresponding to a distance of 40-45 A for an extended polypeptide) either upstream or downstream of a transmembrane segment. The most straightforward interpretation of these results is that the oligosaccharyltransferase active site is positioned 30-40 A above the lumenal membrane surface and that it is oriented such that, when held in the

active site, the Asn-X-Thr/Ser tripeptide is roughly parallel to the membrane. Thisfurther suggests that the dolichol carrier may not be completely buried in the lipid bilayer; rather itmay, while still anchored in themembrane, protrude sufficiently far out intothe lumenal space to reach the active site of the oligosaccharyltransferase. MATERIALS ANDMETHODS

Enzymes and Chemicals-Unless otherwise stated, all enzymes were from Promega. T7 DNA polymerase wasfrom Pharmacia. Protease K was from Merck. [35S]Metwas from Amersham. Ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, andthe cap analog m7G(5’)ppp(5’)G were from Pharmacia. Oligonucleotides were produced using an Applied Biosystems synthesizer 380B followed by NAP-5 (Pharmacia) purification. Spermidine, phenylmethylsulfonyl fluoride, bovine serum albumin, creatine phosphate, and creatine phosphokinase were from Sigma. The glycosylation acceptor and the nonacceppeptide N-benzoyl-Asn-Leu-Thr-N-methylamide tor peptide N-benzoyl-Asn-Leu-(a1lo)Thr-N-methylamide were synthesized according to Erickson and Merrifield (7). Plasmid pGEMl was from Promega. DNA Technique-Site-specific mutagenesis was performed according to the method of Kunkel (a), asmodified by Geisselsoder et al. (9). All mutants were confirmed by DNA sequencing of singlestranded M13 DNA using T7 DNA polymerase. For cloning into and expression from the pGEMl plasmid, the 5’ end of the lep gene was modified, first, by the introduction of an XbaI site and, second, by changing the context 5’ to the initiator ATGcodon to a “Kozak consensus” sequence (10). Thus, the 5’ region of the gene was modified to: . . .ATAACCCTCTAGAGCCACCEGCGAATATG.. . (XbaI site and initiator codon underlined). The XbaI-SmaI fragment carrying lep was cloned into pGEMl behind the SP6promoter. In Vitro Transcription and Translation-Synthesis of RNA by SP6 RNA polymerase and translation inreticulocyte lysate in the presence of dog pancreas microsomes was performed as described (11).Translocation of polypeptides to the lumenal side of the microsomes was assayed by resistance to exogenously added protease K and by prevention of N-linked glycosylation through competitive inhibition by addition of the glycosylation acceptor tripeptide. RESULTS

Design of Oligosacchuryltransferme Substrate MoleculesWe have chosen to use a protein normally found in the inner membrane of Escherichia coli, leader peptidase (Lep), as a scaffold for presenting potential Asn-Ser-Thr acceptor sites to the oligosaccharyltransferase and have assayed its glycosylation in vitrousing dog pancreas microsomes. Weconsider Lep a good model for a number of reasons. From the point of * This work was supported by grants from the Swedish Natural viewof glycosylation, it is a “virgin” molecule that has not Sciences Research Council andthe Swedish National Board for been under any selective pressure to evolve a structure that Industrial and Technical Development (to G. von Heijne). The costs either promotes or prevents glycosylation at specific sites. It of publication of this article were defrayed in part by the payment of has two transmembrane segments (Fig. 1; Refs. 12-14) and page charges. This article must therefore be hereby marked “aduer- both the N and C terminus are located in the periplasmic tisement” in accordance with 18U.S.C. Section 1734 solelyto indicate space (and thusface the lumen when the protein is integrated this fact. $ To whom correspondence should be addressed. Fax: 46-8-774-55- into microsomes, see below). This allows the introduction of potential glycosylation sites either N- or C-terminally to the 38.

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FIG.2. Integration of wild type leader peptidase (top) and mutant N214Q (bottom) into dog pancreas microsomes. Lep was expressedin reticulocyte lysate in the absence (lane I ) or presence (lanes 2-7) of dog pancreas microsomes ( R M ) . Protease sensitivity was assessed by protease K treatment in theabsence and presence of Triton X-100 (P-K, lanes 3, 6, and 7). N-Linked glycosylation on Asn2I4in the P2 domain was probed by addition of acceptor (Ac; lane 4 ) or nonacceptor(N-Ac;lane 5 ) peptide to the translation mix. Rand MA*ALI. ..LI*SFIYPFQ.. a, glycosylated Lep; band b, non-glycosylated Lep; band c , glycosylated FIG.1. Orientation of Lep in the inner membraneof E. coli protease-resistant P2 fragment; band d, non-glycosylated proteaseand inmicrosomes. The twohydrophobic transmembrane segments resistant P2 fragment. H1 and H2, the cytoplasmic loop P1, the large periplasmic (lumenal) domain P2, and the Am2“glycosylation site are indicated. The wild lated forms of the protein canbe easily distinguished. type aminoacid sequence of the N-terminal part upstreamof H1 and C-terminal Acceptor Sites (the Minimum Distance Is 12-13 of a segment immediately downstream of H2 are also given; the Residues)-To measure the minimum distance from the luboldface residues M4 (converted to Glu in the N-terminal glycosylation menal end of the second transmembrane domain to a funcmutants, see Table I) and R,? arethe reference points for the calculation of distances between potential glycosylation acceptor sites tional acceptor site, we introduced the tripeptide Asn-SerThr at various positions downstream of H2 in the N214Q and the transmembranesegments.

.

mutant (Table I). When the Asn residue was placed 5 or 11 lumenal end of a transmembrane segment. Finally, we have residues downstream of Arg” that ends the hydrophobic H2 recently been able to map therelative orientation of the two domain, no glycosylation was observed (Fig. 3, mutants C5, transmembrane segments,’ and thus have a fairly good idea Cl la, and Cllb).However, when the distance from H2 was 13, 14, 15, or 20 residues, the acceptor site was efficiently of their position in the membrane. Lep was cloned into plasmid pGEMl under the SP6 pro- glycosylated (mutants C13-C20), and glycosylation could be moter. When expressed in vitro in the absenceof microsomes, inhibited by acceptor but not by nonacceptor peptide (data a product with the same mobility as Lep produced in E. coli not shown). The P2 domain was translocated in all cases, as that could be immunoprecipitated by Lepantiserum was evidenced by the appearance of a (glycosylated or non-glycoi.e. obtained (data not shown). In the presence of microsomes, a sylated) protease-protected fragment (data not shown); more slowly migrating form was produced (Fig. 2, compare the results were similar to those shown above for wild type of this productwas largely inhibited Lep and the N214Q mutant, respectively. T o rule out the lanes I and 2); formation by the acceptor peptide N-benzoyl-Asn-Leu-Thr-N-methy-possibility that the transition from non-glycosylated to glylamide, a competitive inhibitor of N-linked glycosylation (lane cosylated forms resulted fromlocal context effects rather than 4 ) , but not by the nonacceptor control peptide N-benzoyl- from the increased distance from the transmembrane segAsn-Leu-(al1o)Thr-N-methylamide(lane 5 ) . Fortuitously, a ment, the Asn-Ser-Thr acceptor site was positioned around single potentialacceptorsiteforN-linked glycosylation the “critical distance” bothby direct point mutations (mutants C l l a (non-glycosylated) and C13 (glycosylated)) and by the (Asn214-Glu-Thr) ispresentinthe large C-terminalperiplasmic P2 domain. Protease K treatment of the microsomes introduction of extra “spacer” residues (mutants Cll b (nonleft a large protected fragment (lane 3 ) of the size expected glycosylated) and C14 (glycosylated)). It is thus clear that mobility of this glycosylation is possible only when the distance from the Cfor cleavage in the P1 domain,andthe fragment was increased when acceptor peptide was included terminal end of the transmembrane H2 segment to the Asn (lane 7), proving unambiguously that it represents theglyco- acceptor is 212-13 residues. N-terminal Acceptor Sites (the Minimum Distance Is 14-15 sylated P2domain. When themicrosomes were first disrupted Residues)-We also measured the minimum distance between by Triton X-100, no part of the molecule remained resistant to protease K (lane 6). For comparison, Fig. 2 also presents a functional acceptor site and the N-terminal (lumenal) end the results for a mutant (N214Q) where Asn214was changed of theH1transmembrane segment. Inthis case, spacers to Gln to prevent glycosylation. Note that, in this case, the composed of Ser, Gly, and Gln residues and ending with a protease-protected fragment (lane 3 ) had the same mobility Glu were introduced near the N terminus of Lep, replacing as the fragment obtained from wild type Lep assayed in the Met4 (Table I). Since residue 3 of the wild type sequence is Asn, this createsa potential acceptor site Asn-Ser-Thrat the presence of acceptor peptide. We conclude that the P2 domain efficiently is translocated N-terminal end of the spacers. When the Asn in the acceptor site was placed 13 residues into the lumen of dog pancreas microsomes where it is glycosylated on Asn214and that glycosylated and non-glycosy- from the beginning of H1 (taken asresidue Phe6 in the wild type sequence, which in the mutants is next to theGlu ending the spacers), no glycosylation was observed (Fig. 3, mutant P. Whitley and G . von Heijne, manuscript in preparation.

Mapping the Oligosaccharyltransferase Active Site

5800

TABLE I Mutants are discussed in the text. The amino acid sequences of the relevant regions are given (see Fig. 1). Potential glycosylation sites and inserted spacers are underlined. The mutants are designated by either "C" or "N" (C- and N-terminal acceptor sites, respectively) followed by the number of amino acids between the Asn residue in the acceptor site and the end of the transmembrane segment. Mutant N214Q is derived from wildtype Lep by an Asn + Gln replacement that destroys the naturally occurring Asn'"-Glu-Thr acceptor site. Sequence

Mutant

N214Q c5 Clla Cllb C13 C14 C15 c20 N214Q N13 N15 N17 N19 N25

. .R77SFINSTFQIPSGSMMPTLLIGD.. . . .R77SFIYEPFQINSTSMMPTLLIGD.. .

..R77SQISQISFINSTFQIPSGSMMPTLLIGD...

..R-mSFIYEPFOIPSNSTMPTLLIGD. .. . .R;;SQISQIS~~ISFKS~SFQIPSGSMMPTLLIGD. . . ..R77SFIYEPFQIPSGSNSTTLLIGD...

..R77SFIYEPFQIPSGS"PTLm.. W F A L I L V I ...

MANNSQGSQAPGSQGEFALILVI...

... ...

MANNSQGSQAPVAGSQGEFALILVI MANNSQGSQAPVAQGGSQGEFALILVI MANNSQGSQPINAQAAPVAQGGSQGEFALILVI

R FIG.3. Glycosylation of potential . placed C-terminally " acceptor sites (top) and N-terminally (bottom) to a transmembranesegmentinthe presence of dog pancreas microsomes (RM). See Table I for descrip-

N N Y Y Y

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tion of the mutants. Rand a,glycosylated Lep; band b, non-glycosylated Lep.

N13). In contrast, mutantswhere the distancewas 15,17, 19, and 25 residues were allefficientlyglycosylated (mutants N17-N25). In this case, the protease-protected fragmentsall had the same mobility irrespective of whether the proteinwas glycosylated or not, as expected if glycosylation was N-terminal to the P1 domain; furthermore, glycosylation was inhibited by acceptor but notby nonacceptor peptide (data not shown). Thus, the N terminus of H1 is indeed translocated to thelumenal side of themembrane(as expectedfrom its when periplasmic location in E. coli), and the critical distance the acceptor site is placed N-terminally to theH1 transmembrane segment is 14-15 residues.

values for the N- and C-terminal constructs can be directly compared. The precise location of the Hl-H2 helical bundle in the membrane is unknown, but since sidethe chain of Arg77 is unlikely to beembedded within the apolar part of the membrane, it would seem that Arg77 (and thus also Met4) should be positioned very Fear to or within the headgroup region, i.e. a t most 5-10 A below themembrane surface. Finally, since the results are the same both in the presence and absence of spacers rich in Ser, Gly, and Gln between the membrane and the acceptor site and thus independent of sequence context, it islikely that thepolypeptide chain is in a flexible extended conformation when glycosylation takes place. The simplest interpretation of the data is that oligosacthe DISCUSSION charyltransferase active site is located a t a distance above the We have measured the minimum distance from the lumenal headgroup region corresponding to 1:-13 residues in an exend of a transmembrane segment required for efficient gly- tended conform@ion (Fig. 4); with 3.5 Alresidue, this amounts cosylation; when the acceptor site is downstream of the H2 to some 40-45 A. Furthermore, since the N- and C-terminal transmembrane segment in our model protein (Lep) the Asn distances are nearly the same, it would seem that the active residue must be 212-13 residues away; when upstream of the site mustbe oriented approximately parallel to membrane. the H1 transmembrane segment a distance 214-15 residues is Other orientations would require the chain to turn back on necessary. We have recently determined the relative orienta- itself in either the N- or C-terminalcase, and the difference tion of the H1 and H2 transmembrane segments using a between the two distances would be expected to be substandisulfide mapping technique? In the resulting model, Met4 in tially larger (Fig. 4). We note that this model is compatible H1 (which is replaced by a Glu in the N-terminal mutants) yith the dimensions-of the dolichol carrier, which is 90-100 from A long: some 45-65 A could be buried in the membrane orbe and Arg77in H2 are at approximately the same distance the membranesurface, and as those are reference the positions wound around the transmembrane segment of ribophorin I used to calculate the minimum distances,we believe that the (6), whereas the remainderwould extend above the membrane surface, perhaps within the oligosaccharyltransferase com'P. Whitley and G . von Heijne, unpublished data. plex, to reach the active site.

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Mapping the Oligosaccharyltransferase Active Site GOOH

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cytoplasm FIG. 4. Proposed location of the acceptor tripeptide in the oligosaccharyltransferaaeactive site (left).Note that if the active site (oual) was oriented perpendicular to the membrane (middle and right), the difference between the minimum number (dm) of N- and Cterminal residues required for glycosylation would be substantially different from what is observed.

Other more complicated possibilities are that the active site is within a deep cleft and cannot be reached if the potential acceptor site is too closely tethered to themembrane, or that glycosylation is possible only when the nascent chain is still held within the translocation complex (the "translocon") and that thestructure of this complex does not allow the oligosaccharyltransferase to approach closer than some 30-40 A. The latter possibility seems rather unlikely in view of the fact that synthetic acceptor tripeptides canbe glycosylatedwhen added to cells in culture (3) and thus need not be presented in the context of the translocon. We have searched the literature and protein sequence databanks for cases of documented glycosylation sites located close to the lumenal end of transmembrane segments and have found none that is closer than -10 residues. Examples in this category are the yeast plasma membrane ATPase (15), the mouse nicotinic acetylcholine receptor (16), the cyclic AMP receptor from Dictyostelium discoideum (17), the rotavirus NS28 protein (18),the hamster 3-hydroxy-3-methylglutaryl-CoA reductase (19), and engineered variants of the asialoglycoprotein receptor (20). It may be noted that secretory proteins are known with a glycosylated Asn in position +6 (21); presumably, in such cases the modification can take place only after the signal peptide has been cleaved off. In summary, we have found that there is a sharp distance threshold for N-linked glycosylation of membrane-bound proteins and that thecritical distance, d,,,, from the lumenal end of a transmembrane segment is nearly the same when the glycosylation site is either upstream (dm= 14-15 residues) or downstream (dm = 12-13 residues) of the transmembrane segment. This defines the position of the oligosaccharyltransferase active site with considerable precision; earlier studies could only place it at a distance corresponding t o 55 5 25

amino acids from the ribosomal exit site (22). We favor a model where the oligosaccharyltransferase active site is located 30-40 above, and is oriented parallel to, the membrane, suggesting that thedolichol carrying the high mannose oligosaccharide has to extend a considerable distance above the membrane to reach the active site.

A

Acknowledgments-Oligonucleotide synthesis was done by Zekiye Cansu at the Karolinska Institute Center for Biotechnology. The acceptor and nonacceptor peptides were gifts from Dr. Henrik Garoff (Huddinge, Sweden). REFERENCES 1. Kaplan, H. A,, Welply, J. K., and Lennarz,W. J. (1987) Biochim. Biophys. Acta 906, 161-173 2. Hubbard, S. C., and Ivatt R. J. (1981) Annu. Reu. Biochem. 50,555-583 3. Welply J. K. ShenbagaGurthi P., Lennarz, W. J., and Naider, F. (1983) J . E ~ O LC d m . 258,11856-11'863 4. Gavel, Y., and van Heijne, G. (1990) Protein Eng. 3,433-442 5. Avanov, A. Y. (1991) Mol. Biol. (Engl. Traml. Mol. Biol.1 (Mosc.) 25, 237250 6. Kelleher, D. J., Kreibich, G., and Gilmore, R. (1992) Cell 69, 55-65 7. Erickson, B. W., and Merrifield, R. B. (1976) in The Proteins (Hill, R. L., and Neurath, H., eds) Academic Press, London 8. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82.488-492 9. Geisselsoder, J., Witney, F., and Yuckenberg, P. (1987) BioTechniques 5,

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lR6-741

10. Kozak M. (1989) Mol. Cell. Biol. 9 5073-80 11. Liljestkom, P., and Garoff, H.(199i) J. Virol. 65,147-154 12. Wolfe, P. B., Wickner, W., and Goodman, J. M. (1983) J. Biol. Chem. 258, 1 ~ n.m-1 "-

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13. Moore K. E. and Miura S. (1987) J. Biol. Chem. 262,8806-8813 14. Lee, J.'L, Kuhn, A,, and Dalbe R E (1992) J. Biol. Chem. 267,938-943 15. Serrano, R., Kielland-Brandt, $I. C., and Fink, G. R. (1986) Nature 319, 689-693 16. Chavez, R. A., and Hall, Z. W. (1991) J. Biol. Chem. 266,15532-15538 17. Klein, P. S., Sun, T. J., Saxe, C. L., Kimmel, A. R., Johnson, R. L., and 241.1467-1472 Devreotes. P. N. (1988) , ~. ScLence .~~ . . ---, ~ 18. Bergmann, C. C., Maass, D., Poruchynsky, S.,Atkinson, P. H., and Bellamy, A. R. (1989) EMBO J. 8. ~~.~ 1695-1- 703 .. 19. Olender,'E. H:,~and$imoni, R. D. (1992) J.Biol. Chem. 267,4223-4235 20. Wessels, H.P., and Spiess, M. (1988) Cell 55, 61-70 21. Frangione, ,B., Rosenwasser, E., Prelli, F., andFranklin, E. C. (1980) Biochemcst 19,4304-4308 22. Glabe, C. G., ganover, J. A., and Lennarz,W. J. (1980)J . Biol. Chem. 255, 9236-9242 ~

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