Unraveling the Mechanism of Protein N-Glycosylation (2004)

Asparagine-linked glycosylation is the most ubiquitous protein co-translational modification in the endoplasmic reticulum (ER). 1The abbreviations used are: ER, endoplasmic reticulum; OT, oligosaccharyl transferase. 1The abbreviations used are: ER, endoplasmic reticulum; OT, oligosaccharyl transferase. The enzyme that catalyzes this process is called oligosaccharyl transferase (OT). It catalyzes the transfer of an oligosaccharyl moiety (Glc3Man9GlcNAc2) from the dolichol-linked pyrophosphate donor to the side chain of Asn within a consensus sequence of Asn-X-Thr/Ser, where X can be any amino acid residue except for Pro (1Welply J.K. Shenbagamurthi P. Lennarz W.J. Naider F. J. Biol. Chem. 1983; 258: 11856-11863Abstract Full Text PDF PubMed Google Scholar, 2Knauer R. Lehle L. Biochim. Biophys. Acta. 1999; 1426: 259-273Crossref PubMed Scopus (171) Google Scholar, 3Silberstein S. Gilmore R. FASEB J. 1996; 10: 849-858Crossref PubMed Scopus (207) Google Scholar). This modification serves as a primary determinant for specific molecular recognition as well as protein folding and stability (4Dempski Jr., R.E. Imperiali B. Curr. Opin. Chem. Biol. 2002; 6: 844-850Crossref PubMed Scopus (100) Google Scholar, 5Helenius A. Aebi M. Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1618) Google Scholar) and therefore is an essential and highly conserved protein modification pathway in eukaryotic cells. It has been established in both yeast and higher eukaryotic organisms that this enzyme exists as a heteromeric, multisubunit complex in the ER membrane. In the last decade, because of the facility of yeast genetics, genes encoding 9 OT subunits in Saccharomyces cerevisiae have been cloned and identified (for review see Ref. 2Knauer R. Lehle L. Biochim. Biophys. Acta. 1999; 1426: 259-273Crossref PubMed Scopus (171) Google Scholar and earlier studies cited therein). Among them, the five genes encoding Ost1p, Ost2p, Stt3p, Wbp1p, and Swplp are essential for the viability of the cell; the OST4 gene is essential for growth of the cell at 37 °C but not at 25 °C; Ost3p, Ost5p, and Ost6p subunits are not essential for the viability of the yeast cell but are required for maximal enzyme activity. At present, many mammalian OT subunit proteins have also been identified, and some of them share high sequence identity and similarity with yeast homologs. Four of them were isolated from highly purified and active enzyme fractions: ribophorin I (homolog of yeast Ost1p), ribophorin II (homolog of yeast Swp1p), OST48 (homolog of yeast Wbp1p), and DAD1 (homolog of yeast Ost2p) (6Kelleher D.J. Kreibich G. Gilmore R. Cell. 1992; 69: 55-65Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 7Kelleher D.J. Gilmore R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4994-4999Crossref PubMed Scopus (159) Google Scholar, 8Kumar V. Heinemann F.S. Ozols J. J. Biol. Chem. 1994; 269: 13451-13457Abstract Full Text PDF PubMed Google Scholar, 9Kumar V. Korza G. Heinemann F.S. Ozols J. Arch. Biochem. Biophys. 1995; 320: 217-223Crossref PubMed Scopus (24) Google Scholar, 10Breuer W. Bause E. Eur. J. Biochem. 1995; 228: 689-696Crossref PubMed Scopus (57) Google Scholar). Recently STT3-A and STT3-B (homologs of yeast Stt3p), N33 and implantation-associated protein (homologs of yeast Ost3p and Ost6p, respectively), as well as OST4 (homolog of yeast Ost4p) have been characterized by genome-wide searches (11Kelleher D.J. Karaoglu D. Mandon E.C. Gilmore R. Mol. Cell. 2003; 12: 101-111Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). These proteins have been demonstrated to be assembled together with ribophorin I, ribophorin II, OST48, and DAD1 into a multimeric complex similar to the yeast OT (11Kelleher D.J. Karaoglu D. Mandon E.C. Gilmore R. Mol. Cell. 2003; 12: 101-111Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Although genetic studies have yielded considerable information on this enzyme complex, one of the fundamental questions, the enzymatic mechanism of N-glycosylation, has remained unanswered. Specifically, the substrate recognition and/or catalytic sites, the role of each of the subunits, and how they interact structurally has continued to be obscure. In this review, we focus on explorations developed within the past 5 years to clarify the mechanism of this highly conserved protein modification pathway as well as the function of each of the subunits. The concept that OT functions as an enzyme complex originated from the earlier biochemical studies where it was demonstrated that in a variety of organisms multiple components were detected in the purified fraction that exhibited highest specific OT activity (6Kelleher D.J. Kreibich G. Gilmore R. Cell. 1992; 69: 55-65Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 8Kumar V. Heinemann F.S. Ozols J. J. Biol. Chem. 1994; 269: 13451-13457Abstract Full Text PDF PubMed Google Scholar, 9Kumar V. Korza G. Heinemann F.S. Ozols J. Arch. Biochem. Biophys. 1995; 320: 217-223Crossref PubMed Scopus (24) Google Scholar, 12Kelleher D.J. Gilmore R. J. Biol. Chem. 1994; 269: 12908-12917Abstract Full Text PDF PubMed Google Scholar, 13Kumar V. Heinemann F.S. Ozols J. Biochem. Mol. Biol. Int. 1995; 36: 817-826PubMed Google Scholar, 14Pathak R. Imperiali B. Arch. Biochem. Biophys. 1997; 338: 1-6Crossref PubMed Scopus (18) Google Scholar). Thus far, it is not certain how these subunits interact to form a catalytically active complex in vivo. Genetic screens to characterize a particular gene with respect to its capability of suppressing the phenotype caused by a null or by a mutation in another gene have provided a few clues to the interrelationship of some of the OT subunits (15te Heesen S. Knauer R. Lehle L. Aebi M. EMBO J. 1993; 12: 279-284Crossref PubMed Scopus (106) Google Scholar, 16Zufferey R. Knauer R. Burda P. te Stagljar I. Heesen S. Lehle L. Aebi M. EMBO J. 1995; 14: 4949-4960Crossref PubMed Scopus (170) Google Scholar, 17Karaoglu D. Kelleher D.J. Gilmore R. J. Biol. Chem. 1997; 272: 32513-32520Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Combining these genetic findings with the results of biochemical co-immunoprecipitation experiments, a concept was developed that OT is composed of three subcomplexes: Ost1p-Ost5p, Ost3p-Stt3p-Ost4p, and Ost2p-Wbp1p-Swp1p (17Karaoglu D. Kelleher D.J. Gilmore R. J. Biol. Chem. 1997; 272: 32513-32520Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). However, recently application of a cross-linker, dithiobissuccinimidylpropionate, which bears a spacer arm of 12 Å, to study the interrelationship of any two of all of the yeast OT subunits has yielded a model that is not completely consistent with the above "subcomplex" model (18Yan A. Ahmed E. Yan Q. Lennarz W.J. J. Biol. Chem. 2003; 278: 33078-33087Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In this study, we found that Ost3p and Ost6p cannot co-immunoprecipitate with each other suggesting that they do not exist in the same complex, but they showed exactly the same interaction pattern with all other OT subunits. This finding combined with a recent split ubiquitin study led to our proposal that the two isoforms of OT complex exist in the ER membrane and differ only in the presence of either Ost3p or Ost6p. 2A. Yan, E. Wu, and W. Lennarz, manuscript in preparation. Fig. 1 demonstrates the structural interrelationship of the eight OT subunits in the complex containing Ost3p. Ost6p is proposed to be located in the corresponding position in another form of the OT complex that lacks Ost3p. In this model, five essential gene products in OT complex were found to be within a distance of 12 Å of each other. Two low molecular weight subunits, Ost4p and Ost5p, were shown to interact with only a restricted number of subunits and were proposed to locate closely to Stt3p and Ost1p, respectively. Most interestingly, Ost1p was found to be cross-linked to all of the other eight components (18Yan A. Ahmed E. Yan Q. Lennarz W.J. J. Biol. Chem. 2003; 278: 33078-33087Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) and therefore was placed in the core of the OT complex, probably playing an important role in the assembly of the enzyme complex (Fig. 1). It is clear that each of the nine gene products of yeast OT contribute to the efficiency of the enzymatic glycosylation process because null or mutations on each of these genes caused reduction or loss of OT activity in vivo and in vitro (2Knauer R. Lehle L. Biochim. Biophys. Acta. 1999; 1426: 259-273Crossref PubMed Scopus (171) Google Scholar). Why does the OT enzyme require so many different subunits? Co-translational glycosylation of polypeptides is a very complex process. Among the mechanistic complexities are the requirements to ensure the active site recognizes glycosylatable consensus sequences in the rapidly growing polypeptide chain, to activate the Asn amide nitrogen for nucleophilic attack, and to ensure the rapid product discharge from the active site. In addition to these catalytic events of N-glycosylation per se, it is likely that the active site of OT must be directed close to the translocon, and there must be a system to regulate the selective glycosylation of the -Asn-X-Thr/Ser-sites. Therefore the prevailing view is that multiple subunits of the OT are required to accomplish the above requirements. The functions of the various domains of three single transmembrane containing proteins (Ost1p, Wbp1p, and Ost4p) have been studied by site-directed mutagenesis and polyleucine replacement. Both Ost1p and Wbp1p have short cytosolic tails, and deletion of this tail has no effect on the growth phenotype of the yeast cell, indicating they are not essential for the function of Ost1p and Wbp1p (19Yan Q. Lennarz W.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15994-15999Crossref PubMed Scopus (17) Google Scholar, 20Li G. Yan Q. Oen H.O. Lennarz W.J. Biochemistry. 2003; 42: 11032-11039Crossref PubMed Scopus (12) Google Scholar). With respect to their transmembrane domains, the specific transmembrane sequence of Ost1p is not required for its function; instead, it appears only to be important in anchoring the protein to the ER membrane (19Yan Q. Lennarz W.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15994-15999Crossref PubMed Scopus (17) Google Scholar). In contrast, the specific sequences of the transmembrane domains in Ost4p and Wbp1p play a critical role in mediating their physical association with their partners. Further mutagenesis studies revealed that the part of the transmembrane domain of Wbp1p that is close to the ER lumen (residues 398–406) plays an important role in incorporation of Wbp1p into the OT complex, whereas the other half that faces the cytoplasm (residues 407–414) does not (20Li G. Yan Q. Oen H.O. Lennarz W.J. Biochemistry. 2003; 42: 11032-11039Crossref PubMed Scopus (12) Google Scholar). In the case of Ost4p, however, it is the segment closest to the cytosolic face of Ost4p that is crucial for its association with Stt3p and Ost3p (21Kim H. Park H. Montalvo L. Lennarz W.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1516-1520Crossref PubMed Scopus (30) Google Scholar), because mutations on this segment abolished their interaction and the resulting strains no longer grew at the elevated temperature (37 °C) (21Kim H. Park H. Montalvo L. Lennarz W.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1516-1520Crossref PubMed Scopus (30) Google Scholar). It was later shown by NMR structural analysis that this particular segment actually localizes in the second half of the transmembrane domain of Ost4p, the α2 helix (residues 15–28) (Fig. 2) (22Zubkov S. Lennarz W.J. Mohanty S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3821-3826Crossref PubMed Scopus (29) Google Scholar). The three-dimensional structure of Ost4p also revealed that this α2 helix is connected to another helix segment, the α1 helix formed by residues 10–14, via a kink of 37° between residues Phe-14 and Gly-15. Because all of the OT subunit proteins must be ER membrane-bound in yeast and in mammals to carry out the co-translational protein glycosylation, it is likely that one or more of the subunits may function to direct the whole complex to the site where it functions. Examination of the sequences of the S. cerevisiae OT subunits demonstrated that only Wbp1p was found to contain the "KKXX" motif, which is believed to function as the ER retention signal of type I membrane proteins. However, deletion of this segment did not lead to a significant growth defect in yeast cells, suggesting that it does not function to retain the OT complex in the ER (20Li G. Yan Q. Oen H.O. Lennarz W.J. Biochemistry. 2003; 42: 11032-11039Crossref PubMed Scopus (12) Google Scholar, 23te Gaynor E.C. Heesen S. Graham T.R. Aebi M. Emr S.D. J. Cell Biol. 1994; 127: 653-665Crossref PubMed Scopus (224) Google Scholar). In contrast, in the mammalian system it has been shown (24Fu J. Kreibich G. J. Biol. Chem. 2000; 275: 3984-3990Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 25Fu J. Pirozzi G. Sanjay A. Levy R. Chen Y. De Lemos-Chiarandini C. Sabatini D. Kreibich G. Eur. J. Cell Biol. 2000; 79: 219-228Crossref PubMed Scopus (20) Google Scholar) that when one of the components of OT complex was mislocated by mutations in its own retention signal, it could be retained in the ER via its interaction with other subunits. Three mammalian OT subunits, ribophorin I, ribophorin II, and OST48 (mammalian homologs of yeast Ost1p, Swp1p, and Wbp1p, respectively), were found to contain specific ER localization signals. Replacement of two Lys residues in OST48 by two serine residues (OST48ss) resulted in the mislocalization of the protein to the plasma membrane. However, if it is co-expressed with ribophorin I, ribophorin II, or chimeras that contain domains of ribophorin I, ribophorin II, and the Tac antigen (interleukin-2 receptor α chain precursor), which by themselves do not exit from the ER, OST48ss could be retained in the ER. Similar observations were found with ribophorin II (24Fu J. Kreibich G. J. Biol. Chem. 2000; 275: 3984-3990Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 25Fu J. Pirozzi G. Sanjay A. Levy R. Chen Y. De Lemos-Chiarandini C. Sabatini D. Kreibich G. Eur. J. Cell Biol. 2000; 79: 219-228Crossref PubMed Scopus (20) Google Scholar). Therefore, it is possible that ribophorin I, ribophorin II, and OST48 may play a critical role in maintaining the mammalian OT complex in the ER. In the context of the mechanistic complexities, some of the OT subunits have been found to participate in processes other than catalyzing protein N-glycosylation per se. Stt3p was recently found to be involved in biosynthesis of the cell wall β-1,6-glucan of S. cerevisiae by means of its interaction with two KRE gene products, Kre5p and Kre9p (26Chavan M. Suzuki T. Rekowicz M. Lennarz W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15381-15386Crossref PubMed Scopus (28) Google Scholar). It was also proposed to interact with the protein kinase C cascade components through its N-terminal domain based on the distinctive staurosporine sensitivity caused by mutations in one of the cytosolic oriented loops near its N terminus (amino acids 158–168) (27Chavan M. Rekowicz M. Lennarz W. J. Biol. Chem. 2003; 278: 51441-51447Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Moreover, Koiwa et al. (28Koiwa H. Li F. McCully M.G. Mendoza I. Koizumi N. Manabe Y. Nakagawa Y. Zhu J. Rus A. Pardo J.M. Bressan R.A. Hasegawa Cell. 2003; PubMed Scopus Google Scholar) found that one of the two in is involved in to in the cell and has been proposed to or play a critical role in growth and/or function based on the sequence and structure similarity of proteins has that Ost3p and Ost6p may activity N. M. J. Sci. 10: PubMed Scopus Google Scholar). However, there is no so to this In to the activity of the of were N. M. J. Sci. 10: PubMed Scopus Google Scholar). In contrast, very information is the role of and was proposed to with Ost1p based on observations that it could be cross-linked with Ost1p (18Yan A. Ahmed E. Yan Q. Lennarz W.J. J. Biol. Chem. 2003; 278: 33078-33087Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) and 2) of but not of other OT subunits the phenotype of an G. Heesen S. Gilmore R. R. Aebi M. EMBO J. 1997; PubMed Scopus Google Scholar). In the case of the essential gene product Ost2p, its has been shown to be required for the structural of the OT complex and is also involved in D.J. Gilmore R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4994-4999Crossref PubMed Scopus (159) Google Scholar, E. J. Kreibich G. J. Cell Biol. 2002; PubMed Scopus Google Scholar, A. J. Kreibich G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), but no possible similar function has been found in the case of in In the case of OT complex, the of the catalytic mechanism is which of the subunits recognizes sequences in the polypeptide In an earlier Yan et al. Q. Lennarz W.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) found that an was to to the domain of a Ost1p was to be the subunit that is for substrate However, the site in Ost1p was to a 9 amino acid segment (19Yan Q. Lennarz W.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15994-15999Crossref PubMed Scopus (17) Google Scholar), it was found that various mutations on this did not significant in or glycosylation of membrane and in vivo suggesting that the part of Ost1p is in close to the substrate and/or the catalytic but is not involved in substrate recognition (19Yan Q. Lennarz W.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15994-15999Crossref PubMed Scopus (17) Google Scholar). Stt3p as a to be involved in the process based on studies in which which contain in the form of residues at various were as the Q. Lennarz W.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). analysis of the products, it was found that Ost1p, Stt3p, and Ost3p were Because Ost3p is not an essential gene product and mutations on the segment of Ost1p where the no significant on the growth the focus Stt3p, a highly conserved protein from yeast to that is also found in certain M. D. M. M. A. Aebi M. 2002; PubMed Scopus Google Scholar). mutagenesis demonstrated that mutations on the sequence from residues to in Stt3p caused of the cell and the of glycosylatable to Ost1p suggesting that this segment play a critical role in the or catalytic activity of OT Q. Lennarz W.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). possible of Yan and Q. Lennarz W.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) was the of a substrate of a is well enzymatic very rapidly in and it is to the the recognition or the process of product discharge from the It be to this in which a may when the enzyme one of the in has been to be the case for OT based on D. Kelleher D.J. Gilmore R. Biochemistry. PubMed Scopus Google Scholar). Moreover, of the polypeptide that is may not the process as it in vivo. these and I. Kelleher D.J. Y. Y. Kreibich G. Gilmore R. G. J. Cell Biol. 2003; PubMed Scopus Google Scholar) a study a growing polypeptide chain that bears glycosylation as a In this system the glycosylation on the polypeptide were to a of the and the was into the side chain of the Lys residue in the the polypeptide was cross-linked only to the subunit of mammalian OT and the was by the presence of a substrate of This study has provided for the that the active site of OT is located in Stt3p I. Kelleher D.J. Y. Y. Kreibich G. Gilmore R. G. J. Cell Biol. 2003; PubMed Scopus Google Scholar). Although both of these studies Q. Lennarz W.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, I. Kelleher D.J. Y. Y. Kreibich G. Gilmore R. G. J. Cell Biol. 2003; PubMed Scopus Google Scholar) provided on the critical role of Stt3p in N-glycosylation, a of the is that the residue was to the X which can be by any amino acid except for Because at this position are when amino the residue at this position must not play a critical role in This is consistent with the that the OT subunit which was by a at this Ost1p, is not the subunit where the site the NMR structural analysis B. Biochemistry. PubMed Scopus Google Scholar) of a of that contain the sequence and as OT in vitro has revealed that the X position is actually oriented to the side chain of Asn and which an shown in Fig. in a formed by the consensus to ensure the in the side chain of is oriented close to the amide in the side chain of the side chain of the X amino acid is actually oriented these In the case of a where X is as shown in Fig. the of the is by the structure of This results in of the which led to the of to face from the side chain of and the is These in the X position the glycosylation of B. Biochemistry. PubMed Scopus Google Scholar). the at all the Stt3p as the subunit that plays a role in catalyzing protein N-glycosylation by In there are studies suggesting that Stt3p is of and the of both the substrate and substrate (11Kelleher D.J. Karaoglu D. Mandon E.C. Gilmore R. Mol. Cell. 2003; 12: 101-111Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, M. D. M. M. A. Aebi M. 2002; PubMed Scopus Google Scholar, D. Kelleher D.J. Gilmore R. Biochemistry. PubMed Scopus Google Scholar). N-glycosylation system in which the chain is structurally different from that in eukaryotic was recently found in a M. D. M. M. A. Aebi M. 2002; PubMed Scopus Google Scholar). It was found that the a protein with sequence similarity to Stt3p in eukaryotic plays a critical role in catalyzing the N-glycosylation process in this and that the highly conserved amino acid located on the domain of oriented the is essential for the activity M. D. M. M. A. Aebi M. 2002; PubMed Scopus Google Scholar). These results are consistent with the earlier that the same is essential in yeast Stt3p Q. Lennarz W.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Because no other genes were found in C. with sequence similarity with the other OT subunits in eukaryotic it has been the protein bears the activity on by the whole OT complex in it likely that a single protein subunit bears the to interact and the of its two In this however, it is very to other essential gene products are required in higher eukaryotic there are more studies in of the possible role of Stt3p, which is involved in recognition of the other substrate in the catalytic process. on on yeast OT, Gilmore and D. Kelleher D.J. Gilmore R. Biochemistry. PubMed Scopus Google Scholar) proposed that Stt3p may be involved in the recognition and of the dolichol-linked substrate that bears different of in Recently two isoforms of OT complex were found in cells, and the complex which STT3-B has been shown to be more active and a more to between donor with different chain than the complex containing STT3-A (11Kelleher D.J. Karaoglu D. Mandon E.C. Gilmore R. Mol. Cell. 2003; 12: 101-111Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). on all these it is to that protein plays a critical role in the catalytic mechanism of OT subunit proteins is they themselves are does the play a role in glycosylation of other protein Li and G. Yan Q. A. Lennarz W.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) have this and found that only Ost1p, Wbp1p, and Stt3p are proteins. Among them, the glycosylation of Ost1p and Wbp1p are not essential for their functions in vivo and in In contrast, mutation of two glycosylation and of Stt3p and led to of the cell, indicating an important function of the in Further mutagenesis studies as well as analysis were out to the defect was caused by the of a moiety or was a of a to the amino acid per se. The was that it is the on the residue on the site of that is essential for the function of Although the glycosylation site consensus sequence is a of only selective in a are R.A. 2004; 14: PubMed Scopus Google Scholar). to this is that in addition to the consensus the amino acids probably also play a role in et al. L. Biochemistry. PubMed Scopus Google Scholar) this by the as substrate to study the of the amino acid in the glycosylation of The results demonstrated that the residue at position can have a or effect on core glycosylation of Similar results were in the case of developed by Imperiali and L. M. Imperiali B. Chem. 2000; 10: PubMed Scopus Google Scholar, Imperiali B. Biochemistry. 1999; PubMed Scopus Google Scholar, C. Imperiali B. Biochemistry. 1997; 36: PubMed Scopus Google Scholar, M. Imperiali B. Chem. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, E. Imperiali B. Chem. 2003; PubMed Scopus Google Scholar). of and have been found to as of Among them, the most developed was a with a of 37 L. M. Imperiali B. Chem. 2000; 10: PubMed Scopus Google Scholar, C. Imperiali B. Biochemistry. 1997; 36: PubMed Scopus Google Scholar). study of the amino acid in and 5 revealed that these residues participate in the of the to the active site. was found to be other amino acid residues at position and was at position These observations that the substrate site on the enzyme recognizes a particular by amino acids than only the two specific amino acid and It has been demonstrated that of can as the donor substrate of OT in vivo Proc. Natl. Acad. Sci. U. S. A. 1983; PubMed Scopus Google Scholar) and in vitro Lehle L. W. Eur. J. Biochem. PubMed Scopus Google Scholar). Because OT the assembled donor to the polypeptide chain in of the between the various as the assembled dolichol-linked substrate be to into the mechanism by OT to the donor substrate and F. J. Biol. Chem. Full Text PDF PubMed Google Scholar) found that the in vitro transfer for and differ by the for the two donor were suggesting that donor substrate cannot be by the in a substrate mechanism has been proposed recently by Gilmore and based on D. Kelleher D.J. Gilmore R. Biochemistry. PubMed Scopus Google Scholar). study revealed a from enzyme when assembled was used as the donor This could be by an substrate the assembled donor substrate to a site on the enzyme and a at the catalytic site. This in to a more of the donor substrate at the catalytic site and the enzymatic It was proposed that this mechanism may the donor substrate of OT D. Kelleher D.J. Gilmore R. Biochemistry. PubMed Scopus Google Scholar). The was also in the case of yeast OT in a study developed to the of a substrate and the corresponding product yeast OT M. Imperiali B. Chem. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). with the of substrate for yeast OT, as a very low for the the efficiency of protein N-glycosylation and substrate However, formed by the dolichol-linked and the substrate in which the Asn residue was by as and acid similar or analysis that it is the of the in these that them to their to be in the OT whereas the amide in the product a very and is therefore from the site. it was that the and/or catalytic site of OT is M. Imperiali B. Chem. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). This of more complexities in the process of has been in the mechanism of the highly conserved protein N-glycosylation process within the past 5 Most Stt3p has been shown to be an essential in a variety of our the mechanism is and to be where is the site on Stt3p that is for substrate subunit recognizes and the dolichol-linked donor subunit the selective N-glycosylation of the The to these lead to our of the mechanism and of oligosaccharyl which has been to be the to the pathway (4Dempski Jr., R.E. Imperiali B. Curr. Opin. Chem. Biol. 2002; 6: 844-850Crossref PubMed Scopus (100) Google Scholar). of as well as all of for critical of the manuscript and