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The Oligosaccharyltransferase Complex from Saccharomyces cerevisiae

1999; Elsevier BV; Volume: 274; Issue: 24 Linguagem: Inglês

10.1074/jbc.274.24.17249

ISSN

1083-351X

Autores

Roland Knauer, Ludwig Lehle,

Tópico(s)

Fungal and yeast genetics research

Resumo

The key step of N-glycosylation of proteins, an essential and highly conserved protein modification, is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST). So far, eight genes have been identified in Saccharomyces cerevisiae that are involved in this process. Enzymatically active OST preparations from yeast were shown to be composed of four (Ost1p, Wbp1p, Ost3p, Swp1p) or six subunits (Ost2p and Ost5p in addition to the four listed). Genetic studies have disclosed Stt3p and Ost4p as additional proteins needed for N-glycosylation. In this study we report the identification and functional characterization of a new OST gene, designated OST6, that has homology to OST3 and in particular a strikingly similar membrane topology. Neither gene is essential for growth of yeast. Disruption of OST6 orOST3 causes only a minor defect inN-glycosylation, but an Δost3Δost6double mutant displays a synthetic phenotype, leading to a severe underglycosylation of soluble and membrane-bound glycoproteinsin vivo and to a reduced OST activity in vitro. Moreover, each of the two genes has also a specific function, since agents affecting cell wall biogenesis reveal different growth phenotypes in the respective null mutants. By blue native electrophoresis and immunodetection, a ∼240-kDa complex was identified consisting of Ost1p, Stt3p, Wbp1p, Ost3p, Ost6p, Swp1p, Ost2p, and Ost5p, indicating that probably all so far identified OST proteins are constituents of the OST complex. It is also shown that disruption of OST3 and OST6 leads to a defect in the assembly of the complex. Hence, the function of these genes seems to be essential for recruiting a fully active complex necessary for efficient N-glycosylation. The key step of N-glycosylation of proteins, an essential and highly conserved protein modification, is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST). So far, eight genes have been identified in Saccharomyces cerevisiae that are involved in this process. Enzymatically active OST preparations from yeast were shown to be composed of four (Ost1p, Wbp1p, Ost3p, Swp1p) or six subunits (Ost2p and Ost5p in addition to the four listed). Genetic studies have disclosed Stt3p and Ost4p as additional proteins needed for N-glycosylation. In this study we report the identification and functional characterization of a new OST gene, designated OST6, that has homology to OST3 and in particular a strikingly similar membrane topology. Neither gene is essential for growth of yeast. Disruption of OST6 orOST3 causes only a minor defect inN-glycosylation, but an Δost3Δost6double mutant displays a synthetic phenotype, leading to a severe underglycosylation of soluble and membrane-bound glycoproteinsin vivo and to a reduced OST activity in vitro. Moreover, each of the two genes has also a specific function, since agents affecting cell wall biogenesis reveal different growth phenotypes in the respective null mutants. By blue native electrophoresis and immunodetection, a ∼240-kDa complex was identified consisting of Ost1p, Stt3p, Wbp1p, Ost3p, Ost6p, Swp1p, Ost2p, and Ost5p, indicating that probably all so far identified OST proteins are constituents of the OST complex. It is also shown that disruption of OST3 and OST6 leads to a defect in the assembly of the complex. Hence, the function of these genes seems to be essential for recruiting a fully active complex necessary for efficient N-glycosylation. Asparagine-linked glycosylation is one of the most common types of eukaryotic protein modifications (1Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3750) Google Scholar, 2Herscovics A. Orlean P. FASEB J. 1993; 7: 540-550Crossref PubMed Scopus (437) Google Scholar, 3Lehle L. Tanner W. Montreuil J. Schachter H. Vliegenthart J.F.G. Glycoproteins: New Comprehensive Biochemistry. 29a. Elsevier Science, Netherlands1995: 475-509Google Scholar). N-Glycans are essential for cell viability (4Huffaker T.C. Robbins P.W. J. Biol. Chem. 1982; 257: 3203-3210Abstract Full Text PDF PubMed Google Scholar, 5Ioffe E. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 728-732Crossref PubMed Scopus (357) Google Scholar, 6Metzler M. Gertz A. Sarkar M. Schachter H. Schrader J.W. Marth J.D. EMBO J. 1994; 13: 2056-2065Crossref PubMed Scopus (310) Google Scholar) and can have a profound role in the biological function and physicochemical properties of many secreted and integral membrane proteins (7Varki A. Glycobiology. 1993; 3: 97-130Crossref PubMed Scopus (4962) Google Scholar). The key step of this pathway is theen bloc transfer of the core oligosaccharide Glc3Man9GlcNAc2 from dolichyl pyrophosphate to selected asparagine residues in an Asn-X-Ser/Thr consensus sequon of nascent polypeptides, where X can be any amino acid except proline (8Marshall R.D. Biochem. Soc. Symp. 1974; 40: 17-26PubMed Google Scholar, 9Bause E. Biochem. J. 1983; 209: 331-336Crossref PubMed Scopus (515) Google Scholar, 10Lehle L. Bause E. Biochim. Biophys. Acta. 1984; 799: 246-251Crossref Scopus (81) Google Scholar, 11Roitsch T. Lehle L. Eur. J. Biochem. 1989; 181: 525-529Crossref PubMed Scopus (78) Google Scholar, 12Gavel Y. von Heijne G. Protein Eng. 1990; 3: 433-442Crossref PubMed Scopus (631) Google Scholar, 13Mellquist J.L. Kasturi L. Spitalnik S.L. Shakin-Eshleman S.H. Biochemistry. 1998; 37: 6833-6837Crossref PubMed Scopus (198) Google Scholar). The reaction is catalyzed by the ER 1The abbreviations used are: ER, endoplasmic reticulum; OST, oligosaccharyltransferase; ORF, open reading frame; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; Dol, dolichol; BN, blue native; Bistris, bis(2-hydroxyethyl)iminotris(hydroxy-methyl)methane; Tricine, N-tris(hydroxymethyl)methylglycine; HPLC, high pressure liquid chromatography; CPY, carboxypeptidase Y; DPAP, dipeptidyl aminopeptidase; CFW, Calcofluor White1The abbreviations used are: ER, endoplasmic reticulum; OST, oligosaccharyltransferase; ORF, open reading frame; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; Dol, dolichol; BN, blue native; Bistris, bis(2-hydroxyethyl)iminotris(hydroxy-methyl)methane; Tricine, N-tris(hydroxymethyl)methylglycine; HPLC, high pressure liquid chromatography; CPY, carboxypeptidase Y; DPAP, dipeptidyl aminopeptidase; CFW, Calcofluor White -resident enzymeN-oligosaccharyltransferase (OST) (for review see Refs. 14Silberstein S. Gilmore R. FASEB J. 1996; 10: 849-858Crossref PubMed Scopus (206) Google Scholarand 15Knauer R. Lehle L. Biochim. Biophys. Acta. 1999; 1426: 259-273Crossref PubMed Scopus (170) Google Scholar).Previous attempts to purify and characterize this enzyme have met with limited success due to its lability upon solubilization (16Sharma C.B. Lehle L. Tanner W. Eur. J. Biochem. 1981; 116: 101-108Crossref PubMed Scopus (102) Google Scholar, 17Kaplan H.A. Welply J.K. Lennarz W.J. Biochim. Biophys. Acta. 1987; 906: 161-173Crossref PubMed Scopus (135) Google Scholar, 18Das R.C. Heath E.C. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3811-3815Crossref PubMed Scopus (59) Google Scholar) and probably also due to the fact that it is a multimeric membrane protein complex. Meanwhile, however, OST complexes have been purified from different sources, such as dog pancreas (19Kelleher D.J. Kreibich G. Gilmore R. Cell. 1992; 69: 55-65Abstract Full Text PDF PubMed Scopus (223) Google Scholar, 20Kelleher D.J. Gilmore R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4994-4999Crossref PubMed Scopus (158) Google Scholar), yeast (21Knauer R. Lehle L. FEBS Lett. 1994; 344: 83-86Crossref PubMed Scopus (59) Google Scholar, 22Kelleher D.J. Gilmore R. J. Biol. Chem. 1994; 269: 12908-12917Abstract Full Text PDF PubMed Google Scholar, 23Pathak R. Parker C.S. Imperiali B. FEBS Lett. 1995; 362: 229-234Crossref PubMed Scopus (22) Google Scholar), hen oviduct (24Kumar V. Heinemann S. Ozols J. J. Biol. Chem. 1994; 269: 13451-13457Abstract Full Text PDF PubMed Google Scholar), and human (25Kumar V. Korza G. Heinemann F.S. Ozols J. Arch. Biochem. Biophys. 1995; 320: 217-223Crossref PubMed Scopus (24) Google Scholar) and pig (26Breuer W. Bause E. Eur. J. Biochem. 1995; 228: 689-696Crossref PubMed Scopus (57) Google Scholar) liver. The subunit composition of the various isolated complexes and the protein sequences, so far obtained, reveal in part a high conservation of the structural organization of this enzyme throughout evolution. Independent OST purifications from Saccharomyces cerevisiaehave yielded active complexes consisting of four polypeptides (Ost1p (64/62 kDa), Wbp1p (47 kDa), Ost3p (34 kDa), and Swp1p (30 kDa)) (21Knauer R. Lehle L. FEBS Lett. 1994; 344: 83-86Crossref PubMed Scopus (59) Google Scholar,23Pathak R. Parker C.S. Imperiali B. FEBS Lett. 1995; 362: 229-234Crossref PubMed Scopus (22) Google Scholar) or of six subunits (Ost1p, Wbp1p, Ost3p, Swp1p, Ost2p (16 kDa), and Ost5p (9.5 kDa)) (22Kelleher D.J. Gilmore R. J. Biol. Chem. 1994; 269: 12908-12917Abstract Full Text PDF PubMed Google Scholar). Cloning and functional analysis ofOST1 (23Pathak R. Parker C.S. Imperiali B. FEBS Lett. 1995; 362: 229-234Crossref PubMed Scopus (22) Google Scholar, 27Silberstein S. Collins P.G. Kelleher D.J. Rapiejko P.J. Gilmore R. J. Cell Biol. 1995; 128: 525-536Crossref PubMed Scopus (60) Google Scholar), WBP1 (28te Heesen S. Janetzky B. Lehle L. Aebi M. EMBO J. 1992; 11: 2071-2075Crossref PubMed Scopus (118) Google Scholar), SWP1 (29te Heesen S. Knauer R. Lehle L. Aebi M. EMBO J. 1993; 12: 279-284Crossref PubMed Scopus (106) Google Scholar), and OST2 (30Silberstein S. Collins P.G. Kelleher D.J. Gilmore R. J. Cell Biol. 1995; 131: 371-383Crossref PubMed Scopus (106) Google Scholar) have indicated that these genes are essential for the vegetative growth of the yeast cell and reveal significant homology to components of the canine complex: to ribophorin I (27Silberstein S. Collins P.G. Kelleher D.J. Rapiejko P.J. Gilmore R. J. Cell Biol. 1995; 128: 525-536Crossref PubMed Scopus (60) Google Scholar), OST48 (31Silberstein S. Kelleher D.J. Gilmore R. J. Biol. Chem. 1992; 267: 23658-23663Abstract Full Text PDF PubMed Google Scholar), to the C-terminal half of ribophorin II (22Kelleher D.J. Gilmore R. J. Biol. Chem. 1994; 269: 12908-12917Abstract Full Text PDF PubMed Google Scholar), and DAD1 (defender against apoptotic cell death) (20Kelleher D.J. Gilmore R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4994-4999Crossref PubMed Scopus (158) Google Scholar), respectively. In contrast, OST3 (32Karaoglu D. Kelleher D.J. Gilmore R. J. Cell Biol. 1995; 130: 567-577Crossref PubMed Scopus (71) Google Scholar) and OST5 (33Reiss G. te Heesen S. Gilmore R. Zufferey R. Aebi M. EMBO J. 1997; 16: 1164-1172Crossref PubMed Scopus (63) Google Scholar) coding for the 34- and 9.5-kDa subunits, respectively, are not essential, but their deletion yields glycosylation defects and reduces OST activity in vitro. In addition to these six proteins, genetic screens have identified two other loci, OST4 (34Chi J.H. Roos J. Dean N. J. Biol. Chem. 1996; 271: 3132-3140Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) and STT3(35Zufferey R. Knauer R. Burda P. Stagljar I. te Heesen S. Lehle L. Aebi M. EMBO J. 1995; 14: 4949-4960Crossref PubMed Scopus (169) Google Scholar), that are required for optimal OST function in vivo andin vitro. Recent evidence indicates now that the derived proteins are indeed part of the complex (36Spirig U. Glavas M. Bodmer D. Reiss G. Burda P. Lippuner V. te Heesen S. Aebi M. Mol. Gen. Genet. 1997; 256: 628-637Crossref PubMed Scopus (56) Google Scholar, 37Karaoglu D. Kelleher D.J. Gilmore R. J. Biol. Chem. 1997; 272: 32513-32520Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). OST4encodes an unusually small, hydrophobic polypeptide of 3.6 kDa; its deletion leads to underglycosylation of N-glycoproteins and a temperature-sensitive growth phenotype. STT3 encodes a 78-kDa transmembrane protein with the highest conservation among the proteins associated with OST function. The essential Stt3p was found to be necessary for stability or assembly of the complex, and its lack affects the substrate specificity for the lipid-linked oligosaccharide donor (35Zufferey R. Knauer R. Burda P. Stagljar I. te Heesen S. Lehle L. Aebi M. EMBO J. 1995; 14: 4949-4960Crossref PubMed Scopus (169) Google Scholar).So far, the specific function of the various enzyme subunits is obscure and remains to be defined. In addition, the question must be answered which subunit is a bona fide constituent of the complex or serves only an auxiliary function. In this report, we describe the isolation and functional characterization of a new, not essential gene, designated OST6, that has sequence homology and in particular a very similar membrane topology to OST3. Disruption of OST6 causes only a minor defect inN-glycosylation, both in vivo and in vitro. However, a Δost6Δost3 double mutant exhibits a synthetic phenotype with a strong underglycosylation of soluble and membrane-bound glycoproteins as well as a defect in complex formation. By blue native electrophoresis, we demonstrate that the OST complex has a molecular mass of about 240 kDa and consists of all hitherto defined OST subunits (except for the small 3.6-kDa Ost4p, for which no antibody is available for detection).DISCUSSIONWe have described the identification and functional characterization of OST6, a novel gene encoding a subunit of the OST from yeast. Ost6p is a 32-kDa transmembrane protein not disclosed in any of the previously purified OST complexes. It is homolog to Ost3p, the 34-kDa γ-subunit of yeast OST (32Karaoglu D. Kelleher D.J. Gilmore R. J. Cell Biol. 1995; 130: 567-577Crossref PubMed Scopus (71) Google Scholar). The identity between both proteins with 21% is rather low, but they display a strikingly similar membrane topology containing four predicted C-terminal membrane-spanning domains. Such a hydropathy profile and the same degree of identity (23%) is also found for aCaenorhabditis elegans ORF of unknown function and a human candidate tumor suppressor. The relationship of OST6 andOST3 to a neoplastic phenotype is obscure, but altered glycosylation of cell surface proteins is a well known feature of tumor cell lines (66Dennis J.W. Fukuda M. Cell Surface Carbohydrates and Development. CRC Press, London1992: 161-194Google Scholar, 67Kobata A. Montreuil J. Schachter H. Vliegenthart J.F.G. Glycoproteins and Disease: New Comprehensive Biochemistry. 30. Elsevier Science, Netherlands1996: 211-228Google Scholar).Disruption of OST3 was reported (32Karaoglu D. Kelleher D.J. Gilmore R. J. Cell Biol. 1995; 130: 567-577Crossref PubMed Scopus (71) Google Scholar), and is confirmed in the present study, to cause an only moderate underglycosylation of proteins in vivo and a small decrease in OST activity measured in vitro. In the case of disruption ofOST6 the underglycosylation defect is even less. However, anΔost3Δost6 double mutation leads to a synthetic phenotype with a severe underglycosylation, both in vivo andin vitro (Fig. 2; Table I), as is the case for tsmutations of essential OST genes (27Silberstein S. Collins P.G. Kelleher D.J. Rapiejko P.J. Gilmore R. J. Cell Biol. 1995; 128: 525-536Crossref PubMed Scopus (60) Google Scholar, 28te Heesen S. Janetzky B. Lehle L. Aebi M. EMBO J. 1992; 11: 2071-2075Crossref PubMed Scopus (118) Google Scholar, 30Silberstein S. Collins P.G. Kelleher D.J. Gilmore R. J. Cell Biol. 1995; 131: 371-383Crossref PubMed Scopus (106) Google Scholar). Surprisingly, however, growth is not impaired at temperatures up to 37 °C. Overexpression of Ost6p not only complements the Δost6 knockout, but also rescues the Δost3 underglycosylation effect. Although we have not performed the reverse experiment (due to the very mildΔost6 defect), it seems that both genes have in part a redundant function and are able to partially replace each other. On the other hand, they also reveal specific effects (cf. Fig. 6). We observed that upon stressing the cells by compounds interfering with cell wall biogenesis, like Calcofluor White, caffeine, or SDS, anΔost6 mutant behaves differently compared with anΔost3. Even though no distinct target reaction can be given for these findings, they may eventually help to unravel the complexity of formation and function of N-linked saccharide chains.Previous biochemical investigations of the yeast OST suggested that the enzyme consists of four (Ost1p, Wbp1p, Ost3p, Swp1p) (21Knauer R. Lehle L. FEBS Lett. 1994; 344: 83-86Crossref PubMed Scopus (59) Google Scholar, 23Pathak R. Parker C.S. Imperiali B. FEBS Lett. 1995; 362: 229-234Crossref PubMed Scopus (22) Google Scholar), five (68Pathak R. Imperiali B. Arch. Biochem. Biophys. 1997; 338: 1-6Crossref PubMed Scopus (18) Google Scholar), or six subunits (Ost1p, Wbp1p, Ost3p, Swp1p, Ost2p, Ost5p) (22Kelleher D.J. Gilmore R. J. Biol. Chem. 1994; 269: 12908-12917Abstract Full Text PDF PubMed Google Scholar). Genetic screens have identified in addition STT3 (35Zufferey R. Knauer R. Burda P. Stagljar I. te Heesen S. Lehle L. Aebi M. EMBO J. 1995; 14: 4949-4960Crossref PubMed Scopus (169) Google Scholar) andOST4 (34Chi J.H. Roos J. Dean N. J. Biol. Chem. 1996; 271: 3132-3140Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In particular the lack of the essential and highly conserved Stt3p in all OST preparations was puzzling, and it was hypothesized that Stt3p could be a substoichiometric assembly factor (35Zufferey R. Knauer R. Burda P. Stagljar I. te Heesen S. Lehle L. Aebi M. EMBO J. 1995; 14: 4949-4960Crossref PubMed Scopus (169) Google Scholar). We have shown now by more sensitive probing with antibodies that the "tetrameric," enzymatically active complex isolated by us (21Knauer R. Lehle L. FEBS Lett. 1994; 344: 83-86Crossref PubMed Scopus (59) Google Scholar) also contains Stt3p, Ost2p, Ost5p, and the newly discovered Ost6p. The present study, and also recent experiments employing affinity purification of tagged Stt3 protein, or analysis of co-immunoprecipitates of in vivo radiolabeled subunits clearly identify Stt3p (36Spirig U. Glavas M. Bodmer D. Reiss G. Burda P. Lippuner V. te Heesen S. Aebi M. Mol. Gen. Genet. 1997; 256: 628-637Crossref PubMed Scopus (56) Google Scholar, 37Karaoglu D. Kelleher D.J. Gilmore R. J. Biol. Chem. 1997; 272: 32513-32520Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) and also Ost4p (37Karaoglu D. Kelleher D.J. Gilmore R. J. Biol. Chem. 1997; 272: 32513-32520Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) as components of the complex. Moreover, an estimation of the amount of radioactivity incorporated into the subunits of the co-immunoprecipitates (37Karaoglu D. Kelleher D.J. Gilmore R. J. Biol. Chem. 1997; 272: 32513-32520Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) is compatible with the notion that the eight subunits identified therein (Stt3p, Ost1p, Wbp1p, Ost3p, Swp1p, Ost2p, Ost5p, and Ost4p) may be present in equimolar amounts. Thus, the underrepresentation of some subunits in the various complex preparations seems to be due to their depletion during isolation rather than to a real substoichiometric participation in the in vivo complex. This interpretation is also in agreement with our analysis of the native complex composition using the method of blue native electrophoresis. A complex in the range of ∼240 kDa was found, which agrees with a calculated molecular mass assuming all 9 subunits are present in equimolar amounts. Nevertheless, the isolation of an active "tetrameric" complex may indicate that for the actual catalysis of the glycosylation reaction less subunits are sufficient, and the additional components may be essential or important only for in vivo function.OST3 and OST6 are not essential for OST activity, but their simultaneous lack drastically decreases glycosylationin vitro and in vivo. Therefore, one could envisage that the products of these genes are needed for optimal OST activity either directly by interacting with and regulating the catalytic subunit, or indirectly by affecting the assembly of an optimal complex, or being involved in proper positioning the OST to the polypeptide at the translocation site or to the site of formation of the lipid-linked oligosaccharide precursor. Analysis of the subunit composition of the affinity-purified complex in theΔost3Δost6 double mutant by Western analysis clearly indicates a severe defect in the structural organization of the complex (Fig. 7).Studies demonstrating genetic interactions among different OST genes, either by using a high copy number suppression approach or by constructing double mutants with a synthetic phenotype, have led to the suggestion that OST subunits can be sorted into three groups: I, Ost1p-Ost5p; II, Wbp1p-Swp1p-Ost2p; and III, Stt3p-Ost4p-Ost3p (15Knauer R. Lehle L. Biochim. Biophys. Acta. 1999; 1426: 259-273Crossref PubMed Scopus (170) Google Scholar, 36Spirig U. Glavas M. Bodmer D. Reiss G. Burda P. Lippuner V. te Heesen S. Aebi M. Mol. Gen. Genet. 1997; 256: 628-637Crossref PubMed Scopus (56) Google Scholar,37Karaoglu D. Kelleher D.J. Gilmore R. J. Biol. Chem. 1997; 272: 32513-32520Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The structural similarity of Ost3p and Ost6p, their synthetic interaction, as well as the suppression of Δost3 byOST6 overexpression suggests a grouping of Ost6p to (III). Complementary biochemical evidence indicates a direct physical interaction between Wbp1p and Swp1p (29te Heesen S. Knauer R. Lehle L. Aebi M. EMBO J. 1993; 12: 279-284Crossref PubMed Scopus (106) Google Scholar) and Ost2p, 2R. Knauer and L. Lehle, unpublished results. as well as between Stt3p, Ost3p, and Ost4p (37Karaoglu D. Kelleher D.J. Gilmore R. J. Biol. Chem. 1997; 272: 32513-32520Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), supporting the idea that these groups may represent discrete subcomplexes. In this context, it is interesting to note that upon simultaneous disruption of the OST3 andOST6 genes, Ost5p and Ost1p (subcomplex I) and Stt3p (subcomplex III) are decreased, whereas Wbp1p, Swp1p, and Ost2p (subcomplex II) are not affected in their amount (Fig. 7). Since glycosylation still occurs in the Δost3Δost6 strain, albeit reduced, one could speculate that Wbp1p, Swp1p, and Ost2p represent the catalytic core unit of the complex.At present it is not clear, however, whether the proposed subcomplexes comprise intermediates in the assembly of a fully functional complex, or whether they are autonomous in vivo pools that may assemble into OST complexes with slightly different composition and function. Recent findings indicate the occurrence of multiple pathways of protein translocation into the ER (69Rapoport T.A. Jungnickel B. Kutay U. Annu. Rev. Biochem. 1996; 65: 271-303Crossref PubMed Scopus (492) Google Scholar, 70Finke K. Plath K. Panzer S. Prehn S. Rapoport T.A. Hartmann E. Sommer T. EMBO J. 1996; 15: 1482-1494Crossref PubMed Scopus (144) Google Scholar, 71Ng D.T.W. Brown J.D. Walter P. J. Cell Biol. 1996; 134: 269-278Crossref PubMed Scopus (372) Google Scholar, 72Rothe C. Lehle L. Eur. J. Biochem. 1998; 252: 16-24Crossref PubMed Scopus (33) Google Scholar), one dependent on the signal-recognition particle and the other independent. The identified translocation complexes share in part subunit components. The fact that no distinct OST subcomplexes can be detected in wild-type cells by blue native polyacrylamide gel electrophoresis makes it less likely that OSTs of different composition are associated with the respective translocation machineries. In agreement with this idea is also our observation that in the Δost3Δost6 mutant proteins are underglycosylated irrespective whether they use the SRP-dependent (e.g. invertase, DPAP B) or the SRP-independent (e.g. CPY) sorting pathway. Nevertheless, this view does not exclude specific interactions of particular OST subunits and/or not yet identified auxiliary proteins with the different targeting pathways.The combination of biochemistry and powerful yeast genetic methods has advanced tremendously our knowledge of the N-glycosylation of proteins. Now that the composition of one of the most complex enzymes in nature as well as some of the interactions between its nine subunits has been defined, further work needs to concentrate on a number of intriguing issues. These include the specific functions of the various subunits of the complex, the regulation of the enzyme and the coupling of OST to protein translocation and protein folding. Finally, the results obtained in yeast may also lead to the isolation of the homologous mammalian proteins, not yet identified in respective complexes. Asparagine-linked glycosylation is one of the most common types of eukaryotic protein modifications (1Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3750) Google Scholar, 2Herscovics A. Orlean P. FASEB J. 1993; 7: 540-550Crossref PubMed Scopus (437) Google Scholar, 3Lehle L. Tanner W. Montreuil J. Schachter H. Vliegenthart J.F.G. Glycoproteins: New Comprehensive Biochemistry. 29a. Elsevier Science, Netherlands1995: 475-509Google Scholar). N-Glycans are essential for cell viability (4Huffaker T.C. Robbins P.W. J. Biol. Chem. 1982; 257: 3203-3210Abstract Full Text PDF PubMed Google Scholar, 5Ioffe E. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 728-732Crossref PubMed Scopus (357) Google Scholar, 6Metzler M. Gertz A. Sarkar M. Schachter H. Schrader J.W. Marth J.D. EMBO J. 1994; 13: 2056-2065Crossref PubMed Scopus (310) Google Scholar) and can have a profound role in the biological function and physicochemical properties of many secreted and integral membrane proteins (7Varki A. Glycobiology. 1993; 3: 97-130Crossref PubMed Scopus (4962) Google Scholar). The key step of this pathway is theen bloc transfer of the core oligosaccharide Glc3Man9GlcNAc2 from dolichyl pyrophosphate to selected asparagine residues in an Asn-X-Ser/Thr consensus sequon of nascent polypeptides, where X can be any amino acid except proline (8Marshall R.D. Biochem. Soc. Symp. 1974; 40: 17-26PubMed Google Scholar, 9Bause E. Biochem. J. 1983; 209: 331-336Crossref PubMed Scopus (515) Google Scholar, 10Lehle L. Bause E. Biochim. Biophys. Acta. 1984; 799: 246-251Crossref Scopus (81) Google Scholar, 11Roitsch T. Lehle L. Eur. J. Biochem. 1989; 181: 525-529Crossref PubMed Scopus (78) Google Scholar, 12Gavel Y. von Heijne G. Protein Eng. 1990; 3: 433-442Crossref PubMed Scopus (631) Google Scholar, 13Mellquist J.L. Kasturi L. Spitalnik S.L. Shakin-Eshleman S.H. Biochemistry. 1998; 37: 6833-6837Crossref PubMed Scopus (198) Google Scholar). The reaction is catalyzed by the ER 1The abbreviations used are: ER, endoplasmic reticulum; OST, oligosaccharyltransferase; ORF, open reading frame; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; Dol, dolichol; BN, blue native; Bistris, bis(2-hydroxyethyl)iminotris(hydroxy-methyl)methane; Tricine, N-tris(hydroxymethyl)methylglycine; HPLC, high pressure liquid chromatography; CPY, carboxypeptidase Y; DPAP, dipeptidyl aminopeptidase; CFW, Calcofluor White1The abbreviations used are: ER, endoplasmic reticulum; OST, oligosaccharyltransferase; ORF, open reading frame; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; Dol, dolichol; BN, blue native; Bistris, bis(2-hydroxyethyl)iminotris(hydroxy-methyl)methane; Tricine, N-tris(hydroxymethyl)methylglycine; HPLC, high pressure liquid chromatography; CPY, carboxypeptidase Y; DPAP, dipeptidyl aminopeptidase; CFW, Calcofluor White -resident enzymeN-oligosaccharyltransferase (OST) (for review see Refs. 14Silberstein S. 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The subunit composition of the various isolated complexes and the protein sequences, so far obtained, reveal in part a high conservation of the structural organization of this enzyme throughout evolution. Independent OST purifications from Saccharomyces cerevisiae

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