Alg14 Recruits Alg13 to the Cytoplasmic Face of the Endoplasmic Reticulum to Form a Novel Bipartite UDP-N-acetylglucosamine Transferase Required for the Second Step of N-Linked Glycosylation

Abstract

N-linked glycosylation requires the synthesis of an evolutionarily conserved lipid-linked oligosaccharide (LLO) precursor that is essential for glycoprotein folding and stability. Despite intense research, several of the enzymes required for LLO synthesis have not yet been identified. Here we show that two poorly characterized yeast proteins known to be required for the synthesis of the LLO precursor, GlcNAc2-PP-dolichol, interact to form an unusual hetero-oligomeric UDP-GlcNAc transferase. Alg13 contains a predicted catalytic domain, but lacks any membrane-spanning domains. Alg14 spans the membrane but lacks any sequences predicted to play a direct role in sugar catalysis. We show that Alg14 functions as a membrane anchor that recruits Alg13 to the cytosolic face of the ER, where catalysis of GlcNAc2-PP-dol occurs. Alg13 and Alg14 physically interact and under normal conditions, are associated with the ER membrane. Overexpression of Alg13 leads to its cytosolic partitioning, as does reduction of Alg14 levels. Concomitant Alg14 overproduction suppresses this cytosolic partitioning of Alg13, demonstrating that Alg14 is both necessary and sufficient for the ER localization of Alg13. Further evidence for the functional relevance of this interaction comes from our demonstration that the human ALG13 and ALG14 orthologues fail to pair with their yeast partners, but when co-expressed in yeast can functionally complement the loss of either ALG13 or ALG14. These results demonstrate that this novel UDP-GlcNAc transferase is a unique eukaryotic ER glycosyltransferase that is comprised of at least two functional polypeptides, one that functions in catalysis and the other as a membrane anchor. N-linked glycosylation requires the synthesis of an evolutionarily conserved lipid-linked oligosaccharide (LLO) precursor that is essential for glycoprotein folding and stability. Despite intense research, several of the enzymes required for LLO synthesis have not yet been identified. Here we show that two poorly characterized yeast proteins known to be required for the synthesis of the LLO precursor, GlcNAc2-PP-dolichol, interact to form an unusual hetero-oligomeric UDP-GlcNAc transferase. Alg13 contains a predicted catalytic domain, but lacks any membrane-spanning domains. Alg14 spans the membrane but lacks any sequences predicted to play a direct role in sugar catalysis. We show that Alg14 functions as a membrane anchor that recruits Alg13 to the cytosolic face of the ER, where catalysis of GlcNAc2-PP-dol occurs. Alg13 and Alg14 physically interact and under normal conditions, are associated with the ER membrane. Overexpression of Alg13 leads to its cytosolic partitioning, as does reduction of Alg14 levels. Concomitant Alg14 overproduction suppresses this cytosolic partitioning of Alg13, demonstrating that Alg14 is both necessary and sufficient for the ER localization of Alg13. Further evidence for the functional relevance of this interaction comes from our demonstration that the human ALG13 and ALG14 orthologues fail to pair with their yeast partners, but when co-expressed in yeast can functionally complement the loss of either ALG13 or ALG14. These results demonstrate that this novel UDP-GlcNAc transferase is a unique eukaryotic ER glycosyltransferase that is comprised of at least two functional polypeptides, one that functions in catalysis and the other as a membrane anchor. Asparagine (N)-glycosylation is an essential modification that regulates protein folding and stability. Prior to its attachment to protein, the oligosaccharide Glu3Man9GlcNAc2 is assembled on the lipid carrier, dolichyl pyrophosphate (dol-PP), in the ER 2The abbreviations used are:ERendoplasmic reticulumLLOlipid-linked oligosaccharideALGasparagine-linked glycosylationdoldolicholORFopen reading frameHAhemagglutininCPYcarboxypeptidase YGFPgreen fluorescent proteinPPpyrophosphate (see Refs. 1Burda P. Aebi M. Biochim. Biophys. Acta. 1999; 1426: 239-257Crossref PubMed Scopus (528) Google Scholar, 2Abeijon C. Hirschberg C.B. Trends Biochem. Sci. 1992; 17: 32-36Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 3Kornfeld R. Kornfeld S. Ann. Rev. of Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3776) Google Scholar, 4Herscovics A. Orlean P. FASEB J. 1993; 7: 540-550Crossref PubMed Scopus (440) Google Scholar for review). The earliest steps of this lipid-linked oligosaccharide (LLO) synthesis begin on the cytoplasmic face of the ER. Seven sugars, (two N-acetylglucosamines and five mannoses) are sequentially added to dol-P to form Man5GlcNAc2-PP-dol by enzymes that have their catalytic domain on the cytosolic side of the ER membrane and use sugar nucleotide substrates (2Abeijon C. Hirschberg C.B. Trends Biochem. Sci. 1992; 17: 32-36Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 5Snider M.D. Rogers O.C. Cell. 1984; 36: 753-761Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 6Perez M. Hirschberg C.B. J. Biol. Chem. 1986; 261: 6822-6830Abstract Full Text PDF PubMed Google Scholar). The enzymes that catalyze addition of the next seven sugars (four mannoses and three glucoses) do so within the lumen of the ER and use dolichol-linked sugar substrates (see Ref. 1Burda P. Aebi M. Biochim. Biophys. Acta. 1999; 1426: 239-257Crossref PubMed Scopus (528) Google Scholar for review). Once assembled, this core oligosaccharide is transferred to protein by oligosaccharyltransferase through an N-glycosidic bond to an asparagine that is part of the Asn-X-(Ser/Thr) consensus sequence (7Yan A. Lennarz W.J. J. Biol. Chem. 2005; 280: 3121-3124Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Protein-linked oligosaccharide is immediately modified by the removal of glucoses and mannose by ER glucosidases and mannosidases. Failure to properly synthesize, transfer, or modify the N-linked glycan results in glycoproteins that are recognized by the quality control systems that restrict these aberrant proteins from exiting the ER to the Golgi and target their degradation (for review see Refs. 8Helenius A. Aebi M. Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1620) Google Scholar and 9Hebert D.N. Garman S.C. Molinari M. Trends Cell Biol. 2005; 15: 364-370Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). endoplasmic reticulum lipid-linked oligosaccharide asparagine-linked glycosylation dolichol open reading frame hemagglutinin carboxypeptidase Y green fluorescent protein pyrophosphate In the yeast, Saccharomyces cerevisiae, many of the glycosyltransferases that catalyze synthesis of the core oligosaccharide have been identified. All of the ALG genes (asparagine-linked glycosylation) identified thus far (ALG1-12) encode membrane-spanning glycosyltransferases that have their catalytic domains on one or the other side of the ER membrane, consistent with the topological constraints of this pathway (10Couto J.R. Huffaker T.C. Robbins P.W. J. Biol. Chem. 1984; 259: 378-382Abstract Full Text PDF PubMed Google Scholar, 11Jackson B.J. Kukuruzinska M.A. Robbins P.W. Glycobiology. 1993; 3: 357-364Crossref PubMed Scopus (55) Google Scholar, 12Aebi M. Gassenhuber J. Domdey H. te Heesen S. 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Glycobiology. 1999; 9: 617-625Crossref PubMed Scopus (80) Google Scholar). In all organisms studied thus far, ALG mutations affecting the cytosolic reactions leading to Man5GlcNAc2-PP-dol synthesis result in lethality or very severe phenotypes (19Cipollo J.F. Trimble R.B. Chi J.H. Yan Q. Dean N. J. Biol. Chem. 2001; 276: 21828-21840Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 21Huffaker T.C. Robbins P.W. J. Biol. Chem. 1982; 257: 3203-3210Abstract Full Text PDF PubMed Google Scholar, 22Huffaker T.C. Robbins P.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7466-7470Crossref PubMed Scopus (187) Google Scholar). Mutations affecting the later, luminal intermediates of LLO synthesis manifest as a range of phenotypes, from undetectable in S. cerevisiae (12Aebi M. Gassenhuber J. Domdey H. te Heesen S. Glycobiology. 1996; 6: 439-444Crossref PubMed Scopus (115) Google Scholar, 13Reiss G. te Heesen S. Zimmerman J. Robbins P.W. Aebi M. Glycobiology. 1996; 6: 493-498Crossref PubMed Scopus (85) Google Scholar, 15Stagljar I. te Heesen S. Aebi M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5977-5981Crossref PubMed Scopus (93) Google Scholar, 16Burda P. te Heesen S. Brachat A. Wach A. Dusterhoft A. Aebi M. Proc. Natl. Acad. Sci. 1996; 93: 7160-7165Crossref PubMed Scopus (91) Google Scholar, 18Burda P. Aebi M. Glycobiology. 1998; 8: 455-462Crossref PubMed Scopus (94) Google Scholar, 22Huffaker T.C. Robbins P.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7466-7470Crossref PubMed Scopus (187) Google Scholar, 23te Heesen S. Lehle L. Weissmann A. Aebi M. Eur. J. Biochem. 1994; 224: 71-79Crossref PubMed Scopus (96) Google Scholar) to the heterogenous phenotypes associated with congenital disorders of glycosylation (CDG) in humans (24Aebi M. Hennet T. Trends Cell Biol. 2001; 11: 136-141Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 25Freeze H.H. Biochim. Biophys. Acta. 2002; 1573: 388-393Crossref PubMed Scopus (67) Google Scholar). All of the ALG genes are highly conserved among eukaryotes, although are a J. S. P. J. R. Robbins P.W. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). These organisms lipid-linked that are that is in S. cerevisiae and in the of and LLO mannose and the glycan by all that have been is Man5GlcNAc2-PP-dol J. S. P. J. R. Robbins P.W. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). The and of the proteins Man5GlcNAc2-PP-dol the of this glycan and the reactions that on the cytosolic face of the ER that to its Despite its the of several of the enzymes that catalyze the reactions on the cytosolic face of the ER a a two S. cerevisiae and identified that are to the UDP-GlcNAc glycosyltransferase in synthesis and that are in a role in N-linked glycosylation I. J. A. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). of either of these genes the of lipid-linked oligosaccharide the addition of a to that GlcNAc2-PP-dol on the cytosolic face of the ER I. J. A. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). any other eukaryotic ER we as Alg13, is predicted to a consensus glycosyltransferase catalytic domain but lacks any predicted membrane-spanning domains. The other we as contains several predicted membrane-spanning domains but lacks any sequences that are predicted to in sugar Alg13 contains a domain that is to the catalytic domain in other and a that its in LLO to play a direct role in UDP-GlcNAc The role of Alg14 is glycosyltransferases are among In this we have the that Alg13 and Alg14 interact on the ER face to form a functional hetero-oligomeric UDP-GlcNAc glycosyltransferase membrane-spanning domain and catalytic domain are on We demonstrate that these enzymes physically interact with one on the ER and that Alg14 is required to Alg13 from the to the ER. although of the human Alg13 and Alg14 orthologues can interact with their yeast partners, when co-expressed these proteins can functionally complement the loss of ALG13 or ALG14. We that this is an unusual hetero-oligomeric comprised of at least two polypeptides, both of are required for LLO and used in this are in is the for all the used in and of or A. A. C. 1993; PubMed Scopus Google Scholar) the of as A. Wach A. Brachat A. P. J.R. 1998; PubMed Scopus Google Scholar). The a of by M. yeast conditions, and used C. PubMed Scopus Google used in this in and in and in and in and in and in and in and in and in and in and in and in a In and the ALG14 and ALG13 sequences the and the by the by a and the with the an of the and its sequence at the is an essential that to ALG13 but that is from the of We that of the ALG13 with the with normal a the and of the P. PubMed Google and at the in by the ALG13 with a ALG13 with the S. 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Glycobiology. 2004; PubMed Scopus Google Scholar) to the its sequence by with and and of the and and at in of for at and in of with at for of to and with The in of and by with of a on and by at for to a The and at for the and the The at for to the and with and and in a of and protein by protein of by and transferred to the membrane that used in and of yeast by with in and at for to and The at for in a The with and in of The in of by and transferred to the used in yeast by with in and at for to and The and used for to the of carboxypeptidase Y of human ALG13 and ALG14 genes the or glycosylation ALG13 or ALG14 the human ALG13 or ALG14 genes and to in with with or with to of the ALG13 and ALG14 and for at three of on or and protein as under by and with an The of the and of or N-linked are B.J. R. PubMed Google The protein by a to and by and used to and with by with a or to by yeast at an of and to with from of in of that and as Dean N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). proteins in with or by transferred to the the membrane with a to the protein, the membrane with a by a to of and the localization of and to an of in the with the and the on an Alg13 and Alg14 a role for Alg13 as a glycosyltransferase in GlcNAc2-PP-dol synthesis requires its with the ER membrane, lacks any membrane-spanning domains. ALG14 an membrane protein that when leads to phenotypes to that of I. J. A. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). These a in these proteins pair with one on the cytosolic face of the ER to form a functional hetero-oligomeric UDP-GlcNAc transferase that the of core oligosaccharide synthesis on the ER. Alg13 and Alg14 physically this we a that Alg13 and Alg14 contains an ALG14 by the but this is when this is on of these are at when on and both the glycosylation and of the and not protein from these and by at to any protein (see Alg14 with the by and with to Alg13 with this we that Alg13 with Alg14 with and of Alg13 with Alg14 on the of and of the of with in the of with In we evidence for the of other membrane proteins and not that this is a these results demonstrate that these proteins physically Alg13 and Alg14 at the Alg13 and Alg14 be on ER pair to form the UDP-GlcNAc transferase that the of GlcNAc2-PP-dol on the cytosolic face of the ER. this we that Alg13 and Alg14 at to their localization in and see by both of these at least in in a that is of the yeast ER The of this by the with an ER protein, and contains the sequence to a ER see in ER by this the in and to a of with demonstrating that both Alg13 and Alg14 are associated with the ER. the of the Alg13 evidence for its ER the that Alg13, lacks any predicted in the this the the of Alg13 by that an of ALG14 and a of ALG14 that proteins at from these to to ER from cytosolic proteins (see at to ER The at to membrane the Golgi and from cytosolic proteins that in the of protein from of these the and by and with and to Alg13 and Alg14 In with the of we that the with the ER, with the ER protein, and from a Golgi associated with ER but in the The of Alg13 associated with from to but at least a in the with the these results demonstrate that the of both Alg14 and Alg13 are associated with the ER membrane. Alg14 and to Alg13 to the the of any predicted membrane-spanning our results show that at Alg13 is associated with ER a of localization in a under the or and not of and in the of in the and result that overproduction of Alg13 can its localization so that of in the on our demonstration of the interaction Alg13 and Alg14 one for this result is that the localization of Alg13 to the ER is its with that when with Alg13 leads to its cytosolic Alg14 is required for the of Alg13 to the ER, of Alg14 from the ER result in the of Alg13 to the this we the localization of Alg13 as a of Alg14 ALG14 under the control of the and in a of by its These under and at added to ALG14 of and from these to and by and with to the of Alg13 In this at Alg13 in the membrane and with a loss of ALG14 the of Alg13 that in the cytosolic when of the of Alg14 not of Alg13 in both the membrane and cytosolic and of Alg14 results in the cytosolic partitioning of Alg13, the ER localization of Alg13 is on We Alg14 is sufficient to Alg13 to the ER. this we a in ALG14 can the Alg13 cytosolic We the localization of Alg13 in a yeast both its normal ALG14 and a ALG14 that is in the of thus normal of ALG14 when on but ALG14 when in The of ALG14 on Alg13 localization by from in or the membrane and proteins by a and the of in by in when ALG14 is not Alg13 in both the and in the protein at a In of ALG14 by in in of with the Alg13 undetectable in the under these result is consistent with our to any Alg13 in the on and demonstrate that of ALG14 can to the ER and the cytosolic localization of Alg14 is both necessary and sufficient for the of Alg13 to the ER. of and by demonstrate that both Alg13 and Alg14 have been conserved among eukaryotes, as predicted for proteins that are in the steps of LLO The and of the yeast and human orthologues are in The human and yeast Alg13 proteins but sequence their and and are of a the yeast protein is predicted to have a of the human Alg13 protein is The Alg14 orthologues are in their sequence and the sequence of the Alg14 orthologues in the of the the of three predicted membrane-spanning domains in These Alg14 proteins are predicted to be the yeast Alg14 is and the human Alg14 protein is We the human ALG13 and ALG14 (see and for functional of the yeast and yeast of these a yeast that their under the control of the yeast ALG13 or ALG14 and These in or and on with either or to of the yeast this we that of these human genes complement the of the to complement be of the of of the human proteins to pair properly with their yeast to sequence this the human ALG13 and ALG14 genes co-expressed in either the or and for of lethality in the of In with our we that when the human ALG13 and ALG14 genes on to levels. We of these genes the glycosylation that from the of either or in We the of as a for N-linked glycosylation in the ER. protein N-linked in the ER, but the to a normal LLO a reduction in the with the oligosaccharide is transferred from the lipid to the N-linked glycosylation in the ER results in a on of that or the yeast ALG13 or the human genes in or for the yeast and or from through three and with with results of I. J. A. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google of either Alg13 or Alg14 in of The in the with is to Alg14 protein in three in Alg14 is Alg13 not of the human ALG13 and ALG14 genes in either the or in a in the of that in these when on and and and although the glycosylation not to normal levels. these demonstrate that the human Alg13 and Alg14 proteins interact with their yeast but can pair with one to form a functional UDP-GlcNAc transferase in Alg13 of the of the that the of LLO oligosaccharide contains a consensus is required for the synthesis of GlcNAc2-PP-dol I. J. A. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google a severe when and is conserved among other its of a membrane-spanning domain the of can be to the cytosolic face of the ER where this occurs. results demonstrate that the ER localization of Alg13 is through its interaction with an essential ER protein that as a membrane anchor. a novel topological for this glycosyltransferase that contains at least two Alg13, sugar transfer, and as a membrane anchor. our this is the of a eukaryotic We have that Alg13 and Alg14 interact by a of other ER and catalyze several steps of LLO on the cytosolic face of the ER have been to interact as hetero-oligomeric that are for their glycosyltransferase A. Dean N. Glycobiology. 2004; PubMed Scopus Google Scholar). We have not the of Alg13 and Alg14 within the have we the that be Alg13 and these or other ER proteins that modify the of the UDP-GlcNAc transferase our results demonstrate that Alg14 is both necessary and sufficient for the localization of Alg13 to the ER. The of Alg14 results in loss of Alg13 ER as does Alg13 The of this by of ALG14 that are consistent with Alg14 the for Alg13 Further for the relevance of the comes from our of the ALG13 and ALG14 human we that of either the or when both human genes are of these is that the of these human proteins to properly pair with their yeast been through but that these human proteins can pair with one to form a functional hetero-oligomeric UDP-GlcNAc transferase. The use of and that contains three domains. that one of the protein the the other the that the of Alg14 is within the lumen and the is in the the and domains of Alg14 are predicted to be very and a topological in the is within the lumen and the is in the result in Alg14 domains the a interaction with Alg13 on the cytosolic face of the ER, where Alg13 to catalyze the of Alg13 is to the of the glycosyltransferase Alg14 to the this comes from our of a functional protein, of Alg13 to the of that can complement loss of either ALG13 or ALG14. and N. result that sequences in the domains of Alg14 in its interaction with Alg13. The of the membrane-spanning domain of Alg14 is not but one is that this of the protein not to Alg13 to the ER membrane but in the of dolichol in the membrane. The of the human Alg13 and Alg14 proteins to interact with their yeast that in the cytosolic domains of Alg14 within the Alg13 protein have to the interaction with their yeast of the within these domains that the interaction Alg13 and a of eukaryotic organisms identified that ER glycosyltransferases and thus N-linked demonstrating a the of N-linked and the ALG genes required for their synthesis J. S. P. J. R. Robbins P.W. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). a to be an is all identified glycosyltransferases dolichol transferase that of and N-linked that of GlcNAc2-PP-dol J. S. P. J. R. Robbins P.W. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). a eukaryotic that lacks and does not any of the identified ALG glycosyltransferase for the role of both Alg13 and Alg14 in the of the N-linked glycosylation pathway that both ALG13 and ALG14 and that with this of the demonstrate the of highly ALG13 and ALG14 but genes in the are of eukaryotic glycosyltransferases that as part of see C.L. Cell Biol. 2004; PubMed Scopus (93) Google are any of eukaryotic Alg13, the catalytic of the protein, and the membrane a novel of a eukaryotic glycosyltransferase within this of the of Alg13 and Alg14 to the protein, an of these two that have two proteins where the of a the eukaryotic that we are is that a cytosolic of the N-linked glycosylation one that in the pathway as Alg13, as a for the of the In with this we that Alg13 is a protein and of ALG13 is not by The of LLO synthesis and is by a of affecting normal and by The of this the of been as the of LLO M.A. Glycobiology. PubMed Scopus Google and is evidence that this precursor is by the of GlcNAc2-PP-dol N. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the enzymes required for synthesis of GlcNAc2-PP-dol for the of the N-linked glycosylation of ALG13 and ALG14 the to the regulates the of these oligosaccharide We for and for the in We to for and reading of the We A. of and for to H. and N. of and H. of for this