1 Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, NY 11794-5215, and 2 Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1 Higashi, Tsukuba 305-8566, Japan
Received on January 26, 2004; revised on March 2, 2004; accepted on March 2, 2004
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Abstract |
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Key words: ALG1, ALG2, ALG11 / endoplasmic reticulum / mannosyltransferase / N-glycosylation / S. cerevisiae
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Introduction |
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Since the pioneering work of Robbins and colleagues, who initially isolated and characterized yeast mutants defective in lipid-linked oligosaccharide assembly (asparagine linked glycosylation or alg mutants) (Huffaker and Robbins, 1982, 1983
), many of the genes affecting lipid-linked oligosaccharide synthesis in the ER have now been identified (for review see Burda and Aebi, 1999
). With few exceptions, mutations in any one of the ALG genes result in accumulation of defined oligosaccharide intermediates. This phenotype is consistent with the notion that the lipid-linked oligosaccharide biosynthetic pathway involves a series of steps that lead to the synthesis of a branched and structurally complex oligosaccharide. Furthermore there is good evidence to support the hypothesis that this ordered assembly arises at least in part from differences in the substrate specificities of the various glycosyltransferases that participate in these reactions (Burda et al., 1999
).
During this ordered assembly, an important distinction can be made between the reactions that occur on the cytosolic face of the ER and those that occur in the lumen. A deletion of any gene affecting synthesis or translocation of Man5GlcNAc2-PP-Dol on the cytosolic face of the ER leads to death or an extremely severe phenotype. For instance, alg1, alg2
, and alg11
mutants, required for addition of the first, third, and fourth or fifth mannose, respectively, of Man5GlcNAc2-P-P-Dol are inviable or grow very slowly (Albright and Robbins, 1990
; Cipollo et al., 2001
; Jackson et al., 1993
). In contrast, mutations in genes affecting lumenal sugar additions result in little if any growth phenotypes (Burda et al., 1999
). These observations suggest that although N-linked glycosylation is an essential modification, the cytosolic Man5GlcNAc2 core structure is the minimal intermediate whose synthesis and translocation across the membrane is required for the successful execution of N-linked glycosylation. The Man4GlcNAc2 core and earlier intermediates may be poor substrates for lumenal flipping or transfer to protein or, alternatively, for outer chain elongation by the Golgi mannosyltransferases.
Despite the fact that many of the enzymes involved in lipid-linked oligosaccharide biosynthesis have been identified, the regulation and coordination of their activities is poorly understood. Here we show that three of the enzymes that participate in Man5GlcNAc2-PP-Dol synthesis, namely, Alg1, Alg2, and Alg11, exist in oligomeric protein complexes. Two distinct complexes that are distinguished by the presence of Alg2 or Alg11 can be identified, both of which contain Alg1. These results suggest that the physical association of the functionally related catalytic enzymes involved in lipid-linked oligosaccharide synthesis provides an additional level of regulation that assures proper construction of this complex oligosaccharide.
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Results |
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To explore this idea, we tested whether Alg1 interacts with any other mannosyltransferase known to catalyze lipid-linked oligosaccharide synthesis on the cytosolic face of the ER, namely, Alg2 and Alg11. Alg2 is required for addition of the third 1,6-linked mannose. Loss of ALG2 is lethal, and alg2 mutants accumulate Man2GlcNAc2-PP-Dol (Jackson et al., 1993
). Alg11 is required for addition of the fourth and/or fifth
1,2-linked mannose to Man3GlcNAc2-PP-Dol. Loss of ALG11 results in a severe growth phenotype and temperature sensitive lethality (Cipollo et al., 2001
). To determine if Alg2 or Alg11 interact with Alg1, we used a coimmunoprecipitation assay. Yeast strains were constructed that coexpressed different HA- and myc-tagged ALG genes. The chromosomal ALG1, ALG2, or ALG11 coding sequences were replaced with the corresponding myc-tagged alleles. In each of these strains, an additional copy of an HA-tagged triose phosphoisomerase (TPI) promoter-driven ALG1 gene was integrated in the chromosome (see Materials and methods). Strains solely expressing epitope-tagged ALG1, ALG2, or ALG11 display no obvious growth or glycosylation defects and each of the epitope-tagged genes can complement the corresponding mutant allele, suggesting that these additional sequences do not affect normal protein function (data not shown).
Extracts from each of these strains were prepared in buffer containing the nonionic detergent digitonin to maintain oligomeric interactions between membrane proteins, and these extracts were subjected to coimmunoprecipitation assays. Myc-tagged Alg1, Alg2, or Alg11 proteins were precipitated from these extracts with anti-myc antibody, and the immunoprecipitates were fractionated by SDSPAGE. The relative steady state levels of myc-tagged Alg1, Alg2, and Alg11 proteins in the same extracts used for the immunoprecipitations were determined by western blot analysis of aliquots removed prior to immunoprecipitation and were found to be similar (Figure 2B). HA-tagged Alg1 that coprecipitated with myc-tagged Alg1, Alg2, or with Alg11 was detected by blotting with anti-HA antibody (Figure 2A). The result of this experiment demonstrated that Alg1 coprecipitated with itself, Alg2, and Alg11 (Figure 2A, lanes 1, 2, 3) at comparable levels. Similar amounts of Alg1-, Alg2-, and Alg11-myc were precipitated with anti-myc antibody, determined by stripping these blots of anti-HA antibody and reprobing with anti-myc antibody (Figure 2A, lanes 610). Coprecipitation of Alg1-HA occurred in the presence of several other nonionic detergents, including octylglucoside, Triton X-100, and CHAPS, though less efficiently than digitonin (not shown). Therefore we used buffers containing digitonin in all our further analyses of these protein complexes.
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Alg2 and Alg11 proteins do not interact with each other
If Alg1, Alg2, and Alg11 proteins interact with one another in one large complex, as suggested by the results of the immunoprecipitation assay, all three proteins would be predicted to cofractionate when separated by gel filtration chromatography. To test this idea, digitonin extracts were prepared from a yeast strain (XGY32) that simultaneously expresses HA-tagged ALG1, ALG2, and ALG11 and size fractionated by FPLC. In this strain, each of the normal genes has been replaced by an epitope-tagged allele whose expression is controlled by the corresponding endogenous promoter. This strain grows normally even though it solely produces epitope-tagged Alg1, Alg2, and Alg1 proteins, further suggesting that their normal function is not altered by the presence of the C-terminal tags. Western blot analysis of extracts from this strain suggested that Alg1-HA, Alg2-HA, and Alg11-HA accumulated in a 2:1:1 ratio, respectively, demonstrating that at steady state, Alg1 is roughly twice as abundant as Alg2 or Alg11 (Figure 3, lane marked Load).
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A simple model to explain the different chromatographic behaviors of Alg2 and Alg11, as well as their stable interactions with Alg1, envisages two Alg1-containing complexes, one containing Alg2 and the other Alg11. If this model were correct, Alg2 and Alg11 would not be predicted to interact. To test this model, we used coimmunoprecipitation assays to examine the interaction between Alg2 and Alg11. Yeast strains were constructed that coexpressing myc-tagged ALG2 and HA-tagged ALG1 or ALG11(see Materials and methods). Digitonin extracts were prepared from these strains, Alg1-HA or Alg11-HA proteins were precipitated with anti-HA antibody, and precipitates were fractionated by SDSPAGE and blotted with anti-myc to quantitate the amount of associated Alg2-myc. By this assay, we found no evidence for an interaction between Alg2 and Alg11, although, as shown, Alg2 associated stably with Alg1 (Figure 3B, lanes 1 and 2). This failure to detect an interaction between Alg2 and Alg11 was not due to a low level of Alg11-HA in these extracts. Western blot analysis of these extracts prior to immunoprecipitation demonstrated the levels of Alg11 and Alg1were comparable (Figure 3B, lanes 3 and 4). The lack of interaction between Alg2 and Alg11 observed by this coimmunoprecipitation assay was in agreement with their distinct chromatographic elution profiles (Figure 3). Together, these results suggest that both Alg2 and Alg11 interact with Alg1, but they do not interact with each other.
Alg1, but not Alg2 and Alg11, forms homo-oligomers
As a first step toward addressing the stoichiometry of each of these components in the oligomeric complexes, we examined the homomeric interactions between Alg1, Alg2, and Alg11 more closely. Specifically, we asked if Alg2 or Alg11 could self-assemble, as shown for Alg1 (Figure 2). To address this question, we used the immunoprecipitation assay, using extracts from yeast strains that coexpress HA- and myc-tagged Alg2 or Alg11 proteins. Proteins in digitonin extracts prepared from these strains were precipitated with anti-myc antibodies and fractionated by SDSPAGE; the ability of each of these proteins to homo-oligomerize was assayed by western blotting with anti-HA antibodies (Figure 4). By this assay, we found that although Alg1 homo-oligomerizes very efficiently, neither Alg2 nor Alg11 dimerize. The amounts of Alg11-HA or Alg2-HA that self-assembled were undetectable (Figure 4, compare lanes 1 with 2 and 3), despite the fact that western blot analysis demonstrated that Alg1, Alg2, and Alg11 proteins were found at similar levels in these extracts (Figure 4, lanes 4, 5, 6). These results demonstrate that although Alg1efficiently homo-oligomerizes, neither Alg2 and Alg11 can self-assemble.
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alg1-1 was identified previously as a temperature sensitive allele (Huffaker and Robbins, 1982). To determine the molecular basis for this effect, the alg1-1 locus was isolated by polymerase chain reaction (PCR) of genomic DNA from a mutant strain carrying this allele (see Materials and methods). Sequence analysis demonstrated the presence of a single opal nonsense mutation affecting W434, resulting in the truncation of the C-terminal 16 amino acids of Alg1 in a domain predicted by several algorithms to face the cytoplasm (Figure 5A).
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Identification of catalytically inactivating mutations in ALG1
The model that these ER mannosyltransferase complexes function in vivo strongly predicts that overexpression of mutant alg alleles that affect sugar transfer but not proteinprotein interactions should lead to a dominant negative phenotype. In contrast to Alg1, neither Alg2 nor Alg11 accumulated at high steady state levels when over expressed on a 2µ plasmid, for reasons that are not apparent (data not shown). Therefore, to test the idea that these complexes are important in vivo, mutations were introduced into ALG1 that were predicted to affect catalysis. To obtain such mutations, we chose to alter amino acid residues that are (1) conserved among related retaining glycosyltransferases and (2) predicted to face the cytoplasm as would be required for utilization of the cytosolic GDP-mannose substrate (Figure 5A). Based on criteria used by several different databases, Alg1, Alg2, and Alg11 have structural features that place them as members of various glycosyltransferase families. All three proteins are members of the PFAM glycosyltransferase 1 group (GT1-PFAM accession number PF0053), whose members transfer nucleotide sugars to a variety of substrates, including polyprenol lipid carriers. In contrast only Alg2 and Alg11 are found in family 4 of the CaZY database, which notably excludes Alg1. The majority of members of both families contain a C-terminal EX7E domain, whose first glutamic acid residues has been proposed to participate directly in catalysis (Bourne and Henrissat, 2001) (see Figure 5A for an alignment of a small subset of members of these families). In the case of one family member, the bacterial
-mannosyltransferase AceA, this glutamic acid has been demonstrated to be critical for enzymatic activity (Abdian et al., 2000
). Despite other similarities with members of this family, Alg1 notably lacks the EX7E, and instead, contains DX6D (D363-X6-D370) in this region. In addition, at the position corresponding to E278 of Alg1, most members of the PFAM GT1 and all members of CaZY family 4 contain a lysine that has been proposed to function in nucleotide sugar binding and which is also critical for AceA activity (Abdian et al., 2000
). Residues in highly conserved regions that lie in the predicted C-terminal cytoplasmic domain correspond to E278, G310, D363, and D370 of Alg1 (see Figure 5A for a schematic diagram). Each D of the DX6D (D363 and D370) was replaced with alanine or residues that would alter the charge. In addition E278 was changed to the more highly conserved K and the conserved G310 and H356 were replaced with alanine.
Each of these mutant alg1 alleles was first tested for complementation of the lethality caused by loss of ALG1 (Figure 6A). Each mutant allele was introduced on a CEN-containing plasmid into a strain in which the expression of ALG1 is controlled by the glucose-repressible GAL1 promoter and assayed for the ability to support growth after repression of ALG1 expression by growth in glucose containing media. Neither D363 nor D370 of the DX6D domain are essential because mutations that alter either of these residues complemented loss of ALG1, although it should be noted that the alg1-D363A mutant displayed a reduced growth rate (Figure 6A). Therefore, despite the conservation of the Alg1 DX6D domain with the EX7E catalytic domain of other PFAM glycosyltransferases, these results demonstrate that the aspartates of the Alg1 DX6D motif do not play an important role in catalysis. On the other hand, the E278K, G310D and H356Q mutant alleles completely failed to support growth on medium containing glucose, demonstrating that these are null alleles (Figure 6A).
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To determine the phenotypic consequences of their overexpression, these mutations, either singly or in combination, were introduced on 2µ plasmids into an otherwise normal strain. We observed no detrimental effect of single-copy expression, but overexpression of both alg1-E278K/H356Q and the alg1-G310D/H356Q alleles displayed both temperature and hygromycin B sensitive growth (Figure 7). These phenotypes were not observed in a strain overexpressing the wild-type ALG1 gene, demonstrating that the dominant negative phenotype in each case is a direct result of the alg1 mutation. One interpretation of this dominant negative affect is that catalytically inactive Alg1 maintains the ability to oligomerize, but these Alg1 mutantcontaining hetero-oligomeric complexes are nonfunctional. When overexpressed, the catalytically inactive Alg1 protein competitively inhibits its wild-type counterpart, which leads to the growth defects we observed. These results thus provide strong genetic evidence for the importance of Alg1-containing complexes in vivo.
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Discussion |
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Mutational analysis of ALG1 identified several important residues that lie in positions conserved among related glycosyltransferases. Surprisingly, alteration of the aspartates of the DX6D, which aligns with the predicted EX7E catalytic motif of other Alg1-related glycosyltransferases (including Alg2 and Alg11), does not result in a severe growth or dominant negative phenotype (Figure 6). Thus despite its similarity with other members of the PFAM GT1 family of glycosyltransferases, Alg1 does not utilize this DX6D motif for catalysis. On the other hand, alteration of several other residues conserved in this family (E278, G310, H356) lead to complete loss of Alg1 function, without having an effect on protein stability or proteinprotein interactions (Figure 6). Inexplicably, overexpression of either ALG2 or ALG11 does not lead to comparably high steady state, as is observed by ALG1 overexpression (data not shown). A test of the prediction that overexpression of catalytic alg2 and alg11 mutants should result in similar dominant negative phenotypes awaits the identification of experimental conditions that allow overexpression of these genes.
How would the association of these proteins in oligomeric complexes benefit the cell? One obvious advantage is that such a nondissociating arrangement of enzymes that act to catalyze sequential reactions limits the distance through which the substrate must diffuse. In the case of the Alg1/Alg2-containing complex, this arrangement could speed up the rate with which Man3GlcNAc2-PP-Dol is produced. This explanation is far less satisfying for the role of the Alg1/Alg11 complex. In this case, the benefits are less clear, because the reactions catalyzed by each of these two proteins, that is, addition of the first and the fourth or fifth mannose, are further removed from one another. One other possibility is brought to mind by the observation that Alg2 and Alg11 bear significant similarity to each other along their entire lengths (31% identity/48% similar/p = 2.6e06). On the basis of their structural similarities, both proteins are classified in the same glycosyltransferase family, according to the algorithms used by CAZy (http://afmb.cnrs-mrs.fr/CAZY/GT_intro.html) that are not based on predicted activity or substrate usage (Bourne and Henrissat, 2001). Alg2, Alg11, and YPL175W (involved in glycosylphosphatidylinositol anchor synthesis) are the only three Saccharomyces cerevisiae proteins that are members of this particular family, which notably excludes Alg1.
The observation that Alg2 and Alg11 are structurally similar but interact with a common partner, Alg1, raises the possibility that this interaction may facilitate or even modify their respective activities. For instance, Alg1 may perform dual functions in both sugar transfer and in the recognition of dolichol-phosphatederived substrates. An association between Alg1 with both the dolichol anchor as well as with Alg2 or Alg11 could facilitate their activities toward the lipid-linked oligosaccharide. The need for a dolichol recognition mechanism by glycosyltransferases of the ER has been appreciated for many years. A predicted membrane-embedded peptide sequence, conserved among several enzymes (including Alg1) was implicated in dolichol recognition (Albright et al., 1989). Although there is evidence that Alg1 does not require its transmembrane domain or even a dolichol-linked substrate for substrate recognition when expressed in Escherichia coli or in vitro (Revers et al., 1994
), whether or not this membrane-spanning domain plays a role in the modulation of mannosyltransferase activity in vivo is unknown. Although the idea that an interaction with Alg1 may affect the activity of Alg2 and Alg11 is purely speculative, it suggests both a common mechanism for dolichol recognition by these glycosyltransferases and a possible advantage for their association. This idea also predicts that Alg1 may be associated with other as yet unidentified mannosyltransferases that catalyze addition of the second
-1,3-linked and perhaps the fourth
-1,2- linked mannose on the cytosolic face of the ER. Further work will be required to test the validity of such an idea.
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Materials and methods |
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Construction of plasmids
Standard molecular biology techniques were used for all plasmid constructions (Sambrook et al., 1989). pSKALG1-HA contains an epitope-tagged allele of ALG1 that encodes Alg1 fused to three copies of the HA epitope at the C-terminus (Neiman et al., 1997
). To construct the yeast integrative plasmid pTiALG1-HA, an EcoRI/SacI fragment from pSKALG1-HA containing the ALG1-HA open reading frame was cloned into a derivative of p aO (Gao et al., 2001
), which places ALG1-HA expression under the constitutive TPI promoter. Linearization of this plasmid with XhoI within the URA3 gene targets integration at the ura3-1 locus in W303a. To generate pRS305TiALG1-HA, a fragment containing TPI-ALG1-HA from pTiALG1-HA was cloned into the LEU2, integrative plasmid pRS305 (Sikorski and Hieter, 1989
). Linearization of this plasmid with AflII within the LEU2 gene targets integration at the leu2 locus. Identical constructions, but containing the triple myc tag at the C-terminus are pSKALG1-myc, pTiALG1-myc, and pRS305TiALG1-myc. A series of epitope-tagged ALG2 and ALG11 plasmids, including pTiALG2-HA, pTiALG11-HA, pTiALG2-myc, pTiALG11-myc, pRS305TiALG2-myc, and pRS305TiALG11-myc, were constructed with exactly the same strategy as used to make the ALG1 derivatives already described.
The alg1-1 open reading frame was amplified by PCR from the genomic DNA isolated from alg1-1 strain, PRY55 (from P. Robbins), and sequenced. This alg1-1 gene was epitope-tagged and cloned into p O as described for TiALG1-HA, to generate pTialg1-1-HA.
Single missense mutations in ALG1were generated by PCR mutagenesis using pTiALG1-myc as a template and verified by DNA sequence analysis. This created myc-tagged alg1 alleles that produce Alg1 proteins that contain the following amino acids changes: E278K, G310D, H356Q, D363A, and D370A. These mutant alg1 alleles were over expressed by placing each under control of the TDH3 promoter, in the 2µ-containing plasmid, YEp352GAP.
Preparation of detergent extracts
Exponentially growing yeast cells were harvested at an OD600 of 13 and converted to spheroplasts with lyticase (Poster and Dean, 1996). To prepare detergent extracts that were used for both FPLC analysis and the coimmunoprecipitation assays, spheroplasts from 56 OD units of cells were resuspended in 500 µl ice cold lysis buffer (150 mM NaCl, 10 mM HEPES-KOH [pH 7.5], 5 mM MgCl2) that contained protease inhibitors and 1.0% digitonin as described (Gao and Dean, 2000
). Extracts were clarified by centrifugation at 100 kg in a Beckman TLA 100 rotor prior to chromatographic fractionation or immunoprecipitation to remove protein aggregates.
Coimmunoprecipitation and western immunoblotting
Epitope-tagged proteins in digitonin extracts were immunoprecipitated with anti-HA 12CA5 monoclonal antibodies, anti-HA Y-11 rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), anti-myc 9E10 monoclonal antibodies, or anti-myc A-14 rabbit polyclonal antibodies (Santa Cruz Biotechnology) and protein A-Sepharose (Pharmacia, Uppsala, Sweden). After fractionation by 10% SDSPAGE, immunoprecipitated proteins were detected by chemiluminescence (ECL, Amersham, Little Chalfont, U.K.) as described previously (Gao and Dean, 2000). Antibody combinations in immunoprecipitations used the rabbit polyclonal antibodies in the primary incubation and mouse monoclonal antibodies for subsequent blotting to eliminate cross-reactivity between the rabbit IgG used for the immunoprecipitation and the anti-mouse IgGhorseradish peroxidase used for chemiluminescence detection.
Gel filtration chromatography
Detergent extracts containing 1.0% digitonin were prepared as described from yeast expressing the HA-tagged ALG1, ALG2, or ALG11 alleles. After clarification at 100 kg, 200 µl extract were fractionated over a gel filtration column (Superose 12 HR 10/30, Pharmacia) equilibrated with 150 mM NaCl, 50 mM HEPES-KOH (pH 7.5), 5 mM MgCl2, 1.0% digitonin, 1 mM phenylmethylsulfonyl fluoride using the FPLC system (Pharmacia). FPLC was performed at a flow rate of 0.2 ml/min, and 400 µl was collected for each fraction. Fractions were analyzed for the presence of HA-tagged Alg1, Alg2, and Alg11 by SDSPAGE, followed by immunoblotting with HA-specific antibodies. For molecular weight determination, proteins that range from 29,000 to 700,000 MW (Pharmacia) were fractionated in parallel and detected after staining with Coomassie brilliant blue.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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