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Physical Interactions Between the Alg1, Alg2, and Alg11 Mannosyltransferases of the Endoplasmic Reticulum

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Physical Interactions Between the Alg1, Alg2, and Alg11 Mannosyltransferases of the Endoplasmic Reticulum Glycobiology vol. 14 no. 6 pp. 559±570, 2004 DOI: 10.1093/glycob/cwh072 Advance Access publication on March 24, 2004 Physical interactions between the Alg1, Alg2, and Alg11 mannosyltransferases of the endoplasmic reticulum Xiao-Dong Gao2, Akiko Nishikawa1, and Neta Dean1 begins on the cytosolic face of the ER, where seven sugars (two N-acetylglucoseamines and five mannoses) are added 1Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, sequentially to dolichyl phosphate on the outer leaflet of NY 11794-5215, and 2Research Center for Glycoscience, National the ER, using nucleotide sugar donors (Abeijon and Institute of Advanced Industrial Science and Technology, Tsukuba Hirschberg, 1992; Perez and Hirschberg, 1986; Snider and Downloaded from https://academic.oup.com/glycob/article/14/6/559/638968 by guest on 30 September 2021 Central 6, 1-1 Higashi, Tsukuba 305-8566, Japan Rogers, 1984). After a ``flipping'' or translocation step, the Received on January 26, 2004; revised on March 2, 2004; accepted on last seven sugars (four mannoses and three glucoses) are March 2, 2004 added within the lumen of the ER, using dolichol-linked sugar donors (Burda and Aebi, 1999). Once assembled, the The early steps of N-linked glycosylation involve the synthesis oligosaccharide is transferred from the lipid to nascent of a lipid-linked oligosaccharide, Glc3Man9GlcNAc2-PP- protein in a reaction catalyzed by oligosaccharyltransferase. dolichol, on the endoplasmic reticulum (ER) membrane. After removal of terminal glucoses and a single mannose, Prior to its lumenal translocation and transfer to nascent nascent glycoproteins bearing the N-linked Man8GlcNAc2 glycoproteins, mannosylation of Man5GlcNAc2-PP-dolichol core can exit the ER to the Golgi, where this core may is catalyzed by the Alg1, Alg2, and Alg11 mannosyltrans- undergo further carbohydrate modifications. The structure ferases. We provide evidence for a physical interaction and assembly pathway of the lipid-linked oligosaccharide between these proteins. Using a combination of biochemical has been highly conserved among almost all eukaryotes, as and genetic assays, two distinct complexes that contain multi- expected in light of its pivotal role during glycoprotein ple copies of Alg1 were identified. The two Alg1-containing maturation and exit from the ER (Helenius and Aebi, complexes differ from one another in that one complex con- 2001). tains Alg2 and the other contains Alg11. Alg1 self-assembles Since the pioneering work of Robbins and colleagues, through a C-terminal domain that is distinct from the region who initially isolated and characterized yeast mutants required for its association with Alg2 or Alg11. Missense defective in lipid-linked oligosaccharide assembly (aspara- mutations affecting catalysis but not Alg1 protein stability gine linked glycosylation or alg mutants) (Huffaker and or assembly with Alg2 or Alg11 were also identified. Over- Robbins, 1982, 1983), many of the genes affecting lipid- expression of these catalytically inactive alleles resulted in linked oligosaccharide synthesis in the ER have now been dominant negative phenotypes, providing genetic evidence for identified (for review see Burda and Aebi, 1999). With few functional Alg1-containing complexes in vivo. These data exceptions, mutations in any one of the ALG genes result in suggest that an additional level of regulation that ensures accumulation of defined oligosaccharide intermediates. the fidelity of complex oligosaccharide structures involves This phenotype is consistent with the notion that the lipid- the physical association of the related catalytic enzymes in linked oligosaccharide biosynthetic pathway involves a the ER membrane. series of steps that lead to the synthesis of a branched and structurally complex oligosaccharide. Furthermore there is Key words: ALG1, ALG2, ALG11/endoplasmic reticulum/ good evidence to support the hypothesis that this ordered mannosyltransferase/N-glycosylation/S. cerevisiae assembly arises at least in part from differences in the sub- strate specificities of the various glycosyltransferases that participate in these reactions (Burda et al., 1999). Introduction During this ordered assembly, an important distinction can be made between the reactions that occur on the cyto- Glycosylation is an essential modification important for solic face of the ER and those that occur in the lumen. protein folding and stability. The early steps that lead to A deletion of any gene affecting synthesis or translocation synthesis of an asparagine (N )-linked glycan start in the of Man5GlcNAc2-PP-Dol on the cytosolic face of the ER encoplasmic reticulum (ER) and involve synthesis of a lipid- leads to death or an extremely severe phenotype. For linked oligosaccharide precursor that is subsequently trans- instance, alg1D, alg2D, and alg11D mutants, required for ferred to selected asparagines on nascent glycoproteins (for addition of the first, third, and fourth or fifth mannose, review see Herscovics and Orlean, 1993; Kornfeld and respectively, of Man5GlcNAc2-P-P-Dol are inviable or Kornfeld, 1985; Tanner and Lehle, 1987). Synthesis of this grow very slowly (Albright and Robbins, 1990; Cipollo lipid-linked oligosaccharide, Glc3Man9GlcNAc2-PP-Dol, et al., 2001; Jackson et al., 1993). In contrast, mutations in genes affecting lumenal sugar additions result in little if any 1To whom correspondence should be addressed; e-mail: growth phenotypes (Burda et al., 1999). These observations [email protected] suggest that although N-linked glycosylation is an essential Glycobiology vol. 14 no. 6 # Oxford University Press 2004; all rights reserved. 559 X.-D. Gao et al. modification, the cytosolic Man5GlcNAc2 core structure is 1000 the minimal intermediate whose synthesis and translocation Apoferritin(443) across the membrane is required for the successful execution α-Amylase(200) of N-linked glycosylation. The Man4GlcNAc2 core and earlier intermediates may be poor substrates for lumenal 100 BSA(66) 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 10 lipid-linked oligosaccharide biosynthesis have been identi- Fraction 2324 25 26 27 28 29 30 31 3332 34 fied, the regulation and coordination of their activities is Alg1-HAp poorly understood. Here we show that three of the enzymes that participate in Man5GlcNAc2-PP-Dol synthesis, Fig. 1. Gel filtration chromatography of yeast extracts containing Alg1- namely, Alg1, Alg2, and Alg11, exist in oligomeric protein HA. 1.0% digitonin extracts from an ALG1-HA yeast strain (XGY25) complexes. Two distinct complexes that are distinguished were prepared as described in Materials and methods. Samples were clarified by centrifugation at 100 kg and fractionated by Fast by the presence of Alg2 or Alg11 can be identified, both of Downloaded from https://academic.oup.com/glycob/article/14/6/559/638968 by guest on 30 September 2021 which contain Alg1. These results suggest that the physical Performance Liquid Chromatography (FPLC) over a Superose 12 column. Aliquots of fractions were subjected to SDS±PAGE and western association of the functionally related catalytic enzymes blotted with anti-HA antibodies. The panel above the western blot involved in lipid-linked oligosaccharide synthesis provides indicates the mobility of protein molecular weight markers run in parallel an additional level of regulation that assures proper under identical conditions. construction of this complex oligosaccharide. rapid kinetics is that lipid-linked oligosaccharide biosynthe- Results sis is not a diffusion-limited process but rather involves the physical association of enzymes that catalyze each of the The Alg1 protein exists in oligomeric complexes that sequential mannose additions, which would in turn limit contain Alg2 or Alg11 diffusion of the acceptor. Second, an increasingly large The Alg1 mannosyltransferase catalyzes addition of the number of glycosyltransferases have been shown to func- ~ first b1,4-linked mannose on GlcNAc2-PP-Dol, producing tion as homo- or hetero-oligomers (e.g., Jungmann and ManGlcNAc2-PP-Dol (Couto et al., 1984). To investigate Munro, 1998; Marks et al., 1999; Sasai et al., 2001), suggest- the physical properties of the Alg1 protein, a yeast strain ing a precedent for this mechanism in N-linked glycan was constructed whose chromosomal ALG1 locus was synthesis. replaced with an allele that encodes an hemaglutinnin To explore this idea, we tested whether Alg1 interacts (HA)-tagged Alg1 protein. An ALG1-HA strain that solely with any other mannosyltransferase known to catalyze produces Alg1-HA grows at rates comparable to the par- lipid-linked oligosaccharide synthesis on the cytosolic face ental wild type and exhibits no detectable glycosylation of the ER, namely, Alg2 and Alg11. Alg2 is required for abnormalities, suggesting that addition of the HA epitope addition of the third a~1,6-linked mannose. Loss of ALG2 is to Alg1 does not alter its normal behavior (data not shown). lethal, and alg2 mutants accumulate Man2GlcNAc2-PP- To determine the native molecular weight of Alg1, we used Dol (Jackson et al., 1993). Alg11 is required for addition gel filtration chromatography. Detergent extracts were pre- of the fourth and/or fifth a~1,2-linked mannose to Man3- pared from this ALG1-HA strain, clarified by centrifuga- GlcNAc2-PP-Dol. Loss of ALG11 results in a
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