ALG9 mannosyltransferase is involved in two different steps of lipid-linked oligosaccharide biosynthesis

N-linked protein glycosylation follows a conserved pathway in eukaryotic cells. The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for the oligosaccharyltransferase (OST), is catalyzed by different glycosyltransferases located at the membrane of the endoplasmic reticulum (ER). The substrate specificity of the different glycosyltransferase guarantees the ordered assembly of the branched oligosaccharide and ensures that only completely assembled oligosaccharide is transferred to protein. The glycosyltransferases involved in this pathway are highly specific, catalyzing the addition of one single hexose unit to the lipid-linked oligosaccharide (LLO). Here, we show that the dolichylphosphomannose-dependent ALG9 mannosyltransferase is the exception from this rule and is required for the addition of two different alpha-1,2-linked mannose residues to the LLO. This report completes the list of lumen-oriented glycosyltransferases required for the assembly of the LLO.     

The ALG10 locus of Saccharomyces cerevisiae encodes the alpha-1,2 glucosyltransferase of the endoplasmic reticulum: the terminal glucose of the lipid-linked oligosaccharide is required for efficient N-linked glycosylation

The biosynthesis of the lipid-linked oligosaccharide substrate for N- linked protein glycosylation follows a highly conserved pathway at the membrane of the endoplasmic reticulum. Based on the synthetic growth defect in combination with a reduced oligosaccharyltransferase activity (wbp1), we have identified alg10 mutant strains which accumulate lipid- linked Glc2Man9GlcNAc2. We cloned the corresponding wild-type gene and show in a novel in vitro assay that Alg10p is a dolichyl-phosphoglucose- dependent glucosyltransferase which adds the terminal alpha-1,2 glucose to the lipid-linked Glc2Man9GlcNAc2 oligosaccharide. Hypoglycosylation of secreted proteins in alg10 deletion strains demonstrates that the terminal alpha-1,2-linked glucose residue is a key element in substrate recognition by the oligosaccharyltransferase. This ensures that primarily completely assembled oligosaccharide is transferred to protein.      

Biosynthesis of lipid-linked oligosaccharides. I. Preparation of lipid-linked oligosaccharide substrates.

In order to purify the glycosyltransferases involved in the assembly of lipid-linked oligosaccharides and to be able to study the acceptor substrate specificity of these enzymes, methods were developed to prepare and purify a variety of lipid-linked oligosaccharides, differing in the structure of the oligosaccharide moiety. Thus, Man9 (GlcNAc)2-pyrophosphoryl-dolichol was prepared by isolation and enzymatic synthesis using porcine pancreatic microsomes, while Glc3Man9(GlcNAc)2-PP-dolichol was isolated from Madin-Darby canine kidney cells. Treatment of these oligosaccharide lipids with a series of selected glycosidases led to the preparation of Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6(Man alpha 1,3)Man alpha 1,6]Man beta 1,4GlcNAc beta 1,4GlcNAc-PP-dolichol; Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6]Man beta 1,4GlcNAc beta 1, 4GlcNac-PP-dolichol; and Man alpha 1,6(Man alpha 1,3)Man alpha 1, 6[Man alpha 1,3]Man beta 1,4GlcNAc-beta 1,4GlcNAc-PP-dolichol. The preparation, isolation, and characterization of each of these lipid-linked oligosaccharide substrates are described.     

Transmembrane movement of oligosaccharide-lipids during glycoprotein synthesis

The transport of sugar residues into the endoplasmic reticulum (ER) during glycoprotein synthesis was studied by examining the transmembrane orientations of the oligosaccharide-lipid precursors of asparagine-linked oligosaccharides. Using the lectin concanavalin A, the lipid-linked oligosaccharides Man3-5GlcNAc2 were found on the cytoplasmic side of ER-derived vesicles in vitro while lipid-linked Man6-9GlcNAc2 and Glc1-3Man9GlcNAc2 were found facing the lumen. These results suggest that Man5GlcNAc2-lipid is synthesized on the cytoplasmic side of the ER membrane and then translocated to the luminal side. Glc3Man9GlcNAc2-lipid is then completed on the luminal side where it serves as the donor in peptide glycosylation. Translocation of Man5GlcNAc2-lipid offers a mechanism for the export of sugar residues from the cytoplasm during glycoprotein synthesis. This translocation may be the reason for the participation of lipid-linked mono- and oligosaccharides in glycoprotein synthesis.     

Large-scale isolation of dolichol-linked oligosaccharides with homogeneous oligosaccharide structures: determination of steady-state dolichol-linked oligosaccharide compositions

The dolichol-linked oligosaccharide donor (Glc(3)Man(9)GlcNAc(2)-PP-Dol) for N-linked glycosylation of proteins is assembled in a series of reactions that initiate on the cytoplasmic face of the rough endoplasmic reticulum and terminate within the lumen. The biochemical analysis of the oligosaccharyltransferase and the glycosyltransferases that mediate assembly of dolichol-linked oligosaccharides (OS-PP-Dol) has been hindered by the lack of structurally homogeneous substrate preparations. We have developed an improved method for the preparative-scale isolation of dolichol-linked oligosaccharides from vertebrate tissues and yeast cells. Preparations that were highly enriched in either Glc(3)Man(9)GlcNAc(2)-PP-Dol or Man(9)GlcNAc(2)-PP-Dol were obtained from porcine pancreas and a Man(5)GlcNAc(2)-PP-Dol preparation was obtained from an alg3 yeast culture. Chromatography of the OS-PP-Dol preparations on an aminopropyl silica column was used to obtain dolichol-linked oligosaccharides with defined structures. A single chromatography step could achieve near-baseline resolution of dolichol-linked oligosaccharides that differed by one sugar residue. A sensitive oligosaccharyltransferase endpoint assay was used to determine the concentration and composition of the OS-PP-Dol preparations. Typical yields of Glc(3)Man(9)GlcNAc(2)-PP-Dol, Man(9)GlcNAc(2)-PP-Dol, and Man(5)GlcNAc(2)-PP-Dol ranged between 5 and 15 nmol per chromatographic run. The homogeneity of these preparations ranged between 85 and 98% with respect to oligosaccharide composition. Purification of dolichol-linked oligosaccharides from cultures of alg mutant yeast strains provides a general method to obtain authentic OS-PP-Dol assembly intermediates of high purity. The analytical methods described here can be used to accurately evaluate the steady-state dolichol-linked oligosaccharide compositions of wild-type and mutant cell lines.     

Glycoengineering of the methylotrophic yeast Hansenula polymorpha for the production of glycoproteins with trimannosyl core N-glycan by blocking core oligosaccharide assembly

The initial lipid-linked oligosaccharide Glc(3)Man(9)GlcNAc(2)-dolichyl pyrophosphate (Dol-PP) for N-glycan is synthesized and assembled at the membrane of the endoplasmic reticulum (ER) and subsequently transferred to a nascent polypeptide by the oligosaccharide transferase complex. We have identified an ALG3 homolog (HpALG3) coding for a dolichyl-phosphate-mannose dependent alpha-1,3-mannosyltransferase in the methylotrophic yeast Hansenula polymorpha. The detailed analysis of glycan structure by linkage-specific mannosidase digestion showed that HpALG3 is responsible for the conversion of Man5GlcNAc(2)-Dol-PP to Man(6)GlcNAc(2)-Dol-PP, the first step to attach a mannose to the lipid-linked oligosaccharide in the ER. The N-glycosylation pathway of H. polymorpha has been remodeled by deleting the HpALG3 gene in the Hpoch1 null mutant strain blocked in the yeast-specific outer mannose chain synthesis and by introducing an ER-targeted Aspergillus saitoi alpha-1,2-mannosidase gene. This glycoengineered H. polymorpha strain produced glycoproteins mainly containing trimannosyl core N-glycan (Man(3)GlcNAc(2)), which is the common core backbone of various human-type N-glycans. The results demonstrate the high potential of H. polymorpha to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.     

The ins(ide) and out(side) of dolichyl phosphate biosynthesis and recycling in the endoplasmic reticulum.

The precursor oligosaccharide donor for protein N-glycosylation in eukaryotes, Glc3Man9GlcNAc(2)-P-P-dolichol, is synthesized in two stages on both leaflets of the rough endoplasmic reticulum (ER). There is good evidence that the level of dolichyl monophosphate (Dol-P) is one rate-controlling factor in the first stage of the assembly process. In the current topological model it is proposed that ER proteins (flippases) then mediate the transbilayer movement of Man-P-Dol, Glc-P-Dol, and Man5GlcNAc(2)-P-P-Dol from the cytoplasmic leaflet to the lumenal leaflet. The rate of flipping of the three intermediates could plausibly influence the conversion of Man5GlcNAc(2)-P-P-Dol to Glc3Man(9)GlcNAc(2)-P-P-Dol in the second stage on the lumenal side of the rough ER. This article reviews the current understanding of the enzymes involved in the de novo biosynthesis of Dol-P and other polyisoprenoid glycosyl carrier lipids and speculates about the role of membrane proteins and enzymes that could be involved in the transbilayer movement of the lipid intermediates and the recycling of Dol-P and Dol-P-P discharged during glycosylphosphatidylinositol anchor biosynthesis, N-glycosylation, and O- and C-mannosylation reactions on the lumenal surface of the rough ER.     

Physical interactions between the Alg1, Alg2, and Alg11 mannosyltransferases of the endoplasmic reticulum

The early steps of N-linked glycosylation involve the synthesis of a lipid-linked oligosaccharide, Glc(3)Man(9)GlcNAc(2)-PP-dolichol, on the endoplasmic reticulum (ER) membrane. Prior to its lumenal translocation and transfer to nascent glycoproteins, mannosylation of Man(5)GlcNAc(2)-PP-dolichol is catalyzed by the Alg1, Alg2, and Alg11 mannosyltransferases. We provide evidence for a physical interaction between these proteins. Using a combination of biochemical and genetic assays, two distinct complexes that contain multiple copies of Alg1 were identified. The two Alg1-containing complexes differ from one another in that one complex contains Alg2 and the other contains Alg11. Alg1 self-assembles through a C-terminal domain that is distinct from the region required for its association with Alg2 or Alg11. Missense mutations affecting catalysis but not Alg1 protein stability or assembly with Alg2 or Alg11 were also identified. Overexpression of these catalytically inactive alleles resulted in dominant negative phenotypes, providing genetic evidence for functional Alg1-containing complexes in vivo. These data suggest that an additional level of regulation that ensures the fidelity of complex oligosaccharide structures involves the physical association of the related catalytic enzymes in the ER membrane.     

Potential regulation of N-glycosylation precursor through oligosaccharide-lipid hydrolase action and glucosyltransferase-glucosidase shuttle.

The potential role of degradative mechanisms in controlling the level of the dolichyl pyrophosphate-linked Glc3Man9GlcNAc2 required for protein N-glycosylation has been explored in thyroid slices and endoplasmic reticulum (ER) vesicles, focusing on cleavage of the oligosaccharide from its lipid attachment and on the enzymatic removal of peripheral monosaccharide residues. Vesicle incubations demonstrated a substantial release of free Glc3Man9GlcNAc2 (at 30 min approximately 35% of that transferred to protein) which was inhibited in the presence of exogenous peptide acceptor and was sensitive to disruption of membrane integrity by detergent. In thyroid slices glucosylated oligosaccharides terminating in the di-N-acetylchitobiose sequence were also noted and these continued to be formed even during inhibition by puromycin of both protein synthesis and the attendant N-glycosylation. These observations indicated that the oligosaccharide originated from the lipid donor and suggested, together with previously reported similarities in substrate specificity and cofactor requirements, that the oligosaccharyltransferase can carry out in vivo both the hydrolytic and transfer functions. In addition to the release of the intact Glc3Man9GlcNAc2, we also obtained evidence that the lipid-linked oligosaccharide can be modified by the in vivo action of ER glycosidases. Since radiolabeling of the oligosaccharide-lipid in thyroid slices indicated a preferential turnover of the glucose residues, the possible existence of a glucosyltransferase-glucosidase shuttle was explored with the use of castanospermine. In the presence of this glucosidase inhibitor, the formation of under-glucosylated and nonglucosylated oligosaccharides was not observed, even under conditions of energy deprivation in which they accumulate. Glucosidase inhibition in ER vesicle incubations likewise prevented the appearance of incompletely glucosylated oligosaccharide-lipids. Studies employing the mannosidase inhibitor 1-deoxymannojirimycin in thyroid slices furthermore indicated that in vivo removal of at least one mannose residue from the dolichyl pyrophosphate-linked oligosaccharide can occur.     

Release of glucose-containing polymannose oligosaccharides during glycoprotein biosynthesis. Studies with thyroid microsomal enzymes and slices

Incubations of thyroid microsomes with radiolabeled dolichyl pyrophosphoryl oligosaccharide (Glc3Man9-GlcNAc2) under conditions optimal for the N-glycosylation of protein resulted in the release, by apparently independent enzymatic reactions, of two types of neutral glucosylated polymannose oligosaccharides which differed from each other by terminating either in an N-acetylglucosamine residue (Glc3Man9GlcNAc1) or a di-N-acetylchitobiose moiety (Glc3Man9GlcNAc2). The first mentioned oligosaccharide, which was released in a steady and slow process unaffected by the addition of EDTA, appeared to be primarily the product of endo-beta-N-acetylglucosaminidase action on newly synthesized glycoprotein and such an enzyme with a neutral pH optimum capable of hydrolyzing exogenous glycopeptides and oligosaccharides (Km = 18 microM) was found in the thyroid microsomal fraction. The Glc3Man9GlcNAc2 oligosaccharide, in contrast, appeared to originate from the oligosaccharide-lipid by a rapid hydrolysis reaction which closely paralleled the N-glycosylation step, progressing as long as oligosaccharide transfer to protein occurred and terminating when carbohydrate attachment ceased either due to limitation of lipid-saccharide donor or addition of EDTA. There was a striking similarity between oligosaccharide release and transfer to protein with lipid-linked Glc3Man9GlcNAc2 serving as a 10-fold better substrate for both reactions than lipid-linked Man9-8GlcNAc2. The coincidence of transferase and hydrolase activities suggest the possibility of the existence of one enzyme with both functions. The physiological relevance of oligosaccharide release was indicated by the formation of such molecules in thyroid slices radiolabeled with [2-3H]mannose. Large oligosaccharides predominated (12 nmol/g) and consisted of two families of components; one group terminating in N-acetylglucosamine, ranged from Glc1Man9GlcNAc1 to Man5GlcNAc1 while the other contained the di-N-acetylchitobiose sequence and included Glc3Man9GlcNAc2, Glc1Man9GlcNAc2, and Man9GlcNAc2.     

Malectin: a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein N-glycosylation

N-Glycosylation starts in the endoplasmic reticulum (ER) where a 14-sugar glycan composed of three glucoses, nine mannoses, and two N-acetylglucosamines (Glc(3)Man(9)GlcNAc(2)) is transferred to nascent proteins. The glucoses are sequentially trimmed by ER-resident glucosidases. The Glc(3)Man(9)GlcNAc(2) moiety is the substrate for oligosaccharyltransferase; the Glc(1)Man(9)GlcNAc(2) and Man(9)GlcNAc(2) intermediates are signals for glycoprotein folding and quality control in the calnexin/calreticulin cycle. Here, we report a novel membrane-anchored ER protein that is highly conserved in animals and that recognizes the Glc(2)-N-glycan. Structure determination by nuclear magnetic resonance showed that its luminal part is a carbohydrate binding domain that recognizes glucose oligomers. Carbohydrate microarray analyses revealed a uniquely selective binding to a Glc(2)-N-glycan probe. The localization, structure, and binding specificity of this protein, which we have named malectin, open the way to studies of its role in the genesis, processing and secretion of N-glycosylated proteins.     

Arabidopsis thaliana alpha1,2-glucosyltransferase (ALG10) is required for efficient N-glycosylation and leaf growth

Assembly of the dolichol-linked oligosaccharide precursor (Glc(3) Man(9) GlcNAc(2) ) is highly conserved among eukaryotes. In contrast to yeast and mammals, little is known about the biosynthesis of dolichol-linked oligosaccharides and the transfer to asparagine residues of nascent polypeptides in plants. To understand the biological function of these processes in plants we characterized the Arabidopsis thaliana homolog of yeast ALG10, the α1,2-glucosyltransferase that transfers the terminal glucose residue to the lipid-linked precursor. Expression of an Arabidopsis ALG10-GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed a reticular distribution pattern resembling endoplasmic reticulum (ER) localization. Analysis of lipid-linked oligosaccharides showed that Arabidopsis ALG10 can complement the yeast Δalg10 mutant strain. A homozygous Arabidopsis T-DNA insertion mutant (alg10-1) accumulated mainly lipid-linked Glc(2) Man(9) GlcNAc(2) and displayed a severe protein underglycosylation defect. Phenotypic analysis of alg10-1 showed that mutant plants have altered leaf size when grown in soil. Moreover, the inactivation of ALG10 in Arabidopsis resulted in the activation of the unfolded protein response, increased salt sensitivity and suppression of the phenotype of α-glucosidase I-deficient plants. In summary, these data show that Arabidopsis ALG10 is an ER-resident α1,2-glucosyltransferase that is required for lipid-linked oligosaccharide biosynthesis and subsequently for normal leaf development and abiotic stress response.     

STT3, a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo.

N-linked glycosylation is a ubiquitous protein modification, and is essential for viability in eukaryotic cells. A lipid-linked core-oligosaccharide is assembled at the membrane of the endoplasmic reticulum and transferred to selected asparagine residues of nascent polypeptide chains by the oligosaccharyl transferase (OTase) complex. Based on the synthetic lethal phenotype of double mutations affecting the assembly of the lipid-linked core-oligosaccharide and the OTase activity, we have performed a novel screen for mutants in Saccharomyces cerevisiae with altered N-linked glycosylation. Besides novel mutants deficient in the assembly of the lipid-linked oligosaccharide (alg mutants), we identified the STT3 locus as being required for OTase activity in vivo. The essential STT3 protein is approximately 60% identical in amino acid sequence to its human homologue. A mutation in the STT3 locus affects substrate specificity of the OTase complex in vivo and in vitro. In stt3-3 cells very little glycosyl transfer occurs from incomplete lipid-linked oligosaccharide, whereas the transfer of full-length Glc3Man9GlcNAc2 is hardly affected as compared with wild-type cells. Depletion of the STT3 protein results in loss of transferase activity in vivo and a deficiency in the assembly of OTase complex.     

Characterization of lipid-linked octa- through undecasaccharides implicated in the biosynthesis of Saccharomyces cerevisiae mannoproteins.

The lipid-linked oligosaccharide Glc3-Man9(GlcNAc)2 (Glc, glucose; Man, mannose; GlcNAc, N-acetylglucosamine) serves as a precursor for the biosynthesis of the inner core portion of the asparagine-linked polysaccharide of Saccharomyces cerevisiae mannoproteins. It has been shown previously that incubation of a microsomal preparation from this organism with UDP-N-acetylglucosamine and GDP-[14C]mannose gives rise to a series of lipid-linked oligosaccharides of the general structure Mann(GlcNAc)2, with n from 1 to 9. A structural characterization of Man1- to Man5(GlcNAc)2 oligosaccharides indicated that the major structures among these were identical to the intermediates proposed for the biosynthesis of animal glycoproteins (C. Prakash and I. K. Vijay, Biochemistry 21:4810-4818, 1982). In the present study, the structural characterization of the Man6- through Man9(GlcNAc)2 species was conducted. The Man6- through Man8(GlcNAc)2 species have two isomers, whereas Man9(GlcNAc)2 is monoisomeric. One isomer each of Man6- through Man8(GlcNAc)2 and the monoisomeric Man9(GlcNAc)2 are identical to the intermediates for the biosynthesis of asparagine-linked glycoproteins in animal systems. It is proposed that the steps of the lipid-linked assembly of the carbohydrate precursor for S. cerevisiae mannoproteins are identical to those of the major pathway in animal systems. A lack of acceptor substrate specificity by the mannosyltransferases, as observed with in vitro studies with animal systems, also might be responsible for the biosynthesis of multiple isomers reported here.     

Allosteric regulation provides a molecular mechanism for preferential utilization of the fully assembled dolichol-linked oligosaccharide by the yeast oligosaccharyltransferase

The oligosaccharyltransferase (OST) preferentially utilizes the fully assembled dolichol-linked oligosaccharide Glc(3)Man(9)GlcNAc(2)-PP-Dol as the donor for N-linked glycosylation of asparagine residues in N-X-T/S consensus sites in newly synthesized proteins. A wide variety of assembly intermediates (Glc(0-2)Man(0-9)GlcNAc(2)-PP-Dol) can serve as the donor substrate for N-linked glycosylation of peptide acceptor substrates in vitro or of nascent glycoproteins in mutant cells that are defective in donor substrate assembly. A kinetic mechanism that can account for the selection of the fully assembled donor substrate from a complex mixture of dolichol-linked oligosaccharides (OS-PP-Dol) has not been elucidated. Here, the steady-state kinetic properties of the OST were reinvestigated using a proteoliposome assay system consisting of the purified yeast enzyme, near-homogeneous preparations of a dolichol-linked oligosaccharide (Glc(3)Man(9)GlcNAc(2)-PP-Dol or Man(9)GlcNAc(2)-PP-Dol) and an (125)I-labeled tripeptide as the acceptor substrate. The K(m) of the OST for the acceptor tripeptide was only slightly enhanced when Glc(3)Man(9)GlcNAc(2)-PP-Dol was the donor substrate relative to when Man(9)GlcNAc(2)-PP-Dol was the donor substrate. Evaluation of the kinetic data for both donor substrates showed deviations from typical Michaelis-Menten kinetics. Sigmoidal saturation curves, Lineweaver-Burk plots with upward curvature, and apparent Hill coefficients of about 1.4 suggested a substrate activation mechanism involving distinct regulatory (activator) and catalytic binding sites for OS-PP-Dol. Results of competition experiments using either oligosaccharide donor as an alternative substrate were also consistent with this hypothesis. We propose that binding of either donor substrate to the activator site substantially enhances Glc(3)Man(9)GlcNAc(2)-PP-Dol occupancy of the enzyme catalytic site via allosteric activation.     

The effect of mannosamine on the formation of lipid-linked oligosaccharides and glycoproteins in canine kidney cells

Madin-Darby canine kidney (MDCK) cells normally form lipid-linked oligosaccharides having mostly the Glc3Man9GlcNAc2 oligosaccharide. However, when MDCK cells are incubated in 1 to 10 mM mannosamine and labeled with [2-3H]mannose, the major oligosaccharides associated with the dolichol were Man5GlcNAc2 and Man6GlcNAc2 structures. Since both of these oligosaccharides were susceptible to digestion by endo-beta-N-acetylglucosaminidase H, the Man5GlcNAc2 must be different in structure than the Man5GlcNAc2 usually found as a biosynthetic intermediate in the lipid-linked oligosaccharides. Methylation analysis also indicated that this Man5GlcNAc2 contained 1----3 linked mannose residues. Since pulse chase studies indicated that the lesion was in biosynthesis, it appears that mannosamine inhibits the in vivo formation of lipid-linked oligosaccharides perhaps by inhibiting the alpha-1,2-mannosyl transferases. Although the lipid-linked oligosaccharides produced in the presence of mannosamine were smaller in size than those of control cells and did not contain glucose, the oligosaccharides were still transferred in vivo to protein. Furthermore, the oligosaccharide portions of the glycoproteins were still processed as shown by the fact that the glycopeptides were of the complex and hybrid types and were labeled with [3H]mannose or [3H]galactose. In contrast, control cells produced complex and high-mannose structures but no hybrid oligosaccharides were detected. The inhibition by mannosamine could be overcome by adding high concentrations of glucose to the medium.     

Two yeast mutations in glucosylation steps of the asparagine glycosylation pathway

Two complementing mutations in lipid-linked oligosaccharide biosynthesis have been isolated following a [3H]mannose suicide enrichment. Rather than making the wild type precursor oligosaccharide, Glc3man9Glc-NA2-P-P-dolichol, the mutants, alg5-1 and alg6-1, accumulate Man9GlcNAc2-P-P-dolichol as their largest lipid-linked oligosaccharide in vivo and in vitro. When UDP-[3H]Glc was added to microsomal membranes of each mutant, neither could elongate Man9GlcNAc2-P-P-dolichol and only alg6-1 could synthesize dolichol-phosphoglucose. When dolicholphospho[3H]glucose was added to microsomes from alg5-1, alg6-1, or the parental strain, only alg5-1 and the parental strain made glucosylated lipid-linked oligosaccharides. These results indicate that alg5-1 cells are unable to synthesize dolichol phosphoglucose while alg6-1 cells are unable to transfer glucose from dolichol phosphoglucose to the unglucosylated lipid-linked oligosaccharide. We also present evidence that both mutants transfer Man9GlcNAc2 to protein.     

The yeast ALG11 gene specifies addition of the terminal alpha 1,2-Man to the Man5GlcNAc2-PP-dolichol N-glycosylation intermediate formed on the cytosolic side of the endoplasmic reticulum

The initial steps in N-linked glycosylation involve the synthesis of a lipid-linked core oligosaccharide followed by the transfer of the core glycan to nascent polypeptides in the endoplasmic reticulum (ER). Here, we describe alg11, a new yeast glycosylation mutant that is defective in the last step of the synthesis of the Man(5)GlcNAc(2)-PP-dolichol core oligosaccharide on the cytosolic face of the ER. A deletion of the ALG11 gene leads to poor growth and temperature-sensitive lethality. In an alg11 lesion, both Man(3)GlcNAc(2)-PP-dolichol and Man(4)GlcNAc(2)-PP-dolichol are translocated into the ER lumen as substrates for the Man-P-dolichol-dependent sugar transferases in this compartment. This leads to a unique family of oligosaccharide structures lacking one or both of the lower arm alpha1,2-linked Man residues. The former are elongated to mannan, whereas the latter are poor substrates for outerchain initiation by Ochlp (Nakayama, K.-I., Nakanishi-Shindo, Y., Tanaka, A., Haga-Toda, Y., and Jigami, Y. (1997) FEBS Lett. 412, 547-550) and accumulate largely as truncated biosynthetic end products. The ALG11 gene is predicted to encode a 63.1-kDa membrane protein that by indirect immunofluorescence resides in the ER. The Alg11 protein is highly conserved, with homologs in fission yeast, worms, flies, and plants. In addition to these Alg11-related proteins, Alg11p is also similar to Alg2p, a protein that regulates the addition of the third mannose to the core oligosaccharide. All of these Alg11-related proteins share a 23-amino acid sequence that is found in over 60 proteins from bacteria to man whose function is in sugar metabolism, implicating this sequence as a potential sugar nucleotide binding motif.     

A new yeast mutation in the glucosylation steps of the asparagine-linked glycosylation pathway. Formation of a novel asparagine-linked oligosaccharide containing two glucose residues

We have isolated and characterized a new yeast mutation in the glucosylation steps of lipid-linked oligosaccharide biosynthesis, alg8-1. Cells carrying the alg8-1 mutation accumulate Glc1Man9GlcNAc2-lipid both in vivo and in vitro. We present evidence showing that the alg8-1 mutation blocks addition of the second alpha 1,3-linked glucose. alg8-1 cells transfer Glc1Man9GlcNAc2 to protein instead of the wild type oligosaccharide, Glc3Man9GlcNAc2. Pulse-chase studies indicate that the Glc1Man9GlcNAc2 transferred is processed more slowly than the wild type oligosaccharide. The yeast mutation gls1-1 lacks glucosidase I activity (Esmon, B., Esmon, P.C., and Schekman, R. (1984) J. Biol. Chem. 259, 10322-10327), the enzyme responsible for removing the alpha 1,2-linked glucose residues from protein-linked oligosaccharides. We demonstrate that gls1-1 cells contain glucosidase II activity (which removes alpha 1,3-linked glucose residues) and have constructed the alg8-1 gls1-1 haploid double mutant. The Glc1Man9GlcNAc2 oligosaccharide was trimmed normally in these cells, demonstrating that the alg8-1 oligosaccharide contained an alpha 1,3-linked glucose residue. A novel Glc2 compound was probably produced by the action of the biosynthetic enzyme that normally adds the alpha 1,2-linked glucose to lipid-linked Glc2Man9GlcNAc2. This enzyme may be able to slowly add alpha 1,2-linked glucose residue to protein-bound Glc1Man9GlcNAc2. The relevance of these findings to similar observations in other systems where glucose residues are added to asparagine-linked oligosaccharides and the possible significance of the reduced rate of oligosaccharide trimming in the alg mutants are discussed.