Localization of alpha 1----3-linked mannoses in the N-linked oligosaccharides of Saccharomyces cerevisiae mnn mutants

Neutral and phosphorylated N-linked oligosaccharides were isolated from Saccharomyces cerevisiae mnn9 and mnn9 gls1 mutant mannoproteins and separated into homologues that differed in the number of terminal alpha 1----3-linked mannoses. In each type of oligosaccharide, the addition of such mannose was shown to occur in an ordered rather than a random fashion. The results confirm and extend an earlier report that dealt with the N-linked oligosaccharides from yeast invertase [Trimble, R.B., & Atkinson, P.H. (1986) J. Biol. Chem. 261, 9815-9824], and they suggest that the postulated processing pathway can be generalized to include phosphorylated and glucose-containing N-linked oligomannosides. We conclude that this processing pathway is identical for the analogous oligosaccharides from the mnn9 and wild-type strains of S. cerevisiae. Analysis of the mnn2 mnn10 mannoprotein revealed that a similar modification occurred at the branched terminus of the outer chain as well as in the core in this mutant.     

Effect of oligosaccharides and chloride on the oligomeric structures of external, internal, and deglycosylated invertase

External invertase exists in an oligomeric equilibrium of dimer, tetramer, hexamer, and octamer, the concentrations of which vary with pH, time, and concentration of enzyme [Chu, F.K., Watorek, W., & Maley, F. (1983) Arch. Biochem. Biophys. 223, 543-555; Tammi, M., Ballou, L., Taylor, A., & Ballou, C.E. (1987) J. Biol. Chem. 262, 4395-4401]. To assess the influence of carbohydrate on this equilibrium, we investigated the self-association of external invertase (10 oligosaccharides per subunit), deglycosylated external invertase (2 oligosaccharides per subunit), and internal invertase (no carbohydrate) under various conditions. In addition, the effect of carbohydrate on the interaction of the subunits of these various invertases to form heterooligomers was studied. Chloride ion was found to promote subunit association in the various invertases irrespective of their glycosylation status. However, external invertase was less responsive to chloride ion relative to the internal and deglycosylated invertases. The higher oligomers of deglycosylated invertase were stable at 47 degrees C whereas those of external invertase dissociated rapidly into dimers, suggesting that the additional oligosaccharides in external invertase destabilize subunit interaction. Hybridization experiments with the various invertases showed that the dimers of internal invertase formed heterooligomers with either external or deglycosylated invertase. By contrast, the monomers of external and internal invertases formed their respective homodimers, but not heterodimers. These results suggest that the oligosaccharide content of invertase not only influences the extent of self-association but also affects heterooligomer formation.     

Structure of the phosphorylated N-linked oligosaccharides from the mnn9 and mnn10 mutants of Saccharomyces cerevisiae

The N-linked oligosaccharides, from Saccharomyces cerevisiae mnn1 mnn9 mutant mannoprotein extracted from the cells in hot citrate buffer, were separated by ion exchange into a monophosphate diester, a monophosphate monoester, a diphosphate diester, and a diphosphate monoester diester. The structures of the major components with diesterified phosphate were assigned as follows (where M = mannose), according to a recently revised oligosaccharide structure for the mnn mutants (Hernandez, L. M., Ballou, L., Alvarado, E., Gillece-Castro, B. L., Burlingame, A. L., and Ballou, C. E. (1989) J. Biol. Chem. 264, 11849-11856). formula; see text The monoester derivatives were mixtures of the possible isomers produced by removal of one or the other phosphoglycosyl-linked mannose units, and they were shown to arise by chemical degradation during isolation. The mnn1 mnn2 mnn10 acidic oligosaccharide fraction contained a mono- and a diphosphate ester. The monophosphate consisted predominantly of a single isomer with a mannosyl phosphate unit located at the end of the outer chain in an oligosaccharide with the following structure, where x may range from 2 to 12. The diphosphate had a mannosyl phosphate in this formula; see text position as well as one on the terminal alpha 1----6-linked mannose in the core. The presence in the mnn1 mnn9 or mnn1 mnn2 mnn10 background of the mnn4 or mnn6 mutations, which are known to regulate phosphorylation in yeast, reduced phosphorylation by 90% but did not eliminate it. AI-12522     

Diverse properties of external and internal forms of yeast invertase derived from the same gene

It has been shown by genetic analysis that the external and internal invertases from Saccharomyces cerevisiae share a common structural gene [Taussig, R., & Carlson, M. (1983) Nucleic Acids Res. 11, 1943-1954]. However, the only amino acid composition of these two forms of invertase reported to date has revealed extensive differences [Gascon, S., Neumann, N.P., & Lampen, J.O. (1968) J. Biol. Chem. 243, 1573-1577]. We have found from amino acid analyses of both enzymes and sodium dodecyl sulfate-polyacrylamide gel analysis of their cyanogen bromide peptides that they are most likely identical in their amino acid sequence. However, the invertases exhibit dramatically different physical properties, particularly in their stability. The most striking difference was in their renaturation following guanidine treatment where it was shown that inactivated external invertase could be renatured completely. Endo-beta-N-acetylglucosaminidase H treated external invertase was restored to 40% of its original activity while internal invertase remained completely inactive. The observed differences may be attributed to the presence and absence of the oligosaccharide moiety in the external and internal invertases, respectively.     

Requirements of cleavage of high mannose oligosaccharides in glycoproteins by peptide N-glycosidase F

Peptide N-glycosidase from Flavobacterium meningosepticum cleaves complex as well as neutral glycoproteins (Plummer, T.H., Jr., Elder, J.H., Alexander, S., Phelan, A.W., and Tarentino, A.L. (1984) J. Biol. Chem. 259, 10700-10704). Examples of neutral glycoprotein substrates include ribonuclease B (one high mannose oligosaccharide chain) and yeast external invertase (nine chains/invertase subunit). The rate of deglycosylation by the glycosidase was greatly enhanced if the glycoprotein substrate was denatured prior to enzyme treatment, from a low of 11-fold for external invertase to a high of 844-fold for ribonuclease B. Peptide N-glycosidase F was unable to cleave the asparaginyl-N-acetylglucosamine bond in endo-beta-N-acetylglucosaminidase H-modified external invertase or ribonuclease B, although that in similarly modified glycopeptide substrate was cleaved. Ribonuclease B was digested sequentially with various exoglycosidases to produce an oligosaccharide chain of varied length. Using the resulting forms of ribonuclease B as substrates for peptide N-glycosidase F, the minimum oligosaccharide chain for cleavage was the di-N-acetyl-chitobiosyl core unit.     

Protein glycosylation defects in the Saccharomyces cerevisiae mnn7 mutant class. Support for the stop signal proposed for regulation of outer chain elongation

Total cell mannoprotein was isolated from Saccharomyces cerevisiae X2180 mutants that have defects in elongation of the outer chain attached to the N-linked core oligosaccharides (mnn7, mnn8, mnn9, and mnn10) (Ballou, L., Cohen, R. E., and Ballou, C. E. (1980) J. Biol. Chem. 255, 5986-5991). Comparison of the oligosaccharides released by endoglucosaminidase H digestion confirmed that the mnn9 mutation eliminates all but two mannoses of the outer chain, whereas the mnn8 and mnn10 strains produce outer chains of variable but similar lengths. The isolate designated mnn7 was found to be allelic with mnn8. Haploid mutants of the type mnn8 mnn9 or mnn9 mnn10 had the mnn9 phenotype, which established that the mnn9 defect is dominant and presumably acts at a processing step prior to the steps affected by mnn8 and mnn10. Analysis of the mnn1 mnn2 mnn10 oligosaccharides revealed that the heterogeneous outer chain contained 6-16 alpha 1----6-linked mannose units and each was terminated by a single alpha 1----2-linked mannose unit, whereas the core lacked one such unit that was present in the mnn9 oligosaccharide. The results are consistent with and support the hypothesis (Gopal, P. K., and Ballou, C. E. (1988) Proc. Natl. Acad. Sci. U.S.A. 84, 8824-8828) that addition of such a side-chain mannose unit is associated with termination of outer chain elongation in these mutants and may serve as a stop signal that regulates outer chain synthesis in the parent wild-type strain.     

Carbohydrate structure of yeast invertase. Demonstration of a form with only core oligosaccharides and a form with completed polysaccharide chains.

Invertase, extracted from broken cells of Saccharomyces cerevisiae X-2180 mm2 mannan mutant, was separated into a fraction insoluble in 75% ammonium sulfate (P75 invertase, 36% carbohydrate) and a soluble fraction (S75 invertase, 53% carbohydrate). The latter reacted with antibodies specific for the alpha 1 leads to 6-linked mannose of the mannoprotein outer chain, whereas the P75 invertase failed to react with this antiserum although it did react with serum against terminal alpha 1 leads to 3-linked mannose units that are characteristic of the mannoprotein core. A bacterial endo alpha 1 leads to 6-mannanase removed the outer chains from the S75 invertase and converted it to a form that was similar in electrophoretic and immunochemical properties to the P75 invertase, whereas the endomannanase had little effect on the latter invertase. The results suggest that the P75 invertase is a form of the enzyme to which only the core oligosaccharide units had been added, and the S75 invertase represents an enzyme fraction to which the polysaccharide outer chains were also attached. A strong anomeric PMR signal for unsubstituted alpha 1 leads to 6-linked mannose in the S75 invertase, and a much reduced signal in the P75 invertase and endomannanase-digested S75 invertase, support these conclusions. Endo-N-acetyl-beta-glucosaminidase digestion of the S75 and P75 invertases, as well as of a purified wild type yeast invertase, produced an apparently identical series of 3 to 4 carbohydrate-containing proteins that were separable by polyacrylamide gel electrophoresis in sodium dodecyl sulfate but that migrated as a single band on isoelectric focusing. The bands ranged from about 63,000 to 69,000 daltons and differed by the size of one or more carbohydrate core units each of 15 mannoses and 1 N-acetylglucosamine. The results suggest that the external invertase molecules contain some core units without attached outer chains, and that the cells contain a precursor form of the enzyme to which only the core units have been added. In support of this conclusion, PMR spectra and chromatographic patterns show that the core fragments from the P75, S75, and wild type invertases are essentially identical.     

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.     

Effects of mannoprotein mutations on Saccharomyces cerevisiae core oligosaccharide structure

By the combined actions of an endo-alpha-1 leads to 6-mannanase and an endo-beta-N-acetylglucosaminidase, the core oligosaccharides can be released from Saccharomyces cerevisiae X2180 mnn2 mannoproteins. The effects of various mannoprotein mutations were evaluated by structural comparison of these core oligosaccharides with those prepared from double mutant strains with the genotypes mnn1 mnn2, mnn2 mnn3, mnn2 mnn4, and mnn2 mnn5. The results indicate that only the mnn1 lesion has a major effect on the mannoprotein core structure. Whereas the mnn2 mannoprotein yields a core composed of 6 fragments that differ in size from each other by single mannose units, only the two smallest species predominate in the mnn1 mnn2 preparation. This change is correlated with a loss of terminal alpha 1 leads to 3-mannosyl residues, an effect on the mnn1 lesion that is found also in the polysaccharide outer chain and hydroxyamino acid-linked mannooligosaccharides. The mnn3 and mnn5 mutations also had slight effects on the core size, but clear differences in linkage composition were not apparent. The results suggest that core oligosaccharides have an average composition of Man11GlcNAc, whereas Man9GlcNAc is the major oligosaccharide in strains containing the mnn1 defect. These values are 2 to 3 sugars less than those estimated previously (Nakajima, T., and Ballou, C. E. (1975) Biochem. Biophys. Res. Commun. 66, 870-879). Detailed analysis of the major core oligosaccharide from the mnn1 mnn2 mutant revealed that the two mannoses in alpha 1 leads to 3 linkage to the backbone were adjacent to each other and that the oligosacccharide is nearly identical with one isolated from chinese hamster ovary cell membranes (Li, E., and Kornfeld, S. (1979) J. Biol. Chem. 254, 1600-1605). This finding provides strong evidence for the evolutionary conservation of this structural feature of the high mannose core oligosaccharides.     

Man8GlcNAc2糖基化的酿酒酵母菌株的构建

酿酒酵母糖蛋白的N-糖基化经过高尔基体的修饰后形成聚合度约150-200的甘露寡糖,高尔基体N-糖基化的糖基转移酶Mnn1p和Och1p在甘露寡糖的形成过程中起关键作用。通过同源重组置换敲除了酵母中的MNN1和OCH1基因阻断高尔基体N-糖基化修饰,分离纯化了mnn1 och1突变株中的N-糖蛋白,糖酰胺酶PNGaseF酶解释放的N-糖链经过2-氨基吡啶衍生后,利用HPLC和MALDITOF/MS结合的方法分析了突变株糖蛋白上的N-糖链。结果显示mnn1 och1突变株中的糖蛋白的N-糖链为结构单一的糖链,分子量为1794.66,推测为Man₈GlcNAc₂。     

The mnn2 mutant of Saccharomyces cerevisiae is affected in phosphorylation of N-linked oligosaccharides

We studied the phosphorylation of the inner core region of N-linked oligosaccharides in the mannan defective mutant Saccharomyces cerevisiae mnn2 which was described as unable to synthesize branches on the outer chain. We performed structural studies of the N-oligosaccharides synthesized by the strains mnn2, mnn1mnn2mnn9 and mnn1mnn9ldb8, and the results are compared with previously published structural data of mnn1mnn2mnn10 and mnn1mnn9 [Hernández, L.M., Ballou, L., Alvarado, E., Tsai, P.-K. and Ballou, C.E. (1989) J. Biol. Chem. 264, 13648-13659]. We conclude that the mnn2/ldb8 mutation is responsible for the inhibition of incorporation of phosphate to mannose A(3) (see below), a particular phosphorylation site of the inner core, while phosphorylation at the other possible site (mannose C(1)) is allowed, although it is also reduced. *Phosphorylation sites in mnn1mnn9. (see structure below)     

Expression of Pichia anomala INV1 gene in Saccharomyces cerevisiae results in two different active forms of hypoglycosylated invertase

The Pichia anomala invertase gene (INV1) was introduced at different copy numbers into a sucrose-nonfermenting mutant of Saccharomyces cerevisiae and expressed from its own promoter sequences. The level reached in the production of invertase by the transformants (up to 540 units/10(10) cells) was in agreement with the INV1 gene dosage. Two forms of multimeric active and glycosylated invertase displaying different subcellular locations and molecular masses could be detected in the transformants. One was found to be present in the culture medium and in the periplasm, and the other could only be detected inside the cell. Each of the two heterologous forms of invertase was shown to be an oligomer composed of identical subunits. The difference found in the apparent molecular masses of their monomers (81.5 and 78.3 kDa, respectively) seems to be due to the size of their N-linked oligosaccharide chains (on average 2.4 and 1.9 kDa, respectively), since the number of sugar chains (9) and the molecular mass of the protein moiety (60.5 kDa) are identical in both forms. The shorter size of their oligosaccharides must also be the reason for the lower apparent molecular masses of the heterologous invertases when compared with the enzyme purified from P. anomala. The hypoglycosylated invertase accumulated within the cells of the transformants to an unusual level (up to 130 units/10(10) cells). Such accumulation of active enzyme inside the cells, as well as its underglycosylation, could be due to intrinsic properties of the P. anomala invertase that are determined by the particular primary structure of its protein moiety.     

Hypoglycosylation in the alg12delta yeast mutant destabilizes protease A and causes proteolytic loss of external invertase

The Saccharomyces cerevisiae alg12delta mutant accumulates oligosaccharide lipid with a Man(7)GlcNAc(2) oligosaccharide. To determine the N-glycan structures present on S. cerevisiae glycoproteins in the alg12delta strain, we made attempts to purify external invertase, a highly glycosylated secreted protein. These efforts revealed that, in the alg12delta background, external invertase was mildly hypoglycosylated and rapidly destroyed proteolytically. Although secreted alg9delta invertase was more severely hypoglycosylated than the alg12delta form, it was paradoxically stable during purification. The loss of periplasmic invertase was prevented by addition of pepstatin A to the cell cultures, suggesting that aspartyl proteases were active. We found that during overexpression of invertase in alg12delta yeast, sufficient protease A was mistargeted to the periplasmic space, where it hydrolyzed the invertase. Even though alg9delta invertase is underglycosylated in comparison to the alg12delta form, it is more stable because in this genetic background much less protease A is secreted compared to alg12delta cells. These observations may be relevant to studies using other extracellular proteins (e.g., mating factors, alpha-glucosidase) as probes when characterizing glycosylation defects in yeast.     

Oligosaccharide structures of the major exoglucanase secreted by Saccharomyces cerevisiae.

We have determined the structures of the N-linked carbohydrate chains, released by endo H, of exoglucanase II that are secreted by wild-type Saccharomyces cerevisiae and by the mnn1 mnn9 and mnn1 glycosylation mutants. The mnn9 mutation does not significantly affect N-linked oligosaccharides of exoglucanase II since we found almost identical structures in both mutant strains consisting of a slightly enlarged core with the basic structure shown in A (where M = mannose). Most of the molecules (77%) were phosphorylated on one of the starred mannoses (34%) or on both (43%) with a diesterified (alpha M-->P-->) or monoesterified phosphate group. In addition, some of the molecules apparently escape normal processing and retain the alpha-(1-->2)-linked mannose (italicized) and/or the three glucoses that are characteristic of the lipid-linked precursor (structure B). In the wild type, we found the same basic structure but more [formula; see text] than 90% of the molecules were modified with one to four alpha-(1-->3)-linked mannoses, which were absent in the strains bearing the mnn1 mutation (structure C). The proportion of acidic components was similar to that found in the mutants (78%), although, in this case, the monophosphorylated forms were more abundant (50%) than the diphosphorylated ones (28%). Most of the phosphate groups (69%) were diesterified by a disaccharide (alpha M-->3 alpha M-->P-->) instead of the single mannose found when the mnn1 mutation was present. In both mnn1 and wild type 10-15% of the oligosaccharides had an extra alpha-(1-->6)-linked mannose in the outer chain, a structure described in the recently isolated vrg1 mutant [Ballou, L., Hitzeman, R.A., Lewis, M. S., & Ballou, C. E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3209-3212].     

Invertase Activity in Normal and Mutant Maize Endosperms during Development

A bound invertase and two soluble invertases are found in the developing endosperm of maize (Zea mays L.). The two soluble invertases can be separated on diethylaminoethyl-cellulose and Sephadex columns and distinguished by their kinetic constants. One soluble invertase, invertase I, is present from the 10- to 28-day stages of endosperm development with maximal activity per normal endosperm at the 12-day stage. In two endosperm mutant lines, shrunken-1 and shrunken-2, there is a second increase in invertase I activity later in development which could be a secondary effect caused by the abnormal metabolism in these lines. Another soluble invertase, invertase II, is present in the embryo upon germination and is also found in the very young developing endosperm (6-day stage). The third form of invertase, bound invertase, is present in the endosperm by the 6-day stage, and its activity remains approximately constant during development.     

The stability of yeast invertase is not significantly influenced by glycosylation

Yeast invertase exists in two different forms. The cytoplasmic enzyme is nonglycosylated, whereas the external invertase contains about 50% carbohydrate of the high mannose type. The protein moieties of both enzymes are identical. The two invertases have been used previously as a model system to investigate the influence of covalently linked carbohydrate chains on the stability of large glycoproteins, and controversial results were obtained. Here, we measured thermal and denaturant-induced unfolding by various probes, such as the loss of enzymatic activity, and by the changes in absorbance and fluorescence. The ranges of stability of the two invertases were found to be essentially identical, indicating that the presence of a high amount of carbohydrate does not significantly contribute to the stability of external invertase. Earlier findings that invertase is stabilized by glycosylation could not be confirmed. The stability of this glycoprotein is apparently determined by the specific interactions of the folded polypeptide chain. Unlike the glycosylated form, the carbohydrate-free invertase is prone to aggregation in the denatured state at high temperature and in a partially unfolded form in the presence of intermediate concentrations of guanidinium chloride.     

Biosynthesis of yeast mannan. Isolation of Kluyveromyces lactis mannan mutants and a study of the incorporation of N-acetyl-D-glucosamine into the polysaccharide side chains.

One side chain in the cell wall mannan of the yeast Kluyveromyces lactis has the structure (see article). (Raschke, W. C., and Ballou, C. E. (1972) Biochemistry 11, 3807). This (Man)4GNAc unit (the N-acetyl-D-glucosamine derivative of mannotetroase) and the (Man)4 side chain, aMan(1 yields 3)aMan(1 yields 2)aMan(1 yields 2)Man, are the principle immunochemical determinants on the cell surface. Two classes of mutants were obtained which lack the N-acetyl-D-glucosamine-containing determinant. The mannan of one class, designated mmnl, lacks both the (Man)4GNAc and (Man)4 side chains. Apparently, it has a defective alpha-1 yields 3-mannosyltransferase and the (Man)4 unit must be formed to serve as the acceptor before the alpha-1 yields 2-N-acetyl-glucosamine transferase can act. The other mutant class, mnn2, lacks only the (Man)4GNAc determinant and must be defective in adding N-acetylglucosamine to the mannotetrasose side chains. Two members of this class were obtained, one which still showed a wild type N-acetylglucosamine transferase activity in cell-free extracts and the other lacking it. They are allelic or tightly linked, and were designated mnn2-1 mnn2-2. Protoplast particles from the wild type cells catalyzed a Mn2+-dependent transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the mannotetraose side chain of endogenous acceptors. Exogenous mannotetraose also served as an acceptor in a Mn2+-dependent reaction and yielded (Man)4GNAc. Related oligosaccharides with terminal alpha (1 yields 3)mannosyl units were also good acceptors. The product from the reaction with alphaMan(1 yields 3)Man had the N-acetylglucosamine attached to the mannose unit at the reducing end, which supports the conclusion that the cell-free glycosyltransferase activity is identical with that involved in mannan synthesis. The reaction was inhibited by uridine diphosphate. Protoplast particles from the mmnl mutants showed wild type N-acetylglucosamine transferase activity with exogenous acceptor, but they had no endogenous activity because the endogenous mannan lacked acceptor side chains. Particles from the mnn2-1 mutant failed to catalyze N-acetylglucosamine transfer. In contrast, particles from the mnn2-2 mutant were indistinguishable from wild type cells in their transferase activity. Some event accompanying cell breakage and assay of the mnn2-2 mutant allowed expression of a latent alpha-1 yields 2-N-acetylglucosamine transferase with kinetic properties similar to those of the wild type enzyme.     

Stability, quaternary structure, and folding of internal, external, and core-glycosylated invertase from yeast.

The role of carbohydrate chains for the structure, function, stability, and folding of glycoproteins has been investigated using invertase as a model. The protein is encoded by several different genes, and its carbohydrate moiety is heterogeneous. Both properties complicate physicochemical comparisons. Here we used the temperature-sensitive sec18 secretion mutant of yeast with a single invertase gene (SUC2). This mutant produces the carbohydrate-free internal invertase, the core-glycosylated form, and, at the permissive temperature, the fully glycosylated external enzyme, all with identical protein moieties. The core-glycosylated enzyme resembles the nascent glycoprotein chain that folds in the endoplasmic reticulum. Therefore, it may be considered a model for the in vivo folding of glycoproteins. In addition, because of its uniform glycosylation, it can be used to investigate the state of association of native invertase. Glycosylation is found to stabilize the protein with respect to thermal denaturation and chaotropic solvent components; the stabilizing effect does not differ for the external and the core-glycosylated forms. Unlike the internal enzyme, the glycosylated forms are protected from aggregation. Native internal invertase is a dimer (115 kDa) whereas the core-glycosylated enzyme is a mixture of dimers, tetramers, and octamers. This implies that core-glycosylation is necessary for oligomerization to tetramers and octamers. Dimerization is required and sufficient to generate enzymatic activity; further association does not alter the specific activity of core-glycosylated invertase, suggesting that the active sites of invertase are not affected by the association of the dimeric units. Reconstitution of the glycosylated and nonglycosylated forms of the enzyme after preceding guanidine denaturation depends on protein concentration. The maximum yield (approximately 80%) is obtained at pH 6-8 and protein concentrations < or = 4 micrograms/mL for the nonglycosylated and < or = 40 for the glycosylated forms of the enzyme. The lower stability of the internal enzyme is reflected by a narrower pH range of reactivation and enhanced aggregation. As indicated by the sigmoidal reactivation kinetics at low protein concentration both folding and association are rate-determining.     

A new Saccharomyces cerevisiae mnn mutant N-linked oligosaccharide structure

We find that the N-linked Man8GlcNAc2- core oligosaccharide of Saccharomyces cerevisiae mnn mutant mannoproteins is enlarged by the addition of the outer chain to the alpha 1----3-linked mannose in the side chain that is attached to the beta 1----4-linked mannose rather than by addition to the terminal alpha 1----6-linked mannose. This conclusion is derived from structural studies on a phosphorylated oligosaccharide fraction and from mass spectral fragment analysis of neutral core oligosaccharides.