Structure of Saccharomyces cerevisiae alg3, sec18 mutant oligosaccharides.

M F Verostek,P H Atkinson,R B Trimble

doi:10.1016/s0021-9258(19)67629-5

M F Verostek, P H Atkinson + Show 1 more

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https://doi.org/10.1016/s0021-9258(19)67629-5

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Abstract

Asparagine-linked oligosaccharides are synthesized by transfer of Glc3Man9GlcNAc2 from dolichol pyrophosphate to nascent polypeptides. Assembly of the precursor proceeds by highly ordered sequential addition of mannose and glucose to form Glc3Man9GlcNAc2-P-P-dolichol. Yeast mutants in asparagine-linked glycosylation (alg), generated by an 3H-Man suicide technique, were assigned to eight complementation groups which define steps in oligosaccharide-lipid synthesis (Huffaker, T.C., and Robbins, P.W. (1982) J. Biol. Chem. 257, 3203-3210). Alg3 invertase oligosaccharides are resistant to endo-beta-N-acetylglucosaminidase H, and the lipid-oligosaccharide pool yields Man5Glc-NAc2, suggesting its structure may be that from mammalian cells lacking Man-P-dolichol (Chapman, A., et al. (1980) J. Biol. Chem. 255, 4441-4446). To test this supposition, the endoplasmic reticulum form of invertase derepressed in alg3,sec18 yeast at 37 degrees C was isolated as a source of oligosaccharides whose processing beyond glucose and/or mannose trimming, if involved, would be prevented. Man8GlcNAc2 and Man5GlcNAc2 were released by peptide-N-glycosidase F from alg3,sec18 invertase in a 1:5 molar ratio. 1H NMR spectroscopy revealed Man8GlcNAc2 to be the alpha 1,2-mannosidase-trimming product described earlier (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666), while Man5GlcNAc2 was Man alpha 1, 2Man alpha 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc beta 1, 4GlcNAc. This provides a structural proof for the lipid-linked Man5GlcNAc2 originally proposed from enzymatic and chemical analyses of the radiolabeled mammalian precursor. Experimental evidence indicates that, unlike the mammalian cell mutants which are unable to synthesize Man-P-dolichol, alg3 yeast accumulate Man5GlcNAc2-P-P-dolichol due to a defective alpha 1,3-mannosyltransferase required for the next step in oligosaccharide-lipid elongation.

Highlights

Yeast mutants iansparagine-linkedglycosylationwere generated by a 3H-Man suicide technique [5]
SDS-PAGEanalysis of invertase derepressed in alg3,seclS yeas3t 7a“t C was invertase synthesized inalg3or alg3,sec18-1 cells at 37 “Chas isolated as a source of oligosaccharides whose process- shown that the majority of attendant oligosaccharides are ing beyond glucose and/or mannose trimming, if in- resistant to hydrolysis by Endo H [6]
NMR spectroscopy revealed MansGlcNAcz to be the quent to any glucose and/or mannose trimming, but prior to al,2-mannosidase-trimmingproduct described earlier outerchain elongationby Golgi mannosyltransferases [2]. 01

Summary

RESULTS

Al,3-mannosyltransferase required for the step The one-dimensional NMR spectra for MansGlcNAcs and in oligosaccharide-lipid elongation. The asparagine-linked glycosylation pathway is similar in for C1-, C2-, and C3-H protons and integration of selected yeast and animalcells up to the first stagoefs glycan processing [1,2]. This observation has allowed yeast t o be utilized as.

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DISCUSSION

Methods