GLYCOPROTEIN-BIOSYNTHESIS IN THE ALG3 SACCHAROMYCES-CEREVISIAE MUTANT.2. STRUCTURE OF NOVEL MAN6-10GLCNAC2 PROCESSING INTERMEDIATES ON SECRETED INVERTASE

Alg3 yeast mutants synthesize endoglycosidase H-resistant oligosacchar ides whose precursor for elongation is n1alpha-->2Man1alpha-->2Man1alp ha-->3(Man1alpha--> 6) Man1beta-->4GlcNAc2 (Verostek, M. F., Atkinson, P. H., and Trimble, R. B. (1991) J. Biol. Chem. 266, 5547-5551). To c haracterize alg3 glycan elongation in vivo, oligosaccharides on alg3,s ec18 invertase synthesized and secreted at 26-degrees-C were released with peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase and puri fied by Bio-Gel P-4 chromatography. Large (Man>30GlcNAc2) and intermed iate (Man5-10GlcNAc2) sized oligosaccharides were pooled separately, a nd the smaller ones were exchanged with (H2O)-H-2 for one- and two-dim ensional DQF-COSY H-1 NMR analyses at 500 MHz. Although there was no d etectable substitution of the terminal alpha1,6-core-linked mannose, a ddition of alpha1,6-, alpha1,2-, and alpha1,3-mannoses to the alpha1,3 -linked core branch of a majority of the Man5 precursor was analogous to core-filling reactions seen on wild type invertase glycans (Trimble , R. B., and Atkinson, P. H. (1986) J. Biol. Chem. 261, 9815-9824). Tw o additional types of oligosaccharide structures were found; those whi ch retained glucose and those consistent with mannan elongation. Gluco se retention appeared to be due to inefficient trimming from minor glu cosylated intermediates, while mannan elongation was by extension of a new alpha1,6-linked branch from the alpha1,3-core-linked residue as s een in wild-type core oligosaccharides (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) or mnnl,mnn2,mnn10 process ing intermediates (Ballou, L., Alvarado, E., Tsai, P-k., Dell, A., and Ballou, C. E. (1989) J. Biol. Chem. 264, 11857-11864). Thus, the alph a1,6-linked branch additions which form Man9GlcNAc2-PP-dolichol from M an5GlcNAc2-PP-dolichol appear to provide important structural informat ion enabling efficient recognition by the endoplasmic reticulum-glucos yltransferases forming oligosaccharide-lipid as well as the glucosidas es involved in early trimming reactions, but the alg3 mutant documents that they are unnecessary for normal yeast man-nan elongation.