GLYCOPROTEIN-SYNTHESIS IN YEAST - EARLY EVENTS IN N-LINKED OLIGOSACCHARIDE PROCESSING IN SCHIZOSACCHAROMYCES-POMBE

Oligosaccharide-lipid precursors and glycoprotein N-linked oligosaccha rides isolated from the fission yeast, Schizosaccharomyces pombe, were compared with those from the budding yeast, Saccharomyces cerevisiae. Bio-Gel P-4 chromatography of oligosaccharide intermediates showed th at Glc(3)Man(9)GlcNAc(2)-PP-dol synthesis, transfer of glycan to prote in, and glucose removal to yield Man(9)GlcNAc(2) proceeded in S. pombe as in S. cerevisiae. Two series of oligosaccharides were released fro m S. pombe glycoproteins by endo-beta-N-acetylglu cosaminidase H; larg e ''mannan-like'' structures and smaller precursor or ''core-filling'' species. Unexpectedly, the smallest S. pombe N-linked glycan was Man( 9)GlcNAc, confirmed by 500 MHz H-1 NMR spectroscopy to be the lipid-li nked isomer. No endoplasmic reticulum Man(9)-alpha 1,2-mannosidase act ivity was detected in S. pombe, thus identifying Man(9)GlcNAc as the m inimum precursor for oligosaccharide elongation in contrast to the Man (8)GlcNA(2), intermediate identified in S. cerevisiae (Byrd, J.C., Tar entino, A.L., Maley, F., Atkinson, P.H., and Trimble, R.B. (1982) J. B iol. Chem. 257, 14657-14666). S. pombe Hex(10)GlcNAc was at least four isomers by high pH anion-exchange chromatography with pulsed amperome tric detection. Compositional analyses identified two of the major spe cies as GalMan(9)GlcNac and GlcMan(9)GlcNAc, the latter of which sugge sts that glycan trimming may be attenuated in the S. pombe endoplasmic reticulum. Hex(13)GlcNAc from S. pombe was homogeneous by mass spectr ometry but yielded 12 species by high pH anion-exchange chromatography . Compositional analyses, alpha-galactosidase digestion, and lectin af finity chromatography on Griffonia simplicifolia lectin I-agarose indi cated these to be a family of Gal(x) Man(13-x)GlcNAc isomers (X = 1-4 residues). The absence of Man(9)GlcNAc(2), to Man(8)GlcNAc(2), trimmin g in S. pombe and elongation of the lipid precursor of Man(9)GlcNAc wi th both Man and Gal to form ''galactomannans'' provides a novel system for N-linked glycoprotein processing studies.