PURIFICATION TO HOMOGENEITY OF UDP-GLUCOSE-GLYCOPROTEIN GLUCOSYLTRANSFERASE FROM SCHIZOSACCHAROMYCES-POMBE AND APPARENT ABSENCE OF THE ENZYME FROM SACCHAROMYCES-CEREVISIAE

The UDP-Glc:glycoprotein glucosyltransferase was purified to homogenei ty from the fission yeast Schizosaccharomyces pombe. The enzyme has be en recently suggested to be involved in the mechanism by which unfolde d, partially folded, or misfolded glycoproteins are retained in the en doplasmic reticulum. The pure yeast glucosyltransferase formed protein -linked Glc(1)Man(9)GlcNAc(2),Glc(1)Man(8)GlcNAc(2), and Glc(1)Man(7)G lcNAc(2) when incubated with UDP-Glc and denatured thyroglobulin. The same compounds were formed upon glucosylation of endogenous accepters by crude microsomes. The enzyme was a soluble microsomal protein that required Ca2+ for activity, used UDP-Glc and not TDP-Glc, ADP-Glc, or UDP-Gal as sugar donor, had an almost neutral optimum pH value, and as the glucosyltransferase obtained from rat liver, glucosylated denatur ed but not native glycoproteins or glycopeptides. A similar enzymatic activity could not be detected in Saccharomyces cerevisiae microsomes and transient glucosylation of glycoproteins (addition of a single glu cose unit to glucose-free oligosaccharides by the glucosyltransferase followed by its removal by glucosidase II) could not be detected in in tact S. cerevisiae cells. These are the only eukaryotic cells describe d so far in which these processing reactions of the endoplasmic reticu lum do not occur. Availability of the pure S. pombe enzyme will eventu ally allow testing the possible involvement of the glucosyltransferase in sensing glycoprotein tertiary structures in the endoplasmic reticu lum.