Purification to homogeneity of UDP-glucose:glycoprotein glucosyltransferase from Schizosaccharomyces pombe and apparent absence of the enzyme fro Saccharomyces cerevisiae.
The UDP-Glc:glycoprotein glucosyltransferase was purified to homogeneity from the fission yeast Schizosaccharomyces pombe. The enzyme has been recently suggested to be involved in the mechanism by which unfolded, partially folded, or misfolded glycoproteins are retained in the endoplasmic reticulum. The pure yeast glucosyltransferase formed protein-linked Glc1-Man9GlcNAc2,Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2 when incubated with UDP-Glc and denatured thyroglobulin. The same compounds were formed upon glucosylation of endogenous acceptors 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 glucosyl-transferase obtained from rat liver, glucosylated denatured 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 glucose unit to glucose-free oligosaccharides by the glucosyltransferase followed by its removal by glucosidase II) could not be detected in intact S. cerevisiae cells. These are the only eukaryotic cells described so far in which these processing reactions of the endoplasmic reticulum do not occur. Availability of the pure S. pombe enzyme will eventually allow testing the possible involvement of the glucosyltransferase in sensing glycoprotein tertiary structures in the endoplasmic reticulum.
Fernández
F S
FS
Instituto de Investigaciones Bioquímicas Fundación Campomar, Buenos Aires, Argentina.
Trombetta
S E
SE
Hellman
U
U
Parodi
A J
AJ
D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005964:Glucosyltransferases / Q000032:analysis* / Q000302:isolation & purification* / Q000378:metabolism; D006020:Glycopeptides / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012568:Schizosaccharomyces / Q000201:enzymology*; D013045:Species Specificity
D006020:Glycopeptides; D006023:Glycoproteins; D009844:Oligosaccharides; D005964:Glucosyltransferases; C072423:mannosylglycoprotein 1,3-glucosyltransferase