A misfolded protein conformation is not a sufficient condition for in vivo glucosylation by the UDP-Glc:glycoprotein glucosyltransferase.
A key element in the quality control of glycoprotein folding is the UDP-Glc:glycoprotein glucosyltransferase (GT), which in cell-free assays exclusively glucosylates misfolded glycoproteins. In order to test if such a protein conformation is a sufficient condition for in vivo glucosylation of all N-linked oligosaccharides by GT, a Schizosaccharomyces pombe double mutant (gls2/alg6) was constructed. With this mutant, Man9GlcNAc2 is transferred to proteins and no removal of glucose units added by GT occurs as it lacks glucosidase II. The same proportion of glucosylated (Glc1Man9GlcNAc2) and unglucosylated (Man9GlcNAc2 and Man8GlcNAc2) endoplasmic reticulum (ER)-specific compounds was produced when cells were pre-incubated for 10, 20 or 30 min and further incubated with [14C]glucose for 10 min at 28 degrees C with or without 5 mM dithiothreitol (DTT), thus indicating not only that DTT did not affect protein glucosylation but also that no increased glucosylation of glycoproteins occurred in the presence of the drug. Monitoring Golgi-specific modifications of oligosaccharides after pulse-chase experiments performed in the presence or absence of 5 mM DTT showed that exit of the bulk of glycoproteins synthesized from the ER and thence their proper folding had been prevented by the drug. Cells pulse-chase labeled at 37 degrees C in the absence of DTT also yielded glucosylated and unglucosylated protein-linked oligosaccharides without Golgi-specific modifications. It was concluded that a misfolded protein conformation is not a sufficient condition for in vivo glucosylation of all N-linked oligosaccharides by GT.
Fernández
F
F
Instituto de Investigaciones Bioquímicas Fundación Campomar, Antonio Machado 151, 1405 Buenos Aires, Argentina.
D'Alessio
C
C
Fanchiotti
S
S
Parodi
A J
AJ
D004229:Dithiothreitol / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D018869:Heat-Shock Response; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism
D009844:Oligosaccharides; D005964:Glucosyltransferases; C072423:mannosylglycoprotein 1,3-glucosyltransferase; D005947:Glucose; D004229:Dithiothreitol