Free oligosaccharides to monitor glycoprotein endoplasmic reticulum-associated degradation in Saccharomyces cerevisiae.
In eukaryotic cells, N-glycosylation has been recognized as one of the most common and functionally important co- or post-translational modifications of proteins. "Free" forms of N-glycans accumulate in the cytosol of mammalian cells, but the precise mechanism for their formation and degradation remains unknown. Here, we report a method for the isolation of yeast free oligosaccharides (fOSs) using endo-beta-1,6-glucanase digestion. fOSs were undetectable in cells lacking PNG1, coding the cytoplasmic peptide:N-glycanase gene, suggesting that almost all fOSs were formed from misfolded glycoproteins by Png1p. Structural studies revealed that the most abundant fOS was M8B, which is not recognized well by the endoplasmic reticulum-associated degradation (ERAD)-related lectin, Yos9p. In addition, we provide evidence that some of the ERAD substrates reached the Golgi apparatus prior to retrotranslocation to the cytosol. N-Glycan structures on misfolded glycoproteins in cells lacking the cytosol/vacuole alpha-mannosidase, Ams1p, was still quite diverse, indicating that processing of N-glycans on misfolded glycoproteins was more complex than currently envisaged. Under ER stress, an increase in fOSs was observed, whereas levels of M7C, a key glycan structure recognized by Yos9p, were unchanged. Our method can thus provide valuable information on the molecular mechanism of glycoprotein ERAD in Saccharomyces cerevisiae.
Glycometabolome Team, Systems Glycobiology Research Group, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan.
Hiroto
H
Hirayama
Junichi
J
Seino
Toshihiko
T
Kitajima
Yoshifumi
Y
Jigami
Tadashi
T
Suzuki
D001483:Base Sequence; D002240:Carbohydrate Sequence; D002352:Carrier Proteins / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D017353:Gene Deletion; D005800:Genes, Fungal; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D008954:Models, Biological; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013312:Stress, Physiological; D043323:alpha-Mannosidase / Q000378:metabolism
D002352:Carrier Proteins; D017931:DNA Primers; D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C495027:Yos9 protein, S cerevisiae; D006026:Glycoside Hydrolases; D043323:alpha-Mannosidase; C020105:endo-1,6-beta-glucanase; C468820:PNG1 protein, S cerevisiae; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase