Free Oligosaccharides to Monitor Glycoprotein Endoplasmic Reticulum-associated Degradation in Saccharomyces cerevisiae

Hiroto Hirayama,Junichi Seino,Toshihiko Kitajima,Yoshifumi Jigami,Tadashi Suzuki

doi:10.1074/jbc.m109.082081

Abstract

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.

Highlights

Ferase using, in most cases, dolicholpyrophosphate-linked Glc3Man9GlcNAc2 as a donor substrate [1, 2]
Use of Endo-1,6-␤-glucanase to Establish the Method for Isolation of Yeast free oligosaccharides (fOSs)—Several reports have shown that higher eukaryotic organisms generate fOSs, and extensive studies have revealed that these organisms generate a diverse range of fOS structures, including fOS-GN2 (M6-8GN2) and fOS-GN1 (M2-9GN1 and G1M9GN1), in the cytosol [28, 33,34,35, 40, 51]
Recent studies showed that the M7C form generated by Htm1p was key for the recognition of misfolded glycoprotein substrates for endoplasmic reticulum (ER)-associated degradation (ERAD) [11, 14], raising a question about the source of fOSs in yeast

Summary

EXPERIMENTAL PROCEDURES

Growth Conditions, and Gene Disruption—We used a BL21(DE3) strain of Escherichia coli (Novagen, Madison, WI) to produce His6-tagged endo-1,6-␤-glucanase (His6-Neg). 100 A600 units of yeast cells were washed twice (all centrifugation steps in this protocol were carried out at 4 °C); resuspended in 500 ␮l of fOSs extraction buffer containing mannosidase inhibitors (20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1 mM 1-deoxymannojirimycin (Calbiochem, Darmstadt, Germany), 0.5 mM swainsonine (Calbiochem), and protease inhibitor mixture (Roche Applied Science)); and disrupted with glass beads using Multi-beads Shocker௡ (Yasui Kikai, Osaka, Japan), according to the manufacturer’s protocol. Ten A600 units of cells were harvested, washed twice with water, resuspended in 100 ␮l of TEG buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, and CompleteTM protease inhibitor mixture (Roche Applied Science)), and disrupted with glass beads using a Multi-beads Shocker௡ (Yasui Kikai). Immunoreactive bands were visualized using LAS3000-mini (Fujifilm Co., Tokyo, Japan) with Immobilon Western reagents (Millipore)

RESULTS

A Part of Misfolded Glycoproteins

DISCUSSION