Free oligosaccharides to monitor glycoprotein endoplasmic reticulum-associated degradation in Saccharomyces cerevisiae

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.

FIGURE 1.

Two generation and processing pathways for fOSs in mammalian cells. A, PNGase-dependent fOS generation and processing pathway. B, fOSs derived from Dol-PP-OS-processing pathways. These fOSs are further processed by cytosol-localized hydorolases, endo-β-N-acetylglucosaminidase, and α-mannosidase. P and MP, protein and misfolded protein, respectively. Regions circled by a dotted line indicate processing reactions occurring in the cytosol.

FIGURE 2.

Endo-β-1,6-glucanase treatment of PA-oligosaccharides prepared from wild-type cells. A, size fractionation HPLC profile of glycosidase-treated PA-oligosaccharides isolated from yeast cytosol. Profiles of non-treated control oligosaccharides (top), oligosaccharides treated with endo-β-1,6-glucanase (middle), and oligosaccharides treated with both endo-β-1,6-glucanase and JB mannosidase (bottom) are shown. B, magnification of the middle and lower charts in A. Peaks a–f indicate N-glycan-derived fOSs. The arrowheads indicate the elution position of PA-isomaltooligosaccharides (glucose oligomer) for the elution standard.

FIGURE 3.

The deletion of PNG1 causes complete reduction of fOSs generation. Size fractionation HPLC profile of fOSs derived from wild-type cells (upper chart) and png1Δ cells (lower chart) that were digested with endo-1,6-β-glucanase as described in the legend to Fig. 2. *, nonspecific peak, which is resistant to JB mannosidase treatment.

FIGURE 4.

Several fOSs are further modified by Golgi-resident mannosyltransferase, Och1p. A, the structure of the N-linked glycan chain in S. cerevisiae. The mannose residue added by Och1p is marked by an asterisk. B–E, unassigned peaks (c3, d2, e1, and f1) were digested with α-1,2-mannosidase alone or with both α-1,2-mannosidase and α-1,6-mannosidase. M5–M9 in the size fractionation HPLC charts represent elution positions of authentic standards of M5GN2-PA-M9GN2-PA. The elution positions of the authentic standards (M8H, M8I, and M8J) are indicated by closed arrowheads (C and D, lower chart).

FIGURE 5.

Ams1p is involved in fOSs processing in the cytosol. A, expression of Ams1p was significantly elevated at stationary phase. Wild-type (BY4741) and YME2028 (AMS1::AMS1-HA strain) cells were grown to mid-log or stationary phase. Equal cell numbers were lysed, and total protein extracts were analyzed by immunoblotting using an anti-HA antibody. The immunoblot was subsequently probed with anti-Pgk1p antibody as a protein loading control. B, size fractionation HPLC profile of fOSs derived from wild-type, ams1Δ, and atg19Δ cells. Hex3–Hex12, Hex₃HexNAc₂–Hex₁₂HexNAc₂, respectively. The mass was determined by MALDI-TOF MS analysis. *, nonspecific peak, which is resistant to JB mannosidase treatment. C, quantitation of PA-labeled fOSs by size fractionation HPLC. Each peak was quantitated with PA-Glc₆, using a PA-glucose oligomer (TaKaRa; 2 pmol/μl) as a reference. Error bars, mean ± S.D. from three independent experiments.

FIGURE 6.

fOSs structures in ams1Δ cells reflect N-glycan structures on misfolded glycoproteins. A, scheme of ER stress induction. ams1Δ cells were inoculated and grown to mid-log phase at 30 °C. Then 2 mm DTT or 2 μg/ml Tm was added to the medium as described previously (86), and cells were further incubated at 30 °C for 90 min. Samples were then collected, and fOSs were analyzed. B, reverse transcription-PCR analysis of HAC1 mRNA splicing. HAC1^u and HAC1ⁱ are uninduced and induced HAC1 by the UPR, respectively. C, size fractionation HPLC profile of fOSs derived from ams1Δ cells treated with or without UPR-induced reagents. D, quantitation of fOSs observed in C. Hex7–Hex12, Hex₇HexNAc₂–Hex₁₂HexNAc₂, respectively. Error bars, mean ± S.D. from three independent experiments. E, quantitation of fOSs separated by reversed phase HPLC. Error bars, mean ± S.D. from three independent experiments.

FIGURE 7.

Predicted scheme for overall N-glycan processing on misfolded glycoproteins subjected to ERAD. A, a current model for N-glycan processing in glycoprotein ERAD (14). N-Glycans on misfolded glycoproteins are further trimmed by glucosidases (Gls1p and Gls2p) and mannosidases (Mns1p and Htm1p), which can generate M7C glycan. Yos9p, a lectin-like ERAD component, recognizes the α-1,6-terminal mannose residue of M7C. Several misfolded glycoproteins may traffic between ER and Golgi before their degradation, as depicted by downward arrows. B, hypothetical scheme of N-glycan processing on misfolded glycoproteins, based on our fOS analysis. The numbers (pmol/10 mg protein) on N-glycans are based on fOSs analysis in ams1Δ cells. C, when UPR is induced by DTT, yeast cells facilitate the generation of M8B glycans on misfolded proteins and transport misfolded glycoproteins to the Golgi. Thick arrows indicate pathways up-regulated by UPR.