Department of Biochemistry and Cell Biology and the Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA
Received on March 31, 1999; revised on July 22, 1999; accepted on July 23, 1999.
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Abstract |
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Key words: endoplasmic reticulum/N-glycosylation/de-N-glycosylation/glycosidase inhibitors/quality control
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Introduction |
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In vitro studies in mammalian cells and in S. cerevisiae have shown that small N-glycosylatable tripeptides, following their glycosylation, undergo retro-translocation back out of the ER to the cytosol and then are de-N-glycosylated by PNGase (Römisch and Schekman, 1992; Römisch and Ali, 1997
; Suzuki et al., 1998b
). This finding suggested that these small peptides could serve as potential model substrates for studying the translocation/de-N-glycosylation processes. In the previous studies, several methods were used to detect N-glycosylated and de-N-glycosylated peptides (Römisch and Ali, 1997
; Suzuki et al., 1998b
). For example, concanavalin A (Con A)-resin was used to quantitate the amount of N-glycosylated peptide, and anion-exchange or thin layer chromatography after desalting was used to detect de-N-glycosylated peptide. However, none of these methods are convenient when dealing with a large number of samples. Accordingly, we have developed a simple method to detect N-glycosylated and de-N-glycosylated peptides using paper chromatography and paper electrophoresis, respectively. This method does not require desalting of samples, and is suitable for processing many samples. With these assays we showed a clear-cut difference in the subcellular distribution of N-glycosylated and de-N-glycosylated peptides, which further supports the idea that de-N-glycosylation occurs after the N-glycosylated peptide is exported out of the ER to the cytosol (Römisch and Ali, 1997
; Suzuki et al., 1998a
,b). Further, we have confirmed and extended the observations that ATP is only required for the glycopeptide export step, but not for peptide import and/or N-glycosylation (Römisch and Ali, 1997
). We also found that, in contrast to export of misfolded proteins from the ER to the cytosol which is believed to be affected by oligosaccharide structure (Knop et al., 1996
; Jakob et al., 1998
), the structure of oligosaccharide chain of the glycopeptide does not affect its export. This implies that although the process of degradation of glycopeptides and misfolded glycoproteins may share common features, there may be differences in the export process.
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Results |
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To confirm that the glycosidase inhibitors altered the structures of the oligosaccharides on glycoprotein without affecting the structures of the lipid-linked oligosaccharides, analysis of the oligosaccharides isolated from both lipid- and protein-linked oligosaccharides was carried out. After 30 min incubation of the permeabilized spheroplasts in the presence and absence of glycosidase inhibitors, [3H]-mannose-labeled lipid-linked oligosaccharides and protein-linked oligosaccharides were isolated and analyzed using Bio-Gel P4 column. As shown in Figure 5, it was found that in the presence of glycosidase inhibitors a drastic change occurred in the structure of the protein-linked oligosaccharides. Upon analysis of the protein-linked oligosaccharides released by PNGase F digestion, we found that in DNM-treated cells, two major peaks were observed at a position corresponding to a oligosaccharide that was 3 to 4 glucose units larger than the major oligosaccharides from control (Figure 5A,C). Furthermore, in the presence of DMM the major oligosaccharide was found to be 1 glucose unit larger than the major oligosaccharides from the control cells (Figure 5B,C). These results clearly show that in the presence of the glycosidase inhibitors, major protein-linked oligosaccharide chains was altered due to the inhibition of glycosidase processing. In sharp contrast, when we analyzed the oligosaccharide released from lipid-linked oligosaccharides from each sample, no apparent difference in the size of the oligosaccharide chain was observed on the sizing column. The major product was found to be the putative triglucosylated species (approx. elution position = 420 min) (data not shown).
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Discussion |
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To study the mechanism of glycopeptide export and its relationship to the process utilized for misfolded (glyco)proteins, the establishment of a simple assay method was critical. Therefore, we developed a simple N-glycosylation/de-N-glycosylation assay using 3H-labeled glycosylatable tripeptide and two simple analytical methods. Using this assay we found that de-N-glycosylated peptide was found exclusively in the cytosol, supporting the idea that de-N-glycosylation occurs after the export of glycopeptide takes place. In addition, this assay has provided some further insight into the four steps: peptide import, N-glycosylation, export, and de-N-glycosylation, in that it was shown that ATP is not required for the first two steps, peptide import and N-glycosylation in the ER. Moreover, since yeast soluble PNGase does not require the presence of ATP for activity (Suzuki and Lennarz, unpublished observations), it is unlikely to be involved in the fourth step, de-N-glycosylation. Therefore, we conclude that the ATP requirement is localized to the translocation event per se.
It is believed that export of misfolded carboxypeptidase Y in yeast is glycan structure-dependent (but calnexin-independent) because the half-life of the protein was substantially prolonged when glucosidase or mannosidase processing was blocked (Knop et al., 1996; Jakob et al., 1998
). Assuming similarity in the export of misfolded (glyco)proteins with that of small glycopeptides from the ER into the cytosol, we expected that the structure of the glycan chains would affect glycopeptide export. By analyzing the size of lipid- and protein-linked oligosaccharides in the presence and absence of glycosidase inhibitors, we confirmed that the inhibitors are effective in this in vitro system, but their effect is limited to glycans linked to peptides and proteins; no effect was observed on synthesis of lipid-linked oligosaccharides or on peptide import (N-glycosylation). However, unexpectedly the modifications of the oligosaccharide chains by mannosidase or glucosidase inhibitors had no significant effect on export of the glycopeptide. We also obtained a similar result using mutants which are deficient in glucosidase I (gls1) or ER mannosidase (
mns1) (Suzuki and Lennarz, unpublished results), further confirming the lack of effect of glycan chain structure on glycopeptide export. These findings suggest that in yeast, even though retrograde export of glycopeptide and misfolded glycoprotein share a number of features in common (requirement of ATP, Sec61p-dependence), there is a clear difference in some aspects of the process with these two classes of molecules. Although both the glycan and the polypeptide chain are recognition elements for retrograde export of misfolded proteins this is not the case for glycopeptides. Clearly, a next step will be to define precisely how glycopeptides are targeted for export out of the ER.
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Materials and methods |
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Yeast strains and culture conditions
The yeast strain used in this study was W3031a (MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100). Cells were grown at 25°C in 1% Bacto-yeast extract, 2% Bacto-peptone, 2% dextrose, and 0.004% adenine sulfate (YPAD). Unless noted, 10 ml of cells were grown with shaking in a 50 ml centrifuge tube.
Preparation of permeabilized cells
Preparation of permeabilized spheroplasts was carried out as described (Baker et al., 1988; Suzuki et al., 1998b
). The cells prepared were resuspended to a A600 of 150/ml in B88 buffer (20 mM HEPES-KOH buffer (pH 6.8), 150 mM potassium acetate, 5 mM magnesium acetate, and 250 mM sorbitol) with 10 mM dithiothreitol. Aliquots (200 µl) of the cell suspension were transferred to an Eppendorf tube, quickly frozen under liquid N2, and stored at 80°C until use.
N-Glycosylation/de-N-glycosylation assay
A tripeptide substrate for N-glycosylation/de-N-glycosylation assay, [3H]acetyl-Asn-Bpa-Thr-amide (10.6 µCi/nmol), where Bpa represents p-benzoylphenylalanine, was kindly provided by Ms. Qi Yan, SUNY at Stony Brook, and prepared as described previously (Yan et al., 1999). To 200 µl of the permeabilized cell suspension, the following compounds were added from 100-fold concentrated stock solutions except for creatine phosphate, which was a 25-fold stock: ATP (final concentration, 1 mM), GDP-Man (50 µM), creatine phosphate (40 mM), and creatine phosphate kinase (concentrated solution was made fresh; final concentration, 200 µg/ml). Reactions were started by adding 2 x 106 d.p.m. (87 pmol) of [3H]acetyl-Asn-Bpa-Thr-amide, and incubated on a rocker at room temperature with gentle shaking. Reactions were stopped by putting the reaction mixture on ice, followed by the immediate addition of the following compounds from 100-fold stock solutions: ATP-
S (final concentration, 1 mM), N-methylmaleimide (2 mM), and EDTA (5 mM). Unless specified, samples were incubated for 20 min for the N-glycosylation assay and for 90 min for the de-N-glycosylation assay.
Analytical methods
The N-glycosylation reaction was measured using ascending paper chromatography carried out essentially as described (Suzuki et al., 1998b). Briefly, 1020 µl samples were spotted 1 cm from the bottom of the paper in a band of 1.3 cm. A 0.7 cm margin was left between samples. Elution was with 1-butanol/acetic acid/water (2/1/1; v/v/v) at room temperature. For general assays, the paper was developed until the solvent front migrated up to 8 cm, dried, and then cut into 1 cm segments from the origin to the solvent front. The radioactivity in each segment was quantitated by liquid scintillation counter. Initially, for comparison, N-glycosylated peptide was also quantitated by binding to Con A-agarose as described previously (Roos et al., 1994
; Suzuki et al., 1998b
).
De-N-glycosylated peptide was quantitated using paper electrophoresis (model EF-200, Advantec Toyo Kaisha Ltd., Tokyo, Japan). A second aliquot of the samples (1020 µl) was spotted onto Whatman 3MM paper 7 cm from anode in a band 1 cm in width with a 1 cm margin between samples. Electrophoresis was carried out for 3 h with 10 mA constant current in pyridine/acetic acid/water = 5/0.2/95, pH 6.5. After air drying, the pattern was analyzed by cutting 1.0 cm segments from the origin for quantitation or 0.5 cm segment from the origin for more detailed analysis of the electrophoretic pattern. Each segment was quantitated using a liquid scintillation counter.
For comparison, several reference samples were prepared: Purified N-glycosylated peptide ([3H]acetyl-Asn(CHO)-Bpa-Thr-amide), was prepared as described previously (Suzuki et al., 1998b). The reaction product resulting from incubation of the N-glycopeptide with yeast lysate was prepared by incubation of 10,000 d.p.m. purified glycopeptide with 10 µl of yeast lysate (100 µg of total protein) and 10 µl of 200 mM HEPES-NaOH buffer (pH 7.0) containing 5 mM dithiothreitol at room temperature for 16 h. The method for preparation of the yeast lysate was reported previously (Suzuki et al., 1998b
). Authentic PNGase F-deglycosylated peptide ([3H]acetyl-Asp-Bpa-Thr-amide) was prepared by digestion of 100,000 d.p.m. N-glycosylated peptide with 5 units of PNGase F in 50 µl of 50 mM TrisHCl buffer (pH 8.0) at 37°C for 2 h. Endo H-deglycosylated peptide ([3H]acetyl-Asn(GlcNAc)-Bpa-Thr-amide) was prepared by digestion of glycopeptide (20,000 d.p.m.) with 5 mU of Endo H in 20 µl of 50 mM Mes-NaOH buffer (pH 6.0) at 37°C for 2 h.
The activities of N-glycosylation (or de-N-glycosylation) were expressed as the percent of the cleaved substrate; this was calculated as the amount of labeled N-glycosylated (or de-N-glycosylated) peptide formed divided by total radioactivity recovered from the entire chromatographic lane.
Subcellular fractionation of the reaction products
When required, after the N-glycosylation/de-N-glycosylation reaction, the samples were separated into MSP (mid-speed pellet) fraction, MSS (mid-speed supernatant) fraction, HSP (high-speed pellet) fraction, and cytosol fraction. MSP and HSP were obtained essentially as described before (Rexach and Schekman, 1991). This MSP fraction consists of the nucleus and the ER, and the HSP contains other vesicles such as the Golgi. For 40 µl of samples, MSP were obtained with brief (~10 sec) centrifugation in an Eppendorf microfuge, and the pellet was once washed by 40 µl of B88 buffer, and resuspended with 20 µl of B88 buffer containing 1% of a detergent, NP-40, prior to paper electrophoresis/paper chromatography analysis. To obtain the HSP fraction, the MSS fraction was centrifuged at 48,000 r.p.m. in a TLA 100.3 rotor (Beckman Instruments Inc., Fullerton, CA) for 15 min at 4°C. The pellet was washed with 20 µl of B88 buffer, and resuspended with 20 µl of the same buffer containing 1% of NP-40.
Effect of ATP and glycosidase inhibitors on N-glycosylation and/or glycopeptide transport from the ER to the cytosol
To examine the effect of glycosidase inhibitors on glycopeptide transport from the ER, N-glycosylation reaction was carried out in the presence of 500 µM of 1-deoxynojirimycin or 1-deoxymannojirimycin, which was added from the 100-fold stock solution. Samples were preincubated for 10 min prior to the addition of the 3H-tripeptide. After 5, 10, 20, and 30 min incubation, 40 µl were taken from 200 µl samples, inactivated, and fractionated into the ER (MSP) and the cytosol (MSS) fractions as described above. The amount of N-glycosylated peptide in each fraction was quantitated as described above. In the case of glycosidase inhibitors, formation of de-N-glycosylated peptide was also examined, and deglycosylated peptide on the cytosol (MSS) fraction was quantitated at the time of 60 min and 120 min incubation.
For examining the effect of ATP on peptide import and N-glycosylation, assays were carried out with ATP or without both ATP and an ATP-regenerating system (creatine phosphate and creatine phosphate kinase), a condition that is known to impair the export of glycopeptide from the ER to the cytosol. In the condition without ATP, 2-deoxyglucose (final concentration; 10 mM) and antimycin A (50 µM) was added from 100-fold and 20-fold stock solution, respectively, to deplete endogenous ATP.
Analysis of lipid- and protein-linked oligosaccharides synthesized by permeabilized spheroplasts in the presence and absence of glycosidase inhibitors
To examine the effect of glycosidase inhibitors on the structure of both lipid- and protein-linked oligosaccharides, we added 100 µCi of [3H]-mannose (30 Ci/mmol) in place of UDP-mannose to 100 µl of permeabilized spheroplasts to label oligosaccharides. Unless otherwise noted, the conditions of incubation with glycosidase inhibitors were same as described above. After 30 min incubation, the reaction was stopped by adding 5 volumes of chloroform/methanol (3/2, v/v), and protein and dolichylpyrophosphoryl oligosaccharide fractions were isolated according to the method described earlier (Zufferey et al., 1995). Dolichol-linked oligosaccharide fraction was resuspended with 100 µl of 80% tetrahydrofuran/0.1 N HCl and oligosaccharides were released by heating at 65°C for 30 min. After neutralizing the solution by adding 1 N NaOH, the solution was evaporated to dryness, resuspended with 10 mM TrisHCl (pH 8.0), and was applied for Bio-Gel P4 column (1.25 x 100 cm). Elution was carried out with water, and flow rate was 170 µl/min. Fractions were collected every 5 min and radioactivity in each fraction was monitored using liquid scintillation counter. For reference, elution positions of glucose oligomers were also determined. Protein fraction, on the other hand, was resuspended in 100 µl if 50 mM of TrisHCl buffer (pH 8.0), digested with 5 U PNGase F for 37°C for 16 h, and released oligosaccharides were recovered from supernatant after adding 3 volumes of ethanol for precipitation of proteins. The supernatant was evaporated to dryness, resuspended with 10 mM TrisHCl buffer (pH 8.0), and was analyzed by Bio-Gel P4 column.
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Acknowledgments |
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Footnotes |
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