In yeast the export of small glycopeptides from the endoplasmic reticulum into the cytosol is not affected by the structure of their oligosaccharide chains

Tadashi Suzuki and William J. Lennarz1

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.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
A "quality control" system associated with the endoplasmic reticulum (ER) that discriminates between misfolded proteins and correctly folded proteins is present in a variety of eukaryotic cells, including yeast. Recently, it has been shown that misfolded proteins that are N-glycosylated in the lumen of the ER are transported out of the ER, de-N-glycosylated by a soluble peptide:N-glycanase (PNGase) and degraded by action of the proteasome. It also has been shown that small N-glycosylatable peptides follow a fate similar to that of misfolded proteins, i.e., glycosylation in the lumen of the ER, transport out of the ER, and de-N-glycosylation in the cytosol. These processes of retrograde glycopeptide transport and de-N-glycosylation have been observed in mammalian cells, as well as in yeast cells. However, little is known about the mechanism involved in the movement of glycopeptides from the ER to the cytosol. Here we report a simple method for assaying N-glycosylation/de-N-glycosylation by simple paper chromatographic and electrophoretic techniques using an N-glycosylatable 3H-labeled tripeptide as a substrate. With this method, we confirmed the cytosolic localization of the de-N-glycosylated peptide, which supports the idea that de-N-glycosylation occurs after the export of the glycopeptide from the lumen of the ER to the cytosol. Further, we found that the variations in the structure of the oligosaccharide chain on the glycopeptide did not cause differences in the export of the glycopeptide. This finding suggests that the mechanism for the export of small glycopeptides may differ from that of misfolded (glyco)proteins.

Key words: endoplasmic reticulum/N-glycosylation/de-N-glycosylation/glycosidase inhibitors/quality control


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Recent evidence has shown that the endoplasmic reticulum (ER) has a quality control system for the efficient routing of correctly folded proteins out of the ER (Hammond and Helenius, 1995Go). In this system, proteins that fail to fold correctly and/or oligomerize properly are retained in the ER by various lumenal chaperones that assist in their folding and appropriate subunit assembly. However, those proteins that remain misfolded are subsequently degraded by a mechanism that was formerly called "ER degradation" (Klausner and Sitia, 1990Go). With respect to this mechanism, much progress has been made in the past several years, and it is now evident that the major site for the degradation of these misfolded proteins is the cytosol (Brodsky and McCracken, 1997Go; Cresswell and Hughes, 1997Go; Kopito, 1997Go; Ploegh, 1997Go; Sommer and Wolf, 1997Go; Suzuki et al., 1998aGo). This degradation initially involves the translocation of membrane-associated and/or secretory proteins from the ER into the cytosol, after which they are degraded by the proteasome. In mammalian cells, N-glycosylated proteins are known to be de-N-glycosylated by the action of peptide:N-glycanase (PNGase) during this degradation process (Wiertz et al., 1996aGo,b; Halaban et al., 1997Go; Hughes et al., 1997Go; Huppa and Ploegh, 1997Go; Yu et al., 1997Go; Bebok et al., 1998Go; deVirgilio et al., 1998Go; Mosse et al., 1998Go; Yang et al., 1998Go).

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, 1992Go; Römisch and Ali, 1997Go; Suzuki et al., 1998bGo). 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, 1997Go; Suzuki et al., 1998bGo). 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, 1997Go; Suzuki et al., 1998aGo,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, 1997Go). 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., 1996Go; Jakob et al., 1998Go), 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
N-Glycosylation assay using paper chromatography
The presence or absence of oligosaccharide chains on acceptor peptides brings about a marked change in their hydrophobicity, and we have recently shown that this characteristic is useful in identifying PNGase-deglycosylated peptides (Suzuki et al., 1998bGo). For quantitation of N-glycosylated peptide, Con A–agarose or Con A–Sepharose precipitation is widely used (Römisch and Schekman, 1992Go; Roos et al., 1994Go; Römisch and Ali, 1997Go; Suzuki et al., 1998bGo; Yan et al., 1999Go). Paper chromatography is simple, rapid and more practical for analyzing large numbers of samples. Therefore, we quantitatively compared the extent of N-glycosylation of acceptor peptides using the Con A-agarose method (Roos et al., 1994Go) and paper chromatography. As shown in Figure 1, essentially no significant difference in these two quantitation methods is present; therefore we routinely used paper chromatography for quantitation of N-glycosylated peptide. In general, we measure the N-glycosylation after incubation with yeast lysate for 20 min, because at this time point we can not detect any de-N-glycosylation, which causes an underestimate of the extent of N-glycosylation reaction (Römisch and Ali, 1997Go; Suzuki et al., 1998bGo).



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Fig. 1. Comparison of quantitation of N-glycosylated peptide by paper chromatographic and concanavalin A-binding methods. Permeabilized yeast spheroplasts (200 µl) were incubated with 87 pmol (2 x 106 d.p.m.) of 3H-tripeptide. At the indicated time, the MSS fraction was recovered as described in Materials and methods, and N-glycosylated peptide formed was quantitated either by paper chromatography (PC) or Concanavalin A-agarose (Con A). The values shown are the average of four separate assays.

 
Identification of reaction products using paper electrophoresis
Previously we detected de-N-glycosylated peptide by anion exchange chromatography, since the de-N-glycosylation reaction by PNGase results in the introduction of a negative charge in the peptide by conversion of the glycosylated Asn residue into an Asp residue (Suzuki et al., 1998bGo). Another study showed that thin layer chromatography is another useful method for identification of de-N-glycosylated peptide (Römisch and Ali, 1997Go), although both of methods require desalting of samples prior to the analysis. Since paper electrophoresis does not require desalting of samples and the reaction products can be applied directly, we used this method to assay de-N-glycosylation by this method. To validate this method, we incubated N-glycosylated peptide with yeast lysate. Then we compared the elution position of the product formed (which has shown to be derived by a PNGase mediated reaction (Suzuki et al., 1998bGo)), with several reference samples. As shown in Figure 2, upon electrophoresis we observed a marked change in mobility in the reaction product and, as expected, the migration position was identical to that of the authentic PNGase F-deglycosylated peptide, and was clearly distinguishable from other related compounds. Thus, this method provides a simple de-N-glycosylation assay and should be applicable to any other systems. However, caution should be taken in terms of the origin of the putative de-N-glycosylated peptide; it is also important to establish if the formation of the putative charged de-N-glycosylated peptide requires prior N-glycosylation and is not merely the result of direct deamidation of the original peptide (cf. Suzuki et al., 1998bGo).



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Fig. 2. Comparison of the electrophoretic migration of reaction products formed by N-glycosylated peptide incubated with yeast lysate or reference compounds. Purified glycopeptide ([3H]acetyl-Asn(CHO)-Bpa-Thr-amide, 10,000 d.p.m.) was incubated with yeast lysate and then analyzed by paper electrophoresis. The migration position was compared with that of the following reference peptides: PNGase F-deglycosylated peptide ([3H]acetyl-Asp-Bpa-Thr-amide, 5000 d.p.m.); Endo H-deglycosylated peptide ([3H]acetyl-Asn(GlcNAc)-Bpa-Thr-amide, 10,000 d.p.m.); unglycosylated peptide ([3H]acetyl-Asn-Bpa-Thr-amide, 10,000 d.p.m.).

 
Subcellular distribution of N-glycosylated and de-N-glycosylated peptide
Based on previous observations, it was proposed that de-N-glycosylation by PNGase occurred in the cytosol after the translocation of N-glycosylated protein or peptides (Wiertz et al., 1996aGo). Consistent with this hypothesis, most of the PNGase activity could be recovered in the soluble (cytosolic) fraction (Suzuki et al., 1993Go, 1994, 1997, 1998b; Kitajima et al., 1995Go; Römisch and Ali, 1997Go), although the precise localization of the enzyme still has not been rigorously established. To further study the localization of PNGase activity, we examined the distribution of de-N-glycosylated peptide, the reaction product of the action of PNGase, and compared it with that of N-glycosylated peptide. Using the assays described herein, we found that after a 90 min incubation of permeabilized cells with 3H-tripeptide in the presence of ATP, 44.3% of the added peptide was recovered as N-glycosylated peptide, while 7.3% was detected as de-N-glycosylated peptide. To examine the subcellular distribution of these peptides, permeabilized cells were prepared as described in Materials and methods and separated into three fractions according to the previously described method (Rexach and Schekman, 1991Go): the nucleus and the ER (mid-speed pellet (MSP)), vesicle components other than the ER, including the Golgi apparatus (high-speed pellet (HSP)), and the cytosol (high-speed supernatant (HSS)). Under these fractionation conditions, over 90% of oligo­saccharyl transferase activity, an ER marker enzyme, was recovered in the MSP fraction, while the remaining 10% was recovered in the HSP fraction consisting of vesicles (data not shown). These observations confirm that the MSP fraction contains most of the ER. As indicated in Table I, most of the de-N-glycosylated peptide (96%) was recovered in the cytosol fraction, while 34% of the N-glycosylated peptide was still associated with the ER. These data support the idea that de-N-glycosylation takes place in the cytosol after translocation of the N-glycosylated peptide from the ER to the cytosol.


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Table I. Subcellular distribution of N-glycosylated and de-N-glycosylated peptides
 
Effect of ATP on the import/N-glycosylation of 3H-tripeptide
Previously, it has been shown that glycopeptide release from the ER is dependent on the presence of ATP (Römisch and Schekman, 1992Go), suggesting that the ATP is required for the export of glycopeptides from the ER to the cytosol. However, it was not known if ATP also effected peptide import and/or N-glycosylation, so we compared the formation of N-glycosylated peptide in the presence and absence of ATP and an ATP-regenerating system. The glycopeptide formed at various times was quantitated by paper chromatography. The results in Figure 3 revealed that the overall N-glycosylation efficiency was not affected during the first 10 min of incubation even under conditions in which a severe defect of glycopeptide export was observed (see Figure 4A,B). After 10 min a modest decrease in activity was observed, possibly caused by the depletion of either oligosaccharyl pyrophosphoryl dolichol or acceptor substrate (unglycosylated 3H-tripeptide in the ER). Indeed, even at 30 min in the absence of ATP the N-glyco­sylation activity was still 69% of that observed in the presence of ATP (Figure 3). This clearly shows that while, as demonstrated previously (Römisch and Schekman, 1992Go), ATP affects the export of glycopeptides, it has little if any effect on the import and the glycosylation of peptide.



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Fig. 3. The effect of ATP on the N-glycosylation reaction in permeabilized yeast spheroplasts. Values were expressed as the % glycopeptide formed and calculated as the amount of total glycopeptide divided by the total radiolabeled probe recovered in the presence (open circles) or the absence (solid circles) of ATP.

 


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Fig. 4. Effect of glycosidase inhibitors on glycopeptide transport out of the ER. [3H]-Glycopeptide ([3H]acetyl-Asn(CHO)-Bpa-Thr-amide; 87 pmol) was incubated with 200 µl of permeabilized yeast spheroplasts in the complete system containing ATP ({sigma}o{lambda}{iota}{delta} {chi}{iota}{rho}{chi}{lambda}{varepsilon}{sigma}), plus 1-deoxymannojirimycin (DMM) (o{pi}{varepsilon}{nu} {chi}{iota}{rho}{chi}{lambda}{varepsilon}{sigma}) or 1-deoxynojirimycin (DNM) (o{pi}{varepsilon}{nu} {tau}{rho}{iota}{alpha}{nu}{gamma}{lambda}{varepsilon}{sigma}) or in the absence of ATP and an ATP-regenerating system (x) as indicated in Materials and methods. At the indicated times, aliquots were removed and the MSS fraction, which contains cytosol, and MSP fraction, which contains ER membranes, were prepared. The N-glycosylated peptide in the MSS fraction (A) and the MSP fraction (B) was quantitated using paper chromatography. Values were expressed as the % glycopeptide formed and calculated as the amount of total glycopeptide divided by the total radiolabeled probe recovered.

 
Effect of glycosidase inhibitors on the export of N-glycosylated peptide
Previously, it has been reported that the structure of the oligosaccharide chain affected export of a mutant form of carboxypeptidase Y (CPY*) that does not fold correctly. Cells that were devoid of the ER processing mannosidase (Mns1p) exhibited reduced degradation of CPY* (Knop et al., 1996Go; Jakob et al., 1998Go). Further, degradation of CPY* with glucosylated N-linked oligosaccharide residues was also shown to be less efficient (Jakob et al., 1998Go). To ask if the export of glycopeptides, like glycoproteins, was affected by the structure of the oligosaccharide chain, we analyzed the effect of two glycosidase inhibitors (Kaushal and Elbein, 1994Go), 1-deoxy­nojirimycin (DNM) for glucosidase, and 1-deoxymanno­jirimycin (DMM) for mannosidase, on glycopeptide transport. After the incubation of 3H-tripeptide with permeabilized spheroplasts in the presence of these inhibitors, we compared the amount and the distribution of glycosylated peptide in the cytosol (MSS) fraction and the nucleus/ER (MSP) fraction. The distribution of radioactivity recovered in the nucleus/ER (MSP) fraction and the cytosol (MSS) fraction was essentially unchanged in the presence of these glycosidase inhibitors (data not shown). Unlike misfolded glycoproteins (Knop et al., 1996Go; Jakob et al., 1998Go), it can be clearly seen in Figure 4A that the amount of glycopeptide released into the cytosol fraction was not affected in the presence of these inhibitors. For comparison, we also examined the effect of ATP, which is necessary for the export of glycopeptide (Römisch and Schekman, 1992Go). We confirmed that impairment of glycopeptide export occurred in the absence of ATP and an ATP-regenerating system (Figure 4A). However, the rate of glycopeptide formation in the ER fraction was not changed in the presence of glycosidase inhibitors (Figure 4B), suggesting that peptide import/N-glycosylation was not affected by these inhibitors. In the absence of ATP, a rapid accumulation of glycopeptide in the nucleus/ER fraction occurred, presumably due to the impairment of its export (Figure 4B).

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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Fig. 5. Glycosidase inhibitors altered the protein-linked oligosaccharide structures in permeabilized yeast spheroplast cells. After incubation of cells with [3H]-mannose the oligosaccharides were released by PNGase digestion. [3H]-Labeled oligosaccharides synthesized in 1-deoxynojirimycin-treated (A), 1-deoxymannojirimycin-treated (B), or control permeabilized spheroplast cells (C) were applied to a Bio-Gel P4 column (1.25 x 100 cm). Fractions were collected every 5 min and radioactivity was monitored in each fraction. G15 to G10 represent the elution positions of glucose oligomers on this column.

 
Finally, we compared the appearance of de-N-glycosylated peptide in the cytosol (MSS) fraction with or without the two inhibitors. Essentially no difference was observed: the percentage of de-N-glycosylated peptide was 15.2% for control cells, 14.9% for DMM-treated, and 14.3% DNM-treated cells at 120 min incubation.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Recent studies have established that eukaryotic cells export misfolded or unassembled proteins from the ER using the same components used for protein import, i.e., the Sec61p-complex (Wiertz et al., 1996bGo; Pilon et al., 1997Go; Plemper et al., 1997Go). Small glycopeptides appear to share this retrograde transport route (Römisch and Schekman, 1992Go; Römisch and Ali, 1997Go; Suzuki et al., 1998bGo), and utilize Sec61p for this process (Gillece et al., 1998Go). It also is clear that ATP plays an important role in the export of both misfolded proteins and glycopeptides (Römisch and Schekman, 1992Go; McCracken and Brodsky, 1996Go; Qu et al., 1996Go; Wiertz et al., 1996bGo). However, while a number of other protein components that are involved in export of misfolded proteins have been identified in yeast (Sommer and Wolf, 1997Go), it is not known if components other than Sec61p are involved in the export of glyco­peptides.

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., 1996Go; Jakob et al., 1998Go). 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 ({Delta}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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Materials
Adenine, ATP, ATP-{gamma}S, antimycin A, 1-deoxymannojirimycin, 2-deoxyglucose, GDP-Man, N-ethylmaleimide, and tetrahydrofuran were obtained from Sigma Chemical Co. (St. Louis, MO). Creatine phosphate, creatine phosphate kinase, dithiothreitol, HEPES, PNGase F, and Endo H were from Boehringer Mannheim Corp. (Indianapolis, IN). Bacto-yeast extract, Bacto-peptone were from Difco Laboratories (Detroit, MI). Whatman 3MM paper was from Whatman LabSales (Hillsboro, OR). NP-40 detergent and 1-deoxynojirimycin were purchased from Calbiochem-Novabiochem Intl. (La Jolla, CA). All other chemicals were either from Fisher Scientific Co. (Pittsburgh, PA) or J. T. Baker Inc. (Phillipsburg, NJ). Bio-Gel P4 column was from Bio-Rad Laboratories (Hercules, CA). [2-3H(N)]-Mannose was from ICN Pharmaceuticals, Inc. (Costa Mesa, CA).

Yeast strains and culture conditions
The yeast strain used in this study was W303–1a (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., 1988Go; Suzuki et al., 1998bGo). 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., 1999Go). 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-{gamma}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., 1998bGo). Briefly, 10–20 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., 1994Go; Suzuki et al., 1998bGo).

De-N-glycosylated peptide was quantitated using paper electrophoresis (model EF-200, Advantec Toyo Kaisha Ltd., Tokyo, Japan). A second aliquot of the samples (10–20 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., 1998bGo). 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., 1998bGo). 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 Tris–HCl 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, 1991Go). 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., 1995Go). 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 Tris–HCl (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 Tris–HCl 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 Tris–HCl buffer (pH 8.0), and was analyzed by Bio-Gel P4 column.


    Acknowledgments
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
We thank Dr. Neta Dean for the use of an ultracentrifuge, Dr. Robert S.Haltiwanger for the use of Bio-Rad P4 column, Dr. Hangil Park for various technical comments, Ms. Qi Yan for providing 3H-labeled tripeptide, the members of our laboratory for discussions, and Ms. Lorraine Conroy for helping to prepare the manuscript. T.S. is a Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellow for research abroad. This research is supported by NIH Grant GM33184 to W.J.L.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
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