Glycobiology, 2002, Vol. 12, No. 12 803-811
© 2002 Oxford University Press
Glycopeptide export from the endoplasmic reticulum into cytosol is mediated by a mechanism distinct from that for export of misfolded glycoprotein
2 Undergraduate Program for Bioinformatics and Systems Biology, Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo and PRESTO, Japan Science and Technology Corporation (JST), Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan; and 3 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 11794-5215, USA
Received on March 29, 2002; revised on July 5, 2002; accepted on July 31, 2002
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When glycoproteins formed in the endoplasmic reticulum (ER) are misfolded, they are generally translocated into the cytosol for ubiquitination and are subsequently degraded by the proteasome. This system, the so-called ER-associated glycoprotein degradation, is important for eukaryotes to maintain the quality of glycoproteins generated in the ER. It has been established in yeast that several distinct proteins are involved in this translocation and degradation processes. Small glycopeptides formed in the ER are exported to the cytosol in a similar manner. This glycopeptide export system is conserved from yeast to mammalian cells, suggesting its basic biological significance for eukaryotic cells. These two export systems (for misfolded glycoproteins and glycopeptides) share some properties, such as a requirement for ATP and involvement of Sec61p, a central membrane protein presumably forming a dislocon channel for export of proteins. However, the machinery of glycopeptide export is poorly understood. In this study, various mutants known to have an effect on export/degradation of misfolded glycoproteins were examined for glycopeptide export activity with a newly established assay method. Surprisingly, most of the mutants were found not to exhibit a defect in glycopeptide export. The only gene that was found to be required on efficient export of both types of substrates was PMR1, the gene encoding the medial-Golgi Ca2+/Mn2+-ion pump. These results provide evidence that although the systems involved in export of misfolded glycoproteins and glycopeptides share some properties, they have exhibited distinct differences.
Key words: de-N-glycosylation/endoplasmic reticulum/glycopeptide export/quality control
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Introduction |
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Recent evidence has shown that the endoplasmic reticulum (ER) has a quality control system for efficient routing of correctly folded proteins out of the ER (Ellgaard and Helenius, 2001
). 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 called ER-associated degradation (ERAD) (McCracken and Brodsky, 1996). Moreover, studies suggest that there is an evolutionally conserved system dedicated for N-linked glycoproteins, named glycoprotein ERAD (Helenius and Aebi, 2001; Cabral et al., 2001). As to the mechanism of protein translocation from the ER to the cytosol, it has been suggested that Sec61p is also involved in the retrotranslocation processes (Wiertz et al., 1996; Plemper et al., 1997; Pilon et al., 1997). In yeast, there are more than 10 proteins identified to be involved in export of misfolded proteins (Suzuki et al., 1998a; Römisch, 1999; Plemper and Wolf, 1999; Brodsky and McCracken, 1999; Hampton, 2000). To identify these proteins, a point mutation of carboxypeptidase Y (CPY*) that causes this glycoprotein to remain unfolded has been mainly used as a model substrate (Knop et al., 1995; Hiller et al., 1996; Biederer et al., 1997; Plemper et al., 1997, 1999; Bordallo et al., 1998; Dürr et al., 1998; Casagrande et al., 2000).
In vitro studies using mammalian cells and Saccharomyces cerevisiae have shown that small N-glycosylatable tripeptides following their glycosylation undergo retrotranslocation out of the ER to the cytosol in a similar manner as that of misfolded glycoproteins (Römisch and Schekman, 1992; Römisch and Ali, 1997; Suzuki et al., 1998b; Gillece et al., 2000; Suzuki and Lennarz, 2000; Ali and Field, 2000; Ali et al., 2000). Although the involvement of Sec61p in this process has been established in yeast (Gillece et al., 2000), other components of glycopeptide export system are poorly understood compared with that of misfolded glycoprotein export. Unlike the case for misfolded glycoproteins (Knop et al., 1996; Jakob et al., 1998, 2001; Nakatsukasa et al., 2001), the structure of the oligosaccharide chains was found to have no effect on the rate of export of glycopeptide in yeast as well as in mammalian cells (Suzuki and Lennarz, 2000; Ali and Field, 2000), suggesting that the mechanism for the export of small glycopeptide may differ, at least in some respects, from that of glycoproteins.
Peptide:N-glycanase (PNGase) is a deglycosylating enzyme that acts on glycoproteins/glycopeptides to release intact N-glycans. Cytoplasmic PNGase has been suggested to be involved in degradation of misfolded glycoproteins (Suzuki et al., 2002). The same activity has also been found to be responsible for release of N-linked glycans from glycopeptides in the cytosol, making it difficult to detect glycopeptides exported from the ER to the cytosol of mammalian cells (Römisch and Ali, 1997). Recently we identified a gene encoding the cytosolic PNGase (PNG1) by genetic mapping (Suzuki et al., 2000). Because PNGase activity was not required in the export of glycopeptide (Ali et al., 2000), the png1-deletion strains were expected to have an advantage for the assay of glycopeptide export because it would avoid underestimation of the quantitation of cytosolic glycopeptides due to lack of degradation.
In this study, various mutant strains that have been implicated in misfolded glycoprotein (mainly CPY*) export/degradation were studied to identify factors common to these two export processes. Surprisingly, most mutant strains had a normal rate of glycopeptide export, further supporting the idea that two export processes were mediated by quite different mechanisms. PMR1, a gene encoding the medial-Golgi Ca2+/Mn2+ ion pump was found to be required for efficient export of both glycoproteins (CPY*) and small glycopeptides. The possible significance of this observation is discussed.
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Glycopeptide export assay using yeast permeabilized cells
Previously, an in vitro assay for glycopeptide export from the ER membrane to the cytosol has been established (Römisch and Schekman, 1992
). In this study, we prepared a member of isogenic mutants that have been implicated in export/degradation process of CPY*, and examined their effects on the export of small glycopeptides. Because we found that the integrity of the ER membrane varied widely among strains, a direct comparison between strains was made difficult (data not shown). To solve this problem, we took advantage of measuring the rate of N-glycosylation as an internal control to indicate the membrane integrity. This assumption was based on the experimental observation that membrane vesicles that are not sealed correctly are not capable of N-glycosylating small peptides (Yan et al., 1999). In this method, the rate of glycopeptide export (glycopeptide export index, GEI) was defined as follows: (1) Glycopeptide export index (GEI) = (rate of accumulation of N-glycosylated peptide in the soluble [cytosol] fraction)/ (rate of formation of N-glycosylated peptide in total preparation)
First, the export rate of the png1
strain with that of the isogenic wild type was compared using this method. As shown in Figure 1, although the rate of N-glycosylated peptide export into the soluble (cytosolic) fraction was different, as expected the GEI value was found quite similar between these two strains, suggesting that PNG1 was not required for the process of glycopeptide export (Figures 1A and 1B). On the other hand, when an ATP-regenerating system was absent, the GEI value was significantly lower (Figure 1C). These results indicated that the activity of glycopeptide export on various mutants could be assessed by measuring the GEI value. To carry out this assay, it is important to note that the N-glycosylation efficiency should be similar to that of wild type; in this study none of the strains used had a defect in glycosylation judging from the in vitro efficiency of the N-glycosylation of small peptide (data not shown).
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It is known that de-N-glycosylation by PNGase can cause an underestimation of the export rate of glycopeptide (Römisch and Ali, 1997
; Suzuki et al., 1998b). As shown in Figure 1, the png1 strain was as effective as wild type in glycopeptide export. This result allowed us to use png1
ns as a background to avoid a potential underestimation of export rate. Therefore we have made a set of isogenic double mutants of CPY* degradation-defective strains that also had a png1-deletion (Table I), and carried out export assays in the png1-deletion background.
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The effect of various compounds on glycopeptide export
To further validate this new assay system, compounds that have been studied by others (Römisch and Ali, 1997
; Ali and Field, 2000) were examined for their effect on glycopeptide export. Each compound was added 30 min before the addition of the [3H]-tripeptide, and the GEI was obtained by comparing total glycosylation rate with the amount of glycopeptide in the cytosol. In mammalian cells, an unidentified microsomal GTPase was reported to be required for glycopeptide export from the ER (Ali et al., 2000); in the ATP-regenerating system (Ali et al., 2000), GDP-Man was routinely included, and it could have served as the source for GTP, which is required for the action of microsomal GTPase, because the assay was carried out in ATP-regenerating system (Ali et al., 2000). In our hand, glycopeptide export was unchanged even in the absence of GDP-Man. However, when 5 mM GTP-
S was added to the reaction mixture, significant impairment of export was observed (Figure 2A). This would suggest that GTP hydrolysis might be required in this process, as is the case for mammalian cells. However, as noted previously (Ali et al., 2000), the presence of only ATP and GTP did not allow efficient glycopeptide export in the absence of cytosol fraction (data not shown), suggesting that additional factor(s) are required for efficient glycopeptide export in yeast.
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It was previously suggested that dislocation of a protein substrate from the ER to the cytosol and its proteolysis by the proteasome might be coupled (Mayer et al., 1998
). To check the effect of proteasome activity on glycopeptide export, two distinct proteasome inhibitors were examined (50 µM lactacystin; 200 µM MG-132). As shown in Figure 2A, a small but reproducible inhibitory effect on glycopeptide export was observed. However, when mutants of the 26S proteasome subunit were examined for export activity, no effect was observed (Figure 2B). Therefore the effect of proteasome activity on the retrograde export of a glycopeptide is negligible.
Neither Kar2p nor Pdi1p are involved in glycopeptide export
Although it is known that glycopeptide was exported from the ER into the cytosol in a Sec61p-dependent manner (Gillece et al., 2000), there was little information about other factors that might be involved in glycopetide export. Especially interesting was the question if there are any peptide-recognizing component(s) that would be responsible for the export process into the cytosol. So far, Kar2p (Plemper et al., 1997) and Pdi1p (Gillece et al., 1999) were known to affect export/degradation of CPY*. We therefore utilized various kar2 and pdi1 alleles for their effect on glycopeptide export. For kar2 alleles, two alleles that affect its peptide-binding (kar2-1; kar2-133) (Scidmore et al., 1993; Brodsky et al., 1999) and two alleles that affect ATPase activity (kar2-113; kar2-159) (Scidmore et al., 1993; Brodsky et al., 1999) were used in this study. We found these mutants had a similar efficiency of glycopeptide export with equivalent wild types (Figure 3A, B). This result was consistent with the fact that BiP/Kar2p does not bind well to peptides smaller than tetrapeptides (Flynn et al., 1991; Spee and Neefjes, 1997).
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The Pdi1p homolog in hen oviduct was initially thought to be a glycosylation site-binding protein (Geetha-Habib et al., 1988
). Mammalian PDI protein was found to be important for the export of toxin protein from the ER to the cytosol (Tsai et al., 2001). Moreover, a mutant of Pdi1p, Pdi1252–277, was found to affect both peptide binding and degradation of misfolded cysteine-free secretory proteins (Gillece et al., 1999). On the other hand, an active site mutant of Pdi1p (S2S6 mutant) was also found to cause the retention of vacuolar CPY in the ER (Luz and Lennarz, 1998). To examine unequivocally the effect of Pdi1p on the export of small glycopeptide, we determined the GEI on these three different pdi1 mutants. As shown in Figure 3A, no differences were observed in the rate of glycopeptide export.
The structure of the glycan as well as the absence of the calnexin homolog (Cne1p) had no effect on the transport efficiency
Several lines of evidence suggested that glycan structure is important for the export of misfolded glycoproteins from the ER to the cytosol (Cabral et al., 2001). Although this effect seemed to occur widely in eukaryotic cells, glycan structure was found not to be critical for glycopeptide export (Suzuki and Lennarz, 2000; Ali and Field, 2000). To confirm these observations, several mutants were used in this study.
There are mainly two proteins that are known to affect glycan-dependent ERAD: ER alpha-mannosidase (Mns1p), which converts Man9GlcNAc2 to Man8GlcNAc2, and Htm1p/Mnl1p, a putative lectin that recognizes the specific glycan structure (Man8GlcNAc2) formed by Mns1p. For CPY*, deletion of either protein was found to cause a significant delay in degradation (Knop et al., 1996; Jakob et al., 1998, 2001; Nakatsukasa et al., 2001). As shown in Figure 3A, deletion of neither protein had a critical effect on glycopeptide export. In sharp contrast to the case for misfolded glycoproteins, these results clearly confirmed the earlier observation that glycan structure did not affect the kinetics of glycopeptide export.
Previously, Cne1p, the only calnexin homolog found in S. cerevisiae (Parlati et al., 1995) was shown to be important for the degradation of mutant alpha-preprofactor (McCracken and Brodsky, 1996), although this protein was not required for the degradation of CPY* (Knop et al., 1996). Deletion of the CNE1 gene also was found not to have any effect on glycopeptide export (Figure 3A).
Deletion of PMR1 affected glycopeptide export activity in yeast
Next we examined effects of the other genes that had been shown to cause delay in the degradation of CPY* proteins. The genes examined were PMR1 (Dürr et al., 1998), DER1 (Knop et al., 1995), HRD1/DER3 (Hampton et al., 1996; Plemper et al., 1999), HRD3/EKS1 (Hampton et al., 1996; Saito et al., 1999), UBC6 (Hiller et al., 1996), CUE1 (Biederer et al., 1997), and IRE1 (Casagrande et al., 2000). As already mentioned, only one of these ERAD-related genes was combined with the png1-deletion strain (see Table I), and the glycopeptide export activity was examined for each strain. As shown in Figure 4, only one of the strains showed an effect on glycopeptide export, the pmr1-deletion. It is noteworthy that in our assay system, 5 mM of magnesium acetate is routinely included in the export buffer (B88). To examine if addition of various divalent cations could affect the export of glycopeptide, semi-intact cells from TSY172 (pmr1::HIS3 png1::URA3) were prepared, and glycopeptide export activity was assayed according to the method of Römisch and Schekman (1992)). Addition of 5 mM CaCl2, MgCl2, MnCl2, and ZnCl2 did not result in recovery of peptide export activity. These results were consistent with previous results obtained using rat liver microsomes (Ali et al., 2000).
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Discussion |
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In higher eukaryotes, export of small peptides was suggested to be necessary to reduce competition between these peptides and nascent proteins for chaperone proteins that helps folding of these nascent chains (Lehner et al., 2000
). Previously it was known that the export system for small glycopeptides shared some properties with that for misfolded proteins, such as an ATP requirement and involvement of Sec61p (Römisch and Schekman, 1992; Gillece et al., 2000), implying that the same export machinery might be used in these two processes. On the other hand, the glycan structure of small glycopeptides, in sharp contrast to export of misfolded glycoproteins, were found to have no effect on rate of export, indicating the difference in components required for these two processes (Suzuki and Lennarz, 2000; Ali and Field, 2000). In this study, we aimed to gain more insight into the mechanism of glycopeptide export from the ER using permeabilized yeast spheroplasts. To examine the similarities and differences between these two processes, various mutants that are known to cause the delay on degradation of misfolded glycoproteins (mainly CPY*) were prepared.
As mentioned, the rate of the glycosylation reaction was measured as an internal control of membrane integrity and glycopeptide export activity was calculated as the ratio of the relative rate of export to that of the rate of glycosylation rate (GEI). Moreover, we utilized strains with a png1 deletion as background to avoid any postexport de-N-glycosylation of glycopeptide in the cytosol, which could cause an underestimation of glycopeptide export activity (Römisch and Ali, 1997; Suzuki et al., 1998b). The png1-deletion strain showed no effect on glycopeptide export activity. This is consistent with the experimental observation in mammalian cells, where PNGase activity was found not to be required for export activity (Ali et al., 2000).
It has been shown that glycan structure of glycopeptide does not affect the rate for small glycopeptide export (Suzuki and Lennarz, 2000; Ali and Field, 2000). This study clearly supports these conclusion, because deletion of neither Mns1p (
-mannosidase) or Htm1p/Mnl1p (a putative lectin) had any effect on glycopeptide export process, whereas these proteins were shown to be required for export/degradation of CPY* (Knop et al., 1996; Jakob et al., 1998, 2001; Nakatsukasa et al., 2001). In sharp contrast to the misfolded glycoproteins, there is a lack of evidence of involvement of lumenal chaperone proteins for this export process. The lack of effect of two pdi1 mutants (252–277 and S2S6) was especially surprising because hen oviduct protein disulfide isomerase (homolog of Pdi1p) was previously identified as a major glycosylation site-binding protein (Geetha-Habib et al., 1988). Whether there are no lumenal chaperones involved in this process or other unknown chaperones may be responsible remains to be determined. Lack of effect of deletion of components of ubiquitin-conjugation pathway on glycopeptide export was also found in this study (ubc6, der3/hrd1, and cue1).
It should be noted that misfolded proteins that do not contain lysine residues have the same kinetics of export/degradation as wild type proteins with lysine residues (Yu et al., 1997). Although the ubiquitination machinery itself was found to be required for dislocation as well as targeting the substrate to the proteasome (Yu and Kopito, 1999), the result indicated that ubiquitin modification of the misfolded protein is not required for its degradation. Therefore, although the peptide we used did not have a lysine residue and polyubiquitination could not occur, the result still contrasted with the case of misfolded proteins in this regard.
It is known that free oligosaccharide formed in the ER can also be exported from the ER to the cytosol (Moore, 1999). Recently it was observed that free oligosaccharide export was moderately inhibited by a proteasome inhibitor (Karaivanova and Spiro, 2000). Using similar chemical inhibitors, we also observed minor but reproducible inhibition effects, although mutants of proteasome subunits did not have any effect. This effect of proteasome inhibition therefore could be a secondary effect of the proteasome inhibitor. Alternatively, because the mutations in the strains used in this study were in the regulatory subunits, they might not impair glycopeptide export. In any case, our study clearly shows that the proteasome does not contribute a major role in the export of glycopeptide from the lumen of the ER.
Further glycopeptide export experiments identified the pmr1 deletion as causing significant inhibition of glycopeptide export. As summarized in Table II, it is clear that there is a drastic difference in export systems between small glycopeptides and misfolded glycoprotein. It is interesting to note that Mg2+, not Ca2+, was previously reported to be required for the export of glycopeptide in mammalian cells (Ali and Field, 2000). Pmr1p is a Ca2+/Mn2+-ATPase localized in the yeast medial Golgi (Antebi and Fink, 1992), and its deletion causes a delay in ERAD using CPY* as a substrate (Dürr et al., 1998). Pmr1p is well conserved throughout eukaryotes (Ton et al., 2002), and inactivation of this allele has been linked to Hailey-Hailey disease in human, whose symptoms involve a loss of keratinocyte cohesion (Hu et al., 2000; Sudbrak et al., 2000). Except for sec61, which is required for forming the export channel pmr1, is the only gene among those tested that was found to have effects on export of both glycopeptides and misfolded glycoproteins. The mechanism by which the delay of glycoprotein and glycopeptide export occurs in pmr1 strains remains unclear.
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At present it is not clear whether a very low level of Pmr1 protein remaining in the ER plays a role in export, or whether Golgi Pmr1p can regulate the distribution of cations in other organelles, including the ER. The Pmr1p-mediated ion transport system affects processes hosted in early secretory compartments in addition to those in the Golgi, where Pmr1p is primarily localized (Dürr et al., 1998
). For example, pmr1-null mutant was known to display mislocalization of several proteins in the secretory pathway (Okorokov and Lehle, 1998). Therefore the defect of export in both glycoproteins and glycopeptides may be due to the mislocalization of required proteins rather than the direct involvement of Pmr1p in this process. Alternatively (but not mutually exclusive), abnormal cation distribution in intralumenal compartments could impair the function of the basic export machinery on the ER membrane used for both glycopeptides and glycoproteins. Last, it should also be noted that Pmr1p catalyzes ATP hydrolysis for its cation-transport activity (Okorokov et al., 1993). The ATP requirement for glycopeptide export may be at least partly related to the catalytic function of Pmr1p.
Interestingly, pmr1-deletion mutants were also known to be sensitive to various treatments that promote accumulation of unfolded proteins in the ER (Dürr et al., 1998). We have found that pmr1 is more sensitive to these treatments than other ERAD-related mutants (unpublished data). Moreover, we have found that level of unfolded protein response (Urano et al., 2000; Mori, 2000; Patil and Walter, 2001) in the pmr1-deletion strain is much higher than the isogenic wild-type strain, suggesting that the pmr1 strain accumulate unfolded proteins even under the unstressed conditions (unpublished data). Given that the pmr1 strain accumulates high levels of unfolded proteins under noninduced conditions, further stress may not be tolerated by the pmr1 strain. Recently, requirement of components for degradation was found to vary between different substrates, suggesting specific factors on a subset of misfolded (glyco)proteins (Plemper et al., 1998; Wilhovsky et al., 2000). Therefore, the greater sensitivity of the pmr1 strain toward drugs may imply that loss of Pmr1p causes a defect in the basic export machinery from the ER. Such export machinery may also be responsible for glycopeptide export. Further studies are necessary to define the relationship of Pmr1p function and the ER to cytosol protein/peptide export system.
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Materials and methods |
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Materials
ATP-
S, GTP-S, and GDP-Man were obtained from Sigma (St. Louis, MO). Creatine phosphate, creatine phosphate kinase, dithiothreitol, and HEPES were obtained from Roche (Palo Alto, CA). Bacto-yeast extract and Bacto-peptone were purchased from Difco Laboratories (Detroit, MI). Whatman 3MM paper was from Whatman LabSales (Hillsboro, OR). Lactacystin and MG-132 were obtained from Calbiochem-Novabiochem (La Jolla, CA). pBluescript SK vector was from Stratagene (La Jolla, CA). All other chemicals were either from Fisher Scientific (Pittsburgh, PA) or J.T. Baker (Phillipsburg, NJ). A beta-galactosidase assay kit was obtained from Pierce (Rockford, IL).
Plasmid constrution
DNA manipulations were performed according to Sambrookand Russell (2001). For construction of the png1 deletion strain, a disruption plasmid, HpaI/XbaI fragment of URA3 gene from YEp352 vector (Hill et al., 1993) was inserted into the BlnI/NruI sites of pRS316-PNG1 (Suzuki et al., 2000). The EcoRI/XhoI fragment of the disruption allele was further cloned into EcoRI/XhoI site of pBluescript SK to generate pBS-png1::URA3.
Yeast strains and culture conditions
The wild-type yeast strain used in this study was W303–1a (MATa ade2-1 ura3-1 his3-11,15, trp1-1, leu2-3,112 can1-100). Standard yeast media and genetic technique were used (Rose et al., 1990; Sherman, 1991; Elble, 1992). The list of cells used was summarized in Table I. The following cells were the generous gifts; cne1 (Parlati et al., 1995) from Dr. David Y. Thomas, McGill University; MS10/MS193/MS1111 (Scidmore et al., 1993) from Dr. Mark D. Rose, Princeton University; K609 (Cunningham and Fink, 1994) from Drs. Yoko Takita and Ryle Cunningham, Johns Hopkins University; and JC147 (Cox et al., 1993) from Dr. Davis T.W. Ng, Pennsylvania State University. Integration plasmids for kar2 alleles (kar2-113 and kar2-159) (Scidmore et al., 1993) were also the generous gift of Dr. Mark D. Rose, Princeton University and Dr. Dieter H. Wolf, Universitat Stuttgart. These kar2 alleles were introduced in yeast using the two-step gene replacement method (Rothstein, 1991). CMY763, CMY765, and their congenic wild type (YPH499) were kind gifts from Dr. Carl Mann, Centre d’Etudes de Saclay (Gif-sur-Yvette, France) (Ghislain et al., 1993). The disruption plasmid for EKS1/HRD3 allele (Saito et al., 1999) was a kind gift from Dr. Akihiko Nakano, RIKEN Institute (Wako, Japan). The other deletion-mutant cells for genes involved in ERAD were made by direct gene replacement method, either by using S. pombe his5+ gene or KanR gene (Longtine et al., 1998) and correct integration was confirmed by colony polymerase chain reaction (PCR). For PDI1 mutant strains, MLY200 (LaMantia and Lennarz, 1993) were used as the original strain and pPDI1(S2S6) (Luz and Lennarz, 1998) and pPDI(252–277) (Gillece et al., 1999) were used as mutant alleles for Pdi1p. After swapping the plasmid with wild type, TSY183 and TSY187 was isolated following sporulation of diploids made of mutant cells and TSY151 followed by the isolation following the haploid segregants of the appropriate phenotype.
TSY151 was made by direct transfomation of EcoRI/XhoI digests of pBS-png1::URA3. Ura+ colonies were tested for correct integration by PNGase activity assay (Suzuki et al., 1994, 1998b) as well as colony PCR. To introduce png1::URA3 allele to various mutant cells, direct transformation of EcoRI/XhoI digests of pBS-png1::URA3 was carried out. In some cases, sporulation of diploids made of mutant cells and TSY151 followed by the isolation of the haploid segregates of the appropriate phenotype was performed to obtain mutants of interest.
Glycopeptide export assay
For preparation of permeabilized yeast cells, 500 ml of cells were grown with shaking in a 2-L Erlenmeyer flask for overnight at 30°C. For temperature sensitive strains (kar2-113, kar2-159), incubation was performed at 25°C. Preparation of permeabilized cells was carried out as described elsewhere (Baker et al., 1988; Suzuki et al., 1998b). N-glycosylation of tripeptide export of glycopeptide and quantitation of glycopepetide in this assay was performed as described previously (Suzuki and Lennarz, 2000). Briefly, 4.3 pmol of tritiated glycotripeptide (1 x 105 dpm of [3H]acetyl-Asn-Bpa-Thr-amide, where Bpa represents p-benzoylphenylalanine (Yan et al., 1999) were added to 100 µl of the permeabilized cells with an ATP-regenerating system to initiate the reaction at room temperature. At indicated time, 20 µl of samples were taken and added to the tube containing ATP-S (final concentration 1 mM) and were separated into two aliquots. One aliquot is directly assayed for glycopeptide formation by paper chromatography. That amount represents the total glycopeptides in the preparation. The other half was separated into a midspeed pellet) fraction that contains ER membrane and midspeed supernatant fraction by brief centrifugation (Rexach and Schekman, 1991). The midspeed pellet obtained were washed once with 10 µl of B88 buffer, and another brief spin was carried out. The supernatant obtained was combined with the midspeed supernatant fraction. The combined soluble fraction was regarded as the soluble (cytosolic) fraction, and glycopeptide amount in this fraction was also quantitated.
To examine the effect of divalent cation on glycopeptide export, permeabilized cell fractions with or without cytosol were prepared and the export assay was performed essentially as described earlier (Römisch and Schekman, 1992).
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Acknowledgments |
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We thank Drs. Hangil Park, Stefan Tafrov, and David Zappulla (SUNY Stony Brook) for technical comments on this study. We also appreciated Drs. Kyle Cunningham (Johns Hopkins University), Carl Mann (Centre d’Etudes de Saclay, Gif-sur-Yvette, France), Akihiko Nakano (RIKEN Institute, Wako, Japan), Davis T.W. Ng (Pennsylvania State University), Mark D. Rose (Princeton University), David Y. Thomas (McGill University), and Dieter H. Wolf (Universtät Stuttgart) for providing various strains/plasmids. We thank the members of Lennarz’s lab for discussion and Miki Suzuki for editing this manuscript. This research is supported by NIH grant no. 33184 to W.J.L. T.S. is supported by a grant from the Mizutani Foundation for Glycosciences.
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Abbreviations |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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CPY, carboxypeptidase Y; ER, endoplasmic reticulum, ERAD, endoplasmic reticulum-associated degradation; GEI, glycopeptide export index; PCR, polymerase chain reaction; PNGase, peptide:N-glycanase.
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Footnotes |
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1 To whom correspondence should be addressed; E-mail: wlennarz@notes.cc.sunysb.edu
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References |
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