ACCELERATED PUBLICATION
Mnl1p, an alpha -Mannosidase-like Protein in Yeast Saccharomyces cerevisiae, Is Required for Endoplasmic Reticulum-associated Degradation of Glycoproteins*

Kunio NakatsukasaDagger , Shuh-ichi NishikawaDagger , Nobuko Hosokawa§, Kazuhiro Nagata§, and Toshiya EndoDagger ||

From the Dagger  Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, the § Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, and  Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), Japan

Received for publication, January 17, 2001, and in revised form, January 31, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The endoplasmic reticulum (ER) has a mechanism to block the exit of misfolded or unassembled proteins from the ER for the downstream organelles in the secretory pathway. Misfolded proteins retained in the ER are subjected to proteasome-dependent degradation in the cytosol when they cannot achieve correct folding and/or assembly within an appropriate time window. Although specific mannose trimming of the protein-bound oligosaccharide is essential for the degradation of misfolded glycoproteins, the precise mechanism for this recognition remains obscure. Here we report a new alpha -mannosidase-like protein, Mnl1p (mannosidase-like protein), in the yeast ER. Mnl1p is unlikely to exhibit alpha 1,2-mannosidase activity, because it lacks cysteine residues that are essential for alpha 1,2-mannosidase. However deletion of the MNL1 gene causes retardation of the degradation of misfolded carboxypeptidase Y, but not of the unglycosylated mutant form of the yeast alpha -mating pheromone. Possible roles of Mnl1p in the degradation and in the ER-retention of misfolded glycoproteins are discussed.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The endoplasmic reticulum (ER)1 is the site of entry for proteins destined for the secretory pathway. The ER provides an optimized environment for correct maturation, including correct folding, oligomerization, N- and O-linked glycosylation, and disulfide bond formation of proteins imported into the ER (1, 2). Several components, including molecular chaperones and folding enzymes that mediate these processes, have been identified in the ER. Shortage or defects of these components as well as mutations in secretory proteins and environmental stress tend to result in failure and therefore misfolding of secretory proteins. The misfolded proteins are retained in the ER and are subjected to retrial for correct maturation with the aid of molecular chaperones and folding enzymes. However if this process is not successful, prolonged retention of the misfolded proteins in the ER eventually leads to their degradation (3, 4). The ER-associated degradation (ERAD) requires the retrotranslocation of misfolded proteins through the Sec61 channel from the ER lumen to the cytosol and subsequent degradation by the 26 S proteasome located in the cytosol (5-9).

For ERAD of aberrant proteins, the question of how proteins that are misfolded and to be degraded are identified and targeted for the retrotranslocation system has not been resolved. In yeast Saccharomyces cerevisiae, slow removal of alpha 1,2-mannose from the middle branch of the protein-bound oligosaccharide, or formation of Man8GlcNAc2, has been shown to be critical for the degradation of misfolded carboxypeptidase Y (CPY), a vacuolar glycoprotein (10). This is consistent with the reports that inhibition of alpha -mannosidase trimming stabilizes specific misfolded glycoproteins in the mammalian ER (11, 12). On the basis of these observations, it has been proposed that the trimming of the glycoprotein-bound oligosaccharide may well function as the biological timer for the onset of the glycoprotein degradation that prevents permanent residence of misfolded glycoproteins in the ER (2). This naturally suggests the presence of a Man8GlcNAc2-binding lectin involved in ERAD of misfolded glycoproteins, which remains to be identified (10).

In the present study, we identified a new alpha -mannosidase-like protein, Mnl1p (mannosidase-like protein), in the yeast ER. Although it shows some homology with Mns1p, yeast alpha 1,2-mannosidase, Mnl1p is unlikely to exhibit the alpha 1,2-mannosidase activity, because it lacks cysteine residues that are essential for the alpha 1,2-mannosidase activity (13). However deletion of the MNL1 gene resulted in retardation of the degradation of misfolded CPY, but not of the unglycosylated mutant form of the yeast alpha -mating pheromone (Delta Gpalpha F). Possible roles of Mnl1p in ERAD and the ER retention of misfolded glycoproteins will be discussed.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids, Strains, and Culturing Conditions-- Standard recombinant techniques were employed using an Escherichia coli strain TG1 (supE hsdDelta 5 thiDelta (lac-proAB) F' [traDelta 36 proAB+ lacIq lacZDelta M15]). Yeast strains used in this study were SEY6210 (MATalpha ura3 leu2 trp1 his3 lys2 suc2) (14) and KYSC1 (MATalpha prc1-1 ura3 leu2 trp1 his3 lys2 suc2).2 Yeast strain BJ3505 (MATa pep4::HIS3 prb1 lys2 trp1 ura3 gal2 can1) was used for preparation of the cytosol (9). Cells were grown in YPD medium containing 1% yeast extract, 2% polypeptone, and 2% glucose. A sulfate-free synthetic minimal medium (15) was used for metabolic labeling of yeast cells. The gene for the C-terminally HA (influenza hemagglutinin) epitope-tagged version of Mnl1p was cloned by PCR from the yeast genomic DNA. The amplified DNA was subcloned into a yeast multicopy plasmid pYO326 (16) to generate pKNM1, which expresses the Mnl1p-HA fusion protein. A null allele of MNL1 was constructed by the PCR-based gene disruption using the HIS3 gene of Candida glabrata (a gift from Dr. Satoshi Harashima) as described previously (17, 18). The Delta mnl1 strains derived from SEY6210 and KYSC1 were named SNY1079 and SNY1080, respectively.

ERAD Assays-- Metabolic labeling of yeast cells with Tran35S-label (ICN) and preparation of cell extracts were performed as described previously (19). Immunoprecipitation was performed as described by Nishikawa et al. (20). Yeast microsome fractions were prepared from wild-type cells and Delta mnl1 cells (9) and the yeast cytosol S100 fraction from yeast BJ3505 cells. 35S-Labeled mutant prepro-alpha factor lacking the three consensus glycosylation sites (Delta Gppalpha F) (9) was synthesized in vitro using a yeast cell-free translation. The in vitro ERAD assay was performed as described previously (9).

Fluorescence Microscopy-- Immunofluorescent staining of yeast cells was performed as described previously (20) with minor modifications. Incubation with the primary and the secondary antibodies was performed as follows: 1) 1:250 dilution of the rabbit anti-BiP polyclonal antibodies (21) and 1:250 dilution of the 16B12 mouse monoclonal antibody and 2) 1:100 dilution of the rhodamine-conjugated goat anti-rabbit IgG antibody, 1:100 dilution of the fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibody.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mnl1p Is an ER Protein-- MNL1 (YHR204W) on yeast chromosome VIII encodes an alpha -mannosidase-like protein, which is 796 amino acids long, with a calculated molecular weight of 92,206. The predicted amino acid sequence of Mnl1p shows 25% identity with Mns1p, yeast alpha 1,2-mannosidase that converts glycosyl residues from Man9GlcNAc2 to Man8GlcNAc2 in the ER (Fig. 1A). Mns1p is a type II ER transmembrane glycoprotein with its N-terminal hydrophobic segment functioning as a signal-anchor sequence. The question of whether the segment of hydrophobic residues 1-21 (Fig. 1A) functions as a signal-anchor sequence to make Mnl1p a type-II membrane protein should await future studies. The amino acid residues involved in the substrate binding, Ca2+ binding, and catalytic activity in Mns1p (22) are conserved in Mnl1p except for the two cysteine residues (Cys340 and Cys385 in Mns1p) that form a disulfide bridge and are essential for the alpha -mannosidase activity (13, 22). The lack of the essential cysteine residues in Mnl1p suggests that Mnl1p is unlikely to have the alpha -mannosidase activity. The C-terminal part of Mnl1p shows no homology with any proteins deposited in the data base.


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Fig. 1.   Properties of Mnl1p. A, predicted amino acid sequence of yeast Mnl1p aligned to the sequence of a homologous yeast alpha -mannosidase, Mns1p. Identical amino acids are boxed. Hydrophobic segments are indicated with horizontal bars and putative N-linked glycosylation sites with dots. Two conserved cysteine residues that are essential for the alpha -mannosidase activity are indicated with asterisks. B, localization of Mnl1p by immunofluorescence microscopy. Cells of SEY6210/pKNM1 were grown in SCD medium (minimal medium containing 2% (w/v) glucose and 0.5% (w/v) casamino acids) lacking uracil at 30 °C and analyzed by double label immunofluorescence microscopy using anti-BiP polyclonal antibodies and the 16B12 monoclonal antibody. Panels a, b, and c show the same field of the fluorescent images stained with the anti-BiP antibodies, and the 16B12 antibody, and 4',6-diamidino-2-phenylindole, respectively. Bar, 2 µm.

To assess the functions of Mnl1p, we first analyzed its subcellular location by indirect immunofluorescent microscopy. Mnl1p was tagged at the C terminus with the HA epitope for recognition by the monoclonal antibody 16B12. Cells that express the HA-tagged version of Mnl1p from a multicopy plasmid were fixed, permeabilized, and subjected to staining with the anti-HA antibody. Staining with the anti-HA antibody showed perinuclear staining with tubular extensions in the cytoplasm (Fig. 1B, panel b). This staining is typical for yeast ER proteins, and nearly identical staining was observed with the anti-BiP antibodies (Fig. 1B, panel a). Cells that contain a multicopy plasmid without expression of the HA-tagged Mnl1p did not show such staining (data not shown). These results indicate that Mnl1p resides exclusively in the ER.

Deletion of MNL1 Causes a Defect in ERAD of CPY*-- The trimming of N-linked oligosaccharides is critical for proteasome-dependent ERAD of misfolded glycoproteins. For example in yeast, removal of alpha 1,2-glucose by glucosidase I and glucosidase II and that of alpha 1,2-mannnose by ER-alpha 1,2-mannosidase, Mns1p, to yield Man8GlcNAc2 are essential for the efficient degradation of CPY*, mutated and therefore misfolded carboxypeptidase Y (10, 23). Although Mnl1p is not expected to have the alpha -mannosidase activity, its homology with Mns1p raises the possibility that Mnl1p is involved in the recognition of specific oligosaccharide structures and/or ERAD of misfolded glycoproteins. We thus analyzed the effects of deletion of the MNL1 gene on ERAD of a model misfolded protein, CPY*, in vivo. The Delta mnl1 null mutant strain did not show any detectable growth phenotypes as reported previously (24).

CPY is known to receive distinct posttranslational modifications in different cellular compartments on its transport pathway to the vacuole (25, 26). On translocation across the ER membrane, a prepro form of CPY (prepro-CPY) receives proteolytic cleavage of the signal sequence and addition of four N-linked oligosaccharide chains to generate a 67-kDa ER form, p1CPY. In the Golgi complex, p1CPY is converted to a 69-kDa form, p2CPY, by addition of mannose to the N-linked oligosaccharide chains (26). After reaching the vacuole, p2CPY becomes a 61-kDa mature form, mCPY (26, 27). When wild-type cells or Delta mnl1 cells were metabolically labeled with 35S-containing amino acids for 5 min at 30 °C, the p1 form of labeled CPY* was recovered as pellets by immunoprecipitation (Fig. 2A, upper panel, chase 0 min). When the fate of labeled p1CPY* in wild-type cells was followed, the amount of p1CPY* decreased rapidly without changing its molecular mass during the chase period. This indicates that misfolded p1CPY* was prevented from its exit from the ER for the Golgi complex and was degraded efficiently with a half-life of 18 min at 30 °C (Fig. 2, A and B). In contrast, the p1 form of labeled CPY* in Delta mnl1 cells was degraded 3-fold more slowly than in wild-type cells (Fig. 2B). This suggests that Mnl1p is, whether directly or indirectly, involved in ERAD of CPY*.


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Fig. 2.   Deletion of MNL1 causes retardation of the degradation of CPY*. A, wild-type cells (KYSC1, wt) and Delta mnl1 cells (SNY1080, Delta mnl1) were pulse-labeled with 35S-containing amino acids for 5 min at 30 °C and chased for the indicated times. Cell extracts were prepared from the labeled cells and subjected to immunoprecipitation with the anti-CPY antibodies. The immunoprecipitated proteins were incubated with (+) or without (-) 0.17 unit/ml endoglycosidase H (EndoH) at 37 °C for 22 h and analyzed by SDS-PAGE and radioimaging with a Storm 860 image analyzer (Molecular Dynamics). p1, p1CPY*; p1', p1'CPY*; Delta Gp1, a deglycosylated form of p1CPY* and p1'CPY*. B, quantification of the results shown in A (- EndoH). The amount of CPY* at 0-min chase is set to 100%. Data represent the average of the results of two independent assays.

We also noticed that p1CPY* in Delta mnl1 null mutant cells was converted to a higher molecular weight form, which was designated as p1'CPY* and was observed as a smear on the SDS-PAGE gel, during the chase period (Fig. 2A). When p1'CPY* was treated with endoglycosidase H, it was converted to a deglycosylated form with the same electrophoretic mobility as that for p1CPY* (Fig. 2A). This means that p1'CPY* arose from glycosyl modifications of p1CPY*. As a control, we confirmed that the deletion of MNL1 does not affect the exit of p1CPY from the ER for the Golgi complex and for the vacuole (not shown).

A possible explanation for the glycosyl modifications of p1CPY* in Delta mnl1 cells is that a part of misfolded p1CPY* escaped the ER and received addition of alpha 1right-arrow6- and/or alpha 1right-arrow3-linked mannose to core oligosaccharides in the Golgi complex (25, 28). To test if p1'CPY* had been modified by the Golgi enzymes, reactivity of p1'CPY* to the anti-alpha 1right-arrow6 mannose and the anti-alpha 1right-arrow3 mannose antibodies was examined. Wild-type cells expressing CPY or CPY* and Delta mnl1 cells expressing CPY* were metabolically labeled for 5 min and chased, and the CPY or CPY* species were immunoprecipitated with the anti-CPY antibodies from the cell extracts. The immunoprecipitated proteins were subjected to the second-round immunoprecipitation with the antibodies against CPY, alpha 1right-arrow6 mannose, or alpha 1right-arrow3 mannose. As shown in Fig. 4, p1'CPY* in Delta mnl1 cells was immunoprecipitated with the anti-alpha 1right-arrow6 mannose antibodies but not with the anti-alpha 1right-arrow3 mannose antibodies (Fig. 3, lanes 14 and 15), whereas p1CPY* was not recognized by these antibodies (Fig. 3, lane 13). p1CPY* in wild-type cells, which was retained in the ER and therefore not glycosylated by the Golgi enzymes, was not precipitated with the anti-alpha 1right-arrow6 mannose or anti-alpha 1right-arrow3 mannose antibodies (Fig. 3, lanes 9 and 10). As a control, it was confirmed that mCPY in wild-type cells, which had received carbohydrate modification in the Golgi complex, were precipitated with these antibodies (Fig. 3, lanes 4 and 5). These results indicate that p1'CPY* was modified by the early Golgi enzymes in Delta mnl1 cells and that it is the intermediate form of the mannose addition in the Golgi complex. In other words, deletion of the MNL1 gene allowed exit of a fraction of p1CPY*, which is strictly retained in the ER in wild-type cells, from the ER for at least the early Golgi cisternae (29).


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Fig. 3.   CPY* becomes an early-Golgi glycosylated form in Delta mnl1 cells. Wild-type cells (KYSC1, wild-type) expressing CPY (CPY/MNL1), wild-type cells expressing CPY* (CPY*/MNL1), and Delta mnl1 cells (SNY1080, Delta mnl1) expressing CPY* (CPY*/Delta mnl1) were pulse-labeled with 35S-containing amino acids for 5 min at 30 °C and chased for the indicated times. Cell extracts were prepared from the labeled cells and subjected to immunoprecipitation with the anti-CPY antibodies (1st antibody). The immunocomplexes were solubilized, and aliquots were subjected to the second round of immunoprecipitation with the indicated antibodies (2nd antibody; the 1-6 and 1-3 antibodies are specific for the alpha 1, 6-, and alpha 1,3-mannose linkages). The amounts of cell extracts for lanes 2-5, 7-10, and 12-15 were twice as much as those for lanes 1, 6, and 11, respectively. The immunoprecipitated proteins were analyzed by SDS-PAGE and radioimaging. p1, p1CPY; p1', p1'CPY*; p2, p2CPY; m, mCPY.

Mnl1p Is Not Involved in ERAD of Delta Gpalpha F-- We next asked if Mnl1p is involved in ERAD of misfolded but unglycosylated proteins. For this purpose, we performed in vitro export/degradation assays with an unglycosylated mutant form (Delta Gpalpha F) of the yeast alpha -mating pheromone, pro-alpha -factor (palpha F), as a substrate (8, 9, 30, 31). A radiolabeled precursor form of Delta Gpalpha F (Delta Gppalpha F) was translocated into microsomes prepared from the Delta mnl1 strain or from the isogenic wild-type strain. Upon signal sequence cleavage, the resulting product, Delta Gpalpha F, becomes an ERAD substrate when the washed vesicles are incubated in the presence of the cytosol and ATP (9). When this assay was performed using microsomes prepared from Delta mnl1 cells, degradation of Delta Gpalpha F was as efficient as that with wild-type microsomes at 30 °C (Fig. 4A).


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Fig. 4.   Deletion of MNL1 does not affect the degradation of Delta Gpalpha F either in vitro or in vivo. A, in vitro ERAD assays for radiolabeled Delta Gppalpha F. 35S-Labeled Delta Gppalpha F was translocated into microsomes prepared from wild-type (SEY6210, wt) and Delta mnl1 (SNY1079, Delta mnl1) cells at 20 °C for 50 min. Microsomes containing Delta Gpalpha F were collected by centrifugation, washed once with buffer 88 (20 mM HEPES-KOH, pH 6.8, 250 mM sorbitol, 150 mM KOAc, 5 mM Mg(OAc)2) (9), and resuspended in buffer 88. The microsomes were further incubated in buffer 88 containing an ATP-regenerating system (1 mM ATP, 50 µM GDP-mannose, 40 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase) and the yeast cytosol (5 mg of protein/ml) at 30 °C. At the indicated time points, proteins were precipitated with trichloroacetic acid and subjected to SDS-PAGE in the presence of 4 M urea. The upper band is Delta Gppalpha F, which is bound to the cytosolic surface of the microsomes and escaped ERAD. B, quantification of the results shown in A. The amount of Delta Gpalpha F at 0-min incubation was set to 100%. Data represent the average of the results of two independent assays. C, in vivo ERAD assays for radiolabeled palpha F. Wild-type (SEY6210, wt) cells and Delta mnl1 (SNY1079, Delta mnl1) cells were pulse-labeled with 35S-containing amino acids for 5 min at 30 °C in the presence of 10 µg/ml of tunicamycin, chased for the indicated times, and subjected to immunoprecipitation with the anti-alpha F antibodies. D, quantification of the results shown in C. The amount of Delta Gpalpha F at 0-min chase was set to 100%. Data represent the average of the results of two independent assays.

In parallel with these in vitro degradation assays, we followed the degradation of palpha F in vivo by pulse-chase experiments. The previous study showed that tunicamycin, an inhibitor of the N-linked glycosylation in the ER, causes formation of the unglycosylated and therefore misfolded alpha F precursor (32).2 The unglycosylated palpha F is rapidly degraded in a Sec61p-dependent manner, suggesting that it is a substrate for ERAD in vivo ((32); Fig. 4C). Wild-type cells and Delta mnl1 cells were metabolically labeled for 5 min in the presence of tunicamycin, and palpha F was recovered as pellets by immunoprecipitation by the anti-alpha -factor antibodies. As shown in Fig. 4B, unglycosylated palpha F was rapidly degraded in Delta mnl1 cells with nearly the same kinetics as that for wild-type cells. Taken together, these results of the in vivo as well as the in vitro degradation assays strongly suggest that Mnl1p is not required for ERAD of unglycosylated proteins.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we have characterized the functions of Mnl1p, an alpha -mannosidase-like protein in the ER. Deletion of the MNL1 gene led to retardation of ERAD of CPY*, a misfolded glycoprotein, but not of Delta Gpalpha F, a misfolded unglycosylated protein, suggesting that Mnl1p plays important roles in ERAD of misfolded proteins with N-linked oligosaccharides. Then what is the role of Mnl1p in ERAD? The lack of two cysteine residues strongly suggests that Mnl1p does not have the alpha -mannosidase activity. Mnl1p may be involved in the recognition of oligosaccharide structures, including Man8GlcNAc2, which is essential for ERAD substrates, of misfolded proteins. Indeed, a recently identified mammalian counterpart of Mnl1p does not have the mannosidase activity and probably functions as a lectin that specifically recognizes the oligosaccharide structure of Man8GlcNAc2 (34).

The fate of CPY* was followed in detail when its degradation was impaired by the deletion of the MNL1 gene. Whereas CPY* was partly retained as p1CPY*, the ER form to be degraded, a part of p1CPY* molecules were converted to the early-Golgi form, p1'CPY*, containing alpha 1right-arrow6 mannose. This indicates that at least some p1CPY* was released from the ER to the Golgi complex. However, since alpha 1right-arrow3 mannose was not attached to p1'CPY* even after the prolonged chase, CPY* most likely undergoes recycling through the early-Golgi cisternae into the ER. Deletion of DER1 encoding an ER protein involved in ERAD also leads to escape of some CPY* from the ER for the early-Golgi compartment (33). Therefore ER-resident proteins that are involved in ERAD are also responsible for retaining their substrates in the ER probably by their affinity for misfolded proteins.

    ACKNOWLEDGEMENTS

We thank Dr. Tohru Yoshihisa in our laboratory for discussions; Dr. Akihiko Nakano for the antisera against palpha F, alpha 1right-arrow3 mannose, and alpha 1right-arrow6 mannose; Dr. Jeffrey L. Brodsky for the plasmid for in vitro translation of Delta Gppalpha F; and Dr. Satoshi Harashima for the HIS3 gene of C. glabrata.

    FOOTNOTES

* This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan. Tel.: 81-52789-2490; Fax: 81-52789-2947; E-mail: endo@biochem.chem.nagoya-u.ac.jp.

Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.C100023200

2 S. Nishikawa, S. W. Fewell, Y. Kato, J. L. Brodsky, and T. Endo, unpublished results.

    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; CPY, carboxypeptidase Y; CPY*, a mutated version of carboxypeptidase Y; palpha F, pro-alpha factor; ppalpha F, prepro-alpha factor; Delta Gpalpha F, an unglycosylated mutant form of prepro-alpha factor; Delta Gppalpha F, an unglycosylated mutant form of prepro-alpha factor; HA, hemagglutinin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Hammond, C., and Helenius, A. (1995) Curr. Opin. Cell Biol. 7, 523-529[CrossRef][Medline] [Order article via Infotrieve]
2. Ellgaard, L., Molinari, M., and Helenius, A. (1999) Science 286, 1882-1888[Abstract/Free Full Text]
3. Brodsky, J. L., and McCracken, A. A. (1999) Semin. Cell Dev. Biol. 10, 507-513[CrossRef][Medline] [Order article via Infotrieve]
4. Plemper, R. K., and Wolf, D. H. (1999) Trends Biochem. Sci. 24, 266-270[CrossRef][Medline] [Order article via Infotrieve]
5. Romisch, K. (1999) J. Cell Sci. 112, 4185-4191[Abstract/Free Full Text]
6. Sommer, T., and Wolf, D. H. (1997) FASEB J. 11, 1227-1233[Abstract/Free Full Text]
7. Wiertz, E. J., Tortorella, D., Bogyo, M., Yu, J., Mothes, W., Jones, T. R., Rapoport, T. A., and Ploegh, H. L. (1996) Nature 384, 432-438[CrossRef][Medline] [Order article via Infotrieve]
8. Pilon, M., Schekman, R., and Romisch, K. (1997) EMBO J. 16, 4540-4548[Abstract/Free Full Text]
9. McCracken, A. A., and Brodsky, J. L. (1996) J. Cell Biol. 132, 291-298[Abstract]
10. Jakob, C. A., Burda, P., Roth, J., and Aebi, M. (1998) J. Cell Biol. 142, 1223-1233[Abstract/Free Full Text]
11. Su, K., Stoller, T., Rocco, J., Zemsky, J., and Green, R. (1993) J. Biol. Chem. 268, 14301-14309[Abstract/Free Full Text]
12. Liu, Y., Choudhury, P., Cabral, C. M., and Sifers, R. N. (1999) J. Biol. Chem. 274, 5861-5867[Abstract/Free Full Text]
13. Lipari, F., and Herscovics, A. (1996) J. Biol. Chem. 271, 27615-27622[Abstract/Free Full Text]
14. Robinson, J. S., Klionsky, D. J., Banta, L. M., and Emr, S. D. (1988) Mol. Cell. Biol. 8, 4936-4948[Medline] [Order article via Infotrieve]
15. Rothblatt, J. A., Deshaies, R. J., Sanders, S. L., Daum, G., and Schekman, R. (1989) J. Cell Biol. 109, 2641-2652[Abstract]
16. Qadota, H., Ishii, I., Fujiyama, A., Ohya, Y., and Anraku, Y. (1992) Yeast 8, 735-741[Medline] [Order article via Infotrieve]
17. Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F., and Cullin, C. (1993) Nucleic Acids Res. 21, 3329-3330[Medline] [Order article via Infotrieve]
18. Kitada, K., Yamaguchi, E., and Arisawa, M. (1995) Gene (Amst.) 165, 203-206[CrossRef][Medline] [Order article via Infotrieve]
19. Nishikawa, S., and Nakano, A. (1991) Biochim. Biophys. Acta 1093, 135-143[CrossRef][Medline] [Order article via Infotrieve]
20. Nishikawa, S., Hirata, A., and Nakano, A. (1994) Mol. Biol. Cell 5, 1129-1143[Abstract]
21. Nishikawa, S., and Endo, T. (1997) J. Biol. Chem. 272, 12889-12892[Abstract/Free Full Text]
22. Vallée, F., Lipari, F., Yip, P., Sleno, B., Herscovics, A., and Howell, P. L. (2000) EMBO J. 19, 581-588[Abstract/Free Full Text]
23. Knop, M., Hauser, N., and Wolf, D. H. (1996) Yeast 12, 1229-1238[CrossRef][Medline] [Order article via Infotrieve]
24. Winzeler, E. A., Shoemaker, D. D., Astromoff, A., Liang, H., Anderson, K., Andre, B., Bangham, R., Benito, R., Boeke, J. D., Bussey, H., Chu, A. M., Connelly, C., Davis, K., Dietrich, F., Dow, S. W., El Bakkoury, M., Foury, F., Friend, S. H., Gentalen, E., Giaever, G., Hegemann, J. H., Jones, T., Laub, M., Liao, H., Davis, R. W., et al.. (1999) Science 285, 901-906[Abstract/Free Full Text]
25. Stevens, T. H., Rothman, J. H., Payne, G. S., and Schekman, R. (1986) J. Cell Biol. 102, 1551-1557[Abstract]
26. Deshaies, R. J., and Schekman, R. (1987) J. Cell Biol. 105, 633-645[Abstract]
27. Hemmings, B. A., Zubenko, G. S., Hasilik, A., and Jones, E. W. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 435-439[Abstract]
28. Franzusoff, A., and Schekman, R. (1989) EMBO J. 8, 2695-2702[Abstract]
29. Herscovics, A., and Orlean, P. (1993) FASEB J. 7, 540-550[Abstract/Free Full Text]
30. Caplan, S., Green, R., Rocco, J., and Kurjan, J. (1991) J. Bacteriol. 173, 627-635[Medline] [Order article via Infotrieve]
31. Werner, E. D., Brodsky, J. L., and McCracken, A. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13797-13801[Abstract/Free Full Text]
32. Zhou, M., and Schekman, R. (1999) Mol. Cell 4, 925-934[Medline] [Order article via Infotrieve]
33. Knop, M., Finger, A., Braun, T., Hellmuth, K., and Wolf, D. H. (1996) EMBO J. 15, 753-756[Abstract]
34. Hosokawa, N., Wada, I., Hasegawa, K., Yorihuzi, T., Tremblay, L. O., Herscovics, A., and Nagata, K. (2001) EMBO Rep., in press


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