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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Abstract |
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Key words: de-N-glycosylation/endoplasmic reticulum/glycopeptide export/quality control
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Introduction |
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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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Results |
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(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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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 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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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 (
252277 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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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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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 W3031a (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 dEtudes 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(
252277) (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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Abbreviations |
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Footnotes |
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References |
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Ali, B.R.S., Tjernberg, A., Chait, B.T., and Field, M.C. (2000) A microsomal GTPase is required for glycopeptide export from the mammalian endoplasmic reticulum. J. Biol. Chem., 275, 3322233230.
Antebi, A. and Fink, G.R. (1992) The yeast Ca2+-ATPase homologue, PMR1, is required for normal Golgi function and localizes in a novel Golgi-like distribution. Mol. Biol. Cell, 3, 633654.[Abstract]
Baker, D., Hicke, L., Rexach, M., Schleyer, M., and Schekman, R. (1988) Reconstitution of SEC gene product-dependent intercompartmental protein transport. Cell, 54, 335344.[Medline]
Biederer, T., Volkwein, C., and Sommer, T. (1997) Role of Cue1p in ubiquitination and degradation at the ER surface. Science, 278, 18061809.
Bordallo, J., Plemper, R.K., Finger, A., and Wolf, D.H. (1998) Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins. Mol. Biol. Cell., 9, 209222.
Brodsky, J.L. and McCracken, A.A. (1999) ER protein quality control and proteasome-mediated protein degradation. Semin. Cell Dev. Biol., 10, 507513.[CrossRef][ISI][Medline]
Brodsky, J.L., Werner, E.D., Dubas, M.E., Goeckeler, J.L., Kruse, K.B., and McCracken, A.A. (1999) The requirement for molecular chaperones during endoplasmic reticulum-associated protein degradation demonstrates that protein export and import are mechanistically distinct. J. Biol. Chem., 274, 34533460.
Cabral, C.M., Liu, Y., and Sifers, R.N. (2001) Dissecting glycoprotein quality control in the secretory pathway. Trends Biochem. Sci., 26, 619624.[CrossRef][ISI][Medline]
Casagrande, R., Stern, P., Diehn, M., Shamu, C.E., Osario, M., Zuniga, M., Brown, P.O., and Ploegh, H.L. (2000) Degradation of proteins from the ER of S. cerevisiae requires an intact unfolded protein response pathway. Mol. Cell, 5, 729735.[ISI][Medline]
Cox, J.S., Shamu, C.E., and Walter, P. (1993) Transcriptional induction of gene encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell, 73, 11971206.[ISI][Medline]
Cunningham, K.W. and Fink, G.R. (1994) Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases. J. Cell Biol., 124, 351363.[Abstract]
Dürr, G., Strayle, J., Plemper, R., Elbs, S., Klee, S.K., Catty, P., Wolf, D.H., and Rudolph, H.K. (1998) The medial-Golgi ion pump Pmr1 supplies the yeast secretory pathway with Ca2+ and Mn2+ required for glycosylation, sorting, and endoplasmic reticulum-associated protein degradation. Mol. Biol. Cell, 9, 11491162.
Elble, R. (1992) A simple and efficient procedure for transformation of yeasts. Biotechniques, 13, 1820.[ISI][Medline]
Ellgaard, L. and Helenius, A. (2001) ER quality control: towards an understanding at the molecular level. Curr. Opin. Cell Biol., 13, 431437.[CrossRef][ISI][Medline]
Flynn, G.C., Pohl, J., Flocco, M.T., and Rothman, J.E. (1991) Peptide-binding specificity of the molecular chaperone BiP. Nature, 353, 726730.[CrossRef][ISI][Medline]
Geetha-Habib, M., Noiva, R., Kaplan, H.A., and Lennarz, W.J. (1988) Glycosylation site binding protein, a component of oligosaccharyl transferase, is highly similar to three other 57 kd luminal proteins of the ER. Cell, 54, 10531060.[ISI][Medline]
Ghislain, M., Udvardy, A., and Mann, C. (1993) S. cerevisiae 26S protease mutants arrest cell division in G2/metaphase. Nature, 366, 358362.[CrossRef][ISI][Medline]
Gillece, P., Luz, J.M., Lennarz, W.J., de La Cruz, F.J., and Römisch K. (1999) Export of a cysteine-free misfolded secretory protein from the endoplasmic reticulum for degradation requires interaction with protein disulfide isomerase. J. Cell Biol., 147, 14431456.
Gillece, P., Pilon, M., and Römisch K. (2000) The protein translocation channel mediates glycopeptide export across the endoplasmic reticulum membrane. Proc. Natl Acad. Sci. USA, 97, 46094614.
Hampton, R.Y. (2000) ER stress response: getting the UPR hand on misfolded proteins. Curr. Biol., 10, R518R521.[CrossRef][ISI][Medline]
Hampton, R.Y., Gardner, R.G., and Rine, J. (1996) Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein. Mol. Biol. Cell, 7, 20292044.[Abstract]
Helenius, A. and Aebi, M. (2001) Intracellular functions of N-linked glycans. Science, 291, 23642369.
Hill, J.M., Myers, A.M., Koerner, T.J., and Tzagoloff, A. (1993) Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast, 2, 163167.
Hiller, M.M., Finger, A., Schweiger, M., and Wolf, D.H. (1996) ER degradation of a misfolded luminal protein by the cyotosolic ubiquitin-proteasome pathway. Science, 273, 17251728.
Hu, Z., Bonifas, J.M., Beech, J., Bench, G., Shigihara, T., Ogawa, H., Ikeda, S., Mauro, T., and Epstein, E.H. (2000) Mutations in ATP2C1, encoding a calcium pump, cause Hailey-Hailey disease. Nature Genet., 24, 6165.[CrossRef][ISI][Medline]
Jakob, C.A., Burda, P., Roth, J., and Aebi, M. (1998) Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure. J. Cell Biol., 142, 12231233.
Jakob, C.A., Bodmer, D., Spirig, U., Battig, P., Marcil, A., Dignard, D., Bergeron, J.J.M., Thomas, D.Y., and Aebi, M. (2001) Htm1p, a mannodisase-like protein, is involved in glycoprotein degradation in yeast. EMBO Rep., 2, 423430.
Karaivanova , V.K. and Spiro, R.G. (2000) Effect of proteasome inhibitors on the release into the cytosol of free polymannose oligosaccharides from glycoproteins. Glycobiology, 10, 727735.
Knop, M., Finger, A., Braun, T., Hellmuth, K., and Wolf, D.H. (1995) Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast. EMBO J., 15, 753763.[Abstract]
Knop, M., Hauser, N., and Wolf, D.H. (1996) N-glycosylation affects endoplasmic reticulum degradation of a mutated derivative of carboxypeptidase yscY in yeast. Yeast, 12, 12291238.[CrossRef][ISI][Medline]
LaMantia, M.L. and Lennarz, W.J. (1993) The essential function of yeast protein disulfide isomerase does not reside in its isomerase activity. Cell, 74, 899908.[ISI][Medline]
Lehner, P.J., Hewitt, E.W., and Römisch, K. (2000) Antigen presentation: peptides and proteins scramble for the exit. Curr. Biol., 10, R839R842.[CrossRef][ISI][Medline]
Longtine, M.S., McKenzie, I.A. Damarini, D.J., Shah, N.G., Wach, A., Brachat, A., Phillipsen, P., and Pringle, J.R. (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast, 14, 953961.[CrossRef][ISI][Medline]
Luz, J.M. and Lennarz, W.J. (1998) The nonactive site cysteine residues of yeast protein disulfide isomerase are not required for cell viability. Biochem. Biophys. Res. Commun., 248, 621627.[CrossRef][ISI][Medline]
Mayer, T.U., Braun T., and Jentsch, S. (1998) Role of the proteasome in membrane extraction of a short-lived ER-transmembrane protein. EMBO J., 15, 32513257.[CrossRef]
McCracken, A.A. and Brodsky, J.L. (1996) Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin and ATP. J. Cell Biol., 132, 291298.[Abstract]
Moore, S.E.H. (1999) Oligosaccharide transport: pumping waste from the ER into lysosome. Trends Cell Biol. 9, 441446.[CrossRef][ISI][Medline]
Mori, K. (2000) Tripartite management of unfolded proteins in the endoplasmic reticulum. Cell, 101, 451454.[ISI][Medline]
Nakatsukasa, K., Nihiskawa, S., Hosokawa, N., Nagata, K., and Endo T. (2001) Mnl1p, a alpha-mannosidase-like protein in yeast Saccharomyces cerevisiae, is required for endoplasmic-associated degradation of glycoproteins. J. Biol. Chem., 2776, 86358638.[CrossRef]
Okorokov, L.A. and Lehle, L. (1998) Ca2+-ATPases of Saccharomyces cerevisiae: diversity and possible role in protein sorting. FEMS Microbiol. Lett., 162, 8391.[CrossRef][ISI][Medline]
Okorokov, L.A., Tanner, W., and Lehle, L. (1993) A novel primary Ca2+-dependent system from Saccharomyces cerevisiae. Eur. J. Biochem., 216, 573577.[Abstract]
Parlati, F., Dominguez, M., Bergeron, J.J.M., and Thomas, D.Y. (1995) Saccharomyces cerevisiae CNE1 encodes an endoplasmic reticulum (ER) membrane protein with sequence similarity to calnexin and calreticulin and functions as a constituent of the ER quality control apparatus. J. Biol. Chem., 270, 244253.
Patil, C. and Walter, P. (2001) Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals. Curr. Opin. Cell Biol., 13, 349355.[CrossRef][ISI][Medline]
Pilon, M., Schekman, R., and Römisch, K. (1997) Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation. EMBO J., 16, 45404548.
Plemper, R.K. and Wolf, D.H. (1999) Retrograde protein translocation: eradication of secretory proteins in health and disease. Trends Biochem. Sci., 24, 266270.[CrossRef][ISI][Medline]
Plemper, R.K., Bohmler, S, Bordallo, J, Sommer, T., and Wolf DH. (1997) Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation. Nature, 388, 891895.[CrossRef][ISI][Medline]
Plemper, R.K., Enger, R., Kuchler, K., and Wolf, D.H. (1998) Endoplasmic reticulum degradation of a mutated ATP-binding cassette transporter Pdr5 proceeds in a concerted action of Sec61 and the proteasome. J. Biol. Chem., 273, 3284832456.
Plemper, R.K., Bordallo, J., Deal, P.M., Taxis, C., Hitt, R., and Wolf, D.H. (1999) Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a retro-translocation complex mediating protein transport for ER degradation. J. Cell Sci., 112, 41234134.
Rexach, M.F. and Schekman, R.W. (1991) Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles. J. Cell Biol., 114, 219229.[Abstract]
Römisch, K. (1999) Surfing the Sec61 channel: bidirectional protein translocation across the ER membrane. J. Cell Sci., 112, 41854191.
Römisch, K. and Ali, B.R.S. (1997) Similar processes mediate glycopeptide export from the endoplasmic reticulum in mammalian cells and Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA, 94, 67306734.
Römisch, K. and Schekman, R. (1992) Distinct processes mediate glycoprotein and glycopeptide export from the endoplasmic reticulum in Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA, 89, 72277231.[Abstract]
Rose, M., Winston, F., and Hieter, P. (1990) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Rothstein, R. (1991) Targetting, disruption, replacement and allele rescue integrative transformation of yeast. Methods Enzymol., 194, 281301.[ISI][Medline]
Saito, Y., Yamanushi, T., Oka, T., and Nakano, A. (1999) Identification of SEC12, SED4, truncated SEC16, and EKS1/HRD3 as multicopy suppressors of ts mutants of Sar1 GTPase. J. Biochem. (Tokyo), 125, 130137.[Abstract]
Sambrook, J. and Russell D.W. (2001) Molecular cloning. A laboratory manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Scidmore, M.A., Okamura, H.H., and Rose, M.D. (1993) Genetic interaction between KAR2 and SEC63, encoding eukaryotic homologues of DnaK and DnaJ in the endoplasmic reticulum. Mol. Biol. Cell, 4, 11451159.[Abstract]
Sherman, F. (1991) Getting started with yeast. Methods Enzymol., 194, 321.[ISI][Medline]
Spee, P. and Neefjes, J. (1997) TAP-translocated peptides specifically bind proteins in the endoplasmic reticulum, including gp96, protein disulfide isomerase and calreticulin. Eur. J. Immunol., 27, 24412449.[ISI][Medline]
Sudbrak, R., Brown, J., Dobson-Stone, C., Carter, S., Ramser, J., White, J., Healy, E., Dissanayake, M., Larregue, M., Perrussel, M., and others. (2000) Hailey-Hailey disease is caused by mutations in ATP2C1 encoding a novel Ca(2+) pump. Hum. Mol. Genet., 9, 11311140.
Suzuki, T. and Lennarz, W.J. (2000) In yeast the export of small glycopeptides from the endoplasmic reticulum into the cytosol is not affected by the structure of their oligosaccharide chains. Glycobiology, 10, 5158.
Suzuki, T., Seko, A., Kitajima, K., Inoue Y., and Inoue S. (1994) Purification and enzymatic properties of peptide:N-glycanase from C3H mouse-derived L-929 fibroblast cells. Possible widespread occurrence of post-translational remodification of proteins by N-deglycosylation. J. Biol. Chem., 269, 1761117618.
Suzuki, T., Yan, Q., and Lennarz, W.J. (1998a) Complex, two-way traffic of molecules across the membrane of the endoplasmic reticulum. J. Biol. Chem., 273, 1008310086.
Suzuki, T., Park, H., Kitajima, K., and Lennarz, W.J. (1998b) Peptides glycosylated in the endoplasmic reticulum of yeast are subsequently deglycosylated by a soluble peptide:N-glycanase activity. J. Biol. Chem., 273, 2152621530.
Suzuki, T., Park, H., Hollingsworth, N.M., Sternglanz, R., and Lennarz, W.J. (2000) PNG1, a yeast gene encoding a highly conserved peptide:N-glycanase. J. Cell Biol., 149, 10391052.
Suzuki, T., Park, H., and Lennarz, W. J. (2002) Cytoplasmic Peptide:N-glycanase; occurrence, primary structure and potential functions. FASEB J., 16, 635641.
Ton, V.-K., Mandal, D., Vahadji, C., and Rao, R. (2002) Functional expression in yeast of the human secretory pathway Ca2+, Mn2+-ATPase defective in Hailey-Hailey disease. J. Biol. Chem., 277, 64226427.
Tsai, B., Rodighiero, C., Lencer, W.I., and Rapoport, T.A. (2001) Protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin. Cell, 104, 937948.[ISI][Medline]
Urano, F., Bertolotti , A., and Ron, D. (2000) IRE1 and efferent signaling from the endoplasmic reticulum. J. Cell Sci., 113, 36973702.
Wiertz, E.J.H.J., Tortorella, D., Bogyo, M., Yu, J., Mothes, W., Jones, T.R., Rapoport, T.A., and Ploegh, H.L. (1996) Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction. Nature, 384, 432438.[CrossRef][ISI][Medline]
Wilhovsky, S., Gardner, R., and Hampton, R. (2000) HRD gene dependence of endoplasmic reticulum-associated degradation. Mol. Biol. Cell, 11, 16971708.
Yan, Q., Prestwich, G.D., and Lennarz, W.J. (1999) The Ost1p subunit of yeast oligosaccharyl transferase recognizes the peptide glycosylation site sequence, -Asn-X-Ser/Thr-. J. Biol. Chem., 274, 50215025.
Yu, H. and Kopito, R. R. (1999) The role of multiubiquitination in dislocation and degradation of the subunit of the T cell antigen receptor. J. Biol. Chem., 274, 3685236858.
Yu, H., Kaung, G., Kobayashi, S., and Kopito, R.R. (1997) Cytosolic degradation of T-cell receptor chains by the proteasome. J. Biol. Chem., 272, 2080020804.