Report |
Address correspondence to Dr. Elizabeth Sztul, Dept. of Cell Biology, MCLM Room 668, 1530 3rd Ave. South, Birmingham, AL 35294. Tel.: (205) 934-1465. Fax: (205) 975-9131. E-mail: esztul{at}uab.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: ER sorting; proteasomal degradation; CFTR; ERAD; yeast
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The exact pathway for degradation of transmembrane ERAD substrates from the ER is unclear, but there is evidence to suggest that at least some retained proteins are sorted into specialized ER subdomains before degradation (Kamhi-Nesher et al., 2001; Kiser et al., 2001; Zhang et al., 2001). It is unknown what molecular mechanisms regulate sequestration of ERAD substrates, and whether such sequestration is required for ER retrotranslocation and proteasomal degradation. To explore these questions, we used the yeast Saccharomyces cerevisiae and mutant strains defective in the Sar1p/COPII sorting machinery and a known ERAD substrate, CFTR, as our model system.
Sar1p/COPII-mediated selection of ER proteins for secretory traffic is one of the best understood mechanisms for selective recruitment of proteins in the ER (Antonny and Schekman, 2001). COPII sorting is initiated by Sar1p, a small GTPase (Veldhuisen et al., 1997) that is activated by Sec12p-mediated guanine-nucleotide exchange. Sar1p in its GTP-bound state associates with the ER membrane and recruits the Sec23p/Sec24pCOPII complex and subsequently the Sec13p/Sec31pCOPII complex to the membrane (for review see Barlowe, 2000). The assembly of the COPII coat on ER budding structures is coupled to the selection of transmembrane proteins into the nascent budding structures that will eventually bud from the ER as transport vesicles. COPII function has been examined to date exclusively in the context of secretory traffic.
CFTR is a chloride channel present on the apical surface of epithelial cells lining the respiratory, intestinal, and exocrine tissues (Kirk, 2000). In mammalian cells, only 20% of newly synthesized wild-type CFTR folds correctly and is transported from the ER to the plasma membrane through the secretory pathway, whereas the remaining
80% of CFTR is degraded from the ER through the ubiquitin-proteasome pathway (Jensen et al., 1995; Ward et al., 1995; Moyer et al., 1998; Riordan, 1999). In mammalian cells, CFTR is not sequestered into ER subdomains before degradation because inhibition of proteasomal activity by chemical inhibitors or overtaxing the proteolytic capacity by overexpressing CFTR leads to retrotranslocation of CFTR from the ER and its accumulation in cytosolic aggresomes (Johnston et al., 1998). In yeast, the majority (if not all) of newly synthesized CFTR is also degraded through the ubiquitin-proteasome pathway, but in contrast to mammalian cells, CFTR in yeast is not delivered to aggresomes, but appears sequestered in ER subdomains before degradation (Kiser et al., 2001; Zhang et al., 2001).
Here, we report that the Sar1p/COPII machinery functions in sorting CFTR into ER subdomains before proteasomal degradation, and that such sorting is required for CFTR entry into the degradative pathway. This function of Sar1p/COPII does not involve ER-Golgi traffic. Our results support a model in which the Sar1p/COPII machinery participates in sorting proteins to both the anterograde secretory pathway and the degradative pathway. These findings raise the possibility that all newly synthesized proteins are subjected to a Sar1p/COPII sorting mechanism, irrespective of their ultimate secretory or degradative fate.
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
EGFP-CFTR is sorted to subcompartments of ER before degradation
Previous studies have shown that CFTR tagged at the COOH terminus with GFP (Kiser et al., 2001) or HA (Zhang et al., 2001) is degraded as an integral membrane protein and colocalizes with the ER chaperone Kar2p in punctate ER structures before degradation. To analyze our EGFP-CFTR construct, we first tested its association with membranes. As shown in Fig. 2 A, EGFP-CFTR is detected exclusively in the membrane pellet fraction after medium speed centrifugation, indicating that it is degraded as a membrane-associated form.
|
EGFP-CFTR is detected in relatively large punctate structures that contain Kar2p (Fig. 2 B). The punctate Kar2p distribution in EGFP-CFTRexpressing yeast is distinct from its normal cage-like perinuclear localization in control yeast (Paddon et al., 1996). The effects of EGFP-CFTR expression on intracellular morphology were analyzed by electron microscopy of yeast transformed with either empty or EGFP-CFTRcontaining plasmids. As shown in Fig. 2 C, normal nuclear membrane and subplasma membrane ER structures are evident in the control cell (arrows), in agreement with published images (Zhang et al., 2001). In contrast, EGFP-CFTR cell shows clusters of membranous structures in proximity to the perinuclear ER (arrowheads). Higher magnification shows them to be accumulations of tubular and vesicular elements, morphologically analogous to ER extensions observed in yeast expressing CFTR (Zhang et al., 2001). The relative size of such clusters (average diameter 0.51 µm) corresponds to the approximate size of the fluorescent Kar2p/EGFP-CFTR puncta. Significantly, membrane amplification induced by CFTR (Zhang et al., 2001) or EGFP-CFTR (this work) is distinct from multi-layered ER "karmellae" induced by expression of HMG-CoA reductase (Koning et al., 1996). Together, the data suggest that EGFP-CFTR localizes to subdomains of the ER network that represent penultimate stations before degradation. The results also imply that sequestration into the ER domains is more efficient than subsequent removal by degradation.
Functional Sar1p/COPII machinery is required for sorting EGFP-CFTR to ER subdomains
To determine if the Sar1p/COPII machinery participates in sorting EGFP-CFTR, we examined its localization when COPII function is disrupted. First, we tested the localization of EGFP-CFTR in wild-type, ubc6, and sec181 yeast strains when grown at 24°C (permissive temperature for strains defective in COPII components) and when grown at 39°C (restrictive temperature for strains defective in COPII components). As shown in Fig. 2 D, EGFP-CFTR in the wild-type strain remains as punctate spots at both temperatures. Similarly, the pattern of EGFP-CFTR in the ubc6
strain defective for proteasomal degradation is analogous to that in the wild-type strain. This strongly suggests that EGFP-CFTR is sorted into the punctate structure and maintained there before it is targeted for ubiquitination and degradation. Our data, together with the finding that CFTR remains sequestered in Kar2p-containing subdomains in yeast strain (pre11pre22) defective for proteasomal degradation (Zhang et al., 2001), suggest that the catalytic activity of ubiquitin-conjugating enzyme Ubc6p is not sufficient to remove EGFP-CFTR from ER subdomains, and that ubiquitination and proteasomal degradation are tightly coupled. The same punctate EGFP-CFTR pattern is observed in sec181 strain grown at either temperature, indicating that block in vesicular fusion during ER-Golgi protein transport does not significantly affect EGFP-CFTR sequestration.
Analysis of EGFP-CFTR localization in the sec231ts strain (Kuehn et al., 1998) showed significant temperature-dependent changes (Fig. 2 D). At the permissive temperature, EGFP-CFTR is sequestered into ER subdomains, but at the restrictive temperature, EGFP-CFTR is diffusely distributed throughout the ER in a pattern characteristic of ER proteins (compare enlargement with Kar2p staining in inset; Fig. 2 C). Similar results were obtained when yeast strains mutant in other components of the Sar1p/COPII machinery (described below) were analyzed (unpublished data).
Functional Sar1p/COPII machinery is required for EGFP-CFTR degradation
To uncover whether the Sar1p/COPII machinery is also involved in CFTR degradation, we compared the degradation rate of EGFP-CFTR at permissive (24°C) and restrictive (39°C) temperature in yeast mutant in Sar1p/COPII components. In wild-type strain, EGFP-CFTR is degraded rapidly at both temperatures, with a half-life of 1015 min (Fig. 3 A). The temperature-sensitive strain, sec124ts, is defective in catalyzing GDP/GTP exchange on Sar1p at the restrictive temperature due to a P73L mutation (Barlowe and Schekman, 1993). EGFP-CFTR is degraded rapidly in the sec124ts strain at permissive temperature; with a half-life (10 min) analogous to that in wild-type strain (Fig. 3 B). In contrast, the degradation rate is delayed significantly when the yeast is shifted to the restrictive temperature, with the half-life extending to 4045 min, a threefold increase compared with the permissive temperature. The temperature-sensitive strains sec231ts and sec131ts (Salama et al., 1997) are defective at the restrictive temperature due to an S382L mutation (Yoshihisa et al., 1993), and a mutation that has not yet been identified, respectively. In both cases, COPII function is compromised. EGFP-CFTR is degraded rapidly in both sec231ts and sec131ts strains at the permissive temperature, with half-lives of <15 min (Fig. 3, C and D). In contrast, the degradation rates are significantly delayed in both strains at the restrictive temperatures, with a half-life of
50 min. The significant inhibition of EGFP-CFTR degradation suggests that functional COPII machinery is required for proteasomal degradation of EGFP-CFTR.
|
That Sar1p/COPII is likely to participate in the secretory and the degradative pathways is supported by data obtained from whole genome microarray analysis where many genes involved in secretory traffic were found to be UPR targets (Travers et al., 2000). Initially, the up-regulation was viewed as means to increase traffic in the ER-Golgi recycling degradative pathway for soluble ERAD substrates (Caldwell et al., 2001). However, our work suggests that the up-regulation is also a means to increase components required for ER sequestration of transmembrane ERAD substrates. Specifically relevant to our analysis is the up-regulation of SEC12, SEC13, and SEC24. Thus, it may be that the UPR-mediated up-regulation of these genes may not be a cellular strategy to remove protein from the ER by exporting them, but additionally might promote sorting of retained proteins toward degradation.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
To generate an inducible construct expressing GFP-tagged CFTR in yeast, pEGFP-CFTR plasmid (Moyer et al., 1998) was first digested with SacII, and then treated with Klenow fragment followed by digestion with NheI. The pRSETB/EGFP plasmid (provided by Dr. David Bedwell) was first digested with HindIII and then treated with Klenow fragment followed by digestion with NheI. The EGFP-containing fragment from pRSETB/EGFP and CFTR-containing fragment from pEGFP-CFTR were gel-purified, ligated, and transformed into Eschericia coli. The resulting plasmid was then digested with NheI and EcoRV, and treated with Klenow fragment. The EGFP-CFTRcontaining fragment was gel-purified and cloned into the SmaI site of pCU426CUP1 (Labbe and Thiele, 1999). The final construct was sequenced to ensure the correct open reading frame.
Yeast strains
CTY182 (MATa, ura352, his3200, lys280), CTY252 (MATa, ura352, sec124ts), CTY253 (MATa, ura352, sec131ts), and CTY260 (MAT
, ura352, leu23, 112, sec231ts) were a gift from Dr. Vytas Bankaitis. YHI29/W (MATa, ura3, leu23, 112, his311, 15, cans, GAL1), YHI29/1 (MATa, pre11, ura3, leu23, 112, his311, 15, cans, GAL1), and YWO0346 (MAT
, ura31, leu23, 112, his311, 15, ade21 ocre, trp11, can1100, ubc6::LEU2) were a gift from Dr. Dieter Wolf (Universitat Stuttgart, Stuttgart, Germany). SEY6210 (MAT
ura352 leu23, 112 his3-
200 trp1-
901 lys2801 suc2-
9) and SEY5186 (MAT
sec181 ura352 leu23, 112 GAL+) were a gift from Dr. David Bedwell.
Yeast media were prepared as described previously (Rose et al., 1990). Unless specified, in all experiments, cultures were grown for a minimum of 56 generations to an A600 of no more than 1.0.
Fractionation, immunofluorescence, and electron microscopy
Yeast expressing EGFP-CFTR was fractionated as described previously (Paddon et al., 1996). In brief, cells grown to exponential phase were converted to spheroplasts using yeast lytic enzyme (ICN Biomedicals) in the presence of 20 mM potassium phosphate buffer, pH 7.0, and containing 1.2 M sorbitol. The spheroplasts were washed and disrupted using a Dounce homogenizer in 25 mM Hepes-NaOH buffer, pH 7.4, plus protease inhibitors (Sigma-Aldrich). Cell lysates were centrifuged at 4°C at 500 g for 10 min, and the resulting supernatant was spun at 25,000 g for 15 min. The resulting supernatant was centrifuged at 150,000 g for 1 h. Pellet from each spin was washed with Hepes buffer and saved as P1, P2, and P3, respectively. Protein concentration was measured using a Detergent Compatible Protein Assay kit (Bio-Rad Laboratories).
For immunofluorescent studies, yeast were grown in synthetic complete medium lacking uracil to an OD600 of no more than 1.0. Yeast were fixed by adding formaldehyde to 3% and incubating at either 24°C or 39°C for 30 min. Cells were harvested and washed twice with 0.1 M potassium phosphate, pH 6.5, and once with potassium phosphate buffer containing 1.2 M sorbitol. The cell walls were digested by adding ß-mercaptoethanol to 0.1% and zymolase 20T (U.S. Biological) to 20 µg/ml. After incubating at 30°C for 1 h, cells were washed twice with sorbitol buffer, applied to polylysine-coated coverslips, and were allowed to settle at RT for 20 min. The cells were refixed with 3% formaldehyde for 5 min followed by quenching with 50 mM of NH4Cl for 5 min. Cells were permeabilized with 0.1% Triton X-100 for 10 min and washed twice for 5 min each with blocking buffer (PBS containing 0.2% Tween 20 and 1 mg/ml BSA). Cells were incubated overnight with primary antibody diluted 1:5,000 for anti-Kar2p antibody and 1:2,000 for anti-Kex2p antibody. Coverslips were washed three times with blocking buffer. Cells were incubated with secondary antibody for 1 h and washed as above. Coverslips were mounted on slides in 9:1 glycerol/PBS with 0.1% p-phenylenediamine. Images were acquired with an inverted microscope (Axiovert 30; Carl Zeiss MicroImaging Inc.). IpLab Spectrum software (Signal Analytics Corp.) was used to control image acquisition.
Yeast containing either an empty plasmid or EGFP-CFTRcontaining plasmid were grown to an A600 of 0.5, induced with copper for 2 h, and processed for electron microscopy as described previously (Kaiser and Schekman, 1990). Grids were examined on an electron microscope (model 100CX; JEOL USA, Inc.).
Pulse-chase labeling and immunoprecipitation
Yeast were grown overnight to log phase at 24°C in synthetic complete medium supplemented with appropriate amino acids. EGFP-CFTR expression was induced with 100 µM of CuSO4 for 2 h (Labbe and Thiele, 1999). A total of 32 OD600 U of cells was harvested (3 OD600 U for each time point) and resuspended in 4 ml synthetic complete medium plus 100 µM CuSO4. Each culture was separated into two parts and was incubated with shaking at 24°C or 39°C for 10 min. Cells were then labeled with 200 µCi of [35S]methionine (Trans35S label from ICN Biomedicals) for 20 min. The label was chased with 40 µl of chasing mixture (1 mg/ml methionine, 1 mg/ml cysteine, and 15% yeast extract). 0.4-ml samples were collected at 0, 15, 30, 60, and 90 min and added to 25 µl of 100% TCA (5% final TCA concentration). Samples were mixed, placed on ice for 20 min, and washed twice with ice-cold acetone. Cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.5% SDS, and protease inhibitors) and broken down by vortexing with glass beads. EGFP-CFTR was immunoprecipitated from total cell lysate using a polyclonal anti-NBD1 CFTR antibody (1:250 dilutions). Immunoprecipitates were separated on 6% SDS-PAGE and analyzed by PhosphorImager (Molecular Dynamics, Inc.). EGFP-CFTR signal was quantified using ImageQuant Software (Molecular Dynamics, Inc.), and relative amounts were calculated and plotted using Excel software (Microsoft). For CPY analysis, pulse-chase was done as above, and lysates were immunoprecipitated with anti-CPY antibodies. Immunoprecipitates were resolved on 10% SDS-PAGE and analyzed as above.
SDS-PAGE and immunoblotting
Samples were resolved on 6% SDS-PAGE as described previously (Bebok et al., 1998). Some gels were electrotransferred to nitrocellulose membrane and immunoblotted with antiCOOH-terminal CFTR mAb as described previously (Bebok et al., 1998).
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
Submitted: 15 October 2002
Revised: 6 December 2002
Accepted: 9 December 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antonny, B., and R. Schekman. 2001. ER export: public transportation by the COPII coach. Curr. Opin. Cell Biol. 13:438443.[CrossRef][Medline]
Bannykh, S.I., G.I. Bannykh, K.N. Fish, B.D. Moyer, J.R. Riordan, and W.E. Balch. 2000. Traffic pattern of cystic fibrosis transmembrane regulator through the early exocytic pathway. Traffic. 1:852870.[CrossRef][Medline]
Barlowe, C. 2000. Traffic COPs of the early secretory pathway. Traffic. 1:371377.[CrossRef][Medline]
Barlowe, C., and R. Schekman. 1993. SEC12 encodes a guanine-nucleotide-exchange factor essential for transport vesicle budding from the ER. Nature. 365:347349.[CrossRef][Medline]
Bebok, Z., C. Mazzochi, S.A. King, J.S. Hong, and E.J. Sorscher. 1998. The mechanism underlying cystic fibrosis transmembrane conductance regulator transport from the endoplasmic reticulum to the proteasome includes Sec61beta and a cytosolic, deglycosylated intermediary. J. Biol. Chem. 273:2987329878.
Bordallo, J., and D.H. Wolf. 1999. A RING-H2 finger motif is essential for the function of Der3/Hrd1 in endoplasmic reticulum associated protein degradation in the yeast Saccharomyces cerevisiae. FEBS Lett. 448:244248.[CrossRef][Medline]
Brodsky, J.L., and A.A. McCracken. 1999. ER protein quality control and proteasome-mediated protein degradation. Semin. Cell Dev. Biol. 10:507513.[CrossRef][Medline]
Brodsky, J.L., E.D. Werner, M.E. Dubas, J.L. Goeckeler, K.B. Kruse, and A.A. McCracken. 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.
Caldwell, S.R., K.J. Hill, and A.A. Cooper. 2001. Degradation of endoplasmic reticulum (ER) quality control substrates requires transport between the ER and Golgi. J. Biol. Chem. 276:2329623303.
Ellgaard, L., and A. Helenius. 2001. ER quality control: towards an understanding at the molecular level. Curr. Opin. Cell Biol. 13:431437.[CrossRef][Medline]
Gelman, M.S., E.S. Kannegaard, and R.R. Kopito. 2002. A principal role for the proteasome in endoplasmic reticulum-associated degradation of misfolded intracellular cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 277:1170911714.
Hampton, R.Y., R.G. Gardner, and J. Rine. 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]
Jensen, T.J., M.A. Loo, S. Pind, D.B. Williams, A.L. Goldberg, and J.R. Riordan. 1995. Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell. 83:129135.[Medline]
Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. Aggresomes: a cellular response to misfolded proteins. J. Cell Biol. 143:18831898.
Kaiser, C.A., and R. Schekman. 1990. Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway. Cell. 61:723733.[Medline]
Kamhi-Nesher, S., M. Shenkman, S. Tolchinsky, S.V. Fromm, R. Ehrlich, and G.Z. Lederkremer. 2001. A novel quality control compartment derived from the endoplasmic reticulum. Mol. Biol. Cell. 12:17111723.
Katzmann, D.J., E.A. Epping, and W.S. Moye-Rowley. 1999. Mutational disruption of plasma membrane trafficking of Saccharomyces cerevisiae Yor1p, a homologue of mammalian multidrug resistance protein. Mol. Cell. Biol. 19:29983009.
Kirk, K.L. 2000. New paradigms of CFTR chloride channel regulation. Cell. Mol. Life Sci. 57:623634.[Medline]
Kiser, G.L., M. Gentzsch, A.K. Kloser, E. Balzi, D.H. Wolf, A. Goffeau, and J.R. Riordan. 2001. Expression and degradation of the cystic fibrosis transmembrane conductance regulator in Saccharomyces cerevisiae. Arch. Biochem. Biophys. 390:195205.[CrossRef][Medline]
Knop, M., A. Finger, T. Braun, K. Hellmuth, and D.H. Wolf. 1996. Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast. EMBO J. 15:753763.[Abstract]
Koning, A.J., C.J. Roberts, and R.L. Wright. 1996. Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations. Mol. Biol. Cell. 7:769789.[Abstract]
Kuehn, M.J., J.M. Herrmann, and R. Schekman. 1998. COPII-cargo interactions direct protein sorting into ER-derived transport vesicles. Nature. 391:187190.[CrossRef][Medline]
Labbe, S., and D.J. Thiele. 1999. Copper ion inducible and repressible promoter systems in yeast. Methods Enzymol. 306:145153.[Medline]
Loayza, D., A. Tam, W.K. Schmidt, and S. Michaelis. 1998. Ste6p mutants defective in exit from the endoplasmic reticulum (ER) reveal aspects of an ER quality control pathway in Saccharomyces cerevisiae. Mol. Biol. Cell. 9:27672784.
McCracken, A.A., and J.L. Brodsky. 1996. Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP. J. Cell Biol. 132:291298.[Abstract]
Moyer, B.D., J. Loffing, E.M. Schwiebert, D. Loffing-Cueni, P.A. Halpin, K.H. Karlson, I.I. Ismailov, W.B. Guggino, G.M. Langford, and B.A. Stanton. 1998. Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in madin-darby canine kidney cells. J. Biol. Chem. 273:2175921768.
Nishikawa, S.I., S.W. Fewell, Y. Kato, J.L. Brodsky, and T. Endo. 2001. Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation. J. Cell Biol. 153:10611070.
Paddon, C., D. Loayza, L. Vangelista, R. Solari, and S. Michaelis. 1996. Analysis of the localization of STE6/CFTR chimeras in a Saccharomyces cerevisiae model for the cystic fibrosis defect CFTR delta F508. Mol. Microbiol. 19:10071017.[CrossRef][Medline]
Pind, S., J.R. Riordan, and D.B. Williams. 1994. Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 269:1278412788.
Plemper, R.K., S. Bohmler, J. Bordallo, T. Sommer, and D.H. Wolf. 1997. Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation. Nature. 388:891895.[CrossRef][Medline]
Preuss, D., J. Mulholland, A. Franzusoff, N. Segev, and D. Botstein. 1992. Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy. Mol. Biol. Cell. 3:789803.[Abstract]
Rabinovich, E., A. Kerem, K.U. Frohlich, N. Diamant, and S. Bar-Nun. 2002. AAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation. Mol. Cell. Biol. 22:626634.
Riordan, J.R. 1999. Cystic fibrosis as a disease of misprocessing of the cystic fibrosis transmembrane conductance regulator glycoprotein. Am. J. Hum. Genet. 64:14991504.[CrossRef][Medline]
Rose, M.D., F. Winston, and P. Hieter. 1990. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Salama, N.R., J.S. Chuang, and R.W. Schekman. 1997. Sec31 encodes an essential component of the COPII coat required for transport vesicle budding from the endoplasmic reticulum. Mol. Biol. Cell. 8:205217.[Abstract]
Shenkman, M., M. Ayalon, and G.Z. Lederkremer. 1997. Endoplasmic reticulum quality control of asialoglycoprotein receptor H2a involves a determinant for retention and not retrieval. Proc. Natl. Acad. Sci. USA. 94:1136311368.
Schekman, R., and L. Orci. 1996. Coat proteins and vesicle budding. Science. 271:15261533.[Abstract]
Taxis, C., F. Vogel, and D.H. Wolf. 2002. ER-Golgi traffic is a prerequisite for efficient ER degradation. Mol. Biol. Cell. 13:18061818.
Travers, K.J., C.K. Patil, L. Wodicka, D.J. Lockhart, J.S. Weissman, and P. Walter. 2000. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell. 101:249258.[Medline]
Vashist, S., W. Kim, W.J. Belden, E.D. Spear, C. Barlowe, and D.T. Ng. 2001. Distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding. J. Cell Biol. 155:355368.
Veldhuisen, G., M. Saloheimo, M.A. Fiers, P.J. Punt, R. Contreras, M. Penttila, and C.A. van den Hondel. 1997. Isolation and analysis of functional homologues of the secretion-related SAR1 gene of Saccharomyces cerevisiae from Aspergillus niger and Trichoderma reesei. Mol. Gen. Genet. 256:446455.[CrossRef][Medline]
Vida, T.A., and S.D. Emr. 1995. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128:779792.[Abstract]
Wang, Q., and A. Chang. 1999. Eps1, a novel PDI-related protein involved in ER quality control in yeast. EMBO J. 18:59725982.
Ward, C.L., S. Omura, and R.R. Kopito. 1995. Degradation of CFTR by the ubiquitin-proteasome pathway. Cell. 83:121127.[Medline]
Yoshihisa, T., C. Barlowe, and R. Schekman. 1993. Requirement for a GTPase-activating protein in vesicle budding from the endoplasmic reticulum. Science. 259:14661468.[Medline]
Zhang, Y., G. Nijbroek, M.L. Sullivan, A.A. McCracken, S.C. Watkins, S. Michaelis, and J.L. Brodsky. 2001. Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast. Mol. Biol. Cell. 12:13031314.