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Vol. 18, Issue 9, 3398-3413, September 2007

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Inhibiting Endoplasmic Reticulum (ER)-associated Degradation of Misfolded Yor1p Does Not Permit ER Export Despite the Presence of a Diacidic Sorting Signal

Silvere Pagant^*, Leslie Kung^*, Mariana Dorrington, Marcus C.S. Lee, and Elizabeth A. Miller

Department of Biological Sciences, Columbia University, New York, NY 10027-6902

Submitted January 22, 2007; Revised June 22, 2007; Accepted June 25, 2007
Monitoring Editor: Jeffrey Brodsky

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Capture of newly synthesized proteins into endoplasmic reticulum (ER)-derived coat protomer type II (COPII) vesicles represents a critical juncture in the quality control of protein biogenesis within the secretory pathway. The yeast ATP-binding cassette transporter Yor1p is a pleiotropic drug pump that shows homology to the human cystic fibrosis transmembrane conductance regulator (CFTR). Deletion of a phenylalanine residue in Yor1p, equivalent to the major disease-causing mutation in CFTR, causes ER retention and degradation via ER-associated degradation. We have examined the relationship between protein folding, ERAD and forward transport during Yor1p biogenesis. Uptake of Yor1p into COPII vesicles is mediated by an N-terminal diacidic signal that likely interacts with the "B-site" cargo-recognition domain on the COPII subunit, Sec24p. Yor1p-F is subjected to complex ER quality control involving multiple cytoplasmic chaperones and degradative pathways. Stabilization of Yor1p-F by inhibiting its degradation does not permit access of Yor1p-F to COPII vesicles. We propose that the ER quality control checkpoint engages misfolded Yor1p even after it has been stabilized by inhibition of the degradative pathway.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Eukaryotic cells maintain a strict policy of protein quality control to prevent the improper deployment of aberrant proteins within the cell, which may impair cellular function and ultimately result in proteotoxicity. This quality control checkpoint is of particular importance with respect to secretory proteins, which enter the endoplasmic reticulum (ER) as linear, unfolded polypeptides and generally must attain the correct quaternary structure before gaining transport competence to be delivered to downstream compartments (Ellgaard and Helenius, 2001; Bukau et al., 2006). Misfolded and aberrant proteins are disposed of by ER-associated degradation (ERAD), which targets proteins for reverse translocation out of the ER, ubiquitination, and destruction by the cytoplasmic proteasome (Ahner and Brodsky, 2004). Thus, the ER plays a fundamental role in coordinating protein synthesis, folding, degradation and transport to ensure accurate and efficient delivery of proteins to the compartments of the secretory pathway.

With recent progress in defining many of the components that participate in these processes, the complexity of these interrelated pathways is beginning to be understood. For example, ERAD is now recognized to be a multifaceted program that uses multiple machineries to degrade misfolded proteins with differently located lesions (Ahner and Brodsky, 2004; Ismail and Ng, 2006). Substrates within the ER lumen or ER membrane each have distinct topological requirements in terms of both lesion detection and protein extraction before the common endpoint of proteasomal degradation. Thus, rather than being a simple protein detection and degradation service, ERAD is attuned to dispose of proteins from different environments. Despite the advanced state of knowledge with respect to the destructive side of protein quality control, the precise nature of the interplay between the biosynthetic and degradative pathways remains poorly understood. Specifically, the mechanisms by which a single protein is diverted according to its particular folding status are largely unknown.

One critical juncture in the process of protein quality control is the uptake of newly synthesized proteins into ER-derived transport vesicles, known as coat protomer type II (COPII) vesicles for the cytoplasmic coat proteins that sculpt vesicles from the ER membrane and populate them with the appropriate cargo (Lee et al., 2004). Membrane proteins are generally captured into COPII vesicles via cytoplasmic ER export signals that bind directly to the COPII subunit Sec24p, which is responsible for efficiently recruiting cargo into nascent vesicles as they form from the ER membrane (Miller et al., 2002). Similarly, soluble secretory proteins bind to receptors that in turn likely contain Sec24p-binding sites (Otte and Barlowe, 2004). Misfolded proteins seem to be largely excluded from COPII vesicles, but the mechanism by which these proteins fail to enter vesicles is not clear.

Recent experiments using chimeric ERAD substrates demonstrated that degradation of a misfolded protein can be overcome when a strong ER export motif is appended to the protein (Kincaid and Cooper, 2007). This suggests that rather than using an active ER retention mechanism, quality control may result from a competitive interaction between ERAD and forward transport. Misfolded proteins may be rapidly destroyed by the ERAD machinery, leaving little opportunity to engage the COPII coat. Thus, disabling the degradative pathway might be expected to stabilize a misfolded protein sufficiently to drive forward transport. Indeed, inhibiting ERAD can allow rescue of forward transport, and, in some cases, the deployment of a functional protein (Kota et al., 2007). One mechanism that may play a role in establishing a hierarchy of degradation over forward transport is the presentation of an ER export motif; misfolded proteins may simply fail to present an appropriate signal, resulting in their default degradation. This may be especially relevant to soluble secretory proteins, which contain ER export motifs that mediate interaction with their transmembrane receptors (Otte and Barlowe, 2004). Simple failure to engage the receptor would ultimately cause degradation. However, misfolded membrane proteins, which interact directly with Sec24p, present a more difficult quality control problem (Mossessova et al., 2003). Where the misfolding lesion is located in a cytosolic domain, it is easy to imagine how structural changes may obscure an ER export motif. This is thought to be true of the human cystic fibrosis transmembrane receptor (CFTR); the most common disease-causing mutation, F508, is located in the same cytosolic domain as the ER export signal, potentially disrupting interaction with Sec24p (Wang et al., 2004). Conversely, where the misfolding lesion is in a lumenal or transmembrane region, or in a cytosolic domain distinct from that containing the ER export signal, interaction with Sec24p may be preserved. How these proteins are excluded from COPII vesicles remains unclear, but lumenal or cytosolic chaperones that might bind the misfolded domain may play a role by actively retaining the protein or obscuring an interaction with Sec24p. Finally, the discovery that some misfolded proteins require a round-trip to the Golgi before ERAD suggests that misfolded proteins may not be entirely absent from COPII vesicles (Vashist et al., 2001). As for ERAD, the mechanism that excludes a protein from COPII vesicles may be unique for each specific aberrant protein.

We study the close relationship between protein folding and forward transport through the secretory pathway in the budding yeast, Saccharomyces cerevisiae. We describe here the biogenesis of a plasma membrane protein, Yor1p, which is an ATP-binding cassette (ABC) transporter that functions as a pleiotropic drug pump to clear toxic substances from the yeast cytoplasm (Katzmann et al., 1999). ABC transporters are a large family of proteins that contain distinct arrangements of membrane spanning domains (MSDs), which form a channel in the lipid bilayer, and nucleotide-binding domains (NBDs) that provide the driving force for substrate translocation across the membrane. The arrangement of MSDs and NBDs in Yor1p places it in the same class of ABC transporters as human CFTR (Riordan et al., 1989). Deletion of a phenylalanine residue in NBD1 of Yor1p (F670), equivalent to the F508 mutation in CFTR, causes ER retention and proteasomal degradation of Yor1p (Katzmann et al., 1999). In CFTR, this mutation results in a channel that is capable of conducting chloride (Drumm et al., 1991; Li et al., 1993), but it is recognized by the ER quality control machinery and retained in the ER where it is degraded via ERAD instead of being deployed to the apical membrane (Cheng et al., 1990; Denning et al., 1992; Ward et al., 1995). Extensive work elucidating the mechanisms of destruction of aberrant CFTR have revealed that it is a complex process involving multiple cytoplasmic chaperone complexes (Zhang et al., 2001; Youker et al., 2004; Younger et al., 2004, 2006), but the relationship between the degradative and forward transport itineraries remains unclear.

We aimed to better define the protein folding, degradation, and forward transport pathways accessed by both the wild-type and F forms of Yor1p to gain insight into how the intracellular fate of aberrant ABC transporters is controlled. Specifically, we have defined the ER export motif used by Yor1p in gaining access to ER-derived COPII vesicles and determined the site on Sec24p that likely recognizes this signal. Furthermore, we have used yeast mutants in several chaperones and the ERAD machinery to test how perturbation of these key processes directly impacts the intracellular itineraries of newly synthesized Yor1p. Because many components of the ER folding, degradation and export pathways are remarkably conserved among all eukaryotes, increasing our basic understanding of the regulation of these processes in yeast is likely to be directly applicable to protein folding problems in mammalian systems.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Yeast Strains and Media
S. cerevisiae strains used in this study are listed in Table 1. Strains bearing mutant forms of SEC24 as the sole copy of this essential gene were created by transforming YTB1 (sec24::LEU2 with wild-type SEC24 borne on a URA-containing plasmid) with either wild-type or mutant forms of SEC24 contained on a HIS-marked plasmid. Cells were cured of the wild-type SEC24::URA plasmid by growth on 5-fluoroorotic acid (0.1% final concentration), leaving the plasmid-borne copy as the sole copy of SEC24. UBC7 was deleted in various strain backgrounds by transformation with a polymerase chain reaction (PCR) product composed of the ubc7::KANMX disruption cassette amplified from the EUROSCARF (Frankfurt, Germany) deletion strain collection. The allelic replacement of UBC7 was confirmed by PCR. LMY314 (sel1::KanMX), LMY285 (hrd1::KanMX), and LMY115 (doa10:: KanMX) were constructed by integration of the respective disruption cassettes into HLJ1/YDJ1. Disruption cassettes were previously generated by PCR amplification from the pUG6 plasmid (Guldener et al., 1996). Retrieval of the KanMX gene in LMY285 (hdr1::KanMX) was performed by cre/lox-mediated excision (Guldener et al., 1996), and the doa10::KanMX disruption cassette was introduced to create LMY313 (hrd1 doa10::KanMX). All allelic replacements were confirmed by PCR. Cultures were grown at 30°C in standard rich media (YPD: 1% yeast extract, 2% peptone, and 2% glucose) or synthetic complete media (SC: 0.67% yeast nitrogen base and 2% glucose, supplemented with amino acids appropriate for auxotrophic growth). For testing sensitivity to oligomycin, strains were grown to saturation, and then they were diluted to an OD₆₀₀ of 0.5 and fourfold serial dilutions were applied to YPEG plates (1% yeast extract, 2% peptone, 3% ethanol, and 3% glycerol), that were supplemented with oligomycin (Sigma-Aldrich, St. Louis, MO) from a 1 mg/ml stock in ethanol. Drug sensitivities of cells expressing mutant forms of Sec24p were assayed by streaking the strains indicated onto YPEG plates supplemented with 0.1 µg/ml oligomycin, YPD plates supplemented with 400 µg/ml rhodamine B (Sigma-Aldrich) or onto YPD plates supplemented with 10 µg/ml methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate (benomyl, 95%; Sigma-Aldrich).

Protein Purification
COPII proteins Sar1p, Sec23p/24p, and Sec13/31p were prepared as described previously (Barlowe et al., 1994).

In Vitro Vesicle Budding
Microsomal membranes were purified from RSY620 cells expressing Yor1p- HA or Yor1pF-HA as described previously (Wuestehube and Schekman, 1992). In vitro vesicle budding was performed essentially as described previously (Miller et al., 2002), except the urea wash was omitted. Briefly, membranes were washed twice with buffer B88 (20 mM HEPES, pH 6.8, 250 mM sorbitol, 160 mM potassium acetate, and 5 mM magnesium acetate), and 125 µg of membranes per reaction were incubated with COPII proteins (10 µg/ml Sar1p, 10 µg/ml Sec23p/24p, and 20 µg/ml Sec13/31p) either in the presence of 0.1 mM GTP with a 10x ATP regeneration system or 0.1 mM GDP. Vesicles were separated from donor membranes by centrifugation at 16,000 rpm for 5 min, and the vesicle fraction was further concentrated by high-speed centrifugation at 55,000 rpm for 20 min. Vesicle pellets were resuspended in SDS sample buffer and heated at 55°C for 5 min before separation by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes and analyzed by immunoblotting. HA-tagged Yor1p was detected with a monoclonal anti-HA antibody (Covance, Princeton, NJ), and control proteins Erv46p and Sec22p were detected with polyclonal antibodies, gifts from C. Barlowe (Dartmouth Medical School) and R. Schekman (U. C. Berkeley), respectively.

Radiolabeled semi-intact cells were prepared essentially as described previously (Kuehn et al., 1996). Briefly, cells were grown to mid-log phase in synthetic complete medium, and a total of 5.0 OD₆₀₀ of cells were harvested and starved of methionine/cysteine for 10 min before addition of Express protein labeling mix (70µCi/OD₆₀₀ of cells; MP Biomedicals, Irvine, CA). Cells were labeled for 15 min at 30°C, and then they were metabolically killed and converted to spheroplasts. Cells were gently lysed, washed once with low acetate B88 (20 mM HEPES, pH 6.8, 250 mM sorbitol, 50 mM potassium acetate, and 5 mM magnesium acetate) and twice with B88 before incubation with COPII proteins (10 µg/ml Sar1p, 10 µg/ml Sec23p/24p, and 20 µg/ml Sec13/31p) in a final reaction that contained 2.5 OD of cells either in the presence of 0.1 mM GTP with a 10x ATP regeneration system or 0.1 mM GDP. Vesicles were separated from donor membranes by centrifugation at 16,000 rpm for 5 min, solubilized with 1% SDS (final concentration), and diluted with immunoprecipitation (IP) buffer (50 mM Tris, pH 7.5, 160 mM NaCl, 1% Triton X-100, and 2 mM NaN₃). Proteins were immunoprecipitated using monoclonal anti-HA antibodies (Covance) precoupled to protein G-Sepharose beads (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom), or polyclonal antibodies against Gas1p, Sec22p or Vph1p (gifts from R. Schekman) coupled to protein A-Sepharose beads (GE Healthcare). Immune complexes were separated by SDS-PAGE and analyzed by PhosphorImage analysis using a Storm PhosphorImager (GE Healthcare). Proteins were quantified using ImageQuant software (GE Healthcare).

Blue Native Gel Electrophoresis (BNGE)
In total, 5.0 OD₆₀₀ of cells expressing wild-type HA-tagged Yor1p or HA-tagged mutant Yor1p were collected during mid-log phase, washed with 250 µl of lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1 mM dithiothreitol [DTT]), and lysed by vortexing with glass beads for 10 min at 4°C. The lysate was transferred to a fresh tube, membranes collected by centrifugation at 15,000 rpm for 5 min and proteins solubilized with 100µl of lysis buffer with 1% digitonin. After incubation on ice for 30 min, the soluble fraction was separated from the insoluble material by centrifugation at 5000 rpm for 2 min, and the supernatant was mixed with 10x BNP sample buffer (5% Coomassie G250, 10% 6-aminocaprioic acid, 50 mM Bis-Tris, and 10% glycerol). Samples were run on a 7% acrylamide Bis-Tris gel (50 mM Bis-Tris, 6.55% 6-aminocaproic acid, and 13.9% glycerol) by using a two-buffer system consisting of a cathode buffer (50 mM Tricine, 15 mM Bis-Tris, and 0.02% Coomassie G250) and anode buffer (50 mM Bis-Tris). Proteins were transferred to PVDF overnight at 20 V and analyzed by immunoblotting with a monoclonal anti-HA antibody (Covance).

Limited Proteolysis
Cells expressing either wild-type Yor1p-HA or HA-tagged Yor1p mutants were harvested during mid-log phase. In total, 10.0 OD₆₀₀ of cells were collected, resuspended in 200 µl of B88 buffer, and glass bead lysed at 4°C for 5 min. Cell lysates were subjected to centrifugation at 16,000 rpm for 5 min, and the membrane pellet was resuspended in 100 µl of B88 and divided into four 25-µl reactions (2.5 OD₆₀₀/reaction). Each reaction was treated with a final concentration of 0, 25, 50, or 100 ng/µl trypsin (Sigma-Aldrich) for 10 min on ice. Digestion was terminated by addition of 0.2 µg/ml (final concentration) soybean trypsin inhibitor (Sigma-Aldrich) to all reactions and incubated on ice for 15 min. Proteins were separated by SDS-PAGE, transferred to PVDF, and the pattern of Yor1p fragments analyzed by immmunoblot by using an anti-HA antibody.

For cells bearing thermosensitive alleles, radiolabeled semi-intact cells were used as the source of membranes. Cells were grown to mid-log phase in complete synthetic medium at 30°C, and then they were harvested, resuspended in fresh medium lacking methionine/cysteine to an OD₆₀₀ of 5, and incubated for 10 min with gentle shaking at 37°C. Cells were metabolically labeled at 37°C for 10 min by adding 60 µCi of Express protein labeling mix (MP Biomedicals) per OD₆₀₀ unit of cells. Spheroplasts prepared from these labeled cells were resuspended in 100 µl of B88 and divided into four 25-µl reactions. Each reaction was treated with a final concentration of 0, 100, 200, or 400 ng/µl trypsin for 10 min on ice. Digestion was terminated by addition of soybean trypsin inhibitor to all reactions followed by incubation on ice for 15 min and by two washes with B88. After solubilization with SDS (1% final concentration) and heating to 55°C for 5 min, the resulting protein extracts were diluted with 5 volumes of IP buffer (50 mM Tris, pH 7.5, 160 mM NaCl, 1% Triton X-100, and 2 mM NaN₃) and cleared by centrifugation. Yor1p fragments were immunoprecipitated from the cleared supernatant and analyzed by SDS-PAGE and PhosphorImage analysis as described above.

Cross-linking of TM Domains
Cells expressing cysteine-substituted forms of Yor1p-HA were grown to mid-log phase, and then they were harvested and converted to spheroplasts. Spheroplasts were washed twice in 20 mM HEPES, pH 7.4, and incubated with increasing concentrations of M8M (prepared as a 100x stock in dimethyl sulfoxide) as indicated. Cells were cross-linked for 15 min at room temperature, and then they were harvested, resuspended in 100 µl of 1% SDS, 50 µl of 3x SDS sample buffer without -mercaptoethanol or DTT. Cells were disrupted by glass bead lysis (15 min; 4°C), heated to 55°C for 5 min, and proteins were separated by SDS-PAGE followed by immunoblot analysis using anti-HA antibodies.

Pulse-Chase Analysis of Yor1p Stability
Cells were grown to mid-log phase in complete synthetic medium, and then they were harvested, resuspended in fresh medium lacking methionine/cysteine, and incubated for 15 min with gentle shaking at either 30°C (for cells lacking a temperature-sensitive allele) or at 37°C (for cells bearing a temperature-sensitive allele). Cells were metabolically labeled for 5 min by adding 30 µCi of Express protein labeling mix (MP Biomedicals) per OD₆₀₀ unit of cells. A 10x chase solution (10 mM L-cysteine, 50 mM L-methionine, 4% yeast extract, and 2% glucose) was added and 2 OD aliquots of cells harvested at different times. At each time point, cells were transferred to chilled tubes, and sodium azide was added to a final concentration of 20 mM. Cells were washed once with 20 mM sodium azide and resuspended in 100 µl of 1% SDS. Glass beads were added, and cells were lysed by vortexing for 15 min at 4°C. Cell lysates were heated at 55°C for 5 min, diluted with 5 volumes of IP buffer (50 mM Tris, pH 7.5, 160 mM NaCl, 1% Triton X-100, and 2 mM NaN₃), and cleared by centrifugation. Yor1p and Gas1p were immunoprecipitated from the cleared lysate and analyzed by SDS-PAGE and PhosphorImage analysis as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The yeast ABC transporter Yor1p is a polytopic membrane protein that functions at the plasma membrane as a pleiotropic drug pump to clear toxins from the cytosol. A mutant form of Yor1p, Yor1p-F, which carries a deletion of a phenylalanine residue at position 670, is trapped in the ER, destabilized in vivo, and degraded by the cytoplasmic ubiquitin/proteasome system (Katzmann et al., 1999). To independently confirm that ER-export of Yor1p-F is impaired, we used an in vitro vesicle budding assay that recapitulates COPII vesicle formation from purified ER membranes. Microsomal membranes were isolated from cells expressing an HA-tagged copy of either wild-type Yor1p or Yor1p-F. These purified membranes were incubated with the COPII components Sar1p, Sec23p/Sec24p, and Sec13p/Sec31p in the presence of either GTP or GDP. This incubation is sufficient to generate transport vesicles, which can be separated from donor membranes by differential centrifugation and specific capture of cargo proteins into vesicles analyzed by immunoblotting (Barlowe et al., 1994). Whereas wild-type Yor1p was efficiently packaged into COPII vesicles, very little Yor1p-F was detected in the vesicle fraction (Figure 1B). Two proteins that cycle between the ER and Golgi, Sec22p and Erv46p served as positive controls to demonstrate efficient vesicle biogenesis in this assay. The specific absence of Yor1p-F in COPII vesicles generated in vitro is consistent with its in vivo ER localization and suggests that the mutant protein has engaged the ER quality control checkpoint that prevents deployment of aberrant cellular proteins.

View larger version (38K): [in this window] [in a new window]   Figure 1. Yor1p-F is excluded from COPII vesicles and is misfolded. (A) Schematic diagram of the yeast ABC transporter, Yor1p, which, like CFTR, has two membrane-spanning domains (MSD1 and MSD2), two nucleotide binding domains (NBD1 and NBD2), and an analogous phenylalanine residue (F670) in NBD1 that is necessary for protein stability and trafficking. Yor1p also has two previously identified terminal diacidic motifs (D71E73 and D1472E1474) and two additional acidic motifs in NBD1 (D691D693 and D729) that may interact with COPII coat machinery in facilitating ER export. (B) Capture of Yor1p into ER-derived COPII vesicles was examined in an in vitro vesicle budding assay by using microsomal membranes expressing HA-tagged forms of Yor1p as the donor membranes. Membranes were incubated with COPII proteins in the presence of GTP (+) or GDP (–), and vesicles were separated from the total membrane (T) fraction by centrifugation. Cargo proteins were detected by immunoblot analysis. (C) Yor1p and Yor1p-F were analyzed by BNGE after solubilization with either digitonin or SDS as indicated followed by immunoblotting to detect HA-tagged forms of Yor1p. The amount of material loaded following solubilization with SDS represents a 1/5 dilution compared with that loaded for digitonin-solubilized samples. (D) Microsomal membranes expressing HA-tagged forms of Yor1p were subjected to limited proteolysis by treating membranes with increasing amounts of trypsin followed by SDS-PAGE and immunoblotting to detect HA-tagged proteolytic fragments. (E) Membranes expressing HA-tagged forms of cysteine-modified Yor1p were cross-linked with increasing concentrations of M8M before SDS-PAGE and immunoblot analysis. Yor1p expressed from two separate plasmids, each bearing single cysteine substitutions showed no cross-linked species (left), whereas Yor1p containing both F481C and L1162C mutations was readily cross-linked into a higher-molecular-weight species (middle). Yor1p-F containing the same substitutions instead presented as a very high-molecular-weight aggregate, with neither the wild-type nor cross-linked species detectable (right).

As a separate assay to probe the folding and assembly of Yor1p, we subjected membranes containing either wild-type or mutant Yor1p to limited proteolysis. Microsomal membranes were treated with increasing concentrations of trypsin and analyzed by immunoblotting. Yor1p presented a persistent 160-kDa band and a single major cleavage product of 60 kDa with increasing amounts of trypsin (Figure 1D, left). The pattern of Yor1p-F cleavage products was markedly different, with both the 160- and 60-kDa bands further digested into smaller products with increasing trypsin (Figure 1D, right). The increased susceptibility of Yor1p-F to trypsinolysis supports the hypothesis that this mutant protein is improperly assembled, thereby exposing additional trypsin cleavage sites that are obscured in the correctly folded protein.

Finally, we developed a cross-linking assay to probe the assembly of the transmembrane domains (TMDs) of Yor1p. Previous studies on the related ABC transporters P-glycoprotein and CFTR had used cysteine residues, substituted within specific transmembrane domains, to probe the ability to form cross-linked disulfide bonds (Chen et al., 2004). Misfolding mutations, including the F508 lesion, resulted in an inability to form specific cross-links, suggesting that these transmembrane domains fail to assemble correctly. We introduced cysteine residues at specific positions in the 6th and 12th TM domains of Yor1p, equivalent to the sites used for CFTR. Membranes expressing wild-type Yor1p that contained cysteine substitutions at F₄₈₁ (TM6) and L₁₁₆₂ (TM12) were exposed to a methanethiosulfonate cross-linker with a 13-Å spacer arm and the mobility of the protein monitored by nonreducing SDS-PAGE and anti-HA immunoblotting (Figure 1E). On exposure to cross-linker, a species with reduced mobility was detected, similar to that observed for CFTR. This cross-linked protein was also detected with a L₄₇₉C/L₁₁₆₂C substitution pair, analogous to a second cross-linking pair that was used to probe transmembrane domain assembly in CFTR (Pagant, unpublished data). The cross-linked species likely represents an intramolecular modification, because when the individual F₄₈₁C and L₁₁₆₂C substitutions were introduced separately on two different plasmids and cotransformed into cells, no cross-linked proteins were detected (Figure 1E, left). When the paired F₄₈₁C/L₁₁₆₂C substitutions were introduced into Yor1p-F, no cross-linked species were detected; instead, the unmodified protein disappeared and the majority of the protein presented as a very-high-molecular weight aggregate that largely failed to enter the resolving gel. Similar higher order aggregation was also detected for CFTR-F, and it is thought to represent nonspecific cross-linking of exposed cysteine residues (Chen et al., 2004). Thus, like CFTR-F, Yor1p-F seems to contain improperly assembled transmembrane domains.

Identification of the ER Export Motif Used by Yor1p
To gain a better understanding of how Yor1p engages the ER export pathway, a clear definition of the ER export signals used by Yor1p is required. Two independent diacidic motifs have been identified in Yor1p; when either the N-terminal D₇₁XE₇₃ or C-terminal D₁₄₇₂XE₁₄₇₄, were mutated, Yor1p was trapped in the ER, causing cells to become oligomycin sensitive (Epping and Moye-Rowley, 2002). Similarly, ER exit of CFTR is dependent upon the presence of a diacidic motif, although the putative signal in CFTR is contained within NBD1 (Wang et al., 2004). We investigated the potential roles of several diacidic motifs in mediating ER exit of Yor1p, focusing on the two terminal DXE motifs and two potential diacidic motifs in NBD1 of Yor1p: D₆₉₁XD₆₉₃ and D₇₂₉XY₇₃₁. The latter signal is spatially homologous to the aspartic acid residue of the DXE CFTR export signal, but it only contains a single acidic residue (Figure 1A). We generated HA-tagged mutants where each potential motif was replaced with alanine, and we examined in vivo phenotypes and in vitro capture into COPII vesicles of each of the mutant proteins (Figure 2).

View larger version (95K): [in this window] [in a new window]   Figure 2. Identification of an N-terminal diacidic motif as the ER exit signal for Yor1p. (A) Four-fold serial dilutions of yeast expressing HA-tagged forms of Yor1p were tested for sensitivity to increasing concentrations of oligomycin (1, 2.5, and 5 µg/ml). Cells expressing wild-type Yor1p were able to grow under all conditions, whereas cells containing the plasmid alone (pRS316) were sensitive to very low levels of oligomycin. The various diacidic mutants showed a range of oligomycin sensitivities, whereas the ER-retained Yor1p-F was completely oligomycin sensitive. (B) Packaging of Yor1p mutants into COPII vesicles was examined by an in vitro vesicle budding assay. Cells were radiolabeled with [35S]methionine/cysteine for 15 min, permeabilized, and incubated with COPII proteins in the presence of either GTP (+) or GDP (–). HA-tagged wild-type and mutant forms of Yor1p were immunoprecipitated and analyzed by SDS-PAGE and PhosphorImage analysis. The percentage of Yor1p found in the vesicle fraction was quantified relative to the total Yor1p (T) present in the donor membranes. Packaging of D71XE73A was severely impaired, whereas that of the D691XD693A, D729A, and D1472XE1474 mutants was similar to that of wild-type Yor1p. An independent cargo protein, Vph1, was also examined to indicate efficient generation of vesicles in the in vitro assay. (C) Wild-type cells coexpressing pRS315–PDR1-3 and Yor1p–GFP fusions as indicated were examined by epifluorescence microscopy. Only the N-terminal di-acidic mutant showed perinuclear staining consistent with ER retention. Wild-type Yor1p and the C-terminal diacidic mutant both localized to the plasma membrane. (D) Folding and stability of Yor1p and the N-terminal diacidic mutant were analyzed by limited proteolysis. Whole cell lysates containing HA-tagged wild-type or mutant forms of Yor1p were incubated with increasing amounts of trypsin, subjected to SDS-PAGE and Yor1p fragments detected by immunoblotting with an anti-HA antibody. (E) The arrangement of transmembrane domains in the N-terminal di-acidic mutant was probed by cysteine cross-linking as described in Figure 1E.

To better define the signal responsible for ER export of Yor1p, we examined uptake of each of the mutants into COPII vesicles generated in vitro from radiolabeled permeabilized cells. Wild-type Yor1p was efficiently packaged into COPII vesicles, and of all the diacidic mutants that we tested, only the N-terminal D₇₁AXE₇₃A mutant was impaired in its capture into COPII vesicles (Figure 2B). A control cargo protein, Vph1p, was equivalently packaged in each of these experiments, indicative of general budding efficiency. We further tested the localization of Yor1p mutants by microscopy by using Yor1p–GFP fusions (Figure 2C). As reported previously, wild-type Yor1p fused to GFP was found at the plasma membrane, whereas the N-terminal (D₇₁AXE₇₃A) diacidic mutant showed a perinuclear localization and cortical ER localization, consistent with its retention in the ER (Epping and Moye-Rowley, 2002). Conversely, the localization of the C-terminal (D₁₄₇₂AXE₁₄₇₄A) diacidic mutant more closely resembled that of the wild-type protein, with strong plasma membrane fluorescence and no detectable internal perinuclear staining. This is consistent with our in vitro budding data, but it conflicts with a previous report that used sucrose gradients to show that Yor1p-D₁₄₇₂AXE₁₄₇₄A was ER retained (Epping and Moye-Rowley, 2002). We note that the oligomycin sensitivity phenotype of this C-terminal mutant is intermediate between wild-type Yor1p and the N-terminal mutant, suggesting some in vivo impairment of protein delivery or function. It is possible that the C-terminal mutant protein is turned over more rapidly at the plasma membrane, rendering cells slightly more sensitive to oligomycin. Such destabilization may create a steady-state distribution such that the mutant protein is located both at the plasma membrane and in the ER; the ER pool may be undetectable by GFP fluorescence but more easily resolved by cell fractionation. Consistent with this model, destabilization of CFTR by truncation of the C terminus causes the protein to be rapidly internalized from the plasma membrane and cycled through an endosomal compartment before lysosomal destruction (Sharma et al., 2004).

To confirm that the impaired trafficking of the D₇₁AXE₇₃A mutant results from an abrogated exit signal and not a folding defect, we examined the folding status of the mutant protein (Figure 2D). Yor1p-D₇₁AXE₇₃A showed a similar cleavage profile to that of wild-type Yor1p with a persistent band at 160 kDa and another at 60 kDa. Similar experiments with BNGE also indicated that each of the mutants was properly folded, with migration patterns similar to wild-type Yor1p (Lee, unpublished data). Finally, we probed the arrangement of transmembrane domains of the di-acidic mutant by using cysteine cross-linking, which revealed a similar cross-linking pattern to that of the wild-type protein (Figure 2E). Together, these data confirm that the N-terminal diacidic motif, D₇₁XE₇₃, is the ER export signal for Yor1p, and that the ER export defects observed when this motif is altered are not caused by misfolding of Yor1p. The other diacidic mutants were also correctly folded, as determined by trypsin sensitivity and BNGE, suggesting that the oligomycin sensitivity of these proteins may stem from defects in protein function rather than impaired protein trafficking or gross protein misfolding.

The Diacidic ER Export Motif of Yor1p Uses the B-Site on Sec24p
ER export signals function by interacting with Sec24p, which contains three known cargo-binding sites: the A-, B- and C-sites that each recognize distinct motifs. The observation that Yor1p uses a diacidic export motif (DxE) suggests the B-site of Sec24p as the likely binding site (Mossessova et al., 2003). However, because the superficially similar diacidic motif of Gap1 (DID) does not use the B-site, we also explored a potential role for either the A-site or C-site in Yor1p uptake (Miller et al., 2003). We tested seven defined mutants of Sec24p by examining various drug sensitivities of strains expressing each of these mutants as the sole copy of Sec24p, anticipating that disrupting the Sec24p–Yor1p interaction would result in increased oligomycin sensitivity. Indeed, B-site mutants of Sec24p that displayed normal growth on YPEG (Figure 3A) were unable to grow in the presence of low concentrations (0.1 µg/µl) of oligomycin (Figure 3B), but viability could be rescued by introducing an episomal copy of Yor1p (Figure 3C). Similar sensitivity was seen in the presence of rhodamine (400 µg/ml; Figure 3D), which is cleared from the cell predominantly by another ABC transporter, the pleiotropic drug pump Pdr5p, but can also use Yor1p (Rogers et al., 2001). Neither the Sec24p A-site nor C-site mutants were sensitive to oligomycin or rhodamine. All of the Sec24p mutants were able to grow in the presence of benomyl at concentrations that result in lethality of mutants with impaired microtubule function (10 µg/ml; Figure 3E), suggesting that the sensitivity of the B-site mutants to oligomycin and rhodamine is not a general drug intolerance but results from impaired trafficking of specific transporters. These observations suggest that an intact B-site on Sec24p is critical for the trafficking of Yor1p out of the ER and is most likely the site that binds the N-terminal D₇₁XE₇₃ motif of Yor1p.

View larger version (94K): [in this window] [in a new window]   Figure 3. Identification of the Sec24p B-site as the cargo capture site for Yor1p. Strains expressing WT Sec24p or various A-, B-, or C-site Sec24p mutants as the sole copy of Sec24p were tested for growth defects in the presence of various compounds as indicated. (A) All strains grew normally on YPEG plates. (B) B-site mutants were unable to grow on plates containing oligomycin whereas A- and C-site mutants grew normally. (C) Oligomycin sensitivity of the B-site mutants could be rescued by transforming these strains with an extra episomal copy of Yor1p. (D) The B-site mutants also showed growth defects in the presence of rhodamine. (E) All strains grew normally in the presence of benomyl.

View larger version (103K): [in this window] [in a new window]   Figure 4. Yor1p-GFP accumulates in the ER in a Sec24-B mutant. Yor1p-GFP was introduced into strains expressing either wild-type Sec24p or A-, B- or C-site Sec24p mutants as indicated. Localization of Yor1p was examined by epifluorescence microscopy (left) and cell integrity monitored by differential interference contrast microscopy (right). In wild-type cells as well as in the A- and C-site mutants, Yor1p-GFP was located at the plasma membrane, whereas in B-site Sec24p mutant cells, the protein showed perinuclear localization consistent with ER-retention in these cells.

View larger version (53K): [in this window] [in a new window]   Figure 5. Yor1p and Yor1p-F are destabilized in a hsp90 mutant strain. (A) Wild-type (HSP82), hsp82ts (hsc82/HSP82G313N), or hsp82ts/ubc7 strains expressing an HA-tagged form of Yor1p were grown at 30°C, pulse labeled for 5 min with [35S]methionine/cysteine at 37°C, and chased at 37°C for the times indicated. Cells were subjected to immunoprecipitation for both Yor1p and Gas1p, and the abundance and maturation of these proteins were quantified by SDS-PAGE and PhosphorImage analysis. The results shown are the average of three independent experiments. (B) Wild-type and hsp82 mutants expressing HA-tagged Yor1p-F were analyzed as described in A. (C) The conformation of Yor1p was probed by limited proteolysis. Cells expressing Yor1p were grown at 30°C and pretreated for 25 min at 37°C before pulse labeling with [35S]methionine/cysteine. Cells were permeabilized, and the washed membranes were incubated on ice for 10 min with increasing concentrations of trypsin (0, 100, 200, and 400 ng/µl). Yor1p fragments were immunoprecipitated and visualized by SDS-PAGE and PhosphorImage analysis. (D) Packaging of Yor1p into COPII vesicles was examined by an in vitro vesicle budding assay from cells that had been radiolabeled with [35S]methionine/cysteine at 37°C, permeabilized and incubated with COPII proteins in the presence of either GTP (+) or GDP (–). HA-tagged Yor1p was immunoprecipitated and analyzed by SDS-PAGE and PhosphorImage analysis. The percentage of Yor1p found in the vesicle fraction was quantified relative to the total Yor1p (T) present in the donor membranes. An independent cargo protein, Vph1p, was also examined to indicate efficient generation of vesicles in the in vitro assay.

Yor1p-F Uses Multiple ERAD Pathways
Having probed some of the mechanisms of protein folding and ER export of wild-type Yor1p, we determined which ERAD pathway is used in the degradation of the aberrant Yor1p-F. We expressed Yor1p-F in the ERAD mutants, ubc7 and sel1/ubx2, both of which are common to all known ERAD pathways; Ubc7p is the E2 ligase responsible for ubiquitin modification, and Sel1p/Ubx2p is the membrane anchor for the protein extraction apparatus Cdc48p. We examined the stability of Yor1p by pulse-chase analysis in each of these mutants (Figure 6). As reported by Katzmann et al. (1999), mutation of Ubc7p resulted in a significant stabilization of Yor1p-F, restoring the protein to essentially wild-type Yor1p levels (Figure 6A). Similarly, deletion of Sel1p caused Yor1p-F to be degraded more slowly than in wild-type cells (Figure 6A). We note that the half-life that we observe for Yor1p-F is significantly longer than that described by Katzmann et al. (1999). Because our experimental approaches are largely the same, we suspect that these differences result from the distinct strain backgrounds used in the two studies. In each case, disruption of Ubc7p resulted in the stabilization of Yor1p-F to essentially wild-type Yor1p levels.

View larger version (22K): [in this window] [in a new window]   Figure 6. Yor1p-F uses multiple ERAD pathways. HA-tagged Yor1p or Yor1p-F was expressed in either a wild-type strain or strains deleted for a variety of ERAD machineries as indicated. Cells were pulse-labeled for 5 min with [35S]methionine/cysteine and chased for the times indicated. Yor1p was immunoprecipitated from cell lysates and analyzed by SDS-PAGE and PhosphorImage analysis. The results shown are the average of three independent experiments.

Stabilization of Yor1p-F in a ubc7 Mutant Does Not Permit Packaging into COPII Vesicles
We next investigated a possible mechanism by which Yor1p- F might be prevented from entering into COPII vesicles: misfolded or aberrant proteins may be disposed of by ERAD so rapidly that they do not have time to engage the ER export machinery. Moreover, either ubiquitination itself, or simple engagement of the ERAD machinery may mask ER export motifs that would otherwise drive forward transport. We tested this hypothesis by using the ubc7 mutant to block the ER ubiquitination of Yor1p-F and by asking whether this stabilization could rescue uptake into COPII vesicles (Figure 7). Stabilization of Yor1p-F did not result in improved folding of Yor1p-F, because newly synthesized Yor1p-F remained highly susceptible to trypsin cleavage in both wild-type and ubc7 cells (Figure 7A). Similarly, the ability of Yor1p-F to form transmembrane domain cross-links was not restored in a ubc7 strain (Figure 7B). We next tested capture of this stabilized pool of Yor1p-F into COPII vesicles. Loss of UBC7 did not result in uptake of Yor1p-F into COPII vesicles; the vacuolar protein Vph1p served as a positive control to demonstrate efficient vesicle formation (Figure 7C). These data suggest that neither the ubiquitination of Yor1p-F nor the engagement of ERAD components downstream of Ubc7p is responsible for the ER retention of Yor1p-F.

View larger version (52K): [in this window] [in a new window]   Figure 7. Biogenesis and trafficking of Yor1p-F in a ubc7 mutant strain. (A) Permeabilized cells from wild type and ubc7 strains expressing HA-tagged Yor1p or Yor1p-F were used in an in vitro vesicle budding assay. Total membranes (T) and budded vesicles were collected from reactions that contained either GTP (+) or GDP (–). Yor1p and Vph1 packaging into vesicles was quantified by immunoprecipitation followed by SDS-PAGE and PhosphorImage analysis. (B) Radiolabeled permeabilized cells from a wild-type strain and a ubc7 strain containing HA-tagged Yor1p or Yor1p-F were incubated on ice for 10 min with increasing concentrations of trypsin (0, 100, 200, and 400 ng/µl). Yor1p fragments were immunoprecipitated from cell lysates and analyzed by SDS-PAGE and PhosphorImage analysis. (C) Whole cell lysates from wild-type or ubc7 strains expressing cysteine-substituted forms of Yor1p or Yor1p-F were cross-linked with increasing concentrations of M8M as described in Figure 1E. Stabilization of Yor1p-F in the ubc7 strain did not result in the appearance of the wild-type cross-linked species. (D) Yor1p function at the plasma membrane was tested phenotypically by assessing growth in the presence of oligomycin. Strains bearing deletions in YOR1 or YOR1 and UBC7 were transformed with the plasmids indicated and serial dilutions spotted onto YPEG or YPEG supplemented with oligomycin.

Stabilization of Yor1p-F in hsp40 Mutants Rescues Assembly but Does Not Permit Uptake into COPII Vesicles
To investigate whether blocking the ERAD pathway upstream of Ubc7p could lead to export of Yor1p-F from the ER, we analyzed the intracellular fate of Yor1p-F in cells mutated for the ER localized Hsp40s, a class of chaperones known to play roles in protein folding and degradation. Ydj1p is the yeast homologue of Hdj-2, which has been shown to be involved in the ER quality control of mammalian CFTR by assisting in polyubiquitination before degradation (Younger et al., 2004). However, a second yeast ER-localized Hsp40, Hlj1p, is functionally redundant with Ydj1p in the degradation of human CFTR expressed in yeast cells (Youker et al., 2004). We therefore analyzed the biogenesis of Yor1p in the temperature-sensitive double mutant hlj1 ydj1-151. Pulse-chase experiments at restrictive temperature showed that Yor1p-F was stabilized in the absence of Hsp40 function in the ER membrane (Figure 8A), consistent with the model that ER-localized Hsp40s divert misfolded proteins for proteosomal degradation. Maturation of the cell wall protein, Gas1p, was identical in wild-type and hlj1 ydj1-151 cells, demonstrating that loss of chaperone function does not lead to general defects in secretory protein biogenesis. Interestingly, the trypsin sensitivity of Yor1p-F synthesized at restrictive temperature in the hlj1 ydj1-151 strain more closely resembled that of the wild-type protein, suggesting that cytoplasmic Hsp40s participate in the unfolding of Yor1p-F before degradation (Figure 8B). However, despite the stabilization and at least partial refolding of Yor1p-F in the hlj1 ydj1-151 mutant, this mutant protein was not packaged into COPII vesicles in an in vitro vesicle budding assay (Figure 8C). We used cysteine cross-linking to probe the assembly of the membrane domains of Yor1p-F in the Hsp40 mutants, which revealed that despite the conversion of Yor1p-F to a trypsin-resistant form, the TMDs did not show a wild-type configuration (Figure 8D). Together, these data suggest that Hsp40-containing chaperone systems actively unfold the cytoplasmic domains of Yor1p before degradation without affecting the arrangement of TMDs, and that even after stabilization of aberrant Yor1p, the ER quality control checkpoint is still enforced.

View larger version (55K): [in this window] [in a new window]   Figure 8. Yor1p-F is stabilized in a hsp40 mutant strain. (A) Wild-type and hsp40 (hlj1/ydj1-151) strains expressing HA-tagged Yor1p or Yor1p-F were grown at 30°C and pretreated for 25 min at 37°C. Then, strains were pulse-labeled for 5 min with [35S]methionine/cysteine at 37°C and chased at 37°C for the times indicated. Yor1p and Gas1p were immunoprecipitated from cell lysates and analyzed by SDS-PAGE and PhosphorImage analysis. The results shown are the average of three independent experiments. (B) Permeabilized cells from wild-type and hsp40 (hlj1/ydj1-151) strains that had been pretreated at 37°C before labeling were used in an in vitro vesicle budding assay. Total membranes (T) and budded vesicles were collected from reactions that contained either GTP (+) or GDP (–). Yor1p and Vph1 packaging into vesicles was quantified by immunoprecipitation followed by SDS-PAGE and PhosphorImage analysis. (C) Radiolabeled permeabilized cells from wild type and hsp40 (hlj1/ydj1-151) strains expressing HA-tagged Yor1p or Yor1p-F at restrictive temperature were incubated on ice for 10 min with increasing concentrations of trypsin (0, 100, 200, and 400 ng/µl). Fragments were immunoprecipitated from cell lysates and analyzed by SDS-PAGE and PhosphorImage analysis. (D) Radiolabeled permeabilized cells from wild-type and hsp40 (hlj1/ydj1-151) strains expressing cysteine-substituted forms of Yor1p or Yor1p-F at restrictive temperature were subjected to cross-linking with M8M before solublization and immunoprecipitation. Stabilization of Yor1p-F in the hsp40 mutant did not result in the appearance of the typical cross-linked species.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We used limited proteolysis to demonstrate that Yor1p-F showed enhanced susceptibility to proteolytic attack, consistent with a partially folded or conformationally destabilized structure similar to that seen for the F form of CFTR (Du et al., 2005). Biochemical and structural analyses of CFTR NBD1 suggest that the F508 mutation does not dramatically alter the fold of the isolated domain but that it may instead disrupt surface topology. This in turn may perturb an interaction between NBD1 and MSD1, indirectly affecting the interactions between transmembrane helices (Lewis et al., 2005; Thibodeau et al., 2005). Furthermore, F508 in CFTR also contributes to the posttranslational folding of NBD2, likely through interdomain interactions (Du et al., 2005). Because the epitope tag with which we detected Yor1p is located at the C terminus of the protein, trypsinolysis specifically probes the conformational state of the second half of the protein. Thus, the stable 65-kDa proteolytic fragment observed for wild-type Yor1p likely corresponds to a cleavage event between TM8 and TM9, with the F670 mutation likely destabilizing NBD2, similar to the situation observed for CFTR. We also used cysteine substitution and cross-linking analysis to probe the arrangement of transmembrane domains in Yor1p. The inability of Yor1p-F to form cross-linkable interactions between membrane spanning domains suggests that, like CFTR, multiple interdomain interactions are impaired by this misfolding lesion (Cui et al., 2007).

Together, these in vitro assays suggest that Yor1p-F presents multiple assembly defects: improperly arranged transmembrane segments as well as a destabilized cytoplasmic domain (NBD2). The presence of multiple misfolding lesions is consistent with its redundant use of at least two disposal pathways. Both the Hrd1p-dependent pathway, which likely disposes of proteins with membrane-localized lesions in addition to ERAD-L clients (Carvalho et al., 2006), and the Doa10p-dependent pathway, which degrades ERAD-C substrates, can divert Yor1p-F for degradation; only when both pathways are disabled is Yor1p-F stabilized. Because this stabilization is only partial, the possibility remains that additional machinery also contributes in vivo to the efficient destruction of aberrant Yor1p. The specific misfolding lesions detected by each of these degradation systems remain to be defined, but they likely result from the exposure of multiple domain interfaces that are usually buried in the native protein. Our evidence that both NBD2 and the transmembrane domains of Yor1p-F are improperly assembled suggests that both cytosolic and intramembrane lesions are potential signals for degradation by the ERAD-C and ERAD-L/M pathways, respectively. The dual disposal mechanism we see for Yor1p-F is similar to that used by the amino acid permease, Gap1p, which is rapidly degraded in the absence of its chaperone, Shr3p (Kota et al., 2007). Again, the precise nature of the conformational abnormality presented by Gap1p in the absence of Shr3p is not known, but based on our data for Yor1p-F, it seems likely that Gap1p must present multiple folding defects to access both the ERAD-C and ERAD-L/M pathways.

Although misfolded Gap1p and Yor1p-F share the same redundant degradation pathways, these two proteins differ in their ability to be rescued by inhibiting degradation. Blocking ERAD of Yor1p-F did not permit capture into COPII vesicles, nor did it restore oligomycin resistance. However, when misfolded Gap1p was stabilized by introducing ERAD mutations into an shr3 strain, partial recovery of amino acid transport function was observed (Kota et al., 2007). This difference in protein recovery may stem from the nature of the specific folding defects associated with each individual protein. Aberrant Gap1p produced in the absence of its chaperone may represent a mixed population of folding intermediates, some of which can attain a folding state compatible with COPII engagement if the degradation pathways are impaired. Conversely, the specific genetic lesion that causes misfolding of Yor1p-F may render it incapable of proper assembly even given prolonged residence time in the ER. Thus, the ability of an aberrant protein to be rescued by impeding its destruction seems likely to be dependent on the specific nature of the misfolding defect, which will in turn influence the folding, degradative and trafficking pathways that each specific protein can access. Indeed, different misfolding variants of a single protein may behave distinctly; a variety of transthyretin mutants showed distinct secretion phenotypes that were not entirely correlated with the degree of global protein stability, even in the absence of stabilization through ERAD inhibition (Sekijima et al., 2005). Furthermore, even when considering a single protein, different mechanisms of stabilization may result in different outcomes. Attempts to rescue CFTR-F by inhibiting its destruction have met with mixed results; early experiments using proteasome inhibitors did not result i