HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 14, 11709-11714, April 5, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/14/11709    most recent M111958200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gelman, M. S. Articles by Kopito, R. R. Search for Related Content PubMed PubMed Citation Articles by Gelman, M. S. Articles by Kopito, R. R. Social Bookmarking                      What's this?

A Principal Role for the Proteasome in Endoplasmic Reticulum-associated Degradation of Misfolded Intracellular Cystic Fibrosis Transmembrane Conductance Regulator^*

Marina S. Gelman, Elisa S. Kannegaard, and Ron R. Kopito^§

From the Department of Biological Sciences, Stanford University, Stanford, California 94305-5020

Received for publication, December 14, 2001, and in revised form, January 16, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Endoplasmic reticulum-associated degradation of misfolded cystic fibrosis transmembrane conductance regulator (CFTR) protein is known to involve the ubiquitin-proteasome system. In addition, an ATP-independent proteolytic system has been suggested to operate in parallel with this pathway and become up-regulated when proteasomes are inhibited (Jensen, T. J., Loo, M. A., Pind, S., Williams, D. B., Goldberg, A. L., and Riordan, J. R. (1995) Cell 83, 129-135). In this study, we use two independent techniques, pulse-chase labeling and a noninvasive fluorescence cell-based assay, to investigate the proteolytic pathways underlying the degradation of misfolded CFTR. Here we report that only inhibitors of the proteasome have a significant effect on preventing the degradation of CFTR, whereas cell-permeable inhibitors of lysosomal degradation, autophagy, and several classes of protease had no measurable effect on CFTR degradation, when used either alone or in combination with the specific proteasome inhibitor carbobenzoxy-L-leucyl-leucyl-L-leucinal (MG132). Our results suggest that ubiquitin-proteasome-mediated degradation is the dominant pathway for disposal of misfolded CFTR in mammalian cells and provide new mechanistic insight into endoplasmic reticulum-associated degradation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The endoplasmic reticulum (ER)¹ is the site of synthesis of membrane and secretory proteins and the site of their conformational maturation and assembly into correctly folded functional molecules. "Quality control" refers to the system that monitors the folding and assembly of proteins in the ER, ensuring that only correctly folded proteins can mature to later compartments of the secretory pathway (1, 2). Nascent proteins that fail to fold or assemble in the ER are degraded by a process known as ER-associated degradation (ERAD) (3). Studies in mammalian cells and in yeast have led to the formulation of a general model for ERAD in which substrates are degraded by cytoplasmic proteasomes following retrotranslocation (i.e. "dislocation") across the ER membrane by a process that requires the Sec61 translocon and functional ubiquitylation machinery at the cytosolic face of the ER membrane (4-6). Dislocation of ERAD substrates across the ER membrane is kinetically coupled to their degradation by proteasomes, and in many cases, cytosolic intermediates of degradation are hard to detect (7, 8). Although a central role for proteasomes in ERAD is widely accepted, a number of studies have suggested that other, nonproteasomal, proteolytic systems may also contribute to the elimination of misfolded proteins from the ER (9-12).

The polytopic membrane protein cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel expressed at the apical surface of polarized epithelial cells and one of the first integral membrane proteins demonstrated to be a substrate of the ubiquitin-proteasome system (9, 13). Mutations in the CFTR gene cause the recessively inherited fatal disease cystic fibrosis. Although nearly 1000 mutations have been linked to cystic fibrosis, the majority of Caucasian cystic fibrosis patients have at least one copy of the F508 mutation, a temperature-sensitive allele that is unable to fold correctly at physiological temperature. As a consequence, F508 CFTR (F508) is not deployed to the plasma membrane and is instead rapidly degraded without being processed in the Golgi apparatus (14, 15).

Several lines of evidence support a role for the ubiquitin-proteasome pathway in F508 turnover. First, CFTR undergoes both co-translational and posttranslational ubiquitylation when expressed in a cell-free system (16, 17). Second, inhibition of proteasome function with chemical inhibitors or with a dominant negative form of ubiquitin leads to accumulation of multiubiquitylated, undegraded forms of CFTR and F508 (13). Third, degradation of F508 in a cell-free system is sensitive to inhibitors of both the 19 S and 20 S subunits of the proteasome (17, 18). Curiously, despite this compelling evidence in support of a role for proteasomes in the degradation of misfolded CFTR, the effect of proteasome inhibition on the disappearance of F508 from pulse-chase studies is minimal (9, 13). This observation has led some investigators to conclude that other proteolytic systems are primarily responsible for the ERAD of misfolded CFTR molecules (9). However, no other proteases or proteolytic systems have been convincingly demonstrated to contribute to the degradation of CFTR or other ERAD substrates. Therefore, the intracellular fate of F508 and the identity of the proteases that participate in its degradation remain unresolved.

In this study, we used a reporter (GFP-F508) consisting of a fusion of GFP with F508 (19) to assess the participation of proteasomes and other proteolytic systems in F508 degradation. Our data establish that proteolytic activities of the proteasome are responsible for vast majority of intracellular degradation of misfolded CFTR molecules.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- Most chemicals (with the exception of some proteasome inhibitors) were obtained from Sigma Chemical Co. Epoxomicin and carbobenzoxy-L-leucyl-leucyl-L-leucinal (MG132) were obtained from Calbiochem. YU102 was generously provided by Dr. Graig Crews (Yale University, New Haven, CT). Polyclonal anti-GFP antibody was a kind gift from Dr. Michael Rexach (Stanford University).

Constructs and Cell Lines-- pGFP-CFTR in a pEGFP-C1 vector (CLONTECH) was obtained from Dr. B. Stanton (Dartmouth Medical School, Hanover, NH) and subsequently subcloned into pcDNA3.1 vector (Invitrogen) using NheI and EcoRV. F508 mutation was introduced by replacing the [Pml1-Blp1] cassette within the CFTR sequence. For selection of stable CHO cell lines, GFP-F508/pcDNA3 plasmid was linearized and transfected into CHO cells by calcium phosphate precipitation. The cells were subjected to selection in G418-containing medium. Stable transfectants were expanded in culture and enriched for GFP-F508-expressing cells by fluorescence-activated cell sorting of highly fluorescent cells after overnight incubation of these cells with the proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. Cells were then cloned into 96-well dishes at a density of 1 cell/well. Clonal cell lines were expanded and analyzed for full-length GFP-F508 expression by immunoblotting, and a single clone (clone 2) was selected for this study. Generation of human embryonic kidney HEK293 cells stably expressing GFP^u was described elsewhere (20).

³⁵S Labeling and Immunoprecipitation-- CHO clone 2 cells were plated on 10 × 10-cm dishes and treated with 5 mM butyrate overnight to increase GFP-F508 expression. The next day, cells were starved in cysteine/methionine-free media for 30 min and then labeled with [³⁵S]cysteine/methionine (specific activity, 1 mCi/ml) for 30 min in the presence of 5 mM butyrate. Two 10-cm dishes were harvested immediately after the pulse (t = 0 h), whereas the remaining dishes were chased in the presence of the protein synthesis inhibitor emetine (75 µM) or emetine and MG132 (25 µM) together. At specified time intervals, cells were solubilized in buffer A (10 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.05% deoxycholate) containing a Complete^TM protease inhibitor mixture (Roche Diagnostics GmbH) for 30 min. Cell lysates were precleared with protein A-Sepharose beads and incubated with anti-GFP antibody and protein A-Sepharose. The immunoprecipitates were washed extensively with buffer A and fractionated by 4-15% gradient SDS-PAGE (Bio-Rad). Quantitation of radioactive bands was done using PhosphorImager SI and ImageQuant software (Molecular Dynamics).

Fluorescence-activated Cell-sorting Assay-- Analysis of GFP-F508 CFTR fluorescence by flow cytometry was performed using Coulter^® Epics^® XL-MCL Flow Cytometer and EXPO v.2 cytometer software. Cells were typically incubated with 5 mM butyrate overnight to increase GFP-F508 CFTR expression or with 10 µg/ml N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal for 3 h to increase GFP^u steady-state level. After the addition of emetine with or without protease inhibitors, cultures were incubated for different time intervals before analysis. Data from 10,000 cells were collected, and the mean fluorescence of the cell population was used as a measure of GFP-F508 levels after each treatment.

ATP Depletion-- CHO cl.2 cells expressing GFP-F508 and HEK293 cells expressing GFP^u were ATP-depleted by incubating the cells in glucose-free medium (Dulbecco's modified Eagle's medium; Invitrogen) containing the metabolic inhibitors cyanide (5 mM) and 2-deoxy-D-glucose (5 mM) as described previously (21). This procedure reduces ATP content below 10% of the baseline value within 10 min and sustains this low level of ATP content (22). Based on this determination, the initial fluorescence measurement in ATP-depleted cells (t = 0 h) was taken at 10 min after exposure of cells to metabolic inhibitors.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effect of Proteasome Inhibitors on GFP-F508 Degradation in Metabolic Pulse-chase Analysis-- To validate the use of GFP-F508 as a model of F508 degradation, we performed pulse-chase analysis in a clonal CHO cell line (cl.2) stably expressing this fusion protein. cl.2 cells were selected for low basal expression levels to avoid accumulation of GFP-F508 in detergent-insoluble aggresomes, which are highly refractory to degradation (23). Before use, the cells were treated with the transcriptional activator sodium butyrate to increase GFP-F508 expression. Under these conditions, aggregation of GFP-F508 was undetectable by either immunoblot or fluorescence microscopy (data not shown). Immunoprecipitation of metabolically labeled cell lysates with anti-GFP antibody revealed a major M_r ~170,000 band corresponding to core-glycosylated GFP-F508 and high molecular weight (HMW) species migrating near the top of the gel (Fig. 1A). During the chase, the M_r 170,000 band disappeared with first-order kinetics (t_1/2 = 50 min), consistent with previous determinations of F508 (9, 24, 25) and GFP-F508 half-life (23). In the presence of MG132, a specific proteasome inhibitor (26), the half-life of this species was only modestly extended (t_1/2 = 1.5 h) (Fig. 1B, top left panel). A minor band migrating at M_r ~50,000 probably corresponds to an amino-terminal fragment of the GFP-508 fusion protein comprising M_r ~20,000 of CFTR in addition to the GFP moiety because it reacted with antibody to GFP, but not with an antibody raised against the carboxyl terminus of CFTR (data not shown). Disappearance of the M_r 50,000 fragment paralleled that of the M_r 170,000 band (t_1/2 = 1.1 h) and was also only modestly inhibited by MG132 (t_1/2 = 1.5 h) (Fig. 1B, bottom left panel). HMW forms of GFP-F508 were detected immediately after the pulse (top band in Fig. 1A). These complexes disappeared rapidly when the chase was conducted in the absence of proteasome inhibitor (t_1/2 = 0.9 h), but in contrast to the core-glycosylated protein and the M_r 50,000 fragment, they were strongly stabilized by the presence of MG132 (t_1/2 = 6.6 h) (Fig. 1B, top right panel).

View larger version (59K): [in this window] [in a new window]   Fig. 1.   Pulse-chase analysis of GFP-F508 CFTR degradation. A, butyrate-treated CHO cells were labeled with [35S]cysteine/methionine and chased for the indicated time intervals in the presence or absence of MG132 (25 µM). Cells were then solubilized, and lysates were immunoprecipitated with anti-GFP antibody as described under "Experimental Procedures." B, quantitation of individual bands and total radioactivity in each lane was performed using ImageQuant software. Filled symbols represent cells that were chased without MG132, and open symbols represent cells chased in the presence of 25 µM MG132. The insets show semilog plots of these data. In the insets, solid and broken lines represent cells chased without or with MG132, respectively.

The HMW complexes could represent either polyubiquitylated forms of monomeric GFP-F508 (16, 17) or detergent-insoluble aggregates of CFTR (23). To distinguish between these possibilities, we sought to determine whether the HMW material that accumulated during the chase in the presence of MG132 could be degraded after washout of the inhibitor. Our results (data not shown) indicate that the HMW material was soluble in nonionic detergent and competent for degradation, strongly suggesting that the increase in molecular weight was due to covalent modification with Ub, as observed previously for CFTR in cell-free extracts (17).

Quantification of the pulse-chase data indicates that MG132 was only marginally effective at stabilizing the M_r 170,000 F508 band. Even when the total amount of GFP-immunoreactive material is considered (the sum of all electrophoretic species, Fig. 1B, bottom right panel), only 40% of the initial amount of GFP-F508 was stable at the end of a 4-h chase, despite proteasome inhibition. This observation suggests several plausible explanations. First, as suggested by Jensen et al. (9), in addition to proteasomes, other proteolytic systems could also contribute to F508 ERAD. Alternatively, it is possible that some proteolytic activities within the proteasome remain uninhibited in the presence of MG132 and continue to degrade the substrate. Finally, immunoprecipitation of radiolabeled material from detergent extracts of pulse-labeled cells may underestimate the amount of stable F508 if some of the protein becomes insoluble, if the epitope used for immunoprecipitation becomes inaccessible, or if the increased molecular weight (due to polyubiquitylation and/or aggregation) of the protein prevents it from being well resolved by SDS-PAGE. We reasoned that a noninvasive method of monitoring the fluorescence of GFP-F508 in intact cells using fluorescence-activated cell-sorting analysis may be a more accurate way to measure protein stability because it does not rely upon efficient recovery of radiolabeled protein from cells or upon the detection of a resolvable electrophoretic species. This would in turn allow us to better evaluate the effect of protease inhibitors on prevention of CFTR degradation.

Fluorescence-based Determination of GFP-F508 Degradation-- To circumvent the limitations of the pulse-chase approach, we developed a fluorescence-based assay to measure degradation of GFP-F508. Inhibition of GFP-F508 synthesis in butyrate-treated cells with emetine, an inhibitor of translational elongation, caused a rapid decrease in mean fluorescence, as measured by flow cytometry (Fig. 2). Similar results were obtained with other translation inhibitors including cycloheximide and puromycin (data not shown). Emetine does not affect the degradation kinetics of CFTR or F508 (13). The decrease in GFP-F508 fluorescence fitted a first-order exponential with a t_1/2 = 60 min, in good agreement with the GFP-F508 half-life measured in pulse-chase experiments (Fig. 2). In a control experiment, CHO cells expressing GFP alone were incubated with emetine for up to 10 h without any loss of total fluorescence (data not shown), consistent with GFP being a stable protein (20) and indicating that emetine treatment does not cause detectable cell lysis during the course of the experiment. Together, these results suggest that the decline in the fluorescence of cl.2 cells upon exposure to protein synthesis inhibitor faithfully reflects the degradation of F508 and constitutes a valid and convenient method for characterization of this degradation process.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Fluorescence-based measurement of F508 CFTR degradation rate. Butyrate-treated cells were incubated with 75 µM emetine for the indicated time intervals, and cellular fluorescence was measured by flow cytometry. The mean fluorescence of 10,000 cells was determined at each time point and represented as a percentage of initial fluorescence. The inset shows a semilog plot of fluorescence decline versus time in emetine, from which the half-time of fluorescence decline was calculated.

Inhibition of GFP-F508 Degradation by Proteasome Inhibitors-- To assess the effect of inhibition of different proteolytic systems on the degradation of mutant CFTR using the fluorescence assay, we evaluated the effect of a panel of protease inhibitors on the decline in GFP-F508 fluorescence in butyrate-treated cl.2 cells in the presence of emetine (Fig. 3A). Among the inhibitors tested, only those that are known to inhibit the proteasome (N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal, clasto-lactacystin -lactone, and MG132) significantly slowed the degradation of GFP-F508. In contrast, inhibitors of serine and cysteine proteases (TPCK, N-tosyl-Lys-chloromethyl ketone, and phenylmethylsulfonyl fluoride), calpains (EDTA), lysosomal cathepsins ((2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, chloroquine, and NH₄Cl), and autophagy (3-methyladenine and wortmannin) had no measurable effect on the degradation process, either alone (Fig. 3A) or in combination with MG132 (25 µM) (data not shown). These data confirm a central role for proteasomes in the degradation of F508 and suggest that other major proteolytic systems do not contribute significantly to this process.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Effect of protease inhibitors on GFP-F508 fluorescence. A, CHO cl.2 cells were treated with 5 mM butyrate overnight and incubated for 4 h with emetine alone (75 µM) or with emetine and one of the following protease inhibitors: N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (ALLN; 10 µg/ml), (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (EST; 10 µM), -lactone (25 µM); EDTA (2 mM), chloroquine (150 µM), NH4Cl (10 mM), MG132 (25 µM), N-tosyl-Lys-chloromethyl ketone (TLCK; 2 µg/ml), 3-methyladenine (3-MA; 10 mM), TPCK (2 µg/ml), and wortmannin (100 nM). Fluorescence at the end of 4 h was expressed as a percentage of initial fluorescence at t = 0, after subtraction of background fluorescence (CHO cell autofluorescence). B, CHO clone 2 cells expressing GFP-F508 (solid line) or HEK293 cells expressing GFPu (broken line) were incubated with 75 µM emetine ( or , respectively) or with 75 µM emetine and 25 µM MG132 ( or , respectively) for the indicated time intervals. Mean cellular fluorescence was expressed as a percentage of initial fluorescence at t = 0 h, after subtraction of background fluorescence.

In the presence of MG132 (25 µM), the fluorescence of GFP-F508 first increased, reaching almost 120% of the initial fluorescence after 1 h of emetine chase (Fig. 3B). This increase probably reflects slow fluorogenesis of the GFP moiety (t_1/2 = 30-90 min; 27, 28) in the absence of CFTR degradation. Indeed, this transient increase in fluorescence is also observed with GFP^u, a GFP molecule that is targeted to the ubiquitin-proteasome system for degradation via a short carboxyl-terminal degron (20) (Fig. 3B). However, in contrast to GFP^u, which was completely stabilized by 25 µM MG132, the fluorescence of GFP-F508 started to decline after the first hour and dropped to about 70% of initial fluorescence after 4 h of emetine chase. The half-time of the decline of GFP-F508 fluorescence in the presence of MG132 (25 µM) was 6.8 h, indicating that proteasome inhibition results in substantial stabilization of GFP-F508.

To determine whether the continued decline in GFP-F508 fluorescence in the presence of MG132 was due to degradation by the proteasome, we examined the effect of increased inhibitor concentration on the fluorescence remaining at the 4 h chase point (Fig. 4A). These data suggest that GFP-F508 degradation is inhibited by MG132 biphasically with a high-affinity component (IC₅₀  10 µM) and a low-affinity component that we were not able to saturate, even at concentrations as high as 300 µM.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Dose dependence of proteasome inhibitors in the fluorescence assay. A, fluorescence of butyrate-treated CHO cl.2 cells was measured at 4 h after the addition of emetine and the indicated concentrations of MG132 and expressed as a percentage of the initial fluorescence at t = 0 h. Values represent the average of three experiments. B, CHO cl.2 cells were incubated with emetine alone () or emetine plus the following concentrations of epoxomicin: 1 µM (), 5 µM (), 10 µM (), 20 µM (), and 50 µM (). Inset, rate constants of GFP-F508 degradation were calculated and plotted as a function of epoxomicin concentration. C, CHO cl.2 cells were incubated with increasing concentrations of epoxomicin in the absence () or presence () of 500 µM YU102. Values represent the average of three separate experiments + S.E.

To further investigate the role of proteasomes in GFP-F508 degradation, we performed a titration of epoxomicin, a highly specific and potent proteasome inhibitor (29). Like MG132, epoxomicin inhibited GFP-F508 fluorescence decay biphasically with a high-affinity component (IC₅₀ < 1 µM) and a low-affinity component (Fig. 4B). Although epoxomicin can inhibit all three of the characterized activities of the proteasomes, it is 66- and 500- fold more effective at inhibiting the chymotrypsin-like (CT-L) activity than the trypsin-like or peptidyl-glutamyl hydrolyzing activities, respectively (30). To investigate whether the low-affinity component of the MG132 and epoxomicin titrations was due to non-CT-L activities, we performed a titration of epoxomicin in the presence of 500 µM YU102, an ','-epoxyketone inhibitor that is selective for the peptidyl-glutamyl hydrolyzing activity of the proteasome (30). This treatment resulted in a small (10-15%) but significant increase in the degree of inhibition of GFP-F508 fluorescence decay at all epoxomicin concentrations (Fig. 4C). These results suggest that the CT-L and peptidyl-glutamyl hydrolyzing activities of the proteasome may contribute independently to the degradation of GFP-F508.

A hallmark of proteolysis by the Ub-proteasome system is its dependence on ATP hydrolysis. ATP is required for at least two steps in Ub-dependent degradation: activation of Ub, and substrate unfolding (31, 32). However, previous studies suggested that the fraction of CFTR degradation that is insensitive to proteasome inhibitors is independent of ATP (9). To assess the dependence of GFP-F508 degradation on ATP, we assayed GFP-F508 fluorescence in ATP-depleted cl.2 cells (Fig. 5A). These data demonstrate that GFP-F508 degradation is absolutely dependent on the presence of cellular ATP. The degradation of GFP^u was similarly dependent upon ATP (Fig. 5B).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effect of ATP depletion on GFP-F508 and GFPu degradation. CHO cl.2 cells expressing GFP-F508 (A) or HEK293 cells expressing GFPu (B) were subjected to emetine chase for up to 4 h in either high-glucose medium () or glucose-free medium containing 5 mM cyanide and 5 mM 2-deoxy-D-glucose (). Each data point represents an average of three separate experiments + S.E.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ER-associated degradation is a central element of the quality control pathways that ensure that only correctly folded and assembled proteins are deployed in the secretory pathway of eukaryotic cells. Although many studies have confirmed a primary role of the Ub-proteasome system in ERAD, the apparently minimal effect of highly potent proteasome inhibitors on the degradation of CFTR has remained an enigma and has suggested that other ATP-independent proteolytic systems also contribute to ERAD. In this study, we have used a fluorescent substrate to re-evaluate the role of the UPS in ERAD. Our data establish the primacy of the UPS in the degradation of ER-associated misfolded CFTR and suggest that the different proteolytic functionalities of the proteasome, associated with the three active -subunits, may operate independently in the degradation of an ERAD substrate (GFP-F508), but apparently not in the degradation of a cytoplasmic substrate (GFP^u). Three lines of evidence support our conclusion that the UPS is the principal pathway for degradation of misfolded CFTR. First, proteasome inhibitors, but not inhibitors of serine or cysteine proteases or inhibitors of lysosomal or autophagic pathways, strongly stabilize GFP-F508 fluorescence. Second, GFP-F508 degradation was inhibited by MG132 and epoxomicin in a dose-dependent fashion, with biphasic apparent IC₅₀ values in the expected range for CT-L and non-CT-L activities (30). Third, GFP-F508 degradation was strictly dependent on ATP, a hallmark of UPS-mediated proteolysis (31, 32). The possibility that other proteases initiate the clipping of the CFTR molecule while it is situated in the ER membrane, followed by proteasome-mediated proteolysis, is also unlikely because simultaneous addition of protease and proteasome inhibitors did not protect CFTR from degradation. Although our results do not rule out participation in CFTR degradation of protease classes for which cell-permeable inhibitors are not available (i.e. aspartic proteases, aminopeptidases, and so forth) or of substrate-specific proteases (e.g. colligin is a specific inhibitor of procollagen degradation (33)), these data establish that inhibition of proteolytic activities of the proteasome alone is sufficient to prevent the degradation of the majority of F508 molecules in living cells.

In previous studies using metabolic pulse-chase labeling and immunoprecipitation, proteasome inhibitors including MG132 and lactacystin had a very modest effect at inhibiting the decay of the electrophoretic species corresponding to core-glycosylated F508 degradation (9, 13). In the present work, we show that at least part of radiolabel lost from core-glycosylated GFP-F508 could be recovered as high molecular weight material. Because this HMW material remains competent for degradation, our data suggest that in the absence of proteasome function, a significant fraction of radiolabeled GFP-F508 molecules are converted to multiubiquitylated forms. Accumulation of HMW forms in the presence of proteasome inhibition is not observed for most other ERAD substrates such as the -subunit of the T-cell receptor (7, 34) and soluble proteins such as IgG light chains (8), which tend to accumulate as core-glycosylated detergent-soluble monomers in the ER after proteasome inhibition. It is likely that this difference is a consequence of the topology of CFTR, in which most of the mass of the protein is accessible to the cytoplasmic face of the ER and hence to components of the ubiquitin conjugation system. A recent study reported that inhibition of proteasome function leads to accumulation of a substantial fraction of CFTR in reconstituted cell-free extracts as high molecular weight multiubiquitylated protein (17).

In our studies, no proteasome inhibitor or combination thereof was able to completely suppress the decay of GFP-F508 fluorescence. It is unlikely that this apparent degradation (a ~20-30% decrease of fluorescence after a 4-h chase in the presence of proteasome inhibitors) is the result of cell death due to toxic effects of simultaneous exposure to emetine and proteasome inhibitor because >98% of cells were able to exclude propidium iodide staining at the end of a 4-h chase. Alternatively, the decrease in GFP-F508 fluorescence could, in principle, be due to self-quenching of GFP fluorescence in closely packed aggregates of GFP-F508, which are known to form upon chronic exposure to proteasome inhibitors (23). Such self-quenching is a form of fluorescence resonance energy transfer (fluorescence resonance energy transfer homotransfer). Because we do not observe heterotransfer between the compatible fluorescence resonance energy transfer heterotransfer pair CFP-F508 and YFP-F508, even under conditions designed to maximize aggregation,² it is unlikely that homotransfer between adjacent GFP-F508 molecules could account for the observed decrease in fluorescence. It is also formally possible that the small decrease in GFP-F508 fluorescence could be due to partial unfolding of the GFP moiety by the ATPase activity of the 19 S proteasome cap, without actual degradation of CFTR itself. However, the simplest explanation may be that none of the proteasome inhibitors used, alone or in combination, are able to fully inhibit all of the three proteasomal -subunits. This explanation is consistent with conversion of a similar fraction (~30%) of labeled GFP-F508 as high molecular weight multiubiquitin conjugates in the presence of MG132 in both our pulse-labeling experiments (Fig. 1) and in the cell-free experiments of Oberdorf et al. (18). It is likely, therefore, that the proteolytic activities of the proteasome associated with individual -subunits do not act in a strictly cooperative fashion in ERAD. Our findings with GFP-F508 in vivo are consistent with those of Oberdorf et al. (18), who found that complete inhibition of the 5-subunit (associated with CT-L activity) led to only a 40% reduction in cell-free CFTR degradation, and with those of Myung et al. (30), who reported recently that selective peptidyl-glutamyl hydrolyzing inhibition is insufficient to inhibit protein degradation in vivo. These data all strongly indicate that the catalytic sites of the proteasome function independently. It is not clear why, in our studies, the degradation of GFP^u, a small soluble substrate of the UPS, is completely inhibited by MG132, whereas degradation of GFP-F508 is not. Perhaps this discrepancy is a reflection of functional differences in the mechanisms by which proteasomes engage substrates delivered from the cytosol and those that are dislocated across the ER membrane.

	ACKNOWLEDGEMENTS

We thank Neil Bence for providing HEK293 cells expressing GFP^u and Karim Helmy for help with epoxomicin and ATP depletion experiments. We are grateful to Dr. Craig Crews for a kind gift of YU102. We also thank members of the Kopito laboratory for helpful comments.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant R01 DK43994 (to R. R. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported in part by a postdoctoral training grant from the National Institutes of Health.

^§ To whom correspondence should be addressed: Dept. of Biological Sciences, Stanford University, 371 Serra Mall, Stanford, CA 94305-5020. Tel.: 650-723-7581; Fax: 650-723-8475; E-mail: kopito@stanford.edu.

Published, JBC Papers in Press, January 25, 2002, DOI 10.1074/jbc.M111958200

² R. Rajan and R. R. Kopito, unpublished data.

	ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; UPS, ubiquitin-proteasome system; TPCK, tosylphenylalanyl chloromethyl ketone; CHO, Chinese hamster ovary, CFTR, cystic fibrosis transmembrane conductance regulator; GFP, green fluorescent protein; cl.2, clone 2; HMW, high molecular weight; Ub, ubiquitin; CT-L, chymotrypsin-like.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				M. H. Brush and S. Shenolikar Control of Cellular GADD34 Levels by the 26S Proteasome Mol. Cell. Biol., December 1, 2008; 28(23): 6989 - 7000. [Abstract] [Full Text] [PDF]

				L. Cebotaru, N. Vij, I. Ciobanu, J. Wright, T. Flotte, and W. B. Guggino Cystic Fibrosis Transmembrane Regulator Missing the First Four Transmembrane Segments Increases Wild Type and {Delta}F508 Processing J. Biol. Chem., August 8, 2008; 283(32): 21926 - 21933. [Abstract] [Full Text] [PDF]

				S. M. Kelly, J. K. VanSlyke, and L. S. Musil Regulation of Ubiquitin-Proteasome System mediated Degradation by Cytosolic Stress Mol. Biol. Cell, November 1, 2007; 18(11): 4279 - 4291. [Abstract] [Full Text] [PDF]

				N. S. Sabeva, E. J. Rouse, and G. A. Graf Defects in the Leptin Axis Reduce Abundance of the ABCG5-ABCG8 Sterol Transporter in Liver J. Biol. Chem., August 3, 2007; 282(31): 22397 - 22405. [Abstract] [Full Text] [PDF]

				M. W. Wendeler, O. Nufer, and H.-P. Hauri Improved maturation of CFTR by an ER export signal FASEB J, August 1, 2007; 21(10): 2352 - 2358. [Abstract] [Full Text] [PDF]

				Z. E. V. Worthington and N. H. Carbonetti Evading the Proteasome: Absence of Lysine Residues Contributes to Pertussis Toxin Activity by Evasion of Proteasome Degradation Infect. Immun., June 1, 2007; 75(6): 2946 - 2953. [Abstract] [Full Text] [PDF]

				L. Catley, E. Weisberg, T. Kiziltepe, Y.-T. Tai, T. Hideshima, P. Neri, P. Tassone, P. Atadja, D. Chauhan, N. C. Munshi, et al. Aggresome induction by proteasome inhibitor bortezomib and {alpha}-tubulin hyperacetylation by tubulin deacetylase (TDAC) inhibitor LBH589 are synergistic in myeloma cells Blood, November 15, 2006; 108(10): 3441 - 3449. [Abstract] [Full Text] [PDF]

				D. Ito, I. Koshino, N. Arashiki, H. Adachi, M. Tomihari, S. Tamahara, K. Kurogi, T. Amano, K.-i. Ono, and M. Inaba Ubiquitylation-independent ER-associated degradation of an AE1 mutant associated with dominant hereditary spherocytosis in cattle J. Cell Sci., September 1, 2006; 119(17): 3602 - 3612. [Abstract] [Full Text] [PDF]

				T. E. Machen Innate immune response in CF airway epithelia: hyperinflammatory? Am J Physiol Cell Physiol, August 1, 2006; 291(2): C218 - C230. [Abstract] [Full Text] [PDF]

				K. Harada, T. Okiyoneda, Y. Hashimoto, K. Ueno, K. Nakamura, K. Yamahira, T. Sugahara, T. Shuto, I. Wada, M. A. Suico, et al. Calreticulin Negatively Regulates the Cell Surface Expression of Cystic Fibrosis Transmembrane Conductance Regulator J. Biol. Chem., May 5, 2006; 281(18): 12841 - 12848. [Abstract] [Full Text] [PDF]

				J. Oberdorf, E. J. Carlson, and W. R. Skach Uncoupling proteasome peptidase and ATPase activities results in cytosolic release of an ER polytopic protein J. Cell Sci., January 15, 2006; 119(2): 303 - 313. [Abstract] [Full Text] [PDF]

				M. V. Soldovieri, P. Castaldo, L. Iodice, F. Miceli, V. Barrese, G. Bellini, E. M. del Giudice, A. Pascotto, S. Bonatti, L. Annunziato, et al. Decreased Subunit Stability as a Novel Mechanism for Potassium Current Impairment by a KCNQ2 C Terminus Mutation Causing Benign Familial Neonatal Convulsions J. Biol. Chem., January 6, 2006; 281(1): 418 - 428. [Abstract] [Full Text] [PDF]

				Z. Bebok, J. F. Collawn, J. Wakefield, W. Parker, Y. Li, K. Varga, E. J. Sorscher, and J. P. Clancy Failure of cAMP agonists to activate rescued {Delta}F508 CFTR in CFBE41o- airway epithelial monolayers J. Physiol., December 1, 2005; 569(2): 601 - 615. [Abstract] [Full Text] [PDF]

				J. K. VanSlyke and L. S. Musil Cytosolic Stress Reduces Degradation of Connexin43 Internalized from the Cell Surface and Enhances Gap Junction Formation and Function Mol. Biol. Cell, November 1, 2005; 16(11): 5247 - 5257. [Abstract] [Full Text] [PDF]

				C. M. Farinha and M. D. Amaral Most F508del-CFTR Is Targeted to Degradation at an Early Folding Checkpoint and Independently of Calnexin Mol. Cell. Biol., June 15, 2005; 25(12): 5242 - 5252. [Abstract] [Full Text] [PDF]

				M. Sugita, H. Kongo, and Y. Shiba Molecular Dissection of the Butyrate Action Revealed the Involvement of Mitogen-Activated Protein Kinase in Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis Mol. Pharmacol., November 1, 2004; 66(5): 1248 - 1259. [Abstract] [Full Text] [PDF]

				R. T. Youker, P. Walsh, T. Beilharz, T. Lithgow, and J. L. Brodsky Distinct Roles for the Hsp40 and Hsp90 Molecular Chaperones during Cystic Fibrosis Transmembrane Conductance Regulator Degradation in Yeast Mol. Biol. Cell, November 1, 2004; 15(11): 4787 - 4797. [Abstract] [Full Text] [PDF]

				X. Wang, J. Matteson, Y. An, B. Moyer, J.-S. Yoo, S. Bannykh, I. A. Wilson, J. R. Riordan, and W. E. Balch COPII-dependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code J. Cell Biol., October 11, 2004; 167(1): 65 - 74. [Abstract] [Full Text] [PDF]

				G. A. Graf, J. C. Cohen, and H. H. Hobbs Missense Mutations in ABCG5 and ABCG8 Disrupt Heterodimerization and Trafficking J. Biol. Chem., June 4, 2004; 279(23): 24881 - 24888. [Abstract] [Full Text] [PDF]

				J. P. Richardson, S. S. Mohammad, and G. D. Pavitt Mutations Causing Childhood Ataxia with Central Nervous System Hypomyelination Reduce Eukaryotic Initiation Factor 2B Complex Formation and Activity Mol. Cell. Biol., March 15, 2004; 24(6): 2352 - 2363. [Abstract] [Full Text] [PDF]

				T. Okiyoneda, K. Harada, M. Takeya, K. Yamahira, I. Wada, T. Shuto, M. A. Suico, Y. Hashimoto, and H. Kai {Delta}F508 CFTR Pool in the Endoplasmic Reticulum Is Increased by Calnexin Overexpression Mol. Biol. Cell, February 1, 2004; 15(2): 563 - 574. [Abstract] [Full Text] [PDF]

				J. M. Wright, P. L. Zeitlin, L. Cebotaru, S. E. Guggino, and W. B. Guggino Gene expression profile analysis of 4-phenylbutyrate treatment of IB3-1 bronchial epithelial cell line demonstrates a major influence on heat-shock proteins Physiol Genomics, January 15, 2004; 16(2): 204 - 211. [Abstract] [Full Text] [PDF]

				P. L. Zeitlin, D. B. Gail, and S. Banks-Schlegel Protein Processing and Degradation in Pulmonary Health and Disease Am. J. Respir. Cell Mol. Biol., November 1, 2003; 29(5): 642 - 645. [Full Text]

				D. W. Miller, R. Ahmad, S. Hague, M. J. Baptista, R. Canet-Aviles, C. McLendon, D. M. Carter, P.-P. Zhu, J. Stadler, J. Chandran, et al. L166P Mutant DJ-1, Causative for Recessive Parkinson's Disease, Is Degraded through the Ubiquitin-Proteasome System J. Biol. Chem., September 19, 2003; 278(38): 36588 - 36595. [Abstract] [Full Text] [PDF]

				S. Kalra, N. Li, S. Seetharam, D. H. Alpers, and B. Seetharam Function and stability of human transcobalamin II: role of intramolecular disulfide bonds C98-C291 and C147-C187 Am J Physiol Cell Physiol, July 1, 2003; 285(1): C150 - C160. [Abstract] [Full Text] [PDF]

				J. L. Webb, B. Ravikumar, J. Atkins, J. N. Skepper, and D. C. Rubinsztein {alpha}-Synuclein Is Degraded by Both Autophagy and the Proteasome J. Biol. Chem., June 27, 2003; 278(27): 25009 - 25013. [Abstract] [Full Text] [PDF]

				B. Drisaldi, R. S. Stewart, C. Adles, L. R. Stewart, E. Quaglio, E. Biasini, L. Fioriti, R. Chiesa, and D. A. Harris Mutant PrP Is Delayed in Its Exit from the Endoplasmic Reticulum, but Neither Wild-type nor Mutant PrP Undergoes Retrotranslocation Prior to Proteasomal Degradation J. Biol. Chem., June 6, 2003; 278(24): 21732 - 21743. [Abstract] [Full Text] [PDF]

				R. C. Zangar, T. A. Kocarek, S. Shen, N. Bollinger, M. S. Dahn, and D. W. Lee Suppression of Cytochrome P450 3A Protein Levels by Proteasome Inhibitors J. Pharmacol. Exp. Ther., June 1, 2003; 305(3): 872 - 879. [Abstract] [Full Text] [PDF]

				Y. Xu, J. C. Clark, B. J. Aronow, C. R. Dey, C. Liu, J. L. Wooldridge, and J. A. Whitsett Transcriptional Adaptation to Cystic Fibrosis Transmembrane Conductance Regulator Deficiency J. Biol. Chem., February 21, 2003; 278(9): 7674 - 7682. [Abstract] [Full Text] [PDF]

				W.-J. Wang, S. Mulugeta, S. J. Russo, and M. F. Beers Deletion of exon 4 from human surfactant protein C results in aggresome formation and generation of a dominant negative J. Cell Sci., February 15, 2003; 116(4): 683 - 692. [Abstract] [Full Text] [PDF]

				L. Fu and E. Sztul Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator J. Cell Biol., January 21, 2003; 160(2): 157 - 163. [Abstract] [Full Text] [PDF]

				L. Garrigue-Antar, N. Hartigan, and K. E. Kadler Post-translational Modification of Bone Morphogenetic Protein-1 Is Required for Secretion and Stability of the Protein J. Biol. Chem., November 1, 2002; 277(45): 43327 - 43334. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/14/11709    most recent M111958200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gelman, M. S. Articles by Kopito, R. R. Search for Related Content PubMed PubMed Citation Articles by Gelman, M. S. Articles by Kopito, R. R. Social Bookmarking                      What's this?