From the Department of Molecular and Cellular Engineering and Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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The ubiquitin-proteasome pathway has been
implicated in the degradation of newly synthesized, misfolded and
unassembled proteins in the endoplasmic reticulum (ER). Using a
cell-free reticulocyte lysate system we have examined the relationship
between biosynthesis and ER-associated degradation of the cystic
fibrosis transmembrane conductance regulator (CFTR), a polytopic
protein with 12 predicted transmembrane segments. Our results provide
direct evidence that full-length, glycosylated and membrane-integrated
CFTR is a substrate for degradation and that degradation involves
polyubiquitination and cytosolic proteolytic activity. CFTR
ubiquitination was both temperature- and ATP-dependent.
Degradation was significantly inhibited by EDTA, apyrase, and the
proteasome inhibitors hemin and MG132. Degradation was inhibited to a
lesser extent by clasto-lactacystin The rough endoplasmic reticulum
(ER)1 facilitates
translocation, membrane integration, folding, and oligomeric assembly
of most proteins found in the secretory pathway of eukaryotic cells (1-4). The ER also functions to recognize misfolded or unassembled proteins and prevent their exit to the Golgi complex (5-7) through a
process termed ER-associated degradation (ERAD) (8). Substrates for
ERAD, like other secretory and transmembrane proteins, are initially
targeted to and translocated across the ER membrane via the Sec61
translocon complex (1, 2). However, aberrantly folded proteins are
selectively translocated back into the cytosol where they undergo
ubiquitination and degradation by cytosolic proteases (9-11). Certain
ERAD substrates such as major histocompatibility complex class I heavy
chains (10, 12, 13) and unglycosylated prepro- The ubiquitin-proteasome pathway is also involved in the ER-associated
degradation of polytopic proteins. Yeast mutants with defective
ubiquitin-conjugating enzymes or proteasome subunits show decreased
degradation of mutant forms of Sec61p (16, 17), a resident ER protein
with 10 predicted transmembrane segments (18). ERAD may also play a
role in ensuring proper protein levels in the ER as suggested by
mevalonate-mediated regulation of 3-hydroxy-3-methylglutaryl-coenzyme A
reductase (19-21). For the cystic fibrosis transmembrane conductance regulator (CFTR), a plasma membrane protein, approximately 80% of wild
type protein and 100% of common mutant forms are degraded prior to
reaching the Golgi complex (22, 23). Recent studies have demonstrated
that ER-associated degradation of CFTR also requires an intact
ubiquitin conjugation pathway and likely involves the 26 S proteasome
(24, 25). While the underlying reasons for CFTR degradation remain
unknown, it has been proposed that the majority of wild type CFTR, like
mutant CFTR, is recognized by ER quality control machinery, because it
fails to fold properly (26-29).
Degradation of polytopic proteins by cytosolic proteases poses a
particular problem in that multiple transmembrane (TM) segments must be
removed from the membrane. It is unclear whether polytopic proteins,
like secretory and bitopic transmembrane proteins, proceed through
cytosolic intermediates prior to degradation. One possibility is that
the proteasome might degrade only cytosolic peptide loops, leaving
lumenal and/or transmembrane segments to be degraded by other proteases
(7, 30). Alternatively, degradation might be coupled to the process of
retrograde translocation. To distinguish among these possibilities, it
will be necessary to define the temporal relationship between
recognition, retrograde translocation, and degradation events involved
in polytopic protein ER degradation. In this regard, a cell-free system
that reconstitutes ERAD is attractive, because it is readily amenable
to physical and pharmacological manipulation. Using such a system, Sato
et al. (31) recently demonstrated that CFTR could be
ubiquitinated cotranslationally, indicating that initial recognition
events in ER degradation might occur very early in the lifetime of a
polytopic protein. In the current study we use a similar cell free
system to demonstrate that CFTR ubiquitination can also occur after
protein synthesis, N-linked glycosylation, and membrane
integration have been completed. We have also been able to separate the
process of ubiquitination from that of degradation and now show that
ubiquitinated CFTR remains tightly associated with the ER membrane
until it is degraded into trichloroacetic acid-soluble peptide
fragments. CFTR degradation is therefore physically localized to the
ER. Our results are consistent with a model in which cytosolic
degradation machinery is recruited to the ER membrane by
polyubiquitinated substrates and suggest that degradation occurs
coincident with polypeptide removal from the lipid bilayer.
Materials--
ALLN, TPCK, TLCK, hemin, bovine ubiquitin,
anti-bovine ubiquitin antibody, and 14C-methylated lysozyme
were purchased from Sigma. The protease inhibitor mixture,
(4-(2-aminoethyl)benzenesulfonyl fluoride, aprotinin, E-64, EDTA, and
leupeptin), MG132, lactacystin, and clasto-lactacystin
Rabbit reticulocyte lysate for translation was prepared from fresh
rabbit reticulocytes induced by acetylphenylhydrazine treatment as
described previously (32) and stored at
Methylated ubiquitin was prepared by reductive methylation as described
previously (34). Bovine ubiquitin was incubated at room temperature for
20 h in 12 mM formaldehyde and 20 mM
cyanoborohydride followed by an additional hour in fresh reagents. The
reaction was dialyzed for 48 h in 10 mM HEPES, 5 mM MgCl2, 3 mM DTT and concentrated
to 10 mg/ml using a centricon (number 3) filter.
Cell-free Transcription/Translation--
CFTR mRNA was
transcribed in vitro at 40 °C for 1 h in reactions
containing 0.4 mg/ml plasmid DNA (plasmid pSPCFTR(35)), 40 mM Tris (pH 7.5), 6.0 mM MgAc2, 2 mM spermidine, 0.5 mM each of ATP, CTP, UTP,
0.1 mM GTP, 0.5 mM GpppG (Amersham Pharmacia
Biotech), 10 mM DTT, 0.2 mg/ml bovine calf tRNA, 0.75 unit/ml RNase inhibitor (Promega, Madison, WI), and 0.4 unit/ml SP6 RNA
polymerase (New England Biolabs, Beverly, MA). Transcript was stored
frozen at Carbonate Extraction--
Translation products were incubated in
0.1 M NaC03 (pH 11.5) or in 0.25 M
sucrose, 0.1 M Tris (pH 7.5) for 30 min on ice as described
(37). Samples were centrifuged at 180,000 × g for 30 min, and membrane pellets were dissolved directly in SDS loading buffer. Supernatants were precipitated in 20% trichloroacetic acid,
pelleted at 16,000 × g for 15 min, washed with
acetone, and dissolved in SDS loading buffer.
Immunoprecipitation--
RRL aliquots were diluted directly into
1 ml of ice-cold Buffer A (0.1 M NaCl, 1% Triton X-100, 2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride,
and 0.1 M Tris (pH 8.0)) and preincubated with antisera for
10-30 min prior to addition of 5 µl of protein A Affi-Gel (Bio-Rad).
CFTR antisera and nonimmune antisera were used at 1:1000 dilution.
Ubiquitin antibody was used at 1:200 dilution (similar amounts of Ig
were present in all reactions). Samples were mixed at 4 °C for 6-10
h, washed three times with Buffer A, twice with 0.1 M NaCl,
0.1 M Tris (pH 8.0), and taken up in SDS loading buffer.
Degradation Assay--
The translation mixture was layered onto
a 0.5 M sucrose buffer (0.5 M sucrose, 50 mM HEPES (pH 7.5), 0.1 M KCl, 5 mM
MgCl2, and 1 mM DTT) and centrifuged at
180,000 × g for 10 min. Membranes were resuspended in
ice-cold 0.1 M sucrose buffer (0.1 M sucrose, 50 mM HEPES (pH 7.5), 0.1 M KCl, 5 mM MgCl2, and 1 mM DTT) at one-half
the original translation volume. The degradation reaction, assembled on
ice, included 20% resuspended membranes, 72% reticulocyte lysate, and
a final concentration of 10 mM Tris (pH 7.5), 1 mM ATP, 5 mM MgCl2, 12 mM creatine phosphate, 3 mM DTT, and 80 µg/ml of creatine kinase. Where indicated, RRL was replaced with water, and
proteasome inhibitors were added. For ATP-depleted RRL, ATP and
creatine phosphate were omitted. The degradation mixture was incubated
at 37 °C, and aliquots were added directly to loading buffer for
SDS-PAGE, EnHance (NEN Life Sceince Products) fluorography and
autoradiography. Autoradiograms were scanned on an AGFA studioscan II
transmission scanner using Adobe Photoshop software. Quantitation of
autoradiograms was performed using an Amersham Pharmacia Biotech DTS
scanning densitometer precalibrated with a Kodak photographic step
tablet (Eastman Kodak Co.). Trichloroacetic acid-soluble counts were
quantitated by precipitating degradation mixture in 20%
trichloroacetic acid, incubating on ice for 30 min, pelleting at
16,000 × g for 15 min, and counting supernatants in 10 volumes of Ready Safe scintillation fluid (Beckman, Fullerton, CA) in a
Beckman LS6500 scintillation counter.
Vesicle Pelleting and Floatation--
Microsomal membranes from
translation mixture were pelleted, resuspended, and incubated in ATP-
containing RRL in the presence of 40 µM hemin as
described above. For pelleting experiments, aliquots of this RRL/hemin
mixture were taken at specified time points; KCl or Triton X-100 was
added as indicated; the mixture was incubated on ice for 30 min and
pelleted through 0.5 M sucrose buffer as above. Equivalent
fractions of pellet and supernatant (resuspended directly in SDS
loading buffer) were analyzed by SDS-PAGE. For floatation experiments,
aliquots of the RRL/hemin mixture were diluted into 10 volumes of 2.2 M sucrose, 0.1 M KCl, 10 mM Tris
(pH 7.5), 5 mM MgCl2, 1 mM DTT, or
into 2.2 M sucrose, 0.6 M KCl, 10 mM Tris (pH 7.5), 1 mM DTT, and 10 mM EDTA. 100-µl samples were incubated on ice for 30 min
and overlaid with 300 µl of 1.8 M sucrose, 0.1 M KCl, 4 mM MgCl2, 10 mM Tris (pH 7.5), and 1 mM DTT and then
overlaid with 200 µl of 0.5 M sucrose, 0.1 M
KCl, 10 mM Tris (pH 7.5), 5 mM
MgCl2, and 1 mM DTT. Solutions were centrifuged
at 350,000 × g for 2 h in a TLA 100.2 rotor. 55-µl aliquots were taken from top to bottom, mixed with 180 µl of
0.1 M sucrose buffer, and samples were repelleted at
170,000 × g for 10 min prior to addition of SDS
loading buffer and autoradiography.
In Vitro CFTR Biogenesis--
To examine the steps of CFTR
degradation in native ER membranes, we first characterized expression
of full-length CFTR protein in a cell-free RRL system. Translation of
plasmid pSPCFTR (35) in the absence of ER-derived microsomal membranes
generated a polypeptide migrating at ~160 kDa (Fig.
1A). This translation product
shifted 6 kDa in size when rough microsomes were added at the start of
translation, consistent with the addition of two N-linked
core carbohydrates at the predicted consensus sites in the fourth
extracytoplasmic loop. The presence of full-length CFTR was confirmed
by immunoprecipitation with peptide-specific antisera raised against N
terminus, R domain, and C terminus epitopes. Carbonate extraction of
translation products demonstrated that in vitro synthesized
CFTR was fully integrated into the ER membrane. Thus the RRL system is
capable of efficiently reconstituting the early events of CFTR
biosynthesis and processing, in agreement with results reported by Sato
et al. (31). To test the stability of newly synthesized CFTR
in the ER membrane, cyclohexamide was added after 2 h of
translation, and samples were incubated for an additional 4 h
(Fig. 1B). At 24 °C, CFTR was remarkably stable, whereas
at 37 °C CFTR was rapidly converted into a high molecular weight
(HMW) complex with an estimated size of >450 kDa
(T1/2 <30 min). For convenience, this large complex
will subsequently be referred to as the HMW complex, and the ~170 kDa
band will be referred to as full-length CFTR.
ATP-dependent Conversion of CFTR into a HMW
Complex--
To determine the requirements for conversion of CFTR into
the HMW complex, CFTR was translated in RRL, and microsomal membranes were collected by pelleting through a sucrose cushion (Fig.
2A). Microsomes were then
resuspended and incubated at 37 °C in sucrose buffer containing ATP
(lanes 1-5), RRL supplemented with an ATP regeneration
system (lanes 6-10), ATP-depleted RRL (lanes
11-15), or ATP-depleted RRL supplemented with additional ATP
(lanes 15-20). At specified time points samples were
analyzed directly by SDS-PAGE. In the presence of buffer alone, or in
ATP-depleted RRL, >70% of full-length CFTR remained intact after
4 h of incubation (Fig. 2B). However, when microsomal
membranes were incubated in fresh RRL containing ATP, or in
ATP-depleted RRL supplemented with additional ATP, full-length CFTR was
rapidly converted into the HMW complex (T1/2 < 30 min) that subsequently disappeared over the next 4 h. These results are consistent with a
two-step process in which full-length CFTR is initially converted into
a HMW complex via an ATP-dependent mechanism, and the HMW complex is then a substrate for degradation.
The CFTR HMW Complex Is Polyubiquitinated--
We noted that
degradation of the CFTR HMW complex in Fig. 2 contrasted with results
in Fig. 1B where the complex accumulated over time. One
difference between these two experiments is that CFTR translation is
performed in the presence of hemin, a required co-factor for protein
expression in RRL (38), and aurin tricarboxylic acid, an inhibitor of
protein synthesis initiation. Both of these compounds inhibit
ATP-dependent degradation of ubiquitinated substrates in
RRL. These results, together with the requirement for ATP and cytosol,
suggested that the HMW complex might represent polyubiquitinated CFTR.
Microsomal membranes containing newly synthesized CFTR were therefore
incubated in fresh RRL supplemented with bovine ubiquitin and 40 µM hemin. Aliquots were then immunoprecipitated with
anti-bovine ubiquitin antisera and analyzed by SDS-PAGE at specified
time points. As shown in Fig.
3A, the HMW complex, but not
full-length CFTR, was reactive against anti-ubiquitin antisera
(lanes 11-15). No reactivity was observed when
immunoprecipitation was performed with nonimmune sera (NIS,
lanes 6-10) or when bovine ubiquitin was omitted from the
lysate (data not shown). To further confirm CFTR ubiquitination,
microsomes containing newly synthesized CFTR were incubated in RRL in
the presence of methylated ubiquitin, which blocks the sequential
addition of ubiquitin molecules by preventing isopeptide bond formation
at ubiquitin residue Lys-48. Under these conditions we observed a
dose-dependent inhibition in the rate of HMW complex
formation (Fig. 3B). Taken together, these data demonstrate
that the sequential, covalent addition of multiple ubiquitin molecules
contributes to the formation of the CFTR HMW complex.
Inhibition of CFTR HMW Complex Degradation by Proteasome
Inhibitors--
We next examined the ability of protease inhibitors to
influence the disappearance of the CFTR HMW complex. Inhibitors of serine, cysteine, and metalloproteases had essentially no effect on the
rate of disappearance of either full-length CFTR or the HMW complex
(Fig. 4A). In contrast, hemin
and MG132, both known inhibitors of the proteasome, resulted in
significant delay in the disappearance of the HMW complex particularly
at early time points (Fig. 4B). Quantitation of
autoradiograms revealed that hemin effectively blocked 60% of HMW
complex degradation. In contrast, MG132, ALLN, and TPCK slowed but did
not prevent HMW complex disappearance. Surprisingly, little inhibition
was observed for lactacystin or TLCK. These results suggested either
that certain proteasome inhibitors might be relatively inactive in the
RRL system, or alternatively, that degradation of ubiquitinated CFTR
might involve additional protease activities. To distinguish between
these possibilities, we therefore examined the effect of proteasome
inhibitors on the degradation of methylated lysozyme, a known substrate
of the 26 S proteasome. For these and subsequent experiments the rate
and extent of protein degradation was quantitated by measuring the conversion of CFTR and/or lysozyme into trichloroacetic acid-soluble peptide fragments.
As shown in Fig. 5, 26% of
[14C]lysozyme and 65% of newly synthesized CFTR were
degraded into trichloroacetic acid-soluble peptide fragments within
3 h in RRL. Lysozyme degradation was inhibited by approximately
90% in the presence of 40 µM hemin or 10 mM
EDTA consistent with previous studies (39). Hemin also resulted in formation of a HMW complex containing [14C]lysozyme (data
not shown). In contrast, the specific proteasome inhibitor lactacystin,
as well as its active metabolite clasto-lactacystin Ubiquitinated CFTR Remains Tightly Associated with the ER
Membrane--
Sixty-five percent of in vitro synthesized
CFTR protein was degraded to trichloroacetic acid-soluble fragments
within 3 h of incubation in RRL (Fig. 5). Degradation could not be
restricted solely to large cytosolic peptide loops (i.e. N
terminus, nucleotide binding domains, and the R-domain), as these loops
contain only 47% of total methionine residues (40). Rather, our data
indicate that proteolytic machinery also gains access to small
cytosolic loops and possibly lumenal and/or transmembrane regions of
the protein. Thus prior to or coincident with degradation, some or all
of CFTR is likely to be extracted from the membrane. We therefore tested whether the ubiquitinated HMW complex, which has already been
targeted for degradation by ubiquitin-conjugating enzymes, might
undergo retrograde translocation to a cytosolic intermediate. Ubiquitinated CFTR complexes were generated by incubation in RRL (in
the presence of hemin), and vesicles were collected by pelleting through a sucrose cushion. As shown in Fig.
6A, both CFTR and the CFTR HMW
complex were nearly quantitatively recovered in the microsome pellet in
the absence of detergent. In addition, preincubation of CFTR-containing
microsomes in high salt (up to 1.1 M KCl) failed to extract
full-length CFTR or the HMW complex into the supernatant fraction (Fig.
6B). Thus ubiquitinated CFTR does not behave as a cytosolic
or peripheral membrane protein.
Results from Fig. 6 do not rule out the possibility that HMW
ubiquitinated CFTR might also be pelleted as a large,
detergent-sensitive protein complex. We therefore tested whether the
HMW complex was physically associated with the ER by membrane
floatation. Aliquots of RRL containing newly synthesized CFTR and/or
polyubiquitinated CFTR were diluted into 2 M sucrose,
layered beneath a discontinuous sucrose gradient, and centrifuged for
2 h as described under "Experimental Procedures" (Fig.
7A). For half of the samples,
ribosomes were first stripped from membranes by incubation in KCl/EDTA
(Fig. 7B). The secretory protein bovine prolactin (41) was
used as a standard to determine the location of microsomal membranes in the gradient. Both full-length CFTR and the polyubiquitinated complex
exhibited the same floatation pattern as the secretory control. Thus in
the absence of degradation, CFTR remained membrane-associated long
after ubiquitination had occurred. Following ribosome stripping with
EDTA/KCl microsomes more uniformly partitioned into the lighter fractions. However ribosome removal had no detectable effect on dislocation of ubiquitinated CFTR from the ER membrane.
Degradation of Membrane-bound, Ubiquitinated CFTR--
To test
whether membrane-bound CFTR remained a substrate for degradation and
not simply a protease resistant aggregate, CFTR-containing microsomes
were incubated in RRL in the presence of hemin to allow accumulation of
the ubiquitinated intermediate. Vesicles were isolated at sequential
time points, reincubated in the presence or absence of fresh RRL, and
degradation of CFTR was monitored by release of trichloroacetic
acid-soluble counts. As shown in Fig.
8A, 70% of total CFTR was
degraded into trichloroacetic acid soluble counts following 3 h
incubation in ATP-containing RRL. When vesicles were collected after 20 min of preincubation in hemin-containing RRL and resuspended in fresh
RRL lacking hemin, 56% of the total CFTR was subsequently degraded.
This degradation remained sensitive to both hemin and EDTA. Incubation
of CFTR in hemin-containing RRL for longer periods of time
(i.e. 1-2 h) resulted in the near complete conversion
(~90%) of full-length protein into the HMW complex as in prior
experiments (data not shown). Even after 2 h of preincubation,
57% of total CFTR protein was still able to be degraded upon removal
of hemin. In each of these experiments, the degradation of
polyubiquitinated CFTR was thus 80% as efficient as the degradation of
newly synthesized protein. Furthermore, with longer preincubation times
the reversible component of CFTR degradation became progressively more
sensitive to inhibition such that after 2 h of preincubation,
hemin and EDTA each blocked degradation of polyubiquitinated CFTR by
approximately 90%. When microsomes containing pre-ubiquitinated CFTR
were similarly resuspended and incubated in the presence of buffer
alone (Fig. 8B), no degradation of the HMW polyubiquitinated
CFTR was observed.
Together these results demonstrate that: (i) the inhibitory effects of
hemin on CFTR degradation are reversible, (ii) most, but not all, of
the membrane-bound ubiquitinated CFTR remains a substrate for
degradation, (iii) CFTR degradation, like ubiquitination, requires the
presence of both ATP and cytosolic factors, and (iv) degradation is
physically localized to the ER membrane and likely occurs coincident
with removal of at least a portion of CFTR from the lipid bilayer.
The ER is a primary site of cellular quality control involved in
the recognition and disposal of misfolded and unassembled proteins (5).
While it was initially proposed that ER proteases were responsible for
degrading ER substrates, recent evidence indicates that ERAD is
mediated largely by cytosolic proteases including the 26 S proteasome
(6, 7). This topologic disparity between substrate and degradation
machinery has resulted in a reevaluation of protein movement into and
out of the ER compartment (6). It now appears that translocation across
the ER membrane is a bidirectional process that is regulated, in part,
by the folded state of a given protein substrate. Whereas, nascent
secretory and transmembrane proteins are targeted to, translocated
across, and/or integrated into the ER membrane by ER translocation
machinery (2, 42), proteins that fail to fold properly may also be recognized by ER quality control machinery and transported out of the
ER and back to the cytosol (6, 9, 10, 15). These observations have
raised important questions regarding the process of retrograde
transport across the ER membrane. At what stage of biogenesis are
protein substrates identified? How do recognition events target
proteins back to the translocon and/or facilitate movement into the
cytosol? And how is this retrograde transport coupled to the process of
degradation? In the current study, we address these issues by
developing an in vitro system that reconstitutes de
novo synthesis and ER-associated degradation of the polytopic protein CFTR. Full-length, glycosylated and membrane-integrated CFTR
was a substrate for ATP-dependent ubiquitination. In
addition, polyubiquitinated CFTR remained tightly associated with the
ER membrane until it was degraded into trichloroacetic acid-soluble peptide fragments. Thus ubiquitination precedes retrograde
translocation, and degradation of polytopic proteins such as CFTR is
physically localized to the ER membrane rather than the cytosol as
proposed for some secretory and transmembrane substrates (8, 10,
13).
Our results are consistent with a model in which CFTR degradation is
tightly coupled to removal of the polypeptide from the lipid bilayer.
At least 65% of [35S]methionine-labeled CFTR was
degraded into trichloroacetic acid-soluble fragments, consistent with
proteasomal degradation in which the resulting polypeptides are 6-9
residues in length (43). Because a small fraction of full-length CFTR
(approximately 10-15%) remained intact in nearly all experiments, the
extent of degradation for individual CFTR polypeptides actually
approached 80%. Thus CFTR degradation could not be limited to large
cytosolic regions containing the N terminus, nucleotide binding
domains, and R domain, which together comprise 61% of the total mass
but only 47% of methionine residues (40). If all cytosolic loops
within 8-15 residues from the membrane were cleaved, then degradation
of all full-length CFTR polypeptide chains would liberate only 60%
total CFTR methionine into the trichloroacetic acid-soluble fraction
(40). It therefore seems likely that proteolytic machinery such as the
proteasome might participate in removal of CFTR from the lipid bilayer
to gain access to transmembrane segments and/or lumenal peptide
regions. This possibility is supported by the observation that: (i)
proteasome-like particles are physically localized to the cytosolic
surface of the ER membrane (44) and (ii) our results that CFTR
degradation occurs via a membrane-bound, rather than cytosolic,
intermediate. If retrograde translocation of CFTR occurred through the
Sec61 translocon, then extraction from the lipid bilayer could
potentially involve the reverse process of membrane integration, namely
lateral movement of TM segments from the membrane back into the
translocation channel and into the cytosol (45, 46).
While this study provides additional support that the
ubiquitin-proteasome pathway is involved in ER-associated degradation of CFTR, it is unclear why different proteasome inhibitors exhibited different effects on CFTR degradation. Lactacystin and
clasto-lactacystin The most potent inhibitors of CFTR degradation were EDTA, apyrase, and
hemin. EDTA and hemin have been shown previously to inhibit the
degradation of other proteasome substrates in RRL by stabilizing HMW
ubiquitin conjugates (39, 52, 53). Hemin, a heme precursor, exerts
pleiotropic effects in RRL. Its stimulatory effect on protein
translation (38) is proposed to be mediated by inactivation of the
translation factor eif-2, possibly by inhibiting an endogenous RRL
kinase (54, 55). In addition, hemin directly inhibits ATPase activities
of the purified 19 S proteasome regulatory complex (PA700) as well as
the 26 S proteasome itself (56). Because the catalytic core of the 20 S
proteasome cannot accommodate folded or ubiquitinated polypeptides, it
has been proposed that ATPases contained within the 19 S regulatory
subunit are required for substrate unfolding as well as
deubiquitination (43, 48, 56-59). It is therefore likely that hemin
inhibits proteasome-mediated CFTR degradation by blocking ATPase
activity and thereby preventing CFTR unfolding and/or deubiquitination.
If CFTR unfolding were required for removal from the bilayer, such a
mechanism would explain the accumulation of membrane-bound,
polyubiquitinated CFTR observed in the current study.
Our results do not rule out the possibility that additional peptidase
activities either in the ER lumen or within the membrane also
participate in CFTR degradation as has been suggested previously (25,
60). One such candidate is signal peptidase complex, an ER-localized
protease that is capable of cleaving transmembrane proteins (61) and
free signal peptides (30) at sites located within the plane of the
lipid bilayer. Interestingly, CFTR contains cryptic signal peptidase
cleavage sites within or adjacent to TM segments of the N terminus
hydrophobic domain (62, 63). If similar proteases facilitated CFTR
degradation, then TM segments and small peptide loops could potentially
be released from the membrane. Such a mechanism might explain why a
significant amount of CFTR degradation was ATP-independent and hemin
insensitive. It is also possible that the membrane-bound HMW complex
might actually consist of polyubiquitinated CFTR cleavage products that are then degraded by the ubiquitin-proteasome pathway.
Cell-free systems have significantly enhanced our understanding of
protein folding (64) and translocation (2). However we do not yet know
whether the folded state of newly synthesized CFTR in the RRL system is
the same as that found in the ER of cells or whether the mechanism of
CFTR recognition by ER quality control machinery in vitro is
the same as in vivo. Processing in the RRL system is limited
to early events that occur at the ER membrane, and it is possible that
CFTR might require specialized cellular components that are limiting in
RRL and/or canine pancreas microsomes. Recently, RRL and yeast-derived
cell-free systems have been used to examine the degradation of other
secretory and transmembrane proteins (8, 51), including CFTR (31). Our observation that 85-90% of newly synthesized wild type CFTR is degraded in the RRL system is consistent with studies that demonstrate most wild type CFTR is also degraded in the ER membrane of intact cells
(22, 23). In addition, preliminary experiments in our laboratory have
demonstrated that other polytopic proteins expressed in the RRL system,
such as human P-glycoprotein and aquaporin water channels, are much
more stable than CFTR.2 These
studies indicate that rapid CFTR degradation in vitro is not
simply a cell free artifact, but rather reflects specific structural
features recognized by ER quality control machinery. In
vitro systems such as RRL will thus likely provide a versatile tool for investigating the events and cellular machinery involved in
ER-associated degradation. In the case of CFTR, they may also provide
screening assays to identify compounds that improve folding and
trafficking efficiency.
-lactone, ALLN, and
N
-tosyl-L-phenylalanine
chloromethyl ketone and was relatively unaffected by lactacystin and
N-tosyl lysyl chloromethyl ketone. In the presence of
hemin, polyubiquitinated CFTR remained tightly associated with ER
microsomes. However, membrane-bound ubiquitinated CFTR could be
subsequently degraded into trichloroacetic acid-soluble fragments upon
incubation in hemin-free, ATP-containing lysate. Thus ER-associated
degradation of CFTR occurs via a membrane-bound, rather than cytosolic,
intermediate and likely involves recruitment of degradation machinery
to the ER membrane. Our data suggest a model in which the degradation
of polytopic proteins such as CFTR is coupled to retrograde
translocation and removal of the polypeptide from the lipid bilayer.
INTRODUCTION
Top
Abstract
Introduction
References
factor undergo
complete retrograde translocation into a soluble, cytosolic form prior
to degradation (8). In contrast, mutant forms of carboxypeptidase Y and
unassembled T cell receptor
subunits translocate back to the
cytosol but remain associated with cytoplasmic face of the ER membrane
(14, 15). In both cases the generation of cytosolic intermediates is
thought to occur via retrograde transport through the Sec61 translocon
complex (9-11).
EXPERIMENTAL PROCEDURES
-lactone were purchased from Calbiochem. Nucleotide triphosphates,
creatine phosphate, creatine kinase, and dithiothreitol (DTT) were
purchased from Boehringer Mannheim. Anti-CFTR antisera was raised
against synthetic peptides corresponding to residues 45-65 (N
terminus), 680-700 (R domain), and 1458-1476 (C terminus) (BAbCo,
Berkeley, CA). Other regents were purchased from Sigma unless otherwise stated.
80 °C. Prior to freezing,
hemin was added to a final concentration of 40 µM. For degradation experiments, RRL was either purchased from Green Hectare Farm (Oregon, WI) or prepared as described previously (32) but without
addition of hemin. Although degradation efficiency varied between
different preparations of RRL, no consistent differences were observed
between different RRL sources. To prepare ATP-depleted RRL,
reticulocytes were washed three times with ice-cold phosphate-buffered saline, resuspended in Krebs-Ringer phosphate medium lacking glucose, and incubated in 0.2 mM 2,4-dinitrophenol and 20 mM 2-deoxyglucose at 37 °C for 90 min (33).
Reticulocytes were then washed twice with phosphate-buffered saline and
lysed by adding 1.5 volume of 1.0 mM DTT. Samples were
centrifuged at 15,000 rpm for 30 min (Beckman JA17 rotor), and the
supernatant (i.e. RRL) was stored at
80 °C.
80 °C. Translation was performed at 24-25 °C for
2 h in a reaction containing 20% transcription mixture, 40%
nuclease-treated rabbit reticulocyte lysate, 20% Emix, and a final
concentration of 400 µg/ml creatine kinase, 0.1 mg/ml tRNA, 0.2 µl
of RNAse inhibitor (Promega), 10 mM Tris (pH 7.5), 100 mM KOAc, 2 mM MgCl2, and 2 mM DTT. Emix contains 5 mM ATP, 5 mM GTP, 60 mM creatine phosphate, 0.2 mM each of 19 essential amino acids, except methionine, and 5 µCi/µl of Tran35S-label (ICN Pharmaceuticals, Irvine
CA). Canine pancreas microsomal membranes, prepared as described
elsewhere (36), were added at the start of translation to a final
concentration of A280 8.0. For all experiments
except those in Fig. 1A, aurin tricarboxylic acid (0.1 mM) was added to the translation mixture after 15 min to
synchronize translation and reduce synthesis of partial length chains.
For membrane pelleting and floatation experiments puromycin HCl (final
concentration 1 mM) was added at T = 110 min to ensure that nascent chains were released from ribosomes.
RESULTS
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Fig. 1.
Expression of CFTR in rabbit
reticulocyte lysate. A, CFTR cDNA (plasmid
pSPCFTR(35)) was translated in a transcription-linked RRL translation
system at 24 °C for 90 min as described under "Experimental
Procedures." Canine pancreas microsomal membranes were present during
translation in all lanes except lane 1. Lanes 1,
2, and 7 represent total translation products
analyzed by SDS-PAGE. Translation products were immunoprecipitated with
nonimmune sera (lane 6) or with peptide-specific antisera
raised against the N terminus (lane 3), R domain (lane
4) or C terminus (lane 5) of CFTR. For carbonate
extraction, translation products (lane 7) were incubated at
pH 7.5 (Tris) or at pH 11.5 (Carb), and
supernatant (S) and pellet (P) fractions were
analyzed directly. B, CFTR translation was
carried out as in A except that aurin tricarboxylic acid
(0.1 mM) and cyclohexamide were added 15 min and 2 h
after the start of translation, respectively. Following addition of
cyclohexamide, aliquots were removed at the specified time points and
analyzed directly by SDS-PAGE.
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Fig. 2.
In vitro degradation of CFTR via a
HMW intermediate. A, CFTR was translated for 2 h
in RRL (as in Fig. 1B), after which microsomal membranes
were pelleted through a 0.5 M sucrose cushion and
resuspended in 0.1 M sucrose buffer as described under
"Experimental Procedures." Membranes were then incubated at
37 °C in buffer (lanes 1-5), RRL containing an ATP
regeneration system (lanes 6-10), ATP-depleted RRL
(lanes 11-15), or ATP-depleted RRL containing an ATP
regeneration system (lanes 16-20). At specified time
points, aliquots were removed from each tube and analyzed directly by
SDS-PAGE. B, autoradiograms were digitized, and band
intensities of full-length CFTR and the HMW complex were quantitated,
normalized to the amount of full-length CFTR present at
T = 0, and plotted for each time point.
Closed and open symbols represent CFTR and the
HMW complex, respectively.
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Fig. 3.
In vitro ubiquitination of
CFTR. Microsomal membranes containing newly synthesized CFTR were
resuspended in 0.1 M sucrose buffer and incubated at
37 °C in RRL containing 40 µM hemin and 0.4 mg/ml of
bovine ubiquitin (A) or methylated ubiquitin (B).
At times indicated, samples were immunoprecipitated with nonimmune sera
(NIS; A, lanes 6-10), anti-ubiquitin antisera
(A, lanes 11-15), or analyzed directly
(A, lanes 1-5 and B, all
lanes).
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Fig. 4.
Effect of protease inhibitors on CFTR
degradation. Microsomal membranes containing newly synthesized
CFTR were collected and incubated in RRL in the presence of a protease
inhibitor mixture consisting of 500 µM
4-(2-aminoethyl)benzenesulfonyl fluoride, 150 nM aprotinin,
1 µM E-64, 0.5 mM EDTA and 1 µM
leupeptin (A) or in individual proteasome inhibitors at the
indicated concentrations (B). At each time point, aliquots
were mixed directly with SDS loading buffer and analyzed by PAGE.
C, CFTR and the HMW complex were quantitated at each time
point by densitometry, normalized to the amount of CFTR present at
T = 0, and plotted as a function of time. Data shown
are representative of results from multiple experiments.
-lactone, had little effect on lysozyme degradation (<10%
inhibition). Under these same conditions CFTR degradation was inhibited
by approximately 80% in the presence of EDTA or the ATP hydrolyzing enzyme, apyrase. Hemin inhibited CFTR degradation by 56%, while lactacystin and clasto-lactacystin
-lactone inhibited
CFTR degradation by only 20 and 35%, respectively. In these latter
experiments, lactacystin-based inhibitors were preincubated with RRL
for 30-60 min prior to addition of CFTR-containing microsomal
membranes. These experiments demonstrate that specific
lactacystin-based proteasome inhibitors are relatively inactive at
blocking degradation of these ubiquitinated substrates in RRL. It
should also be noted that a significant amount of CFTR degradation in
RRL (35-40%) appears to proceed through a hemin insensitive pathway
distinct from that utilized by lysozyme.
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Fig. 5.
Degradation of lysozyme and CFTR.
Degradation mixtures were assembled as described in the legend to Fig.
2 and added to resuspended microsomal membranes containing CFTR or
[14C]lysozyme (0.5 µCi/ml). Hemin (40 µM), EDTA (10 mM), or apyrase (0.1 unit/µl)
was added at T = 0 as indicated. Lactacystin (100 µM) or clasto-lactacystin -lactone (100 µM) were preincubated with RRL for 30-60 min prior to
assembling the reaction. Aliquots were precipitated in 20%
trichloroacetic acid, and soluble counts were calculated by the
following formula: % counts released = (Cn
C0)/(T
C0), where Cn = soluble
counts at each time point, C0 = soluble counts
at T = 0, and T = total counts per
aliquot. Results represent the average (±S.E.) of two to six separate
experiments.
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Fig. 6.
HMW complex pelleting. CFTR-containing
microsomes were incubated in RRL in the presence of hemin.
A, at times specified, aliquots were analyzed directly
(lanes 1-4), pelleted through 0.5 M sucrose
buffer (lanes 5-9), or incubated on ice in 1% Triton X-100
for 30 min prior to pelleting (lanes 10-14). Pellets were
dissolved directly into SDS loading buffer. B, aliquots of
RRL degradation mixture were taken at specified times and analyzed
directly in SDS loading buffer (lanes 1-3) or incubated for
30 min on ice following addition of KCl to the final concentrations
indicated (lanes 4-21). Samples were then pelleted through
0.5 M sucrose buffer, and equal fractions of pellet and
supernatant were analyzed by SDS-PAGE.
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Fig. 7.
HMW complex floatation. Microsomal
membranes containing newly synthesized CFTR or bovine prolactin were
incubated at 37 °C in RRL degradation mixture for 0, 20, or 120 min
in the presence of hemin. A, samples were mixed to a final
concentration of 2 M sucrose, layered beneath a
discontinuous sucrose gradient, and centrifuged at 350,000 × g for 2 h as described under "Experimental
Procedures." B, EDTA (10 mM) and KCl (0.6 M) were added prior to centrifugation. Aliquots were taken
from top to bottom of the gradient (lanes 1-10,
respectively). The HMW complex, CFTR, and prolactin control are
indicated.
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Fig. 8.
Degradation of preubiquitinated CFTR.
CFTR-containing microsomal membranes were incubated in RRL in the
presence of hemin as indicated. A, at each time point,
samples were pelleted through 0.5 M sucrose buffer and
resuspended in fresh RRL in the presence or absence of hemin or EDTA.
Aliquots were precipitated in 20% trichloroacetic acid at the
indicated times, and percent soluble counts released was determined by
scintillation counting. B, samples were pelleted through 0.5 M sucrose buffer in the presence or absence of 1 mM ATP and resuspended in 10 mM Tris, 5 mM MgCl2, 3 mM DTT, and 80 µg/ml
creatine kinase, with or without 1 mM ATP/12 mM
creatine phosphate. Time scale for each graph is based on
T = 0 for the starting RRL degradation mixture. For
T = 20, 60, and 120 min graphs, the base line soluble
counts reflect the percent of counts released from RRL in the presence
of hemin. Total trichloroacetic acid-soluble counts plotted therefore
represent the counts released during initial incubation plus the counts
released following resuspension and reincubation of microsomes.
DISCUSSION
-lactone, which covalently inactivate
the active site threonine residue on the
5 subunit of
the 20 S proteasome (subunit X of the mammalian proteasome) (47, 48),
had relatively minor effects on CFTR degradation as determined either
by disappearance of the CFTR HMW complex (Fig. 4) or generation of
trichloroacetic acid-soluble fragments (Fig. 5). It is interesting that
clasto-lactacystin
-lactone, the active form of
lactacystin, is inactivated by glutathione in cells (49, 50). Our
observation that neither lactacystin nor its
-lactone form inhibited
the degradation of lysozyme, a known proteasome substrate, suggests
that RRL is capable of rendering these agents inactive, even when
preincubated in the absence of exogenous reducing agents. We do not
know why the results reported here differ from those of Qu et
al. (51) that demonstrated 20 µM lactacystin
inhibited the degradation of both
1-antitrypsin and
lysozyme in a similar RRL system. However, our results were consistent
for two different preparations of lactacystin and at multiple inhibitor
concentrations up to 100 µM. It is possible that these
differences reflect varying amounts of proteasome in different RRL preparations.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. S. Marks and members of the Skach laboratory for their helpful comments.
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FOOTNOTES |
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* This work was supported by Grants GM53457 and DK51818 from the National Institutes of Health and by the North American Cystic Fibrosis Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Present address:
Division of Molecular Medicine, 3181 SW Sam Jackson Park Rd., NRC-3,
Oregon Health Sciences University, Portland, OR 97201. Tel.:
503-494-7322; Fax: 503-494-7368; E-mail: skachw{at}ohsu.edu.
The abbreviations used are:
ER, endoplasmic
reticulum; ERAD, ER associated degradation; CFTR, cystic fibrosis
transmembrane conductance regulator; DTT, dithiothreitol; HMW, high
molecular weight; RRL, rabbit reticulocyte lysate; ALLN, N-acetyl-L-leucyl-L-leucinyl-norleucinal; TPCK, N-tosyl-L-phenylalanine
chloromethyl ketone; TLCK, N-tosyl lysyl chloromethyl
ketone; TM, transmembrane; PAGE, polyacrylamide gel electrophoresis.
2 E. Chong and W. R. Skach, unpublished observations.
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REFERENCES |
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