From the Department of Biological Sciences, Stanford University, Stanford, California 94305-5020
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ABSTRACT |
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Ubiquitination is a covalent protein modification that can target proteins in eukaryotic cells for degradation by the 26 S proteasome. Substrates for this degradation pathway include abnormal proteins that arise from misfolding and/or mutation. How and when the ubiquitination machinery recognizes misfolded proteins and targets them for degradation remains largely unknown. We have previously shown that cystic fibrosis transmembrane conductance regulator (CFTR), is rapidly degraded in a ubiquitin-dependent fashion, without any detectable lag following its synthesis (Ward, C. L., and Kopito, R. R. (1994) J. Biol. Chem. 269, 25710-25718), suggesting that ubiquitination and protein synthesis may be temporally linked. In the present study, we have investigated the timing of CFTR ubiquitination relative to its translation in reticulocyte lysates containing 125I-ubiquitin. In synchronized, proteasome-inhibited lysates, translation of full-length CFTR chains was completed in approximately 30 min, whereas modification of CFTR with [125I]ubiquitin was evident by 20 min, indicating that ubiquitination precedes the completion of full-length polypeptide chains. Moreover, ubiquitin was also found to be transferred to nascent CFTR chains while attached to ribosomes. Together, these data establish that ubiquitination, which is widely assumed to be a post-translational event, can occur cotranslationally and suggest a role for ubiquitination early in protein biosynthesis.
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INTRODUCTION |
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Ubiquitin is a highly conserved 76-amino acid polypeptide that plays a central role in determining protein turnover (reviewed in Refs. 1-3). Covalent attachment of ubiquitin serves as signal that targets proteins for degradation by the 26 S proteasome. Substrates for this degradation pathway include potentially toxic, aggregation-prone misfolded proteins that can be synthesized de novo as a result of mutation or of errors in translation or transcription. Although the enzymatic mechanism by which ubiquitin is conjugated to degradation substrates has been well characterized (reviewed in Ref. 4), the processes by which misfolded protein substrates are initially recognized by the Ub1 conjugation machinery remain largely unknown.
Genetic and biochemical studies suggest that some substrate specificity
resides in the enzymatic machinery of the ubiquitin coupling system,
particularly the ubiquitin-conjugating enzymes (E2s) and
ubiquitin-protein ligases (E3s), which function in transferring thiol-activated ubiquitin intermediates to lysine -amino groups on
the target polypeptide (2, 3). However, attachment of a single
ubiquitin is not sufficient to mark a protein for degradation (5).
Efficient degradation by the 26 S proteasome appears to require the
presence on the target protein of "multiubiquitin" polymers often
composed of more than 20 ubiquitins (5, 6). Proteasomal degradation of
a protein depends therefore not only on the presence of covalently
attached ubiquitin but also on the length of the multiubiquitin chain
and possibly the type of internal ubiquitin-ubiquitin linkage. An
additional level of specificity is suggested by the presence in cells
of multiple ubiquitin isopeptidases that appear to "edit"
multiubiquitin chains and suggest that the ultimate fate of a protein
may be determined by kinetic partitioning between ubiquitination and
de-ubiquitination (7, 8).
Recent data implicate the ubiquitin-proteasome pathway in the
degradation of misfolded integral membrane proteins (9) including CFTR
(10, 11). It is well established that CFTR folding is an inefficient
process in vivo (12, 13). The majority (50-75%) of newly
synthesized wild type CFTR molecules are rapidly degraded by a pathway
that involves both ubiqutination and proteaseome activity (10, 11). The
common F508 mutation increases the ineffienciency of CFTR folding
(to greater than 99%) but does not change the kinetics or ubiquitin
dependence of its degradation (12, 13). CFTR and
F508 degradation
in vivo occur without a detectable lag following
biosynthesis (12). Moreover, the apparent rate of CFTR synthesis
in vivo (assessed by the incorporation of
[35S]Met) is increased 2-3-fold in the presence of
proteasome inhibitors.2
Together these data suggest that recognition of CFTR misfolding by the
ubiquitin-conjugating machinery may be closely linked to translation
and may not be easily reconciled with the generally held assumption
that ubiquitination is a "post-translational modification." To
clarify this point, we address in this paper when nascent CFTR chains
are tagged with ubiquitin by employing a cell-free system. Our data
indicate that nascent CFTR polypeptides can become ubiquitinated prior
to release from the translation apparatus. These data establish that
ubiquitin-conjugating machinery can recognize nascent polypeptides during their translation and suggest that the destiny of nascent polypeptides may be established by ubiquitination, whereas the polypeptide chains are still elongating on ribosomes.
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MATERIALS AND METHODS |
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In Vitro Transcription, Translation, and
Immunoprecipitation--
HA-CFTR and HA-F508 cDNA were
generated by introducing the HA epitope (YPYDVPDYA) between codons 109 and 110 of human CFTR and
F508 cDNA, respectively, by polymerase
chain reaction mutagenesis. RNA was transcribed from linearized HA-CFTR
and HA-
F508 templates using T7 polymerase (Ambion Inc.). Because
native CFTR contains 5' sequences that interfere with translational
initiation in vitro, the 5' leader and the first 18 codons
of CFTR were replaced with the 5' region of a viral internal ribosomal
entry sequence and an S-tag antigen (pCITE-4b; Novagen). RNA was
translated in 60% RRL (Promega) supplemented with 20 µM
amino acid (except Met), [35S]Met (0.4 mCi/ml), 0.75 mM magnesium acetate, 90-110 mM KCl, 2 mM dithiothreitol in the presence or the absence of canine
pancreas microsomes (RM) (Promega; 2.5 A280
units) at 30 °C. Where indicated, translations were synchronized by
addition of RNA (following 5 min of preincubation at 30 °C) followed
after 5-10 min by addition of 75 µM aurintricarboxylic
acid. Translation was terminated either by adding ice-cold 2× SDS-PAGE
sample buffer or by dilution with ice-cold buffer A (50 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5%
sodium deoxycholate, 0.1% SDS). In some cases, RM were pelleted by
centrifugation through 0.5 M sucrose cushion at
100,000 × g for 15 min (TLA 100.2, Beckman). To assess
the integration of HA-CFTR, RM was resuspended in 0.2 M
Na2CO3 buffer (pH 11.5), 2% (v/v) glycerol, 10 mM dithiothreitol essentially as described (14). For
immunoprecipitation, RRL diluted in buffer A was incubated with HA
monoclonal antibody (HA11, BAbCo) overnight at 4 °C, followed by
incubation with
-Bind-plus-Sepharose (Pharmacia) for 2 h. The
beads were washed twice in buffer A, once in buffer B (10 mM Tris-HCl (pH 7.5), 0.1% Nonidet P-40, 1 mM
EDTA, 0.15 M NaCl), once in buffer C (10 mM
Tris-HCl (pH 7.5), 2 mM EDTA, 0.05% SDS), and once in
buffer D (10 mM Tris-HCl (pH 7.5), 2 mM EDTA).
Immunoprecipitated chains were released by heating at 65 °C in
SDS-PAGE sample buffer.
Ubiquitination Assay-- Bovine ubiquitin (Sigma) was labeled with [125I] to 4-6 × 105 cpm/µg using Iodogen (Pierce) and purified by gel filtration. For in vitro ubiquitination in RRL, translation reactions (50 µl) were conducted as above (except that [35S]Met was replaced with unlabeled Met) and in the presence of [125I]Ub (2-5 × 104 cpm/µl).
Separation of Ribosome-bound Polypeptide Chains-- Run-off translation of HA-CFTR RNA (truncated at codon 920) was terminated with cycloheximide (2 mM) to stabilize the ribosome nascent chain complex or puromycin (2 mM) to release the polypeptide from ribosomes together with an ATP depletion mixture containing hexokinase (0.4 mg/ml) and D-glucose (5 mM). Samples to which puromycin was added were further incubated at 30 °C for 5 (for translation without RM) or 30 min (for translation with RM) as indicated. The RM fraction was sedimented as above and solubilized in 3% digitonin in buffer E (50 mM HEPES-KOH (pH 7.5), 500 mM potassium acetate, 5 mM magnesium acetate, 0.5 mM EDTA) for 30 min on ice. The solubilized fraction was precleared by 10 min of centrifugation at 14,000 × g at 4 °C and then applied onto a 1.2-ml sucrose gradient (15-50%) in 3% digitonin/buffer E and centrifuged for 90 min in TLA 100.2 at 100,000 × g. The gradient was fractionated (0.1 ml/fraction), and one-fifth of each fraction was used to monitor truncated HA-CFTR polypeptide by immunoblotting. The remainder was diluted in buffer A and immunoprecipitated with anti-HA antibody. For purification of ribosome-polypeptide in the absence of RM, translation mixture was precleared and subjected to sedimentation on a sucrose density gradient in buffer E without detergent.
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RESULTS AND DISCUSSION |
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To monitor the fate of nascent CFTR polypeptide chains, an HA
epitope tag was inserted near the N terminus of CFTR and F508 (Fig.
1a). This construct,
designated HA-CFTR (or HA-
F508) was efficiently translated in
vitro in a RRL supplemented with canine pancreas microsomes (RM),
giving rise to a single predominant polypeptide of molecular mass of
140 kDa (Fig. 1b) that was identified as full-length HA-CFTR
because it was immunoprecipitated by antibodies both to the extreme C
terminus of CFTR and to the N-terminal HA epitope. Most HA-CFTR
translated in the presence of RM bound to concanavalin A-Sepharose,
suggesting that it is glycosylated (data not shown). Moreover, the
mobility of HA-CFTR translated in the absence of RM was ~2-4 kDa
lower than its mobility in the presence of RM, consistent with the
presence of 1-2 core N-linked oligosaccharide chains (Fig.
1c). Finally, HA-CFTR was not extracted from RM membranes at
pH 11.5 under conditions sufficient to extract the lumenal protein
-lactamase (Fig. 1d). Together, these data demonstrate that full-length HA-CFTR is efficiently translocated, glycosylated, and
integrated into RM membranes in vitro and establish that
this cell-free system is a suitable model for the early stages of CFTR biosynthesis.
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To study ubiquitination of nascent CFTR polypeptides in
vitro, HA-CFTR or HA-F508 were translated in the presence of
[125I]Ub, and HA-CFTR polypeptide chains were purified
from the translation mixture by immunoprecipitation with anti-HA
antibody. The presence of hemin (20 µM), a proteasome
inhibitor, in this translation-optimized RRL ensured that proteins
could be ubiquitinated by the extract but remain undegraded by the
proteasome (15). Ubiquitinated protein was detected as an intense smear
on autoradiograms of SDS-PAGE separated proteins from RRL programmed
with either HA-CFTR or HA-
F508 mRNA but not when translation was
conducted in the absence of mRNA (Fig.
2a), suggesting that Ub can be
transferred onto newly synthesized HA-CFTR polypeptides. The high
molecular weight of 125I label relative to unmodified CFTR
could be due to multiubiquitination at a single lysine or to
monoubiquitination at multiple lysines, although the data do not
discount the possibility that conjugation of single Ub could promote
CFTR aggregation. There was no significant difference in the degree of
ubiquitination between HA-CFTR and HA-
F508 (Fig. 2a), in
agreement with previous data indicating that both
F508 and the
~75% of CFTR that fails to fold are ubiquitinated in vivo
(10). By contrast, significantly more label was incorporated into HA
immunoprecipitates when the translation was conducted in the absence of
RM, consistent with the expectation that CFTR, a hydrophobic polytopic
integral membrane protein, is likely to be severely misfolded when
translated in the absence of membranes. To test if the
125I-labeled smear associated with HA-CFTR is due to the
small fraction of CFTR molecules that fail to be translocated and to
become membrane integrated, we isolated RM. As expected, the majority
of core-glycosylated CFTR (Fig. 2b, lower panel)
together with the majority of the 125I label (Fig.
2b, upper panel) cosedimented with RM. When we
tested for glycosylation in detergent extracts of RM from
HA-CFTR-programmed lysates, we found that most of the 125I
label was adsorbed with concanavalin A-Sepharose (data not shown). Together, these data strongly suggest that RRL can transfer Ub onto
both glycosylated membrane-associated and untranslocated CFTR
polypeptides.
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To precisely determine the timing of ubiquitination relative to translation, we monitored the kinetics of [125I]Ub incorporation into a cohort of HA-CFTR molecules synthesized in a synchronized RRL extract. 10 min after translation was initiated by the addition of HA-CFTR mRNA, the translation reaction was synchronized with aurintricarboxylic acid to prevent re-initiation (16). Full-length HA-CFTR monitored by immunoblotting (Fig. 3a, lower panel) or by [35S]Met incorporation in parallel translations (data not shown) was first detectable after 30 min, corresponding to a rate of CFTR synthesis of 50-70 amino acid residues/min, comparable with previously reported rates (17). By contrast, high molecular weight, [125I]ubiquitinated HA-immunoreactive polypeptide was evident in the autoradiograms after 20 min (Fig. 3a, upper panel), suggesting that ubiquitination of the translating polypeptide precedes the completion of full-length HA-CFTR (similar results were obtained with a 3-5-min initiation window; data not shown). To further examine this conclusion, we investigated whether [125I]Ub label was associated with incomplete HA-CFTR chains that were immunoprecipitated with anti-HA antibody following immunodepletion of full-length CFTR chains with antibody to the extreme C terminus (Fig. 3b). These data indicate that incomplete HA-CFTR chains are ubiquitinated in the translation system.
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Incomplete ubiquitinated chains could arise from cotranslationally ubiquitinated nascent HA-CFTR molecules or from polypeptide chains that were released prematurely from the translational apparatus. To discriminate between these two possibilities, we assessed the incorporation of [125I]Ub into nascent, ribosome-bound HA-CFTR polypeptides translated from a truncated mRNA construct lacking a stop codon and then fractionated on sucrose density gradients (Fig. 4). When the translation was conducted in the absence of RM (Fig. 4a), virtually all of the truncated HA-CFTR chains of the expected size (110 kDa), assessed by immunoblotting, cosedimented in the heavier fractions near the bottom of the gradient together with 125I-labeled protein. The presence of 125I label in the lighter fractions (2-5) that lacked significant 110-kDa HA immunoreactivity suggests that some HA-CFTR chains that had been prematurely released from ribosomes were ubiquitinated. Treatment of the lysates with puromycin B (Fig. 4b), which releases nascent chains from ribosomes, shifted most of the 125I label and 110-kDa HA-CFTR to lighter fractions, indicating that the majority of the ubiquitinated HA-CFTR polypeptides in fractions 7-10 were ribosome associated. Although the release of nascent chains from membrane-bound ribosomes was inefficient, when truncated HA-CFTR was translated in the presence of RM, a fraction of ubiquitinated truncated HA-CFTR chains were shifted to lighter fractions by puromycin treatment (data not shown), suggesting that nascent membrane-integrated chains that are attached to ribosomes are also modified by Ub. Taken together, these data show that ribosome-associated nascent CFTR polypeptides chains can be modified cotranslationally with ubiquitin.
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Protein folding is the process by which the linear information in the genetic code is converted into the three-dimensional structures of proteins. Because incorrectly folded proteins expose hydrophobic surfaces and therefore have a strong tendency to form insoluble potentially toxic aggregates, eukaryotic cells possess "quality control" mechanisms that monitor the progress of protein folding and selectively degrade misfolded proteins (reviewed in Ref. 18). Intermediates in the normal pathway of protein folding also transiently expose aggregation-prone hydrophobic surfaces; the formation of aggregated, off-pathway products in cells is minimized by sequential, transient interaction of nascent, partially folded proteins with molecular chaperones (19).
How the cellular quality control machinery discriminates between bona
fide intermediates in the folding pathway and aggregation-prone products that lie off of the folding pathway is not known. Misfolded F508 CFTR molecules appear to remain associated with the molecular chaperones calnexin (20) and Hsp70 (21) until they are degraded, suggesting that molecular chaperones may contribute to quality control.
However, because these chaperones also lie on the folding pathway, it
is unlikely that chaperone binding is the sole signal for the
degradation machinery. The existence of an editing isopeptidase recently identified as a component of the 26 S proteasome (7) raises
the possibility that multiubiquitination is reversible and that
Ub-dependent proteasomal targeting is not sufficient for
efficient degradation.
The data in this paper are the first evidence demonstrating that protein ubiquitination can occur while the nascent polypeptide chain is still attached to the ribosome. Cotranslational ubiquitination could occur because of early recognition of a misfolding/ubiquitination signal by the cellular quality control apparatus. Alternatively, the reversible conjugation of Ub to nascent chains might itself serve a chaperone-like role where the choice between folding and degradation would be determined by the competition between net elongation or shortening of a Ub chain. Future studies will be needed to discriminate between these two possibilities.
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FOOTNOTES |
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* This work was supported by Grant DK43994 from the National Institutes of Health.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.
Recipient of a Human Frontier Science Program Long-term
Fellowship. Present address: Research Center for Infectious Diseases, CHUL, Laval University, Quebec G1V 4G2, Canada.
§ Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Biological Sciences, Stanford University, Stanford, CA 94305-5020. Tel.: 650-723-7581; E-mail: kopito{at}stanford.edu.
1 The abbreviations used are: Ub, ubiquitin; CFTR, cystic fibrosis transmembrane conductance regulator; HA, influenza hemagglutinin epitope tag; RRL, rabbit reticuolcyte lysate; RM, canine pancreas rough microsomes; PAGE, polyacrylamide gel electrophoresis.
2 C. L. Ward and R. R. Kopito, unpublished observations.
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REFERENCES |
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