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J. Biol. Chem., Vol. 278, Issue 37, 34774-34782, September 12, 2003
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¶ 
From the
Department of Cell Biology, Harvard
Medical School, Boston, Massachusetts 02115, the
Howard Hughes Medical Institute and Department
of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, and the
||Department of Cellular and Molecular Physiology,
Penn State University College of Medicine, Hershey, Pennsylvania 17033
Received for publication, April 1, 2003 , and in revised form, May 16, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Most substrates destined to be degraded are polyubiquitinated while undergoing retrotranslocation. Our previous results showed that polyubiquitination is not only required for degradation by the proteasome but also for retrotranslocation per se (8). Lysine 48-linked polyubiquitin chains are required for protein degradation by the proteasome (9, 10), but given that other linkages are required for other processes (11–15), it would be important to know which kind of polyubiquitin chains are involved in retrotranslocation. The role of polyubiquitination in retrotranslocation also remained unclear. In one model, polyubiquitin provides the driving force for moving the substrate into the cytosol by acting as a ratcheting molecule (for discussion, see Ref. 3). The attachment of the bulky polyubiquitin moiety to a polypeptide segment on the cytosolic side of the membrane would bias random movements of the substrate across the ER membrane by preventing it from sliding back into the ER lumen. This model would be analogous to the one describing posttranslational translocation in the forward direction. In this case, the binding of the luminal chaperone BiP prevents the translocating polypeptide chain from moving back into the cytosol, eventually resulting in its complete transport into the ER lumen (16). If a polyubiquitin-mediated ratcheting mechanism functioned in a similar way in retrotranslocation, polyubiquitination alone should be sufficient to move a substrate into the cytosol. Alternatively, it is possible that the polyubiquitin chain serves as a recognition signal for a downstream component. One candidate for this downstream component is the ATPase p97 (called Cdc48 in yeast), which together with its cofactor Ufd1-Npl4 has been implicated in moving polypeptides from the ER membrane into the cytosol (17–21).
In this study, we have addressed the role of polyubiquitination in retrotranslocation. We show that polyubiquitination is not sufficient to move a substrate into the cytosol. Rather, a subsequent ATP-dependent step is required, during which polyubiquitin in a lysine 48 linkage serves as a recognition signal. Our in vitro experiments suggest that the downstream component may be the p97-Ufd1-Npl4 complex.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Lysate Preparation and Immunoprecipitations—The lysates were
made from samples corresponding to 1–2 x 106 cells in
0.5% Nonidet P-40 (Igepal; CA-630; Sigma), 50 mM Tris, pH 8, 150
mM NaCl, and 10 mM MgCl2. The buffer also
contained 1 mM phenylmethylsulfonyl fluoride and the previously
mentioned protease inhibitor mix. The samples were agitated for 20 min at 4
°C and then clarified by centrifuging in a microcentrifuge at full speed
for 10 min. The resulting supernatant was used for immunoprecipitation after
SDS and dithiothreitol were added to final concentrations of 0.1% and 0.2
mM, respectively. Denaturing SDS lysates were made by resuspending
cell pellets or cell fractions in 100 µl of 2% SDS and 5 mM
dithiothreitol. The samples were heated to 95 °C for 5 min, cooled to room
temperature, agitated vigorously, and diluted into Nonidet P-40 buffer so that
the final concentrations in the lysate used for immunoprecipitation were
0.2% SDS, 0.5 mM dithiothreitol, 0.5% Igepal, 50 mM
Tris, pH 8, 150 mM NaCl, and 10 mM MgCl2. All
of the immune complexes were recovered by precipitation with fixed
Staphylococcus aureus bacteria (Staph A).
Anti-heavy chain serum and antibodies against bovine ubiquitin were described previously (7). The monoclonal antibody 12CA5, which recognizes the influenza hemagglutinin (HA) epitope, was purified from tissue culture cell supernatants by standard methods (23) or purchased from Sigma. Immunoprecipitation with heavy chain antibodies and reprecipitation with ubiquitin antibodies were done as described (7).
Ubiquitin Depletion—Ubiquitin was depleted from cow liver cytosol as described, using a recombinant GST-tagged Ubc2 that carries a serine instead of a cysteine in the active site (8). The depletion reaction mixture contained 16 µM GST-tagged Ubc2Ser and an ATP-regenerating system (24). The reaction was allowed to proceed for 10 min at 37 °C. The reaction mixture was subsequently transferred to glutathione-Sepharose 4B beads and incubated at 4 °C for 15–30 min. The non-bound material was frozen at –80 °C.
Ubiquitin Reagents—Bovine ubiquitin was purchased from Sigma. For the experiment in Fig. 2, ubiquitin was labeled using the following procedure. Bovine ubiquitin was iodinated using Iodogen Iodination Reagent (Pierce). Each 90-µl iodination reaction mixture contained 8.9 mg/ml ubiquitin and 22.2 mCi/ml Na125I (PerkinElmer Life Sciences) in 44.4 mM potassium phosphate, pH 7.6. The reaction tube was coated with 100 µl of 1 mg/ml Iodogen in chloroform. The reaction was carried out on ice for 30 min, after which the reaction was stopped by the addition of 100 µl of 0.4 mg/ml tyrosine and 16 µl of 0.1 M nonradioactive NaI. Iodinated ubiquitin was purified by passing it through a Sephadex G-25 spin column equilibrated with PB. The final concentration was estimated at 300 µM, labeled to 16,500 cpm/pmol. 125I-Ubiquitin was added to the cow liver cytosol at a concentration of 20 µM.
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The pGEX-Ubiquitin plasmids coding for GST-ubiquitin and GST-K48R were
provided by T. Sommer. All GST-ubiquitin proteins were expressed in BL21 (DE3)
Escherichia coli. The cells were grown to an A value of
1.0 and induced with 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside for 6 h. They were
lysed and centrifuged at 42,000 rpm in an ultracentrifuge to remove nonsoluble
material. The fusion protein was purified from the supernatant by glutathione
affinity chromatography.
Binding of p97 to Heavy Chains in Permeabilized Cells—The experiments were performed essentially as described (18). The cell fractions were solubilized in 1% Deoxy Big CHAP ([N,N'-bis(3-D-gluconamidopropyl)deoxycholamide]), 30 mM Tris/HCl pH 7.4, 150 mM potassium acetate, 4 mM magnesium acetate, 1 mM ATP, and protease inhibitors. Immunoprecipitations were carried out with His antibodies followed by a second precipitation with HA antibodies.
Binding of Polyubiquitin and Poly-GST-ubiquitin to Ufd1-Npl4
— Plasmids encoding mammalian C-terminally His-tagged Ufd1, Npl4,
and Npl4
ZF, lacking the zinc finger domain, were described previously
(25). The plasmids pGEX-gp78c
and pGEX-MmUBC7 were provided by A. Weissman. p97, GST-gp78c, and GST-Ubc7
were purified as described
(18).2
Polyubiquitin chains were synthesized at 37 °C with 4 µM
Ubc7, 1 µM GST-gp78c in 25 mM Tris/HCl, pH 7.4, 2
mM magnesium/ATP, 0.1 mM dithiothreitol, 110
nM E1, and 20 µM ubiquitin. The binding experiments
were carried out at 4 °C in 0.3 ml of 50 mM Hepes, pH 7.3, 150
mM potassium chloride, 2.5 mM magnesium chloride, 5%
glycerol, 2 mM -mercaptoethanol, 0.1% Triton X-100, 1 mg/ml
bovine serum albumin. 20-µl samples of polyubiquitination reactions were
incubated with 1 µg of the various purified recombinant proteins. The
proteins were then precipitated with specific antibodies, and bound ubiquitin
chains were detected by immunoblotting with ubiquitin antibodies.
Miscellaneous Methods—For ATP depletion, the cytosol was incubated for 20 min at 37 °C with 0.1 unit/µl hexokinase and 20 mM glucose. Where indicated, 3 mM AMP-PNP and 3 mM MgOAc were added during the chase period. Wild-type HA/A2 and K-R HA/A2 constructs and cell lines were described previously (7).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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HC), and analyzed by SDS-PAGE and autoradiography
(Fig. 1A, lanes
9–12). As reported previously
(5,
7), the heavy chains were
degraded during the chase period (lane 10 versus lane 9). In
addition, the characteristic deglycosylated, faster migrating heavy chain
species, which fractionated with the cytosol, appeared (lane 11 versus
lane 12). To detect polyubiquitinated heavy chains, a portion of the
samples was subjected to a second round of immunoprecipitation with ubiquitin
antibodies (lanes 29–32). Polyubiquitinated heavy chains,
appearing as a smear of high molecular mass species, accumulated over time
(lane 30 versus lane 29). Most of the polyubiquitinated heavy chains
were found in the cytosolic fraction (lane 31 versus lane 32),
indicating that they had been released from the ER membrane. In control cells
not expressing US11, the heavy chains were stable (lanes 1–8),
and no polyubiquitination was observed (lanes 21–28). It should
be noted that previous experiments showed that polyubiquitination is required
for retrotranslocation (8),
implying that the majority of the retrotranslocated heavy chains, visible in
the cytosolic fraction as the deglycosylated species, must have undergone
deubiquitination, even in the presence of 1 µM
ubiquitin-aldehyde.
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When permeabilized cells expressing US11 were incubated with ATP-depleted
cow liver cytosol (ATP), the degradation of the heavy chains was
greatly reduced, and the majority of the material stayed on the membrane
(lane 16 versus lane 15). As expected from the ATP requirement of the
ubiquitin-activating enzyme, no polyubiquitination was observed (lanes
33–36). The addition of AMP-PNP to the ATP-depleted sample did not
increase the degradation of heavy chains (lanes 17–20), but it
partially restored polyubiquitination (lane 38), in agreement with
the expectation that the ubiquitin-activating enzyme can utilize the ATP
analog because it hydrolyzes ATP into AMP and PPi
(27,
28). The polyubiquitinated
chains that were formed fractionated with the membrane rather than with the
cytosol (lane 40 versus lane 39). The apparent incomplete restoration
of polyubiquitination by AMP-PNP may in part be due to deubiquitination
occurring during sample preparation (a band at the approximate size of
unmodified heavy chains (indicated by an asterisk) was consistently
generated even in the presence of protease inhibitors).
To show that this polyubiquitination occurs in a domain of the heavy chain that was originally in the ER lumen, we employed a mutant (K-R) heavy chain, in which the lysines in the C-terminal tail were mutated to arginines. Because ubiquitination can only take place on free amino groups (internal lysine residues or the extreme N terminus), the cytoplasmic domain of this mutant heavy chain cannot be ubiquitinated. For these experiments, we used stable cell lines that express HA-tagged MHC class I heavy chains in addition to US11. In the presence of ATP, a fraction of both the wild-type and the mutant (K-R) heavy chains were deglycosylated during the chase period (Fig. 1B, lane 2 versus lane 1 and lane 10 versus lane 9) and appeared in the cytosolic fraction (lanes 3 and 11). Polyubiquitinated heavy chains accumulated during the chase period (lane 18 versus lane 17 and lane 26 versus lane 25) and were mostly released into the cytosol (lanes 19 and 27). In the presence of AMP-PNP, however, retrotranslocation was blocked (lane 7 versus lane 8 and lane 15 versus lane 16). Polyubiquitinated heavy chains were generated (lanes 22 and 30) but remained in the membrane fraction (lanes 24 and 32).
To detect polyubiquitin chains directly, 125I-ubiquitin was
added to cow liver cytosol and incubated with permeabilized astrocytoma cells
expressing US11. The samples were subjected to immunoprecipitation with heavy
chain antibodies and analyzed by SDS-PAGE and autoradiography
(Fig. 2A).
Polyubiquitinated heavy chains accumulated over time (lane 2 versus lane
1), and the majority was found in the cytosolic fraction (lane 3
versus lane 4). When ATP was depleted (ATP), polyubiquitination
was significantly reduced (lanes 5–8). The residual modified
chains were found in the membrane rather than in the cytosolic fraction
(lane 8 versus lane 7). When AMP-PNP was added to the ATP-depleted
sample, polyubiquitination was restored (lane 10), and essentially
all modified chains fractionated with the membranes (lane 12 versus lane
11). When similar experiments were performed with US11 cells expressing
the HA-tagged wild-type or K-R mutant heavy chains, we observed that in the
presence of ATP polyubiquitination occurred on both the wild-type and mutant
heavy chains, with most modified chains appearing in the cytosolic fraction
(Fig. 1B, lane 4
versus lane 3 and lane 12 versus lane 11). When
polyubiquitination was performed with AMP-PNP, the chains failed to be moved
into the cytosol and remained associated with the membrane (lane 8 versus
lane 7 and lane 16 versus lane 15). These chains appear to have
a higher molecular mass than in the presence of ATP and ATP-regenerating
system (lanes 5–8 versus lanes 1–4 and lanes
13–16 versus lanes 9–12), which may reflect the existence of
an ATP-dependent deubiquitinating step. Taken together, these results indicate
that polyubiquitination occurs on a segment that was originally in the ER
lumen and that modification of these residues alone is insufficient to move
the substrate into the cytosol. An additional ATP-dependent step that requires
hydrolysis of the 
-phosphate of ATP appears to be involved.
To provide further evidence that an additional ATP-dependent step is required for the release of heavy chains into the cytosol, we performed experiments in the absence of AMP-PNP, thus excluding the possibility that the ATP analog blocked a step following polyubiquitination. Permeabilized cells labeled with [35S]methionine and [35S]cysteine were resuspended in cow liver cytosol in the absence of ATP and chase-incubated for 20 min. Then either buffer or 0.1 mM ATP was added, and the incubation was continued for another 10 min (30 min total). The polyubiquitinated heavy chains formed during the labeling period stayed on the membrane in the absence of ATP (Fig. 3, lanes 1–7, P). However, when ATP was added, about half of the polyubiquitinated chains were released into the cytosol (lane 13 versus lane 14). In the presence of ATP, some shorter polyubiquitinated species appeared in the membrane fraction, either because existing chains were deubiquitinated or because new polyubiquitin chains were formed. These results confirm that polyubiquitination alone is insufficient and that ATP is required for the release of modified substrate from the ER membrane into the cytosol.
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Lysine 48 Linkage of Polyubiquitin Chains Is Required for
Retrotranslocation—Different lysines in the ubiquitin molecule can
serve for chain elongation, resulting in polyubiquitin chains with distinct
roles in the cell. For example, polyubiquitin chains with lysine 48 linkages
are required for proteasomal degradation
(9), whereas chains with lysine
63 linkages are implicated in nonproteolytic events
(11,
13–15).
We therefore wished to determine whether a specific linkage is required for
retrotranslocation. US11 cells were pulse-labeled with
[35S]methionine/[35S]cysteine, permeabilized, and
resuspended in ubiquitin-depleted cytosol replenished with buffer, wild-type
ubiquitin, or ubiquitin mutants lacking specific attachment sites (K48R or
K63R ubiquitin). The cells were incubated for 30 min at 37 °C, and then a
portion of the samples was analyzed directly, whereas the other was separated
into a membrane pellet and a cytosolic supernatant fraction. After
solubilization in detergent, the samples were subjected to immunoprecipitation
with HC and analyzed by SDS-PAGE and autoradiography. In the absence of
ubiquitin (Fig. 4, lanes
1–4), MHC class I heavy chains were retained in the membrane
(lane 4). In the presence of wild-type ubiquitin (lanes
5–8), the deglycosylated heavy chain species appeared in the
cytosolic fraction (lane 7 versus lane 8). When the K48R mutant was
present (lanes 9–12), however, hardly any deglycosylated
species was observed in the cytosolic fraction (lane 11). The K63R
mutant behaved like wild-type ubiquitin (lanes 13–16 versus lane
5–8). These results indicate that polyubiquitin chains with lysine
48 linkages are required for retrotranslocation.
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Poly-GST-Ubiquitination of MHC Class I Heavy Chain Prevents Its Retrotranslocation—Because polyubiquitination with a specific linkage is required for retrotranslocation but on its own is insufficient, we tested whether polyubiquitin chains may function as a recognition signal. Specifically, we investigated whether modification of polyubiquitin chains by the addition of a GST moiety to the N-terminal part of ubiquitin would influence retrotranslocation. We reasoned that GST-ubiquitin molecules may still be linked with one another to form poly-GST-ubiquitinated heavy chains but that these chains may no longer be recognized by a downstream component.
We incubated 35S-labeled permeabilized cells with cow liver cytosol supplemented with GST-ubiquitin. Compared with control cow liver cytosol, the appearance of deglycosylated heavy chains in the cytosol was reduced (Fig. 5A, lanes 16–20 versus lanes 11–15). In addition, a high molecular mass species appeared during the chase period, which was not seen in the absence of GST-ubiquitin. The high molecular mass species were GST-ubiquitinated heavy chains as demonstrated by reimmunoprecipitation with either ubiquitin antibodies (bottom panel, lanes 41–45) or GST antibodies (Fig. 5B, lanes 13–16). In most experiments, a fraction of 15–20% of the total population of heavy chains present at the beginning of the chase period were poly-GST-ubiquitinated, but occasionally up to 55% was modified. This is a much higher percentage of modification than seen with wild-type ubiquitin (see lanes 11–15 and 36–40), which may suggest that deubiquitination is impaired by GST-ubiquitin. A significant fraction of GST-ubiquitinated heavy chains were membrane-associated (lane 20 versus lane 19 and lane 25 versus lane 24). Some deglycosylated chains were released into the cytosol even in the presence of GST-ubiquitin (lane 19), and some poly-GST-ubiquitinated molecules could be immunoprecipitated from the cytosolic fraction (lane 44). This is probably due to chains containing endogenous ubiquitin in addition to GST-ubiquitin. Indeed, when ubiquitin was depleted from the cytosol (8) before the addition of GST-ubiquitin, the appearance of the deglycosylated species was completely blocked, and essentially all modified chains remained in the membrane (lanes 21–25). The maximum amount of GST-ubiquitinated heavy chains was reached after 20–30 min of incubation (Fig. 5B), and these adducts were stable for at least 2 h. Similar results were obtained with both the wild-type and K-R mutant heavy chains (data not shown). The GST-ubiquitin modified heavy chains were sensitive to protease treatment (data not shown), as expected from the fact that ubiquitination is a cytosolic event. These results show that the attachment of multiple GST-ubiquitin molecules to heavy chains is not sufficient for their release into the cytosol. They suggest that poly-GST-ubiquitin prevents retrotranslocation by disrupting a recognition signal for a downstream component. Apparently, the GST moiety also prevents recognition by the deubiquitinating enzymes.
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Poly-GST-Ubiquitin Is Not Recognized by a Ubiquitin-binding Domain in the Ufd1-Npl4 Cofactor of the ATPase p97—One of the candidates for a downstream component is the ATPase p97, which has been shown to function together with its cofactor Ufd1-Npl4 in retrotranslocation (17–21). We first tested whether poly-GST-ubiquitination of MHC class I heavy chains would abrogate substrate recognition by p97, which would provide an explanation why these chains can no longer be moved into the cytosol. To determine substrate binding by p97, astrocytoma cells expressing US11 and HA-tagged wild-type heavy chains were treated with proteasome inhibitor, labeled, and permeabilized. The endogenous cytosol was replaced with cow liver cytosol that was either depleted of ubiquitin as described (8) or first depleted and then replenished with ubiquitin or GST-ubiquitin (Fig. 6A). Where indicated, His-tagged recombinant p97 protein was added, and the samples were chase-incubated for 30 min and then separated into pellet and supernatant fractions. Immunoprecipitation of the heavy chains showed that in the presence of ubiquitin, the deglycosylated species appeared in the supernatant, regardless of whether p97 was added or not (upper panel, lanes 4 and 8). In the absence of ubiquitin, retrotranslocation was blocked (lanes 9–12). In the presence of GST-ubiquitin, retrotranslocation was also reduced, and the characteristic high molecular adducts appeared in the membrane fraction (lanes 13–16). To detect p97-associated heavy chains, the samples were first immunoprecipitated with His antibodies and then with HA antibodies (lanes 17–32). Binding of heavy chains was seen regardless of whether or not ubiquitin was present during the incubation (lanes 21–24 versus lanes 25–28), demonstrating that p97 can interact with nonmodified substrate. In the absence of His-p97, no material was immunoprecipitated (lanes 17–20). p97 also interacted with poly-GST-ubiquitinated heavy chains (lanes 29–32). Thus, the fact that poly-GST-ubiquitinated heavy chains cannot be released into the cytosol cannot be explained by disruption of substrate recognition by p97.
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Because previous experiments implicated the cofactor Ufd1-Npl4 in the
recognition of the polyubiquitin chains,2 we next tested whether
poly-GST-ubiquitin may not be recognized by this cofactor, providing a
possible explanation for why this modification disrupts retrotranslocation.
Polyubiquitin chains were synthesized in vitro using purified
recombinant proteins. The assay contained ubiquitin, the E1, the E2, and a GST
fusion to a cytosolic fragment of the E3. We employed Ubc7 and gp78 because
they are known to be involved in the ER degradation of at least some proteins
(29–32).
When all of the components were incubated together in the presence of ATP,
polyubiquitin chains were synthesized (Fig.
6B, upper panel, lane 1). These chains were
tested for interaction with the Ufd1-Npl4 (U/N) complex. The mammalian
cofactor complex has two polyubiquitin-binding domains: one in Ufd1 and one in
a zinc finger domain of Npl4. Because the Npl4 ubiquitin-binding domain is
lacking in yeast (33) and the
complex likely functions analogously to the mammalian one, the binding site in
Ufd1 appears to be most important in retrotranslocation. We therefore tested
the binding of both wild-type Ufd1-Npl4 and of a mutant complex that lacked
the zinc finger domain in Npl4 (U/NZF). Polyubiquitin chains were
incubated with these complexes, and binding was assessed by
immunoprecipitation with Ufd1 antibodies followed by immunoblotting with
ubiquitin antibodies. With the wild-type complex, a significant fraction of
the polyubiquitin chains was bound (Fig.
6B, upper panel, lane 3; the lower
panel demonstrates coprecipitation of Ufd1 and Npl4). As expected, the
mutant complex (U/NZF), containing only the Ufd1 binding site for
polyubiquitin, had reduced but still significant binding activity (lane
4). Next, we performed binding experiments with poly-GST-ubiquitin
instead of wild-type polyubiquitin. The polymerization reaction with
GST-ubiquitin was less efficient, but high molecular mass adducts were
generated (lane 5). The poly-GST-ubiquitin chains bound to U/N but
not to U/NZF as demonstrated with both ubiquitin and GST blots
(lanes 7 and 8 versus lanes 3 and 4 in upper and
lower panels). The generated poly-GST-ubiquitin chains were linked
through lysine 48 of ubiquitin, as demonstrated by the fact that
poly-GST-ubiquitin chains could not generated with GST-K48R or GST-K0
ubiquitin (which lacks all 7 lysines in ubiquitin)
(Fig. 6C, compare
lane 4 with lanes 5 and 6). Taken together, these
results show that poly-GST-ubiquitin chains are not recognized by the Ufd1
ubiquitinbinding domain of the cofactor Ufd1-Npl4, even though the chains have
the correct ubiquitin linkage. On the other hand, intact Ufd1-Npl4 can
interact with poly-GST-ubiquitin, likely through the zinc finger domain in
Npl4. Our data are consistent with the hypothesis that poly-GST-ubiquitinated
heavy chains may not be recognized by the cofactors of p97 during
retrotranslocation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-phosphate of ATP. Because
polyubiquitination alone was insufficient to move a polypeptide into the
cytosol, we conclude that the driving force for retrotranslocation is not
provided by a simple ratcheting mechanism. This is in contrast to
posttranslational translocation in the forward direction, in which a secretory
protein can be moved completely into the ER lumen by a ratcheting mechanism
(16). Although
polyubiquitination does not suffice to move a polypeptide chain completely
into the cytosol, it may still prevent movements back into the ER lumen. The
initial steps of retrotranslocation must be independent of polyubiquitination
of the substrate, because the modification occurs on segments that were
originally in the ER lumen. It may not be too surprising that
polyubiquitination does not provide a simple ratcheting mechanism, because the
distances between consecutive lysine residues, the usual attachment sites in a
polypeptide chain, would make the ratchet in general inefficient. In addition,
some proteins contain few or even no lysines. Our data not only indicate the existence of an ATP-dependent step following polyubiquitination but also suggest that polyubiquitin serves as a recognition signal for a downstream component, because a substrate that is poly-GST-ubiquitinated could not be moved into the cytosol. This idea of polyubiquitin as a recognition signal is strengthened by the fact that a specific lysine 48 linkage within the polyubiquitin chain is required for retrotranslocation, the same linkage that is required for the proteasome that acts subsequent to retrotranslocation in substrate degradation.
The simplest model is that a downstream component recognizes polyubiquitin and functions in an ATP-dependent process. Previously, the ATPase p97 and its cofactor Ufd1-Npl4 have been implicated in retrotranslocation. Our data suggest that this complex may be involved in the ATP- and polyubiquitin-dependent step. A requirement for ATP hydrolysis by p97 would explain why AMP-PNP did not allow retrotranslocation. Furthermore, a p97 mutant defective in ATP hydrolysis leads to a similar phenotype as AMP-PNP, i.e. the accumulation of polyubiquitinated substrate on the ER membrane.2 The binding of polyubiquitin to the p97 complex is also consistent with it being the immediate downstream component. The binding occurs to an N-terminal domain of Ufd1 in the cofactor complex.2 Although the zinc finger domain in Npl4 can also bind polyubiquitin (25), this interaction is likely not important for retrotranslocation. It does not discriminate between lysine 48 and lysine 63 linkages,2 and the yeast homolog of Npl4 lacks the zinc finger domain (33) altogether but likely functions analogously to the mammalian protein in retrotranslocation. The fact that this domain can even interact with mono-ubiquitin (34) may explain the interaction we observe with poly-GST-ubiquitin. In contrast, the ubiquitin-binding domain in Ufd1 is specific for lysine 48 linkages in polyubiquitin chains and does not recognize lysine 63 linkages,2 consistent with our finding that lysine 48 is required for retrotranslocation. We have found that Ufd1-Npl4 lacking the zinc finger domain in Npl4 does not bind poly-GST-ubiquitin, even though the chains have the correct lysine 48 linkages. The lack of interaction of poly-GST-ubiquitin with the ubiquitin-binding domain in Ufd1 could explain why this modification blocks the movement of MHC class I heavy chains into the cytosol. However, it is possible that other polyubiquitin-binding proteins, such as the proteasome or deubiquitinating enzymes, are also prevented from binding and cause the retrotranslocation defect. For example, deubiquitinating enzymes may be important to remove the polyubiquitin chains from polypeptides so that they can be moved through the relatively narrow pore in the double-barrel structure of the p97 ATPase (35). This function would be similar to the role suggested for deubiquitinating enzymes in the degradation of proteins by the proteasome. The inhibitory effect of AMP-PNP on deubiquitination seen in our experiments could be explained by an enzyme similar to that found on the proteasome (26, 36).
Interestingly, p97 itself can bind to both nonubiquitinated and polyubiquitinated substrate molecules. This suggests that there are two consecutive interactions of the p97-Ufd1-Npl4 complex with retrotranslocating heavy chains. It first binds to nonubiquitinated segments of the substrate and then, following ubiquitination, to the polyubiquitin chain. How exactly the polypeptide substrate would subsequently be moved into the cytosol remains unclear, and our data also do not exclude that a component other than the p97-Ufd1-Npl4 complex could be involved. However, one of the possibilities is that the interaction of the cofactor with the polyubiquitin chain activates the ATPase p97 to "pull" the polypeptide chain out of the membrane.
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FOOTNOTES |
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¶ Supported by the Helen Hay Whitney postdoctoral fellowship.
** Supported by National Institutes of Health Grant R01 GM62194.
Supported by a grant from the National Institutes of Health. Howard Hughes
Medical Institute Investigator. To whom correspondence should be addressed.
E-mail:
tom_rapoport{at}hms.harvard.edu.
1 The abbreviations used are: ER, endoplasmic reticulum; MHC, major
histocompatibility complex; HA, hemagglutinin; GST, glutathione
S-transferase; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier
protein; E3, ubiquitin-protein isopeptide ligase; AMP-PNP, adenosine
5'-(,-imino)triphosphate; HC, heavy chain
antibodies; Ub, ubiquitin antibodies; HA, HA antibodies.
2 Ye, Y., Meyer, H. H., and Rapoport, T. A. (2003) J. Cell Biol.
162, 71–84.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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X. Wang, R. A. Herr, W.-J. Chua, L. Lybarger, E. J.H.J. Wiertz, and T. H. Hansen Ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce ERAD of MHC-I by viral E3 ligase mK3 J. Cell Biol., May 21, 2007; 177(4): 613 - 624. [Abstract] [Full Text] [PDF]
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