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J. Biol. Chem., Vol. 277, Issue 5, 3258-3267, February 1, 2002
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From the Department of Pathology, Harvard Medical School,
Boston, Massachusetts 02115
Received for publication, October 9, 2001, and in revised form, November 12, 2001
Human cytomegalovirus encodes two glycoproteins,
US2 and US11, that target major histocompatibility complex (MHC) class
I heavy chains for proteasomal degradation. We have developed a mRNA-dependent cell-free system that recapitulates US2-
and US11-mediated degradation of MHC class I heavy chains. Microsomes
support the degradation of MHC class I heavy chains in the presence of
US2 or US11 in a cytosol-dependent manner. In
vitro, the glycosylated heavy chain is exported from the
microsomes. A deglycosylated breakdown intermediate of the heavy chain
identical to that generated in intact cells accumulates in soluble form
in the presence of proteasome inhibitors. Microsomes derived from the
U373 astrocytoma cell line are far more effective than canine-derived
membranes in supporting this US2- or US11-dependent
reaction. In contrast, the HIV-encoded Vpu membrane protein can
cause the destruction of CD4 from either human- or canine-derived
membranes. Using the in vitro system, we show that a
truncation mutant of US2 that lacks the cytosolic domain is unable to
catalyze degradation, whereas a similar truncation of US11 continues to
catalyze degradation of class I heavy chains. Therefore, US2 requires
both transmembrane and cytosolic interactions to trigger dislocation of
heavy chains, whereas US11 relies on the transmembrane domain to target
heavy chains. US2 and US11 thus utilize different targeting mechanisms for class I degradation.
The mammalian immune system relies on the presentation of cell
surface glycoproteins to alert lymphocytes to the presence of
intracellular pathogens. One class of these glycoproteins, MHC1 class I molecules, is
assembled in the endoplasmic reticulum (ER) as a trimeric complex
consisting of a 43-kDa heavy chain in association with a 12-kDa light
chain ( The ER is a major site of quality control for secretory proteins in all
eukaryotes. Many glycoproteins that lack the ability to fold properly
or associate with partner polypeptides are retained in the ER and are
eventually targeted for degradation by the proteasome (4, 5). The
involvement of the proteasome in the destruction of ER proteins
necessitates their access to the cytosol where proteasomes are located.
The mechanism by which aberrant proteins are targeted for destruction
and dislocated from the ER remains unresolved and there may in fact be
multiple pathways (6-8). US2 and US11 have co-opted this degradation
process to interfere with the expression of MHC class I molecules and
therefore provide a model system for the study of ER-to-cytosol transport.
Genetic and cell-free systems in yeast have identified molecules that
participate in the recognition, dislocation, and degradation steps of
the process. Chaperones such as BiP and calnexin participate in
recognition of some substrates (9, 10). Several mutations in the Sec61p
translocon compromise degradation and suggest that this pore may
mediate dislocation into the cytosol, in addition to the import of
nascent polypeptides (11). Ubiquitin-conjugating enzymes also play a
role in the degradation of most ER-to-cytosol substrates in yeast,
consistent with ubiquitination as a major signal for proteasomal
destruction (4).
Mammalian cell-free systems have confirmed some of the observations
made in yeast and intact cells. Ubiquitination and degradation of the
cystic fibrosis transmembrane conductance regulator has been observed
in canine microsomes (12, 13). The degradation of CD4 by HIV Vpu has
also been observed in canine microsomes (14). Export of a misprocessed
glycosylphosphatidylinositol-anchored protein from microsomes was shown
to be cytosol dependent (15). The release of Our objective was to recapitulate the steps of early MHC class I
synthesis and recognition by US2/US11 in vitro. Dislocation and degradation in a cell-free environment would facilitate dissection of the components required for these events, but no such integrated system has been reported. Synthesis and assembly of MHC class I
molecules within microsomes has been demonstrated (18). Furthermore, interactions between in vitro translated assembled class I
molecules with US2 and US11 in canine pancreas-derived microsomes have
likewise been shown, but in vitro dislocation by US2 or US11
has not been demonstrated (19, 20).
The use of the system presented here reveals a heterogeneity in the
membrane-bound components required for dislocation of heavy chains by
US2 and US11. The intermediates that occur in the course of degradation
by US2 and US11 in vitro are biochemically indistinguishable
from the deglycosylated, cytosolic intermediates observed in intact
cells. However, US2 and US11 do not rely equally on cytosolic domains
to trigger degradation. Further, the mechanism for US2/US11 and class I
appears to be distinct from that used by Vpu for the degradation of
CD4. Here we also show that microsomes of human origin support US2/US11
degradation, whereas canine pancreas microsomes do not. Our data are
consistent with wide divergence in the manner by which proteins are
exported from the ER for cytosolic degradation.
Cell Lines and Cell Culture--
U373-MG astrocytoma cells
(control), US2 transfectants (US2+), and US11 transfectants
(US11+) cell lines were maintained as described (21).
DNA Constructs and Transfection--
The HLA-A2, US2, US11,
Antibodies and Reagents--
Polyclonal rabbit anti-HC, which
recognizes unfolded heavy chains, has been described (24). W6/32, a
mouse monoclonal antibody that recognizes properly folded MHC class I
molecules in association with
The anti-US2 polyclonal antibody was generated by immunizing rabbits
with peptides corresponding to amino acids 20-38, 92-110, and
146-164 of US2. The Sec61 In Vitro Transcription and Translation--
In vitro
transcription was performed as described (22). In vitro
translations were performed for 45 min (except where otherwise noted)
at 30 °C in a reaction mixture containing 17.5 µl of Flexi rabbit
reticulocyte lysate (RRL) (Promega), 0.8 µl of KCl (2.5 M), 0.5 µl of amino acid mixture minus methionine (1 mM, Promega), 2.5 µl of [35S]methionine (10 mCi/ml, translation grade; PerkinElmer Life Sciences), and 0.5 µl human astrocytoma or 1 µl canine pancreas microsomal membranes,
where indicated.
Preparation of Microsomes--
Canine pancreas microsomes were
prepared as described (28).
Astrocytoma microsomes were prepared from U373-MG control cells, US2,
and US11 transfectants. Astrocytoma cells were detached by trypsin
treatment, resuspended in homogenization buffer (10 mM
HEPES, pH 7.4, 1 mM EDTA, 0.25 M sucrose, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 0.25 mM
phenylmethylsulfonyl fluoride) at 25-35 × 106
cells/ml, and homogenized by repeated passage through 25-, 27-, and
30-gauge syringe needles. Unbroken cells and nuclei were pelleted by
centrifugation (1,500 × g for 10 min at 4 °C). The
1,500 × g supernatant was then centrifuged at
75,000 × g for 60 min at 4 °C. The membrane pellet
was resuspended in homogenization buffer (approximately 100 × 106 cells/ml of homogenization buffer) and added to
in vitro translations as a source of microsomes. The
supernatant was discarded.
In Vitro Degradation Assay--
In vitro translations
of HLA-A2 and/or other polypeptides were performed in the presence of
microsomes (0.5 µl of astrocytoma microsomes/21.5 µl of translation
reaction, 1 µl of canine pancreas microsomes/21.5 µl of translation
reaction). Translation reactions were centrifuged at 20,000 × g for 15 min. The supernatant was removed, and microsomes
were resuspended in 5 µl of homogenization buffer and 15 µl of
Flexi RRL containing 1 mg/ml RNase (Roche Molecular Biochemicals) and
50 µM MG132 where indicated. Cytosol extracts containing
an ATP-regenerating system (29) were added where indicated in place of
RRL. Reactions were incubated at 30 °C, and aliquots were withdrawn
at indicated time points. Microsomal pellet was separated from RRL
supernatant by centrifugation at 20,000 × g for 15 min. Reducing sample buffer was added to supernatant and pellet
fractions followed by separation by SDS-PAGE, or samples were mixed
with lysis buffer (see below) for immunoprecipitation where indicated.
Pulse-Chase Analysis, Immunoprecipitation, and Endoglycosidase
Digestion--
Pulse-chase analysis, immunoprecipitation, and
endoglycosidase H digestion was performed as described (24). Peptide
N-glycanase (PNG) (New England Biolabs) digestion was
performed as described by the manufacturer.
Gel Electrophoresis and Immunoblotting--
SDS-PAGE,
one-dimensional isoelectric focusing (IEF), fluorography (30), and
immunoblotting (31) were performed as described.
Microsomes Derived from Human Astrocytoma Cells Support Proper
Insertion, Signal Sequence Cleavage, Glycosylation, and Folding of in
Vitro Translated Membrane Glycoproteins--
The HCMV US2 and US11
gene products induce proteasomal degradation of MHC class I heavy
chains. To generate a cell-free system with which to study US2- and
US11-mediated degradation, microsomes were isolated from U373-MG cells,
a human astrocytoma cell line that supports rapid degradation when
transfected with US2 or US11. We first characterized the ability of
these microsomes to insert and process glycoproteins in the course of
in vitro translation reactions (Fig.
1). The addition of membranes to an
in vitro translation reaction of HLA-A2 yielded a
polypeptide with a slower mobility than the product translated in the
absence of membranes (Fig. 1A, compare lanes
1 and 3). This product was sensitive to digestion with endoglycosidase H (EH), indicating the presence of an
N-linked glycan (Fig. 1A, lane
2). The faster mobility of the EH-treated microsomal species
relative to the HLA-A2 polypeptide translated in the absence of
microsomes indicates that the signal sequence had been cleaved from the
microsomal HLA-A2. This HLA-A2 polypeptide was identical in molecular
size to HLA-A2 translated into canine pancreas microsomes (Fig. 7). To
ascertain proper assembly of HLA-A2 molecules in astrocytoma
microsomes, HLA-A2 was co-translated with mRNA that encodes the
light chain, Microsomes Derived from Human Astrocytoma Cells Support US2- and
US11-mediated Degradation of Class I Molecules--
We next examined
the ability of the membranes to induce US2/US11-mediated degradation of
MHC class I heavy chains. HLA-A2 was translated into microsomes derived
from control, US2-expressing, or US11-expressing astrocytoma cells
(Fig. 2A). RNase was added after 45 min to terminate ongoing translation, and reactions were sampled at 0, 30, 60, and 90 min after addition of RNase. Microsomal fractions from each time point were isolated by centrifugation and
analyzed directly by SDS-PAGE. The amount of the HLA-A2 polypeptide recovered decreased over time in the US2 and US11 microsomes (Fig. 2A, lanes 5-12), whereas it was
stable in control microsomes over 90 min (Fig. 2A,
lanes 1-4). Amounts of HLA-A2 were quantitated and plotted as the percentage remaining relative to time 0. Approximately 60% of HLA-A2 molecules were degraded in US2 and US11
microsomes after 90 min, with a more rapid decline in HLA-A2 levels in
US2 microsomes at earlier time points. Additionally, less HLA-A2 was visible in US2 microsomes at time 0 relative to control and US11 microsomes (Fig. 2A, compare lanes 1,
5, and 9). These results have been observed
consistently in three experiments and indicate that US2-mediated
degradation of HLA-A2 was active during the translation period. US2
targets HLA-A2 for degradation at an earlier step during HLA-A2
maturation than US11.
Next, we modified the assay by performing translations in the presence
of microsomes, followed by isolation of the microsomes by
centrifugation, removal of the translation supernatant, and resuspension of microsomes in a fresh batch of cytosol containing RNase
to terminate translation and start the chase. HLA-A2 was translated
into control or US2 microsomes, and CD4 was co-translated to serve as a
control (Fig. 2B). CD4 is a protein of a size comparable with that of MHC class I products, and likewise a member of the Ig
superfamily, yet not a substrate for US2 or US11. After translation for
45 min at 30 °C, the microsomes were pelleted by centrifugation and
resuspended in a small volume of homogenization buffer. RRL containing
an ATP-regenerating system and RNase was added to resuspended microsomes as chase cytosol. The mixture was then incubated at 30 °C
and sampled at 0, 30, and 60 min after the addition of cytosol. The
membrane fractions were analyzed directly by SDS-PAGE (Fig. 2B). Whereas the amount of radiolabeled HLA-A2 polypeptide
present in the US2 microsomes decreased over the chase period as
compared with the control microsomes, CD4 was equally stable over 60 min in US2 and control microsomes (Fig. 2B, lanes
1-3 and 7-9). No significant loss of HLA-A2 or CD4
occurred in control microsomes (Fig. 2B, lanes
1-6). Therefore, US2 specifically destabilized HLA-A2.
US2/11-mediated degradation is sensitive to agents that alter redox
potential (21). Similar to what is observed in intact cells, no
dislocation of HLA-A2 was observed from US2 microsomes when microsomes
were resuspended in RRL containing 10 mM
N-ethylmaleimide (NEM), to alkylate free sulfhydryls (Fig.
2B, lanes 4-6 and 10-12). The addition of 1 mM NEM or another oxidant, diamide,
blocked dislocation by more than 50% in RRL as opposed to 100% in
intact cells.2 The lower
sensitivity to NEM and diamide in the in vitro reactions may
be attributable to the high (100-200 mg/ml) protein concentration of
RRL.
Proteasome Inhibition Reveals Accumulation of Soluble Heavy Chain
Intermediates Released from US2 Microsomes--
When US2-expressing
cells are treated with proteasome inhibitors, deglycosylated MHC class
I heavy chains accumulate in the cytosol (3). To determine whether the
same intermediates can be observed in vitro, the dislocation
assay was performed with proteasome inhibitors added to the RRL chase
mix. Membrane-associated and soluble class I products were analyzed
separately (Fig. 3). When the supernatant
fraction was analyzed, two distinct polypeptide species were observed
in inhibitor-treated reactions, but at later chase points only (Fig.
3A, lane 8). These species were
detected in incubations only in the presence of the proteasome
inhibitor, MG132, and when US2 was present in the microsomes. Identical
species were observed in supernatants recovered from US11-containing
microsomes (Fig. 6A, lanes 9 and
12) but not from control microsomes.2 Other
specificity controls confirm that these intermediates were a product of
functional US2/US11-mediated degradation only (see below). The loss of
HLA-A2 from microsomes was unaffected by the inclusion of MG132 in the
chase cytosol (Fig. 3A, lanes 1-4). The amount of soluble intermediates that can be recovered was always
less than the amount lost from the microsomes, indicating incomplete
inhibition of proteasome activity or the involvement of other proteases
present in rabbit reticulocyte lysate. Release of glycosylated CD4 into
the supernatants was not observed in the presence of US2 (Fig.
3A, lanes 5-8). Immunoblotting of membrane and
supernatant fractions for Sec61
To establish the identity of the class I-derived polypeptides and to
verify that they represent the deglycosylated species, immunoprecipitations were performed and analyzed by both SDS-PAGE and
IEF (Fig. 3B). The polypeptides found in the supernatants of
the in vitro reactions in the presence of MG132 (Fig.
3B, lane 6) were immunoprecipitable
with anti-heavy chain antiserum and co-migrate with HLA-A2 recovered
from US2 cells treated with MG132 (Fig. 3B, lane
2). Furthermore, the mobility of the faster-migrating species was identical to the mobility of HLA-A2 treated with PNG both
on SDS-PAGE and on IEF gels (Fig. 3B, lanes
4 and 8).
The heavy chain intermediate that accumulates in US2- and
US11-expressing cells in the presence of proteasome inhibitors is generated by the removal of the N-linked glycan by an
N-glycanase and therefore has a faster mobility on SDS-PAGE
(2, 3). Deglycosylated heavy chains acquire an additional negative
charge, because of the conversion of asparagine to aspartic acid upon hydrolysis of the glycoamide linkage by N-glycanase, and
therefore have a distinct mobility on IEF gels (3). This identification was confirmed by digestions of immunoprecipitates with EH and recombinant PNG (Fig. 3B, IEF gel). The latter is
particularly relevant, as the time-dependent conversion of
glycosylated HLA-A2 in the in vitro reactions to the more
acidic species identifiable by IEF paralleled that seen in US2 cells
(except that less of the material was recovered from the in
vitro chase supernatants (lane 6) than from
intact cells (lane 2)) (Fig. 3B).
Thus, biochemical analysis confirmed that the intermediates generated
in this system were identical to the HLA-A2 intermediates produced in
intact cells. In the absence of a source of the reportedly cytosolic mammalian N-glycanase that could be added to the in
vitro system described here, the role of N-glycanase in
the reaction remains to be explored.
In Vitro Degradation of MHC Class I Molecules by US2 Is
Allele-specific--
For intact cells, the recognition of heavy chains
by US2 and US11 is to a large extent specific for particular MHC locus
products. To establish the specificity of the in vitro
reactions, degradation assays were performed in US2 microsomes with
another MHC class I allele, HLA-C. Unlike HLA-A2, HLA-C was stable in
US2 microsomes and no intermediates of HLA-C were released from the
microsomes (Fig. 4, lanes
7 and 8), consistent with the resistance of the C-locus product to degradation in intact cells (20). Furthermore, HLA-E
and HLA-G, when analyzed in the in vitro system, were
stable, again consistent with observations in intact cells (20,
32).2 We conclude that the in vitro system
faithfully recapitulates the specificity of dislocation described in
living cells.
Ablation of the Cytosolic Domain Ablates US2 Function but Not US11
Function--
Microsomes derived from US2 and US11 transfectants
contain high levels of the viral protein. The ability of the
pre-existing viral protein in these microsomes to target MHC class I
heavy chains for dislocation shows that US2 and US11 need not be
translated simultaneously with heavy chains to trigger dislocation. Do
in vitro translated US2 and US11 catalyze the degradation of
heavy chains? If so, this would facilitate the analysis of US2 and US11 mutants by translation. Detection of radiolabeled viral protein would
assist in future identification of interacting proteins in the system.
We translated HLA-A2 into control microsomes, divided the translation
mixture in half, and added either wild type US2 mRNA or US2-186
mRNA, which lacks the putative cytosolic domain. Translation was
continued for 30 min, after which the assay was continued as above with
membrane and supernatant fraction sampled at the indicated times.
HLA-A2 was unstable in the presence of co-translated US2 but not
US2-186 (Fig. 5A,
lanes 1-6), indicating that the cytosolic
portion of US2 is critical for inducing heavy chain degradation.
Additionally, deglycosylated HC intermediates appeared in the
supernatants of reactions that contained wild type US2 but not US2-186
(Fig. 5A, lanes 7-12). A portion of
each reaction was immunoblotted for Sec61 Canine Pancreas Microsomes Do Not Support Degradation of HLA-A2 in
the Presence of Cotranslated US2--
We were able to observe
US2/US11-specific destabilization of HLA-A2 in the astrocytoma-derived
microsomes. We examined canine pancreas as a more readily available
source of microsomes to support US2-mediated degradation. Degradation
assays were performed with HLA-A2 and US2 co-translated in control
astrocytoma or canine pancreas membranes (Fig.
7). Whereas the HLA-A2 polypeptide was unstable in the presence of co-translated US2 in the astrocytoma microsomes (Fig. 7, lanes 1-3), we were unable to observe
destabilization of HLA-A2 in the canine microsomes (Fig. 7,
lanes 4-6). No deglycosylated species were
released from the canine microsomes in the presence of
US2.2 One possible explanation would be the inability of
preexisting canine
To examine whether canine-derived membranes were competent to dislocate
another ER-resident protein susceptible to degradation, we performed
in vitro assays with the CD4 and Vpu polypeptides. Vpu is an
accessory protein encoded by HIV that causes ER-resident CD4 to be
destroyed by the proteasome in a manner similar to MHC class I by US2,
although many of the details of Vpu- and US2/US11-mediated degradation
remain to be worked out (34, 35). CD4 was translated into control or
canine pancreas membranes and mRNA encoding Vpu or a mutant of Vpu
lacking the ability to catalyze CD4 degradation (23) was added (Fig.
8). CD4 was destabilized by wild type Vpu in both types of microsomes (Fig. 8, lanes 1-3
and 7-9), although the destabilization was more pronounced
in the human microsomes. However, CD4 was stable in the presence of
mutant Vpu in both sets of microsomes, indicating that the instability
of CD4 was not the result of a nonspecific property of the membranes or
the presence of an additional in vitro translated
polypeptide (Fig. 8, lanes 4-6 and
10-12). Degradation of CD4 did not result in the
accumulation of soluble intermediates in this system, consistent with
what has been observed in vitro and in intact cells (14, 36). We conclude that the inability of canine microsomes to support
US2-mediated degradation is specific to US2. This observation points to
the existence of multiple mechanisms for recognition and/or dislocation
of type I membrane proteins, with a species-specific component to the
host membrane protein involved.
Degradation of Heavy Chains in Vitro Is Dependent on
Cytosol--
To examine the requirements for in vitro
degradation, polypeptides were translated into US2 microsomes and these
microsomes were then further incubated in either RRL, cow liver cytosol
with an ATP-regenerating system, or homogenization buffer with an
ATP-regenerating system. Cytosol is required to induce the loss of
heavy chains from the microsomes as well as the appearance of the
deglycosylated intermediate in the cytosolic supernatant (Fig.
9). Both rabbit and cow liver cytosol
supported heavy chain degradation from US2 microsomes (Fig. 9,
lanes 3-6). Cytosol prepared from astrocytoma cells also supported degradation and formation of the deglycosylated intermediate (data not shown). No disappearance of HLA-A2 was seen in
from US2 microsomes chased in buffer (+ATP regenerating system) (Fig.
9, lanes 1 and 2). Therefore, although
species-specific membrane factors may be required to interact with US2
and US11 in the membrane, the cytosolic factors required for
dislocation are less species-specific. RRL was more effective than cow
liver cytosol in extraction of HLA-A2 from membranes but equal amounts of intermediate were observed in the supernatant (Fig. 9,
lanes 10 and 12). Either proteasome
inhibition was more effective in the less concentrated cow liver
cytosol, or RRL contained proteases not inhibited by proteasome
inhibitors, that also destroy HLA-A2.
Here we describe a cell-free system that duplicates the
ER-to-cytosol dislocation and degradation of MHC class I molecules triggered by the HCMV US2 and US11 proteins in a specific manner. We
use this in vitro system to dissect the dislocation process. First, we demonstrate that membranes prepared from a human cell line
perform comparably to canine microsomal membranes in the insertion and
processing of glycoproteins. Next, we show that class I heavy chains
are unstable in astrocytoma-derived membranes that contain US2 or US11.
The half-life of heavy chains in US2 and US11 microsomes is ~40-60
min compared with less than 5 min in intact cells. This is comparable
with the delayed kinetics for the degradation of CD4 by Vpu in
microsomes (14). For the Vpu-CD4 co-translation, the degradation
in vitro occurred three times more slowly than in intact
cells. The US2-mediated degradation in vitro is ~8-10
times slower than in intact cells. The relative amounts of US2 present
in microsomes prepared from US2 cells is high, as the cell line
overexpresses US2, and therefore a shortage of US2 is unlikely to be
the reason for the delay. Additionally, supplementation of US2
microsomes with additional US2 by in vitro translation does
not enhance degradation.2 Many factors may explain the
reduced rate of degradation in the in vitro system. A
specialized subcompartment of the ER in which US2 rapidly triggers
dislocation may be disrupted in the microsomes used. The microsomal
vesicles most proficient in import of in vitro translated
polypeptides may not support efficient dislocation. Local
concentrations or assembly of membrane or cytosolic factors that drive
extraction may be limiting. Although energy in the form of ATP is
required, it remains to be established that ATP is in fact the high
energy intermediate directly powering the reaction and therefore
metabolic steps that may occur in vitro may be
rate-limiting.
Proteasome inhibition permits the accumulation of a breakdown
intermediate that is identical to the breakdown intermediate generated
in intact US2/US11 cells. It is sufficient to treat the cytosol with
proteasome inhibitors to observe this effect in vitro. The
appearance of glycosylated and non-glycosylated heavy chains released
from the microsomes suggests that fully glycosylated material is
exported through the ER membrane and that this material is released
from the membrane prior to exposure to N-glycanase. This
observation required the use of the in vitro system
described here. In US2/US11 cells, only deglycosylated material is
detected in cytosolic fractions. N-Glycanase may be significantly more active in intact cells than in RRL. Alternatively, glycosylated material that has exited may not be immediately accessible to N-glycanase and the steps that render it accessible may
be less efficient in RRL. The molecular identity of the
N-glycanase involved in this reaction in mammalian cells
remains to be established.
The ER-luminal portion of US2 contains an immunoglobulin-like fold that
mediates the interaction with folded MHC class I molecules but is not
itself sufficient to induce degradation. Therefore elements in the
transmembrane and cytoplasmic tail regions of US2 must be important for
its function (37). Alignment of the US11 sequence with US2 suggests
that US11 contains a similar Ig-like fold, which is also likely to
dictate association with MHC class I (37). Surprisingly, deletion of
the cytosolic tail ablated US2 function but did not ablate US11
function, a result verified both in vitro and in intact
cells. Therefore, the two molecules may use different machinery to
target heavy chains, or may depend to a different degree on regions of
the heavy chain cytosolic tail to stimulate extraction. Despite the
proposed Ig fold for both molecules, there is only 25% amino acid
similarity between US2 and US11, very little of which maps to the
transmembrane and tail regions. A difference in mechanism may also
explain different kinetics of degradation between US2 and US11 in
vitro.
The ability of newly translated US2 to target class I molecules for
degradation further strengthens the hypothesis that US2 recruits,
rather than induces, factors that trigger dislocation. The mammalian ER
thus contains all of the factors required for US2 to function. Several
studies in yeast show that the degradation process is assisted by the
induction of an unfolded protein response (38, 39). Up-regulation of
chaperones during an unfolded protein response is thought to assist
protein folding and/or facilitate recognition of misfolded proteins for
dislocation. The mechanism used by US2/US11 may bypass this requirement
by recognizing the target soon after synthesis. An important
distinction between US2/US11-mediated degradation and the degradation
of misfolded proteins is the difference in kinetics; class I molecules
are rapidly degraded in US2/US11-expressing cells, whereas misfolded proteins may be retained in the ER for hours before being recognized and disposed of. Additionally, the ability of newly translated US2 to
target pre-existing heavy chains suggests that US2 recruits factors to
stimulate dislocation rather than triggering dislocation of heavy
chains while they are still in the Sec61 pore.
The difference between human astrocytoma microsomes and canine pancreas
microsomes to enable US2/US11 degradation indicates that
membrane-associated factors play a role in steps subsequent to
recognition of class I heavy chains. Whether this is the result of a
species-specific or tissue-specific factor is unclear at present.
Degradation of HLA molecules by vaccinia virus-expressed US2/US11 in a
porcine endothelial cell line has been observed (20). Vpu activity may
not be as dependent on membrane components because it directly
interacts with a component of an ubiquitin-protein isopeptide ligase
complex, Another observation from this work addresses the potential
heterogeneity of the ER. Canine pancreas is often used as a convenient and reliable source of microsomes that will support all manner of
ER-associated reactions. Generally, the results obtained by in
vitro translation using canine pancreas have compared well with
results obtained in living cells, where relevant. However, the exocrine
portion of the pancreas is a tissue that may well display highly
tissue-specific specializations and limitations. Not necessarily all
ER-dependent reactions may be catalyzed with equal
efficiency. Assembly and processing reactions for multimeric glycoproteins are supported well, but as we show here, not all aspects
of the dislocation reaction can be recapitulated as effectively in
canine microsomes.
The dislocation reaction observed in vitro is
specific. First, HLA-A2, but not HLA-C locus products (as well as HLA-E
and HLA-G)2 are dislocated. Second, a mutant of US2 that
lacks only 13 residues of the cytoplasmic tail is inactive. Third, we
observe no dislocation of the CD4 glycoprotein, also a member of the Ig
superfamily. Conversely, the HIV-encoded Vpu protein is capable of
extracting CD4 from the ER in vitro. Thus, CD4 is not
uniquely resistant to dislocation in vitro. The cytosol
dependence of ER-to-cytosol translocation and degradation is well
established (10, 15) and is observed in this system also.
Interestingly, the species dependence of the US2/US11-mediated process
is specific to the membranes but not to the cytosol, as cow liver and
rabbit cytosols both support the reaction.
Ubiquitinated class I heavy chains have been detected in US11 and US2
cells (41), and ubiquitination is required for dislocation of heavy
chains in US11 cells (42, 43). A similar requirement for ubiquitination
during US2-mediated dislocation is likely, as no differences in
cytosolic requirements between US2 and US11 have been noted thus far,
other than the presence of cytosolic domains of the US2 molecules (see
above) and class I molecules2 themselves. The functional
relevance of ubiquitinated heavy chains as an obligate intermediate in
the extraction process remains unclear. Ubiquitination of the
cytoplasmic tail of heavy chains is not required for their disposal, as
removal of all lysines from the tail prevents neither ubiquitination
nor degradation of the class I molecules (41). ER-resident class I
heavy chains remain resistant to protease in the absence of a
functional ubiquitin system, suggesting that they are not partially
dislocated (42, 43). Therefore, it remains unclear how luminal regions
are ubiquitinated and direct extraction. We have so far not observed
ubiquitinated heavy chains in this in vitro system. Because
we always observed less soluble intermediates than the amount degraded
from the microsomes, a fraction of the intermediates may be in a form
not readily observable by SDS-PAGE. Heterogeneous polyubiquitinated
species would fall into this category. It is estimated that less than
10-20% of heavy chains are ubiquitinated in semipermeable,
proteasome-inhibitor-treated US11 cells (41). Given the complexity of
the ubiquitin conjugation machinery and the diversity of ubiquitin
ligases, identification of the enzymes that are active during the
dislocation of heavy chains represents a major undertaking. An in
vitro system such as this one that recapitulates the dislocation
reaction may be helpful in such endeavors.
We thank Klaus Strebel and Ulrich
Schubert for providing reagents.
*
This work was supported in part by National Institutes of
Health Grants 5R37-AI33456 and P01 AI42257.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.
§
A Charles A. King research fellow.
Published, JBC Papers in Press, November 20, 2001, DOI 10.1074/jbc.M109765200
2
M. H. Furman, H. L. Ploegh, and D. Tortorella, unpublished data.
The abbreviations used are:
MHC, major
histocompatibility complex;
ER, endoplasmic reticulum;
Membrane-specific, Host-derived Factors Are Required for US2- and
US11-mediated Degradation of Major Histocompatibility Complex Class I
Molecules*
, and
ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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INTRODUCTION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-microglobulin (2m)) and an
antigenic peptide of 8-10 amino acids. Presentation of viral peptides
complexed with MHC class I molecules on the surface of infected cells
leads to recognition and destruction by cytotoxic T lymphocytes. Human
cytomegalovirus (HCMV) encodes two proteins, the products of the US2
and US11 genes, which interfere with MHC class I expression by inducing
rapid degradation of newly synthesized heavy chains (1-3). The
destruction of heavy chains by US2 and US11 presumably enables evasion
of cytotoxic T lymphocyte recognition early after infection or
reactivation from latency, allowing the virus more time to replicate
unnoticed. The degradation of MHC class I heavy chains by US2 and US11
is similar to the destruction of misfolded proteins that arises as a
consequence of imperfections in the normal translation and folding
reactions. US2 and US11 recognize the class I molecules within the
lumen of the ER and dislocate them into the cytosol where they are
destroyed by the proteasome (2, 3).
2m from
microsomes is suppressed by export of short (8-10 residues) peptides,
suggesting that such peptides and glycoproteins may both exit through
the Sec61 translocon (16, 17).
EXPERIMENTAL PROCEDURES
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2m, CD4, and HLA-C constructs used to generate mRNA
have been described previously (19, 20, 22). The US2-186 and US11-201
were amplified from full-length US2 and full-length US11 cDNA using
PCR. The 3' primer introduced a stop codon after amino acid 186 in US2
and 201 in US11 and contained a restriction site for subcloning into
pCDNA3.1 (Invitrogen). The pSP9-Vpu and pSP9-U2/6 plasmids encoding
Vpu and a phosphorylation mutant of Vpu, respectively, were kindly
provided by Klaus Strebel (23). Transfection of US2-186 and US11-201
was performed with LipofectAMINE (Invitrogen) as described
(24).
2m, has been described
(25). MG132 was synthesized in the laboratory as described (26).
antibody was prepared as described (27).
Cow liver cytosol was kindly provided by C. Shamu.
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DISCUSSION
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2m (Fig. 1B).
Immunoprecipitations with a rabbit polyclonal antiserum that recognizes
unfolded MHC class I heavy chains, or with W6/32, a monoclonal antibody
that specifically recognizes properly folded MHC class I molecules in
association with 2m (Fig. 1B,
lanes 2 and 3) showed that proper assembly of translated HLA-A2 and 2m readily occurred.
Therefore folded and unfolded species can be immunoprecipitated from
the translation reactions, indicating that these microsomes fully support protein folding.
View larger version (42K):
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Fig. 1.
Human astrocytoma microsomes support
insertion, signal sequence cleavage, and folding of MHC class I
molecules. A, HLA-A2 mRNA was translated in the
absence (lane 1) or in the presence (lanes 2 and
3) of human astrocytoma cell microsomes. For translations
with microsomes, microsomes were isolated by centrifugation and treated
with EH (lane 2) or mock-treated (lane 3).
Translation products were analyzed by SDS-PAGE (10%). Note that the
radiolabeled species in lane 1 with the slow mobility
corresponds to charged [35S]methionine tRNA in the
translation reactions. This species is not present in the microsomal
samples analyzed in lanes 2 and 3. B, HLA-A2 and 2m mRNA were co-translated
in the presence of astrocytoma microsomes. The microsomal pellet was
analyzed directly (lane 1) or immunoprecipitated with W6/32,
a monoclonal antibody that recognizes folded MHC class I heavy chains
in association with 2m (lane 2), or with a
polyclonal antiserum that recognizes free MHC class I heavy chains
(lane 3). Samples were analyzed by SDS-PAGE (12.5%).
View larger version (31K):
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Fig. 2.
Human astrocytoma microsomes support US2- and
US11-mediated degradation of MHC class I heavy chains.
A, HLA-A2 mRNA was translated into microsomes derived
from control (lanes 1-4), US2-transfected (lanes
5-8) or US11-transfected (lanes 9-12) U373 cells for
30 min. RNase was added to each reaction, and aliquots were withdrawn
at 0, 30, 60, and 90 min after the addition of RNase. Microsomes were
analyzed directly by SDS-PAGE (12.5%). Autoradiograms were quantitated
with ALPHAIMAGER software (Alpha Innotech, San Leandro, CA). Band
intensities were plotted as percentage of HLA-A2 remaining relative to
intensity at 0 min chase point. B, CD4 and HLA-A2 mRNAs
were co-translated into control microsomes (lanes 1-6) or
US2 microsomes (lanes 7-12). The degradation assay was
performed in the presence (lanes 4-6 and 10-12)
or absences (lanes 1-3 and 7-9) of 10 mM NEM added to chase cytosol. Reactions were incubated at
30 °C and sampled at 0, 30, and 60 min. Microsomes were recovered by
centrifugation and analyzed directly by SDS-PAGE (12.5%).
Autoradiograms were quantitated as in A, and HLA-A2 and CD4
band intensities were plotted as percentage remaining relative to time
0.
, an integral ER protein, shows that
supernatants did not contain ER-membrane proteins, and therefore the
release of HC into the supernatant cannot be attributed to a failure to
retrieve small microsomal vesicles (see below; Fig. 5A).
View larger version (50K):
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Fig. 3.
Deglycosylated intermediates are released
from US2 microsomes in the presence of proteasome inhibitors.
A, CD4 and HLA-A2 mRNAs were translated into US2
microsomes for 45 min, followed by the degradation assay in the
presence (lanes 3, 4, 7, and
8) or absence (lanes 1, 2,
5, and 6) of 50 µM MG132. Reactions
were sampled at 0 and 60 min. Microsomes (US2+
ms. pellet) (lanes 1-4) and supernatant
(lanes 5-8) were separated by centrifugation and analyzed
by SDS-PAGE (12.5%). B, to compare intermediates generated
in vitro with intermediates found in intact cells, US2 cells
preincubated with 50 µM MG132 for 60 min were
metabolically labeled with [35S]methionine for 5 min and
chased up to 20 min. MHC class I molecules were recovered from cell
lysates with anti-HC serum (lanes 1-4). HC
immunoprecipitates were digested with EH (lane 3) or PNG
(lane 4) prior to analysis. In vitro degradation
reactions were performed in US2 microsomes (as described above, with
HLA-A2 mRNA, chased in the presence of MG132), and heavy chains
were recovered from the microsomal fraction of the reactions
(M) at time 0 (lanes 5, 7, and
8) or supernatant fraction (S) recovered after 60 min (lane 6) with anti-HC serum. Microsomal HC
immunoprecipitates were digested with EH (lane 7) or PNG
(lane 8). All samples were analyzed by SDS-PAGE (10%) and
one-dimensional IEF. +CHO and 
CHO refer to the
presence or absence of an N-linked glycan, respectively; *
indicates the more acidic species generated by N-glycanase
and PNG digestion.
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Fig. 4.
In vitro degradation of MHC class
I molecules is allele-specific. CD4 mRNA was cotranslated with
HLA-A2 mRNA or HLA-C mRNA in the presence of US2 microsomes.
The degradation assay was performed in the presence of 50 µM MG132 (as in Fig. 3A). The microsomal
pellet (lanes 1-4) and supernatant fractions (lanes
5-8) were analyzed by SDS-PAGE (10%). Lanes
1-4 were exposed for 4 days, and lanes 5-8 were
exposed for 14 days.
(Fig. 5A). The
Sec61 polypeptide was observed only in pellet fractions, indicating
that the appearance of HLA-A2 in the supernatant fractions was not
because of an inability to retrieve membrane proteins. The luminal
domain of US2 is sufficient for interaction with HLA-A2 (33), and
therefore the lack of dislocation by US2-186 is not attributable to an
inability to bind to HLA-A2 molecules. Pulse-chase analysis of
proteasome-inhibitor treated US2-186 astrocytoma transfectants
confirmed the inactivity of this mutant also in intact cells, as no
conversion of HC from glycosylated to deglycosylated species was
observed (Fig. 5B, lanes 1 and
2). A corresponding set of experiments was done with US11.
When wild type US11 or US11-201, which lacks the putative cytosolic
domain, were translated into microsomes which contain radiolabeled
HLA-A2 and chased (as described for Fig. 5) (Fig. 6A),
deglycosylated intermediates accumulated in the supernatant from both US11- and US11-201-containing reactions (Fig. 6A,
lanes 7-12). These HLA-A2 intermediates migrated at
positions identical to intermediates released from US2-containing
microsomes.2 Similarly, MHC class I intermediates found in
US11- and US2-expressing cells are biochemically indistinguishable.
Significant loss of HLA-A2 was not apparent from microsome pellets
(Fig. 6A, lanes 1-6), as US11
degradation is less efficient in vitro and quantities of
in vitro translated US11 were less than the amounts present in microsomes derived from US11 transfectants. Again, the ability of
both molecules to degrade HLA-A2 was identical to the result observed
in intact US11/US11-201 transfectants (Fig. 6B). HC
immunoprecipitations indicated that rapid conversion to deglycosylated
HC was observed in both wild type US11 and US11-201 transfectants
(Fig. 6B, lanes 1-4). The apparent
increase of wild type US11/US11-201 over the chase was caused by
delayed cleavage of the US11 signal sequence (Fig. 6B,
lanes 1-4) (24). Both wild type US11 and
US11-201 are stable in intact cells and in microsomes. Therefore the
stability of these molecules does not correlate with their ability to
degrade MHC class I. We conclude that US2 requires its cytosolic domain for function whereas US11 does not, suggesting that the two
polypeptides utilize distinct mechanisms to facilitate degradation of
the MHC class I molecules.
View larger version (46K):
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Fig. 5.
US2 lacking the cytosolic domain is unable to
degrade MHC class I molecules. A, HLA-A2 mRNA was
translated into control astrocytoma microsomes for 30 min followed by
the addition of wild type (w.t.) US2 or US2-186 mRNA
and continued translation for 30 min. The degradation assay was
performed in the presence of 50 µM MG132 and sampled at
0, 30, and 60 min. Pellet (lanes 1-6) and supernatant
fractions (lanes 7-12) were analyzed by SDS-PAGE (12.5%)
(upper panel). Western blot analysis was
performed on a portion of each reaction using anti-Sec61 antiserum
(lower panel). B, astrocytoma cells
stably transfected with wild type US2 or US2-186 (pretreated with 50 µM MG132 for 60 min) were metabolically labeled with
[35S] methionine for 10 min and chased for up to 30 min.
MHC class I HC and US2 molecules were recovered from cell lysates with
anti-HC and anti-US2 serum and analyzed by SDS-PAGE (12.5%).
View larger version (48K):
[in a new window]
Fig. 6.
US11 lacking the cytosolic domain is able to
degrade MHC class I molecules. A, HLA-A2 mRNA was
translated into control microsomes for 30 min followed by the addition
of wild type US11 or US11-201 mRNA and continued translation for
30 min. The degradation assay was performed in the presence of MG132
and sampled at 0, 30, and 60 min. Pellet (lanes 1-6) and
supernatant (lanes 7-12) fractions were analyzed by
SDS-PAGE (12.5%). Lanes 1-6 were exposed for 15 h,
and lanes 7-12 were exposed for 72 h. B,
astrocytoma transfectants of US11 or US11-201 were starved for 60 min
in 50 µM MG132, metabolically pulsed with
[35S]methionine for 10 min, and chased with unlabeled
medium for 30 min. MHC class I and US11 molecules were recovered from
cell lysates with anti-HC and anti-US11 serum and analyzed by SDS-PAGE
(12.5%).
2m to support assembly of HLA-A2.
However, addition of human 2m mRNA to translation
reactions did not enable HLA-A2 degradation from canine membranes even
though complete assembly of the HLA-A2 molecules was readily
demonstrable.2
View larger version (33K):
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Fig. 7.
Canine microsomes do not support US2-mediated
degradation in vitro. HLA-A2 mRNA was
translated into control astrocytoma microsomes or canine pancreas
microsomes for 30 min. US2 mRNA was added and translations
continued for 30 min. Microsomes were pelleted and resuspended in RRL + RNase + MG132 and sampled at 0, 30, and 60 min. Astrocytoma pellet
fractions (lanes 1-3) and canine pellet fractions
(lanes 4-6) were analyzed directly by SDS-PAGE (12.5%).
Autoradiograms were quantitated and plotted as in Fig. 2.
View larger version (78K):
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Fig. 8.
Canine pancreas and human astrocytoma
microsomes support the degradation of CD4 by HIV Vpu in
vitro. CD4 mRNA was translated into canine or
astrocytoma microsomes for 30 min, followed by the addition of Vpu or
Vpu2/6 mRNA to each, and translations were continued for 30 min.
The degradation assay was performed in the presence of MG132 and
sampled at 0, 30, and 60 min. Pellet fractions from each time point
were analyzed by SDS-PAGE (12.5%).
View larger version (37K):
[in a new window]
Fig. 9.
Degradation of heavy chains in
vitro is dependent on cytosol. HLA-A2 mRNA was
translated into US2 microsomes for 45 min. The degradation assay was
performed with homogenization buffer (lanes 1, 2,
7, and 8), RRL (lanes 3, 4,
9, and 10), or cow liver cytosol (lanes
5, 6, 11, and 12) as chase
cytosol. (buffer and cow liver cytosols had an ATP-regenerating system
added; all included RNase and MG132). Reactions were sampled at 0 and
60 min, and pellet and supernatant fractions were analyzed by SDS-PAGE
(10%). Lanes 1-6 were exposed for 1 day, and lanes
7-12 were exposed for 4 days.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-TRCP, which is proposed to recruit Skp1 and direct
ubiquitination of CD4 (40). This model does not address the mechanism
by which CD4 is physically extracted from the ER, but it is notable
that no cytosolic intermediates of CD4 are detected in vitro
or in cells in the presence of proteasome inhibitors (14, 36). The
interactions between translocating heavy chains or CD4 and the Sec61
complex need to be examined. The soluble heavy chain intermediates
generated in vitro are found in a compartment in which no
Sec61 is detected (Fig. 5A). It appears that fully
glycosylated material is released from the microsomes. Therefore, a
fraction of the glycosylated membrane-associated heavy chains in US2
microsomes may be associated with translocation channels. The delayed
kinetics of dislocation in this system and the ability to manipulate
heavy chains after translation and before dislocation may allow us to
trap molecules in association with these channels in future experiments.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Harvard Medical
School, 200 Longwood Ave., Armenise Bldg., Rm. 137, Boston, MA 02115. Tel.: 617-432-4777; Fax: 617-432-4775; E-mail:
ploegh@hms.harvard.edu.
ABBREVIATIONS
2m, 2-microglobulin;
HCMV, human
cytomegalovirus;
NEM, N-ethylmaleimide;
HLA-A2, HLA-A*0201
or human leukocyte antigen subtype A2;
HC, MHC class I heavy chain;
EH, endoglycosidase H;
PNG, peptide N-glycanase;
RRL, rabbit
reticulocyte lysate;
IEF, isoelectric focusing;
HIV, human
immunodeficiency virus.
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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B. M. Baker and D. Tortorella Dislocation of an Endoplasmic Reticulum Membrane Glycoprotein Involves the Formation of Partially Dislocated Ubiquitinated Polypeptides J. Biol. Chem., September 14, 2007; 282(37): 26845 - 26856. [Abstract] [Full Text] [PDF]
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 B. Mueller, B. N. Lilley, and H. L. Ploegh SEL1L, the homologue of yeast Hrd3p, is involved in protein dislocation from the mammalian ER J. Cell Biol., October 23, 2006; 175(2): 261 - 270. [Abstract] [Full Text] [PDF]
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G. C. Hassink, M. T. Barel, S. B. Van Voorden, M. Kikkert, and E. J. Wiertz Ubiquitination of MHC Class I Heavy Chains Is Essential for Dislocation by Human Cytomegalovirus-encoded US2 but Not US11 J. Biol. Chem., October 6, 2006; 281(40): 30063 - 30071. [Abstract] [Full Text] [PDF]
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N. R. Hegde, M. S. Chevalier, T. W. Wisner, M. C. Denton, K. Shire, L. Frappier, and D. C. Johnson The Role of BiP in Endoplasmic Reticulum-associated Degradation of Major Histocompatibility Complex Class I Heavy Chain Induced by Cytomegalovirus Proteins J. Biol. Chem., July 28, 2006; 281(30): 20910 - 20919. [Abstract] [Full Text] [PDF]
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K. Oresic, V. Noriega, L. Andrews, and D. Tortorella A Structural Determinant of Human Cytomegalovirus US2 Dictates the Down-regulation of Class I Major Histocompatibility Molecules J. Biol. Chem., July 14, 2006; 281(28): 19395 - 19406. [Abstract] [Full Text] [PDF]
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 N. T. Pande, C. Powers, K. Ahn, and K. Fruh Rhesus Cytomegalovirus Contains Functional Homologues of US2, US3, US6, and US11 J. Virol., May 1, 2005; 79(9): 5786 - 5798. [Abstract] [Full Text] [PDF]
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 Y. Zou, W. Bresnahan, R. T. Taylor, and P. Stastny Effect of Human Cytomegalovirus on Expression of MHC Class I-Related Chains A J. Immunol., March 1, 2005; 174(5): 3098 - 3104. [Abstract] [Full Text] [PDF]
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 E. Fiebiger, D. Tortorella, M.-H. Jouvin, J.-P. Kinet, and H. L. Ploegh Cotranslational endoplasmic reticulum assembly of Fc{varepsilon}RI controls the formation of functional IgE-binding receptors J. Exp. Med., January 18, 2005; 201(2): 267 - 277. [Abstract] [Full Text] [PDF]
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M. T. Barel, M. Ressing, N. Pizzato, D. van Leeuwen, P. Le Bouteiller, F. Lenfant, and E. J. H. J. Wiertz Human Cytomegalovirus-Encoded US2 Differentially Affects Surface Expression of MHC Class I Locus Products and Targets Membrane-Bound, but Not Soluble HLA-G1 for Degradation J. Immunol., December 15, 2003; 171(12): 6757 - 6765. [Abstract] [Full Text] [PDF]
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M. H. Furman, J. Loureiro, H. L. Ploegh, and D. Tortorella Ubiquitinylation of the Cytosolic Domain of a Type I Membrane Protein Is Not Required to Initiate Its Dislocation from the Endoplasmic Reticulum J. Biol. Chem., September 12, 2003; 278(37): 34804 - 34811. [Abstract] [Full Text] [PDF]
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N. R. Hegde and D. C. Johnson Human Cytomegalovirus US2 Causes Similar Effects on Both Major Histocompatibility Complex Class I and II Proteins in Epithelial and Glial Cells J. Virol., September 1, 2003; 77(17): 9287 - 9294. [Abstract] [Full Text] [PDF]
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 B. N. Lilley, D. Tortorella, and H. L. Ploegh Dislocation of a Type I Membrane Protein Requires Interactions between Membrane-spanning Segments within the Lipid Bilayer Mol. Biol. Cell, September 1, 2003; 14(9): 3690 - 3698. [Abstract] [Full Text] [PDF]
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M. S. Chevalier and D. C. Johnson Human Cytomegalovirus US3 Chimeras Containing US2 Cytosolic Residues Acquire Major Histocompatibility Class I and II Protein Degradation Properties J. Virol., April 15, 2003; 77(8): 4731 - 4738. [Abstract] [Full Text] [PDF]
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B. Tirosh, M. H. Furman, D. Tortorella, and H. L. Ploegh Protein Unfolding Is Not a Prerequisite for Endoplasmic Reticulum-to-Cytosol Dislocation J. Biol. Chem., February 21, 2003; 278(9): 6664 - 6672. [Abstract] [Full Text] [PDF]
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M. H. Furman, N. Dey, D. Tortorella, and H. L. Ploegh The Human Cytomegalovirus US10 Gene Product Delays Trafficking of Major Histocompatibility Complex Class I Molecules J. Virol., October 11, 2002; 76(22): 11753 - 11756. [Abstract] [Full Text] [PDF]
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M. S. Chevalier, G. M. Daniels, and D. C. Johnson Binding of Human Cytomegalovirus US2 to Major Histocompatibility Complex Class I and II Proteins Is Not Sufficient for Their Degradation J. Virol., July 17, 2002; 76(16): 8265 - 8275. [Abstract] [Full Text] [PDF]
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R. S. Tirabassi and H. L. Ploegh The Human Cytomegalovirus US8 Glycoprotein Binds to Major Histocompatibility Complex Class I Products J. Virol., June 5, 2002; 76(13): 6832 - 6835. [Abstract] [Full Text] [PDF]
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B. E. Gewurz, H. L. Ploegh, and D. Tortorella US2, a Human Cytomegalovirus-encoded Type I Membrane Protein, Contains a Non-cleavable Amino-terminal Signal Peptide J. Biol. Chem., March 22, 2002; 277(13): 11306 - 11313. [Abstract] [Full Text] [PDF]
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