The EMBO Journal Vol. 21 No. 5 pp. 1041±1053, 2002
Visualization of the ER-to-cytosol dislocation
reaction of a type I membrane protein
Edda Fiebiger, Craig Story, Hidde L.Ploegh1
and Domenico Tortorella
Department of Pathology, Harvard Medical School, 200 Longwood
Avenue, Armenise Building, Boston, MA 02115, USA
1
Corresponding author
e-mail: [email protected]
The human cytomegalovirus gene products US2 and
US11 induce proteasomal degradation of MHC class I
heavy chains. We have generated an enhanced green
¯uorescent protein±class I heavy chain (EGFP±HC)
chimeric molecule to study its dislocation and degradation in US2- and US11-expressing cells. The
EGFP±HC fusion is stable in control cells, but is
degraded rapidly in US2- or US11-expressing cells.
Proteasome inhibitors induce in a time-dependent
manner the accumulation of EGFP±HC molecules
in US2- and US11-expressing cells, as assessed biochemically and by cyto¯uorimetry of intact cells.
Pulse±chase analysis and subcellular fractionation
show that EGFP±HC proteins are dislocated from the
endoplasmic reticulum and can be recovered as deglycosylated ¯uorescent intermediates in the cytosol.
These results raise the possibility that dislocation of
glycoproteins from the ER may not require their full
unfolding.
Keywords: dislocation/endoplasmic reticulum/HCMV
US2/HCMV US11/proteasomal degradation
Introduction
Misfolded proteins, a consequence of imperfections in
protein folding, are unavoidable by-products of protein
biosynthesis, regardless of the destination of the translation product (Schubert et al., 2000). Polypeptides that do
not acquire their proper conformation or quaternary
structure usually fail to reach their target and are destroyed
(Bonifacino and Weissman, 1998). The endoplasmic
reticulum (ER) is the point of insertion of most secretory
and membrane proteins and serves as an important station
for quality control (Ellgaard et al., 1999). In the ER, the
requirements for protein folding and sensing of the
misfolded state are not easily reconciled with the coincident degradation of mis®ts. The distinctions between a
nascent polypeptide that has yet to reach its ®nal
conformation, and a protein that has exhausted all attempts
at proper folding defy a simple description.
In living cells, folding and degradation of ER-inserted
proteins are carefully segregated in space. The ER
provides an environment that is conducive to protein
folding, usually catalyzed by chaperones that include
calnexin and calreticulin as well as oxidoreductases such
as PDI and ERp57 (Frand et al., 2000; High et al., 2000).
ã European Molecular Biology Organization
Proteolysis of unwanted polypeptides is segregated from
the folding environment by the ER membrane and can
occur in the cytosol. This arrangement requires the transfer
of misfolded proteins from the ER to the cytosol, a process
known as dislocation or retrograde translocation (Wiertz
et al., 1996a). Based on genetic experiments in yeast and
biochemical analysis in mammalian cells, the dislocation
reaction is carried out via the translocon or Sec61 complex
(Wiertz et al., 1996b; Pilon et al., 1997; Plemper et al.,
1997, 1998; Bebok et al., 1998; Zhou and Schekman,
1999). Some lumenal chaperones, such as calnexin, BIP
and PDI, have also been implicated in this reaction
(McCracken and Brodsky, 1996; Plemper et al., 1997;
Gillece et al., 1999; Nishikawa et al., 2001). In addition,
ER membrane proteins as well as cytosolic proteins are
involved in the degradation process (Hampton et al., 1996;
Hiller et al., 1996; Knop et al., 1996; Biederer et al., 1997;
Bordallo et al., 1998; Tiwari and Weissman, 2001; Zhang
et al., 2001). However, most of the proteins in the latter
category are associated with ubiquitylation events, usually
a necessary step for proteasomal degradation (Hirsch and
Ploegh, 2000; Pickart, 2000).
Virus-infected cells must avoid detection by the
immune system if they are to continue to produce
infectious virus (Tortorella et al., 2000). The Herpes
viruses are particularly adept at avoiding immune recognition. To achieve temporary invisibility, they have coopted the mechanisms of protein turnover to selectively
eliminate major histocompatibility complex (MHC) class I
molecules involved in antigen presentation (Tortorella
et al., 2000). The human cytomegalovirus (HCMV)encoded US2 and US11 proteins accelerate destruction of
MHC class I heavy chains (Wiertz et al., 1996a; Huppa
and Ploegh, 1997; Tortorella et al., 1998; Shamu et al.,
1999). By conferring speci®city for class I molecules and
by accelerating the rate constant of degradation, the
HCMV-encoded US2 and US11 proteins manage to
selectively reduce the half-life of the class I heavy chains
from hours to minutes (Wiertz et al., 1996a,b). Since the
presence of US2 or US11 is suf®cient to observe enhanced
degradation, these viral glycoproteins must somehow have
adopted the machinery used for turnover of ER proteins.
Does removal of glycans, when present, precede extraction
or is it con®ned to the cytosol, and is it always catalyzed in
the same manner? Is ubiquitin conjugation obligatorily
required, and if so, where does this happen, and in what
type of linkage is ubiquitin attached (Shamu et al., 1999)?
Is reduction of intra- and inter-chain disul®de bonds a
prerequisite for dislocation (Tortorella et al., 1998)? Can
folded proteins be extracted, or is complete unfolding
required? The answers to these questions will require a
detailed analysis of a number of substrates susceptible to
this mode of degradation.
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E.Fiebiger et al.
Fig. 1. The EGFP±HC reporter construct is a properly folded
EGFP±class I fusion protein. U373EGFP±HC cells were pulse-labeled
with [35S]methionine for 15 min and chased up to 90 min. Cells were
lysed in NP-40 lysis buffer and immunoprecipitated with W6/32, a
mAb that recognizes properly folded class I molecules only. The
immunoprecipitates were analyzed by SDS±PAGE (12.5%). The
endogenous class I heavy chains (HC) and EGFP±HC associated with
b2m were recovered from cell lysates (lanes 1±3). Half of the
immunoprecipitates were digested with Endo H (lanes 4±6). The
positions of migration of class I molecules that contain an N-linked
glycan (+CHO) or lack an N-linked glycan (±CHO) are indicated.
Molecular weight markers are indicated on the right in kDa.
Here we used an enhanced green ¯uorescent protein±
class I heavy chain fusion (EGFP±HC) as a model protein
to study US2- and US11-mediated dislocation and degradation. Characterization of EGFP±HC expressed in
control U373-MG cells that do not express US2 or US11
shows that this reporter construct largely behaves like its
endogenous counterpart. We show that the chimeric
molecules and the endogenous class I heavy chains are
dislocated and degraded in a similar manner in the
presence of HCMV US2 and US11. Fluorescence microscopy and spectroscopy data are consistent with the
possibility that dislocation does not require complete
unfolding of the dislocation substrate.
Results
The EGFP±HC chimeric molecule is inserted into
the membrane and folds properly
In the course of US2-/US11-induced class I degradation,
ER-resident class I molecules are dislocated into the
cytosol for degradation by the proteasome. To visualize
the dislocation of class I molecules in US2- and US11expressing cells, we generated a reporter construct comprised of an N-terminal signal sequence, followed by the
EGFP moiety fused to the N-terminus of the HLA-A2
heavy chain (EGFP±HC). Because GFP is a stable
molecule with a compact tertiary structure, the ¯uorescent
properties of this reporter should be helpful in addressing
the unfolding requirements of the EFGP/HC chimera prior
to degradation.
1042
For a simple GFP fusion protein, folding is inferred
from the emergence of green ¯uorescence. In the case of
the EGFP±HC fusion protein, we may use as an additional
criterion the acquisition of the W6/32 antibody epitope,
present on properly folded class I molecules only. We ®rst
examined the biosynthesis of EGFP±HC in a pulse±chase
experiment in U373-MG cells stably transfected with
EGFP±HC (U373EGFP±HC) (Figure 1). U373EGFP±HC cells
were metabolically labeled for 15 min and chased up to
90 min. The product encoded by the EGFP±HC fusion
construct was directed to the ER by the H2-Kb signal
sequence fused to the N-terminus of the chimera. The
ability of the fusion protein to fold and associate with b2microglobulin (b2m) was assessed by its reactivity with
the monoclonal antibody, W6/32 (Figure 1, lanes 1±6).
Both the fusion protein and the endogenous MHC class I
molecules acquired the W6/32 epitope, indicative of
correct folding and assembly, although the EGFP±HC is
less effective at acquiring the W6/32 epitope.
Does the EGFP±HC traf®c to the cell surface? We ®rst
addressed biochemically whether the EGFP±HC molecules exit the ER. We found that a population of
EGFP±HC molecules and endogenous class I molecules
that were precipitated with W6/32 are resistant to
endoglycosidase H (Endo H) treatment (Figure 1,
lanes 4±6). Resistance to Endo H is indicative of
N-linked glycan modi®cations that occur in the Golgi.
Therefore, the EGFP±HC molecules that associate with
b2m can exit the ER although we have not determined
whether EGFP±HC travels to the surface as ef®ciently as
its endogenous counterpart. We conclude that a signi®cant
population of EGFP±HC molecules is properly folded. For
the remainder of our experiments, we focus on the events
in the ER and cytosol.
Fluorescence microscopy was used to further analyze
the distribution of the fusion protein in intact cells. The
localization of the green ¯uorescent species, EGFP±HC,
relative to the red staining pattern derived from W6/32,
shows properly folded EGFP±HC at the cell surface and
intracellularly, presumably in the ER (Figure 2A). Partial
ER localization of EGFP±HC was con®rmed by staining
with antibodies against ER markers calnexin (Figure 2B)
and PDI (Figure 2C). These results establish that the
EGFP±HC is found throughout the secretory pathway and
at the cell surface.
The EGFP±HC fusion molecule is a substrate for
US2- and US11-mediated degradation
Is the EGFP±HC fusion molecule a substrate for the
HCMV-encoded US2 and US11 proteins? We generated
stable cotransfectants of EGFP±HC and US2 or US11 in
U373-MG cells (US2EGFP±HC and US11EGFP±HC). The
levels of expression of EGFP±HC, as assessed by pulse
labeling, were similar to those in U373EGFP±HC cells. The
fate of EGFP±HC in both cell lines was examined by
pulse±chase analysis (Figure 3). The EGFP±HC molecules
were recovered from SDS-treated cell lysates using a
polyclonal anti-GFP serum (aGFP). The class I heavy
chains were recovered using a polyclonal anti-heavy chain
serum (aHC) that recognizes unfolded class I heavy
chains. The immunoprecipitates were then analyzed by
SDS±PAGE. Only in US2EGFP±HC and US11EGFP±HC cells
did we observe a rapid loss of EGFP±HC and endogenous
Dislocation and degradation of EGFP±HC by US2/US11
Fig. 2. Intracellular distribution of EGFP±HC is similar to that of endogenous class I molecules. U373EGFP±HC were grown on chamber slides over
night, prior to ®xation, immunohistochemistry and confocal laser scanning microscopy. Double-positivity of the green ¯uorescent EGFP±HC molecule
with W6/32 corresponds to properly folded class I complexes that contain the chimeric molecule. These are found at the cell surface (A) and in the
ER. ER localization of EGFP±HC is demonstrated by staining with the ER markers calnexin (mAb AF8) (B) and PDI (anti-PDI) (C). For each
antibody the green EGFP±HC (left column), the respective second staining in red (middle column) and a merge image (right column) are shown, as
indicated for the individual panels.
HC over the chase period (Figure 3A, lanes 4±9). These
results are consistent with degradation of class I induced
by US2 or US11 (Tortorella et al., 1998). Given the
structural information available for the class I±US2
complex, we suggest that the ability of the EGFP±HC
fusion to be degraded in a US2-dependent manner is
further evidence of a correctly folded state for EGFP±HC
(Gewurz et al., 2001a). Even though EGFP±HC and
endogenous HC molecules are degraded with similar
kinetics in US2EGFP±HC and US11EGFP±HC cells
(Figure 3B), different amounts of endogenous HC are
recovered at the 0 chase period (Figure 3A, compare
lanes 13 and 16). This suggests that the presence of
EGFP±HC molecules in US2 cells affects the kinetics of
class I degradation, and slows degradation of endogenous
class I heavy chains.
A pulse±chase analysis of EGFP±HC in US2EGFP±HC
and US11EGFP±HC cells was then conducted in the presence
of the proteasome inhibitor, ZL3VS. The EGFP±HC and
endogenous heavy chains were recovered from cell lysates
using aHC serum (Figure 4). A precursor±product relationship between the EGFP±HC molecule and a faster
moving polypeptide was observed during the chase period
(Figure 4A, lanes 1±6). This intermediate is absent from
cells not exposed to proteasome inhibitors (Figure 3). The
presence of a biosynthetic intermediate of reduced molecular weight is consistent with the loss of the single
N-linked glycan (Figure 4A, lanes 7±9) present in both the
endogenous class I heavy chains and the fusion product.
Indeed, the intermediate is resistant to digestion with
puri®ed N-glycanase. The deglycosylated intermediate is
thus the product of an N-glycanase reaction, as con®rmed
by an isoelectric focusing experiment (Wiertz et al.,
1996a; data not shown). The rate of appearance of the
deglycosylated EGFP±HC intermediates in pulse±chase
experiments was only moderately delayed when compared
with the endogenous class I heavy chain intermediates.
The presence of a compact folded EGFP moiety is likely to
effect the rate of discharge of the fusion protein from the
ER. In the presence of the proteasome inhibitor, minor
quantities of additional polypeptides were detected over
the course of the chase period (* and **, Figure 4A).
Subcellular fractionation followed by immunoprecipitation shows that these polypeptides are cytosolic
(Figure 4B). The slower migrating polypeptide (*)
(Figure 4A) reacts with both aHC and aGFP, while the
faster migrating polypeptide (**) (Figure 4A) reacts only
with aHC (data not shown). Therefore, these fragments
correspond to heavy chain-derived digestion products
generated from cytosolic deglycosylated intermediates. At
steady state, these products are not detectable (Figure 4C).
In keeping with our earlier observations on the
endogenous MHC class I products, the deglycosylated
fusion product fractionated with the cytosolic compartment, unlike the transferrin receptor analyzed in the same
experiment (Figure 4B). The transferrin receptor was
recovered quantitatively in the particulate fraction obtained by differential centrifugation, whereas the deglycosylated MHC class I intermediate remained soluble
(Figure 4B, lanes 4±6). We conclude that the EGFP±HC
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E.Fiebiger et al.
fusion protein behaves like the endogenous class I heavy
chain as far as US2 and US11-mediated dislocation and
degradation are concerned.
Steady-state expression levels of EGFP±HC in
U373EGFP±HC and US11EGFP±HC cells were analyzed by
aGFP western blots (Figure 4C). Lysates prepared in
SDS from equal numbers of untreated (Figure 4C, lanes 1
and 2) and ZL3VS-treated (Figure 4C, lanes 3 and 4)
cells were subjected to SDS±PAGE and immunoblotting. U373EGFP±HC cells (Figure 4C, lanes 1 and 3)
express equivalent levels of EGFP±HC, independent of
the presence of the proteasome inhibitor. In contrast,
EGFP±HC is barely detectable in untreated US11EGFP±HC
cells (Figure 4C, lane 2), but appears as the deglycosylated
intermediate when proteasomal degradation is inhibited
(Figure 4C, lane 4). No other aGFP-reactive degradation
products were detected at steady state, in agreement with
the results from pulse±chase experiments.
Time-dependent accumulation of EGFP±HC in
US2EGFP±HC and US11EGFP±HC upon inhibition of
proteasomal degradation
Fig. 3. EGFP±HC is degraded in an US2- and US11-dependent
manner. (A) US11EGFP±HC and US2EGFP±HC cells were pulse-labeled
with [35S]methionine for 15 min and chased up to 90 min. Cells were
lysed in 1% SDS, then diluted to 0.07% SDS with NP-40 lysis mix
followed by immunoprecipitation with anti-GFP serum (aGFP)
(lanes 1±9) and anti-class I heavy chain serum (aHC) (lanes 10±18).
The immunoprecipitates were analyzed by SDS±PAGE (12.5%). The
positions of migration of the EGFP±HC and endogenous class I heavy
chain (HC) polypeptides are indicated. (B) The amount of EGFP±HC
and HC polypeptides recovered from U373EGFP±HC cells (black bar),
US2EGFP±HC cells (gray bar) and US11EGFP±HC cells (white bar) were
quanti®ed by Phosphoimager analysis. The remaining class I molecules
recovered at each chase point are given as a percentage of the class I
molecules recovered at the 0 chase time.
1044
We used the EGFP±HC reporter in FACS analysis,
¯uorimetric emission analysis and ¯uorescence microscopy experiments to explore the dislocation reaction in
intact cells. Co-expression of either US2 or US11 with
EGFP±HC essentially abolished all ¯uorescence, a further
indication of the ability of the fusion protein to serve as a
substrate for US11/US2-mediated degradation (Figure 5A).
We quanti®ed the appearance of green ¯uorescence
EGFP±HC in US2EGFP±HC and US11EGFP±HC cells by
FACS analysis when proteasome activity is blocked.
Treatment of the cells with ZL3VS induces the accumulation of EGFP±HC in a time-dependent manner in
US2EGFP±HC and US11EGFP±HC cells, with the half-maximal value attained after ~1 h (Figure 5A). Untransfected
U373-MG cells did not show any ¯uorescence accumulation (Figure 5A). In the absence of US11 and US2, the
levels of EGFP±HC fusion increased only slightly upon
proteasomal inhibition (Figure 5A). These results were
corroborated using ¯uorimetric emission analysis of
NP-40 lysates from U373-MG, U373EGFP±HC and
US11EGFP±HC cells (Figure 5B). The EGFP ¯uorescence
from lysates of US11EGFP±HC cells was signi®cantly
greater when cells had been treated with the proteasome
inhibitor, ZL3VS. As expected, there was no signi®cant
difference in the EGFP ¯uorescent observed from lysates
of ZL3VS treated and non-treated U373EGFP±HC cells.
Next, we examined the intracellular localization of
ZL3VS-induced EGFP±HC by ¯uorescence microscopy in
®xed cells. Cells grown on glass cover slips were
pretreated with ZL3VS for 3±5 h prior to immunohistochemistry. In US11EGFP±HC cells, we observed co-localization of green ¯uorescence with W6/32 (Figure 5C) or
HC10 (Figure 5C) reactive material. Co-localization of
EGFP±HC with W6/32 reactive material shows that
properly formed class I complexes include the
EGFP±HC fusion protein. A small population of W6/32
reactive EGFP±HC can be observed at the cell surface
(Figure 5C). However, this co-distribution is substantially
less than that seen for EGFP±HC and HC10 (Figure 5C,
compare the ®rst two rows). EGFP±HC co-localizes
perfectly with HC10, a monoclonal anti-class I reagent
Dislocation and degradation of EGFP±HC by US2/US11
that reacts with free (unfolded) class I heavy chains (Stam
et al., 1986). Direct examination of EGFP species with
anti-GFP serum (Seedorf et al., 1999), does not show a
signi®cant population of non-folded, non-¯uorescent
EGFP±HC molecules (Figure 5C, bottom row). Similar
results were obtained when these experiments were
performed in US2EGFP±HC cells (data not shown). We
conclude that the green ¯uorescent signal observed in our
cells is indeed derived from EGFP±HC chimeric molecules, fully consistent with the result from the biochemical analysis (Figure 3).
Cytosolic EGFP±HC does not form aggresomes
Cells respond to the production of high levels of certain
misfolded protein by transport of the aggregated material
to a perinuclear inclusion region and the formation of
structures referred to as aggresomes (Johnston et al.,
1998). Formation of these inclusion bodies was initially
described for the cystic ®brosis transmembrane conductance regulator (CFTR) (Johnston et al., 1998), presenilin
(Wigley et al., 1999) and a GFP-fusion protein (GarciaMata et al., 1999) as a general cellular response to
misfolded unassembled proteins. Aggresomes presumably
result when cytoplasmic degradation cannot clear the
misfolded material in a timely manner. Does inhibition of
proteasomal degradation induce such cytosolic structures
for EGFP±HC? Aggresomes have been de®ned by their
location close to the centrosome and an attendant redistribution of the intermediate ®lament protein vimentin into
a halo-like cage around the aggregated proteins. Antivimentin immunostaining was performed on U373EGFP±HC
and US11EGFP±HC cells (Figure 6). Vimentin ®laments of
U373EGFP±HC cells (Figure 6A) were compared with the
ZL3VS-treated US11EGFP±HC cells (Figure 6B). No difference in the distribution of vimentin was apparent. The
presence of US11 did not affect the vimentin pattern as
compared with control cells, regardless of the inclusion of
proteasome inhibitors. Vimentin distribution was equally
unaffected by ZL3VS treatment of U373EGFP±HC cells
(data not shown). Similar results were observed with
US2EGFP±HC cells. Furthermore, some of the perinuclear
EGFP±HC must be ER-resident (Figure 2, middle row;
Figure 7A). The lack of clearly de®ned vimentin cages and
the broad distribution of the accumulated EGFP±HC in
proteasome inhibitor-treated cells argues against the
presence of aggresomes in our experimental model
(Kopito, 2000).
Visualization of EGFP±HC dislocation
Direct visualization of the dislocation of proteins in
live cells has not been possible so far. We used the
Fig. 4. EGFP±HC is dislocated from the ER into the cytosol.
(A) US11EGFP±HC and US2EGFP±HC cells were pulse-labeled with
[35S]methionine for 15 min and chased up to 90 min in the presence
of the proteasome inhibitor, ZL3VS. Cells were lysed in 1% SDS,
then diluted to 0.07% SDS with NP-40 lysis mix followed by
immunoprecipitation with anti-class I heavy chain serum (aHC).
The immunoprecipitates were analyzed by SDS±PAGE (12.5%).
The EGFP±HC (lanes 1±9) and endogenous class I molecules (HC)
(lanes 10±15) were recovered from US2EGFP±HC and US11EGFP±HC cell
lysates. Some class I degradation intermediates (* and **) were present
in the US2 and US11 cell lysates. Half of the immunoprecipitates from
US11 cells were digested with N-glycanase (PNGase) (lanes 7±9
and 16±18). (B) US11EGFP±HC cells were pulse-labeled with
[35S]methionine for 15 min and chased up to 90 min in the presence
of the proteasome inhibitor, ZL3VS. Cells were homogenized and
subjected to fractionation as described in Materials and methods. The
EGFP±HC, endogenous HC (aHC; lanes 1±9) and transferrin receptor
(aTfr; lanes 10±18), were recovered from the whole-cell lysate (Whole
Cell), from the 100 000 g supernatant (100Kg-sup) and the 100 000 g
pellet (100Kg-pellet). The immunoprecipitates were analyzed by
SDS±PAGE (12.5%). (C) Steady-state levels of EGFP±HC in
U373EGFP±HC and US11EGFP±HC cells. Immunoblots of SDS lysates
from equal numbers of untreated (lanes 1 and 2) and ZL3VS-treated
(3 h; 50 mM; lanes 3 and 4) cells (0.25 3 106) were performed with a
polyclonal anti-GFP serum. U373EGFP±HC cells (lanes 1 and 3) express
equal levels of EGFP±HC, independent of the proteasome inhibitor
treatment. Inhibition of proteasomal degradation results in
accumulation of EGFP±HC in US11EGFP±HC cells (lanes 2 and 4)
mostly as the deglycosylated intermediate. Molecular weight markers
are indicated on the left in kDa.
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E.Fiebiger et al.
Fig. 5. Inhibition of proteasomal degradation in US11EGFP±HC and US2EGFP±HC cells induces accumulation of ¯uorescent EGFP±HC. (A) Flow
cytometric quanti®cation of the induction of green reporter ¯uorescence. Cells were incubated with ZL3VS (50 mM) for the indicated time periods.
The mean ¯uorescence intensity was measured by FACS: untransfected U373 cells (open squares), U373EGFP±HC (closed squares), US11EGFP±HC (open
circles) and US2EGFP±HC (open diamonds). The results shown are representative of three experiments. (B) Fluorimetric emission quanti®cation of
NP-40 lysates from U373 cells, U373EGFP±HC and US11EGFP±HC cells. Fluorescent units [490 nm(excitation)/515 nm(emission)] of EGFP±HC were
measured from cell lysates of cells treated for 5 h with Zl3VS (black bars) or without Zl3VS (white bars). (C) Immuno¯uorescence of US11EGFP±HC
cells. Immunostaining followed by confocal laser scanning microscopy was carried out as described in Materials and methods. For each antibody the
green EGFP±HC (left column), the respective second staining in red (middle column) and a merge image (right column) are shown, as indicated for
the individual panels. Double-positivity of EGFP±HC with W6/32 shows the localization and the co-localization of properly folded class I complexes
and their presence at the cell surface (top row). Almost complete co-localization of EGFP±HC with HC10, the monoclonal antibody reactive with
class I independent of its folding, shows that this ¯uorescence is indeed derived from a green class I fusion protein (middle row). Staining with the
aGFP reagent depicts no additive GFP-tagged population that for some reason (unfolding) failed to acquire green ¯uorescence (anti-GFP; bottom row).
EGFP±HC fusion to address two types of question. First,
where exactly do cells accumulate EGFP±HC induced by
inhibition of proteasomal degradation? Secondly, can we
follow the fate of a distinct EGFP±HC subpopulation
within a cell? We ®rst examined the intracellular distribution of the EGFP±HC in detail by confocal laser
scanning microscopy. US11EGFP±HC cells treated with
ZL3VS were stained for the ER markers concanavalin A
(Figure 7A, top row), calnexin (Figure 7A, middle row)
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and PDI (Figure 7A, bottom row) to identify the ERresident fraction of EGFP±HC. In both US11EGFP±HC and
US2EGFP±HC (data not shown) cells, a signi®cant population of EGFP±HC co-localizes with these ER markers
(Figure 7A), demonstrating that properly folded ¯uorescent EGFP±HC molecules are detected in the ER.
We next designed an experiment that would allow the
accumulation of EGFP±HC in the ER by blockade of
proteasomal proteolysis, followed by removal of inhibition
Dislocation and degradation of EGFP±HC by US2/US11
Fig. 6. Inhibition of proteasomal degradation in US11EGFP±HC cells does not induce the formation of aggresomes. Immunostaining with anti-vimentin
mAb V9 and confocal laser scanning microscopy was performed to study the distribution of vimentin ®laments in untreated U373EGFP±HC cells (A)
and US11EGFP±HC cells treated with ZL3VS (50 mM; 3 h) (B). No signi®cant difference between the vimentin distribution was detected for either cell
type. The green EGFP±HC (left column), the anti-vimentin staining in red (middle column) and a merge image (right column) are shown for each
panel.
and ensuing dislocation and degradation of EGFP±HC. For
this purpose we used the reversible inhibitor ZL3-aldehyde
(MG132) (Lee and Goldberg, 1996): proteasomal activity
resumes upon removal of the inhibitor. By blocking de
novo protein synthesis by the addition of cycloheximide
(CHX), the egress of the pre-existing, ER-localized
population of EGFP±HC can be followed during the
dislocation process without the confounding effect of its
de novo synthesis.
In Figure 7B (top row), EGFP±HC molecules colocalized with ER markers in MG132-treated US11EGFP±HC
cells are represented as yellow pixels. This yellow signal
corresponds to the merged image of the green EGFP±HC
and in red the ER staining with concanavalin A, anticalnexin antibody or anti-PDI antiserum, respectively
(Figure 7B). The fraction of immunostaining that does not
colocalize is rendered on a gray scale. Examination of
US11EGFP±HC cells 1.5 h after the addition of CHX sharply
reduces colocalization in cells stained with the respective
ER markers (Figure 7B, lower panels). This implies that
the initially ER-localized population of EGFP±HC has
now left the ER. Likewise, co-localization of W6/32 and
EGFP±HC is no longer observed in ER compartments of
such treated cells (data not shown). These results demonstrate that a properly folded ¯uorescent EGFP±HC species
can be targeted for dislocation and subsequent degradation
in an US11-dependent manner.
To examine the degradation kinetics of ¯uorescent
EGFP±HC molecules, we stopped the degradation of
EGFP±HC in US11EGFP±HC cells by blocking proteasomal
function with MG132 for 3 h (Figure 8A). The inhibitor
was then removed and the mean ¯uorescence intensity of
accumulated EGFP±HC was monitored over a 3 h time
period in the presence of the protein synthesis inhibitor
CHX. FACS analysis shows that the initial accumulation
of green ¯uorescence is comparable to that seen in cells
treated with the irreversible inhibitor ZL3VS (Figure 5A).
Within 3 h after the washout of the proteasome inhibitor,
this accumulation phase is followed by a decline of signal
to background levels (Figure 8A). CHX treatment of cells
that did not express US11 failed to show this rapid loss of
green ¯uorescence (Figure 8A). Similar results were
obtained from US2EGFP±HC cells (data not shown).
Judged from the decay of EGFP signal in FACS analysis,
we determined that a period of 1 h after imposition of the
CHX block was optimal to obtain samples for subcellular
fractionation and ¯uorimetric emission analysis of the
cytosolic fractions. We detected ¯uorescent EGFP±HC
molecules in the cytosol of ZL3VS-treated US11EGFP±HC
cells and US2EGFP±HC cells (Figure 8B). After a 1 h CHX
chase, there was an increase in recovery of cytosolic
¯uorescent EGFP±HC molecules. In the absence of
proteasome inhibitor, US2EGFP±HC and US11EGFP±HC
cells showed very little ¯uorescence in their cytosolic
fractions (data not shown). We conclude that EGFP±HC
molecules can exist in the cytosol as ¯uorescent species
and accumulate if dislocation is allowed to proceed in the
presence of proteasome inhibitor. EGFP ¯uorescence
implies a properly folded state at least for the EGFP
moiety. This raises the question whether EGFP±HC needs
to unfold for dislocation to proceed, and if so, whether an
unfolded EGFP±HC molecule could re-acquire ¯uorescent
properties.
Hysteresis in unfolding of EGFP±HC and
EGFP molecules
We addressed the stability of EGFP±HC chimeric molecules by examining in vitro their ¯uorescent properties in
the presence of a denaturant. All ¯uorescence of
EGFP±HC molecules from U373EGFP±HC cells, ZL3VStreated US2EGFP±HC and ZL3VS-treated US11EGFP±HC
cells is extinguished in a guanidine hydrochloride
(GuHCl)-concentration dependent manner (Figure 8C).
The EGFP±HC molecules are denatured at similar
concentrations of GuHCl as free, cytosolic EGFP molecules in U373-MG cells (U373EGFP) (Figure 8C).
Can an unfolded EGFP±HC chimera refold into
a ¯uorescent species? EGFP±HC molecules from
U373EGFP±HC cells denatured by addition of 5.0 M
GuHCl (Figure 8D; open square) do not re-acquire green
¯uorescence upon dilution to 0.3 M GuHCl (closed
square) (Figure 8D). In contrast, when cytosolically
expressed, free EGFP molecules were subjected to the
same treatment [5.0 M GuHCl (open circle) to 0.3 M
1047
E.Fiebiger et al.
Fig. 7. Direct visualization of EGFP±HC dislocation with intact cells.
(A) A signi®cant subpopulation of EGFP±HC is detected in the
endoplasmic reticulum. Immunostaining followed by confocal laser
scanning microscopy was used to analyze subcellular distribution of the
accumulated EGFP±HC. US11EGFP±HC cells were incubated with
ZL3VS (50 mM) for 3 h, ®xed, and stained. ER localization of
EGFP±HC is demonstrated by co-staining with the ER markers
concanavalin A (top row), calnexin (anti-calnexin, mAb AF8; middle
row) and PDI (anti-PDI; bottom row), as indicated. For each stain the
green EGFP±HC (left column), the respective second staining in red
(middle column) and a merge image (right column) are shown. Similar
results were obtained in four independent experiments. (B) EGFP±HC
traf®cs from the ER to the cytosol. The co-localization program from
Bio-Rad was used for evaluation of the confocal laser scanning
microscopy. The program depicts colocalization green and red signals
from the merge picture (data not shown) and reproduces them as
yellow pixels. Single color signals are shown in gray. Stainings were
performed as in (A). In the presence of inhibitor, a large proportion of
EGFP±HC colocalizes with concanavalin A (top row, left image),
calnexin (top row, middle image) and PDI (top row, right image).
Following wash-out of the proteasome inhibitor and a 90 min chase in
the presence of cycloheximide (CHX) (bottom row), EGFP±HC is no
longer detected in concanavalin A- (bottom row, left image), calnexin(bottom row, middle image) and PDI-positive (bottom row, right
image) compartments, yet is readily visible in the cytosol (see also
Figure 8). Similar results were obtained in four independent
experiments.
GuHCl (closed circle)], recovery of EGFP ¯uorescence
was essentially complete (Figure 8D). The EGFP moiety
itself can thus refold into a ¯uorescent species after
denaturation, in agreement with published data (Battistutta
et al., 2000; Fukuda et al., 2000). Taken together, the
results imply that the ¯uorescent EGFP±HC molecules
detected in the cytosol do not necessarily arise from an
unfolded cytosolic EGFP±HC species that refolds and
requires ¯uorescent properties, but instead may originate
1048
from EGFP±HC molecules dislocated in a ¯uorescent
state.
Discussion
To investigate ER dislocation and proteasomal degradation, we use a reporter protein composed of an EGFPmoiety fused at its C-terminus to the HLA-A2 heavy chain
(EGFP±HC) preceded by a cleavable N-terminal signal
Dislocation and degradation of EGFP±HC by US2/US11
Fig. 8. Dislocated EGFP±HC is a cytosolic green ¯uorescent protein and does not, once unfolded, reacquire ¯uorescence. (A) FACS analysis shows
that accumulated EGFP±HC induced by proteasome inhibition can be degraded. U373EGFP±HC (closed squares) and US11EGFP±HC (open circles) were
incubated for 3 h with the reversible proteasome inhibitor MG132 (50 mM). After removal of MG132, the accumulated EGFP±HC was chased for an
additional 3 h in the presence of cycloheximide (CHX). Similar results were obtained in three independent experiments. (B) Fluorimetric emission
quanti®cation of EGFP±HC from cytosolic fractions of U373EGFP±HC, US2EGFP±HC and US11EGFP±HC cells treated with the proteasome inhibitor,
ZL3VS (white and black bars). The ¯uorescence emitted by cytosolic EGFP±HC molecules was examined for US2EGFP±HC and US11EGFP±HC cells
after addition of proteasome inhibitor and cycloheximide (CHX) for 1 h (black bars). Cytosolic fractions were prepared as described in Materials and
methods. The ¯uorescence emission of the cytosolic population of EGFP±HC was plotted as a percentage of signal calculated for unfractionated
homogenates. (C) Fluorimetric quanti®cation of the EGFP±HC molecules after exposure to the denaturant guanidine hydrochloride (GuHCl). The
¯uorescence emission of lysates from U373EGFP±HC (open squares), ZL3VS-treated US2EGFP±HC (open diamonds), ZL3VS-treated US11EGFP±HC
(asterisks) and U373EGFP (open circles) cells incubated with increasing concentrations of GuHCl were examined. The percentage of EGFP±HC
¯uorescence intensity was plotted against GuHCl concentration. The mean ¯uorescence of EGFP±HC at 0 M GuHCl was used as 100% ¯uorescence.
(D) Renaturation of denatured EGFP±HC and EGFP molecules. EGFP±HC molecules from U373EGFP±HC cells and EGFP from U373EGFP cells were
denatured with 5.0 M GuHCl (open square and circle, respectively) followed by dilution with PBS to 0.3 M GuHCl (closed square and circle,
respectively). The percentage of green ¯uorescent signal was plotted against GuHCl concentration. The mean green ¯uorescence signal at 0 M GuHCl
was used as 100% ¯uorescence.
sequence. We show that this protein folds properly, as
assessed by the acquisition of the appropriate antibody
epitopes and the display of green ¯uorescence.
Furthermore, we demonstrate that the EGFP±HC, like
endogenous class I heavy chains, is readily recognized by
the US2 and US11 proteins. This interaction leads to
degradation of the fusion protein, a reaction accompanied
by the accumulation of the diagnostic deglycosylation
intermediate when proteasomal proteolysis is inhibited,
both in pulse±chase analysis and at steady state. In
addition to endogenous class I heavy chains, the only
intermediate(s) of the breakdown reaction are the cytosolic
deglycosylated forms of the EGFP±HC. Cytochemical and
biochemical analysis at steady state failed to reveal the
presence of obvious cleavage products or of a free EGFPmoiety that could have confounded further morphological
analysis.
Examination of EGFP±HC in US11EGFP±HC and
US2EGFP±HC by ¯uorescence microscopy showed an
almost complete lack of ¯uorescence, unless proteolysis
was inhibited by the proteasome inhibitors ZL3VS or
MG132. Block of degradation with either proteasomal
inhibitor results in the accumulation of the dislocation
substrate. This accumulation is not con®ned only to the
cytosol, but extends to the ER as well. We interpret the
latter result as the consequence of feedback inhibition: the
accumulation of an intermediate in the cytosol affects the
reaction(s) immediately upstream, which include(s) the
removal from the ER (dislocation) itself. This accumulation is reversible once proteasome inhibitors are removed.
When degradation resumes, extraction of ¯uorescent
material formerly arrested in the ER can now occur. This
is best visualized when de novo protein synthesis is
blocked, and no new material is inserted into the ER. In
1049
E.Fiebiger et al.
other words, EGFP±HC is inserted into the ER where it
folds, but can still be effectively dislocated thereafter and
degraded by proteasomes.
Vimentin ®laments do not collapse upon cytosolic
accumulation of EGFP±HC in US11EGFP±HC and
US2EGFP±HC cells. This ®nding, in combination with the
broad distribution of green ¯uorescence throughout the
cell, argues against the formation of aggresomes for the
EGFP±HC substrate, de®ned as inclusion bodies surrounded by a vimentin cage (Garcia-Mata et al., 1999;
Kopito, 2000). Furthermore, EGFP±HC accumulation is
reversible and EGFP±HC remains in a cellular compartment that is readily extractable with NP-40 (this work).
The formation of aggresomes, as pointed out by Kopito
(2000), might be a highly substrate- and cell type-speci®c
process, with certain cell types more prone to aggresome
formation than others. Likewise, the type I membrane
protein EGFP±HC might behave differently when compared with multi membrane-spanning proteins, such as
CFTR or presenilin (12 and eight transmembrane domains,
respectively), commonly used to document aggresome
formation (Johnston et al., 1998).
How is EGFP±HC dislocated from the ER into the
cytosol in a manner consistent with its ¯uorescent
properties in both the ER and the cytosol? As the ®rst of
two possible scenarios we propose the following: the ERresident EGFP±HC molecule is initially denatured by ER
chaperones, followed by its dislocation into the cytosol as
a completely or partially unfolded molecule. We have
observed that the disul®de bonds that stabilize the
conformation of class I heavy chains are reduced prior to
loss of the single N-linked glycan, brought about by the
action of a cytosolic N-glycanase (Tortorella et al., 1998).
A catalyst speci®cally involved in this redox reaction
remains to be identi®ed, and perhaps is not even required
when a disul®de-bonded protein reaches the cytosol. In the
presence of proteasome inhibitors, the EGFP±HC molecule would then refold partially to accumulate as a
deglycosylated, ¯uorescent species. In other words, the
folded EGFP±HC molecule would unfold in the ER and at
least the EGFP moiety refold in the cytosol. What detracts
from this possibility is the fact that the GFP molecule is
rather resistant to denaturation. In addition, only in the
presence of 6 M GuHCl, but not 6 M urea, is GFP
denatured and loses its ¯uorescent properties (Weber-Ban
et al., 1999). Complete unfolding of the EGFP±HC
molecule also requires a high concentration of GuHCl
(5.0 M) (Figure 8C). A fully unfolded free EGFP molecule
can refold in vitro upon the removal of the denaturants, as
judged by the re-emergence of ¯uorescent properties
of GFP (Figure 8D) (Battistutta et al., 2000), but the
EGFP±HC fusion does not (Figure 8D). The extent to
which such unfolding occurs in living cells is not easily
assessed. However, the in vitro renaturation of partially or
completely unfolded EGFP±HC molecules to a ¯uorescent
species was not observed upon dilution of denaturant
(Figure 8D). Clearly, the attachment of the class I moiety
affects the folding properties of EGFP in the fusion
protein: free EGFP refolds upon removal of denaturant,
whereas EGFP fused to the HC does not. We suggest that
in living cells the refolding of the unfolded EGFP±HC
fusion protein might be likewise compromised. If correct,
our data would be consistent with dislocation of
1050
EGFP±HC reporter in a conformation that retains at least
the fold for ¯uorescent EGFP. In the presence of the ClpA
unfoldase, ¯uorescent GFP is unfolded in vitro but only if
equipped with a degradation signal (Weber-Ban et al.,
1999). A 20% decrease in GFP ¯uorescence was observed
in the presence of ClpA. It could be argued that the
attached heavy chain moiety is in fact a degradation signal
for EGFP±HC that acts in a manner similar to the ssrA
sequence used to target GFP to the ClpA unfoldase
(Weber-Ban et al., 1999). However, the fact remains that
the ubiquitin conjugation apparatus and the proteasome
cannot access the EGFP±HC protein until it is exposed to
the cytosol.
An additional complication of a model that requires
unfolding of the EGFP±HC complex prior to dislocation is
the following: the three-dimensional structure of the
US2±class I complex has been determined for the lumenal
domains (Gewurz et al., 2001a). This complex is quite
stable and survives native gel electrophoresis, as well as
several chromatography steps used in the puri®cation of
the complex (Gewurz et al., 2001b). If unfolding were a
prerequisite for dislocation, the unfolding reaction would
presumably occur in the same compartment where the
complex between US2 and US11 was formed in the ®rst
place, the ER. The remarkable ef®cacy of the entire
dislocation process, perhaps not more than 2 min between
completion of the polypeptide chain and the dislocation
event (Wiertz et al., 1996a,b), raises important questions
as to the nature of this presumed unfolding and the
catalysts involved, given the stability of the US2±class I
complex. In summary, one model consistent with our data
postulates unfolding immediately prior to escape of
EGFP±HC from the ER, followed by re-acquisition of
¯uorescent properties of the EGFP moiety upon arrival in
the cytosol. The inhibition of the proteasome is essential
for this reaction to yield EGFP±HC in detectable amounts.
An alternative model for dislocation of the EGFP±HC
molecule through the ER membrane is that it is dislocated
as a folded structure, a suggestion not entirely without
precedent and perhaps more consistent with the remarkable stability of the US2±class I complex. Nonetheless, we
cannot formally exclude the possibility that the unfolded
GFP moiety attached to the class I heavy chain may refold
upon arrival in the cytosol. However, we note the
following examples that may serve as precedents for the
transport of folded molecules across a membrane.
Unfolding of cytosolic peroxisomal precursor proteins is
not required for import into the peroxisome (Subramani
et al., 2000). Cytosolic proteins stabilized by crosslinking
or by pharmacological means were ef®ciently translocated
into peroxisomes and glycosomes respectively (Walton
et al., 1995; Hausler et al., 1996). In fact, multimeric
proteins are also transported into the peroxisomal matrix.
In yeast, a trimer of chloramphenicol acetyltransferase
containing a peroxisomal targeting sequence as well as a
dimer of peroxisomal thiolase are imported into peroxisomes as oligomeric complexes (Glover et al., 1994;
McNew and Goodman, 1994). Such import sets a precedent for the ability of folded or even multimeric proteins to
cross membranes when assisted by the appropriate partners. In view of the shared ancestry of the peroxisomal
membrane and the ER (Titorenko and Rachubinski, 2001),
the requirement for unfolding, in contrast to the transport
Dislocation and degradation of EGFP±HC by US2/US11
of a multimeric protein complex, is an issue that deserves
further study in the context of the dislocation reaction. If
transport of a multimeric protein complex across the
peroxisomal membrane is possible (Smith and Schnell,
2001), there is in principle no reason why such transport
should be denied to proteins destined to leave the ER for
the cytosol.
The Sec61 complex is believed to be the conduit via
which proteins are dislocated from the ER. The inner
diameter of the translocon pore has been estimated to
Ê in its inactive state (Menetret et al.,
¯uctuate between 15 A
Ê during protein
2000; Beckmann et al., 2001) to 40±60 A
translocation (Johnson and van Waes, 1999). Can GFP
pass through the pore of the Sec61 complex as a folded
Ê long with a diameter of
molecule? GFP is a cylinder 42 A
Ê
24 A (Ormo et al., 1996). The estimated pore size of the
translocon during its active state could therefore allow the
passage of the folded GFP molecule. Even if the pore size
Ê when fully active,
of the translocon is maximally 60 A
further rearrangements of the translocon subunits could
produce different results. Relatively modest adjustments in
the angle of connexin subunits within the membrane can
create a completely closed or an open state for gap
junctions (Simon and Goodenough, 1998). In a similar
event, re-arrangements of translocon subunits or an
adjustment of their stoichiometry initiated from within
the ER could modulate pore size.
Materials and methods
Cell lines and antibodies
U373-MG astrocytoma cells transfected with the US2 or US11 cDNA
were prepared as described (Jones et al., 1995; Kim et al., 1995), cells
were maintained in DME medium as described (Rehm et al., 2001).
EGFP±HC (U373EGFP±HC), US2-EGFP±HC (US2EGFP±HC), US11EGFP±HC (US11EGFP±HC) and EGFP (U373EGFP) cells were maintained
in DME medium supplemented with 5% FCS, 5% calf serum and 0.5 mg/
ml of Geneticin (Gibco, Fredrick, MD). The aHC serum was generated
by immunizing rabbits with the bacterially expressed lumenal fragment of
HLA-A2 and HLA-B27 heavy chain (Tortorella et al., 1998). aGFP was
generated by immunizing rabbits with the bacterially expressed GFP
(Seedorf et al., 1999). mAbs AF8 (Schreiber et al., 1994), HC10 (Stam
et al., 1986), W6/32 (Parham et al., 1979) and anti-transferrin receptor
(van de Rijn et al., 1983) were used as described. Anti-vimentin mAb V9
(Sigma, St Louis, MO) was used for the characterization of aggresomes
(Heath et al., 2001). Polyclonal anti-PDI serum was generated with
bacterially expressed human PDI.
Metabolic labeling of cells, pulse±chase analysis,
immunoprecipitation and immunoblotting
Cells were detached by trypsin treatment, followed by starvation in
methionine-/cysteine-free DME for 45 min at 37°C. Cells were
metabolically labeled with 500 mCi/ml of [35S]methionine/cysteine
(1200 Ci/mM; NEN-Dupont, Boston, MA) at 37°C for the times
indicated. Pulse±chase experiments, cell lysis and immunoprecipitations
were performed as described previously (Rehm et al., 2001). The
immunoprecipitates were analyzed by SDS±PAGE followed by
¯uorography (Ploegh, 1995). aGFP immunoblots were performed with
SDS lysates of inhibitor-treated or untreated U373EGFP±HC and
US11EGFP±HC cells as described (Fiebiger et al., 2001).
cDNA, transfection and Endo H digestion
The EGFP±HC molecule was cloned with the murine class I molecule
H2-Kb signal sequence at its N-terminal end and the class I heavy chain
HLA-A2 allele (amino acids 25±365) at the C-terminal end. The H2-Kb
signal sequence directs the chimeric molecule to the ER. The chimeric
construct was inserted into pCDNA 3.1 (Invitrogen, Carlsbad, CA)
(Swann et al., 2001). The EGFP±HC construct was transfected into
U373-MG, US2 and US11 cells using a liposome-mediated transfection
protocol (4 mg of DNA/20 ml of lipofectamine/10 cm dish; Lipo-
fectamine, Gibco, Fredrick, MD). The cytosolic EGFP construct was
cloned into pCDNA 3.1 and transfected into U373-MG cells as above.
Endo H and N-glycanase (New England Biolabs, Beverly, MA)
digestions were performed as described by the manufacturer.
Subcellular fractionation
Subcellular fractionation of metabolically labeled US11EGFP±HC cells was
performed as described (Tortorella et al., 1998).
Fluorimetric analysis and folding studies
Whole-cell NP-40 lysates and 100 K supernatants obtained by subcellular
fractionation of U373EGFP±HC, US2EGFP±HC, US11EGFP±HC and U373EGFP
cells, respectively, were analyzed for the ¯uorescence emission of
EGFP±HC chimeric molecules using a ISSâ KS Multifrequency Phase
Fluorimeter (ISSâ Fluorescence Analytical Instrumentation, Champaign,
IL) at 490 nm excitation and 515 nm emission. The lysates were
incubated with increasing concentrations of guanidine hydrochloride
(GuHCl) for at least 1 h at 25°C. Refolding experiments were performed
by the addition of PBS to GuHCl containing samples and incubate at 25°C
for 1 h. Results were shown directly as ¯uorescent units or as a percentage
of the signal from unfractionated homogenates. For the folding studies,
the ¯uorescent signal obtained from samples not treated with GuHCl were
taken as 100%.
Flow cytometry analysis
Quantitative ¯ow cytometry analysis of accumulation of the green
¯uorescent reporter construct after the inhibition of proteasomal
degradation in living cells was performed by FACS (FACS Calibur;
Beckton Dickinson, Mountain View, CA) supported by CellQuest
software (Beckton Dickinson). Mean ¯uorescence intensity correlates
to the amount of intracellular EGFP±HC and is monitored under the
conditions indicated.
Immunostaining and confocal microscopy
Immuno¯uorescence experiments were performed essentially as described (Driessen et al., 1999) with minor modi®cations. Cells were
allowed to attach to slides overnight before inhibitor incubation with
[ZL3VS (Wiertz et al., 1996b) or the peptide aldehyde inhibitor
carboxybenzyl-leucyl-leucyl-leucinal, MG132, (Lee and Goldberg, 1996);
50 mM ®nal from a DMSO stock]. DMSO was used as solvent control.
Where indicated, protein de novo synthesis was inhibited by the addition
of cycloheximide (CHX) (50 mg/ml, ®nal). After ®xation with 3.7%
paraformaldehyde for 20 min at room temperature immunohistochemistry
was performed in a 0.5% saponin/3% BSA/PBS solution to de®ne the
intracellular location of the EGFP±HC fusion protein. Alexa Fluorâ 594conjugated concanavalin A (Molecular Probes, Eugene, OR), anticalnexin (mAb A8) and anti-PDI (polyclonal serum, rabbit anti-human)
staining were performed to de®ne ER structures. mAbs W6/32 and HC10
were used to de®ne localization of properly folded and total MHC class I
reactivity, respectively. aGFP serum depicts the EGFP moiety. Antimouse Alexa Fluorâ 568 (Molecular Probes) and anti-rabbit CyÔ3
(Jackson Laboratory, Bar Harbor, ME) were used as the ¯uorescent
probe. Further analysis were performed with a Bio-Rad MRC1024
confocal laser scanning microscope. The merge images were analyzed for
the presence of EGFP±HC in the ER with the colocalization program
from Bio-Rad (Driessen et al., 1999).
Acknowledgements
We thank Rebecca Tirabassi for generating the U373EGFP cell line. We
thank Kristina Cunningham (Laboratory of R.J.Collier) for her assistance
with the ¯uorescence measurements. During the course of this study E.F.
was supported by a Max Kade Fellowship of the Max Kade Foundation,
NY and the Austrian Academy of Sciences. D.T. is a Charles King Trust
(Boston, MA) fellow. This study was supported by the NIH grant 5R37AI33456.
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Received August 31, 2001; revised and accepted January 14, 2002
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