2217
Journal of Cell Science 111, 2217-2226 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
JCS4513
Removal and degradation of the free MHC class II β chain in the endoplasmic
reticulum requires proteasomes and is accelerated by BFA
Simone Dusseljee1, Richard Wubbolts1, Desiree Verwoerd1, Abraham Tulp1, Hans Janssen2, Jero Calafat2
and Jacques Neefjes1,*
Divisions of 1Tumor Biology and 2Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The
Netherlands
*Author for correspondence (e-mail: [email protected])
Accepted 21 May; published on WWW 15 July 1998
SUMMARY
We have studied the degradation of the free major
histocompatibility complex (MHC) class II β subunit in the
ER. Domain swapping experiments demonstrate that both
the intra- and extracellular domain determine the rate of
degradation. Recently, it has been shown that some ERretained proteins are exported from the ER by the
translocon followed by deglycosylation and degradation in
the cytosol by proteasomes. Degradation of the β chain
follows a different route. The proteasome is involved but
inhibition of the proteasome by lactacystin does not result
in deglycosylation and export to the cytosol. Instead, the β
chain is retained in the ER implying that extraction of the
β chain from the ER membrane requires proteasome
activity. Surprisingly, brefeldin A accelerates the
degradation of the β chain by the proteasome. This suggests
that various processes outside the ER are involved in ERdegradation. The ER is the site from where misfolded class
II β chains enter a proteasome-dependent degradation
pathway.
INTRODUCTION
et al., 1997). However, in vitro degradation of the unglycosylated
pro-alpha factor in yeast derived vesicles requires cytosol and
ATP (McCracken and Brodsky, 1996).
Recent studies have implicated the proteasome in the
degradation of an ER-retained CFTR mutant. This mutant
polypeptide becomes ubiquitinated, a modification that targets
proteins to proteasomes. Indeed, degradation was inhibited by
proteasome inhibitors (Jensen et al., 1995; Ward et al., 1995).
Similarly, the degradation of an ER-luminal carboxypeptidase
mutant required proteasome activity (Hiller et al., 1996). How
the proteasome may mediate ER-degradation became clear by
analysing the degradation of newly synthesized MHC class I
molecules by the human cytomegalo virus (HCMV) proteins
US11 and US2 (Wiertz et al., 1996a,b). In this system, class I
H-chains are, very rapidly after translation, released from the
translocon to enter the cytosol where they are deglycosylated
and degraded by cytosolic proteasomes. Analysis of yeast
mutants has more definitively shown the involvement of
translocon subunits in ER degradation (Pilon et al., 1997).
Whether this scheme is generally applicable for ER
degradation, is unclear.
The half-life of various (unassembled) polypeptides that are
retained in the ER varies for reasons poorly understood.
Bonifacino et al. (1990) have shown that the transmembrane
regions of the TCR α and β chain contain charged residues that
influence the rate of ER degradation. The type of N-linked glycan
present on an ER-retained molecule can also affect its half-life
Multimeric surface or secreted proteins are assembled in the
endoplasmic reticulum (ER). Proper assembly is usually required
for release from the ER, which implies the existence of a ‘quality
control mechanism’ (Hurtley and Helenius, 1989). Failure to
assemble properly typically results in retention in the ER and cisGolgi reticulum and ultimately in degradation (Klausner and
Sitia, 1990; Bonifacino, 1996). Degradation of free subunits or
partially assembled complexes in an early biosynthetic
compartment has been studied for a number of proteins including
T cell receptor (TCR) subunits (Bonifacino et al., 1990; Chen et
al., 1988; Lippincott-Schwartz et al., 1988; Wileman et al., 1993;
Huppa and Ploegh, 1997), MHC class I (Hsu et al., 1991; Raposo
et al., 1995; Wiertz et al., 1996a,b) and II subunits (Koppelman
and Cresswell, 1990; Cotner and Pious, 1995), the asialoglycoprotein receptor (Wikström and Lodish, 1991) and the
cystic fibrosis transmembrane conductance regulator CFTR
(Jensen et al., 1995; Ward et al., 1995). Degradation of ERretained proteins is independent of lysosomal activity, continues
in the presence of Brefeldin A (BFA) and is inhibited at
temperatures below 15°C. Degradation of the TCR α chain
occurs in ER-derived vesicles and streptolysin O-permeabilized
cells without requirement for ATP. This suggested that the ER
contains all the components required for the proteolysis of the
TCR α chain (Stafford and Bonifacino, 1991). At least some ER
chaperones appear to be rate limiting in ER-degradation (Plemper
Key words: MHC class II molecule, ER-degradation, Brefeldin A,
Proteasome, Antigen presentation
2218 S. Dusseljee and others
since persistence of Glc3Man9 carbohydrates stabilizes the TCR
α chain (Kearse et al., 1994) and VSV-G (Hammond et al., 1994).
Here, we have followed the rate of degradation of the free
MHC class II subunits in the ER. Free α and β subunits of MHC
class II human leucocyte antigen (HLA)-DR1 molecules and
chimeric constructs of these chains containing the
transmembrane and cytoplasmic domain of HLA-B27 were
analysed. The free class II chains are associated with
immunoglobulin binding protein (BiP; Nijenhuis and Neefjes,
1994). We show that both the intra- and extracellular domains
of these chains determine their half-life. The degradation of the
free HLA-DR1 β chain was studied in detail. The majority of
β chains is located in the ER with only a minor amount present
in the cis-Golgi reticulum (cGR). Degradation of the β chain is
strongly accelerated by BFA, which coincides with trimming,
but not removal, of the attached N-linked glycan. Inhibiting the
proteasome with the specific inhibitor lactacystin (Fenteany et
al., 1995; Dick et al., 1996) protected the β chain from
degradation. Also BFA-accelerated degradation was inhibited
by lactacystin, although trimming of the carbohydrate chain
continued. When cells were cultured for prolonged periods with
lactacystin, the β chain (as well as the GFP-tagged β chain) does
not accumulate in the cytosol or cGR but in the ER, suggesting
that extraction of the β chain from the ER membrane is coupled
to proteasomal activity, possibly by the small portion of
proteasomes that we found cofractionating with ER vesicles.
Our data suggest that degradation of the β chain is regulated
by processes in the ER and Golgi. However, the removal from
the ER-membrane and degradation is finally executed by
cytosolic proteasomes that may be associated with ER
membranes. A coupled ER membrane extraction/proteasomal
degradation process suggests a second mechanism for ERdegradation in which the free β chain does not have to enter
the cytosol before degradation by proteasomes.
MATERIALS AND METHODS
Antibodies and reagents
The following antibodies were used: rabbit anti-human class II α
chain serum and rabbit anti-human class II β chain serum (Neefjes et
al., 1990), anti-class II mAb Tü36 (Shaw et al., 1985), rabbit antihuman p53 serum ERGIC-53 (Schweitzer et al., 1991), mouse antihuman proteasome α subunit iota mAb IB5 (Organon Teknica, Oss,
The Netherlands), mouse anti-human calnexin mAb (Hochstenbach et
al., 1992), mouse anti-human class I H-chain mAb HC-10 (Stam et
al., 1986). Lactacystin was obtained from E. J. Corey, Harvard
University and was stored as a 10 mM solution in sterile water at 4°C.
Brefeldin A was obtained from Sigma and used at a concentration of
5 µg/ml.
Cell lines and transfectants
Human embryonal kidney 293 cells (CRL 1573, American Type
Culture Collection, Rockville, MD) were transfected with cDNAs
encoding the class II α or β chain or the chimeric products consisting
of an extracellular α or β chain and transmembrane and cytoplasmic
region of HLA-B27, as described (Nijenhuis and Neefjes, 1994). 293
cells transfected with the β-GFP chimera, with GFP attached to the
cytoplasmic tail of the β chain, were made and selected as for the
other single chain transfectants. The construction of the β-GFP
chimera has been described (Wubbolts et al., 1996). The cells were
maintained in DMEM supplemented with 7.5% FCS and 500 µg/ml
G418.
293 cells transfected with the class II α, β and ∆15Ii chains were
generated as described (Nijenhuis et al., 1994) and maintained in DMEM
supplemented with 7.5% FCS, 500 µg/ml G418 and 1 µM ouabain.
Confocal analysis and immuno-electronmicroscopy
Living 293 cells expressing the β-GFP chimera were analysed in a
tissue culture chamber at 37°C, using a Bio-Rad MRC 600 CLSM
(Wubbolts et al., 1996).
For electronmicroscopy, 293 cells transfected with the class II β
chain were fixed in a mixture of 4% (w/v) paraformaldehyde and 0.5%
(v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) and
embedded in 10% (w/v) gelatin in PBS. Alternatively, transfectants
were cultured for 20 hours in the presence of 10 µM lactacystin prior
to fixation. Ultrathin cryosections were incubated with the rabbit anticlass II β chain serum (diluted 1:100) followed by gold-conjugated
goat anti-rabbit IgG. Both incubations were for 1 hour at room
temperature. In double labeling experiments involving rabbit anti-β
chain serum and rabbit anti-p53 serum (at a dilution of 1:50), the
procedure was as follows: the first incubation was with rabbit anti-β
chain serum followed by gold-conjugated goat anti-rabbit IgG. Crossreactivity was blocked by treatment with 1% glutaraldehyde for 10
minutes. The second antibody (anti-p53 serum) was added followed
again by goat anti-rabbit IgG coupled to gold. After immunolabeling,
the cryosections were embedded in a mixture of methylcellulose and
uranyl acetate. Sections were examined with an electron microscope
(CM10; Philips Electronic Instruments, Eindhoven, The Netherlands).
Biochemistry
Pulse chase analysis
The transfectants were cultured in separate dishes for each timepoint.
Each dish was labeled for 30 minutes with a mixture of
[35S]methionine and cysteine (NEN) after starvation for 0.5 hour in
Cys/Met-free RPMI 1640 supplemented with 10% FCS. Further
incorporation of radioactivity was stopped by addition of nonradioactive methionine and cysteine to a final concentration of 1 mM,
followed by chasing the cells for the times indicated. The cells were
lysed in Triton X-100-containing lysis buffer (50 mM Tris-HCl pH 7.4,
5 mM MgCl2 and 0.5% (v/v) Triton X-100). The respective proteins
were isolated from equal amounts of TCA-precipitable radioactivity
and analyzed by SDS-PAGE. Gels were dried and exposed to Kodak
XAR5 film. 1-deoxymannojirimycin (dMM) or castanospermine (1
mM) were present during the biosynthetic labeling whereas BFA (5
µg/ml; Sigma) or cycloheximide (200 µM; Sigma) were added to the
cells at the beginning of the chase. Pulse chase experiments in the
presence of lactacystin were performed by preculturing the cells in the
presence of 10 µM lactacystin for 18 hours. 10 µM lactacystin was
also present during the pulse and the chase.
Endo H treatments were performed on immunoprecipitates by
incubating overnight at 37°C with 2 mU Endoglycosidase H
(Boehringer), according to the manufacturer’s instructions.
Subcellular fractionation
Separation by DGE
293 cells transfected with the class II β chain were incubated with HRP
for 30 minutes to label the early and late endosomes. Post-nuclear
supernatant (PNS) was generated as described and separated, after a
mild trypsin digestion, in an electric field by DGE (Tulp et al., 1996).
The position of the endosomes and lysosomes was determined by
measuring the HRP- and β-hexosaminidase activity respectively (Tulp
et al., 1996). Proteins were concentrated by TCA-precipitation,
separated by SDS-PAGE and transferred to nitrocellulose.
Subsequently, the position of the class II β chain, the proteasome α
subunit iota and calnexin was determined with specific antibodies.
Separation by density gradient centrifugation
PNS of 293 cells transfected with the class II β chain was adjusted to
2.0 M sucrose and layered at the bottom of a linear 0.6 to 1.8 M
ER-degradation 2219
sucrose gradient. The gradient was centrifuged at 20,000 rpm (70,000
g) for 18 hours in a SW27 rotor and fractions were collected from the
top to the bottom. Proteins were precipitated by TCA, separated by
SDS-PAGE and transferred to nitrocellulose. The position of the class
II β chain, the proteasome α subunit iota, the class I H-chain and
calnexin was visualized with specific antibodies.
RESULTS
Intra- and extracellular portions of the ER-retained
class II subunits determine the rate of degradation
In order to follow ER degradation of free protein subunits, we
transfected human embryonic kidney 293 cells with either the
HLA-DR1 α or β chain. The same cells were also transfected
with chimeric molecules composed of the extracellular domain
of the α or β chain coupled to the transmembrane and
cytoplasmic region of class I HLA-B27 molecules. We have
previously shown that these chains associate with the ER-resident
protein BiP in 293 cells (Nijenhuis and Neefjes, 1994). The
stability of ER-retained molecules appears to vary considerably
and it is unclear whether the intracellular, extracellular or
transmembrane (TM) segments of these molecules determine the
stability in the ER. We therefore determined the half-life of the
free class II subunits and their chimeric counterparts by pulse
labeling the respective transfectants, followed by isolation of the
free subunits and analysis by SDS-PAGE (Fig. 1). The class II α
chain has a half-life similar to the class II β chain. However, when
the TM and cytoplasmic region of these chains are exchanged by
the same region of class I HLA-B27, the half-life of the β/B27
chimera is markedly increased compared to the β chain whereas
the half-life of the α/B27 chimera is decreased compared to the
α chain. Thus the TM and/or cytoplasmic tail affect the half-life
of ER-retained molecules, but not in an unidirectional fashion.
The α/B27 chimera differs from that of the β/B27 chimera only
in its extracellular portion. Since the half-life of these chimers
differs considerably, we conclude that the extracellular or luminal
part of ER-retained molecules influences their half-life as well.
We studied the intracellular location and degradation of the
free class II β chain in further detail. Transfectants expressing
the free class II β chain were analyzed by immunoelectronmicroscopy and stained for the β chain and the cisGolgi reticulum marker p53 (Schweitzer et al., 1991) (Fig. 2).
The free β chain is exclusively found in the ER and is not
detected at the cell surface, in Golgi stacks, or in other
intracellular compartments. Whereas the vast majority of the
free β chains is associated with the ER, double labeling with
anti-p53 antibodies revealed that a minor portion of the β
chains is located in the cGR (Fig. 2).
The role of Golgi processes in ER-degradation
BiP cycles between the ER and cis-Golgi and the BiPassociated β chain may cycle in concert. The β chains mainly
reside in the ER and are observed at only very low quantities
in the cis-Golgi reticulum (Fig. 2). This distribution may be a
consequence of BiP binding. To analyse whether entry of the
β chain in post-ER compartments had an effect on degradation,
transfectants were biosynthetically labeled and chased in the
presence or absence of BFA, followed by immunoprecipitation
of the β chain and analysis by SDS-PAGE (Fig. 3A, lower
panel). BFA-treatment induces fusion of the Golgi with the ER
(Lippincott-Schwartz et al., 1989). In the presence of BFA, the
β chain is rapidly trimmed to a smaller molecular mass product
followed by a dramatic acceleration in degradation. Similar
findings are observed for the other single chain transfectants
used in this study (not shown). To analyse whether the β chain
was deglycosylated prior to degradation, cells were labeled for
30 minutes and chased for the times indicated in the presence
or absence of BFA (Fig. 3B). The β chain was
immunoprecipitated and one half was subjected to EndoH
digestion. Again, BFA induces rapid degradation of the β chain
which coincides with an increased mobility by SDS-PAGE.
Apparently, the carbohydrate chain is trimmed but can still be
removed by Endo H treatment. Glucosidase and mannosidase
I activities are involved in the BFA-induced trimming of the β
chain, since trimming can be inhibited by the glucosidase I
inhibitor castanospermine and partially by the mannosidase I
inhibitor dMM (data not shown). However, glycosidase action
is not a prerequisite for BFA-induced degradation, since
accelerated degradation of the free β chain by BFA is not
affected by these inhibitors (data not shown).
To exclude the possibility that BFA does result in non-specific
ER proteolysis, we biosynthetically labelled 293 cells expressing
properly folded class II molecules and chased the cells in the
presence or absence of BFA. MHC class II complexes were
recovered and analyzed by SDS-PAGE (Fig. 3A, upper panel).
Class II molecules are rapidly transported in control cells as they
migrate more slowly on SDS-gels. BFA treatment results in
trimming of the glycans of the class II complex in a similar
fashion to that of the free chains. However, the class II complex
remains stable over the period analysed. Note that the amount
Fig. 1. The rate of ER-degradation depends
on intra- and extracellular protein segments.
The half-lives of the α chain, the α chain
with the TM and cytoplasmic tail of HLAB27 (α/B27), the β chain and the β chain
with the TM and cytoplasmic tail of HLAB27 (β/B27) were analysed by
biosynthetically labeling the respective
transfectants for 0.5 hour followed by
culture for the times indicated above the
figure. Free chains were
immunoprecipitated from equal amounts of
TCA-precipitable radioactivity and
analyzed by 12% SDS-PAGE. The TM/cytoplasmic tail of HLA-B27 decreases the half-life of the α chain and increases that of the β chain.
Because free α/B27 and β/B27 chains differ in half-life, the extracellular region also affects stability.
2220 S. Dusseljee and others
Fig. 2. The free class II β chain is localised in the ER and the cGR. Sections of 293 cells transfected with the β chain were stained for the β
chain (5 nm gold in a and 10 nm gold in b) and for the cis-Golgi reticulum marker p53 (10 nm gold in a and 5 nm gold in b). p53 is observed in
vesicles and tubules (shown by arrow) and localized on one side of the Golgi. The β chain is mainly located in the ER and only small amounts
are visualised in p53 positive vesicles (arrows in a). G, Golgi; n, nucleus. Bars, 100 nm.
ER-degradation 2221
A
C
D
B
Fig. 3. The effect of BFA and lactacystin on the degradation of the β chain. (A) BFA selectively accelerates degradation of misfolded β chain.
293 cells transfected with the β chain or with class II αβ∆15 complexes were biosynthetically labeled for 30 minutes and chased in the
presence or absence of BFA for the times indicated. The class II complex and the free β chain were analysed as in Fig. 1. Whereas the class II
complex remains stable, the β chain is rapidly degraded in the presence of BFA. Note that BFA causes a small reduction in the molecular mass
of both the class II complex and the β chain. (B) The β chains remain EndoH sensitive in control and BFA-treated cells. The β chain
transfectant was labeled for 30 minutes and chased in the presence or absence of BFA for the times indicated. The β chain was isolated from
equal amounts of incorporated radioactivity and one half of the isolate was subjected to EndoH digestion as indicated. The samples were
analysed by 12% SDS-PAGE. The β chain remains EndoH sensitive even when degradation is accelerated by BFA. However, the glycan is still
modified by BFA action. (C) Degradation of the β chain involves the proteasome. The β chain transfectant was precultured in the presence or
absence of the proteasome inhibitor lactacystin followed by labeling for 30 minutes and culture for the times indicated in the absence or
presence of lactacystin. The β chain was immunoprecipitated from equal amounts of TCA-precipitable radioactivity and one half was subjected
to Endo H digestion. Analysis was by 12% SDS-PAGE. Lactacystin inhibits degradation of the β chain which remains EndoH sensitive.
(D) Lactacystin inhibits BFA-accelerated degradation. The β chain transfectant was precultured in the presence or absence of the proteasome
inhibitor lactacystin, labeled for 30 minutes and cultured for the times indicated in the presence or absence of BFA and/or lactacystin. The β
chain was immunoprecipitated and analysed as in Fig. 1. BFA accelerates degradation which is inhibited by lactacystin. Note that trimming of
the N-linked glycan continues when BFA-accelerated degradation is inhibited by lactacystin.
of radioactive ∆15Ii associated with class II increases with time
in the BFA-treated samples due to the continuing association
with class II after the pulse labeling. Normally ∆15Ii is degraded
in a post-ER step (in the lysosomes) (Nijenhuis et al., 1994).
The β chain is degraded by the proteasome
To study the effect of the proteasome on degradation of the β
chain, the transfectants were cultured in the presence or absence
of lactacystin, a specific inhibitor of the proteasome (Fenteany
et al., 1995; Dick et al., 1996). Cells were pulse labeled for 30
minutes and chased for the times indicated. The β chain was
isolated and one half was treated with Endo H (Fig. 3C).
Degradation of the β chain is clearly inhibited by lactacystin.
Moreover, the β chain remains a substrate for EndoH and is
therefore not deglycosylated. Note the small shift in molecular
mass at 8 hours as the result of carbohydrate trimming.
Apparently the β chain is still a substrate to glycosidases.
Similar data were obtained for the free class II α chain (data
not shown). A similar experiment was performed to determine
whether BFA-induced acceleration of degradation was also
dependent on proteasome activity (Fig. 3D). The transfectants
were cultured in the presence or absence of lactacystin followed
by a 30 minute pulse and then chased in the presence or absence
of BFA, as indicated. The lactacystin-pretreated cells were
pulsed and chased in the presence of lactacystin. BFAaccelerated degradation was completely abolished by
lactacystin. Note that trimming of the β chain carbohydrate
continued in the cells treated by BFA and lactacystin. However,
2222 S. Dusseljee and others
Fig. 4. Subcellular fractionation
A
of proteasomes.
(A) Fractionation by charge.
Transfectants expressing the β
chain were cultured with HRP
for 30 minutes to label
endosomes. PNS was generated
(lane PNS) and mildly treated
with trypsin (lane DGE) before
separation in an electric field by
DGE. Fractions were analysed
for HRP- and β-hexosaminidase
activity, which was plotted as
the percentage of maximal
activity. In addition, proteins
were collected from the
fractions, separated by 12%
SDS-PAGE and transferred to
nitrocellulose. The position of
B
the β chain, calnexin and the
proteasome α subunit iota was
determined with specific
antibodies. Trypsin treatment
affects the molecular mass of
calnexin (compare lane ‘PNS’
to lane ‘DGE’) but not the
proteasome iota subunit or the β
chain. The β chain and calnexin
comigrate in higher fractions
than the endosomes and
lysosomes (HRP- and β-hex
activity, respectively). Whereas
most proteasomes migrate at the
position of the free protein pool
(fraction 44-54), a small
fraction migrates at a position
overlapping the β chain and
calnexin. (B) Fractionation by
density. PNS from the β
transfectant adjusted to 2.0 M sucrose was loaded at the bottom of a linear 0.6 to 1.8 M sucrose gradient. After centrifugation, the lightest
fractions were first collected. Proteins were collected by TCA-precipitation, separated by 12% SDS-PAGE and transferred to nitrocellulose. The
position of the class II β chain (panel β), the proteasome iota subunit (panel IB5), the class I H-chain (panel HC10) and calnexin was
determined. Most of the proteasome migrates at the highest density fractions 45-47 (the free proteins) but a small amount migrates at a lower
density and comigrates with the β chain and with calnexin. A small peak of calnexin is seen at a low density (fraction 11-17). Most class I
molecules migrate in fractions lighter than the β chain.
the complete glycan chain was not removed since the β chain
remained susceptable to EndoH (data not shown).
Intracellular location of proteasomes
If proteasomes are involved in degradation of the β chain, the
above experiments suggest that at least some proteasomes
should be associated with ER membranes. To study this, PNS
was generated from the β chain transfectant that had been
cultured with HRP for 30 minutes to load the endocytic
pathway. Vesicles were separated in an electric field using
density gradient electrophoresis (DGE; Tulp et al., 1996). To
obtain a homogeneous vesicle preparation, PNS was briefly
incubated with trypsin. This treatment affected the molecular
mass of the ER-marker calnexin whereas the β chain as well
as the proteasome subunit iota were unchanged (Fig. 4A;
compare lane PNS and DGE). The β-hexosaminidase as well
as the HRP-activity was measured in the uneven samples to
position the lysosomes (fractions 25-39) and endosomes
(fractions 10-37), respectively. The position of the β chain,
calnexin and the proteasome was determined by western
blotting with specific antibodies. The class II β chain
comigrated with calnexin in fractions 30-44 (the ER). Most
proteasomes are found in fractions 44-54, at the expected
position of free proteins. A small fraction of proteasomes is
observed in fractions 26-42, which overlaps with the position
of the ER, although it is more broadly distributed. Since
endosomes and lysosomes peak around fraction 30, a position
where hardly any proteasomes are observed, these should be
vesicles other than the endosomes, lysosomes or the ER.
Density gradient electrophoresis (DGE) does not readily
separate ER, Golgi and plasma membranes. To further
determine the identity of proteasome-associated vesicles, PNS
of the β chain transfectants was layered at the bottom of a linear
0.6 to 2.0 M sucrose gradient and vesicles were separated by
density. Free protein will remain at the highest density whereas
vesicles have a lower density and will migrate to lighter
ER-degradation 2223
Fig. 5. ER-retention of the β chain and the
GFP-tagged β chain upon inhibition of the
proteasome. (a,b) 293 cells transfected with
the class II β chain were incubated with
lactacystin for 20 hours before fixation and
processing for immuno-electronmicroscopy.
Sections were labeled with antibodies
against the class II β chain. Abundant gold
labeling in ER sheets (arrows) is observed
whereas the cytosol, nucleus (n) and plasma
membrane are devoid of gold labeling. Bars,
300 nm. (c) 293 cells were stably transfected
with a chimeric construct in which GFP is
attached to the cytoplasmic tail of the class II
β chain. The upper panel shows a pulse
chase labeling of cells cultured in the
absence or presence of lactacystin, as
indicated. Cells were chased for the
timepoints indicated and the isolated β-GFP
chain was analysed by SDS-PAGE.
Proteasome inhibition prevents degradation
and deglycosylation of β-GFP. Bottom panel:
living transfectants were then analysed by
confocal microscopy after overnight culture
in the presence or absence of lactacystin.
Lactacystin-treatment does not alter the
distribution of the β-GFP chain in the
nuclear envelope and membranous sheets.
c
2224 S. Dusseljee and others
fractions. The ER will migrate at a higher density than the
Golgi, plasma membrane and endosomes, due to the association
with ribosomes. The positions of the β chain, the proteasome α
subunit iota (IB5), class I molecules (HC-10; a marker for the
plasma membrane and the ER) and calnexin (ER and cGR) are
determined by western blotting (Fig. 4B). The class II β chain
comigrates with a small fraction of proteasomes and the
majority of calnexin. Most proteasomes, however, remain at the
site of loading at the highest density (fraction 45,47). The ER
is well separated from the plasma membrane (panel HC-10) and
the cGR (as marked by the small amount of calnexin detected
in a second peak between fraction 11 and 17). Proteasomes are
not detected at this position. Thus a small fraction of
proteasomes cofractionates with the ER when separated by two
different physical parameters. However, proteasomes do not
exclusively associate with ER vesicles, as is apparent in the
DGE separation, and the majority migrates as free protein.
The site of degradation of ER-retained free β and βGFP
Many intracellular locations have been proposed as the site
where ER substrates meet the degradation machinery,
including the ER (Finger et al., 1993; Otsu et al., 1995), the
cis-Golgi (Raposo et al., 1996) or post-ER compartments
(Amitay et al., 1992) and the cytosol (Wiertz et al., 1996a,b).
We reasoned that this site could be visualised by analysing
where the β chain would accumulate after prolonged culture of
the transfectants in the presence of the proteasome inhibitor
lactacystin. Lactacystin-treated cells were fixed, sectioned and
stained with anti-class II β chain antibodies prior to analysis
by immuno-electronmicroscopy (Fig. 5a,b). Extensive β chain
labeling was seen in ER structures and not in the cytosol or
cGR. Sometimes the β chain was observed in ER sheets
incorporated in autophagosomal-like structures which are
probably the result of accumulation of the β chain in the ER
after prolonged inhibition of degradation (not shown).
To follow the fate of the class II β chain in 293 cells in an
antibody-independent fashion and in living cells, GFP was
attached to the cytoplasmic tail of the β chain and stable
transfectants in 293 cells were generated. Pulse chase
experiments in the presence or absence of lactacystin confirmed
that degradation of β-GFP could be inhibited by lactacystin
without removal of the N-linked glycan (Fig. 5c, top panel), as
for the free β and free α chain. CLSM analysis of living cells
at 37°C showed fluorescence in reticular membraneous
structures and the nuclear envelope indicating retention in the
ER (Fig. 5c, bottom). Overnight incubation with lactacystin did
not affect the distribution and fluorescence does not appear in
the cytosol, which would have resulted in an even distribution
of fluorescence over the cell (Reits et al., 1997).
DISCUSSION
The ER represents a site where proteins are synthesized, folded
and assembled into functional complexes. Failure to fold or
assemble correctly usually results in retention of the protein in
the ER and sometimes in the cis-Golgi reticulum. These
molecules are then degraded by a process that does not involve
lysosomes. Degradation of ER-retained molecules is selective
and occurs with different kinetics. With the exception of the T
cell receptor α chain in which the transmembrane domain
determines its half-life (Bonifacino et al., 1990), the reasons for
different kinetics are unclear unless the degradation is initiated
by other proteins. Here, we have examined which domains of
ER-retained molecules are involved in determining their fate.
We have studied degradation of free class II α and β chains
because these chains are likely to be similar in structure, based
on the three-dimensional structure of class II HLA-DR1 (Brown
et al., 1993). Furthermore, we have analysed chimeric proteins
that contain the transmembrane and cytoplasmic region of
MHC class I HLA-B27 molecules and the luminal part of either
the class II α or β chain. Following the half-life of the respective
free chains we observed that every segment affects their halflife in the ER, although not in an unidirectional manner. The
cytoplasmic and transmembrane portion of HLA-B27 increases
the rate of degradation of the α chain whereas it decreases the
rate of degradation of the β chain.
How is the rate of degradation of the ER-retained free class
II subunits controlled? Degradation of ‘misfolded’ molecules in
the ER should necessarily be limited because of the continous
insertion of newly synthesized, but still (partly) unfolded,
proteins. Unfolded proteins or protein subunits usually associate
with accessory proteins like BiP and calnexin. This interaction
may protect unfolded molecules from destruction, and their
release from, or alteration of, accessory molecules may be a
prerequisite for degradation. This is indeed suggested for
degradation of the free TCR α chain and for the VSV-G protein
which are degraded faster when they fail to associate with
calnexin as a result of inhibition of glucosidase I-activity with
castanospermine (Kearse et al., 1994; Hammond et al., 1994).
The rate of β chain degradation is not influenced by inhibition
of glycan trimming by the glycosidase inhibitors
castanospermine, MedNM or dMM. This is not unexpected
since, in our transfectants, these molecules associate not with
calnexin but with BiP (Nijenhuis and Neefjes, 1994), which
does not require glycans for binding (Flynn et al., 1991).
However, the yeast BiP homologue Kar2p has been shown to
be instrumental in ER degradation (Plemper et al., 1997), either
through direct targeting or by affecting the accessability of the
pore of the translocon (Hamman et al., 1998). Stress conditions
affecting the relative concentration of chaperones may further
determine unfolding and/or targeting for degradation.
The free β chain is probably released from accessory ER
proteins before it can dock to the site where it is released from
the ER membrane and entering the proteolytic machinery. BFA
treatment of cells may introduce components into the ER that
accelerate this release. Factors introduced by BFA into the ER
include additional glucosidase and mannosidase activity,
although they are not instrumental in rapid degradation of the β
chain. How BFA acts in enhancing ER degradation by the
proteasome is thusfar unclear. It may be that cycling of the free
β chain through post-ER compartments marks these molecules
for degradation and acts as a molecular clock defining the halflife of ER-proteins. BFA-treatment would then introduce a ratedetermining factor into the ER and hence accelerate degradation.
Degradation of the free β chain is unlikely to occur in post-ER
compartments since it continues in isolated vesicles (not shown)
and is clearly retained in the ER after prolonged times of
proteasome inhibition. Degradation of the TCR α chain also
continues in streptolysin O-permeabilized cells and in ERderived vesicles (Stafford and Bonifacino, 1991), but clearly
requires the proteasome for ER degradation (Huppa and Ploegh,
ER-degradation 2225
1997). In vitro ER degradation requires ATP and cytosol
(McCracken and Brodsky, 1996). The cytosolic multicatalytic
proteasome has been implicated in degradation of various ER
proteins like a CFTR mutant and class I H-chains (Wiertz et al.,
1996a,b; Ward et al., 1995; Jensen et al., 1995). We showed that
degradation of the β chain can also be inhibited by the specific
proteasome inhibitor lactacystin (Fenteany et al., 1995; Dick et
al., 1996) and V3S (Bogyo et al., 1997; data not shown). To
visualize whether the β chain first enters the cytosol before
becoming a substrate for the proteasome, as shown for various
other proteins (e.g. Wiertz et al., 1996a,b), we analyzed the
intracellular location of the β chain or the β-GFP chimera in cells
cultured with lactacystin. If the β chain enters the cytosol before
degradation by the proteasome, we would expect to find the β
chain accumulating in the cytosol. If the cGR were the site of
contact of ER proteins with the proteasomal degradation
machinery, as suggested for class I H chains in murine thymic
epithelial cells (Raposo et al., 1996), accumulation at this site
should be observed. However, the β chain accumulates in the ER.
In addition, under these conditions the β chain is still a substrate
for glucosidase and mannosidase action, especially when cultured
in the presence of BFA. This suggests that the N-linked glycan
of the β chain is still exposed to the ER lumen and that
proteasomal activity is required for removal of the glycosylated
β chain from the ER membrane for degradation. This mechanism
is very recently confirmed by Yang et al. (1998) for the TCR delta
chain. A process of ‘ER-removal coupled to proteasomal
degradation’ may be mediated by the ER-associated yeast
component of the ubiquitin-transfering machinery Cue1p which
has been shown to be essential for ER degradation and release
from the ER membrane of misfolded ER proteins (Biederer et al.,
1997). Here, the step preceeding proteasomal degradation is
required for ER release. Both mechanisms differ from US2 and
US11 mediated degradation of the class I H-chain which is first
transported to the cytosol in a state lacking ubiquitin moieties,
followed by deglycosylation before proteasome-mediated
degradation (Wiertz et al., 1996a,b). The class I H-chain therefore
accumulates in a deglycosylated form in the cytosol whereas the
β chain remains in the ER and N-glycosylated after prolonged
incubation with the proteasome inhibitor lactacystin.
Degradation of the β chain may either be orchestrated by the
free pool of rapidly diffusing proteasomes or the small pool of
vesicle-associated proteasomes (Yang et al., 1995; Palmer et al.,
1996; Reits et al., 1997). The latter pool constitutes less than 8%
of the total pool of proteasomes (Reits et al., 1997). We showed
by subcellular fractionation, using two different physical
principles (density and charge), that a small pool of proteasomes
(visualised by the α subunit iota) cofractionates with the ER.
Yang et al. (1995) and Palmer et al. (1996) also showed the
association of intact proteasomes to microsomal vesicles
although they did not perform extensive further purifications to
formally show it to be the ER. Separation by DGE also showed
that proteasomes are associated with other, as yet unidentified,
membranes. The ER-associated proteasomes probably degrade
the β chain. It is possible that the subunit composition of these
proteasomes differs from the free cytosolic proteasomes but it is
unclear whether this affects the specificity of degradation or their
intracellular location (Yang et al., 1995; Palmer et al., 1996).
Interestingly, some ubiquitin conjugating enzymes, UBC6 and
Cue1p, have been found associated with yeast ER membranes
(Sommer and Jentsch, 1993; Biederer et al., 1997). Hence
various components for (ubiquitin-mediated) degradation by
proteasomes are ER-associated. Unlike CFTR (Ward et al.,
1995; Jensen et al., 1995), no ubiquitination of the β chain or
class I subunits (Wiertz et al., 1996) was observed under
conditions of proteasome inhibition.
The β chain is likely to use a pore for ER-membrane departure
and this channel could be the docking site for the proteasome on
the ER. The translocon has been suggested to be involved in US2
and US11-driven removal of the class I H-chain from the ER
(Wiertz et al., 1996a,b) and degradation of misfolded pro-alpha
factor in yeast (Pilon et al., 1997). This degradation is very fast,
and the class I H-chains may not have been properly inserted
into the lipid layer of the ER membrane. It is unknown whether
proteins that are more slowly degraded use the translocon as a
channel for removal from the ER. Since removal of the β chain
is coupled to proteasome activity, the above scheme would
suggest that the ribosome and the proteasome ‘compete’ for
binding to the translocon. Whether a complex of proteasome and
translocon is indeed involved in the extraction from the ER
membrane of stably inserted proteins remains to be established.
We have shown that both cytosolic and post-ER processes
are involved in the degradation of ER-retained proteins. The
removal from the ER membrane is coupled to subsequent
degradation of free class II β chains by the proteasome.
We thank Dr H.-P. Hauri for anti-p53 antibodies, A. Benham and
C. Vos for critically reading the manuscript. This research was
supported by research grant NKB 93-525 from the Netherlands
Cancer Society and a Pioneer grant from the Dutch Society of
Sciences (NWO).
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