From the Laboratory of Immune Cell Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-1152
Received for publication, August 22, 2000, and in revised form, January 18, 2001
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ABSTRACT |
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Degradation of proteins from the endoplasmic
reticulum is fundamental to quality control within the secretory
pathway, serves as a way of regulating levels of crucial proteins, and
is utilized by viruses to enhance pathogenesis. In yeast two
ubiquitin-conjugating enzymes (E2s), UBC6p and UBC7p are implicated in
this process. We now report the characterization of murine homologs of
these E2s. MmUBC6 is an integral membrane protein that is anchored via its hydrophobic C-terminal tail to the endoplasmic reticulum. MmUBC7,
which is not an integral membrane protein, shows significant endoplasmic reticulum colocalization with MmUBC6. Overexpression of
catalytically inactive MmUBC7 significantly delayed degradation from
the endoplasmic reticulum of two T cell antigen receptor subunits, In eukaryotes, a primary means by which proteins are targeted for
degradation is by their modification with chains of ubiquitin (Ub).1 Ubiquitinated proteins
are recognized and degraded by the multicatalytic 26 S proteasome.
Attachment of Ub to proteins involves a process in which one of a
number of different Ub-conjugating enzymes (UBCs or E2s) accept Ub from
activated E1 enzyme in a transthiolation reaction and subsequently
catalyze the formation of an isopeptide bond between Ub and substrate,
either with or without the involvement of an Ub-protein ligase (E3)
(1). Proteasomal degradation is not limited to proteins native to the
nucleus and cytosol where proteasomes reside. Many transmembrane and
lumenal proteins of the secretory pathway are degraded from the
endoplasmic reticulum (ER) by proteasomes. The processes that
ultimately result in the proteasomal degradation of these proteins are
referred to as ERAD (ER-associated degradation). Steps involved in ERAD
can include trimming of N-linked glycans, ubiquitination,
retrograde movement through the ER membrane, deglycosylation, and
degradation in the cytosol by proteasomes (2). The temporal and
mechanistic relationships between retrograde movement, conjugation with
Ub, and possible chaperone-like functions of proteasomes appear to
differ based on the nature of the substrate. This might be expected
given the varied substrates, which include luminal proteins, proteins
having a single membrane-spanning domain, and complex polytopic
proteins. Moreover, these substrates may be either mutated misfolded
proteins, otherwise normal proteins that have failed to assemble in a
complex, or, as is the case with HMGCoA reductase, a normal protein
whose activity is regulated by ERAD (3). Consistent with different requirements for degradation of these varied substrates, a genetic analysis of yeast mutants that are defective in ERAD of HMG-CoA reductase led to the identification of HRD genes (4), and a differential dependence among ERAD substrates on yeast HRD genes was
recently demonstrated (5).
In yeast, a number of ERAD substrates are multiubiquitinated, examples
include mutant forms of Sec61p (6) and carboxypeptidase Y (CPY*) (7) as
well as HMGCoA-reductase (3). Genetic analysis has implicated two yeast
E2s, UBC6p and UBC7p, in ERAD. Deletion of UBC6 and UBC7 stabilizes
mutant Sec61p, Sss1p, CPY, Pdr5, and uracil permease (6-9). UBC6p is a
C-terminal anchored membrane protein whose catalytic site faces the
cytosol (10). Unlike UBC6p, UBC7p lacks a membrane anchor but
associates with an ER-bound protein, Cue1p (11).
In mammalian cells ERAD substrates such as cystic fibrosis
transmembrane conductance regulator and apoB are ubiquitinated in a
cotranslational fashion in vitro (12, 13). Subunits of the T
cell antigen receptor (TCR), when not assembled into complexes capable
of exiting the ER, are also degraded from the ER. In T lymphocytes
multiubiquitinated forms of TCR- Despite a clear role for ERAD in mammals, no specific E2s have been
implicated in this process. We now report characterization of mammalian
E2s homologous to yeast UBC6p and UBC7p, establish that these proteins
are ER membrane proteins, and provide evidence that a murine UBC7p
homolog, MmUBC7, plays a role in the degradation of unassembled TCR
subunits from the ER.
Cells and Reagents--
Cos-7 (number CRL1651; American
Tissue Culture Collection, Manassas, VA) and HEK-293 (number
CRL1573; American Tissue Culture Collection) cells were maintained in
complete Dulbecco's modified Eagle's medium and transfected using the
calcium-phosphate method as described (16). Anti-CD3- Plasmids--
Lysineless TCR-
MmUBC6 and MmUBC7 were also cloned into pGEX-KG (20) to generate
N-terminal glutathione S-transferase fusions. For some experiments, glutathione S-transferase moieties were cleaved
by thrombin treatment and residual thrombin was removed by
benzamidine-Sepharose beads according to the manufacturer's protocol
(Amersham Pharmacia Biotech).
In Vitro Transcription and Translation--
Coupled in
vitro transcription and translation was done in rabbit
reticulocyte lysates (Promega, Madison, WI) in the presence or absence
of canine pancreas microsomal membranes. Membranes were isolated by
centrifugation at 100,000 × g for 30 min in an air
centrifuge. For urea extraction, the membranes were resuspended in 2.5 M urea for 15 min at room temperature followed by
centrifugation and separation of supernatant and membrane. The
supernatant was precipitated with 10% trichloroacetic acid and the
pellet resuspended in 0.1 N NaOH.
Immunofluorescence Analysis--
Cos-7 cells were grown on
coverslips and transfected using Superfect (Qiagen, Valencia, CA)
according to manufacturer's protocol. Cells were fixed with 2%
formaldehyde in phosphate-buffered saline (PBS) for 10 min then washed
twice with PBS. This was followed by incubation with 12CA5 or 9E10
antibody diluted 1:5 in PBS containing 0.1% saponin and 0.1% bovine
serum albumin for 30 min followed by washing with PBS and incubation
with secondary antibody coupled to Cy3. For double staining, coverslips
were then incubated with second primary antibody conjugated to biotin
followed by staining with streptavidin coupled to Alexa 488. Images
were obtained on a Leica confocal microscope using 63× and 100×
objectives. The 488-nm line of the Argon laser and 568-nm line of
the Krypton laser were used to excite Alexa 488 and Cy3, respectively.
Emission was collected between 520 and 530 nm for Alexa 488 and between 610 and 700 nm for Cy3.
Pulse-Chase Analysis--
HEK-293 cells were transfected using
calcium-phosphate method with 2.5 µg of 2B4 TCR- Immunoprecipitations and Western Blotting--
Cells were lysed
in Triton X-100 lysis buffer containing protease inhibitors as
described (14). Immunoprecipitations were carried out at 4 °C using
protein A-Sepharose beads prebound to indicated antibodies. Beads were
washed with 50 mM Tris, pH 7.4, 300 mM NaCl,
0.1% Triton X-100. In some cases 0.05% SDS was included in the wash
buffer. SDS-polyacrylamide gel electrophoresis and Western transfer
were done according to standard protocols. Blots were developed using
either 125I-labeled protein A or chemiluminescence (Pierce).
Proteinase K Protection and Subcellular Fractionation--
Cells
were lysed in triethanolamine buffer and broken as described (14).
Unbroken cells and nuclei were removed by centrifugation at 1000 × g for 5 min. The supernatant was divided in two, and one
half was treated with proteinase K as described (14) Samples were
centrifuged at 100,000 × g for 45 min at 4 °C.
Pellets were washed once with triethanolamine buffer and solubilized in
lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl,
1% digitonin). To the supernatant, 2× digitonin lysis buffer was
added. All samples were centrifuged again at 10,000 × g for 10 min, and the supernatant was immunoprecipitated
with H28 antibody.
N-Glycanse Treatment--
Treatment with N-glycanase
(PNGase F, New England Biolabs Inc., Beverly, MA) was carried out as
specified by the manufacturer.
Sequence Analysis of MmUBC6--
To identify mammalian orthologs
of yeast UBC6p, the GenBankTM expressed sequence tag data
base was searched and homologous murine cDNAs obtained and
sequenced. One such clone included a putative start codon that was in a
good context for translation initiation (21) with a stop codon 5' to
this site in the same reading frame (GenBankTM accession
number AF 296656). The open reading frame predicts a protein of 262 amino acids, 12 amino acids more than UBC6p (Fig. 1). This putative protein is referred to
hereafter as MmUBC6 for the murine homolog of yeast UBC6p based on the
convention that E2s are named for the corresponding Saccharomyces
cerevisiae member preceded by a two-letter genus/species
abbreviation. Homology with UBC6p is most striking in the region
surrounding the core E2 domain (amino acids 8-166 of UBC6p), where
there is 59% identity and 72% similarity. However, beyond amino acid
166 of the mouse protein there is less than 11% identity. MmUBC6 was
determined to be an active E2 by the ability of recombinant MmUBC6 to
form thiol ester bonds with Ub in the presence of E1 (data not shown). This activity was abolished by mutation of the predicted active site
cysteine (amino acid 94).
UBC6p is unique among E2s in that it is a type IV membrane protein,
defined as such by its C-terminal membrane anchor that allows for its
ER membrane insertion in yeast (10) and when ectopically expressed in
mammalian cells (22). MmUBC6 similarly has a C-terminal hydrophobic
domain, although it is highly divergent from yeast UBC6p.
Hydrophilicity analysis using MacVector (Oxford Molecular Group,
Oxford, UK) and GCG (Genetics Computer Group Inc., Madison, WI)
programs suggests that this domain begins at amino acid 228 and extends
at least to amino acid 246 and possibly to amino acid 249. Notably a
Schizosaccharomyces pombe E2, SpUBC25K, which
exhibits overall homology to UBC6p, also shows very little homology in
the C-terminal hydrophobic region. However, a human expressed sequence
tag clone homologous to yeast UBC6p was also sequenced and was found to
be 94% identical to MmUBC6 with a single amino acid substitution in
the C-terminal hydrophobic region (GenBankTM Accession
number AF 296658). This clone will be referred to as encoding HsUBC6.
It should be noted, however, that HsUBC6 is not a member of the unique
mammalian UBCH6 family of E2 enzymes (23).
Membrane Association and Subcellular Localization of
MmUBC6--
To determine whether MmUBC6 is a membrane protein, it was
translated in rabbit reticulocyte lysate in the presence of canine microsomes. This yielded a ~31-kDa protein (Fig.
2), of which substantially more than 50%
was consistently found in the membrane fraction (Fig. 2A,
compare lanes 1 and 2). Membrane-associated MmUBC6 was resistant to urea extraction (Fig. 2A,
lanes 3 and 4) and released into the soluble
fraction upon extraction with Triton X-100 (Fig. 2A,
lanes 5 and 6). A truncation lacking the C-terminal hydrophobic domain (MmUBC6d) partitioned almost entirely into the soluble fraction (Fig. 2A, lanes 7 and
8). Thus, MmUBC6 is also a C-terminal anchored, integral
membrane protein. Other C-terminal anchored membrane proteins have been
reported to be inserted in membranes post-translationally. To determine
whether this is the case for MmUBC6, it was translated in
vitro without microsomes and then assessed for membrane insertion
by incubation with canine micosomes followed by separation of membrane
and soluble fractions by centrifugation. When membranes were added,
~40% of MmUBC6 partitioned into the membrane fraction (Fig.
2B, c and d) and was largely resistant
to urea extraction (Fig. 2B, e and f).
In contrast MmUBC6d was almost entirely recovered in the soluble fraction (Fig. 2B). When ectopically expressed in mammalian
cells, increasing the C-terminal hydrophobic domain of yeast UBC6p from 17 to 21 amino acids results in a redistribution along the secretory pathway from the ER to the Golgi (22). The predicted hydrophobic domain
of MmUBC6 is longer than its yeast counterpart. However, its
subcellular localization by immunofluorescence revealed a lacelike ER
pattern (Fig. 3, C and
G) and colocalization with UBC6p (Fig. 3, D-F).
Thus, MmUBC6 is also an ER membrane protein.
Identification of a Murine Homolog of UBC7p--
Rat and
human homologs of UBC7p have been reported (24, 25); however, no
evidence for physical or functional association with the ER has been
demostrated. A murine UBC7p homolog was identified in the expressed
sequence tag data base that encodes an open reading frame predicting
a protein of 168 amino acids that shares 62% identity and 73%
similarity with yeast UBC7p, and 96% identity and similarity with its
human homolog (GenBankTM accession number AF 296657). We
refer to this protein as MmUBC7. In vitro translation
results in an 18-kDa protein, consistent with the deduced amino acid
sequence. The capacity of this E2 to form thiol ester bonds with Ub was
confirmed (data not shown).
Membrane Association of MmUBC7--
MmUBC7 lacks a hydrophobic
domain and is therefore predicted to be a soluble protein. However,
incubation of MmUBC7 with microsomal membranes after in
vitro translation resulted in ~15% of MmUBC7 becoming membrane
associated (Fig. 4A), whereas
another core E2, UbcH5B (26) did not bind to membranes (Fig.
4A). Further evaluation of the membrane association of
MmUBC7 was undertaken by transient expression of Myc-epitope-tagged
MmUBC7. MmUBC7 was found both in the cytosolic and microsomal
membrane fractions (Fig. 4B, upper panel,
lanes 1 and 3). In contrast all of the
anti-UbcH5B immunoreactivity was in the cytosolic fraction (Fig.
4B, lower panel, lanes 1 and 2).
MmUBC7 Colocalizes with MmUBC6--
When evaluated by
confocal immunofluorescence microscopy, MmUBC7 appears to be diffusely
expressed, but a discrete underlying ER pattern is evident (Fig.
5A). To confirm this ER
association, it was coexpressed with MmUBC6. Fig. 5 (B-D)
shows confocal images from the same cell expressing both HA-MmUBC6 (5B)
and Myc-MmUBC7 (5C). A significant colocalization of Myc-MmUBC7 with
HA-MmUBC6 to the ER was observed when (5B) and (5C) were overlaid (Fig. 5D, yellow shows colocalization).
Overexpression of Inactive MmUBC7 Inhibits Degradation of T Cell
Receptor Subunits--
Previous studies have established that ERAD of
TCR-
To confirm that the effect observed with overexpression of
C89SMmUBC7 was due to inhibition of degradation, TCR- Majority of TCR-
As already noted, the faster migrating species observed when proteasome
function is inhibited are consistent with previous reports for TCR-
To determine whether the inhibitory effects on TCR- C89SMmUBC7 Has Dominant Negative Effect on CD3-
To further evaluate CD3-
Unlike TCR- This study provides evidence that two mammalian E2s localize to
the ER membrane. For MmUBC6 its C-terminal hydrophobic domain provides
a basis for its membrane insertion. MmUBC7 lacks a membrane anchor that
would allow for direct membrane insertion. In yeast, interaction with a
C-terminal anchored protein, Cue1p, provides a molecular explanation
for ER localization of UBC7p (11). Although mammalian Cue1p homologs
have not been reported, it seems likely that an analogous protein may
play a role in tethering of MmUBC7 to the ER membrane.
Previous studies in cells expressing a temperature-sensitive ubiquitin
activating enzyme (E1) have shown that a functional Ub pathway is
required for degradation of TCR- For proteins such as CD3- An important question is whether it is the ubiquitination of TCR- Another issue that arises is the nature of the E3s with which MmUBC7
interacts. In yeast HRD1/DER3 is implicated in epistasis analysis along
the same pathway as UBC7 in the degradation of several yeast proteins
including CPY* and HMGCoA reductase (5). Whether mammalian homologs of
HRD1 exist remains to be determined. Another ubiquitin ligase component
implicated in degradation from the ER is the F-box protein and CD3-
, and suggests a role for the ubiquitin
conjugating system at the initiation of retrograde movement from the
endoplasmic reticulum. These findings also implicate, for the first
time, a specific E2 in degradation from the endoplasmic reticulum in mammalian cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and the TCR CD3-
subunit are
associated with the ER membrane, suggesting that their ubiquitination
occurs while still membrane-bound (14). In initial studies on ERAD of
major histocompatability complex class I proteins, evidence for
ubiquitination was lacking. However, more recent analyses have provided
evidence for ubiquitinated major histocompatability complex class I
molecules as degradation intermediates (15). Collectively, these
finding suggest that in mammals, as in yeast, components of the Ub
conjugating machinery functionally interact with substrates at the ER membrane.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(R9) (17);
anti-ubiquitin (18); and anti-TCR-
, H28 (19) have all been
described. Anti-HA (12CA5) and anti-Myc (9E10) monoclonal antibodies
were from culture supernatants. Anti-GRP78 (BiP) antibody was from
StressGen Biotech. (Victoria, Canada). Anti-MDM2 was from Oncogene
Science (Cambridge, MA).
(K
RTCR-
/pcDNA3.1) was
a gift from Dr. Ron Kopito. Wild type 2B4 TCR-
in pCDM8 (Invitrogen,
Carlsbad, CA) and CD3-
in pCI (Promega, Madison, WI) were obtained
from Dr. Juan Bonifacino. MDM2/pCINeo was a gift from Dr. Shengyun
Fang. GenBankTM expressed sequence tag data bases were
searched using the amino acid sequences of yeast UBC6p and UBC7p.
Murine cDNA clones that were homologous to yeast sequences
(referred to as MmUBC6 and MmUBC7) were obtained and sequenced.
MmUBC6 was cloned into pCI vector and tagged with HA epitope at the
N-terminal by polymerase chain reaction using the primers
5'-ATAGAATTCACCATGGCCTACCCATACGACGTCCCAGACTACGCTCCCGGGCCCGAGATTAGCAATAAC-3' and 5'-GCCACCTTCATAAGGAGTCATC-3'. The polymerase chain reaction product
was digested with EcoRI and SacI and cloned into
pCI digested with the same enzymes. The construct was confirmed by
sequencing. To generate MmUBC6 lacking the C-terminal tail, nucleotides
encoding the last 52 amino acids were removed by restriction enzyme
digestion, and the remainder of the cDNA was subcloned into
pCDNA3 (Invitrogen, Carlsbad, CA). MmUBC7 was tagged with Myc
epitope at the N terminus using primers
5'-ATATGAATTCATGGAGCAGAAGCTGATTTCCGAGGAGGACCTGAACCTCAAATTGGCGGGGACGGCGTTGAAG-3' and 5'-AACGACGGCCAGTGCCAAGC-3'. The polymerase chain reaction product
was digested with EcoRI and NotI and cloned
into pcDNA3. The construct was confirmed by sequencing.
Site-directed point mutations were created using QuickChange
Mutagenesis kit (Stratagene, La Jolla, CA). The mutations were
confirmed by sequencing.
/pCDM8 and 5 µg each of either wild type or mutant HA-MmUBC6 or Myc-MmUBC7 or both
together. The total amount of plasmid was equalized with pCDNA3.
Cells were harvested 36 h post-transfection and incubated for 45 min in methionine-free medium and then labeled for 20 min with
250 µCi of [35S]methionine/ml (ICN Biomedicals, Costa
Mesa, CA). Cells were then washed twice at 4 °C with complete
medium followed by incubation in complete medium at 37 °C. Cells
were harvested at indicated times, and immunoprecipitations were
performed as described below followed by SDS-polyacrylamide gel
electrophoresis and autoradiography.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequence comparison of MmUBC6 with
S. cerevisiae UBC6p. Dark gray boxes
represent identical amino acids, and light gray boxes show
similarity. Active site Cys is indicated with an asterisk.
Hydrophobic C-terminal domains are underlined.
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Fig. 2.
MmUBC6 is a type IV membrane protein.
A, MmUBC6 and MmUBC6 lacking the C-terminal 52 amino acids
(MmUBC6d) were translated in vitro using rabbit reticulocyte
lysate and microsomal membranes. Translated protein was fractionated
into soluble (S) and pellet (P) fractions
(lanes 1, 2, 7, and 8) by
centrifugation. The pellet was further extracted with 2.5 M
urea (lanes 3 and 4) or Triton X-100 (lanes
5 and 6) and again separated into soluble and pellet
fractions. B, MmUBC6 and MmUBC6d were translated in
vitro in the absence of membranes followed by fractionation into
soluble (S) and membrane (P) fractions either
without incubation with membranes (a and b) or
after incubation with membranes (c and d). A
duplicate of the pellet from d was extracted with 2.5 M urea and separated in soluble and pellet fractions.
MmUBC6d was treated similar to samples in c and
d.
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Fig. 3.
Confocal immunofluorescence localization of
MmUBC6 and ScUBC6p. Cos-7 cells were transfected with HA-MmUBC6
and Myc-ScUBC6p. A and B show control cells
stained with anti-HA and anti-Myc antibodies, respectively.
C, MmUBC6; G, enlarged view of MmUBC6
staining. A cell cotransfected with MmUBC6 and ScUBC6p is shown
in D-F where MmUBC6 is shown in D, ScUBC6p is
shown in E, and the overlay of both is shown in
F. Colocalization of the two proteins is in
yellow.
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Fig. 4.
Membrane association of MmUBC7.
A, UbcH5b and MmUBC7 were translated in vitro in
the absence of membranes (lanes 1 and 2 show 20%
of the input used in lanes 3 and 4). Membranes
were incubated with translated material followed by isolation and
washing of membranes by centrifugation (lanes 3 and
4). B, Cos-7 cells transfected with
MycMmUBC7/pCDNA3 (lanes 1 and 3) or
pCDNA3 empty vector (lanes 2 and 4) were
lysed without detergent, and cytosolic and membrane fractions were
prepared as described under "Experimental Procedures." After
solubilization with detergent, these fractions were resolved by
SDS-polyacrylamide gel electrophoresis, and blotted with anti-Myc
antibody. The same blot was stripped and probed with anti-UbcH5b
antibody (lower panel).
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Fig. 5.
Colocalization of MmUBC7 and
MmUBC6 to endoplasmic reticulum. Cos-7 cells were transfected
with Myc-MmUBC7/pCDNA3 alone (A) or together with
HA-MmUBC6/pCI (B-D). B and C show
fluorescence from the same cell for MmUBC6 (B) and MmUBC7
(C). The overlay of the two is shown in D where
colocalization of the two proteins appears in yellow.
, a type I transmembrane protein with a short cytoplasmic tail
of 5 amino acids, is dependent on proteasome function. To evaluate
whether mammalian homologs of UBC6p and UBC7p are involved in targeting TCR-
for degradation, catalytically inactive forms of these E2s were
coexpressed with TCR-
in HEK-293 cells. Levels of TCR-
were
evaluated by immunoblotting with anti-TCR-
. Although overexpression of a mutant of MmUBC6 in which the active site Cys was converted to Ser
(C94SMmUBC6) did not substantially affect TCR-
levels, the analogous catalytically inactive MmUBC7 (C89SMmUBC7)
resulted in marked increase in TCR-
levels (Fig.
6, A and B).
Coexpression of the two inactive E2s did not result in a further
increase (Fig. 6A). In contrast, overexpression of inactive
MmUBC7 did not result in accumulation of Mdm2, which is a non-ER
protein that is ubiquitinated and degraded by proteasomes (Fig.
6C). Neither wild type MmUBC6 nor MmUBC7 had any significant
or reproducible effect on TCR-
(Fig. 6, A and
B).
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Fig. 6.
Effect of MmUBC6 and MmUBC7 on steady state
levels of TCR- . A, HEK-293
cells transfected with TCR-
/pCDM8 alone (lane 1) or with
wild type or mutant MmUBC6/pCI (lanes 2 and 3) or
MmUBC7/pCDNA3 (lanes 4 and 5) or both
together (lanes 6 and 7) were immunoprecipitated
(IP) with H28 antibody and blotted with the same antibody.
Mutant MmUBC6 and MmUBC7 are indicated by
C94S and
C89S, respectively. Cells were also
cotransfected with GFP, and 10 µl of whole cell lysate was blotted
with anti-GFP to check the transfection efficiency. 50 µl of whole
cell lysate was blotted with anti-HA and anti-Myc antibodies for MmUBC6
and MmUBC7 expression, respectively. B, a separate
experiment similar to A. C, HEK-293 cells were
transfected with MDM2 with or without C89SMmUBC7.
Lane 1, vector transfected control; lanes 2 and 3, MDM2 alone without and with lactacystin
treatment; lane 4, MDM2 cotransfected with
C89SMmUBC7. Upper panel shows immunoblotting
(IB) with anti-MDM2 antibody, and lower panel
shows anti-GFP blot for loading control.
half-life was directly determined by pulse-chase metabolic labeling.
TCR-
consistently exhibited a 45-65% increase in half-life when
C89SMmUBC7 was coexpressed (Table
I). Concomitant overexpression of
inactive MmUBC6 did not further increase TCR-
survival.
Half-life of TCR- in HEK-293 cells calculated from three independent
experiments
Is Largely Membrane-bound and Proteinase
K-resistant--
Previous studies in non-T cells have suggested that
when proteasome function is inhibited, a significant amount of the
accumulated TCR-
has undergone retrotranslocation from the ER to the
cytosol accompanied by deglycosylation (29, 30). To determine the location of TCR-
that accumulates with inactive MmUBC7, cells expressing TCR-
were subjected to proteinase K (PnK) digestion followed by separation of cytosolic and membrane fractions (Fig. 7). When inactive MmUBC7 was coexpressed,
increased amount of TCR-
were seen (compare lanes 1-4,
9-12, and 17-21). All of this accumulated
TCR-
was found in the membrane fraction and was resistant to PnK
digestion and therefore has not undergone retrotranslocation through
the ER membrane. When proteasome function was inhibited (lanes
5-8, 13-16, and 21-24) levels of
accumulated TCR-
increased, still the large majority of the
immunoreactive material was found in the membrane fraction resistant to
PnK. However, a small fraction of the accumulated TCR-
was
PnK-sensitive and included full-length and more rapidly migrating
species (open arrow) that represent deglycosylated forms
(see below and Fig. 7B) in the cytosolic fraction as well as
similar rapidly migrating species in the membrane fractions. Although
these PnK-sensitive forms were observed in all proteasome-treated
samples, when compared with the levels of full-length PnK-resistant
TCR-
, retrotranslocated forms were found at a proportionally lower
level in cells expressing inactive MmUBC7. The findings that inactive
MmUBC7 results in the accumulation of fully protected
membrane-associated TCR-
and that there is a proportional decrease
in cytoplasmically disposed species when proteasome function is
inhibited implicates MmUBC7 in playing a role in ERAD prior to
retrograde movement through the ER membrane.
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Fig. 7.
PnK protection and subcellular localization
of TCR- . A, HEK-293 cells
transfected with TCR-
alone or with wild type or mutant MmUBC7 were
treated with or without lactacystin (LCN) and lysed without
detergent. After pelleting unbroken cells and nuclei, the lysate was
treated with PnK followed by separation of cytosolic (C) and
membrane (M) fractions. Samples were immunoprecipitated
(IP) with H28 antibody and blotted with the same antibody.
After immunoprecipitation with H28, 50 µl of supernatant was taken
from the membrane fractions and blotted with anti-BiP antibody to check
membrane integrity. Solid and open arrows show
the full-length and faster migrating forms of TCR-
. An
asterisk indicates antibody heavy chain. B,
faster migrating forms represent deglycosylated TCR-
. Cells were
transfected with TCR-
and treated with lactacystin (LCN).
PNGase treatment was done after immunoprecipitation of TCR-
.
Long and short arrows show the full-length and
deglycosylated forms of TCR-
, respectively. IB,
immunoblotting.
where cleavage of N-linked oligosaccharides is observed
concomitant with retrotranslocation to the cytosol. Consistent
with this, TCR-
treated with N-glycanase comigrates with
the lower molecular weight forms of TCR-
(Fig. 7B).
degradation
observed with C89SMmUBC7 are dependent on ubiquitination of
TCR-
on lysine residues, we evaluated a previously reported lysine-less form of TCR-
(27). This form of TCR-
is degraded from
the ER in a proteasome-dependent manner despite a lack of potential sites for ubiquitination. As is evident, lysine-less TCR-
also accumulated when inactive MmUBC7 is coexpressed (Fig. 8A). Treatment with
lactacystin results in accumulation of lysine-less TCR-
, including
lower molecular weight forms. As with wild type TCR-
, appearance of
these forms is decreased when C89SMmUBC7 is coexpressed
(Fig. 8B), and, as would be predicted, these species were
PnK-sensitive (data not shown).
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Fig. 8.
Effect of inactive MmUBC7 on levels of
lysine-less TCR- . A, HEK-293
cells were transfected with Lysine-less (K
R) TCR-
with or without
C89SMmUBC7. Immunoprecipitation (IP) was carried
out with anti-TCR-
antibody followed by immunoblotting
(IB) with the same antibody. Blots were developed using
125I-labeled protein A. B, HEK-293 cells were
transfected as in A, and cells in lanes 3 and
5 were treated with lactacystin. Immunoprecipitation and
blotting was done as in A. Faster migrating forms are
indicated with an arrow. Lower panel shows immunoblot with
anti-GFP antibody for control of transfection efficiency.
Degradation--
The CD3-
subunit of the TCR is also a substrate
for ERAD when it fails to assemble with other TCR components and exit
the ER (14). This protein differs from TCR-
in having a substantial cytoplasmic domain and only a single charge in its transmembrane domain. To assess whether MmUBC7 is also involved in the degradation of
this protein, pulse-chase analysis of CD3-
was carried out. When
CD3-
was expressed alone in HEK-293 cells, it was rapidly degraded
(Fig. 9A). However, as with
TCR-
, its loss was significantly inhibited when coexpressed with
catalytically inactive MmUBC7. Inactive MmUBC6 did not significantly
affect CD3-
degradation (not shown).
View larger version (44K):
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Fig. 9.
Effect of C89SMmUBC7 on
half-life of CD3- . Pulse-chase analysis
of CD3-
alone or cotransfected with Myc-C89SMmUBC7.
Lower panel shows sequential immunoprecipitation
(IP) with anti-Myc antibody for MmUBC7 expression. Graph
shows the degradation curve for CD3-
from this experiment with 100%
representing CD3-
at 0 h time point. Open circles,
CD3-
; closed circles, CD3-
cotransfected with
C89SMmUBC7. B, HEK-293 cells transfected with
HA-CD3-
/pCI alone or with Myc-C89SMmUBC7 were treated
with lactacystin (LCN) for 12 h and immunoprecipitated
with anti-HA antibody and blotted with anti-CD3-
antibody.
Lower panel shows anti-Myc and anti-GFP blots of the whole
cell lysate to show MmUBC7 expression and loading control,
respectively. C, Cells transfected as in B were
treated with lactacystin (LCN) and lysed without detergent.
After pelleting unbroken cells and nuclei, cytosolic (C) and
membrane (M) fractions were prepared. Samples were
immunoprecipitated with anti-HA antibody and blotted with anti-CD3-
antibody. IB, immunoblotting.
a C-terminal HA-tagged form was generated.
After determining by pulse-chase metabolic labeling that it was
degraded with kinetics indistinguishable from wild type CD3-
(data
not shown), it was used to assess the effects of catalytically inactive
MmUBC7 on steady state levels of CD3-
by Western blotting. As
expected, steady state levels of CD3-
increased either when catalytically inactive MmUBC7 was overexpressed or when proteasome function was inhibited (Fig. 9B).
, for which retrotranslocated forms are detectable when
proteasome function is inhibited, our previous studies in T cells had
not noted any evidence of retrotranslocation of endogenous CD3-
(14). Consistent with this, regardless of whether or not inactive
MmUBC7 was coexpressed with CD3-
, forms that accumulated in the
presence of proteasome inhibitor were limited in distribution to the
membrane fraction. These results are in accord with the model of
coupled membrane extraction and proteasomal activity previously
suggested for CD3-
and suggest that, as with TCR-
, MmUBC7
is acting prior to retrograde movement through the ER membrane (Fig.
9C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
from the ER (27). Data presented
herein provides the first evidence implicating a specific E2, MmUBC7,
in degradation from the ER in mammalian cells. Overexpression of
catalytically inactive MmUBC7 results in decreased degradation of both
the TCR-
and the CD3-
subunits of the TCR. In contrast there is
no evidence of a role for MmUBC6 in degradation of TCR-
. Similarly,
no effect of mutant MmUBC6 was observed on degradation of
CD3-
.2 The negative data
obtained with inactive MmUBC6 is consistent with deletion analyses in
yeast where UBC7p is the predominant E2 in ERAD, whereas UBC6p has
partial effect on degradation of certain proteins, including Sec61 (6)
and CPY* (7), and no effect on Vph1 (28).
, which have cytoplasmically disposed
lysines, it is easy to envisage models for ERAD that include ubiquitination of cytoplasmic lysines, recognition of ubiquitinated species by proteasomes, and dislocation and destruction from the ER
facilitated by chaperone-like functions of proteasome. The lack of
discernable retrotranslocation observed for CD3-
in the presence of
lactacystin with or without coexpression of inactive MmUBC7 is
consistent with such a model and extends previous observations from our
laboratory made on endogenous CD3-
in T cells. Less obvious is how
components of the Ub-conjugating system function in the
retrotranslocation of lumenal proteins and of transmembrane proteins
lacking cytoplasmic sites for ubiquitination, such as TCR-
. The N
terminus of this protein is in the ER lumen, and it has no lysines in
its cytoplasmic tail. When evaluated by pulse-chase analyses in T
cells, TCR-
undergoes a discrete degree of
proteasome-independent retrograde translocation that should allow
exposure of potential sites of ubiquitination (14). However, similar
evidence for partial retrograde movement is not obvious in our
experiments in HEK-293 cells. Studies from other groups carried out in
non-T cells have provided evidence that when expressed ectopically some level of complete retrograde translocation and accompanying
deglycosylation of TCR-
occurs in the absence of proteasome function
(29, 30). Our observations corroborate these findings. However, even
after 16 h of proteasome inhibition, the amount of cytoplasmically
disposed TCR-
represents only a small fraction of the total
accumulated material, suggesting a continued requirement for proteasome
function for efficient complete retrotranslocation and degradation from the ER, as has been suggested for an engineered model substrate (31)
and for Pdr5 (8) in yeast. Similarly, a requirement for proteasome
function in retrotranslocation has been reported for unassembled
soluble Ig subunits (32) and CD4 (33, 34) in mammalian cells. Results
with TCR-
demonstrate that the relative amounts of cytoplasmically
disposed material is consistently decreased when inactive MmUBC7 is
coexpressed, even when proteasome function is inhibited, suggesting
that MmUBC7 may play a role in the process leading to
retrotranslocation upstream of the involvement of proteasomes. For
CD3-
the absence of detectable cytoplasmic forms under any circumstances precludes statements as to whether MmUBC7 act upstream of
the proteasome. But it is evident that, as with TCR-
, this E2 is
affecting the fate of CD3-
prior to its removal from the ER
membrane. Results obtained with both of these transmembrane TCR
components are consistent with findings in yeast for a nontransmembrane ER protein, CPY*, where deletion of UBC7 resulted in its accumulation in the ER lumen (35).
itself by mammalian UBC7 homologs that is required for retrograde
movement. Although this is the simplest explanation, and TCR-
clearly is a substrate for ubiquitination (14), the fact that TCR-
does not include cytoplasmic primary amines that could serve as sites
of ubiquitination argues against this. Also against such a model is the
finding that accumulation of retrotranslocated forms of lysine-less
TCR-
in response to lactacystin is also inhibited by catalytically
inactive MmUBC7. This therefore leads to the consideration of more
complex models in which MmUBC7 enhances TCR-
loss indirectly,
perhaps by ubiquitination of another protein that then facilitates
translocation of TCR-
out of the ER. Applying such a model to other
ERAD substrates would resolve the topological conundrum implicit in
postulating ubiquitination of lumenal proteins such as CPY* as a
primary event allowing for retrograde movement from the ER (35).
TRCP.
This protein forms part of an SCF E3 complex and is implicated in the
degradation of CD4 from the ER with phosphorylated HIV-1 Vpu
functioning as an adaptor (33, 36). At least for nonmembrane proteins,
SCF complex components are known to function with the yeast E2 CDC34
and its mammalian homologs. Whether
TRCP-containing SCF takes
advantage of ER membrane-bound MmUBC7 or perhaps MmUBC6 when targeting
CD4 for degradation from the ER now awaits determination.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr.
Jim McNally for assistance with confocal microscopy, Dr. Juan
Bonifacino and Dr. Ron Kopito for generously providing us with TCR-
and lysineless-TCR-
, respectively, Dr. Mei Yang for help in
generating HA-tagged CD3-
, and Dr. Lawrence Samelson for
anti-CD3-
. We thank Drs. Alessandra Magnifico, Shengyun Fang, Kevin
Lorick, and Jane Jensen for helpful discussions and critical
reading of the manuscript.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF 296656, AF 296657, and AF 296658.
To whom correspondence should be addressed. E-mail:
amw@nih.gov.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M007640200
2 S. Tiwari and A. M. Weissman, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Ub, ubiquitin; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum associated degradation; TCR, T cell antigen receptor; CPY, carboxypeptidase Y; PnK, proteinase K; HA, hemagglutinin; PBS, phosphate-buffered saline; GFP, green fluorescent protein.
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