1 The Max-Delbrück-Centrum für Molekulare Medizin,
Robert-Rössle-Str. 10, 13092 Berlin, Germany
2 Department of Biological Sciences, Stanford University, Stanford, CA
94305-5020, USA
3 Universität Lübeck, Institut für Biologie, Ratzeburger Allee
160, 23538 Lübeck, Germany
* Present address: Odyssey Pharmaceuticals Inc., 4550 Norris Canyon Road, Suite
140, San Ramon, CA 94583, USA
Author for correspondence (e-mail:
tsommer{at}mdc-berlin.de
)
Accepted 10 May 2002
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Summary |
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Key words: Endoplasmic reticulum, Ubiquitin, Proteolysis
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Introduction |
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Formation of ubiquitin conjugates requires three classes of enzymes, the
ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2s or Ubcs)
and ubiquitin-protein ligases (E3s)
(Hershko and Ciechanover,
1998). Yeast Ubc6p is the first Ubc demonstrated to be involved in
ERAD. It is the only integral membrane member of the yeast Ubc family. Its
active ubiquitin-conjugating domain (Ubc domain) is located at the N-terminus
while the C-terminus comprises a charged tail containing a stretch of
hydrophobic amino acids. This transmembrane domain localizes Ubc6p in the ER
membrane with the active site facing the cytosol. Because of this topology and
the lack of an N-terminal signal sequence Ubc6p belongs to the family of
tail-anchored membrane proteins (Sommer
and Jentsch, 1993
). Both genetic and biochemical evidence suggests
that Ubc6p is required for ERAD of mutant Sec61p, an integral component of the
ER membrane, as well as for mutant carboxypeptidase Y (CPY*), an
ER-lumenal protein (Biederer et al.,
1996
; Hiller et al.,
1996
). In addition, Ubc6p is required for degradation of some
cytoplasmic substrates (Chen et al.,
1993
). Restriction of Ubc6p to the ER membrane is essential for
these proteolytic functions (Sommer and
Jentsch, 1993
; Chen et al.,
1993
). Surprisingly, Ubc6p is not only a component of the ERAD
system but also a short-lived substrate of this system
(Walter et al., 2001
). A
second ubiquitin-conjugating enzyme involved in ERAD, Ubc7p, is also localized
to the ER membrane but lacks a membrane anchor. This Ubc is recruited to the
ER surface through interaction with its membrane-bound receptor Cue1p
(Biederer et al., 1997
).
Involvement of Ubc7p in ERAD has been demonstrated for several substrates
(Biederer et al., 1996
;
Hiller et al., 1996
;
Hampton and Bhakta, 1997
).
Recently, Ubc1p has also been implicated in ERAD of CPY*
(Friedlander et al., 2000
;
Bays et al., 2001a
), and Ubc1p
and Ubc4p in the turnover of unassembled Vph1p
(Hill and Cooper, 2000
).
Although there has been considerable progress in the identification and
functional characterization of Ubcs required for ERAD in yeast, little is
known about ERAD-related Ubcs in mammalian cells.
Here we describe the identification and functional characterization of two
distinct families of Ubc6 orthologues in mammals. Both families share
significant sequence similarity with the yeast enzyme and display a similar
structural organization. Although both share similar topological arrangement
in the ER membrane with yeast Ubc6p, neither mammalian protein is capable of
complementing deficiencies in yeast. Interestingly, overexpression of
wild-type and mutant forms of members of both families of Ubc6p-related
proteins appear to exert a dominant-negative phenotype on the degradation of
two well-characterized mammalian ERAD substrates, F508-CFTR and
TCR
.
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Materials and Methods |
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The mouse cDNA 423319 contained a single 777 bp ORF and has also been deposited in GenBank (accession no. U93242). This sequence encodes a protein of 259 amino acids with 58% identity to yeast Ubc6p and was therefore termed MMUBC6. The human cDNA 417998 contained a 3' sequence, which aligned to mouse cDNA 423319. The assumed ORF encodes C-terminal 197 amino acids with 93% identity to mmUbc6p.
To generate epitope-tagged versions of hsUbc6ep and mmUbc6p the
corresponding DNA sequences were subcloned into pSL 1190. Then a PCR fragment
containing three successive myc-epitopes
(Schneider et al., 1995) was
ligated in-frame to the N-terminus of both sequences. PCR-mutagenesis was used
to introduce the functional mutations C91S into HSUBC6e and myc-HSUBC6e, and
C94S into MMUBC6 and myc-MMUBC6.
All generated versions of HSUBC6e and MMUBC6 (wild-type, myc-tagged wild-type, Cys/Ser-mutants and myc-tagged Cys/Ser-mutants) were subcloned into the following yeast expression vectors: pRS426 (2µ), pRS414 (ARS-CEN) and pDP83 (ARS-CEN). Expression was driven by the CUP1 (pRS426), GAL10 (pRS414) and the endogenous yeast UBC6 (pDP83) promoter sequences.
For the expression in HEK 293 cells, HSUBC6e, HSUBC6e-C91S, myc-HSUBC6e,
myc-HSUBC6e-C91S, MMUBC6, MMUBC6-C94S, myc-MMUBC6 and myc-MMUBC6-C94S were
subcloned into pCMVPLD, a pCMV (Stratagene)-derived eukaryotic expression
vector. HEK 293 cells were maintained in complete DMEM medium and were
transiently transfected using calcium phosphate-DNA precipitates formed in
Hepes (Ausubel et al.,
1989).
The cDNA corresponding to 2B4 TCR
(Saito et al., 1987
), a kind
gift from J. Bonifacino (NIH, Bethesda, MD), was subcloned into pCDNA3.1
(Stratagene) for expression in HEK cells. HEK cells were grown in DME
containing 10% fetal bovine serum, penicillin and streptomycin at 37°C and
5% CO2. The cells (
2x106) were transiently
cotransfected by calcium phosphate precipitation as described previously
(Ward and Kopito, 1994
) with 3
µg TCR
.
Expression analysis, localization and pulse chase analysis
Microsomes of pCMVPLDmyc-HSUBC6e- or pCMVPLDmyc-MMUBC6-transfected HEK 293
cells were prepared as follows: 24 hours post transfection cells were scraped,
then washed once in PBS and once in HB-buffer (50 mM Tris/HCl pH 7.5, 250 mM
saccharose, 2 mM EDTA, 150 mM KCl, 1 mM DTT). The cell suspensions were
carefully dounced on ice for 5 minutes and then centrifuged at 3000
g for 10 minutes at 2°C. These crude homogenates were used
for protease protection assays (see below). Membranes were prepared by
centrifugation of the homogenates at 10,000 g for 15 minutes.
The supernatants were then centrifuged at 75,000 g for 60
minutes and the pellets were resuspended in membrane buffer (150 mM sucrose,
50 mM Hepes pH 7.5, 2.5 mM MgOAc, 50 mM KOAc). Expression of
myc-tagged Ubc6 homologues was checked by SDS/PAGE of membranes and
supernatants followed by immunoblotting using an anti-myc antibody
(Santa Cruz Biotechnology). Protease protection assays were done according to
Sommer and Jentsch (Sommer and Jentsch,
1993). Crude HEK 293 microsomes remained untreated, treated with
proteinase K (50 µg/ml) or treated with proteinase K (50 µg/ml) and 1%
Triton X-100 on ice.
For pulse-chase experiments, 48 hours following transfection, HEK cells
were starved in Cys/Met-free DME for 30 minutes, pulse-labeled with 500
µCi/ml [35S]Met/Cys (>1,000 Ci/mmol, NEN), and chased in
complete growth media with 75 µM emetine (Sigma). The cells were washed and
lysed in IPB+ (10 mM Tris, pH 7.5, 5 mM EDTA, 0.15 M NaCl, 1% NP-40, 0.5%
deoxycholate, 2 mg/ml BSA) supplemented with a protease inhibitor cocktail (1
mM PMSF, 100 µM TLCK, 200 µM TPCK, 10 µg/ml ALLN). The lysate was
cleared of insoluble material by centrifugation at 16,000 g.
The resulting detergent-insoluble pellet was sonicated in 50 µl 10 mM Tris,
pH 7.5 and 1% SDS then diluted in 0.95 ml IPB+. Samples were pre-cleared with
50 µl Gammabind Sepharose beads (Pharmacia) for 2 hours at 4°C.
Immunoprecipitation was performed using monoclonal anti-TCR antibody
H28-7-10 (Becker et al., 1989
)
and Gammabind Plus Sepharose overnight. The Gammabind beads were washed
sequentially with WB1 (10 mM Tris, pH 7.5, 0.1% NP-40, 0.15 M NaCl, 1 mM
EDTA), WB2 (10 mM Tris, pH 7.5, 2 mM EDTA, 0.05% SDS), and WB3 (10 mM Tris, pH
7.5, 2 mM EDTA). The beads were boiled for 3 minutes and the
immunoprecipitates fractionated in 11% SDS-polyacrylamide gel, and analyzed by
fluorography or phosphorimager analysis (Molecular Dynamics).
FACS analysis
HEK 293 cells expressing GFPu, grown on 10 cm dishes, were
transfected with 8 µg of vector alone (mock-transfected shown as control),
wild-type hsubc6 or hsubc6eser using calcium phosphate
precipitation. 48 hours after transfection, GFP fluorescence of transfected
cells was measured using Coulter Epics XL-MCL Flow Cytometer and EXPO v.2
cytometer software.
Immunofluorescence microscopy
HEK 293 cells were grown adhered on coverslips in complete DMEM medium and
transfected with myc-tagged expression constructs. 24 hours post-transfection,
coverslips were washed in PBS and cells were fixed in methanol/acetone for 5
minutes at -20°C. Then coverslips were air dried, cells were rehydrated in
PBS and primary antibodies were incubated for 90 minutes at room temperature.
Coverslips were washed in PBS, incubated for 20 minutes with biotin-labeled
secondary antibody, washed in PBS again and then transferred to
Streptavidine-FITC labeled antibody solution for 20 minutes. After a final
washing procedure with PBS, coverslips were embedded in mounting solution and
prepared for fluorescence microscopy, which was performed using a Zeiss
Axioplan 1 microscope equipped with a MC 100 SPOT camera (Zeiss, Oberkochen,
Germany).
Yeast experiments
Yeast minimal media (SD) were prepared as described and standard
transformation techniques were employed
(Ausubel et al., 1989). The
following yeast strains were used: YWO1 [MAT
, trp1-1(am),
his3-
200, ura3-52, lys2-801, leu2-3,-112
(Seufert et al., 1990
)],
YTX150 (MAT
, sec61-2, trp1-1(am), his3-
200, ura3-52,
lys2-801, leu2-3,-112), YTX140 [MATa, prc1-1, trp1-1(am),
his3-
200, ura3-52, lys2-801, leu2-3,-112
(Biederer et al., 1997
)], and
K700 [MAT
, trp1-1, can1-100, ura3, leu2-3,-112, his3-11,-15,
ssd1-d (K. Nasmyth, IMP, Vienna, Austria)]. YTX150 was constructed by
cloning the sec61-2 allele [YFP338
(Biederer et al., 1996
)] into
pRS406 and subsequent integration into YWO1 by the plasmid shuffle technique
(Ausubel et al., 1989
). The
integration vector was sequenced (E.H. and T.S., unpublished) and correct
integration into YWO1 was confirmed by testing for temperature sensitivity.
Ubc6p and the active site mutant Ubc6serp were overexpressed from a
high-copy plasmid (pSEY8) as described
(Sommer and Jentsch, 1993
).
The expression vector pUB23P, which encodes for the N-end rule substrate
Pro-ß-galactosidase (Bachmair et al.,
1986
), was a kind gift of J. Dohmen (Cologne).
Pulse chase experiments were basically conducted as described previously
(Biederer et al., 1996), except
that total lysates were prepared for anti-CPY* and Ub-Pro-ßGal
immuno-precipitation (Friedlander et al.,
2000
).
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Results |
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All sequences harbored an N-terminal Ubc domain that differed significantly in its primary sequence from that of their closest homologues, the Ubc4/5 protein family. Sequence comparison between the Ubc domain of yeast and human Ubc6p revealed an identity of 55.5%, while human Ubc4p and Ubc6p showed only 23.5% identity. All Ubc6 homologues contained a tail region consisting of many charged amino acids following the Ubc domain. The extreme C-terminus contained stretches of hydrophobic amino acids, which may serve as membrane anchors. In no case was there evidence of an N-terminal signal sequence. Thus, all members of the Ubc6p family appear to belong to the class of tail-anchored (type IV) membrane proteins.
The Ubc6p family could be further divided in two different sub-families (Fig. 1A,B): the first group comprised cDNAs encoding proteins with a high degree of overall sequence identity with Ubc6p (approximately 40%) and a similar calculated molecular weight (28 kDa). The second group displayed less similarity to yeast Ubc6p (approximately 25% identical residues) and contained a longer C-terminal tail region containing the transmembrane segment. The first group was named Ubc6p and the latter sub-family, Ubc6ep. Sequence comparison of the two subtypes derived from one organism make it unlikely that the two proteins are generated by alternative splicing (data not shown). In both groups the sequence identity in the Ubc domain was higher than in the tail region: comparison of human Ubc6ep and yeast Ubc6p revealed 25% overall sequence identity and 36% identity in the Ubc domain. In contrast, 40% overall identity was found in a comparison of yeast and human Ubc6p, while in the Ubc domain the conservation was even higher (55%).
For functional analysis, cDNAs corresponding to mammalian representatives of each sub-family were obtained from the German Resource Center for Genome Research (RZPD) and their identity to the corresponding proposed open reading frame was verified by sequencing. A sequence alignment of these proteins, mmUbc6p, a member of the highly conserved class of Ubc6 homologues, and hsUbc6ep, a member of the larger class, with yeast Ubc6p is shown (Fig. 1B).
While members of the Ubc6p family seem to be present in all eukaryotic kingdoms, including fungi, plant, protists and metazoa, a representative of the Ubc6ep family was not found in yeast or D. melanogaster, but was in plants (including the green algae Chlamydomonas rheinhardii) and in many groups of the metazoa (worms, vertebrates). Several protists also contain open reading frames that display striking similarity to Ubc6ep; however, at present it is not clear whether these genes code for Ubc6ep-like membrane-anchored proteins or whether they represent another gene orthologue to Ubc6p that codes for a soluble protein. The existence of both families in many metazoa indicates that the function of the single Ubc6p found in the yeast Saccharomyces cerevisiae may be subsumed by two distinct membrane-bound ubiquitin-conjugating enzymes in higher eukaryotic cells.
Ubc6 homologous proteins can be divided into two families
We were interested whether members of both families of proteins exhibit the
same membrane topology and are located in the same cellular membrane as yeast
Ubc6p. For this purpose we fused a c-myc epitope to the N-terminus of mmUbc6p,
and to hsUbc6ep, a human member of the Ubc6e-family. The tagged versions were
expressed in HEK 293 cells from plasmids under control of the CMV-promotor.
Signals corresponding to myc-mmUbc6p and myc-hsUbc6ep were detectable with an
anti-myc antibody in immunoblots of total membrane preparations from these
transfectants but not in mock-transfected cells
(Fig. 2A). This observation,
together with the finding that relatively little Ubc6p immunoreactivity
partitions in cytosolic fractions (data not shown), suggest that these
proteins, like their yeast counterpart are membrane-associated.
Immunfluorescence microscopy revealed that myc-tagged mmUbc6p and hsUbc6ep
were distributed in a classical reticular pattern similar to that observed for
an ER membrane protein marker, TRAP
(Fig. 2B). Taken together,
these results suggest that members of both families of mammalianUbc6p-related
proteins are associated with ER membranes.
|
To assess the nature of the interaction of Ubc6 homologues with membranes,
microsomes from transient transfectants were subjected to extraction with
inorganic chaotropes or detergent (Fig.
3A). Similar to the known ER membrane protein TRAP, the
mammalian Ubc6 homologues were resistant to extraction by chemical chaotropes
but were extracted in non-ionic detergent. Since the membrane anchors of all
the proteins investigated were at the extreme C-terminus, we performed
protease protection assays using microsomal vesicles prepared from cells
expressing the epitope-tagged Ubcs in order to assess the topology of the
Ub-conjugation domain with respect to the bilayer
(Fig. 3B). Both myc-mmUbc6p and
hsUbc6ep are susceptible to digestion by proteinase K in the absence and, of
course, also in the presence of detergent. At the same time, the lumenal
protein GRP78 was protected from the protease, indicating that the membranes
were sealed. These data suggest that, like yeast scUbc6p, both hsUbc6ep and
mmUbc6p are oriented in the ER membrane with their epitope-tagged N-termini
exposed to the cytosol.
|
The mammalian Ubc6 homologues are tail-anchored ER-membrane
proteins
In order to test a possible involvement in ERAD, we expressed hsUbc6ep and
mmUbc6p in yeast mutants lacking scUbc6p and bearing the sec61-2
allele. Cells expressing the conditional lethal allele sec61-2 are
unable to form colonies at 37°C, because its gene product is degraded in a
scUbc6p-dependent manner at elevated temperature
(Biederer et al., 1996).
Loss-of-function mutants of scUbc6p exhibit reduced ERAD capacity and thus are
suppressors of the temperature sensitivity of the sec61-2 allele
(Sommer and Jentsch, 1993
).
HsUbc6ep and mmUbc6p are unable to replace yeast scUbc6p in this assay, since
the temperature-sensitive phenotype of sec61-2
ubc6 was not
restored upon expression of the mammalian homologues. In parallel, controls
with yeast scUbc6p were performed and the expression of the mammalian
Ubc6p-related proteins was verified by immunoblotting (data not shown).
However, the expression level of Ubc6p might be critical for normal
ERAD-function, as it was reported that overexpression of wild-type scUbc6p
also suppresses the temperature-sensitive growth of a sec61-2 mutant
(Sommer and Jentsch, 1993
).
Therefore we tested different expression levels of hsUbc6ep and mmUbc6p for
complementation. Irrespective of whether we overexpressed the proteins
(multi-copy vector, induced CUP promotor, galactose induced
GAL10-promotor) or whether we expressed them at low levels from single-copy
vector, neither mmUbc6p nor hsUbc6ep restored the temperature sensitivity of
sec61-2
ubc6 cells at elevated temperature. Even when we
expressed both mammalian enzymes under control of the weak yeast UBC6
promotor, complementation could not be achieved (data not shown).
In contrast to the results with the mammalian homologues, we found that overexpression of yeast scUbc6p (from a multi-copy plasmid) exerts a dominant inhibitory effect on ERAD. Elevated levels of yeast scUbc6p restored growth of sec61-2 cells at non-permissive temperature, indicative of stabilization of Sec61-2p. This effect was even more pronounced when a non-functional version of scUbc6p was used (Fig. 4A). In this mutant (scUbc6pser), the active-site cysteine residue was replaced by serine, thereby abolishing ubiquitin-conjugating activity. We also tested the effect of overexpresion of scUbc6p and scUbc6pser on the degradation of mutant carboxypeptidase Y (CPY*) (prc1-1). In agreement with the genetic experiments using sec61-2, we observed that the half-life of CPY* was prolonged from 17.5 minutes to 31.5 minutes in scUbc6p-overexpressing cells. Elevated levels of scUbc6pser further increased the half-life of CPY* to 60 minutes (Fig. 4B). These data suggest that overexpressed scUbc6p, and to a greater extent scUbc6pser, is a dominant inhibitor of ERAD in yeast. To exclude that this effect is due to a general delay in ubiquitin-dependent proteolysis, we measured the turnover of an N-end rule substrate, Ub-Pro-ßGal, in parallel. Turnover of this cytosolic substrate was unaffected and thus we concluded, that specifically ER-associated proteolysis was affected (Fig. 4C).
|
Dominant-negative alleles of Ubc6 interfere with ERAD in yeast and
mammals
To assess the role of Ubc6p in mammalian ERAD we overexpressed wild-type
mmUbc6p, hsUbc6ep and the corresponding mutants in the active site Cys (C94S
for mmUbc6pser and C91S for hsUbc6epser) together with
ERAD substrates. Pulse-chase labeling was used to assess the influence of
these Ubc6 constructs on the stability of TCR or
F508
transiently co-transfected into HEK cells
(Fig. 5A). Similar to the
results in yeast, overexpression of either mammalian wild-type Ubc6p isoform
induced a small, but reproducible stabilization of both substrates. Likewise,
overexpression of the mammalian active site mutants resulted in significant
additional stabilization of TCR
(
50-80 minutes) and of
F508
(
75-140 minutes). These results do not simply reflect a nonspecific
effect of protein overexpression since the Ubc6 data were compared with cells
in which equal amounts of GFP plasmid were cotransfected. In addition, a
short-lived cytosolic substrate, GFPu
(Bence et al., 2001
) was
investigated. Its steady state level was not altered by overexpression of
wild-type hsUbc6ep and the corresponding active site mutant hsUbc6e-C91S
(Fig. 5B). Since the steady
state level of GFPu rises rapidly if its turnover is reduced, we concluded
that the half-life of GFPu was not affected by Ubc6p
overexpression. In parallel experiments, an increased GFPu level
was observed upon overexpression of ubiquitin K48R, a dominant inhibitor of
ubiquitin-dependent proteolysis (data not shown). Thus, similar to the
situation in yeast, the delay in turnover is be limited to proteolytic events
at the ER membrane.
|
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Discussion |
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We observed that overexpression of wild-type scUbc6p in S.
cerevisae delays turnover of the soluble ERAD substrate CPY*
and restores growth of sec61-2 mutant cells. These effects were even
more pronounced when a catalytically inactive version of scUbc6p is
overexpressed. Similar to the observation in yeast, we noticed that
overexpression of wild-type and mutant version of mmUbc6p and hsUbc6ep delay
the turnover of TCR and
F508CFTR in mammalian cells. However, in
both yeast and mammals the slowed decay rates are limited to ERAD substrates,
since the turnover short-lived cytosolic substrates remained unaffected. It is
likely that this dominant effect of Ubc6p overexpression, which has not been
hitherto reported, reflects titration of a limiting component in the ERAD
pathway. Our results differ from previous studies
(Tiwari and Weissman, 2001
)
reporting that overexpression of murine Ubc6pser did not influence
the turnover of TCR
. However, under the experimental conditions used by
Tiwari et al., the half-life of TCR
in the absence of any Ubc6p
overexpression was between 3.5 and 4.1 hours. This is significantly
slower than all other previous reports of TCR
, which put the half-life
between 30 and 60 minutes, independent of the particular cell line or
expression level (Lippincott-Schwartz et
al., 1988
; Bonifacino et al.,
1989
; Yu et al.,
1997
; Huppa and Ploegh,
1997
; Yang et al.,
1998
). Thus stabilizing effects caused by Ubc6p overexpression
might have been hardly detectable under these conditions.
Our results suggest that in mammalian cells, like in yeast cells, the
expression level of Ubc6p is critical for normal ERAD. The complete absence of
Ubc6p in yeast leads to a slight delay in ERAD of CPY* and Sec61-2p
but has no effect on HmgR degradation
(Biederer et al., 1996;
Hiller et al., 1996
;
Friedlander et al., 2000
). As
shown here, elevated levels of scUbc6p also lead to a decrease in the turnover
of some ERAD substrates. Thus, it is feasible to speculate that Ubc6p
functions as a modulator of the ERAD activity. In this context, it is
interesting to note that scUbc6p is itself a short-lived protein, the
degradation of which is dependent on membranebound Ubc7p
(Walter et al., 2001
). In
mammalian cells, elevated levels of wild-type and active site center mutants
of Ubc6p have comparable effects on the mammalian ERAD substrates TCR
and
F508CFTR. Therefore, not only the sequence of Ubc6p is highly
conserved during evolution, but also its function seems to be preserved from
yeast to mammals.
In conclusion, the data reported here identify two mammalian homologues of yeast scUbc6p that are residents of the ER membrane, and suggest that these ubiquitin conjugating enzymes may contribute to quality control ER-associated degradation by the ubiquitin-proteasome system. We were unable to find conditions in which either of the mammalian enzymes could replace the function of yeast scUbc6p. This might be due to the fact that it is critical to adjust the expression level in a way that allows ERAD to proceed normally or that the mammalian homologues are unable to interact with other components of the yeast ERAD machinery. An alternative explanation is that the function of the single yeast Ubc6p is split between the two mammalian homologues. Future studies will be needed to discriminate among these possibilities.
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Acknowledgments |
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