E2-25K mediates US11-triggered retro-translocation
of MHC class I heavy chains in a permeabilized
Dennis Flierman*, Catherine S. Coleman*, Cecile M. Pickart†‡, Tom A. Rapoport§¶, and Vincent Chau*¶
*Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033;†Department of Biochemistry
and Molecular Biology, The Johns Hopkins University, Baltimore, MD 21205; and§The Howard Hughes Medical Institute and Department of Cell Biology,
Harvard Medical School, Boston, MA 02115
Contributed by Tom A. Rapoport, June 21, 2006
In cells expressing human cytomegalovirus US11 protein, newly
synthesized MHC class I heavy chains (HCs) are rapidly dislocated
from the endoplasmic reticulum (ER) and degraded in the cytosol,
a process that is similar to ER-associated degradation (ERAD), the
pathway used for degradation of misfolded ER proteins. US11-
triggered movement of HCs into the cytosol requires polyubiquiti-
nation, but it is unknown which ubiquitin-conjugating and ubiq-
uitin-ligase enzymes are involved. To identify the ubiquitin-
conjugating enzyme (E2) required for dislocation, we used a
permeabilized cell system, in which endogenous cytosol can be
replaced by cow liver cytosol. By fractionating the cytosol, we
show that E2-25K can serve as the sole E2 required for dislocation
of HCs in vitro. Purified recombinant E2-25K, together with com-
ponents that convert this E2 to the active E2-ubiquitin thiolester
form, can substitute for crude cytosol. E2-25K cannot be replaced
by the conjugating enzymes HsUbc7?Ube2G2 or Ube2G1, even
though HsUbc7?Ube2G2 and its yeast homolog Ubc7p are known
to participate in ERAD. The activity of E2-25K, as measured by
ubiquitin dimer formation, is strikingly enhanced when added to
permeabilized cells, likely by membrane-bound ubiquitin protein
ligases. To identify these ligases, we tested RING domains of
RING domains of gp78?AMFR, a ligase previously implicated in
ERAD, and MARCHVII?axotrophin, a ligase of unknown function,
greatly enhanced the activity of E2-25K. We conclude that in
permeabilized, US11-expressing cells polyubiquitination of the HC
substrate can be catalyzed by E2-25K, perhaps in cooperation with
the ligase MARCHVII?axotrophin.
cytomegalovirus ? ubiquitin-mediated proteolysis ? ubiquitin-conjugating
enzyme ? ubiquitin protein ligase
polyubiquitin chains and subsequently degraded by the 26S pro-
teasome (1, 2). Ubiquitin-mediated proteolysis also functions in
protein quality control, resulting in the degradation of misfolded or
damaged proteins. A particularly well studied quality-control sys-
tem is found in the endoplasmic reticulum (ER) (3, 4). A large
number of diseases are known in which mutant proteins fail to fold
properly in the ER and are degraded. Examples include the CFTR
protein in cystic fibrosis, ?1-antitrypsin in childhood liver disease
and adult emphysema, low-density lipoprotein receptor in familial
hypercholesterolemia and myeloperoxidase deficiency, and insulin
receptor in type A insulin resistance (1, 5). It was initially believed
that protein degradation occurs inside the ER (6), but it is now
accepted that misfolded proteins are transported back into the
cytosol, a process termed dislocation or retro-translocation, before
they are degraded by the proteasome (4, 7). In this ER-associated
degradation (ERAD) pathway most substrates are polyubiquiti-
nated before being moved into the cytosol (8, 9).
Polyubiquitination of proteins requires the concerted action of a
ubiquitin-conjugating enzyme (E2) and a ubiquitin protein ligase
he expression level of a large set of proteins in eukaryotes is
regulated by proteolysis, in which proteins are modified with
(E3). Ubiquitin is linked via its C-terminal carboxyl group to a
cysteine in an E2 to form a thiolester, a reaction that is catalyzed
by the ubiquitin-activating enzyme (E1) in the presence of ATP.
This E2-ubiquitin thiolester complex interacts with an E3 that is
bound to a substrate, leading to the transfer of ubiquitin to the
substrate protein. Our understanding of the specificity of E2–E3
interactions is incomplete, but available evidence indicates that
most individual E3 enzymes use a specific cognate E2 enzyme. In
Saccharomyces cerevisiae, Ubc7p is the E2 that plays the most
prominent role in ERAD, although Ubc1p and, to a lesser extent,
Ubc6p can also participate (10–17). Although the mammalian
homolog of Ubc7p, HsUbc7?Ube2G2, has been demonstrated to
function in ERAD (18–20), it is not clear whether it has a similar
more E2 enzymes than yeast, and the functions of most ubiquitin-
conjugating enzymes have not yet been clarified.
cells infected with the human CMV, newly synthesized MHC class
I heavy chains (HCs) are rapidly dislocated from the ER and
degraded by the proteasome, in a manner resembling the disloca-
of either US2 or US11, two small virally encoded proteins that are
inserted into the host ER membrane, is sufficient for this process,
indicating that the dislocation of HCs from the ER membrane uses
the US11-triggered pathway. It begins with the recognition of HC
by US11. US11 probably delivers the substrate to the US11-
interacting, multispanning membrane protein Derlin-1, postulated
cytosolic side of the ER membrane, a polyubiquitin chain is
attached to a part of the HC that was previously in the ER lumen
(22). The polyubiquitin chain is subsequently recognized by an
ATPase complex (25–27), consisting of the AAA ATPase p97 and
a cofactor (Ufd1p-Npl4p). It is thought that the ATPase complex
moves the substrate into the cytosol in a process that requires ATP
hydrolysis (4, 25, 27). Much of our current understanding of the
US11-dependent dislocation pathway comes from the use of a
permeabilized cell system (8) in which astrocytoma cells, stably
expressing US11, are permeabilized with digitonin and the cytosol
is exchanged or manipulated.
One of the most important steps in the US11-dependent dislo-
cation pathway is polyubiquitination, and yet this process is only
poorly understood, particularly because neither the E2 nor the E3
Conflict of interest statement: No conflicts declared.
Freely available online through the PNAS open access option.
Abbreviations: HC, heavy chain; ER, endoplasmic reticulum; ERAD, ER-associated degrada-
tion; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin
protein ligase; f-Ub, Oregon green-labeled ubiquitin.
‡Deceased April 5, 2006.
¶To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
© 2006 by The National Academy of Sciences of the USA
August 1, 2006 ?
vol. 103 ?
no. 31 ?
enzymes involved have been identified. In the present study, we
have used the permeabilized cell system to identify a ubiquitin-
conjugating enzyme (E2-25K) that, together with ubiquitin, ATP,
and E1, is sufficient to replace crude cytosol in the HC dislocation
assay. We also identify E3 enzymes that activate the activity of
E2-25K and are thus candidates for playing a role in the dislocation
We previously described a permeabilized cell system to follow
US-11 mediated dislocation of MHC class I HCs (8). HC disloca-
tion in such permeabilized cells relies on exogenously added
cytosol, which can be manipulated to address the role of cytosolic
factors in this reaction. Because polyubiquitinated HC is an inter-
mediate in the dislocation reaction (8, 22, 27), it is reasonable to
expect that the required cytosolic factors include proteins involved
in HC polyubiquitination. Because most known E2 enzymes are
cytosolic proteins, we first concentrated on the identification of an
E2 enzyme involved in the dislocation of HCs.
US11-expressing astrocytoma cells were pulse-incubated with
[35S]methionine and [35S]cysteine and permeabilized in digitonin,
and the cytosol was removed by sedimentation. The cells were
resuspended in cow liver cytosol and incubated for an additional 30
showed that a large fraction of HC was converted into a faster
migrating species (Fig. 1A, lane 6 versus 5). Previous experiments
have shown that the conversion is caused by the removal of a
carbohydrate chain from HC, which is catalyzed by a cytosolic
N-glycanase, and by deubiquitination of previously polyubiquiti-
nated HC (8, 22). The gel mobility shift is thus an indication of
dislocation of HC from the ER lumen to the cytosol. Upon
fractionation of the sample by low-speed centrifugation, deglyco-
sylated HC appeared in the supernatant (Fig. 1A, lane 7), whereas
residual, nondislocated material was found in the pellet (Fig. 1A,
lane 8). We also observed some discrete species with slower
mobility that likely corresponds to ubiquitinated HC (Fig. 1A, lane
7). When cytosol-depleted cells were incubated with purified ubiq-
uitin and E1, instead of cytosol, no shift in gel mobility of HC was
observed and no material appeared in the supernatant fraction
(Fig. 1A, lanes 1–4), indicating that these proteins alone are
insufficient to support dislocation of HC.
As part of our initial effort to identify the relevant E2, we
subdivided cow liver cytosol into two fractions, one that was
retained on an anion-exchange gel matrix (FII) and another that
was not (FI). Both fractions contain E2 enzymes and FI contains
ubiquitin, whereas FII is depleted of ubiquitin (data not shown).
When added to permeabilized cells, complementation was found
with FII (Fig. 1A, lanes 13–16), but not with FI (Fig. 1A, lanes
9–12), and combining FI and FII did not increase the efficiency of
a well established procedure to further enrich all E2 enzymes from
FII (28, 29). The proteins in this fraction were incubated with
ubiquitin-coupled gel beads, which led to the binding of all E2
enzymes via a thiolester bond linkage. Elution was performed with
a reducing agent that cleaves the thiol bonds (28, 29). Because E1
eluate. When this E1?E2-enriched fraction was added together
with ubiquitin to cytosol-depleted permeabilized cells, dislocation
of HC, and the appearance of slower migrating species character-
istic of ubiquitinated HC, were observed (Fig. 1B, lanes 3 and 4).
lane 4), indicating that the E1?E2-enriched fraction may be defi-
cient in N-glycanase activity.
To identify the E2 enzyme, the E1?E2-enriched protein mixture
was bound to a Mono-Q column and eluted with a linear salt
gradient. Twenty-five fractions were collected and tested for the
presence of E2 enzymes by the generation of E2-ubiquitin thio-
lesters. Twelve fractions contained one or more distinct E2s (data
not shown). When these fractions were tested for HC dislocation
with cytosol-depleted permeabilized cells, E1, and ubiquitin, frac-
tion 9 was found to be most active (f9, Fig. 2A, lanes 5 and 6, and
with fluorescently labeled ubiquitin, two E2 enzyme activities were
apparent molecular masses of 35 and 27 kDa (Fig. 2B, lane 2). The
35-kDa thiolester was most prominent in fraction 9 (Fig. 2B, lanes
1–3), indicating that the corresponding E2 enzyme shows a good
correlation with the activity in the dislocation assay. Fraction 9
labeled as A and C, have molecular masses consistent with E2s that
could form ubiquitin-thiolesters of 35 and 27 kDa, respectively.
The bands corresponding to proteins A and C were excised and
treated with trypsin, and the resulting peptides were subjected to
MALDI-TOF analysis. The peptide mass data were used to search
the protein database and identified proteins A and C as the
ubiquitin-conjugating enzymes E2-25K and Ubc13, respectively.
Band D was identified in a similar way as UEV2, an E2 variant that
functions with Ubc13 in a heterodimer (30, 31). The identity of
tryptic peptides from each protein, which in all cases yielded
fragmentation patterns consistent with the expected peptide se-
quence. Thus, the two E2 activities seen in f9 are caused by the
presence of E2-25K and Ubc13, and E2-25K is the better candidate
for being involved in the dislocation of HC.
To directly test a role for E2-25K in HC dislocation, we used
purified recombinant protein made in Escherichia coli. When
purified E2-25K, ubiquitin, and E1 were added to cytosol-depleted
permeabilized cells, efficient dislocation of HC was observed (Fig.
added cytosolic proteins. Cells expressing the human cytomegalovirus US11
protein were pulse-labeled with [35S]methione?cysteine, permeabilized, pel-
leted, and washed to deplete their cytosol. Dislocation reactions were initi-
ated by the addition of ATP together with cytosol or cytosolic proteins as
specified. HC immunoprecipitates were analyzed by SDS?PAGE and autora-
diography. Radiolabeled proteins corresponding to glycosylated HC and deg-
lycosylated HC are marked as HC?CHO and HC?CHO, respectively. (A) Reac-
tions contained either 1 ?M E1 (lanes 1–4), cow liver cytosol (lanes 5–8), FI
(supplemented with 1 ?M E1, lanes 9–12), or FII (lanes 13–16). (B) Reactions
contained 1 ?M E1 (lanes 1 and 2), enriched E1?E2 fraction (lanes 3 and 4), or
FII from cow liver cytosol (lanes 5 and 6). Ubiquitin was added to all reactions
at 20 ?M.
HC dislocation in permeabilized cells initiated with exogenously
www.pnas.org?cgi?doi?10.1073?pnas.0605215103Flierman et al.
2C, lanes 5–8). Sequential immunoprecipitation with antibodies
against HC and ubiquitin demonstrated directly that HC is ubiqui-
tinated in the presence of added E2-25K (Fig. 2C, lanes 13–16), but
not with E1 alone (Fig. 2C, lanes 9–12). Thus, we conclude that
E2-25K can act as the sole E2 for polyubiquitination and subse-
quent dislocation of HC.
Of the 11 E2s in the yeast S. cerevisiae, E2-25K most closely
resembles Ubc1p, both in the core UBC domain sequence and in
having a C-terminal UBA domain (32, 33). In yeast, Ubc1p and
Ubc7p may have overlapping functions in ERAD (10–12, 14, 17).
We therefore tested whether the mammalian orthologue of yeast
Ubc7p, HsUbc7?Ube2G2, which has been reported to function in
assay. In contrast to recombinant E2-25K, neither recombinant
enzymes were active in a ubiquitin-thiolester formation assay (data
an enzyme known to function in ERAD. Recombinant human
Ubc2b and Ubc3b, two other E2s present in FII, were also unable
to support HC dislocation (data not shown), providing additional
support that the activity seen with E2-25K is specific. A C-terminal
truncation mutant of E2-25K lacking the UBA domain could
UBA domain is not essential for the function of E2-25K in HC
Next, we wanted to identify potential ubiquitin protein ligase
partners of E2-25K, based on the assumption that they would
stimulate the activity of the E2. To measure the activity of E2-25K
we used the fact that it has the ability to form ubiquitin dimers by
transfer of thiolester-linked ubiquitin to free ubiquitin. Additional
ubiquitin moieties can then be attached, leading to formation of
higher-order ubiquitin oligomers with distributive kinetics (34, 35).
Although E2-25K on its own gave rise to only a small amount of
ubiquitin dimers and trimers, the addition of cytosol-depleted
permeabilized cells greatly stimulated the reaction (Fig. 4A, lane 4
versus 2). The addition of ubiquitin aldehyde, an inhibitor of
deubiquitinating enzymes, had only a small effect on dimer forma-
tion, but boosted the formation of trimers (Fig. 4A, lane 3). The
addition of E1 alone had also a small effect, presumably because
some E2 enzyme is still present in the permeabilized cells (Fig. 4A,
lanes 5–8). Taken together, these data suggest that a membrane-
exogenously added E2-25K.
Further identification of a potential ligase was based on the
previous observation that a similar ubiquitin dimer formation
activity of HsUbc7?Ube2G2 is activated by its interaction with the
RING domain of its known ubiquitin protein ligase partners Hrd1
or TEB4 (36, 37). We therefore tested whether E2-25K could also
be activated by RING domains of ubiquitin protein ligases. The
RING domains of several membrane-bound human ubiquitin
protein ligases were expressed in E. coli and purified. These
included the RING domains of the ligases Hrd1, gp78?AMFR,
TEB4?MARCH-VI, TRC8, MARCH-I, which activate HsUbc7?
Ube2G2 (36, 37), and the RING domains of the ligases MARCH-
II, III, IV, VII, VIII, and IX (38), which do not activate HsUbc7?
Ube2G2 (unpublished results). When tested with E2-25K, a large
stimulation of ubiquitin dimer formation was seen with the RING
domains of gp78?AMFR or MARCH-VII (Fig. 4B, lanes 4 and 8).
nation is supplied by E2-25K. (A) SDS-gel
analysis of HC immunoprecipitates from
reactions using permeabilized cells with
added E1?E2-enriched mixture (lanes 1
and 2), 1 ?M E1 (lanes 3 and 4), or f9
(supplemented with 1 ?M E1, lanes 5 and
6). All reactions were done in the pres-
ence of 20 ?M ubiquitin. (B) Assay of
E2-ubiquitin thiolester. In lanes 1–3, frac-
tions (f8–f10) from Mono-Q anion-
exchange separation of the E1?E2 mix-
ture were incubated with Oregon green-
labeled ubiquitin (f-Ub), E1, and ATP for
10 min. Proteins were separated on SDS-
gels and visualized by fluorescence. In
lanes 4 and 5, proteins from fractions 8
and 9 were separated on SDS-gels and
stained with Sypro Ruby Protein Stain
and visualized by fluorescence. (C) HC im-
munoprecipitates from reactions with
permeabilized cells supplemented with 1
?M E1 and 20 ?M ubiquitin alone, or
together with a recombinant E2-25K (5
?M) were either analyzed directly (lanes
1–8) or precipitated further with ubiq-
uitin-specific (?Ub) antibodies (lanes
9–16) and then analyzed by SDS?PAGE
and autoradiography. The band denoted
by*has a mobility similar to HC. (D)
Comparison of HC dislocation in assays
containing E1, cow liver cytosol, E1?E2-enriched fraction, recombinant HsUbc7, Ube2G1, or E2-25K. Reactions contained equivalent amounts of
permeabilized cells, and E2s were used at 5 ?M, E1 at 1 ?M, and ubiquitin at 25 ?M. Analysis was done by SDS?PAGE and autoradiography.
The E2 activity for HC ubiquiti-
Reactions containing radiolabeled permeabilized cells were supplemented
with 1 ?M E1, 25 ?M ubiquitin alone, or in the presence of 5 ?M either
in Materials and Methods). HC was recovered by immunoprecipitation with
anti-HC (?HC). Analysis was done by SDS?PAGE and autoradiography.
E2-25K does not require its UBA domain for dislocation of HC.
Flierman et al.
August 1, 2006 ?
vol. 103 ?
no. 31 ?
Representative time courses of this reaction with and without the
other RING domains, including TEB4, the proposed homolog of
Ssm4p?Doa10p, which has also been implicated in ERAD (39)
E2s (E2-25K and HsUbc7?Ube2G2) are activated by gp78?
VII. The two identified ligases are prime candidates to be involved
in the US11-triggered dislocation of HC.
We have identified a ubiquitin-conjugating enzyme, E2-25K,
that together with ubiquitin and the ubiquitin-activating enzyme,
can replace crude cytosol in generating polyubiquitinated HC,
allowing its subsequent dislocation from the ER into the cytosol.
Our results suggest that this E2 enzyme is involved in the
US11-triggered degradation of MHC class I HCs, but further
experiments are required to test its function in vivo. E2-25K was
first discovered by the Pickart laboratory (33, 34) by tracing an
intrinsic activity of this enzyme that allows the formation of
ubiquitin dimers linked through Lys-48 (K48). Although syn-
thetic K48-specific polyubiquitin chains made with the help of
this enzyme have been instrumental in our understanding of how
polyubiquitinated proteins are recognized by the proteasome
(40–42), a biological function for this enzyme had not been
established previously to our knowledge. The formation of
K48-linked polyubiquitin chains by E2-25K is consistent with our
previous observation that these types of chains are required for
interaction with the p97 ATPase complex that functions down-
stream by moving polyubiquitinated HC into the cytosol (25, 26).
Most of the ATPase complex is tightly bound to ER membranes
and thus present in cytosol-depleted permeabilized cells, ex-
plaining why purified E2, E1, and ubiquitin can satisfy the
cytosol requirement in our assay. Although we have shown that
purified E2-25K is sufficient to replace crude cytosol, we cannot
exclude the possibility that other E2 enzymes with redundant
function were inefficiently recovered during the purification
procedure. Also, it is possible that E2 enzymes are involved that
are already associated with the ER membrane. In yeast, ERAD
mediated by Doa10p requires Ubc6p, an E2 enzyme that is
anchored to the ER membrane via a C-terminal transmembrane
domain and Ubc7p that is recruited to the ER membrane via
binding to Cue1p (39). Thus, it remains to be determined
whether HC ubiquitination may have an additional requirement
for a membrane-associated E2.
The possibility that E2-25K functions in ERAD is further
suggested by its resemblance to S. cerevisiae Ubc1p, which among
This similarity includes the presence of a UBA domain not found
in other members of this family. However, it is not known
whether E2-25K provides the equivalent function(s) in mammals
or whether E2-25K can complement a UBC1 deletion in S.
cerevisiae. In yeast, Ubc1p functions with Hrd1p, an ER-resident
ubiquitin protein ligase that mediates the degradation of ER
proteins with misfolded luminal or intramembrane domains (12,
of these two enzymes can interact with the RING domain of
Hrd1p (12, 14, 17, 43), and HRD1-deletion phenotypes are
recapitulated in a UBC1?UBC7 double-deletion mutant, but
only partially in UBC1 or UBC7 single-deletion mutants (17).
Similar to the dual utilization of Ubc1p and Ubc7p by Hrd1p, we
have observed that both E2-25K and HsUbc7?Ube2G2 can be
activated by the RING domain of gp78?AMFR, a ligase that is
sequence-related to Hrd1p. Previous studies have indeed impli-
cated gp78?AMFR and HsUbc7 in the regulated degradation of
HMG-CoA reductase and the destruction of unassembled T cell
receptor (20, 44, 45). Our results thus raise the possibility that,
as in yeast, E2-25K might function interchangeably with HsUbc7
in these ERAD events. Many other aspects of the ERAD
pathways are conserved between yeast and mammals, but the
mammalian system is clearly more complex, with often more
than one homolog for a given yeast component. For example,
there are two mammalian homologs of yeast Ubc7p (HsUbc7?
Ube2G2 and Ube2G1) and yeast Hrd1p (Hrd1 and gp78?
AMFR), and it is possible that they function in the polyubiq-
uitination of distinct substrates.
E2-25K cannot be replaced by HsUbc7?Ube2G2 in its func-
tion in US11-triggered HC dislocation, but our data do not
exclude that other E2s could also function in HC polyubiquiti-
nation; the residual levels of E2 in cytosol-depleted permeabil-
ized cells make the assay rather insensitive and it is therefore
some activity. Nevertheless, the homologs of yeast Ubc1p and
Ubc7p are certainly the most obvious candidates for a function
in ERAD and HC dislocation. It is therefore surprising that only
the Ubc1p homolog E2-25K, and not the Ubc7p homolog,
functions in HC polyubiquitination. Apparently, the ubiquitin
protein ligase involved in HC polyubiquitination is more selec-
tive than gp78?AMFR, which interacts with both E2-25K and
formation activity in E2-25K. (A) Immunoblot show-
ing ubiquitin oligomer formation by E2-25K. Reac-
tion mixtures containing 1 ?M E1 and 100 ?M ubiq-
uitin with or without 5 ?M E2-25K were added to
Parallel reactions were also carried out in the ab-
sence of permeabilized cells. Where indicated, per-
meabilized cells were pretreated with ubiquitin C-
terminal aldehyde (Ubal). (B) Ubiquitin dimer assay
in the absence and presence of the specified RING
single turnover conditions as described in Materials
and Methods. Preformed E2-25K-f-Ub was then in-
cubated for 5 min at 4°C without and with 15 ?M
indicated RING domain and 10 ?M Ub74. Analysis
was done by SDS-gel separation of proteins and vi-
dimer formation was carried out with preformed
Ub74, in the absence or presence of 5 ?M RING
domain from MARCH-VII?axotrophin.
Activation of an intrinsic ubiquitin dimer
www.pnas.org?cgi?doi?10.1073?pnas.0605215103 Flierman et al.
the Ubc7p homolog. For this reason, gp78?AMFR is unlikely to
be the relevant E3 in HC ubiquitination. The only other tested
RING domain that activated E2-25K was the one derived from
MARCH-VII?axotrophin, and this RING domain did not acti-
vate HsUbc7?Ube2G2 (unpublished result). MARCH-VII is
therefore a prime candidate to be the ubiquitin protein ligase
involved in the polyubiquitination of HC. MARCH-VII has two
putative transmembrane sequences, but its subcellular localiza-
tion is unknown. Whether this ligase has a function in US11-
triggered HC dislocation remains to be determined.
Materials and Methods
Pulse–Chase Analysis with Permeabilized Cells. Control and US11-
expressing U373-MG astrocytoma cells (46) were cultured as
described (21). The cells were detached from tissue culture
flasks with trypsin and incubated in suspension in methionine-
and cysteine-free DMEM for 1 h at 37°C. Cells were then
centrifuged at 1,000 ? g for 5 min and resuspended at 1 ?
107?ml. They were pulse-labeled for 3–5 min at 37°C in 290
?Ci?ml [35S]methionine and -cysteine (35S-Protein Express La-
beling Mix; New England Nuclear, Wellesley, MA), after which
for 15 s at 18,000 ? g in a microfuge and resuspended at the same
cell density in PBS supplemented with 0.9 mM CaCl2. They were
then centrifuged as described, resuspended at 1.6 ? 107cells?ml
in PB (25 mM Hepes, pH 7.3?115 mM potassium acetate?5 mM
sodium-acetate?2.5 mM MgCl2?0.5 mM EGTA) containing
0.04% digitonin (Merck, Whitehouse Station, NJ, purified as
described in ref. 47), and incubated on ice for 10 min with gentle
agitation. The digitonin-permeabilized cells were pelleted by
centrifugation for 15 min at 18,000 ? g in a microfuge, resus-
pended at the same density in PB without digitonin, and pelleted
again by centrifugation as described.
The chase reaction was initiated by resuspending the perme-
abilized cell pellets in a buffer of 25 mM Tris?HCl, pH 7.6,
containing 10 ?M proteasome inhibitor PS-341, 1 mM ATP, 1
mM magnesium acetate, an ATP-regenerating system (48), and
other specified protein components. The samples were incu-
bated at 37°C, and aliquots were withdrawn at specified time
points. Withdrawn samples were divided into two equal parts.
One part was placed directly into NET lysis buffer, consisting of
0.5% Nonidet P-40 (Igepal; CA-630; Sigma, St. Louis, MO), 50
mM Tris?HCl (pH 7.6), 150 mM NaCl, 5 mM EDTA, a mam-
malian protease inhibitor mix (Sigma), and 0.2 mg?ml BSA. The
other part was centrifuged for 15 min at 18,000 ? g in a
microfuge, to obtain supernatant and pellet fractions, which
were then separately placed into NET lysis buffer. Lysates were
made by agitation on a rotator for 20 min at 4°C and subse-
quently centrifuged in a microfuge at 18,000 ? g for 10 min. The
resulting supernatant was used for immunoprecipitation after
SDS and DTT were added to final concentrations of 0.1% and
0.2 mM, respectively. The lysates made from samples corre-
sponded to 1–1.5 ? 106cell equivalents. Immunoprecipitations
were carried out as described (22).
Expression and Purification of Recombinant Proteins. E2-25K was
either expressed as a GST fusion (33, 49) or a nontagged protein
(49, 50) and purified accordingly. For the GST fusion, the GST
moiety was removed by thrombin cleavage, which left behind a
13-aa sequence of RRASVGSHMPMGD fused to the N termi-
nus of E2-25K. The UBA domain deletion mutant of E2-25K
(E2-25K?UBA) was created by changing the codon for residue
156 into a stop codon, expressed as a GST fusion, and purified
with the GST removed. HsUbc7?Ube2G2 was obtained by
expression as a tobacco etch virus protease-cleavable polyHis-
tagged protein and purified as described (51). The purified
HsUbc7 contains an extra two-residue sequence of Gly-His at its
N terminus. HsUbe2G1 was expressed as a GST fusion and
purified with the GST moiety removed but with an extra
two-residue sequence of Gly-Ser at its N terminus.
RING domain constructs for Hrd1 and gp78?AMFR were
expressed as histidine-tagged fusions. The coding sequences for
the RING domains were inserted between the NdeI and HindIII
sites of the pT7 plasmid. The inserted sequence for Hrd1
encodes residues 272–342 and encodes residues 322–394 for
gp78?AMFR. All other proteins were expressed as GST fusions,
where the RING domain coding sequences were inserted be-
tween the BamH1 and EcoRI sites in pGEX-4T1. The inserted
sequence encodes residues 529–599 for TRC8, 46–102 for
MARCH-I, 47–120 for MARCH-II, 52–127 for MARCH-III,
145–221 for MARCH-IV, 1–80 for MARCH V, 537–619 for
MARCH-VII, 62–137 for MARCH-VIII, and 91–168 for
MARCH IX. The GST-TEB4?MARCH-VI fusion was as de-
A truncated ubiquitin (Ub74) that lacks the C-terminal Gly-
Gly sequence was obtained by expressing the human sequence
that encodes the first 74 residues of the protein.
Fractionation of Cow Liver Cytosol. FI and FII were isolated from
cow liver cytosol by using a procedure described for reticulocyte
lysate (28). Briefly, 50 ml of cow liver cytosol (1.5 g protein) in
25 mM Tris?HCl, pH 7.6 was absorbed onto a 200 ml Q-
Sepharose (Amersham Pharmacia, Piscataway, NJ) column. The
to contain 90% ammonium sulfate to yield FI. Bound proteins
were eluted with 25 mM Tris?HCl (pH 7.6) and 0.6 M sodium
chloride and precipitated similarly to yield FII. The precipitated
proteins were dialyzed against 25 mM Tris?HCl, pH 7.6, con-
taining 0.1 mM DTT and stored at ?80°C until used. FI
contained 72 mg?ml and FII contained 31 mg?ml protein.
Ubiquitin Affinity Column Chromatography. E1 and E2 were ob-
tained from FII as described (28, 29). Ubiquitin-coupled gel
ml of activated CH-Sepharose (Amersham Pharmacia) accord-
ing to the manufacturer’s protocol. To obtain an E1 and
E2-enriched fraction from FII, 50 mg of FII proteins, supple-
mented with 5 mM ATP and 10 mM magnesium chloride, was
applied to the ubiquitin-coupled gel beads that were previously
equilibrated with 25 mM Tris?HCl, pH 7.6, containing 5 mM
proteins by washing with equilibration buffer, the gel beads were
treated with 25 mM Tris?HCl (pH 9) and 25 mM DTT to
facilitate the cleavage of thiolester bond that leads to release of
E1 and E2 enzymes from the gel beads. Eluted proteins from this
condition were dialyzed against 25 mM Tris?HCl (pH 7.6) and 1
mM DTT and stored at ?80°C until use.
E2-Ubiquitin-Thiolester Assays. In vitro assays were carried out at
room temperature in a reaction mixture containing 25 mM
Tris?HCl (pH 7.6), 5 mM ATP, 10 mM magnesium chloride, E1
enzyme (0.1 ?M), and Oregon green-labeled ubiquitin (f-Ub)
(22). Reactions were carried out for 5 min, and proteins in the
reaction mixture were separated by SDS?PAGE under nonre-
ducing conditions. Fluorescence of the Oregon green label was
Dynamics, Piscataway, NJ).
Ubiquitin Dimer Assays. Reactions were carried out under single-
turnover conditions, where ubiquitin in the preformed ubiquitin-
E2-25K thiolester complex is transferred to a free ubiquitin in
the reaction mixture (34). The thiolester complex was obtained
by incubating 4 ?M E2-25K for 5 min at 25°C in a mixture
containing 50 mM Tris?HCl (pH 7.6), 1 mM ATP, 1 mM
magnesium chloride, E1 enzyme (0.1 ?M), and f-Ub (?1 ?M).
At the end of the incubation period, 10 mM EDTA was added
Flierman et al.
August 1, 2006 ?
vol. 103 ?
no. 31 ?
to chelate magnesium to block further thiolester formation. Download full-text
Dimer formation reactions were initiated upon addition of the
preformed E2-thiolester to a mixture containing 25 mM
Tris?HCl (pH7.6) and 25 mM C-terminally truncated ubiquitin
incubated at 4°C for 5 min (Fig. 4B), or during a time course for
the times indicated (Fig. 4C). The reaction was stopped by
addition of sample buffer without reducing agents. Samples were
analyzed by SDS?PAGE and a fluorimager.
Core facility at the Pennsylvania State University College of
Medicine on a fee-for-service basis. Database searches were
performed with the program MASCOT (52).
Materials. Human ubiquitin was expressed and purified from the
bacterial strain AR58 (53). Oregon green-labeled ubiquitin (8)
and ubiquitin-aldehyde were synthesized as described (54, 55).
E1 was purified from rabbit reticulocyte lysate (29) as described.
Cow liver cytosol was prepared as described (8). Anti-HC serum
and antibodies against bovine ubiquitin were as described (22).
We dedicate this work to Cecile M. Pickart, who died during the
preparation of this manuscript. We thank Y. Ye (National Institute of
Diabetes and Digestive and Kidney Diseases, Bethesda, MD) for HC
antibodies and C. N. Gon ˜i for experimental help. This work was
supported by National Institutes of Health Grants GM 62194 (to V.C.)
and GM52286 (to T.A.R.) and the Pennsylvania Department of Health
Tobacco Settlement Fund (V.C.).
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www.pnas.org?cgi?doi?10.1073?pnas.0605215103Flierman et al.