(Received for publication, September 4, 1995; and in revised form, November 20, 1995)
From the
A novel member of the ubiquitin carrier protein family,
designated E2, has been cloned by our laboratory and
expressed in a bacterial system in an active form. Ubiquitin carrier
proteins, or E2s, catalyze one step in a multistep process that leads
to the covalent conjugation of ubiquitin to substrate proteins. In this
paper, we show that recombinant E2
catalyzes
auto/multiubiquitination, the conjugation of multiple ubiquitin
molecules to itself. Multiubiquitination has been shown previously to
be required for targeting of a substrate protein for rapid degradation.
Using a rabbit reticulocyte lysate system, E2
was shown
to support the degradation of a model substrate in an ATP- and
ubiquitin-dependent fashion. In contrast to a previous study which
showed that selective protein degradation in one system is dependent
upon multiubiquitination via the lysine 48 residue of ubiquitin,
multiubiquitination, and proteolytic targeting by E2
was
shown here to be independent of the lysine 48 multiubiquitin linkage.
This functional characterization of E2
revealed a
combination of features that distinguishes this enzyme from all
previously characterized members of the ubiquitin carrier protein
family. These results also suggest several possible autoregulatory
models for E2
involving auto- and multiubiquitination.
Ubiquitin, a highly conserved 76-amino acid polypeptide, is
present in all eukaryotic cells either in a free state or covalently
conjugated to various cellular proteins(1, 2) .
Ub-protein conjugates are formed through an isopeptide linkage between
the carboxyl-terminal glycine residue of ubiquitin and -amino
groups of substrate proteins (3, 4) . Ub-protein
conjugates typically account for about 50% of total cellular ubiquitin (5) and have been localized to various cellular compartments
including the cytosol(6) , nucleus(7) , cell
surface(8) , and mitochondrion(6) . Ubiquitin
conjugation has been implicated in a variety of cellular functions. The
best understood of these is the targeting of the substrate protein for
selective degradation via a nonlysosomal pathway(9) . Other
cellular processes mediated by the ubiquitin conjugation system include
DNA repair(10) , cell cycle progression(11) ,
regulation of chromatin structure(12) , cell surface
recognition(13) , and regulation of transcription factors, e.g. NF
B(14) .
The formation of
ubiquitin-protein conjugates involves a three-step
process(15, 16) . The first step is the ATP-dependent
activation of ubiquitin by the 105-kDa ubiquitin activating (E1) ()enzyme (15, 17) involving the formation
of a thiol ester linkage between the ubiquitin carboxyl terminus and a
thiol group of E1. In the second step, ubiquitin is transferred to the
active site cysteine residue of a ubiquitin carrier protein (or E2). In
the last step, an isopeptide bond is formed between the carboxyl
terminus of ubiquitin and a lysyl
-amino group within a substrate
protein, a reaction catalyzed either directly by the E2 enzyme or via a
third enzyme designated isopeptide ligase (E3). Enzymes designated
ubiquitin isopeptidases have also been described which deubiquitinate
conjugates(18) , consistent with evidence for the dynamic
balance governing ubiquitin adduct pools(5) .
E2s exist as a
family of isozymes that exhibit variability in terms of molecular
weight, physiological function, substrate specificity, and dependence
on E3 in in vitro systems(2, 19, 20) . For example, in the
yeast Saccharomyces cerevisiae, eight genes (designated
UBC1-8) encoding distinct E2s have been
described(2, 21) . The RAD6 (UBC2) protein functions
in DNA repair, sporulation, and induced mutagenesis(10) . CDC34
(UBC3) is an E2 of 24 kDa that is essential for G/S
transition during mitosis(11) . The basic structure of E2s
consists of a 153-amino acid core domain containing an active site
cysteine within a highly conserved random coil segment. Additional
carboxyl-terminal extension domains present on many isozymes are often
acidic and generally show a high degree of sequence divergence,
suggesting that they may play a role in substrate specificity during
E3-independent conjugation or may contribute to the specificity of
binding to cognate E3 isoforms.
A subset of E2s has been shown to
support multiubiquitination, a process in which successive ubiquitin
molecules are linked by an isopeptide bond between a side chain amino
group of one ubiquitin and the terminal carboxyl group of a second
ubiquitin molecule. In vitro studies have demonstrated that
E3-independent multiubiquitination by several E2 isozymes is sufficient
for degradative targeting by the 26 S multicatalytic protease
complex(22) . In contrast, polyubiquitination refers to the
conjugation of multiple ubiquitin molecules directly to different
lysine residues within a single substrate molecule. Studies show that
RAD6, CDC34, and the rabbit reticulocyte E2 catalyze
E3-indepedent multiubiquitination and support E3-dependent ubiquitin
conjugation; however, these enzymes differ in their linkage specificity
for multiubiquitination, with CDC34 and E2
using Lys-48 (20) and RAD6 utilizing Lys-6(20) . (
)Multiubiquitination via lysine 48 of ubiquitin has also
been demonstrated in the E2
of wheat germ (23) and E2
from calf thymus(24) .
Formation of branched, multiubiquitin adducts have been shown to be
involved in targeting of the substrate protein for selective
degradation by an ATP-dependent protease complex(25) . Chau and
co-workers (26, 27) have shown that proteolytic
targeting can be inhibited by preventing multiubiquitination via the
lysine 48 residue of ubiquitin.
A novel member of the E2 protein
family, E2, has recently been cloned from human
keratinocytes(28) . E2
is unique among E2s in
that it contains a highly basic carboxyl-terminal extension domain. The
E2
transcript was also shown to encode an antigenic
polypeptide recognized by autoantibodies from pemphigus foliaceus
patients. E2
is therefore the first member of the E2
enzyme family to be implicated in a disease process. The present paper
reports the detailed enzymatic characterization of recombinant
E2
, revealing a set of functional properties that
distinguishes this isozyme from all other characterized E2s. E2
is shown to exhibit auto- and multiubiquitination activities. We
further show that this E2 supports the ubiquitin-dependent protein
degradation pathway in the absence of Lys-48 multiubiquitination. The
latter observation indicates that multiubiquitination by linkage other
than Lys-48 are competent degradative intermediates, supporting a role
for subpopulations of ubiquitin having different linkage specificities
within the overall pathway of ATP, ubiquitin-dependent protein
degradation.
Ubiquitin, rcmBSA (reduced, carboxymethylated form of BSA),
UbK48R (site-directed mutant of Ub in which the lysine residue at
position 48 has been replaced by arginine), and rmUb (reductively
methylated form of ubiquitin in which all free amino groups are blocked
by methyl groups) were prepared as described previously(20) .
Rabbit reticulocyte E1 and E2 were purified to
homogeneity by a combination of affinity and high performance
chromatography and then quantitated by stoichiometric activity
assays(29) . Recombinant yeast RAD6 was expressed and purified
as described previously(20) . Protein concentrations were
determined by the Bio-Rad dye binding assay using BSA as a standard.
All other proteins and reagents were purchased from Sigma unless
otherwise indicated.
Isolation of E2 (cDNA-encoded polypeptide without the amino-terminal GST moiety)
was accomplished as follows. The immobilized fusion protein was
incubated with a highly purified preparation of thrombin (4000 NIH
units/mg of protein; Sigma) at a concentration of 30 NIH units/ml in TD
buffer resulting in elution of the E2
while the GST
moiety of the fusion protein remained bound to the column. Optimal
digestion conditions were 1 unit of thrombin per 5 µg of fusion
protein at 25 °C for 1-2 h. Longer incubations resulted in
partial degradation of E2
(data not shown). To remove
minor contaminants including thrombin, undigested fusion protein, and
GST, the eluted fraction was subjected to further purification by anion
exchange chromatography(29) . Prior to chromatography, the
sample was filtered through a 0.2-µm membrane. The filtrate was
chromatographed at 4 °C on a Mono Q HR 5/10 anion exchange column
equilibrated with solution TD using a Pharmacia Biotech Inc. fast
protein liquid chromatography system(29) . Flow rate was
maintained at 1 ml/min during sample loading and gradient elution.
After sample injection, the column was washed with solution TD and
eluted with a linear 0-0.5 M NaCl gradient having a
slope of 12.5 mM/min. Eluted fractions were assayed by both
SDS-PAGE and ubiquitin thiol ester formation(29) . E2
consistently eluted as a single peak at 125 mM NaCl. A
typical yield of E2
based on the thiol ester formation
assay was approximately 10 nmol/liter bacterial culture. Purified
E2
was stored at -80 °C. Under these
conditions, E2
retained full activity for over 8 months
and after two cycles of freeze thawing.
Quantification of E2 activity was accomplished using a modification of the above thiol
ester assay. One-minute incubations containing different amounts of
E2
were analyzed by electrophoresis and autoradiography.
Thiol ester bands were cut from the gel and quantified by
counting on an automatic
counter (Micromedic 4/600 Plus, ICN
Micromedic Systems Inc.). The absolute content of thiol ester was
calculated based on the specific activity of
I-ubiquitin(32) .
A similar approach
was used to assay the conjugation of ubiquitin to endogenous
reticulocyte proteins. Assays contained 50 mM Tris-Cl (pH
7.5), 2 mM ATP, 10 mM MgCl, 0.5 mM DTT, 20 IU/ml inorganic pyrophosphatase, 5 µM
I-ubiquitin (4-8
10
cpm/pmol), 10 nM E1, indicated concentrations of E2s,
and the depleted fraction II (158 µg) as a source of both E3 and
substrates.
Figure 1:
Purification of recombinant
E2. Purity of GST-E2
fusion protein and
E2
was determined by SDS-PAGE analysis followed by silver
staining. Lane 1, total protein extract (43 µg of protein)
of E. coli DH5
harboring pGEXEPF5-ORF2B and induced with
isopropyl-1-thio-
-D-galactopyranoside; lane 2,
GST-E2
fusion protein (1 µg) eluted from a
glutathione-agarose affinity column; lane 3, recombinant
E2
(1 µg) after thrombin digestion and FPLC Mono Q
column. The marker proteins were rabbit muscle phosphorylase b (97 kDa), BSA (66 kDa), ovalbumin (43 kDa), bovine carbonic
anhydrase (31 kDa), and soybean trypsin inhibitor (22
kDa).
The
GST-E2 fusion protein is catalytically active in forming
the corresponding
I-ubiquitin thiol ester in the presence
of E1(28) . Fig. 2shows that recombinant E2
processed from GST by thrombin and subsequently purified by Mono
Q FPLC is also active in ubiquitin thiol ester formation (lane
5). The amount of thiol ester formed to free E2
,
determined by quantification of
I radioactivity
associated with the corresponding band in lane 5 of Fig. 2(20) , agreed with that predicted from the mass of
E2
protein determined as described under ``Materials
and Methods.'' The
I-ubiquitin thiol ester adducts
of both E2
and GST-E2
formed in incubations
parallel to those of Fig. 2were quantitatively labile to
reducing conditions in the presence of 2-mercaptoethanol (not shown),
confirming that the associated
I-ubiquitin was in thiol
ester linkage(20) . We consistently noted that, at equimolar
concentrations, free E2
formed more ubiquitin thiol ester
than did the GST-E2
fusion protein (Fig. 2, lanes 4 and 5). The amount of thiol ester formed to
the fusion protein did not increase on longer incubation and thus did
not result from a lower rate of transthiolation from the E1 ternary
complex (not shown). The lower level of GST-E2
thiol
ester also did not result from inactivation since the predicted amount
of free E2
thiol ester, based on protein determination,
was observed if the fusion protein was first incubated with thrombin
(not shown). These results indicate that the presence of GST at the
amino terminus of E2
sterically alters the equilibrium
constant rather than the rate of thiol ester formation from the E1
ternary complex.
Figure 2:
Ubiquitin thiol ester formation with
GST-E2 and E2
. Thiol ester formation of
radioiodinated ubiquitin was performed at 37 °C for 1.5 min in the
presence or absence of rabbit reticulocyte E1 (2.5 pmol),
GST-E2
fusion protein (1 pmol), or E2
(1
pmol). The reaction products were analyzed by 12% SDS-PAGE under
nonreducing conditions. Lane 1, E1 alone; lane 2,
GST-E2
alone; lane 3, E2
alone; lane 4, E1 plus GST-E2
; lane 5, E1 plus
E2
.
Figure 3:
Time course of E2-dependent
multiubiquitination. Conjugation of
I-ubiquitin to
E2
was allowed to proceed for the amount of time
indicated at 37 °C in incubations of 25 µl containing 2.5 pmol
of E1 and 1 pmol of E2
. Conjugation reactions were
quenched with 25 µl of sample buffer, and 20-µl aliquots were
resolved by 12% SDS-PAGE under either nonreducing (A) or
reducing conditions (B). Lane c shows the products of
the above reaction in the absence of E2. Migration positions for E1
thiol ester and E2-Ub1 are indicated by the upper and lower arrows, respectively, shown at the left of each
panel.
Figure 4:
Characterization of ubiquitin linkages
formed by E2. E3-independent multiubiquitination was
conducted at 37 °C for 8 min with E2
and for 30 min
with CDC34. Incubations of 50 µl contained 4 pmol of liver E1, 4
pmol of either E2
or yeast CDC34, and 5.0 µM
I-Ub (lanes 1 and 4),
I-rmUb (lanes 2 and 5), or
I-UbK48R (lanes 3 and 6). The products
were resolved by 10% SDS-PAGE under reducing conditions and visualized
by autoradiography. Sample volumes were adjusted to correct for slight
variations in specific activities of the three radioiodinated
proteins.
Figure 5:
E3-dependent ubiquitin conjugation
supported by E2. Conjugation reactions were carried out
in the presence of
I-ubiquitin and depleted fraction II
of reticulocyte extract as described under ``Materials and
Methods'' with the indicated combinations of E1 (5 pmol) and E2 (2
pmol). The reactions were incubated for 5 min at 37 °C and
terminated by the addition of an equal volume of SDS-PAGE sample buffer
containing
-mercaptoethanol. The reaction products were analyzed
by 12% SDS-PAGE followed by autoradiography. Lane 1, no E1 or
E2; lane 2, E1 alone; lane 3, E2
alone; lane 4, E1 plus E2
; lane 5, E2
alone; lane 6, E1 plus
E2
.
When similar experiments were conducted using
recombinant E2, this isoform also was shown to stimulate
I-rcmBSA degradation but only when the incubations were
also supplemented with pure E1 (Table 1, Experiment C). Thus, in
the in vitro system, E2
can function in
targeting a substrate protein for selective degradation in an energy-
and ubiquitin-dependent fashion. This targeting appears to be dependent
on E3 activity since in the absence of a reticulocyte extract BSA does
not function as an E2
substrate (not shown). Table 1also shows that the efficiency of E2
in
supporting degradation approaches that of the cognate E2
at equimolar concentrations. The degradation rate of E2
is approximately 75% of that exhibited by E2
, which
agrees favorably with the difference in initial rates of E3-dependent
conjugation observed between E2
and E2
in Fig. 5(lanes 4 and 6).
In this paper, we report the functional characterization of a
new member of the E2 enzyme family, E2, from human
epidermis. Sequence comparison with all previously characterized E2s
revealed that E2
exhibited the highest degree of homology
with yeast UBC4, with a 60% similarity and a 38% identity (28) . UBC4 and UBC5 are central components of the
ubiquitin-mediated proteolytic pathway in yeast (35) and are
categorized as class I E2s (2) defined as those members of the
E2 protein family that lack adjunct sequences extending from the E2
core structure. In contrast, class II E2s contain carboxyl-terminal
extensions that are highly divergent and are thought to play a role in
substrate specificity(12, 36) . For example, RAD6 and
CDC34, which have polyacidic carboxyl-terminal tails, catalyze
ubiquitination of the highly basic histone proteins(20) .
E2
has a polybasic carboxyl tail with a primary structure
that is unique among the E2 family. This keratinocyte enzyme may
therefore have a highly restricted substrate specificity, possibly
limited to certain acidic proteins. A variety of model proteins (such
as histones, lysozyme, cytochrome c, myoglobin, hemoglobin,
and BSA) have been assayed with E2
in the in vitro E3-independent ubiquitin conjugation assay, but none has been
found to function as a substrate in this system. The identification of
E2
-specific substrate(s) will be an important step in
characterizing the physiological role of this enzyme.
It has been
demonstrated that reticulocyte E2, E2
,
RAD6, and CDC34 are bifunctional enzymes capable of catalyzing both
E3-independent and E3-dependent ubiquitin
ligation(20, 29, 33) . A recent study has
shown that ubiquitination and selective degradation of some proteins is
dependent upon specific association with
E3(20, 33, 37) . The results presented in
this report show that E2
is also bifunctional. Fig. 3and Fig. 4demonstrate that E2
can
catalyze ubiquitination in the absence of an E3. Evidence that
E2
also conjugates ubiquitin to substrate proteins in an
E3-dependent manner came from the results of the in vitro degradation assay (Table 1). Radioiodinated rcmBSA, which is
not a substrate for E2
in the absence of E3, was shown to
be targeted for degradation by E2
in a
ubiquitin-dependent manner using a reticulocyte lysate containing E3.
We speculate that E2
may normally conjugate ubiquitin to
a restricted set of specific substrate proteins in the absence of E3
and may function in an E3-dependent pathway with a more general range
of substrate proteins.
Several E2s, including RAD6, CDC34, rabbit
E2, and bovine E2
have been shown to
support the formation of multiubiquitin
chains(20, 25) . In the present study, we demonstrate
that human E2
supports both auto- and multiubiquitination (Fig. 3) via sequential addition of ubiquitin to the growing
multiubiquitin chain. Although rigorous kinetic studies have not yet
been done, it appears that the rates of mono- and diubiquitinations are
much slower than those of the subsequent elongation of ubiquitin chains
to form higher order conjugates. Auto-multiubiquitination has also been
documented in yeast CDC34(20) . Unlike CDC34,
multiubiquitination by E2
does not appear to be
restricted to the ubiquitin lysine 48 linkage since substitution of
ubiquitin with UbK48R did not significantly alter the resulting
conjugation pattern (Fig. 4). Parallel work with Lys to Arg
ubiquitin mutants has been used subsequently to identify novel linkage
specificities catalyzed by several E2 isozymes(38) .
E2-supported targeting of a substrate protein for
proteolysis is dependent on the formation of branched ubiquitin chains
since substitution of ubiquitin with reductively methylated ubiquitin
resulted in inhibition of proteolysis. These results agree with those
of a previous study involving the characterization of the Ub-dependent
degradation of a test protein
-galactosidase(26) .
Proteolytic targeting by reticulocyte lysate E2 isoforms was further
shown to be dependent on multiubiquitination via the lysine 48
linkage(26, 27) . In contrast, however, E2
was shown here to support selective proteolysis in the absence of
lysine 48-mediated multiubiquitination, indicating that E2
and the E2 isoforms present in reticulocyte lysate may represent
members of two distinct subgroups supporting ubiquitin-dependent
proteolysis but differing in E3-dependent linkage specificity.
Substitution of ubiquitin with UbK48R did result in a 50% decrease in
E2
-dependent proteolysis of the model substrate as shown
in Table 2. This difference could be accounted for by a partial
inhibition of E2
activity by UbK48R or by the possibility
that both lysine 48 and other ubiquitin residues may be used equally by
E2
for targeting protein degradation.
The 225-residue
E2 protein contains 17 lysine residues (28) , 9
of which are clustered near the polybasic carboxyl terminus (within the
last 30 residues). Which of these lysine residue(s) in E2
are used for multiubiquitination remains unknown at present.
Preliminary evidence indicates that E2
in which the
polybasic carboxyl terminus has been deleted is still capable of thiol
ester formation and auto-multiubiquitination. (
)In contrast,
deletion of the carboxyl-terminal 81 residues of CDC34 prevents
auto-multiubiquitination(39) . Therefore, the site of
E2
autoubiquitination may reside within the catalytic
core of the protein, although at this time we cannot exclude secondary
effects of carboxyl-terminal extension deletion. It is thus important
to confirm the preferential auto-ubiquitination site on E2
since this assignment may offer useful information related to the
question of how E2
catalyzes both E3-dependent and
E3-independent ubiquitin conjugation. This site may be involved in the
recognition of specific substrate proteins (E3-independent pathway) and
in the interaction with specific E3(s) which, in turn, determine
substrate specificity (E3-dependent pathway).
The demonstration that
E2 catalyzes auto- and multiubiquitination and targets
substrate proteins for selective degradation suggests several possible
autoregulatory models for E2
. In one model, E2
could down-regulate its own activity by targeting itself for
degradation using the ubiquitin-dependent pathway. Alternatively, the
ubiquitin conjugation activity of E2
may be determined by
the conjugation state of the enzyme (native, mono-, or
multiubiquitinated). But, in either case, the possibility that
autoubiquitination of E2
in vivo may be coupled
to an additional signal, e.g. phosphorylation or interaction
with other cellular proteins, cannot be ruled out. The enzymatic
properties of E2
documented in this report provide a
useful model system to address these important issues regarding
autoregulation of this keratinocyte E2.