THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
Vol. 258, No. 13, Issue of July 10, pp. 8206-8214, 1983
Printed I n U.S.A
Components of Ubiquitin-Protein Ligase System
RESOLUTION, AFFINITY PURIFICATION, AND ROLE IN PROTEIN BREAKDOWN*
(Received for publication, December 27, 1982)
Avram HershkoS, Hannah Heller, Sarah Elias, and Aaron Ciechanover
From the Unitof Biochemistry, Faculty of Medicine, Technwn-Israel Institute of Technology, Haifa, Israel
in part by the payment of page charges. This article must therefore
be hereby marked "aduertisement" inaccordance with 18 U.S.C.
Section 1734 solely to indicate thisfact.
$ Supported by United States Public Health Service Grant A M 25614 and a grant from the United States-Israel Binational Science
Foundation.
MATERIALS AND METHODS
Ubiquitin was purified from human erythrocytesby a modification
(17) of a previously describedmethod (5). Ubiquitin andbovine serum
albumin (Pentex) were radiolabeled with NalWI (Nuclear Research
Center, Negev, Israel) by the chloramine-T method,as described (7).
8206
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By affinity chromatography of a crude reticulocyte
The energy dependence of the degradation of intracellular
extract on ubiquitin-Sepharose,threeenzymes
re- proteins has been recognized a long time (1,2), but itsmechquired for the conjugation of ubiquitin with proteins
anisms remained unknown. Recent studies on the mode of
have been isolated. Oneis the ubiquitin-activating en- action of an ATP-dependent proteolytic system for reticulozyme (E,), which is covalently linked to the affinity cytes led to the identificationof a pathway of protein breakcolumn in the presenceof ATP and can bespecifically down. The system iscomposed of several essentially required
eluted withAMP and pyrophosphate (Ciechanover,
A., components (3),including a small, heat-stable polypeptide (4,
Elias, S., Heller, H., and Hershko, A. (1982) J. Biol. 5). The polypeptide was subsequently identified as ubiquitin,
Chem. 257,2537-2542). A second enzyme, designated a universally occurring protein of previously unknown funcEz,is bound to theubiquitin column when E, and ATP tion (6). Ubiquitin is covalently bound toreticulocyte proteins
are present, and is eluted with a thiol compound at
process,
high concentration. The third enzyme, designated E3, or exogenous protein substrates in an ATP-requiring
in
which
several
molecules
of
the
polypeptide
are
conjugated
is adsorbed to the affinitycolumn by noncovalent into the substrate protein
by amide linkages (7,8).A model was
teractions and can be eluted with high
salt
or increased
pH. The presence of all three enzymes is absolutely proposed accordingto which the conjugation of ubiquitin with
signal eventinprotein
breakdown
required for the conjugation
of '251-~biq~itin
with pro- proteinsistheinitial
teins. All three affinity-purified enzymesare also re- (reviewed in Ref. 9). The relationshipbetween ubiquitin conquired for the breakdownof 1251-albuminto acid-sol- jugation and protein breakdown was corroborated by experiuble material in the presence of ubiquitin, ATP, and ments with intact cells, in which striking correlations were
observed between the rapid degradationof abnormal proteins
the unadsorbed fractionof the affinitycolumn.
and increased formation of ubiquitin-protein conjugates (10,
The following observations indicate that the function
site of 11).
of E2is the transferof activated ubiquitin to the
conjugation in the form of an Ez-ubiquitin thiol ester
Our knowledge of theintermediarystepsintheATPintermediate. (a)E2is rapidly inactivatedby iodoacet- ubiquitin proteolytic pathway is still rudimentary. The conamide, but can be protected against inactivation by a jugation of ubiquitin with proteins is apparently initiated by
prior incubation with El, ATP, and ubiquitin. This
a specific ubiquitin-activatingenzyme,first
identifiedby
suggests an El-mediated transfer
of activated ubiquitin ubiquitin-dependent PP,:ATP and AMP:ATP
exchange reto an iodoacetamide-sensitive thiol site of E2. (b) The actions, and by the binding of activated ubiquitin toenzyme
requirements for the binding of Ez to the ubiquitin in a thiolester linkage (12). A mechanism involving the
column and the mode of its elution, cited above, are formation of ubiquitin adenylate and its transfer to a thiol
consistent with the notion that a covalent linkage is site of the enzyme was proposed (12) and substantiated by
formed betweenE, and Sepharose-bound ubiquitin.
(c)
direct evidence (13, 14). Ubiquitin is activated at its COOHUpon the incubationof '251-ubiquitin with E, and ATP,
terminal glycine (15), which is inaccord with the observation
followed by the addition of purified E,, activated
ubiquitin is transferred fromEl to several low molec- that this residue is bound by isopeptidelinkage t o e N H 2
ular weight forms
of Ez,as analyzed bysodium dodecyl lysine in a ubiquitin-histone conjugate (16).
The ubiquitin-activating enzyme was purified to near hosulfate-polyacrylamide gel electrophoresis. The linkage of ubiquitin to all these forms has
the characteris- mogeneity by a covalent affinity chromatography procedure,
tics of a thiol ester bond. In a further incubation with in which the enzyme is first bound to ubiquitin-Sepharose in
the presence of ATP asa thiol ester intermediate, and is then
E3 and a protein substrate for conjugation, activated
ubiquitin was transferred from the different forms
of specifically eluted with AMP and pyrophosphate (17). The
Ez-ubiquitintostableubiquitin-protein
conjugates. activating enzyme does not form ubiquitin-protein conjugates
of the ligasesystem. by itself (12, 17), but it isa donor for conjugate formation in
Thus, E3is involved in the last step
the presence of a crude reticulocyte extract (13). In the present
report, we describe two further enzymes which participate in
the conjugation of ubiquitin with proteins. Evidence is presented which indicates the involvement of the three compo* This work was supported by United States Public HealthService nents of the ubiquitin-protein ligase system in protein degGrant AM-21811 and funds from the Institute for Cancer Research
radation.
to Irwin A. Rose. The costsof publication of this articlewere defrayed
Ubiquitin-Protein Ligase System
The abbreviations used are: DTT, dithiothreitol; SDS, sodium
dodecyl sulfate; E , , ubiquitin-activating enzyme.
CF-25 Centriflo membrane cones (Amicon Corp., Lexington, MA),
and the buffers were changed by three successive 10-fold dilutions
with 20 mM Tris-HCI(pH 7.2) containing 1 mM dithiothreitol,
followedby ultrafiltration inthe cone. The final volume ofthe column
eluates was brought to 5% of the starting volumeof Fraction 11.
Protein concentration of the various fractions was determined by the
method of Lowry et al. (20). Preparations were stored at -80 "C in
small samples. The ubiquitin-Sepharose column was regenerated by
washing with 10 column volumes of 50 mM Tris-HC1 (pH 9.0) containing 1 M KCl, followed by 10 column volumes of 50 mM Tris-HC1
(pH 7.2) containing 0.02% NaN3. The column was stored in the last
buffer at 4 " C and was reused many times over a period of 2 years.
The uhiquitin-activating enzyme used in this study was further
purified from the AMP-PP, eluate by gel filtration chromatography
on a column (0.9 X 60 cm) of Sephacryl S-200 (Pharmacia) equilibrated with 20 mM Tris-HCI (pH 7.2), 1 mM dithiothreitol, and 1
mg/ml of ovalbumin. The peak of enzyme activity (located by the
ubiquitin-dependent 3'PPi-ATP exchange assay (17)) was collected
and stored at -80 'C in small samples. Ovalbumin was included in
the elution buffer for enzyme stabilization, since it is arelatively inert
protein for the ubiquitin-ATP system. In concentrations up to 2 mg/
ml, ovalbumin does not inhibit the degradation of 1251-albumin,it
does not form conjugates with '251-~biq~itin, and
1z51-ovalbuminis
not broken down significantly by the reticulocyte proteolytic system
(data not shown).
Assay of Protein Breakdown-The breakdown of '251-labeledbovine
serum albumin ('%I-albumin) to acid-soluble material was determined
essentially as described (3). The reaction mixture contained, in a
final volume of 50 pl, 50 mM Tris-HC1 (pH 7.6), 5 mM M & ~ z ,3 mM
dithiothreitol, 0.5 mM ATP, 10 mM creatinephosphate,5
pg of
creatine phosphokinase, 4 pg of ubiquitin, 1-2 pg of Iz5I-albumin(510 X IO6 cpm), and enzyme fractions as indicated in the legends.
Following incubation at 37 "C for 2 h, the reaction was terminated
by the addition of 0.8 ml of 5% trichloroacetic acid in the presence of
10 mg of carrier bovine serum albumin. The samples were centrifuged
for 3 min in an Eppendorf microcentrifuge, and radioactivity in a 0.5ml sample of the supernatant was estimated by y counting. Acidsoluble radioactivity present inzero time samples was subtracted,
and the results are expressed as the percentage of '251-albumindegraded to acid-soluble material. '2511-albumin was
chosen as the substrate for protein breakdown because it is not attacked significantly
by non-ATP-dependent proteases present in reticulocyte extracts,
and thus its degradation is completely dependent upon the supplementation of ATP and ubiquitin (3).
Assay of Conjugation of Ubiquitin-Since the determination of the
conjugation of '"I-ubiquitin by SDS-polyacrylamide gel electrophoresis (8) is time consuming and laborious, a rapid quantitative assay
wasdeveloped. The assay is based on the observation that free
ubiquitin, which has a neutral isoelectric point, is not adsorbed on
either anion or cation exchange resins a t neutral pH (7). On the other
hand, ubiquitin-protein conjugates are adsorbed on such resins, presumably via their protein moieties. The reaction mixture contained,
in a final volume of 50 pl, 50 mM Tris-HC1 (pH 7.2), 2 mM ATP, 5
mM MgClZ, 2 mM dithiothreitol, 0.04 unit of inorganic pyrophosphatase, 20 pg of oxidized RNase, 50 pmol of '9-ubiquitin (about 20,000
cpm), and enzyme preparations as indicated. Oxidized RNase was
included since it is a good substrate for conjugation, in contrast to
native RNase.' Following incubation a t 37 "Cfor 30 min, the reaction
was stopped by the addition of 10 pl of 0.5 N NaOH in the presence
of 20pgof carrier unlabeled ubiquitin. Treatment with alkali was
required to release '%I-ubiquitin from thiol ester enzyme intermediates (see "Results"), which would be
adsorbed to resin in the following
step. The addition of carrier ubiquitin was necessary to prevent
nonspecific adsorption of tracer amounts of '9-ubiquitin. Following
incubation a t 37 "C for 5 min, the samples were neutralized by the
addition of 10 $1 of 0.5 N HCI. To each sample, 100 p1 of a 50% (v/v)
suspension of DE52 and 30 p1 of a similar suspension ofCM52
(Whatman) were added. Both resins had been equilibrated with 10
mM potassium phosphate (pH 7.0) prior to use. The samples were
agitated on a Vortex mixer for 10 s, the resins were washed twice
with 2-ml portions of 10 mM phosphate buffer (pH 7.0), and resinbound radioactivity was estimated with a y counter. Radioactivity
adsorbed to resin in a parallel incubation without enzymes (not more
than 10% of total radioactivity) was subtracted, and the results were
calculated as picomoles of IZ5I-ubiquitinincorporated into conjugates.
A. Hershko and H. Heller, unpublished results.
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RNase A (bovine pancreas, type I-A) was purchased from Sigma and
was subjected to performic acid oxidation as described by Hirs (18).
Creatine phosphokinase (150 units/mg) and yeast inorganic pyrophosphatase (550 units/mg) were purchased from Sigma, and yeast
hexokinase (140 units/mg) from Boehringer Mannheim. Crystallized
hen ovalbumin was obtained from Worthington.
Preparation of Reticulocyte Fractions-Reticulocyte extracts were
prepared by a modification of previously described procedures (3,4).
Briefly, reticulocyte-rich blood (70-90% reticulocytes) was obtained
from rabbits following injections of phenylhydrazine (19). The cells
were washed twice with phosphate-buffered saline (150 mM NaC1, 10
mM potassium phosphate (pH 7.4)), suspended in an equal volume of
Krebs-Ringer phosphate medium lacking glucose (19), and incubated
at 37 "C for 90 min with 0.2 mM 2,4-dinitrophenol and 20 mM 2deoxyglucose. This treatment of ATP depletion is required to release
ubiquitin from endogenous ubiquitin-protein conjugates. All subsequent operations were carried out at 0-4 "C. The cells were washed
twice with phosphate-buffered saline, lysed with 1.5 volumes of 1 mM
dithiothreitol, and centrifuged at 80,000 X g for 90 min to remove
particulate material. Crude lysates could be stored at -80 'C for over
a year without loss of activity.
Lysates were fractionated on a column of DEAE-cellulose (Whatman DE52) equilibrated with 3 mM potassium phosphate (pH 7.0)
and 1 mM dithiothreitol, a t a ratio of lysate to resin of 1.5:l (by
volume). Unadsorbed material (Fraction I, containing ubiquitin (4))
was collected and the column was washed with 2.5 column volumes
of a buffer containing 3 mM potassium phosphate (pH 7.0), 1 mM
dithiothreitol, and 20 mM KCl. Proteins adsorbed to the resin (Fraction 11) were eluted with 2.5 column volumes of a solution consisting
of 0.5 mM KCl, 20 mM Tris-HC1 (pH 7.2), and 1 mM dithiothreitol.
The eluate was concentrated by ammonium sulfate precipitationand
dialyzed as described (4). Following dialysis, some insoluble material
was removedby centrifugation (20,000 X g, 15 min). The final volume
of Fraction I1 was usually one-fifth of the starting volume of crude
lysate, and its protein concentration was in the range of 20-30 mg/
ml. Fraction I1 contains all the enzymes required for ATP-uhiquitindependent proteinbreakdown (3,4) andfor the formation and breakdown of uhiquitin-protein conjugates (7,8). Itcan be stored at -80 "C
for a t least a year without loss of proteolytic activity, provided that
ATP (0.5 mM) is added to protect an ATP-stabilized factor (3).
Without ATP, Fraction I1 was stored a t -80 "C in small samples,
thawed only once, and used within 2-3 weeks of preparation.
Affinity Chromatography-Affinity chromatography of the components of the ubiquitin-protein ligase system was performed by an
extension of the procedure described previously for the purification
of ubiquitin-activating enzyme (17). Ubiquitin was coupled to activated CH-Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ)
as described previously (17), except that the concentration of Sepharose-bound ubiquitin was approximately 20 mg/ml of swollen gel.
This high concentration of Sepharose-bound ubiquitin was required
for efficient binding of E3 (see "Results"), whereas Eland E, were
completely hound to columns containing much less ubiquitin (around
5 mg/ml of gel). Column operations were performed at room temperature, but enzyme fractions were collected on ice. A 6-ml column of
ubiquitin-Sepharose was equilibrated with 5 column volumes of a
buffer consisting of 50 mM Tris-HC1 (pH 7.2), 2 mM ATP, 5 mM
MgClz, and 0.2 mM dithiothreitol (Buffer A). Fraction I1 from reticulocytes (6 ml) was adjusted to 50 mM Tris-HC1 (pH 7.2), 5 mM ATP,
10 mM MgCI,, and 0.2 mM dithiothreitol and applied to the column
a t a flow rate of 0.5 ml/min. The unadsorbed fraction was collected
until the end of the yellowish concentrated protein color; the protein
concentration of the unadsorbed fraction was diluted about 1.5-fold
relative to thatof Fraction 11. The column was washed with 3 column
volumes of Buffer A and then sequentially eluted with the following
solutions: 1 M KC1 containing 50 mM Tris-HCI, pH 7.2 (KC1 eluate);
the above Tris buffer, to remove salt; 2mM AMP and 0.04 mM sodium
pyrophosphate in the above Tris buffer (AMP-PPi eluate), to elute
ubiquitin-activating enzyme (17); 10 mM dithiothreitol in the same
Tris buffer at pH 7.2 (DTT' eluate); and 50 mM Tris-HCI (pH 9.0)
containing 2 mM dithiothreitol (pH 9 eluate). Each elution was with
3 column volumes of the respective buffer, except for the KC1 elution
which was with 6 column volumes. The pH 9 eluate was neutralized
with 100 mM Tris-HCI (pH 7.2) immediately following elution. All
column eluates were concentrated by centrifuge ultrafiltration with
8207
8208
Ubiquitin-Protein Ligase System
The assay could be used
for estimation of activity of each ofthe three
enzymes of the ligase system (see “Results”),provided that the other
two were in excess. All
assays were performed in the range of linearity
with respect to enzyme concentration, which was usually up to 15
pmol of ‘9-ubiquitin conjugated with proteins. Oneunit of enzyme
activity is defined as the amount of enzyme required for the incorporation of 1 @molof ‘251-uhiquitininto conjugates/min under the
conditionsemployed. For the case of the ubiquitin-activatingenzyme,
this is different from the previously defined unit (17) which was by
32PPi-ATPexchange activity. Withthe purified activatingenzyme, it
was estimated that 1 unit of conjugating activity is equivalentto 41
units of PP;-ATP exchange activity.
W
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part of column-bound activthe unadsorbed fraction. Another
ity, which was not displaced by high salt, could be eluted by
the subsequent wash at pH 9. Control experiments indicated
that neither the high salt eluate nor the pH 9 eluate had
significant proteolyticactivity bythemselves, withoutthe
unadsorbedfraction(datanotshown).
A furthercontrol
showed that no activity was bound to a Sepharose column
which had no ubiquitin attached.
Although the high salt eluateof the affinity column restored
protein breakdown in the unadsorbed materialof Fraction I1
applied to the column in the absence
of ATP, it was not
RESULTS
sufficient to do so with the unadsorbed fraction of chromaIsolation of the Three Factorsof the ATP-dependent Proteo- tography performed in the presenceof ATP (Fig. 2 B ) . This
lytic System by Affinity Chromatography-In a previous study, indicated that in the presence of ATP, some further factors
we have described a n affinity procedure for the isolation of of the proteolytic system are bound to ubiquitin-Sepharose.
ubiquitin-activating enzyme, whichwas based on the covalent A likelycandidate was the ubiquitin-activatingenzyme, which
is covalently bound to the ubiquitin column and would be
binding of the enzyme to ubiquitin-Sepharose in the presence
removed from Fraction I1 under these conditions. However,
of ATP (17). We next examined whether other components
of the ATP-ubiquitin proteolytic system can alsobe isolated the addition of purified ubiquitin-activating enzyme was not
by the affinity column. In the experiment. shown in Fig. 1, sufficient to restore protein breakdown in the presenceof the
KC1 eluate (TableI, Experiment
reticulocyte Fraction II was appliedto ubiquitin-Sepharose in unadsorbed fraction and the
1).
This
suggested
the
ATP-dependent
binding of a further
the presence or absence of ATP, and the activity
of the
unadsorbed fraction to degrade 1251-albumin(in the presence factor, in addition to the ubiquitin-activatingenzyme. Since
of binding suggested the formationof a
of ATP and ubiquitin) was examined. There was a complete the ATP requirement
between an enzyme andcolumnloss of proteolytic activity in the unadsorbed fraction when thiolesterintermediate
(17),
we
attempted
to elute this factor with
a
bound
ubiquitin
the extractwas applied in the presence
of ATP. In the absence
a
thiol
compound.
Reticulocyte
Fraction
high
concentration
of
of ATP, therewas also a considerable decrease,although some
of ATP,
residual activity remained. In different experiments, this re- I1 was applied to the affinity column in the presence
and
bound
material
was
serially
eluted
with
high
salt,
AMP
sidual activity varied between 10 and 50% of the activity of
untreated Fraction I1 (maximal variation). Since the ubiqui- + PP, (to elute ubiquitin-activating enzyme (17)), abuffer
tin-activating enzyme is bound to the
affinity column only in containing a high concentration of dithiothreitol, anda wash
the presence of ATP (17),these results indicated that some at p H 9 (see “Materials and Methods”).As shown in Table I,
Experiment 1, the DTT eluate
of the affinity column restored
other factor(s) of the proteolytic system are
alsoremoved
the
activity
of
the
proteolytic
system in the presence of the
from Fraction I1 by the ubiquitin column, presumably by a
KC1 eluate and the unadsorbed fraction. In this reconstituted
noncovalent interaction.
In an attemptto recover the factor(s) bound to the affinity
column in the absence of ATP, the column was sequentially
30 AB.
eluted with high salt and then by raising the pH to 9.0. As
D
Applied-ATP
shown in Fig. 2 4 , the high salt eluate of the affinity column
a
KC1 eluate
restored the activityof the proteolytic system, when added to
Untreated
Q,
Applied+ATP
0
Column eluate ( p g )
Applied-ATP
Applied+ATP
100
200
300
Fraction added ( p g of protein)
FIG. I. Binding of components of the proteolytic system to
ubiquitin-Sepharose in the presence or absence of ATP. 1-ml
portions of Fraction I1 from reticulocytes were applied to 1-ml columns (0.5 X 5 em) of ubiquitin-Sepharose in the presence of ATP
(A),as described under “Materials and Methods,” or under similar
conditions but with ATP omitted (A). Samples of the unadsorbed
fractions or of untreated Fraction I1 (0)were assayed for the degradation of ‘?%albuminin the presence of ubiquitinandATP, as
described under“Materials and Methods.”
100
200
Breakthrough
fraction
(pg)
FIG. 2. Elution of factorb) bound to the affinity column in
the absence of ATP. A, reconstitution of activity by eluates of high
salt or pH 9. Fraction I1 was applied to ubiquitin-Sepharose in the
absence of ATP as described in the legend to Fig. 1 and the column
was washed with 3 column volumes of Buffer A (see “Materials and
Methods”) lackingATP.Thecolumn
was eluted with 6 column
volumes of 1 M KC1 containing 50 mM Tris-HC1buffer (pH 7.2),
followed by 3 column volumes of 50 mM Tris-HC1(pH 9.0) containing
2 mM dithiothreitol. The degradation of ‘251-albuminwas determined
in the presence of the unadsorbed fraction of the same column (120
pg of protein) and the indicated amountsof the KC1 eluate (0)or pH
9 eluate (0).B, lack of reconstitution by KC1 eluate with the unadsorbed fraction of extract chromatographed in the presence of ATP.
Fraction I1 was applied to ubiquitin-Sepharose columns in the presence or absence of ATP, as described in the legend to Fig. 1, and
increasing amountsof the unadsorbed (Breakthrough) fractions were
assayed in the presence of 23 pg of KC1 eluate.
miquitin-Protein Ligase System
8209
4B)*The molecular size Of
derived from the
Of
theaffinity column, as determined bygel filtration, was
similar to that derived from the PH9 eluate (data not shown).
Role of the Three Affinity-purified Factors in. the Conjugation of Ubiquitin with Proteim-Trying to identify the functions of E2 and E, we considered the possibility that they may
be required for the ubiquitin-protein conjugation process. As
E3
purified factors of the proteoiytic system
Conjugation of 1251-UbiqUitinwas determined as described under
“Materials and Methods.’’ Where indicated, enzymes were added at
the following amounts: E,, 3.6 nanounits; E2 (pooled peak fractions
from Fig. 3), 10 pl; E3 (pooled peak fractions from Fig. 4B),3 pl, In
Experiment 2, E3 was treated with iodoacetamide as described in the
legend to Fig. 3.
Additions
’“I-Ubiquitin conjugated
pmol
Experiment 1
TABLE
I
E,
Restoration of uctiuity of the proteolytic system by factors bound to
the affinity column in the presence of ATP
Fraction I1 was applied to ubiquitin-Sepharose in the presence of
ATP, and affinity chromatography was carried
asout described under
“Materials and Methods.” The degradation of 1251-albumin
was
determined as described under
“Materials
and Methods,” in the presence of 171 pgof the unadsorbed fraction. Where indicated, the
following amounts of affinity column eluates were added (micrograms
of protein): KC1 eluate, 17.5; DTT eluate, 5.7; and pH 9 eluate, 5.2.
Purified E , was supplemented at 14.6 nanounits, and purified E2(cf.
Fig. 3) at 1.6 nanounits.
E2
Additions
Experiment 1
KC1 eluate
KC1 eluate + El
KC1 eluate + DTT eluate
pH 9 eluate
pH 9 eluate + E,
Experiment 2
KC1 eluate + E ,
KC1 eluate + E,
KC1 eluate + E, + E2
‘“I-Albumin degraded
%
5.4
4.7
23.9
4.7
11.0
4.2
5.8
24.4
E3
+ 2‘
E2 + E3
+ E3
+ E2 + E3
Experiment
’3,
iodoacetamide-treated
E37 iodoacetamide-treated +
Ea,iodoacetamide-treated + E2
Es, iodoacetamide-treated + E, + E2
0.7
0.7
1.3
2.4
4.3
5.3
12.1
0.3
1.o
1.2
7.1
shown Table
in
11, when El and E2 were incubated with 1251ubiquit,in and ATP,little if any formationof ubiquitin-protein
conjugates could be detected. However, upon the supplementation of all three factors (purified by affinity chromatography
and gel filtration), significant conjugation of ubiquitin with
proteins was observed. partial
The
requirement for E , and E2
in the presence of E3 is presumably due to contamination with
E1 and incomplete separation of the high molecular weight
form of E , from E3 on gel filtration (Fig. 4). However, E3
could be freed of residual E2 and El activities by treatment
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system too, there was a complete requirement of protein
breakdown for the presence of ubiquitin and ATP (data not
shown). For convenience of reference, we shall term the three
30
factors eluted from the affinity column E, (ubiquitin-activating enzyme), E2 (factor eluted by DTT), and E3 (eluted with
high salt or high pH). It seemed that some of the affinity
column eluates contained mixtures of at least two of these
factors. The lack of requirement for added El in the presence
of the DTT eluate (Table I, Experiment 1) can be explained
by the presence of considerable amounts of E, in thisfraction,
Elution Volume tml)
E
which was not completely removed by the prior elution with
AMP and PPI (cf. Table 111). On theotherhand,
in the ‘E lo
presence of the pH9 eluate, El stimulated protein breakdown
without DTT eluate (Table I, Experiment l),suggesting that
the pH 9 eluate contains E, as well as E3.
To obtain a better separation between the three factors,
eluates of the affinitycolumn were subjected to gel filtration
F r a c t i m Mumbn
chromato@aphy. When the DTT e’uate was separated On a
FIG. 3. Gel filtration chromatography of factor eluted with
Cohmn of Sepharose 6B and column fractions were assayed dithiothreitol. 570 p1 of DTT eluate (containing 532 nanounits of
for E, activity (protein breakdown in the presence of El, E a , E, activity) were applied to a column (0.9 x 58 cm) of Sepharose 6B
and the unadsorbed fraction), a single peak of an apparent (Pharmacia) equilibrated with 20 mM Tris-HC1 (pH 7 3 , 1 mM
molecular weight of around 35,000 was found (Fig. 3). By dithiothreitol, and 1 rng/ml of ovalbumin. Elution was with the above
and fractions of 0.74 ml were collected at 4 “C. Protein breakcontrast, whenthe p~ 9
was separated on a similar buffer
down (0)
was assayed in fraction samples of 5 p1 in the presence of
column, two peaks of E2 activity were observed; in addition 180 pg of unadsorbed fraction, 33 pg of KC1eluate, and 7.3 nanounits
to the M r = 35,000 enzyme, there is a higher molecular weight of purified
Conjugation of l251-~biquitin(0)was assayed as deform (MI
E 250,000) of apparently similar activity (Fig. a).
scribed under “Materials and Methods,” in samples of 2 pl in the
Since the lower molecular weight form of E2is well separated presence of 3.6 nanounits of El and 4.6 p g of iodoacetamide-treated
from E , (M,= 210,000, Ref. 17) by gel filtration, this form pH 9 eluate. The pH 9 eluate was treated with iodoacetamide ( 5 mM)
was mainly used for subsequent studies. With purified E2, at 25 “c for 10 min, followed by the addition of excess (8 mM)
dithiothreitol. Inset, estimationof molecular weight. Marker proteins:
there was a nearly complete requirement of protein break- 1,alcohol dehydrogenase (M, = 150,000);2, hemoglobin (M, = 64,000);
down for El, when assayed in the Presence of KC1 eluate and 3, ovalbumin (M, = 43,000); 4, myoglobin (M,= 17,000). The arrow
the unadsorbed fraction (Table I, Experiment 2). Assay of indicates the elution position of the enzyme.
Sepharose 6B column fractions of the pH 9 eluate for E3
activity (protein breakdown in the presence of excess E l , E,,
the
and
unadsorbed material) showed a single peak with an
TABLE
I1
apparent molecular weight of approximately 3oo,000 ( F ~ ~ .Conhwtion of ubiquitin with proteins requires the three affinity-
Ubiquitin-Protein Ligase System
A.
Fa
Cat ADH
Hb
1
4
I
4
Yb
4
I
4
Y
R
7
lh
Minutes
116
FIG. 5. Effect of iodoacetamide on the activity of the three
enzymes. El (purified bygel filtration from AMP-PPi eluate, see
“Materials and Methods”), E2 (purified by gel filtration from DTT
eluate, Fig. 31, and E3 (purified bygel filtration from pH 9 eluate,
Fig. 4 8 ) were diluted in 20 mM Tris-HC1 (pH 7.2) containing 1 mg/
ml of ovalbumin and 0.1 mMof dithiothreitol to a concentration of
75 nanounits/ml. Following the addition of iodoacetamide (5 mM,
final concentration),the samples were incubated at 37 ‘C. At various
time intervals, aliquots of 5 pl were transferred to thereaction mixture
of the conjugation assay (see “Materials and Methods”), which contained an %fold excess of dithiothreitol over iodoacetamide. The
ubiquitin-conjugating activity of Ez was determined as described in
the legend to Fig. 3, and thatof E3 as described in the legend to Fig.
4B. The activity of E, was determined in the presence of0.67
nanounits of purified E2 and 2.3 yg of pH 9 eluate.
Downloaded from www.jbc.org at Univ of Ottawa - OCUL on December 7, 2007
B.
I
6
activity of each of the threeenzymes, provided that the other
two are inexcess. As shown in Fig. 3, the ubiquitin-conjugating activityof E2derived from the DTT eluate of the affinity
column coincided exactly with the proteolytic activityof this
enzyme, assayed in fractions of the same Sepharose 6B column. A similar coincidence between the ubiquitin conjugation
andprotein breakdown activities of the two forms of E,
derived from the pH 9 eluate (Fig. 4A) and of EBfrom the
same eluate (Fig. 4B)were observed. Moreover, when E , was
separated ona Sephacryl S-200 column, the peak
of its activity
assayed by ubiquitin-dependent PPI-ATP exchange reaction
(17) coincided with the activity of this enzyme to stimulate
protein breakdown (data not shown). These
data indicate the
identity of the three enzymes of the ubiquitin-protein ligase
system with the corresponding factors participating in protein
breakdown.
Purification of E2and E3-The ubiquitin conjugation assay
(which is more accurate and sensitive than the proteolytic
assay) wasused to determine the extent
of purification of E,
with iodoacetamide. El is rapidly inactivated by iodoaceta- and EB by the affinity chromatography procedure. In Table
mide (13), and so is E2(Fig. 5 ) . By contrast, E3is much more 111, the distribution of Ez in various fractions of the affinity
of E,. It may be seen that most
resistant to thissulfhydryl blocking agent (Fig. 5). As shown column is compared with that
in Table 11, Experiment 2, following treatment of E3 with of eluted E, activity distributed about equally between the
eluate. However, the specific activity
iodoacetamide, a virtually complete requirement for El and DTT eluate and the 9pH
was about 2-fold higher than that in
EP for ubiquitin conjugationwas observed. Iodoacetamide- of E, in the DTT eluate
treated E, (derivedfrom the pH 9 eluate)also showed a the pH 9 eluate, and a more than 90-fold purification was
complete requirement for E, and E2 in the stimulation of achieved in the former fraction. It shouldbe mentioned that
some EPactivity eluted in other fractionswell,
as most notably
protein breakdown (data not shown).
the of El). Therefore,
To examinewhether
thethreeenzymesrequired
for in the AMP-PP, eluate (containing bulk
ubiquitin-protein conjugation are indeed similar to those par-we used E, purified on a Sephacryl S-200 gel filtration column
ticipating in protein breakdown, the gel filtration profiles of (which separates it from residual low molecular weight EP)
the proteolytic and conjugating activities
were compared. The throughout this study (see “Materials and Methods”).
Table IV shows the purification of E3 and its distribution
ubiquitin conjugation assaycan be used to determine the
FIG.4. Gel filtration chromatography of factors eluted at
pH 9. A , elution profile of E2. 380 pl of pH 9 eluate (containing933
nanounits of Ez activity) were applied to a Sepharose 6B column
under conditions identical with those described in the legend to Fig.
3. The breakdown of 1251-albumin(0)was assayed in samples of 15
pl in the presence of 180 pgof unadsorbed fraction, 44pgofKC1
eluate, and 7.3 nanounits of purified E,. Conjugation of 1251-ubiquitin
(0)was assayed as described under “Materials and Methods” in
fraction samples of 3 pl in the presence of 3.6 nanounits of E, and
2.3 pg of iodoacetamide-treated pH 9 eluate (see Fig. 3). B, elution
profile of E3. 330 pl of pH 9 eluate (containing 535 nanounits of E,
activity) were separated on Sepharose 6B under conditions identical
with those described above. Protein breakdown was assayed in samples of 20 pl, in the presence of 110 pg of unadsorbed fraction, 7.0 pg
of DTT eluate, and 7.3 nanounits of E,. Conjugation of 1251-ubiquitin
was determined in samples of 2 p l , in the presence of 3.6 nanounits
of E , and 0.7 pg of DTT eluate. Markers (arrows):Fe, ferritin ( M , =
480,000); Cat, catalase (M,
= 240,000); ADH, alcohol dehydrogenase
( M , = 150,000);Hb, hemoglobin (M,
= 64,000); Mb, myoglobin ( M , =
17,000).
8211
Ubiquitin-Protein Ligase System
TABLE
111
Distribution of E , and E2 in fractions of the affinity column
22 ml of Fraction I1 from reticulocytes were subjected to affinity chromatography as described under “Materials
and Methods.” The activity of E2 was determined by the lZ5I-ubiquitinconjugation assay (see “Materials and
Methods”) as described in the legend to Fig. 3, and theactivity of E , as described in the legend to Fig. 5.
Total activity
Fraction
Total protein
El
Fraction I1
KC1 eluate
AMP-PPi eluate
DTT eluate
DH 9 eluate
562
15.4
0.79
0.72
1.72
%
3,975
75
163
584
605
35,060
935
7,104
1,990
533
w
Fraction I1
Unadsorbed fraction
KC1 eluate
AMP-PPi eluate
DTT eluate
pH 9 eluate
235
195
8.1
0.41
0.57
0.77
Total Recov- Specific Purifiactivity
ery
activity cation
units
%
6,167
2,888
440
22
74
501
100
46.8
7.1
0.4
1.2
8.1
u?Z,&
26.2
14.8
54.3
53.6
130
651
-fold
1
2.1
1
144
36.7
94.5
49.4
TABLE
V
Binding of E, to ubiquitin-Sepharose requiresE , and ATP
0.5-ml portions of E2 (low molecular weight enzyme from the
Sepharose 6B column of the pH 9 eluate (Fig. U ) )containing
,
73.3
nanounits, were adjusted to 20 mM Tris-HC1 (pH 7.2), 1 mg/ml of
ovalbumin, 0.2 mM dithiothreitol, and 5 m M MgCl,. Where indicated,
2 mM ATP or 350 nanounits of E , were added. The samples were
applied to 1-ml columns (0.5 X 5 cm) of ubiquitin-Sepharose equilibrated with buffers of identical composition to the corresponding
samples. Affinity chromatography was carried out as described under
“Materials and Methods,” except that all solutions contained ovalbumin (1 mg/ml), to minimize inactivation of the dilute enzyme. E2
activity was determined by the 1251-uhiquitinconjugation assay (see
“Materials and Methods”), as described in the legend to Fig. 3.
Additions
Ezactivity recovered
24.8
infractions of the affinity column. About one-half of Ea
activity remained in the unadsorbed fraction in this preparation, and column-bound E3 eluted mainly in the KC1 eluate
and pH 9 eluate fractions. However, the KC1 eluate contains
a considerable amount of protein, apparently bound nonspecifically to the ubiquitin-Sepharose column. Therefore, the
extent of purification of E3 in the high salt eluate is relatively
low, in contrast toa nearly 25-fold purification of E3achieved
in the pH 9 eluate (TableIV).
Although considerable purification is achieved by the present procedure, the preparationsof E2 and E3 obtained are not
homogenous. SDS-polyacrylamide gel electrophoresis of E2
(DTT eluate further purified by gel filtration on Sephadex G100) showed four Coomassie blue staining bands of M, =
28,000, 20,000, 16,000, and <10,000, while the preparation of
E, (pH 9 eluate purified on Sepharose 6B) had three major
protein bands (Mr = 200,000, 100,000, and 64,000) and numerous minor bands(not shown). The presence of these
impurities presumably accounts for the observation that even
with the purified system, about one-half of ubiquitin conjugates formed are derived from endogenous protein substrates.
Both preparationswere free of detectable nonspecific protease
and ATPase activities. However, the preparation of E3 contained some enzyme activity which degrades ubiquitin-protein
conjugates. The latter activity is inhibited by sulfhydryl reagents, and thus E3 can be freed of the conjugate-degrading
enzyme by treatment with iodoacetamide.
Requirements for the Binding of E2 and E3 to the Affinity
Column-We next asked in what form are E2 and E3 bound
to the affinity column. The general characteristics of the
binding of E2, i.e. the requirement for ATP, lack of displacement of column-bound enzyme by high salt, and its elution
by high concentrations of DTT or increased pH, resemble
those of El, which is bound to theaffinity column as a covalent
thiol ester intermediate (17). However, E2does not activate
ubiquitin sincepurified E, had no ATP-PPI exchange activity
1 7.1
4.9
206
671
351
62.3
60.7
8,992
2,287
310
100
1.9
4.1
14.6
15.2
E2
(-fold)
E~
ATP
+
+
-
++
‘:;%:Fd
KCI eluate
DTT eluate p~ geluate
% of applied
3.6
59.0
40.0
11.7
0
3.8
36.0
0
2.3
17.1
0
4.2
TABLE
VI
to ubiquitin-Sephurose in the presence orabsence of
A TP
25O-pl portions of EBfrom the Sepharose 6B separation of the pH
9 eluate (Fig. 4B), containing 43.5 nanounits of activity, were applied
to 1-ml ubiquitin-Sepharose columns in the presence or absence of
ATP and E,, under conditions identical with those described in the
legend Table V. Activity of E3 was assayed by the lZI-ubiquitin
conjugation method, as described in the legend to Fig. 4B.
Binding of
E3
Ea activity recovered
Additions
U
~
~
~ KCI
: eluate
~ d
zuz
p~ 9 eluate
% of applied
None
51.5
+ ATP and
43.3E ,
13.3
18.0
0
0
40.4
62.6
in the presence of ubiquitin (data not shown). Therefore, we
tested whether the re-binding of E2 (purified through the
affinity and gel filtration columns) to the ubiquitin column
requires E, as well as ATP. As shown in TableV, this indeed
was found to be the case. In the presence of El and ATP,
most E2was bound to the column and recovered in the DTT
and pH 9 eluates, whereas upon the omission of either El or
ATP there was no significant binding of Ez, and most of the
recovered activity was in the unadsorbed fraction. Similar
results were obtained with the high molecular weight form of
E2 (data not shown). These results suggest an E,-mediated
binding of Ez to the ubiquitin column as a covalent intermediate (see “Discussion”).
In contrast toE2, the binding of purified E, to the affinity
column did not require El or ATP (Table VI), suggestive of
noncovalent interactions. It is notable, however, that only a
Downloaded from www.jbc.org at Univ of Ottawa - OCUL on December 7, 2007
Total protein
Purification
EI
Ez
nanounits/mg
100
2.7
20.2
5.7
1.5
TABLEIV
Distribution of E, in fractions of the affinity column
10ml of Fraction I1 from reticulocytes were subjected to affinity
chromatography as described under “Materials and Methods.” The
activity of E, was determined by the ‘?-ubiquitin conjugation assay
(see “Materials and Methods”), as described in the legend to Fig. 4B.
Fraction
EI
E2
E1
nanounits
mg
Specific activity
Recovery
E2
8212
Ubiquitin-Protein Ligase System
Downloaded from www.jbc.org at Univ of Ottawa - OCUL on December 7, 2007
part of column-bound E3 can be eluted with high salt, while
TABLE
VI1
another part is eluted with high pH (Table IV), suggesting
Protection of E, and E2 against inactivation by iodoacetamide
different types of interactions. To examine whether these
For Experiment 1purified Et (see "Materials and Methods") was
represent two different species of E3, the fraction of enzyme diluted to a concentration of 66.7 nanounits/ml in a solution of 50
eluted at pH 9 was applied again to ubiquitin-Sepharose and mM Tris-HCI (pH 7.61, 1 mM dithiothreitol, 1 mg/ml of ovalbumin,
its elution pattern was determined. As shown in Table VI, 50 mMM&12, and 2 units/ml ofinorganicpyrophosphatase, in a
final volume of50 pl. Where indicated, 2 mM ATP or 1 p~ unlabeled
again a partition of E, between the high salt and pH9 eluate ubiquitin
was supplemented. The mixtures were incubated at 25 "C
was observed. Moreover, with E, derived from the high salt for 5 min beforethe addition of iodoacetamide (4 mM) and incubation
eluate, a similarpartitioning between the above two fractions for a further 10 min. Samples of 2 pl were then transferred to the
was found (data not shown). The results indicate, therefore, reaction mixture of the 12SI-ubiquitinconjugation assay (see "Matethat this elution pattern is not due to distinct forms of the rials and Methods"), which contained a 25-fold excess dithiothreitol
enzyme, but possibly represents different types
of interactions overiodoacetamide. The reaction mixtures wereadjusted to equal
of ubiquitin (54 pmol), so that the specific radioactivity of
of E3 with column-bound ubiquitin. It should be noted that amounts
125
I-ubiquitin was identical in all samples. Activity of E , was assayed
ubiquitin is possibly bound heterogenously to thecolumn both in the presence of 0.67 nanounits of purified E2 (Fig. 3) and 4.6 pg of
with regard to the site of attachment of lysine residues of iodoacetamide-treatedpH 9 eluate (Fig. 3). Results are expressed as
the percentage of the activity of a control sample which was treated
ubiquitin and also relative to matrix structure.
similarly, except that iodoacetamide was mixed with a IO-fold molar
Transfer of Activated Ubiquitin fromE, to E,-Attempting
to clarify the roles of E2and E3 in the ligation of ubiquitin excess of dithiothreitol prior to its addition. For Experiment 2, a
purified preparationof E2 (see Fig. 3) was diluted to a concentration
with proteins, we considered the possibility that E2may have of 56 nanounits/ml and incubated under conditions similar to those
a function inthe transferof activated ubiquitin to the siteof describedfor Experiment 1. Where indicated, 2 mM ATP, 1 y M
amide bond formation. Such a possibility was initially sug- ubiquitin, or 150 nanounits/ml of purified E1 were supplemented to
gested by the observation that thebinding of E2to ubiquitin- the incubation priorto the addition of iodoacetamide. E2 activity was
Sepharose requires El as well as ATP (Table V). Further assayed in the presence of 3.6 nanounits of E , and 4.6 pgof iodoevidence for the transfer of ubiquitin from E, to a thiol site acetamide-treatedpH 9 eluate. For Experiment 3, experimental conditions were similar to those of Experiment 2, except that the low
on E2 was provided by the characteristics of the protection of molecular weight peakof E2 from the gel filtration column of the pH
these enzymes against inactivation by iodoacetamide. E, can 9 eluate (see Fig. 4A) was used, at a concentration of 76.7 nanounits/
be protected againstiodoacetamide by a prior incubationwith ml.
ATP and ubiquitin (Table VII, Experiment 1). This is preActivity remained
Additions
sumably due to the formation of the thiol ester linkage beE1
El
tween ubiquitin andthe thiol site of El. E, can also be
% of control
protectedagainst iodoacetamide inactivation, butthis re- Experiment 1
quires a preincubation with El, in addition to ubiquitin and
El + ATP
6
ATP (Table VII, Experiments 2 and 3). This suggests the 19 E , + ubiquitin
transfer of ubiquitin, activated by E, in the presence of ATP,
E,+ATP+Ub
82
Experiment 2
to an iodoacetamide-sensitive thiol site of E2.
E2 + E, + ATP
3
The thiol esterEl-ubiquitin is sufficiently stable to be
E, + E, + ubiquitin
0
separated by SDS-polyacrylamide gel electrophoresis when
E2 + E , + ATP + ubiquitin
96
electrophoresis is performed at 4 "C (17). We therefore Experiment 3
searched for a possible transfer of lZ5I-ubiquitin from
E1 to E2
El + ATP + ubiquitin
5
on gels run under similar conditions. In theexperiment shown
E, + ATP + ubiquitin + E ,
86
was first incubated with El
in Fig. 6 , lanes 1-3, 1251-~biq~itin
in the presence of ATP (Fig. 6, lane I ) , resultingin the
formation of El-ubiquitin thiol ester (subunit size, M , = of E2 was more labile than that of ubiquitin-protein conju105,000, Ref. 17). The specific radioactivity of residual free gates. When the sample was boiled in thepresence of mercapubiquitin was then lowered 1,000-fold by the addition of a toethanol priorto electrophoresis, ubiquitin was released from
large excess of unlabeled ubiquitin, following which E2 was all bands (Fig. 6, lane 7). This is in contrast to the stability
added to theincubation. As shown in Fig. 6, lane 2, there was of the amide linkage of ubiquitin-protein conjugates in such
a marked decrease in the amount of El-bound l2'1-ubiquitin, treatment (7). In addition, ubiquitin was also released from
with the appearance of four new bands of lower molecular its linkage to E2by treatment with 0.1 N NaOH (but notwith
weight. When unlabeled ubiquitin was added before El and 1 N formic acid) or by treatments with 1 M hydroxylamine at
E2, none of the bands was significantly labeled (Fig. 6 , lane pH 8 or 1%mercuric acetate (data not shown), under condi3 ) , indicating that the isotopic dilution of lZ5I-ubiquitinwas tions identicalwith those used to characterize the E1-ubiquitin
adequate, and thus the new bands of Ez originate from El- thiol ester linkage (12). These data indicate that activated
bound "'I-ubiquitin. Similar transfer of activated ubiquitin ubiquitin is transferred from El-ubiquitin to thiol ester interfrom E,-ubiquitin to thefour bands of E2 was observed when mediates of ubiquitin with E,. In experiments of similar
ATP was removed with hexokinase and glucose prior to the design, no transfer of activatedubiquitin from E1 to E3
addition of E2 (data not shown). The apparent molecular (without E2)was observed.
Transfer of Activated Ubiquitin to Conjugate Formation in
weight of the four bands of E,, designated bands 1-4 in
increasing molecular size, are 21,500, 23,000, 32,000, and the Presence of E3-We next asked whetherthe EP-ubiquitin
34,000, respectively. In addition, a region of diffuse radioac- thiol esters can be donors for conjugate formation. In the
tivity between band 1 and free ubiquitin can be seen, which experiment shown in Fig. 6, lanes 4-6,E, was first incubated
may represent ubiquitin released from enzyme-bound forms with '9-ubiquitin, ATP, and a smaller amount of El. AS
during electrophoresis. All four bands of EPwere present in shown in Fig. 6 , lane 4 , the expected thiol esters of EI and EP
different preparations of E2, although in some preparations were formed. ATP was then removed with hexokinase and
bands 1 and 2 were much more prominent than bands 3 and glucose, and E3was added for a further 10-min incubation (in
the presence of oxidized RNase as theconjugation substrate).
4.
The linkage of ubiquitin to thelow molecular weight bands As seen in Fig. 6 , lane 5, there was a marked loss of lZ5I-
Ubiquitin-Protein Ligase System
78
Cont.
P
\
8213
quantitation, the various lanes were cut into4-mm piecesand
radioactivity in the different bands was estimated by y counting. A small amount of high molecular weight contaminants,
present in this preparation of "'1-ubiquitin, was subtracted
from the corresponding positions. In this experiment, the
totalamount of 1251-ubiquitin-proteinconjugates was0.40
pmol, as compared to 0.06 pmol of ubiquitin lost from E,ubiquitin, and 0.36 pmol lost from all fourforms of EZubiquitin. This indicates thatthe different forms of E2ubiquitin were the main donors for conjugate formation in
the presence of E3.
DISCUSSION
ubiquitin from bands 1 and 2 and a partial decrease in bands
3-4, concomitant with the appearance of numerous high molecular weight bands. These new bands are ubiquitin-protein
conjugates, as shown by the finding that they are resistantto
boiling in the presence of SDS and mercaptoethanol (Fig. 6,
lane 8).A control (Fig. 6, lane 6 ) showed that theaddition of
a similar amount of hexokinase and glucose prior to the
addition of all three enzymes prevented the formation of
conjugates, indicating that ATP was sufficiently removed
under these conditions in the transfer experiment.
It should be noted that activated ubiquitin bound to E,, as
well as to the
different forms of E2-ubiquitin, were lost during
Protein- Ub
the formation of ubiquitin-protein conjugates (Fig. 6, compare Ub*ATP
€2- S-Ub
Conjugate
lanes 4 and 5). The question arose which enzyme-ubiquitin
FIG.7. Proposed sequence of events inthe ubiquitin-protein
thiol ester is the main source for conjugate formation. For ligase system. See the text. Ub,ubiquitin.
AMp*wixE;i;+- "")
:c
~
Downloaded from www.jbc.org at Univ of Ottawa - OCUL on December 7, 2007
FIG.6. Transfer of activated ubiquitin from Elto Ezand to
conjugate formation in thepresence of Es.All incubations contained, in a final volume of 20 pl, 50 mM Tris-HCI (pH 7.2), 5 mM
MgCI2, 0.1 mM ATP, 0.2 mM dithiothreitol, 0.1 unit of inorganic
pyrophosphatase, 10 pgof oxidized RNase, and 1.84 pmol of '%Iubiquitin (9900cpm/pmol). Lanes 1-3, transfer of activated ubiquitin
from El to E2.Lune 1, incubated with E, (0.46 nanounits) a t 37 "C
for 5 min. Lane 2, incubated with El as in lane 1 , then 2000 pmol of
unlabeled ubiquitin were added, followed by the addition of purified
E2 (0.6 nanounits), and a further incubation of 5 min. Lane 3, 2000
pmol of unlabeled ubiquitin were added before the addition of the
above amounts of El and E2,and the mixture was incubated for 10
min. Lanes 4-8, transfer of E,-bound ubiquitin to conjugate formation
in the presence of En. Lane 4, incubated a t 37 "C for 5 min in the
presence of E, (0.073 nanounits) and E2 (0.6 nanounits); lane 5,
incubated as in lane 4, then hexokinase (1 unit) and 2-deoxyglucose
(10 mM) were added for a further 3-min incubation. This was followed
by the addition of 0.46 nanounits of En(from the Sepharose 6B peak
of the pH 9 eluate, treated with 5 mM iodoacetamide for 15 min a t
37 "C) and incubation was continued for a further 5 min. Lane 6,
hexokinase and deoxyglucose, at theabove amounts, were incubated
with the reaction mixture for 3 min before the supplementation of
E,, E2, and E3(at theabove amounts) and a further incubation of 10
min. Samples 1-6 were treated with 0.5% SDS a t 0 "C for 30 min
before electrophoresis. Lanes 7 and 8, incubations identical with lanes
4 and 5,respectively, but thesamples were boiled for 3 min, with 2%
SDS and 3% mercaptoethanol prior to electrophoresis. SDS-polyacrylamide gel electrophoresis was performed as described previously
(17)on 12.5% polyacrylamide running gel and 6%stacking gel a t 30
mA for 3.5 h a t 4 "C. The gel was stained, destained, dried, and
radioautographed as described (7). EI-Ub,El-ubiquitin thiol ester;
Cont., contaminations in the preparation of '&I-ubiquitin; 1-4, different E2-bound forms of 9-ubiquitin.
The present study was initiated by an examination of the
components of the ubiquitin-ATP system which can be isolated by affinity chromatography on ubiquitin-Sepharose.
Since several enzymes in this pathway may have specificsites
for ubiquitin, it was expected that further components, in
addition to the ubiquitin-activating enzyme (17), may bind to
the affinity column. In fact, two further factors of the proteolytic system were isolated by this method: E2,which binds to
the affinity column in the presence of ATP, and ES, the
binding of which does not require ATP. We then found that
E2and En,in concert with E,, are participating in the conjugation of ubiquitin with proteins (Table 11).We propose to
designate the three enzymes as components of the ubiquitinprotein ligase system. The identity of the components of the
ligase system with the factors of the proteolytic system was
indicated by the coincidence of the corresponding activities
across gel filtration columns (Figs. 3 and 4). These results
provide further support for the role of the conjugation of
ubiquitin in protein breakdown.
Since the purified ubiquitin-activating enzyme does not
carry out conjugation by itself (18),the existence of a further
enzyme (which would catalyze amide bond formation between
ubiquitin and proteins) was expected, but the observed requirement for twodistinct additional enzymes wassurprising.
The present data indicate that the role of E2 might be the
transfer of activated ubiquitin to the site of amide bond
formation, and the proposed sequence of events is depicted in
Fig. 7. According to this scheme, activated ubiquitin bound
via its COOH terminus to the thiol site of E, is first transferred to another sulfhydryl site onE2.The first clue for such
a transfer process was the observation that thebinding of E2
to the ubiquitin column requires ATP, as well as E , (Table
V). This canbe explained by the assumption that E2replaces
column-bound El in a thiol ester linkage, although the possibility that E2 is bound through E, could not be ruled out. A
more direct proof for the involvement of thiol groups on Ez
in this process is the finding that protection of E2 against
inactivation by iodoacetamide requires preincubation with E,,
in the presence of ubiquitin and ATP (Table VII). Finally,
transfer of activated "'I-ubiquitin from E, to the different
forms of E2can be directly demonstrated by polyacrylamide
gel electrophoresis (Fig. 6), and thelinkage of ubiquitin to all
forms of E2 has the stability characteristics of a thiol ester
bond. It should be noted that thiol transesterification of
itin-Protein8214
such as the specificity and control of the ubiquitin-ligase
system, remain for future investigation.
Acknowledgments-A part of this work was done during the stay
of A. H. atthe Institute for CancerResearch, Philadelphia. We thank
Dr.Irwin A. Rose for heipful suggestions. The expert technical
assistance of Clara Segal is gratefully acknowledged.
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activated carboxyl residues occurs in otherbiosynthetic procmore closely
esses such as in fatty acid synthesis (21) or in the
analogous synthesis of peptide antibiotics (22). In both of
these cases, reversible transfer of thiol esters is between
enzyme-bound cysteine and 4'-phosphopanthetheineresidues. It may well bethat a pantetheine group has ananalogous
role in theubiquitin-ligase system.
We find that ubiquitin is further transferred from E,-bound
thiol esters to stable conjugates in the presence of E3(Fig. 6).
The function of EBmay thus be the catalysis of amide bond
formation between ubiquitin and proteins. It is possible, however, that E3 has an essentially required structural role, in
which case the ligase function may reside in one o f the earlier
components. Another unsolved problem is the significance of
the multiple forms of E,. The high molecular weight form of
E, does not seem to be an E2.E3 complex, since its molecular
weight is slightly lower than that of E3 (Fig. 4). It might be a
multimer oflow molecular weight E,, or an isoenzyme that
carries out asimilarfunction.
In addition, low molecular
weight E, is further composed of several different proteins
which bind ubiquitin (Fig. 6). The different E,-ubiquitin
bands do not seem to be incompletely dissociated subunits of
a single enzyme, since their treatment with increasing concentrations of SDS or incubation with 0.5% SDS a t 37 "C for
prolonged time periods did not convert the higher molecular
weight bands to thelower bands, but rather a uniform loss of
'251-ubiquitin from all bands of E, was observed (data not
shown). In addition, the apparent molecular weight of the
different E,-ubiquitin bands does not fit the assumption that
they consist of increasing numbers of ubiquitin residues bound
to a single enzyme subunit. It is possible that some of the
bands are nonspecific thiol esters of ubiquitin with proteins
that contaminate the preparations of E,. This may be the
case with bands 3 and 4, which are presentin variable amounts
and are only partially transferred toconjugates in the presence
of E3 (Fig. 6). It is also possible, however, that the different
subspecies of E , represent a family of enzymes of related
function, but of different specificities. For example, the attachment of successive molecules of ubiquitin to the protein
substrate may occur at different sites of the ligase, each
specific for a particular type of lysine residue; the different
subspecies of E, may then transfer ubiquitin to the corresponding specific ligation sites. These and other questions,
Ligase System