From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
Received for publication, November 4, 2002, and in revised form, January 2, 2003
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ABSTRACT |
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Rate studies have been employed as a
reporter function to probe protein-protein interactions within a
biochemically defined reconstituted N-end rule ubiquitin ligation
pathway. The concentration dependence for E1-catalyzed
HsUbc2b/E214kb transthiolation is hyperbolic
and yields Km values of 102 ± 13 nM and 123 ± 19 nM for high affinity
binding to rabbit and human E1/Uba1 orthologs. Competitive inhibition
by the inactive substrate and product analogs HsUbc2bC88A
(Ki = 104 ± 15 nM) and
HsUbc2bC88S-ubiquitin oxyester (Ki = 169 ± 17 nM), respectively, indicates that the ubiquitin moiety contributes little to E1 binding. Under conditions of
rate-limiting E3 The majority of short-lived cellular proteins are targeted for
degradation by the 26 S proteasome in response to assembly of
degradation signals on their surface comprising chains of ubiquitin moieties covalently linked through specific lysine residues (1). Target
protein specificity for this process is determined in part by a large
family of diverse ubiquitin-protein isopeptide ligases (E3)1 that recognize specific
features of the native structure (2-4), transposable
trans-acting amino acid sequences (5-7), or exposed regions of non-native conformation (8). The activated ubiquitin required to drive isopeptide bond formation is donated by specific ubiquitin carrier proteins (E2/Ubc), also termed ubiquitin-conjugating enzymes, in which the ubiquitin carboxyl terminus is bound as a
thiolester to a conserved cysteine (9, 10). The apparent specificity of
different isopeptide ligases for recognizing a single or limited number
of E2 isozymes accounts for the large cohort of related carrier protein
isoforms (9-11). Some E2 moieties may contribute to the substrate
specificity of their cognate E3 isozyme because they are able to
associate with targets in the absence of ligase (12-14). In addition,
a subset of E2 isozymes catalyzes formation of polyubiquitin
degradation signals on model protein substrates in the absence of their
cognate E3 (15-18). The E3-independent conjugation reactions catalyzed
by these selected isoforms presumably reflect catalytic roles within
their respective ligase-dependent substrate targeting mechanisms.
Ubiquitin thiolester formation to the different E2 isozymes occurs by
transfer of this moiety from a ternary complex of ubiquitin-activating enzyme (E1) containing two forms of activated ubiquitin polypeptide: a
tightly bound ubiquitin adenylate intermediate and a covalent E1-ubiquitin thiolester (19, 20). Plants inexplicably contain multiple
E1 isozymes (21), whereas other eukaryotes possess a single gene that
is transcribed into a 3.5-kb message subject to translation at
alternate start sites to yield cytoplasmic and nuclear isoforms of 110 and 117 kDa, respectively (22). The additional amino-terminal 40 residues of the 117-kDa E1 contain a nuclear localization signal and
multiple phosphorylation sites that promote sequestering of this
isozyme within the nucleus (22). Because each ligase family recognizes
a cognate E2 or its orthologs, rates of conjugation through the
different pathways and, therefore, their contribution to degradative
targeting, depend on efficient loading of these enzymes by the E1
ternary complex (9).
We have shown recently that ubiquitin ligation is amenable to detailed
kinetic analysis in biochemically defined in vitro assays
(23, 24). In the present work, we extend these studies to
protein-protein interactions among the components of the N-end rule
targeting pathway for ubiquitin conjugation. This pathway requires the
E3 isozyme Ubr1 in Saccharomyces cerevisiae (2) and the
19.7-kDa E2 isozyme
Rad6/ScUbc22 (25). In rabbit
reticulocytes the corresponding pathway is catalyzed by E3 Bovine ubiquitin, creatine phosphokinase, yeast inorganic
pyrophosphatase, yeast hexokinase, and human Generation and Purification of Wild Type and Mutant
HsUbc2b--
The human/rabbit HsUbc2b coding sequence was excised from
plasmid pECO10, derived from pKK223-3 (30), and cloned into plasmid pET11d to generate pET-HsUbc2b. Mutation of the active site cysteine 88 of human HsUbc2b to either alanine or serine was achieved by the
PCR-based overlap extension protocol of Ho et al. (31) to generate pET-HsUbc2bC88A and pET-HsUbc2bC88S, respectively. The complete HsUbc2b coding regions of these constructs were sequenced using a Sequenase kit (U. S. Biochemical Corp.) to confirm the desired
base change(s) and the absence of secondary mutations introduced by the
PCR steps.
A single colony of Escherichia coli BL21 (DE3) strain
harboring the appropriate plasmid was grown in 10 ml of LB medium
containing 100 µg/ml ampicillin for 6 h at 37 °C, and then 50 µl was used to inoculate a 250-ml overnight culture of the same
medium. The next morning 10 liters of LB containing 100 µg/ml
ampicillin were inoculated with 100 ml of the overnight culture and
then grown at 37 °C to an A600 of 0.8-1.0 in
a VirTis 20-liter fermenter. Expression was induced by adding
isopropyl-1-thio-
The resulting lysate was then centrifuged at 105 × g for 1 h after which the high speed supernatant was
adjusted to pH 7.5 with 1 M NaOH followed by bulk
absorption to 250 ml of DEAE-cellulose (Whatman DE52) equilibrated in
Buffer A. The DEAE slurry was stirred gently at 4 °C for 1 h to
allow complete adsorption of protein and then washed on a Buchner
funnel with 3 bed volumes of Buffer A, followed by batch elution with 2 bed volumes of Buffer A containing 0.5 M NaCl. Protein in
the eluate was precipitated by adjusting to 80% saturated ammonium
sulfate then recovered by centrifugation for 20 min at 104 × g. The pellet was suspended in 50 ml of 50 mM
Tris-HCl (pH 7.5) containing 1 mM DTT (Buffer B) and then
dialyzed overnight against 2 liters of the same. The dialysate was
loaded onto a 5 × 10-cm Q-Sepharose Fast Flow (Amersham
Biosciences) column equilibrated with Buffer B and fitted to an
Amersham Biosciences FPLC system. Protein was eluted with a linear
0-0.5 M NaCl gradient (1.7 mM/min) in Buffer B
at a flow rate of 2 ml/min. Fractions containing HsUbc2b activity (0.18 M elution position) were pooled and adjusted to a final
concentration of 1.8 M ammonium sulfate and then applied to
a 3 × 25-cm phenyl-Sepharose Fast Flow (Amersham Biosciences)
column equilibrated with 1.8 M ammonium sulfate in Buffer
B. Protein was eluted with a negative linear gradient of 1.8-0
M ammonium sulfate (7.2 mM/min) at a flow rate
of 2 ml/min. Wild type and mutant HsUbc2b enzymes eluted at ~0.5
M ammonium sulfate. Aliquots from the phenyl-Sepharose
column containing HsUbc2b were pooled and dialyzed against 1 liter of
Buffer B before being loaded onto a Mono Q HR 5/10 FPLC column
(Amersham Biosciences) equilibrated with Buffer B. Protein was eluted
from the Mono Q column with a linear 0-0.5 M NaCl gradient
(12.5 mM/ml) at a flow rate of 1 ml/min. Mono Q fractions
with HsUbc2b activity were pooled and concentrated to less than 5 ml on
an Amicon cell fitted with a PM10 membrane and then resolved on a
10 × 30-cm Superose-12 FPLC column (Amersham Biosciences) column
equilibrated with 50 mM Tris-HCl (pH 7.5) containing 1 mM DTT and 50 mM NaCl at a flow rate of 1 ml/min.
The isolated HsUbc2b proteins were greater than 99% pure as assessed
by SDS-PAGE followed by Coomassie Blue R-250 staining. Absolute protein
concentrations were calculated spectrophotometrically using an
empirical Direct Kinetic Assay of HsUbc2b-125I-Ubiquitin
Thiolester Formation--
Initial rates of 125I-ubiquitin
thiolester formation on HsUbc2b were measured in 175-µl reactions
containing 50 mM Tris-HCl (pH 7.5), 2 mM ATP,
10 mM MgCl2, 10 mM creatine
phosphate, 1 mM DTT, 10 IU/ml creatine phosphokinase, 5 µM 125I-ubiquitin (about 3,000 cpm/pmol), 0.7 nM rabbit reticulocyte E1, and the indicated concentrations
of HsUbc2b. Aliquots of 25 µl were removed at 30-s intervals over the
first 1.5 min and quenched with an equal volume of SDS sample buffer.
Thiolesters were then resolved from free 125I-ubiquitin by
12% (w/v) SDS-PAGE under nonreducing conditions at 4 °C (29). Bands
containing E2 thiolesters, visualized by autoradiography of the dried
gels, were excised and quantitated by Coupled Kinetic Assay of E1-HsUbc2b Interactions--
Binding
interactions between the E1 ternary complex and HsUbc2b were
quantitated kinetically as the initial net forward rate of E1-catalyzed
ubiquitin transthiolation to HsUbc2b by directly coupling this step to
E3
The interaction of HsUbc2bC88A or HsUbc2bC88S-ubiquitin oxyester with
the E1 ternary complex was measured by the
concentration-dependent inhibition of wild type
HsUbc2b/E3 Kinetic Assay of HsUbc2b-E3 Cell Culture--
Confluent cultures of IMR90, A549, and Caco-2
cells were maintained at 37 °C in a constant atmosphere of 5%
CO2 in Eagle's minimum essential medium supplemented with
5 mM glutamine and 10% fetal calf serum. Two days after
the addition of fresh supplemented medium, triplicate 100-mm plates
were washed three times with 10 ml of 25 mM
phosphate-buffered saline (pH 7.4) and then harvested by scraping into
0.5 ml of ice-cold homogenizing buffer containing 50 mM
Tris-HCl (pH 7.5), 0.25 M sucrose, and 1 mM
DTT. Cells were then lysed by brief sonication and clarified by
centrifugation for 10 min at 16,000 × g. Thiolester
assays of a 50-µl final volume containing 50 mM Tris-HCl
(pH 7.5), 2 mM ATP, 10 mM MgCl2, 10 mM creatine phosphate, 1 mM DTT, 10 IU/ml
creatine phosphokinase, 5 µM 125I-ubiquitin,
68 nM rabbit liver E1, and varying amounts of cell extract
were incubated for 5 min at 37 °C before quenching with an equal
volume of SDS sample buffer from which E1-catalyzed Formation of HsUbc2b-Ubiquitin
Thiolester--
Incubation of the E1 ternary complex with an equimolar
concentration of E2 results in rapid stoichiometric transthiolation to
form the corresponding E2-ubiquitin thiolester (29, 34). In principle,
the dependence of initial rate for E2-ubiquitin thiolester formation
versus HsUbc2b concentration can be used to examine the
kinetics of ubiquitin transfer in the key step linking the
half-reactions of ubiquitin activation and ligation under conditions
for which [E1]o
Nonstoichiometric transthiolation is a function of the
[HsUbc2b]o/[E1]o ratio rather than a
concentration-dependent change in oligomerization state of
the activating enzyme because the predicted amount of
HsUbc2b-125I-ubiquitin thiolester was formed when
incubations contained 0.7 nM E1 but only 54 nM
HsUbc2b (Fig. 1B). Because the E1 present in these
incubations formed the expected amount of ternary complex even at high
[HsUbc2b]o/[E1]o ratios (not shown), the
results of Fig. 1 were not the result of inhibition of E1-catalyzed
steps by the E2 isozyme. However, the data suggest that the
nonstoichiometric formation of the high energy HsUbc2b intermediate
results from equilibration between the E1 ternary complex and
HsUbc2b-ubiquitin thiolester at high
[E2]o/[E1]o ratios, particularly because
previous studies have shown that transthiolation is readily reversible (29, 34). This conclusion is supported by subsequent control experiments demonstrating that high concentrations of HsUbc2bC88A or
HsUbc2bC88S-ubiquitin oxyester inhibited the rate but not the final end
point for wild type HsUbc2b-125I-ubiquitin thiolester
formation (not shown).
Kinetics of E1-HsUbc2b Transthiolation--
Although the
stoichiometry of E2-ubiquitin thiolester formation is
underestimated at high
[HsUbc2b]o/[E1]o ratios, the kinetics of
transthiolation can be quantitated for the isolated activation half-reaction by measuring the initial rate for approach to equilibrium in the absence of ligase (36). At 0.7 nM E1, initial rates
for HsUbc2b-125I-ubiquitin thiolester formation determined
during the first 1.5 min followed hyperbolic kinetics below 0.5 µM HsUbc2b, as shown by the linearity of the
corresponding reciprocal plot (Fig. 2). Hyperbolic kinetics requires the initial formation of a Michaelis complex between uncharged HsUbc2b and the E1 ternary complex prior to
intramolecular transthiolation. Nonlinear least squares fitting of the
data in Fig. 2 within the linear region of the reciprocal plot yielded
a Km of 102 ± 13 nM for
HsUbc2b binding and a kcat of 4.8 ± 0.2 s
At the highest concentrations of HsUbc2b tested, the initial rates for
thiolester formation consistently exhibited substrate inhibition (Fig.
2). This was not a consequence of underestimating vo because rates were linear over at least three
time points encompassing the first 1.5 min of the reactions. Linearity
during the measurement interval for the kinetic studies also ruled out
equilibration of the E1 ternary complex with
HsUbc2b-125I-ubiquitin thiolester observed in Fig. 1,
although an equilibrium end point was reached at longer incubation
times (not shown). We have subsequently observed analogous substrate
inhibition with members of other E2 families (not shown), including the
rabbit Ubc8 ortholog E220K (37) and the human Ubc4/5
orthologs HsUbc5A/B/C (38). Substrate inhibition of E1-catalyzed
transthiolation is consistent with either of two models. In the most
plausible model, uncharged E2 can bind two distinct conformations of
the E1 ternary complex for which one bound conformer (presumably of
lower affinity than the productive binding complex) represents a dead
end species. An alternative model in which the E2 carrier protein forms
a nonproductive alternate conformation having a low affinity for the E1
ternary complex is much less likely because NMR studies find that the E2 core domain fold is relatively static (39).
Kinetics of E1-HsUbc2b Interactions during
E3 Kinetics of HsUbc2b Transthiolation Catalyzed by Human
E1--
Recently, Wee et al. (40) reported similar
transthiolation kinetics for recombinant human E1 using ubiquitin
modified at lysine 6 with the fluorescent label Oregon green. Wee and
co-workers report substrate inhibition by HsUbc4 at micromolar
concentrations similar to that found for HsUbc2b in the present work
(Fig. 2). However, their results differ in several key aspects from
those reported here for the activation of ubiquitin by rabbit
reticulocyte E1. Because rabbit E1 is 96.5% identical to its human
ortholog (41), we extended our transthiolation studies to E1 isolated from human erythrocytes to resolve whether these apparent discrepancies reflected significant differences in catalytic properties of rabbit versus human E1 orthologs, differences in the experimental
approaches, or both.
In our hands, human erythrocyte-activating enzyme exhibits the same
equilibration between the E1 ternary complex and HsUbc2b-ubiquitin thiolester as found for the rabbit ortholog in Fig. 1 (not shown). As
with the rabbit ortholog, we were able to examine the kinetics for
transthiolation under initial velocity conditions for approach to
equilibrium (36), the results of which are summarized in Table
I. Human E1 exhibits values of
Km for ATP and ubiquitin of 7.0 ± 1.1 µM and 0.8 ± 0.2 µM, respectively,
which are in good agreement with the values of 4.7 ± 1.0 µM and 0.17 ± 0.03 µM, respectively,
reported by Wee et al. (40). In addition, human E1-catalyzed
transthiolation kinetics yields a Km for HsUbc2b of 123 ± 19 nM and kcat of
4.5 ± 0.3 s
The nearly 100-fold difference in kcat values
for E2 transthiolation suggests that alkylation of ubiquitin with
Oregon green at lysine 6 (40) significantly alters the kinetics of
transthiolation. This is consistent with the marked difference between
wild type ubiquitin and Oregon green-modified polypeptide in their
binding to E1 (40) as well as our observation that the values of
Km and kcat for HsUbc5A,
a member of the same E2 family as HsUbc4 (9), are nearly identical to
those of HsUbc2b (not shown). Finally, comprehensive point mutagenesis
of ubiquitin has shown that lysine 6 is not required for the initial
binding of the polypeptide to human E1 but is essential for subsequent
binding of the transition state during formation of the enzyme-bound
ubiquitin adenylate.4
Therefore, derivatization of lysine 6 likely effects a change in
rate-limiting step from E2 transthiolation to ubiquitin adenylate formation.
Kinetics of E3 Generation of Nonfunctional HsUbc2b Analogs--
Modification of
the active site cysteine 88 renders the point mutant HsUbc2bC88A
incapable of accepting the ubiquitin thiolester from the E1 ternary
complex; therefore, HsUbc2bC88A represents a nonfunctional analog of
the uncharged carrier protein (for review, see Ref. 9). In contrast,
previous studies show that mutation of the conserved active site
cysteine to serine in Arabidopsis thaliana Ubc1 (43) and
S. cerevisiae Rad6/ScUbc2 (44) results in E2 polypeptides
capable of accepting activated ubiquitin from the E1 ternary complex to
form the corresponding E2-ubiquitin oxyester but incapable of
supporting subsequent ligase-catalyzed isopeptide bond formation,
presumably because of the relative stability of the corresponding
ubiquitin oxyester. The analogous ubiquitin oxyester adduct of
HsUbc2bC88S is a potential nonfunctional analog of the wild type
E2-ubiquitin thiolester. Therefore, these HsUbc2 active site point
mutants represent important potential reagents for examining the
affinity of the E1 ternary complex for uncharged and charged forms of
the ubiquitin carrier protein in carefully designed competitive
inhibition assays.
Because HsUbc2bC88A cannot be assayed by functional
125I-ubiquitin thiolester or oxyester assays, the absolute
concentration of this point mutant could only be accurately estimated
spectrophotometrically in homogeneous preparations using the empirical
280 nm extinction coefficient for wild type HsUbc2b (see "Materials
and Methods"). Such quantitation reasonably assumes that the C88A
point mutation has no effect on the folding of the polypeptide. To
confirm the latter assumption, spectral fitting of circular dichroism
data for wild type HsUbc2b and HsUbc2bC88A was employed using the
self-consistent method of Sreerama and Woody (45). The percentages of
The autoradiogram of Fig. 5 (0 min
lanes) demonstrates that HsUbc2bC88S is also capable of accepting
activated 125I-ubiquitin from the E1 ternary complex to
form the corresponding HsUbc2bC88S-125I-ubiquitin oxyester
adduct. In a parallel rate study, oxyester formation followed first
order kinetics with a ko of 1.7 × 10
Under the conditions of Fig. 5, HsUbc2bC88S formed 72% of the oxyester
predicted from the total mutant concentration calculated spectrophotometrically. This value was confirmed by quantitation of the
shift in mobility of the oxyester in a parallel gel stained with
Coomassie Blue (not shown). The difference between predicted and
observed oxyester formation likely reflects the presence of denatured
mutant in the preparation; therefore, the active mutant concentration
was determined by stoichiometric 125I-ubiquitin oxyester
formation, analogous to the method for quantitating 125I-ubiquitin thiolester (29).
HsUbc2bC88A and HsUbc2bC88S-Ubiquitin Oxyester Are Competitive
Inhibitors of E1-catalyzed Transthiolation--
The kinetic study of
Fig. 3 was repeated in the presence of HsUbc2bC88A, which served as an
analog of uncharged wild type E2. Preliminary experiments
measuring vo versus
[HsUbc2b]o at various constant concentrations of
HsUbc2bC88A yielded a series of reciprocal plots characteristic of
competitive inhibition by the active site mutant (not shown); however,
the cumulative measurement error in the resulting secondary plot of
apparent Km versus
[HsUbc2bC88A]o precluded exact estimates of
Ki for the E2 mutant. Therefore, to minimize
experimental error and to allow more precise estimates of
Ki, independent data were collected at a
constant concentration of HsUbc2b, whereas [HsUbc2bC88A]o
was varied. The data were evaluated according to a rearranged
Michaelis-Menten equation (Equation 1) for which
Ki was calculated directly as the ratio of
y intercept/slope for experiments in which the competitive
inhibitor was varied. For these experiments, the concentration of wild
type HsUbc2b was held constant at 180 nM. The value of
Vmax was estimated at 1 µM HsUbc2b
(about 10 Km) from a parallel incubation
Data for vo versus
[HsUbc2bC88A]o at constant [HsUbc2b]o were
plotted according to Equation 1 and yielded the predicted linear relationship (Fig. 6A,
closed circles with solid line) from which Ki for mutant binding to the E1 ternary complex
could be calculated as 104 ± 15 nM. The intrinsic
Km determined from the y intercept (85 ± 9 nM) was statistically indistinguishable from
that determined in the absence of inhibitor. Good agreement between the
value of Ki for HsUbc2bC88A binding to the E1
ternary complex and the Km of 102 ± 13 nM for wild type HsUbc2b obtained by direct transthiolation kinetics (Fig. 2) suggests that the structure of the C88A mutant reasonably approximates that of wild type carrier protein. This conclusion is consistent with the comparable CD spectra and calculated secondary structure composition for wild type versus mutant
HsUbc2b (previous section). Therefore, the corresponding kinetically
determined Km values approximate the equilibrium
dissociation constant (Kd) for binding of
uncharged HsUbc2b to the E1 ternary complex.
Similar studies using HsUbc2bC88S-ubiquitin oxyester also exhibited
competitive inhibition (not shown). Plots of appropriate data according
to the modified Michaelis-Menten equation (Equation 1) conformed to the
expected linear relationship (Fig. 6A, open circles and dashed line) from which a value for
Ki = 169 ± 17 nM was
calculated. As before, the intrinsic Km
determined from the y intercept (119 ± 9 nM) was statistically indistinguishable from that
determined in the absence of inhibitor. The data suggest that the E1
ternary complex displays a small but statistically significant affinity
preference for binding uncharged HsUbc2b. The difference in binding
affinity between HsUbc2b and HsUbc2b-ubiquitin thiolester is much less
than the orders of magnitude differences in affinities typical of most
substrates versus products. Under some experimental
conditions the E2 mutants can potentially serve as competitive
inhibitors of the E3 HsUbc2bC88A and HsUbc2bC88S-Ubiquitin Oxyester Are Competitive
Inhibitors of E3
In these experiments, HsUbc2bC88S-ubiquitin oxyester serves as a
nonfunctional analog of wild type HsUbc2b-ubiquitin thiolester, the
actual cosubstrate for E3 Determination of Cellular E1 and HsUbc2b Concentrations--
The
previous kinetic results define affinities for protein-protein
interactions among the three components of the N-end
rule-dependent targeting pathway. To interpret these
affinities within a cellular context, the content of endogenous Ubc2
within selected cell types was quantitated in fresh tissue culture cell
extracts by its stoichiometric formation of 125I-ubiquitin
thiolester (29), a method used previously to monitor the coordinated
induction of E1 and E2 isoforms that accompany the programmed cell
death of Manduca sexta intersegmental muscles (48). Extracts
were prepared from confluent monolayer cultures and assayed as
described under "Materials and Methods." The autoradiogram of Fig.
7 shows typical data from one such
thiolester assay in which exogenous rabbit liver E1 (odd numbered
lanes) was added to ensure quantitative loading of Ubc2 present in
the cell extracts. Separate incubations from which exogenous E1 was
omitted were used to quantitate endogenous activating enzyme (not
shown). In Fig. 7, thiolesters resolved under nonreducing conditions
(odd numbered lanes) are distinguished from the small
amounts of conjugates revealed in parallel reducing gels (even
numbered lanes) which are formed during the brief incubation (29).
Fig. 7 demonstrates that a 125I-ubiquitin thiolester
species comigrating with authentic in vitro formed
HsUbc2b-125I-ubiquitin is the major E2 isoform present in
these cells under normal culture conditions, although several other
lower abundance E2 thiolester bands are also observed which correspond
to E2EPF (49, 50) and HsUbc5/UbcH5 isoforms (38, 51), the
human orthologs of S. cerevisiae Ubc4/5 isozymes
(51) and A. thaliana Ubc8 (52).
Radioactivity associated with thiolester bands for endogenous Ubc2 were
quantitated by In the present studies we have exploited rate measurements as
reporter functions for probing protein-protein interactions within a
biochemically defined reconstituted N-end rule ligation pathway.
Despite the complexity in the overall mechanism for ubiquitin conjugation, such studies demonstrate that the approach provides a
facile means of examining this pathway in detail, particularly when
combined with genetic manipulation of the interacting partners. In
addition, the precision with which the concentrations of E1 and E2
components can be determined by the stoichiometric formation of their
respective 125I-ubiquitin thiolesters provides a means of
accurately determining kinetic constants without the need for
assumptions regarding the relative content of active protein (19, 20).
These technical advantages have allowed us, for the first time, to
address details of a ubiquitin ligation pathway not otherwise
accessible to other techniques.
The rabbit reticulocyte E1 exhibits a relatively high affinity for
binding uncharged HsUbc2b and yields a Km of
102 ± 13 nM when measured directly by monitoring the
kinetics of transthiolation (Fig. 2). Excellent agreement between the
value for Km determined directly and the
Ki of 104 ± 15 nM determined
for the nonfunctional competitive inhibitor HsUbc2bC88A (Fig.
6A) requires that the Km reflect the
intrinsic binding affinity (Kd) for the
interaction between E1 and the uncharged carrier protein. Side chain
interactions between HsUbc2b and its E1 binding site must be largely
maintained during the catalytic cycle of transthiolation, with little
additional contribution from residues present on the ubiquitin moiety,
because the Km for uncharged HsUbc2b
approximates the Ki of 169 ± 17 nM found for HsUbc2bC88S-ubiquitin oxyester, the isosteric
competitive inhibitor of HsUbc2b-ubiquitin thiolester (Fig.
6A). Therefore, there is relatively little difference in
affinity ( In contrast, E3 These observations have important implications for correctly
interpreting in vivo studies based on overexpression of E2
dominant negative mutants. Frequently, overexpression of active site
C In the present studies we have also examined the net forward kinetics
of HsUbc2b transthiolation catalyzed by the human ortholog of the
ubiquitin activating enzyme (Table I). The Km
for ATP and ubiquitin of 7.0 ± 1.1 µM and 0.8 ± 0.2 µM, respectively, agree favorably with the values
of 20 µM and 0.9 µM, respectively, determined previously in equilibrium studies (55). In addition, the
Km values for binding of HsUbc2b by rabbit and
human E1 orthologs of 102 ± 13 nM (Fig. 2) and
123 ± 19 nM (Table I), respectively, show remarkable
conservation that reflects the high degree of homology between the two
enzymes (41). The marked similarity between the rabbit and human E1
orthologs is also reflected in their comparable
kcat values for HsUbc2b transthiolation of
4.8 ± 0.2 s Overall, the correspondence between values of Km
determined kinetically and Kd (determined either
directly from earlier equilibrium studies (55) or as
Ki from inhibitor studies with nonfunctional HsUbc2b mutants) for the three substrates of the E1 reaction indicates that the kinetics of ubiquitin activation approximates a
pre-equilibrium rather than the steady-state mechanism proposed earlier
based on similar kinetic studies using fluorescently labeled ubiquitin (40). The radioiodinated ubiquitin used in the present studies has been
validated previous as functionally equivalent to the wild type
polypeptide (19, 20); therefore, the apparent discrepancy between the
present data and earlier studies of Wee et al. (40) can be
best explicated by invoking steric hindrance from the fluorescent label
used in the latter work. The marked difference in reported kcat for E2 transthiolation of 4.8 ± 0.2 s We have quantitated the content of E1 and Ubc2 within selected cell
lines by exploiting the ability of these enzymes to form stoichiometric
125I-ubiquitin thiolesters (29, 48) (Table II). Several
general trends emerge from our analysis of E1 and Ubc2 content within the selected cell lines. The Ubc2 isoforms represent the major E2
family present in the four nonerythroid cell lines examined (Fig. 7).
Although several of the E2 families have quite similar molecular
weights, their corresponding 125I-ubiquitin thiolesters can
be resolved by nonreducing SDS-PAGE, allowing their unambiguous
quantitation (29, 48, 56). The intracellular concentration of Ubc2 is
consistently within the micromolar range, which is saturating with
respect to the Km of 102 ± 13 nM for uncharged HsUbc2b binding to E1 and the
Km of 54 ± 18 nM for
HsUbc2b-ubiquitin thiolester binding to E3 Previous work from our laboratory has quantitated steady-state pools of
free and conjugated ubiquitin in various cell lines (for review, see
Ref. 58). Comparison of ubiquitin pools with the intracellular content
of E1 and Ubc2 suggests that a significant fraction of the "free"
ubiquitin is actually present as thiolester intermediates of the
ligation machinery. For example, confluent IMR90 fibroblasts contain a
total ubiquitin content of 135 pmol/106 cells, of which the
free ubiquitin represents 54 pmol/106 cells (58, 59).
Because IMR90 cells contain 12 and 17.4 pmol/106 cells of
Ubc2 and E1, respectively (Table II), the actual pool of free ubiquitin
is about 7 pmol/106 cells, representing an intracellular
concentration of about 4 µM. The latter value remains
saturating with respect to the Km of 0.8 ± 0.2 µM for ubiquitin determined with human E1 (Table I).
This predicts that significant induction in E2 levels should be
accompanied by a coordinated induction of ubiquitin to avoid depletion
of free intracellular ubiquitin pools.
The present studies provide the first comprehensive examination of
protein-protein interactions within a ubiquitin ligation pathway and
the relationship between the resulting binding constants and
intracellular concentrations of the targeting components. The results
demonstrate that detailed features of the ubiquitin ligation mechanism
can be resolved by appropriately designed rate studies.
-catalyzed conjugation to human
-lactalbumin,
HsUbc2b-ubiquitin thiolester exhibits a Ki of
54 ± 18 nM and is competitively inhibited by the
substrate analog HsUbc2bC88S-ubiquitin oxyester
(Ki = 66 ± 29 nM). In
contrast, the ligase product analog HsUbc2bC88A exhibits a
Ki of 440 ± 55 nM with
respect to the wild type HsUbc2b-ubiquitin thiolester, demonstrating
that ubiquitin binding contributes to the ability of E3
to
discriminate between substrate and product E2. A survey of E1 and E2
isoform distribution in selected cell lines demonstrates that Ubc2
isoforms are the predominant intracellular ubiquitin carrier protein.
Intracellular levels of E1 and Ubc2 are micromolar and approximately
equal based on in vitro quantitation by stoichiometric
125I-ubiquitin thiolester formation. Comparison of
intracellular E1 and Ubc2 pools with the corresponding ubiquitin pools
reveals that most of the free ubiquitin in cells is present as
thiolesters to the components of the conjugation pathways. The present
data represent the first comprehensive analysis of protein interactions within a ubiquitin ligation pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, the
mammalian Ubr1 ortholog, in concert with HsUbc2/E214kb (23, 26, 27), the 17.3-kDa mammalian
E2 ortholog of S. cerevisiae Rad6 (9). We have
employed rate studies to determine the affinity for binding of
HsUbc2b3 to E1 and of the
corresponding HsUbc2b-ubiquitin thiolester to E3
. In addition,
active site mutants of the E2 have been used to estimate affinities for
product binding to E1 and E3
. The latter results have cautionary
implications for the unambiguous interpretation of in vivo
observations based on overexpression of dominant negative E2 mutants.
Finally, activity assays involving 125I-ubiquitin
thiolester formation have been exploited to quantitate intracellular
concentrations of E1 and Ubc2 within selected cell lines. Comparison of
these binding constants with the concentrations of the components
required for N-end rule-dependent ligation suggests that
the three enzymes of this pathway minimally form dynamic binary
complexes within the cell.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-lactalbumin were purchased from Sigma. The yeast inorganic pyrophosphatase and human
-lactalbumin were further purified to apparent homogeneity (23). The
ubiquitin was also further purified to homogeneity (15) then
radioiodinated by the chloramine-T procedure (28). Restriction
endonucleases and other DNA-modifying enzymes were purchased from New
England Biolabs or Amersham Biosciences. Carrier-free Na125I and [2,8-3H]ATP were purchased from
PerkinElmer Life Sciences. Rabbit liver, rabbit reticulocyte, and human
erythrocyte E1 enzymes were purified to apparent homogeneity by
adapting previously reported affinity chromatography and FPLC methods
(29), then quantitated by 125I-ubiquitin thiolester assay
and confirmed by the stoichiometric formation of ubiquitin
[3H]adenylate (19, 29). Unless otherwise indicated,
rabbit reticulocyte E1 was used in all kinetic studies for which the
activation step was kinetically isolated, whereas the more abundant but
otherwise identical liver enzyme was used for studies in which it
served only as a reagent for generating E2 thiolester. Human
recombinant HsUbc2b (23) was purified to apparent homogeneity using
previously established methods and then quantitated by its
stoichiometric formation of 125I-ubiquitin thiolester
formation in the presence of E1 (29). Rabbit reticulocyte Fraction II
was prepared by phenylhydrazine induction (28). Rabbit reticulocyte and
liver E3
were purified from Fraction II by E2-ligand affinity
chromatography (23). Rabbit reticulocyte E3
was used in all kinetic
studies for which the ligase step was rate-limiting, whereas the more
abundant liver E3
was used in coupled assays as a reagent ligase in
all other rate studies.
-D-galactopyranoside to a final
concentration of 0.4 mM. Following expression for 1.5 h, cells were collected by centrifugation at 4,000 × g
for 25 min and then resuspended in 50 mM Tris-HCl (pH 7.5)
containing 5 mM EDTA and 5 mM DTT (Buffer A) to
~30% (w/v) before lysing by French press. All subsequent steps were
carried out at 4 °C unless otherwise stated. Recombinant wild type
HsUbc2b and HsUbc2bC88S were assayed by their stoichiometric formation
of 125I-ubiquitin thiolester or oxyester, respectively, in
brief incubations (29). Recombinant HsUbc2bC88A was assayed by Western
blot using an affinity-purified rabbit anti-HsUbc2 antibody and
recombinant HsUbc2b standards (32, 33).
280 of 1.71 (mg/ml)
1 for native
HsUbc2b, determined by measuring the absorbance at 280 nm of a solution
of apparently homogeneous recombinant HsUbc2b for which the absolute
protein concentration had been determined by amino acid analysis.
Typical final yields ranged from 1 to 1.5 mg of HsUbc2b protein/liter
of culture and were usually greater than 90% active by comparing the
stoichiometric formation of 125I-ubiquitin thiolester to
the absolute protein concentration. Isolated proteins were stored at
80 °C for several months without loss of activity.
counting.
-catalyzed conjugation of human
-lactalbumin under E1-limiting
conditions. Incubations of 25 µl contained 50 mM Tris-HCl
(pH 7.5), 2 mM ATP, 10 mM MgCl2, 10 mM creatine phosphate, 1 mM DTT, 10 IU/ml
creatine phosphokinase, 5 µM 125I-ubiquitin,
0.34 nM rabbit reticulocyte E1, ~1.5 µg
affinity-purified rabbit liver E3
, and the indicated concentrations
of human wild type recombinant HsUbc2b. Samples were incubated for 25 min at 37 °C, quenched with an equal volume of SDS sample buffer
containing 4% (v/v)
-mercaptoethanol, and boiled for 5 min.
Radiolabeled conjugates were then resolved from free
125I-ubiquitin by 12% (w/v) SDS-PAGE and visualized by
autoradiography of the dried gels. Radioactive conjugates from each
sample were excised from the gel and quantitated by
counting
(28).
-catalyzed 125I-ubiquitin conjugation of human
-lactalbumin under conditions identical to those for the HsUbc2b/E1
assays except that [HsUbc2b]o was held constant at 180 nM. Maximum velocity in the absence of inhibitor was
determined in a parallel reaction containing saturating wild type
HsUbc2b (1.0 µM). The HsUbc2bC88S-ubiquitin oxyester adduct used in the inhibitor studies was stoichiometrically formed during a 30-min incubation at 37 °C in a 100-µl final volume
containing 50 mM Tris-HCl (pH 7.5), 2 mM ATP,
10 mM MgCl2, 1 mM DTT, 10 IU/ml PPi, 20 nM rabbit liver E1, 40 µM
125I-ubiquitin, and 34 µM HsUbc2bC88S. The
resulting HsUbc2bC88S-125I-ubiquitin oxyester adduct was
resolved from other reaction components by gel exclusion chromatography
using a 1 × 30-cm analytical Superdex 75 FPLC column (Amersham
Biosciences) equilibrated in 50 mM Tris-HCl (pH 7.5) and 50 mM NaCl. The concentration of
HsUbc2bC88S-125I-ubiquitin oxyester was determined from the
associated radioactivity of peak fractions quantitated by
counting
and confirmed by direct determination of oxyester following nonreducing
SDS-PAGE.
Interactions--
Binding
interactions between the 125I-ubiquitin thiolesters of wild
type recombinant HsUbc2b and affinity-purified E3
were quantitated under E3
-limiting conditions identical to those in the preceding section with the exception that incubations contained 65 nM
rabbit liver E1 and the indicated concentrations of HsUbc2b and E3
. Inhibition studies were conducted under identical conditions with the
exception that wild type [HsUbc2b]o was held constant at
25 nM (HsUbc2bC88A study) or 16 nM (HsUbc2bC88S
study), and the concentration of either HsUbc2bC88A or
HsUbc2bC88S-125I-ubiquitin oxyester was varied. Maximum
velocity in the absence of inhibitor was determined in a parallel
incubation containing 1.0 µM wild type HsUbc2b.
-mercaptoethanol had been
omitted. An aliquot of each sample was adjusted to 2% (v/v)
-mercaptoethanol then boiled for 5 min to correct for limited conjugate formation during the incubation. All samples were then resolved from free 125I-ubiquitin by 12% SDS-PAGE. Bands
corresponding to E1- and Ubc2-125I-ubiquitin thiolester
were excised from dried gels and the associated radioactivity
quantitated by
counting (29).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
[HsUbc2b]o (35).
However, preliminary range studies in which [E1]o was
decreased at a constant HsUbc2b concentration of 1 µM
revealed a progressive decline in the end point formation of E2
thiolester, as illustrated in Fig. 1A. At 34 nM
rabbit reticulocyte E1 (closed circles), the final amount of
HsUbc2b thiolester formed was 90% of that determined from a parallel
incubation for which E1 and HsUbc2b were present at equivalent
concentrations of 1 µM (dashed line), whereas
at 0.7 nM activating enzyme the end point for HsUbc2b
thiolester formation was 45% of that predicted (open
circles). At still lower concentrations of E1 we consistently
observed an end point for HsUbc2b-125I-ubiquitin thiolester
formation only about 50% of the stoichiometric amount found at
equimolar E1 and HsUbc2b (dashed line).
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Fig. 1.
End point HsUbc2b-125I-ubiquitin
thiolester formation is E1-dependent. The formation of
HsUbc2b-125I-ubiquitin thiolester was determined as
described under "Materials and Methods." A, incubations
contained 5 µM 125I-ubiquitin, 1 µM HsUbc2b, and either 34 nM (closed
circles) or 0.7 nM (open circles) rabbit
reticulocyte E1. B, incubations identical to A
contained 54 nM HsUbc2b and 0.7 nM rabbit
reticulocyte E1. In both panels the dashed line
represents the stoichiometric HsUbc2b-125I-ubiquitin end
point determined in separate control experiments for which
[E1]o was present at the same concentration as
HsUbc2b.
1, the latter value being calculated as
Vmax/[E1]o.
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Fig. 2.
Dependence of E1-catalyzed transthiolation on
HsUbc2b concentration. Initial rates for
HsUbc2b-125I-ubiquitin thiolester formation were determined
at the indicated substrate concentrations as described under
"Materials and Methods" for incubations containing 0.7 nM rabbit reticulocyte E1.
-dependent Conjugation--
The previous
transthiolation assay monitored rates of
HsUbc2b-125I-ubiquitin thiolester directly. However, the
kinetics can also be measured by coupling E2-ubiquitin thiolester
formation to the subsequent ligase-catalyzed conjugation step to
examine potential effects of ligase on the activation half-reaction. To
extend the kinetic studies of E1-HsUbc2b transthiolation, the initial
net forward rate of E1-catalyzed HsUbc2b-125I-ubiquitin
thiolester formation was coupled to conjugation of the model N-end rule
substrate human
-lactalbumin in an E3
-dependent in vitro assay (23). The step of E1-catalyzed
transthiolation is kinetically isolated under conditions for which E1
is rate-limiting, defined by the linearity of vo
versus [E1]o and the independence of
vo on [E3
]o. The coupled assay
provided the additional advantage of increased sensitivity compared
with that of HsUbc2b thiolester formation in Fig. 2. Under E1-limiting
conditions, the initial rates of E3
-catalyzed
125I-ubiquitin ligation followed normal hyperbolic kinetics
with respect to HsUbc2b concentration (Fig.
3). Computer fitting of the data from
Fig. 3 yielded a Km of 72 ± 13 nM for uncharged HsUbc2b binding to the E1 ternary complex,
in reasonably good agreement with the value of 102 ± 13 nM determined directly in Fig. 2, and an apparent
kcat of 4.4 ± 0.2 s
1
calculated as Vmax/[E1]o.
Correspondence between the kinetic constants determined directly by
transthiolation (Fig. 2) and indirectly by the E3
-coupled reaction
(Fig. 3) indicates that the presence of the ligase does not
significantly alter either the affinity or catalytic competence of the
E1 ternary complex-HsUbc2b interaction. In addition, substrate
inhibition by HsUbc2b noted in direct transthiolation assays (Fig. 2)
was not observed when this step was coupled to
E3
-dependent
-lactalbumin conjugation (not shown),
indicating that the former effect was an artifact of the isolated
E1-catalyzed half-reaction.
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Fig. 3.
Dependence of E1-catalyzed transthiolation on
HsUbc2b concentration during E3 -mediated
conjugation. Initial rates for E3
-catalyzed conjugation of
human
-lactalbumin with 125I-ubiquitin were determined
under E1-limiting conditions as described under "Materials and
Methods." Incubations contained 0.34 nM rabbit
reticulocyte E1, 60 µg/ml ligand affinity-purified rabbit liver
E3
, and the indicated concentrations of recombinant HsUbc2b.
1 (Table I) which are statistically
indistinguishable from the corresponding values of 102 ± 13 nM and 4.8 ± 0.2 s
1 found for rabbit E1
(Fig. 2). In contrast, Wee and coworkers report a
Km of 1.9 ± 0.5 µM and a
kcat of 0.07 ± 0.02 s
1 for
HsUbc4 (40).
Transthiolation kinetic constants for human E1
-HsUbc2b-Ubiquitin Thiolester Interactions during
E3
--
dependent Conjugation
The rate study of Fig. 3 could
additionally be used to probe the step of HsUbc2b-ubiquitin thiolester binding to human E3
by kinetically isolating the step of ubiquitin conjugation. This was achieved by increasing the concentration of E1 to
64 nM to make the assay rate-limiting with respect to [E3
]o, as evidenced by the linear dependence of
initial velocity for 125I-ubiquitin conjugation on
[E3
]o but independence of the observed initial rate on
the concentration of E1 (not shown). Under these conditions the
concentration dependence of [HsUbc2b]o on the initial rate of human
-lactalbumin conjugation is hyperbolic, as
demonstrated by the linearity of the corresponding Lineweaver-Burk plot
(Fig. 4). Hyperbolic kinetics requires
that E3
bind the HsUbc2b-125I-ubiquitin thiolester
cosubstrate prior to the catalytic step of target protein-ubiquitin
isopeptide bond formation. This conclusion is consistent with earlier
qualitative evidence from Reiss et al. (42) for binding of
rabbit reticulocyte OcUbc2 to E3
. Nonlinear least squares fitting of
the data from Fig. 4 yielded a value for Km of
54 ± 18 nM, representing the binding of
HsUbc2b-125I-ubiquitin thiolester to E3
, and a
Vmax of 0.30 ± 0.03 pmol/min. Unambiguous
interpretation of the kinetic data in Fig. 4 assumes that the
HsUbc2b-125I-ubiquitin cosubstrate for E3
be formed
stoichiometrically in this E1-coupled reaction. We confirmed the
validity of this assumption in parallel incubations resolved by
SDS-PAGE under nonreducing conditions for which the steady-state amount
of HsUbc2b-125I-ubiquitin thiolester was stoichiometric
with the theoretical amount of carrier protein present in the reaction
(not shown). Finally, because there is no analogous stoichiometric
assay for functional E3
, we have no means of reliably estimating
kcat from the maximum velocity.
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Fig. 4.
Dependence of
E3 -catalyzed conjugation on HsUbc2b
concentration. Initial rates for conjugation of human
-lactalbumin were determined under E3
-limiting conditions as
described under "Materials and Methods." Incubations were identical
to those of Fig. 3 except that the rabbit erythrocyte E1 concentration
was increased to 64 nM for each incubation to yield
E3
-dependent initial velocities.
-helix,
-sheet, and hydrogen-bonded turn (36, 16, and 22%,
respectively) were identical between wild type HsUbc2b and HsUbc2bC88A,
within the experimental error of the method (45), and comparable with that predicted from the crystal structure of the S. cerevisiae Rad/ScUbc2 ortholog (37, 18, and 21%,
respectively) based on Kabasch-Sander secondary structure analysis from
the crystal structure of Rad/ScUbc2 (46, 47). The excellent agreement
between the calculated and predicted secondary structures for
HsUbc2bC88A indicates that the mutant exists predominantly in the
native form. In addition, convergence of the spectral fit precludes
significant contributions from non-native conformers, as discussed
previously (45).
3 s
1 at 172 nM HsUbc2bC88S
and the 20 nM E1 ternary complex, considerably slower than
the lower limit of 5 s
1 estimated for wild type HsUbc2b
transthiolation under identical conditions (not shown). Transthiolation
is readily reversible for several isozymes, including HsUbc2b, when ATP
is depleted, and the E1 ternary complex is quantitatively reversed by
addition of excess AMP and PPi (29). In contrast, the
HsUbc2bC88S-125I-ubiquitin oxyester is stable under
conditions for which the E1 ternary complex and wild type HsUbc2b
thiolesters are readily reversible (Fig. 5, 10 min lanes).
The inability to reverse the oxyester indicates that formation of
the adduct is irreversible. Other studies confirmed that the HsUbc2b
oxyester is incapable of supporting the E3
-catalyzed ligation of
125I-ubiquitin to
-lactalbumin compared with wild type
thiolester (not shown), consistent with earlier studies of
125I-ubiquitin oxyester adducts of E2 isozymes (9).
Therefore, HsUbc2bC88S-125I-ubiquitin oxyester is
catalytically inert once formed and thus meets the criteria for a
potential analog of the corresponding thiolester intermediate. The
relative stability of the HsUbc2bC88S-125I-ubiquitin
oxyester permitted us to generate reagent quantities of the adduct (see
"Materials and Methods") for subsequent use in inhibition studies
(below).
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Fig. 5.
E1-catalyzed formation of
HsUbc2bC88S-125I-ubiquitin thiolester is irreversible.
Parallel 10-min incubations similar to those of Fig. 3 but containing
20 nM E1 and 1 µM HsUbc2b (wt) or
HsUbc2bC88S (C88S) were used to form the corresponding
125I-ubiquitin thiolester or oxyester, respectively
(0 min lanes). Aliquots of these incubations were adjusted
to 12 mM 2-deoxyglucose, 10 IU/ml yeast hexokinase, 1.5 mM AMP, and 0.15 mM PPi then
incubated an additional 10 min at 37 °C to reverse the E1-catalyzed
reaction (10 min lanes). Incubations were then analyzed by
nonreducing SDS-PAGE and autoradiography (19, 29). Migration of the E1
thiolester (E1S~Ub), the
HsUbc2b-125I-ubiquitin
thiolester/HsUbc2bC88S-125I-ubiquitin oxyester
(HsUbc2bX~Ub), and free 125I-ubiquitin are
shown on the right. The HsUbc2b thiolester/oxyester migrates
as two bands on nonreducing gels because of partial unfolding, as
described previously (29).
in the absence of mutant E2.
(Eq. 1)
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Fig. 6.
Kinetic characterization of HsUbc2bC88A and
HsUbc2bC88S-ubiquitin oxyester. The effects of the HsUbc2bC88A and
HsUbc2bC88S active site mutants on the E1- (A) and
E3 -dependent (B) rates of
125I-ubiquitin conjugation were analyzed according to
Equation 1. A, initial rates of E1-catalyzed HsUbc2b
transthiolation were measured similar to Fig. 3 in the presence of 180 nM HsUbc2b and the indicated concentrations of either
HsUbc2bC88A (closed circles and solid line) or
HsUbc2bC88S-125I-ubiquitin thiolester (open
circles and dashed line). The y
intercepts yielded estimates for wild type HsUbc2b
Km of 85 ± 9 nM (closed
circles and solid line) and 119 ± 9 nM (open circles and dashed line).
B, direct rates of E3
-limiting conjugation of
-lactalbumin were measured as described in Fig. 4 in the presence of
25 nM (HsUbc2bC88A study) or 16 nM (HsUbc2bC88S
study) wild type HsUbc2b and the indicated concentrations of either
HsUbc2bC88A (closed circles and solid line) or
HsUbc2bC88S-125I-ubiquitin thiolester (open
circles and dashed line). The y intercepts
yielded estimates for wild type HsUbc2b Km of
84 ± 22 nM (closed circles and solid
line) and 78 ± 14 nM (open circles
and dashed line).
-catalyzed step used for coupling HsUbc2b-ubiquitin thiolester formation (confirmed below); however, the
linearity of the plots in Fig. 6A argue against this
possibility under the conditions of the experiment. In addition,
separate control studies (not shown) demonstrated that the rate assays remained E1-limiting even at the highest inhibitor concentrations tested in Fig. 6A because the observed initial velocities
were linearly dependent on [E1]o. Finally, the inhibitors
had no effect on the steady-state formation of the E1 ternary complex,
measured by 125I-ubiquitin thiolester formation to the
activating enzyme (not shown).
-catalyzed Conjugation--
When kinetic studies
similar to those of Fig. 4 were repeated in the presence of either
Ubc2bC88A or HsUbc2bC88S-ubiquitin oxyester, competitive inhibition of
the E3
-limiting step was observed (not shown). Analysis of
appropriate data by Equation 1 yielded the predicted linear
relationships for the concentration dependence of HsUbc2bC88A (Fig.
6B, closed circles with solid line)
and HsUbc2bC88S-ubiquitin oxyester (Fig. 6B, open
circles with dashed line) on the initial rates for
conjugation. These plots yielded values for Ki
of 440 ± 55 nM for HsUbc2bC88A and 66 ± 29 nM for HsUbc2bC88S-ubiquitin oxyester. As in Fig.
6A, the Km values calculated from the
y intercepts of Fig. 6B were statistically
identical to those determined from Fig. 4 in the absence of inhibitor
and corresponded to values of 84 ± 18 nM (HsUbc2bC88A
study) and 78 ± 14 nM (HsUbc2bC88S study). Separate control experiments confirmed that assays remained E3
-limiting, as
demonstrated by the independence of the initial rates on
[E1]o at the highest inhibitor concentrations tested (not shown).
; in contrast, HsUbc2bC88A mimics binding
of the wild type uncharged HsUbc2b product to the ligase. Good
agreement between the Ki for
HsUbc2bC88S-ubiquitin oxyester binding to the ligase (66 ± 29 nM) and the Km for wild type
HsUbc2b-ubiquitin thiolester (54 ± 18 nM) indicates
that the nonfunctional oxyester serves as a reasonable structural
analog of the wild type intermediate. Comparison of these values with
the Ki of 439 ± 55 nM for
binding of the HsUbc2bC88A product analog indicates that E3
has
nearly a 10-fold discrimination in favor of the thiolester substrate. This requires E3
to interact with both the ubiquitin and E2 moieties of the thiolester, although the majority of the binding energy must
reside in the carrier protein.
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Fig. 7.
E2-125I-ubiquitin thiolester
profiles of selected cell lines. Extracts were prepared from
confluent cultures of the indicated cell lines and endogenous E2 levels
determined by their stoichiometric formation of
125I-ubiquitin thiolester as described under "Materials
and Methods." Samples were resolved under nonreducing (odd
numbered lanes) or reducing (even numbered lanes)
conditions to distinguish 125I-ubiquitin thiolesters from
the trace formation of 125I- ubiquitin conjugates,
respectively.
counting to calculate the absolute content of each
intermediate from the specific radioactivity of 125I-ubiquitin (29). Values for Ubc2 present in the various
cell lines tested, as well as published values from other cell types, are summarized in Table II. The cellular
content of Ubc2 varies widely among the cell lines when expressed per
106 cells; however, when normalized to cell volume, the
resulting concentrations were all within the micromolar range. The
content of Ubc2 was consistently lower in erythroid cells
(reticulocytes and mouse erythroleukemia cells) than in the other
epithelioid and fibroblast-like cell lines (Table II). This observation
is consistent with an expanded dependence on alternate targeting pathways in erythroid cells which is inferred from the greater variety
and content of other E2 family isozymes in reticulocytes (29, 34). The
Ubc2 activity reported here for rabbit reticulocytes agrees with
earlier estimates by immunological methods (53, 54), confirming the
validity of Ubc2 quantitation by direct thiolester formation.
E1 and Ubc2 content in selected cell lines
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Go = 0.3 kcal/mol) between the
uncharged E2 substrate and ubiquitin-charged E2 product of this
half-reaction. The nearly identical affinities for substrate HsUbc2b
versus product HsUbc2b-ubiquitin thiolester contrasts with
most enzymes that exhibit significantly attenuated binding of product
as a means of promoting the net forward reaction by favoring
dissociation of product. Subsequent product utilization by the E3
ligases appears to drive the E1-catalyzed charging of E2 with ubiquitin thiolester.
exhibits a marked discrimination in binding affinity
between the HsUbc2b-ubiquitin thiolester cosubstrate and the uncharged
HsUbc2b product. The Km for HsUbc2b under E3
limiting conditions for human
-lactalbumin conjugation is 54 ± 18 nM (Fig. 4). Because the empirically determined
conditions for these rate studies were set to assure that HsUbc2b was
present stoichiometrically as the 125I-ubiquitin
thiolester, this Km reflects the affinity for
binding of the corresponding thiolester to the ligase. This
interpretation is consistent with the Kd of
66 ± 29 nM, measured as Ki,
for the HsUbc2bC88S-ubiquitin oxyester as a competitive inhibitor of
E3
-catalyzed
-lactalbumin conjugation (Fig. 6A),
indicating that the Km reflects the intrinsic
binding affinity of the wild type E2-ubiquitin thiolester. Prior NMR
studies reveal negligible differences between the structures for
uncharged and thiolester forms of HsUbc2b (39). Therefore, the
ubiquitin moiety of the HsUbc2b thiolester must contribute to binding
within the E3
active site because the uncharged E2 analog
HsUbc2bC88A yields a Kd, measured as
Ki, of 440 ± 55 nM (Fig. 6),
representing a nearly 10-fold reduction in affinity (
Go = 1.2 kcal/mol). Because HsUbc2bC88A
exhibits a Kd for binding to E1 which is
statistically identical to the Km for wild type HsUbc2b and the secondary structure content of the inactive mutant determined by CD spectroscopy approximates that of the native polypeptide (see "Materials and Methods"), we can preclude
significant denaturation of the point mutant as an alternative
explanation for the larger Ki.
A or C
S mutants of E2/Ubc paralogs has been exploited as a
genetic approach for probing the cellular functions of various E2
families (for review, see Ref. 9). Stabilization of short lived
proteins following overexpression of specific E2 dominant negative
mutants has been interpreted to indicate a role for those E2 isoforms in their ubiquitin-dependent targeting to the proteasome.
However, the present studies suggest that the consequences of
overexpressing these dominant negative mutants instead potentially
reflects indirect inhibition of global E1-catalyzed charging of all E2
isoforms rather than specific E3-dependent effects.
Therefore, studies predicated on overexpression of E2 dominant negative
mutants should be interpreted cautiously and be accompanied by
additional control experiments precluding more general effects of the
mutants on global E2 thiolester formation.
1 (Fig. 2) and 4.5 ± 0.3 s
1 (Table I), respectively. The observed
kcat for the net forward reaction of rabbit
reticulocyte E1-catalyzed HsUbc2b transthiolation is below the
empirical lower limit for all internal steps of the E1 catalytic cycle,
based on the reported kcat for ATP:AMP exchange of 9 s
1 (20). This requires that transfer of the
ubiquitin thiolester from the E1 ternary complex to HsUbc2b represents
the rate-limiting step for HsUbc2b charging rather than an internal
step of the E1 mechanism. Therefore, convergence in the
kcat values for human versus rabbit
E1-catalyzed transthiolation requires a conserved geometry for the
transition state for ubiquitin thiolester transfer from the E1 ternary
complex to cysteine 88 of HsUbc2b.
1 (Fig. 2) versus 0.07 ± 0.02 s
1 (40) likely reflects a change in the rate-limiting
step in the latter study from E1-E2 transthiolation to ubiquitin
adenylate formation. A shift to an earlier rate-limiting step because
of steric hindrance by the lysine 6-linked fluorescent label would manifest as an overestimation of the Km for
binding in subsequent steps. The Km of 1.9 ± 0.5 µM for HsUbc4 reported for alkylated ubiquitin
(40) versus that of 102 ± 13 nM (Fig. 2)
is consistent with the latter prediction, assuming that HsUbc4 binds
with similar affinity as
HsUbc2b.5
. This suggests minimally
that Ubc2 exists intracellularly as E1 and E3
heterodimers. The data
do not address whether stable E1·Ubc2·E3
heterotrimers may also
be present; however, we have consistently failed to immunoprecipitate
in vitro complexes composed of E1 and E3
using an
affinity-purified rabbit anti-HsUbc2b polyclonal antibody (not shown).
Within IMR90 human lung fibroblasts the concentration of E1 is
approximately stoichiometric with Ubc2 and in the range of the
concentration of activating enzyme found in rabbit reticulocytes. Near
equivalence of E1 and Ubc2 levels in cells appears to hold for other
cell lines because we have qualitatively observed similar amounts of
their corresponding 125I-ubiquitin thiolesters in fresh
cell extracts (not shown). Rabbit reticulocytes and mouse
erythroleukemia cells have atypically low Ubc2 levels (Table II) and
differ from the other cell types in possessing significant levels of
other E2 families (29, 53, 57). However, in rabbit reticulocytes the
total E2 content is approximately equivalent to that of E1 (29). This
suggests a mechanistic requirement for maintaining equivalence between
E1 and total E2 levels within cells, presumably to maintain the
ubiquitin carrier proteins in their thiolester forms to preclude
potential competitive inhibition of the ubiquitin activation and
ligation half-reactions observed with HsUbc2bC88A (Fig. 6).
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant GM34009 (to A. L. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Genetics, Howard Hughes Medical
Institute, University of Pennsylvania, Philadelphia, PA 19104.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-8768; Fax: 414-456-6510; E-mail: arthaas@mcw.edu.
Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M211240200
2 In this and subsequent manuscripts we will use the empirical functional/phylogenetic family classification and systematic nomenclature for the E2/Ubc isoforms presented earlier (9).
3 Human HsUbc2b is identical to rabbit OcUbc2b, previously termed E214kb and HHR6B (9).
4 E. Laleli-Sahin and A. L. Haas, manuscript in preparation.
5 A survey of HsUba1-catalyzed transthiolation of human E2 paralogs shows that the Km values for Ubc4/5 orthologs are significantly less than the corresponding Km for HsUbc2b (T. J. Siepmann and A. L. Haas, manuscript in preparation).
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ABBREVIATIONS |
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The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; DTT, dithiothreitol; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein, also termed ubiquitin-conjugating enzyme (Ubc); FPLC, fast protein liquid chromatography.
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