(Received for publication, August 21, 1995)
From the
The biological effect of type 1 interferons is proposed to arise
in part from the conjugation of ubiquitin cross-reactive protein
(UCRP), the ISG15 gene product, to intracellular target
proteins in a process analogous to that of its sequence homolog
ubiquitin, a highly conserved 8.6-kDa polypeptide whose ligation marks
proteins for degradation via the 26 S proteasome. Inclusion of
CoCl during the purification of recombinant UCRP blocks the
proteolytic inactivation of the polypeptide occurring by cleavage of
the carboxyl-terminal glycine dipeptide required for activation and
subsequent ligation. Intact UCRP supports a low rate of
ubiquitin-activating enzyme (E1)-dependent ATP:PP
exchange
but fails to form a stoichiometric E1-UCRP thiol ester or undergo
transfer to ubiquitin carrier protein (E2). The binding affinity of E1
for UCRP is significantly diminished relative to that of ubiquitin.
These results suggest that UCRP conjugation proceeds through an enzyme
pathway distinct from that of ubiquitin, at least with respect to the
step of activation. This was confirmed for an in vitro conjugation assay in which
I-UCRP could be ligated
in an ATP-dependent reaction to proteins present within an A549 human
lung carcinoma cell extract and could be competitively inhibited by
excess unlabeled UCRP but not ubiquitin. Other results demonstrate that
I-UCRP conjugation is significantly increased in cell
extracts after 24 h of incubation in the presence of interferon-
,
consistent with the late induction of UCRP conjugating activity. Thus,
interferon-responsive cells contain a pathway for UCRP ligation that is
parallel but distinct from that of ubiquitin.
The interferons exert their biological effects through induction
of a subset of cellular genes whose patterns of expression define the
cell- and tissue-specific responses characteristic of these cytokines.
Several of these interferon-induced genes have been the subject of
considerable study (reviewed in (1) ); however, the mechanisms
by which many of these proteins contribute to the interferon response
remain poorly understood. One such protein within the latter group is
the 15-kDa polypeptide originally identified by Farrell et al.(2) and later characterized by Knight and
co-workers(3, 4) . Interferon-induced expression of
the 15-kDa protein is regulated by an upstream interferon-stimulated
response element typical of all early genes induced by type 1
interferons, IFN- (
)and IFN-
(5) .
Subsequently, the sequence of the 15-kDa protein was noted to possess
significant homology to a tandem diubiquitin sequence, accounting for
its cross-reaction with affinity-purified anti-ubiquitin antibodies (6) . The carboxyl-terminal LRLRGG sequence of ubiquitin
essential for its conjugation to cellular proteins is conserved within
the carboxyl terminus of the ubiquitin cross-reactive protein (UCRP),
leading to the proposal that UCRP contributes to the type 1 interferon
response through an analogous conjugation reaction(6) . More
recently, high molecular weight UCRP conjugates have been detected
constitutively and within interferon-induced cultured cell lines using
anti-UCRP-specific antibodies(7, 8) .
Ubiquitin is
one of the most highly conserved proteins found widely distributed
among eucaryotes. The best studied function of ubiquitin is to target
cellular proteins for degradation through a post-translational
modification wherein the carboxyl terminus of ubiquitin is covalently
linked via isopeptide bond to primary amines on target
proteins(9) . The resulting conjugates are degraded by a
multienzyme ATP-dependent pathway requiring the 26 S multicatalytic
protease complex (proteasome)(10) . Conjugation of ubiquitin to
cellular proteins proceeds through a three-step pathway, reviewed in
Hershko and Ciechanover (10) and Pickart(11) . The
ubiquitin-activating enzyme (E1) catalyzes an ATP-coupled activation of
the carboxyl-terminal glycine of ubiquitin to generate an enzyme-bound
ubiquitin adenylate intermediate and free PP(12) .
Transfer of activated ubiquitin to an active site cysteine of E1
releases AMP and generates a covalent E1-ubiquitin thiol
ester(12) . In the second step, ubiquitin is transferred by
transacylation to a cysteine residue conserved among all members of a
family of ubiquitin carrier proteins, E2(13, 14) . The
third step involves aminolysis of these E2 thiol esters to form
isopeptide bonds between the carboxyl-terminal glycine of ubiquitin and
-amino groups of lysine residues on target proteins in both E3
(ubiquitin:protein ligase) -dependent and -independent
mechanisms(15) .
A persistent question has been whether the regulatory pathway of ubiquitin ligation is unique within the cell or if other similar pathways exist. Discovery of UCRP and its conjugates suggests such ligation events represent a general regulatory strategy within cells. However, a remaining unresolved question is whether ubiquitin and UCRP ligation mechanisms share a common set of enzymes or proceed through parallel but distinct pathways. In order to address the latter question, we have examined the ability of recombinant human UCRP to support the reaction catalyzed by ubiquitin-activating enzyme. The results suggest that conjugation of ubiquitin and UCRP proceed through distinct enzymes for their initial activation, a conclusion supported by competition studies utilizing an in vitro UCRP ligation assay.
Bovine ubiquitin and yeast inorganic pyrophosphatase were
purchased from Sigma. In all studies the absolute concentration of
ubiquitin was determined using an empirically determined of 0.16 ml/mg
cm(16) . Carboxypeptidase B was
purchased from Boehringer Mannheim. Recombinant ubiquitin
carboxyl-terminal hydrolase (L3) was a generous gift from Dr. Keith D.
Wilkinson (Emory University Medical School). Human recombinant
interferon bearing a C17S mutation to enhance stability (IFN-
) was
supplied by Triton Biosciences. Carrier-free Na
I,
[2,8-
H]ATP, and
Na
[
P]PP
were obtained
from DuPont NEN. Ubiquitin and UCRP were labeled with
I
by the chloramine-T (17) and the IODO-GEN methods(18) ,
respectively. Precast isoelectric focusing gels having a broad pH range
(3.5-9.5) were obtained from Pharmacia Biotech Inc. The
ubiquitin-activating enzyme (E1) was purified by modification of the
published procedure (14) from rabbit reticulocytes, rabbit
liver, and human erythrocytes.
The
stoichiometry of E1-bound adenylate intermediate formation to either
ubiquitin or UCRP was determined by measuring trichloroacetic
acid-precipitable radioactivity in the presence of
[2,8-H]ATP(12, 20) . Incubations
were conducted at 37 °C in a final volume of 50 µl containing
50 mM Tris-Cl (pH 7.6), 1 mM DTT, 10 mM MgCl
, 1 IU of inorganic pyrophosphatase, 1 mg/ml
carrier bovine serum albumin, the indicated concentrations of
[
H]ATP (2.5
10
cpm/pmol), and
either ubiquitin or UCRP. Pyrophosphatase was omitted from equilibrium
reactions used to determine internal E1 equilibrium constants in which
PP
was present at the indicated concentrations. Data were
corrected for nonspecific adsorption by subtracting radioactivity
contained in control incubations from which E1 was omitted.
The
stoichiometry of I-ubiquitin or
I-UCRP
thiol ester formation on E1 was determined after resolving incubations
by SDS-PAGE under nonreducing conditions, then determining
I-associated radioactivity in the thiol ester band by
counting(12, 20) . Incubations were identical to
those for measuring adenylate formation except that 2 mM unlabeled ATP was used in place of the radiolabeled nucleotide.
Five pmol of rabbit liver E1 and 32 µM recombinant UCRP
were used in the reaction at 37 °C for 5 min. SDS-PAGE sample
buffer containing 4% SDS and 5 M urea was used to quench the
reaction.
-Mercaptoethanol was excluded from the sample buffer.
Due to covalent aggregation of
I-UCRP during iodination
and the low specific activity of radiolabeled polypeptide, Western
blotting was used for the detection of E1-bound UCRP thiol
ester(21) . Blots were immunostained with 10 µg/ml
affinity-purified anti-UCRP antibody followed by
I-protein A and visualized by autoradiography. Control
reactions were included in which E1 was quenched prior to addition of
UCRP. A 7% gel was used to increase transfer efficiency and resolve the
difference in electrophoretic mobility between the ubiquitin and UCRP
thiol esters formed with E1.
Mature forms of ubiquitin and UCRP share a common LRLRGG carboxyl terminus(6) . Previous work has shown that the carboxyl-terminal glycine dipeptide of ubiquitin is acutely sensitive to cleavage by trypsin-like activities(22, 23) . The resulting des-Gly-Gly ubiquitin retains a pI characteristic of native ubiquitin(22, 23) . However, removal of the carboxyl-terminal glycine dipeptide exposes Arg-74 which is subject to cleavage by carboxypeptidase B to yield a product exhibiting a pI shift to 5.4(22, 23) . The specificity of carboxypeptidase B for lysine and arginine precludes a similar pI shift for intact ubiquitin in which Arg-74 is masked by the glycine dipeptide (22) . When a similar experiment was conducted with recombinant UCRP isolated by our published protocol(22) , the polypeptide exhibited a shift in pI from the value of 6.7 characteristic of UCRP to 5.4 upon incubation with carboxypeptidase B (Fig. 1, lanes 3 and 4). This indicated that the carboxyl-terminal glycine dipeptide was absent from UCRP.
Figure 1:
Analysis
of the carboxyl-terminal sequence of UCRP. Recombinant UCRP (5 µg)
purified in the absence (lanes 3 and 4) or presence (lanes 1 and 2) of CoCl was incubated in
the absence (odd-numbered lanes) or presence (even-numbered lanes) of 2 units of carboxypeptidase B at 37
°C for 15 min, then resolved by isoelectric focusing gel
electrophoresis.
The pI shift upon incubation of
ubiquitin with carboxypeptidase B was used as a convenient assay to
test for carboxyl-terminal glycine dipeptide processing in BL21
extracts. These experiments revealed that the inactivating enzyme was a
periplasmic carboxypeptidase similar in specificity to carboxypeptidase
A; that is, the activity readily cleaved the glycine dipeptide from
ubiquitin but failed to remove the resulting Arg-74 carboxyl terminus.
Similar inactivating activity was found in all expression strains of E. coli tested, except for that of AR58 used in the
heat-inducible expression of recombinant ubiquitin(24) .
However, the AR58 expression system was precluded for UCRP since the
polypeptide fails to fold into a soluble, native conformation at the
elevated temperature (42 °C) required for induction(19) .
Various inhibitors were unsuccessfully screened for their ability to
block ubiquitin carboxyl-terminal inactivation by BL21 extracts. In
contrast, 5 mM EDTA stimulated the rate of ubiquitin
inactivation, suggesting that endogenous divalent metal(s) present in
the lysates acted as natural inhibitors of the activity. Among the
metal salts subsequently tested, CoCl was the most
effective in inhibiting ubiquitin inactivation; therefore, 5
mM CoCl
was included in all buffers used in the
isolation of recombinant UCRP, since this concentration quantitatively
blocks ubiquitin and UCRP glycine dipeptide excision.
Recombinant
UCRP purified in the presence of CoCl failed to show a pI
shift upon incubation with carboxypeptidase B (Fig. 1, lanes
1 and 2), suggesting the presence of an intact native
carboxyl terminus. However, the pI of UCRP shifted from 6.7 to 5.4
following successive incubation with carboxypeptidase A, to remove the
glycine dipeptide, followed by carboxypeptidase B (not shown). This
latter control experiment precluded the possibility that residual
CoCl
present in the UCRP preparation directly inhibited the
activity of carboxypeptidase B. Recombinant UCRP purified in the
presence of 5 mM CoCl
was used in all subsequent
enzymatic studies reported here.
Subsequent rate studies using UCRP
preparations having variable amounts of intact carboxyl terminus
yielded comparable values of k but different
values for K
that were inversely proportional to
the fraction of total UCRP possessing an intact carboxyl terminus, as
judged by relative Coomassie staining intensity in the pI shift assay
following incubation with carboxypeptidase B (not shown). The latter
observations are consistent with the inability of des-Gly-Gly UCRP to
support the E1-catalyzed reaction. The dependence of ubiquitin
concentration on E1-catalyzed ATP:PP
exchange exhibits
pronounced substrate inhibition at high concentrations, characteristic
of the obligatory ordered addition of substrates for which ATP binds
prior to ubiquitin(12) . However, no substrate inhibition was
observed at the highest concentration of UCRP tested (50
µM), presumably because this concentration remains below
the zone of inhibition for the polypeptide. Insolubility of UCRP at
higher concentrations prevented testing for substrate inhibition by
UCRP.
Recent studies by Burch and Haas (24) have shown that
mutagenesis of specific ubiquitin residues sufficiently compromises
binding of the resulting adenylate within the E1 active site to allow
dissociation of the intermediate. The free but not the bound pool of
ubiquitin [H]adenylate is sensitive to cleavage
by ubiquitin carboxyl-terminal hydrolase(24, 25) .
Considering the anticipated structural differences between ubiquitin
and UCRP, the UCRP [
H]adenylate measured by
trichloroacetic acid precipitation could represent a similar
equilibrium between bound and free forms since this method is otherwise
unable to distinguish between the pools, as has been discussed
previously(24) . The amount of UCRP
[
H]adenylate formed was unaffected by incubation
in the presence of 0.4 IU/ml recombinant ubiquitin carboxyl-terminal
hydrolase (Table 2), precluding the existence of a free pool of
intermediate. Separate control studies demonstrated that the UCRP
[
H]adenylate was completely cleaved by the
hydrolase if the intermediate was first dissociated from E1 by
trichloroacetic acid precipitation then resolubilized in the presence
of 0.2 M triethanolamine-Cl (pH 8.0), not shown. These results
indicate that nonconservative sequence differences between ubiquitin
and the carboxyl-terminal ubiquitin-like domain of UCRP affect the
equilibrium formation of UCRP adenylate but not the tight binding
characteristic of this intermediate.
Figure 2:
Thiol ester formation between E1 and UCRP.
Thiol ester formed between UCRP (Panel A) or I-ubiquitin (Panel B) and 5 or 1 pmol of rabbit
liver E1, respectively, was detected as described under
``Materials and Methods.'' Lane 1 in each panel
represent a control in which the E1 was quenched prior to the
reaction.
Western blotting and detection with
anti-UCRP antibody proved a facile means of routinely detecting E1-UCRP
thiol ester formation; however, the method was inadequate for
determining the stoichiometry of this step. Conversely, the low
specific activity of I-UCRP and covalent aggregation
during radiolabeling made direct detection of
I-UCRP
thiol esters difficult except at elevated levels of E1. Using 10 pmol
of E1 in the standard thiol ester incubation (see ``Materials and
Methods'') we were able to detect
I-UCRP thiol ester
formation with E1 at a level corresponding to 5% of that formed with
I-ubiquitin (not shown) following a 15-min incubation. In
these experiments, the specific activity of
I-UCRP was
determined by estimating the amount of radiolabeled 15-kDa band by
Coomassie staining relative to that of a series of known amounts of
unlabeled UCRP determined spectrophotometrically using the empirically
determined extinction coefficient at 280 nm (see ``Materials and
Methods''). The amount of
I-UCRP thiol ester
formation did not increase on prolonged incubation, indicating that the
20-fold lower stoichiometry for
I-UCRP formation was an
equilibrium rather than a kinetic effect.
Table 3compares the interaction of ubiquitin and UCRP with
affinity-purified E1 isolated from rabbit liver and human erythrocytes.
Values for the K of ATP, ubiquitin, and PP
binding show good agreement between rabbit liver and human
erythrocyte E1 and with the values previously reported for rabbit
reticulocyte enzyme(12) . These results with enzyme isolated
from both a different rabbit tissue and species/tissue confirm earlier
preliminary conclusions that ubiquitin activation is saturated with
respect to intracellular ATP concentrations normally
encountered(12) . The results of Table 3also indicate
that ubiquitin activation is saturating with respect to
polypeptide(12) , based on quantitative estimates of free
ubiquitin pools in a number of cultured cell lines and
tissues(21, 26) . In addition, the equilibrium
constants for formation of enzyme-bound ubiquitin adenylate of 0.2 and
0.13 for liver and erythrocyte E1 enzymes, respectively, agree very
well with the value of 0.16 reported for the rabbit reticulocyte
form(12) .
The results of Table 3also demonstrate
that the amino acid substitutions present in UCRP have significant
effects on the interaction of the polypeptide with ubiquitin-activating
enzyme. The K for binding of UCRP is 80-fold
higher than that of ubiquitin for liver E1 and 19-fold higher for
erythrocyte enzyme. In addition, the equilibrium constant for formation
of enzyme-bound UCRP adenylate is approximately 20-fold lower for both
forms of E1, accounting in part for the diminished stoichiometry for
equilibrium formation of this intermediate (Table 2). The
increase in K
for UCRP binding and the decrease in
equilibrium constant for formation of the adenylate intermediate is
consistent with earlier proposals that tight binding of the polypeptide
is functionally coupled to this catalytic step compared to predicted
formation constants based on model organic reactions(12) . The
significant decrease in K
for ATP and
PP
binding in the presence of UCRP observed for both
enzymes is puzzling since such an effect cannot be accounted for
directly by the presently accepted mechanism for E1(12) . This
effect on binding of cosubstrate and product suggests that binding of
UCRP, and presumably ubiquitin, is also coupled to those of ATP and
PP
.
The significantly diminished affinity of ubiquitin-activating enzyme for UCRP suggests that the enzyme does not normally function within the conjugation pathway of the ubiquitin homolog. This conclusion is supported by the argument that free ubiquitin, normally present at 5-10 µM within cells(21, 26) , would effectively competitively block a kinetically significant rate of UCRP activation, since free concentrations of the latter are constitutively present at 0.1 µM and accumulate to only about 1 µM after 24 h induction with saturating concentrations of type 1 interferons(7) . Therefore, it is likely that cells contain a separate enzyme for the activation of UCRP.
Figure 3:
In vitro conjugation of UCRP to
cellular proteins. Cell free extracts were prepared from A549 cultures
grown 24 h in the absence (-IFN) or presence
(+IFN) of 10 IU/ml of IFN-
, then assayed
for in vitro conjugation of
I-UCRP as described
under ``Materials and Methods.'' Briefly, extracts were
supplemented with ATP, MgCl
, and a creatine
phosphokinase/creatine phosphate ATP-regenerating system, and
I-UCRP, then incubated at 37 °C for 90 min (90 min)
or quenched with SDS sample buffer before addition of radiolabeled
polypeptide (0 min). To test the specificity of
I-UCRP
conjugation, parallel reactions were incubated in the presence of a
10-fold excess of unlabeled UCRP (+UCRP) or ubiquitin
(+Ub).
For the incubations of Fig. 3, I-UCRP was added to a final concentration 50-fold greater
than that of endogeneous UCRP present in the extracts to obviate
effects of isotope dilution. Radioiodinated UCRP exhibits a low but
measurable rate of conjugation to cellular proteins present in extracts
obtained from uninduced cells (-IFN) after 90 min of
incubation. The rate of
I-UCRP conjugation is
significantly greater in extracts from parallel A549 cultures induced
for 24 h in the presence of 1000 IU/ml interferon-
(+IFN). Enhanced in vitro conjugation of
I-UCRP agrees with the previous results demonstrating the
late induction of UCRP conjugating activity by IFN-
(7, 19) Conjugation of
I-UCRP in both
extracts is absolutely dependent on the presence of ATP and a creatine
phosphate/creatine phosphokinase ATP-regenerating system (not shown).
That the conjugation of
I-UCRP is specific for the
homolog is demonstrated by the marked inhibition by isotope dilution
when an excess of unlabeled UCRP (+UCRP) but not
ubiquitin (+Ub) is included in parallel incubations. The
latter observations suggest that conjugation of UCRP and ubiquitin
proceed through distinct ligation pathways, consistent with the earlier
conclusion that UCRP activation is unlikely to require
ubiquitin-activating enzyme.
The significant number of additional
low molecular weight radiolabeled bands obvious at 0 min and persisting
after 90 min of incubation is not due to contaminating proteins present
in the recombinant UCRP preparation since the polypeptide was >99%
pure (see ``Materials and Methods'') but, rather, results
from covalent aggregation of the radioiodinated polypeptide during the
labeling reaction. ()Preliminary studies indicate that this
aggregation results in part from disulfide dimerization of UCRP through
the single cysteine residue present in the sequence under the
nonreducing conditions required for radioiodination.
Identification of ubiquitin conjugation as a required step in targeting proteins for ATP-dependent degradation by the 26 S proteasome poses the question of whether this novel post-translational modification is unique or represents the first example of a general regulatory strategy within cells by which ligation of low molecular weight polypeptides serves distinct signaling functions. Recognition that UCRP, the ISG15 gene product, is evolutionarily derived from ubiquitin(6) , and the subsequent immunochemical identification of UCRP conjugates within interferon responsive cells (7, 8) has supported a role for ligation of this polypeptide in mediating the diverse cellular effects of the cytokine. In addition, identification of low constitutive levels of free and conjugated UCRP within cells not exposed to type 1 interferons suggests UCRP ligation also functions in normal cellular regulation(7, 8) .
Previous sequence analysis (6) reveals that UCRP contains two tandem ubiquitin-like
domains predicted to retain the basic ubiquitin folding motif, based on
the positions of homology between the domains and those of ubiquitin
relative to the 1.8 Å crystal structure of the latter (27) . In addition, the carboxyl-terminal domain of UCRP that
is anticipated to interact most directly with enzyme(s) required for
its ligation bears substantially greater homology to ubiquitin than
does the amino-terminal domain, including retention of the
characteristic ubiquitin LRLRGG carboxyl terminus involved in
ATP-dependent activation and subsequent isopeptide bond
formation(6, 12, 24) . These facts would
argue for a common ligation pathway for the two polypeptides or
distinct pathways sharing some components. The present results indicate
that the UCRP and ubiquitin ligation pathways are distinct, at least
with respect to activation of their respective carboxyl termini. This
conclusion is supported by the inability of ubiquitin to compete with I-UCRP during in vitro conjugation under
conditions for which unlabeled UCRP effectively blocks addition of the
radiolabeled polypeptide (Fig. 3). Although intact recombinant
UCRP supports a modest rate of E1-dependent activation (Table 1),
the affinity for UCRP is considerably less than that for ubiquitin (Table 3). Because of this difference in affinity between the two
proteins, it is unreasonable to assume that UCRP could compete with
ubiquitin for activation by E1, particularly since intracellular
constitutive concentrations of UCRP within uninduced cells is
approximately 50-100-fold lower than that of ubiquitin and only
accumulate to 10% of the free pool of ubiquitin following interferon
induction(7) . Differences in affinity and concentration thus
necessitate a distinct enzyme for UCRP activation.
Recent
structure-function studies exploiting site-directed mutagenesis of
ubiquitin provide some insight into the sequence differences that may
in part account for the inability of UCRP efficiently to support the
E1-catalyzed reaction. Position Arg-72 of ubiquitin, retained as
Arg-152 in UCRP(7) , represents a major contribution in the
initial binding of polypeptide to E1 and is absolutely required for
defining the ordered addition of substrates(24) . However, the
additional significant binding contribution of the ubiquitin Arg-54
site (24) is lost through mutation at the corresponding Leu-134
of UCRP(7) . This sequence difference probably accounts in part
for the decreased binding of UCRP to E1, although the magnitude of the
effect is greater than predicted from this sequence change
alone(24) , indicating that other nonconserved sites or steric
factors associated with the increased size of UCRP must also contribute
to diminished binding. For ubiquitin, the single His-68 residue is
essential for function since mutation at this site blocks the ability
of the polypeptide to support ATP-dependent degradation(28) .
We have recently found that ubiquitin H68N duplicates this effect and
results from an impaired rate of conjugation resulting from a kinetic
defect in formation of the E1-ubiquitin thiol ester. ()It is
likely that a similar effect in UCRP, for which His-68 is replaced by
Phe-148(7) , accounts for the observed low steady state level
of enzyme-bound E1-UCRP thiol ester (Fig. 2); however,
additional steric effects cannot be ruled out.
In the course of these studies we compared the ability of ubiquitin and UCRP to support the E1-catalyzed reaction using enzymes isolated from two different sources (Table 3). A similar analysis had only been previously reported for E1 isolated from rabbit reticulocytes(12) . The new data support and extend the previous conclusions that E1 is saturating with respect to both ATP and free ubiquitin within cells (12) . Therefore, the rate-limiting step for ubiquitin ligation cannot normally reside in the activation step and changes in steady state levels of ubiquitin conjugates within cells cannot be directly explained by a substrate dependence on ubiquitin concentration, as has been discussed previously(26) .
The present results argue that UCRP ligation represents a parallel but distinct pathway to that of ubiquitin conjugation. In spite of the sequence similarities between the two polypeptides, they most likely utilize different activating enzymes. Interestingly, several recent cDNA sequences have recently been reported that are similar to those of the ubiquitin-activating enzyme(29, 30, 31) . Whether these sequences represent the putative UCRP-dependent E1 is uncertain at present. Also uncertain is whether other steps in the ubiquitin and UCRP ligation mechanisms share common components. Nonetheless, the ubiquitin and UCRP conjugation pathways represent an unparalleled opportunity for comparative biochemical studies of related processes.