(Received for publication, August 24, 1995; and in revised form, October 13, 1995)
From the
Degradation of a protein via the ubiquitin system involves two
discrete steps, conjugation of ubiquitin to the substrate and
degradation of the adduct. Conjugation follows a three-step mechanism.
First, ubiquitin is activated by the ubiquitin-activating enzyme, E1. Following activation, one of several E2 enzymes
(ubiquitin-carrier proteins or ubiquitin-conjugating enzymes, UBCs)
transfers ubiquitin from E1 to the protein substrate that is
bound to one of several ubiquitin-protein ligases, E3s. These
enzymes catalyze the last step in the process, covalent attachment of
ubiquitin to the protein substrate. The binding of the substrate to E3 is specific and implies that E3s play a major role
in recognition and selection of proteins for conjugation and subsequent
degradation. So far, only a few ligases have been identified,
and it is clear that many more have not been discovered yet. Here, we
describe a novel ligase that is involved in the conjugation and
degradation of non ``N-end rule'' protein substrates such as
actin, troponin T, and MyoD. This substrate specificity
suggests that the enzyme may be involved in degradation of muscle
proteins. The ligase acts in concert with E2-F1, a previously
described non N-end rule UBC. Interestingly, it is also involved in
targeting lysozyme, a bona fide N-end rule substrate that is recognized
by E3 and E2-14 kDa. The novel ligase
recognizes lysozyme via a signal(s) that is distinct from the
N-terminal residue of the protein. Thus, it appears that certain
proteins can be targeted via multiple recognition motifs and distinct
pairs of conjugating enzymes. We have purified the ligase
200-fold
and demonstrated that it is different from other known E3s,
including E3
/UBR1, E3
, and E6-AP.
The native enzyme has an apparent molecular mass of
550 kDa and
appears to be a homodimer. Because of its unusual size, we designated
this novel ligase E3L (large). E3L contains an -SH
group that is essential for its activity. Like several recently
described E3 enzymes, including E6-AP and the ligase
involved in the processing of p105, the NF-
B precursor, the novel
ligase is found in mammalian tissues but not in wheat germ.
The ubiquitin pathway is involved in the processing and
degradation of key regulatory short-lived proteins. Among these are
mitotic cyclins and cyclin-dependent kinase inhibitors, the NF-B
p105 precursor and the inhibitor I
B
, p53 and the
transcriptional activator AP-1, and major histocompatibility complex
class I antigens (reviewed in (1, 2, 3) ).
Degradation of a protein via the ubiquitin pathway involves two
distinct steps: signaling of the protein by covalent attachment of
multiple molecules of ubiquitin and degradation of the targeted protein
by the 26 S proteasome complex. Conjugation of ubiquitin involves a
three-step mechanism. Following activation of the C-terminal Gly of
ubiquitin by E1, one of several E2 enzymes transfers
ubiquitin to the substrate that is specifically bound to a member of
the ubiquitin-protein ligase family, E3. The ligase catalyzes
the last step in the conjugation process, formation of an isopeptide
bond between the activated Gly residue of ubiquitin, and an
-NH
group of a Lys residue in the substrate or in the
previously conjugated ubiquitin moiety.
The structure of the
ubiquitin system appears to be hierarchial: a single E1
carries out, most probably, activation of ubiquitin required for all
modifications. Several major species of E2 enzymes were
characterized in mammalian cells, plants, and
yeast(4, 5, 6) . Yeast UBC2 (RAD6) and
mammalian E2-14 kDa are involved in degradation of
``N-end rule''
substrates(4, 5, 7, 8) . UBC4 and
UBC5 are involved in the degradation of short-lived and abnormal
proteins in yeast(9) . UBC8, (E2-15 kDa; (6) ) from Arabidopsis thaliana and its rabbit (E2-F1; (10) ) and human (UbcH5; (11) )
homologs are involved in ubiquitination of certain non N-end rule
substrates such as p53 (11, 12, 13) , the
NF-B precursor, p105(14) , and glyceraldehyde-3-phosphate
dehydrogenase(10) . All these E2 enzymes act in
concert with E3s, and it appears that each E2 enzyme
can act with one or more E3 proteins.
Five E3
enzymes have been described so far. (a) Mammalian E3 (UBR1 in yeast) recognizes protein substrates via
their free basic or bulky hydrophobic N-terminal amino acid residues
(N-end rule; reviewed in (7) and (15) ). (b)
Similarly, E3
recognizes N-end rule substrates via their
free small uncharged N-terminal residues(16) . (c) E6-AP is involved in the recognition of
p53(12, 17, 18) . Recently, a series of
unique E6-AP homologous proteins have been
identified(19) ; however, their role as E3 enzymes and
the identity of their putative substrates is still obscure. (d) E3-C is involved in cell cycle-dependent
conjugation and degradation of mitotic cyclins. The ligase is a
component of a large particle (
1,500 kDa), the cyclosome (20) or the anaphase-promoting complex(21) , that
regulates the programmed destruction of mitotic cyclins containing the
``destruction box'' motif RAALGNISEN(22) . E3-C is inactive during interphase and is activated during M
phase, most probably by phosphorylation. The cyclosome contains also
CDC16, CDC27 (21, 23) , CDC23, and CSE1(24) .
It is not clear whether E3 is one of these components, or
whether it is a distinct component of the complex. (e)
Processing of p105, the NF-
B transcriptional activator precursor,
involves yet a novel ligase(14) ; however, the enzyme and its
mode of action have not been characterized.
An important, yet unresolved, problem involves the hierarchial relationships between the ligases and their cognate substrates. It is unlikely that a single E3 enzyme recognizes a specific, single substrate. Rather, it is conceivable that each ligase recognizes similar, but not necessarily identical, shared motifs in a subset of protein substrates. These motifs can be either primary, or generated post-translationally. Secondary targeting signals can be, for example, cell cycle- or signal transduction-induced phosphorylations.
N-end rule substrates are
recognized by a ``pair'' of defined E2-E3
enzymes, the mammalian E2-14 kDa and its yeast homolog
UBC2/RAD6, and E3 and its yeast homolog
UBR1(7, 15) . The mode of recognition of non-N-end
rule substrates is more elaborate: the recognition motifs are poorly
understood and the conjugating enzymes do not necessarily consist of
defined pairs. Recognition of some substrates is mediated by E2 enzymes that are homologous to the yeast UBC4 and UBC5.
These enzymes can be either the A. thaliana UBC8(6, 12) , the rabbit E2-F1(10) , or the human UbcH5(11) . However,
the conjugation reactions are catalyzed by distinct ligases. For
example, the ligase that is involved in processing of p105 (14) is clearly different from E6-AP, the p53
conjugating enzyme(12, 17) . Thus, it appears that E2-F1 or UbcH5 can each act in concert with several species of E3s. For the N-end rule pair, it has been shown that E3
/UBR1 has a binding site for its cognate E2
enzyme, E2-14
kDa/UBC2-RAD6(8, 25, 26) . It is not known
whether E2-F1 or UbcH5 associates with their different
ligases, although the necessity to preserve the activation energy
suggests that such interaction does exist. Despite this complexity, it
is assumed that a single substrate follows a single degradation
pathway: it is recognized via a single recognition motif and by one
pair of conjugating enzymes. The repressor MAT
2 appears to be one
exception to this rule. It has two degradation signals (27) and
is recognized by four E2 enzymes (28; see
``Discussion'').
Here we demonstrate that lysozyme is
conjugated and degraded by two distinct, however ubiquitin-dependent,
pathways. The protein is conjugated by E2-14
kDa-E3 and recognized via its basic N-terminal residue,
Lys(29) . In addition, it is also conjugated and degraded by a
non-N-end rule pathway consisting of E2-F1 and a novel, yet
unidentified, species of E3 (designated E3L). The
recognition motif, that is clearly distinct from the N-terminal Lys,
has not been identified.
Figure 1:
Effect of Arg-Ala and E2-F1 on
the degradation of I-labeled lysozyme (A) and
oxRNase A (B) in crude reticulocyte lysate and
ubiquitin-supplemented Fraction II. Degradation of lysozyme (A) and oxRNase A (B) was assayed in the presence of
the indicated concentrations of Arg-Ala in the following systems: crude
reticulocyte lysate (
), ubiquitin-supplemented Fraction II
(
), and ubiquitin-supplemented Fraction II in the presence of
purified E2-F1 (
). Degradation assays were carried out
as described under ``Experimental Procedures.'' Following
incubation at 37 °C for 2 h, release of
I-labeled
material into 20% trichloroacetic acid-soluble fraction was
determined.
Figure 2:
Effect of Arg-Ala and E2-F1 on
the conjugation of I-labeled lysozyme (A) and
oxRNase A (B and C) in crude reticulocyte lysate and
ubiquitin-supplemented Fraction II. Conjugation of lysozyme in whole
lysate and in Fraction II (A) and of oxRNase A in whole lysate (B) and Fraction II (C) was monitored as described
under ``Experimental Procedures.'' A, lane
1, conjugation in crude reticulocyte lysate in the absence of
ATP
S; lane 2, as lane 1, but in the presence of
ATP
S; lane 3, as lane 2, but in the presence of
5 mM Arg-Ala; lane 4, conjugation in Fraction II in
the absence of ATP
S; lane 5, same as lane 4, but
in the presence of ATP
S; lane 6, as lane 5, but
in the presence of 5 mM Arg-Ala; lane 7, as lane
6, but in the presence of 0.5 µg of E2-F1. B, lane 1, conjugation in whole lysate in the absence
of ATP
S; lane 2, as lane 1, but in the presence
of ATP
S; lane 3, as lane 2, but in the presence
of 5 mM Arg-Ala; lane 4, as lane 3, but in
the presence of 0.5 µg of purified E2-F1; lane 5,
as lane 4, but in the presence of 2.0 µg of E2-F1. C, lane 1, conjugation in Fraction II
in the absence of ATP
S; lane 2, as lane 1, but
with ATP
S; lane 3, as lane 2, but with 5 mM Arg-Ala; lane 4, as lane 2, but with 0.5 µg
of E2-F1; lane 5, as lane 4, but with 2.0
µg of E2-F1. Molecular mass markers are: 200.0, myosin;
97.4, phosphorylase b; 68.0, bovine serum albumin; 46.0,
ovalbumin; 30.0, carbonic anhydrase; 21.5, soybean trypsin inhibitor;
14.3, lysozyme. Conj. denotes conjugates of the labeled
substrate.
I-Lyso. denotes
I-lysozyme. Ori. and D.F. denote origin
and dye front, respectively.
Figure 3:
A novel ubiquitin-protein ligase, E3, is involved in conjugation of lysozyme via N-end
rule-independent pathway. 2 ml of Fraction IIA (40 mg of protein;
prepared as described under ``Experimental Procedures'') were
loaded onto a HiLoad Superdex 200 gel filtration chromatography column
and resolved as described under ``Experimental Procedures''
(determination of the molecular mass of E3L). Aliquots (2
µl) from the concentrated fractions were assayed for ligase
activity in the presence of E1 and E2 enzymes as
described under ``Experimental Procedures.'' 1, E2-14 kDa-dependent conjugation of
I-lysozyme in the absence (A) or presence (B) of Arg-Ala (5 mM). 2, E2-F1-dependent conjugation of
I-lysozyme in the
absence (C) or presence (D) of Arg-Ala. Lanes
A, incubation in the presence of E1 and E2. Lanes B, same as lanes A, but with E3
(0.6 microunits; (29) ). Lanes C, as lanes A,
but with 1 µl of Fraction IIA. Conj. denote conjugates of
labeled lysozyme. Ori. denotes origin of
gel.
Figure 4: Purification of E3L. E3L was partially purified via a 7-step purification procedure as described under ``Experimental Procedures.''
In parallel, the protein profile of the different fractions was
followed by SDS-PAGE and Coomassie Brilliant Blue staining. The peak of
activity eluted from all columns co-migrates in SDS-PAGE with a protein
of 270 kDa. Fig. 5and Fig. 6demonstrate the peak
of activity recovered from the two last purification steps,
hydroxylapatite and Mono Q anion exchange chromatographies, along with
the profile of the proteins resolved by electrophoresis. Although the E3 enzyme has not been purified to homogeneity, it is highly
likely that the 270-kDa protein is indeed E3L: the activity
and the protein band co-migrate (see also below for additional
functional assay).
Figure 5:
Activity and protein profile of E3L resolved via hydroxylapatite chromatography, the fifth
purification step. Active fractions from the hydrophobic HIC methyl
chromatography were further resolved on a hydroxylapatite cartridge as
described under ``Experimental Procedures.'' The eluted
fractions were analyzed for E3L activity by monitoring
conjugation of labeled lysozyme (A) and for protein
composition (SDS-PAGE, 6%; B) as described under
``Experimental Procedures.'' Cr. denotes reaction
carried out in the presence of ``crude'' fraction (the
hydrophobic column). Molecular mass markers are prestained myosin
(250.0), myosin (200.0), and -galactosidase (116.0). Lanes
12-22 (A) and 14-21 (B)
represent aliquots derived from the respective fractions. Arrows denote peak fraction. Other notes are as described in the legend
to Fig. 2.
Figure 6:
Activity and protein profile of E3L resolved via anion exchange chromatography over Mono Q,
the sixth (and last) purification step. Active fractions from the HTP
column (Fig. 5) were further resolved on a Mono Q anion exchange
chromatography column as described under ``Experimental
Procedures.'' The eluted fractions were analyzed for E3L
activity by monitoring conjugation of labeled lysozyme (A) and
for protein composition (SDS-PAGE, 6%; B) as described under
``Experimental Procedures.'' Cr. denotes reaction
carried out in the presence of crude fraction (the HTP peak). BT denotes reaction carried out in the presence of the unadsorbed
material (breakthrough, BT). M denotes multicolored
standard molecular mass marker from Novel Experimental Technology
(myosin, 250 kDa). Other molecular mass markers are myosin (200.0) and
-galactosidase (116.0). Lanes 12-23 (A)
and 15-22 (B) represent aliquots derived from the
respective fractions. Arrows denote peak fraction. Other notes
are as described in the legend to Fig. 2.
To strengthen further the notion that the 270-kDa
band is indeed E3L, we examined its ability, via chemical
cross-linking, to recognize and bind labeled lysozyme. As can be seen
in Fig. 7, preincubation of E3L (derived from the last
purification step) in the presence of labeled lysozyme followed by the
addition of the chemical cross-linker results in the formation of a
specific, high molecular mass cross-linking product. The molecular mass
of the product corresponds to the approximate sum of masses of the
270-kDa E3L subunit and lysozyme (the migration of the
cross-linking product was compared with that of unreacted E3L;
not shown). As a control, we carried out a similar reaction in the
presence of E3. It is known that this enzyme binds
specifically to some of its substrates, lysozyme and
-lactoglobulin for example(29) . Indeed, we were able to
demonstrate the cross-linking product between this ligase and labeled
lysozyme. Addition of excess unlabeled lysozyme prior to the addition
of the labeled protein inhibited formation of the labeled cross-linking
products, indicating that the association between the enzymes and the
substrate is specific.
Figure 7:
Cross-linking of I-lysozyme
to E3L and E3
. Labeled lysozyme was cross-linked
to Mono Q-purified E3L (product of the last purification step)
and to purified E3
as described under ``Experimental
Procedures.'' Unlabeled lysozyme was added to the reaction mixture
prior to the addition of the labeled protein as indicated. Arrows point to the cross-linking products of the ligases. Molecular mass
markers are as follows: 250.0, prestained myosin; 148.0, prestained
-galactosidase; 97.6, phosphorylase b.
Figure 8:
E3L has an active -SH group.
Conjugation of ubiquitin to lysozyme was monitored as described under
``Experimental Procedures.'' All reaction mixtures contained E1, E2-F1, and partially purified E3L
(derived from the last purification step, step 7, Mono Q anion exchange
chromatography; purification steps are described under
``Experimental Procedures'' and in Fig. 4). Lane
1, complete reaction mixture; lanes 2-4, same as lane 1, but the E3L was preincubated for 10 min at 25
°C in the presence of 10 mM NEM (lane 2),
iodoacetamide (lane 3), and p-hydroxy mercuribenzoate (lane 4). Following preincubation, the alkylating agent was
neutralized with the addition of 8 mM DTT, and the treated
enzyme was added to the reaction mixture. Lane 5, same as lane 2, but DTT was added to E3L prior to the
addition of NEM. Lane 6, E2-F1 was treated with NEM
following neutralization with DTT. Ori. and D.F.
denote origin of gel and dye front, respectively. I-Lyso. denotes
I-lysozyme. Conj. denotes ubiquitin conjugates. Molecular mass markers and
notes are as described in the legend to Fig. 2.
Figure 9:
Conjugation of actin and troponin T is
ubiquitin- and E2-F1-dependent. Conjugation of I-actin (A) and
I-troponin T (B) was carried out in reticulocyte lysate and Fraction II as
described under ``Experimental Procedures.'' Lanes
1, conjugation in the presence of crude reticulocyte lysate
without ATP; lanes 2, same as lane 1, but with
ATP
S; lanes 3, conjugation in the presence of Fraction
II; lanes 4, same as lane 3, but with the addition of
ubiquitin; lanes 5, same as lane 4, but with the
addition of E2-F1. Notes and molecular mass markers are as
described in the legend to Fig. 2.
Figure 10: Conjugation of actin and troponin T by partially purified E3L. Fractions from the last E3L purification procedure (Mono Q column; see ``Experimental Procedures'' and Fig. 4and Fig. 6) were analyzed for conjugation of actin (A) and troponin T (B) in the presence of purified E1 and E2-F1 as described under ``Experimental Procedures.'' Lanes A, reaction mixtures incubated in the presence of E1 and E2-F1; lanes B, same as lane A, but with a crude fraction of E3L (derived from step 6 of the purification procedure, hydroxylapatite chromatography). Lanes 13-22 (A) and lanes 12-21 (B), reactions carried out in the presence of aliquots from the respective fractions. HMW Conj. and LMW. Conj. denote high and low molecular mass conjugates, respectively. Arrows indicate the fraction with the highest E3L activity. Other notes and molecular mass markers are as described in the legend to Fig. 2.
Figure 11:
Conjugation of MyoD is E2-F1-
and E3L-dependent. MyoD was expressed in bacteria and purified
as described(33) . 1.4 µg of MyoD were added to the
conjugation assay as described under ``Experimental
Procedures.'' Following incubation, reaction mixtures were
resolved via SDS-PAGE. Proteins were transferred to nitrocellulose
(Western blot) that was incubated in the presence of affinity-purified
rabbit polyclonal anti-MyoD antibody. Visualization was carried out
using the ECL detection system (Amersham). Lane 1, conjugation
in the presence of E1 and E2-14 kDa; lane
2, as lane 1, but with E3; lane 3,
conjugation with E1 and E2-F1; lane 4, as lane 3, but with E3L (fraction 18 from the Mono Q
column; Fig. 6); lane 5, conjugation in the presence of E1 and E3L. lane 6, as lane 5, but
with E2-14 kDa; lane 7, as lane 4.
Notes and molecular mass markers are as described in the legend to Fig. 2.
Figure 12:
E3L is present in reticulocytes
but not in wheat germ extract. Conjugation assay was carried out in
complete wheat germ extract in the presence or absence of ATPS and E3L (40 units) derived from the last purification step (Mono Q
anion exchange chromatography) as described under ``Experimental
Procedures.'' Arg-Ala (5 mM) was added to all reaction
mixtures to inhibit background activity of the N-end rule pathway.
Notes and molecular mass markers are as described in the legend to Fig. 2.
It is well established now that recognition of proteins for conjugation and subsequent degradation by the ubiquitin system is carried out by E3 enzymes. Except for the N-end rule enzymes, few other ligases have been identified, and their mode of recognition is still obscure. Because of their crucial role in substrate recognition, it is important to identify additional E3s and study their mode of action.
In this study, we identified, characterized, and partially purified a novel E3 (E3L) enzyme involved in recognition of substrates in an N-end rule-independent mode. It is different from all other known ligases, both structurally and functionally. The enzyme is found, although not exclusively, in muscle extracts, and recognizes the muscle proteins actin, troponin T, and MyoD. Although the ubiquitin system is probably involved in certain pathophysiological processes unique to muscle(38) , it is not clear that E3L is indeed a muscle-specific enzyme. It is present in reticulocytes and other mammalian tissues (not shown), but not in wheat germ. A more extensive substrate survey will be necessary in order to better define the specificity of this novel ligase.
An interesting finding relates to
the targeting of lysozyme by E3L. Lysozyme is a bona fide
N-end rule substrate that has a destabilizing amino acid, Lys, at the
N-terminal residue(7) . It is clear that recognition of
lysozyme by E3L does not traverse the rule, as Arg-Ala does
not inhibit the conjugation reaction. Thus, lysozyme is recognized by
at least two distinct signals, the N-terminal amino acid residue and a
new, yet unidentified, ``body'' motif. The two motifs are
identified by two distinct pairs of conjugating enzymes, E2-14 kDa-E3 and E2-F1-E3L. Quantitative analysis reveals that the
N-end rule pathway contributes
30% and the rule-independent
pathway
70% to the overall proteolysis of the labeled substrate ( Fig. 1and data not shown). We hypothesized that each of the
different pathways recognizes and targets for degradation a distinct
part of the molecule. To test this hypothesis, we attempted to identify
a processed product of lysozyme following incubation in a proteolytic
mixture that contains only the N-end rule pathway enzymes, a
ubiquitin-supplemented Fraction II that does not contain E2-F1. However, we were not able to identify any proteolytic
intermediate, suggesting that each pathway can degrade the substrate
completely to free amino acids. Thus, interpretation of the finding
that the activity of the two pathways is additive, is still missing. It
should be noted that degradation in the cell-free system is, in most
cases, limited, and only a fraction (
20-30%) of the
substrate is degraded. It is possible that the system is gradually
inactivated during incubation and what one measures is the sum of
proteolytic yields of each of the pathways during the period in which
it is active: removal of one pathway decreases the overall proteolysis
that can be observed in a system in which the two pathways are active
(crude lysate).
Lysozyme is not the first substrate that is targeted
via two distinct domains and different conjugating enzymes. The
MAT2 repressor is signalled for degradation by two independent
signals, DEG1 and DEG2(1, 27) , and is targeted by two
distinct pairs of E2 enzymes, UBC4 and UBC5, and UBC6 and UBC7
(28; see introduction). However, the case of lysozyme and E3L
appears to be different. First, it is not clear whether DEG2 is at all
a ubiquitin degradation signal. DEG1 is probably recognized by a
complex between UBC6 and UBC7, whereas the role of UBC4 and UBC5 in the
process remains obscure. The two signals in lysozyme are clearly
targeted by the ubiquitin system. They are independent and each of them
is sufficient to target the protein to complete degradation. Also, the
identity of the E3s that are involved in the degradation of
the repressor is not known. The existence of dual and independent
proteolytic pathways within the ubiquitin system appears, at first
glance, to be redundant. Yet, it raises interesting problems related to
the evolution of the system and its physiological roles. It may well be
that for key regulatory proteins, nature evolved a ``safety''
mechanism to ensure their prompt removal even under conditions that one
pathway is inactivated. It is clear that lysozyme is only a model
substrate. The discovery that it has two degradation signals and it
shares two proteolytic pathways is the result of fortuity, that however
reflects the complexity of selective degradation of specific cellular
proteins.