From the Department of Chemistry, Bates College, Lewiston, Maine 04240
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ABSTRACT |
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The amino acid sequence LLVRGRTLVV, which is probably
located in a strand-turn-strand structure, has been identified as a protein destruction signal in the rapidly degraded encephalomyocarditis virus 3C protease. Mutations within this sequence reduced the susceptibility of the 3C protease toward ubiquitination and degradation in rabbit reticulocyte lysate. This signal is transferable, since poliovirus 3C protease, which is a poor ubiquitin-mediated proteolytic system substrate, was found to be ubiquitinated and degraded when the
signal sequence was either generated at an internal location in the
protein or fused to the N terminus. An evaluation of the behavior of 3C
protease proteins containing mutations in the signal region indicates
that considerable variability in the primary structure is tolerated,
although the conservation of certain features appears to be required
for signal function. Two E3 ubiquitin-protein ligases that recognize
the encephalomyocarditis virus 3C protease as a substrate were also
partially purified. One of these was identified as the previously
described E3 Evidence accumulated to date has shown that proteins degraded by
the ubiquitin (Ub)1-mediated
proteolytic system (1, 2) contain structural features that serve as
recognition signals for Ub system factors. These signals generally
appear to be composed of short primary structure elements. A conserved
nine-amino acid sequence, for example, in the A and B cyclins (3, 4),
as well as in certain other proteins (5, 6), is required for these
molecules to be recognized as substrates for Ub attachment and for
degradation. Short regions rich in proline, glutamate, serine, and
threonine, or PEST sequences, have been postulated to serve as protein
degradation signals (7), and it now appears that some proteins
containing these regions are substrates for the Ub-mediated proteolytic
system (8-14). Proteins with certain N-terminal amino acids are
subject to Ub-mediated proteolysis (15-18). Still other degradation
signals have been demonstrated to exist, and in some cases their
locations have been at least partially mapped (19-25). Given the small
number of destruction signal elements whose locations have been
precisely determined, it is difficult to conclude whether it is only
the primary structures of these signals that are important for their recognition or whether defined, higher order structural features come
into play. The destruction signal of the MAT Very few of the protein destruction signals that have been mapped so
far have been shown to be recognized by a particular Ub system factor
or factors. It is likely that the recognition of substrate proteins
occurs primarily through the binding of a Ub-protein ligase, or E3, to
the destruction signal elements (2). Several E3 proteins or protein
complexes have been identified to date. As characterized by function,
these can be divided into two classes (26). Some, such as SCF complexes
composed of SKP1, CDC53, and an F-box protein (27), appear to operate
as docking proteins by binding to both the substrate to be
ubiquitinated and an E2 Ub-conjugating protein, the latter of which
transfers the Ub molecule to the target. Other E3 proteins actually
catalyze the attachment of Ub to the target, with a ubiquitinated E2
serving as a Ub donor. These include the mammalian E3 We have recently identified the 3C proteases of two picornaviruses as
substrates for Ub-mediated degradation (35, 36). The 3C proteases are
cysteine proteases of approximately 21-24 kDa which exhibit a high
degree of substrate specificity (37). These proteins are initially
synthesized within large polyproteins encoded by the positive stranded
RNA viral genomes. They are responsible for most of the proteolytic
processing of the polyproteins, which leads to the generation of
individual functional proteins, including free, mature 3C proteases
(37, 38). The mature 3C protease of the encephalomyocarditis virus
(EMCV) has been shown to be rapidly degraded in vivo and
in vitro (39, 40). Indirect evidence indicates that the
mature 3C protease of the hepatitis A virus (HAV) is short lived
in vivo (41), and it has been directly proven to be quickly
degraded in rabbit reticulocyte lysate (36). The destruction of both of
these proteins requires both the synthesis of Ub-3C protease conjugates
and the action of the proteasome (35, 36). Neither of these 3C
proteases possesses a destabilizing N-terminal amino acid or homology
with any reported sequences suspected of being required for recognition
by the Ub-mediated proteolytic system.
In order to understand how the short lived 3C proteases are selected
for rapid destruction and to facilitate studies of what role this
destruction might play in the viral infectious cycles, we have
attempted to identify structural features of the EMCV 3C protease that
are required for recognition by the Ub-mediated proteolytic system.
Because we have recently demonstrated that at least one Ub-conjugating
system component in rabbit reticulocyte lysate recognizes both the EMCV
and the HAV 3C proteases (36), and because of the difficulties
associated with detecting unstable 3C proteases in vivo, we
have employed the reticulocyte lysate system in our search. Here we
report the identification and characterization of a 10-amino acid
destruction signal sequence in the EMCV 3C protease. An analogous
sequence is demonstrated to exist in the HAV 3C protease as well. In
addition, we have identified two E3 Ub-protein ligases, one being
E3 Construction of in Vitro Transcription Plasmids Containing
3C Protease Wild Type, Mutated, and Fusion Coding Sequences--
pE3C
(36) or pE3B'CD'* (35) were employed in the construction of all
in vitro transcription plasmids containing mutated EMCV 3C
protease coding sequences. pETP3C (36) or pT7-1 (42) were used as the
starting points for the construction of all plasmids containing mutated
poliovirus 3C protease coding sequences. pE5P-P3 (43) was used as the
source of sequences to construct transcription vectors encoding mutated
HAV 3C protease coding sequences. All insert DNA fragments were
prepared by PCR using PfuI DNA polymerase (Stratagene). All
synthetic primers were obtained from Biosynthesis. Site-specific
mutagenesis was accomplished using both coding and non-coding strand
mutagenic primers. The large number of synthetic primers utilized in
this study precludes listing the sequences here, and these may be
obtained from the authors upon request. The 3C protease coding regions
of all DNA constructs were sequenced with an Applied Biosystems
automated DNA sequencer.
The inserts for pENPC and pPNEC, plasmids carrying the coding sequences
for fused N- and C-terminal halves of the EMCV 3C and poliovirus 3C
proteases, were prepared by two rounds of PCR. In the first round, the
N- and C-terminal coding halves of each sequence were amplified from
pE3C and pETP3C using primers containing the common 5' ...
TTCAGAGAC ... 3' (FRD encoding) region and flanking primers
containing the appropriate restriction endonuclease sites. In the
second round, the sequences were fused in frame via the common
aforementioned sequence, using the appropriate flanking primers. The
NcoI- and EcoRI-treated insert for pENPC was
inserted into modified pGEM-3Z (35), and the NcoI- and
BamHI-treated insert for pPNEC was inserted into pET3d (44).
Inserts for pE3C(N), pE3C(C), pE3C-5, pE3C-15, pE3C-26, and pE3C-46
were prepared by amplifying the sequence to be retained with
NcoI-containing coding strand deletion primers and
EcoRI-containing non-coding strand flanking primers.
Restriction endonuclease-treated inserts were ligated into modified
pGEM-3Z. For constructs carrying codon mutations in the suspected EMCV
3C protease destruction signal sequence region or for constructs
carrying EMCV 3C protease coding sequences with one or two lysine to
arginine codon changes, the appropriate mutagenic primers and flanking
primers containing NcoI and EcoRI sites were
employed to amplify sequences from pE3C or pE3B'CD'*. Plasmids
containing additional lysine to arginine codon changes in the EMCV 3C
coding sequence (pE3CK(1-5)R, pE3CK(8-12)R, pE3CK(6-12)R, and pE3CK(1-12)R) were prepared
from multiple rounds of PCR with mutagenic and flanking primers. The restriction endonuclease-treated inserts were ligated into modified pGEM-3Z.
The insert for pETP3C-DS, which carries the sequence coding for the
poliovirus 3C-DS protein, was prepared by two rounds of PCR
amplification of the poliovirus 3C encoding DNA carried in pT7-1.
Mutagenic primers and flanking primers containing NcoI or
BamHI sites were employed. The NcoI- and
BamHI-treated fragment was ligated into pET3d. pP3C-NDS,
which carries the poliovirus 3C protease wild type coding sequence
fused with a sequence encoding the N-terminal addition MGLLVRGRTLVV,
was prepared as follows. A 75-base coding strand primer containing an
NcoI site, the sequence coding for MGLLVRGRTLVV, and a
sequence that overlaps the N-terminal encoding wild type sequence was
used in combination with a non-coding strand flanking primer containing
an EcoRI site to amplify the poliovirus 3C encoding sequence
from pT7-1. Following treatment with NcoI and
EcoRI, the resulting DNA fragment was ligated into modified
pGEM-3Z. A modified version of this plasmid, pP3C-NDSI, was generated
by using the mutagenic primers and PCR to synthesize a DNA insert in
which the codons for the LVV sequence in the N-terminal extension were
changed to codons for AAA. This insert was also ligated into modified
pGEM-3Z. For the construction of pP3CA35L and
pP3CA35L,I36L, the appropriate mutagenic primers and
flanking primers containing NcoI or EcoRI sites
were employed to amplify sequences from pETP3C. The restriction
endonuclease-treated inserts were ligated into modified pGEM-3Z.
For the construction of pHAV3CL32A,V34A and
pHAV3CLLV(39-41)AAA, which carry the HAV 3C protease
coding sequence with two or three codon mutations, respectively, the
appropriate mutagenic primers and flanking primers containing
NcoI or EcoRI sites were employed to amplify
sequences from pE5P-P3. The restriction endonuclease-treated inserts
were ligated into modified pGEM-3Z.
Cloning, Expression, and Purification of EMCV 3C Protease and
Mutated Protease Proteins--
The preparation of Escherichia
coli cells that express the wild type EMCV 3C protease has already
been described (35). The mutated 3C coding sequence carried in
pE3CLVV(41-43)AAA (see "Results") was amplified by PCR
using primers containing NcoI or BamHI. The
restriction endonuclease-treated fragment was ligated into pET-3d. This
construct was used to transform E. coli BL21 (pLysS) cells.
Expression, refolding, and purification of the protein were carried out
as described previously (35).
Evaluation of the Susceptibility of the Mutated 3C Protease
Proteins toward Conjugation with Ub and Degradation--
The in
vitro transcriptions and translations in reticulocyte lysate were
carried out as described previously (40). The ability of the
35S-labeled 3C protease proteins to be conjugated with MeUb
(35, 45) was tested in reaction mixtures containing 20 mM
HEPES-KOH, pH 7.5, 100 mM
KC2H3O2, 1 mM
Mg(C2H3O2)2, 1 mM DTT, 0.1 mM MeUb, and 0.1 mg/ml
cycloheximide in 50% (v/v) nuclease-treated reticulocyte lysate
containing an ATP-regenerating system (35). The reaction mixtures were
incubated at 30 °C, and 1.5-µl aliquots were removed at the
indicated times and analyzed by 12 or 16% SDS-PAGE and autoradiography
as described previously (40).
The degradation of the 35S-labeled proteins in the
reticulocyte lysate system was monitored in reaction mixtures similar
to that described above, except MeUb was replaced with 0.1 mM bovine Ub (Sigma). In some reactions 0.1 µM Ub aldehyde (46), kindly provided by K. Wilkinson, was
used in place of Ub. When lactacystin (Boston Biomedica) was included,
it was added to give a final concentration of 100 nM. These
reaction mixtures were incubated at 30 °C, and 1.5-µl aliquots
were removed at the indicated times and analyzed by 12% SDS-PAGE and
autoradiography. In some cases the amount of radioactivity present in
the 35S-labeled 3C protease proteins was measured by liquid
scintillation counting (40).
Evaluation of the Catalytic Activity of the Mutated EMCV 3C
Protease Proteins--
The activity of the in vitro
synthesized EMCV 3C protease proteins was tested using
35S-labeled EMCV LVP0 polyprotein as a substrate as
described previously (40).
Protein Purifications and Reactions with E1, E2, and E3
Preparations--
Partially purified rabbit Ubc5 and purified rabbit
E2-14K and E1 were kindly donated by C. Pickart. Human E3
UbcH5 was purified from expressing E. coli cells kindly
provided by P. Howley (see Ref. 47). The UbcH5 purification procedure was adapted from procedures developed by L. Mastrandrea and C. Pickart.2 After induction with
0.08 mM isopropyl-
A 0-30% (NH4)2SO4 precipitated
fraction of fraction II (DEAE-bound; Ref. 48) protein from rabbit
reticulocyte lysate, generously provided by C. Pickart, was used as the
starting material for the partial purification of E3 Ub-protein ligases
that recognize the EMCV 3C protease. The Ubc5-dependent E3
activity was partially purified as follows. Eighty mg of 0-30%
(NH4)2SO4 precipitated rabbit
reticulocyte fraction II protein was loaded onto 4 ml of Q-Sepharose
equilibrated with 50 mM Tris-HCl, pH 7.6, 1 mM
DTT, and 0.1 mM EDTA (TDE buffer). Following washing with
24 ml of TDE buffer, bound protein was eluted with a 45-ml linear
gradient of 0.1-0.5 M NaCl in TDE. Fractions containing
the E3 activity peak (eluting between about 0.30 and 0.35 M
NaCl) were pooled and concentrated. This material (17 mg of protein)
was loaded onto an 80 × 1-cm column of Sephadex S-200
equilibrated with TDE buffer. The E3 activity eluted between 27 and 32 ml. These fractions were pooled, concentrated, and then dialyzed
against 1.0 M (NH4)2SO4 in 20 mM sodium phosphate, pH 7.4, and 2 mM DTT
(PD buffer), followed by dialysis against the same buffer containing
0.6 M (NH4)2SO4. The
dialyzed material was loaded onto a column of 5 ml of methyl-HIC resin
(Bio-Rad) equilibrated in PD buffer containing 0.6 M
(NH4)2SO4. The column was washed
with 2 volumes of the same buffer, and bound protein was eluted with a
25-ml linear gradient of 0.6-0.3 M
(NH4)2SO4 in PD buffer. The
fractions were desalted by multiple rounds of concentration and
dilution in TDE buffer containing 0.1 mg/ml ovalbumin. The E3 activity
eluted between 0.55 and 0.45 M
(NH4)2SO4.
The typical reconstitution assay reaction mixtures consisted of 50 mM Tris-HCl, pH 7.6, 10 mM creatine phosphate,
5 mM MgCl2, 4 mM ATP, 0.6 units/ml
creatine phosphokinase, 0.6 units/ml inorganic pyrophosphatase, either
1 or 9 µM EMCV 3C or 3C[LVV(41-43)AAA] proteases, 0.2 mM Ub or MeUb, 0.1 µM E1, approximately 1-2
µM UbcH5 or E2-14K, and up to 2 µg of E3-containing
preparation, in a final volume of 8 or 12 µl. The mixtures were
incubated at 37 °C. Two-µl aliquots were removed from the mixtures
at the indicated times and were subjected to 12% SDS-PAGE. 3C protease
and Ub-3C protease conjugates were detected by Western blotting, using
affinity purified antibodies against EMCV 3C protease (35) as the
primary antibody. Blot development was accomplished using secondary
antibody-conjugated alkaline phosphatase (for 9 µM 3C
protease reactions) or the enhanced chemiluminescence (ECL) detection
system (Amersham Pharmacia Biotech; for 1 µM 3C protease reactions).
Identification of a Signal Required for the Conjugation of Ub to
the EMCV 3C Protease--
In order to determine whether the
recognition of the EMCV 3C protease by the Ub-conjugating system occurs
because of a discrete, identifiable structural feature within the
substrate protein, several fusion and deletion mutants were prepared
and evaluated. Portions of the EMCV 3C protease were first fused with
portions of the poliovirus 3C protease, which is known to be a poor
substrate for the Ub-mediated proteolytic system (36). Although the
poliovirus protein does not share extensive sequence homology with the
EMCV 3C protease (49, 50), the two do contain the centrally placed common sequence FRD, located at positions 92-94 in the EMCV 3C protease and at positions 83-85 in the poliovirus 3C protease. This
shared sequence was utilized to construct in vitro
transcription plasmids carrying either the sequence coding for the
N-terminal half of the EMCV 3C protease fused with the C-terminal half
of the poliovirus 3C protease (ENPC protein) or the sequence coding for
the N-terminal half of the poliovirus 3C protease fused with the
C-terminal half of the EMCV 3C protease (PNEC protein). The ability of
proteins encoded by these plasmids (Fig.
1A) to become incorporated into
conjugates in reticulocyte lysate in the presence of methylated Ub
(MeUb) was evaluated. Although reticulocyte lysate contains endogenous
Ub, which can become incorporated into polymeric Ub chains conjugated
to substrate proteins, the inclusion of a high concentration of MeUb
(45) in the reaction mixtures results in the synthesis of more easily
detected and relatively stable mono-Ub conjugates, at least with
full-length wild type 3C protease proteins (35, 36). The use of MeUb in
this system facilitates the detection of even low levels of
ubiquitination, and it allows comparisons to be easily made between the
abilities of proteins to serve as substrates for the initial Ub
conjugation event. As shown in Fig. 1B, only the ENPC
protein serves as a substrate for Ub attachment, demonstrating that the
N-terminal 94 amino acids of the EMCV 3C protease include at least some
elements required for recognition by the Ub system. This was confirmed
by testing the ability of the N- or the C-terminal regions of the EMCV
3C protease alone (Fig. 1A) to serve as substrates for
ubiquitination. A polypeptide composed of amino acids 1-101 (E3C(N))
was found to be readily conjugated with at least four MeUb molecules,
whereas a polypeptide composed of amino acids 102-205 (E3C(C)) was a
poor substrate for conjugate formation (Fig. 1C). A further
evaluation of the N-terminal portion of the EMCV 3C protease by
deletion mutagenesis revealed that the elimination of the N-terminal 5, 15, or 26 amino acids from the wild type protein had little effect on
ubiquitination (Fig. 1A). Elimination of the N-terminal 46 amino acids, however, completely abolished all Ub conjugate formation (Fig. 1D). This suggests that the Ub system recognizes a
structural feature located within the region which includes amino acids
27-46.
Although EMCV 3C protease shares little sequence homology with HAV 3C
protease (50, 51), the fact that the two proteins are recognized by a
common Ub-conjugating system component in rabbit reticulocyte lysate
(36) led us to compare amino acid positions 27-46 in the EMCV 3C
protease with the similar region in the HAV 3C protease. Fig.
2 shows the sequence of this region of the
EMCV 3C protease, aligned with the analogous regions in the HAV and
poliovirus 3C proteases (52). Based upon amino acid R group similarity
and the location of the conserved catalytic histidine, the LLV sequence
at positions 34-36 in the EMCV 3C protease can be aligned with the LGV
sequence at positions 32-34 in the HAV 3C protease, and the LVV
sequence at positions 41-43 in the EMCV 3C protease can be matched
with the LLV sequence at positions 39-41 in the HAV 3C protease. The
poliovirus 3C protease contains an LGV sequence (positions 28-30) in
this region as well, but it contains an AIL sequence at positions
35-37, instead of either LVV or LLV. The crystal structures of the
HAV, poliovirus, and rhinovirus 3C proteases have been determined
(53-55), and all three have very similar secondary and tertiary
structural arrangements. Amino acids 32-41 of the HAV protein and
amino acids 28-37 of the poliovirus protein are contained within
similarly folded structures in which the central four, primarily
hydrophilic, amino acids comprise a surface-exposed loop that connects
two antiparallel
In order to test whether the LLVRGRTLVV sequence is involved in the
recognition of the EMCV 3C protease for ubiquitination and degradation,
the amino acids comprising the LLV and LVV triplets, at positions
34-36 and 41-43, respectively, were substituted with alanine
residues. Whereas the incubation of in vitro synthesized non-mutated 3C protease in reticulocyte lysate in the presence of MeUb
resulted in the synthesis of an easily detected mono-Ub conjugate, a
much smaller fraction of the mutated proteins was incorporated into
these conjugates during the incubations (Fig. 3A). In addition, the presence of
endogenous Ub in the lysate resulted in the incorporation of a portion
of the non-mutated 3C protease into poly-Ub conjugates during its
synthesis. This is revealed by the ladder of bands, previously shown to
represent Ub-3C protease conjugates (35), above the primary translation product in lane 1 (0-min sample) of Fig. 3A.
These high molecular weight polymeric conjugates are difficult to
detect in the newly synthesized mutated 3C protease samples
(lanes 4 and 7). The mutated proteins were
degraded considerably more slowly than the non-mutated protein (Fig.
3B), with the initial rates of destruction being about
one-third that measured for the non-mutated 3C protease. The addition
of Ub aldehyde to the reticulocyte lysate system, at a concentration
known to inhibit isopeptidases (46), did not result in an increase in
the rates at which the non-mutated or the mutated EMCV 3C proteases
were degraded (data not shown). This indicates that the effects of the
mutations are due to an inhibition of the conjugation of Ub to the 3C
protease and not to an increased rate of Ub removal from Ub-3C protease
conjugates. It appears, then, that the LLV and LVV triplets in the
proposed signal sequence are indeed involved in the attachment of Ub to the EMCV 3C protease and are required for the rapid destruction of the
protein.
Further evidence that the LLVRGRTLVV region in the EMCV 3C protease
functions as a destruction signal was obtained by mutating the
poliovirus 3C protease sequence shown in Fig. 2. Alterations were made
in this section of the sequence to increase its homology with the EMCV
3C protease sequence. A mutated poliovirus protein, designated 3C-DS
(for destruction signal), containing the sequence LGVRDRTLVV in place
of LGVHDNVAIL was prepared. This new sequence is identical to the
putative EMCV 3C protease destruction signal, except that Gly-29 and
Asp-32 were left unchanged. These residues also occur in the HAV 3C
protease sequence (Fig. 2), so we predicted they would allow the signal
sequence to function. The poliovirus 3C-DS protein was found to be
subject to conjugation with MeUb (Fig.
4A, lanes
1-6). Analysis of the in vitro translation
reactions in which the 3C-DS protein was synthesized revealed the
presence of poly-Ub-conjugated protein products (Fig. 4A, lanes
7 and 8), which were almost absent in the non-mutated
poliovirus 3C protease synthesis reactions. In addition, although the
wild type poliovirus 3C protease was quite stable in the reticulocyte
system, the mutated protein was degraded (Fig. 4B). The rate
of this degradation was reduced by lactacystin (Fig. 4C;
ref. 56), implicating the involvement of the proteasome.
An important question is whether the proposed EMCV 3C protease
destruction signal sequence can also function if it is located somewhere else in a substrate protein. In order to answer this, a DNA
sequence coding for the EMCV 3C protease destruction signal was fused
to the wild type poliovirus 3C protease coding sequence. This allowed
for the synthesis of the poliovirus 3C protease with the sequence
MGLLVRGRTLVV fused to the N-terminal proline residue of the poliovirus
protein (designated the 3C-N-terminal destruction signal or 3C-NDS
protein). The presence of the new N-terminal MG results from the
construction of the transcription plasmid. The analysis of the in
vitro translation product of the RNA coded for by this plasmid
revealed the presence of high molecular weight labeled species,
consistent with the formation of poly-Ub conjugates in the reticulocyte
system (Fig. 4D, lanes 1 and 2). The incubation of the poliovirus 3C-NDS protein in the presence of MeUb resulted in
the synthesis of a detectable mono-Ub-3C-NDS conjugate (Fig. 4D,
lanes 3-5). The 3C-NDS protein was also degraded at a rate similar to that of the poliovirus 3C-DS protein shown in Fig. 4B,
lanes 5-8. These results indicate that the fusion of the signal sequence to the N terminus of the poliovirus 3C protease leads to the
recognition of the protein as a substrate for ubiquitination. In order
to ascertain whether the effects of the N-terminally located
destruction sequence are specifically due to the signal itself, and not
simply the presence of the extra N-terminal amino acids, the signal
sequence was mutated to MGLLVRGRTAAA (poliovirus 3C-NDSI). This is
analogous to the EMCV 3C protease mutant with the LVV sequence at
positions 41-43 replaced with AAA (Fig. 3). The poliovirus 3C-NDSI
protein was incorporated into conjugates with MeUb less readily than
was the 3C-NDS protein (Fig. 4D, lanes 6-8), indicating
that the transfer of susceptibility toward conjugation with Ub results,
at least primarily, from the attachment of the destruction signal sequence.
Additional Characterization of the Destruction Signal and the
Location of Ub Attachment Sites--
An effort was made to
systematically evaluate which features in the EMCV 3C protease
destruction signal are important for recognition by the Ub system.
Several mutations, the majority being single alanine substitutions,
were generated in the destruction signal sequence. The mutated proteins
that were synthesized, and the relative susceptibility of these
proteins toward conjugation with Ub in the reticulocyte lysate system,
are summarized in Table I. The effects of the
mutations were determined by comparing the fractions of the proteins
that were incorporated into mono-Ub conjugates during a 15-min
incubation in the presence of MeUb, with the extent of ubiquitination
observed with the non-mutated 3C protease. None of the mutated proteins
demonstrated a greater susceptibility toward recognition by the Ub
system than the non-mutated EMCV 3C protease. The relative rates at
which these proteins were degraded correlated with their relative
susceptibilities toward conjugation with MeUb. The results indicate few
of the single amino acid changes strongly reduced the ability of the
destruction signal sequence to function. One exception to this is the
first amino acid in the sequence, Leu-34, which is conserved in the analogous positions in the HAV and poliovirus 3C proteases. The substitution of an alanine or methionine residue for this leucine, which would not be expected to induce drastic structural changes, strongly reduced the efficiency of Ub conjugation, as did the substitution of a valine. Whereas the replacement of the arginine at
position 39 with an alanine or lysine residue negatively affected the
efficiency of ubiquitination of the EMCV 3C protease, the presence of
an aspartate in this position resulted in an even more poorly
ubiquitinated substrate. This suggests that signal functionality is
dependent upon more than simply the presence of polar amino acids in
this region. In addition, the simultaneous replacement of both
arginines in the central region of the signal with alanine residues
virtually eliminated the susceptibility of the 3C protease toward
conjugation with ubiquitin. Besides causing an obvious reduction in the
hydrophilic character of this part of the signal sequence, these
mutations may result in significant higher order structural
alterations. The strongly inhibitory effects of eliminating a residue
from (
The severe attenuation of the conjugation of Ub to the EMCV
3C[LLV(34-36)AAA]- and 3C[LVV(41-43)AAA] proteins (Fig.
3A) and the absence of leucines or valines in positions 35 and 36 of the wild type poliovirus 3C protease (Fig. 2) raise the
possibility that a high density of leucine and valine residues in the
three positions on both ends of the destruction signal sequence are necessary for the signal to function. Support for this hypothesis was
obtained by testing the ability of poliovirus 3C protease proteins
containing A35L or A35L plus I35L substitutions to serve as substrates
for the Ub-mediated proteolytic system. Both the single and double
leucine substitutions allowed the protein to become ubiquitinated and
to be degraded at rates comparable to that observed for the poliovirus
3C-DS protein, shown in Fig. 4, A and B (data not shown).
Given that the EMCV 3C protease undergoes conjugation with Ub primarily
at a single lysine (35), an attempt was made to determine if a specific
lysine in the EMCV 3C protease, perhaps because of its location
relative to that of the destruction signal, is exclusively utilized as
the Ub attachment site. All 12 of the lysine residues in the 3C
protease were replaced with arginine residues, either individually or
in pairs. The extent to which all of these substitution mutants were
incorporated into MeUb-3C protease conjugates in the reticulocyte
lysate system was only slightly less than that observed for the
non-mutated protein (data not shown). Additional mutagenesis was
performed to prepare in vitro transcription vectors that
code for the 3C protease with lysine to arginine substitutions at
positions 10, 14, 68, 74, and 78 (3C[K(1-5)R] protease), positions
151, 156, 171, and 172 (3C[K(8-12)R] protease), positions 101, 130, 151, 156, 171, and 172 (3C[K(6-12)R] protease), and at all 12 lysine
positions (3C[K(1-12)R] protease). The results of incubating these
proteins in reticulocyte lysate in the presence of MeUb are shown in
Fig. 5A. Only the 3C[K(1-12)R]
protease protein was found to be completely resistant to
ubiquitination, although the other mutated proteins were less susceptible to conjugation with MeUb than was the non-mutated 3C
protease. A comparison of the stabilities of these proteins is shown in
Fig. 5B. Whereas the 3C[K(1-5)R), 3C[K(8-12)R), and 3C[K(6-12)R] proteins were degraded less rapidly than was the non-mutated EMCV 3C protease, presumably a reflection of their reduced
susceptibility toward ubiquitination, the 3C[K(1-12)R] protease was
found to be degraded quite slowly. This no lysine version of the 3C
protease was degraded at a rate about one-fourth that measured for the
non-mutated protein, and this degradation was not inhibited by
lactacystin (Fig. 5C). The ubiquitin and proteasome-independent destruction of the EMCV 3C protease is likely
due to one or more other proteases present in the reticulocyte lysate.
We have previously observed, for example, that other EMCV proteins,
including the LVP0 polyprotein and the 3D polymerase are degraded in
the reticulocyte system, although at rates that are considerably slower
than that of the mature 3C protease (40). This turnover does not appear
to require the ubiquitination of these proteins (35). All of the lysine
to arginine mutants retained levels of catalytic activity comparable to
that of the non-mutated EMCV 3C protease. The retention of the ability
of the EMCV 3C[K(1-12)R] protease to cleave the leader (L) protein
from the EMCV polyprotein capsid precursor LVP0 (40) is shown in Fig.
5D. This suggests that the mutations do not have a major
effect on the higher order structure of the protein. These results
demonstrate that more than one lysine in the EMCV 3C protease can be
used as the site for the initial attachment of Ub to the protein in
reticulocyte lysate.
Confirmation That the HAV 3C Protease Also Contains a Destruction
Signal--
As discussed above, we predicted that the HAV 3C protease
contains a destruction signal sequence, presumably the 10-amino acid
sequence that is aligned with the EMCV 3C protease signal sequence in
Fig. 2, i.e. LGVKDDWLLV. This prediction was tested by
evaluating the ability of HAV 3C protease with mutations in the LGV and
LLV triplets to serve as substrates for conjugation with Ub. The
results of incubating the in vitro synthesized proteins containing either L32A plus V34A or L39A, L40A, and V41A mutations in
reticulocyte lysate in the presence of MeUb is shown in Fig. 6A. The ability of both mutants to
become incorporated into mono-Ub conjugates was sharply attenuated. The
rates at which both mutated proteins are degraded was also reduced
(Fig. 6B). These results confirm that the HAV 3C protease
LGVKDDWLLV sequence comprises at least part of a destruction signal
sequence.
Partial Purification and Identification of an E3 Ub-Protein Ligase
That Recognizes the EMCV 3C Protease Destruction Signal--
By having
identified a sequence in the EMCV 3C protease required for the
attachment of Ub to occur, we attempted to at least partially purify
the E3 Ub-protein ligase that interacts with this signal.
Reconstitution experiments with rabbit reticulocyte lysate fraction II
(48) and a crude rabbit Ubc5 E2 preparation revealed that Ubc5 can
support the ubiquitination of the EMCV 3C protease, presumably by
functioning in a pathway with the E1-activating enzyme and an E3
present in fraction II. A screening of
(NH4)2SO4 precipitate fractions
revealed that this E3 activity resides in the 0-30%
(NH4)2SO4 fraction. This material
was used as a starting point for a small scale purification of the E3
activity. The progress of the purification was monitored using a
reconstitution mixture composed of purified wild type EMCV 3C protease,
Ub, purified rabbit E1, human UbcH5 purified from expressing E. coli cells (47), and E3 ligase-containing fractions.
The details of the purification scheme are described under
"Experimental Procedures." The 0-30%
(NH4)2SO4 precipitate material was
further fractionated by anion exchange chromatography on a column of
Q-Sepharose, size exclusion chromatography on a column of Sephadex
S-200, and hydrophobic interaction chromatography on a column of
methyl-HIC resin. The composition of the most active fraction recovered
from the methyl-HIC resin is shown in Fig. 7A. Six major proteins are
visible, along with several minor ones. A demonstration of the
catalytic activity of this E3 fraction is shown in the Western blot,
probed with anti-3C protease antibodies, in Fig. 7B, lanes
1-4. The formation of mono- and poly-Ub conjugates of the EMCV 3C
protease is apparent, and the synthesis of these products requires the
E3 preparation.
Several other E2 enzymes were tested for their ability to function with
the E3 preparation, in place of UbcH5. As Fig. 7B, lane 6, shows, E2-14K from rabbit reticulocytes supported the formation of
Ub-EMCV 3C protease conjugates in the presence of the E3 preparation.
These results were something of a surprise, since E2-14K appears to
work exclusively with E3
These results demonstrate that the Ub-protein ligase E3 Although considerable understanding of how the Ub-mediated
proteolytic system functions to selectively degrade proteins has been
gained in recent years, the mechanism by which proteins are recognized
as substrates for this system has largely remained undefined. We have
identified a previously unknown protein destruction signal in the
rapidly degraded EMCV 3C protease, a protein that is crucial for viral
replication. The sequence LLVRGRTLVV, located at positions 34-43, has
been demonstrated here to contribute, at least in vitro, to
the recognition of the EMCV 3C protease for ubiquitination and to be
necessary for the rapid destruction of the protein. Evidence was also
obtained which suggests that the HAV 3C protease contains a destruction
signal sequence (LGVKDDWLLV) in a location homologous with that of the
EMCV 3C protease.
An examination of the crystal structures of the picornavirus 3C
proteases that have been determined to date reveal that they are
conserved (53-55). The putative HAV 3C protease destruction signal
sequence coincides with segments of two antiparallel An analysis of the effects of mutations in the EMCV 3C protease signal
sequence indicates that the signal can tolerate considerable sequence
variability and remain functional, although certain definable characteristics appear to contribute to its ability to function. The
data, along with the comparisons between the EMCV, HAV, and poliovirus
3C protease sequences, suggest a leucine must be present in the first
position of the signal sequence and that the three positions on either
end of the signal sequence must contain a high density of amino acids
with branched aliphatic R groups (leucines and valines). The absence of
these residues in the C-terminal triplet of the poliovirus 3C protease
sequence which aligns with the EMCV 3C and putative HAV 3C protease
destruction signals was shown to be a major contributing factor to the
stability of the poliovirus protein. These results are also consistent
with observations made in studies with artificial substrates (58, 59),
which indicate that short sequences of bulky hydrophobic amino acids can cause proteins to be recognized for Ub-mediated degradation. Given
the strand-turn-strand structure in which the signal appears to reside,
the four amino acids that occupy the center of the signal may also
comprise an another important feature, since these residues would
contribute to the formation of the hydrophilic loop. Although the data
obtained shows that sequence in this region can be highly variable, any
combination of polar amino acids does not support the generation of a
functional destruction signal. Other experiments with EMCV 3C protease
proteins containing lysine to arginine substitutions revealed that the
location of the destruction signal does not restrict the initial
attachment of Ub to a specific lysine residue. The absence of unique
ubiquitination sites has been reported to exist for other substrates of
the Ub-mediated proteolytic system (19, 60, 61).
Two E3 Ub-protein ligases were shown to be involved in the
ubiquitination of the EMCV 3C protease. One of these was identified as
E3 The second E3 shown to be capable of catalyzing the ubiquitination of
the EMCV 3C protease functions with the E2 Ubc5. This may, therefore,
be an E3 that belongs to the hect family of Ub-protein ligases (32).
This E3 was found not to require the LLVRGRTLVV destruction signal
sequence to recognize the 3C protease. It is unclear what structural
features are recognized by the Ubc5-dependent E3. The fact
that no ubiquitination was observed to occur in reticulocyte lysate
with proteins containing only the C-terminal 113 amino acids or with
N-terminal deletion mutants lacking the first 46 amino acids suggests
the N-terminal region contains a recognition element necessary for this
E3 to interact with the 3C protease. It appears that, in reticulocyte
lysate, at least, the Ubc5-dependent E3 is responsible for
a much smaller fraction of the Ub-3C protease conjugate synthesis than
is E3 It is likely that the protein destruction signal identified here also
exists in one or more cellular proteins, since the ability of the
ubiquitin system to recognize this signal would be expected to have
evolved to meet the needs of cells not infected with picornaviruses. Because of the apparent degeneracy of the signal sequence, and because
the secondary structure of the signal region may contribute to the
efficiency with which it functions, meaningful searches of protein data
bases for other examples of the signal are difficult. A search of the
SWISS-PROT data base, however, revealed the existence of at least 17 eukaryotic cellular and 5 (in addition to the EMCV and HAV 3C
proteases) mammalian viral proteins with candidate destruction signal
sequences. These proteins all contain the sequence Leu-(Gly, Leu, or
Val)-(Leu or Val)-X4-(Leu or Val)3,
where X represents amino acids frequently found in reverse turns.
It remains to be seen if the identified destruction signal functions
in vivo the same way it functions in vitro.
Similarities between the kinetics of the EMCV 3C protease degradation
in vitro and in vivo (35, 39, 40) suggest similar
mechanisms of destruction occur in both systems. Detailed studies of
the behavior of destruction signal mutants of the 3C protein in
vivo will be required to answer this question. It is also not yet
clear what function, if any, the rapid degradation of the EMCV and HAV
3C proteases serves during the replication of these viruses. In the
case of EMCV, at least, the amount of 3C protease present reaches a
maximum 2-3 h into the infectious cycle and then rapidly declines to
near undetectable levels by the time the cells lyse (39). It may be
that the presence of 3C protease activity during the later phases of
the infectious cycle is detrimental to virus reproduction. It is also
unclear why the 3C protease of poliovirus has evolved to be relatively
stable. The ability to now generate catalytically active, relatively
stable versions of the EMCV and HAV 3C proteases will allow future
studies into whether the rapid and selective degradation of these
proteins is biologically important.
, and this was shown to require the destruction signal
sequence to catalyze efficiently the ubiquitination of the 3C protease.
The other is a Ubc5-dependent E3 that appears to recognize
a different, unidentified feature of the 3C protease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 repressor, for
example, has been hypothesized to contain an amphipathic
-helix (24).
and its yeast
homologue Ubr1 (28-31) and the family of hect Ub-protein ligases (32). E3
and Ubr1 are known to ubiquitinate N-end rule substrates (29, 30), and Ubr1 has recently been found to participate in the ubiquitination of the yeast proteins G
and Cup9 (18, 23), neither of
which contains a destabilizing N-terminal amino acid. In the case of
G
, the Ubr1 recognition element has been mapped to a 60-amino acid
region near the N terminus (25), and the recognition of Cup9 requires
the C-terminal two-thirds of the protein (23). The only other
definitively known connection between a protein destruction signal and
a Ub-protein ligase is that between the cyclin destruction signal
sequence and the cyclosome complex (33, 34).
, that recognize the EMCV 3C protease and consequently catalyze
the conjugation of Ub to the protein.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, purified
by affinity chromatography using an E2-14K column, was the generous gift of A. Haas.
-D-thiogalactopyranoside for 5 h at 30° C, 10 g of pelleted cells were
recovered and lysed by freezing and thawing in 2 volumes of 50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 2 mM DTT, 1 mM p-toluenesulfonyl
fluoride, 0.1 mM EDTA, 10 µg/ml leupeptin, and 10 µg/ml
DNase I. Following removal of cell debris by centrifugation, the
50-80% (NH4)2SO4 precipitate fraction was recovered and dialyzed against 10 mM Tris-HCl,
pH 7.6, 1 mM DTT, and 0.1 mM EDTA. The material
was filtered through 20 ml of Q-Sepharose (Amersham Pharmacia Biotech)
equilibrated with 50 mM Tris-HCl, 2 mM DTT, 0.1 mM EDTA, 0.1 mM p-toluenesulfonyl fluoride, and 2 µg/ml leupeptin. The flow-through was concentrated and loaded onto a 44 × 1-cm column of Sephadex S-200 (Amersham Pharmacia Biotech) equilibrated in 50 mM Tris-HCl, 2 mM DTT, and 0.1 mM EDTA. The 16-kDa UbcH5
eluted between about 25 and 30 ml. The UbcH5-containing fractions were
stored in aliquots with 0.1 mg/ml added ovalbumin.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Fusion proteins and deletion mutants employed
to locate the destruction signal region in the EMCV 3C protease.
A, diagrams of fusion proteins and deletion mutants employed
and the results of incubating the in vitro synthesized
proteins in the reticulocyte lysate system in the presence of MeUb.
Open boxes indicate EMCV 3C protease (E3C)
sequences, and hatched boxes indicate poliovirus 3C protease
(P3C) sequences. The numbers indicate the amino
acid positions in the EMCV or the poliovirus 3C proteases. The
identities of the three amino acids located at each end of the proteins
are shown. Methionine residues added to the N termini of the wild type
sequences to allow translation initiation are indicated by M
outside the boxes. B, autoradiograph
of the SDS-PAGE analysis of aliquots removed from reaction mixtures
containing the ENPC and PNEC fusion proteins. Lane 1, ENPC,
0 min; lane 2, ENPC, 60 min; lane 3, PNEC, 0 min;
lane 4, PNEC, 60 min. C, autoradiograph of the
SDS-PAGE analysis of aliquots removed from reaction mixtures containing
the E3C(N) and E3C(C) proteins. Lane 1, E3C(N), 0 min;
lane 2, E3C(N), 60 min; lane 3, E3C(C), 0 min;
lane 4, E3C(C), 60 min. D, autoradiograph of the
SDS-PAGE analysis of aliquots removed from reaction mixtures containing
the non-mutated EMCV (E3C) and E3C-46 3C proteins. Lane 1,
E3C, 0 min; lane 2, E3C, 60 min; lane 3, E3C-46,
0 min; lane 4, E3C-46, 60 min. * indicates the location of
MeUb conjugates.
-sheet strands. There is no reason not to expect
that the aligned EMCV 3C protease sequence also exists within a
strand-turn-strand structure. Based upon these comparisons, we
hypothesized that the 10-amino acid sequence LLVRGRTLVV in the EMCV 3C
protease might comprise a protein destruction signal that is recognized by a Ub-mediated proteolytic system component. We also predicted that,
if this is the case, then the LGVKDDWLLV segment in the HAV 3C protease
could serve this function as well.
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Fig. 2.
The amino acid sequence of the EMCV 3C
protease from positions 27 to 46 and the aligned sequences from the HAV
and poliovirus 3C proteases. The catalytic histidine common to all
three proteins is shown in bold.
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Fig. 3.
Evaluation of the ability of the EMCV
3C[LLV(34-36)AAA] and 3C[LVV(41-43)AAA] proteins to serve as
substrates for the reticulocyte Ub-mediated proteolytic system.
A, autoradiograph of the SDS-PAGE analysis of aliquots of
reaction mixtures in which in vitro synthesized non-mutated
and mutated EMCV 3C protease proteins were incubated in the presence of
MeUb. Lanes 1-3, non-mutated EMCV 3C protease; lanes
4-6, EMCV 3C[LLV(34-36)AAA] protease; lanes 7-9,
EMCV 3C[LVV(41-43)AAA] protease. Aliquots were removed from the
reaction mixtures at 0 min (lanes 1, 4, and 7),
15 min (lanes 2, 5, and 8), and 60 min
(lanes 3, 6, and 9). * indicates the location of
mono-Ub conjugates. B, autoradiograph of the SDS-PAGE
analysis of aliquots of reaction mixtures in which in vitro
synthesized non-mutated and mutated EMCV 3C protease proteins were
incubated in the presence of added Ub. Lanes 1-4,
non-mutated EMCV 3C protease; lanes 5-8, EMCV
3C[LLV(34-36)AAA] protease; lanes 9-12, EMCV
3C[LVV(41-43)AAA] protease. Aliquots were removed from the reaction
mixtures at 0 min (lanes 1, 5, and 9), 30 min
(lanes 2, 6, and 10), 90 min (lanes 3, 7, and 11), and 180 min (lanes 4, 8, and
12).
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Fig. 4.
Evaluation of the ability of the poliovirus
3C-DS, 3C-NDS, and 3C-NDSI proteins to serve as substrates for the
reticulocyte Ub-mediated proteolytic system. A,
autoradiograph of the SDS-PAGE analysis of aliquots of reaction
mixtures in which in vitro synthesized poliovirus 3C and
3C-DS proteins were incubated in the presence of MeUb. Lanes
1-3, poliovirus 3C protease; lanes 4-6, poliovirus
3C-DS protease. Aliquots were removed from the reaction mixtures at 0 min (lanes 1 and 4), 15 min (lanes 2 and 5), and 60 min (lanes 3 and 6).
Lanes 7 and 8 show the analysis of aliquots of
in vitro translation reaction mixtures used to prepare the
non-mutated 3C and 3C-DS proteins, respectively. The time for the
autoradiography of these samples was extended to enhance visualization
of poly-Ub conjugates. * indicates the location of mono-Ub conjugates.
B, autoradiograph of the SDS-PAGE analysis of aliquots of
reaction mixtures in which in vitro synthesized poliovirus
3C and 3C-DS proteins were incubated in the presence of added Ub.
Lanes 1-4, poliovirus 3C protease; lanes 5-8,
poliovirus 3C-DS protease. Aliquots were removed from the reaction
mixtures at 0 min (lanes 1 and 5), 30 min
(lanes 2 and 6), 90 min (lanes 3 and
7), and 180 min (lanes 4 and 8).
C, autoradiograph of the SDS-PAGE analysis of aliquots of
reaction mixtures in which in vitro synthesized poliovirus
3C-DS protein was incubated in the absence or presence of lactacystin
and added Ub. Aliquots were removed from the reaction mixtures at 0 min
(lanes 1), 10 min (lanes 2), 30 min (lanes
3), 60 min (lanes 4), and 120 min (lanes 5).
D, autoradiograph of the SDS-PAGE analysis of aliquots of
reaction mixtures in which in vitro synthesized poliovirus
3C-NDS and 3C-NDSI proteins were incubated in the presence of MeUb.
Lanes 1 and 2 show a comparison of aliquots of
in vitro translation reaction mixtures used to prepare
non-mutated poliovirus 3C and 3C-NDS proteins, respectively. The time
for the autoradiography of these samples was extended to enhance
visualization of poly-Ub conjugates. Lanes 3-5, poliovirus
3C-NDS protein incubated with MeUb; lanes 6-8, poliovirus
3C-NDSI protein incubated with MeUb. Aliquots were removed from the
reaction mixtures at 0 min (lanes 3 and 6), 15 min (lanes 4 and 7), and 60 min (lanes
5 and 8). * indicates the location of mono-Ub
conjugates.
Gly-38), or adding a residue to (+Ala-38), the sequence
indicates that a number of residues in the sequence, or at least in the
central region, may also be important. All of the mutated proteins
listed in Table I, as well as the triple alanine substitutions
described above (Fig. 3), were tested for catalytic activity. Except
for the 3C[LLV(34-36)AAA] protein, all retained the ability to
catalyze the cleavage of the leader (L) protein from the EMCV
polyprotein capsid precursor LVP0 (40). For these mutated proteins, at
least, the retention of catalytic activity is indicative of minimal
alterations in the overall higher order structures.
Effects of single amino acid changes in the destruction signal sequence
on the conjugation of Ub to the EMCV 3C protease.
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Fig. 5.
Evaluation of the ability of lysine to
arginine mutants of the EMCV 3C protease to serve as substrates for the
reticulocyte Ub-mediated proteolytic system. A,
autoradiograph of the SDS-PAGE analysis of aliquots of reaction
mixtures in which in vitro synthesized EMCV 3C protease
proteins with multiple lysine to arginine substitutions were incubated
in the presence of MeUb. Lane 1, non-mutated EMCV 3C
protease, 0 min; lane 2, non-mutated EMCV 3C protease, 60 min; lane 3, EMCV 3C[K(1-5)R] protease, 0 min; lane
4, EMCV 3C[K(1-5)R] protease, 60 min; lane 5, EMCV
3C[K(8-12)R] protease, 0 min; lane 6, EMCV
3C[K(8-12)R] protease, 60 min; lane 7, EMCV
3C[K(6-12)R] protease, 0 min; lane 8, EMCV
3C[K(6-12)R] protease, 60 min; lane 9, EMCV
3C[K(1-12)R] protease, 0 min; and lane 10, EMCV
3C[K(1-12)R] protease, 60 min. * indicates the location of mono-Ub
conjugates. B, autoradiograph of the SDS-PAGE analysis of
aliquots of reaction mixtures in which in vitro synthesized
non-mutated and mutated EMCV 3C protease proteins were incubated in the
presence of added Ub. Lanes 1-3, non-mutated EMCV 3C
protease; lanes 4-6, EMCV 3C[K(1-5)R] protease;
lanes 7-9, EMCV 3C[K(8-12)R] protease; lanes
10-12, EMCV 3C[K(6-12)R] protease; lanes 12-15,
EMCV 3C[K(1-12)R] protease. Aliquots were removed from the reaction
mixtures at 0 min (lanes 1, 4, 7, 10, and 13), 30 min (lanes 2, 5, 8, 11, and 14), and 180 min
(lanes 3, 6, 9, 12, and 15). The smaller, minor
product that migrates below the 22-kDa 3C protease proteins is the
result of translation initiation at a methionine codon in a Kozak
consensus context (35), located a short distance downstream of the
primary initiation site. This is easily visible here because of the
size of the gels utilized in the analysis. C, autoradiograph of the
SDS-PAGE analysis of aliquots of reaction mixtures in which in
vitro synthesized EMCV 3C and 3C[K(1-12)R] protease proteins
were incubated in the absence or presence of lactacystin and added Ub.
Lanes 1-5, non-mutated EMCV 3C protease; lanes
6-10, EMCV 3C[K(1-12)R] protease. Aliquots were removed from
the reaction mixtures at 0 min (lanes 1 and 6),
10 min (lanes 2 and 7), 30 min (lanes
3 and 8), 60 min (lanes 4 and 9),
and 120 min (lanes 5 and 10). D,
autoradiograph of the SDS-PAGE analysis of reaction mixtures containing
in vitro synthesized labeled EMCV LVP0 polyprotein capsid
precursor and unlabeled EMCV 3C or 3C[K(1-12)R] protease proteins.
Lanes 1-4, LVP0 polyprotein only; lanes 5-7,
LVP0 polyprotein plus non-mutated EMCV 3C protease; lanes
8-10, LVP0 polyprotein plus EMCV 3C[K(1-12)R] protease.
Aliquots were removed from the reaction mixtures at 0 min (lane
1), 10 min (lanes 2, 5, and 8), 30 min
(lanes 3, 6, and 9), and 60 min (lanes 4, 7, and 10). The locations of the LVP0 polyprotein
precursor and the product VP0 diprotein and leader (L)
protein are indicated.
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Fig. 6.
Evaluation of the ability of the HAV
3C[LGV(32-34)AGA]- and 3C[LVV(39-41)AAA] protein mutants to serve
as substrates for the reticulocyte Ub-mediated proteolytic system.
A, autoradiograph of the SDS-PAGE analysis of aliquots of
reaction mixtures in which in vitro synthesized non-mutated
and mutated HAV 3C protease proteins were incubated in the presence of
MeUb. Lanes 1-3, non-mutated HAV 3C protease; lanes
4-6, HAV 3C[LGV(32-34)AGA] protease; lanes 7-9,
HAV 3C[LVV(39-41)AAA] protease. Aliquots were removed from the
reaction mixtures at 0 min (lanes 1, 4 and 7), 15 min (lanes 2, 5 and 8), and 60 min (lanes
3, 6 and 9). * indicates the location of mono-Ub
conjugates. B, autoradiograph of the SDS-PAGE analysis of
aliquots of reaction mixtures in which in vitro synthesized
non-mutated and mutated HAV 3C protease proteins were incubated in the
presence of added Ub. Lanes 1-4, non-mutated HAV 3C
protease; lanes 5-8, HAV 3C[LGV(32-34)AGA] protease;
lanes 9-12, HAV 3C[LVV(39-41)AAA] protease. Aliquots
were removed from the reaction mixtures at 0 min (lanes 1, 5, and 9,), 30 min (lanes 2, 6 and
10), 90 min (lanes 3, 7, and 11), and
180 min (lanes 4, 8, and 12).
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Fig. 7.
E3 Ub-protein ligase activities that catalyze
the ubiquitination of the EMCV 3C protease. A, SDS-PAGE
and Coomassie stain analysis of a partially purified E3 preparation
derived from rabbit reticulocyte fraction II. Lane 1,
molecular mass standards; lane 2, 15 µg of E3 preparation
eluted from a methyl-HIC column. B, demonstration of the
activity of the partially purified E3 in catalyzing the synthesis of
Ub-3C protease conjugates. All reaction mixtures were incubated for 60 min and included in addition to 9 µM EMCV 3C protease and
Ub: lane 1, nothing; lane 2, E1 and UbcH5;
lane 3, E1, and the E3 preparation; lane 4, E1,
UbcH5, and the E3 preparation; lane 5, E1 and E2-14K;
lane 6, E1, E2-14K, and the E3 preparation. C,
demonstration of the activity of affinity purified E3 in catalyzing
the synthesis of Ub-3C protease conjugates. The reaction mixtures were
incubated for 60 min and included in addition to 9 µM
EMCV 3C protease and Ub: lane 1, E1 and E2-14K; lane
2, E1, E2-14K, and E3
. The results shown in B and
C were generated using Western blotting and development with
alkaline phosphatase-conjugated secondary antibody.
(57). The E3 preparation contains a protein
that migrates in SDS-PAGE gels with a mass consistent with that of a
subunit of E3
, which was about 180 kDa (Ref. 28; Fig.
7A). These observations support the notion that the
preparation contains E3
, which is capable of recognizing the EMCV 3C
protease as a substrate for ubiquitination. In order to confirm this,
affinity purified human E3
was tested in the reconstituted system.
This E3, in combination with E2-14K, was indeed found to catalyze the
synthesis of Ub-EMCV 3C protease conjugates (Fig.
7C).
recognizes the EMCV 3C protease as a substrate for ubiquitination. A
second, Ubc5-dependent E3 protein also appears to be
capable of participating in the synthesis of Ub-3C protease conjugates. The two E3 activities apparently co-precipitated in the same
(NH4)2SO4 fraction and at least
partially co-eluted from each of the chromatography matrices employed
in the purification procedure. In order to determine directly whether
the recognition of the EMCV 3C protease by one, or both, of these E3
proteins requires the identified destruction signal, experiments were
carried out using a purified 3C protease with a mutated destruction
signal sequence. The EMCV 3C[LVV(41-43)AAA] protease, which retains
catalytic activity but is a poor substrate for conjugation with Ub
(Fig. 3), was expressed in E. coli cells and purified. When
wild type EMCV 3C protease or the 3C[LVV(41-43)AAA] protease was
incubated in the reconstituted system with MeUb, in the presence of
either affinity purified E3
plus E2-14K or the partially purified
E3 preparation plus UbcH5, the results shown by the Western blots in
Fig. 8 were obtained. E3
catalyzed the
ubiquitination of the wild type protease much more rapidly than it did
the 3C protease with the mutation in the destruction signal (Fig.
8A). This result provides a direct link between the integrity of the destruction signal sequence and the ability of E3
to catalyze the ubiquitination of the EMCV 3C. The
Ubc5-dependent E3, however, catalyzed the attachment of Ub
to both the wild type and the mutated proteins at similar rates (Fig.
8B). This E3 does not, therefore, utilize the identified
destruction signal or at least not the C-terminal portion of the signal
sequence.
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Fig. 8.
Evaluation of the ability of the Ub-protein
ligase preparations to catalyze the ubiquitination of the EMCV 3C
protease containing a mutated destruction signal sequence.
A, demonstration of the activity of purified E3 in
catalyzing the synthesis of Ub-3C protease conjugates. Lanes
1-4 show the analysis of reaction mixtures containing 1 µM 3C protease, MeUb, E1, E2-14K, and E3
. Lanes
5-8 show the analysis of reaction mixtures containing 1 µM 3C[LVV(41-43)AAA] protease, MeUb, E1, E2-14K, and
E3
. Aliquots were removed at 0 min (lanes 1 and
5), 10 min (lanes 2 and 6), 20 min
(lanes 3 and 7), and 60 min (lanes 4 and 8). B, demonstration of the activity of the
partially purified Ubc5-dependent E3 in catalyzing the
synthesis of Ub-3C protease conjugates. Lanes 1-4 show the
analysis of reaction mixtures containing 1 µM 3C
protease, MeUb, E1, UbcH5, and the E3 preparation. Lanes
5-8 show the analysis of reaction mixtures containing 1 µM 3C[LVV(41-43)AAA] protease, MeUb, E1, UbcH5, and
the E3 preparation. Aliquots were removed at 0 min (lanes 1 and 5), 10 min (lanes 2 and 6), 20 min
(lanes 3 and 7), and 60 min (lanes 4 and 8). The results shown were generated using Western
blotting and development with ECL.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet strands
connected by a surface-exposed turn. The internal sequence changes made
in the poliovirus 3C protease, which converted it to a substrate for
the Ub system, are located in an identically placed structure. It is
likely that the EMCV 3C protease signal sequence is placed in a similar
location as well. This would mean that the central four, mostly
hydrophilic, amino acids in the EMCV 3C protease signal exist in a
surface-exposed loop which connects two strands of an antiparallel
-sheet. The leucine/valine-rich hydrophobic triplets would occupy
the strand portions of the signal. The comparable strand sequences in
the HAV and poliovirus 3C protease strand segments are not exposed on
the surfaces of the proteins (53, 55), but it is possible to imagine
them becoming accessible though a partial unfolding of the protein
structure. It may be that the secondary structure of the signal region
in the native protein contributes to its identity as a recognition
element for the Ub-mediated proteolytic system. That the EMCV 3C
protease signal sequence linked to the N terminus of the poliovirus 3C protease was observed to bring about the ubiquitination of the poliovirus protein may, however, indicate that the strand-turn-strand motif is not an absolute requirement for the signal to function. The
folding of the signal sequence in this context may be different than
that which occurs in the internal location in the native EMCV 3C protease.
, and this was demonstrated by reconstitution experiments to
require the identified destruction signal sequence to participate in
the attachment of Ub to the 3C protease protein. Although the most
straightforward interpretation of these results is that E3
binds to
the EMCV 3C protease signal during initial substrate recognition, it is
possible that a necessary interaction between E3
and the signal
occurs subsequently to this step. Preliminary experiments have
demonstrated that E3
can also participate in the ubiquitination of
the HAV 3C protease,3 presumably
by interacting with the destruction signal identified in that protein.
E3
has been thought to recognize only proteins with certain
N-terminal amino acids (29, 30). Its yeast homologue, Ubr1, has,
however, recently been found to participate in the ubiquitination of
yeast proteins G
and Cup9, both of which have internally located
destruction signals (18, 23, 25). A search of the reported signal
regions for both of these proteins did not reveal the presence of a
sequence that mimics the EMCV or HAV 3C protease destruction signal
sequences. This may indicate that mammalian E3
and yeast Ubr1
recognize dissimilar internally located signal elements or that these
E3 proteins are capable of interacting with more than one type of
internal signal feature.
. This is indicated by the sharply attenuated rate of
ubiquitination and degradation that was observed for some of the EMCV
3C protease proteins containing mutations in the E3
destruction
signal sequence.
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ACKNOWLEDGEMENTS |
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We are deeply indebted to Cecile Pickart and Art Haas for valuable discussions, advice, and the provision of enzyme preparations.
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FOOTNOTES |
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* This work was supported by National Science Foundation RUI Award MCB-9505810 and by a Henry Dreyfus Teacher-Scholar Award.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.
To whom correspondence should be addressed. Tel.: 207-786-6293;
Fax: 207-786-8336; E-mail: tlawson{at}bates.edu.
2 L. Mastrandrea and C. Pickart, personal communication.
3 T. G. Lawson, D. L. Gronros, P. E. Evans, M. C. Bastien, K. M. Michalewich, J. K. Clark, J. H. Edmonds, K. H. Graber, J. A. Werner, B. A. Lurvey, and J. M. Cate, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: Ub, ubiquitin; DTT, dithiothreitol; EMCV, encephalomyocarditis virus; HAV, hepatitis A virus; HIC, hydrophobic interaction chromatography; MeUb, methylated ubiquitin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase.
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