From the School of Biomedical Science, University of St. Andrews, St. Andrews, Fife KY169ST Scotland
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ABSTRACT |
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The ubiquitin-like protein SUMO-1 is conjugated
to a variety of proteins including Ran GTPase-activating protein 1 (RanGAP1), I Covalent linkage of one protein to another represents an important
means of generating protein conjugates with unique properties. Although
the best characterized example of such a modification is the addition
of ubiquitin to proteins destined for proteolysis, it is now recognized
that a number of other small protein molecules can be linked to target
proteins in a similar fashion. Ubiquitin addition is accomplished via a
thioester cascade with ubiquitin first being activated by a unique
E11 enzyme that utilizes ATP
to adenylate the C-terminal glycine of ubiquitin. Release of AMP
accompanies the formation of a thioester bond between the C terminus of
ubiquitin and a cysteine residue in the E1 protein. In a
transesterification reaction, the ubiquitin is transferred from the
ubiquitin-activating enzyme to an E2 ubiquitin-conjugating enzyme,
which may in turn transfer the ubiquitin to an E3 ubiquitin protein
ligase. In many cases it is this enzyme that recognizes the protein
substrate and catalyzes formation of an isopeptide bond between the C
terminus of ubiquitin and the To fully characterize the SUMO-1 modification reaction, we have
purified the SUMO-1-activating enzyme and shown that it contains subunits of 38 and 72 kDa. cDNAs corresponding to these subunits (SUMO-1-activating enzyme (SAE)1 and SAE2) are homologous to enzymes involved in the activation of ubiquitin, Smt3p, and Rub1p. In the
presence of recombinant SAE1/SAE2, Ubch9, and ATP, SUMO-1 was
efficiently conjugated to the protein substrate I Antibodies--
The SV5 Pk tag 336 monoclonal antibody (25) was
obtained from R. E. Randall, University of St. Andrews and was
used to immunodetect and immunoprecipitate SAE1 containing an
N-terminal SV5 epitope tag.
SUMO-1 Affinity Chromatography--
Five mg of
SUMOGG-1 (1-97 amino acids) purified as described (15)
were coupled to a 1-ml N-hydroxysuccinimide-activated Hi-Trap column (Amersham Pharmacia Biotech) according to the
manufacturer's instructions. 40% of the added protein was coupled to
the beads. After the affinity chromatography procedure, the column was
regenerated by washing with 10 column volumes of a buffer containing 50 mM Tris/HCl, pH 9.0, 1 M KCl, 2 mM
dithiotheitrol, followed by 10 column volumes of 50 mM
Tris/HCl, pH 7.2, buffer containing 0.02% NaN3. The
affinity column was stored at 4 °C.
HeLa cell extracts were prepared and fractionated on a Q-Sepharose
column into Fr I and Fr II.1 to Fr II.5 as described previously (26).
Fr II.4 and Fr II.5 (eluted with 0.4 M and 0.5 M of KCl) containing SUMO-1-activating enzyme activity (15)
were dialyzed overnight against 50 mM Tris, pH 6.8, 0.2 mM DTT, and affinity chromatography was carried out
essentially as described for ubiquitin (27). After dialysis, fractions
were adjusted to 50 mM Tris, pH 6.8, 2 mM ATP,
5 mM MgCl2, 0.2 mM DTT, 2.5 units/ml yeast inorganic pyrophosphatase, 10 mM creatine
phosphate, 2 units/ml creatine kinase and applied at a flow rate of
approximately 0.5 ml/min to the SUMOGG-1-Sepharose column
pre-equilibrated with 3 column volumes of a buffer containing 50 mM Tris/HCl, pH 6.8, 2 mM ATP, 5 mM
MgCl2, 0.2 mM DTT (Buffer A). The column was
washed with 3 column volumes of Buffer A followed by 5 column volumes
of 50 mM Tris/HCl, pH 7.5, 1 M KCl, and then 50 mM Tris/HCl, pH 7.5. SUMO-1 E1 activity was eluted with 3 column volumes of 50 mM Tris/HCl, pH 7.5, 0.2 mM DTT, 2 mM AMP, 2 mM sodium
pyrophosphate (AMP eluate) and finally with 3 column volumes of 50 mM Tris/HCl, pH 9.0, containing 10 mM DTT and 1 M NaCl. Column eluates were 10-fold-concentrated using
Centricon 30 microconcentrators that had been pretreated with bovine
serum albumin to reduce nonspecific adsorption. The buffers were
changed by 3 successive 10-fold dilutions in 50 mM Tris/HCl, pH 7.5, 1 mM DTT followed by 10-fold
reconcentration using the same microconcentrator. The final volume of
the column eluates was brought to 10% of the starting volume of
loading fraction. Column operations were carried out at room
temperature, but all fractions were collected on ice. The enzymatic
activity present in each fraction was determined using a thioester
assay as described below.
N-terminal Peptide Sequencing--
The concentrated AMP eluate
was fractionated by SDS-PAGE using freshly prepared
acrylamide:piperazine diacrylamide solution (30:0.8) and with the
addition of sodium thioglycolate to 0.1 mM in the upper
electrophoresis buffer. Proteins were electrophoretically transferred
to polyvinylidene difluoride membrane (Amersham Pharmacia Biotech), and
proteins were stained with 0.1% Amido Black in 40% methanol, 1%
acetic acid, for 30 s before bands were excised. Excised membrane
bands were extensively washed in distilled water, and the peptide
sequence was determined using a Procise microsequencer (Applied
Biosystems) with on-line phenylthiohydantoin analysis.
"In-gel" Trypsin Digestion--
Samples to be digested in
the gel were fractionated by SDS-PAGE as described for N-terminal
peptide sequencing. The gel was stained in 0.1% Coomassie Brilliant
Blue R-250, 20% methanol, 0.5% acetic acid and then destained in 30%
methanol until the bands were visible above a clear background. After
excision, gel slices were washed for at least 1 h in 100 mM NH4HCO3, pH 8.0, and 30 min at
60 °C in 100 mM NH4HCO3, pH 8, 3 mM DTT. Iodoacetamide was added to a final concentration of
6 mM, and after 30 min of incubation at room temperature in
the dark, gel slices were washed with 50% acetonitrile,100
mM NH4HCO3, pH 8.0, for 1 h
with shaking. Acetronitrile was added to shrink gel pieces, and after
10-15 min of incubation, the solvent was removed, and the samples were dried in a rotatory evaporator. Gel slices were reswollen with 25 mM NH4HCO3, pH 8.0, containing
modified trypsin (Promega) and incubated for 4 h at 37 °C. The
supernatant was acidified by adding trifluoroacetic acid to a final
concentration of 1%, and peptides were extracted from gel slices twice
with 60% acetonitrile,0.1% trifluoroacetic acid for 20 min. All
supernatants were combined, and after evaporation to near dryness,
peptides fragments were reconstituted in 20 µl of 0.1%
trifluoroacetic acid and separated by HPLC using a microbore HPLC
system. The amino acid sequence of selected peptides was determined as
described for N-terminal peptide sequencing.
cDNA Cloning--
Three and 8 peptides were sequenced from
the 38-kDa and 72-kDa species, respectively. Each peptide sequence was
searched for mammalian homologues using BLAST similarity search program
and EST public data base. ATCC clones were obtained for some of the peptides (AA236737 for 38 kDa and AA 373795 for 72 KDa). Tentative of
Human Consensus (THC) 183945 for the 38-kDa and THC 167372 for the
72-kDa protein were obtained when the sequences were used for search in
the data base of THC. Analysis of DNA sequencing data allowed us to set
up upstream and downstream primers flanking the coding sequence.
A cDNA encoding the complete open reading frame of SAE1 was
obtained as a single fragment by reverse transcription followed by PCR
(RT-PCR) using Boehringer TitanTM one-tube RT-PCR system. The following
primer containing an EcoRI restriction site was used
as the downstream primer for reverse transcription
(5'-GCGGGAATTCTCACTTGGGGCCAAGGCACTCCACAA-3'). The upstream primer used
for PCR amplification (5'-AACGGATCCATGGTGGAGAAGGAGGAGGCTGGC-3') contains a BamHI site at the 5' end. PCR products were
cloned as a BamHI/EcoRI insert in pGEX-2T
(Amersham Pharmacia Biotech) or in pcDNA3 (Invitrogen) with an
N-terminal Pk-SV5 tag that is recognized by the 336 monoclonal antibody
(25). The DNA sequence encoding the Pk-SV5 peptide (IPNPLLGLE) was
inserted into pcDNA3 using KpnI and BamHI
cloning sites and the following oligonucleotides: 5'-CATGGGAAAGCCGATCCCAAACCCTTTGCTGGGATTGGACTCCACCG-3' and
5'-GATCCGGTGGAGTCCAATCCCAGCAAAGGGTTTGGGATCGGCTTTCCCATGGTAC-3'.
The cDNA of SAE2 was obtained from two different RT-PCR reactions
and a final PCR. An intermediate upstream and downstream primer
(5'-GATATCAAATCAATGGCAGGGAAC-3' and 5'-GTTCCCTGCCATTGATTTGATATC-3') were used in each RT-PCR reaction to generate two different fragments that were later used in the final PCR reaction as template with the
following primers: 5'-GAGGAATTCATGGCACTGTCGCGGGGGCTG-3' and GAGGAATTCTCAATCTAATGCTATGACATC-3', both containing an
EcoRI restriction site. The final PCR fragment was inserted
into the EcoRI restriction site of pcDNA3. The DNA
sequence of all plasmids was determined by Alex Houston of the
University of St. Andrews DNA sequencing facility (ABI377).
In Vitro Transcription Translation--
In vitro
transcription/translation was performed using 1-2 µg of plasmid DNAs
and a TNT-coupled wheat germ extract system (Promega) according to the
instructions provided by the manufacturer. [35S]Methionine (Amersham Pharmacia Biotech) was used in
the reactions to generate radiolabeled proteins.
Expression and Purification of Recombinant
Proteins--
I Cell Culture and Transfections--
COS7 cells were maintained
in exponential growth in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum. 1-2 µg of plasmid DNAs were
transfected for 14 h in subconfluent 75-cm2 flasks using
LipofectAMINETM according to instructions provided by the manufacturer
(Life Technologies, Inc.). After 36 h of expression, cells were
washed in phosphate-buffered saline, and extracts were prepared by
lysis in 20 mM sodium phosphate buffer, pH 7.5, 50 mM NaF, 2 mM EDTA, 0.5% Nonidet P-40, 5 mM tetrasodium pyrophosphate, 1 mM sodium
orthovanadate, 10 mM Immunoprecipitations--
Extracts from COS 7-transfected cells
or in vitro transcribed/translated SAE proteins were
incubated for 1 h at 4 °C with 336 anti-SV5 antibody either in
lysis buffer or 50 mM Tris, pH 7.5, 10 mM
MgCl2 buffer containing 1 mM ATP and complete®
protease inhibitor mixture, respectively. Antigen-antibody complexes
were collected by adding 20 µl of protein A-Sepharose beads, and
incubation was continued for an additional 2 h on a rotating
shaker. The beads were collected by brief centrifugation, and after
extensive washing with ice-cold 50 mM Tris/HCl, pH 7.5, 10 mM MgCl2 containing complete® protease
inhibitor mixture, antigen-antibody complexes were used as the source
of SUMO-1-activating enzyme in thioester or conjugation assays.
Protein Interaction Assays--
GST or GST-SAE1 beads were
incubated with [35S]methionine-labeled in
vitro transcribed/translated SAE2 for 1 h at room
temperature. Beads were collected by centrifugation and washed 4 times
with 50 mM Tris/HCl, pH 7.5, 10 mM
MgCl2, 1 mg/ml bovine serum albumin containing complete®
protease inhibitor mixture and 0.5% Nonidet P-40. Washing buffer was
removed, and beads, after resuspension in 3× disruption buffer (5%
SDS, 0.15 M Tris/HCl, pH 6.7, 30% glycerol, 0.72 M Thioester Assay--
Recombinant SUMOGG-1 was
acetylated and radiolabeled with carrier-free Na125I
(Amersham Pharmacia Biotech) by the chloramine T method as described (15). To detect the activity of SUMO-1-activating enzyme, formation of
thioester adducts between either immunoprecipitated or
affinity-purified SAE and SUMOGG-1 was determined
essentially as described (29). Immunoprecipitated protein A beads or
200 ng of affinity-purified SAE were incubated with 1 unit of inorganic
pyrophosphatase (Sigma), 0.5 µg of
125I-SUMOGG-1 in the presence or absence of 0.6 µg of recombinant Ubch9 in a final volume of 20 µl of 50 mM Tris/HCl, pH 7.5, 10 mM MgCl2, 1 mM ATP. Reactions were incubated at 30 °C for 10 min and
terminated either by boiling for 5 min in the presence of 2% (w/v)
SDS, 4% (v/v) 2-mercaptoethanol, and 50 mM DTT (+DTT) or
by incubating the samples at 30 °C for 20 min in the same buffer containing 4 M urea instead of DTT and mercaptoethanol.
Samples were subjected to SDS-PAGE (10%), and dried gels were analyzed by phosphorimaging (Fujix BAS1500, MacBAS software).
In Vitro SUMO-1 Conjugation Assay--
SUMO-1 conjugation using
recombinant proteins was accomplished in a 20-µl reaction containing
50 mM Tris, pH 7.6, 5 mM MgCl2, 2 mM ATP, 10 mM creatine phosphate, 3.5 units/ml
creatine kinase, and 0.6 units/ml inorganic pyrophosphatase and 0.6 µg of human recombinant I Purification of SUMO-1-activating Enzyme--
The purification
procedure for the SAE used an affinity procedure previously described
(27, 30) for isolation of ubiquitin-activating enzymes based on
covalent binding of the enzyme to ubiquitin-Sepharose beads in the
presence of ATP. FrII.4 containing SUMO-1-activating activity from HeLa
cells (15) was thus applied to SUMO-1-Sepharose beads in the presence
of ATP and inorganic pyrophosphatase to suppress the reverse reaction.
Under these conditions, SAE activates the immobilized SUMO-1, forming a
thioester bond that retains the SAE on the column. The affinity column
was washed sequentially with buffers containing ATP, 1 M
KCl, Tris buffer alone, and AMP + pyrophosphate. Column eluates were
assayed for their ability to form a thioester with
125I-SUMO-1 (Fig. 1). SAE
activity was detected in the column load and was substantially reduced
in the column flow-through, indicating that the activity was bound to
the column (Fig. 1A). The bulk of the SAE activity was
eluted with AMP and pyrophosphate, which reverses the activation
reaction (Fig. 1A). Eluates were fractionated by SDS-PAGE,
and proteins were detected by staining with Coomassie Brilliant Blue.
Two polypeptide species with apparent molecular weights of 40,000 and
90,000 were detected in the AMP eluate in addition to bovine serum
albumin, which was added to the fractions to prevent nonspecific
adsorption to surfaces during concentration (Fig. 1B).
cDNA Cloning of Human SUMO-1-activating Enzyme--
Proteins
present in the AMP eluate were fractionated by SDS-PAGE and transferred
to polyvinylidene difluoride membrane, and the location of bound
species was detected by Amido Black staining. Both the 90-kDa and
40-kDa species were excised and subjected to direct N-terminal
sequencing. Although the N terminus of the 90-kDa species appeared to
be blocked, a unique sequence was obtained from the N terminus of the
40-kDa species. To obtain sequences from the 90-kDa species and
additional sequences from the 40-kDa species, peptides generated by
in-gel trypsin digestion were fractionated by microbore HPLC.
Unambiguous sequence was obtained from 3 40-kDa- and 8 90-kDa-derived
peptides (Fig. 2A,
underlined sequences). BLAST searching of the EST public
data base allowed the identification of ATCC clones, which were used in
combination with RT-PCR to construct full-length cDNAs for each
species. The 40-kDa species was designated as SAE1 and has a predicted
molecular mass of 38 kDa, whereas the 90-kDa species was designated as
SAE2 and has a predicted molecular mass of 72 kDa (Fig.
2A).
Amino acid sequence alignments of different E1s demonstrate that SAE is
similar to Smt3p (Aos1p/Uba2p) and Rub1p (Ula1p/Uba3p)-activating enzymes (16, 22). Although SAE1 displays significant similarities to
the N terminus of human and S. cerevisiae Uba1, the SAE2
protein contains the putative active-Cys (Cys-173) and is homologous to the C terminus of Uba1. The apparent molecular mass of the
125I-SUMO-SAE thioester conjugate of 100 kDa (Fig.
2A) confirms the presence of the active-site cysteine in
SAE2. Most of the sequence similarity (Fig. 2B) is
concentrated in a limited number of previously identified domains (16,
22).
SUMO-1 Is Activated by an E1-like Enzyme Containing Two
Subunits--
cDNA corresponding to SAE1 and SAE2 obtained by
RT-PCR was cloned into a eukaryotic expression vector permitting
proteins to be produced and 35S-labeled by coupled in
vitro transcription and translation (Fig. 3A). SAE1 was fused at the N
terminus to an epitope from simian virus 5 that is recognized by the
previously characterized SV5 Pk Tag (336) monoclonal antibody (25).
Although SAE1 and SAE2 copurified in equimolar amounts (Fig.
1B) during SUMO-1 affinity chromatography, it was not clear
that they were associated. To obtain direct evidence for such an
interaction, different mixtures of 35S-labeled in
vitro transcribed-translated proteins were immunoprecipitated with
a specific (336) and a nonspecific (214) monoclonal antibody. In the
presence of SV5-tagged SAE1, SAE2 was co-immunoprecipitated with
anti-SV5 antibody (336). Labeled SAE2 protein was not
immunoprecipitated with either the control antibody (214) or when SAE2
was in vitro transcribed-translated in the absence of SAE1
(Fig. 3C). Confirmation of the interaction between SAE1 and
SAE2 was obtained when glutathione S-transferase SAE1 beads
were used to pull down 35S-labeled in vitro
transcribed-translated SAE2. (Fig. 3B).
To demonstrate that SAE1 and SAE2 are required for SUMO-1 activation
in vitro, its ability to form a stable adduct with
radioactively labeled SUMO-1 in an ATP-dependent reaction
was tested. SAE proteins were either in vitro
transcribed-translated or expressed in transfected COS7 cells. In both
situations, the activity was immunoprecipitated using anti-SV5 protein
A beads. The ability of recombinant SAE to form a thioester with
125I-SUMO-1 in presence of MgATP was analyzed by SDS-PAGE
under nonreducing conditions. This immunoprecipitated activity was
capable of catalyzing formation of a Ubch9-125I-SUMO-1
thioester when recombinant Ubch9 was added to the reaction (Fig.
4, A and B). The
linkages formed between SAE2 and 125I-SUMO-1 or Ubch9 and
125I-SUMO-1 were labile to reducing agents such as DTT
(Fig. 4A), indicating that they are likely to be thioester
bonds. Neither SAE1 nor SAE2 alone were capable of forming a thioester
complex with SUMO-1. Some activity is detected when SAE1 is
immunoprecipitated from COS7 SAE1-transfected extracts, probably
because of endogenous SAE2 activity (Fig. 4B). These results
together suggest that the SUMO-1-activating enzyme is composed of
SAE1/SAE2.
Role of SAE in Conjugation of SUMO-1 to I Targeting of proteins for ubiquitin-mediated proteolysis is an
irrevocable decision, and as such, the process needs to be highly
specific and tightly regulated. This specificity appears to be
accomplished by a combination of E2 ubiquitin-conjugating enzymes and
E3 ubiquitin protein ligases. In many cases, the E3 appears to consist
of a multiprotein complex that recognizes the substrate and brings it
in to intimate contact with the E2, which catalyzes the addition of
ubiquitin to the substrate. Because the E1, ubiquitin-activating enzyme
is unique, it does not appear to play a role in selecting protein
substrates for ubiquitination. However, ubiquitin co-exists with a
number of ubiquitin-like molecules, and the E1 enzymes must distinguish
between these molecules. Because distinct E1 activities have been
described for ubiquitin (31), Smt3p (16), and Rub1p (21, 22), we
undertook the isolation and characterization of the SAE.
By taking advantage of the mechanism of ubiquitin-activating and
-conjugating enzymes, which involves formation of a thioester intermediate with ubiquitin, we have used SUMO-1 affinity
chromatography to isolate a novel enzyme that catalyzes the
ATP-dependent activation of SUMO-1, the first step in the
conjugation pathway. Furthermore this enzyme could also transfer
activated SUMO-1 to Ubch9, the conjugating enzyme involved in this
process (11, 15, 18, 19) (Fig. 4). Although the E1 activity for
ubiquitin is contained within a single large polypeptide, the E1
activity of SUMO-1, like that of Smt3p and Rub1p, is partitioned
between two smaller polypeptides, SAE1 and SAE2. Sequence comparisons
between the E1 enzymes indicates that SAE1 is homologous to Aos1p,
Ula1p, and the N terminus of the ubiquitin-activating enzymes (Fig. 2), whereas SAE2 is homologous to Uba2p, Uba3p, and the C terminus of the
ubiquitin-activating enzymes. The association between SAE1 and SAE2,
confirmed by immunoprecipitations and GST pull-downs (Fig. 3), brings
together conserved domains present in each subunit. Because purified
SAE contains equimolar amounts of SAE1 and SAE2 and the two proteins
associate in vitro, it is probable that, like the Smt3p E1,
the activating enzyme is a heterodimer. Each SAE subunit contains a
conserved nucleotide binding motif, GXGXXG (positions 24-29 in SAE2 and positions 43-48 in SAE1, domain
I, Fig. 2B). The putative cysteine (Cys-173),
which forms a thioester bond with the C-terminal glycine of SUMO-1, is
in an active-site consensus sequence
(KXXPZCTXXXXP) found in conserved domain III. Conserved domain II is present in SAE1, whereas conserved domain IV is
found in SAE2. The function of conserved domains II and IV has yet to
be determined. The C-terminal extension of SAE2 contains a region that
matches with two consensus sequences for nuclear localization signals
(RKRK, 610-613, and RKRKLDEKENLSAKRSR, 610-626), which are also
present in the C-terminal region of Uba2p (32).
To further investigate the enzymatic properties of the
SUMO-1-activating enzyme, recombinant SAE protein was tested for the ability to activate SUMO-1 in a purified in vitro SUMO-1
conjugation system together with an ATP regenerating system, Ubch9, and
a recombinant I Within the cell it appears that virtually all of the SUMO-1 is present
in protein conjugates, and there is a very low concentration of free
SUMO-1(7). Thus it is likely that the availability of free SUMO-1 is
tightly controlled by a dynamic equilibrium between SAE/Ubc9-mediated
conjugation of SUMO-1 and deconjugation mediated by the highly active
but as yet uncharacterized SUMO-1-deconjugating and processing enzymes.
However the cellular signals that regulate this process have yet to be defined.
The large number of ubiquitin-specific proteases and ubiquitin
C-terminal hydrolases (UCH) already identified (33) suggest that they
may be involved in the recognition of different types of ubiquitin
conjugates, but little is known about their biological roles. It is
likely that some of the known ubiquitin-specific proteases and
ubiquitin C-terminal hydrolases will be responsible for processing of
ubiquitin-like proteins. UCH-L3, a putative ubiquitin C-terminal
hydrolase, was recently identify as a NEDD8 (23)-interacting protein,
able to cleave the C terminus of NEDD8 but not bind to sentrin-1,
sentrin-2, or sentrin-3 (34). The availability of the genes for the
SUMO-1-activating and -conjugating enzymes, SAE1/SAE2 and Ubc9, will
facilitate further biochemical and cell biological studies aimed at
defining the role of these proteins in vivo.
B
, and PML. SUMO-1-modified proteins display altered
subcellular targeting and/or stability. We have purified the
SUMO-1-activating enzyme from human cells and shown that it contains
two subunits of 38 and 72 kDa. Isolation of cDNAs for each subunit
indicates that they are homologous to ubiquitin-activating enzymes and
to the Saccharomyces cerevisiae enzymes responsible for
conjugation of Smt3p and Rub-1p. In vitro, recombinant
SAE1/SAE2 (SUMO-1-activating enzyme) was capable of catalyzing the
ATP-dependent formation of a thioester linkage between
SUMO-1 and SAE2. The addition of the SUMO-1-conjugating enzyme Ubch9
resulted in efficient transfer of the thioester-linked SUMO-1 from SAE2
to Ubch9. In the presence of SAE1/SAE2, Ubch9, and ATP, SUMO-1 was
efficiently conjugated to the protein substrate I
B
. As SAE1/SAE2,
Ubch9, SUMO-1, and I
B
are all homogeneous, recombinant proteins,
it appears that SUMO-1 conjugation of I
B
in vitro
does not require the equivalent of an E3 ubiquitin protein ligase activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino group of lysine in the target
protein. Proteins destined for degradation via the proteasome are
coupled to multiple copies of ubiquitin by formation of further
isopeptide bonds between additional ubiquitin molecules and lysine
residues in the bound ubiquitin (1). In some instances protein
ubiquitination functions not as a signal for degradation but to alter
the properties of the linked protein. Thus histone ubiquitination
alters chromatin structure (2), whereas ubiquitination of a plasma
membrane receptor modifies ligand-stimulated endocytosis (3). Whereas
addition of multiple copies of ubiquitin targets proteins for
degradation, it is now widely recognized that covalent attachment of
other ubiquitin-related molecules does not result in degradation of the
modified protein. The protein UCRP, which contains two ubiquitin-like
domains, is conjugated to a number of intracellular proteins by a
series of reactions that are separate from ubiquitination (4, 5). Recently a small ubiquitin-like protein variously known as sentrin, GMP1, SUMO-1, UBL1, and PIC1 has been found covalently linked to Ran
GTPase-activating protein 1 (RanGAP1) and associated with a variety of
other proteins (6-10). Covalent modification of RanGAP1 appears to be
necessary for its interaction with the Ran-GTP-binding protein RanBP2
at the cytoplasmic face of the nuclear pore complex (8, 11), whereas
SUMO-1 modification of PML targets the protein to PML nuclear bodies
(12, 13). SUMO-1 modification of I
B
takes place on the same
residues used for ubiquitination, thus rendering the protein resistant
to signal-induced degradation and consequently blocking
NF-
B-dependent transcriptional activation (14). SUMO-1
and Smt3p, a yeast homologue of SUMO-1, are conjugated to target
proteins by a pathway that is distinct from, but analogous to,
ubiquitin conjugation. A separate E1-like enzyme is responsible for
SUMO-1 modification (15), and in yeast, the enzyme responsible for
Smt3p activation has been shown to consist of a heterodimer of Uba2p
and Aos1p (16). Also found in the complex between SUMO-1-modified RanGAP1 and RanBP2 is a protein designated Ubc9, which is homologous to
the E2 class of ubiquitin-conjugating enzymes (11). In yeast, Ubc9 is
essential for cell cycle progression (17), and mammalian homologues
have been isolated repeatedly from yeast two-hybrid screens in
association with a wide variety of proteins. In both yeast and human
cells, the product of the Ubc9 gene acts as the SUMO-1/Smt3p
E2-conjugating activity (11, 15, 18, 19). Recently an additional
protein modification pathway involving the ubiquitin-like protein Rub1p
has been characterized in Saccharomyces cerevisiae (21, 22).
A major substrate for Rub1p is CDC53/cullin, which is a common
component of SCF (Skp1, CDC53, F-box) ubiquitin ligase complexes. In this system the products of the ULA1 and UBA3
genes act as a heterodimeric E1 enzyme, whereas the product of the
UBC12 gene acts as the E2-conjugating enzyme. It also appears that this
conjugation system is active in higher eukaryotes with the Rub1p
homologue Nedd8 also being conjugated to CDC53 (23). In all of the
above cases, the protein conjugated has been similar to ubiquitin.
However, a separate protein conjugation system, which is required for
autophagy in S. cerevisiae, involves conjugation of a
protein unrelated to ubiquitin. In this case, the C terminus of Apg12,
a 186-residue protein, is conjugated to a lysine side chain in
Apg5. Although Apg12 is unrelated to ubiquitin, it appears that it is
activated by Apg7, which is homologous to ubiquitin E1 enzymes,
and conjugated by Apg10, an E2 equivalent (24).
B
. Thus it
appears that SUMO-1 conjugation in vitro does not require
the equivalent of an E3 protein ligase activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
, Ubch9, and SUMOGG-1 were expressed
and purified as described previously (15, 28). GST-SAE1 was expressed
in Escherichia coli B834 and purified as described for
glutathione S-transferase (GST)-I
B
(28).
-glycerophosphate containing complete® protease inhibitor mixture (Boehringer Mannheim). The lysates were cleared by centrifugation, and the supernatants were used
for immunoprecipitations. 10 µl of each lysate was fractionated on a
10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane
(Sigma). Protein expression was checked by Western blotting using the
SV5 Pk tag monoclonal antibody with an ECL detection system and
horseradish peroxidase-conjugated anti-mouse IgG (Amersham Pharmacia
Biotech) as secondary antibody.
-mercaptoethanol) were fractionated in a 10% polyacrylamide gel. Dried gels were exposed to a phosphorimaging screen
to detect 35S radioactivity. In vitro
transcribed/translated SAE proteins were incubated for 1 h at
4 °C with 336 anti-SV5 in 50 mM Tris, pH 7.5, 10 mM MgCl2 containing 1 mM ATP and
complete® protease inhibitor mixture. Antigen-antibody complexes were
collected on protein A-Sepharose beads and analyzed as described for
GST pull-downs.
B
(or mutants), 0.5 µg of
125I-SUMOGG-1, 0.6 µg of Ubch9, and either
recombinant SAE-immunoprecipitated protein A beads (rSAE) or 90 ng of
affinity-purified SAE. Reactions were incubated at 37 °C for 2 h and terminated by boiling in 3× disruption buffer. Samples were
subjected to SDS-PAGE (10%), and dried gels were analyzed by phosphorimaging.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification of SUMO-1-activating enzyme by
SUMO-1 covalent affinity chromatography. HeLa cell extracts were
initially fractionated on a Q-Sepharose column. FrII.4, containing SAE
activity, was supplemented with ATP and inorganic pyrophosphatase and
fractionated on a SUMOGG-1 Hi-trap column (see
"Experimental Procedures"). A, equivalent amounts of
SUMO-1 column load (Fr II.4, lanes 1 and
2), flow-through (F/T, lanes 3 and
4), and AMP eluate (lanes 5 and 6)
were assayed for the ability to form thioester adducts with
125I-SUMO-1 either alone ( ) or in the presence of
recombinant Ubch9. After 10 min at 30 °C, reactions were stopped,
and products were subjected to SDS-PAGE (12.5%) under nonreducing
conditions. Dried gels were analyzed by phosphorimaging. The positions
of 125I-SUMO-1 and thioester adducts with SAE and Ubch9 are
indicated. B, fractions indicated in A were
fractionated by SDS-PAGE (10%) and stained with Coomassie Blue. Before
concentration, the AMP eluate was supplemented with bovine serum
albumin, which was also loaded on lane 6 as a control. The
molecular weight of protein markers (M) and SAE components
are indicated.
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Fig. 2.
SAE1/SAE2 sequence analysis.
A, protein sequences of SAE1 and SAE2. The putative
active-site cysteine residue (Cys-173) in SAE2 is shown in
bold. Peptides sequences obtained either by direct Edman
degradation N-terminal sequencing or after in-gel trypsin digestion are
shown underlined. B, schematic representation of
homologous domains in human ubiquitin E1 enzyme, hUba1 (31); S. cerevisiae (S.c.) ubiquitin E1, Uba1p (20); S. cerevisiae Smt3p-activating enzymes, Aos1p (16) and Uba2p (32);
RUB1-activating enzymes, Ula1p and Uba3p (22); and SUMO-1-activating
enzymes, SAE1 and SAE2. The domains shown correspond to those of
Johnson et al. (16).
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Fig. 3.
Interaction of SAE1 with SAE2.
A, SV5-tagged SAE1 (lane 1) and SAE2 (lane
2) were either separately in vitro transcribed,
translated (IVTT), and labeled with [35S]methionine or
labeled cotranslationally (lane 3). B, GST or
GST-SAE1 beads were used to pull down
[35S]methionine-labeled IVTT SAE2. After extensive
washing, bound proteins were analyzed in a 10% SDS-PAGE, and
35S radioactivity in the dried gel was detected by
phosphorimaging. C, [35S]methionine-labeled
IVTT SV5-tagged SAE1, SAE2, or cotranslated SV5-tagged SAE1 and SAE2
were immunoprecipitated (I.P.) either with 336 anti-SV5
monoclonal antibody or with an unrelated antibody (214). After
extensive washing with 50 mM Tris/HCl, pH 7.5, 10 mM MgCl2, 0.5% Nonidet P-40
immmunoprecipitates were analyzed as described in panel B.
The IVTT protein present and antibody used for the immunoprecipitation
are shown for each reaction. [35S]Methionine-labeled
SAE1tag and SAE2 proteins are indicated.
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Fig. 4.
Both SAE1 and SAE2 are required for SUMO-1
activating activity. A, IVTT SV5-tagged SAE1 or
cotranslated SV5-tagged SAE1 and SAE2 were incubated with 336 anti-SV5tag monoclonal antibody and immunoprecipitated
(I.P.) with protein A beads. The ability of these complexes
to form a thioester bond with 125I-SUMO-1 was tested either
in the absence ( ) or presence of 0.6 µg of Ubch9. Samples treated
with 100 mM DTT before SDS-PAGE fractionation are indicated
(+DTT). B, COS7 cells were either transfected
with control pcDNA3, SV5-tagged SAE1, SAE2, or co-transfected with
SV5-tagged SAE1 and SAE2. After 36 h of expression, cell extracts
were prepared and immunoprecipitated with anti-336 SV5 monoclonal
antibody. Immunoprecipitates were assayed for the ability to form
thioester adducts with 125I-SUMO-1 either alone (
) or in
the presence of 0.6 µg of Ubch9. SUMO-1 column AMP fraction was
tested in the same thioester assay as a positive control.
A.Mix represents the assay mix containing labeled substrate
but lacking any additional protein components. Reactions products were
analyzed as described in the legend to Fig. 1A.
B
--
To
demonstrate that the cloned SAE is functional in an
Ubch9/ATP-dependent SUMO-1 conjugation reaction, its
ability to mediate SUMO-1 conjugation to recombinant I
B
was
investigated. Although SUMO-1 conjugation of I
B
is dependent on
Ubch9, ATP, and FrII.4 (14), a complete description of the biochemical
activities present in the HeLa cell fraction or in the in
vitro transcribed-translated substrate was lacking. The SAE was
affinity-purified from FrII.4, but by analogy with ubiquitin
modification, it is not clear if other activities such as an E3
equivalent would be required. In the presence of recombinant,
immunoprecipitated SAE, Ubch9, and ATP, 125I-SUMO-1 was
efficiently conjugated to recombinant I
B
(Fig. 5A). This reaction was
dependent on SAE, Ubch9, ATP, and I
B
substrate (Fig.
5B). The enzymatic properties of rSAE were identical to
those of the protein isolated from HeLa FrII.4. rSAE formed a thioester
adduct with 125I-SUMO-1 (Fig. 4) and produced similar
I
B
SUMO-1 conjugates under identical reactions conditions (Fig.
5). Because SUMO-1, Ubch9, and I
B
are all homogeneous recombinant
proteins expressed in bacteria and the SAE is either highly purified
(Fig. 5B) or recombinant anti-SV5-immunoprecipitated (Fig.
5A), it appears that SUMO-1 conjugation of I
B
in
vitro does not require an equivalent E3 activity and that after
SUMO-1 activation by SAE, the SUMO-1-conjugating enzyme Ubch9 is
capable of recognizing, then transferring, SUMO-1 onto I
B
. To
demonstrate specificity in vitro, two deleted forms of
I
B
and GST were used as substrates in reactions also containing purified SAE, Ubch9, 125I-SUMO-1, and ATP. SUMO-1 is
efficiently conjugated to I
B
and I
B
C (lacks the
C-terminal 61 amino acids) but is not conjugated to either
N
I
B
(lacks the N-terminal 70 amino acids) or GST (Fig.
5C). That the lysine involved in SUMO-1 modification is present in wild type I
B
and I
B
C but absent in
N
I
B
indicates that the purified conjugation system is displaying
the expected specificity.
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Fig. 5.
SUMO-1 modification of
I B
with purified
proteins. SAE1/SAE2 and Ubch9 are necessary and sufficient for
conjugation of SUMO-1 to I
B
. Recombinant I
B
was incubated
at 37 °C for 2 h with 125I-SUMO-1 in the presence
(+) or absence (
) of either recombinant immunoprecipitated SAE1/SAE2
(rSAE, panel A) or affinity-purified SAE (panel
B), ATP, and Ubch9. Reactions products were fractionated by
SDS-PAGE and analyzed by phosphorimaging. C, substrate
specificity in the purified SUMO-1 conjugation system. Purified
recombinant I
B
, an N-terminal-deleted I
B
(
N I
B
)
and a C-terminal-deleted I
B
(I
B
C) were assayed for
conjugation to 125I-SUMO-1 in a reaction containing ATP,
Ubch9, and SAE1/SAE2. Recombinant GST protein was analyzed as negative
control. Reactions were incubated at 37 °C for 2 h, and the
products were analyzed by SDS-PAGE and phosphorimaging as before.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
substrate. Under the conditions employed in this assay, SUMO-1 was efficiently conjugated to I
B
, indicating that conjugation does not require the presence of an E3-like protein ligase
activity. However, we cannot rule out the possibility that in
vivo, such proteins may increase the efficiency of the conjugation process. Because our initial yeast two-hybrid screen demonstrated a
protein-protein interaction between Ubch9 and I
B
(15), it is
likely that substrate specificity is achieved by Ubch9. A diverse range
of proteins have been shown to interact with Ubc9 in yeast two-hybrid experiments, and this may be a direct consequence of substrate recognition by Ubc9. Again it is not possible to rule out the participation of yeast proteins in these interactions.
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ACKNOWLEDGEMENTS |
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We would like to thank Ellis Jaffray for large scale growth of HeLa cells, Paul Talbot for protein sequencing, and Alex Houston for DNA sequencing.
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FOOTNOTES |
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* This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC), the Medical Research Council (MRC), and the European Union Concerted Action BIOMED II (ROCIO II project).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF110956 (SUMO-activating enzyme 1 (SAE1)) and AF110957 (SAE2).
Supported by JNICT-Praxis XXI (Portugal).
§ To whom correspondence should be addressed: School of Biomedical Science, BMS Bldg., University of St. Andrews, St. Andrews, Fife KY169ST Scotland. Tel.: 44-1334-463396; Fax: 44-1334-462595; E-mail: rth{at}st-and.ac.uk.
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ABBREVIATIONS |
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The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin protein ligase; SAE, SUMO-1-activating enzyme; rSAE, recombinant SAE; RanGAP1, Ran GTPase-activating protein 1; RanBP2, Ran-binding protein 2; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; RT-PCR, reverse transcription-polymerase chain reaction; GST, glutathione S-transferase; UCH, ubiquitin C-terminal hydrolase; IVTT, in vitro transcribed and translated.
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