The Small Ubiquitin-like Modifier-1 (SUMO-1) Consensus
Sequence Mediates Ubc9 Binding and Is Essential for SUMO-1
Modification*
Deborah A.
Sampson,
Min
Wang, and
Michael J.
Matunis
From the Johns Hopkins University, School of Hygiene and Public
Health, Department of Biochemistry and Molecular Biology,
Baltimore, Maryland 21205
Received for publication, January 2, 2001, and in revised form, March 12, 2001
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ABSTRACT |
SUMO-1 is an ubiquitin-related protein that is
covalently conjugated to a diverse assortment of proteins. The
consequences of SUMO-1 modification include the regulation of
protein-protein interactions, protein-DNA interactions, and protein
subcellular localization. At present, very little is understood about
the specific mechanisms that govern the recognition of proteins as substrates for SUMO-1 modification. However, many of the proteins that
are modified by SUMO-1 interact directly with the SUMO-1 conjugating
enzyme, Ubc9. These interactions suggest that Ubc9 binding may play an
important role in substrate recognition as well as in substrate
modification. The SUMO-1 consensus sequence (SUMO-1-CS) is a motif of
conserved residues surrounding the modified lysine residue of most
SUMO-1 substrates. This motif conforms to the sequence
"
KXE," where
is a large hydrophobic residue, K
is the lysine to which SUMO-1 is conjugated, X is any amino acid, and E is glutamic acid. In this study, we demonstrate that the
SUMO-1-CS is a major determinant of Ubc9 binding and SUMO-1 modification. Mutating residues in the SUMO-1-CS abolishes both Ubc9
binding and substrate modification. These findings have important implications for how SUMO-1 substrates are recognized and for how
SUMO-1 is ultimately transferred to specific lysine residues on these substrates.
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INTRODUCTION |
SUMO-11 is a member of a
family of ubiquitin-like proteins that are post-translationally
conjugated to other proteins (1). The specific effects of SUMO-1
modification appear to be substrate dependent, but they are clearly
distinct from the effects of ubiquitination in mediating protein
degradation. In a number of cases, SUMO-1 modification regulates the
subcellular localization of specific substrates. For example, SUMO-1
modification targets RanGAP1 from the cytoplasm to the nuclear pore
complex (2, 3) and PML from the nucleoplasm to PML nuclear bodies (4).
SUMO-1 modification of certain other substrates may play a role in
antagonizing ubiquitin-mediated proteolysis. I
B
and MDM2, for
example, are both modified by SUMO-1 on lysine residues that also
function as sites for ubiquitination (5, 6). SUMO-1 modification of
these lysines has been proposed to stabilize the substrates by blocking
ubiquitin modification. For a growing list of other substrates, the
exact effects of SUMO-1 modification remain to be determined. A
majority of these substrates, including p53 (7-9), c-Jun (7), and
topoisomerases I and II (10, 11) are nuclear proteins that function in
regulating transcription or chromatin structure.
Immunofluorescence analysis and cell fractionation studies further
indicate that the majority of proteins modified by SUMO-1 are nuclear
and that they correspond to only a small subfraction of all cellular
proteins (12). The specific subfraction of proteins modified by SUMO-1
also varies throughout the cell cycle (13), and possibly in response to
cellular growth conditions, indicating that SUMO-1 modification and
de-modification are dynamic processes. However, the precise mechanisms
involved in substrate selection and in regulating the timing of
modification or demodification are poorly defined.
Many steps involved in SUMO-1 modification parallel those involved in
ubiquitination. Like ubiquitin, SUMO-1 is synthesized as a precursor
that is proteolytically processed to generate the mature, active
polypeptide (13, 14). Once processed, SUMO-1 is activated in an
ATP-dependent reaction that creates a thioester intermediate between the active-site cysteine of the SUMO-1 activating enzyme (E1) and the carboxyl terminus of SUMO-1. The SUMO-1 E1 enzyme,
a heterodimer consisting of Aos1 and Uba2, is structurally and
functionally related to the ubiquitin E1 enzyme (14-16). Following activation, SUMO-1 is transferred from the E1 enzyme to Ubc9, a protein
similar in structure and function to ubiquitin E2 enzymes (17-21). How
SUMO-1 is subsequently transferred from Ubc9 to specific protein
substrates is the most poorly defined step in the SUMO-1 conjugation
pathway. Although SUMO-1 modification must be quite specific by virtue
of the limited number of cellular proteins that are modified, there is
very little, if any, homology among the currently known substrates.
Probably all ubiquitination reactions involve E3 ligases, factors that
mediate the transfer of ubiquitin from E2 enzymes to specific protein
substrates (22, 23). In general, E3s function in substrate recognition
and are responsible for the high degree of specificity that is
characteristic of most ubiquitination reactions. Currently no E3-like
factors have been identified for SUMO-1 conjugation, so how specific
proteins are recognized as substrates for SUMO-1 conjugation remains unknown.
Relative to ubiquitin-specific E2 enzymes, an unusual feature of
Ubc9 is that it interacts directly with many SUMO-1 substrates (1).
These interactions suggest that Ubc9 may play a direct role in
recognizing SUMO-1 substrates, as well as in modifying them. In this
study, we demonstrate that Ubc9 binds to the SUMO-1 consensus sequence
(SUMO-1-CS), a motif of conserved residues surrounding the modified
lysine of many SUMO-1 substrates. We further demonstrate that the
binding of Ubc9 to the SUMO-1-CS is essential for SUMO-1 modification.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructions--
Expression vectors coding for
wild-type mouse RanGAP1, C
23, N
419/PK, and N
502/PK were
constructed as previously described (3). Site-directed mutagenesis was
performed using the GeneEditor Mutatgenesis System (Promega Corp.,
Madison, WI) and mutations were verified by DNA sequencing. A cDNA
coding for human Ubc9 was obtained from a fetal liver cDNA library
using the polymerase chain reaction and cloned into the GST
expression vector, pGEX-5X-1 (Amersham Pharmacia Biotech, Piscataway,
NJ). The carboxyl-terminal domain of mouse RanGAP1 (N
419: amino
acids 420-589) was similarly amplified from a cDNA clone by
polymerase chain reaction and subcloned into pGEX-5X-1.
Protein Expression, Fractionation, and SUMO-1
Modification--
In vitro transcription and translation of
RanGAP1 and RanGAP1-pyruvate kinase fusion proteins were performed in
rabbit reticulocyte lysate in the presence of
[35S]methionine as described by the manufacturer (Promega
Corp., Madison, WI). GST-N
419 and GST-Ubc9 fusion proteins were
expressed in bacteria, purified by affinity chromatography on
glutathione-Sepharose beads, and cleaved from the beads by Factor Xa as
outlined by the manufacturer (Amersham Pharmacia Biotech).
Gel filtration analysis of the carboxyl-terminal domain of RanGAP1 and
Ubc9 was performed on a Amersham Pharmacia Biotech Superdex 75 chromatography column. The column was equilibrated and proteins were
fractionated with buffer containing 110 mM potassium acetate, 2 mM magnesium acetate, 20 mM HEPES
(pH 7.3), and 1 mM dithiothreitol. 20 µg of each protein
was loaded either individually or together following mixing and
incubation for 30 min at room temperature. 0.5-ml fractions were
collected, trichloroacetic acid precipitated, and analyzed by
SDS-PAGE.
In Vitro Protein Binding Assays--
GST-Ubc9, or GST alone, was
bound to 20 µl of glutathione-Sepharose beads (1 mg of protein/ml
beads) in phosphate-buffered saline containing 1 mM
dithiothreitol. Nonspecific protein-binding sites were blocked by
incubation with 2% bovine serum albumin for 60 min at 4 °C. An
equivalent amount (radioactive counts) of each in vitro
translated protein was incubated with the beads in 100 µl of binding
buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 0.1%
Tween 20) for 30 min at room temperature. Beads were washed three times
with binding buffer followed by elution of the bound proteins with
SDS-PAGE sample buffer. Binding was analyzed by SDS-PAGE, or by
quantifying counts in a liquid scintillation counter. The Ubc9 binding
observed for each mutant protein relative to that of wild-type was
determined from the ratio of GST-Ubc9 bound radioactive counts
(following subtraction of counts bound to GST alone). Competition
studies using SUMO-1 as a competitor were performed as described above
except that translated proteins were incubated with immobilized Ubc9 in
the presence of the indicated amounts of recombinant SUMO-1.
Quantification of the relative amounts of modified and unmodified
proteins bound to Ubc9 was determined by PhosphorImager analysis after
separation of the proteins by SDS-PAGE.
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RESULTS |
Ubc9 Binding Is Required for SUMO-1 Modification--
The
carboxyl-terminal domain of RanGAP1 is modified by SUMO-1 at lysine
residue 526 (3, 24). We have previously shown that modification at this
site is dependent on a ~120-amino acid domain of RanGAP1 extending
from residue 470 to the carboxyl terminus (3) (summarized in Fig.
1). The ability of this domain to specify SUMO-1 modification was demonstrated by in vitro translation
in rabbit reticulocyte lysate, where SUMO-1 modification is mediated by
endogenous Aos1/Uba2 and Ubc9 activities (3) (Fig.
2, lanes 1 and
7).

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Fig. 1.
A 120-amino acid domain near the carboxyl
terminus of RanGAP1 mediates SUMO-1 modification. A schematic
representation of RanGAP1 and pyruvate kinase fusion proteins used to
delineate a minimal SUMO-1 modification domain in RanGAP1. Leucine-rich
repeats in RanGAP1 are indicated by black boxes, the acidic
domain by a hatched box, and the carboxyl-terminal domain by
dark gray boxes. Pyruvate kinase is indicated by light
gray boxes. Results of in vitro SUMO-1 modification
assays and in vitro Ubc9 binding assays are indicated at the
right.
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Fig. 2.
A 120-amino acid domain near the carboxyl
terminus of RanGAP1 mediates Ubc9 binding. Wild-type RanGAP1
(lanes 1-3), the carboxyl-terminal deletion mutant C 23
(lanes 4-6), and the pyruvate kinase fusion proteins
N 419/PK (lanes 7-9), and ND502/PK (lanes
10-12) were transcribed and translated in rabbit reticulocyte
lysates in the presence of [35S]methionine and incubated
with immobilized GST-Ubc9 or GST. Bound proteins were eluted with SDS
sample buffer and analyzed by SDS-PAGE followed by autoradiography. The
amount of protein loaded in "input" is equivalent to 40% of the
amount of protein assayed in each binding reaction. Molecular mass
standards are indicated on the left and asterisks
indicate SUMO-1-modified proteins. Unmodified RanGAP1 and pyruvate
kinase fusion protein translation products all appear as triplets
following separation by SDS-PAGE, possibly due to initiation of
translation at internal start sites or to phosphorylation.
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A significant fraction of the proteins modified by SUMO-1 are known to
interact directly with the SUMO-1 conjugating enzyme, Ubc9 (1).
Although RanGAP1 is one of the best characterized SUMO-1 substrates,
its interactions with Ubc9 have not been analyzed previously. To
investigate the functional relevance of Ubc9-substrate interactions, we
assayed for the ability of Ubc9 to bind to RanGAP1 and also to the
various mutant and heterologous proteins summarized in Fig. 1. GST-Ubc9
(or GST alone as a control) was immobilized on glutathione-Sepharose
beads and incubated with 35S-labeled proteins produced by
translation in rabbit reticulocyte lysate. Bound proteins were eluted
with SDS sample buffer and analyzed by SDS-PAGE. Full-length RanGAP1
interacted specifically with Ubc9 (Fig. 2, lanes 1-3), as
did N
419/PK (Fig. 2, lanes 7-9). Significantly, both of
these proteins are also modified by SUMO-1. Notably, both unmodified
and SUMO-1-modified RanGAP1 and N
419/PK interacted with Ubc9. In
contrast, C
23 (Fig. 2, lanes 4-6) and N
502/PK (Fig.
2, lanes 10-12) did not interact with Ubc9. As apparent in
lanes 4 and 10, these same two proteins also
failed to be modified by SUMO-1. These results indicate a correlation
between the ability of these proteins to interact with Ubc9 and their
ability to be modified by SUMO-1.
The binding reactions described above were done in the presence of
rabbit reticulocyte lysate, making it possible that the observed
interaction between Ubc9 and RanGAP1 was indirect. To investigate
whether Ubc9 and the carboxyl-terminal domain of RanGAP1 could form a
complex in the absence of other factors, we analyzed the proteins by
gel filtration chromatography either alone or together after mixing and
incubating at room temperature. Bacterially expressed Ubc9 and N
419
were purified and individually fractionated by gel filtration
chromatography on a Superdex 75 column (Amersham Pharmacia Biotech).
Under these conditions, both proteins eluted as apparent monomers with
calculated molecular masses of 20 kDa (Fig.
3). When Ubc9 and N
419 were incubated
together and subsequently fractionated on the same column they
co-eluted as an apparent heterodimer with a calculated molecular mass
of 40 kDa (Fig. 3). This result demonstrates that Ubc9 and the
carboxyl-terminal domain of RanGAP1 interact directly to form a complex
and that the complex is sufficiently stable to allow purification by
gel filtration chromatography. This result also demonstrates that Ubc9
can bind to RanGAP1 prior to forming a thiol ester with SUMO-1.

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Fig. 3.
The carboxyl-terminal domain of RanGAP1 and
Ubc9 interact directly to form a 40-kDa heterodimer. N 419 and
Ubc9 were expressed in bacteria and purified. The purified proteins
were analyzed individually on a Superdex 75 gel filtration column
(top two panels) or analyzed together following
preincubation for 30 min at room temperature (bottom panel).
Column fraction numbers are indicated at the top. The
elution positions of standard protein molecular mass markers are also
indicated.
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Residues Surrounding the SUMO-1 Modification Site Are Essential for
Modification--
The precise lysine residues modified by SUMO-1 have
been identified in approximately a dozen known substrates (1). The majority of these modification sites conform to a consensus sequence that we refer to as the SUMO-1 consensus sequence, or SUMO-1-CS. The
SUMO-1-CS is defined by four amino acids with the sequence "
KXE," were
is a large hydrophobic amino acid, K
is the lysine residue modified by SUMO-1, X is any amino
acid, and E is glutamic acid (Fig. 4).
The conservation of residues surrounding the modified lysine suggests
that they may be critical for substrate recognition and/or for the
selection of the specific lysine residue for SUMO-1 modification.
Nearly all of the substrates containing the SUMO-1-CS also interact
with Ubc9, suggesting that the SUMO-1-CS may play a role in Ubc9
binding. To address this possibility, we mutated residues surrounding
lysine 526 of RanGAP1, including the highly conserved residues
conforming to the consensus sequence (Fig. 5A). Individual mutants were
analyzed for their ability to be modified by SUMO-1 following
translation in rabbit reticulocyte lysate and separation by SDS-PAGE
(Fig. 5B). Approximately one-third of the wild-type protein
was converted to the SUMO-1-modified form when translated in rabbit
reticulocyte lysate (Fig. 5B, lane 1). As expected, mutating
lysine 526 to arginine or alanine completely inhibited SUMO-1
modification (Fig. 5B, lanes 5 and 6). Mutating histidine 521 to alanine had a noticeable, but less dramatic effect on
SUMO-1 modification (Fig. 5B, lane 2), whereas substituting alanine for lysine 530 had no effect on SUMO-1 modification (Fig. 5B, lane 8). Most notably, alanine substitutions of leucine
residues 524 and 525 (Fig. 5B, lanes 3 and 4),
and glutamic acid 528 (Fig. 5B, lane 7) completely inhibited
SUMO-1 modification. These results demonstrate that conserved residues
surrounding the SUMO-1 modification site are critical for efficient
SUMO-1 modification.

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Fig. 4.
The SUMO-1-CS defines the SUMO-1 modification
site of many SUMO-1 substrates. Defined SUMO-1 modification sites
in the indicated proteins were aligned, revealing the SUMO-1-CS:
KXE (where is a large hydrophobic amino acid, K is
the SUMO-1 modified lysine residue, X is any amino acid, and
E is glutamic acid). Positive interactions with Ubc9 are indicated.
References: RanGAP1 (2, 3), PML (4, 26, 37), Sp100 (28), p53 (7-9,
34), I B (5, 42), c-Jun (7, 35), IE2 (43), HSF2 (45), and androgen
receptor (AR) (44).
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Fig. 5.
Conserved residues of the SUMO-1-CS are
essential for SUMO-1 modification. Mutations in the SUMO-1-CS were
introduced into the RanGAP1 pyruvate kinase fusion protein N 419/PK.
Wild-type and mutant proteins were assayed for their ability to be
modified by SUMO-1 by transcription and translation in rabbit
reticulocyte lysates. Reaction products were analyzed by SDS-PAGE
followed by autoradiography. A, amino acid sequences
surrounding the SUMO-1-CS of RanGAP1. B, SDS-PAGE
analysis of modification assays for wild-type N 419/PK (lane
1) and various N 419/PK mutant proteins containing point
mutations in and around the SUMO-1-CS (lanes 2-8). Protein
molecular mass standards are indicated on the left and the
bracket on the right indicates SUMO-1-modified
proteins.
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Residues Surrounding the SUMO-1 Modification Site Mediate Ubc9
Binding--
As indicated above, we observed a correlation between the
ability of proteins to interact with Ubc9 and their ability to be modified by SUMO-1. We therefore assayed for whether mutations of the
conserved residues surrounding the SUMO-1 modification site of RanGAP1
affected Ubc9 binding. GST-Ubc9 was immobilized on glutathione beads
and incubated with 35S-labeled proteins produced by
in vitro translation. Bound proteins were eluted with SDS
sample buffer and analyzed by SDS-PAGE, or by quantification using a
scintillation counter. Four mutations were found to dramatically affect
the interaction with Ubc9: leucine 524 to alanine (Fig.
6, lanes 7-9), leucine 525 to
alanine (Fig. 6, lanes 10-12), lysine 526 to alanine (Fig.
6, lanes 13-15), and glutamic acid 528 to alanine (Fig. 6,
lanes 19-21). Each of these mutations reduced the
interaction with Ubc9 to less than 10% of that observed with the
wild-type protein. Mutations of histidine 521 (Fig. 6, lanes
4-6) and lysine 530 (Fig. 6, lanes 22-24) to alanine
had only modest affects on Ubc9 binding, reducing the interaction by
~50 and 30%, respectively. Mutating the actual modification site,
lysine 526, to arginine (Fig. 6, lanes 16-18) had no
noticeable affect on Ubc9 binding, possibly due to the conservative
nature of this amino acid substitution. These results again demonstrate
a correlation between Ubc9 binding and SUMO-1 modification: proteins
that retained their ability to interact with Ubc9 were modified by
SUMO-1 (with the exception of K526R), whereas those that did not bind
Ubc9 were not modified by SUMO-1. These results further demonstrate
that conserved residues surrounding the SUMO-1 modification site are
essential for Ubc9 binding.

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Fig. 6.
Conserved residues of the SUMO-1-CS are
essential for Ubc9 binding. Amino acid substitutions were
introduced in and around the SUMO-1-CS of RanGAP1. Wild-type and mutant
N 419/PK fusion proteins were translated in rabbit reticulocyte
lysates and incubated with immobilized GST-Ubc9 or GST. Total
translation reactions (lanes 1, 4, 7, 10, 13, 16, 19, and
22) or proteins bound to GST-Ubc9 (lanes 2, 5, 8, 11, 14, 17, 20, and 23) and GST (lanes 3, 6, 9, 12, 15, 18, 21, and 24) were analyzed by SDS-PAGE followed
by autoradiography. The amount of protein loaded in input is equivalent
to 40% of the amount of protein assayed in each binding reaction.
Molecular mass standards are indicated on the left.
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SUMO-1 Contributes to Interactions between Ubc9 and SUMO-1-modified
RanGAP1--
Despite the apparent role of the SUMO-1-CS (including the
targeted lysine) in mediating Ubc9 binding, we observed that SUMO-1 modified RanGAP1 bound to Ubc9 as well as, and possibly better than,
unmodified RanGAP1. To investigate whether SUMO-1 itself, after
being conjugated to RanGAP1, could contribute to Ubc9 binding, we
assayed for the binding of N
419/PK to Ubc9 in the presence of
increasing concentrations of free SUMO-1. Surprisingly, we found that
free SUMO-1 had a significant effect on the binding of SUMO-1-modified
RanGAP1 to Ubc9, but had only a modest effect on the binding of
unmodified RanGAP1 (Fig. 7). We observed
an ~60% reduction in the binding of SUMO-1-modified RanGAP1 when binding assays were performed in the presence of 3.25 mg/ml SUMO-1 (corresponding to an ~15-fold excess over immobilized Ubc9) (Fig. 7,
lanes 1 and 5). In the same assay, the binding of
unmodified RanGAP1 was reduced by less than 20% (Fig. 7, lanes
1 and 5). These findings indicate that Ubc9 interacts
with SUMO-1 modified RanGAP1 through SUMO-1, possibly independent of
direct interactions with RanGAP1.

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Fig. 7.
Ubc9 interacts with the SUMO-1 moiety of
modified RanGAP1. Wild-type ND419/PK was translated in rabbit
reticulocyte lysate and incubated with immobilized GST-Ubc9 in the
presence of increasing concentrations of free SUMO-1 (lane
1, 0 mg/ml; lane 2, 0.2 mg/ml; lane 3, 1 mg/ml; lane 4, 2 mg/ml; lane 5, 3.25 mg/ml).
Quantitative comparison of modified and unmodified proteins in
lanes 1 and 5 using PhosphorImager analysis
indicates a 57% reduction in SUMO-1-modified RanGAP1 and only an 18%
reduction in unmodified RanGAP1 binding to Ubc9.
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DISCUSSION |
SUMO-1 modification modulates protein functions by altering
protein-protein interactions, protein-DNA interactions, protein subcellular localization, and possibly by directly altering protein activity. It is anticipated that SUMO-1 modification is highly regulated, with substrates being selectively recognized and modified in
response to specific cellular signals. Several studies, for example,
have demonstrated that SUMO-1-modified proteins vary with changes in
the cell cycle (13) and in response to cellular growth conditions (10,
25). However, the mechanisms by which proteins are selectively
recognized as substrates for SUMO-1 modification or de-modification and
how such mechanisms might be regulated are not yet understood.
We have begun to investigate how proteins are recognized as substrates
for SUMO-1 modification and present evidence that direct interaction
with the E2 enzyme, Ubc9, is an important part of this process. Using a
domain derived from a well characterized SUMO-1 substrate, RanGAP1, we
have demonstrated that SUMO-1 modification correlates with the ability
to directly interact with Ubc9. This domain of RanGAP1 contains a
consensus sequence, the SUMO-1-CS, which is found in nearly all known
SUMO-1 substrates. The SUMO-1-CS contains the lysine residue to which
SUMO-1 is covalently attached, and several highly conserved residues
that flank this lysine. Although recognized by several other groups
(26-28), the functional significance of the SUMO-1-CS was not
previously characterized. To investigate its role in SUMO-1
modification, we made alanine substitutions of the conserved residues
in the SUMO-1-CS. These mutations were found to inhibit both Ubc9
binding and SUMO-1 modification. These findings indicate that the
SUMO-1-CS plays a direct role in mediating the binding of Ubc9 to
SUMO-1 substrates and that this binding is essential for substrate modification.
The actual lysine acceptor in the SUMO-1-CS of RanGAP1 is important for
the interaction with Ubc9, as an alanine substitution at this position
inhibits Ubc9 binding. In apparent contradiction to this finding, it
was also observed that SUMO-1-modified RanGAP1 and free RanGAP1
interact equally well with Ubc9. Competition experiments using excess
free SUMO-1, however, indicated that modified RanGAP1 likely interacts
with Ubc9 through the SUMO-1 moiety. It remains to be determined
whether the binding of modified RanGAP1 to Ubc9 is mediated solely
through interactions with SUMO-1 or through a combination of
interactions with RanGAP1 and SUMO-1. Previous studies have indicated
that Ubc9 and free SUMO-1 can form direct, noncovalent interactions
(29). It will be interesting to determine whether other SUMO-1-modified
substrates interact similarly with Ubc9, or whether this interaction is
specific for modified RanGAP1. In vivo, Ubc9 is concentrated
at the nuclear envelope at sites that overlap with the localization of
SUMO-1-modified RanGAP1 (19). Although Ubc9 has been shown to interact
with the nucleoporin Nup358 (18), it remains to be determined whether this interaction is direct or mediated by SUMO-1-modified RanGAP1.
In addition to demonstrating a role for the SUMO-1-CS in Ubc9 binding,
we also found that ~50 amino acids on either side of the consensus
sequence were required for Ubc9 binding and SUMO-1 modification.
Comparison of the amino acid sequence of this larger domain with other
SUMO-1 substrates reveals no obvious homologies outside of the
SUMO-1-CS. It therefore remains to be determined whether additional
residues outside of the SUMO-1-CS are directly involved in Ubc9
binding, or whether they indirectly affect binding by influencing the
proper folding and exposure of a smaller domain containing the
SUMO-1-CS. Evidence that the precise position of the SUMO-1 CS within a
protein can be an important determinant of its ability to function in
SUMO-1 modification is provided by analysis of the heat shock
transcription factor, HSF2. HSF2 contains three putative SUMO-1-CS
motifs, but is modified at a lysine residue in only one of these motifs
(45). The precise position of the SUMO-1-CS within a protein is likely
to determine its accessibility and, therefore, its potential to
interact with Ubc9. The presence of a SUMO-1-CS in a protein is
therefore not an absolute indicator of whether that protein will be a
substrate for SUMO-1 modification. Further analysis of the amino acid
residues surrounding the SUMO-1-CS and their contributions to Ubc9
binding will be needed to refine the definition of a SUMO-1 substrate.
In this particular study, we examined the SUMO-1-CS of RanGAP1, but we
propose that the SUMO-1-CS of other SUMO-1 substrates will function
similarly to mediate Ubc9 binding and facilitate SUMO-1 modification.
Ubc9-binding domains have been partially mapped in a large number of
known and putative SUMO-1 substrates. Many of these domains contain
SUMO-1-CS motifs, including the Ubc9-binding domains found in WT1 (30),
TEL (31), ETS-1 (32), Dorsal (33), p53 (34), c-Jun (35), glucocorticoid
receptor (35), and poly(ADP-ribose) polymerase (36). Other
motifs in addition to the SUMO-1-CS may also mediate Ubc9 binding,
however. Most notably, Ubc9 also interacts with the RING domain of PML (26). The functional significance of this interaction remains unclear
as mutations that disrupt interactions between Ubc9 and the PML RING
domain have very little effect on the SUMO-1 modification of PML (26,
37). It has also recently been proposed that PEST sequences may play a
role in regulating the interaction of Ubc9 with at least some SUMO-1
substrates (1, 38). Although PEST sequences are best known for their
involvement in ubiquitin-mediated proteolysis (39), they are found in
more than half of the known SUMO-1 substrates.
The binding of Ubc9 to the SUMO-1-CS argues against a direct
requirement for E3 ligases in SUMO-1 modification. However, it is
possible that the modification of substrates with a SUMO-1-CS may be
regulated or facilitated by the action of E3-like factors in
vivo. Regardless of this consideration, a more detailed analysis of the direct interactions between Ubc9 and the SUMO-1-CS will provide
important insights into the mechanisms involved in the ultimate
transfer of SUMO-1 to specific substrates. Despite crystallographic studies of ubiquitin E2-E3 complexes (40, 41), the exact mechanism that
underlies the transfer of ubiquitin from upstream factors to protein
substrates remains poorly defined. Given the high degree of homology
between Ubc9 and ubiquitin E2 enzymes, it is likely that the transfer
of SUMO-1 to protein substrates will be very similar to the process of
ubiquitination. Detailed characterization of the interactions between
Ubc9 and the SUMO-1-CS should, therefore, provide important insights
into the mechanisms of both ubiquitination and SUMO-1 modification. It
will also be important to further characterize the role of amino acids
surrounding the SUMO-1-CS, to determine whether they function
indirectly to present an exposed consensus sequence, or whether they
are also involved in significant interactions with Ubc9.
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ACKNOWLEDGEMENTS |
We thank Janet Cronshaw, Richard Rogers,
Maria Vassileva, and Cecile Pickart for comments and suggestions during
the course of this work.
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FOOTNOTES |
*
This work was supported by the National Institutes of Health
Grant GM60980 (to M. J. M.).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.: 410-614-6878;
Fax: 410-955-2926; E-mail: mmatunis@jhsph.edu.
Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M100006200
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ABBREVIATIONS |
The abbreviations used are:
SUMO-1, small
ubiquitin-like modifier-1;
SUMO-1-CS, SUMO-1 consensus sequence;
Ubc9, SUMO-1 conjugating enzyme;
RanGAP1, Ran GTPase activating protein 1;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel
electrophoresis;
PML, promyelocytic leukemia protein;
E1, ubiquitin
activating enzyme;
E2, ubiquitin carrier protein;
E3, ubiquitin-protein
isopeptide ligase..
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