From the School of Biology, Biomolecular Sciences Building,
University of St. Andrews, St. Andrews, Fife KY169ST, United Kingdom
and the Laboratoire de Transport
Nucléocytoplasmique, Institut Jacques Monod, CNRS UMR
7592, Tour 43, 2 Place Jussieu, 75251 Paris Cedex 05, France
Received for publication, October 17, 2000, and in revised form, December 13, 2000
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ABSTRACT |
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SUMO-1 is a small ubiquitin-related modifier that
is covalently linked to many cellular protein targets. Proteins
modified by SUMO-1 and the SUMO-1-activating and -conjugating enzymes
are located predominantly in the nucleus. Here we define a transferable sequence containing the SUMO-11 is a small
ubiquitin-related modifier (also known as sentrin, GMP1, UBL1, PIC1, or
SMT3 in yeast) that has been found covalently conjugated to various
cellular proteins (for reviews see Refs. 1-3). Several substrates for
SUMO-1 have been reported: the RanGTPase-activating protein (RanGAP1)
(4, 5) and Ran-binding protein 2 (6) implicated in
nucleocytoplasmic trafficking; the promyelocytic leukemia protein (PML)
and Sp100 (7) found in subnuclear structures known as PML oncogenic
domains or PODs; the I SUMO-1 is conjugated to a target protein by a pathway that is distinct
from but analogous to ubiquitin conjugation. Like ubiquitin, SUMO-1 is
proteolytically processed to expose its mature C terminus by recently
described SUMO-1-specific proteases variously called Ulp1 and Ulp2 in
yeast (11, 12) or SENP1 and SUSP-1 in vertebrates (13-15). Ulp1, Ulp2,
and SENP1, but not SUSP-1, are capable of both deconjugating SUMO-1
from modified proteins and removing four amino acids from the C
terminus of the 101-amino acid SUMO-1 precursor to generate the mature
97-amino acid form. SUMO-1 addition is accomplished by a thioester
cascade, with SUMO-1 first being activated by a heterodimeric
SUMO-1-activating enzyme (SAE) that adenylates the C-terminal glycine
of SUMO-1 (16-19) before catalyzing the formation of a thioester bond
between the C terminus of SUMO-1 and a cysteine residue in SAE. In a
transesterification reaction SUMO-1 is transferred from the SAE to the
SUMO-1-conjugating enzyme Ubc9, which catalyzes the formation of an
isopeptide bond between the C terminus of SUMO-1 and the Here, we demonstrate that a short sequence containing the consensus
Cell Culture and Transfections--
HeLa cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum. Cells were transfected by electroporation as described
previously (25). For immunofluorescence analysis 2 µg of plasmid were
transfected in 1 × 106 HeLa cells. For nickel bead
purification, 10 µg of each plasmid DNA encoding pyruvate
kinase (PK) fusions and His6-SUMO-1 were transfected in 1 × 107 HeLa cells. To increase efficiency of protein
expression, no DNA carrier was used in cotransfections. After
transfection, cells were seeded in 75 cm2 flasks.
One-twentieth of transfected cells were seeded in a separated plate (to
control protein input), and incubation was continued for 24 h.
Plasmids and DNA Manipulations--
Plasmids encoding
His6-SUMO-1, HA-SUMO-1, SV5-SAE1, HA-SAE2, and SV5-Ubc9 were
reported previously (8, 9, 17). pcDNA3 plasmids encoding Myc-tagged
PK and NLS-PK were described previously (26). cDNA encoding the
1-26 fragment of I Immunofluorescence Microscopy--
Indirect immunofluorescence
analysis was performed as described previously (28). Monoclonal
antibodies anti-HA (Babco), anti-SV5 (Dr. R. Randall, University of St.
Andrews), and anti-Myc (9E10) were applied for 30 min followed by a
30-min incubation with fluorescein isothiocyanate-conjugated donkey
anti-mouse IgG (Jackson). Coverslips were mounted in Mowiol (Hoechst,
Frankfurt, Germany). Images were acquired on a DMRB fluorescence
microscope (Leica) with a CCD camera (Princeton).
In Vitro SUMO-1 Assay--
In vitro
transcription/translation (Promega) and SUMO-1 conjugation assays were
performed as reported (8).
Preparation of Cell Extracts and Immunoblotting--
Cells were
harvested in 100 µl of lysis buffer for Western blot analysis (8).
His6-SUMO-1 conjugates were purified as described (9). Proteins were
resolved by electrophoresis in 8.5% polyacrylamide gels containing
SDS, transferred to polyvinylidene difluoride membranes (Sigma) by
electroblotting, and processed for Western blotting as reported
previously (9). Primary monoclonal antibody anti-Myc (9E10) was
obtained from Dr. R. E. Randall. Horseradish peroxidase-conjugated
anti-mouse IgG was purchased from Amersham Pharmacia Biotech. An
enhanced chemiluminescence detection system was used to detect specific
antigen-antibody interactions (Amersham Pharmacia Biotech).
Nuclear Distribution of the SUMO-1 Conjugation Pathway
Enzymes--
It has been previously reported that Ubc9 promotes the
nuclear localization of a Ubc9- Sequence Requirement for SUMO-1 Modification--
To test the
proposition that SUMO-1 modification takes place in the nucleus, we
designed an experiment strategy in which a minimal SUMO-1 modification
site, fused to a heterologous protein, is located in either the nucleus
or the cytoplasm by virtue of the presence or absence of an NLS.
Constructs were designed such that these could be tested for SUMO-1
conjugation in vitro, where there is no influence of
compartmentalization, or expressed in vivo either in the
nucleus or the cytoplasm. Analysis of the sequence of SUMO-1
conjugation sites in multiple proteins indicates that a short motif
SUMO-1 Conjugation in Vivo Requires Nuclear Targeting--
In most
cells, SUMO-1 is found in conjugates with target proteins, and as such
the pool of free SUMO-1 is limiting (8). At present, most cellular
substrates reported to be conjugated with SUMO-1 show a nuclear
distribution (PML, Sp100, and p53), shuttle between the nucleus and the
cytoplasm (I Intrinsic Substrate Activity of SUMO-1 Modification
Motifs--
Although a short motif can direct SUMO-1 modification when
transferred to a heterologous protein, the efficiency of SUMO-1 modification will be a combination of the intrinsic substrate activity
of the motif and the environment of the motif in the native protein. To
investigate the intrinsic substrate activity of a range of SUMO-1
modification motifs, we generated additional PK constructs (Fig.
4A) encoding amino acids
485-495 of PML (31, 37) and amino acids 99-109 of the human
adenoviral protein E1B,2
which contain the lysine residue that is the site of SUMO-1
conjugation (Fig. 4A). 35S-labeled
PK-RanGAP1-(519-529), PK-p53-(381-391), PK-I Mutational Analysis of the Post-translational protein modifications modulate protein function
by altering protein activity or the ability to interact with ligands or
by changing subcellular localization of the modified protein.
Conjugation with SUMO-1 has been proposed to regulate protein function
through all these mechanisms. Identification of a short amino acid
sequence motif required for the transfer of the capacity to be
conjugated with SUMO-1 to a heterologous protein indicates that this
motif is necessary for recognition by the SUMO-1 modification enzymes.
Most of the targets for SUMO-1 modification are Ubc9-interacting
proteins, and it is likely that substrate specificity is achieved by
Ubc9. The C-terminal region of Ubc9, which is thought to be involved in
substrate binding, lies close to the catalytic site and favors the
direct transfer of SUMO-1 to substrate proteins (38). The most
important amino acids in the consensus sequences are Lys and Glu.
Whereas the acceptor Lys residue cannot be substituted, Glu can be
replaced by Asp to generate a recognition motif that, although
functional, is poorly conjugated with SUMO-1 (Fig. 4). To date, the
only reported modification site containing Asp rather than Glu in a
natural protein is in the yeast septin Cdc3, although it was not clear how efficiently this site was utilized for modification (32). In
addition to the Lys and Glu residues, the large hydrophobic residue
contributes substantially to the efficiency of SUMO-1 modification.
Short transferable sequences from various protein substrates modified
with SUMO-1 show different capacities to be conjugated with SUMO-1. The
best conjugated short sequence contains the sequence IKME from PML,
whereas the sequence that is least efficiently modified contains
the sequence LKSE from RanGAP1. Surprisingly, RanGAP1 is one of the
best cellular substrates for conjugation with SUMO-1 (4, 5), suggesting
that other cis or trans factors may influence its efficiency of
conjugation. When fused to the C terminus of a heterologous protein,
the 7-amino acid sequence containing the The observed predominant nuclear distribution of SUMO-1, SAE, and Ubc9,
as well as the exclusive SUMO-1 modification of NLS-PK substrates,
indicates that SUMO-1 modification requires nuclear targeting. However,
because Ubc9 is concentrated at the nuclear envelope, we cannot
determine whether SUMO-1 modification with SUMO-1 takes place after
transport of substrates into the nucleus or whether modification occurs
during docking at the nuclear envelope and/or translocation into the
nucleus. It has previously been noted that mutations or deletions of
the nuclear localization signals of PML, RanGAP1, and Sp100 that
resulted in cytoplasmic accumulation of these proteins also resulted in
loss of SUMO-1 modification in vivo (31, 33, 35). In
addition, a 97-kDa isoform of PML, which, for unknown reasons, is
located in the cytoplasm, also fails to undergo SUMO-1 modification
in vivo. The same protein, however, is efficiently modified
by SUMO-1 in vitro (31).
Thus, newly synthesized unmodified substrates would be targeted to the
nucleus, where they would be modified by the combination of
SAE1, SAE2, and Ubc9. Once modified, these proteins may be incorporated into subnuclear structures such as PODs. In particular, SUMO-1 modification of the POD component PML is required for both formation of the PODs (40) and for recruitment of other proteins such
as Daxx into the PODs (41). Alternatively, newly SUMO-1-modified proteins could, as in the case of RanGAP1, be targeted to the nuclear pore complex (4, 5) or shuttle between the nucleus and the
cytoplasm like I Although nuclear targeting appears to be required for SUMO-1
modification of most substrates, we cannot rule out the possibility that some proteins may be modified in other cellular compartments. This
may be the case for the glucose transporters GLUT1 and GLUT4, which are
targeted to the cell membrane and yet appear to be SUMO-1-modified (42). One possibility is that newly synthesized SAE and Ubc9 are
recruited to a cytoplasmic complex containing GLUT1 and GLUT4, where
modification takes place. This is consistent with the observation that
GLUT1 and GLUT4 both interact directly with Ubc9. If the NLSs of
SAE and Ubc9 are occluded in this complex, then this would allow
a small proportion of the predominantly nuclear SAE and Ubc9 to remain
in the cytoplasm tightly associated with their substrate.
The two recently described SMT3-specific proteases Ulp1 and Ulp2 (11,
12) accumulate a different pattern of SMT3-conjugated proteins in
their mutants, indicating that deconjugation of substrates can
be achieved and regulated by multiple SMT3 proteases with different
specificities. Because SUMO-1 is a limiting factor for conjugation of
substrates, deconjugation of SUMO-1 may be a dual mechanism to decrease
(or increase) protein activity of a deconjugated protein and increase
(or decrease) the activity of a newly conjugated target, when released
SUMO-1 is available. However the cellular sites at which this process
takes place are not known, because the cellular localization of
endogenous SUMO-1-specific proteases has yet to be determined. Thus
SUMO-1 modification of most proteins appears to be regulated by the
requirement of the substrate to be targeted to the nucleus and by the
possession of a SUMO-1 recognition motif displayed on the surface of
the target protein. It is likely that SUMO-1 modification emerges as an
important control mechanism that regulates the activity of many nuclear proteins.
KXE motif, where
represents
a large hydrophobic amino acid, that confers the ability to be
SUMO-1-modified on proteins to which it is linked. Whereas addition of
short sequences from p53 and I
B
, containing the
KXE motif, to a carrier protein is sufficient for
modification in vitro, modification in vivo requires the additional presence of a nuclear localization signal. Thus, protein substrates must be targeted to the nucleus to undergo SUMO-1 conjugation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
inhibitor of the transcription factor
nuclear factor
B, implicated in the control of immune and
inflammatory responses (8); and the tumor suppressor protein p53 (9,
10). Consequently, "SUMOylation" plays a role in multiple vital
cellular processes such as oncogenesis, cell cycle control, apoptosis,
and response to virus infection.
-amino
group of a lysine residue of the target protein (6, 20-23). Ubc9 is
required for cell cycle progression in yeast (24). Unlike ubiquitin
conjugation, SUMO-1 modification of target proteins in vitro
is not dependent on the equivalent of an E3 protein ligase (17,
19).
KXE, where
represents a large hydrophobic amino
acid, constitutes a transferable signal that confers the ability
to be modified with SUMO-1 on proteins to which it is linked. The predominantly nuclear localization of both subunits of the SAE, Ubc9
and SUMO-1, suggest that SUMOylation is a nuclear process. We
demonstrate that heterologous proteins carrying the SUMO-1 consensus
modification sequences present in I
B
and p53 are only conjugated
to SUMO-1 in vivo when a nuclear localization signal (NLS)
is also present. These data suggest that protein substrates must be
targeted to the nucleus to undergo SUMO-1 conjugation and allow us to
propose that this modification may be involved in regulating multiple
processes in the nucleus.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
was obtained by polymerase chain
reaction using as template I
B
wild type and I
B
K21R,K22R-encoding plasmids (27) to generate
PK-I
B
-(1-26) and PK-I
B
-(1-26)-KR. cDNAs
encoding 361-393 and 361-393 KR of p53 were subcloned from previously
described constructs (9). Polymerase chain reaction fragments and
synthetic oligonuclotides encoding the 381-391 fragment of p53, the
16-26 fragment of I
B
, the 519-529 fragment of human RanGAP1,
the 99-109 fragment of adenovirus type 2 E1B, and the 485-495
fragment of PML, as well as Lys to Arg versions, were cloned in
BamHI and XbaI restriction sites of the PK
vector. The same cDNAs were subcloned into BamHI and
XbaI restriction sites of the NLS-PK vector that contains a
polylinker,
KpnI-BamHI-EcoRV-XbaI, to
generate NLS-PK versions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase fusion protein in
Saccharomyces cerevisiae (24). Moreover, Ubc9 has been
localized predominantly in the nucleus of T cells (29). To analyze the
subcellular distribution of all the components of the SUMO-1
conjugation pathway, we transiently transfected HeLa cells with
HA-tagged versions of SUMO-1 and SAE-2 and SV5-tagged versions of Ubc9
and SAE-1 (Fig. 1). As expected, SUMO-1
displayed nuclear distribution and accumulated in nuclear dot-like
structures (30) (Fig. 1a). Using paraformaldehyde fixation, Ubc9 was localized mainly in the nucleus but also in the cytoplasm of
transfected cells (Fig. 1b). However, when cells were fixed with 1:1 methanol/acetone, Ubc9 immunoreactive material was
concentrated at the nuclear envelope (data not shown and Ref. 23). The
SAE-1 and SAE-2 (Fig. 1, c and d, respectively)
subunits of the SAE presented an exclusively nuclear distribution that
was not changed when both subunits were coexpressed or when cells were
fixed by different methods (data not shown). Thus, the predominant
nuclear localization of enzymes implicated in the SUMO-1 modification pathway suggests that this ubiquitin-like modification may take place
in the nucleus.
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Fig. 1.
Nuclear distribution of SUMO-1 and the SUMO-1
conjugation pathway enzymes. HeLa cells were transfected with
plasmids encoding HA-SUMO-1 (a), SV5-Ubc9 (b),
SV5-SAE1 (c), and HA-SAE2 (d). Indirect
immunofluorescence was carried out with monoclonal antibodies
recognizing HA and SV5 tags followed by fluorescein
isothiocyanate-conjugated anti-mouse IgG.
KXE represents the primary site of SUMO-1 modification (8, 9, 31-33). To further define the sequence required for conjugation
with SUMO-1, we designed a series of constructs containing I
B
N-terminal and p53 C-terminal modification sites fused to the C
terminus of either a Myc-tagged version of PK or an equivalent construct containing the SV40 NLS (NLS-PK) (Fig.
2). [35S]Met-labeled PK and
NLS-PK fusions generated by in vitro transcription and
translation were assayed for SUMO-1 conjugation in vitro
using the previously described assay (8). PK and NLS-PK fused to amino
acids 1-26 and 16-26 of I
B
or amino acids 361-393 and 381-391
of p53 were conjugated with SUMO-1, whereas PK or NLS-PK alone were not
(Fig. 2, A and B). When lysine residues 21 of
I
B
and 386 of p53 were changed to arginine (KR constructs),
SUMO-1-modified forms of PK-I
B
-KR and PK-p53-KR fusions were not
detected, indicating that SUMO-1 was conjugated specifically to the
previously described lysine residues (8-10). To determine whether the
11-amino acid sequence required for conjugation with SUMO-1 could be
further reduced, a series of synthetic oligonucleotides that specifies the human RanGAP1 519-529 (11 amino acids), 520-528 (9 amino acids), 521-527 (7 amino acids), and 522-526 (5 amino acids) amino acid sequences was fused to the C terminus of PK to generate the
corresponding PK-RanGAP1 constructs (Figs. 2C and
4A). Efficient SUMO-1 conjugation was observed with PK
fusion encoding 11, 9, and 7 amino acids, whereas the efficiency of
conjugation was reduced but still detectable with only 5 amino acids
(Fig. 2C). Thus, SUMO-1 modification requires a core
recognition motif of five amino acids, although flanking residues
influence the efficiency of conjugation.
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Fig. 2.
Sequence requirement for SUMO-1
modification. In vitro expressed
35S-labeled PK or NLS-PK constructs containing 1-26,
1-26KR, 16-26, and 16-26KR sequences of I B
(A) or
361-393, 361-393KR, and 381-391 sequences of p53 (B) were
tested for substrate activity in an in vitro SUMO-1
conjugation assay. Reaction products were fractionated by
SDS-polyacrylamide gel electrophoresis, and the dried gel was analyzed
by phosphorimaging. SUMO-1-conjugated forms are indicated by an
asterisk. C, in vitro SUMO-1
conjugation of 35S-labeled PK constructs containing amino
acids 519-529, 520-528, 521-527, or 522-526 of human RanGAP1.
Reaction products were processed as above.
B
) (34, 35), or are associated to the nuclear
pore complex (RanGAP1 and Ran-binding protein 2) (4-6). Cell
fractionation analysis indicates that 80-90% of endogenous
SUMO-1-conjugated proteins have a nuclear distribution (7, 36, and data
not shown). To determine whether SUMO-1 conjugation requires nuclear
targeting, the PK-I
B
and PK-p53 constructs were compared with
NLS-PK-I
B
and NLS-PK-p53 constructs for SUMO-1 conjugation
in vivo. The ability of the NLS-PK constructs to be
conjugated with SUMO-1 in vitro is identical to the PK
fusion counterpart (Fig. 2, A and B). As
expected, all PK constructs were localized in the cytoplasm, and all
NLS-PK constructs were localized in the nucleus of transfected cells (Fig. 3A). To detect forms of
PK and NLS-PK modified by SUMO-1 in vivo, constructs
specifying these proteins were cotransfected into HeLa cells with an
expression plasmid for His6-SUMO-1 (9). His6-SUMO-1-conjugated proteins
were isolated on nickel beads, and eluted proteins were analyzed by
Western blotting with a monoclonal antibody recognizing the Myc tag.
NLS-PK-I
B
-(1-26) and -(16-26) as well as NLS-PK-p53-(361-393)
and -(381-391) are efficiently conjugated with SUMO-1 (Fig. 3,
B and C, top). Under the same conditions, KR mutants were not modified. In contrast to NLS-PK constructs, PK counterparts were not modified. These results clearly indicate that nuclear localization is required for conjugation of
proteins with SUMO-1.
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Fig. 3.
SUMO-1 conjugation in vivo
requires nuclear targeting. A, HeLa cells were
transfected with Myc-tagged PK (a-e) or NLS-PK
(f-j) constructs containing no insert (a and
f), I B
-(1-26) (b and g),
I
B
-(16-26) (c and h), p53-(361-393)
(d and i), and p53-(381-391) (e and
j). Indirect immunofluorescence analysis was carried out
with a monoclonal antibody recognizing the Myc tag followed by
fluorescein isothiocyanate-conjugated anti-mouse IgG. B and
C, HeLa cells were cotransfected with plasmids encoding
His6-SUMO-1 and Myc-tagged PK or NLS-PK constructs containing
I
B
-(1-26), I
B
-(1-26)-KR, I
B
-(16-26), and
I
B
-(16-26)-KR (B) or p53-(361-393),
p53-(361-393)-KR, and p53-(381-391) (C). After 24 h
of expression, cells were lysed in buffer containing guanidinium-HCl.
Proteins linked to His6-SUMO-1 were purified by nickel beads, and,
after being washed, were eluted with 200 mM
imidazole. Eluted proteins were fractionated by SDS-polyacrylamide gel
electrophoresis and transferred to a polyvinylidene difluoride
membrane. His6-SUMO-1 conjugates were detected by Western blotting
using the 9E10 anti-Myc monoclonal antibody. To control protein input,
a fraction of each lot of transfected cells was lysed in buffer
containing SDS and analyzed as above.
B
-(16-26), PK-AdE1B-(99-109), and PK-PML-(485-495) were used as substrates for
SUMO-1 conjugation in vitro (Fig. 4B). A range of
substrate activities is evident (Fig. 4B), with PK-AdE1B and
PK-PML being modified efficiently, PK-I
B
and PK-p53 being
modified less efficiently, and PK-RanGAP1 being modified poorly. Those
differences were also observed in vivo when comparing NLS-PK
fusions encoding PML, I
B
, and p53 sequences (Fig. 4C)
in conditions where PK counterparts were poorly modified or not
modified at all (data not shown). The differences observed in the
conjugation of SUMO-1 to this range of substrates indicates that the
precise sequence of the SUMO-1 modification motif, when isolated from
its native environment, defines the efficiency of SUMO-1
conjugation.
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Fig. 4.
Intrinsic substrate activity of SUMO-1
modification motifs. 35S-labeled
PK-RanGAP1-(519-529), PK-p53-(381-391), PK-I B
-(16-26),
PK-AdE1B-(99-109), and PK-PML-(485-495) (A) were tested as
substrates for SUMO-1 conjugation in vitro (B).
Reaction products were fractionated by SDS-polyacrylamide gel
electrophoresis, and the dried gel was analyzed by phosphorimaging.
Different levels of conjugation with SUMO-1 are indicated in
A. We arbitrarily considered PK-PML conjugation as 100%.
+++, 100-67%; ++, 66-33%; and +,
<33%. C, HeLa cells were cotransfected with plasmids
encoding His6-SUMO-1 and NLS-PK or NLS-PK constructs encoding
PML-(485-495), I
B
-(16-26), and p53-(381-391) amino acid
sequences. Transfected cells were processed as in Fig. 3. Purified
His6-SUMO-1 conjugates and protein input were detected by Western
blotting using an anti-Myc antibody.
KXE Motif--
To evaluate the role
of each amino acid within the
KXE motif in SUMO-1
conjugation, a series of PK constructs encoding alanine mutations in
the FKTE sequence of p53 were generated (9). Mutations of the
X residue T did not affect conjugation with SUMO-1 (Fig. 5A). When residue
was
changed to alanine, conjugation with SUMO-1 was reduced (Fig.
5A). As expected, mutation of the strictly conserved Lys and
Glu residues abrogates conjugation with SUMO-1 (Fig. 5A). These results confirm that Lys and Glu are the most important residues
of the consensus and suggest that the
and X residues may
influence the efficiency of SUMO-1 conjugation. To investigate this
point, a series of PK-RanGAP1 molecules was generated in which the
residue (Leu) of the poorly modified PK-RanGAP1 was changed to each of
the possible hydrophobic residues, and the efficiency of conjugation
was determined in vitro. Whereas changing the Leu to either
Ile or Val increased the efficiency of SUMO-1 conjugation, changes to
Ala, Pro, or Trp substantially reduced the efficiency of conjugation.
Substitutions of Leu with Phe or Met did not alter the efficiency of
conjugation (Fig. 5B). To evaluate the role of the
X amino acid, the Met residue at this position in PK-PML was
changed to either Ala, Lys, Ile, Ser, or Asp. Whereas substitution of
Met with Asp caused a small decrease in substrate activity, all of the
other modifications at this position were without consequence. Although
most described modification sites in naturally occurring proteins
contain the sequence
KXE, it was of interest to determine
whether a Glu residue was absolutely required for activity. The
conservative Glu to Asp change was therefore made within the context of
the PK-PML construct, and the substrate activity was determined. The
altered substrate is modified very poorly when compared with a
substrate containing the wild type motif (Fig. 5B). Thus,
SUMO-1 modification is directed by a
KXE motif, where the
nature of the hydrophobic amino acid preceding the acceptor lysine
residue has a major influence on the efficiency with which SUMO-1 is
conjugated to the target protein.
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Fig. 5.
Mutational analysis of the
KXE motif. A, the
in vitro SUMO-1 conjugation assay was performed with
35S-labeled PK-p53-(381-391) (WT),
PK-p53-(381-391) (K386A), PK-p53-(381-391)
(E386A), PK-p53-(381-391) (T387A), and
PK-p53-(381-391) (F385A). Asterisks indicate
SUMO-1-conjugated forms. B, 35S-labeled
PK-RanGAP1-(519-529)-wild type (L),
PK-PML-(485-495)-wild type (M), and indicated mutants were
tested as substrates for SUMO-1 conjugation in vitro.
Different levels of conjugation with SUMO-1 are indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
KXE motif acts
as an efficient substrate for SUMO-1 modification in vitro.
However, when an amino acid is removed from each end of this sequence
to generate a five-amino acid sequence containing the
KXE
motif, its substrate activity is reduced. Although the shortest
transferable sequence we have tested contains five amino acids,
extensive additional mutagenesis failed to identify any elements
outside the
KXE motif that contributed to substrate
activity. It is likely that the seven-amino acid sequence is a more
efficient substrate than the five-amino acid sequence, because the
sequences were added to the C-terminal end of a carrier protein such
that the Glu in the
KXE motif is now the C terminus of
the protein. Thus the
KXE motif needs to be flanked by at
least one additional C-terminal amino acid for optimal recognition by
the SUMO-1 modification machinery. One important factor is how the
recognition motif is presented to the surface of the protein. If it is
located in an exposed loop, then it may be highly accessible to the
modification machinery. Mutational analysis of RanGAP1 indicates that
sequences flanking the domain containing the acceptor lysine (Lys-526)
are required for efficient SUMO-1 modification in vitro,
although the function of these additional domains has yet to be defined
(35). Alternatively, additional binding sites for Ubc9 in the target
protein may result in tighter binding of Ubc9 to the protein substrate,
with a consequent increase in the efficiency of conjugation. This may
be the case with PML, in which the RING finger is required for
interaction with Ubc9 in a yeast two-hybrid analysis. PML proteins
containing mutations that disrupt the RING finger are poorly modified
in vivo but efficiently modified in vitro (31).
Whereas SUMO-1 conjugation of RanGAP1 and I
B
can be catalyzed
in vitro simply with SAE and Ubc9 (17, 19), it is possible
that additional protein factors may influence the efficiency of
conjugation. The importance of how the modification motif is presented
is exemplified by the case of the splicing protein p32, which contains
a perfect match to the consensus recognition site for SUMO-1
modification and yet is not modified by SUMO-1 in
vitro.3 Moreover,
inspection of the structure of p32 (39) reveals that the recognition
motif is not surface-exposed and would not be accessible to the
modification machinery.
B
and p53, although there is as yet no data
indicating whether this is the case for the SUMO-1-modified form of
these proteins (8-10).
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ACKNOWLEDGEMENTS |
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We thank Alex Houston, University of St. Andrews, for DNA sequencing. We thank Joana Desterro, Ellis Jaffray, and Magali Prigent for technical advice and support.
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FOOTNOTES |
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* This work was supported by the Medical Research Council, the Biotechnology and Biology Research Council, and the Fondation pour la Recherche Medicale.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.: 44-1334-463396; Fax: 44-1334-462595; E-mail: rth@st-and.ac.uk.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M009476200
2 A. Errico and R. T. Hay, unpublished data.
3 R. T. Hay, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are:
SUMO-1, small
ubiquitin-like modifier 1;
RanGAP, RanGTPase-activating protein;
PML, promyelocytic leukemia protein;
POD, PML oncogenic domain;
IB
, inhibitor of (nuclear factor)
B
;
SAE, SUMO-1-activating
enzyme;
NLS, nuclear localization signal;
PK, pyruvate kinase;
HA, hemagglutinin;
SV5, simian virus type 5.
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