Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York 10021
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Abstract |
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RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.
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Introduction |
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POSTTRANSLATIONAL protein modifications are required for a variety of cell functions, regulating protein interactions, enzymatic activity, subcellular localization, and stability. Ubiquitination is a posttranslational
modification that involves the covalent attachment of ubiquitin (Ub),1 itself a 76 amino acid protein, to lysine residues of targeted substrates (for review see Wilkinson,
1995; Hochstrasser, 1996
). Ub can be considered to be a
posttranslationally added signal that targets its substrates
to specific fates. Among other factors, different metabolic
fates can depend on the number of Ub molecules conjugated to a particular substrate, with mono-ubiquitinated proteins being relatively stable (such as histone H2A;
Goldknopf and Busch, 1977
), and multiubiquitinated
proteins being relatively unstable. Covalent attachment of
Ub to intracellular proteins has effects on a wide range of
cell functions, including gene expression, cell division,
DNA repair, programmed cell death, peroxisome biogenesis, mitochondrial protein import, and ribosome assembly. While the precise mechanisms underlying the roles of Ub in many of these processes are not fully understood,
the best characterized function of the Ub signal is to target
proteins for ATP-dependent proteolysis by the 26S proteasome (Wilkinson, 1995
; Hochstrasser, 1996
). Targeting
by Ub is mediated by direct interactions between 26S proteasome subunits and Ub itself (Deveraux et al., 1994
).
It has long been suspected that Ub modification may
have other consequences for targeted substrates, and recently ubiquitination was shown to function as a signal for
ligand-induced receptor endocytosis and lysosomal targeting (Hicke and Riezman, 1996; Strous et al., 1996
), as well
as in activation of the I
B
protein kinase (Chen et al.,
1996
). The question of whether there may be related ubiquitin systems using novel, ubiquitin-like proteins as modifiers has also been raised. The first example of such a system was described for the interferon inducible protein,
UCRP (ubiquitin cross-reactive protein), a 15-kD protein
containing two Ub-related domains that are 43 and 62%
homologous to Ub, respectively (Haas et al., 1987
). UCRP
is covalently ligated to a heterogeneous set of proteins by
a pathway that is parallel to, but also distinct from, ubiquitination (Narasimhan et al., 1996
). Like Ub, UCRP also
appears to function as a posttranslationally added signal, targeting modified substrates to intermediate filaments in
the cytoplasm (Loeb and Haas, 1994
).
Recently, a novel family of Ub-like proteins, termed
small ubiquitin-like modifiers (SUMOs), has been described
that, while sharing only 18% identity with Ub, also appear
to be processed and covalently ligated to protein substrates by mechanisms similar to ubiquitination (Boddy et
al., 1996; Mannen et al., 1996
; Matunis et al., 1996
; Okura et
al., 1996
; Shen et al., 1996a
; Mahajan et al., 1997
). The first
member of this family of proteins, SUMO-1 (previously
called GMP1, PIC1, UBL1, and Sentrin), was identified as
a covalent modification of the Ran GTPase-activating protein, RanGAP1 (Matunis et al., 1996
; Mahajan et al., 1997
).
SUMO-1 was also identified in two-hybrid screens with the
Fas/APO-1 receptor (Okura et al., 1996
), PML (Boddy et
al., 1996
), and Rad51 and Rad52 (Shen et al., 1996a
), although
it remains to be determined whether SUMO-1 is covalently
ligated to these proteins. While RanGAP1 remains the only
confirmed substrate for SUMO-1 modification, SUMO-1
is conjugated to a limited number of predominantly nuclear, high molecular mass proteins (Matunis et al., 1996
;
Kamitani et al., 1997
; Mahajan et al., 1997
). As predicted
from conserved elements in its primary sequence, reactions
involved in SUMO-1 processing and ligation parallel ubiquitination. First, SUMO-1 contains a COOH-terminal extension of four amino acids (HSTV) that is proteolytically
removed to expose the mature COOH terminus ending
with a double glycine (Kamitani et al., 1997
). This double glycine is invariant in all known Ub and SUMO proteins.
Secondly, the exposed COOH-terminal glycine residue is
essential for subsequent conjugation reactions, similar to
the COOH-terminal glycine of Ub (Kamitani et al., 1997
).
And finally, SUMO-1 modification is reversible, as is ubiquitination (Matunis et al., 1996
; Mahajan et al., 1997
). Unlike Ub, SUMO-1 does not appear to form polymeric chains (at least when attached to RanGAP1), and as will
be discussed below, SUMO-1 modification has novel functions distinct from those of ubiquitination.
Saccharomyces cerevisiae contains a single SUMO protein, encoded by the SMT3 gene, which is 40% identical to
SUMO-1 (Meluh and Koshland, 1995). The SMT3 gene
was first identified in a genetic screen as a suppressor of
MIF2, a centromere-associated protein (Meluh and Koshland, 1995
). Smt3p also functions as a Ub-like modifier,
and considerable progress has recently been made in characterizing its processing and conjugation reactions. After proteolytic processing, Smt3p is activated by a heterodimeric enzyme consisting of Uba2p, a protein homologous to the
COOH terminus of Ub-activating enzymes (E1s), and Aos1p,
a protein homologous to the NH2 terminus of E1s (Johnson
et al., 1997
). The second step in the pathway, Smt3p conjugation, is mediated by Ubc9p, a protein homologous to
Ub-conjugating enzymes, or E2s (Johnson and Blobel, 1997
).
While the enzymes involved in conjugating the vertebrate
SUMOs have not been identified to date, it can be anticipated that they will be homologous to those required for
Smt3p.
As indicated, SUMO-1 was identified as a covalent modification of the Ran GTPase-activating protein, RanGAP1
(Matunis et al., 1996; Mahajan et al., 1997
). Ran is a small
nuclear ras-like GTPase required for the bidirectional
transport of proteins and ribonucleoproteins across the
nuclear pore complex (NPC) (Melchior et al., 1993
; Moore
and Blobel, 1993
; for review see Rush et al., 1996
). RanGAP1 is the only known GTPase-activating protein for Ran (Bischoff et al., 1994
, 1995a
,b), and its subcellular localization
is, therefore, an important indicator of where RanGTP hydrolysis is required during transport through the NPC (for
recent reviews of nuclear transport see Corbett and Silver,
1997
; Nigg, 1997
). In vertebrate cells, unmodified RanGAP1
is present in the cytoplasm and SUMO-1-modified RanGAP1
is associated with the cytoplasmic fibers of NPCs (Matunis et al., 1996
; Mahajan et al., 1997
). This observation led us
to propose that SUMO-1 modification functions to target
RanGAP1 to the NPC. Here, we provide evidence for this
proposal by demonstrating that mutations in RanGAP1
that block SUMO-1 modification also prevent RanGAP1
from localizing at the NPC. We have also investigated the
mechanism by which SUMO-1 modification targets RanGAP1
to the NPC. Our results indicate that SUMO-1 modification exposes, or creates, a binding site in the COOH-terminal domain of RanGAP1 that binds to a region in the
COOH terminus of Nup358, a nucleoporin associated
with the cytoplasmic fibers of the NPC (Wu et. al., 1995;
Yokoyama et al., 1995
). Finally, we have identified a nuclear localization signal in the COOH-terminal domain of
RanGAP1, raising the interesting possibility that RanGAP1
may shuttle between the nucleus and the cytoplasm.
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Materials and Methods |
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Plasmid Constructions
The cDNA coding for mouse RanGAP1 was kindly provided by Dr. Mark
Rush (New York University, New York; Ren et al., 1995). The expression
vector coding for myc-tagged, wild-type RanGAP1 was constructed as follows: a synthetic 5
PCR primer complementary to the NH2 terminus of
RanGAP1 and containing an EcoRI site was used in conjunction with a 3
primer complementary to the COOH terminus and containing a NotI site
to prime PCR using the mouse RanGAP1 cDNA as template. The amplified fragment was digested with EcoRI and NotI and ligated into EcoRI-NotI-digested pcDNA3-mycPK, replacing the insert coding for pyruvate
kinase. pcDNA3-mycPK was kindly provided by Dr. Haru Siomi (University of Pennsylvania, Philadelphia, PA) and contains an insert cloned into
the EcoRI and NotI sites coding for pyruvate kinase, and an insert cloned into the BstXI and EcoRI sites that codes for the sequence MEQKLISEEDL in frame with pyruvate kinase (Siomi and Dreyfuss, 1995
). This sequence is the epitope recognized by the anti-myc monoclonal antibody,
9E10 (Evan et al., 1985
). COOH- and NH2-terminal deletions of
RanGAP1 were generated using a similar strategy, using PCR primers
complementary to appropriate coding sequences. Point mutations in the
COOH terminus of RanGAP1 were generated using the Altered Sites II
Mammalian Mutagenesis System (Promega Corp., Madison, WI). The
cDNA coding for myc-tagged RanGAP1 was excised from pcDNA3 using
KpnI and NotI and ligated into KpnI-NotI-digested pAlter-Max. Oligonucleotide-directed point mutations were generated according to instructions provided by the manufacturer. Vectors for expressing pyruvate kinase-RanGAP1 fusion proteins were synthesized as follows: 5
primers
complementary to the appropriate coding sequences of RanGAP1 and
containing EcoRI sites were used in conjunction with 3
primers complementary to the COOH terminus of RanGAP1 and containing XhoI sites
to prime PCR using mouse RanGAP1 cDNA as template. The amplified
fragments were digested with EcoRI and XhoI and ligated into EcoRI-XhoI-digested pcDNA1-mycPK. pcDNA1-mycPK was kindly provided
by Dr. Matthew Michael (University of San Diego, San Diego, CA) and
contains an insert coding for myc-tagged pyruvate kinase cloned into the
BamHI and EcoRI sites of pcDNA1 (Michael et al., 1995
). The expression
vector for the SUMO-1-pyruvate kinase fusion protein was made by PCR
amplification of a fragment of SUMO-1 coding for amino acids 1-95. The
resulting fragment, containing a BamHI site at the 5
-end and a BstXI site
at the 3
-end, was digested and cloned into the BamHI and BstXI sites of
pcDNA3-mycPK.
Transfections and Immunofluorescence Localization
HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were grown on glass coverslips in
35-mm dishes and transfected with 1 µg of plasmid using lipofectamine
(GIBCO-BRL, Gaithersburg, MD), as described by the manufacturer. 36 h
after transfection, the cells were washed twice with PBS, fixed for 30 min
at room temperature with 2% formaldehyde in PBS, and permeabilized
with 20°C acetone for 3 min. After rinsing three times with PBS, the
cells were incubated with the anti-myc monoclonal antibody, 9E10 (diluted in PBS containing 2% BSA), for 1 h at room temperature, followed
by three more washes with PBS. Cells were subsequently incubated with
fluorescein-conjugated goat anti-mouse (diluted in PBS containing 2%
BSA) for 30 min at room temperature. After three washes with PBS, coverslips were mounted in buffer containing 80% glycerol, 50 mM Tris-HCl,
pH 8.0, and 0.1% p-phenylenediamine and analyzed with a Zeis Axiophot fluorescence microscope (Thornwood, NY).
Gel Electrophoresis and Immunoblot Analysis
HeLa cells transfected as described above were lysed in SDS sample
buffer 36 h after transfection. Proteins were separated by SDS-PAGE and
transferred to nitrocellulose membrane as previously described (Dreyfuss
et al., 1984). Membranes were blocked in 5% nonfat dry milk in PBST
(PBS containing 0.1% Tween-20), followed by incubation with monoclonal antibody 9E10 (diluted 1:1,000 in PBST containing 2% BSA). Antibodies were detected using luminol-based chemiluminescence.
Expression and Purification of Recombinant Proteins
DNA fragments coding for specific regions of Nup358 were generated by
PCR amplification using appropriate complementary primers. These fragments were cloned into pGEX-2TL (pGEX-2T with a modified multiple
cloning site; generously provided by Dr. Hui Ge, National Institute of
Child Health and Human Development, National Institutes of Health,
Bethesda, MD), and the plasmids were transformed into DH5 cells. Protein expression was induced with 0.1 mM IPTG, and proteins were purified by affinity chromatography using glutathione Sepharose 4B as described by the manufacturer (Pharmacia Biotech Inc., Piscataway, NJ).
In Vitro Protein Binding Assay
RanGAP1 substrates were expressed in rabbit reticulocyte transcription/
translation extracts in the presence of [35S]methionine as described by the
manufacturer (Promega Corp.). Incorporation of radioactive label was determined by TCA precipitation followed by filter binding and counting in
a liquid scintillation counter. Binding assays were performed as follows: 1 µg
of each purified protein was diluted into 100 µl of PBS and bound to the wells of a microtiter plate overnight at 4°C. The wells were subsequently blocked with 150 µl of assay buffer (2% BSA, 20 mM Hepes-KOH, pH
7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA,
0.05% Tween-20) for 1 h at room temperature. In vitro translated RanGAP1 substrates were diluted into 100 µl of assay buffer and incubated with the bound proteins for 1 h at room temperature. After five
washes with 150 µl of assay buffer, proteins were eluted with SDS sample
buffer. Quantification of N419 and N
470 binding was as follows: assays
were performed as described above in triplicate, loading equivalent
amounts of N
419 and N
470 to the assays (based on radioactive incorporation during in vitro translation). Radioactive counts bound in each
binding assay were determined using a liquid scintillation counter, and
counts bound to glutathione-S-transferase (GST) were subtracted from
each assay as background. N
419 binding to Nup358 fragment 2500-C was
arbitrarily set at 100%, and percentages bound in all other assays were
calculated relative to this reaction. Approximately 20% of the SUMO-1-modified N
419 added to the assays was bound by 2500-C. 2500-C is estimated to be at ~200-fold molar excess over SUMO-1-modified N
419.
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Results |
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Lysine 526 Is Required for SUMO-1 Modification of RanGAP1
To identify elements of RanGAP1 required for SUMO-1 modification, we developed a rapid and simple in vitro assay using rabbit reticulocyte transcription and translation extracts. When a cDNA coding for myc-tagged RanGAP1 was transcribed and translated in an vitro extract, two products were observed, migrating with relative molecular masses of 75 and 95 kD (Fig. 1, lane 1). Both products appeared as doublets, possibly as a result of initiation of translation from an internal methionine. Their 20-kD difference suggested that the larger product represented SUMO-1-modified RanGAP1, which was confirmed by immunopurification with antibodies specific for SUMO-1 (data not shown). These results indicate that rabbit reticulocyte extracts contain all of the necessary factors for SUMO-1 modification of RanGAP1.
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We used this in vitro assay to analyze a series of NH2-
and COOH-terminal deletions of RanGAP1 and found
that the COOH-terminal domain of RanGAP1 is essential
for SUMO-1 modification (data not shown; also see Fig. 3
B). To identify specific amino acid residues required for
SUMO-1 modification, lysines in the COOH terminus of
RanGAP1 were systematically mutated to arginine, based
on the assumption that SUMO-1 conjugation would occur
via a lysine, similar to ubiquitination (Wilkison, 1995;
Hochstrasser, 1996). Substitution of arginine for lysine residues at positions 567, 555, 532, and 530 had no effect on
RanGAP1 modification in vitro (data not shown). However, when lysine 526 was mutated to arginine, RanGAP1
was no longer modified by SUMO-1 (Fig. 1, lane 2). This
finding identifies lysine 526 of RanGAP1 as essential for
modification and implicates this residue as the acceptor
for SUMO-1 conjugation.
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SUMO-1 Modification Is Required for Localization of RanGAP1 at the NPC
To analyze the effects of SUMO-1 modification on RanGAP1 localization, we next transfected cells with plasmids coding for either wild-type RanGAP1 or for mutant RanGAP1 with the lysine to arginine substitution at residue 526 (K/R 526). Both proteins were designed to contain a myc epitope tag at their NH2 terminus, which was used for immunolocalization and for immunoblot analysis. Transiently expressed wild-type protein showed a pattern of localization similar to the endogenous RanGAP1; it was detected both in the cytoplasm and concentrated at the nuclear envelope (Fig. 2 A, a). Discontinuous rim staining at the equatorial plane of the nuclear envelope, and punctate staining on the surface indicated association with NPCs. In cells expressing increasing amounts of RanGAP1, NPC labeling remained constant while the cytoplasmic signal increased, indicating that NPC binding sites were saturated. In contrast to wild-type RanGAP1, the K/R 526 mutant form of RanGAP1 was confined strictly to the cytoplasm and showed no evidence of association with the nuclear envelope or NPCs even at the lowest levels of expression (Fig. 2 A, b). No signal was detected in untransfected cells (data not shown).
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To determine whether the transiently expressed forms of RanGAP1 were modified in vivo as predicted, immunoblot analysis was performed on lysates prepared from transfected cells. Consistent with the in vitro translation assays, modified RanGAP1 was detected in cells transfected with wild-type RanGAP1 (Fig. 2 B, lane 1), but not in cells expressing RanGAP1 with the K/R 526 mutation (Fig. 2 B, lane 2). The relative ratio of modified to unmodified RanGAP1 was very low, indicating that SUMO-1 conjugation to RanGAP1 is closely regulated. These results confirm that lysine 526 is required for SUMO-1 modification of RanGAP1 in vivo, and they provide further evidence that SUMO-1 modification is required for RanGAP1 localization at the NPC.
The COOH-terminal Domain of RanGAP1 Specifies SUMO-1 Modification and NPC Localization
We next designed a series of constructs to assay for the
minimal sequence in RanGAP1 required to specify
SUMO-1 modification (Fig. 3 A). Either RanGAP1 with
COOH-terminal deletions or pyruvate kinase fused to regions of the COOH-terminal domain of RanGAP1 were
assayed for SUMO-1 modification using the in vitro assay described above. When RanGAP1 lacking the COOH-terminal 23 amino acids (C23) was translated in rabbit reticulocyte lysate, modified RanGAP1 was not detected, indicating that the extreme COOH terminus of RanGAP1 is
required for SUMO-1 modification (Fig. 3 B, lane 2).
Pyruvate kinase fused with amino acids 420-589 of
RanGAP1 (N
419/PK), as well as amino acids 471-589
(N
470/PK), were modified with efficiencies similar to
wild-type RanGAP1 (Fig. 3 B, lanes 1, 3, and 4), indicating
that the first 470 amino acids of RanGAP1 could be deleted without affecting SUMO-1 modification. When pyruvate kinase was fused to amino acids 503-589 of RanGAP1
(N
502/PK), however, no modification was detected, indicating that amino acid residues between 471 and 502 are
also required for SUMO-1 modification (Fig. 3 B, lane 5).
Together, these data indicate that SUMO-1 modification
of RanGAP1 requires a domain extending from amino acid 470 to the COOH terminus.
To investigate the determinants specifying NPC localization further, the constructs described above were transfected into HeLa cells, and the transiently expressed proteins were localized by indirect immunofluorescence. As
evident in Fig. 4 a, pyruvate kinase was localized in the cytoplasm and showed no detectable nuclear envelope staining. C23, which is not a substrate for SUMO-1 modification, was also strictly cytosolic (Fig. 4 b). In contrast,
N
419/PK was concentrated at the nuclear envelope in a
pattern consistent with NPC binding and also in the nucleus (Fig. 4 c). N
470/PK was also localized to the nucleus but showed no evidence of NPC binding, despite the
fact that it is predicted to be modified by SUMO-1 (Fig. 4 d).
N
502/PK showed a similar intranuclear localization and
no NPC staining (Fig. 4 e). These results indicate that
SUMO-1 modification is required, but not sufficient, for
RanGAP1 localization at the NPC. This conclusion is further supported by the finding that pyruvate kinase, expressed as a fusion protein with SUMO-1, was strictly cytosolic (Fig. 4 f). The difference in localization between
N
419 and N
470 indicate that the 50 amino acid residues
between 419 and 470 are also required for localization of
RanGAP1 at the NPC, in addition to SUMO-1 modification.
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To determine whether the proteins transiently expressed in vivo were modified as expected, lysates from
transfected cells were analyzed by immunoblot analysis
using an antibody to the myc epitope tag. In cells expressing N419/PK and N
470/PK, SUMO-1-modified forms
of the proteins were detected, with approximately half of the proteins present in the modified form (Fig. 5, lanes 2 and 3). This ratio of modified to unmodified protein was
noticeably higher than that observed with wild-type
RanGAP1, where a relatively small amount of modified
protein was detected (see Fig. 2, lane 1). Surprisingly,
N
502/PK was also modified in vivo, contrary to the in
vitro translation result, with the ratio of modified to unmodified forms again being ~50% (Fig. 5, lane 4). SUMO-1 modification of the transiently expressed fusion proteins
was confirmed by immunoblot analysis with an antibody
specific for SUMO-1 (data not shown).
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RanGAP1 Contains a Functional Nuclear Localization Signal and Nine Putative Nuclear Export Signals
The localization of the pyruvate kinase fusion proteins
(Fig. 4, c-e) indicated that the COOH terminus of
RanGAP1 contains a nuclear localization signal (NLS).
This was further mapped using additional pyruvate kinase
fusion proteins. When fused to amino acids 541-589 of
RanGAP1 (N540/PK), pyruvate kinase was again targeted to the nucleus (Fig. 6 a). However, when fused with
amino acids 556-589 of RanGAP1 (N
555/PK), pyruvate
kinase was detected only in the cytosol (Fig. 6 b). These
results indicated that amino acids 541-589 of RanGAP1
can function as an NLS. A database search with the NLS
of RanGAP1 (shown in Fig. 7 B) revealed no significant homologies with other proteins and no homologies to
other known NLSs. However, the nine amino-terminal
leucine-rich repeats of RanGAP1 were each found to have
striking homology with the leucine-rich nuclear export sequence motif (Fig. 7 C), first described for the HIV REV
protein and the cAMP-dependent protein kinase inhibitor (Fischer et al., 1995
; Wen et al., 1995
). The presence of potential nuclear import and export signals raises the interesting possibility that RanGAP1 may shuttle between the
nucleus and the cytoplasm.
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SUMO-1-modified RanGAP1 Binds to a COOH-terminal Region of Nup358
SUMO-1-modified RanGAP1 interacts with Nup358 (Mahajan et al., 1997; Saitoh et al., 1997
), a nucleoporin that is
a component of the cytoplasmic fibers of the NPC (Wu et
al., 1995
; Yokoyama et al., 1995
). To characterize this interaction in more detail, we used an in vitro binding assay
using bacterially expressed regions of Nup358 and radiolabeled RanGAP1 produced in rabbit reticulocyte extracts.
Purified GST fusion proteins corresponding to various domains of Nup358 were bound to the wells of a microtiter plate and incubated with in vitro-translated RanGAP1.
After incubation, the wells were washed, and bound proteins were eluted with SDS sample buffer. No binding of
either modified or unmodified RanGAP1 was detected to
regions of Nup358 corresponding to the NH2-terminal leucine-rich region, the Ran-binding domains, or the cyclophilin homologous domain (data not shown). RanGAP1
binding was, however, detected with a region of Nup358
extending from amino acid 2500 to the COOH terminus
(Fig. 8 B, lane 2). Binding was specific for SUMO-1-modified RanGAP1, with no unmodified RanGAP1 being detected. The specificity of the binding was further demonstrated by the absence of interactions with GST (Fig. 8 B,
lane 5). The region of Nup358 from amino acid 2500 to the
COOH terminus contains two Ran-binding domains (domains three and four) separated by a 470-amino acid segment, and the cyclophilin homology domain. The 470-
amino acid segment between the two Ran-binding domains contains direct repeats of ~40 amino acids (Yokoyama et
al., 1995
). Because our results showed that the Ran-binding
domains and cyclophilin homology domain did not interact with RanGAP1, we assayed more specifically for binding to the region separating Ran-binding domains three
and four. This domain is flanked by clusters of FXFG repeats, and fragments with (amino acids 2503-2893) and
without (amino acids 2550-2837) these repeats were tested
in the binding assay. SUMO-1-modified RanGAP1 bound
specifically to both of these fragments (Fig. 8 B, lanes 3 and 4). However, interaction with the fragment lacking
FXFG repeats was of lower affinity relative to the fragment containing FXFG repeats, based on the four- to fivefold reduction in observed binding (see Fig. 9). Again, unmodified RanGAP1 did not bind to these fragments.
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Based on the in vivo NPC targeting data, it was anticipated that the COOH terminus of RanGAP1, from amino
acid 420 to 589 (N419), would be sufficient for interaction with Nup358. We therefore translated this domain in
vitro (without pyruvate kinase) and assayed for its ability
to bind to the regions of Nup358 found to interact with
full-length, modified RanGAP1. Both SUMO-1-modified and unmodified forms of N
419 were produced when
translated in vitro (Fig. 8 B, lane 6), and as observed with
full-length RanGAP1, the modified protein bound to the
region of Nup358 between Ran-binding domains three
and four, whereas the unmodified protein did not (Fig. 8
B, lanes 7-9). Similar to full-length RanGAP1, N
419 had a lower affinity for the domain lacking FXFG repeats,
based on a fivefold reduction in binding compared with
the domain containing these repeats (Fig. 8 B, lanes 8 and
9; Fig. 9). We next assayed for binding with the region of
RanGAP1 between amino acids 471 and 589 (N
470),
which is SUMO-1 modified but does not have NPC targeting activity in vivo. N
470 also showed the same binding specificity as full-length RanGAP1 and N
419, with only
the SUMO-1-modified form binding to the region of
Nup358 between Ran-binding domains three and four
(Fig. 8 B, lanes 12-14). However, when their relative binding properties were compared, it was found that N
470
had a much lower affinity for Nup358, as evidenced by a
six- to sevenfold reduction in the observed binding compared with N
419 (Fig. 9). These results are consistent
with the in vivo localization data and indicate that the region of RanGAP1 between amino acids 420 and 470 stabilizes the interaction between RanGAP1 and the COOH terminus of Nup358. As previously reported, competition
with free SUMO-1 had little if any effect on the binding of
SUMO-1-modified RanGAP1 to Nup358 (data not shown;
Mahajan et al., 1997
).
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Discussion |
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We have further characterized the ubiquitin-related protein, SUMO-1, and its effects on RanGAP1 localization. Our results, based on several independent lines of analysis, indicate that SUMO-1 modification is required for the association of RanGAP1 with the NPC. First, we have previously shown by cellular fractionation and immunoelectron microscopy that unmodified RanGAP1 is strictly cytoplasmic, whereas SUMO-1-modified RanGAP1 is primarily associated with the NPC. While this result was in itself suggestive, SUMO-1 modification as a cause for RanGAP1 association with the NPC, or an effect of RanGAP1 association with the NPC, could not be distinguished. Here, we have mapped the domain in RanGAP1 specifying SUMO-1 modification and show that a single amino acid substitution in this domain that prevents SUMO-1 modification also prevents NPC targeting. In addition, we have demonstrated that the binding of RanGAP1 to a COOH-terminal domain in Nup358 is positively regulated by SUMO-1 modification. Together, these data indicate that SUMO-1 modification functions to target RanGAP1 to the NPC through regulation of its interaction with Nup358.
Our results indicate that SUMO-1 modification is not
the only element required for targeting RanGAP1 to the
NPC. This conclusion is based on the observations that
neither direct fusion of SUMO-1 to pyruvate kinase, nor
fusion of the minimal domain of RanGAP1 that specifies
SUMO-1 modification of pyruvate kinase, is sufficient to
target RanGAP1 to the NPC. This result is not completely
unexpected, as RanGAP1 is the only SUMO-1 modified
substrate associated with the NPC, the remaining substrates being located primarily in the nucleus (Matunis et al.,
1996; Mahajan et al., 1997
). Differences in the subcellular
localization of the constructs N
419/PK and N
470/PK indicate that the 50 amino acids between residues 420 and
470 of RanGAP1 are also required for NPC targeting. In
vitro binding studies with these regions of RanGAP1 demonstrate that this 50-amino acid domain is required for efficient binding to Nup358. When expressed as a fusion
protein with pyruvate kinase, this 50-amino acid domain
(and several larger domains) was not sufficient to mediate
Nup358 binding or NPC localization (data not shown).
This result suggests that SUMO-1 is either required to
form part of the Nup358 binding site or that the binding
site overlaps with elements that inhibit binding in the absence of modification. Based on these observations, we
propose that SUMO-1 functions to target RanGAP1 to
the NPC by exposing, or possibly creating, a Nup358 binding site. In this capacity, SUMO-1 is different from Ub and
UCRP, which appear to mediate the interactions between their substrates and their intracellular targets directly (Deveraux et al., 1994
; Loeb et al., 1994). Rather, SUMO-1
modification of RanGAP1 appears to be more analogous
to a phosphorylation event that exposes a masked signal
sequence.
The most significant mutation affecting RanGAP1 modification was a lysine to arginine substitution at residue
526. The effect of this single conservative substitution on
RanGAP1 strongly implicates lysine 526 as the acceptor
for SUMO-1 ligation, although protein sequence analysis
of modified RanGAP1 will be required for definitive proof. In addition to lysine 526, deletion analysis indicated that a region from amino acid 470 to the COOH terminus
is required for SUMO-1 modification in vitro. (In vivo results suggest that this region may actually be smaller.) This
region is likely to contain elements required for recognition of RanGAP1 by the SUMO-1-conjugating enzyme
(E2) and possibly SUMO-1 ligase (E3), if one is required. Alignment of the region between amino acids 470 and the
COOH terminus of RanGAP1 with sequences in the protein data bases revealed a surprising homology with the
Ran-binding motifs of the yeast nucleoporins, Nup2 and
Nup36 (data not shown; Loeb et al., 1993; Noguchi et al.,
1997
). Whether this region in RanGAP1 binds Ran, and
whether yeast Nup2 and Nup36 are substrates for SUMO-1
modification, remain to be determined. The yeast homologue of RanGAP1, Rna1p (Traglia et al., 1989
; Bischoff
et al., 1995a
), lacks the entire COOH-terminal domain
found in vertebrate RanGAP1 and is therefore not likely to be a substrate for SUMO-1 modification.
While other substrates for SUMO-1 have not yet been
confirmed, a number have been implicated in both yeast
and vertebrates based on genetic analysis and two-hybrid
screens. The yeast homologue of SUMO-1, Smt3p, was
first identified as a suppressor of a temperature-sensitive allele of MIF2, which codes for a centromere binding protein (Meluh and Koshland, 1995). In addition, SUMO-1
was found to interact with the death domain of the Fas/
APO-1 receptor, which is involved in apoptosis (Okura et al.,
1996
), and with Rad51 and Rad52, proteins involved in
DNA recombination and repair (Shen et al., 1996a
). Finally, SUMO-1 was also identified in a two-hybrid screen
with PML, a protein normally localized to discrete nuclear
foci, called PML nuclear bodies (Boddy et al., 1996
). PML
fusion with the retinoic acid receptor, as a result of chromosomal translocation, leads to a redistribution of PML
throughout the nucleus and acute promyelocytic leukemia.
The functional relevance of the interactions involving each
of these proteins, and whether they are substrates for
SUMO-1 modification, remains to be determined.
A significant body of evidence now indicates that the E2
enzyme for SUMO-1 conjugation is Ubc9p (Seufert et al.,
1995), including biochemical and genetic evidence that
Ubc9p is essential for Smt3p modification in yeast (Johnson
and Blobel, 1997
). In addition, vertebrate Ubc9p has been
found to interact with SUMO-1 by two-hybrid analysis
(Shen et al., 1996b
) and to be present in a complex with Nup358 and modified RanGAP1 in Xenopus egg extracts
(Saitoh et al., 1997
). The association of Ubc9p with Nup358
raises the interesting question of where RanGAP1 is modified in vivo. Immunoblot analysis of whole cell extracts indicates that approximately half of the endogenous RanGAP1
is modified by SUMO-1 and that most, if not all, of this is
associated with the NPC (Matunis et al., 1996
; Mahajan et
al., 1997
). In this study, we found that overexpression of
RanGAP1 leads to a relatively small fraction of RanGAP1
being modified and localized to the NPC, with the majority of the protein accumulating unmodified and in the cytoplasm. These observations suggest that SUMO-1 modification is sensitive to the association of RanGAP1 with the
NPC and can be explained by a number of models. First,
modified RanGAP1 in the cytoplasm may negatively regulate SUMO-1-conjugating enzymes once sites at the NPC
are saturated. Alternatively, unmodified RanGAP1 may
associate with the NPC transiently, being modified after
this association by Nup358-bound Ubc9p. Modification, as
we have shown, would allow RanGAP1 to form a stable
complex with Nup358. A third possibility is that RanGAP1
could be modified by SUMO-1 in the cytoplasm, with the
modification being rapidly removed by a SUMO-1 demodifying activity. According to this model, interaction with
Nup358 would stabilize modified RanGAP1 by preventing removal of SUMO-1. Better characterization and subcellular localization of the SUMO-1 conjugating and demodifying enzymes will be required to test these models. Interestingly, we also observed that SUMO-1 modification of
several nuclear pyruvate kinase fusion proteins (N
419,
N
470, and N
502) appeared to be less strictly regulated compared with modification of cytoplasmic RanGAP1.
This finding suggests that there may be differences in the
regulation of SUMO-1 modification in different subcellular compartments. The modification of N
502 in vivo, but
not in vitro, could also be indicative of SUMO-1 conjugating activities with different specificities.
Our data indicate that the COOH-terminal domain of
RanGAP1 also contains a novel nuclear localization signal. This NLS was mapped to amino acids 541-589 by virtue of its ability to target pyruvate kinase to the nucleus,
although a minimal domain remains to be defined. Similar
fusions with pyruvate kinase have been used to identify
the NLSs of a number of proteins, including the SV-40
large T antigen (Kalderon et al., 1984), p53 (Dang and
Lee, 1989), and hnRNP A1 (Siomi and Dreyfuss, 1995
). At
this time, we have no evidence that the NLS in RanGAP1
actually functions to target RanGAP1 to the nucleus, and
immunofluorescence data actually indicates that there is
little if any intranuclear RanGAP1 (Matunis et al., 1996
;
Mahajan et al., 1997
). However, the presence of nine potential nuclear export signals in its NH2 terminus raises the
interesting possibility that RanGAP1 may shuttle between
the nucleus and the cytoplasm. Consistent with this possibility, we previously found ~10% of the NPC-associated
RanGAP1 to be located on the nucleoplasmic side of the
pore complex (Matunis et al., 1996
). RanGAP1 could conceivably shuttle to regulate RanGTP hydrolysis on the nucleoplasmic side of the NPC, where RanGTP has been
proposed to bind to karyopherin
1 (importin
) to release
import substrates into the nucleoplasm (Görlich et al.,
1996
). It appears almost certain that RanGTP hydrolysis
will occur at the cytoplasmic filaments of the NPC, where
we have localized SUMO-1-modified RanGAP1. We have
mapped the binding site for RanGAP1 to a region on
Nup358 that is flanked by Ran-binding domains. The Ran-binding domains in Nup358 are homologous to the Ran-binding domain of RanBP1 (Coutavas et al., 1993
), and
like RanBP1, these domains costimulate GTP hydrolysis
by Ran in the presence of RanGAP1 (Beddow et al.,
1995
). RanGTP hydrolysis at the cytoplasmic filaments
likely serves the dual purpose of preventing free RanGTP
from entering the cytoplasm, where it could prematurely
dissociate karyopherin/substrate complexes (Rexach and
Blobel, 1995
), and providing RanGDP, which has been proposed to be the active form of Ran required for nuclear
import (Nehrbass and Blobel, 1995; Görlich et al., 1996
;
Weis et al., 1996
). RanGTP hydrolysis could also be involved in recycling RanGTP-bound karyopherin
1 (Floer
et al., 1997
), and the nuclear export receptor, CRM1 (Fornerod et al., 1997
; Stade et al., 1997
). We found that the
FXFG and FG repeats affected RanGAP1 binding, suggesting that RanGAP1 may have some affinity for these
repeats, or that there may be interactions between RanGAP1
and other factors bound to these repeats (Moroianu et al.,
1995
; Radu et al., 1995
). (Reticulocyte extracts contain,
among other factors, karyopherin
and
1 [Adam and
Adam, 1994
].) It remains to be tested whether the longer, direct repeats in the RanGAP1 binding site on Nup358 are
involved in the interaction with RanGAP1. It is conceivable that each of these repeats serves as a binding site for a
single molecule of RanGAP1, making it possible for two
molecules of RanGAP1 to bind to a single molecule of
Nup358. The binding assays that we have performed to
date do not allow us to address this issue.
In conclusion, our findings indicate that SUMO-1 modification exposes, or creates, a binding site on RanGAP1 that enables it to interact with Nup358. A similar function has not been described previously for Ub or any other Ub-like protein and demonstrates the versatility of this type of posttranslational protein modification. Other effects of SUMO-1 modification on RanGAP1 have not been identified but could possibly include regulation of its GTPase-activating activity or its interactions with additional proteins. It is apparent that SUMO-1 modification will have functions other than that described here for RanGAP1, given that the majority of SUMO-1-modified substrates are not localized at the NPC. Understanding these additional functions awaits identification of other SUMO-1 substrates.
![]() |
Footnotes |
---|
Received for publication 2 October 1997 and in revised form 14 November 1997.
Address all correspondence to Michael Matunis, Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021. Tel.: (212) 327-8101. Fax: (212) 327-7880. E-mail: matunim{at}rockvax.rockefeller.eduWe thank members of the Blobel lab, especially Elias Coutavas, Beatriz Fontoura, and Erica Johnson, for helpful discussions and critical reading of the manuscript. We are also grateful to Dr. Haruhiko Siomi and Dr. Matthew Michael for generously providing us with Myc-tagged expression vectors, and Dr. Hui Ge for providing us with a GST expression vector.
This work was supported by the Howard Hughes Medical Institute. M.J. Matunis is an American Cancer Society-Amgen Fellow (PF-4195), J. Wu is supported by a National Institutes of Health Fellowship (1F32GM16758-01).
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Abbreviations used in this paper |
---|
GST, glutathione-S-transferase; NLS, nuclear localization signal; NPC, nuclear pore complex; SUMO-1, small ubiquitin-like modifier 1; Ub, ubiquitin; UCRP, ubiquitin cross-reactive protein.
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