Identification of the Biochemical Link between RanGAP1 and SUMO-1
RanGAP1 is presently the only characterized target for modification by SUMO-1 (or by any of the other proteins distantly related to ubiquitin). To better understand the mechanism and regulation of this modification, we decided to map the residues involved in the attachment of SUMO-1 to RanGAP1. By analogy to ubiquitin, it seemed plausible that modification of RanGAP1 by SUMO-1 occurred via formation of an isopeptide bond between a COOH-terminal amino acid of SUMO-1 and the ε-amino group of a lysine in RanGAP1 (see introduction). However, considering the extremely low homology of SUMO-1 to ubiquitin (18% identity), alternative links also seemed possible, particularly since ubiquitin itself seems capable of forming alternative nonlysine links (Hodgins et al., 1996
To identify the link between RanGAP1 and SUMO-1, we carried out peptide analysis. For this, modified and unmodified RanGAP1 obtained by immunoprecipitation from rat liver NEs were digested with trypsin, and the tryptic fragments were separated via chromatography on a C8 reversed-phase HPLC column. Fig. A shows a comparison of chromatographic profiles of RanGAP1–SUMO-1 (top line) and unmodified RanGAP1 (bottom line). When chromatographic peaks unique to the RanGAP1–SUMO-1 conjugate were subjected to micropeptide sequencing, we found that one peak that migrated with a retention time of 27 min (Fig. A, arrow) apparently contained two peptides at an equimolar ratio; one from RanGAP1 and one from SUMO-1. The sequences determined for these two peptides are displayed above the arrow in Fig. A, together with additional residues that could not be unambiguously identified (indicated by an X). Since the SUMO-1 peptide was derived from the COOH-terminal end of SUMO-1, it seemed possible that the other peptide might represent the RanGAP1 region to which it was coupled by an isopeptide bond. If that were the case, one might expect to also see disappearance of a peak corresponding to the unconjugated peptide in the unmodified RanGAP1. Although this was not observed in the profiles shown here, after chromatography on a different column (capillary C18 column) we did indeed identify a peptide with the sequence LLIHMGLLK in the tryptic digest of unconjugated RanGAP1 that was absent from conjugated RanGAP1. However, under those conditions the peak containing the potentially conjugated peptide was not resolved.
Figure 1 Identification of a linked peptide between RanGAP1 and SUMO-1. (A) Microbore C8 column profile of tryptic digests of SUMO-1–modified (top profile) and unmodified (bottom profile) RanGAP1, obtained by immunoprecipitation from solubilized rat (more ...)
To test whether the coeluting peptides were indeed linked, we performed mass spectroscopy analysis on the isolated peak fraction (Fig. B). The mass values are significantly larger than those expected for either fragment alone and are consistent with a linked peptide. Our analysis revealed two sets of mass peaks, one with values of 3,634, 3,650, and 3,665 D; and a second with values of 3,878, 3,890, and 3,906 D. The differences in mass among the three peaks in each set suggested that the two larger peaks within a given set (labeled [O] in Fig. B) are methionine-oxidation products of the smaller species that are commonly obtained in gel-purified proteins. By considering the mass values, sequence information, and predicted tryptic products of the two proteins, we determined that the observed mass values correspond to a conjugate containing a single molecule of a SUMO-1 fragment ending at glycine 97 linked via a covalent bond to a RanGAP1 tryptic fragment that includes either residues 518–530 (3,634-D peak) or residues 518–532 (3,877-D peak; Fig. B). Since SUMO-1 is expressed as a 101–amino acid protein that contains four additional COOH-terminal amino acids beyond glycine 97, this demonstrates that SUMO-1 is proteolytically processed before its attachment to RanGAP1. Both RanGAP1 fragments include an internal lysine (K526) that appears to be protected from trypsin digestion, indicating that it may be involved in the formation of an isopeptide bond with SUMO-1. Theoretically, a link could also be formed via an ester bond involving serine 527. However, this possibility was ruled out by the resistance of RanGAP1–SUMO-1 to treatment with either base (pH 12) or 1% hydrazine, conditions that rapidly hydrolyze ester bonds (data not shown).
Taken together, these data strongly suggest that SUMO-1 modifies RanGAP1 via an isopeptide bond between the carboxyl group of glycine 97 of proteolytically processed SUMO-1 and the ε-amino group of lysine 526 of RanGAP1, as depicted in Fig. C.
SUMO-1 Terminating at Glycine 97 Is Fully Competent to Modify RanGAP1 In Vitro
To confirm that the attachment of SUMO-1 to RanGAP1 involves glycine 97 and not valine 101 of SUMO-1, and to determine whether removal of the last four amino acids of SUMO-1 either precedes or is mechanistically coupled to its attachment to RanGAP1, we prepared recombinant wt SUMO-1 along with a mutant SUMO-1 lacking the COOH-terminal four residues (SUMOΔC4). SUMOΔC4 terminates in the glycine–glycine motif corresponding to the COOH-terminal end observed in the peptide analysis (Fig. C), and represents the putative end product of the proteolytically processed protein. First, we tested the ability of the SUMO-1 proteins to modify recombinant Ran GAP1 in vitro by monitoring their ability to shift the 70-kD RanGAP1 to the 90-kD modified form. Recombinant RanGAP1 was added to a digitonin lysate of HeLa cells in the presence of ATP and bacterially expressed SUMO-1 proteins (Fig. ). After 10 min at RT, the reaction products were analyzed by immunoblotting with α-RanGAP1 antibodies. Fig. (lane 2) shows that in the absence of exogenously added SUMO-1, ~30–40% of the recombinant RanGAP1 was converted to the modified 90-kD species by the endogenous SUMO-1. Addition of exogenous recombinant SUMO-1 had only a negligible effect on the amount of RanGAP1 converted (Fig. , lane 3). In contrast, the addition of SUMOΔC4 significantly increased the amount of modified RanGAP1 with a concomitant decrease in the amount of unmodified 70-kD RanGAP1 (Fig. , lane 4). The effect of SUMOΔC4 was even more striking when the shift assays were performed using GST fusions of the two forms of SUMO-1. In this case, the GST-SUMO-1 converts a small amount of RanGAP1 to a unique 115 kD species (Fig. , lane 5). Under the same reaction conditions the GST-SUMOΔC4 converts significantly more of the 70-kD RanGAP1 to the 115-kD form (Fig. , lane 6). These data demonstrate that SUMOΔC4 can be used as a substrate for the modifying enzymes in vitro and indicate that proteolytic processing of SUMO-1 is not mechanistically coupled to the conjugation reaction.
Figure 2 SUMO-1 lacking the last four amino acids (SUMOΔC4) is a better substrate for modification of RanGAP1 in vitro than wt SUMO-1. Digitonin lysates of HeLa cells were mixed with RanGAP1 and ATP, and incubated for 10 min at RT in the absence (lane (more ...)
Moreover, since SUMOΔC4 is conjugated to RanGAP1 much more efficiently than wt SUMO-1, the proteolytic removal of the last four amino acids of SUMO-1 seems significantly slower than the modification reaction. It remains to be seen whether this processing activity is higher in different cell extracts.
Analysis of COOH-terminal Deletions of SUMO-1 In Vivo
To test whether SUMO-1 lacking the last four amino acids (SUMOΔC4) is also an efficient substrate for modification of RanGAP1 and possibly other proteins in vivo, we expressed a series of HA epitope-tagged SUMO-1 constructs in tissue culture cells. HA-tagged wt SUMO-1 was transfected into Cos-7 cells, and the distribution of exogenously expressed SUMO-1 proteins was detected 24 h after transfection by indirect immunofluorescence microscopy using an α-HA monoclonal antibody (Fig. A
, SUMO wt
). HA-tagged SUMO-1 accumulated in intranuclear foci or speckles in addition to a diffuse nucleoplasmic distribution (Fig. A
). A distinct nuclear rim localization, consistent with the localization of SUMO-1–modified RanGAP1 (Matunis et al., 1996
; Mahajan et al., 1997
), could be observed in Cos-7 cells expressing low levels of HA–SUMO-1 (Fig. A, SUMO wt
). The distribution of HA–SUMO-1 was identical to the localization of endogenous SUMO-1 in untransfected cells (Boddy et al., 1996
; Matunis et al., 1996
; data not shown).
Figure 3 SUMO-1 and SUMOΔC4, but not SUMOΔC6, modify multiple substrates in vivo. (A) Immunofluorescence of Cos-7 cells transfected with HA-tagged SUMO-1 constructs and probed with an α-HA monoclonal antibody. Intracellular localization (more ...)
When HA-SUMOΔC4 was transfected into Cos-7 cells, its distribution was indistinguishable from wt HA–SUMO-1 (Fig. A, SUMOΔC4). A strong nuclear signal was detected in >70% of the cells, with staining found diffusely in the nucleoplasm, nuclear rim, and nuclear speckles as observed for HA–SUMO-1. Cos-7 cells were also transfected with an HA-tagged SUMO-1 construct lacking six amino acids at the COOH terminus (SUMOΔC6). This construct lacks the conserved glycine doublet required for conjugation of ubiquitin to its targets and was not expected to be conjugated to target proteins. Under conditions in which >70% of the cells were successfully transfected, SUMOΔC6 expression was consistently barely visible by immunofluorescence microscopy (Fig. A, SUMOΔC6). What protein was detectable was not enriched in nuclear structures, but seemed to be equally distributed throughout the nucleus and cytoplasm. These findings support the notion that the intranuclear accumulation of SUMO-1 is due to SUMO-1 conjugates rather than to free SUMO-1.
This conclusion was further supported by Western blot analysis of transfected Cos-7 cells with an α HA antibody (Fig. B
). As seen in lane 1
of Fig. B
, HA-tagged SUMO-1 in this cell extract was strongly represented by a 90-kD band that comigrated with modified RanGAP1 (data not shown), as well as in a number of higher molecular mass bands. We were also able to detect a band at ~17-kD that presumably represents monomeric HA–SUMO-1. The banding pattern for HA–SUMOΔC4 was very similar to that of the wt SUMO-1, demonstrating that SUMOΔC4 can also be used efficiently by the modification machinery in vivo (Fig. B
, lane 3
). In contrast, HA-SUMOΔC6 was expressed only in its monomeric form (Fig. B
, lane 2
). Similar results were recently reported by Kamitani et al. (1997)
Interestingly, the levels of unconjugated HA–SUMO-1 and HA–SUMOΔC6 in the transfected cell extracts were about the same (Fig. B, bottom panel, lanes 1 and 2), even though cells contain at least an order of magnitude more of HA–SUMO-1 due to its presence in protein conjugates. The lack of higher levels of unconjugated HA– SUMOΔC6 may reflect an instability of the latter, or alternatively could indicate that the levels of unconjugated SUMO-1 are tightly regulated.
A Single Lysine Residue (K526) in RanGAP1 Is Modified by SUMO-1
Our peptide analysis of the modified RanGAP1 pointed to lysine 526 as a potential acceptor site for SUMO-1 in RanGAP1, since it was the only internal lysine residue in the linked peptide (Fig. C). Lysine 526 resides in the 25-kD tail of RanGAP1, a domain that is unique to RanGAP proteins of higher eukaryotes (Fig. ). To unequivocally prove the identity of the acceptor site, we mutagenized the Ran GAP1 cDNA at a single base to convert lysine 526 to arginine, and expressed wt RanGAP1 and mutated K526R RanGAP1 as T7-tagged proteins in bacteria (RanGAP1 derivatives used in this study are shown in Fig. ). We then compared the ability of the recombinant proteins to be modified by SUMO-1 in the in vitro shift assay. For this experiment, bacterial lysates from cells expressing the T7-tagged RanGAP1 proteins (Fig. , lanes 1 and 2) were added to digitonin lysates of HeLa cells in the presence of ATP (Fig. , lanes 4 and 5). After incubation at RT for 10 min, the samples were subjected to Western blot analysis. Under conditions in which ~90% of wt RanGAP1 was modified by SUMO-1 (Fig. , lane 4), RanGAP1 K526R remained completely unmodified (Fig. , lane 5), indicating not only that lysine 526 is the site of modification by SUMO-1, but that it also is the only site in RanGAP1 capable of being modified in this in vitro assay. Western blot analysis of the bacterial extract with α T7-antibodies (Fig. , lanes 1 and 2) revealed the presence of a small amount of a slightly faster migrating species (Fig. , open arrowhead) in addition to full-length RanGAP1 protein. This species is presumably the result of proteolysis at the COOH-terminal portion of the protein, since the T7 epitope resides at the NH2 terminus of the recombinant protein and was still recognized by α T7 antibodies. Interestingly, this faster migrating species was not modified (Fig. , lane 4), suggesting that the extreme COOH terminus of RanGAP1 may contain information essential for the modification.
Figure 5 A single lysine residue (K526) in RanGAP1 is modified by SUMO-1. Bacterial lysates of cells expressing T7-tagged wt RanGAP1 (lanes 1 and 4) or mutant RanGAP1 (lanes 2 and 5) were mixed with a digitonin lysate of HeLa cells in the presence of ATP. (more ...)
SUMO-1 Modification of RanGAP1 at K526 Is Required for Targeting RanGAP1 to the Nuclear Rim In Vivo
To verify that K526 is the only acceptor site for SUMO-1 modification, and to extend upon our previous data indicating a role for SUMO-1 modification in the targeting of RanGAP1 to the NPC (Mahajan et al., 1997
), we transfected Cos-7 cells with HA-tagged wt and K526R RanGAP1 expression plasmids. Analysis of the cells by indirect immunofluorescence microscopy (Fig. A
) and by Western blot analysis (Fig. B
) showed a strikingly different distribution for the two proteins. The wt RanGAP1 was localized primarily to the nuclear rim and was also present in a diffuse cytoplasmic distribution (Fig. A
, wt GAP
), consistent with the localization of endogenous RanGAP1 (Matunis et al., 1996
; Mahajan et al. 1997
). In contrast, RanGAP1 containing the lysine 526 to arginine mutation was unable to localize to the nuclear rim and instead accumulated in the cytoplasm (Fig. A
, K526R GAP
). Western-blot analysis of the transfected cells showed that although both wt RanGAP1 and K526R RanGAP1 were expressed at equivalent levels, only the wt form of RanGAP1 was competent to be modified in vivo to the 90-kD form (Fig. B
). Taken together, these findings demonstrate that in vivo SUMO-1 modification of K526 in RanGAP1 is required to target RanGAP1 to the nuclear envelope.
Figure 6 SUMO-1 modification of RanGAP1 at K526 is required for targeting RanGAP1 to the nuclear rim in vivo. HA-tagged wt RanGAP1 (wt GAP) and mutant RanGAP1 (K526R GAP) were transfected into Cos-7 cells and detected after 24 h. (A) Localization of transfected (more ...)
The Tail Domain of RanGAP1 Contains Both the Modification Site as Well as the Nuclear Rim-targeting Domain
Next, we investigated the nature of the targeting signal provided by the SUMO-1 moiety. Our previous in vitro binding studies did not detect any significant binding of SUMO-1 alone to RanBP2 (Mahajan et al., 1997
) suggesting that SUMO-1 is not simply an adaptor molecule that links RanGAP1 to RanBP2. Thus, SUMO-1 could serve to uncover a binding site in RanGAP1 that is masked in the unmodified protein. Alternatively, modification of RanGAP1 by SUMO-1 could create a composite binding site that involves both proteins. To begin to address this question, we engineered fragments of RanGAP1 (Fig. ) as HA-tagged proteins and transfected them into Cos-7 cells (Fig. ). The RanGAP1 body contained the first 416 NH2
-terminal amino acids of RanGAP1, including both the leucine-rich repeat domain and the acidic stretch that are present in all RanGAP1 proteins from yeast to mammals (Fig. ). Western blot analysis of transfected cells showed that this NH2
-terminal fragment was expressed at high levels and appeared as a single band somewhat larger than the predicted molecular mass of ~43 kD (Fig. B
). This band did not react with α–SUMO-1 antibodies (data not shown), indicating that it is not SUMO-1 modified. Cos-7 cells expressing this construct are shown in the left panels of Fig. A
). This NH2
-terminal fragment was clearly excluded from the nucleus and did not accumulate at the nuclear rim. Digitonin permeabilization before fixation of the cells led to the loss of most of the cytoplasmic staining and again, no accumulation at the NE was observed (see Fig. C, body
). The prominent band observed by Western blot analysis of digitonin-permeabilized cells (Fig. D
) is due to a low level of body that remained in the cytoplasm after permeabilization (visible in longer exposures of Fig. C
; not shown). Taken together these data indicate that the conserved NH2
-terminal part of RanGAP1 does not contain sufficient information for targeting to the NE.
Figure 7 The mammalian-specific tail region of RanGAP1 is both necessary and sufficient for SUMO-1 modification and nuclear rim targeting. (A) Immunofluorescence of Cos-7 cells transfected with HA-tagged RanGAP1 constructs representing the NH2-terminal 416 (more ...)
In contrast, a fragment of RanGAP1 representing the COOH-terminal domain (Fig. , GAP tail) exhibited a distinct nuclear rim localization in addition to intranuclear accumulation (Fig. A, wt Tail). Digitonin permeabilization before fixation of the cells led to loss of the intranuclear staining, whereas the nuclear rim staining remained (see Fig. C, wt Tail). Western blot analysis of transfected cells showed that the tail fragment was expressed at high levels and that a small fraction of the wt tail domain was modified by SUMO-1 (Fig. B, lane 2). Modified tail, but not unmodified tail, was retained in cells after digitonin permeabilization (Fig. D, lane 2), indicating that only SUMO-1–modified tail was localized to the nuclear rim.
A tail domain lacking the SUMO-1 modification site (Fig. , K526R Tail) did not localize to the nuclear rim, but accumulated within the nucleus (Fig. A, K526R Tail). The accumulation of the tail fragments in the nucleus upon overexpression is probably not physiologically relevant, as full-length RanGAP1 is excluded from the nucleus. Western blot analysis of transfected cells showed that the mutant tail fragment was expressed at about the same level as wt tail (Fig. B, lane 3). Digitonin permeabilization before fixation led to a near complete loss of the tail as judged by immunofluorescence microscopy (Fig. C, K526R Tail) and by Western blot analysis (Fig. D, lane 3). From these experiments we concluded that (a) the tail domain contains sufficient information to be recognized by the SUMO-1 modification machinery; (b) the NE-targeting information resides in the tail domain of RanGAP1; and (c) modification with SUMO-1 is required not only for targeting of full-length RanGAP1 but also for targeting of the much shorter tail fragment.
SUMO-1–modified RanGAP1 Remains Stably Bound to the NE During In Vitro Nuclear Protein Import
The finding that the modified tail domain remained stably associated with the NE upon digitonin permeabilization and washing of the cells (Fig. C, wt Tail and D, lane 2) suggested that there was no rapid demodification during the treatment. To test whether this was also true for full-length RanGAP1 under conditions that allow nuclear protein import, Cos-7 cells transfected with full-length HA-tagged RanGAP1 were permeabilized with digitonin and mixed with HeLa cytosol, ATP, and a fluorescent transport substrate (FITC-BSA-NLS), and incubated in the presence or absence of recombinant untagged wt RanGAP1 (Fig. ). After incubation of the mixture at the indicated temperature for 30 min, the cells were fixed and the amount of HA-tagged RanGAP1 in these cells was analyzed quantitatively by flow cytometry. As shown in the top panel of Fig. A, which shows cells that were kept at 0°C, expression of HA-tagged RanGAP1 resulted in a large number of cells that show higher levels of HA-signal compared with background in mock-transfected cells (bottom panel). This is consistent with a transfection efficiency of ~50%, which is also suggested by the indirect immunofluorescence staining shown in Fig. B. Both analysis by flow cytometry and by indirect immunofluorescence showed a high variation of the HA-signal (note the logarithmic scale in Fig. A), indicating a high cell to cell variability in expression levels. Incubation for 30 min at 30°C to carry out nuclear protein import did not alter the HA signal distribution, even in the presence of an excess of recombinant untagged RanGAP1. Fig. B shows analysis of a similar experiment by immunofluorescence. In each field both transfected and untransfected cells are present, indicating that no redistribution of RanGAP1 occurs under conditions that clearly allow many cycles of nuclear protein import (see Discussion). Although the overall cell to cell variation in nuclear import levels was quite high both in transfected and untransfected cells, strong nuclear accumulation of transport substrate occurred in cells that retained high levels of HA-RanGAP1 at the NE (Fig. B, middle and bottom). These findings indicate that the NE-associated RanGAP1–SUMO-1 conjugate is not rapidly turned over under in vitro import conditions, and allows the conclusion that modification and demodification of RanGAP1 is not a required element of nuclear protein import.
Figure 8 Stable association of modified RanGAP1 with the NE during nuclear protein import. (A) Analysis of Cos-7 cells transfected with HA-tagged RanGAP1 by flow cytometry. Cos7 cells transfected with HA-RanGAP1 (A, top three panels), or mock-transfected (more ...)