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 NΔ419/PK and NΔ470/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 (NΔ419, NΔ470, and NΔ502) 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 NΔ502 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 β1 (importin β) 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 β1 (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 α and β1 [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.