SUMO modification is believed to regulate a wide range of cellular activities, many associated with the nucleus. Consistent with a prominent role in the nucleus, a significant number of the known SUMO substrates are nuclear proteins (21
), and the E1 and E2 enzymes that mediate SUMO modification localize to the nucleoplasm (28
). These findings have suggested that SUMO modification itself is predominantly an intranuclear event. Here, we have analyzed the localizations of two factors that control SUMO modification, Ubc9 and SENP2, and have found that they localize to filaments on the cytoplasmic and nucleoplasmic faces of the NPC. The implication of these findings is that SUMO modification and demodification of proteins can occur at the NPC, possibly in connection with nucleocytoplasmic transport.
SENP2 is one of at least seven related SUMO proteases present in vertebrates. We have shown that it is able to cleave the carboxyl-terminal extensions of SUMO-1 and SUMO-3 precursor proteins and reverse the modification of SUMO-1 and SUMO-3 substrates. Although we were unable to observe any obvious substrate specificity for SENP2 in our in vitro studies, the localization of SENP2 at the NPC makes it unique from previously characterized SENPs that have been found to localize to the cytoplasm (13
), nucleoplasm (6
), and nucleolus (25
). The specific functions of the individual SENPs are currently not known; however, it is intriguing to speculate that the unique localizations of these enzymes may contribute to distinct substrate specificities and functions. SENP2 was previously isolated as a protein that interacts with Axin, a regulator of the Wnt signaling pathway (11
). In this study, a truncated version of SENP2 (lacking the fist 71 amino acids) was shown to induce the degradation of β-catenin in cultured cells and to inhibit Wnt-dependent axis duplication in Xenopus
embryos. Consistent with our finding that the first 63 amino acids of SENP2 are required for the targeting of SENP2 to the NPC, the truncated protein used in this study localized exclusively to the cytoplasm (11
). The mislocalization of SENP2 in this study raises questions about the relevance of the effects on the Wnt pathway that were observed. However, another study identified what appears to be an alternatively spliced form of SENP2 that lacks the amino-terminal 47 amino acids and that localizes to the cytoplasm (24
). Because the activation of the Wnt pathway specifically involves the stabilization of β-catenin and its translocation from the cytoplasm to the nucleus (two processes that could involve SUMO modification), it will be particularly important to further investigate the possible roles of SENP2 in regulating the Wnt signaling pathway.
While this manuscript was in preparation, Hang and Dasso also reported that full-length SENP2 localizes to the NPC (8
). Our findings complement theirs in establishing that SENP2 localizes to the nucleoplasmic face of the NPC, likely through interactions with Nup153. One notable difference between their work and ours is the localization of SENP2 containing amino-terminal deletions. Whereas we found that the deletion of 63 amino acids from the amino terminus of SENP2 caused it to localize almost exclusively to the cytoplasm, Hang and Dasso found that similar deletions caused SENP2 to localize to the nucleoplasm. A likely explanation for this difference is the use of different protein tags (myc in our study and green fluorescent protein in the study of Hang and Dasso). The use of the myc epitope tag allowed us to determine that the first 63 amino acids of SENP2 contain signals for both nuclear localization and NPC targeting. We have also extended the findings of Hang and Dasso by showing that SENP2 specifically interacts with the carboxyl-terminal FG repeat domain of Nup153. Interestingly, this domain of Nup153 plays important roles in NLS-mediated nuclear import (36
) and mRNA export (1
). The functions of the Nup153 FG repeat domain have been attributed to its ability to bind to nuclear import and nuclear export receptors. Given our finding that this domain also interacts with SENP2, it will be important to evaluate the potential roles of SENP2 in facilitating protein import and/or RNA export.
Ubc9, the E2 conjugating enzyme for SUMO, plays direct roles in substrate recognition and substrate modification. Ubc9 is unusual in comparison to ubiquitin E2 conjugating enzymes in that it binds directly to a consensus sequence found in most SUMO-modified proteins (2
). Because of this direct recognition of substrates by Ubc9, the role of E3-like factors in SUMO modification remains unclear. Nonetheless, several E3-like factors that stimulate SUMO modification have been identified, including Nup358 (26
). Consistent with Nup358 having a role in SUMO modification, others (26
) and we have shown that Nup358 is able to interact directly with Ubc9. Using immunogold labeling of isolated nuclear envelopes, we have shown, for the first time, that Ubc9 localizes to the cytoplasmic filaments of intact NPCs, a finding that is consistent with Ubc9 interacting with Nup358. We have also shown, for the first time, that SUMO-1-modified RanGAP1 binds directly to Nup358 in the absence of other factors. Although SUMO-1-modified RanGAP1 and Ubc9 interact independently with the same 240-amino-acid domain of Nup358, a synergistic effect on binding was observed when they were incubated with Nup358 together. A likely explanation for the observed synergy is that Ubc9 and SUMO-1-modified RanGAP1 interact simultaneously with each other and with Nup358 to form a stable trimeric complex, a model that is consistent with previous observations that SUMO-1-modified RanGAP1 and Ubc9 form stable interactions (2
). The finding that SUMO-1-modified RanGAP1 is resistant to SENP2 only when complexed with both Nup358 and Ubc9 is also consistent with these proteins forming a stable trimeric complex. Overall, our localization data and protein-protein interaction studies indicate that Ubc9 localizes to the cytoplasmic filaments of the NPC, where it forms a complex with both Nup358 and SUMO-1-modified RanGAP1. These findings are likely to have important implications for the function of Ubc9 at the NPC and for the role of Nup358 as an E3 ligase. We are currently investigating the E3 ligase activity of Nup358 and how it may be affected by interactions with Ubc9 and SUMO-1-modified RanGAP1.
Immunogold labeling of isolated nuclear envelopes also revealed that Ubc9 may interact with the nucleoplasmic filaments of the NPC. Initial assays for interactions between Ubc9 and nucleoporins known to localize to the nucleoplasmic face of the NPC (Tpr, Nup153, and Nup98) revealed no direct binding. Because a significant fraction of Ubc9 localizes to the nucleoplasm, we cannot rule out the possibility that the labeling observed on the nucleoplasmic face of the NPC was nonspecific. However, labeling was clearly specific for NPCs and was not observed on the inner nuclear membrane or lamina. In addition, similar results were obtained with two independently produced antibodies (data not shown).
NPCs are large multiprotein complexes that span the double membrane of the nuclear envelope and mediate the trafficking of macromolecules between the nucleus and the cytoplasm. In general, the NPC is believed to have a relatively passive role in the trafficking process, with transport receptors and their cargos transiently interacting with nucleoporins and translocating across the pore by facilitated diffusion (29
). SENP2 and Ubc9 are unusual in that they are among a very limited number of proteins that are associated with the NPC and that have known biochemical activities. Taken together, our results indicate that SUMO modification and demodification of proteins can occur at the distal ends of the NPC. Proteins associated with the cytoplasmic and nucleoplasmic filaments of the NPC have been proposed to have important roles in determining the directionality of transport through the NPC because of their unique asymmetric localizations (29
). At this point, we can only speculate about the exact functions that Ubc9 and SENP2 may have at the NPC. In the simplest model, SUMO modification and demodification at the NPC may function to alter the activities of specific substrates in a manner that is directly linked to their import or export from the nucleus. Alternatively, the modification and demodification of proteins may be directly related to their vectorial transport through the NPC. The modification of proteins on the cytoplasmic filaments could, for example, serve as a signal that initiates nuclear import. SUMO modification at the cytoplasmic filaments of the NPC could mediate the interaction of substrates with a nuclear import receptor or could allow modified proteins to interact directly with components of the NPC. SUMO-1 modification of RanGAP1, for example, regulates its interaction with Nup358 (18
). Translocation of modified substrates through the NPC would be terminated by their demodification by SENP2 at the nucleoplasmic basket. It is also conceivable that the SUMO modification of nuclear import receptors themselves functions to regulate their interactions with the NPC or with their cargo.
The SUMO modification and demodification of proteins at the NPC could also have a role in regulating RNA and protein export. One possible role for SUMO modification and demodification at the nucleoplasmic basket is to remodel messenger ribonucleoprotein complexes as they are translocated through the NPC or even to alter the conformation of the NPC itself. Analysis of the nuclear export of Balbiani ring transcripts, for example, has revealed dramatic changes in messenger ribonucleoprotein and NPC structures that are coincident with translocation through the pore (14
). Intriguingly, one of the two SUMO proteases in S
(Ulp1) also localizes to NPCs (16
), suggesting that the role of SUMO modification and demodification at the NPC is evolutionarily conserved.