The RanBP2/RanGAP1*SUMO1/Ubc9 complex

Abstract

Posttranslational modification of proteins with SUMO and the RanGTP/GDP cycle are two essential cellular mechanisms contributing directly or indirectly to almost every cellular event. The SUMO E3 ligase RanBP2 (Nup358) and the Ran GTPase activating protein (RanGAP1) are known to form a stable complex throughout the cell cycle suggesting a link between sumoylation of proteins and RanGTP hydrolysis. In a recent study we demonstrated that the stable complex of RanBP2, sumoylated RanGAP1 and Ubc9 (and not RanBP2 by itself) represents the physiologically relevant form of the SUMO ligase. Characterization of the interactions reveals an intricate proximity of two catalytic activities, sumoylation and RanGTP hydrolysis. In this ExtraView we summarize our results and discuss some ideas about a potential coupling of both processes.

Keywords: :

• SUMO
• RanBP2
• Nup358
• RanGAP1
• E3 ligase complex
• RanGTPase

This article refers to:

The regulation and fine-tuning of all cellular processes relies on a number of reversible posttranslational modifications of proteins, among them the attachment of proteins of the ubiquitin-like modifier family. The most intensely studied member in this family besides ubiquitin itself is SUMO, and in almost all eukaryotes the SUMO pathway is essential. All eukaryotic organisms express at least one SUMO, many organisms express several SUMO paralogs with distinct characteristics in respect to target specificity, abundance, the ability to form SUMO chains and involvement in stress responses.^1,2

Following a maturation step to expose a C-terminal Gly-Gly motif, SUMO is conjugated to targets via an enzymatic cascade consisting of a unique E1 enzyme that activates SUMO in an ATP-dependent manner and the single E2 conjugating enzyme Ubc9, which transfers SUMO to a lysine side chain in the target protein with the help of one of several E3 ligases. Like other regulatory modifications, sumoylation is reversible due to specific isopeptidases that remove SUMO from the targets.³

While the functional outcomes of sumoylation are manifold, the most common consequence at the molecular level is a change in protein interactions by either blocking or creating an (additional) binding surface. SUMO interaction motifs (SIM) that allow for noncovalent interaction with SUMO play a major role in SUMO-mediated assembly of protein complexes or subcellular structures like PML bodies,⁴ but have also been shown to contribute to the efficiency and specificity of SUMO modification.^5,6

The recent progress in experimental techniques and tools for the detection of sumoylation in cells provides us with a rapidly growing list of SUMO targets. However, our knowledge about enzymes mediating sumoylation, especially the E3 ligases, is still limited.

Among the few proteins that have unequivocally been demonstrated to act as SUMO E3 ligase in vitro and in vivo is the nucleoporin RanBP2 (Nup358).⁷ This 358 kDa protein constitutes a major component of the cytoplasmic filaments of nuclear pore complexes in interphase;^8,9 after nuclear envelope breakdown it is largely soluble but a pool of it is also found at the kinetochore and mitotic spindle¹⁰ (Fig. 1). RanBP2 is an essential protein playing a role in various cellular processes, but only some of its functions have been linked to its SUMO ligase activity.^11-13 The region in RanBP2 responsible for E3 ligase activity is contained within a C-terminal region harboring two 50 amino acid internal repeats (IR1 and IR2) separated by a 20 amino acid linker. Both repeats can interact with the E2 conjugating enzyme Ubc9, although with different affinities. Due to its higher activity as an isolated protein fragment in vitro, IR1 was assumed to be the RanBP2 region providing E3 ligase activity also in vivo.

Figure 1. Cellular localization and schematic domain structure of RanBP2 with leucine-rich N-terminus (LR), four RanGTP binding domains (RB), an 8-fold zinc finger (ZF), a kinesin binding domain (KBD), a cyclophilin domain (CY), several FG repeats (dashes) and the SUMO E3 ligase region (E3) that is shown in more detail in the lower part. The complex with sumoylated RanGAP1 and Ubc9 is an active SUMO E3 ligase. Two internal repeats (IR) accommodate the binding site for stable interaction with sumoylated RanGAP1 and the structural Ubc9 (IR1) as well as the site for transient interaction with the catalytic Ubc9 (IR2). IR2 is only active in the context of the complex, in free RanBP2 its E3 ligase activity is negligible. The “core” region of the complex is flanked on both sides by RanGTP binding domains (RB) and FG repeats (dashes) that serve as interaction sites for RanGTP and transport receptors.

However, RanBP2 is not an isolated protein, but rather part of a protein complex: it stably associates with the RanGTPase activating protein RanGAP1 throughout the cell cycle, and a prerequisite for this stable interaction is the modification of RanGAP1 with SUMO and the presence of Ubc9 (for references see our original article). Notably, while RanGAP1 sumoylation does not depend on RanBP2, its association with RanBP2 requires RanBP2’s E3 ligase region; structural as well as biochemical analyses even suggested that it may encompass the same determinants as those required for E3 ligase activity and may therefore abrogate its E3 function.¹⁴

In a recent study we provided insights into the questions whether all RanBP2 is integrated into such complexes and whether the RanBP2 complex is still capable to sumoylate other proteins.¹⁵ While it had been evident that a significant pool of RanBP2 is stably engaged with RanGAP1*SUMO1, co-localization analyses combined with a co-depletion experiment revealed that most, if not all RanBP2 is stably incorporated into the complex in cells; RanBP2 in free form however could not be detected. Using biochemical reconstitution as well as competition experiments in cells we found that the stable complex with RanGAP1*SUMO1 required a Ubc9 molecule as molecular glue, and assembled as trimeric complex on the IR1 region of RanBP2. Of note, these observations showed that an existing crystal structure of a RanBP2 fragment in complex with the sumoylated RanGAP1 tail domain and Ubc9¹⁴ describes the interactions between the three proteins in the stable complex.

The obvious question arising from these findings was how sumoylation can still be catalyzed by the RanBP2/RanGAP1*SUMO1/Ubc9 complex when the catalytically most active region in free RanBP2 is masked upon complex formation. A detailed analysis of in vitro reconstituted sumoylation assays revealed that the RanBP2/RanGAP1*SUMO1/Ubc9 complex is indeed active as a SUMO E3 ligase, even on its endogenous substrate Borealin.¹⁶ In line with the existing crystal structure,¹⁴ neither the structural Ubc9 nor RanBP2’s IR1 region was available for catalysis. Instead, our data indicated that upon complex formation a new catalytic center involving RanBP2’s IR2 region is formed that promotes sumoylation with catalytic properties distinct from those of RanBP2’s IR2 region alone. These findings have important implications for the question what is the functional SUMO E3 ligase: our data clearly showed that the RanBP2/RanGAP1*SUMO1/Ubc9 complex rather than free RanBP2 represents the functional entity.

What Makes the RanBP2/RanGAP1*SUMO1/Ubc9 Complex So Fascinating?

RanBP2 is a huge protein of 3224 amino acids (Fig. 1). Some of its domains are currently only poorly characterized: the N-terminus including a leucine-rich domain anchors RanBP2 at the nuclear pore complex and has been implicated in microtubule binding and organization,¹⁷ the 8-fold zinc finger motif was described as interaction site for the export receptor Crm1,¹⁸ a kinesin binding domain mediates interaction with the motor protein kinesin-1¹⁹ and the motor adaptor BicD2,²⁰ and the C-terminal cyclophilin domain binds directly to the HIV-1 capsid.²¹ Better characterized are the already mentioned SUMO E3 ligase region, four RanGTP binding domains and several FG repeats distributed over nearly the whole protein that serve as interaction sites for transport receptors.^8,9 Considering the enormous size of RanBP2 and the variety of cellular processes that RanBP2 may be involved in it is intriguing that only 140 amino acids in the C-terminus are sufficient to accommodate the SUMO E3 ligase core as well as the platform for complex formation with sumoylated RanGAP1. In addition, this area is flanked by two out of the four Ran binding domains. These serve together with RanGAP1 as key regulators of the small GTPase Ran by stimulating its low intrinsic GTPase activity. This places two distinct enzymatic activities, namely the SUMO E3 ligase and the RanGTP hydrolyzing activities, in close proximity within the same protein complex suggesting an intricate link between sumoylation and the Ran GTPase cycle.

This however does not seem to be an evolutionarily conserved feature: plant RanGAP is targeted to the nuclear pore and the mitotic cell plate by a plant specific WWP domain, a mechanism independent of RanBP2 and sumoylation²² suggesting that the precise RanGAP localization is of special importance in higher eukaryotes, yet not necessarily linked to sumoylation. Interestingly, Drosophila dmRanGAP has acquired the capacity to interact with dmRanBP2,²³ however it is unclear whether this interaction involves sumoylation of dmRanGAP and whether dmRanBP2 is a SUMO E3 ligase as the described active site is not discernable by sequence comparison. Since the E3 ligase area is recognizable in Xenopus, one may wonder whether the capacity of the RanBP2-RanGAP complex to also regulate SUMO modification has been acquired to serve vertebrate-specific functions.

In which cellular processes could this play a role? Ran is a well-known key regulator with essential functions throughout the cell cycle, including nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope reformation.²⁴ The common molecular mechanism underlying its diverse functions is the localized assembly and disassembly of transport receptor/cargo complexes depending on its nucleotide-bound state: GTP hydrolysis leads to the disruption of export receptor/cargo/RanGTP complexes, and releases import receptors from inhibitory interactions with RanGTP. Considering that several FG repeats representing interaction sites for transport receptors flank RanBP2’s E3 ligase region in addition to the Ran binding domain, it is likely that transport complexes will encounter the RanBP2 E3 ligase complex. This allows envisioning various scenarios for a dual role of the RanBP2/RanGAP1*SUMO1/Ubc9 complex: (1) Sumoylation may serve to fine-tune the transport machinery; (2) The transport machinery may recruit (cargo) proteins as substrates for sumoylation; (3) Components of the transport machinery may regulate the E3 ligase activity. In the following, we would like to discuss some of these speculative functions in context of RanBP2 biology.

The cellular process for which a direct link between sumoylation and RanGTP hydrolysis has long been anticipated is nucleocytoplasmic transport, due to the identification of RanGAP1 as the first and up to now most prominent SUMO target, and the requirement of sumoylation for RanGAP1 localization at the nuclear pore (see ref. 25 and references therein). While RanBP2’s most essential transport functions have been mapped to the N-terminal half of the protein excluding the E3 ligase region,^13,26 RanBP2-associated RanGAP1 has been found to be important for the efficiency of some nucleocytoplasmic transport processes.²⁷

In a recent study, Rothenbusch and collegues reported that sumoylation serves to regulate the efficiency of a specific transport pathway in yeast: Mms21 dependent sumoylation of the Kap114 import receptor is required to assist RanGTP-mediated cargo release in the nucleus.²⁸ In analogy, RanBP2 complex-dependent sumoylation could influence the stability of nuclear export complexes or may assist in the recycling of import receptors at the nuclear pore.

While a direct involvement of RanBP2’s E3 ligase region in nucleocytoplasmic transport has not yet been demonstrated, it has been found to be important in mitosis: both known SUMO targets of RanBP2, Topoisomerase 2 α and Borealin, are sumoylated in mitosis,^12,16 and the E3 ligase region of RanBP2 is sufficient to alleviate chromosome segregation defects observed upon RanBP2 depletion¹³ that have been linked to Topoisomerase 2 α sumoylation.¹² Interestingly, a fraction of the RanBP2/RanGAP1*SUMO1/Ubc9 complex localizes to kinetochores in dependence of Crm1 and RanGTP,²⁹ raising the possibility that associations with export-like complexes may tether the E3 ligase complex at kinetochores. While there is currently no evidence supporting the notion that kinetochore associations are important for SUMO substrate selection, several centromere/kinetochore proteins can be modified with SUMO,³⁰ and both endogenous RanBP2 substrates can be found at the centromere/kinetochore region. Strikingly, for centromere localization of Survivin, a component of the chromosomal passenger complex also comprising Borealin, Incenp and AuroraB, an interaction with Crm1 is essential.³¹ Whether Crm1 influences Borealin sumoylation remains to be seen.

Finally, while it is tempting to speculate about potential links between sumoylation and the Ran system, the multidomain protein RanBP2 may obviously participate in many more processes than sumoylation and RanGTP hydrolysis. Deciphering the puzzle of RanBP2’s complex functions will be a challenging, but fascinating subject for future studies involving various aspects of cellular biology.

Acknowledgments

We thank Frauke Melchior for continuous support and critical reading of the manuscript and all lab members for sharing advice.