Protein interactions in the sumoylation cascade: lessons from X-ray structures

Sumoylation is a multi-step protein modification reaction in which SUMO (small ubiquitin-like modifier) proteins are covalently attached to lysine residues of substrate proteins. Here, we compare the sequences and structures of modifiers and enzymes involved in sumoylation with those of the related ubiquitination and neddylation cascades. By using available structural data on modifier/enzyme/substrate interactions, we discuss and model sumoylation complexes that include SUMO-1 and the E1 and E2 enzymes Aos1-uba2 and ubc9, or SUMO-1 and E2 together with the E3 ligase RanBP2 and its substrate RanGAP1. Their comparison provides insight into the protein interactions underlying sumoylation, and suggests how SUMO proteins may be translocated between enzymes during the various steps of the protein modification reaction.     

SUMO-1 targets RanGAP1 to kinetochores and mitotic spindles

RanGAP1 was the first documented substrate for conjugation with the ubiquitin-like protein SUMO-1. However, the functional significance of this conjugation has not been fully clarified. We sought to examine RanGAP1 behavior during mitosis. We found that RanGAP1 associates with mitotic spindles and that it is particularly concentrated at foci near kinetochores. Association with kinetochores appeared soon after nuclear envelope breakdown and persisted until late anaphase, but it was lost coincident with nuclear envelope assembly in telophase. A mutant RanGAP1 protein lacking the capacity to be conjugated to SUMO-1 no longer associated with spindles, indicating that conjugation was essential for RanGAP1's mitotic localization. RanBP2, a nuclear pore protein that binds SUMO-1-conjugated RanGAP1 during interphase, colocalized with RanGAP1 on spindles, suggesting that a complex between these two proteins may be involved in mitotic targeting of RanGAP1. This report shows for the first time that SUMO-1 conjugation is required for mitotic localization of RanGAP1, and suggests that a major role of SUMO-1 conjugation to RanGAP1 may be the spatial regulation of the Ran pathway during mitosis.     

SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex

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.     

In vivo identification of human small ubiquitin-like modifier polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy

The length and precise linkage of polyubiquitin chains is important for their biological activity. Although other ubiquitin-like proteins have the potential to form polymeric chains their identification in vivo is challenging and their functional role is unclear. Vertebrates express three small ubiquitin-like modifiers, SUMO-1, SUMO-2, and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical and contain an internal consensus site for sumoylation that is missing in SUMO-1. Combining state-of-the-art mass spectrometry with an "in vitro to in vivo" strategy for post-translational modifications, we provide direct evidence that SUMO-1, SUMO-2, and SUMO-3 form mixed chains in cells via the internal consensus sites for sumoylation in SUMO-2 and SUMO-3. In vitro, the chain length of SUMO polymers could be influenced by changing the relative amounts of SUMO-1 and SUMO-2. The developed methodology is generic and can be adapted for the identification of other sumoylation sites in complex samples.     

Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3

Post-translational modification marked by the covalent attachment of the ubiquitin-like protein SUMO-1/SMT3C has been implicated in a wide variety of cellular processes. Recently, two cDNAs encoding proteins related to SUMO-1 have been identified in human and mouse. The functions and regulation of these proteins, known as SUMO-2/SMT3A and SUMO-3/SMT3B, remain largely uncharacterized. We describe herein quantitative and qualitative distinctions between SUMO-1 and SUMO-2/3 in vertebrate cells. Much of this was accomplished through the application of an antibody that recognizes SUMO-2 and -3, but not SUMO-1. This antibody detected multiple SUMO-2/3-modified proteins and revealed that, together, SUMO-2 and -3 constitute a greater percentage of total cellular protein modification than does SUMO-1. Intriguingly, we found that there was a large pool of free, non-conjugated SUMO-2/3 and that the conjugation of SUMO-2/3 to high molecular mass proteins was induced when the cells were subjected to protein-damaging stimuli such as acute temperature fluctuation. In addition, we demonstrated that SUMO-2/3 conjugated poorly, if at all, to a major SUMO-1 substrate, the Ran GTPase-activating protein RanGAP1. Together, these results support the concept of important distinctions between the SUMO-2/3 and SUMO-1 conjugation pathways and suggest a role for SUMO-2/3 in the cellular responses to environmental stress.     

The RanGAP1-RanBP2 complex is essential for microtubule-kinetochore interactions in vivo

RanGAP1 is the activating protein for the Ran GTPase. Vertebrate RanGAP1 is conjugated to a small ubiquitin-like protein, SUMO-1. This modification promotes association of RanGAP1 with the interphase nuclear pore complex (NPC) through binding to the nucleoporin RanBP2, also known as Nup358. During mitosis, RanGAP1 is concentrated at kinetochores in a microtubule- (MT) and SUMO-1-dependent fashion. RanBP2 is also abundantly found on kinetochores in mitosis. Here we show that ablation of proteins required for MT-kinetochore attachment (Hec1/Ndc80, Nuf2 ) disrupts RanGAP1 and RanBP2 targeting to kinetochores. No similar disruption was observed after ablation of proteins nonessential for MT-kinetochore interactions (CENP-I, Bub1, CENP-E ). Acquisition of RanGAP1 and RanBP2 by kinetochores is temporally correlated in untreated cells with MT attachment. These patterns of accumulation suggest a loading mechanism wherein the RanGAP1-RanBP2 complex may be transferred along the MT onto the kinetochore. Depletion of RanBP2 caused mislocalization of RanGAP1, Mad1, Mad2, CENP-E, and CENP-F, as well as loss of cold-stable kinetochore-MT interactions and accumulation of mitotic cells with multipolar spindles and unaligned chromosomes. Taken together, our observations indicate that RanBP2 and RanGAP1 are targeted as a single complex that is both regulated by and essential for stable kinetochore-MT association.     

SUMO protease SENP1 induces isomerization of the scissile peptide bond

Small ubiquitin-like modifier (SUMO)-specific protease SENP1 processes SUMO-1, SUMO-2 and SUMO-3 to mature forms and deconjugates them from modified proteins. To establish the proteolytic mechanism, we determined structures of catalytically inactive SENP1 bound to SUMO-1-modified RanGAP1 and to unprocessed SUMO-1. In each case, the scissile peptide bond is kinked at a right angle to the C-terminal tail of SUMO-1 and has the cis configuration of the amide nitrogens. SENP1 preferentially processes SUMO-1 over SUMO-2, but binding thermodynamics of full-length SUMO-1 and SUMO-2 to SENP1 and K(m) values for processing are very similar. However, k(cat) values differ by 50-fold. Thus, discrimination between unprocessed SUMO-1 and SUMO-2 by SENP1 is based on a catalytic step rather than substrate binding and is likely to reflect differences in the ability of SENP1 to correctly orientate the scissile bonds in SUMO-1 and SUMO-2.     

Topors acts as a SUMO-1 E3 ligase for p53 in vitro and in vivo

Human Topors, which was originally identified as cellular binding partner of DNA topoisomerase I and of p53, has recently been shown to function as an ubiquitin E3 ligase for p53 in a manner dependent on its N'-terminally located RING finger. Here, we demonstrate that Topors also enhances the conjugation of the small ubiquitin-like modifier 1 (SUMO-1) to p53 in vivo and in a reconstituted in vitro system. The Topors SUMO-1 E3 ligase activity does not depend upon its RING finger motif. In HeLa cells, Topors induced p53 sumoylation was accompanied by an increase in endogenous p53 protein levels. Furthermore, Topors enhances the sumoylation of a variety of other, yet unidentified, cellular proteins.     

Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association

The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101– amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1–SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role.     

A new 30-kDa ubiquitin-related SUMO-1 hydrolase from bovine brain.

SUMO-1 is a ubiquitin-like protein functioning as an important reversible protein modifier. To date there is no report on a SUMO-1 hydrolase/isopeptidase catalyzing the release of SUMO-1 from its precursor or SUMO-1-ligated proteins in mammalian tissues. Here we found multiple activities that cleave the SUMO-1 moiety from two model substrates, (125)I-SUMO-1-alphaNH-HSTVGSMHISPPEPESEEEEEHYC and/or GST-SUMO-1-(35)S-RanGAP1 conjugate, in bovine brain extracts. Of them, a major SUMO-1 C-terminal hydrolase had been partially purified by successive chromatographic operations. The enzyme had the ability to cleave SUMO-1 not only from its precursor but also from a SUMO-1-ligated RanGAP1 but did not exhibit any significant cleavage of the ubiquitin- and NEDD8-precursor. The activity of SUMO-1 hydrolase was almost completely inhibited by N-ethylmaleimide, but not by phenylmethanesulfonyl fluoride, EDTA, and ubiquitin-aldehyde known as a potent inhibitor of deubiquitinylating enzymes. Intriguingly, the apparent molecular mass of the isolated SUMO-1 hydrolase was approximately 30 kDa, which is significantly smaller than the recently identified yeast Smt3/SUMO-1 specific protease Ulp1. These results indicate that there are multiple SUMO-1 hydrolase/isopeptidases in mammalian cells and that the 30-kDa small SUMO-1 hydrolase plays a central role in processing of the SUMO-1-precursor.     

Evidence for Covalent Modification of the Nuclear Dot–associated Proteins PML and Sp100 by PIC1/SUMO-1

PML and Sp100 proteins are associated with nuclear domains, known as nuclear dots (NDs). They were discovered in the context of leukemic transformation and as an autoantigen in primary biliary cirrhosis, respectively. Both proteins are expressed in the form of many COOH-terminally spliced variants, and their expression is enhanced by interferons (IFN). The recent finding that PIC1/SUMO-1, a small ubiquitin-like protein, is covalently linked to the RanGAP1 protein of the nuclear pore complex and also binds PML in yeast cells led us to determine whether PML is covalently modified by PIC1/SUMO-1 and whether the same is true for Sp100. We found an immune reaction of PML and Sp100 proteins with a PIC1/SUMO-1–specific monoclonal antibody by immunoblotting when using cell extracts prepared from stably transfected cells inducibly expressing one isoform of each protein as well as from nontransfected cells. In contrast, both proteins did not react when synthesized in vitro. Immunofluorescence staining showed that PIC1/SUMO-1 colocalized with Sp100 and PML in NDs except in mitotic cells, in which PML and Sp100 are dissociated. Cell fractionation and immunoblotting demonstrated that PIC1/SUMO-1 immunoreactive Sp100 in IFN-treated and untreated cells was exclusively nuclear, whereas nonmodified Sp100 was also found in the cytoplasm. Taken together, these data strongly suggest covalent modification of specific nuclear isoforms of Sp100 and PML by PIC1/SUMO-1. This modification may play a regulatory role in ND structure, composition, and function.     

Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1

The ubiquitin-like protein SUMO-1 is conjugated to a variety of proteins including Ran GTPase-activating protein 1 (RanGAP1), IkappaBalpha, and PML. SUMO-1-modified proteins display altered subcellular targeting and/or stability. We have purified the SUMO-1-activating enzyme from human cells and shown that it contains two subunits of 38 and 72 kDa. Isolation of cDNAs for each subunit indicates that they are homologous to ubiquitin-activating enzymes and to the Saccharomyces cerevisiae enzymes responsible for conjugation of Smt3p and Rub-1p. In vitro, recombinant SAE1/SAE2 (SUMO-1-activating enzyme) was capable of catalyzing the ATP-dependent formation of a thioester linkage between SUMO-1 and SAE2. The addition of the SUMO-1-conjugating enzyme Ubch9 resulted in efficient transfer of the thioester-linked SUMO-1 from SAE2 to Ubch9. In the presence of SAE1/SAE2, Ubch9, and ATP, SUMO-1 was efficiently conjugated to the protein substrate IkappaBalpha. As SAE1/SAE2, Ubch9, SUMO-1, and IkappaBalpha are all homogeneous, recombinant proteins, it appears that SUMO-1 conjugation of IkappaBalpha in vitro does not require the equivalent of an E3 ubiquitin protein ligase activity.     

Bovine papillomavirus E1 protein is sumoylated by the host cell Ubc9 protein

Papillomavirus E1 protein is the replication initiator that recognizes and binds to the viral origin and initiates DNA strand separation through its ATP-dependent helicase activity. The E1 protein also functions in viral DNA replication by recruiting several cellular proteins to the origin, including host DNA polymerase alpha and replication protein A. To identify other cellular proteins that interact with bovine papillomavirus E1, an HeLa cDNA library was screened using a yeast two-hybrid assay. The host cell sumoylating enzyme, Ubc9, was found to interact specifically with E1 both in vitro and in vivo. Mapping studies localized critical E1 sequences for interaction to amino acids 315-459 and strongly implicated leucine 420 as critical for E1.Ubc9 complex formation. In addition to binding E1, Ubc9 catalyzed the covalent linkage of the ubiquitin-like protein, SUMO-1, to E1. An E1 mutant unable to bind Ubc9 showed normal intracellular stability, but was impaired for intranuclear distribution. Failure to accumulate in appropriate nuclear subdomains may account for the previously demonstrated replication defect of a human papillomavirus 16 E1 protein that was also unable to bind Ubc9 and suggests that sumoylation is a functionally important modification with regulatory implications for papillomavirus replication.     

Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1

E2 enzymes catalyze attachment of ubiquitin and ubiquitin-like proteins to lysine residues directly or through E3-mediated reactions. The small ubiquitin-like modifier SUMO regulates nuclear transport, stress response, and signal transduction in eukaryotes and is essential for cell-cycle progression in yeast. In contrast to most ubiquitin conjugation, the SUMO E2 enzyme Ubc9 is sufficient for substrate recognition and lysine modification of known SUMO targets. Crystallographic analysis of a complex between mammalian Ubc9 and a C-terminal domain of RanGAP1 at 2.5 A reveals structural determinants for recognition of consensus SUMO modification sequences found within SUMO-conjugated proteins. Structure-based mutagenesis and biochemical analysis of Ubc9 and RanGAP1 reveal distinct motifs required for substrate binding and SUMO modification of p53, IkappaBalpha, and RanGAP1.