Small ubiquitin-related modifier (SUMO) binding determines substrate recognition and paralog-selective SUMO modification

Small ubiquitin-related modifiers (SUMOs) regulate diverse cellular processes through their covalent attachment to target proteins. Vertebrates express three SUMO paralogs: SUMO-1, SUMO-2, and SUMO-3 (SUMO-2 and SUMO-3 are approximately 96% identical and referred to as SUMO-2/3). SUMO-1 and SUMO-2/3 are conjugated, at least in part, to unique subsets of proteins and thus regulate distinct cellular pathways. However, how different proteins are selectively modified by SUMO-1 and SUMO-2/3 is unknown. We demonstrate that BLM, the RecQ DNA helicase mutated in Bloom syndrome, is preferentially modified by SUMO-2/3 both in vitro and in vivo. Our findings indicate that non-covalent interactions between SUMO and BLM are required for modification at non-consensus sites and that preferential SUMO-2/3 modification is determined by preferential SUMO-2/3 binding. We also present evidence that sumoylation of a C-terminal fragment of HIPK2 is dependent on SUMO binding, indicating that non-covalent interactions between SUMO and target proteins provide a general mechanism for SUMO substrate selection and possible paralog-selective modification.     

SUMO-2/3 modification and binding regulate the association of CENP-E with kinetochores and progression through mitosis

SUMOylation is essential for cell-cycle regulation in invertebrates; however, its functions during the mammalian cell cycle are largely uncharacterized. Mammals express three SUMO paralogs: SUMO-1, SUMO-2, and SUMO-3 (SUMO-2 and SUMO-3 are 96% identical and referred to as SUMO-2/3). We found that SUMO-2/3 localize to centromeres and condensed chromosomes, whereas SUMO-1 localizes to the mitotic spindle and spindle midzone, indicating that SUMO paralogs regulate distinct mitotic processes in mammalian cells. Consistent with this, global inhibition of SUMOylation caused a prometaphase arrest due to defects in targeting the microtubule motor protein CENP-E to kinetochores. CENP-E was found to be modified specifically by SUMO-2/3 and to possess SUMO-2/3 polymeric chain-binding activity essential for kinetochore localization. Our findings indicate that SUMOylation is a key regulator of the mammalian cell cycle, with SUMO-1 and SUMO-2/3 modification of different proteins regulating distinct processes.     

Involvement of SUMO modification in MBD1- and MCAF1-mediated heterochromatin formation

Small ubiquitin-related modifiers, SUMO-2/3 and SUMO-1, are involved in gene regulation and nuclear structures. However, little is known about the roles of SUMO, in heterochromatin formation of mammalian cells. Here we demonstrate that SUMOs directly interact with human MCAF1, which forms complexes with either the methyl-CpG-binding protein MBD1 or SETDB1, which trimethylates histone H3 at lysine 9 (H3-K9) in the presence of MCAF1. Modification of MBD1 with either SUMO-2/3 or SUMO-1 facilitated the interaction between MBD1 and MCAF1, suggesting that SUMOylation links the methylation of DNA and histones. In a cultured human cell line, SUMOs were localized in MBD1- and MCAF1-containing heterochromatin regions that were enriched in trimethyl-H3-K9 and the heterochromatin proteins HP1beta and HP1gamma. Specific knockdown of either SUMO-2/3 or SUMO-1 induced dissociation of MCAF1, trimethyl-H3-K9, and the HP1 proteins from the MBD1-containing heterochromatin foci, suggesting a requirement for SUMOs for heterochromatin assembly. These findings provide insights into the roles of SUMOylation in the regulation of heterochromatin formation and gene silencing.     

Positively charged amino acids flanking a sumoylation consensus tetramer on the 110 kDa tri-snRNP component SART1 enhance sumoylation efficiency

Covalent attachment of Small Ubiquitin-like MOdifiers (SUMOs) to the ε-amino group of lysine residues in target proteins regulates many cellular processes. Previously, we have identified the 110 kDa U4/U6.U5 tri-snRNP component SART1 as a target protein for SUMO-1 and SUMO-2. SART1 contains lysines on positions 94, 141, 709 and 742 that are situated in tetrameric sumoylation consensus sites. Recombinant SART1 was produced in E. coli, conjugated to SUMO-2 in vitro, digested by trypsin and analysed by MALDI-ToF, MALDI-FT-ICR or nanoLC-iontrap MS/MS. We found that Lys⁹⁴ and Lys¹⁴¹ of SART1 were preferentially conjugated to SUMO-2 monomers and multimers in vitro. In agreement with these results, mutation of Lys⁹⁴ and Lys¹⁴¹, but not Lys⁷⁰⁹ and Lys⁷⁴², resulted in a reduced sumoylation of SART1 in HeLa cells. A detailed characterization of the four sumoylation sites of SART1 using full-length recombinant SART1 and a peptide sumoylation approach indicated that positively charged amino acids adjacent to the tetrameric sumoylation consensus site enhance the sumoylation of Lys⁹⁴. These results show that amino acids surrounding the classic tetrameric SUMO consensus site can regulate sumoylation efficiency and validate the use of an in vitro sumoylation-mass spectrometry approach for the identification of sumoylation sites.     

Enzymes of the SUMO modification pathway localize to filaments of the nuclear pore complex

SUMOs are small ubiquitin-related polypeptides that are reversibly conjugated to many nuclear proteins. Although the number of identified substrates has grown rapidly, relatively little is still understood about when, where, and why most proteins are modified by SUMO. Here, we demonstrate that enzymes involved in the SUMO modification and demodification of proteins are components of the nuclear pore complex (NPC). We show that SENP2, a SUMO protease that is able to demodify both SUMO-1 and SUMO-2 or SUMO-3 protein conjugates, localizes to the nucleoplasmic face of the NPC. The unique amino-terminal domain of SENP2 interacts with the FG repeat domain of Nup153, indicating that SENP2 associates with the nucleoplasmic basket of the NPC. We also investigated the localization of the SUMO conjugating enzyme, Ubc9. Using immunogold labeling of isolated nuclear envelopes, we found that Ubc9 localizes to both the cytoplasmic and the nucleoplasmic filaments of the NPC. In vitro binding studies revealed that Ubc9 and SUMO-1-modified RanGAP1 bind synergistically to form a trimeric complex with a component of the cytoplasmic filaments of the NPC, Nup358. Our results indicate that both SUMO modification and demodification of proteins may occur at the NPC and suggest a connection between the SUMO modification pathway and nucleocytoplasmic transport.     

Molecular Cloning,Functional Characterization,and Evolutionary Analysis of Vitamin D Receptors Isolated from Basal Vertebrates

The vertebrate genome is a result of two rapid and successive rounds of whole genome duplication, referred to as 1R and 2R. Furthermore, teleost fish have undergone a third whole genome duplication (3R) specific to their lineage, resulting in the retention of multiple gene paralogs. The more recent 3R event in teleosts provides a unique opportunity to gain insight into how genes evolve through specific evolutionary processes. In this study we compare molecular activities of vitamin D receptors (VDR) from basal species that diverged at key points in vertebrate evolution in order to infer derived and ancestral VDR functions of teleost paralogs. Species include the sea lamprey (Petromyzon marinus), a 1R jawless fish; the little skate (Leucoraja erinacea), a cartilaginous fish that diverged after the 2R event; and the Senegal bichir (Polypterus senegalus), a primitive 2R ray-finned fish. Saturation binding assays and gel mobility shift assays demonstrate high affinity ligand binding and classic DNA binding characteristics of VDR has been conserved across vertebrate evolution. Concentration response curves in transient transfection assays reveal EC₅₀ values in the low nanomolar range, however maximum transactivational efficacy varies significantly between receptor orthologs. Protein-protein interactions were investigated using co-transfection, mammalian 2-hybrid assays, and mutations of coregulator activation domains. We then combined these results with our previous study of VDR paralogs from 3R teleosts into a bioinformatics analysis. Our results suggest that 1, 25D₃ acts as a partial agonist in basal species. Furthermore, our bioinformatics analysis suggests that functional differences between VDR orthologs and paralogs are influenced by differential protein interactions with essential coregulator proteins. We speculate that we may be observing a change in the pharmacodynamics relationship between VDR and 1, 25D₃ throughout vertebrate evolution that may have been driven by changes in protein-protein interactions between VDR and essential coregulators.     

Noncovalent binding of small ubiquitin-related modifier (SUMO) protease to SUMO is necessary for enzymatic activities and cell growth

SUMO proteases possess two enzymatic activities to hydrolyze the C-terminal region of SUMOs (hydrolase activity) and to remove SUMO from SUMO-conjugated substrates (isopeptidase activity). SUMO proteases bind to SUMOs noncovalently, but the physiological roles of the binding in the functions of SUMO proteases are not well understood. In this study we found that SUMO proteases (Axam, SENP1, and yeast Ulp1) show different preferences for noncovalent binding to various SUMOs (SUMO-1, -2, -3, and yeast Smt3) and that the hydrolase and isopeptidase activities of SUMO proteases are dependent on their binding to SUMOs through salt bridge. Expression of Smt3 suppressed the phenotype of yeast mutant lacking smt3, which exhibits growth arrest, and the binding of Ulp1 to Smt3 was essential for this rescue activity. Although expression of an Smt3 mutant (smt3R64E(GG)), which conjugates to substrate but loses the ability to bind to Ulp1, rescued the phenotype of yeast lacking smt3 partially, the mutant cells showed an increment in the doubling time and a delay of desumoylation of Smt3-conjugated Cdc3. These results indicate that the noncovalent binding of SUMO protease to SUMO through salt bridge is essential for the enzymatic activities and that the balance between sumoylation and desumoylation is important for cell growth control.     

The SUMO pathway functions in mouse oocyte maturation

Sumoylation is an important posttranslational modification in which SUMO (small ubiquitin-related modifier) proteins are bonded covalently to their substrates. Studies on the roles of sumoylation in cell cycle regulation have been emerging in both mitosis from yeast to mammals and meiosis in budding yeast, but the functions of sumoylation in mammalian meiosis, especially in oocyte meiotic maturation are not well known. Here, we examined the localization and expression of SUMO-1 and SUMO-2/3, the two basic proteins in the sumoylation pathway and investigated their roles through overexpression of Senp2 during mouse oocyte maturation. Immunofluorescent staining revealed differential patterns of SUMO-1 and SUMO-2/3 localization: SUMO-1 was localized to the spindle poles in prometaphase I, MI and MII stages, around the separating homologues in anaphase I and telophase I stages of first meiosis, while SUMO-2/3 was mainly concentrated near centromeres during mouse oocyte maturation. Immunoblot analysis uncovered the different expression profiles of SUMO-1 and SUMO-2/3 modified proteins during mouse oocyte maturation. Overexpression of Senp2, a SUMO-specific isopeptidase, caused changes of SUMO-modified proteins and led to defects in MII spindle organization in mature eggs. These results suggest that the SUMO pathway may play an indispensable role during mouse oocyte meiotic maturation.Key words: sumoylation, mouse oocyte maturation, overexpression, Senp2, MII spindle     

SUMO modification through rapamycin-mediated heterodimerization reveals a dual role for Ubc9 in targeting RanGAP1 to nuclear pore complexes

SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMO substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification.     

Single-Molecule Studies on PolySUMO Proteins Reveal Their Mechanical Flexibility

Proteins with β-sandwich and β-grasp topologies are resistant to mechanical unfolding as shown by single-molecule force spectroscopy studies. Their high mechanical stability has generally been associated with the mechanical clamp geometry present at the termini. However, there is also evidence for the importance of interactions other than the mechanical clamp in providing mechanical stability, which needs to be tested thoroughly. Here, we report the mechanical unfolding properties of ubiquitin-like proteins (SUMO1 and SUMO2) and their comparison with those of ubiquitin. Although ubiquitin and SUMOs have similar size and structural topology, they differ in their sequences and structural contacts, making them ideal candidates to understand the variations in the mechanical stability of a given protein topology. We observe a two-state unfolding pathway for SUMO1 and SUMO2, similar to that of ubiquitin. Nevertheless, the unfolding forces of SUMO1 (∼130 pN) and SUMO2 (∼120 pN) are lower than that of ubiquitin (∼190 pN) at a pulling speed of 400 nm/s, indicating their lower mechanical stability. The mechanical stabilities of SUMO proteins and ubiquitin are well correlated with the number of interresidue contacts present in their structures. From pulling speed-dependent mechanical unfolding experiments and Monte Carlo simulations, we find that the unfolding potential widths of SUMO1 (∼0.51 nm) and SUMO2 (∼0.33 nm) are much larger than that of ubiquitin (∼0.19 nm), indicating that SUMO1 is six times and SUMO2 is three times mechanically more flexible than ubiquitin. These findings might also be important in understanding the functional differences between ubiquitin and SUMOs.     

Unique binding interactions among Ubc9, SUMO and RanBP2 reveal a mechanism for SUMO paralog selection

The conjugation of small ubiquitin-like modifiers SUMO-1, SUMO-2 and SUMO-3 onto target proteins requires the concerted action of the specific E1-activating enzyme SAE1/SAE2, the E2-conjugating enzyme Ubc9, and an E3-like SUMO ligase. NMR chemical shift perturbation was used to identify the surface of Ubc9 that interacts with the SUMO ligase RanBP2. Unlike known ubiquitin E2-E3 interactions, RanBP2 binds to the beta-sheet of Ubc9. Mutational disruption of Ubc9-RanBP2 binding affected SUMO-2 but not SUMO-1 conjugation to Sp100 and to a newly identified RanBP2 substrate, PML. RanBP2 contains a binding site specific for SUMO-1 but not SUMO-2, indicating that a Ubc9-SUMO-1 thioester could be recruited to RanBP2 via SUMO-1 in the absence of strong binding between Ubc9 and RanBP2. Thus we show that E2-E3 interactions are not conserved across the ubiquitin-like protein superfamily and identify a RanBP2-dependent mechanism for SUMO paralog-specific conjugation.     

Controlled Cre/loxP Site-Specific Recombination in the Developing Brain in Medaka Fish,Oryzias latipes

Background

Genetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain.

Methodology/Principal Findings

To establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0–1 day post fertilization [dpf]) in a thermalcycler (39°C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2–3 dpf embyos compared with 0–1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish.

Conclusions/Significance

We established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages.