SUMO: a ubiquitin-like protein modifier

SUMO (Small Ubiquitin-related Modifier) is a small protein that covalently attaches to a lysine residue of another protein in a reversible fashion. SUMO attachment to its substrate proteins causes changes in the localization, activity, or binding partners of the substrate. SUMO has been shown to play a role in a multitude of processes; these include chromosome segregation, cell cycle progression, and DNA damage recovery. Defects in the SUMO pathway have been demonstrated to affect tumorigenesis and the inflammatory response as well as other human diseases.     

The small ubiquitin-like modifier (SUMO) is essential in cell cycle regulation in Trypanosoma brucei

SUMO, a reversible post-translational protein modifier, plays important roles in many processes of higher eukaryotic cell life. Although SUMO has been identified in many eukaryotes, SUMO and SUMO system are still unknown in some eukaryotic unicellular organisms, such as Trypanosoma brucei (T. brucei). In this study, only one SUMO homologue (TbSUMO) was identified in T. brucei. Expression of TbSUMO was knocked down by using RNA interference technique in procyclic-form T. brucei. The growth of TbSUMO-deficient cells was significantly inhibited. TbSUMO-deficient cells were arrested in G₂/M phase accompanied with an obvious increase of 0N1K cells (zoids), and failed in chromosome segregation. These results indicate that TbSUMO is essential in cell cycle regulation, with one important role in mitosis. Meanwhile, the enrichment of zoids suggests the inhibition of mitosis does not prevent the cell division in procyclic-form T. brucei. HA-tagged TbSUMO was overexpressed in T. brucei and was shown to be localized to the nucleus through the whole cell cycle, further revealing its distinguished functions in nucleus. All these accumulated data imply that a SUMO system essential for regulating cell cycle progression might exist in the procyclic-form T. brucei.     

NSMCE2 suppresses cancer and aging in mice independently of its SUMO ligase activity

The SMC5/6 complex is the least understood of SMC complexes. In yeast, smc5/6 mutants phenocopy mutations in sgs1, the BLM ortholog that is deficient in Bloom's syndrome (BS). We here show that NSMCE2 (Mms21, in Saccharomyces cerevisiae), an essential SUMO ligase of the SMC5/6 complex, suppresses cancer and aging in mice. Surprisingly, a mutation that compromises NSMCE2‐dependent SUMOylation does not have a detectable impact on murine lifespan. In contrast, NSMCE2 deletion in adult mice leads to pathologies resembling those found in patients of BS. Moreover, and whereas NSMCE2 deletion does not have a detectable impact on DNA replication, NSMCE2‐deficient cells also present the cellular hallmarks of BS such as increased recombination rates and an accumulation of micronuclei. Despite the similarities, NSMCE2 and BLM foci do not colocalize and concomitant deletion of Blm and Nsmce2 in B lymphocytes further increases recombination rates and is synthetic lethal due to severe chromosome mis‐segregation. Our work reveals that SUMO‐ and BLM‐independent activities of NSMCE2 limit recombination and facilitate segregation; functions of the SMC5/6 complex that are necessary to prevent cancer and aging in mice.     

SUMO conjugation and deconjugation

Ligation of the ubiquitin-like protein SUMO (Smt3p) to other proteins is essential for viability of the yeast Saccharomyces cerevisiae. Like ubiquitin (Ub), SUMO undergoes ATP-dependent activation by a specific activating enzyme. SUMO-activating enzyme is a heterodimer composed of Uba2p and Aos1p, polypeptides with sequence similarities, respectively, to the C- and N-terminal parts of Ub-activating enzyme. To study the function of SUMO conjugation, we isolated uba2 mutants that were temperature-sensitive for growth. In these mutants conjugation of SUMO to other proteins was drastically reduced, even at the temperature permissive for growth. In a screen for spontaneous suppressors of the temperature-sensitive growth phenotype of the mutant uba2-ts9, we isolated a strain with a null mutation (sut9) in a gene of hitherto unknown function (SUT9/YIL031W/SMT4). This gene encodes a protein with similarities to Ulp1p, a dual-function protease that processes the SUMO precursor and deconjugates SUMO from its substrates. The novel protein was therefore termed Ulp2p. Inactivation of ULP2 in a strain expressing wild-type SUMO-activating enzyme resulted in slow and temperature-sensitive growth, and accumulation of SUMO conjugates. Thus, mutations in SUMO-activating enzyme and mutations in Ulp2p suppress each other, indicating that SUMO conjugation and deconjugation must be in balance for cells to grow normally. Other phenotypes of ulp2 mutants include a defect in cell cycle progression, hypersensitivity to DNA damage, and chromosome mis-segregation. Ulp2p is predominantly located within the nucleus, whereas Ulp1p colocalizes with nuclear pore complex proteins, indicating that the apparently distinct functions of the two SUMO deconjugating enzymes are spatially separated. Received: 1 March 2000 / Accepted: 22 March 2000     

OsSIZ1 Regulates the Vegetative Growth and Reproductive Development in Rice

SAP and MIZ (SIZ) is a small ubiquitin-related modifier (SUMO) E3 ligase that facilitates conjugation of SUMO to protein substrates. Although there have been a number of reports about the functions of SIZ1 in Arabidopsis in the regulation of diverse life processes, no information regarding the role of SIZ in other plants is available yet. In this work, two homologous genes from rice (Oryza sativa) were isolated and designated as OsSIZ1 and OsSIZ2 based on amino acid sequence homology to AtSIZ1 and their phylogenetic relationship. The function in the vegetative growth and reproductive development in rice was investigated using OsSIZ1 mutants containing a T-DNA insertion. The results showed that the mutant Ossiz1 exhibited the significant changes in several growth and developmental parameters, including primary root length, adventitious root number, plant height, leaf and panicle length, flower formation, and seed-setting rate compared with wild type. Taking together these results indicate that OsSIZ1 plays an important role in regulating growth and development in rice.     

Slx8 Removes Pli1-Dependent Protein-SUMO Conjugates Including SUMOylated Topoisomerase I to Promote Genome Stability

The SUMO-dependent ubiquitin ligase Slx8 plays key roles in promoting genome stability, including the processing of trapped Topoisomerase I (Top1) cleavage complexes and removal of toxic SUMO conjugates. We show that it is the latter function that constitutes Slx8''s primary role in fission yeast. The SUMO conjugates in question are formed by the SUMO ligase Pli1, which is necessary for limiting spontaneous homologous recombination when Top1 is present. Surprisingly there is no requirement for Pli1 to limit recombination in the vicinity of a replication fork blocked at the programmed barrier RTS1. Notably, once committed to Pli1-mediated SUMOylation Slx8 becomes essential for genotoxin resistance, limiting both spontaneous and RTS1 induced recombination, and promoting normal chromosome segregation. We show that Slx8 removes Pli1-dependent Top1-SUMO conjugates and in doing so helps to constrain recombination at RTS1. Overall our data highlight how SUMOylation and SUMO-dependent ubiquitylation by the Pli1-Slx8 axis contribute in different ways to maintain genome stability.     

There are two mechanisms of achiasmate segregation in Drosophila females,one of which requires heterochromatic homology

There are numerous examples of the regular segregation of achiasmate chromosomes at meiosis I in Drosophila melanogaster females. Classically, the choice of achiasmate segregational partners has been thought to be independent of homology, but rather made on the basis of availability or similarities in size and shape. To the contrary, we show here that heterochromatic homology plays a primary role in ensuring the proper segregation of achiasmate homologs. We observe that the heterochromatin of chromosome 4 functions as, or contains, a meiotic pairing site. We show that free duplications carrying the 4th chromosome pericentric heterochromatin induce high frequencies of 4th chromosome nondisjunction regardless of their size. Moreover, a duplication from which some of the 4th chromosome heterochromatin has been removed is unable to induce 4th chromosome nondisjunction. Similarly, in the absence of either euchromatic homology or a size similarity, duplications bearing the X chromosome heterochromatin also disrupt the segregation of two achiasmate X chromosome centromeres. Although heterochromatic regions are sufficient to conjoin nonexchange homologues, we confirm that the segregation of heterologous chromosomes is determined by size, shape, and availability. The meiotic mutation Axs differentiates between these two processes of achiasmate centromere coorientation by disrupting only the homology-dependent mechanism. Thus there are two different mechanisms by which achiasmate segregational partners are chosen. We propose that the absence of diplotene-diakinesis during female meiosis allows heterochromatic pairings to persist until prometaphase and thus to co-orient homologous centromeres. We also propose that heterologous disjunctions result from a separate and homology-independent process that likely occurs during prometaphase. The latter process, which may not require the physical association of segregational partners, is similar to those observed in many insects, in Saccharomyces cerevisiae and in C. elegans males. We also suggest that the physical basis of this process may reflect known properties of the Drosophila meiotic spindle. © 1993 Wiley-Liss, Inc.     

ZHP-3 acts at crossovers to couple meiotic recombination with synaptonemal complex disassembly and bivalent formation in C. elegans

Crossover recombination and the formation of chiasmata normally ensure the proper segregation of homologous chromosomes during the first meiotic division. zhp-3, the Caenorhabditis elegans ortholog of the budding yeast ZIP3 gene, is required for crossover recombination. We show that ZHP-3 protein localization is highly dynamic. At a key transition point in meiotic prophase, the protein shifts from along the length of the synaptonemal complex (SC) to an asymmetric localization on the SC and eventually becomes restricted to foci that mark crossover recombination events. A zhp-3::gfp transgene partially complements a null mutation and reveals a separation of function; although the fusion protein can promote nearly wild-type levels of recombination, aneuploidy among the progeny is high, indicating defects in meiotic chromosome segregation. The structure of bivalents is perturbed in this mutant, suggesting that the chromosome segregation defect results from an inability to properly remodel chromosomes in response to crossovers. smo-1 mutants exhibit phenotypes similar to zhp-3::gfp mutants at higher temperatures, and smo-1; zhp-3::gfp double mutants exhibit more severe meiotic defects than either single mutant, consistent with a role for SUMO in the process of SC disassembly and bivalent differentiation. We propose that coordination of crossover recombination with SC disassembly and bivalent formation reflects a conserved role of Zip3/ZHP-3 in coupling recombination with SC morphogenesis.     

ParA of Mycobacterium smegmatis co‐ordinates chromosome segregation with the cell cycle and interacts with the polar growth determinant DivIVA

Mycobacteria are among the clinically most important pathogens, but still not much is known about the mechanisms of their cell cycle control. Previous studies suggested that the genes encoding ParA and ParB (ATPase and DNA binding protein, respectively, required for active chromosome segregation) may be essential in Mycobacterium tuberculosis. Further research has demonstrated that a Mycobacterium smegmatis parB deletion mutant was viable but exhibited a chromosome segregation defect. Here, we address the question if ParA is required for the growth of M. smegmatis, and which cell cycle processes it affects. Our data show that parA may be deleted, but its deletion leads to growth inhibition and severe disturbances of chromosome segregation and septum positioning. Similar defects are also caused by ParA overproduction. EGFP–ParA localizes as pole‐associated complexes connected with a patch of fluorescence accompanying two ParB complexes. Observed aberrations in the number and positioning of ParB complexes in the parA deletion mutant indicate that ParA is required for the proper localization of the ParB complexes. Furthermore, it is shown that ParA colocalizes and interacts with the polar growth determinant Wag31 (DivIVA homologue). Our results demonstrate that mycobacterial ParA mediates chromosome segregation and co‐ordinates it with cell division and elongation.     

PICH regulates the abundance and localization of SUMOylated proteins on mitotic chromosomes

Proper chromosome segregation is essential for faithful cell division and if not maintained results in defective cell function caused by the abnormal distribution of genetic information. Polo-like kinase 1–interacting checkpoint helicase (PICH) is a DNA translocase essential for chromosome bridge resolution during mitosis. Its function in resolving chromosome bridges requires both DNA translocase activity and ability to bind chromosomal proteins modified by the small ubiquitin-like modifier (SUMO). However, it is unclear how these activities cooperate to resolve chromosome bridges. Here, we show that PICH specifically disperses SUMO2/3 foci on mitotic chromosomes. This PICH function is apparent toward SUMOylated topoisomerase IIα (TopoIIα) after inhibition of TopoIIα by ICRF-193. Conditional depletion of PICH using the auxin-inducible degron (AID) system resulted in the retention of SUMO2/3-modified chromosomal proteins, including TopoIIα, indicating that PICH functions to reduce the association of these proteins with chromosomes. Replacement of PICH with its translocase-deficient mutants led to increased SUMO2/3 foci on chromosomes, suggesting that the reduction of SUMO2/3 foci requires the remodeling activity of PICH. In vitro assays showed that PICH specifically attenuates SUMOylated TopoIIα activity using its SUMO-binding ability. Taking the results together, we propose a novel function of PICH in remodeling SUMOylated proteins to ensure faithful chromosome segregation.     

Complement factor H allotype 402H is associated with increased C3b opsonization and phagocytosis of Streptococcus pyogenes

The mechanisms driving bacterial chromosome segregation remain poorly characterized. While a number of factors influencing chromosome segregation have been described in recent years, none of them appeared to play an essential role in the process comparable to the eukaryotic centromere/spindle complex. The research community involved in bacterial chromosome was becoming familiar with the fact that bacteria have selected multiple redundant systems to ensure correct chromosome segregation. Over the past few years a new perspective came out that entropic forces generated by the confinement of the chromosome in the crowded nucleoid shell could be sufficient to segregate the chromosome. The segregating factors would only be required to create adequate conditions for entropy to do its job. In the article by Yazdi et al. ( 2012 ) in this issue of Molecular Microbiology, this model was challenged experimentally in live Escherichia coli cells. A Fis–GFP fusion was used to follow nucleoid choreography and analyse it from a polymer physics perspective. Their results suggest strongly that E. coli nucleoids behave as self‐adherent polymers. Such a structuring and the specific segregation patterns observed do not support an entropic like segregation model. Are we back to the pre‐entropic era?     

SUMO E3 Ligases GmSIZ1a and GmSIZ1b regulate vegetative growth in soybean

SIZ1 is a small ubiquitin‐related modifier (SUMO) E3 ligase that mediates post‐translational SUMO modification of target proteins and thereby regulates developmental processes and hormonal and environmental stress responses in Arabidopsis. However, the role of SUMO E3 ligases in crop plants is largely unknown. Here, we identified and characterized two Glycine max (soybean) SUMO E3 ligases, GmSIZ1a and GmSIZ1b. Expression of GmSIZ1a and GmSIZ1b was induced in response to salicylic acid (SA), heat, and dehydration treatment, but not in response to cold, abscisic acid (ABA), and NaCl treatment. Although GmSIZ1a was expressed at higher levels than GmSIZ1b, both genes encoded proteins with SUMO E3 ligase activity in vivo. Heterologous expression of GmSIZ1a or GmSIZ1b rescued the mutant phenotype of Arabidopsis siz1‐2, including dwarfism, constitutively activated expression of pathogen‐related genes, and ABA‐sensitive seed germination. Simultaneous downregulation of GmSIZ1a and GmSIZ1b (GmSIZ1a/b) using RNA interference (RNAi)‐mediated gene silencing decreased heat shock‐induced SUMO conjugation in soybean. Moreover, GmSIZ1RNAi plants exhibited reduced plant height and leaf size. However, unlike Arabidopsis siz1‐2 mutant plants, flowering time and SA levels were not significantly altered in GmSIZ1RNAi plants. Taken together, our results indicate that GmSIZ1a and GmSIZ1b mediate SUMO modification and positively regulate vegetative growth in soybean.     

Dynein participates in chromosome segregation in fission yeast

Background information. In eukaryotic cells, proper formation of the spindle is necessary for successful cell division. For faithful segregation of sister chromatids, each sister kinetochore must attach to microtubules that extend to opposite poles (chromosome bi‐orientation). At the metaphase—anaphase transition, cohesion between sister chromatids is removed, and each sister chromatid is pulled to opposite poles of the cell by microtubule‐dependent forces. Results. We have studied the role of the minus‐end‐directed motor protein dynein by analysing kinetochore dynamics in fission yeast cells deleted for the dynein heavy chain (Dhc1) or the light chain (Dlc1). In these mutants, we found an increased frequency of cells showing defects in chromosome segregation, which leads to the appearance of lagging chromosomes and an increased rate of chromosome loss. By following simultaneously kinetochore dynamics and localization of the checkpoint protein Mad2, we provide evidence that dynein function is not necessary for spindle‐assembly checkpoint inactivation. Instead, we have demonstrated that loss of dynein function alters chromosome segregation and activates the Mad2‐dependent spindle‐assembly checkpoint. Conclusions. These results show an unexpected role for dynein in the control of chromosome segregation in fission yeast, most probably operating during the process of bi‐orientation during early mitosis.     

SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis

Polo-like kinase 1 (Plk1)-interacting checkpoint helicase (PICH) localizes at the centromere and is critical for proper chromosome segregation during mitosis. However, the precise molecular mechanism of PICH's centromeric localization and function at the centromere is not yet fully understood. Recently, using Xenopus egg extract assays, we showed that PICH is a promiscuous SUMO binding protein. To further determine the molecular consequence of PICH/SUMO interaction on PICH function, we identified 3 SUMO-interacting motifs (SIMs) on PICH and generated a SIM-deficient PICH mutant. Using the conditional expression of PICH in cells, we found distinct roles of PICH SIMs during mitosis. Although all SIMs are dispensable for PICH's localization on ultrafine anaphase DNA bridges, only SIM3 (third SIM, close to the C-terminus end of PICH) is critical for its centromeric localization. Intriguingly, the other 2 SIMs function in chromatin bridge prevention. With these results, we propose a novel SUMO-dependent regulation of PICH's function on mitotic centromeres.     

Achiasmate Segregation of a B Chromosome from the X Chromosome in Two Species of Psyllids (Psylloidea, Homoptera)

The segregation of a B chromosome from the X chromosome was studied in male meiosis in two psyllid species, Rhinocola aceris (L.) and Psylla foersteri (Flor.) (Psylloidea, Homoptera). The frequency of segregation was determined from cells at metaphase II. In R. aceris, the B chromosome was mitotically stable and segregated quite regularly from the X chromosome in four geographically distant populations, while it showed less regular, but preferential segregation in one population. This was attributed to the presence of B chromosome variants that differ in their ability to interact with the X chromosome in segregation. In P. foersteri, the B chromosome was mitotically unstable and segregated preferentially from the X chromosome in spermatocyte cysts, which displayed one B chromosome in every cell. Behaviour of the B chromosome and X chromosome univalents during meiotic prophase and at metaphase I in R. aceris, and during anaphase I in P. foersteri suggested that the regular segregation resulted from the incorporation of B chromosomes in achiasmate segregation mechanisms with the X chromosome in the place occupied by the Y chromosome in species with XY system. The regular segregation of a B chromosome from the X chromosome may obscure the distinction of a B chromosome and an achiasmate Y chromosome in some cases. This revised version was published online in August 2006 with corrections to the Cover Date.     

SMC and the bactofilin/PadC scaffold have distinct yet redundant functions in chromosome segregation and organization in Myxococcus xanthus

In bacteria, ParABS systems and structural maintenance of chromosome (SMC) condensin-like complexes are important for chromosome segregation and organization. The rod-shaped Myxococcus xanthus cells have a unique chromosome arrangement in which a scaffold composed of the BacNOP bactofilins and PadC positions the essential ParB∙parS segregation complexes and the DNA segregation ATPase ParA in the subpolar regions. We identify the Smc and ScpAB subunits of the SMC complex in M. xanthus and demonstrate that SMC is conditionally essential, with Δsmc or ΔscpAB mutants being temperature sensitive. Inactivation of SMC caused defects in chromosome segregation and organization. Lack of the BacNOP/PadC scaffold also caused chromosome segregation defects but this scaffold is not essential for viability. Inactivation of SMC was synthetic lethal with lack of the BacNOP/PadC scaffold. Lack of SMC interfered with formation of the BacNOP/PadC scaffold while lack of this scaffold did not interfere with chromosome association by SMC. Altogether, our data support that three systems function together to enable chromosome segregation in M. xanthus. ParABS constitutes the basic and essential machinery. SMC and the BacNOP/PadC scaffold have different yet redundant roles in chromosome segregation with SMC supporting individualization of daughter chromosomes and BacNOP/PadC making the ParABS system operate more robustly.