Modification in reverse: the SUMO proteases

SUMOs (small ubiquitin-like modifiers) are ubiquitin-related proteins that become covalently conjugated to cellular target proteins that are involved in a variety of processes. Frequently, this modification has a key role in regulating the activities of those targets and, thus, their cellular functions. SUMO conjugation is a highly dynamic process that can be rapidly reversed by the action of members of the Ubl (ubiquitin-like protein)-specific protease (Ulp) family. The same family of enzymes is also responsible for maturation of newly synthesized SUMOs prior to their initial conjugation. Recent advances in structural, biochemical and cell biological analysis of Ulp/SENPs reveal their high degree of specificity towards SUMO paralogs, in addition to discrimination between processing, deconjugation and chain-editing reactions. The dissimilar sub-nuclear localization patterns of Ulp/SENPs and phenotypes of Ulp/SENP mutants further indicate that different Ulp/SENPs have distinct and non-redundant roles.     

The Nup107-160 nucleoporin complex is required for correct bipolar spindle assembly

The Nup107-160 complex is a critical subunit of the nuclear pore. This complex localizes to kinetochores in mitotic mammalian cells, where its function is unknown. To examine Nup107-160 complex recruitment to kinetochores, we stained human cells with antisera to four complex components. Each antibody stained not only kinetochores but also prometaphase spindle poles and proximal spindle fibers, mirroring the dual prometaphase localization of the spindle checkpoint proteins Mad1, Mad2, Bub3, and Cdc20. Indeed, expanded crescents of the Nup107-160 complex encircled unattached kinetochores, similar to the hyperaccumulation observed of dynamic outer kinetochore checkpoint proteins and motors at unattached kinetochores. In mitotic Xenopus egg extracts, the Nup107-160 complex localized throughout reconstituted spindles. When the Nup107-160 complex was depleted from extracts, the spindle checkpoint remained intact, but spindle assembly was rendered strikingly defective. Microtubule nucleation around sperm centrosomes seemed normal, but the microtubules quickly disassembled, leaving largely unattached sperm chromatin. Notably, Ran-GTP caused normal assembly of microtubule asters in depleted extracts, indicating that this defect was upstream of Ran or independent of it. We conclude that the Nup107-160 complex is dynamic in mitosis and that it promotes spindle assembly in a manner that is distinct from its functions at interphase nuclear pores.     

Identification of sex in Cetaceans by multiplexing with three ZFX and ZFY specific primers

We sequenced 540 nucleotides of the last exon in the ZFY/ZFX gene in two males and two females for eight cetacean species; four odontocetes (toothed whales) and four mysticetes (baleen whales). Based upon the obtained nucleotide sequences, we designed two sets of oligonucleotide primers for specific amplification of the ZFX and the ZFY sequence in odontocetes and mysticetes, respectively. Each primer set consisted of three oligonucleotides; one forward-orientated primer, which anneals to the ZFY as well as the ZFX sequence, and two reverse-orientated primers that anneal to either the ZFX or the ZFY sequence. The resulting two amplification products (specific for the ZFY and ZFX sequences) can be distinguished by gel-electrophoresis through 2% NuSieve™. The accuracy of the technique was tested by determination of gender in 214 individuals of known sex. Finally we applied the technique to determine the sex of 3570 cetacean specimens; 2284 humpback whales, 315 fin whales, 37 blue whales, 7 minke whales, as well as 592 belugas, 335 narwhals and 25 harbour porpoises.     

Association of the human SUMO-1 protease SENP2 with the nuclear pore

SUMO-1 is a small ubiquitin-like protein that can be covalently conjugated to other proteins. A family of proteases catalyzes deconjugation of SUMO-1-containing species. Members of this family also process newly synthesized SUMO-1 into its conjugatable form. To understand these enzymes better, we have examined the localization and behavior of the human SUMO-1 protease SENP2. Here we have shown that SENP2 associates with the nuclear face of nuclear pores and that this association requires protein sequences near the N terminus of SENP2. We have also shown that SENP2 binds to Nup153, a nucleoporin that is localized to the nucleoplasmic face of the pore. Nup153 binding requires the same domain of SENP2 that mediates its targeting in vivo. Removal of the Nup153-interacting region of SENP2 results in a significant change in the spectrum of SUMO-1 conjugates within the cell. Our results suggest that association with the pore plays an important negative role in the regulation of SENP2, perhaps by restricting its activity to a subset of the conjugated proteins within the nucleus.     

Phosphorylation of Xenopus p31comet potentiates mitotic checkpoint exit

p31^comet plays an important role in spindle assembly checkpoint (SAC) silencing. However, how p31^comet''s activity is regulated remains unclear. Here we show that the timing of M-phase exit in Xenopus egg extracts (XEEs) depends upon SAC activity, even under conditions that are permissive for spindle assembly. p31^comet antagonizes the SAC, promoting XEE progression into anaphase after spindles are fully formed. We further show that mitotic p31^comet phosphorylation by Inhibitor of nuclear factor κ-B kinase-β (IKK-β) enhances this role in SAC silencing. Together, our findings implicate IKK-β in the control of anaphase timing in XEE through p31^comet activation and SAC downregulation.     

Leaf Development in Lolium temulentum L.: Progressive Changes in Soluble Polypeptide Complement and Isoenzymes

Ougham, Helen J., Jones, Thomas W. A. and Evans, Mair LL. 1987.Leaf development in Lolium temulentum L.: progressive changesin soluble polypeptide complement and isoenzymes.—J. exp.Bot. 38: 1689–1696. The spectrum of soluble polypeptides extracted from segmentsof the developing 4th leaf of Lolium temulentum simplified withincreasing distance from the leaf base. Most of the metabolicallyimportant isoenzymes analysed also exhibited gradients of activitywith respect to distance from the base, and in some cases twoor more contrasting gradients were observed for a given enzyme. Key words: Gradients, isoenzymes, leaves, Lolium temulentum,, soluble polypeptides     

Nucleolar protein B23/nucleophosmin regulates the vertebrate SUMO pathway through SENP3 and SENP5 proteases

Ubiquitin-like protein/sentrin-specific proteases (Ulp/SENPs) mediate both processing and deconjugation of small ubiquitin-like modifier proteins (SUMOs). Here, we show that Ulp/SENP family members SENP3 and SENP5 localize within the granular component of the nucleolus, a subnucleolar compartment that contains B23/nucleophosmin. B23/nucleophosmin is an abundant shuttling phosphoprotein, which plays important roles in ribosome biogenesis and which has been strongly implicated in hematopoietic malignancies. Moreover, we found that B23/nucleophosmin binds SENP3 and SENP5 in Xenopus laevis egg extracts and that it is essential for stable accumulation of SENP3 and SENP5 in mammalian tissue culture cells. After either codepletion of SENP3 and SENP5 or depletion of B23/nucleophosmin, we observed accumulation of SUMO proteins within nucleoli. Finally, depletion of these Ulp/SENPs causes defects in ribosome biogenesis reminiscent of phenotypes observed in the absence of B23/nucleophosmin. Together, these results suggest that regulation of SUMO deconjugation may be a major facet of B23/nucleophosmin function in vivo.     

Effect of inoculum rate on mycorrhizal growth responses in pot-grown onion and clover

Summary White clover and onion plants were grown from seed in pots of sandy loam above pads of mycorrhizal inoculum soil at 0.17–1.40 g/pot (equivalent to 250–2000 kg/ha) and harvested on four occasions. In sterilized soil increasing inoculum rates increased the onset and size of the mycorrhizal growth response of white clover. In unsterilized soil the indigenous mycorrhizal fungi greatly stimulated growth of both clover and onion. Nevertheless, all mycorrhizal inoculum rates further stimulated shoot growth in onion (92% increase over all harvests), while only the highest inoculum rate significantly stimulated clover growth (52% increase).     

Genome informatics: taming the avalanche of genomic data

A report on the fourth Cold Spring Harbor Laboratory/Wellcome Trust Conference on Genome Informatics, Hinxton, UK, 22-26 September 2004.