We have analyzed the abundance of SUMO-conjugated species during the cell cycle in Xenopus egg extracts. The predominant SUMO conjugation products associated with mitotic chromosomes arose from SUMO conjugation of topoisomerase II. Topoisomerase II was modified exclusively by SUMO-2/3 during mitosis under normal circumstances, although we observed conjugation of topoisomerase II to SUMO-1 in extracts with exogenous SUMO-1 protein. Inhibition of SUMO modification by a dominant-negative mutant of the SUMO-conjugating enzyme Ubc9 (dnUbc9) did not detectably alter topoisomerase II activity, but it did increase the amount of unmodified topoisomerase II retained on mitotic chromosomes after high salt washing. dnUbc9 did not disrupt the assembly of condensed mitotic chromosomes or block progression of extracts through mitosis, but it did block the dissociation of sister chromatids at the metaphase–anaphase transition. Together, our results suggest that SUMO conjugation is important for chromosome segregation in metazoan systems, and that mobilization of topoisomerase II from mitotic chromatin may be a key target of this modification.
The Ran GTPase controls multiple cellular processes, including nuclear transport, mitotic checkpoints, spindle assembly and post-mitotic nuclear envelope reassembly. Here we examine the mitotic function of Crm1, the Ran-GTP-binding nuclear export receptor for leucine-rich cargo (bearing nuclear export sequence) and Snurportin-1 (ref. 3). We find that Crm1 localizes to kinetochores, and that Crm1 ternary complex assembly is essential for Ran-GTP-dependent recruitment of Ran GTPase-activating protein 1 (Ran-GAP1) and Ran-binding protein 2 (Ran-BP2) to kinetochores. We further show that Crm1 inhibition by leptomycin B disrupts mitotic progression and chromosome segregation. Analysis of spindles within leptomycin B-treated cells shows that their centromeres were under increased tension. In leptomycin B-treated cells, centromeres frequently associated with continuous microtubule bundles that spanned the centromeres, indicating that their kinetochores do not maintain discrete end-on attachments to single kinetochore fibres. Similar spindle defects were observed in temperature-sensitive Ran pathway mutants (tsBN2 cells). Taken together, our findings demonstrate that Crm1 and Ran-GTP are essential for Ran-BP2/Ran-GAP1 recruitment to kinetochores, for definition of kinetochore fibres and for chromosome segregation at anaphase. Thus, Crm1 is a critical Ran-GTP effector for mitotic spindle assembly and function in somatic cells.
Here we show that the PIASy protein is specifically required for mitotic modification of Topoisomerase-II by SUMO-2 conjugation in Xenopus egg extracts. PIASy was unique among the PIAS family members in its capacity to bind mitotic chromosomes and recruit Ubc9 onto chromatin. These properties were essential, since PIASy mutants that did not bind chromatin or failed to recruit Ubc9 were functionally inactive. We observed that PIASy depletion eliminated essentially all chromosomal accumulation of EGFP-SUMO-2-conjugated species, suggesting that it is the primary E3-like factor for mitotic chromosomal substrates of SUMO-2. PIASy-dependent SUMO-2-conjugated species were highly concentrated on the inner centromere, and inhibition of PIASy blocked anaphase sister chromatid segregation in egg extracts. Taken together, our observations suggest that PIASy is a critical regulator of mitotic SUMO-2 conjugation for Topoisomerase-II and other chromosomal substrates, and that its activity may have particular relevance for centromeric functions required for proper chromosome segregation.
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. INTRODUCTIONCommunication between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC; reviewed in Suntharalingam and Wente, 2003;Hetzer et al., 2005). At mitosis the metazoan nuclear pore disassembles into approximately a dozen subunits. The major subunit, both in size and complexity, is the nine-protein vertebrate Nup107-160 complex, which includes Nup160, Nup133, Nup107, Nup96, Nup85, Nup43, Nup37, Sec13, and Seh1 (Belgareh et al., 2001;Cronshaw et al., 2002;Harel et al., 2003b;Devos et al., 2004;Loiodice et al., 2004). In yeast, a nearly identical complex exists (Siniossoglou et al., 1996(Siniossoglou et al., , 2000Teixeira et al., 1997;Lutzmann et al., 2002), lacking only the vertebrate Nup43 and Nup37 subunits for which there are no yeast counterparts.Nuclear reconstitution in interphase Xenopus egg extracts depleted of the Nup107-160 complex produced nuclear envelopes with enclosed membranes, but completely devoid of nuclear pores (Harel et al., 2003b;Walther et al., 2003a). These studies suggest that the Nup107-160 complex is a central and early determinant in NPC assembly. Genetic studies in yeast indicate the complex plays a critical role in mRNA export (Doye et al., 1994;Heath et al., 1995;Li et al., 1995;Pemberton et al., 1995;Goldstein et al., 1996;Siniossoglou et al., 1996). In human cells, RNA interference (RNAi) and transfection of dominant-negative fragments of individual complex members have revealed that vertebrate mRNA export is similarly dependent upon this complex Boehmer et al., 2003;Harel et al., 2003b;Walther et al., 2003a).The Nup107-160...
Kinetochore protein CENP-I is regulated by SUMO protease SENP6 and RNF4, a ubiquitin ligase which targets polysumoylated proteins for degradation during mitosis.
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