Ran is a small GTPase required for nucleocytoplasmic trafficking, spindle assembly, nuclear assembly and cell cycle control. The nucleotide exchange factor for Ran, RCC1, is a chromatin-associated protein. The GTPase activating protein for Ran, RanGAP1, is cytoplasmic during interphase. During mitosis, the bulk of RanGAP1 is broadly distributed, although a significant fraction of RanGAP1 becomes associated with kinetochores in a SUMO-1 dependent fashion (see Z01 HD001902-10). Ran-GTP nucleotide hydrolysis also requires a family of Ran-GTP binding proteins, which act as RanGAP1 accessory factors. This family includes RanBP1 and RanBP2. The distribution of Ran?s regulators has been widely hypothesized to modulate local concentrations of Ran-GTP within cells, spatially directing processes in which Ran has been implicated. We have been examining the mechanisms through which key Ran regulators are localized within mitotic metazoan cells and the functional consequences to cells when such distribution patterns are disrupted. To look at the mitotic fate of RCC1, we examined its chromosomal association in cycling Xenopus egg extracts. Remarkably, the amount of chromatin-associated RCC1 increased drastically at anaphase onset. In order to determine the significance of this finding, we assayed whether the Ran pathway has a role in mitotic progression or checkpoint control in Xenopus egg extracts. Prior to each anaphase, chromosomes are aligned onto the metaphase through attachment between spindle microtubules and kinetochores, proteinaceous structures that assemble over the centromere of each chromosome. The spindle assembly checkpoint is a cell cycle regulatory pathway that monitors spindle assembly in all eukaryotic cells and prevents the onset of anaphase and the dissolution of sister chromatin cohesion in the presence of unattached on inappropriately attached kinetochores. Remarkably, a five- to seven-fold elevation of RCC1 concentration was sufficient to abrogate spindle checkpoint arrest in extracts containing nuclei plus the microtubule depolymerizing agent nocodazole. While assembly of centromeric structures occurred normally under these circumstances, we found that many checkpoint components were mis-localized away from kinetochores after RCC1 addition, indicating that increased RCC1 levels abolish checkpoint arrest by altering interactions between kinetochores and checkpoint regulators. Together with additional data, these observations suggest that the spindle checkpoint is directly responsive to Ran-GTP levels. Notably, the capacity of RCC1 to reverse spindle checkpoint arrest is specific, since increased RCC1 does not compromise other modes of M phase arrest (e.g. CSF arrest). Our results suggest a model wherein complete chromosome alignment on the metaphase plate triggers the increased binding of RCC1 to chromosomes, resulting in the local elevation of Ran-GTP levels and the ejection of the final population of kinetochore-associated checkpoint components. In parallel to our studies on RCC1, we have investigated the mitotic behavior and function of RanGAP1. In metazoans, RanGAP1 is conjugated with SUMO-1. Studies by this group and others have shown that SUMO-1 modification causes RanGAP1 to associate during interphase with Ubc9 and RanBP2, a large nuclear pore protein with multiple Ran-GTP binding domains and a SUMO E3 ligase domain. Through further investigation of the mitotic behavior and interactions of RanGAP1, we have found that RanGAP1 associates with kinetochores in a SUMO-1 dependent manner. Notably, RanBP2 co-localized with RanGAP1 on spindles and kinetochores. Recently, we have examined the structural requirements for targeting RanGAP1 and RanBP2, as well as their function in mitosis. We found that elimination of RanBP2 expression through RNA interference (RNAi) displaced RanGAP1 from kinetochores, supporting the notion that these proteins target to kinetochores as part of a single complex. Both proteins were displaced after RNAi of integral kinetochore components, suggesting that they require intact kinetochore structures to localize appropriately. By contrast, peripheral kinetochore proteins were not essential for correct targeting of either protein. Cells depleted of RanBP2 show abnormalities in both spindle formation and mitotic progression, substantiating the importance of correct targeting of the RanGAP1/RanBP2 complex during mitosis.
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