The Ran GTPase is required for many cellular functions, including nucleocytoplasmic trafficking, spindle assembly, nuclear assembly and cell cycle control. The sole nucleotide exchange factor for Ran, RCC1, binds chromatin throughout the cell cycle. The GTPase activating protein for Ran, RanGAP1, localizes to the cytosolic face of the nuclear pore complex (NPC) during interphase through association with RanBP2, a large nucleoporin. The interphase distribution of Ran regulators leads to a high concentration of Ran-GTP in nuclei, and low Ran-GTP in cytosol. The major effectors for Ran are a family of Ran-GTP binding proteins that were discovered as nuclear transport receptors. These receptors are collectively called Karyopherins;those that mediate import are called Importins, and those that mediate export are called Exportins. Their cargo loading is governed by Ran-GTP levels: Importins bind to their cargo in the cytoplasm. Import complexes traverse the NPC and dissociate upon Ran-GTP-Importin binding. Exportins bind their cargo inside nuclei in complexes that contain Ran-GTP. After passage through the NPC, export complexes dissociate upon Ran-GTP hydrolysis. To date, two karyopherins have been shown to act as Ran effectors during mitosis: Importin-beta and the exportin Crm1. Proper spindle formation requires a chromatin-based gradient of Ran, with high Ran-GTP levels in the vicinity of mitotic chromosomes. This gradient is established through the activity of RCC1, which remains concentrated on mitotic chromosomes. RanBP1 is a co-activator of RanGAP1, which also forms a stable heterotrimeric complex with RCC1 and Ran (RRR complex) thereby inhibiting RCC1s nucleotide exchange activity. The function of the RRR complex has remained mysterious, however, because RanBP1 is physically separated from RCC1 during interphase. We have found that the RRR complex forms readily in M-phase Xenopus egg extracts (CSF-XEEs). RCC1 binding to chromatin and RRR complex assembly are mutually exclusive, so that promoting RRR complex formation through the addition of recombinant RanBP1 sequestered RCC1 away from chromatin. Consistent with earlier reports, RRR complex assembly inhibits RCC1s RanGEF activity in XEE. Together, these findings suggest that the RRR complex plays a key mitotic role in determining the partitioning of RCC1 between its active chromatin-bound and inactive soluble states, thereby setting both the location and magnitude of mitotic Ran-GTP production. Notably, RCC1s association to mitotic chromatin is dynamic, and there are particularly large changes during the metaphase-to-anaphase window. However, the timing of reported RCC1 modifications suggests that they do not cause such changes. We found that RanBP1 is phosphorylated during anaphase, and that this modification disrupts the RRR complex. Further analysis showed that RanBP1 phosphorylation drove increased RCC1 binding to chromatin in cycling XEE, and thereby indirectly enhanced anaphase Ran-GTP production. This modification may also contribute to Ran pathway function in early interphase, because elevated RCC1 on anaphase chromatin should provide high levels of Ran-GTP to facilitate nuclear re-assembly. Finally, separation of RCC1 and RanBP1 after RRR complex dissociation allows RCC1 sequestration to re-forming nuclei while excluding RanBP1 into the early interphase cytosol. Together, these findings document a novel role of the RanBP1 protein in controlling the localization and activity of Rans nucleotide exchange factor, RCC1. We have shown that phosphorylation of RanBP1 during anaphase drives changes in RCC1 dynamics and allows increased Ran-GTP production. These findings resolve important and long-standing questions within the Ran field regarding the function of the RanBP1/Ran/RCC1 complex and its dynamics. We have also become interested the regulation of IRBIT through its interactions with the Ran pathway. IRBIT is a conserved metazoan protein that has been implicated in a diverse set of functions. IRBIT consists of a putative enzymatic domain that has similarity to S-adenosylhomocysteine hydrolase and an essential N-terminal domain. To identify proteins that bind IRBIT, we performed immunoprecipitation from lysates of HeLa cells, followed by SDS-PAGE and protein staining. We identified prominent co-precipitating proteins and identified them by mass-spectrometry. Ribonucleotide reductase (RNR) was among the most abundant IRBIT-binding proteins, so we have investigated the relationship between these proteins. RNR supplies the balanced pools of deoxynucleotide triphosphates (dNTPs) necessary for DNA replication and maintenance of genomic integrity. RNR is subject to allosteric regulatory mechanisms in all eukaryotes, as well as to control by small protein inhibitors Sml1p and Spd1p in budding and fission yeast, respectively. We found that IRBIT forms a dATP-dependent complex with RNR, stabilizing dATP in the activity site of RNR, and thus inhibiting the enzyme. Formation of the RNR-IRBIT complex is regulated through phosphorylation of IRBIT, and ablation of IRBIT expression in HeLa cells causes imbalanced dNTP pools and altered cell cycle progression. Together, our findings provide a new mechanism for RNR regulation in higher eukaryotes that acts by enhancing allosteric RNR inhibition by dATP.

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13
Fiscal Year
2014
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U.S. National Inst/Child Hlth/Human Dev
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Dasso, Mary (2013) A Mad that wears two hats: Mad1's control of nuclear trafficking. Dev Cell 24:121-2
Bernad, Rafael; Sanchez, Patricia; Rivera, Teresa et al. (2011) Xenopus HJURP and condensin II are required for CENP-A assembly. J Cell Biol 192:569-82
Dasso, Mary (2011) Shedding light on mysterious microtubules. Dev Cell 20:e1
Mishra, Ram Kumar; Chakraborty, Papia; Arnaoutov, Alexei et al. (2010) The Nup107-160 complex and gamma-TuRC regulate microtubule polymerization at kinetochores. Nat Cell Biol 12:164-9
Samoshkin, Alexander; Arnaoutov, Alexei; Jansen, Lars E T et al. (2009) Human condensin function is essential for centromeric chromatin assembly and proper sister kinetochore orientation. PLoS One 4:e6831
Joseph, Jomon; Dasso, Mary (2008) The nucleoporin Nup358 associates with and regulates interphase microtubules. FEBS Lett 582:190-6