Nup2 requires a highly divergent partner, NupA, to fulfill functions at nuclear pore complexes and the mitotic chromatin region.
Bottom Line: These mitotic problems are not caused by overall defects in mitotic NPC disassembly-reassembly or general nuclear import.However, without Nup2 or NupA, although the SAC protein Mad1 locates to its mitotic locations, it fails to locate to NPCs normally in G1 after mitosis.Collectively the study provides new insight into the roles of Nup2 and NupA during mitosis and in a surveillance mechanism that regulates nucleokinesis when mitotic defects occur after SAC fulfillment.
Affiliation: Laboratory of Gene Regulation and Development, National Institute for Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.Show MeSH
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Mentions: Nup2 and its mammalian orthologue Nup50 facilitate importin-α/β–mediated nuclear transport (Solsbacher et al., 2000; Gilchrist et al., 2002; Lindsay et al., 2002; Matsuura and Stewart, 2005; Stewart, 2007; Makise et al., 2012). Consistent with A. nidulans Nup2 playing a similar role, it purified with importins α and β (Figure 1C). To investigate their roles in nuclear transport, we monitored NLS-DsRed to detect whether Δnup2 and ΔnupA mutant nuclei are defective in nuclear protein import. NLS-DsRed was transported into Δnup2 and ΔnupA nuclei during interphase, with little to no cytoplasmic signal apparent (Figure 8, A and B, 0′). This indicates that nuclear transport is active in the absence of Nup2 or NupA. To determine whether the rate of NLS-DsRed nuclear transport is modified without Nup2 and NupA, we followed the rate of dispersal of NLS-DsRed during mitotic entry and then its rate of import during mitotic exit. As cells entered mitosis, NLS-DsRed dispersed from nuclei and was rapidly transported back into daughter G1 nuclei during mitotic exit in wild-type as well as Δnup2 and ΔnupA nuclei (Figure 8B). Note that NLS-DsRed remained mitotically dispersed longer in the mutants than the wild type because they have prolonged SAC-mediated delayed mitosis (Figure 6, A and B, and Supplemental Figure S3). We quantified the fluorescence pixel intensity of the nuclear NLS-DsRed signal at 10-s intervals in comparison to wild type (Figure 8C). Dispersal and import rates of NLS-DsRed in the mutants were comparable to wild type, with the kinetics of both being essentially superimposable between the mutants and wild type. This suggests there are no overall defects in nuclear protein import in the absence of Nup2 or NupA. In addition, because the disassembly and reassembly of NPCs drive release and reimport of NLS-DsRed, respectively, the results further indicate that the rates of mitotic NPC disassembly and reassembly are not affected by the absence of Nup2 or NupA.
Affiliation: Laboratory of Gene Regulation and Development, National Institute for Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.