RCC1 regulates inner centromeric composition in a Ran-independent fashion.

RCC1 associates to chromatin dynamically within mitosis and catalyzes Ran-GTP production. Exogenous RCC1 disrupts kinetochore structure in Xenopus egg extracts (XEEs), but the molecular basis of this disruption remains unknown. We have investigated this question, utilizing replicated chromosomes that possess paired sister kinetochores. We find that exogenous RCC1 evicts a specific subset of inner KT proteins including Shugoshin-1 (Sgo1) and the chromosome passenger complex (CPC). We generated RCC1 mutants that separate its enzymatic activity and chromatin binding. Strikingly, Sgo1 and CPC eviction depended only on RCC1's chromatin affinity but not its capacity to produce Ran-GTP. RCC1 similarly released Sgo1 and CPC from synthetic kinetochores assembled on CENP-A nucleosome arrays. Together, our findings indicate RCC1 regulates kinetochores at the metaphase-anaphase transition through Ran-GTP-independent displacement of Sgo1 and CPC.

Chromatin binding of RCC1 during mitosis is important for its nuclear localization in interphase.

RCC1, a guanine nucleotide exchange factor of the small GTPase Ran, plays various roles throughout the cell cycle. However, the functions of RCC1 in biological processes in vivo are still unclear. In particular, although RCC1 has multifunctional domains, the biological significance of each domain is unclear. To examine each domain of RCC1, we established an RCC1 conditional knockout chicken DT40 cell line and introduced various RCC1 mutants into the knockout cells. We found that nuclear reformation did not occur properly in RCC1-deficient cells and examined whether specific RCC1 mutants could rescue this phenotype. Surprisingly, we found that neither the nuclear localization signal nor the chromatin-binding domain of RCC1 is essential for its function. However, codisruption of these domains resulted in defective nuclear reformation, which was rescued by artificial nuclear localization of RCC1. Our data indicate that chromatin association of RCC1 during mitosis is crucial for its proper nuclear localization in the next interphase. Moreover, proper nuclear localization of RCC1 in interphase is essential for its function through its nucleotide exchange activity.

Analysis of nucleocytoplasmic transport in digitonin-permeabilized cells under different cellular conditions.

The regulation of nucleocytoplasmic transport is crucial not only for basic cellular activities but also for physiological adaptation to specific situation during the cell cycle, development, or stress. Although a wide variety of transport pathways have been identified in eukaryotic cells, the functional significance of their multiplicity remains unclear. The best-characterized nuclear transport receptors (NTRs) are the members of the importin ß family (karyopherin, transportin) whose association with specific cargoes is regulated by the GTPase Ran. In this chapter, we first provide an overview of the various expression vectors used to purify recombinant NTRs. We then describe two sets of recent examples of using well-established digitonin-permeabilized cell-free transport systems in mammalian cells to mimic different cellular conditions in living cells: normal/heat-shock conditions and interphase/mitosis. In the former case, physiological regulation impacts different transport pathways in opposite ways. In the latter case, the importin ß-Ran system is used at different cell-cycle stages but with the same biochemical principle to specify the nuclear localization and chromatin loading of a specific protein, respectively. This in vitro transport assay, when adapted to specific cellular conditions or particular substrates, should help to uncover specific transport pathways or transport factors function under different cellular conditions.

Identification of cargo proteins specific for the nucleocytoplasmic transport carrier transportin by combination of an in vitro transport system and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics.

The human importin-ß family consists of 21 nucleocytoplasmic transport carrier proteins that carry proteins and RNAs across the nuclear envelope through nuclear pores in specific directions. These transport carriers are responsible for the nucleocytoplasmic transport of thousands of proteins, but the cargo allocation of each carrier, which is necessary information if one wishes to understand the physiological context of transport, is poorly characterized. To address this issue, we developed a high-throughput method to identify the cargoes of transport carriers by applying stable isotope labeling by amino acids in cell culture to construct an in vitro transport system. Our method can be outlined in three steps. (1) Cells are cultured in a medium containing a stable isotope. (2) The cell membranes of the labeled cells are permeabilized, and proteins extracted from unlabeled cells are transported into the nuclei of the permeabilized cells. In this step, the reaction system is first depleted of all importin-ß family carriers and then supplemented with a particular importin-ß family carrier of interest. (3) Proteins in the nuclei are extracted and analyzed quantitatively via LC-MS/MS. As an important test case, we used this method to identify cargo proteins of transportin, a representative member of the importin-ß family. As expected, the identified candidate cargo proteins included previously reported transportin cargoes as well as new potential cargoes, which we corroborated via in vitro binding assays. The identified cargoes are predominately RNA-interacting proteins, affirming that cargoes allotted to the same carrier share functional characteristics. Finally, we found that the transportin cargoes possessed at least two classes of signal sequences: the well characterized PY-nuclear localization signals specific for transportin, and Lys/Arg-rich segments capable of binding to both transportin and importin-ß. Thus, our method will be useful for linking a carrier to features shared among its cargoes and to specific nuclear localization signals.

Hikeshi, a nuclear import carrier for Hsp70s, protects cells from heat shock-induced nuclear damage.

During heat shock stress, importin ß family-mediated nucleocytoplasmic trafficking is downregulated, whereas nuclear import of the molecular chaperone Hsp70s is upregulated. Here, we identify a nuclear import pathway that operates during heat shock stress and is mediated by an evolutionarily conserved protein named "Hikeshi," which does not belong to the importin ß family. Hikeshi binds to FG-Nups and translocates through nuclear pores on its own, showing characteristic features of nuclear transport carriers. In reconstituted transport, Hikeshi supports the nuclear import of the ATP form of Hsp70s, but not the ADP form, indicating the importance of the Hsp70 ATPase cycle in the import cycle. In living cells, depletion of Hikeshi inhibits heat shock-induced nuclear import of Hsp70s, reduces cell viability after heat shock stress, and significantly delays the attenuation and reversion of multiple heat shock-induced nuclear phenotypes. Nuclear Hsp70s rescue the effect of Hikeshi depletion at least in part. Thus, Hsp70s counteract heat shock-induced damage by acting inside of the nucleus.

PIASy-dependent SUMOylation regulates DNA topoisomerase IIalpha activity.

DNA topoisomerase IIα (TopoIIα) is an essential chromosome-associated enzyme with activity implicated in the resolution of tangled DNA at centromeres before anaphase onset. However, the regulatory mechanism of TopoIIα activity is not understood. Here, we show that PIASy-mediated small ubiquitin-like modifier 2/3 (SUMO2/3) modification of TopoIIα strongly inhibits TopoIIα decatenation activity. Using mass spectrometry and biochemical analysis, we demonstrate that TopoIIα is SUMOylated at lysine 660 (Lys660), a residue located in the DNA gate domain, where both DNA cleavage and religation take place. Remarkably, loss of SUMOylation on Lys660 eliminates SUMOylation-dependent inhibition of TopoIIα, which indicates that Lys660 SUMOylation is critical for PIASy-mediated inhibition of TopoIIα activity. Together, our findings provide evidence for the regulation of TopoIIα activity on mitotic chromosomes by SUMOylation. Therefore, we propose a novel mechanism for regulation of centromeric DNA catenation during mitosis by PIASy-mediated SUMOylation of TopoIIα.

The 70-kD heat shock cognate protein (hsc70) facilitates the nuclear export of the import receptors.

Transport receptors of the importin beta family continuously shuttle between the nucleus and cytoplasm. We previously reported that the nuclear export of importin beta involves energy-requiring step(s) in living cells. Here, we show that the in vitro nuclear export of importin beta also requires energy input. Cytosol, depleted of ATP-binding proteins, did not support the sufficient nuclear export of importin beta. Further purification revealed that the active component in the absorbed fraction was a 70-kD heat shock cognate protein (hsc70). The addition of recombinant hsc70, but not an ATPase-deficient hsc70 mutant, to the depleted cytosol restored the export activity. In living cells, depletion of hsc70 caused the significant nuclear accumulation of importin beta. These effects of hsc70 were observed in the nuclear export of importin beta, but also for other import receptors, transportin and importin alpha. These results suggest that hsc70 broadly modulates nucleocytoplasmic transport systems by regulating the nuclear export of receptor proteins.

beta-Catenin shows an overlapping sequence requirement but distinct molecular interactions for its bidirectional passage through nuclear pores.

beta-Catenin is an example of a typical molecule that can be translocated bidirectionally through nuclear pore complexes (NPCs) on its own in a facilitated manner. In this work the nuclear import and export of beta-catenin were examined to compare the sequence requirement of this molecule and to determine whether molecular interactions required for its bidirectional NPC passage are distinct or not. Deletion analysis of beta-catenin revealed that armadillo repeats 10-12 and the C terminus comprise the minimum region necessary for nuclear migration activity. Further dissection of this fragment showed that the C terminus tail plays an essential role in nuclear migration. The region of beta-catenin required for export substantially overlapped the region required for import. Therefore, the NPC translocation of beta-catenin is apparently reversible, which is consistent with findings reported previously. However, different translocating molecules blocked nuclear import and export of beta-catenin differentially. The data herein indicate that beta-catenin shows an overlapping sequence requirement for its import and export but that bidirectional movement through the NPC proceeds through distinct molecular interactions.

Heat-shock induced nuclear retention and recycling inhibition of importin alpha.

Heat-shock induces a strong stress response and modifies all aspects of cellular physiology, which involves dynamic changes in the nucleocytoplasmic distributions of a variety of proteins. Many distinct nucleocytoplasmic transport pathways exist in eukaryotic cells, but how a particular transport pathway is regulated under different cellular conditions remains elusive. The finding of this study indicate that conventional nuclear import, which is mediated by importin alpha/beta, is down-regulated, while the nuclear import of 70 kD heat-shock cognate protein is up-regulated in heat-shock cells. Among the factors involved in the mediation of the conventional nuclear import, significant levels of importin alpha accumulate in the nucleus in response to heat-shock. An analysis of the behaviour of importin alpha with fluorescence recovery after photobleaching and fluorescence loss in photobleaching studies show that nuclear importin alpha becomes less mobile and its nucleocytoplasmic recycling is impaired in heat-shock cells. These data coincided well with biochemical and cytological studies. Our present data show that heat-shock induces the nuclear accumulation, nuclear retention, and recycling inhibition of importin alpha, resulting in the suppression of conventional nuclear import. This suggests a new regulatory mechanism for the adaptation of cells to environmental changes, such as heat-shock.

Specific monoclonal antibody against nuclear import factor, importin alpha1/Rch1.

Importin alpha1/Rch1, an importin alpha family member, mediates the nuclear import of karyophilic proteins. The present study reports on a monoclonal antibody (MAb) directed against mammalian importin alpha1/Rch1, which was produced by the hybridization of mouse myeloma cells with lymph node cells of an immunized rat. The MAb 1A6 specifically recognized importin alpha1/Rch1 among the importin alpha isoforms, as evidenced by Western blotting. Furthermore, 1A6 detected importin alpha1/Rch1 in HeLa cells by immunofluorescence. This MAb will be useful in immunolocalization and immunoblotting experiments, conducted on different tissue types, to determine the levels of expression of importin alpha1/Rch1 throughout development, as well as further analyses of the biological function and cellular dynamics of this protein.