During heat shock stress, importin β 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 β 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.

SUMOylation of TopoIIα inhibits its decatenation activity, preventing resolution of tangled DNA at centromeres before anaphase onset.

The human importin-␤ 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. Most genetic processes, including chromosome replication and transcription, occur in the nucleus, and thus the selection of proteins that enter the nucleus and act there is crucial for cellular processes (1). During interphase, all nuclear proteins cross the nuclear envelope via nuclear pores, channels that constitute a selective permeability barrier for macromolecules. Only a fraction of proteins traverse these channels by means of free diffusion; most nuclear proteins are imported into or exported out of the nucleus with the assistance of importin-␤ (Imp-␤) 1 family proteins (Imp-␤s) (2, 3). Imp-␤s interact with the nuclear pore complex in a way that allows them to travel in and out of the nucleus. To drive cargo From the ‡Cellular Dynamics Laboratory, Advanced Science Institute, RIKEN, 2-1 Hirosawa,

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 α α α α /β β β β , is down-regulated, while the nuclear import of 70 kD heatshock 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 α α α α accumulate in the nucleus in response to heat-shock. An analysis of the behaviour of importin α α α α with fluorescence recovery after photobleaching and fluorescence loss in photobleaching studies show that nuclear importin α α α α 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 α α α α , 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.

␤-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 ␤-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 ␤-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 ␤-catenin required for export substantially overlapped the region required for import. Therefore, the NPC translocation of ␤-catenin is apparently reversible, which is consistent with findings reported previously. However, different translocating molecules blocked nuclear import and export of ␤-catenin differentially. The data herein indicate that ␤-catenin shows an overlapping sequence requirement for its import and export but that bidirectional movement through the NPC proceeds through distinct molecular interactions.

Transport receptors of the importin β family continuously shuttle between the nucleus and cytoplasm. We previously reported that the nuclear export of importin β involves energy-requiring step(s) in living cells. Here, we show that the in vitro nuclear export of importin β also requires energy input. Cytosol, depleted of ATP-binding proteins, did not support the sufficient nuclear export of importin β. 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 β. These effects of hsc70 were observed in the nuclear export of importin β, but also for other import receptors, transportin and importin α. These results suggest that hsc70 broadly modulates nucleocytoplasmic transport systems by regulating the nuclear export of receptor proteins.

Aid-based RCC1 conditional knockout cells are established to examine the functional domains of RCC1. Based on an analysis using RCC1-deficient cells, two pathways are proposed for nuclear localization of RCC1: NLS-dependent and NLS-independent pathways.

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.