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.

The interaction between the import carrier Hikeshi and HSP70 is modulated by heat, facilitating the nuclear import of HSP70 under heat stress conditions.

Heat stress strongly triggers the nuclear localization of the molecular chaperone HSP70. Hikeshi functions as a unique nuclear import carrier of HSP70. However, how the nuclear import of HSP70 is activated in response to heat stress remains unclear. Here, we investigated the effects of heat on the nuclear import of HSP70. In vitro transport assays revealed that pretreatment of the test samples with heat facilitated the nuclear import of HSP70. Furthermore, binding of Hikeshi to HSP70 increased when temperatures rose. These results indicated that heat is one of the factors that activates the nuclear import of HSP70. Previous studies showed that the F97A mutation in Hikeshi in an extended loop induced an opening in the hydrophobic pocket and facilitated the translocation of Hikeshi through the nuclear pore complex. We found that nuclear accumulation of HSP70 occurred at a lower temperature in cells expressing the Hikeshi-F97A mutant than in cells expressing wild-type Hikeshi. Collectively, our results show that the movement of the extended loop may play an important role in the interaction of Hikeshi with both FG (phenylalanine-glycine)-nucleoporins and HSP70 in a temperature-dependent manner, resulting in the activation of nuclear import of HSP70 in response to heat stress.

Hikeshi modulates the proteotoxic stress response in human cells: Implication for the importance of the nuclear function of HSP70s.

Hikeshi mediates the heat stress-induced nuclear import of heat-shock protein 70 (HSP70s: HSP70/HSC70). Dysfunction of Hikeshi causes some serious effects in humans; however, the cellular function of Hikeshi is largely unknown. Here, we investigated the effects of Hikeshi depletion on the survival of human cells after proteotoxic stress and found opposite effects in HeLa and hTERT-RPE1 (RPE) cells; depletion of Hikeshi reduced the survival of HeLa cells, but increased the survival of RPE cells in response to proteotoxic stress. Hikeshi depletion sustained heat-shock transcription factor 1 (HSF1) activation in HeLa cells after recovery from stress, but introduction of a nuclear localization signal-tagged HSC70 in Hikeshi-depleted HeLa cells down-regulated HSF1 activity. In RPE cells, the HSF1 was efficiently activated, but the activated HSF1 was not sustained after recovery from stress, as in HeLa cells. Additionally, we found that p53 and subsequent up-regulation of p21 were higher in the Hikeshi-depleted RPE cells than in the wild-type cells. Our results indicate that depletion of Hikeshi renders HeLa cells proteotoxic stress-sensitive through the abrogation of the nuclear function of HSP70s required for HSF1 regulation. Moreover, Hikeshi depletion up-regulates p21 in RPE cells, which could be a cause of its proteotoxic stress resistant.

Leukoencephalopathy and early death associated with an Ashkenazi-Jewish founder mutation in the Hikeshi gene.

BACKGROUND: Leukodystrophies are genetic white matter disorders affecting the formation or maintenance of myelin. Among the recently discovered genetic defects associated with leukodystrophies, several genes converge on a common mechanism involving protein transcription/translation and ER stress response. METHODS: The genetic basis of a novel congenital leukodystrophy, associated with early onset spastic paraparesis, acquired microcephaly and optic atrophy was studied in six patients from three unrelated Ashkenazi-Jewish families. To this end we used homozygosity mapping, exome analysis, western blot (Hikeshi, HSF1-pS326 and b-actin) in patient fibroblasts, indirect immunofluorescence (HSP70 and HSF1) in patient fibroblasts undergoing heat shock stress, nuclear injection of plasmids expressing Hikeshi or EGFP in patient fibroblasts, in situ hybridization and Immunoblot analysis of Hikeshi in newborn and adult mouse brain. RESULTS: All the patients were homozygous for a missense mutation, p.Val54Leu, in C11ORF73 encoding HSP70 nuclear transporter protein, Hikeshi. The mutation segregated with the disease in the families and was carried by 1:200 Ashkenazi-Jewish individuals. The mutation was associated with undetectable level of Hikeshi in the patients' fibroblasts and with lack of nuclear HSP70 during heat shock stress, a phenomenon which was reversed upon the introduction of normal human Hikeshi to the patients cells. Hikeshi was found to be expressed in central white matter of mouse brain. CONCLUSIONS: These data underscore the importance of Hikeshi for HSP70 relocation into the nucleus. It is likely that in the absence of Hikeshi, HSP70 cannot attenuate the multiple heat shock induced nuclear phenotypes, leaving the cells unprotected during heat shock stress. We speculate that the sudden death of three of the six patients following a short febrile illness and the life-threatening myo-pericarditis in the fourth are the result of excess extra-nuclear HSP70 level which initiates cytokine release or provide target for natural killer cells. Alternatively, nuclear HSP70 might play an active role in stressed cells protection.

Nucleocytoplasmic transport under stress conditions and its role in HSP70 chaperone systems.

BACKGROUND: In eukaryotic cells, molecular trafficking between the nucleus and cytoplasm is a highly regulated process related to cellular homeostasis and cellular signaling. However, various cellular stresses induce the perturbation of conventional nucleocytoplasmic transport pathways, resulting in the nucleocytoplasmic redistribution of many functional proteins. SCOPE OF REVIEW: We describe the recent insights into the mechanism and functions of nuclear import of cytosolic chaperone HSP70 under stress conditions and the cellular distribution and functions of its co-chaperones. MAJOR CONCLUSIONS: Hikeshi mediates the nuclear import of the molecular chaperone HSP70. A few of the regulators of the HSP70 chaperone system also accumulate in the nucleus under heat stress conditions. These proteins function collaboratively to protect cells from stress-induced damage and aid in the recovery of cells from stress. GENERAL SIGNIFICANCE: Studies on the regulation of nucleocytoplasmic transport under several cellular stresses should provide new insights into the fundamental principles of protein homeostasis (proteostasis) in both compartments, the nucleus and cytoplasm.

Structural and functional analysis of Hikeshi, a new nuclear transport receptor of Hsp70s.

Hikeshi is a nuclear transport receptor required for cell survival after stress. It mediates heat-shock-induced nuclear import of 70 kDa heat-shock proteins (Hsp70s) through interactions with FG-nucleoporins (FG-Nups), which are proteins in nuclear pore complexes (NPCs). Here, the crystal structure of human Hikeshi is presented at 1.8 Šresolution. Hikeshi forms an asymmetric homodimer that is responsible for the interaction with Hsp70s. The asymmetry of Hikeshi arises from the distinct conformation of the C-terminal domain (CTD) and the flexibility of the linker regions of each monomer. Structure-guided mutational analyses showed that both the flexible linker region and the CTD are important for nuclear import of Hsp70. Pull-down assays revealed that only full-length Hsp70s can interact with Hikeshi. The N-terminal domain (NTD) consists of a jelly-roll/ß-sandwich fold structure which contains hydrophobic pockets involved in FG-Nup recognition. A unique extended loop (E-loop) in the NTD is likely to regulate the interactions of Hikeshi with FG-Nups. The crystal structure of Hikeshi explains how Hikeshi participates in the regulation of nuclear import through the recognition of FG-Nups and which part of Hikeshi affects its binding to Hsp70. This study is the first to yield structural insight into this highly unique import receptor.

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.

Identification of cargo proteins specific for importin-ß with importin-α applying a stable isotope labeling by amino acids in cell culture (SILAC)-based in vitro transport system.

The human importin (Imp)-ß family consists of 21 nucleocytoplasmic transport carrier proteins, which transport thousands of proteins (cargoes) across the nuclear envelope through nuclear pores in specific directions. To understand the nucleocytoplasmic transport in a physiological context, the specificity of cargoes for their cognate carriers should be determined; however, only a limited number of nuclear proteins have been linked to specific carriers. To address this biological question, we recently developed a novel method to identify carrier-specific cargoes. This method includes the following three steps: (i) the cells are labeled by stable isotope labeling by amino acids in cell culture (SILAC); (ii) the labeled cells are permeabilized, and proteins in the unlabeled cell extracts are transported into the nuclei of the permeabilized cells by a particular carrier; and (iii) the proteins in the nuclei are quantitatively identified by LC-MS/MS. The effectiveness of this method was demonstrated by the identification of transportin (Trn)-specific cargoes. Here, we applied this method to identify cargo proteins specific for Imp-ß, which is a predominant carrier that exclusively utilizes Imp-α as an adapter for cargo binding. We identified candidate cargoes, which included previously reported and potentially novel Imp-ß cargoes. In in vitro binding assays, most of the candidate cargoes bound to Imp-ß in one of three binding modes: directly, via Imp-α, or via other cargoes. Thus, our method is effective for identifying a variety of Imp-ß cargoes. The identified Imp-ß and Trn cargoes were compared, ensuring the carrier specificity of the method and illustrating the complexity of these transport pathways.

Lack of Hikeshi activates HSF1 activity under normal conditions and disturbs the heat-shock response.

Hikeshi mediates the nuclear import of the molecular chaperone HSP70 under heat-shock (acute heat stress) conditions, which is crucial for recovery from cellular damage. The cytoplasmic function of HSP70 is well studied, but its nuclear roles, particularly under nonstressed conditions, remain obscure. Here, we show that Hikeshi regulates the nucleocytoplasmic distribution of HSP70 not only under heat-shock conditions but also under nonstressed conditions. Nuclear HSP70 affects the transcriptional activity of HSF1 and nuclear proteostasis under nonstressed conditions. Depletion of Hikeshi induces a reduction in nuclear HSP70 and up-regulation of the mRNA expression of genes regulated by HSF1 under nonstressed conditions. In addition, the heat-shock response is impaired in Hikeshi-knockout cells. Our results suggest that HSF1 transcriptional activity is tightly regulated by nuclear HSP70 because nuclear-localized Hsp70 effectively suppresses transcriptional activity in a dose-dependent manner. Furthermore, the cytotoxicity of nuclear pathologic polyglutamine proteins was increased by Hikeshi depletion. Thus, proper nucleocytoplasmic distribution of HSP70, mediated by Hikeshi, is required for nuclear proteostasis and adaptive response to heat shock.

Live imaging system for visualizing nuclear pore complex (NPC) formation during interphase in mammalian cells.

Nuclear pore complexes (NPCs) are 'supramolecular complexes' on the nuclear envelope assembled from multiple copies of approximately 30 different proteins called nucleoporins (Nups) that provide aqueous channels for nucleocytoplasmic transport during interphase. Although the structural aspects of NPCs have been characterized in detail, NPC formation and its regulation, especially during interphase, are poorly understood. In this study, using the temperature-sensitive RCC1 mutant tsBN2, a baby hamster kidney 21 cell line, we found that a lack of RCC1 activity inhibited NPC formation during interphase, suggesting that RanGTP is required for NPC formation during interphase in mammalian cells. Utilizing the reversible RCC1 activity in tsBN2 cells, we established a live-cell system that allows for the inhibition or initiation of NPC formation by changes in temperature. Our system enables the examination of NPC formation during interphase in living cells. As a lack of RCC1 decreased some Nups containing unstructured phenylalanine-glycine repeats in the NPC structure, we propose that RCC1 is also involved in maintaining NPC integrity during interphase in mammalian cells.

Functional and structural basis of the nuclear localization signal in the ZIC3 zinc finger domain.

Disruptions in ZIC3 cause heterotaxy, a congenital anomaly of the left-right axis. ZIC3 encodes a nuclear protein with a zinc finger (ZF) domain that contains five tandem C2H2 ZF motifs. Missense mutations in the first ZF motif (ZF1) result in defective nuclear localization, which may underlie the pathogenesis of heterotaxy. Here we revealed the structural and functional basis of the nuclear localization signal (NLS) of ZIC3 and investigated its relationship to the defect caused by ZF1 mutation. The ZIC3 NLS was located in the ZF2 and ZF3 regions, rather than ZF1. Several basic residues interspersed throughout these regions were responsible for the nuclear localization, but R320, K337 and R350 were particularly important. NMR structure analysis revealed that ZF1-4 had a similar structure to GLI ZF, and the basic side chains of the NLS clustered together in two regions on the protein surface, similar to classical bipartite NLSs. Among the residues for the ZF1 mutations, C253 and H286 were positioned for the metal chelation, whereas W255 was positioned in the hydrophobic core formed by ZF1 and ZF2. Tryptophan 255 was a highly conserved inter-finger connector and formed part of a structural motif (tandem CXW-C-H-H) that is shared with GLI, Glis and some fungal ZF proteins. Furthermore, we found that knockdown of Karyopherin alpha1/alpha6 impaired ZIC3 nuclear localization, and physical interactions between the NLS and the nuclear import adapter proteins were disturbed by mutations in the NLS but not by W255G. These results indicate that ZIC3 is imported into the cell nucleus by the Karyopherin (Importin) system and that the impaired nuclear localization by the ZF1 mutation is not due to a direct influence on the NLS.

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.

Y-box protein-associated acidic protein (YBAP1/C1QBP) affects the localization and cytoplasmic functions of YB-1.

The Y-box proteins are multifunctional nucleic acid-binding proteins involved in various aspects of gene regulation. The founding member of the Y-box protein family, YB-1, functions as a transcription factor as well as a principal component of messenger ribonucleoprotein particles (mRNPs) in somatic cells. The nuclear level of YB-1 is well correlated with poor prognosis in many human cancers. Previously, we showed that a Y-box protein-associated acidic protein, YBAP1, which is identical to complement component 1, q subcomponent-binding protein (C1QBP, also called gC1qR, hyaluronan-binding protein 1 [HABP1] or ASF/SF2-associated protein p32), relieves translational repression by YB-1. Here we show that the nuclear localization of YB-1 harboring a point mutation in the cold shock domain was inhibited when co-expressed with YBAP1, whereas cytoplasmic accumulation of the wild-type YB-1 was not affected. We showed that YBAP1 inhibited the interaction between YB-1 and transportin 1. In the cytoplasm, YBAP1 affected the accumulation of YB-1 to processing bodies (P-bodies) and partially abrogated the mRNA stabilization by YB-1. Our results, indicating that YBAP1/C1QBP regulates the nucleo-cytoplasmic distribution of YB-1 and its cytoplasmic functions, are consistent with a model that YBAP1/C1QBP acts as an mRNP remodeling factor.

The crystal structure of mouse Nup35 reveals atypical RNP motifs and novel homodimerization of the RRM domain.

The nuclear pore complex mediates the transport of macromolecules across the nuclear envelope (NE). The vertebrate nuclear pore protein Nup35, the ortholog of Saccharomyces cerevisiae Nup53p, is suggested to interact with the NE membrane and to be required for nuclear morphology. The highly conserved region between vertebrate Nup35 and yeast Nup53p is predicted to contain an RNA-recognition motif (RRM) domain. Due to its low level of sequence homology with other RRM domains, the RNP1 and RNP2 motifs have not been identified in its primary structure. In the present study, we solved the crystal structure of the RRM domain of mouse Nup35 at 2.7 A resolution. The Nup35 RRM domain monomer adopts the characteristic betaalphabetabetaalphabeta topology, as in other reported RRM domains. The structure allowed us to locate the atypical RNP1 and RNP2 motifs. Among the RNP motif residues, those on the beta-sheet surface are different from those of the canonical RRM domains, while those buried in the hydrophobic core are highly conserved. The RRM domain forms a homodimer in the crystal, in accordance with analytical ultracentrifugation experiments. The beta-sheet surface of the RRM domain, with its atypical RNP motifs, contributes to homodimerization mainly by hydrophobic interactions: the side-chain of Met236 in the beta4 strand of one Nup35 molecule is sandwiched by the aromatic side-chains of Phe178 in the beta1 strand and Trp209 in the beta3 strand of the other Nup35 molecule in the dimer. This structure reveals a new homodimerization mode of the RRM domain.

Extensive cargo identification reveals distinct biological roles of the 12 importin pathways.

Vast numbers of proteins are transported into and out of the nuclei by approximately 20 species of importin-ß family nucleocytoplasmic transport receptors. However, the significance of the multiple parallel transport pathways that the receptors constitute is poorly understood because only limited numbers of cargo proteins have been reported. Here, we identified cargo proteins specific to the 12 species of human import receptors with a high-throughput method that employs stable isotope labeling with amino acids in cell culture, an in vitro reconstituted transport system, and quantitative mass spectrometry. The identified cargoes illuminated the manner of cargo allocation to the receptors. The redundancies of the receptors vary widely depending on the cargo protein. Cargoes of the same receptor are functionally related to one another, and the predominant protein groups in the cargo cohorts differ among the receptors. Thus, the receptors are linked to distinct biological processes by the nature of their cargoes.

Reconstitution of nucleocytoplasmic transport using digitonin-permeabilized cells.

Nucleocytoplasmic transport is crucial not only for basic cellular activities but also for the physiological adaptation of cells to various environmental stimuli that affect development, cell-fate determination, or disease development. The basic transport mechanisms have been revealed during the past two decades through the identification and biochemical characterizations of factors mediating the transport, dissecting the transport process and examining the function of nuclear pore complexes (NPCs). In this chapter, we describe methods for a nuclear transport reconstitution assay using digitonin-permeabilized mammalian cells. The transport assay can be generally conducted in the lab without special equipment. The assay system is efficient and significantly contributes to the study of nucleocytoplasmic transport.

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.

The Schizosaccharomyces pombe Hikeshi/Opi10 protein has similar biochemical functions to its human homolog but acts in different physiological contexts.

Human Hikeshi (HsHikeshi) is a nuclear import carrier for Hsp70s and is required for cell survival after heat shock. The Hikeshi homolog in Schizosaccharomyces pombe (SpHikeshi/Opi10) localizes to the nuclear rim, interacts with the Hsp70 homolog Ssa2, and mediates its nuclear import in a reconstituted mammalian nuclear transport system. However, SpHikeshi/Opi10 is not required for heat stress response and survival after heat stress. Instead, SpHikeshi/Opi10 is required for the normal expression of stress response genes under optimal conditions and for cell growth during glucose deprivation. Here, the functions of SpHikeshi/Opi10 are discussed and compared to the functions of HsHikeshi.

Heat-shock stress activates a novel nuclear import pathway mediated by Hikeshi.

Cellular stresses significantly affect nuclear transport systems. Nuclear transport pathways mediated by importin ß-family members, which are active under normal conditions, are downregulated. During thermal stress, a nuclear import pathway mediated by a novel carrier, which we named Hikeshi, becomes active. Hikeshi is not a member of the importin ß family and mediates the nuclear import of Hsp70s. Unlike importin ß family-mediated nuclear transport, the Hikeshi-mediated nuclear import of Hsp70s is not coupled to the GTPase cycle of the small GTPase Ran but rather is coupled with the ATPase cycle of Hsp70s. Hikeshi-mediated nuclear import is essential for the attenuation and reversal of the thermal stress response in human cells. The mechanism and functions of this newly identified nuclear import pathway will be discussed.