Shingo Kose

In vivo evidence for involvement of a 58 kDa component of nuclear pore-targeting complex in nuclear protein import.

We recently showed that a nuclear location signal (NLS)‐containing karyophile forms a stable complex with cytoplasmic components for nuclear pore‐targeting The complex, termed nuclear pore‐targeting complex (PTAC), contained two essential proteins of 54 and 90 kDa, respectively, as estimated by electrophoresis. In this study, we found that the 54 kDa component of PTAC is the mouse homologue of Xenopus importin (m‐importin). Cytoplasmic injection of the antibodies raised against recombinant m‐importin showed an inhibitory effect on nuclear import of a karyophile in living mammalian cells. A portion of cytoplasmically injected antibodies migrated rapidly into the nucleus, indicating dynamic movement of this protein across the nuclear envelope. Moreover, the injected antibodies co‐precipitated the karyophile, in an NLS‐dependent manner, with endogenous m‐importin in the cytoplasm. These results provide in vivo evidence that m‐importin is involved in nuclear protein import through association with a NLS in the cytoplasm before nuclear pore binding.

Phosphoproteomics reveals new ERK MAP kinase targets and links ERK to nucleoporin-mediated nuclear transport

Many extracellular signal–regulated kinase (ERK) mitogen-activated protein (MAP) kinase substrates have been identified, but the diversity of ERK-mediated processes suggests the existence of additional targets. Using a phosphoproteomic approach combining the steroid receptor fusion system, IMAC, 2D-DIGE and phosphomotif-specific antibodies, we detected 38 proteins showing reproducible phosphorylation changes between ERK-activated and ERK-inhibited samples, including 24 new candidate ERK targets. ERK directly phosphorylated at least 13 proteins in vitro. Of these, Nup50 was verified as a bona fide ERK substrate. Notably, ERK phosphorylation of the FG repeat region of Nup50 reduced its affinity for importin-β family proteins, importin-β and transportin. Other FG nucleoporins showed a similar functional change after ERK-mediated phosphorylation. Nuclear migration of importin-β and transportin was impaired in ERK-activated, digitonin-permeabilized cells, as a result of ERK phosphorylation of Nup50. Thus, we propose that ERK phosphorylates various nucleoporins to regulate nucleocytoplasmic transport.

Dissociation of heterochromatin protein 1 from lamin B receptor induced by human polyomavirus agnoprotein: role in nuclear egress of viral particles

The nuclear envelope is one of the chief obstacles to the translocation of macromolecules that are larger than the diameter of nuclear pores. Heterochromatin protein 1 (HP1) bound to the lamin B receptor (LBR) is thought to contribute to reassembly of the nuclear envelope after cell division. Human polyomavirus agnoprotein (Agno) has been shown to bind to HP1α and to induce its dissociation from LBR, resulting in destabilization of the nuclear envelope. Fluorescence recovery after photobleaching showed that Agno increased the lateral mobility of LBR in the inner nuclear membrane. Biochemical and immunofluorescence analyses showed that Agno is targeted to the nuclear envelope and facilitates the nuclear egress of polyomavirus‐like particles. These results indicate that dissociation of HP1α from LBR and consequent perturbation of the nuclear envelope induced by polyomavirus Agno promote the translocation of virions out of the nucleus.

Importin-β and the small guanosine triphosphatase Ran mediate chromosome loading of the human chromokinesin Kid

Nucleocytoplasmic transport factors mediate various cellular processes, including nuclear transport, spindle assembly, and nuclear envelope/pore formation. In this paper, we identify the chromokinesin human kinesin-like DNA binding protein (hKid) as an import cargo of the importin-α/β transport pathway and determine its nuclear localization signals (NLSs). Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells. In digitonin-permeabilized mitotic cells, hKid was bound only to the spindle and not to the chromosomes themselves. Surprisingly, hKid bound to importin-α/β was efficiently targeted to mitotic chromosomes. The addition of Ran–guanosine diphosphate and an energy source, which generates Ran–guanosine triphosphate (GTP) locally at mitotic chromosomes, enhanced the importin-β–mediated chromosome loading of hKid. Our results indicate that the association of importin-β and -α with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP–mediated cargo release promotes the accumulation of hKid on chromosomes. Thus, this study demonstrates a novel nucleocytoplasmic transport factor–mediated mechanism for targeting proteins to mitotic chromosomes.

Heat-shock induced nuclear retention and recycling inhibition of importin α

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/β, 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 α accumulate in the nucleus in response to heat‐shock. An analysis of the behaviour of importin α with fluorescence recovery after photobleaching and fluorescence loss in photobleaching studies show that nuclear importin 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, 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.

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 α1/α6 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 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.

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-β 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-β family carriers and then supplemented with a particular importin-β 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-β 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-β. 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-β with importin-α applying a stable isotope labeling by amino acids in cell culture (SILAC)-based in vitro transport system

Background: A limited number of cargoes specific for importin-β family nucleocytoplasmic transport carriers have been reported. We have developed a stable isotope labeling by amino acids in cell culture (SILAC)-based transport method to identify specific cargo. Results: Candidate importin-β (with or without importin-α) cargoes were identified by our method and corroborated with binding assays. Conclusion: New importin-β-specific cargoes were identified. Significance: Our method has the potential to identify cargoes specific for other carriers. The human importin (Imp)-β 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-β, 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-β cargoes. In in vitro binding assays, most of the candidate cargoes bound to Imp-β 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-β cargoes. The identified Imp-β and Trn cargoes were compared, ensuring the carrier specificity of the method and illustrating the complexity of these transport pathways.

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.