Taro Tachibana

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.

Differential Modes of Nuclear Localization Signal (NLS) Recognition by Three Distinct Classes of NLS Receptors

The targeting of karyophilic proteins to nuclear pores is mediated via the formation of a nuclear pore-targeting complex, through the interaction of nuclear localization signal (NLS) with its NLS receptor. Recently, a novel human protein, Qip1, was identified from a yeast two-hybrid system with DNA helicase Q1. This study demonstrates that Qip1 is a novel third class of NLS receptor that efficiently recognizes the NLS of the helicase Q1. Moreover, the data obtained in this study show that the specific interaction between Qip1 and the NLS of the helicase Q1 requires its upstream sequence of the minimal essential NLS. By using purified recombinant proteins alone in the digitonin-permeabilized cell-free transport system, it was demonstrated that the two known human NLS receptors, Rch1 and NPI-1, are able to transport all the tested NLS substrates into the nucleus, while Qip1 most efficiently transports the helicase Q1-NLS substrates, which contain its upstream sequence in so far as we have examined the system. Furthermore, in HeLa cell crude cytosol, it was found that endogenous Rch1 binds to all the tested NLS substrates, while the binding of endogenous NPI-1 is restricted to only some NLSs, despite the fact that NPI-1 itself shows binding activity to a variety of NLSs. These results indicate that at least three structurally and functionally distinct NLS receptors exist in the human single cell population, and suggest that the nuclear import of karyophilic proteins may be controlled in a complex manner at the NLS recognition step by the existence of a variety of NLS receptors with various specificities to each NLS.

A Nuclear Localization Signal of Human Aryl Hydrocarbon Receptor Nuclear Translocator/Hypoxia-inducible Factor 1β Is a Novel Bipartite Type Recognized by the Two Components of Nuclear Pore-targeting Complex

Aryl hydrocarbon receptor nuclear translocator (ARNT) is a component of the transcription factors, aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1, which transactivate their target genes, such as CYP1A1 and erythropoietin, in response to xenobiotic aromatic hydrocarbons and to low O2concentration, respectively. Since ARNT was isolated as a factor required for the nuclear translocation of AhR from the cytoplasm in response to xenobiotics, the subcellular localization of ARNT has been of great interest. In this investigation, we analyzed the subcellular distribution of ARNT using transient expression of a fusion gene with β-galactosidase and microinjection of recombinant proteins containing various fragments of ARNT in the linker region of glutathioneS-transferase/green fluorescent protein. We found a clear nuclear localization of ARNT in the absence of exogenous ligands to AhR, and identified the nuclear localization signal (NLS) of amino acid residues 39–61. The characterized NLS consists of 23 amino acids, and can be classified as a novel variant of the bipartite type on the basis of having two separate regions responsible for efficient nuclear translocation activity, but considerable deviation of the sequence from the consensus of the classical bipartite type NLSs. Like the well characterized NLS of the SV40 T-antigen, this variant bipartite type of ARNT NLS was also mediated by the two components of nuclear pore targeting complex, PTAC58 and PTAC97, to target to the nuclear rim in an in vitro nuclear transport assay.

Essential role of active nuclear transport in apoptosis

Background : Apoptosis is defined by chromatin condensation, nuclear fragmentation and the formation of apoptotic bodies. Because apoptotic signals are transmitted through a common pathway that includes the target steps of death‐driving ICE‐family proteases and anti‐cell death protein Bcl‐2 in the cytoplasm, the signals must be transferred from the cytoplasm to the nucleus, at least to induce the apoptotic manifestation of the nucleus. Small signal molecules might diffuse across nuclear pores, but larger molecules are transported by active mechanisms requiring ATP and GTP hydrolysis. It is not known whether apoptotic signals are transmitted into the nucleus by the mechanisms of active nuclear transport.

Nuclear transport factor p10/NTF2 functions as a Ran–GDP dissociation inhibitor (Ran-GDI)

The cytosolic nuclear transport factor p10/NTF2 is required for the translocation of karyophilic molecules through nuclear pores [1] [2] [3], and the small GTPase Ran is a key regulator of protein transport between the nucleus and cytoplasm [4] [5]. It has been reported that p10/NTF2 interacts directly and specifically with Ran-GDP but not with Ran-GTP [6]. The precise role(s) of p10/NTF2 in the Ran GTP/GDP cycle are thus far unclear, however. In this study, we show that mammalian p10/NTF2 dramatically inhibits the dissociation of [3H]GDP from Ran and the binding of [35S]GTPgammaS to Ran following the dissociation of non-radioactive GDP by RCC1, the only known mammalian guanine nucleotide exchange factor for Ran (Ran-GEF) [7]. In contrast, the dissociation of [35S]GTP gamma S from Ran, which was also catalyzed by RCC1, was not affected by p10/NTF2. Furthermore, the activities of wild-type p10/NTF2 and the mutant forms M84T and D92G in an assay of nuclear protein import in a digitonin-permeabilized cell-free system correlated with their level of inhibition of the dissociation of nucleotide from Ran-GDP. These results suggest that p10/NTF2 acts as a GDP dissociation inhibitor for Ran (Ran-GDI), thereby coordinating the Ran-dependent reactions that underlie nuclear protein import.

Nuclear-Cytoplasmic Shuttling of a RING-IBR Protein RBCK1 and Its Functional Interaction with Nuclear Body Proteins

The intracellular localization of a RING-IBR protein, RBCK1, possessing DNA binding and transcriptional activities, has been investigated. The endogenous RBCK1 was found in both the cytoplasm and nucleus. Particularly in the nucleus, it was localized in the granular structures, most likely nuclear bodies. In contrast, the over-expressed RBCK1 was detected exclusively in the cytoplasm. When the cells were treated with leptomycin B, the over-expressed RBCK1 accumulated in the nuclear bodies. These results suggest that RBCK1 possesses the signal sequences responsible for the nuclearcytoplasmic translocation. Mutational analysis of RBCK1 has indicated that an N-terminal region containing Leu-142 and Leu-145 and a C-terminal one containing the RING-IBR domain serve as the nuclear export and localization signals, respectively. Thus, RBCK1 is a transcription factor dynamically shuttling between cytoplasm and nucleus. Furthermore, RBCK1 was found to interact with nuclear body proteins, CREB-binding protein (CBP), and promyelocytic leukemia protein (PML). Coexpression of RBCK1 with CBP significantly enhanced the transcriptional activity of RBCK1. Although PML per se showed no effect on the transcriptional activity of RBCK1, the CBP-enhanced activity was repressed by coexpression with PML, presumably through the interaction of PML and CBP. Taken together, our data demonstrate that RBCK1 is involved in transcriptional machinery in the nuclear bodies, and its transcriptional activity is regulated by nucleocytoplasmic shuttling.

Nuclear Import of the U1A Splicesome Protein Is Mediated by Importin α/β and Ran in Living Mammalian Cells

Abstract U1A is a component of the uracil-rich small nuclear ribonucleoprotein. The molecular mechanism of nuclear import of U1A was investigated in vivo and in vitro. When recombinant deletion mutants of U1A are injected into the BHK21 cell cytoplasm, the nuclear localization signal (NLS) of U1A is found in the N-terminal half of the central domain (residues 100–144 in mouse U1A). In an in vitro assay, it was found that the U1A-NLS accumulated in only a portion of the nuclei in the absence of cytosolic extract. In contrast, the addition of importin α/β and Ran induced the uniform nuclear accumulation of U1A-NLS in all cells. Furthermore, U1A was found to bind the C-terminal portion of importin α. In addition, the in vitro nuclear migration of full-length U1A was found to be exclusively dependent on importin α/β and Ran. Moreover, in living cells, the full-length U1A accumulated in the nucleus in a Ran-dependent manner, and nuclear accumulation was inhibited by the importin β binding domain of importin α. These results suggest that the nuclear import of U1A is mediated by at least two distinct pathways, an importin α/β and Ran-dependent and an -independent pathway in permeabilized cells, and that the latter pathway may be suppressed in intact cells.

Characterization of the nuclear transport of a novel leucine-rich acidic nuclear protein-like protein

We previously reported that the nuclear localization signal (NLS) peptides stimulate the in vitro phosphorylation of several proteins, including a 34 kDa protein. In this study, we show that this specific 34 kDa protein is a novel murine leucine‐rich acidic nuclear protein (LANP)‐like large protein (mLANP‐L). mLANP‐L was found to have a basic type NLS. The co‐injection of Q69LRan‐GTP or SV40 T‐antigen NLS peptides prevented the nuclear import of mLANP‐L. mLANP‐L NLS bound preferentially to Rch1 and NPI‐1, but not to the Qip1 subfamily of importin α. These findings suggest that mLANP‐L is transported into the nucleus by Rch1 and/or NPI‐1.

Up-regulation of nuclear protein import by nuclear localization signal sequences in living cells

Using an in vivo assay system, nuclear import ability in individual cells was determined by examining the nuclear import rate. It was found that when a small (not excess) amount of SV40 T‐NLS peptides was co‐injected, the nuclear import rate of SV40 T‐NLS‐containing substrates apparently increased. This up‐regulation was reproduced by the co‐injection of peptides containing bipartite type NLS of CBP80, but not mutated non‐functional NLS peptides, which suggests that these phenomena are specific for functional NLSs. It was further shown that although, in growth‐arrested cells, the nuclear import rate was down‐regulated compared to growing cells, the elevation of the functional import rate by co‐injected NLS peptides reached the same level as in proliferating cells. This up‐regulation was abolished by the addition of a protein kinase inhibitor, staurosporine. These results suggest that although potential nuclear import ability does not vary in each cell, the rate of nuclear import may be controlled by the amount of karyophilic proteins, which need to be carried into the nucleus from the cytoplasm, possibly via an NLS‐dependent phosphorylation reaction.

A new technique to co-localise membrane proteins with Homer/vesl.

The minimal requirements were defined as necessary for cluster formation of the group 1 metabotropic glutamate receptor (mGluR), which is regulated by the Homer/vesl family of scaffolding proteins [Curr. Opin. Neurobiol. 10 (2000) 370]. Cluster formation of G-protein-coupled receptors (GPCRs) plays a fundamental role in signal transduction, particularly at the neuronal synapse. To understand the interaction of mGluR with PSD-Zip45, a Homer/vesl family member, we designed a series of chimeric receptor proteins, consisting of C-terminal mGluR1alpha sequences that were fused to endothelin B receptors (ET(B)Rs). In vitro and in vivo studies revealed that an extended 20 amino acid long C-terminal mGluR1alpha peptide, including the proline-rich core motif PPXXF, is sufficient to induce clustering of chimeric ET(B)R/mGluR1alpha receptors by PSD-Zip45. This result is especially important because it constitutes the basis for a new approach to form two-dimensional crystals of membrane proteins in situ, which may render unstable membrane proteins amenable to electron crystallographic structure determination.