Transportin: Nuclear Transport Receptor of a Novel Nuclear Protein Import Pathway

Many nuclear proteins are imported into the cell nucleus by the “classical” nuclear localization signal (NLS)-mediated import pathway. In this pathway, a sequence rich in basic residues in the protein interacts with a heterodimeric complex termed importin and this, along with the GTPase Ran, mediates nuclear import of the NLS-bearing protein. The heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein contains a novel nuclear localization sequence, termed M9, that does not contain any clusters of basic residues. Very recently, we showed that M9 directs import into the nucleus by a novel protein import pathway distinct from the classical NLS pathway. A 90-kilodalton protein termed transportin was identified as a protein that specifically interacts with wild-type M9 but not transport-defective M9 mutants. Transportin and an ATP-regenerating system were found to be necessary and sufficient for import of M9-containing proteins in anin vitroimport assay. In this report, we provide additional evidence that transportin can interact directly with M9-containing proteins and also show that it can mediate import of full-length hnRNP A1. In addition, Ran, or a Ran-binding protein, is identified as a second protein component of this novel nuclear import pathway. Transportin relatives fromSaccharomyces cerevisiaewhich likely serve as additional nuclear transport receptors are described.     

Nuclear Import of Chromatin Remodeler Isw1 Is Mediated by Atypical Bipartite cNLS and Classical Import Pathway

The protein Isw1 of Saccharomyces cerevisiae is an imitation‐switch chromatin‐remodeling factor. We studied the mechanisms of its nuclear import and found that the nuclear localization signal (NLS) mediating the transport of Isw1 into the nucleus is located at the end of the C‐terminus of the protein (aa1079–1105). We show that it is an atypical bipartite signal with an unconventional linker of 19 aa (KRIR X₁₉ KKAK) and the only nuclear targeting signal within the Isw1 molecule. The efficiency of Isw1 nuclear import was found to be modulated by changes to the amino acid composition in the vicinity of the KRIR motif, but not by the linker length. Live‐cell imaging of various karyopherin mutants and in vitro binding assays of Isw1NLS to importin‐α revealed that the nuclear translocation of Isw1 is mediated by the classical import pathway. Analogous motifs to Isw1NLS are highly conserved in Isw1 homologues of other yeast species, and putative bipartite cNLS were identified in silico at the end of the C‐termini of imitation switch (ISWI) proteins from higher eukaryotes. We suggest that the C‐termini of the ISWI family proteins play an important role in their nuclear import.     

细胞因子和生长因子信号转导途径中信号蛋白分子的核转运机制

信号蛋白分子的入核及出核转运是细胞因子和生长因子信号转导途径中的重要环节.核定位序列（NLS）是信号蛋白分子上与入核转运相关的氨基酸序列.核孔复合物（NPC）、核转运蛋白importin和能量供应体Ran/TC4在入核转运过程中也发挥了重要作用.另外,很多细胞因子和生长因子或其受体上所含有的NLS序列也具有核定位功能,并可能通过“伴侣机制”参与其他信号蛋白分子的入核转运.     

A Novel Mechanism by Which Tissue Transglutaminase Activates Signaling Events That Promote Cell Survival

Tissue transglutaminase (tTG) functions as a GTPase and an acyl transferase that catalyzes the formation of protein cross-links. tTG expression is frequently up-regulated in human cancer, where it has been implicated in various aspects of cancer progression, including cell survival and chemo-resistance. However, the extent to which tTG cooperates with other proteins within the context of a cancer cell, versus its intrinsic ability to confer transformed characteristics to cells, is poorly understood. To address this question, we asked what effect the ectopic expression of tTG in a non-transformed cellular background would have on the behavior of the cells. Using NIH3T3 fibroblasts stably expressing a Myc-tagged form of tTG, we found that tTG strongly protected these cells from serum starvation-induced apoptosis and triggered the activation of the PI3-kinase/mTOR Complex 1 (mTORC1)/p70 S6-kinase pathway. We determined that tTG forms a complex with the non-receptor tyrosine kinase c-Src and PI3-kinase, and that treating cells with inhibitors to block tTG function (monodansylcadaverine; MDC) or c-Src kinase activity (PP2) disrupted the formation of this complex, and prevented tTG from activating the PI3-kinase pathway. Moreover, treatment of fibroblasts over-expressing tTG with PP2, or with inhibitors that inactivate components of the PI3-kinase pathway, including PI3-kinase ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) and mTORC1 (rapamycin), ablated the tTG-promoted survival of the cells. These findings demonstrate that tTG has an intrinsic capability to stimulate cell survival through a novel mechanism that activates PI3-kinase signaling events, thus highlighting tTG as a potential target for the treatment of human cancer.     

Mediators of Nuclear Protein Import Target Karyophilic Proteins to Pore Complexes of Cytoplasmic Annulate Lamellae

Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes ofXenopus laevis.In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin β and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central “transporter” and closely resemble those of “transport intermediates” found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.     

Nup124p Is a Nuclear Pore Factor of Schizosaccharomyces pombe That Is Important for Nuclear Import and Activity of Retrotransposon Tf1

The long terminal repeat (LTR)-containing retrotransposon Tf1 propagates within the fission yeast Schizosaccharomyces pombe as the result of several mechanisms that are typical of both retrotransposons and retroviruses. To identify host factors that contribute to the transposition process, we mutagenized cultures of S. pombe and screened them for strains that were unable to support Tf1 transposition. One such strain contained a mutation in a gene we named nup124. The product of this gene contains 11 FXFG repeats and is a component of the nuclear pore complex. In addition to the reduced levels of Tf1 transposition, the nup124-1 allele caused a significant reduction in the nuclear localization of Tf1 Gag. Surprisingly, the mutation in nup124-1 did not cause any reduction in the growth rate, the nuclear localization of specific nuclear localization signal-containing proteins, or the cytoplasmic localization of poly(A) mRNA. A two-hybrid analysis and an in vitro precipitation assay both identified an interaction between Tf1 Gag and the N terminus of Nup124p. These results provide evidence for an unusual mechanism of nuclear import that relies on a direct interaction between a nuclear pore factor and Tf1 Gag.     

Mechanisms and Signals for the Nuclear Import of Proteins

In eukaryotes, the nuclear membrane provides a physical barrier to the passive diffusion of macromolecules from and into the cytoplasm. Nucleocytoplasmic traffic occurs through highly specialized structures known as nuclear pores, and involves the participation of a special class of transport proteins. Active transport across the nuclear pores is an energy-dependent process that relies on the activity of Ran-GTPases both in the nuclear and cytoplasmic compartments. Nuclear import of proteins is an essential step in regulating gene expression and the replication cycle of several viruses. In this review, the key mechanisms, pathways, and models underlying the transport of proteins across nuclear pores are analysed.Key Words: Nuclear pore complex, nuclear localization signal, importin, nuclear transport.     

The Nuclear Pore Complex and Nuclear Transport

Internal membrane bound structures sequester all genetic material in eukaryotic cells. The most prominent of these structures is the nucleus, which is bounded by a double membrane termed the nuclear envelope (NE). Though this NE separates the nucleoplasm and genetic material within the nucleus from the surrounding cytoplasm, it is studded throughout with portals called nuclear pore complexes (NPCs). The NPC is a highly selective, bidirectional transporter for a tremendous range of protein and ribonucleoprotein cargoes. All the while the NPC must prevent the passage of nonspecific macromolecules, yet allow the free diffusion of water, sugars, and ions. These many types of nuclear transport are regulated at multiple stages, and the NPC carries binding sites for many of the proteins that modulate and modify the cargoes as they pass across the NE. Assembly, maintenance, and repair of the NPC must somehow occur while maintaining the integrity of the NE. Finally, the NPC appears to be an anchor for localization of many nuclear processes, including gene activation and cell cycle regulation. All these requirements demonstrate the complex design of the NPC and the integral role it plays in key cellular processes.Taxonomically speaking, all life on earth falls into one of two fundamental groups, the prokaryotes and the eukaryotes. The prokaryotes, the first group to evolve, are single cell organisms bounded by a single membrane. About 1.5 billion years later, a series of evolutionary innovations led to the emergence of eukaryotes. Eukaryotes have multiple inner membrane structures that allow for compartmentalization within the cell, and therefore differentiation of the cell and regulation within it. Ultimately, the greater cellular complexity of eukaryotes allowed them to adopt a multicellular lifestyle, as seen in the plants, fungi and animals of today (reviewed in Field and Dacks 2009).Internal membrane bound structures sequester all genetic material in eukaryotic cells. The most prominent of these structures, which gives the eukaryotes their Greek-rooted name, is the nucleus—the central “kernel” (gr. “karyo-”) of the cell. The nucleus is bounded by a double membrane termed the nuclear envelope (NE), which separates the nucleoplasm and genetic material from the surrounding cytoplasm. However the genetic material in the nucleus is not totally isolated from the rest of the cell. Studded throughout the NE are portals called nuclear pore complexes (NPCs). The NPC is a highly selective, bidirectional transporter for a tremendous range of cargoes. Going into the nucleus, these cargoes include inner nuclear membrane proteins and all the proteins in the nucleoplasm. Going out are RNA-associated proteins that are assembled into ribosomal subunits or messenger ribonucleoproteins (mRNPs). Once transported, the NPC must ensure these cargos are retained in their respective nuclear and cytoplasmic compartments. All the while the NPC must prevent the passage of nonspecific macromolecules, yet allow the free diffusion of water, sugars, and ions. These many types of nuclear transport are regulated at multiple stages, providing a powerful extra level of cellular control that is not necessary in prokaryotes. Assembly, maintenance, and repair of the NPC must somehow occur while maintaining the integrity of the NE. Finally, the NPC appears to be an anchor for localization of many nuclear processes, including gene activation and cell cycle regulation (reviewed in Ahmed and Brickner 2007; Hetzer and Wente 2009). All these requirements demonstrate the complex design of the NPC and the integral role it plays in key cellular processes.     

Physical Factors That Affect In Vitro Autographa californica Nuclear Polyhedrosis Virus Infection

Of the physical parameters tested for in vitro baculovirus infection, multiplicity of infection was most important in governing percent cell infection. Most plaques formed within the first 5 min of incubation. Efficiency of infection, however, was low, and the virus titer did not diminish during prolonged incubation. Efficiency of infection improved markedly when cells or virus were preincubated with selected polyanions and polycations. Precise regulation of the pH, osmotic pressure, and ionic composition of the cell culture medium also promoted maximum in vivo infection.     

Oxidative Stress Inhibits Nuclear Protein Export by Multiple Mechanisms That Target FG Nucleoporins and Crm1

Nuclear transport of macromolecules is regulated by the physiological state of the cell and thus sensitive to stress. To define the molecular mechanisms that control nuclear export upon stress, cells were exposed to nonlethal concentrations of the oxidant diethyl maleate (DEM). These stress conditions inhibited chromosome region maintenance-1 (Crm1)-dependent nuclear export and increased the association between Crm1 and Ran. In addition, we identified several repeat-containing nucleoporins implicated in nuclear export as targets of oxidative stress. As such, DEM treatment reduced Nup358 levels at the nuclear envelope and redistributed Nup98. Furthermore, oxidative stress led to an increase in the apparent molecular masses of Nup98, Nup214, and Nup62. Incubation with phosphatase or β-N-acetyl-hexosaminidase showed that oxidative stress caused the phosphorylation of Nup98, Nup62, and Nup214 as well as O-linked N-acetylglucosamine modification of Nup62 and Nup214. These oxidant-induced changes in nucleoporin modification correlated first with the increased binding of Nup62 to the exporter Crm1 and second with the reduced interaction of Nup62 with other FxFG-containing nucleoporins. Together, oxidative stress up-regulated the binding of Crm1 to Ran and affected multiple repeat-containing nucleoporins by changing their localization, phosphorylation, O-glycosylation, or interaction with other transport components. We propose that the combination of these events contributes to the stress-dependent regulation of Crm1-mediated protein export.