Nuclear Transport Pathways Research Articles

Ubiquitin-Like Proteins

The eukaryotic ubiquitin family encompasses nearly 20 proteins that are involved in the posttranslational modification of various macromolecules. The ubiquitin-like proteins (UBLs) that are part of this family adopt the β-grasp fold that is characteristic of its founding member ubiquitin (Ub). Although structurally related, UBLs regulate a strikingly diverse set of cellular processes, including nuclear transport, proteolysis, translation, autophagy, and antiviral pathways. New UBL substrates continue to be identified and further expand the functional diversity of UBL pathways in cellular homeostasis and physiology. Here, we review recent findings on such novel substrates, mechanisms, and functions of UBLs.

Nucleocytoplasmic Shuttling of Cytoskeletal Proteins: Molecular Mechanism and Biological Significance

Various nuclear functional complexes contain cytoskeletal proteins as regulatory subunits; for example, nuclear actin participates in transcriptional complexes, and actin-related proteins are integral to chromatin remodeling complexes. Nuclear complexes such as these are involved in both basal and adaptive nuclear functions. In addition to nuclear import via classical nuclear transport pathways or passive diffusion, some large cytoskeletal proteins spontaneously migrate into the nucleus in a karyopherin-independent manner. The balance of nucleocytoplasmic distribution of such proteins can be altered by several factors, such as import versus export, or capture and release by complexes. The resulting accumulation or depletion of the nuclear populations thereby enhances or attenuates their nuclear functions. We propose that such molecular dynamics constitute a form of cytoskeleton-modulated regulation of nuclear functions which is mediated by the translocation of cytoskeletal components in and out of the nucleus.

Birth, Death, and Replacement of Karyopherins in Drosophila

Nucleocytoplasmic transport is a broadly conserved process across eukaryotes. Despite its essential function and conserved mechanism, components of the nuclear transport apparatus have been implicated in genetic conflicts in Drosophila, especially in the male germ line. The best understood case is represented by a truncated RanGAP gene duplication that is part of the segregation distorter system in Drosophila melanogaster. Consistent with the hypothesis that the nuclear transport pathway is at the heart of mediating genetic conflicts, both nucleoporins and directionality imposing components of nuclear transport have previously been shown to evolve under positive selection. Here, we present a comprehensive phylogenomic analysis of importins (karyopherins) in Drosophila evolution. Importins are adaptor molecules that physically mediate the transport of cargo molecules and comprise the third component of the nuclear transport apparatus. We find that importins have been repeatedly gained and lost throughout various stages of Drosophila evolution, including two intriguing examples of an apparently coincident loss and gain of nonorthologous and noncanonical importin-α. Although there are a few signatures of episodic positive selection, genetic innovation in importin evolution is more evident in patterns of recurrent gene birth and loss specifically for function in Drosophila testes, which is consistent with their role in supporting host genomes defense against segregation distortion.

Nuclear transporters in a multinucleated organism: functional and localization analyses in Aspergillus nidulans

Nuclear transporters mediate bidirectional macromolecule traffic through the nuclear pore complex (NPC), thus participating in vital processes of eukaryotic cells. A systematic functional analysis in Aspergillus nidulans permitted the identification of 4 essential nuclear transport pathways of a hypothetical number of 14. The absence of phenotypes for most deletants indicates redundant roles for these nuclear receptors. Subcellular distribution studies of these carriers show three main distributions: nuclear, nucleocytoplasmic, and in association with the nuclear envelope. These locations are not specific to predicted roles as exportins or importins but indicate that bidirectional transport may occur coordinately in all nuclei of a syncytium. Coinciding with mitotic NPC rearrangements, transporters dynamically modified their localizations, suggesting supplementary roles to nucleocytoplasmic transport specifically during mitosis. Loss of transportin-SR and Mex/TAP from the nuclear envelope indicates absence of RNA transport during the partially open mitosis of Aspergillus, whereas nucleolar accumulation of Kap121 and Kap123 homologues suggests a role in nucleolar disassembly. This work provides new insight into the roles of nuclear transporters and opens an avenue for future studies of the molecular mechanisms of transport among nuclei within a common cytoplasm, using A. nidulans as a model organism.

Three Dimensional Distinct Nuclear Transport Pathways Illuminated by Single-Molecule Microscopy

The nuclear pore complex (NPC), the sole gateway for nucleocytoplasmic exchange in eukaryotic cells, allows for the passive diffusion of small molecules and the transport-receptor-facilitated translocation of larger molecules. However, whether these two transport modes spatially possess separate or joined pathways and whether they functionally conduct uncoupled or coupled transport remains in dispute. By the single-molecule snapshots of transient diffusion or interactions through native NPCs, we have determined that the three-dimensional transport pathway for each mode is mainly separate. Both passageways can be spatially and functionally regulated by transport receptors. Our data strongly support a transport mechanism in which transport receptors or cargo complexes pave the pathways by interacting with the phenylalanine-glycine repeats at the periphery around a single primary central axial channel for passive diffusion.

Structural Biology of Macromolecular Transport into and out of the Cell Nucleus

Active transport of macromolecules between the nucleus and the cytoplasm is mediated by transport receptors (carriers) that facilitate the passage of specific cargoes through the nuclear pore complexes. All of the nuclear transport pathways are based on a common principle: translocation across the nuclear pore appears to be reversible, and the directionality of transport depends on assembly of cargo-carrier complexes on one side of the nuclear envelope and disassembly of the transport complexes on the other side. This review highlights recent progress in understanding the structural basis of nuclear transport, especially in terms of CRM1-mediated nuclear export.

Specific Cleavage of the Nuclear Pore Complex Protein Nup62 by a Viral Protease

Previous work has shown that several nucleoporins, including Nup62 are degraded in cells infected with human rhinovirus (HRV) and poliovirus (PV) and that this contributes to the disruption of certain nuclear transport pathways. In this study, the mechanisms underlying proteolysis of Nup62 have been investigated. Analysis of Nup62 in lysates from HRV-infected cells revealed that Nup62 was cleaved at multiple sites during viral infection. The addition of purified HRV2 2A protease (2A(pro)) to uninfected HeLa whole cell lysates resulted in the cleavage of Nup62, suggesting that 2A(pro) is a major contributor to Nup62 processing. The ability of purified 2A(pro) to cleave bacterially expressed and purified Nup62 demonstrated that 2A(pro) directly cleaves Nup62 in vitro. Site-directed mutagenesis of putative cleavage sites in Nup62 identified six different positions that are cleaved by 2A(pro) in vitro. This analysis revealed that 2A(pro) cleavage sites were located between amino acids 103 and 298 in Nup62 and suggested that the N-terminal FG-rich region of Nup62 was released from the nuclear pore complex in infected cells. Analysis of HRV- and PV-infected cells using domain-specific antibodies confirmed that this was indeed the case. These results are consistent with a model whereby PV and HRV disrupt nucleo-cytoplasmic trafficking by selectively removing FG repeat domains from a subset of nuclear pore complex proteins.

Characterisation of the passive permeability barrier of nuclear pore complexes

Nuclear pore complexes (NPCs) restrict uncontrolled nucleocytoplasmic fluxes of inert macromolecules but permit facilitated translocation of nuclear transport receptors and their cargo complexes. We probed the passive barrier of NPCs and observed sieve-like properties with a dominating mesh or channel radius of 2.6 nm, which is narrower than proposed earlier. A small fraction of diffusion channels has a wider opening, explaining the very slow passage of larger molecules. The observed dominant passive diameter approximates the distance of adjacent hydrophobic clusters of FG repeats, supporting the model that the barrier is made of FG repeat domains cross-linked with a spacing of an FG repeat unit length. Wheat germ agglutinin and the dominant-negative importin β45-462 fragment were previously regarded as selective inhibitors of facilitated NPC passage. We now observed that they do not distinguish between the passive and the facilitated mode. Instead, their inhibitory effect correlates with the size of the NPC-passing molecule. They have little effect on small species, inhibit the passage of green fluorescent protein-sized objects >10-fold and virtually block the translocation of larger ones. This suggests that passive and facilitated NPC passage proceed through one and the same permeability barrier.

The lactate dehydrogenase-elevating virus capsid protein is a nuclear–cytoplasmic protein

Arteriviruses replicate in the cytoplasm and do not require the nucleus function for virus multiplication in vitro. However, nucleocapsid (N) protein of two arteriviruses, porcine reproductive respiratory syndrome virus and equine arteritis virus, has been observed to localize in the nucleus and nucleolus of virus-infected and N-gene-transfected cells in addition to their normal cytoplasmic distribution. In the present study, the N protein of lactate dehydrogenase-elevating virus (LDV) of mice was examined for nuclear localization. The subcellular localization of LDV-N was determined by tagging N with enhanced green fluorescence protein (EGFP) at the N- and C-terminus. Both N-EGFP and EGFP-N fusion proteins localized to the nucleus and nucleolus of gene-transfected cells. Labeled N also accumulated in the perinuclear region, the site of virus replication. The LDV-N sequence contains a putative ‘pat4’-type nuclear localization signal (NLS) consisting of 38-KKKK. To determine its functional significance, a series of deletion constructs of N were generated and individually expressed in cells. The results showed that the ‘pat4’ NLS was essential for nuclear translocation. In addition, the LDV-N interacted with the importin-α and -β proteins, suggesting that the LDV-N nuclear localization may occur via the importin-mediated nuclear transport pathway. These results provide further evidence for the nuclear localization of N as a common feature within the arteriviruses.

Functional analysis of key nuclear trafficking components reveals an atypical Ran network required for parasite pathogenesis

Protozoan parasites represent major public health challenges. Many aspects of their cell biology are distinct from their animal hosts, providing potential therapeutic targets. Toxoplasma gondii is a protozoan parasite that contains a divergent regulator of chromosome condensation 1 (TgRCC1) that is required for virulence and efficient nuclear trafficking. RCC1 proteins function as a guanine exchange factor for Ras-related nuclear protein (Ran), an abundant GTPase responsible for the majority of nucleocytoplasmic transport. Here we show that while there are dramatic differences from well-conserved RCC1 proteins, TgRCC1 associates with chromatin, interacts with Ran and complements a mammalian temperature-sensitive RCC1 mutant cell line. During the investigation of TgRCC1, we observed several unprecedented phenotypes for TgRan, despite a high level of sequence conservation. The cellular distribution of TgRan is found throughout the parasite cell, whereas Ran in late branching eukaryotes is predominantly nuclear. Additionally, T. gondii tolerates at least low-level expression of dominant lethal Ran mutants. Wild type parasites expressing dominant negative TgRan grew similarly to wild type in standard tissue culture conditions, but were attenuated in serum-starved host cells and mice. These growth characteristics paralleled the TgRCC1 mutant and highlight the importance of the nuclear transport pathway for virulence of eukaryotic pathogens.

Secretory Protein mRNA Finds Another Way Out

Secretory Protein mRNA Finds Another Way Out

Disruption of Oligomerization Induces Nucleocytoplasmic Shuttling of Leukemia-Associated Rho Guanine-Nucleotide Exchange Factor

The rgsRhoGEFs comprise a subfamily of three guanine nucleotide exchange factors, which function in linking heterotrimeric G-proteins to the monomeric RhoGTPase. Here, we reveal the novel finding that oligomerization of leukemia-associated RhoGEF (LARG) functions to prevent nucleocytoplasmic shuttling and to retain LARG in the cytoplasm. We establish that oligomerization is mediated by a predicted coiled-coil sequence (amino acids 1507-1520) in the extreme C terminus of LARG and that substitution of isoleucines 1507/1510 with alanines disrupts homo-oligomerization and leads to nucleocytoplasmic shuttling via the CRM1 nuclear transport pathway. In addition, we demonstrate that induced dimerization of an otherwise nuclear monomeric LARG mutant promotes cytoplasmic localization. Furthermore, we establish that nuclear import of monomeric LARG is mediated by the nuclear localization sequence (29)PTDKKQK(35) in the extreme N terminus. We propose that nucleocytoplasmic shuttling provides a mechanism for spatially regulating the activity of LARG toward its cytoplasmic targets and potentially new nuclear targets.

A Microtubule‐Facilitated Nuclear Import Pathway for Cancer Regulatory Proteins

Nuclear protein import is dependent on specific targeting signals within cargo proteins recognized by importins (IMPs) that mediate translocation through the nuclear pore. Recent evidence, however, implicates a role for the microtubule (MT) network in facilitating nuclear import of the cancer regulatory proteins parathyroid hormone-related protein (PTHrP) and p53 tumor suppressor. Here we assess the extent to which MT and actin integrity may be generally required for nuclear protein import for the first time. We examine 10 nuclear-localizing proteins with diverse IMP-dependent nuclear import pathways, our results indicating that the cytoskeleton does not have a general mechanistic role in nuclear localization sequence-dependent nuclear protein import. Of the proteins examined, only the p110(Rb) tumor suppressor protein Rb, together with p53 and PTHrP, was found to require MT integrity for optimal nuclear import. Fluorescence recovery after photobleaching experiments indicated that the MT-dependent nuclear transport pathway increases both the rate and extent of Rb nuclear import but does not affect Rb nuclear export. Dynamitin overexpression experiments implicate the MT motor dynein in the import process. The results indicate that, additional to IMP/diffusion-dependent processes, certain cancer regulatory proteins utilize an MT-enhanced pathway for accelerated nuclear import that is presumably required for their nuclear functions.

Scientists Share Nuclear Secrets at Jekyll Island

Scientists Share Nuclear Secrets at Jekyll Island

Vesicular Trafficking of Tyrosine Kinase Receptors and Associated Proteins in the Regulation of Signaling and Vascular Function

Receptor tyrosine kinases (RTKs) play a pivotal role in the development and function of the cardiovascular system. Ligand-activated RTKs promote numerous downstream signal transduction pathways that lead to vascular permeability, as well as proliferation, migration, and differentiation of vascular endothelia and smooth muscle cells. Ligand binding also promotes internalization of the activated receptors either to downregulate the signaling via degradation of the ligand/receptor complex or to signal from endosomes. However, the outcomes of receptor internalization via clathrin-dependent or caveolar pathways and trafficking mechanisms are incompletely clarified in vascular systems. Activity modulation through endocytosis and vesicular trafficking significantly impacts downstream targets of RTKs such as endothelial nitric oxide synthase (eNOS) and VE-cadherin. RTKs and their associated targets are also transported to the nucleus, where they may directly impact nuclear signaling. Although the nuclear transport pathways are just beginning to be unraveled, it appears that endocytosis and vesicular trafficking are involved. In this review, we discuss the mechanisms by which activated RTKs and the downstream targets eNOS and VE-cadherin may be internalized and transported to various intracellular compartments. How localization and interacting proteins impact protein function and influence signaling is an important theme, as is the potential for modulating signaling through therapeutic targeting of activated receptors and components of the endocytic machinery.

A selective block of nuclear actin export stabilizes the giant nuclei of Xenopus oocytes

Actin is a major cytoskeletal element and is normally kept cytoplasmic by exportin 6 (Exp6)-driven nuclear export. Here, we show that Exp6 recognizes actin features that are conserved from yeast to human. Surprisingly however, microinjected actin was not exported from Xenopus laevis oocyte nuclei, unless Exp6 was co-injected, indicating that the pathway is inactive in this cell type. Indeed, Exp6 is undetectable in oocytes, but is synthesized from meiotic maturation onwards, which explains how actin export resumes later in embryogenesis. Exp6 thus represents the first example of a strictly developmentally regulated nuclear transport pathway. We asked why Xenopus oocytes lack Exp6 and observed that ectopic application of Exp6 renders the giant oocyte nuclei extremely fragile. This effect correlates with the selective disappearance of a sponge-like intranuclear scaffold of F-actin. These nuclei have a normal G2-phase DNA content in a volume 100,000 times larger than nuclei of somatic cells. Apparently, their mechanical integrity cannot be maintained by chromatin and the associated nuclear matrix, but instead requires an intranuclear actin-scaffold.

Importin 4 Is Responsible for Ligand-independent Nuclear Translocation of Vitamin D Receptor

Vitamin D receptor (VDR) is localized in nuclei and acts as a ligand-dependent transcription factor. To clarify the molecular mechanisms underlying the nuclear translocation of VDR, we utilized an in vitro nuclear transport assay using digitonin-permeabilized semi-intact cells. In this assay, recombinant whole VDR-(4-427) and a truncated mutant VDR-(4-232) lacking the carboxyl terminus of VDR were imported to nuclei even in the absence of ligand. In contrast, VDR-(91-427) lacking the amino-terminal DNA-binding domain was not imported to nuclei in the absence of ligand, and was efficiently imported in its liganded form. These results suggested that there are two distinct mechanisms underlying the nuclear transport of VDR; ligand-dependent and -independent pathways, and that the different regions of VDR are responsible for these processes. Therefore, we performed the yeast two-hybrid screening using VDR-(4-232) as the bait to explore the molecules responsible for ligand-independent nuclear translocation of VDR, and have identified importin 4 as an interacting protein. In the reconstruction experiments where transport factors were applied as recombinant proteins, recombinant importin 4 facilitated nuclear translocation of VDR regardless of its ligand, whereas importin beta failed in transporting VDR even in the presence of ligand. In conclusion, importin 4, not importin beta, is responsible for the ligand-independent nuclear translocation of VDR.

Viral Interactions with the Nuclear Transport Machinery: Discovering and Disrupting Pathways

Viruses have been invaluable tools for discovering key pathways of nucleocytoplasmic transport. Conversely, disruption of specific nuclear transport pathways, are crucial for the productive life cycle of some viruses. The major cellular mRNA export pathway, which uses TAP (NXF1)/p15(NXT) as receptor, was discovered as a result of TAP interaction with CTE-containing RNAs from Mason-Pfizer Monkey Virus. In addition, CRM1 or exportin 1, which is a transport receptor that mediates nuclear export of proteins, snRNAs, rRNAs and a small subset of mRNAs, was discovered as an interacting partner of the Rev protein of HIV1. Viruses may disrupt the nuclear transport machinery to prevent host antiviral response. VSV Matrix (M) protein inhibits mRNA export by forming a complex with the mRNA export factor Rae1 whereas poliovirus inhibits nuclear import of proteins by probably degrading Nup62 and Nup153. Hence, this review focuses on viruses as tools and as disruptors of nucleocytoplasmic trafficking.

A Universal Strategy for Proteomic Studies of SUMO and Other Ubiquitin-like Modifiers

Post-translational modification by the conjugation of small ubiquitin-like modifiers is an essential mechanism to affect protein function. Currently, only a limited number of substrates are known for most of these modifiers, thus limiting our knowledge of their role and relevance for cellular physiology. Here, we report the development of a universal strategy for proteomic studies of ubiquitin-like modifiers. This strategy involves the development of stable transfected cell lines expressing a double-tagged modifier under the control of a tightly negatively regulated promoter, the induction of the expression and conjugation of the tagged modifier to cellular proteins, the tandem affinity purification of the pool of proteins covalently modified by the tagged modifier, and the identification of the modified proteins by LC and MS. By applying this methodology to the proteomic analysis of SUMO-1 and SUMO-3, we determined that SUMO-1 and SUMO-3 are stable proteins exhibiting half-lives of over 20 h, demonstrated that sumoylation with both SUMO-1 and SUMO-3 is greatly stimulated by MG-132 and heat shock treatment, demonstrated the preferential usage of either SUMO-1 or SUMO-3 for some known SUMO substrates, and identified 122 putative SUMO substrates of which only 27 appeared to be modified by both SUMO-1 and SUMO-3. This limited overlapping in the subset of proteins modified by SUMO-1 and SUMO-3 supports that the SUMO paralogues are likely to be functionally distinct. Three of the novel putative SUMO substrates identified, namely the polypyrimidine tract-binding protein-associated splicing factor PSF, the structural microtubular component alpha-tubulin, and the GTP-binding nuclear protein Ran, were confirmed as authentic SUMO substrates. The application of this universal strategy to the identification of the pool of cellular substrates modified by other ubiquitin-like modifiers will dramatically increase our knowledge of the biological role of the different ubiquitin-like conjugations systems in the cell.

Characterization of karyopherin cargoes reveals unique mechanisms of Kap121p-mediated nuclear import.

In yeast there are at least 14 members of the beta-karyopherin protein family that govern the movement of a diverse set of cargoes between the nucleus and cytoplasm. Knowledge of the cargoes carried by each karyopherin and insight into the mechanisms of transport are fundamental to understanding constitutive and regulated transport and elucidating how they impact normal cellular functions. Here, we have focused on the identification of nuclear import cargoes for the essential yeast beta-karyopherin, Kap121p. Using an overlay blot assay and coimmunopurification studies, we have identified 30 putative Kap121p cargoes. Among these were Nop1p and Sof1p, two essential trans-acting protein factors required at the early stages of ribosome biogenesis. Characterization of the Kap121p-Nop1p and Kap121p-Sof1p interactions demonstrated that, in addition to lysine-rich nuclear localization signals (NLSs), Kap121p recognizes a unique class of signals distinguished by the abundance of arginine and glycine residues and consequently termed rg-NLSs. Kap104p is also known to recognize rg-NLSs, and here we show that it compensates for the loss of Kap121p function. Sof1p is also transported by Kap121p; however, its import can be mediated by a piggyback mechanism with Nop1p bridging the interaction between Sof1p and Kap121p. Together, our data elucidate additional levels of complexity in these nuclear transport pathways.