Dissecting the Signaling Events That Impact Classical Nuclear Import and Target Nuclear Transport Factors

Background

Signaling through MEK→ERK1/2 and PI3 kinases is implicated in many aspects of cell physiology, including the survival of oxidant exposure. Oxidants play a role in numerous physiological and pathophysiological processes, many of which rely on transport in and out of the nucleus. However, how oxidative stress impacts nuclear trafficking is not well defined.

Methodology/Principal Findings

To better understand the effect of stress on nucleocytoplasmic trafficking, we exposed cells to the oxidant diethyl maleate. This treatment activated MEK→ERK1/2 as well as PI3 kinase→Akt cascades and triggered the inhibition of classical nuclear import. To define the molecular mechanisms that regulate nuclear transport, we examined whether MEK and PI3 kinase signaling affected the localization of key transport factors. Using recently developed tools for image acquisition and analysis, the subcellular distributions of importin-α, CAS, and nucleoporins Nup153 and Nup88 were quantified in different cellular compartments. These studies identified specific profiles for the localization of transport factors in the nucleus and cytoplasm, and at the nuclear envelope. Our results demonstrate that MEK and PI3 kinase signaling as well as oxidative stress control nuclear trafficking and the localization of transport components. Furthermore, stress not only induced changes in transport factor distribution, but also upregulated post-translational modification of transport factors. Our results are consistent with the idea that the phosphorylation of importin-α, CAS, Nup153, and Nup88, and the O-GlcNAc modification of Nup153 increase when cells are exposed to oxidant.

Conclusions/Significance

Our studies defined the complex regulation of classical nuclear import and identified key transport factors that are targeted by stress, MEK, and PI3 kinase signaling.     

Mechanisms of receptor-mediated nuclear import and nuclear export

Nuclear transport of proteins and RNA occurs through the nuclear pore complex and is mediated by a superfamily of transport receptors known collectively as karyopherins. Karyopherins bind to their cargoes by recognition of specific nuclear localization signals or nuclear export signals. Transport through the nuclear pore complex is facilitated by transient interactions between the karyopherins and the nuclear pore complex. The interactions of karyopherins with their cargoes are regulated by the Ras-related GTPase Ran. Ran is assisted in this process by proteins that regulate its GTPase cycle and subcellular localization. In this review, we describe several of the major transport pathways that are conserved in higher and lower eukaryotes, with particular emphasis on the role of Ran. We highlight the latest advances in the structure and function of transport receptors and discuss recent examples of steroid hormone receptor import and regulation by signal transduction pathways. Understanding the molecular basis of nuclear transport may provide insight into human diseases by revealing how nucleocytoplasmic trafficking regulates protein activity.     

Functional identification of multiple nucleocytoplasmic trafficking signals in the broad-spectrum resistance protein RPW8.2

Nuclear localization signals (NLSs) and nuclear export signals (NESs) are important intramolecular regulatory elements for protein nucleocytoplasmic trafficking. This regulation confers spatial specificity to signal initiation and transduction in eukaryotic cells and thus is fundamental to the viability of all eukaryotic organisms. Here, we developed a simple and rapid method in which activity of putative NLSs or NESs was reported by subcellular localization of two tandem fluorescent proteins in fusion with the respective NLSs or NESs after agroinfiltration-mediated transient expression in leaves of Nicotiana benthamiana (Nb). We further demonstrated that the predicted NES from amino acid residue (aa) 9 to 22 and the NLS from aa91 to 101 in the broad-spectrum disease resistance protein RPW8.2 possess nuclear export and import activity, respectively. Additionally, by testing overlapping fragments covering the full length of RPW8.2, we identified another NLS from aa65 to 74 with strong nuclear import activity and two tandem non-canonical NESs in the C-terminus with strong nuclear export activity. Taken together, our results demonstrated the utility of a simple method to evaluate potential NLSs and NESs in plant cells and suggested that RPW8.2 may be subject to opposing nucleocytoplasmic trafficking forces for its subcellular localization and functional execution.     

Importin-beta family members mediate alpharetrovirus gag nuclear entry via interactions with matrix and nucleocapsid

The retroviral Gag polyprotein orchestrates the assembly and release of virus particles from infected cells. We previously reported that nuclear transport of the Rous sarcoma virus (RSV) Gag protein is intrinsic to the virus assembly pathway. To identify cis- and trans-acting factors governing nucleocytoplasmic trafficking, we developed novel vectors to express regions of Gag in Saccharomyces cerevisiae. The localization of Gag proteins was examined in the wild type and in mutant strains deficient in members of the importin-beta family. We confirmed the Crm1p dependence of the previously identified Gag p10 nuclear export signal. The known nuclear localization signal (NLS) in MA (matrix) was also functional in S. cerevisiae, and additionally we discovered a novel NLS within the NC (nucleocapsid) domain of Gag. MA utilizes Kap120p and Mtr10p import receptors while nuclear entry of NC involves the classical importin-alpha/beta (Kap60p/95p) pathway. NC also possesses nuclear targeting activity in avian cells and contains the primary signal for the import of the Gag polyprotein. Thus, the nucleocytoplasmic dynamics of RSV Gag depend upon the counterbalance of Crm1p-mediated export with two independent NLSs, each interacting with distinct nuclear import factors.     

GAP Activity,but Not Subcellular Targeting,Is Required for Arabidopsis RanGAP Cellular and Developmental Functions

The Ran GTPase activating protein (RanGAP) is important to Ran signaling involved in nucleocytoplasmic transport, spindle organization, and postmitotic nuclear assembly. Unlike vertebrate and yeast RanGAP, plant RanGAP has an N-terminal WPP domain, required for nuclear envelope association and several mitotic locations of Arabidopsis thaliana RanGAP1. A double null mutant of the two Arabidopsis RanGAP homologs is gametophyte lethal. Here, we created a series of mutants with various reductions in RanGAP levels by combining a RanGAP1 null allele with different RanGAP2 alleles. As RanGAP level decreases, the severity of developmental phenotypes increases, but nuclear import is unaffected. To dissect whether the GAP activity and/or the subcellular localization of RanGAP are responsible for the observed phenotypes, this series of rangap mutants were transformed with RanGAP1 variants carrying point mutations abolishing the GAP activity and/or the WPP-dependent subcellular localization. The data show that plant development is differentially affected by RanGAP mutant allele combinations of increasing severity and requires the GAP activity of RanGAP, while the subcellular positioning of RanGAP is dispensable. In addition, our results indicate that nucleocytoplasmic trafficking can tolerate both partial depletion of RanGAP and delocalization of RanGAP from the nuclear envelope.     

Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1.

Proteinase inhibitor 9 (PI-9) is a human serpin present in the cytoplasm of cytotoxic lymphocytes and epithelial cells. It inhibits the cytotoxic lymphocyte granule proteinase granzyme B (graB) and is thought to protect cytotoxic lymphocytes and bystander cells from graB-mediated apoptosis. Following uptake into cells, graB promotes DNA degradation, rapidly translocating to the nucleus, where it binds a nuclear component. PI-9 should therefore be found in cytotoxic lymphocyte and bystander cell nuclei to ensure complete protection against graB. Here we demonstrate by microscopy and subcellular fractionation experiments that PI-9 is present in the nuclei of human cytotoxic cells, endothelial cells, and epithelial cells. We also show that the related serpins, PI-6, monocyte neutrophil elastase inhibitor (MNEI), PI-8, plasminogen activator inhibitor 2 (PAI-2), and the viral serpin CrmA exhibit similar nucleocytoplasmic distributions. Because these serpins lack classical nuclear localization signals and are small enough to diffuse through nuclear pores, we investigated whether import occurs actively or passively. Large (approximately 70 kDa) chimeric proteins comprising PI-9, PI-6, PI-8, MNEI, or PAI-2 fused to green fluorescent protein (GFP) show similar nucleocytoplasmic distributions to the parent proteins, indicating that nuclear import is active. By contrast, CrmA-GFP is excluded from nuclei, indicating that CrmA is not actively imported. In vitro nuclear transport assays show that PI-9 accumulates at a rate above that of passive diffusion, that it requires cytosolic factors but not ATP, and that it does not bind an intranuclear component. Furthermore, PI-9 is exported from nuclei via a leptomycin B-sensitive pathway, implying involvement of the export factor Crm1p. We conclude that the nucleocytoplasmic distribution of PI-9 and related serpins involves a nonconventional nuclear import pathway and Crm1p.     

Starvation promotes nuclear accumulation of the hsp70 Ssa4p in yeast cells

Nuclear import of proteins that are too large to passively enter the nucleus requires soluble factors, energy, and a nuclear localization signal (NLS). Nuclear protein transport can be regulated, and different forms of stress affect nucleocytoplasmic trafficking. As such, import of proteins containing a classical NLS is inhibited in starving yeast cells. In contrast, the hsp70 Ssa4p concentrates in nuclei upon starvation. Nuclear concentration of Ssa4p in starving cells is reversible, and transfer of stationary phase cells to fresh medium induces Ssa4p nuclear export. This export reaction represents an active process that is sensitive to oxidative stress. In starving cells, the N-terminal domain of Ssa4p mediates Ssa4p nuclear accumulation, and a short hydrophobic sequence, termed Star (for starvation), is sufficient to localize the reporter proteins green fluorescent protein or beta-galactosidase to nuclei. To determine whether nuclear accumulation of Star-beta-galactosidase depends on a specific nuclear carrier, we have analyzed its distribution in mutant yeast strains that carry a deletion of a single beta-importin gene. With this assay we have identified Nmd5p as a beta-importin required to concentrate Star-beta-galactosidase in nuclei when cells enter stationary phase.     

The transmembrane nucleoporin NDC1 is required for targeting of ALADIN to nuclear pore complexes

NDC1 is a transmembrane nucleoporin that is required for NPC assembly and nucleocytoplasmic transport. We show here that NDC1 directly interacts with the nucleoporin ALADIN, mutations of which are responsible for triple-A syndrome, and that this interaction is required for targeting of ALADIN to nuclear pore complexes (NPCs). Furthermore, we show that NDC1 is required for selective nuclear import. Our findings suggest that NDC1-mediated localization of ALADIN to NPCs is essential for selective nuclear protein import, and that abrogation of the interaction between ALADIN and NDC1 may be important for the development of triple-A syndrome.     

The Nup153-Nup50 Protein Interface and Its Role in Nuclear Import

Interactions between Nup50 and soluble transport factors underlie the efficiency of certain nucleocytoplasmic transport pathways. The platform on which these interactions take place is important to building a complete understanding of nucleocytoplasmic trafficking. Nup153 is the nucleoporin that provides this scaffold for Nup50. Here, we have delineated requirements for the interaction between Nup153 and Nup50, revealing a dual interface. An interaction between Nup50 and a region in the unique N-terminal region of Nup153 is critical for the nuclear pore localization of Nup50. A second site of interaction is at the distal tail of Nup153 and is dependent on importin α. Both of these interactions involve the N-terminal domain of Nup50. The configuration of the Nup153-Nup50 partnership suggests that the Nup153 scaffold provides not just a means of pore targeting for Nup50 but also serves to provide a local environment that facilitates bringing Nup50 and importin α together, as well as other soluble factors involved in transport. Consistent with this, disruption of the Nup153-Nup50 interface decreases efficiency of nuclear import.     

Reactive Oxygen Species-Mediated DJ-1 Monomerization Modulates Intracellular Trafficking Involving Karyopherin β2

Mutations in DJ-1 are a cause of recessive, early-onset Parkinson''s disease (PD). Although oxidative stress and mitochondrial integrity have been implicated in PD, it is largely unknown why neurons degenerate. DJ-1 is involved in oxidative stress-mediated responses and in mitochondrial maintenance; however, its specific function remains vague. Here we show that DJ-1 exhibits neuronal dynamic intracellular trafficking, with dimeric/monomeric cycling modulated by the oxidative environment. We demonstrate that oxidative stress enhances monomerization of wild-type cytosolic DJ-1, leading to nuclear recruitment. The pathogenic DJ-1/E163K variant is unable to homodimerize but is retained in the cytosol upon wild-type DJ-1 heterodimerization. We found that this wild-type/pathogenic heterodimer is disrupted by oxidative stress, leading to DJ-1/E163K mitochondrial translocation. We further demonstrated that endogenously expressed wild-type DJ-1 is imported into neuronal nuclei as a monomer and that nucleo-cytoplasmic transport is oxidative stress mediated. We identified a novel proline-tyrosine nuclear localization signal (PY-NLS) in DJ-1, and we found that nuclear monomeric DJ-1 import is mediated by an oxidative stress-dependent interaction with karyopherin β2. Our study provides evidence that oxidative stress-mediated intracellular trafficking of DJ-1, mediated by dynamic DJ-1 dimeric/monomeric cycling, is implicated in PD pathogenesis.     

Nuclear Trafficking of the Rabies Virus Interferon Antagonist P-Protein Is Regulated by an Importin-Binding Nuclear Localization Sequence in the C-Terminal Domain

Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP)-recognised nuclear localization sequence in the N-terminal region (N-NLS), the mechanisms underlying nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD) can result in nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro nuclear transport assays to examine in detail the nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on nuclear export, suggesting that nuclear exclusion of mutated P3 involves the P-CTD-localized nuclear export sequence (C-NES). However, assays using cells in which nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 nuclear accumulation and, importantly, prevent nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMPα2/IMPβ1-dependent nuclear import by conferring direct binding to the IMPα2/IMPβ1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPβ-binding autoinhibitory domain (ΔIBB-IMPα2), and IMPβ1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P-protein subcellular localization, consistent with important roles in infection.