Project description:The RanGTPase acts as a master regulator of nucleocytoplasmic transport by controlling assembly and disassembly of nuclear transport complexes. RanGTP is required in the nucleus to release nuclear localization signal (NLS)-containing cargo from import receptors, and, under steady-state conditions, Ran is highly concentrated in the nucleus. We previously showed the nuclear/cytoplasmic Ran distribution is disrupted in Hutchinson-Gilford Progeria syndrome (HGPS) fibroblasts that express the Progerin form of lamin A, causing a major defect in nuclear import of the protein, translocated promoter region (Tpr). In this paper, we show that Tpr import was mediated by the most abundant import receptor, KPNA2, which binds the bipartite NLS in Tpr with nanomolar affinity. Analyses including NLS swapping revealed Progerin did not cause global inhibition of nuclear import. Rather, Progerin inhibited Tpr import because transport of large protein cargoes was sensitive to changes in the Ran nuclear/cytoplasmic distribution that occurred in HGPS. We propose that defective import of large protein complexes with important roles in nuclear function may contribute to disease-associated phenotypes in Progeria.
Project description:Ran, a Ras-like GTPase, has been implicated in controlling the movement of proteins and RNAs in and out of the nucleus. We have constructed strains of Saccharomyces cerevisiae which produce fusion proteins containing glutathione-S-transferase (GST) fused to Gsp1p, which encodes the essential yeast Ran homolog, and a mutant form of Gsp1p that mimics the GTP-bound state. A major protein with the apparent size of 34 kDa co-purifies with the GTP-bound form of Gsp1p. This protein was identified as Yrb1p (Yeast Ran Binding Protein) and stimulates GTP hydrolysis by Gsp1p in the presence of Rna1p, the Gsp1 GTPase activating protein. Yrb1p is located in the cytoplasm with some concentration at the nuclear periphery. Temperature-sensitive yrb1 mutants are defective in nuclear protein import and RNA export. A mutation in the highly conserved Ran binding region of Yrb1p reduces its ability to interact with Gsp1p. These data indicate that Yrb1p functions with Gsp1p and suggest that together they can control transport of macromolecules across the nuclear envelope.
Project description:TMX2 is a thioredoxin family protein, but its functions have not been clarified. To elucidate the function of TMX2, we explored TMX2-interacting proteins by LC-MS. As a result, importin-?, Ran GTPase (Ran), RanGAP, and RanBP2 were identified. Importin-? is an adaptor protein which imports cargoes from cytosol to the nucleus, and is exported into the cytosol by interaction with RanGTP. At the cytoplasmic nuclear pore, RanGAP and RanBP2 facilitate hydrolysis of RanGTP to RanGDP and the disassembly of the Ran-importin-? complex, which allows the recycling of importin-? and reentry of Ran into the nucleus. Despite its interaction of TMX2 with importin-?, we showed that TMX2 is not a transport cargo. We found that TMX2 localizes in the outer nuclear membrane with its N-terminus and C-terminus facing the cytoplasm, where it co-localizes with importin-? and Ran. Ran is predominantly distributed in the nucleus, but TMX2 knockdown disrupted the nucleocytoplasmic Ran gradient, and the cysteine 112 residue of Ran was important in its regulation by TMX2. In addition, knockdown of TMX2 suppressed importin-?-mediated transport of protein. These results suggest that TMX2 works as a regulator of protein nuclear transport, and that TMX2 facilitates the nucleocytoplasmic Ran cycle by interaction with nuclear pore proteins.
Project description:The Ran GTPase regulates nuclear import and export by controlling the assembly state of transport complexes. This involves the direct action of RanGTP, which is generated in the nucleus by the chromatin-associated nucleotide exchange factor, RCC1. Ran interactions with RCC1 contribute to formation of a nuclear:cytoplasmic (N:C) Ran protein gradient in interphase cells. In previous work, we showed that the Ran protein gradient is disrupted in fibroblasts from Hutchinson-Gilford progeria syndrome (HGPS) patients. The Ran gradient disruption in these cells is caused by nuclear membrane association of a mutant form of Lamin A, which induces a global reduction in heterochromatin marked with Histone H3K9me3 and Histone H3K27me3. Here, we have tested the hypothesis that heterochromatin controls the Ran gradient. Chemical inhibition and depletion of the histone methyltransferases (HMTs) G9a and GLP in normal human fibroblasts reduced heterochromatin levels and caused disruption of the Ran gradient, comparable to that observed previously in HGPS fibroblasts. HMT inhibition caused a defect in nuclear localization of TPR, a high molecular weight protein that, owing to its large size, displays a Ran-dependent import defect in HGPS. We reasoned that pathways dependent on nuclear import of large proteins might be compromised in HGPS. We found that nuclear import of ATM requires the Ran gradient, and disruption of the Ran gradient in HGPS causes a defect in generating nuclear ?-H2AX in response to ionizing radiation. Our data suggest a lamina-chromatin-Ran axis is important for nuclear transport regulation and contributes to the DNA damage response.
Project description:Nuclear transport receptors (NTRs) recognize localization signals of cargos to facilitate their passage across the central channel of nuclear pore complexes (NPCs). About 30 different NTRs constitute different transport pathways in humans and bind to a multitude of different cargos. The exact cargo spectrum of the majority of NTRs, their specificity and even the extent to which active nucleocytoplasmic transport contributes to protein localization remains understudied because of the transient nature of these interactions and the wide dynamic range of cargo concentrations. To systematically map cargo-NTR relationships in situ, we used proximity ligation coupled to mass spectrometry (BioID). We systematically fused the engineered biotin ligase BirA* to 16 NTRs. We estimate that a considerable fraction of the human proteome is subject to active nuclear transport. We quantified the specificity and redundancy in NTR interactions and identified transport pathways for cargos. We extended the BioID method by the direct identification of biotinylation sites. This approach enabled us to identify interaction interfaces and to discriminate direct versus piggyback transport mechanisms. Data are available via ProteomeXchange with identifier PXD007976.
Project description:The signal transducer and activator of transcription-3 (STAT3) induces transcription of genes that control differentiation, inflammation, proliferation, and tumor cell invasion. Cytokines such as interleukin-6 and interferon stimulate the specific tyrosine phosphorylation of STAT3, which confers its ability to bind consensus DNA targets. In addition, unphosphorylated STAT3 has been demonstrated to induce specific gene expression. STAT3 must gain entrance to the nucleus to impact transcription, however access to the nucleus is a tightly regulated process. Because nuclear trafficking is critical to the function of STAT3, we investigated the molecular mechanisms by which STAT3 is imported to the nucleus. Live cell imaging techniques were used with STAT3 tagged with green fluorescence protein (GFP) or photoactivatable GFP to follow the cellular dynamics of both unphosphorylated and tyrosine phosphorylated forms. Cytokine activation did not alter the rate of STAT3 nuclear import or nuclear export. In addition, Förster resonance energy transfer experiments revealed homomeric interaction of unphosphorylated STAT3 dependent on its amino terminus, but this dimerization is not necessary for its nuclear import. Previous work demonstrated the adapter importin-?3 binds to STAT3 and is required for nuclear import. To determine whether STAT3 nuclear import is mediated by the importin-?/importin-?1 heterodimer, the effects of siRNA to importin-?1 were evaluated. Results indicate STAT3 nuclear import is dependent on the function of importin-?1. Since the Ran GTPase is necessary to bind importin-?1 in the nucleus for release of importin-?-cargo, the effect of a GTPase deficient mutant of Ran was tested. Expression of the Ran interfering mutant inhibited STAT3 nuclear import. This study defines importin-?/importin-?1/Ran as the molecular mechanism by which STAT3 traffics to the nucleus.
Project description:Lentiviruses, unlike the gammaretroviruses, are able to infect nondividing cells by transiting through nuclear pores to access the host genomic DNA. Several nuclear import and nuclear pore components have been implicated as playing a role in nuclear import, including transportin 3 (TNPO3), a member of the importin-? family of nuclear import proteins. We demonstrated that TNPO3 was required by several lentiviruses, with simian immunodeficiency virus mac239 (SIVmac239) and equine infectious anemia virus (EIAV) the most dependent and human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) the least. Analysis of HIV-1/SIVmac239 chimeric viruses showed that dependence on TNPO3 mapped to the SIVmac239 capsid. Mutation of a single amino acid, A76V in the SIVmac239 capsid, rendered the virus TNPO3 independent and resistant to mCPSF6-358, a truncated splicing factor that prevents HIV-1 nuclear import. Using a complementation assay based on 293T cells that express a TNPO3-targeted short hairpin RNA (shRNA), we showed that the Drosophila TNPO3 homologue can substitute for its human counterpart and that it mapped a key functional domain of TNPO3 to the carboxy-terminal cargo-binding domain. Within the cargo-binding domain, two hydrophobic motifs were required for TNPO3-dependent infection. The mutated TNPO3 proteins maintained their ability to localize to the nucleus, suggesting that their inability to restore lentivirus infection resulted from an inability to bind to a host or viral cargo protein.
Project description:A major question in nuclear import concerns the identity of the nucleoporin(s) that interact with the nuclear localization sequences (NLS) receptor and its cargo as they traverse the nuclear pore. Ligand blotting and solution binding studies of isolated proteins have attempted to gain clues to the identities of these nucleoporins, but the studies have from necessity probed binding events far from an in vivo context. Here we have asked what binding events occur in the more physiological context of a Xenopus egg extract, which contains nuclear pore subcomplexes in an assembly competent state. We have then assessed our conclusions in the context of assembled nuclear pores themselves. We have used immunoprecipitation to identify physiologically relevant complexes of nucleoporins and importin subunits. In parallel, we have demonstrated that it is possible to obtain immunofluorescence localization of nucleoporins to subregions of the nuclear pore and its associated structures. By immunoprecipitation, we find the nucleoporin Nup153 and the pore-associated filament protein Tpr, previously shown to reside at distinct sites on the intranuclear side of assembled pores, are each in stable subcomplexes with importin alpha and beta in Xenopus egg extracts. Importin subunits are not in stable complexes with nucleoporins Nup62, Nup93, Nup98, or Nup214/CAN, either in egg extracts or in extracts of assembled nuclear pores. In characterizing the Nup153 complex, we find that Nup153 can bind to a complete import complex containing importin alpha, beta, and an NLS substrate, consistent with an involvement of this nucleoporin in a terminal step of nuclear import. Importin beta binds directly to Nup153 and in vitro can do so at multiple sites in the Nup153 FXFG repeat region. Tpr, which has no FXFG repeats, binds to importin beta and to importin alpha/beta heterodimers, but only to those that do not carry an NLS substrate. That the complex of Tpr with importin beta is fundamentally different from that of Nup153 is additionally demonstrated by the finding that recombinant beta or beta45-462 fragment freely exchanges with the endogenous importin beta/Nup153 complex, but cannot displace endogenous importin beta from a Tpr complex. However, the GTP analogue GMP-PNP is able to disassemble both Nup153- and Tpr-importin beta complexes. Importantly, analysis of extracts of isolated nuclei indicates that Nup153- and Tpr-importin beta complexes exist in assembled nuclear pores. Thus, Nup153 and Tpr are major physiological binding sites for importin beta. Models for the roles of these interactions are discussed.
Project description:The promyelocytic leukemia (PML) protein participates in a number of cellular processes, including transcription regulation, apoptosis, differentiation, virus defense and genome maintenance. This protein is structurally organized into a tripartite motif (TRIM) at its N-terminus, a nuclear localization signal (NLS) at its central region and a C-terminus that varies between alternatively spliced isoforms. Most PML splice variants target the nucleus where they define sub-nuclear compartments termed PML nuclear bodies (PML NBs). However, PML variants that lack the NLS are also expressed, suggesting the existence of PML isoforms with cytoplasmic functions. In the present study we expressed PML isoforms with a mutated NLS in U2OS cells to identify potential cytoplasmic compartments targeted by this protein.Expression of NLS mutated PML isoforms in U2OS cells revealed that PML I targets early endosomes, PML II targets the inner nuclear membrane (partially due to an extra NLS at its C-terminus), and PML III, IV and V target late endosomes/lysosomes. Clustering of PML at all of these subcellular locations depended on a functional TRIM domain.This study demonstrates the capacity of PML to form macromolecular protein assemblies at several different subcellular sites. Further, it emphasizes a role of the variable C-terminus in subcellular target selection and a general role of the N-terminal TRIM domain in promoting protein clustering.
Project description:Frontotemporal dementia (FTD) is the most common cause of dementia in people under 60 yr of age and is pathologically associated with mislocalization of TAR DNA/RNA binding protein 43 (TDP-43) in approximately half of cases (FLTD-TDP). Mutations in the gene encoding progranulin (GRN), which lead to reduced progranulin levels, are a significant cause of familial FTLD-TDP. Grn-KO mice were developed as an FTLD model, but lack cortical TDP-43 mislocalization and neurodegeneration. Here, we report retinal thinning as an early disease phenotype in humans with GRN mutations that precedes dementia onset and an age-dependent retinal neurodegenerative phenotype in Grn-KO mice. Retinal neuron loss in Grn-KO mice is preceded by nuclear depletion of TDP-43 and accompanied by reduced expression of the small GTPase Ran, which is a master regulator of nuclear import required for nuclear localization of TDP-43. In addition, TDP-43 regulates Ran expression, likely via binding to its 3'-UTR. Augmented expression of Ran in progranulin-deficient neurons restores nuclear TDP-43 levels and improves their survival. Our findings establish retinal neurodegeneration as a new phenotype in progranulin-deficient FTLD, and suggest a pathological loop involving reciprocal loss of Ran and nuclear TDP-43 as an underlying mechanism.