Nuclear Transport Pathways Research Articles

A karyopherin alpha2 nuclear transport pathway is regulated by glucose in hepatic and pancreatic cells.

We studied the role of the karyopherin alpha2 nuclear import carrier (also known as importin alpha2) in glucose signaling. In mhAT3F hepatoma cells, GFP-karyopherin alpha2 accumulated massively in the cytoplasm within minutes of glucose extracellular addition and returned to the nucleus after glucose removal. In contrast, GFP-karyopherin alpha1 distribution was unaffected regardless of glucose concentration. Glucose increased GFP-karyopherin alpha2 nuclear efflux by a factor 80 and its shuttling by a factor 4. These glucose-induced movements were not due to glycolytic ATP production. The mechanism involved was leptomycin B-insensitive, but phosphatase- and energy-dependent. HepG2 and COS-7 cells displayed no glucose-induced GFP-karyopherin alpha2 movements. In pancreatic MIN-6 cells, the glucose-induced movements of karyopherin alpha2 and the stimulation of glucose-induced gene transcription were simultaneously lost between passages 28 and 33. Thus, extracellular glucose regulates a nuclear transport pathway by increasing the nuclear efflux and shuttling of karyopherin alpha2 in cells in which glucose can stimulate the transcription of sugar-responsive genes.

Ca2+-dependent nuclear export mediated by calreticulin.

We have characterized a pathway for nuclear export of the glucocorticoid receptor (GR) in mammalian cells. This pathway involves the Ca2+ -binding protein calreticulin (CRT), which directly contacts the DNA binding domain (DBD) of GR and facilitates its delivery from the nucleus to the cytoplasm. In the present study, we investigated the role of Ca2+ in CRT-dependent export of GR. We found that removal of Ca2+ from CRT inhibits its capacity to stimulate the nuclear export of GR in digitonin-permeabilized cells and that the inhibition is due to the failure of Ca2+-free CRT to bind the DBD. These effects are reversible, since DBD binding and nuclear export can be restored by Ca2+ addition. Depletion of intracellular Ca2+ inhibits GR export in intact cells under conditions that do not inhibit other nuclear transport pathways, suggesting that there is a Ca2+ requirement for GR export in vivo. We also found that the Ran GTPase is not required for GR export. These data show that the nuclear export pathway used by steroid hormone receptors such as GR is distinct from the Crm1 pathway. We suggest that signaling events that increase Ca2+ could positively regulate CRT and inhibit GR function through nuclear export.

β-Catenin's Nuclear Receptor Shuttle

It is unclear how the cytoplasmic protein β-catenin moves into the nucleus to regulate transcription. It lacks a nuclear localization signal, is too large to translocate in a passive manner, and moves independently of the nuclear transport pathways involving importin and Ran. Three studies suggest that transport may occur through association with shuttling nuclear receptors. Yang et al. , Mulholland et al. , and Pawlowski et al. report a direct interaction between β-catenin and the androgen receptor (AR). When activated by ligands such as testosterone, AR moves into the nucleus to stimulate transcription of target genes. AR agonists promoted the translocation and nuclear localization of AR and β-catenin with similar kinetics. Other hormone-bound nuclear receptors, including those for progesterone, estrogen, and glucocorticoid, did not act as shuttles. Moreover, AR antagonists did not induce this effect, indicating that translocation of AR alone is not sufficient to carry β-catenin into the nucleus. The COOH-terminal DNA and ligand-binding domain of the AR and the Armadillo repeats of β-catenin were identified as the associating regions. The Wnt signaling pathway, which controls the balance of nuclear and cytosolic pools of β-catenin in some cells, was not activated by AR in the neuronal cell line examined by Pawlowski et al. Mulholland demonstrated that the translocation did not depend on adenomatous polyposis coli, another potential nuclear shuttle for β-catenin. Yang et al. showed that overexpression of the cell-surface protein E-cadherin in a prostate cancer cell line sequestered β-catenin and reduced AR-mediated transcription. The consequence of AR-β-catenin is uncertain as the reports show either suppression or augmentation of AR-mediated transcription in various reporter assays. Steroid receptors appear to offer another convenient ride for β-catenin's trip to the nucleus. J. E. Pawloski, J. R. Ertel, M. P. Allen, M. Xu, C. Butler, E. M. Wilson, M. E. Wierman, Liganded androgen receptor interaction with β-catenin. J. Biol. Chem. 277 , 20702-20710 (2002). [Abstract] [Full Text] F. Yang, X. Li, M. Sharmu, C. Y. Sasaki, D. L. Longo, B. Lim, Z. Sun, Linking β-catenin to androgen-signaling pathway. J. Biol. Chem. 277 , 11336-11344 (2002). [Abstract] [Full Text] D. J. Mulholland, H. Cheng, K. Reid, P. S. Rennie, C. C. Nelson, The androgen receptor can promote β-catenin nuclear translocation independently of adenomatous polyposis coli. J. Biol. Chem. 277 , 17933-17943 (2002). [Abstract] [Full Text]

-Catenin's Nuclear Receptor Shuttle

It is unclear how the cytoplasmic protein β-catenin moves into the nucleus to regulate transcription. It lacks a nuclear localization signal, is too large to translocate in a passive manner, and moves independently of the nuclear transport pathways involving importin and Ran. Three studies suggest that transport may occur through association with shuttling nuclear receptors. Yang et al., Mulholland et al., and Pawlowski et al. report a direct interaction between β-catenin and the androgen receptor (AR). When activated by ligands such as testosterone, AR moves into the nucleus to stimulate transcription of target genes. AR agonists promoted the translocation and nuclear localization of AR and β-catenin with similar kinetics. Other hormone-bound nuclear receptors, including those for progesterone, estrogen, and glucocorticoid, did not act as shuttles. Moreover, AR antagonists did not induce this effect, indicating that translocation of AR alone is not sufficient to carry β-catenin into the nucleus. The COOH-terminal DNA and ligand-binding domain of the AR and the Armadillo repeats of β-catenin were identified as the associating regions. The Wnt signaling pathway, which controls the balance of nuclear and cytosolic pools of β-catenin in some cells, was not activated by AR in the neuronal cell line examined by Pawlowski et al. Mulholland demonstrated that the translocation did not depend on adenomatous polyposis coli, another potential nuclear shuttle for β-catenin. Yang et al. showed that overexpression of the cell-surface protein E-cadherin in a prostate cancer cell line sequestered β-catenin and reduced AR-mediated transcription. The consequence of AR-β-catenin is uncertain as the reports show either suppression or augmentation of AR-mediated transcription in various reporter assays. Steroid receptors appear to offer another convenient ride for β-catenin's trip to the nucleus.J. E. Pawloski, J. R. Ertel, M. P. Allen, M. Xu, C. Butler, E. M. Wilson, M. E. Wierman, Liganded androgen receptor interaction with β-catenin. J. Biol. Chem. 277, 20702-20710 (2002). [Abstract] [Full Text]F. Yang, X. Li, M. Sharmu, C. Y. Sasaki, D. L. Longo, B. Lim, Z. Sun, Linking β-catenin to androgen-signaling pathway. J. Biol. Chem. 277, 11336-11344 (2002). [Abstract] [Full Text]D. J. Mulholland, H. Cheng, K. Reid, P. S. Rennie, C. C. Nelson, The androgen receptor can promote β-catenin nuclear translocation independently of adenomatous polyposis coli. J. Biol. Chem. 277, 17933-17943 (2002). [Abstract] [Full Text]

Molecular cloning and characterization of Plasmodium falciparum transportin

A cDNA encoding transportin, a protein involved in the nuclear import of M9 nuclear localization signal-bearing proteins, has been cloned from the malaria parasite Plasmodium falciparum. The complete cDNA consists of 3,667 bp encoding 1,136 amino acid residues. Amino acid sequence analysis revealed that Ran-GTP and M9 binding domains are highly conserved in P. falciparum, suggesting that the transportin-mediated nuclear transport pathway exists in this protozoan parasite. Southern blot analysis revealed that the transportin gene exists as a single copy in the malarial genome.

Importins fulfil a dual function as nuclear import receptors and cytoplasmic chaperones for exposed basic domains.

Many nuclear transport pathways are mediated by importin beta-related transport receptors. Here, we identify human importin (Imp) 4b as well as mouse Imp4a, Imp9a and Imp9b as novel family members. Imp4a mediates import of the ribosomal protein (rp) S3a, while Imp9a and Imp9b import rpS7, rpL18a and apparently numerous other substrates. Ribosomal proteins, histones and many other nuclear import substrates are very basic proteins that aggregate easily with cytoplasmic polyanions such as RNA. Imp9 effectively prevents such precipitation of, for example, rpS7 and rpL18a by covering their basic domains. The same applies to Imp4, Imp5, Imp7 and Impbeta and their respective basic import substrates. The Impbeta-Imp7 heterodimer appears specialized for the most basic proteins, such as rpL4, rpL6 and histone H1, and is necessary and sufficient to keep them soluble in a cytoplasmic environment prior to rRNA or DNA binding, respectively. Thus, just as heat shock proteins function as chaperones for exposed hydrophobic patches, importins act as chaperones for exposed basic domains, and we suggest that this represents a major and general cellular function of importins.

Nuclear transport of photosensitizers during photosensitization and oxidative stress

The nuclear transport pathways of the photosensitizers meso-tetra(4-sulfonatophenyl)porphyrin (TPPS 4) and meso-tetra(4-N-methylpyridyl)porphyrin (TMPyP) during photosensitization and oxidative stress were characterized in CT-26 murine colon carcinoma cells using fluorescence microscopy and multi-pixel spectral imaging. Prior to irradiation, TPPS 4 and TMPyP localized mainly in the lysosomes, while irradiation or H 2O 2 treatment induced a relocalization into the nucleus and nucleoli. Flow cytometry analysis of isolated nuclei from the treated cells showed an increase in nuclear fluorescence accompanying the relocalization. Isolation and separation of the nuclear proteins according to molecular weight was performed using a sephadex G-100 column. The protein fractions exhibiting high fluorescence were separated by high performance liquid chromatography. Five major classes of proteins with a retention time of 1, 7, 11, 12 and 15 min were obtained. Each photosensitizer was associated with a distinct class of proteins. While TPPS 4 fluorescence was detected in the protein fraction with a retention time of 11 min, TMPyP fluorescence was associated with a protein fraction having a retention time of 7 min. We conclude that although oxidative stress triggers entry into the nucleus of both TPPS 4 and TMPyP, each sensitizer uses a distinct transport mechanism based on its chemical properties.

Directed Inhibition of Nuclear Import in Cellular Hypertrophy

Each nuclear pore is responsible for both nuclear import and export with a finite capacity for bidirectional transport across the nuclear envelope. It remains poorly understood how the nuclear transport pathway responds to increased demands for nucleocytoplasmic communication. A case in point is cellular hypertrophy in which increased amounts of genetic material need to be transported from the nucleus to the cytosol. Here, we report an adaptive down-regulation of nuclear import supporting such an increased demand for nuclear export. The induction of cardiac cell hypertrophy by phenylephrine or angiotensin II inhibited the nuclear translocation of H1 histones. The removal of hypertrophic stimuli reversed the hypertrophic phenotype and restored nuclear import. Moreover, the inhibition of nuclear export by leptomycin B rescued import. Hypertrophic reprogramming increased the intracellular GTP/GDP ratio and promoted the nuclear redistribution of the GTP-binding transport factor Ran, favoring export over import. Further, in hypertrophy, the reduced creatine kinase and adenylate kinase activities limited energy delivery to the nuclear pore. The reduction of activities was associated with the closure of the cytoplasmic phase of the nuclear pore preventing import at the translocation step. Thus, to overcome the limited capacity for nucleocytoplasmic transport, cells requiring increased nuclear export regulate the nuclear transport pathway by undergoing a metabolic and structural restriction of nuclear import.

Selective disruption of nuclear import by a functional mutant nuclear transport carrier.

p10/NTF2 is a nuclear transport carrier that mediates the uptake of cytoplasmic RanGDP into the nucleus. We constructed a point mutant of p10, D23A, that exhibited unexpected behavior both in digitonin-permeabilized and microinjected mammalian cells. D23A p10 was markedly more efficient than wild-type (wt) p10 at supporting Ran import, but simultaneously acted as a dominant-negative inhibitor of classical nuclear localization sequence (cNLS)-mediated nuclear import supported by karyopherins (Kaps) alpha and beta1. Binding studies indicated that these two nuclear transport carriers of different classes, p10 and Kap-beta1, compete for identical and/or overlapping binding sites at the nuclear pore complex (NPC) and that D23A p10 has an increased affinity relative to wt p10 and Kap-beta1 for these shared binding sites. Because of this increased affinity, D23A p10 is able to import its own cargo (RanGDP) more efficiently than wt p10, but Kap-beta1 can no longer compete efficiently for shared NPC docking sites, thus the import of cNLS cargo is inhibited. The competition of different nuclear carriers for shared NPC docking sites observed here predicts a dynamic equilibrium between multiple nuclear transport pathways inside the cell that could be easily shifted by a transient modification of one of the carriers.

Phosphorylation of the Nuclear Transport Machinery Down-regulates Nuclear Protein Import in Vitro

We have examined whether signal-mediated nucleocytoplasmic transport can be regulated by phosphorylation of the nuclear transport machinery. Using digitonin-permeabilized cell assays to measure nuclear import and export, we found that the phosphatase inhibitors okadaic acid and microcystin inhibit transport mediated by the import receptors importin beta and transportin, but not by the export receptor CRM1. Several lines of evidence, including the finding that transport inhibition is partially reversed by the broad specificity protein kinase inhibitor staurosporine, indicate that transport inhibition is due to elevated phosphorylation of a component of the nuclear transport machinery. The kinases and phosphatases involved in this regulation are present in the permeabilized cells. A phosphorylation-sensitive component of the nuclear transport machinery also is present in permeabilized cells and is most likely a component of the nuclear pore complex. Substrate binding by the importin alpha.beta complex and the association of the complex with the nucleoporins Nup358/RanBP2 and Nup153 are not affected by phosphatase inhibitors, suggesting that transport inhibition by protein phosphorylation does not involve these steps. These results suggest that cells have mechanisms to negatively regulate entire nuclear transport pathways, thus providing a means to globally control cellular activity through effects on nucleocytoplasmic trafficking.

A complex of mammalian ufd1 and npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways.

The AAA-ATPase, p97/Cdc48p, has been implicated in many different pathways ranging from membrane fusion to ubiquitin-dependent protein degradation. Binding of the p47 complex directs p97 to act in the post-mitotic fusion of Golgi membranes. We now describe another binding complex comprising mammalian Ufd1 and Npl4. Yeast Ufd1p is required for ubiquitin-dependent protein degradation whereas yeast Npl4p has been implicated in nuclear transport. In rat liver cytosol, Ufd1 and Npl4 form a binary complex, which exists either alone or bound to p97. Ufd1/Npl4 competes with p47 for binding to p97 and so inhibits Golgi membrane fusion. This suggests that it is involved in another cellular function catalysed by p97, the most likely being ubiquitin-dependent events during mitosis. The fact that the binding of p47 and Ufd1/Npl4 is mutually exclusive suggests that these protein complexes act as adapters, directing a basic p97 activity into different cellular pathways.

Nuclear import of sterol regulatory element-binding protein-2, a basic helix-loop-helix-leucine zipper (bHLH-Zip)-containing transcription factor, occurs through the direct interaction of importin beta with HLH-Zip.

The sterol regulatory element-binding protein-2 (SREBP-2) is produced as a large precursor molecule attached to the endoplasmic reticulum membrane. In response to the sterol depletion, the N-terminal segment of the precursor, which contains a basic helix-loop-helix-leucine zipper domain, is released by two sequential cleavages and is translocated to the nucleus, where it activates the transcription of target genes. The data herein show that released SREBP-2 uses a distinct nuclear transport pathway, which is mediated by importin beta. The mature form of SREBP-2 is actively transported into the nucleus when injected into the cell cytoplasm. SREBP-2 binds directly to importin beta in the absence of importin alpha. Ran-GTP but not Ran-GDP causes the dissociation of the SREBP-2-importin beta complex. G19VRan-GTP inhibits the nuclear import of SREBP-2 in living cells. In the permeabilized cell in vitro transport system, nuclear import of SREBP-2 is reconstituted only by importin beta in conjunction with Ran and its interacting protein p10/NTF2. We further demonstrate that the helix-loop-helix-leucine zipper motif of SREBP-2 contains a novel type of nuclear localization signal, which binds directly to importin beta.

Insulin-like Growth Factor-binding Protein (IGFBP)-3 and IGFBP-5 Share a Common Nuclear Transport Pathway in T47D Human Breast Carcinoma Cells

Insulin-like growth factor-binding proteins (IGFBPs) play an integral role in modifying insulin-like growth factor actions in a wide variety of cell types. Recent evidence suggests that IGFBP-3 and IGFBP-5 also have effects on cell growth that are insulin-like growth factor-independent. In investigating possible mechanisms for this effect, the intracellular trafficking of IGFBP-3 and IGFBP-5, both of which contain sequences with the potential for nuclear localization, was studied in T47D cells. Nuclear uptake of fluorescently labeled IGFBP-3 and IGFBP-5 was observed in a proportion of T47D cells that appeared to be rapidly dividing. IGFBP-1 and IGFBP-2, which do not possess the putative domain for nuclear translocation, were not transported to the nuclei of T47D cells. When T47D cells were preincubated with excess unlabeled IGFBP-3, nuclear localization of labeled IGFBP-3 or IGFBP-5 was not detected, indicating that their nuclear translocation involves a common pathway. Inhibition of receptor-mediated endocytosis did not affect nuclear uptake of IGFBP-3, suggesting that it uses an alternative non-classical import pathway for transport across the plasma membrane. In addition, a variant form of IGFBP-3 with a mutation in the putative nuclear localization sequence was unable to translocate to the nuclei of T47D cells, suggesting that nuclear translocation of IGFBP-3 was dependent on these carboxyl-terminal basic residues.

The Nucleus's Revolving Door

Work done over the past 2 years shows that cells have evolved a highly complex system, involving an intricate division of labor among a host of different proteins that are structurally related, for transporting large molecules such as proteins and mRNAs into and out of the nucleus. Recent work also indicates that a small protein called Ran acts as the dispatcher, coordinating the direction of transport and linking the various nuclear transport pathways with critical events in the life and death of the cell. Because critical processes such as cell division and protein synthesis depend on molecules that have to be carried into or out of the nucleus at exactly the right time, these new findings are causing more and more researchers to take notice of the pivotal role nuclear transport plays in the life of the cell.

HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection.

HIV-1 Vpr promotes nuclear entry of viral nucleic acids in nondividing macrophages and also causes a G2 cell-cycle arrest. Consistent with its role in nuclear transport, we show Vpr localizes to the nuclear envelope in both human and yeast cells. Like the importin-beta subunit of the nuclear import receptor, Vpr also interacts with the yeast importin-alpha subunit and nucleoporins. Moreover, overexpression of either Vpr or importin-beta in yeast blocks nuclear transport of mRNAs. A mutant form of Vpr (Vpr F34I) that does not localize at the nuclear envelope, or bind to importin-alpha and nucleoporins, renders HIV-1 incapable of infecting macrophages efficiently. Vpr F34I, however, still causes a G2 arrest, demonstrating that the dual functions of Vpr are genetically separable. Our data suggest Vpr functionally resembles importin-beta in nuclear import of the HIV-1 pre-integration complex and this function is essential for the role of Vpr in macrophage infection, but not G2 arrest.