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Matthias Kohler - One of the best experts on this subject based on the ideXlab platform.
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nuclear translocation of hypoxia inducible factors hifs involvement of the classical Importin α β pathway
Biochimica et Biophysica Acta, 2008Co-Authors: Reinhard Depping, Beate Friedrich, Enno Hartmann, Susann G Schindler, Amrei Steinhoff, Riku Fagerlund, Eric Metzen, Matthias KohlerAbstract:Hypoxia-inducible factors are the key elements in the essential process of oxygen homeostasis of vertebrate cells. Stabilisation and subsequent nuclear localisation of HIF-alpha subunits results in the activation of target genes such as vegf, epo and glut1. The passage of transcription factors e.g. HIF-1alpha into the nucleus through the nuclear pore complex is regulated by nuclear transport receptors. Therefore nucleocytoplasmic shuttling can regulate transcriptional activity by facilitating the cellular traffic of transcription factors between both compartments. Here, we report on the identification of specific interactions of hypoxia-inducible factors with nuclear transport receptors Importin alpha/beta. HIF-1alpha, -1beta, and HIF-2alpha are binding to Importin alpha1, alpha3, alpha5, and alpha7. The direct interaction of HIF-1alpha to alpha Importins is dependent on a functional nuclear localisation signal within the C-terminal region of the protein. In contrast, the supposed N-terminal NLS is not effective. Our findings provide new insight into the mechanism of the regulation of nuclear transport of hypoxia-inducible factors.
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In Vivo Analysis of Importin α Proteins Reveals Cellular Proliferation Inhibition and Substrate Specificity
Molecular and Cellular Biology, 2004Co-Authors: Christina Quensel, Beate Friedrich, Thomas Sommer, Enno Hartmann, Matthias KohlerAbstract:Macromolecule transport between cell cytosol and nucleus takes place through the nuclear pore complexes (NPC) (2, 41). Import substrates possess nuclear localization signals (NLS), required for recognition by distinct nuclear import factors. The so-called classical nucleocytoplasmic import pathway is mediated by the Importin α/β heterodimer, also known as karyopherin α/β (for reviews, see references 23, 34, and 43). Importin α acts as an adapter by binding both the import substrate and Importin β. The trimeric import complex docks to the NPC via Importin β and translocates into the nucleus. Recently, Npap60/Nup50 was identified as an additional mammalian cofactor for Importin α/β-dependent nuclear protein import (22). While only one Importin β isoform exists, six human α Importins have been described (3, 4, 18, 21, 28, 36, 42). In contrast, the yeast Saccharomyces cerevisiae possesses only one gene for Importin α, which is essential (45). The α Importins are grouped into three subfamilies based on sequence homology. The first subfamily consists of Importin α1/Rch1. Its most closely related homologue, Importin α2, has been found in Xenopus laevis and other vertebrates but not in mammals. Importins α3/Qip1 and α4/hSRP1γ are members of the second subfamily. The third subfamily consists of Importins α5/hSRP1, α6, and α7 (18). The isoforms of one subfamily are highly homologous, showing about 85% sequence identity and differing mostly in regions outside the NLS binding pockets (18, 21, 26). Although the α Importins differ in their cell- and tissue-specific expression patterns, most are expressed ubiquitously (16, 18, 20, 21, 28, 31, 40). Only Importin α6 expression seems to be restricted to the testis (21). The reason for the Importin α diversity in higher eukaryotes, especially in humans and other vertebrates, is unknown. Invertebrates such as Caenorhabditis elegans and Drosophila melanogaster have three Importin α isoforms with about 50% homology to each other. Knockout and knockdown experiments targeting distinct Importin α genes have resulted in severe phenotypes. These studies suggest that Importin α3 is required during development, and it seems to be essential for oogenesis in Drosophila (25) and C. elegans (8). In contrast, Gorjanacz et al. and Mason et al. found that Drosophila Importin α2 (homologue to human Importin α1) seems to be essential for gametogenesis (10, 24). The Adam and the Mattaj laboratories showed the importance of C. elegans α Importins IMA-3 (8) and IMA-2 (1, 9) for embryonic development. Vertebrates as well as humans possess more than three different α Importins. Several in vitro studies have shown that the individual human α Importins are able to import the same target proteins. This state of affairs might argue against specialized roles of distinct α Importins in human cells. Interestingly, many of these studies also provided in vitro evidence that the α Importins differ in their substrate-specific import efficiency (7, 18, 19, 27, 28, 37, 44). Whether or not these results allow conclusions for living cells is unclear. Protein concentrations in these import assays are different from those in vivo, and probably the most important factor is the lack of competing substrates. Two competing substrates added simultaneously in the in vitro assays can change the import capacity of α Importins (18). Whether α Importins can substitute for one another in vivo is still unknown. To address this issue, we conducted a systematic study of the effects of specific α Importin down-regulation in cultured human cells.
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In Vivo Analysis of Importin α Proteins Reveals Cellular Proliferation Inhibition and Substrate Specificity
Molecular and cellular biology, 2004Co-Authors: Christina Quensel, Beate Friedrich, Thomas Sommer, Enno Hartmann, Matthias KohlerAbstract:The "classical" nuclear import pathway depends on Importins alpha and beta. Humans have only one Importin beta, while six alpha Importins have been described. Whether or not distinct alpha Importins are essential for specific import pathways in living human cells is unclear. We used RNA interference technology to specifically down-regulate the expression of ubiquitously expressed human alpha Importins in HeLa cells. Down-regulation of Importins alpha3, alpha5, alpha7, and beta strongly inhibited HeLa cell proliferation, while down-regulation of Importins alpha1 and alpha4 had only a minor effect or no effect. Nucleoplasmin import was not prevented by down-regulation of any alpha Importin, indicating that the Importin alpha/beta pathway was generally not affected. In contrast, Importin alpha3 or alpha5 down-regulation specifically inhibited the nuclear import of the Ran guanine nucleotide exchange factor, RCC1. Coinjection of recombinant alpha Importins and RCC1 into down-regulated cells demonstrated that these transport defects were specifically caused by the limited availability of Importin alpha3 in both cases. Thus, Importin alpha3 is the only alpha Importin responsible for the classical nuclear import of RCC1 in living cells.
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Axoplasmic Importins enable retrograde injury signaling in lesioned nerve
Neuron, 2003Co-Authors: Shlomit Hanz, Matthias Kohler, Eran Perlson, Dianna E. Willis, Jun-qi Zheng, Rada Massarwa, Juan J. Huerta, Martin Koltzenburg, Jan Van-minnen, Jeffery L. TwissAbstract:Axoplasmic proteins containing nuclear localization signals (NLS) signal retrogradely by an unknown mechanism in injured nerve. Here we demonstrate that the Importin/karyopherin alpha and beta families underlie this process. We show that Importins are found in axons at significant distances from the cell body and that Importin beta protein is increased after nerve lesion by local translation of axonal mRNA. This leads to formation of a high-affinity NLS binding complex that traffics retrogradely with the motor protein dynein. Trituration of synthetic NLS peptide at the injury site of axotomized dorsal root ganglion (DRG) neurons delays their regenerative outgrowth, and NLS introduction to sciatic nerve concomitantly with a crush injury suppresses the conditioning lesion induced transition from arborizing to elongating growth in L4/L5 DRG neurons. These data suggest a model whereby lesion-induced upregulation of axonal Importin beta may enable retrograde transport of signals that modulate the regeneration of injured neurons.
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axoplasmic Importins enable retrograde injury signaling in lesioned nerve
Neuron, 2003Co-Authors: Shlomit Hanz, Matthias Kohler, Eran Perlson, Dianna E. Willis, Jun-qi Zheng, Rada Massarwa, Juan J. Huerta, Martin Koltzenburg, Jan Vanminnen, Jeffery L. TwissAbstract:Axoplasmic proteins containing nuclear localization signals (NLS) signal retrogradely by an unknown mechanism in injured nerve. Here we demonstrate that the Importin/karyopherin α and β families underlie this process. We show that Importins are found in axons at significant distances from the cell body and that Importin β protein is increased after nerve lesion by local translation of axonal mRNA. This leads to formation of a high-affinity NLS binding complex that traffics retrogradely with the motor protein dynein. Trituration of synthetic NLS peptide at the injury site of axotomized dorsal root ganglion (DRG) neurons delays their regenerative outgrowth, and NLS introduction to sciatic nerve concomitantly with a crush injury suppresses the conditioning lesion induced transition from arborizing to elongating growth in L4/L5 DRG neurons. These data suggest a model whereby lesion-induced upregulation of axonal Importin β may enable retrograde transport of signals that modulate the regeneration of injured neurons.
Enno Hartmann - One of the best experts on this subject based on the ideXlab platform.
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nuclear translocation of hypoxia inducible factors hifs involvement of the classical Importin α β pathway
Biochimica et Biophysica Acta, 2008Co-Authors: Reinhard Depping, Beate Friedrich, Enno Hartmann, Susann G Schindler, Amrei Steinhoff, Riku Fagerlund, Eric Metzen, Matthias KohlerAbstract:Hypoxia-inducible factors are the key elements in the essential process of oxygen homeostasis of vertebrate cells. Stabilisation and subsequent nuclear localisation of HIF-alpha subunits results in the activation of target genes such as vegf, epo and glut1. The passage of transcription factors e.g. HIF-1alpha into the nucleus through the nuclear pore complex is regulated by nuclear transport receptors. Therefore nucleocytoplasmic shuttling can regulate transcriptional activity by facilitating the cellular traffic of transcription factors between both compartments. Here, we report on the identification of specific interactions of hypoxia-inducible factors with nuclear transport receptors Importin alpha/beta. HIF-1alpha, -1beta, and HIF-2alpha are binding to Importin alpha1, alpha3, alpha5, and alpha7. The direct interaction of HIF-1alpha to alpha Importins is dependent on a functional nuclear localisation signal within the C-terminal region of the protein. In contrast, the supposed N-terminal NLS is not effective. Our findings provide new insight into the mechanism of the regulation of nuclear transport of hypoxia-inducible factors.
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In Vivo Analysis of Importin α Proteins Reveals Cellular Proliferation Inhibition and Substrate Specificity
Molecular and Cellular Biology, 2004Co-Authors: Christina Quensel, Beate Friedrich, Thomas Sommer, Enno Hartmann, Matthias KohlerAbstract:Macromolecule transport between cell cytosol and nucleus takes place through the nuclear pore complexes (NPC) (2, 41). Import substrates possess nuclear localization signals (NLS), required for recognition by distinct nuclear import factors. The so-called classical nucleocytoplasmic import pathway is mediated by the Importin α/β heterodimer, also known as karyopherin α/β (for reviews, see references 23, 34, and 43). Importin α acts as an adapter by binding both the import substrate and Importin β. The trimeric import complex docks to the NPC via Importin β and translocates into the nucleus. Recently, Npap60/Nup50 was identified as an additional mammalian cofactor for Importin α/β-dependent nuclear protein import (22). While only one Importin β isoform exists, six human α Importins have been described (3, 4, 18, 21, 28, 36, 42). In contrast, the yeast Saccharomyces cerevisiae possesses only one gene for Importin α, which is essential (45). The α Importins are grouped into three subfamilies based on sequence homology. The first subfamily consists of Importin α1/Rch1. Its most closely related homologue, Importin α2, has been found in Xenopus laevis and other vertebrates but not in mammals. Importins α3/Qip1 and α4/hSRP1γ are members of the second subfamily. The third subfamily consists of Importins α5/hSRP1, α6, and α7 (18). The isoforms of one subfamily are highly homologous, showing about 85% sequence identity and differing mostly in regions outside the NLS binding pockets (18, 21, 26). Although the α Importins differ in their cell- and tissue-specific expression patterns, most are expressed ubiquitously (16, 18, 20, 21, 28, 31, 40). Only Importin α6 expression seems to be restricted to the testis (21). The reason for the Importin α diversity in higher eukaryotes, especially in humans and other vertebrates, is unknown. Invertebrates such as Caenorhabditis elegans and Drosophila melanogaster have three Importin α isoforms with about 50% homology to each other. Knockout and knockdown experiments targeting distinct Importin α genes have resulted in severe phenotypes. These studies suggest that Importin α3 is required during development, and it seems to be essential for oogenesis in Drosophila (25) and C. elegans (8). In contrast, Gorjanacz et al. and Mason et al. found that Drosophila Importin α2 (homologue to human Importin α1) seems to be essential for gametogenesis (10, 24). The Adam and the Mattaj laboratories showed the importance of C. elegans α Importins IMA-3 (8) and IMA-2 (1, 9) for embryonic development. Vertebrates as well as humans possess more than three different α Importins. Several in vitro studies have shown that the individual human α Importins are able to import the same target proteins. This state of affairs might argue against specialized roles of distinct α Importins in human cells. Interestingly, many of these studies also provided in vitro evidence that the α Importins differ in their substrate-specific import efficiency (7, 18, 19, 27, 28, 37, 44). Whether or not these results allow conclusions for living cells is unclear. Protein concentrations in these import assays are different from those in vivo, and probably the most important factor is the lack of competing substrates. Two competing substrates added simultaneously in the in vitro assays can change the import capacity of α Importins (18). Whether α Importins can substitute for one another in vivo is still unknown. To address this issue, we conducted a systematic study of the effects of specific α Importin down-regulation in cultured human cells.
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In Vivo Analysis of Importin α Proteins Reveals Cellular Proliferation Inhibition and Substrate Specificity
Molecular and cellular biology, 2004Co-Authors: Christina Quensel, Beate Friedrich, Thomas Sommer, Enno Hartmann, Matthias KohlerAbstract:The "classical" nuclear import pathway depends on Importins alpha and beta. Humans have only one Importin beta, while six alpha Importins have been described. Whether or not distinct alpha Importins are essential for specific import pathways in living human cells is unclear. We used RNA interference technology to specifically down-regulate the expression of ubiquitously expressed human alpha Importins in HeLa cells. Down-regulation of Importins alpha3, alpha5, alpha7, and beta strongly inhibited HeLa cell proliferation, while down-regulation of Importins alpha1 and alpha4 had only a minor effect or no effect. Nucleoplasmin import was not prevented by down-regulation of any alpha Importin, indicating that the Importin alpha/beta pathway was generally not affected. In contrast, Importin alpha3 or alpha5 down-regulation specifically inhibited the nuclear import of the Ran guanine nucleotide exchange factor, RCC1. Coinjection of recombinant alpha Importins and RCC1 into down-regulated cells demonstrated that these transport defects were specifically caused by the limited availability of Importin alpha3 in both cases. Thus, Importin alpha3 is the only alpha Importin responsible for the classical nuclear import of RCC1 in living cells.
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Importins fulfil a dual function as nuclear import receptors and cytoplasmic chaperones for exposed basic domains
The EMBO Journal, 2002Co-Authors: Stefan Jakel, Enno Hartmann, Josemanuel Mingot, Petra Schwarzmaier, Dirk GorlichAbstract:Many nuclear transport pathways are mediated by Importin β-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 Impβ and their respective basic import substrates. The Impβ–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.
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Increased Importin α protein expression in diabetic nephropathy
Kidney international, 2001Co-Authors: Matthias Kohler, Enno Hartmann, Igor B. Buchwalow, Gabriele Alexander, Marret Christiansen, Erdenechimeg Shagdarsuren, Vera Samoilova, Eero Mervaala, Hermann HallerAbstract:Increased Importin α protein expression in diabetic nephropathy. Background Importins transport kinases, transcription factors, and viral proteins into the nucleus. Since the expression of several genes is increased in diabetic nephropathy, we tested the hypothesis that Importin protein expression is increased in diabetic kidneys. Methods Immunohistochemistry and Western blotting were used in kidneys from streptozotocin-treated diabetic rats and from spontaneously diabetic Goto-Kakizaki rats. The effects of high glucose and mannose also were tested in cell culture experiments. Results In normal rat kidneys, Importin α isoforms were differentially expressed in glomerular cells and tubular segments, while Importin α1/Rch1 was expressed only in tubules and peritubular cells. In diabetic rat kidneys from both models, the Importin α isoform expression was markedly up-regulated. Western blotting revealed strong up-regulation of Importin α7 and minor up-regulation of other isoforms. Exposure of various cell types to high glucose or mannose (25 mmol/L) led to increased expression of Importins α3, α5/hSRP1, and α7 in different cultured cells, while up-regulation of other Importin α isoforms was less consistent. Conclusions A specific Importin α isoform up-regulation takes place in kidneys of diabetic rats. Diabetes is a stimulus for increased Importin α7 expression. Thus, nuclear transport in diabetes may be increased in glomerular and tubular cells. The signaling pathways appear differentially regulated in glomeruli, proximal, and distal tubules. The enhanced nuclear transport may participate in increased gene expression and nephrosclerosis in diabetes.
David A. Jans - One of the best experts on this subject based on the ideXlab platform.
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nuclear localization of the dystrophin associated protein α dystrobrevin through Importin α2 β1 is critical for interaction with the nuclear lamina maintenance of nuclear integrity
The FASEB Journal, 2015Co-Authors: Areli Aguilar, David A. Jans, Kylie M. Wagstaff, Samuel Zinker, Rocio Suarezsanchez, Bulmaro CisnerosAbstract:Although α-dystrobrevin (DB) is assembled into the dystrophin-associated protein complex, which is central to cytoskeletal organization, it has also been found in the nucleus. Here we delineate the nuclear import pathway responsible for nuclear targeting of α-DB for the first time, together with the importance of nuclear α-DB in determining nuclear morphology. We map key residues of the nuclear localization signal of α-DB within the zinc finger domain (ZZ) using various truncated versions of the protein, and site-directed mutagenesis. Pulldown, immunoprecipitation, and AlphaScreen assays showed that the Importin (IMP) α2/β1 heterodimer interacts with high affinity with the ZZ domain of α-DB. In vitro nuclear import assays using antibodies to specific Importins, as well as in vivo studies using siRNA or a dominant negative Importin construct, confirmed the key role of IMPα2/β1 in α-DB nuclear translocation. Knockdown of α-DB expression perturbed cell cycle progression in C2C12 myoblasts, with decreased acc...
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Nuclear localization of the dystrophin‐associated protein α‐dystrobrevin through Importin α2/β1 is critical for interaction with the nuclear lamina/maintenance of nuclear integrity
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2015Co-Authors: Areli Aguilar, David A. Jans, Kylie M. Wagstaff, Rocío Suárez-sánchez, Samuel Zinker, Bulmaro CisnerosAbstract:Although α-dystrobrevin (DB) is assembled into the dystrophin-associated protein complex, which is central to cytoskeletal organization, it has also been found in the nucleus. Here we delineate the nuclear import pathway responsible for nuclear targeting of α-DB for the first time, together with the importance of nuclear α-DB in determining nuclear morphology. We map key residues of the nuclear localization signal of α-DB within the zinc finger domain (ZZ) using various truncated versions of the protein, and site-directed mutagenesis. Pulldown, immunoprecipitation, and AlphaScreen assays showed that the Importin (IMP) α2/β1 heterodimer interacts with high affinity with the ZZ domain of α-DB. In vitro nuclear import assays using antibodies to specific Importins, as well as in vivo studies using siRNA or a dominant negative Importin construct, confirmed the key role of IMPα2/β1 in α-DB nuclear translocation. Knockdown of α-DB expression perturbed cell cycle progression in C2C12 myoblasts, with decreased acc...
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nuclear import and export inhibitors alter capsid protein distribution in mammalian cells and reduce venezuelan equine encephalitis virus replication
Antiviral Research, 2013Co-Authors: Lindsay Lundberg, David A. Jans, Kylie M. Wagstaff, Chelsea Pinkham, Alan Baer, Moushimi Amaya, Aarthi Narayanan, Kylene KehnhallAbstract:Targeting host responses to invading viruses has been the focus of recent antiviral research. Venezuelan Equine Encephalitis Virus (VEEV) is able to modulate host transcription and block nuclear trafficking at least partially due to its capsid protein forming a complex with the host proteins Importin α/β1 and CRM1. We hypothesized that disrupting the interaction of capsid with Importin α/β1 or the interaction of capsid with CRM1 would alter capsid localization, thereby lowering viral titers in vitro. siRNA mediated knockdown of Importin α, Importin β1, and CRM1 altered capsid localization, confirming their role in modulating capsid trafficking. Mifepristone and ivermectin, inhibitors of Importin α/β-mediated import, were able to reduce nuclear-associated capsid, while leptomycin B, a potent CRM1 inhibitor, confined capsid to the nucleus. In addition to altering the level and distribution of capsid, the three inhibitors were able to reduce viral titers in a relevant mammalian cell line with varying degrees of efficacy. The inhibitors were also able to reduce the cytopathic effects associated with VEEV infection, hinting that nuclear import inhibitors may be protecting cells from apoptosis in addition to disrupting the function of an essential viral protein. Our results confirm that VEEV uses host Importins and exportins during part of its life cycle. Further, it suggests that temporarily targeting host proteins that are hijacked for use by viruses is a viable antiviral therapy.
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Changing subcellular localization of nuclear transport factors during human spermatogenesis.
International journal of andrology, 2011Co-Authors: Penelope Alexandra Falshaw Whiley, David A. Jans, Yoichi Miyamoto, Robert I Mclachlan, Kate L LovelandAbstract:Spermatogenesis requires progressive changes in gene expression mediated by hormonal and local factors. Regulated macromolecular movement between nuclear and cytoplasmic compartments enables these essential responses to changing extracellular cues, and dynamic production of the nucleocytoplasmic transporters and Importin proteins, throughout gametogenesis in rodents implicates them as key mediators of germline differentiation. We examined normal adult human testis expression profiles of six Importins plus five additional proteins involved in nucleocytoplasmic transport. Although most were detected in the nucleus during germline differentiation, Importin α4 was exclusively observed in Sertoli and germ cell cytoplasm. Many proteins were present in round spermatid nuclei (Importins α1, α3, β1, β3; exportin-1, Nup62, Ran, RanBP1, RCC1), and remarkable intense nuclear and/or nuclear-associated signals were detected for Importin α1, Importin α3 and Nup62 in spermatocytes. This study identifies conserved aspects of nucleocytoplasmic transport during spermatogenesis and extends our knowledge of the dynamic presence of these proteins, which indicates that they contribute to germ cell-specific cargo trafficking and potentially to other functions during human spermatogenesis. We also demonstrate for the first time that Importin α3 is nuclear in spermatocytes, when exportin-1 is cytoplasmic, suggesting that nuclear transport is altered during meiosis.
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Subcellular distribution of Importins correlates with germ cell maturation.
Developmental dynamics : an official publication of the American Association of Anatomists, 2007Co-Authors: Cathryn A. Hogarth, David A. Jans, Kate L LovelandAbstract:Importin proteins regulate access to the nucleus by recognizing and transporting distinct cargo proteins. Building on studies in Drosophila and Caenorhabditis elegans, we hypothesized that regulated expression and subcellular localization of specific Importins may be linked to mammalian gonadal differentiation. We identified distinct developmental and cellular localization patterns for Importins β1, α3, α4 and RanBP5 (Importin β3) in fetal and postnatal murine testes using Western blotting and immunohistochemistry. Importin β1 protein is detected in selected germ and somatic cells in fetal gonads, with a striking perinuclear staining evident from embryonic day (E) 14.5 within testicular gonocytes. RanBP5 exhibits age- and gender-specific subcellular localization within fetal gonads. At E12.5, RanBP5 protein is cytoplasmic in gonocytes but predominantly nuclear in oogonia, but by E14.5 RanBP5 appears nuclear in gonocytes and cytoplasmic in oogonia. In postnatal testes, Importin α3 and α4 in spermatocytes, spermatids, and Sertoli cells display cytoplasmic and nuclear localization, respectively. Developmental Dynamics 236: 2311–2320, 2007.© 2007 Wiley-Liss, Inc.
Christina Quensel - One of the best experts on this subject based on the ideXlab platform.
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In Vivo Analysis of Importin α Proteins Reveals Cellular Proliferation Inhibition and Substrate Specificity
Molecular and Cellular Biology, 2004Co-Authors: Christina Quensel, Beate Friedrich, Thomas Sommer, Enno Hartmann, Matthias KohlerAbstract:Macromolecule transport between cell cytosol and nucleus takes place through the nuclear pore complexes (NPC) (2, 41). Import substrates possess nuclear localization signals (NLS), required for recognition by distinct nuclear import factors. The so-called classical nucleocytoplasmic import pathway is mediated by the Importin α/β heterodimer, also known as karyopherin α/β (for reviews, see references 23, 34, and 43). Importin α acts as an adapter by binding both the import substrate and Importin β. The trimeric import complex docks to the NPC via Importin β and translocates into the nucleus. Recently, Npap60/Nup50 was identified as an additional mammalian cofactor for Importin α/β-dependent nuclear protein import (22). While only one Importin β isoform exists, six human α Importins have been described (3, 4, 18, 21, 28, 36, 42). In contrast, the yeast Saccharomyces cerevisiae possesses only one gene for Importin α, which is essential (45). The α Importins are grouped into three subfamilies based on sequence homology. The first subfamily consists of Importin α1/Rch1. Its most closely related homologue, Importin α2, has been found in Xenopus laevis and other vertebrates but not in mammals. Importins α3/Qip1 and α4/hSRP1γ are members of the second subfamily. The third subfamily consists of Importins α5/hSRP1, α6, and α7 (18). The isoforms of one subfamily are highly homologous, showing about 85% sequence identity and differing mostly in regions outside the NLS binding pockets (18, 21, 26). Although the α Importins differ in their cell- and tissue-specific expression patterns, most are expressed ubiquitously (16, 18, 20, 21, 28, 31, 40). Only Importin α6 expression seems to be restricted to the testis (21). The reason for the Importin α diversity in higher eukaryotes, especially in humans and other vertebrates, is unknown. Invertebrates such as Caenorhabditis elegans and Drosophila melanogaster have three Importin α isoforms with about 50% homology to each other. Knockout and knockdown experiments targeting distinct Importin α genes have resulted in severe phenotypes. These studies suggest that Importin α3 is required during development, and it seems to be essential for oogenesis in Drosophila (25) and C. elegans (8). In contrast, Gorjanacz et al. and Mason et al. found that Drosophila Importin α2 (homologue to human Importin α1) seems to be essential for gametogenesis (10, 24). The Adam and the Mattaj laboratories showed the importance of C. elegans α Importins IMA-3 (8) and IMA-2 (1, 9) for embryonic development. Vertebrates as well as humans possess more than three different α Importins. Several in vitro studies have shown that the individual human α Importins are able to import the same target proteins. This state of affairs might argue against specialized roles of distinct α Importins in human cells. Interestingly, many of these studies also provided in vitro evidence that the α Importins differ in their substrate-specific import efficiency (7, 18, 19, 27, 28, 37, 44). Whether or not these results allow conclusions for living cells is unclear. Protein concentrations in these import assays are different from those in vivo, and probably the most important factor is the lack of competing substrates. Two competing substrates added simultaneously in the in vitro assays can change the import capacity of α Importins (18). Whether α Importins can substitute for one another in vivo is still unknown. To address this issue, we conducted a systematic study of the effects of specific α Importin down-regulation in cultured human cells.
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In Vivo Analysis of Importin α Proteins Reveals Cellular Proliferation Inhibition and Substrate Specificity
Molecular and cellular biology, 2004Co-Authors: Christina Quensel, Beate Friedrich, Thomas Sommer, Enno Hartmann, Matthias KohlerAbstract:The "classical" nuclear import pathway depends on Importins alpha and beta. Humans have only one Importin beta, while six alpha Importins have been described. Whether or not distinct alpha Importins are essential for specific import pathways in living human cells is unclear. We used RNA interference technology to specifically down-regulate the expression of ubiquitously expressed human alpha Importins in HeLa cells. Down-regulation of Importins alpha3, alpha5, alpha7, and beta strongly inhibited HeLa cell proliferation, while down-regulation of Importins alpha1 and alpha4 had only a minor effect or no effect. Nucleoplasmin import was not prevented by down-regulation of any alpha Importin, indicating that the Importin alpha/beta pathway was generally not affected. In contrast, Importin alpha3 or alpha5 down-regulation specifically inhibited the nuclear import of the Ran guanine nucleotide exchange factor, RCC1. Coinjection of recombinant alpha Importins and RCC1 into down-regulated cells demonstrated that these transport defects were specifically caused by the limited availability of Importin alpha3 in both cases. Thus, Importin alpha3 is the only alpha Importin responsible for the classical nuclear import of RCC1 in living cells.
Beate Friedrich - One of the best experts on this subject based on the ideXlab platform.
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nuclear translocation of hypoxia inducible factors hifs involvement of the classical Importin α β pathway
Biochimica et Biophysica Acta, 2008Co-Authors: Reinhard Depping, Beate Friedrich, Enno Hartmann, Susann G Schindler, Amrei Steinhoff, Riku Fagerlund, Eric Metzen, Matthias KohlerAbstract:Hypoxia-inducible factors are the key elements in the essential process of oxygen homeostasis of vertebrate cells. Stabilisation and subsequent nuclear localisation of HIF-alpha subunits results in the activation of target genes such as vegf, epo and glut1. The passage of transcription factors e.g. HIF-1alpha into the nucleus through the nuclear pore complex is regulated by nuclear transport receptors. Therefore nucleocytoplasmic shuttling can regulate transcriptional activity by facilitating the cellular traffic of transcription factors between both compartments. Here, we report on the identification of specific interactions of hypoxia-inducible factors with nuclear transport receptors Importin alpha/beta. HIF-1alpha, -1beta, and HIF-2alpha are binding to Importin alpha1, alpha3, alpha5, and alpha7. The direct interaction of HIF-1alpha to alpha Importins is dependent on a functional nuclear localisation signal within the C-terminal region of the protein. In contrast, the supposed N-terminal NLS is not effective. Our findings provide new insight into the mechanism of the regulation of nuclear transport of hypoxia-inducible factors.
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In Vivo Analysis of Importin α Proteins Reveals Cellular Proliferation Inhibition and Substrate Specificity
Molecular and Cellular Biology, 2004Co-Authors: Christina Quensel, Beate Friedrich, Thomas Sommer, Enno Hartmann, Matthias KohlerAbstract:Macromolecule transport between cell cytosol and nucleus takes place through the nuclear pore complexes (NPC) (2, 41). Import substrates possess nuclear localization signals (NLS), required for recognition by distinct nuclear import factors. The so-called classical nucleocytoplasmic import pathway is mediated by the Importin α/β heterodimer, also known as karyopherin α/β (for reviews, see references 23, 34, and 43). Importin α acts as an adapter by binding both the import substrate and Importin β. The trimeric import complex docks to the NPC via Importin β and translocates into the nucleus. Recently, Npap60/Nup50 was identified as an additional mammalian cofactor for Importin α/β-dependent nuclear protein import (22). While only one Importin β isoform exists, six human α Importins have been described (3, 4, 18, 21, 28, 36, 42). In contrast, the yeast Saccharomyces cerevisiae possesses only one gene for Importin α, which is essential (45). The α Importins are grouped into three subfamilies based on sequence homology. The first subfamily consists of Importin α1/Rch1. Its most closely related homologue, Importin α2, has been found in Xenopus laevis and other vertebrates but not in mammals. Importins α3/Qip1 and α4/hSRP1γ are members of the second subfamily. The third subfamily consists of Importins α5/hSRP1, α6, and α7 (18). The isoforms of one subfamily are highly homologous, showing about 85% sequence identity and differing mostly in regions outside the NLS binding pockets (18, 21, 26). Although the α Importins differ in their cell- and tissue-specific expression patterns, most are expressed ubiquitously (16, 18, 20, 21, 28, 31, 40). Only Importin α6 expression seems to be restricted to the testis (21). The reason for the Importin α diversity in higher eukaryotes, especially in humans and other vertebrates, is unknown. Invertebrates such as Caenorhabditis elegans and Drosophila melanogaster have three Importin α isoforms with about 50% homology to each other. Knockout and knockdown experiments targeting distinct Importin α genes have resulted in severe phenotypes. These studies suggest that Importin α3 is required during development, and it seems to be essential for oogenesis in Drosophila (25) and C. elegans (8). In contrast, Gorjanacz et al. and Mason et al. found that Drosophila Importin α2 (homologue to human Importin α1) seems to be essential for gametogenesis (10, 24). The Adam and the Mattaj laboratories showed the importance of C. elegans α Importins IMA-3 (8) and IMA-2 (1, 9) for embryonic development. Vertebrates as well as humans possess more than three different α Importins. Several in vitro studies have shown that the individual human α Importins are able to import the same target proteins. This state of affairs might argue against specialized roles of distinct α Importins in human cells. Interestingly, many of these studies also provided in vitro evidence that the α Importins differ in their substrate-specific import efficiency (7, 18, 19, 27, 28, 37, 44). Whether or not these results allow conclusions for living cells is unclear. Protein concentrations in these import assays are different from those in vivo, and probably the most important factor is the lack of competing substrates. Two competing substrates added simultaneously in the in vitro assays can change the import capacity of α Importins (18). Whether α Importins can substitute for one another in vivo is still unknown. To address this issue, we conducted a systematic study of the effects of specific α Importin down-regulation in cultured human cells.
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In Vivo Analysis of Importin α Proteins Reveals Cellular Proliferation Inhibition and Substrate Specificity
Molecular and cellular biology, 2004Co-Authors: Christina Quensel, Beate Friedrich, Thomas Sommer, Enno Hartmann, Matthias KohlerAbstract:The "classical" nuclear import pathway depends on Importins alpha and beta. Humans have only one Importin beta, while six alpha Importins have been described. Whether or not distinct alpha Importins are essential for specific import pathways in living human cells is unclear. We used RNA interference technology to specifically down-regulate the expression of ubiquitously expressed human alpha Importins in HeLa cells. Down-regulation of Importins alpha3, alpha5, alpha7, and beta strongly inhibited HeLa cell proliferation, while down-regulation of Importins alpha1 and alpha4 had only a minor effect or no effect. Nucleoplasmin import was not prevented by down-regulation of any alpha Importin, indicating that the Importin alpha/beta pathway was generally not affected. In contrast, Importin alpha3 or alpha5 down-regulation specifically inhibited the nuclear import of the Ran guanine nucleotide exchange factor, RCC1. Coinjection of recombinant alpha Importins and RCC1 into down-regulated cells demonstrated that these transport defects were specifically caused by the limited availability of Importin alpha3 in both cases. Thus, Importin alpha3 is the only alpha Importin responsible for the classical nuclear import of RCC1 in living cells.
