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Larry Gerace - One of the best experts on this subject based on the ideXlab platform.
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A GTPase Distinct from Ran Is Involved in Nuclear Protein Import
2013Co-Authors: Deborah J. Sweet, Larry GeraceAbstract:Abstract. Signal-dependent transport of Proteins into the nucleus is a multi-step process mediated by Nuclear pore complexes and cytosolic transport factors. One of the cytosolic factors, Ran, is the only GTPase that has a characterized role in the Nuclear import pathway. We have used a mutant form of Ran with altered nucleotide binding specificity to investigate whether any other GTPases are involved in Nuclear Protein import. D125N Ran (XTP-Ran) binds specifically to xanthosine triphosphate (XTP) and has a greatly reduced affinity for GTP, so it is no longer sensitive to inhibition by nonhydrolyzable analogues of GTP such as guanosine 5'-O-(3-thiotriphosphate) (GTP~/S). Using in vitro transport assays, we have found that Nuclear import supported by XTP-Ran is nevertheless inhibited by the addition of non-hydrolyzable GTP analogues. This in conjunction with the properties of the inhibitory effect indicates that at least one additional GTPase is involved in the import process. Initial characterization suggests that the inhibited GTPase plays a direct role in Protein import and could be a component of the Nuclear pore complex. M OLECULAR transport between the nucleus and cytoplasm plays a fundamental role in eukaryotic cell metabolism. It takes place through Nuclear pore complexes (NPCs) 1, large (~125 MD) supramolecular structures that span the Nuclear envelope (for reviews se
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Nuclear Protein import is decreased by engineered mutants of Nuclear transport factor 2 ntf2 that do not bind gdp ran
Journal of Molecular Biology, 1997Co-Authors: W D Clarkson, Pamela A. Silver, Larry Gerace, Anita H Corbett, Bryce M Paschal, Helen M Kent, Airlie J Mccoy, Murray StewartAbstract:Nuclear transport factor 2 (NTF2) is associated with the translocation stage of Nuclear Protein import and binds both to Nuclear pore Proteins (nucleoporins) containing phenylalanine-rich repeats and to the Ras family GTPase Ran. In this study we probed the role of the NTF2-Ran interaction in Nuclear Protein import using site-directed mutants of NTF2 that interfere with its interaction with GDP-Ran. The design of these mutants was based on the X-ray crystal structure of NTF2 and was concentrated on conserved residues in and around the molecule’s hydrophobic cavity. The mutant NTF2 cDNAs were expressed in Escherichia coli. Purified mutant Proteins retained the interaction with FxFG-repeat nucleoporins, but several mutants in the negatively charged residues that surround the NTF2 cavity or in residues in the cavity itself were unable to bind GDP-Ran in vitro. The crystal structure of the E42K mutant Protein showed significant structural changes only in this side-chain, indicating that it participated directly in the interaction with GDP-Ran. In permeabilised cell Nuclear Protein import assays, only wild-type NTF2 and mutants that bound GDP-Ran were functional. Furthermore, when the NTF2 E42K and D92N/D94N NTF2 mutants that failed to bind GDP-Ran in vitro were substituted for the chromosomal yeast NTF2, the yeast cells became non-viable, whereas yeast substituted with wild-type human NTF2 remained viable. We conclude that interaction between NTF2 and GDP-Ran is important for efficient Nuclear Protein import.
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high levels of the gtpase ran tc4 relieve the requirement for Nuclear Protein transport factor 2
Journal of Biological Chemistry, 1997Co-Authors: Bryce M Paschal, Christian Fritze, Tinglu Guan, Larry GeraceAbstract:The GTPase Ran/TC4 and the 14-kDa Protein Nuclear transport factor 2 (NTF2) are two of the cytosolic factors that mediate Nuclear Protein import in vertebrates. Previous biochemical studies have shown that NTF2 binds directly to the GDP-bound form of Ran/TC4 and to Proteins of the Nuclear pore complex that contain phenylalanine-glycine repeats. In the present study we have used molecular genetic approaches to study the Saccharomyces cerevisiae homologue of NTF2. The scNTF2 gene encodes a Protein that is 44% identical to the human Protein. We found that deletion of the scNTF2 gene is lethal and that repression of NTF2p expression by a regulatable promoter results in gross structural distortions of the Nuclear envelope. In a screen for high copy number suppressors of a scNTF2 deletion, the only gene we isolated other than scNTF2 itself was GSP1, the S. cerevisiae homologue of Ran/TC4. Furthermore, we found that high levels of Ran/TC4 can relieve the requirement for NTF2 in a mammalian-permeabilized cell assay for Nuclear Protein import. These data suggest that certain of the Nuclear Protein import functions of NTF2 and Ran/TC4 are closely linked and that NTF2 may serve to modulate a transport step involving Ran/TC4.
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a gtpase distinct from ran is involved in Nuclear Protein import
Journal of Cell Biology, 1996Co-Authors: Deborah Sweet, Larry GeraceAbstract:Signal-dependent transport of Proteins into the nucleus is a multi-step process mediated by Nuclear pore complexes and cytosolic transport factors. One of the cytosolic factors, Ran, is the only GTPase that has a characterized role in the Nuclear import pathway. We have used a mutant form of Ran with altered nucleotide binding specificity to investigate whether any other GTPases are involved in Nuclear Protein import. D125N Ran (XTP-Ran) binds specifically to xanthosine triphosphate (XTP) and has a greatly reduced affinity for GTP, so it is no longer sensitive to inhibition by nonhydrolyzable analogues of GTP such as guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). using in vitro transport assays, we have found that Nuclear import supported by XTP-Ran is nevertheless inhibited by the addition of non-hydrolyzable GTP analogues. This in conjunction with the properties of the inhibitory effect indicates that at least one additional GTPase is involved in the import process. Initial characterization suggests that the inhibited GTPase plays a direct role in Protein import and could be a component of the Nuclear pore complex.
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mechanisms of Nuclear Protein import
Current Opinion in Cell Biology, 1995Co-Authors: Frauke Melchior, Larry GeraceAbstract:Abstract The past two years have seen a significant increase in our understanding of Nuclear Protein import. Five cytosolic import factors have been identified, two of which have been shown to directly interact with components of the Nuclear pore complex. These findings enable refinement of previous models for steps in the Nuclear import pathway, and provide a framework for future research.
Bryce M Paschal - One of the best experts on this subject based on the ideXlab platform.
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analysis of Nuclear Protein import and export in digitonin permeabilized cells
Cell Biology (Third Edition)#R##N#A Laboratory Handbook, 2006Co-Authors: Ralph H Kehlenbach, Bryce M PaschalAbstract:Publisher Summary This chapter describes Nuclear Protein import and export in digitonin-permeabilized cells. Transport between the nucleus and the cytoplasm is mediated by Nuclear pore complexes (NPCs), specialized channels that are embedded in the Nuclear envelope membrane. Proteins that undergo Nuclear import or Nuclear export usually encode a Nuclear localization signal (NLS) or a Nuclear export signal (NES). The Nuclear export assay is described for HeLa cells expressing a GFP-tagged reporter Protein, the Nuclear factor of activated T cells (GFP-NFAT). NFAT is a transcription factor that contains defined Nuclear localization and Nuclear export signals and that shuttles between the cytoplasm and the nucleus in a phosphorylation-dependent manner. The rabbit reticulocyte lysate contains all the soluble factors necessary to reconstitute import and export in digitonin-permeabilized cells. HeLa cell cytosol is generally subjected to a rapid dialysis step before use in transport reactions. Synthetic peptides containing an NLS can be used to direct the Nuclear import of a variety of fluorescent reporter Proteins.
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identification of an ntf2 related factor that binds ran gtp and regulates Nuclear Protein export
Molecular and Cellular Biology, 1999Co-Authors: Ben E Black, Lyne Levesque, James M Holaska, Todd C Wood, Bryce M PaschalAbstract:Active transport of macromolecules between the nucleus and cytoplasm requires signals for import and export and their recognition by shuttling receptors. Each class of macromolecule is thought to have a distinct receptor that mediates the transport reaction. Assembly and disassembly reactions of receptor-substrate complexes are coordinated by Ran, a GTP-binding Protein whose nucleotide state is regulated catalytically by effector Proteins. Ran function is modulated in a noncatalytic fashion by NTF2, a Protein that mediates Nuclear import of Ran-GDP. Here we characterize a novel component of the Ran system that is 26% identical to NTF2, which based on its function we refer to as NTF2-related export Protein 1 (NXT1). In contrast to NTF2, NXT1 preferentially binds Ran-GTP, and it colocalizes with the Nuclear pore complex (NPC) in mammalian cells. These properties, together with the fact that NXT1 shuttles between the nucleus and the cytoplasm, suggest an active role in Nuclear transport. Indeed, NXT1 stimulates Nuclear Protein export of the NES-containing Protein PKI in vitro. The export function of NXT1 is blocked by the addition of leptomycin B, a compound that selectively inhibits the NES receptor Crm1. Thus, NXT1 regulates the Crm1-dependent export pathway through its direct interaction with Ran-GTP.
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Nuclear Protein import is decreased by engineered mutants of Nuclear transport factor 2 ntf2 that do not bind gdp ran
Journal of Molecular Biology, 1997Co-Authors: W D Clarkson, Pamela A. Silver, Larry Gerace, Anita H Corbett, Bryce M Paschal, Helen M Kent, Airlie J Mccoy, Murray StewartAbstract:Nuclear transport factor 2 (NTF2) is associated with the translocation stage of Nuclear Protein import and binds both to Nuclear pore Proteins (nucleoporins) containing phenylalanine-rich repeats and to the Ras family GTPase Ran. In this study we probed the role of the NTF2-Ran interaction in Nuclear Protein import using site-directed mutants of NTF2 that interfere with its interaction with GDP-Ran. The design of these mutants was based on the X-ray crystal structure of NTF2 and was concentrated on conserved residues in and around the molecule’s hydrophobic cavity. The mutant NTF2 cDNAs were expressed in Escherichia coli. Purified mutant Proteins retained the interaction with FxFG-repeat nucleoporins, but several mutants in the negatively charged residues that surround the NTF2 cavity or in residues in the cavity itself were unable to bind GDP-Ran in vitro. The crystal structure of the E42K mutant Protein showed significant structural changes only in this side-chain, indicating that it participated directly in the interaction with GDP-Ran. In permeabilised cell Nuclear Protein import assays, only wild-type NTF2 and mutants that bound GDP-Ran were functional. Furthermore, when the NTF2 E42K and D92N/D94N NTF2 mutants that failed to bind GDP-Ran in vitro were substituted for the chromosomal yeast NTF2, the yeast cells became non-viable, whereas yeast substituted with wild-type human NTF2 remained viable. We conclude that interaction between NTF2 and GDP-Ran is important for efficient Nuclear Protein import.
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high levels of the gtpase ran tc4 relieve the requirement for Nuclear Protein transport factor 2
Journal of Biological Chemistry, 1997Co-Authors: Bryce M Paschal, Christian Fritze, Tinglu Guan, Larry GeraceAbstract:The GTPase Ran/TC4 and the 14-kDa Protein Nuclear transport factor 2 (NTF2) are two of the cytosolic factors that mediate Nuclear Protein import in vertebrates. Previous biochemical studies have shown that NTF2 binds directly to the GDP-bound form of Ran/TC4 and to Proteins of the Nuclear pore complex that contain phenylalanine-glycine repeats. In the present study we have used molecular genetic approaches to study the Saccharomyces cerevisiae homologue of NTF2. The scNTF2 gene encodes a Protein that is 44% identical to the human Protein. We found that deletion of the scNTF2 gene is lethal and that repression of NTF2p expression by a regulatable promoter results in gross structural distortions of the Nuclear envelope. In a screen for high copy number suppressors of a scNTF2 deletion, the only gene we isolated other than scNTF2 itself was GSP1, the S. cerevisiae homologue of Ran/TC4. Furthermore, we found that high levels of Ran/TC4 can relieve the requirement for NTF2 in a mammalian-permeabilized cell assay for Nuclear Protein import. These data suggest that certain of the Nuclear Protein import functions of NTF2 and Ran/TC4 are closely linked and that NTF2 may serve to modulate a transport step involving Ran/TC4.
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inhibition of Nuclear Protein import by nonhydrolyzable analogues of gtp and identification of the small gtpase ran tc4 as an essential transport factor
Journal of Cell Biology, 1993Co-Authors: Frauke Melchior, Bryce M Paschal, Janice Evans, Larry GeraceAbstract:We have investigated a possible involvement of GTPases in Nuclear Protein import using an in vitro transport system involving digitonin-permeabilized cells supplemented with exogenous cytosol. Transport in this system was measured with a novel ELISA-based assay that allows rapid quantitative analysis. GTP gamma S and other nonhydrolyzable analogues of GTP were found to rapidly inhibit the rate of in vitro Nuclear import. Transport inhibition by GTP gamma S was dependent on the concentrations of permeabilized cells and cytosol, and was strongly enhanced by a cytosolic factor(s). The predominant cytosolic component responsible for this inhibition was found in a 20-30-kD fraction in molecular sieving chromatography. Furthermore, a component(s) of this 20-30-kD fraction was itself required for efficient Nuclear import. Biochemical complementation with bacterially expressed Protein demonstrated that this essential GTP gamma S-sensitive transport factor was Ran/TC4, a previously described GTPase of the Ras superfamily found in both nucleus and cytoplasm. Ran/TC4 and its guanine nucleotide release Protein RCC1 have previously been implicated in DNA replication, cell cycle checkpoint control, and RNA synthesis, processing and export. Our results suggest that Ran/TC4 serves to integrate Nuclear Protein import with these other Nuclear activities.
Richard H Goodman - One of the best experts on this subject based on the ideXlab platform.
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Nuclear Protein cbp is a coactivator for the transcription factor creb
Nature, 1994Co-Authors: Roland P S Kwok, John C Chrivia, James R Lundblad, Jane P Richards, Hans Peter Bachinger, Richard G Brennan, Stefan G E Roberts, Michael R Green, Richard H GoodmanAbstract:The transcription factor CREB binds to a DNA element known as the cAMP-regulated enhancer (CRE). CREB is activated through phosphorylation by Protein kinase A (PKA), but precisely how phosphorylation stimulates CREB function is unknown. One model is that phosphorylation may allow the recruitment of coactivators which then interact with basal transcription factors. We have previously identified a Nuclear Protein of M(r)265K, CBP, that binds specifically to the PKA-phosphorylated form of CREB. We have used fluorescence anisotropy measurements to define the equilibrium binding parameters of the phosphoCREB:CBP interaction and report here that CBP can activate transcription through a region in its carboxy terminus. The activation domain of CBP interacts with the basal transcription factor TFIIB through a domain that is conserved in the yeast coactivator ADA-1 (ref. 8). Consistent with its role as a coactivator, CBP augments the activity of phosphorylated CREB to activate transcription of cAMP-responsive genes.
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phosphorylated creb binds specifically to the Nuclear Protein cbp
Nature, 1993Co-Authors: John C Chrivia, Roland P S Kwok, Ned J C Lamb, Masatoshi Hagiwara, Marc Montminy, Richard H GoodmanAbstract:Cyclic AMP-regulated gene expression frequently involves a DNA element known as the cAMP-regulated enhancer (CRE). Many transcription factors bind to this element, including the Protein CREB, which is activated as a result of phosphorylation by Protein kinase A. This modification stimulates interaction with one or more of the general transcription factors or, alternatively, allows recruitment of a co-activator. Here we report that CREB phosphorylated by Protein kinase A binds specifically to a Nuclear Protein of M(r) 265K which we term CBP (for CREB-binding Protein). Fusion of a heterologous DNA-binding domain to the amino terminus of CBP enables the chimaeric Protein to function as a Protein kinase A-regulated transcriptional activator. We propose that CBP may participate in cAMP-regulated gene expression by interacting with the activated phosphorylated form of CREB.
John C Chrivia - One of the best experts on this subject based on the ideXlab platform.
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Nuclear Protein cbp is a coactivator for the transcription factor creb
Nature, 1994Co-Authors: Roland P S Kwok, John C Chrivia, James R Lundblad, Jane P Richards, Hans Peter Bachinger, Richard G Brennan, Stefan G E Roberts, Michael R Green, Richard H GoodmanAbstract:The transcription factor CREB binds to a DNA element known as the cAMP-regulated enhancer (CRE). CREB is activated through phosphorylation by Protein kinase A (PKA), but precisely how phosphorylation stimulates CREB function is unknown. One model is that phosphorylation may allow the recruitment of coactivators which then interact with basal transcription factors. We have previously identified a Nuclear Protein of M(r)265K, CBP, that binds specifically to the PKA-phosphorylated form of CREB. We have used fluorescence anisotropy measurements to define the equilibrium binding parameters of the phosphoCREB:CBP interaction and report here that CBP can activate transcription through a region in its carboxy terminus. The activation domain of CBP interacts with the basal transcription factor TFIIB through a domain that is conserved in the yeast coactivator ADA-1 (ref. 8). Consistent with its role as a coactivator, CBP augments the activity of phosphorylated CREB to activate transcription of cAMP-responsive genes.
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phosphorylated creb binds specifically to the Nuclear Protein cbp
Nature, 1993Co-Authors: John C Chrivia, Roland P S Kwok, Ned J C Lamb, Masatoshi Hagiwara, Marc Montminy, Richard H GoodmanAbstract:Cyclic AMP-regulated gene expression frequently involves a DNA element known as the cAMP-regulated enhancer (CRE). Many transcription factors bind to this element, including the Protein CREB, which is activated as a result of phosphorylation by Protein kinase A. This modification stimulates interaction with one or more of the general transcription factors or, alternatively, allows recruitment of a co-activator. Here we report that CREB phosphorylated by Protein kinase A binds specifically to a Nuclear Protein of M(r) 265K which we term CBP (for CREB-binding Protein). Fusion of a heterologous DNA-binding domain to the amino terminus of CBP enables the chimaeric Protein to function as a Protein kinase A-regulated transcriptional activator. We propose that CBP may participate in cAMP-regulated gene expression by interacting with the activated phosphorylated form of CREB.
Murray Stewart - One of the best experts on this subject based on the ideXlab platform.
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molecular mechanism of the Nuclear Protein import cycle
Nature Reviews Molecular Cell Biology, 2007Co-Authors: Murray StewartAbstract:The Nuclear import of Proteins through Nuclear pore complexes (NPCs) illustrates how a complex biological function can be generated by a spatially and temporally organized cycle of interactions between cargoes, carriers and the Ran GTPase. Recent work has given considerable insight into this process, especially about how interactions are coordinated and the basis for the molecular recognition that underlies the process. Although considerable progress has been made in identifying and characterizing the molecular interactions in the soluble phase that drive the Nuclear Protein import cycle, understanding the precise mechanism of translocation through NPCs remains a major challenge.
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nup50 npap60 function in Nuclear Protein import complex disassembly and importin recycling
The EMBO Journal, 2005Co-Authors: Yoshiyuki Matsuura, Murray StewartAbstract:Nuclear import of Proteins containing classical Nuclear localization signals (NLS) is mediated by the importin-α:β complex that binds cargo in the cytoplasm and facilitates its passage through Nuclear pores, after which Nuclear RanGTP dissociates the import complex and the importins are recycled. In vertebrates, import is stimulated by nucleoporin Nup50, which has been proposed to accompany the import complex through Nuclear pores. However, we show here that the Nup50 N-terminal domain actively displaces NLSs from importin-α, which would be more consistent with Nup50 functioning to coordinate import complex disassembly and importin recycling. The crystal structure of the importin-α:Nup50 complex shows that Nup50 binds at two sites on importin-α. One site overlaps the secondary NLS-binding site, whereas the second extends along the importin-α C-terminus. Mutagenesis indicates that interaction at both sites is required for Nup50 to displace NLSs. The Cse1p:Kap60p:RanGTP complex structure suggests how Nup50 is then displaced on formation of the importin-α export complex. These results provide a rationale for understanding the series of interactions that orchestrate the terminal steps of Nuclear Protein import.
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interaction between ran and mog1 is required for efficient Nuclear Protein import
Journal of Biological Chemistry, 2001Co-Authors: Rosanna P Baker, Michelle T Harreman, John F Eccleston, Anita H Corbett, Murray StewartAbstract:Abstract Mog1 is a Nuclear Protein that interacts with Ran, the Ras family GTPase that confers directionality to Nuclear import and export pathways. Deletion of MOG1 inSaccharomyces cerevisiae (Δmog1) causes temperature-sensitive growth and defects in Nuclear Protein import. Mog1 has previously been shown to stimulate GTP release from Ran and we demonstrate here that addition of Mog1 to either Ran-GTP or Ran-GDP results in nucleotide release and formation of a stable complex between Mog1 and nucleotide-free Ran. Moreover, MOG1 shows synthetic lethality with PRP20, the Ran guanine nucleotide exchange factor (RanGEF) that also binds nucleotide-free Ran. To probe the functional role of the Mog1-Ran interaction, we engineered mutants of yeast Mog1 and Ran that specifically disrupt their interaction bothin vitro and in vivo. These mutants indicate that the interaction interface involves conserved Mog1p residues Asp62 and Glu65, and residue Lys136in yeast Ran. Mutations at these residues decrease the ability of Mog1 to bind and release nucleotide from Ran. Furthermore, the E65K-Mog1 and K136E-Ran mutations in yeast cause temperature sensitivity and mislocalization of a Nuclear import reporter Protein, similar to the phenotype observed for the Δmog1 strain. Our results indicate that a primary function of Mog1 requires binding to Ran and that the Mog1-Ran interaction is necessary for efficient Nuclear Protein import in vivo.
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Nuclear Protein import is decreased by engineered mutants of Nuclear transport factor 2 ntf2 that do not bind gdp ran
Journal of Molecular Biology, 1997Co-Authors: W D Clarkson, Pamela A. Silver, Larry Gerace, Anita H Corbett, Bryce M Paschal, Helen M Kent, Airlie J Mccoy, Murray StewartAbstract:Nuclear transport factor 2 (NTF2) is associated with the translocation stage of Nuclear Protein import and binds both to Nuclear pore Proteins (nucleoporins) containing phenylalanine-rich repeats and to the Ras family GTPase Ran. In this study we probed the role of the NTF2-Ran interaction in Nuclear Protein import using site-directed mutants of NTF2 that interfere with its interaction with GDP-Ran. The design of these mutants was based on the X-ray crystal structure of NTF2 and was concentrated on conserved residues in and around the molecule’s hydrophobic cavity. The mutant NTF2 cDNAs were expressed in Escherichia coli. Purified mutant Proteins retained the interaction with FxFG-repeat nucleoporins, but several mutants in the negatively charged residues that surround the NTF2 cavity or in residues in the cavity itself were unable to bind GDP-Ran in vitro. The crystal structure of the E42K mutant Protein showed significant structural changes only in this side-chain, indicating that it participated directly in the interaction with GDP-Ran. In permeabilised cell Nuclear Protein import assays, only wild-type NTF2 and mutants that bound GDP-Ran were functional. Furthermore, when the NTF2 E42K and D92N/D94N NTF2 mutants that failed to bind GDP-Ran in vitro were substituted for the chromosomal yeast NTF2, the yeast cells became non-viable, whereas yeast substituted with wild-type human NTF2 remained viable. We conclude that interaction between NTF2 and GDP-Ran is important for efficient Nuclear Protein import.
