scholarly journals Kap120 Functions as a Nuclear Import Receptor for Ribosome Assembly Factor Rpf1 in Yeast

2006 ◽  
Vol 26 (8) ◽  
pp. 3170-3180 ◽  
Author(s):  
Stefanie Caesar ◽  
Markus Greiner ◽  
Gabriel Schlenstedt
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Ribosomal Subunits ◽  
Parallel Import ◽  
Β Protein ◽  
Maturation Factor ◽  
Assembly Factor

ABSTRACT The nucleocytoplasmic exchange of macromolecules is mediated by receptors specialized in passage through the nuclear pore complex. The majority of these receptors belong to the importin β protein family, which has 14 members in Saccharomyces cerevisiae. Nine importins carry various cargos from the cytoplasm into the nucleus, whereas four exportins mediate nuclear export. Kap120 is the only receptor whose transport cargo has not been found previously. Here, we characterize Kap120 as an importin for the ribosome maturation factor Rpf1, which was identified in a two-hybrid screen. Kap120 binds directly to Rpf1 in vitro and is released by Ran-GTP. At least three parallel import pathways exist for Rpf1, since nuclear import is defective in strains with the importins Kap120, Kap114, and Nmd5 deleted. Both kap120 and rpf1 mutants accumulate large ribosomal subunits in the nucleus. The nuclear accumulation of 60S ribosomal subunits in kap120 mutants is abolished upon RPF1 overexpression, indicating that Kap120 does not function in the actual ribosomal export step but rather in import of ribosome maturation factors.

scholarly journals Nuclear Import of IκBα Is Accomplished by a Ran-Independent Transport Pathway

2000 ◽  
Vol 20 (5) ◽  
pp. 1571-1582 ◽  
Author(s):  
Shrikesh Sachdev ◽  
Sriparna Bagchi ◽  
Donna D. Zhang ◽  
Angela C. Mings ◽  
Mark Hannink
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Nuclear Pore ◽  
Transport Pathway ◽  
Repeat Domain ◽  
Nuclear Export Receptor

ABSTRACT The inhibitor of kappa B alpha (IκBα) protein is able to shuttle between the cytoplasm and the nucleus. We have utilized a combination of in vivo and in vitro approaches to provide mechanistic insight into nucleocytoplasmic shuttling by IκBα. IκBα contains multiple functional domains that contribute to shuttling of IκBα between the cytoplasm and the nucleus. Nuclear import of IκBα is mediated by the central ankyrin repeat domain. Similar to previously described nuclear import pathways, nuclear import of IκBα is temperature and ATP dependent and is blocked by a dominant-negative mutant of importin β. However, in contrast to classical nuclear import pathways, nuclear import of IκBα is independent of soluble cytosolic factors and is not blocked by the dominant-negative RanQ69L protein. Nuclear export of IκBα is mediated by an N-terminal nuclear export sequence. Nuclear export of IκBα requires the CRM1 nuclear export receptor and is blocked by the dominant-negative RanQ69L protein. Our results are consistent with a model in which nuclear import of IκBα is mediated through direct interactions with components of the nuclear pore complex, while nuclear export of IκBα is mediated via a CRM1-dependent pathway.

scholarly journals Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran

2000 ◽  
Vol 11 (2) ◽  
pp. 703-719 ◽  
Author(s):  
Susanne M. Steggerda ◽  
Ben E. Black ◽  
Bryce M. Paschal
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Translocation ◽  
Living Cells ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Permeabilized Cells ◽  
Dependent Protein

Nuclear transport factor 2 (NTF2) is a soluble transport protein originally identified by its ability to stimulate nuclear localization signal (NLS)-dependent protein import in digitonin-permeabilized cells. NTF2 has been shown to bind nuclear pore complex proteins and the GDP form of Ran in vitro. Recently, it has been reported that NTF2 can stimulate the accumulation of Ran in digitonin-permeabilized cells. Evidence that NTF2 directly mediates Ran import or that NTF2 is required to maintain the nuclear concentration of Ran in living cells has not been obtained. Here we show that cytoplasmic injection of anti-NTF2 mAbs resulted in a dramatic relocalization of Ran to the cytoplasm. This provides the first evidence that NTF2 regulates the distribution of Ran in vivo. Moreover, anti-NTF2 mAbs inhibited nuclear import of both Ran and NLS-containing protein in vitro, suggesting that NTF2 stimulates NLS-dependent protein import by driving the nuclear accumulation of Ran. We also show that biotinylated NTF2-streptavidin microinjected into the cytoplasm accumulated at the nuclear envelope, indicating that NTF2 can target a binding partner to the nuclear pore complex. Taken together, our data show that NTF2 is an essential regulator of the Ran distribution in living cells and that NTF2-mediated Ran nuclear import is required for NLS-dependent protein import.

scholarly journals Nmd3p Is a Crm1p-Dependent Adapter Protein for Nuclear Export of the Large Ribosomal Subunit

2000 ◽  
Vol 151 (5) ◽  
pp. 1057-1066 ◽  
Author(s):  
Jennifer Hei-Ngam Ho ◽  
George Kallstrom ◽  
Arlen W. Johnson
Keyword(s):  
Nuclear Export ◽  
Ribosomal Subunit ◽  
Specific Inhibitor ◽  
Small Gtpase ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Adapter Protein ◽  
Leptomycin B ◽  
Ribosomal Subunits ◽  
Late Step

In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway.

scholarly journals Formation of Tap/NXT1 Heterodimers Activates Tap-Dependent Nuclear mRNA Export by Enhancing Recruitment to Nuclear Pore Complexes

2002 ◽  
Vol 22 (1) ◽  
pp. 245-256 ◽  
Author(s):  
Heather L. Wiegand ◽  
Glen A. Coburn ◽  
Yan Zeng ◽  
Yibin Kang ◽  
Hal P. Bogerd ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mrna Export ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Protein Functions ◽  
Cellular Mrnas ◽  
Pore Complexes

ABSTRACT The Tap protein has been shown to activate the nuclear export of mRNA species bearing retroviral constitutive transport elements and is also believed to play an essential role in the sequence nonspecific export of cellular mRNAs. However, it has remained unclear how Tap activity is regulated in vivo. Here, we report that the small NXT1/p15-1 protein functions as a critical cofactor for Tap-mediated mRNA export in both human and invertebrate cells. In the absence of NXT1 binding, the Tap protein is unable to effectively interact with components of the nuclear pore complex and both Tap nucleocytoplasmic shuttling and the nuclear export of mRNA molecules tethered to Tap are therefore severely attenuated. Formation of a Tap/NXT1 heterodimer enhances nucleoporin binding both in vitro and in vivo and induces the formation of a Tap/NXT1/nucleoporin ternary complex that is likely to be a key intermediate in the process of nuclear mRNA export. The critical importance of NXT1 for the nuclear export of poly(A)+ RNA is emphasized by the finding that specific inhibition of the expression of the Drosophila homolog of human NXT1, by using RNA interference, results in the nuclear accumulation of poly(A)+ RNA in cultured insect cells. These data suggest that NXT1 may act as a molecular switch that regulates the ability of Tap to mediate nuclear mRNA export by controlling the interaction of Tap with components of the nuclear pore.

005.Efficiency of nuclear import of the chromatin-remodelling factor SRY is critical for sex determination

2005 ◽  
Vol 17 (9) ◽  
pp. 64
Author(s):  
D. A. Jans ◽  
G. Kaur ◽  
I. K. H. Poon ◽  
A. Delluc-Clavieries ◽  
K. M. Wagstaff
Keyword(s):  
Sex Determination ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Sex Reversal ◽  
Nuclear Accumulation ◽  
Missense Mutations ◽  
Nuclear Localization Signals ◽  
Testicular Cells ◽  
Xy Sex Reversal

15% of cases of human XY sex reversal are due to mutations in SRY (sex determining region on the Y chromosome), many of which map to one of SRY’s two independently acting nuclear localization signals (NLSs) flanking its DNA binding domain. The C-terminal NLS (C-NLS) targets SRY to the nucleus through a ‘conventional’ pathway dependent on the nuclear import receptor importin-β (Imp-β). No importin has been shown to bind the N-terminal NLS (N-NLS), but it is known to interact with the Ca2+-binding protein calmodulin (CaM). We examined seven distinct missense mutations in the SRY NLSs from XY sex-reversed human females for effects on nuclear import and ability to interact with CaM/Imp-β1. All mutations were found to result in reduced nuclear localization in transfected testicular cells compared to wild type. The CaM antagonist, calmidazolium chloride (CDZ), was found to significantly reduce SRY nuclear accumulation, indicating a dependence of SRY nuclear import on CaM. Intriguingly, N-NLS mutants were resistant to CDZ’s effects, implying a loss of interaction with CaM; this was confirmed directly by in vitro binding experiments using recombinantly expressed protein. Either impaired CaM or Imp-β1 binding can thus be the basis of sex-reversal in human patients. Our results implicate a CaM-dependent nuclear import pathway for SRY mediated by the N-NLS that, together with the C-NLS, is required to achieve threshold levels of SRY in the nucleus for male sex determination.

scholarly journals A Role for RanBP1 in the Release of CRM1 from the Nuclear Pore Complex in a Terminal Step of Nuclear Export

1999 ◽  
Vol 145 (4) ◽  
pp. 645-657 ◽  
Author(s):  
Ralph H. Kehlenbach ◽  
Achim Dickmanns ◽  
Angelika Kehlenbach ◽  
Tinglu Guan ◽  
Larry Gerace
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Activated T Cells ◽  
Permeabilized Cells ◽  
Binding Domains ◽  
Biochemical Fractionation ◽  
Nuclear Export Receptor ◽  
Gtpase Ran

We recently developed an assay in which nuclear export of the shuttling transcription factor NFAT (nuclear factor of activated T cells) can be reconstituted in permeabilized cells with the GTPase Ran and the nuclear export receptor CRM1. We have now used this assay to identify another export factor. After preincubation of permeabilized cells with a Ran mutant that cannot hydrolyze GTP (RanQ69L), cytosol supports NFAT export, but CRM1 and Ran alone do not. The RanQ69L preincubation leads to accumulation of CRM1 at the cytoplasmic periphery of the nuclear pore complex (NPC) in association with the p62 complex and Can/Nup214. RanGTP-dependent association of CRM1 with these nucleoporins was reconstituted in vitro. By biochemical fractionation and reconstitution, we showed that RanBP1 restores nuclear export after the RanQ69L preincubation. It also stimulates nuclear export in cells that have not been preincubated with RanQ69L. RanBP1 as well as Ran-binding domains of the cytoplasmic nucleoporin RanBP2 promote the release of CRM1 from the NPC. Taken together, our results indicate that RanGTP is important for the targeting of export complexes to the cytoplasmic side of the NPC and that RanBP1 and probably RanBP2 are involved in the dissociation of nuclear export complexes from the NPC in a terminal step of transport.

Arabidopsis At2g40730 encodes a cytoplasmic protein involved in nuclear tRNA export

Botany ◽  
2011 ◽  
Vol 89 (3) ◽  
pp. 175-190 ◽  
Author(s):  
Aaron D. Johnstone ◽  
Robert T. Mullen ◽  
Dev Mangroo
Keyword(s):  
Nuclear Export ◽  
Cell Cycle Progression ◽  
Nuclear Pore ◽  
Cytoplasmic Protein ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Gtpase Ran ◽  
Cytoplasmic Side

Nuclear tRNA export plays an essential role in several key cellular processes, such as regulation of protein synthesis, cell cycle progression, response to nutrient availability and DNA damage, and development. While the overall mechanism of nuclear tRNA export is, in general, poorly understood, the details of specific steps are emerging from studies conducted in different organisms aimed at identifying and characterizing components involved in the process. Here, we report that Arabidopsis thaliana (L.) Heynh At2g40730 encodes CTEXP, a cytoplasmic protein component of the nuclear tRNA export process. CTEXP bound tRNA directly and saturably, and like the nuclear tRNA export receptor PAUSED, overexpression of CTEXP restored export of a nuclear export-defective lysine amber suppressor tRNA in tobacco cells. CTEXP was also found to associate with nucleoporins of the nuclear pore complex (NPC), PAUSED, and the GTPase Ran in vivo. CTEXP interacted directly with PAUSED in vitro and RanGTP, but not RanGDP. Furthermore, a portion of CTEXP appeared to associate with the NPC. Taken together, the data suggest that CTEXP assists with unloading of tRNAs from PAUSED at the cytoplasmic side of the NPC in plant cells.

scholarly journals The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor.

1995 ◽  
Vol 131 (6) ◽  
pp. 1699-1713 ◽  
Author(s):  
M K Iovine ◽  
J L Watkins ◽  
S R Wente
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Structural Basis ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Repetitive Region ◽  
Amino Terminal ◽  
Section Electron ◽  
Rna Export

Nup116p is a member of a family of five yeast nuclear pore complex (NPC) proteins that share an amino terminal region of repetitive tetrapeptide "GLFG" motifs. Previous experiments characterized the unique morphological perturbations that occur in a nup116 null mutant: temperature-sensitive formation of nuclear envelope seals over the cytoplasmic face of the NPC (Wente, S. R., and G. Blobel. 1993. J. Cell Biol. 123:275-284). Three approaches have been taken to dissect the structural basis for Nup116p's role in NPC function. First, deletion mutagenesis analysis of NUP116 revealed that the GLFG region was required for NPC function. This was not true for the other four yeast GLFG family members (Nup49p, Nup57p, Nup100p, and Nup145p). Moreover, deletion of either half of Nup116p's GLFG repeats or replacement of Nup116p's GLFG region with either Nup100p's GLFG region or Nsp1p's FXFG repetitive region abolishes the function of Nup116p. At a semipermissive growth temperature, the cells lacking Nup116p's GLFG region displayed a diminished capacity for nuclear import. Second, overexpression of Nup116p's GLFG region severely inhibited cell growth, rapidly blocked polyadenylated-RNA export, and fragmented the nucleolus. Although it inhibited nuclear export, the overexpressed GLFG region appeared predominantly localized in the cytoplasm and NPC/nuclear envelope structure was not perturbed in thin section electron micrographs. Finally, using biochemical and two-hybrid analysis, an interaction was characterized between Nup116p's GLFG region and Kap95p, an essential yeast homologue of the vertebrate nuclear import factor p97/Imp90/karopherin beta. These data show that Nup116p's GLFG region has an essential role in mediating nuclear transport.

scholarly journals RanBP2/Nup358 Provides a Major Binding Site for NXF1-p15 Dimers at the Nuclear Pore Complex and Functions in Nuclear mRNA Export

2004 ◽  
Vol 24 (3) ◽  
pp. 1155-1167 ◽  
Author(s):  
Daniel Forler ◽  
Gwénaël Rabut ◽  
Francesca D. Ciccarelli ◽  
Andrea Herold ◽  
Thomas Köcher ◽  
...  
Keyword(s):  
Binding Site ◽  
Nuclear Export ◽  
Mrna Export ◽  
Stable Complex ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Cytoplasmic Filaments ◽  
Nuclear Levels ◽  
Pore Complexes

ABSTRACT Metazoan NXF1-p15 heterodimers promote the nuclear export of bulk mRNA across nuclear pore complexes (NPCs). In vitro, NXF1-p15 forms a stable complex with the nucleoporin RanBP2/Nup358, a component of the cytoplasmic filaments of the NPC, suggesting a role for this nucleoporin in mRNA export. We show that depletion of RanBP2 from Drosophila cells inhibits proliferation and mRNA export. Concomitantly, the localization of NXF1 at the NPC is strongly reduced and a significant fraction of this normally nuclear protein is detected in the cytoplasm. Under the same conditions, the steady-state subcellular localization of other nuclear or cytoplasmic proteins and CRM1-mediated protein export are not detectably affected, indicating that the release of NXF1 into the cytoplasm and the inhibition of mRNA export are not due to a general defect in NPC function. The specific role of RanBP2 in the recruitment of NXF1 to the NPC is highlighted by the observation that depletion of CAN/Nup214 also inhibits cell proliferation and mRNA export but does not affect NXF1 localization. Our results indicate that RanBP2 provides a major binding site for NXF1 at the cytoplasmic filaments of the NPC, thereby restricting its diffusion in the cytoplasm after NPC translocation. In RanBP2-depleted cells, NXF1 diffuses freely through the cytoplasm. Consequently, the nuclear levels of the protein decrease and export of bulk mRNA is impaired.

scholarly journals Steady-State Nuclear Localization of Exportin-t Involves RanGTP Binding and Two Distinct Nuclear Pore Complex Interaction Domains

2002 ◽  
Vol 22 (16) ◽  
pp. 5708-5720 ◽  
Author(s):  
Scott Kuersten ◽  
Gert-Jan Arts ◽  
Tobias C. Walther ◽  
Ludwig Englmeier ◽  
Iain W. Mattaj
Keyword(s):  
Steady State ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Dependent Manner ◽  
C Terminus ◽  
Transport Cycle ◽  
Interaction Domains ◽  
Pore Complexes

ABSTRACT Vertebrate tRNA export receptor exportin-t (Xpo-t) binds to RanGTP and mature tRNAs cooperatively to form a nuclear export complex. Xpo-t shuttles bidirectionally through nuclear pore complexes (NPCs) but is mainly nuclear at steady state. The steady-state distribution of Xpo-t is shown to depend on its interaction with RanGTP. Two distinct Xpo-t NPC interaction domains that bind differentially to peripherally localized nucleoporins in vitro are identified. The N terminus binds to both Nup153 and RanBP2/Nup358 in a RanGTP-dependent manner, while the C terminus binds to CAN/Nup214 independently of Ran. We propose that these interactions increase the concentration of tRNA export complexes and of empty Xpo-t in the vicinity of NPCs and thus increase the efficiency of the Xpo-t transport cycle.

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