scholarly journals Cytosolic Hsp70 and co-chaperones constitute a novel system for tRNA import into the nucleus

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Akira Takano ◽  
Takuya Kajita ◽  
Makoto Mochizuki ◽  
Toshiya Endo ◽  
Tohru Yoshihisa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quality Control ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Substrate Recognition ◽  
Structural Features ◽  
Nuclear Accumulation ◽  
Nucleotide Binding Domain ◽  
Trna Import ◽  
Trna Binding

tRNAs are unique among various RNAs in that they shuttle between the nucleus and the cytoplasm, and their localization is regulated by nutrient conditions. Although nuclear export of tRNAs has been well documented, the import machinery is poorly understood. Here, we identified Ssa2p, a major cytoplasmic Hsp70 in Saccharomyces cerevisiae, as a tRNA-binding protein whose deletion compromises nuclear accumulation of tRNAs upon nutrient starvation. Ssa2p recognizes several structural features of tRNAs through its nucleotide-binding domain, but prefers loosely-folded tRNAs, suggesting that Ssa2p has a chaperone-like activity for RNAs. Ssa2p also binds Nup116, one of the yeast nucleoporins. Sis1p and Ydj1p, cytoplasmic co-chaperones for Ssa proteins, were also found to contribute to the tRNA import. These results unveil a novel function of the Ssa2p system as a tRNA carrier for nuclear import by a novel mode of substrate recognition. Such Ssa2p-mediated tRNA import likely contributes to quality control of cytosolic tRNAs.

scholarly journals Nucleocytoplasmic Shuttling of the Cdc42p Exchange Factor Cdc24p

2000 ◽  
Vol 148 (6) ◽  
pp. 1115-1122 ◽  
Author(s):  
Aljoscha Nern ◽  
Robert A. Arkowitz
Keyword(s):  
Cell Cycle ◽  
Actin Cytoskeleton ◽  
Nuclear Import ◽  
Vegetative Growth ◽  
Nuclear Export ◽  
Nuclear Accumulation ◽  
Nucleocytoplasmic Shuttling ◽  
Exchange Factor ◽  
Cycle Dependent ◽  
Necessary And Sufficient

Cdc24p, the GDP/GTP exchange factor for the regulator of actin cytoskeleton Cdc42p, localizes to sites of polarized growth. Here we show that Cdc24p shuttles in and out of the yeast nucleus during vegetative growth. Far1p is necessary and sufficient for nuclear accumulation of Cdc24p, suggesting that its nuclear import occurs via an association with Far1p. Nuclear export is triggered either by entry into the cell cycle or by mating pheromone. As Far1p is degraded upon entry into the cell cycle, cell cycle–dependent export of Cdc24p occurs in the absence of Far1p, whereas during mating similar export kinetics indicate that a Cdc24p–Far1p complex is exported. Our results suggest that the nucleus serves as a store of preformed Cdc24p–Far1p complex which is required for chemotropism.

scholarly journals Nutrient stress does not cause retrograde transport of cytoplasmic tRNA to the nucleus in evolutionarily diverse organisms

2011 ◽  
Vol 22 (7) ◽  
pp. 1091-1103 ◽  
Author(s):  
Shawn C. Chafe ◽  
Jacqueline B. Pierce ◽  
Manoja B. K. Eswara ◽  
Andrew T. McGuire ◽  
Dev Mangroo
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Intracellular Trafficking ◽  
Kluyveromyces Lactis ◽  
Retrograde Transport ◽  
Nutrient Stress ◽  
Hek293 Cells ◽  
H4iie Cells ◽  
Trna Import ◽  
Rat Hepatoma

Intracellular trafficking of tRNA was long thought to be a one-way trip from the site of biogenesis in the nucleus to the translation machinery in the cytoplasm. This view has recently been challenged, however, by the discovery that tRNA can move retrograde from the cytoplasm back to the nucleus in Saccharomyces cerevisiae and rat hepatoma H4IIE cells during nutrient stress and in S. cerevisiae after intron-containing pre-tRNAs are spliced in the cytoplasm. Contrary to studies reported, we present data suggesting that nutrient stress does not cause retrograde transport of cytoplasmic tRNAs to the nucleus in rat hepatoma H4IIE cells, human HeLa and HEK293 cells, and the yeasts Kluyveromyces lactis and S. cerevisiae. However, the efficiency of nuclear re-export of retrograded spliced tRNA was severely affected in S. cerevisiae and two other Saccharomyces species deprived of nutrient. Collectively, the data suggest that nutrient stress does not cause nuclear import of cytoplasmic tRNA; instead, nutrient stress specifically regulates nuclear re-export of retrograded spliced tRNAs but not nuclear export of tRNAs made from intronless pre-tRNAs in Saccharomyces species. Furthermore, we provide evidence suggesting that Mtr10p and the Gsp1pGTP/Gsp1pGDP cycle are not involved in nuclear tRNA import in S. cerevisiae during nutrient stress.

scholarly journals Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Richard Panayiotou ◽  
Francesc Miralles ◽  
Rafal Pawlowski ◽  
Jessica Diring ◽  
Helen R Flynn ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Accumulation ◽  
Subcellular Localisation ◽  
Nuclear Actin ◽  
Inhibitory Function ◽  
Actin Concentration

The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A.

scholarly journals Activation of cyclin B1–Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis

2010 ◽  
Vol 189 (2) ◽  
pp. 247-259 ◽  
Author(s):  
Olivier Gavet ◽  
Jonathon Pines
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Lamina ◽  
Fold Increase ◽  
Cyclin B1 ◽  
Cyclin B ◽  
Nuclear Accumulation ◽  
Animal Cells ◽  
Nuclear Envelope Breakdown ◽  
Prevailing View

The cyclin B–Cdk1 kinase triggers mitosis in most eukaryotes. In animal cells, cyclin B shuttles between the nucleus and cytoplasm in interphase before rapidly accumulating in the nucleus at prophase, which promotes disassembly of the nuclear lamina and nuclear envelope breakdown (NEBD). What triggers the nuclear accumulation of cyclin B1 is presently unclear, although the prevailing view is that the Plk1 kinase inhibits its nuclear export. In this study, we use a biosensor specific for cyclin B1–Cdk1 activity to show that activating cyclin B1–Cdk1 immediately triggers its rapid accumulation in the nucleus through a 40-fold increase in nuclear import that remains dependent on Cdk1 activity until NEBD. Nevertheless, a substantial proportion of cyclin B1–Cdk1 remains in the cytoplasm. The increase in nuclear import is driven by changes in the nuclear import machinery that require neither Plk1 nor inhibition of nuclear export. Thus, the intrinsic link between cyclin B1–Cdk1 activation and its rapid nuclear import inherently coordinates the reorganization of the nucleus and the cytoplasm at mitotic entry.

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 Mechanism Underlying the Iron-dependent Nuclear Export of the Iron-responsive Transcription Factor Aft1p in Saccharomyces cerevisiae

2007 ◽  
Vol 18 (8) ◽  
pp. 2980-2990 ◽  
Author(s):  
Ryo Ueta ◽  
Naoko Fujiwara ◽  
Kazuhiro Iwai ◽  
Yuko Yamaguchi-Iwai
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Intermolecular Interaction ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Iron Homeostasis ◽  
Target Genes ◽  
Critical Role ◽  
Dependent Manner ◽  
Constitutive Activation ◽  
Nuclear Export Receptor

Aft1p is an iron-responsive transcriptional activator that plays a central role in maintaining iron homeostasis in Saccharomyces cerevisiae. Aft1p is regulated primarily by iron-induced shuttling of the protein between the nucleus and cytoplasm, but its nuclear import is not regulated by iron. Here, we have shown that the nuclear export of Aft1p is promoted in the presence of iron and that Msn5p is the nuclear export receptor (exportin) for Aft1p. Msn5p recognizes Aft1p in the iron-replete condition. Phosphorylation of S210 and S224 in Aft1p, which is not iron dependent, and the iron-induced intermolecular interaction of Aft1p are both essential for its recognition by Msn5p. Mutation of Cys291 of Aft1p to Phe, which causes Aft1p to be retained in the nucleus and results in constitutive activation of Aft1-target genes, disrupts the intermolecular interaction of Aft1p. Collectively, these results suggest that iron induces a conformational change in Aft1p, in which Aft1p Cys291 plays a critical role, and that, in turn, Aft1p is recognized by Msn5p and exported into the cytoplasm in an iron-dependent manner.

scholarly journals Identification of Nuclear Import and Export Signals within Fli-1: Roles of the Nuclear Import Signals in Fli-1-Dependent Activation of Megakaryocyte-Specific Promoters

2005 ◽  
Vol 25 (8) ◽  
pp. 3087-3108 ◽  
Author(s):  
Wei Hu ◽  
Alana S. Philips ◽  
Juliana C. Kwok ◽  
Michael Eisbacher ◽  
Beng H. Chong
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Leptomycin B ◽  
Nuclear Localization Signals ◽  
Importin Alpha ◽  
Friend Leukemia ◽  
Important Regulator ◽  
Ets Domain

ABSTRACT The Ets factor Friend leukemia integration 1 (Fli-1) is an important regulator of megakaryocytic (Mk) differentiation. Here, we demonstrate two novel nuclear localization signals (NLSs) within Fli-1: one (NLS1) is located at the N terminus, and another (NLS2) is within the Ets domain. Nuclear accumulation of Fli-1 reflected the combined functional effects of the two discrete NLSs. Each NLS can independently direct nuclear transport of a carrier protein, with mutations within the NLSs affecting nuclear accumulation. NLS1 has a bipartite motif, whereas the NLS2 region contains a nonclassical NLS. Both NLSs bind importin alpha (IMPα) and IMPβ, with NLS1 and NLS2 being predominantly recognized by IMPα and IMPβ, respectively. Fli-1 also contains one nuclear export signal. Leptomycin B abolished its cytoplasmic accumulation, showing CRM1 dependency. We demonstrate that Ets domain binding to specific target DNA effectively blocks IMP binding, indicating that the targeted DNA binding plays a role in localizing Fli-1 to its destination and releasing IMPs for recycling back to the cytoplasm. Finally, by analyzing full-length Fli-1 carrying NLS1, NLS2, and combined NLS1-NLS2 mutations, we conclude that two functional NLSs exist in Fli-1 and that each NLS is sufficient to target Fli-1 to the nucleus for activation of Mk-specific genes.

Heat shock and ethanol stress provoke distinctly different responses in 3′-processing and nuclear export of HSP mRNA in Saccharomyces cerevisiae

2008 ◽  
Vol 414 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Shingo Izawa ◽  
Takeomi Kita ◽  
Kayo Ikeda ◽  
Yoshiharu Inoue
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Transcriptional Activation ◽  
Nuclear Export ◽  
Mrna Export ◽  
Yeast Cells ◽  
Nuclear Accumulation ◽  
Ethanol Stress ◽  
Nuclear Retention ◽  
Eukaryotic Gene

Under conditions of heat shock at 42 °C, mRNAs of HSP (heat shock protein) genes are exported out of the nucleus, whereas bulk poly(A)+ (polyadenylated) mRNA shows a nuclear accumulation in Saccharomyces cerevisiae. Such a selective mRNA export seems an efficacious strategy of yeast cells to adapt rapidly to stress. Although ethanol stress (10%, v/v) as well as heat shock blocks the export of bulk poly(A)+ mRNA, the differences and/or similarity between heat shock and ethanol stress in the mechanisms of selective mRNA export still remain to be clarified. We found that ethanol stress induced transcriptional activation of a subset of yeast HSP genes; however, intriguingly, most such transcripts remained in the nucleus in a hyperadenylated state and, as a consequence, were not translated into HSPs. Elimination of ethanol resulted in a rapid shortening of the poly(A) tails of HSP mRNAs, loss of their nuclear retention, and the coincidental synthesis of the respective HSPs. Since HSP mRNAs are selectively exported from the nucleus in heat-shocked cells, yeast cells respond differently to ethanol stress and heat shock in the 3′-processing and transport of HSP mRNAs. Furthermore, these results also suggest that hyperadenylation and nuclear retention of mRNAs might be used as a means to control eukaryotic gene expression under stressed conditions.

scholarly journals Cellular stresses induce the nuclear accumulation of importin α and cause a conventional nuclear import block

2004 ◽  
Vol 165 (5) ◽  
pp. 617-623 ◽  
Author(s):  
Yoichi Miyamoto ◽  
Takuya Saiwaki ◽  
Junichi Yamashita ◽  
Yoshinari Yasuda ◽  
Ippei Kotera ◽  
...  
Keyword(s):  
Heat Shock ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Stress Conditions ◽  
Nuclear Accumulation ◽  
Nuclear Retention ◽  
Heat Shock Cognate 70 ◽  
Importin Α ◽  
Response To Stress ◽  
Cellular Stresses

We report here that importin α accumulates reversibly in the nucleus in response to cellular stresses including UV irradiation, oxidative stress, and heat shock. The nuclear accumulation of importin α appears to be triggered by a collapse in the Ran gradient, resulting in the suppression of the nuclear export of importin α. In addition, nuclear retention and the importin β/Ran-independent import of importin α also facilitate its rapid nuclear accumulation. The findings herein show that the classical nuclear import pathway is down-regulated via the removal of importin α from the cytoplasm in response to stress. Moreover, whereas the nuclear accumulation of heat shock cognate 70 is more sensitive to heat shock than the other stresses, importin α is able to accumulate in the nucleus at all the stress conditions tested. These findings suggest that the stress-induced nuclear accumulation of importin α can be involved in a common physiological response to various stress conditions.

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