scholarly journals Active Nucleocytoplasmic Shuttling Required for Function and Regulation of Stress-Activated Kinase Spc1/StyI in Fission Yeast

1999 ◽  
Vol 10 (5) ◽  
pp. 1395-1407 ◽  
Author(s):  
Frédérique Gaits ◽  
Paul Russell
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Osmotic Shock ◽  
Nuclear Accumulation ◽  
Nucleotide Exchange ◽  
Nucleocytoplasmic Shuttling ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Complex Forms ◽  
Transcriptional Induction

Transcriptional induction of many stress-response genes is dependent on stress-induced nuclear accumulation of stress-activated protein kinases (SAPKs). In the fission yeastSchizosaccharomyces pombe, nuclear accumulation of the SAPK Spc1 (also known as StyI) requires activating phosphorylation catalyzed by the SAPK kinase Wis1; however, it is unknown whether the localization of Spc1 is regulated by nuclear transport factors. Herein are reported studies that show that Spc1 localization is regulated by active transport mechanisms during osmotic stress. Nuclear import of Spc1 requires Pim1, a homologue of the guanine nucleotide exchange factor RCC1 that is essential for nucleocytoplasmic shuttling of proteins. Nuclear export of Spc1 is regulated by the export factor Crm1. An Spc1–Crm1 complex forms as Spc1 is exported from the nucleus. Wis1 and the tyrosine phosphatases Pyp1 and Pyp2 that inactivate Spc1 are excluded from the nucleus by a Crm1-independent mechanism; hence the nuclear import of Spc1 leads to transient isolation from its regulatory proteins. Thus, active nucleocytoplasmic shuttling is required for both the function and regulation of Spc1 during the osmotic shock response.

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 C3G shows regulated nucleocytoplasmic exchange and represses histone modifications associated with euchromatin

2017 ◽  
Vol 28 (7) ◽  
pp. 984-995 ◽  
Author(s):  
Dhruv Kumar Shakyawar ◽  
Kunal Dayma ◽  
Anesh Ramadhas ◽  
Chavvakula Varalakshmi ◽  
Vegesna Radha
Keyword(s):  
Histone Modifications ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Myogenic Differentiation ◽  
Cell Types ◽  
Sublethal Dose ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Importin Α

C3G (RapGEF1) is a ubiquitously expressed guanine nucleotide exchange factor that functions in signaling pathways regulating cell proliferation, apoptosis, and actin reorganization. It is essential for differentiation and early embryonic development in mice. Overexpressed C3G shows predominant cytoplasmic localization, but endogenous C3G is a component of nuclear fractions in a variety of cell types. Coexpression of importin-α and inhibition of nuclear export by leptomycin B resulted in predominant nuclear localization of C3G. Functional NLSs, NES, and GSK3-β–dependent phosphorylation regulate its dynamic nuclear localization. C3G translocates to the nucleus in response to myogenic differentiation and sublethal dose of cisplatin. C3G is associated with chromatin and nuclear matrix fractions. Cells with C3G localized in the nucleus showed peripheralization of heterochromatin and reduced histone modifications associated with euchromatin. Short hairpin RNA–mediated depletion of C3G in epithelial cells resulted in reduced expression of CDK inhibitors and the histone demethylase KDM5A. Myoblast clones with CRISPR/Cas9-mediated knockout of C3G failed to show repression of histone marks and did not show up-regulation of myosin heavy chain and myotube formation when grown in differentiation medium. Our results document regulated nucleocytoplasmic exchange of C3G in response to physiological stimuli and provide insights into nuclear functions for C3G.

scholarly journals Nuclear Import of the Ran Exchange Factor, Rcc1, Is Mediated by at Least Two Distinct Mechanisms

2000 ◽  
Vol 149 (4) ◽  
pp. 835-850 ◽  
Author(s):  
Michael E. Nemergut ◽  
Ian G. Macara
Keyword(s):  
Nuclear Import ◽  
Nuclear Protein ◽  
Nucleotide Exchange ◽  
Ran Gtpase ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Interphase Cells ◽  
Importin Α

RCC1, the only known guanine-nucleotide exchange factor for the Ran GTPase, is an ∼45-kD nuclear protein that can bind chromatin. An important question concerns how RCC1 traverses the nuclear envelope. We now show that nuclear RCC1 is not exported readily in interphase cells and that the import of RCC1 into the nucleoplasm is extremely rapid. Import can proceed by at least two distinct mechanisms. The first is a classic import pathway mediated by basic residues within the NH2-terminal domain (NTD) of RCC1. This pathway is dependent upon both a preexisting Ran gradient and energy, and preferentially uses the importin-α3 isoform of importin-α. The second pathway is not mediated by the NTD of RCC1. This novel pathway does not require importin-α or importin-β or the addition of any other soluble factor in vitro; however, this pathway is saturable and sensitive only to a subset of inhibitors of classical import pathways. Furthermore, the nuclear import of RCC1 does not require a preexisting Ran gradient or energy. We speculate that this second import pathway evolved to ensure that RCC1 never accumulates in the cytoplasm.

scholarly journals The nucleoporin Nup60p functions as a Gsp1p–GTP-sensitive tether for Nup2p at the nuclear pore complex

2001 ◽  
Vol 154 (5) ◽  
pp. 937-950 ◽  
Author(s):  
Daniel Denning ◽  
Brook Mykytka ◽  
Nadia P.C. Allen ◽  
Lan Huang ◽  
Al Burlingame ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Small Proteins ◽  
Nucleotide Exchange Factor ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
The Stability

The nucleoporins Nup60p, Nup2p, and Nup1p form part of the nuclear basket structure of the Saccharomyces cerevisiae nuclear pore complex (NPC). Here, we show that these necleoporins can be isolated from yeast extracts by affinity chromatography on karyopherin Kap95p-coated beads. To characterize Nup60p further, Nup60p-coated beads were used to capture its interacting proteins from extracts. We find that Nup60p binds to Nup2p and serves as a docking site for Kap95p–Kap60p heterodimers and Kap123p. Nup60p also binds Gsp1p–GTP and its guanine nucleotide exchange factor Prp20p, and functions as a Gsp1p guanine nucleotide dissociation inhibitor by reducing the activity of Prp20p. Yeast lacking Nup60p exhibit minor defects in nuclear export of Kap60p, nuclear import of Kap95p–Kap60p-dependent cargoes, and diffusion of small proteins across the NPC. Yeast lacking Nup60p also fail to anchor Nup2p at the NPC, resulting in the mislocalization of Nup2p to the nucleoplasm and cytoplasm. Purified Nup60p and Nup2p bind each other directly, but the stability of the complex is compromised when Kap60p binds Nup2p. Gsp1p–GTP enhances by 10-fold the affinity between Nup60p and Nup2p, and restores binding of Nup2p–Kap60p complexes to Nup60p. The results suggest a dynamic interaction, controlled by the nucleoplasmic concentration of Gsp1p–GTP, between Nup60p and Nup2p at the NPC.

scholarly journals In Vivo Analysis of Importin α Proteins Reveals Cellular Proliferation Inhibition and Substrate Specificity

2004 ◽  
Vol 24 (23) ◽  
pp. 10246-10255 ◽  
Author(s):  
Christina Quensel ◽  
Beate Friedrich ◽  
Thomas Sommer ◽  
Enno Hartmann ◽  
Matthias Kohler
Keyword(s):  
Nuclear Import ◽  
Cellular Proliferation ◽  
Minor Effect ◽  
Nucleotide Exchange ◽  
Down Regulation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Importin Α ◽  
A Minor

ABSTRACT The “classical” nuclear import pathway depends on importins α and β. Humans have only one importin β, while six α importins have been described. Whether or not distinct α 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 α importins in HeLa cells. Down-regulation of importins α3, α5, α7, and β strongly inhibited HeLa cell proliferation, while down-regulation of importins α1 and α4 had only a minor effect or no effect. Nucleoplasmin import was not prevented by down-regulation of any α importin, indicating that the importin α/β pathway was generally not affected. In contrast, importin α3 or α5 down-regulation specifically inhibited the nuclear import of the Ran guanine nucleotide exchange factor, RCC1. Coinjection of recombinant α importins and RCC1 into down-regulated cells demonstrated that these transport defects were specifically caused by the limited availability of importin α3 in both cases. Thus, importin α3 is the only α importin responsible for the classical nuclear import of RCC1 in living cells.

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