Identification of Novel Saccharomyces cerevisiaeProteins with Nuclear Export Activity: Cell Cycle-Regulated Transcription Factor Ace2p Shows Cell Cycle-Independent Nucleocytoplasmic Shuttling

ABSTRACT Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiaeproteins not previously known to have nuclear export activity. These proteins are the 5′ RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G1-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G1 but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner.

Download Full-text

Nucleocytoplasmic Trafficking of G2/M Regulators in Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e08-03-0286 ◽

2008 ◽

Vol 19 (9) ◽

pp. 4006-4018 ◽

Cited By ~ 25

Author(s):

Mignon A. Keaton ◽

Lee Szkotnicki ◽

Aron R. Marquitz ◽

Jake Harrison ◽

Trevin R. Zyla ◽

...

Keyword(s):

Cell Cycle ◽

Nuclear Import ◽

Nuclear Export ◽

Cell Cycle Regulators ◽

Cyclin Dependent Kinase ◽

Nucleocytoplasmic Trafficking ◽

Nucleocytoplasmic Shuttling ◽

Upstream Regulator ◽

Cellular Compartments ◽

Cdk Regulation

Nucleocytoplasmic shuttling is prevalent among many cell cycle regulators controlling the G2/M transition. Shuttling of cyclin/cyclin-dependent kinase (CDK) complexes is thought to provide access to substrates stably located in either compartment. Because cyclin/CDK shuttles between cellular compartments, an upstream regulator that is fixed in one compartment could in principle affect the entire cyclin/CDK pool. Alternatively, the regulators themselves may need to shuttle to effectively regulate their moving target. Here, we identify localization motifs in the budding yeast Swe1p (Wee1) and Mih1p (Cdc25) cell cycle regulators. Replacement of endogenous Swe1p or Mih1p with mutants impaired in nuclear import or export revealed that the nuclear pools of Swe1p and Mih1p were more effective in CDK regulation than were the cytoplasmic pools. Nevertheless, shuttling of cyclin/CDK complexes was sufficiently rapid to coordinate nuclear and cytoplasmic events even when Swe1p or Mih1p were restricted to one compartment. Additionally, we found that Swe1p nuclear export was important for its degradation. Because Swe1p degradation is regulated by cytoskeletal stress, shuttling of Swe1p between nucleus and cytoplasm serves to couple cytoplasmic stress to nuclear cyclin/CDK inhibition.

Download Full-text

Borna Disease Virus Nucleoprotein Requires both Nuclear Localization and Export Activities for Viral Nucleocytoplasmic Shuttling

Journal of Virology ◽

10.1128/jvi.75.7.3404-3412.2001 ◽

2001 ◽

Vol 75 (7) ◽

pp. 3404-3412 ◽

Cited By ~ 36

Author(s):

Takeshi Kobayashi ◽

Wataru Kamitani ◽

Guoqi Zhang ◽

Makiko Watanabe ◽

Keizo Tomonaga ◽

...

Keyword(s):

Disease Virus ◽

Nuclear Localization ◽

Nuclear Export ◽

Critical Role ◽

Nuclear Export Signal ◽

Nucleocytoplasmic Shuttling ◽

Nuclear Targeting ◽

Borna Disease Virus ◽

Export Activity ◽

Borna Disease

ABSTRACT Nuclear transport of viral nucleic acids is crucial to the life cycle of many viruses. Borna disease virus (BDV) belongs to the orderMononegavirales and replicates its RNA genome in the nucleus. Previous studies have suggested that BDV nucleoprotein (N) and phosphoprotein (P) have important functions in the nuclear import of the viral ribonucleoprotein (RNP) complexes via their nuclear targeting activity. Here, we showed that BDV N has cytoplasmic localization activity, which is mediated by a nuclear export signal (NES) within the sequence. Our analysis using deletion and substitution mutants of N revealed that NES of BDV N consists of a canonical leucine-rich motif and that the nuclear export activity of the protein is mediated through the chromosome region maintenance protein-dependent pathway. Interspecies heterokaryon assay indicated that BDV N shuttles between the nucleus and cytoplasm as a nucleocytoplasmic shuttling protein. Furthermore, interestingly, the NES region overlaps a binding site to the BDV P protein, and nuclear export of a 38-kDa form of BDV N is prevented by coexpression of P. These results suggested that BDV N has two contrary activities, nuclear localization and export activity, and plays a critical role in the nucleocytoplasmic transport of BDV RNP by interaction with other viral proteins.

Download Full-text

Nucleocytoplasmic Shuttling of the Cdc42p Exchange Factor Cdc24p

The Journal of Cell Biology ◽

10.1083/jcb.148.6.1115 ◽

2000 ◽

Vol 148 (6) ◽

pp. 1115-1122 ◽

Cited By ~ 78

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.

Download Full-text

Negative feedback regulation of calcineurin-dependent Prz1 transcription factor by the CaMKK-CaMK1 axis in fission yeast

Nucleic Acids Research ◽

10.1093/nar/gku684 ◽

2014 ◽

Vol 42 (15) ◽

pp. 9573-9587 ◽

Cited By ~ 7

Author(s):

Eugenia Cisneros-Barroso ◽

Tula Yance-Chávez ◽

Ayako Kito ◽

Reiko Sugiura ◽

Alba Gómez-Hierro ◽

...

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Nuclear Export ◽

Mammalian Cells ◽

Calcium Signalling ◽

Feedback Regulation ◽

Negative Feedback Regulation ◽

Cycle Arrest ◽

Cell Cycle Inhibition ◽

Active Transcription

Abstract Calcium signals trigger the translocation of the Prz1 transcription factor from the cytoplasm to the nucleus. The process is regulated by the calcium-activated phosphatase calcineurin, which activates Prz1 thereby maintaining active transcription during calcium signalling. When calcium signalling ceases, Prz1 is inactivated by phosphorylation and exported to the cytoplasm. In budding yeast and mammalian cells, different kinases have been reported to counter calcineurin activity and regulate nuclear export. Here, we show that the Ca2+/calmodulin-dependent kinase Cmk1 is first phosphorylated and activated by the newly identified kinase CaMKK2 homologue, Ckk2, in response to Ca2+. Then, active Cmk1 binds, phosphorylates and inactivates Prz1 transcription activity whilst at the same time cmk1 expression is enhanced by Prz1 in response to Ca2+. Furthermore, Cdc25 phosphatase is also phosphorylated by Cmk1, inducing cell cycle arrest in response to an increase in Ca2+. Moreover, cmk1 deletion shows a high tolerance to chronic exposure to Ca2+, due to the lack of cell cycle inhibition and elevated Prz1 activity. This work reveals that Cmk1 kinase activated by the newly identified Ckk2 counteracts calcineurin function by negatively regulating Prz1 activity which in turn is involved in activating cmk1 gene transcription. These results are the first insights into Cmk1 and Ckk2 function in Schizosaccharomyces pombe.

Download Full-text

14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport

The Journal of Cell Biology ◽

10.1083/jcb.200112059 ◽

2002 ◽

Vol 156 (5) ◽

pp. 817-828 ◽

Cited By ~ 352

Author(s):

Anne Brunet ◽

Fumihiko Kanai ◽

Justine Stehn ◽

Jian Xu ◽

Dilara Sarbassova ◽

...

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Ligand Binding ◽

Nuclear Export ◽

Nuclear Export Signal ◽

Nucleocytoplasmic Transport ◽

Rich Region ◽

Present Evidence ◽

Α Helix ◽

Export Signal

14-3-3 proteins regulate the cell cycle and prevent apoptosis by controlling the nuclear and cytoplasmic distribution of signaling molecules with which they interact. Although the majority of 14-3-3 molecules are present in the cytoplasm, we show here that in the absence of bound ligands 14-3-3 homes to the nucleus. We demonstrate that phosphorylation of one important 14-3-3 binding molecule, the transcription factor FKHRL1, at the 14-3-3 binding site occurs within the nucleus immediately before FKHRL1 relocalization to the cytoplasm. We show that the leucine-rich region within the COOH-terminal α-helix of 14-3-3, which had been proposed to function as a nuclear export signal (NES), instead functions globally in ligand binding and does not directly mediate nuclear transport. Efficient nuclear export of FKHRL1 requires both intrinsic NES sequences within FKHRL1 and phosphorylation/14-3-3 binding. Finally, we present evidence that phosphorylation/14-3-3 binding may also prevent FKHRL1 nuclear reimport. These results indicate that 14-3-3 can mediate the relocalization of nuclear ligands by several mechanisms that ensure complete sequestration of the bound 14-3-3 complex in the cytoplasm.

Download Full-text

Cell Cycle Activation of the Swi6p Transcription Factor Is Linked to Nucleocytoplasmic Shuttling

Molecular and Cellular Biology ◽

10.1128/mcb.23.9.3126-3140.2003 ◽

2003 ◽

Vol 23 (9) ◽

pp. 3126-3140 ◽

Cited By ~ 35

Author(s):

Ethel Queralt ◽

J. Carlos Igual

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Regulatory Subunit ◽

Transcriptional Level ◽

Cell Cycle Regulators ◽

Nucleocytoplasmic Shuttling ◽

Phenotypic Analysis ◽

Cell Cycle Activation ◽

M Phase ◽

Related Transcription Factor

ABSTRACT The control of the subcellular localization of cell cycle regulators has emerged as a crucial mechanism in the regulation of cell division. In the present work, we have characterized the function of the karyopherin Msn5p in the control of the cell cycle of Saccharomyces cerevisiae. Phenotypic analysis of the msn5 mutant revealed an increase in cell size and a functional interaction between Msn5p and the cell cycle transcription factor SBF (composed of the Swi4p and Swi6p proteins), indicating that Msn5p is involved in Start control. In fact, we have shown that the level of Cln2p protein is drastically reduced in an msn5 mutant. The effect on CLN2 expression is mediated at a transcriptional level, Msn5p being necessary for proper SBF-dependent transcription. On the contrary, loss of MSN5 has no effect on the closely related transcription factor MBF (composed of the Mbp1p and Swi6p proteins). Regulation of SBF by Msn5p is exerted by control of the localization of the regulatory subunit Swi6p. Swi6p shuttles between the nucleus and the cytoplasm during the cell cycle, and we have found that Msn5p is required for Swi6p export from the nucleus during the G2-M phase. What is more important, we have demonstrated that export of Swi6p to the cytoplasm is required for SBF activity, providing evidence for a functional switch of Swi6p linked to its nucleocytoplasmic shuttling during the cell cycle.

Download Full-text

Exportin Crm1 is important for Swi6 nucleocytoplasmic shuttling and MBF transcription activation in Saccharomyces cerevisiae

10.1101/2020.06.17.155598 ◽

2020 ◽

Author(s):

Kenneth D. Belanger ◽

William T. Yewdell ◽

Matthew F. Barber ◽

Amy N. Russo ◽

Mark A. Pettit ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Transcription Factor ◽

Nuclear Export ◽

Intracellular Localization ◽

Nuclear Export Signal ◽

Transcription Activation ◽

Cell Phenotype ◽

Export Signal

AbstractThe Swi6 protein acts as a transcription factor in budding yeast, functioning in two different heterodimeric complexes, SBF and MBF, that activate the expression of distinct but overlapping sets of genes. Swi6 undergoes regulated changes in nucleocytoplasmic localization throughout the cell cycle that correlate with changes in gene expression. While the process of Swi6 nuclear import is well understood, mechanisms underlying its nuclear export remain unclear. Here we investigate Swi6 nuclear export and its impact on Swi6 function. We show that the exportin Crm1, in addition to three other karyopherins previously shown to affect Swi6 localization, is important for Swi6 nuclear export and activity. A truncation of Swi6 that removes a putative Crm1 nuclear export signal results in the loss of changes in nucleocytoplasmic Swi6 localization that normally occur during progression through the cell cycle. Mutagenesis of the NES-like sequence or removal of Crm1 activity using leptomycin B results in a similar decrease in nuclear export as cells enter S-phase. Using two-hybrid analysis, we also show that Swi6 associates with Crm1 in vivo. Alteration of the Crm1 NES in Swi6 results in a decrease in MBF-mediated gene expression, but does not affect expression of an SBF reporter, suggesting that export of Swi6 by Crm1 regulates a subset of Swi6 transcription activation activity. Finally, alteration of the Crm1 NES in Swi6 results in cells that are larger than wild type, but not to the extent of those with a complete Swi6 deletion. Expressing a Swi6 NES mutant in combination with a deletion of Msn5, an exportin involved in Swi6 nuclear export and specifically affecting SBF activation, further increases the large cell phenotype, but still not to the extent observed in a Swi6 deletion mutant. These data suggest that Swi6 has at least two different exportins, Crm1 and Msn5, each of which interacts with a distinct nuclear export signal and influences expression of a different subset of Swi6-controlled genes.Summary StatementPrecise intracellular localization is important for the proper activity of proteins. Here we provide evidence that the Swi6 transcription factor important for cell cycle progression shuttles between the cell nucleus and cytoplasm, its nuclear export is important for its activity, and that it contains a nuclear export signal (NES) recognized by the Crm1 nuclear transport factor.

Download Full-text